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1

Running headline Growth improvement of Lactobacillus plantarum by catechin 1

2

3

Improvement of the fermentation performance of Lactobacillus 4

plantarum by the flavanol catechin is uncoupled from its degradation 5

6

Feacutelix Loacutepez de Felipe 1 Joseacute Antonio Curiel 2 amp Rosario Muntildeoz 2 7

8

1 Grupo en Biotecnologiacutea de Bacterias Laacutecticas de Productos Fermentados Instituto del 9

Friacuteo CSIC Jose Antonio de Novaiacutes 10 28040 Madrid Spain 10

2 Departamento de Microbiologiacutea Instituto de Fermentaciones Industriales CSIC Juan 11

de la Cierva 3 28006 Madrid Spain 12

13

Corresponding author Tel +34-91-5492300 Fax +34-91-5493627 14

E-mail address fxlopezifcsices (F Loacutepez de Felipe) 15

Keywords Lactobacillus plantarum flavanol catechin chemically defined medium 16

sugar metabolism malic acid decarboxylation 17

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ABSTRACT 18

19

Aims To determine the influence of the flavanol catechin on key metabolic traits for 20

the fermentation performance of Lactobacillus plantarum strain RM71 in different 21

media and to evaluate the ability of this strain to catabolize catechin 22

23

Methods and Results Growth monitoring and time course of sugar consumption data 24

tracking in chemically defined medium (CDM) revealed that growth of Lact plantarum 25

strain RM71 upon catechin was characterized by a noticeable shorter lag period 26

outcome of earlier sugar consumption and lactic acid production courses Catechin gave 27

rise to higher cell densities compared to controls due to an increased extension of sugar 28

utilization Fermentation of media relevant for practical fermentation processes with 29

Lact plantarum strain RM71 showed that catechin sped up malic acid decarboxylation 30

which besides quicker and extended consumption of several sugars resulted in faster 31

and higher lactic acid production and growth Spectrophotometric evaluation of catechin 32

by HPLC-DAD and the lack of catechin concentration-dependent effects showed that 33

the observed stimulations were uncoupled from catechin catabolism by Lact plantarum 34

35

Conclusions The flavanol catechin stimulated the growth of Lact plantarum strain 36

RM71 by promoting quicker sugar consumption increasing the extension of sugar 37

utilization and stimulating malic acid decarboxylation These stimulations are 38

uncoupled from catechin catabolism since Lact plantarum did not catabolize it during 39

fermentation 40

Significance and Impact of the Study This study for the first time examined the 41

influence of the flavanol catechin on the fermentation performance of a Lact plantarum 42

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strain in several media under different fermentation conditions The information could 43

be relevant to control the production and obtain high-quality food products fermented 44

by this microorganism 45

46

INTRODUCTION 47

48

The flavanols are a subclass of flavonoids widespread in plant foods Free or as 49

basic unit of proanthocyanidins catechin is one of the most common flavanols in the 50

diet and therefore part of our polyphenol intake Catechin is an effective scavenger of 51

reactive oxygen species and plays a role to protect against degenerative diseases 52

(Crespy and Williamson 2004) Before increasing the serum antioxidant capacity 53

catechin either on food or in the GI tract is in contact with autochthonous microbiota 54

The knowledge on this interaction is important since this flavanol could influence the 55

fermentation performance and adaptation of these microorganisms to their respective 56

environmental niches On the other hand the microbial fermentation of catechin could 57

reduce the dietary intake of this flavanol 58

As regards to the interaction between food microorganisms and catechin studies 59

so far have mainly focussed on the lactic acid bacteria Oenococcus oeni and 60

Lactobacillus hilgardii from wine a beverage that is recognized as an important source 61

of catechin for human nutrition (Amarowicz et al 2009) The effect of catechin on the 62

growth of these wine lactic acid bacteria seems to be ambiguous Thus it was reported 63

that catechin stimulated growth of Lact hilgardii 5w (Alberto et al 2001) specially in 64

media containing tomato juice which is a source of polyphenols (Hussein et al 2009) 65

and suggested that catechin consumption caused this effect In apparent contrast and by 66

using a similar medium it was recently described that catechin did not significantly 67

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affect the growth of a different strain Lact hilgardii 5 suggesting that the ability to 68

metabolize flavanols could be strain dependent (Figueiredo et al 2008) In the same 69

work it was reported that similarly to Lact hilgardii catechin did not significantly 70

affect the growth of Oenococcus oeni strain VF however by using a different medium 71

Reguant et al (2000) described that though not metabolized catechin promoted growth 72

as well as malolactic fermentation in Oenococcus oeni strain CECT 4100 73

Among lactic acid bacteria Lactobacillus plantarum was chosen for the present 74

study because it shows ability to adapt to several catechin-containing environmental 75

niches In particular Lact plantarum is involved in the fermentation of plant foods that 76

according to Amarowicz et al (2009) are the most important sources of catechins for 77

human nutrition tea leaves (Okada et al 1996a 1996b) which is the most important 78

source in many countries grapes and wine (Rodas et al 2005) as well as cocoa and 79

chocolate (Kostinek et al 2008) Furthermore Lact plantarum besides its successful 80

adaptation to these plant niches persist and survive in the human gastrointestinal tract 81

(Adlerberth et al 1996) where it also meets with diet flavanols Regarding Lact 82

plantarum as a representative starter of catechin-containing food plants the main goal 83

of this work was to study for first time the interaction between this bacterium and the 84

flavanol catechin On one hand the ability of Lact plantarum to metabolize catechin 85

during growth was evaluated as it could provide primary insight on potential flavanol 86

loss in either the plant material or in the GI tract For this goal a chemically defined 87

medium (CDM) with catechin as the sole flavanol in the formulation was used On the 88

other hand it was studied how catechin affected growth fermentation of several sugars 89

utilization of malic acid and lactic acid production which are common and essential 90

traits for the successful fermentation performance of Lact plantarum in several 91

ecological niches This knowledge could be important to obtain high-quality food 92

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products fermented by this microorganism and to be valuable to understand the survival 93

and persistence of this microorganism in the GI tract For this purpose the influence of 94

catechin on the fermentation performance of Lact plantarum was evaluated in CDM 95

and in media relevant for practical fermentation processes 96

97

98

MATERIAL AND METHODS 99

100

Bacterial strain and catechin stock solutions 101

102

Lactobacillus plantarum RM71 was originally isolated from red wine at the 103

Instituto de Fermentaciones Industriales (CSIC) (Moreno-Arribas et al 2003) and used 104

through this study 105

(+)-Catechin (from now catechin) was obtained from Fluka (Buchs 106

Switzerland) purity was ge 98 A 5 catechin stock solution was prepared in ethanol 107

Appropriate dilutions were prepared to adjust catechin at final concentrations of 100 or 108

200 mg l-1 (final concentration) or omitted as indicated 109

110

Preparation of chemically defined medium Setting up inoculation and incubation of 111

microtiter plates 112

113

The chemically defined medium (CDM) previously reported to support the 114

growth of Lact plantarum WCFS1 strain was prepared as described (Teusink et al 115

2005) and used for bacterial growth Briefly CDM contains K2HPO4 (1 g l-1) KH2PO4 116

(5 g l-1) sodium acetate (1 g l-1) ammonium citrate (06 g l-1) ascorbic ac id (05 g l-1) 117

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strain in several media under different fermentation conditions The information could 43

be relevant to control the production and obtain high-quality food products fermented 44

by this microorganism 45

46

INTRODUCTION 47

48

The flavanols are a subclass of flavonoids widespread in plant foods Free or as 49

basic unit of proanthocyanidins catechin is one of the most common flavanols in the 50

diet and therefore part of our polyphenol intake Catechin is an effective scavenger of 51

reactive oxygen species and plays a role to protect against degenerative diseases 52

(Crespy and Williamson 2004) Before increasing the serum antioxidant capacity 53

catechin either on food or in the GI tract is in contact with autochthonous microbiota 54

The knowledge on this interaction is important since this flavanol could influence the 55

fermentation performance and adaptation of these microorganisms to their respective 56

environmental niches On the other hand the microbial fermentation of catechin could 57

reduce the dietary intake of this flavanol 58

As regards to the interaction between food microorganisms and catechin studies 59

so far have mainly focussed on the lactic acid bacteria Oenococcus oeni and 60

Lactobacillus hilgardii from wine a beverage that is recognized as an important source 61

of catechin for human nutrition (Amarowicz et al 2009) The effect of catechin on the 62

growth of these wine lactic acid bacteria seems to be ambiguous Thus it was reported 63

that catechin stimulated growth of Lact hilgardii 5w (Alberto et al 2001) specially in 64

media containing tomato juice which is a source of polyphenols (Hussein et al 2009) 65

and suggested that catechin consumption caused this effect In apparent contrast and by 66

using a similar medium it was recently described that catechin did not significantly 67

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affect the growth of a different strain Lact hilgardii 5 suggesting that the ability to 68

metabolize flavanols could be strain dependent (Figueiredo et al 2008) In the same 69

work it was reported that similarly to Lact hilgardii catechin did not significantly 70

affect the growth of Oenococcus oeni strain VF however by using a different medium 71

Reguant et al (2000) described that though not metabolized catechin promoted growth 72

as well as malolactic fermentation in Oenococcus oeni strain CECT 4100 73

Among lactic acid bacteria Lactobacillus plantarum was chosen for the present 74

study because it shows ability to adapt to several catechin-containing environmental 75

niches In particular Lact plantarum is involved in the fermentation of plant foods that 76

according to Amarowicz et al (2009) are the most important sources of catechins for 77

human nutrition tea leaves (Okada et al 1996a 1996b) which is the most important 78

source in many countries grapes and wine (Rodas et al 2005) as well as cocoa and 79

chocolate (Kostinek et al 2008) Furthermore Lact plantarum besides its successful 80

adaptation to these plant niches persist and survive in the human gastrointestinal tract 81

(Adlerberth et al 1996) where it also meets with diet flavanols Regarding Lact 82

plantarum as a representative starter of catechin-containing food plants the main goal 83

of this work was to study for first time the interaction between this bacterium and the 84

flavanol catechin On one hand the ability of Lact plantarum to metabolize catechin 85

during growth was evaluated as it could provide primary insight on potential flavanol 86

loss in either the plant material or in the GI tract For this goal a chemically defined 87

medium (CDM) with catechin as the sole flavanol in the formulation was used On the 88

other hand it was studied how catechin affected growth fermentation of several sugars 89

utilization of malic acid and lactic acid production which are common and essential 90

traits for the successful fermentation performance of Lact plantarum in several 91

ecological niches This knowledge could be important to obtain high-quality food 92

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products fermented by this microorganism and to be valuable to understand the survival 93

and persistence of this microorganism in the GI tract For this purpose the influence of 94

catechin on the fermentation performance of Lact plantarum was evaluated in CDM 95

and in media relevant for practical fermentation processes 96

97

98

MATERIAL AND METHODS 99

100

Bacterial strain and catechin stock solutions 101

102

Lactobacillus plantarum RM71 was originally isolated from red wine at the 103

Instituto de Fermentaciones Industriales (CSIC) (Moreno-Arribas et al 2003) and used 104

through this study 105

(+)-Catechin (from now catechin) was obtained from Fluka (Buchs 106

Switzerland) purity was ge 98 A 5 catechin stock solution was prepared in ethanol 107

Appropriate dilutions were prepared to adjust catechin at final concentrations of 100 or 108

200 mg l-1 (final concentration) or omitted as indicated 109

110

Preparation of chemically defined medium Setting up inoculation and incubation of 111

microtiter plates 112

113

The chemically defined medium (CDM) previously reported to support the 114

growth of Lact plantarum WCFS1 strain was prepared as described (Teusink et al 115

2005) and used for bacterial growth Briefly CDM contains K2HPO4 (1 g l-1) KH2PO4 116

(5 g l-1) sodium acetate (1 g l-1) ammonium citrate (06 g l-1) ascorbic ac id (05 g l-1) 117

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affect the growth of a different strain Lact hilgardii 5 suggesting that the ability to 68

metabolize flavanols could be strain dependent (Figueiredo et al 2008) In the same 69

work it was reported that similarly to Lact hilgardii catechin did not significantly 70

affect the growth of Oenococcus oeni strain VF however by using a different medium 71

Reguant et al (2000) described that though not metabolized catechin promoted growth 72

as well as malolactic fermentation in Oenococcus oeni strain CECT 4100 73

Among lactic acid bacteria Lactobacillus plantarum was chosen for the present 74

study because it shows ability to adapt to several catechin-containing environmental 75

niches In particular Lact plantarum is involved in the fermentation of plant foods that 76

according to Amarowicz et al (2009) are the most important sources of catechins for 77

human nutrition tea leaves (Okada et al 1996a 1996b) which is the most important 78

source in many countries grapes and wine (Rodas et al 2005) as well as cocoa and 79

chocolate (Kostinek et al 2008) Furthermore Lact plantarum besides its successful 80

adaptation to these plant niches persist and survive in the human gastrointestinal tract 81

(Adlerberth et al 1996) where it also meets with diet flavanols Regarding Lact 82

plantarum as a representative starter of catechin-containing food plants the main goal 83

of this work was to study for first time the interaction between this bacterium and the 84

flavanol catechin On one hand the ability of Lact plantarum to metabolize catechin 85

during growth was evaluated as it could provide primary insight on potential flavanol 86

loss in either the plant material or in the GI tract For this goal a chemically defined 87

medium (CDM) with catechin as the sole flavanol in the formulation was used On the 88

other hand it was studied how catechin affected growth fermentation of several sugars 89

utilization of malic acid and lactic acid production which are common and essential 90

traits for the successful fermentation performance of Lact plantarum in several 91

ecological niches This knowledge could be important to obtain high-quality food 92

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products fermented by this microorganism and to be valuable to understand the survival 93

and persistence of this microorganism in the GI tract For this purpose the influence of 94

catechin on the fermentation performance of Lact plantarum was evaluated in CDM 95

and in media relevant for practical fermentation processes 96

97

98

MATERIAL AND METHODS 99

100

Bacterial strain and catechin stock solutions 101

102

Lactobacillus plantarum RM71 was originally isolated from red wine at the 103

Instituto de Fermentaciones Industriales (CSIC) (Moreno-Arribas et al 2003) and used 104

through this study 105

(+)-Catechin (from now catechin) was obtained from Fluka (Buchs 106

Switzerland) purity was ge 98 A 5 catechin stock solution was prepared in ethanol 107

Appropriate dilutions were prepared to adjust catechin at final concentrations of 100 or 108

200 mg l-1 (final concentration) or omitted as indicated 109

110

Preparation of chemically defined medium Setting up inoculation and incubation of 111

microtiter plates 112

113

The chemically defined medium (CDM) previously reported to support the 114

growth of Lact plantarum WCFS1 strain was prepared as described (Teusink et al 115

2005) and used for bacterial growth Briefly CDM contains K2HPO4 (1 g l-1) KH2PO4 116

(5 g l-1) sodium acetate (1 g l-1) ammonium citrate (06 g l-1) ascorbic ac id (05 g l-1) 117

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products fermented by this microorganism and to be valuable to understand the survival 93

and persistence of this microorganism in the GI tract For this purpose the influence of 94

catechin on the fermentation performance of Lact plantarum was evaluated in CDM 95

and in media relevant for practical fermentation processes 96

97

98

MATERIAL AND METHODS 99

100

Bacterial strain and catechin stock solutions 101

102

Lactobacillus plantarum RM71 was originally isolated from red wine at the 103

Instituto de Fermentaciones Industriales (CSIC) (Moreno-Arribas et al 2003) and used 104

through this study 105

(+)-Catechin (from now catechin) was obtained from Fluka (Buchs 106

Switzerland) purity was ge 98 A 5 catechin stock solution was prepared in ethanol 107

Appropriate dilutions were prepared to adjust catechin at final concentrations of 100 or 108

200 mg l-1 (final concentration) or omitted as indicated 109

110

Preparation of chemically defined medium Setting up inoculation and incubation of 111

microtiter plates 112

113

The chemically defined medium (CDM) previously reported to support the 114

growth of Lact plantarum WCFS1 strain was prepared as described (Teusink et al 115

2005) and used for bacterial growth Briefly CDM contains K2HPO4 (1 g l-1) KH2PO4 116

(5 g l-1) sodium acetate (1 g l-1) ammonium citrate (06 g l-1) ascorbic ac id (05 g l-1) 117

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and different quantities of several vitamins nucleotides and 18 amino acids Vitamins 118

nucleotides and amino acids were prepared separately as stock solutions and frozen at -119

40 ordmC before thawed prior to use Sodium acetate (1 g l-1) and ammonium citrate (06 g 120

l-1) which are present in the original CDM formulation were added or omitted as 121

indicated The different CDM formulations ie lacking or including citrate andor 122

acetate were adjusted to pH 55 or 65 with 5M NaOH as required and then filter 123

sterilized (022 microm pore size) The media was supplemented with 05 galactose 124

instead of glucose as growth sugar and catechin added at 100 or 200 mg l-1 (final 125

concentration) 126

Lactobacillus plantarum RM71 colonies from MRS plates (de Man et al 1960) 127

were inoculated into fresh complete CDM medium (3 ml) containing 1 glucose After 128

24 h incubation at 30ordm C 1 ml of fully grown cells was centrifuged and washed three 129

times with saline solution (085 NaCl) Stock CDM solutions containing catechin 130

ethanol sodium acetate andor ammonium citrate or none of these acids at the desired 131

concentrations were aseptically prepared Galactose and the bacteria were added to the 132

stock solutions just prior to the incubation step The inoculum size was 1 (vv) 133

Then volumes (100 l each) of the inoculated stock CDM solutions were dispensed on 134

sterile 96-well microtiter plates with lid (Falcon Cajal Madrid Spain) and incubated at 135

30 ordmC Two types of controls were provided control wells containing uninoculated 136

media (to survey contamination and potential OD600 changes due to catechin 137

degradation or precipitation) and inoculated control wells lacking catechin 138

139

Growth experiments and data handling 140

141

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In the experiments carried out in CDM growth curves were recorded at 30degC by 142

measuring the optical density (OD600) of the cultures every hour in a kinetic Bio-Tek 143

KC Junior microplate reader (BioTek Instruments Winooski USA) The data obtained 144

with the reader were exported to and processed with the Excel-MS software Cultures 145

used to follow metabolism of Lact plantarum RM71 were performed in CDM by 146

scaling volumes up to 15 ml The optical densities measured with a microtitre plate 147

reader and with a spectrophotometer were different due to the different optical 148

geometries The lag phase was defined as the time needed to reach an optical density 149

10x that of the initial OD600 The maximum specific growth rate max was determined 150

by calculating the slope of the exponential growth phase (max = ∆ln(OD600)∆t) The 151

results were expressed as the means for lag phases or max calculated from at least two 152

independent experiments153

154

Fermentation in wine-resembling and basal fermentation media 155

156

Fermentations of 15 ml were run at 25ordmC in a wine resembling medium 157

(Ugliano Genovese amp Moio L (2003) with the following composition (g l-1) glucose 158

(2) fructose (2) tartaric acid (5) acetic acid (06) L-malic acid (175) yeast extract 159

(20) NaCl (02) (NH4)2 SO4 (1) MnSO4 (005) MgSO47H2O (02) K2PO4 (1) and 160

ethanol (8 vv) A variant of this medium named basal fermentation medium (BFM) 161

for convenience was also used which lacked of tartaric acid acetic acid and ethanol 162

Galactose (1 ) instead of glucose and fructose was added as sugar substrate in this 163

medium Catechin (100 mg l-1) was added or omitted as indicated The initial pH was 164

adjusted at pH 38 in either medium and then filter sterilized Cells grown in MRS 165

medium were washed three times with saline solution (085 NaCl) and then inoculated 166

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in wine resembling or BFM media The bacterial growth was followed daily by plating 167

100 microl of properly diluted medium on MRS medium (Difco laboratories) adjusted to pH 168

50 before agar addition and sterilization Plates were incubated aerobically at 25 ordmC for 169

three days in a Memmert incubator 170

171

Analysis of catechin degradation by high-performance liquid chromatography-diode 172

array detector (HPLC-DAD) 173

174

Potential catechin degradation by Lactobacillus plantarum RM71 was studied in 175

5 ml of CDM wine resembling or BFM media cultures Supernatants for HPLC-DAD 176

analysis were obtained by filtration (pore size 022 microm Millipore) of three mililiters of 177

inoculated medium or from its cell-free control both after 40 h incubation at 30ordm C 178

Catechin was extracted twice from the supernatants with one third of the reaction 179

volume of ethyl acetate (Lab-Scan Ireland) HPLC-DAD analysis was performed as 180

described previously (Rodriacuteguez et al 2008) Catechin degradation was followed by 181

comparing retention times and spectral data of the peaks obtained in the 182

chromatrograms Commercial catechin was used as standard 183

184

Fermentation analysis by high-performance liquid chromatography (HPLC) 185

186

Supernatants from fermentation experiments were analyzed by HPLC in order to 187

determine the amounts of the sugars galactose glucose and fructose as well as L-lactic 188

and L-malic acids A Thermo (Thermo Electron Corp Waltham MA) chromatograph 189

equipped with a P-4000 Spectra System Pump an AS3000 autosampler a RI-150 190

refraction index and a UV 6000 LP photodiode array detectors were used A 15 mmol l-191

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1 H2SO4 aqueous solution as mobile phase was applied to an Aminexreg HPX-87H 192

column (300 mm x 78 mm) (BIO-RAD) at a 06 mlmin flow rate at 35 ordmC Samples 193

were filtered through a 045-microm polyvinylidene difluoride filter diluted two-fold and 194

injected in duplicated Galactose glucose or fructose detection was performed at 210 195

nm (refraction index detector) and L-lactic and L-malic acids at 380 nm (diode array 196

detector) Sugar and organic acid standards were used for the identification of the HPLC 197

peaks on the chromatograms and for the estimation of their concentration by a 198

calibration curve 199

200

RESULTS 201

202

Growth behaviour of Lactobacillus plantarum RM71 in control CDM medium 203

204

To study the catechin effects on the fermentation performance of Lact 205

plantarum RM71 growth and the time course of sugar consumption were monitored in 206

a chemical defined medium designed for Lact plantarum With the use of CDM it was 207

ensured that catechin was the only flavonoid in the medium and thus the effects on 208

growth could be more easily ascribed to this flavanol Furthermore use of CDM would 209

facilitate the evaluation of potential catechin degradation products As a model growth 210

sugar for CDM experiments we used galactose which is abundant in vegetable fibre and 211

the diet On this sugar cells grow more slowly than ie glucose and the changes 212

produced by catechin could be more properly monitored 213

Before studying catechin effects we firstly examined the growth behaviour of 214

Lact plantarum RM71 in CDM in absence of catechin The single effects of citrate and 215

acetate on growth were studied by using four different CDM variants that were prepared 216

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by addition or omission of one or both of these acids in the formulation The 217

dependence of growth patterns on pH was relevant to subsequently interpret if catechin 218

effects were also pH-dependent Therefore we evaluated this effect by adjusting the 219

initial medium pH at 55 or 65 in absence of catechin 220

The growth patterns of Lact plantarum RM71 in the four CDM variants at pH 221

55 or 65 are shown in Fig 1 Lact plantarum RM71 grew well in absence of both 222

citrate and acetate (panels A and E) The presence of these acids in the medium 223

prolonged the lag phase (panels B and F) and elicited a biphasic growth pattern that did 224

only occur in the pH 55 CDM (panels B and C) It appeared that citrate was the only 225

acid leading to this growth profile because the lag phase prolongation (panels B C F 226

and G) as well as was biphasic growth (panels B and C) were observed only in citrate-227

supplemented CDM regardless of acetate inclusion in the medium 228

229

Catechin modifies growth and metabolism of Lactobacillus plantarum RM71 to improve 230

its fermentation performance in CDM 231

232

Results show that the presence of catechin provided advantages to Lact 233

plantarum RM71 for growing in CDM The two positive effects induced by this 234

flavanol were shorthening of the lag phase and higher final cell densities both occurring 235

regardless of the initial pH set-up (Fig1) 236

Examination of Fig 1 revealed that Lact plantarum RM71 achieved higher cell 237

densities in the catechin-supplemented CDM compared to control This growth 238

advantage was more pronounced in the pH 55 than in the pH 65 CDM The increase in 239

cell density reached in the pH 65 CDM upon catechin was of less magnitude than at pH 240

55 and it became equal to the cell density of the control soon after entering in the 241

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stationary phase (panels E to H) Contrarily in cultures grown at initial pH 55 the 242

higher cell density reached upon catechin was prolonged for a longer time during the 243

stationary phase (panel A) or maintained steadily higher than the control (panels B to 244

D) The increase in cell densitiy upon catechin was not a dose-dependent event 245

Compared to controls growth of Lact plantarum RM71 in catechin-containing 246

medium was quicker upon inoculation in the pH 55 CDM (panels A to E) than in the 247

pH 65 medium (panels E to F) Thus cultures carried out ie in complete pH 55 248

CDM supplemented with 100 mg l-1 of catechin shortened the duration of lag phase by 249

8 h (plusmn 087) compared to control (panel B) while a 45 h (plusmn 054) shorthening was 250

observed at an initial pH 65 (panel F) This quicker growth upon inoculation induced 251

by catechin was however not dose-dependent as the lengths of the lag phases were 252

similar at the two concentrations tested (Fig 1) We also observed that catechin induced 253

a shift in the growth pattern of Lact plantarum RM71 in complete pH 55 CDM This 254

pattern changed from a biphasic growth displayed in absence of catechin to a diauxic 255

pattern in presence of the flavanol (panel B) 256

Two key metabolic traits sugar consumption and lactic acid production were 257

monitored to evaluate the effect of catechin on Lact plantarum RM71 performance in 258

complete pH 55 CDM The time course of galactose consumption as well as of lactic 259

acid formation were compared between cultures grown upon 100 mg l-1 of catechin and 260

control cultures lacking the flavanol (Fig 2) The presence of catechin in CDM gave 261

rise to an increased galactose consumption respect to controls (increase of 39 when 262

compared to the control lacking catechin) A quicker galactose consumption and lactic 263

acid production were also observed upon catechin Thus the galactose consumption rate 264

in the first 48 h was of 555 mmol l-1 d-1 for the catechin supplemented cultures in 265

contrast with 344 mmol l-1 d-1 of the control Catechin had little effect on the maximum 266

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rate of galactose consumption as it was in the period measured and assuming steady-267

state conditions similar in presence ( 105 plusmn 006 mmol l-1 h-1) or in absence (095 plusmn 268

005 mmol l-1 h-1) of the flavanol Growth was improved by the presence of catechin in 269

the medium (Fig 2) confirming the above results obtained in cultures carried out on 270

microtitre plates (Fig 1) (please note that as mentioned in the Material and Methods 271

section the optical densities of experiments displayed in Fig 1 and Fig 2 are different) 272

273

The fermentation performance of Lactobacillus plantarum RM71 in wine-resembling 274

medium and basal fermentation medium is improved by catechin 275

276

To test the effects of catechin in media relevant to practical fermentation 277

processes firstly a wine-resembling medium set up at an initial pH of 38 was 278

fermented with Lact plantarum RM71 strain This fermentation was monitored for two 279

days time needed for this bacterium to achieve its maximum log increase in this 280

medium Sugar consumption malic acid degradation and lactic acid production were 281

assayed daily in catechin-lacking or catechin-supplemented (100 mg l-1) media Lact 282

plantarum RM71 metabolized glucose and fructose simultaneously in both media but 283

the total sugar consumption was higher in the medium supplemented with catechin 284

compared to control (1877 vs 2149 mmol l-1 See Table 1) 285

The presence of catechin clearly accelerated sugar consumption at the initial 286

stage of fermentation (0-24 h) as it could be seen for glucose and fructose (Fig 3) 287

which proceeded about ten and six times faster respectively than their controls (Table 288

1) The mean consumption rates of glucose and fructose per day during the entire 289

fermentation period were of 545 mmol l-1 d -1 for glucose (529 for fructose) upon 290

catechin instead of 45 mmol l-1 d -1 for glucose (48 for fructose) in absence of the 291

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flavanol The fermentation of malic acid was faster than that of sugars and completed in 292

only one day A sample taken at ten hours from the onset of fermentation showed that 293

catechin clearly sped up the consumption of malic acid (Fig 3) Thus at this time (10 294

h) 613 of the total malic acid added (715 mmol l-1) was already consumed by Lact 295

plantarum RM71 in presence of catechin in contrast with the significantly lower 293 296

(335 mmol l-1) malic acid consumption by the control culture 297

Compared to control the presence of catechin gave rise to an increase of lactic 298

acid production as it is reflected by the daily and total amounts produced (Table 1) The 299

difference in lactic acid production was specially marked at the onset of fermentation 300

which could be expected because of the accelerated malic acid and sugar consumption 301

in the catechin supplemented cultures 302

To test if catechin exerted similar effects on Lact plantarum plant-derived 303

fermentations different from wine conditions a medium named BFM was prepared The 304

medium was devoid of wine characteristic components ie tartaric acid ethanol and 305

acetic acid as these were not used by Lact plantarum RM71 in the fermentation of wine 306

resembling medium (not shown) Furthermore glucose and fructose were replaced by 307

galactose (1) to investigate if catechin affected the fermentation of other sugars 308

Galactose is present in the diet and is usually present in dietary fibre from vegetables 309

and fruits such as the arabinogalactan which has been shown to increase the 310

Lactobacillus spp population in the human intestine (Robinson Feirtag amp Slavin 311

(2001) 312

As it can be observed in Table 2 the presence of catechin stimulated the growth 313

of Lact plantarum RM71 in BFM The patterns of malic acid degradation and galactose 314

utilization are shown in Fig 4 The fermentation of malic acid was completed in two 315

days regardless catechin supplementation however the presence of this flavanol clearly 316

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sped up the consumption of this acid Thus during the first day of fermentation 598 317

of the total malic acid added was consumed by Lact plantarum RM71 in presence of 318

catechin in contrast with the significantly lower 137 by the control culture 319

We observed that the extent of sugar utilization by Lact plantarum RM71 was 320

increased upon catechin (176 vs 2914 mmol l-1) In addition catechin promoted a faster 321

galactose consumption respect to controls The mean consumption rate of galactose per 322

day during the entire fermentation period was of 971 mmol l-1 d -1 upon catechin 323

instead of 586 mmol l-1 d -1 in absence of the flavanol Examination of Table 2 revealed 324

that the presence of catechin promoted earlier galactose utilization Thus at the initial 325

stage of fermentation (0-24 h) sugar consumption proceeded at a diminished rate of 326

084 mmol l-1 d-1 in the control cultures compared to a much faster rate of 435 mmol l-1 327

d-1 upon catechin Lactic acid production was also monitored as indicator of the 328

fermentation performance of Lact plantarum RM71 As it occurred in the wine-329

resembling medium in BFM daily and total lactic acid productions (as well as lactic 330

acid yields) were higher upon catechin (Table 2) being these increases more 331

pronounced at the onset of fermentation (0-24h) 332

333

Catechin is not metabolized by Lactobacillus plantarum RM71 334

335

Spectrometric analysis was carried out to search for potential catechin 336

degradationmetabolism resulting from the growth of Lact plantarum RM71 337

Supernatants from stationary phase cultures of Lact plantarum RM71 grown in pH 55 338

CDM wine- resembling medium or BFM upon 100 mg l-1 of catechin were compared 339

with cell-free negative controls incubated under the same conditions Experiments 340

performed in CDM appeared in Fig 5 which displayed practically no differences in the 341

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catechin peak eluting at 1817 min among HPLC chromatograms of cell-free control 342

and culture supernatants after 40 h incubation in CDM 343

344

DISCUSSION 345

346

The effects of phenolics compounds on lactic acid bacteria have been reported to 347

be rather variable (For a recent review see Rodriacuteguez et al 2009) This variability 348

depends on the compound (Vivas et al 1997 Campos et al 2003 Figueiredo et al 349

2008) the strain under study (Figueiredo et al 2008) or the medium used for growth 350

(Alberto et al 2001) Up to date some authors have associated the positive effects of 351

food relevant phenolic compounds on growth of lactic acid bacteria to the degradation 352

of the compound Thus the growth stimulating effects of gallate on O oeni (Vivas et al 353

1997) or Lact hilgardii (Alberto et al 2001) were reported to be dose-dependent and 354

suggested to be linked to gallate degradation In the case of anthocyanins it was 355

suggested that glucose moiety was used as energy source to justify the positive effect on 356

the growth of O oeni (Vivas et al 1997) 357

In this study it was observed that most of the catechin remained in the media 358

after fermentation with Lact plantarum RM71 This result was interpreted as this 359

bacterium did not degrade the largest catechin bulk otherwise the catechin peak would 360

have been disappeared or greatly diminished and new compounds would have been 361

appeared Consequently the metabolic and growth stimulations promoted by catechin 362

can not be ascribed to catechin metabolism by Lact plantarum RM 71 These results 363

are in agreement with those previously reported for O oeni by Reguant et al (2000) 364

who also uncoupled the stimulation of growth triggered by catechin from the 365

metabolism of this flavanol Since O oeni grows better in absence of oxygen these 366

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authors suggested that the oxygen scavenging ability of flavonoids tested including 367

catechin favoured the growth stimulating effects observed Contrarily other authors 368

could not apply the same reasoning as they found that catechin or epicatechin showed 369

neutral effects on the growth of O oeni and Lact hilgardii (Figueiredo et al 2008) It 370

has been recently reported that the radical scavenging capacity of catechins increased 371

with the pH of the medium (Muzolf et al 2008) however in this work the positive 372

effects of catechin on the growth of Lact plantarum were more pronounced at the lower 373

pH and not dependent on the dose of catechin used Therefore the positive influence of 374

catechin on growth found here can hardly be ascribed to its radical scavenging capacity 375

By time course of sugar consumption data tracking experiments it is shown that 376

at least in the case of Lact plantarum RM71 catechin stimulates growth by promoting 377

quicker sugar consumption increasing the extension of sugar utilization and stimulating 378

malic acid decarboxylation Clearly this is a different mode of action of catechin to that 379

suggested by this flavanol on Lact hilgardii where the growth stimulating effects were 380

linked to catechin degradation (Alberto et al 2001 Figueiredo et al 2008) or to that 381

suggested by Reguant et al (2000) for O oeni who justified the stimulating effects on 382

the antioxidant properties of catechin 383

The beneficial effects on growth were observed regardless of the catechin 384

concentration examined which support again the hypotheses that growth stimulation is 385

uncoupled from catechin degradation The lack of dose dependent effects is in 386

agreement with the weak bactericidal activity (Nakayama et al 1998) resulting from the 387

shallow location close to the phospholipidwater interface previously reported for 388

catechin (Caturla et al 2003) Otherwise an increased bactericidal effect would be 389

expected at increased catechin concentrations as it occurs with galloylated catechins 390

which possess a higher insertion capacity in the membrane (Ikagai et al 1993 Caturla 391

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et al 2003) Although the growth advantages were observed regardless the initial pH 392

values they were more pronounced in the pH 55 CDM which could indicate a stronger 393

interaction of catechin with membrane phospholipids at this lower pH than at pH 65 394

According to previous reports (Kajiya et al 2001 Caturla et al 2003) catechin 395

does not enter the cell so it is probable that the effects observed on growth and 396

metabolism were caused by alteration of the biophysical properties of the membrane of 397

Lact plantarum RM71 It is possible that catechin after had caused its biological effect 398

could had left the membrane by passive diffusion along time a process that has been 399

previously described for other flavonoids such as quercetin (Movileanu et al 2000) 400

Thus catechin once located in the membrane could alter the function of proteins 401

associated with the bilayer and affect the transport of materials across it Sugar and 402

malate transporters seem to be affected proteins accordingly to the changes in sugar and 403

malic acid consumption observed here upon catechin The citrate transport system could 404

be affected as well since catechin triggered variations in the growth patterns displayed 405

by Lact plantarum RM71 on citrate supplemented CDM Experiments are currently 406

under way to determine if catechin alters some biophysical properties of the membrane 407

of Lact plantarum and affects these transport systems 408

Citrate prolonged lag phase and induced sequential growth behaviour in CDM 409

The scope of this study was not to investigate this behaviour however the long lag 410

phase observed upon citrate indicates that there might be some limitations on the 411

substrate utilization apparatus at the onset of cultures which is usual when growing in 412

mixed-substrate environments (Daae et al 1998) The biphasic growth could be due to a 413

sequential metabolism of carbon sources since this behaviour was only observed at an 414

initial pH of 55 which is close to the optimum pH range for citrate metabolism in Lact 415

plantarum (Palles et al 1998) This type of sequential metabolism is not unprecedent 416

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for Lactobacillus ssp Thus diauxic growth has been described during growth also 417

under sugar limitation on a mixture of sugars and fructooligosaccharides (FOS) (Goh et 418

al 2007) and glucose and citrate (de Figueroa et al 1996) Catechin shorthened the lag 419

phase so it is probable that this flavanol relieved limitations in substrate utilization 420

Moreover in complete pH 55 CDM catechin replaced the biphasic growth by a diauxic 421

growth where a clear diauxic lag was evident This growth pattern is usually associated 422

to a better control of substrate assimilation (Daae et al 1998) so if catechin as it is 423

suspected inferred a more efficient substrate uptake a diauxic lag would be necessary 424

to fit the enzyme synthesis for the utilization of the second substrate 425

The results obtained in wine resembling medium or BFM revealed that catechin 426

sped-up malolactic fermentation stimulated the consumption of several sugars as well 427

as the lactic acid formation thus providing growth advantages to Lact plantarum 428

RM71 in these media Growth stimulation linked to malolactic fermentation is known to 429

be caused by its coupling to ATP synthesis by either the entry through F(H+)-ATPase 430

of environmental protons generated in the malate decarboxilation process and the 431

malatelactate antiport coupled also to F(H+)-ATPase Accordingly the growth 432

improvement of Lact plantarum RM71 upon catechin should not be surprising in view 433

of the accelerated malolactic fermentation and lactic acid production provided by the 434

flavanol Similar effects have been also described for the growth of O oeni CECT 435

4100T where this flavanol was found to stimulate the malolactic fermentation 436

development (Reguant et al 2000) Although malolactic fermentation is classically 437

associated to winemaking the importance of the growth benefit obtained by Lact 438

plantarum from the utilization of malic acid should not be restricted to the wine 439

environment but extended to other fermented food plants as recently reported (Plumed-440

Ferrer et al 2008) These authors found a significantly higher growth of Lact 441

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plantarum in cucumber juice compared to the rich MRS medium and highlighted the 442

crucial importance of the higher decarboxylation of malic acid in this stimulation 443

In summary the results presented here show that catechin improved malic acid 444

degradation fermentation of several sugars and therefore Lact plantarum RM71 445

growth which are desirable traits for a successful performance of this species as starter 446

in several food plant fermentations These stimulations are not linked to catechin 447

degradation 448

449

Acknowledgements 450

451

This work was supported by grants AGL2005-000470 and AGL2008-001052 (CICYT) 452

and 200670M058 (CAM) J A Curiel is a recipient of a fellowship from Ministerio de 453

Ciencia e Innovacioacuten (MICINN) 454

455

REFERENCES 456

457

Adlerberth I Ahrne S Johansson ML Molin G Hanson LA and Wold AE 458

(1996) A mannose specific adherence mechanism in Lactobacillus plantarum 459

conferring binding to the human colonic cell line HT-29 Appl Environ 460

Microbiol 62 2244-2251 461

462

Alberto MR Fariacuteas ME and Manca de Nadra MC (2001) Effect of gallic acid and 463

catechin on Lactobacillus hilgardii 5w growth and metabolism of organics 464

compounds J Agr Food Chem 49 4359-4363 465

466

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Amarowicz R Carle R Dongowski G Durazzo A Galensa R Kammerer D 467

Maiani G and Piskula MK (2009) Influence of postharvest processing and 468

storage on the content of phenolic acids and flavonoids in foods Mol Nutr Food 469

Res doi 101002mnfr200700486 470

471

Campos FM Couto JA and Hogg TA (2003) Influence of phenolics acids on 472

growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii J Appl 473

Microbiol 94 167-174 474

475

Caturla N Vera-Samper E Villalaiacuten J Reyes Mateo C and Micol V (2003) The 476

relationship between the antioxidant and the antibacterial properties of 477

galloylated catechins and the structure of phospholipids membranes Free Rad 478

Biol Med 34 648-662 479

480

Crespy V and Williamson G (2004) A review of the health effects of green tea 481

catechins in in vivo animal models J Nutr 134 3431-3440 482

483

Daae EB and Ison AP (1998) A simple structured model describing the growth of 484

Streptomyces lividans Biotechnol Bioeng 58 263-266 485

486

de Figueroa R M Benito de Caacuterdenas I L Sesma F Alvarez F de Ruiz Holgado 487

A P and Oliver G (1996) Inducible transport of citrate in Lactobacillus 488

rhamnosus ATCC 7469 J Appl Microbiol 81 348-354 489

490

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de Man J C Rogosa M and Sharpe M E (1960) A medium for the cultivation of 491

lactobacilli J Appl Microbiol 23 130-135 492

493

Figueiredo AR Campos F de Freitas V Hogg T and Couto JA (2008) Effect of 494

phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus 495

oeni and Lactobacillus hilgardii Food Microbiol 25 105-12 496

497

Goh Y J Lee J H and Hutkins R W (2007) Functional analysis of the 498

fructooligosaccharide utilization operon in Lactobacillus paracasei 1195 Appl 499

Environ Microbiol 73 5716-5724 500

501

Hussein L Medina A Barrionuevo A Lammuela-Raventos RM and Andres-502

Lacueva C (2009) Normal distribution of urinary polyphenol excretion among 503

Egyptian males 7-14 years old and changes following nutritional intervention 504

with tomato juice (Lycopersicon esculentum) Int J Food Sci Nutr 60 302 ndash 311 505

506

Ikagai H Nakae T Hara Y and Shimamura T (1993) Bactericidal catechins 507

damage the lipid bilayer Biochim Biophys Acta 1147 132-136 508

509

Kajiya K S Kumazawa and Nakayama T (2001) Steric effects on the interaction of 510

tea catechins with lipid bilayers Biosci Biotechnol Biochem 65 2638-2643 511

512

Kostinek M Ban-Koffi L Ottah-Atikpo M Teniola D Schillinger U Holzapfel 513

WH and Franz C (2008) Diversity of predominant lactic acid bacteria 514

associated with cocoa fermentation in Nigeria Curr Microbiol 56 306-314 515

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In the experiments carried out in CDM growth curves were recorded at 30degC by 142

measuring the optical density (OD600) of the cultures every hour in a kinetic Bio-Tek 143

KC Junior microplate reader (BioTek Instruments Winooski USA) The data obtained 144

with the reader were exported to and processed with the Excel-MS software Cultures 145

used to follow metabolism of Lact plantarum RM71 were performed in CDM by 146

scaling volumes up to 15 ml The optical densities measured with a microtitre plate 147

reader and with a spectrophotometer were different due to the different optical 148

geometries The lag phase was defined as the time needed to reach an optical density 149

10x that of the initial OD600 The maximum specific growth rate max was determined 150

by calculating the slope of the exponential growth phase (max = ∆ln(OD600)∆t) The 151

results were expressed as the means for lag phases or max calculated from at least two 152

independent experiments153

154

Fermentation in wine-resembling and basal fermentation media 155

156

Fermentations of 15 ml were run at 25ordmC in a wine resembling medium 157

(Ugliano Genovese amp Moio L (2003) with the following composition (g l-1) glucose 158

(2) fructose (2) tartaric acid (5) acetic acid (06) L-malic acid (175) yeast extract 159

(20) NaCl (02) (NH4)2 SO4 (1) MnSO4 (005) MgSO47H2O (02) K2PO4 (1) and 160

ethanol (8 vv) A variant of this medium named basal fermentation medium (BFM) 161

for convenience was also used which lacked of tartaric acid acetic acid and ethanol 162

Galactose (1 ) instead of glucose and fructose was added as sugar substrate in this 163

medium Catechin (100 mg l-1) was added or omitted as indicated The initial pH was 164

adjusted at pH 38 in either medium and then filter sterilized Cells grown in MRS 165

medium were washed three times with saline solution (085 NaCl) and then inoculated 166

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in wine resembling or BFM media The bacterial growth was followed daily by plating 167

100 microl of properly diluted medium on MRS medium (Difco laboratories) adjusted to pH 168

50 before agar addition and sterilization Plates were incubated aerobically at 25 ordmC for 169

three days in a Memmert incubator 170

171

Analysis of catechin degradation by high-performance liquid chromatography-diode 172

array detector (HPLC-DAD) 173

174

Potential catechin degradation by Lactobacillus plantarum RM71 was studied in 175

5 ml of CDM wine resembling or BFM media cultures Supernatants for HPLC-DAD 176

analysis were obtained by filtration (pore size 022 microm Millipore) of three mililiters of 177

inoculated medium or from its cell-free control both after 40 h incubation at 30ordm C 178

Catechin was extracted twice from the supernatants with one third of the reaction 179

volume of ethyl acetate (Lab-Scan Ireland) HPLC-DAD analysis was performed as 180

described previously (Rodriacuteguez et al 2008) Catechin degradation was followed by 181

comparing retention times and spectral data of the peaks obtained in the 182

chromatrograms Commercial catechin was used as standard 183

184

Fermentation analysis by high-performance liquid chromatography (HPLC) 185

186

Supernatants from fermentation experiments were analyzed by HPLC in order to 187

determine the amounts of the sugars galactose glucose and fructose as well as L-lactic 188

and L-malic acids A Thermo (Thermo Electron Corp Waltham MA) chromatograph 189

equipped with a P-4000 Spectra System Pump an AS3000 autosampler a RI-150 190

refraction index and a UV 6000 LP photodiode array detectors were used A 15 mmol l-191

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1 H2SO4 aqueous solution as mobile phase was applied to an Aminexreg HPX-87H 192

column (300 mm x 78 mm) (BIO-RAD) at a 06 mlmin flow rate at 35 ordmC Samples 193

were filtered through a 045-microm polyvinylidene difluoride filter diluted two-fold and 194

injected in duplicated Galactose glucose or fructose detection was performed at 210 195

nm (refraction index detector) and L-lactic and L-malic acids at 380 nm (diode array 196

detector) Sugar and organic acid standards were used for the identification of the HPLC 197

peaks on the chromatograms and for the estimation of their concentration by a 198

calibration curve 199

200

RESULTS 201

202

Growth behaviour of Lactobacillus plantarum RM71 in control CDM medium 203

204

To study the catechin effects on the fermentation performance of Lact 205

plantarum RM71 growth and the time course of sugar consumption were monitored in 206

a chemical defined medium designed for Lact plantarum With the use of CDM it was 207

ensured that catechin was the only flavonoid in the medium and thus the effects on 208

growth could be more easily ascribed to this flavanol Furthermore use of CDM would 209

facilitate the evaluation of potential catechin degradation products As a model growth 210

sugar for CDM experiments we used galactose which is abundant in vegetable fibre and 211

the diet On this sugar cells grow more slowly than ie glucose and the changes 212

produced by catechin could be more properly monitored 213

Before studying catechin effects we firstly examined the growth behaviour of 214

Lact plantarum RM71 in CDM in absence of catechin The single effects of citrate and 215

acetate on growth were studied by using four different CDM variants that were prepared 216

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by addition or omission of one or both of these acids in the formulation The 217

dependence of growth patterns on pH was relevant to subsequently interpret if catechin 218

effects were also pH-dependent Therefore we evaluated this effect by adjusting the 219

initial medium pH at 55 or 65 in absence of catechin 220

The growth patterns of Lact plantarum RM71 in the four CDM variants at pH 221

55 or 65 are shown in Fig 1 Lact plantarum RM71 grew well in absence of both 222

citrate and acetate (panels A and E) The presence of these acids in the medium 223

prolonged the lag phase (panels B and F) and elicited a biphasic growth pattern that did 224

only occur in the pH 55 CDM (panels B and C) It appeared that citrate was the only 225

acid leading to this growth profile because the lag phase prolongation (panels B C F 226

and G) as well as was biphasic growth (panels B and C) were observed only in citrate-227

supplemented CDM regardless of acetate inclusion in the medium 228

229

Catechin modifies growth and metabolism of Lactobacillus plantarum RM71 to improve 230

its fermentation performance in CDM 231

232

Results show that the presence of catechin provided advantages to Lact 233

plantarum RM71 for growing in CDM The two positive effects induced by this 234

flavanol were shorthening of the lag phase and higher final cell densities both occurring 235

regardless of the initial pH set-up (Fig1) 236

Examination of Fig 1 revealed that Lact plantarum RM71 achieved higher cell 237

densities in the catechin-supplemented CDM compared to control This growth 238

advantage was more pronounced in the pH 55 than in the pH 65 CDM The increase in 239

cell density reached in the pH 65 CDM upon catechin was of less magnitude than at pH 240

55 and it became equal to the cell density of the control soon after entering in the 241

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stationary phase (panels E to H) Contrarily in cultures grown at initial pH 55 the 242

higher cell density reached upon catechin was prolonged for a longer time during the 243

stationary phase (panel A) or maintained steadily higher than the control (panels B to 244

D) The increase in cell densitiy upon catechin was not a dose-dependent event 245

Compared to controls growth of Lact plantarum RM71 in catechin-containing 246

medium was quicker upon inoculation in the pH 55 CDM (panels A to E) than in the 247

pH 65 medium (panels E to F) Thus cultures carried out ie in complete pH 55 248

CDM supplemented with 100 mg l-1 of catechin shortened the duration of lag phase by 249

8 h (plusmn 087) compared to control (panel B) while a 45 h (plusmn 054) shorthening was 250

observed at an initial pH 65 (panel F) This quicker growth upon inoculation induced 251

by catechin was however not dose-dependent as the lengths of the lag phases were 252

similar at the two concentrations tested (Fig 1) We also observed that catechin induced 253

a shift in the growth pattern of Lact plantarum RM71 in complete pH 55 CDM This 254

pattern changed from a biphasic growth displayed in absence of catechin to a diauxic 255

pattern in presence of the flavanol (panel B) 256

Two key metabolic traits sugar consumption and lactic acid production were 257

monitored to evaluate the effect of catechin on Lact plantarum RM71 performance in 258

complete pH 55 CDM The time course of galactose consumption as well as of lactic 259

acid formation were compared between cultures grown upon 100 mg l-1 of catechin and 260

control cultures lacking the flavanol (Fig 2) The presence of catechin in CDM gave 261

rise to an increased galactose consumption respect to controls (increase of 39 when 262

compared to the control lacking catechin) A quicker galactose consumption and lactic 263

acid production were also observed upon catechin Thus the galactose consumption rate 264

in the first 48 h was of 555 mmol l-1 d-1 for the catechin supplemented cultures in 265

contrast with 344 mmol l-1 d-1 of the control Catechin had little effect on the maximum 266

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rate of galactose consumption as it was in the period measured and assuming steady-267

state conditions similar in presence ( 105 plusmn 006 mmol l-1 h-1) or in absence (095 plusmn 268

005 mmol l-1 h-1) of the flavanol Growth was improved by the presence of catechin in 269

the medium (Fig 2) confirming the above results obtained in cultures carried out on 270

microtitre plates (Fig 1) (please note that as mentioned in the Material and Methods 271

section the optical densities of experiments displayed in Fig 1 and Fig 2 are different) 272

273

The fermentation performance of Lactobacillus plantarum RM71 in wine-resembling 274

medium and basal fermentation medium is improved by catechin 275

276

To test the effects of catechin in media relevant to practical fermentation 277

processes firstly a wine-resembling medium set up at an initial pH of 38 was 278

fermented with Lact plantarum RM71 strain This fermentation was monitored for two 279

days time needed for this bacterium to achieve its maximum log increase in this 280

medium Sugar consumption malic acid degradation and lactic acid production were 281

assayed daily in catechin-lacking or catechin-supplemented (100 mg l-1) media Lact 282

plantarum RM71 metabolized glucose and fructose simultaneously in both media but 283

the total sugar consumption was higher in the medium supplemented with catechin 284

compared to control (1877 vs 2149 mmol l-1 See Table 1) 285

The presence of catechin clearly accelerated sugar consumption at the initial 286

stage of fermentation (0-24 h) as it could be seen for glucose and fructose (Fig 3) 287

which proceeded about ten and six times faster respectively than their controls (Table 288

1) The mean consumption rates of glucose and fructose per day during the entire 289

fermentation period were of 545 mmol l-1 d -1 for glucose (529 for fructose) upon 290

catechin instead of 45 mmol l-1 d -1 for glucose (48 for fructose) in absence of the 291

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flavanol The fermentation of malic acid was faster than that of sugars and completed in 292

only one day A sample taken at ten hours from the onset of fermentation showed that 293

catechin clearly sped up the consumption of malic acid (Fig 3) Thus at this time (10 294

h) 613 of the total malic acid added (715 mmol l-1) was already consumed by Lact 295

plantarum RM71 in presence of catechin in contrast with the significantly lower 293 296

(335 mmol l-1) malic acid consumption by the control culture 297

Compared to control the presence of catechin gave rise to an increase of lactic 298

acid production as it is reflected by the daily and total amounts produced (Table 1) The 299

difference in lactic acid production was specially marked at the onset of fermentation 300

which could be expected because of the accelerated malic acid and sugar consumption 301

in the catechin supplemented cultures 302

To test if catechin exerted similar effects on Lact plantarum plant-derived 303

fermentations different from wine conditions a medium named BFM was prepared The 304

medium was devoid of wine characteristic components ie tartaric acid ethanol and 305

acetic acid as these were not used by Lact plantarum RM71 in the fermentation of wine 306

resembling medium (not shown) Furthermore glucose and fructose were replaced by 307

galactose (1) to investigate if catechin affected the fermentation of other sugars 308

Galactose is present in the diet and is usually present in dietary fibre from vegetables 309

and fruits such as the arabinogalactan which has been shown to increase the 310

Lactobacillus spp population in the human intestine (Robinson Feirtag amp Slavin 311

(2001) 312

As it can be observed in Table 2 the presence of catechin stimulated the growth 313

of Lact plantarum RM71 in BFM The patterns of malic acid degradation and galactose 314

utilization are shown in Fig 4 The fermentation of malic acid was completed in two 315

days regardless catechin supplementation however the presence of this flavanol clearly 316

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sped up the consumption of this acid Thus during the first day of fermentation 598 317

of the total malic acid added was consumed by Lact plantarum RM71 in presence of 318

catechin in contrast with the significantly lower 137 by the control culture 319

We observed that the extent of sugar utilization by Lact plantarum RM71 was 320

increased upon catechin (176 vs 2914 mmol l-1) In addition catechin promoted a faster 321

galactose consumption respect to controls The mean consumption rate of galactose per 322

day during the entire fermentation period was of 971 mmol l-1 d -1 upon catechin 323

instead of 586 mmol l-1 d -1 in absence of the flavanol Examination of Table 2 revealed 324

that the presence of catechin promoted earlier galactose utilization Thus at the initial 325

stage of fermentation (0-24 h) sugar consumption proceeded at a diminished rate of 326

084 mmol l-1 d-1 in the control cultures compared to a much faster rate of 435 mmol l-1 327

d-1 upon catechin Lactic acid production was also monitored as indicator of the 328

fermentation performance of Lact plantarum RM71 As it occurred in the wine-329

resembling medium in BFM daily and total lactic acid productions (as well as lactic 330

acid yields) were higher upon catechin (Table 2) being these increases more 331

pronounced at the onset of fermentation (0-24h) 332

333

Catechin is not metabolized by Lactobacillus plantarum RM71 334

335

Spectrometric analysis was carried out to search for potential catechin 336

degradationmetabolism resulting from the growth of Lact plantarum RM71 337

Supernatants from stationary phase cultures of Lact plantarum RM71 grown in pH 55 338

CDM wine- resembling medium or BFM upon 100 mg l-1 of catechin were compared 339

with cell-free negative controls incubated under the same conditions Experiments 340

performed in CDM appeared in Fig 5 which displayed practically no differences in the 341

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catechin peak eluting at 1817 min among HPLC chromatograms of cell-free control 342

and culture supernatants after 40 h incubation in CDM 343

344

DISCUSSION 345

346

The effects of phenolics compounds on lactic acid bacteria have been reported to 347

be rather variable (For a recent review see Rodriacuteguez et al 2009) This variability 348

depends on the compound (Vivas et al 1997 Campos et al 2003 Figueiredo et al 349

2008) the strain under study (Figueiredo et al 2008) or the medium used for growth 350

(Alberto et al 2001) Up to date some authors have associated the positive effects of 351

food relevant phenolic compounds on growth of lactic acid bacteria to the degradation 352

of the compound Thus the growth stimulating effects of gallate on O oeni (Vivas et al 353

1997) or Lact hilgardii (Alberto et al 2001) were reported to be dose-dependent and 354

suggested to be linked to gallate degradation In the case of anthocyanins it was 355

suggested that glucose moiety was used as energy source to justify the positive effect on 356

the growth of O oeni (Vivas et al 1997) 357

In this study it was observed that most of the catechin remained in the media 358

after fermentation with Lact plantarum RM71 This result was interpreted as this 359

bacterium did not degrade the largest catechin bulk otherwise the catechin peak would 360

have been disappeared or greatly diminished and new compounds would have been 361

appeared Consequently the metabolic and growth stimulations promoted by catechin 362

can not be ascribed to catechin metabolism by Lact plantarum RM 71 These results 363

are in agreement with those previously reported for O oeni by Reguant et al (2000) 364

who also uncoupled the stimulation of growth triggered by catechin from the 365

metabolism of this flavanol Since O oeni grows better in absence of oxygen these 366

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authors suggested that the oxygen scavenging ability of flavonoids tested including 367

catechin favoured the growth stimulating effects observed Contrarily other authors 368

could not apply the same reasoning as they found that catechin or epicatechin showed 369

neutral effects on the growth of O oeni and Lact hilgardii (Figueiredo et al 2008) It 370

has been recently reported that the radical scavenging capacity of catechins increased 371

with the pH of the medium (Muzolf et al 2008) however in this work the positive 372

effects of catechin on the growth of Lact plantarum were more pronounced at the lower 373

pH and not dependent on the dose of catechin used Therefore the positive influence of 374

catechin on growth found here can hardly be ascribed to its radical scavenging capacity 375

By time course of sugar consumption data tracking experiments it is shown that 376

at least in the case of Lact plantarum RM71 catechin stimulates growth by promoting 377

quicker sugar consumption increasing the extension of sugar utilization and stimulating 378

malic acid decarboxylation Clearly this is a different mode of action of catechin to that 379

suggested by this flavanol on Lact hilgardii where the growth stimulating effects were 380

linked to catechin degradation (Alberto et al 2001 Figueiredo et al 2008) or to that 381

suggested by Reguant et al (2000) for O oeni who justified the stimulating effects on 382

the antioxidant properties of catechin 383

The beneficial effects on growth were observed regardless of the catechin 384

concentration examined which support again the hypotheses that growth stimulation is 385

uncoupled from catechin degradation The lack of dose dependent effects is in 386

agreement with the weak bactericidal activity (Nakayama et al 1998) resulting from the 387

shallow location close to the phospholipidwater interface previously reported for 388

catechin (Caturla et al 2003) Otherwise an increased bactericidal effect would be 389

expected at increased catechin concentrations as it occurs with galloylated catechins 390

which possess a higher insertion capacity in the membrane (Ikagai et al 1993 Caturla 391

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et al 2003) Although the growth advantages were observed regardless the initial pH 392

values they were more pronounced in the pH 55 CDM which could indicate a stronger 393

interaction of catechin with membrane phospholipids at this lower pH than at pH 65 394

According to previous reports (Kajiya et al 2001 Caturla et al 2003) catechin 395

does not enter the cell so it is probable that the effects observed on growth and 396

metabolism were caused by alteration of the biophysical properties of the membrane of 397

Lact plantarum RM71 It is possible that catechin after had caused its biological effect 398

could had left the membrane by passive diffusion along time a process that has been 399

previously described for other flavonoids such as quercetin (Movileanu et al 2000) 400

Thus catechin once located in the membrane could alter the function of proteins 401

associated with the bilayer and affect the transport of materials across it Sugar and 402

malate transporters seem to be affected proteins accordingly to the changes in sugar and 403

malic acid consumption observed here upon catechin The citrate transport system could 404

be affected as well since catechin triggered variations in the growth patterns displayed 405

by Lact plantarum RM71 on citrate supplemented CDM Experiments are currently 406

under way to determine if catechin alters some biophysical properties of the membrane 407

of Lact plantarum and affects these transport systems 408

Citrate prolonged lag phase and induced sequential growth behaviour in CDM 409

The scope of this study was not to investigate this behaviour however the long lag 410

phase observed upon citrate indicates that there might be some limitations on the 411

substrate utilization apparatus at the onset of cultures which is usual when growing in 412

mixed-substrate environments (Daae et al 1998) The biphasic growth could be due to a 413

sequential metabolism of carbon sources since this behaviour was only observed at an 414

initial pH of 55 which is close to the optimum pH range for citrate metabolism in Lact 415

plantarum (Palles et al 1998) This type of sequential metabolism is not unprecedent 416

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for Lactobacillus ssp Thus diauxic growth has been described during growth also 417

under sugar limitation on a mixture of sugars and fructooligosaccharides (FOS) (Goh et 418

al 2007) and glucose and citrate (de Figueroa et al 1996) Catechin shorthened the lag 419

phase so it is probable that this flavanol relieved limitations in substrate utilization 420

Moreover in complete pH 55 CDM catechin replaced the biphasic growth by a diauxic 421

growth where a clear diauxic lag was evident This growth pattern is usually associated 422

to a better control of substrate assimilation (Daae et al 1998) so if catechin as it is 423

suspected inferred a more efficient substrate uptake a diauxic lag would be necessary 424

to fit the enzyme synthesis for the utilization of the second substrate 425

The results obtained in wine resembling medium or BFM revealed that catechin 426

sped-up malolactic fermentation stimulated the consumption of several sugars as well 427

as the lactic acid formation thus providing growth advantages to Lact plantarum 428

RM71 in these media Growth stimulation linked to malolactic fermentation is known to 429

be caused by its coupling to ATP synthesis by either the entry through F(H+)-ATPase 430

of environmental protons generated in the malate decarboxilation process and the 431

malatelactate antiport coupled also to F(H+)-ATPase Accordingly the growth 432

improvement of Lact plantarum RM71 upon catechin should not be surprising in view 433

of the accelerated malolactic fermentation and lactic acid production provided by the 434

flavanol Similar effects have been also described for the growth of O oeni CECT 435

4100T where this flavanol was found to stimulate the malolactic fermentation 436

development (Reguant et al 2000) Although malolactic fermentation is classically 437

associated to winemaking the importance of the growth benefit obtained by Lact 438

plantarum from the utilization of malic acid should not be restricted to the wine 439

environment but extended to other fermented food plants as recently reported (Plumed-440

Ferrer et al 2008) These authors found a significantly higher growth of Lact 441

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plantarum in cucumber juice compared to the rich MRS medium and highlighted the 442

crucial importance of the higher decarboxylation of malic acid in this stimulation 443

In summary the results presented here show that catechin improved malic acid 444

degradation fermentation of several sugars and therefore Lact plantarum RM71 445

growth which are desirable traits for a successful performance of this species as starter 446

in several food plant fermentations These stimulations are not linked to catechin 447

degradation 448

449

Acknowledgements 450

451

This work was supported by grants AGL2005-000470 and AGL2008-001052 (CICYT) 452

and 200670M058 (CAM) J A Curiel is a recipient of a fellowship from Ministerio de 453

Ciencia e Innovacioacuten (MICINN) 454

455

REFERENCES 456

457

Adlerberth I Ahrne S Johansson ML Molin G Hanson LA and Wold AE 458

(1996) A mannose specific adherence mechanism in Lactobacillus plantarum 459

conferring binding to the human colonic cell line HT-29 Appl Environ 460

Microbiol 62 2244-2251 461

462

Alberto MR Fariacuteas ME and Manca de Nadra MC (2001) Effect of gallic acid and 463

catechin on Lactobacillus hilgardii 5w growth and metabolism of organics 464

compounds J Agr Food Chem 49 4359-4363 465

466

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Amarowicz R Carle R Dongowski G Durazzo A Galensa R Kammerer D 467

Maiani G and Piskula MK (2009) Influence of postharvest processing and 468

storage on the content of phenolic acids and flavonoids in foods Mol Nutr Food 469

Res doi 101002mnfr200700486 470

471

Campos FM Couto JA and Hogg TA (2003) Influence of phenolics acids on 472

growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii J Appl 473

Microbiol 94 167-174 474

475

Caturla N Vera-Samper E Villalaiacuten J Reyes Mateo C and Micol V (2003) The 476

relationship between the antioxidant and the antibacterial properties of 477

galloylated catechins and the structure of phospholipids membranes Free Rad 478

Biol Med 34 648-662 479

480

Crespy V and Williamson G (2004) A review of the health effects of green tea 481

catechins in in vivo animal models J Nutr 134 3431-3440 482

483

Daae EB and Ison AP (1998) A simple structured model describing the growth of 484

Streptomyces lividans Biotechnol Bioeng 58 263-266 485

486

de Figueroa R M Benito de Caacuterdenas I L Sesma F Alvarez F de Ruiz Holgado 487

A P and Oliver G (1996) Inducible transport of citrate in Lactobacillus 488

rhamnosus ATCC 7469 J Appl Microbiol 81 348-354 489

490

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de Man J C Rogosa M and Sharpe M E (1960) A medium for the cultivation of 491

lactobacilli J Appl Microbiol 23 130-135 492

493

Figueiredo AR Campos F de Freitas V Hogg T and Couto JA (2008) Effect of 494

phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus 495

oeni and Lactobacillus hilgardii Food Microbiol 25 105-12 496

497

Goh Y J Lee J H and Hutkins R W (2007) Functional analysis of the 498

fructooligosaccharide utilization operon in Lactobacillus paracasei 1195 Appl 499

Environ Microbiol 73 5716-5724 500

501

Hussein L Medina A Barrionuevo A Lammuela-Raventos RM and Andres-502

Lacueva C (2009) Normal distribution of urinary polyphenol excretion among 503

Egyptian males 7-14 years old and changes following nutritional intervention 504

with tomato juice (Lycopersicon esculentum) Int J Food Sci Nutr 60 302 ndash 311 505

506

Ikagai H Nakae T Hara Y and Shimamura T (1993) Bactericidal catechins 507

damage the lipid bilayer Biochim Biophys Acta 1147 132-136 508

509

Kajiya K S Kumazawa and Nakayama T (2001) Steric effects on the interaction of 510

tea catechins with lipid bilayers Biosci Biotechnol Biochem 65 2638-2643 511

512

Kostinek M Ban-Koffi L Ottah-Atikpo M Teniola D Schillinger U Holzapfel 513

WH and Franz C (2008) Diversity of predominant lactic acid bacteria 514

associated with cocoa fermentation in Nigeria Curr Microbiol 56 306-314 515

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in wine resembling or BFM media The bacterial growth was followed daily by plating 167

100 microl of properly diluted medium on MRS medium (Difco laboratories) adjusted to pH 168

50 before agar addition and sterilization Plates were incubated aerobically at 25 ordmC for 169

three days in a Memmert incubator 170

171

Analysis of catechin degradation by high-performance liquid chromatography-diode 172

array detector (HPLC-DAD) 173

174

Potential catechin degradation by Lactobacillus plantarum RM71 was studied in 175

5 ml of CDM wine resembling or BFM media cultures Supernatants for HPLC-DAD 176

analysis were obtained by filtration (pore size 022 microm Millipore) of three mililiters of 177

inoculated medium or from its cell-free control both after 40 h incubation at 30ordm C 178

Catechin was extracted twice from the supernatants with one third of the reaction 179

volume of ethyl acetate (Lab-Scan Ireland) HPLC-DAD analysis was performed as 180

described previously (Rodriacuteguez et al 2008) Catechin degradation was followed by 181

comparing retention times and spectral data of the peaks obtained in the 182

chromatrograms Commercial catechin was used as standard 183

184

Fermentation analysis by high-performance liquid chromatography (HPLC) 185

186

Supernatants from fermentation experiments were analyzed by HPLC in order to 187

determine the amounts of the sugars galactose glucose and fructose as well as L-lactic 188

and L-malic acids A Thermo (Thermo Electron Corp Waltham MA) chromatograph 189

equipped with a P-4000 Spectra System Pump an AS3000 autosampler a RI-150 190

refraction index and a UV 6000 LP photodiode array detectors were used A 15 mmol l-191

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1 H2SO4 aqueous solution as mobile phase was applied to an Aminexreg HPX-87H 192

column (300 mm x 78 mm) (BIO-RAD) at a 06 mlmin flow rate at 35 ordmC Samples 193

were filtered through a 045-microm polyvinylidene difluoride filter diluted two-fold and 194

injected in duplicated Galactose glucose or fructose detection was performed at 210 195

nm (refraction index detector) and L-lactic and L-malic acids at 380 nm (diode array 196

detector) Sugar and organic acid standards were used for the identification of the HPLC 197

peaks on the chromatograms and for the estimation of their concentration by a 198

calibration curve 199

200

RESULTS 201

202

Growth behaviour of Lactobacillus plantarum RM71 in control CDM medium 203

204

To study the catechin effects on the fermentation performance of Lact 205

plantarum RM71 growth and the time course of sugar consumption were monitored in 206

a chemical defined medium designed for Lact plantarum With the use of CDM it was 207

ensured that catechin was the only flavonoid in the medium and thus the effects on 208

growth could be more easily ascribed to this flavanol Furthermore use of CDM would 209

facilitate the evaluation of potential catechin degradation products As a model growth 210

sugar for CDM experiments we used galactose which is abundant in vegetable fibre and 211

the diet On this sugar cells grow more slowly than ie glucose and the changes 212

produced by catechin could be more properly monitored 213

Before studying catechin effects we firstly examined the growth behaviour of 214

Lact plantarum RM71 in CDM in absence of catechin The single effects of citrate and 215

acetate on growth were studied by using four different CDM variants that were prepared 216

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by addition or omission of one or both of these acids in the formulation The 217

dependence of growth patterns on pH was relevant to subsequently interpret if catechin 218

effects were also pH-dependent Therefore we evaluated this effect by adjusting the 219

initial medium pH at 55 or 65 in absence of catechin 220

The growth patterns of Lact plantarum RM71 in the four CDM variants at pH 221

55 or 65 are shown in Fig 1 Lact plantarum RM71 grew well in absence of both 222

citrate and acetate (panels A and E) The presence of these acids in the medium 223

prolonged the lag phase (panels B and F) and elicited a biphasic growth pattern that did 224

only occur in the pH 55 CDM (panels B and C) It appeared that citrate was the only 225

acid leading to this growth profile because the lag phase prolongation (panels B C F 226

and G) as well as was biphasic growth (panels B and C) were observed only in citrate-227

supplemented CDM regardless of acetate inclusion in the medium 228

229

Catechin modifies growth and metabolism of Lactobacillus plantarum RM71 to improve 230

its fermentation performance in CDM 231

232

Results show that the presence of catechin provided advantages to Lact 233

plantarum RM71 for growing in CDM The two positive effects induced by this 234

flavanol were shorthening of the lag phase and higher final cell densities both occurring 235

regardless of the initial pH set-up (Fig1) 236

Examination of Fig 1 revealed that Lact plantarum RM71 achieved higher cell 237

densities in the catechin-supplemented CDM compared to control This growth 238

advantage was more pronounced in the pH 55 than in the pH 65 CDM The increase in 239

cell density reached in the pH 65 CDM upon catechin was of less magnitude than at pH 240

55 and it became equal to the cell density of the control soon after entering in the 241

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stationary phase (panels E to H) Contrarily in cultures grown at initial pH 55 the 242

higher cell density reached upon catechin was prolonged for a longer time during the 243

stationary phase (panel A) or maintained steadily higher than the control (panels B to 244

D) The increase in cell densitiy upon catechin was not a dose-dependent event 245

Compared to controls growth of Lact plantarum RM71 in catechin-containing 246

medium was quicker upon inoculation in the pH 55 CDM (panels A to E) than in the 247

pH 65 medium (panels E to F) Thus cultures carried out ie in complete pH 55 248

CDM supplemented with 100 mg l-1 of catechin shortened the duration of lag phase by 249

8 h (plusmn 087) compared to control (panel B) while a 45 h (plusmn 054) shorthening was 250

observed at an initial pH 65 (panel F) This quicker growth upon inoculation induced 251

by catechin was however not dose-dependent as the lengths of the lag phases were 252

similar at the two concentrations tested (Fig 1) We also observed that catechin induced 253

a shift in the growth pattern of Lact plantarum RM71 in complete pH 55 CDM This 254

pattern changed from a biphasic growth displayed in absence of catechin to a diauxic 255

pattern in presence of the flavanol (panel B) 256

Two key metabolic traits sugar consumption and lactic acid production were 257

monitored to evaluate the effect of catechin on Lact plantarum RM71 performance in 258

complete pH 55 CDM The time course of galactose consumption as well as of lactic 259

acid formation were compared between cultures grown upon 100 mg l-1 of catechin and 260

control cultures lacking the flavanol (Fig 2) The presence of catechin in CDM gave 261

rise to an increased galactose consumption respect to controls (increase of 39 when 262

compared to the control lacking catechin) A quicker galactose consumption and lactic 263

acid production were also observed upon catechin Thus the galactose consumption rate 264

in the first 48 h was of 555 mmol l-1 d-1 for the catechin supplemented cultures in 265

contrast with 344 mmol l-1 d-1 of the control Catechin had little effect on the maximum 266

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rate of galactose consumption as it was in the period measured and assuming steady-267

state conditions similar in presence ( 105 plusmn 006 mmol l-1 h-1) or in absence (095 plusmn 268

005 mmol l-1 h-1) of the flavanol Growth was improved by the presence of catechin in 269

the medium (Fig 2) confirming the above results obtained in cultures carried out on 270

microtitre plates (Fig 1) (please note that as mentioned in the Material and Methods 271

section the optical densities of experiments displayed in Fig 1 and Fig 2 are different) 272

273

The fermentation performance of Lactobacillus plantarum RM71 in wine-resembling 274

medium and basal fermentation medium is improved by catechin 275

276

To test the effects of catechin in media relevant to practical fermentation 277

processes firstly a wine-resembling medium set up at an initial pH of 38 was 278

fermented with Lact plantarum RM71 strain This fermentation was monitored for two 279

days time needed for this bacterium to achieve its maximum log increase in this 280

medium Sugar consumption malic acid degradation and lactic acid production were 281

assayed daily in catechin-lacking or catechin-supplemented (100 mg l-1) media Lact 282

plantarum RM71 metabolized glucose and fructose simultaneously in both media but 283

the total sugar consumption was higher in the medium supplemented with catechin 284

compared to control (1877 vs 2149 mmol l-1 See Table 1) 285

The presence of catechin clearly accelerated sugar consumption at the initial 286

stage of fermentation (0-24 h) as it could be seen for glucose and fructose (Fig 3) 287

which proceeded about ten and six times faster respectively than their controls (Table 288

1) The mean consumption rates of glucose and fructose per day during the entire 289

fermentation period were of 545 mmol l-1 d -1 for glucose (529 for fructose) upon 290

catechin instead of 45 mmol l-1 d -1 for glucose (48 for fructose) in absence of the 291

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flavanol The fermentation of malic acid was faster than that of sugars and completed in 292

only one day A sample taken at ten hours from the onset of fermentation showed that 293

catechin clearly sped up the consumption of malic acid (Fig 3) Thus at this time (10 294

h) 613 of the total malic acid added (715 mmol l-1) was already consumed by Lact 295

plantarum RM71 in presence of catechin in contrast with the significantly lower 293 296

(335 mmol l-1) malic acid consumption by the control culture 297

Compared to control the presence of catechin gave rise to an increase of lactic 298

acid production as it is reflected by the daily and total amounts produced (Table 1) The 299

difference in lactic acid production was specially marked at the onset of fermentation 300

which could be expected because of the accelerated malic acid and sugar consumption 301

in the catechin supplemented cultures 302

To test if catechin exerted similar effects on Lact plantarum plant-derived 303

fermentations different from wine conditions a medium named BFM was prepared The 304

medium was devoid of wine characteristic components ie tartaric acid ethanol and 305

acetic acid as these were not used by Lact plantarum RM71 in the fermentation of wine 306

resembling medium (not shown) Furthermore glucose and fructose were replaced by 307

galactose (1) to investigate if catechin affected the fermentation of other sugars 308

Galactose is present in the diet and is usually present in dietary fibre from vegetables 309

and fruits such as the arabinogalactan which has been shown to increase the 310

Lactobacillus spp population in the human intestine (Robinson Feirtag amp Slavin 311

(2001) 312

As it can be observed in Table 2 the presence of catechin stimulated the growth 313

of Lact plantarum RM71 in BFM The patterns of malic acid degradation and galactose 314

utilization are shown in Fig 4 The fermentation of malic acid was completed in two 315

days regardless catechin supplementation however the presence of this flavanol clearly 316

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sped up the consumption of this acid Thus during the first day of fermentation 598 317

of the total malic acid added was consumed by Lact plantarum RM71 in presence of 318

catechin in contrast with the significantly lower 137 by the control culture 319

We observed that the extent of sugar utilization by Lact plantarum RM71 was 320

increased upon catechin (176 vs 2914 mmol l-1) In addition catechin promoted a faster 321

galactose consumption respect to controls The mean consumption rate of galactose per 322

day during the entire fermentation period was of 971 mmol l-1 d -1 upon catechin 323

instead of 586 mmol l-1 d -1 in absence of the flavanol Examination of Table 2 revealed 324

that the presence of catechin promoted earlier galactose utilization Thus at the initial 325

stage of fermentation (0-24 h) sugar consumption proceeded at a diminished rate of 326

084 mmol l-1 d-1 in the control cultures compared to a much faster rate of 435 mmol l-1 327

d-1 upon catechin Lactic acid production was also monitored as indicator of the 328

fermentation performance of Lact plantarum RM71 As it occurred in the wine-329

resembling medium in BFM daily and total lactic acid productions (as well as lactic 330

acid yields) were higher upon catechin (Table 2) being these increases more 331

pronounced at the onset of fermentation (0-24h) 332

333

Catechin is not metabolized by Lactobacillus plantarum RM71 334

335

Spectrometric analysis was carried out to search for potential catechin 336

degradationmetabolism resulting from the growth of Lact plantarum RM71 337

Supernatants from stationary phase cultures of Lact plantarum RM71 grown in pH 55 338

CDM wine- resembling medium or BFM upon 100 mg l-1 of catechin were compared 339

with cell-free negative controls incubated under the same conditions Experiments 340

performed in CDM appeared in Fig 5 which displayed practically no differences in the 341

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catechin peak eluting at 1817 min among HPLC chromatograms of cell-free control 342

and culture supernatants after 40 h incubation in CDM 343

344

DISCUSSION 345

346

The effects of phenolics compounds on lactic acid bacteria have been reported to 347

be rather variable (For a recent review see Rodriacuteguez et al 2009) This variability 348

depends on the compound (Vivas et al 1997 Campos et al 2003 Figueiredo et al 349

2008) the strain under study (Figueiredo et al 2008) or the medium used for growth 350

(Alberto et al 2001) Up to date some authors have associated the positive effects of 351

food relevant phenolic compounds on growth of lactic acid bacteria to the degradation 352

of the compound Thus the growth stimulating effects of gallate on O oeni (Vivas et al 353

1997) or Lact hilgardii (Alberto et al 2001) were reported to be dose-dependent and 354

suggested to be linked to gallate degradation In the case of anthocyanins it was 355

suggested that glucose moiety was used as energy source to justify the positive effect on 356

the growth of O oeni (Vivas et al 1997) 357

In this study it was observed that most of the catechin remained in the media 358

after fermentation with Lact plantarum RM71 This result was interpreted as this 359

bacterium did not degrade the largest catechin bulk otherwise the catechin peak would 360

have been disappeared or greatly diminished and new compounds would have been 361

appeared Consequently the metabolic and growth stimulations promoted by catechin 362

can not be ascribed to catechin metabolism by Lact plantarum RM 71 These results 363

are in agreement with those previously reported for O oeni by Reguant et al (2000) 364

who also uncoupled the stimulation of growth triggered by catechin from the 365

metabolism of this flavanol Since O oeni grows better in absence of oxygen these 366

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authors suggested that the oxygen scavenging ability of flavonoids tested including 367

catechin favoured the growth stimulating effects observed Contrarily other authors 368

could not apply the same reasoning as they found that catechin or epicatechin showed 369

neutral effects on the growth of O oeni and Lact hilgardii (Figueiredo et al 2008) It 370

has been recently reported that the radical scavenging capacity of catechins increased 371

with the pH of the medium (Muzolf et al 2008) however in this work the positive 372

effects of catechin on the growth of Lact plantarum were more pronounced at the lower 373

pH and not dependent on the dose of catechin used Therefore the positive influence of 374

catechin on growth found here can hardly be ascribed to its radical scavenging capacity 375

By time course of sugar consumption data tracking experiments it is shown that 376

at least in the case of Lact plantarum RM71 catechin stimulates growth by promoting 377

quicker sugar consumption increasing the extension of sugar utilization and stimulating 378

malic acid decarboxylation Clearly this is a different mode of action of catechin to that 379

suggested by this flavanol on Lact hilgardii where the growth stimulating effects were 380

linked to catechin degradation (Alberto et al 2001 Figueiredo et al 2008) or to that 381

suggested by Reguant et al (2000) for O oeni who justified the stimulating effects on 382

the antioxidant properties of catechin 383

The beneficial effects on growth were observed regardless of the catechin 384

concentration examined which support again the hypotheses that growth stimulation is 385

uncoupled from catechin degradation The lack of dose dependent effects is in 386

agreement with the weak bactericidal activity (Nakayama et al 1998) resulting from the 387

shallow location close to the phospholipidwater interface previously reported for 388

catechin (Caturla et al 2003) Otherwise an increased bactericidal effect would be 389

expected at increased catechin concentrations as it occurs with galloylated catechins 390

which possess a higher insertion capacity in the membrane (Ikagai et al 1993 Caturla 391

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et al 2003) Although the growth advantages were observed regardless the initial pH 392

values they were more pronounced in the pH 55 CDM which could indicate a stronger 393

interaction of catechin with membrane phospholipids at this lower pH than at pH 65 394

According to previous reports (Kajiya et al 2001 Caturla et al 2003) catechin 395

does not enter the cell so it is probable that the effects observed on growth and 396

metabolism were caused by alteration of the biophysical properties of the membrane of 397

Lact plantarum RM71 It is possible that catechin after had caused its biological effect 398

could had left the membrane by passive diffusion along time a process that has been 399

previously described for other flavonoids such as quercetin (Movileanu et al 2000) 400

Thus catechin once located in the membrane could alter the function of proteins 401

associated with the bilayer and affect the transport of materials across it Sugar and 402

malate transporters seem to be affected proteins accordingly to the changes in sugar and 403

malic acid consumption observed here upon catechin The citrate transport system could 404

be affected as well since catechin triggered variations in the growth patterns displayed 405

by Lact plantarum RM71 on citrate supplemented CDM Experiments are currently 406

under way to determine if catechin alters some biophysical properties of the membrane 407

of Lact plantarum and affects these transport systems 408

Citrate prolonged lag phase and induced sequential growth behaviour in CDM 409

The scope of this study was not to investigate this behaviour however the long lag 410

phase observed upon citrate indicates that there might be some limitations on the 411

substrate utilization apparatus at the onset of cultures which is usual when growing in 412

mixed-substrate environments (Daae et al 1998) The biphasic growth could be due to a 413

sequential metabolism of carbon sources since this behaviour was only observed at an 414

initial pH of 55 which is close to the optimum pH range for citrate metabolism in Lact 415

plantarum (Palles et al 1998) This type of sequential metabolism is not unprecedent 416

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for Lactobacillus ssp Thus diauxic growth has been described during growth also 417

under sugar limitation on a mixture of sugars and fructooligosaccharides (FOS) (Goh et 418

al 2007) and glucose and citrate (de Figueroa et al 1996) Catechin shorthened the lag 419

phase so it is probable that this flavanol relieved limitations in substrate utilization 420

Moreover in complete pH 55 CDM catechin replaced the biphasic growth by a diauxic 421

growth where a clear diauxic lag was evident This growth pattern is usually associated 422

to a better control of substrate assimilation (Daae et al 1998) so if catechin as it is 423

suspected inferred a more efficient substrate uptake a diauxic lag would be necessary 424

to fit the enzyme synthesis for the utilization of the second substrate 425

The results obtained in wine resembling medium or BFM revealed that catechin 426

sped-up malolactic fermentation stimulated the consumption of several sugars as well 427

as the lactic acid formation thus providing growth advantages to Lact plantarum 428

RM71 in these media Growth stimulation linked to malolactic fermentation is known to 429

be caused by its coupling to ATP synthesis by either the entry through F(H+)-ATPase 430

of environmental protons generated in the malate decarboxilation process and the 431

malatelactate antiport coupled also to F(H+)-ATPase Accordingly the growth 432

improvement of Lact plantarum RM71 upon catechin should not be surprising in view 433

of the accelerated malolactic fermentation and lactic acid production provided by the 434

flavanol Similar effects have been also described for the growth of O oeni CECT 435

4100T where this flavanol was found to stimulate the malolactic fermentation 436

development (Reguant et al 2000) Although malolactic fermentation is classically 437

associated to winemaking the importance of the growth benefit obtained by Lact 438

plantarum from the utilization of malic acid should not be restricted to the wine 439

environment but extended to other fermented food plants as recently reported (Plumed-440

Ferrer et al 2008) These authors found a significantly higher growth of Lact 441

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plantarum in cucumber juice compared to the rich MRS medium and highlighted the 442

crucial importance of the higher decarboxylation of malic acid in this stimulation 443

In summary the results presented here show that catechin improved malic acid 444

degradation fermentation of several sugars and therefore Lact plantarum RM71 445

growth which are desirable traits for a successful performance of this species as starter 446

in several food plant fermentations These stimulations are not linked to catechin 447

degradation 448

449

Acknowledgements 450

451

This work was supported by grants AGL2005-000470 and AGL2008-001052 (CICYT) 452

and 200670M058 (CAM) J A Curiel is a recipient of a fellowship from Ministerio de 453

Ciencia e Innovacioacuten (MICINN) 454

455

REFERENCES 456

457

Adlerberth I Ahrne S Johansson ML Molin G Hanson LA and Wold AE 458

(1996) A mannose specific adherence mechanism in Lactobacillus plantarum 459

conferring binding to the human colonic cell line HT-29 Appl Environ 460

Microbiol 62 2244-2251 461

462

Alberto MR Fariacuteas ME and Manca de Nadra MC (2001) Effect of gallic acid and 463

catechin on Lactobacillus hilgardii 5w growth and metabolism of organics 464

compounds J Agr Food Chem 49 4359-4363 465

466

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Amarowicz R Carle R Dongowski G Durazzo A Galensa R Kammerer D 467

Maiani G and Piskula MK (2009) Influence of postharvest processing and 468

storage on the content of phenolic acids and flavonoids in foods Mol Nutr Food 469

Res doi 101002mnfr200700486 470

471

Campos FM Couto JA and Hogg TA (2003) Influence of phenolics acids on 472

growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii J Appl 473

Microbiol 94 167-174 474

475

Caturla N Vera-Samper E Villalaiacuten J Reyes Mateo C and Micol V (2003) The 476

relationship between the antioxidant and the antibacterial properties of 477

galloylated catechins and the structure of phospholipids membranes Free Rad 478

Biol Med 34 648-662 479

480

Crespy V and Williamson G (2004) A review of the health effects of green tea 481

catechins in in vivo animal models J Nutr 134 3431-3440 482

483

Daae EB and Ison AP (1998) A simple structured model describing the growth of 484

Streptomyces lividans Biotechnol Bioeng 58 263-266 485

486

de Figueroa R M Benito de Caacuterdenas I L Sesma F Alvarez F de Ruiz Holgado 487

A P and Oliver G (1996) Inducible transport of citrate in Lactobacillus 488

rhamnosus ATCC 7469 J Appl Microbiol 81 348-354 489

490

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de Man J C Rogosa M and Sharpe M E (1960) A medium for the cultivation of 491

lactobacilli J Appl Microbiol 23 130-135 492

493

Figueiredo AR Campos F de Freitas V Hogg T and Couto JA (2008) Effect of 494

phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus 495

oeni and Lactobacillus hilgardii Food Microbiol 25 105-12 496

497

Goh Y J Lee J H and Hutkins R W (2007) Functional analysis of the 498

fructooligosaccharide utilization operon in Lactobacillus paracasei 1195 Appl 499

Environ Microbiol 73 5716-5724 500

501

Hussein L Medina A Barrionuevo A Lammuela-Raventos RM and Andres-502

Lacueva C (2009) Normal distribution of urinary polyphenol excretion among 503

Egyptian males 7-14 years old and changes following nutritional intervention 504

with tomato juice (Lycopersicon esculentum) Int J Food Sci Nutr 60 302 ndash 311 505

506

Ikagai H Nakae T Hara Y and Shimamura T (1993) Bactericidal catechins 507

damage the lipid bilayer Biochim Biophys Acta 1147 132-136 508

509

Kajiya K S Kumazawa and Nakayama T (2001) Steric effects on the interaction of 510

tea catechins with lipid bilayers Biosci Biotechnol Biochem 65 2638-2643 511

512

Kostinek M Ban-Koffi L Ottah-Atikpo M Teniola D Schillinger U Holzapfel 513

WH and Franz C (2008) Diversity of predominant lactic acid bacteria 514

associated with cocoa fermentation in Nigeria Curr Microbiol 56 306-314 515

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1 H2SO4 aqueous solution as mobile phase was applied to an Aminexreg HPX-87H 192

column (300 mm x 78 mm) (BIO-RAD) at a 06 mlmin flow rate at 35 ordmC Samples 193

were filtered through a 045-microm polyvinylidene difluoride filter diluted two-fold and 194

injected in duplicated Galactose glucose or fructose detection was performed at 210 195

nm (refraction index detector) and L-lactic and L-malic acids at 380 nm (diode array 196

detector) Sugar and organic acid standards were used for the identification of the HPLC 197

peaks on the chromatograms and for the estimation of their concentration by a 198

calibration curve 199

200

RESULTS 201

202

Growth behaviour of Lactobacillus plantarum RM71 in control CDM medium 203

204

To study the catechin effects on the fermentation performance of Lact 205

plantarum RM71 growth and the time course of sugar consumption were monitored in 206

a chemical defined medium designed for Lact plantarum With the use of CDM it was 207

ensured that catechin was the only flavonoid in the medium and thus the effects on 208

growth could be more easily ascribed to this flavanol Furthermore use of CDM would 209

facilitate the evaluation of potential catechin degradation products As a model growth 210

sugar for CDM experiments we used galactose which is abundant in vegetable fibre and 211

the diet On this sugar cells grow more slowly than ie glucose and the changes 212

produced by catechin could be more properly monitored 213

Before studying catechin effects we firstly examined the growth behaviour of 214

Lact plantarum RM71 in CDM in absence of catechin The single effects of citrate and 215

acetate on growth were studied by using four different CDM variants that were prepared 216

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by addition or omission of one or both of these acids in the formulation The 217

dependence of growth patterns on pH was relevant to subsequently interpret if catechin 218

effects were also pH-dependent Therefore we evaluated this effect by adjusting the 219

initial medium pH at 55 or 65 in absence of catechin 220

The growth patterns of Lact plantarum RM71 in the four CDM variants at pH 221

55 or 65 are shown in Fig 1 Lact plantarum RM71 grew well in absence of both 222

citrate and acetate (panels A and E) The presence of these acids in the medium 223

prolonged the lag phase (panels B and F) and elicited a biphasic growth pattern that did 224

only occur in the pH 55 CDM (panels B and C) It appeared that citrate was the only 225

acid leading to this growth profile because the lag phase prolongation (panels B C F 226

and G) as well as was biphasic growth (panels B and C) were observed only in citrate-227

supplemented CDM regardless of acetate inclusion in the medium 228

229

Catechin modifies growth and metabolism of Lactobacillus plantarum RM71 to improve 230

its fermentation performance in CDM 231

232

Results show that the presence of catechin provided advantages to Lact 233

plantarum RM71 for growing in CDM The two positive effects induced by this 234

flavanol were shorthening of the lag phase and higher final cell densities both occurring 235

regardless of the initial pH set-up (Fig1) 236

Examination of Fig 1 revealed that Lact plantarum RM71 achieved higher cell 237

densities in the catechin-supplemented CDM compared to control This growth 238

advantage was more pronounced in the pH 55 than in the pH 65 CDM The increase in 239

cell density reached in the pH 65 CDM upon catechin was of less magnitude than at pH 240

55 and it became equal to the cell density of the control soon after entering in the 241

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stationary phase (panels E to H) Contrarily in cultures grown at initial pH 55 the 242

higher cell density reached upon catechin was prolonged for a longer time during the 243

stationary phase (panel A) or maintained steadily higher than the control (panels B to 244

D) The increase in cell densitiy upon catechin was not a dose-dependent event 245

Compared to controls growth of Lact plantarum RM71 in catechin-containing 246

medium was quicker upon inoculation in the pH 55 CDM (panels A to E) than in the 247

pH 65 medium (panels E to F) Thus cultures carried out ie in complete pH 55 248

CDM supplemented with 100 mg l-1 of catechin shortened the duration of lag phase by 249

8 h (plusmn 087) compared to control (panel B) while a 45 h (plusmn 054) shorthening was 250

observed at an initial pH 65 (panel F) This quicker growth upon inoculation induced 251

by catechin was however not dose-dependent as the lengths of the lag phases were 252

similar at the two concentrations tested (Fig 1) We also observed that catechin induced 253

a shift in the growth pattern of Lact plantarum RM71 in complete pH 55 CDM This 254

pattern changed from a biphasic growth displayed in absence of catechin to a diauxic 255

pattern in presence of the flavanol (panel B) 256

Two key metabolic traits sugar consumption and lactic acid production were 257

monitored to evaluate the effect of catechin on Lact plantarum RM71 performance in 258

complete pH 55 CDM The time course of galactose consumption as well as of lactic 259

acid formation were compared between cultures grown upon 100 mg l-1 of catechin and 260

control cultures lacking the flavanol (Fig 2) The presence of catechin in CDM gave 261

rise to an increased galactose consumption respect to controls (increase of 39 when 262

compared to the control lacking catechin) A quicker galactose consumption and lactic 263

acid production were also observed upon catechin Thus the galactose consumption rate 264

in the first 48 h was of 555 mmol l-1 d-1 for the catechin supplemented cultures in 265

contrast with 344 mmol l-1 d-1 of the control Catechin had little effect on the maximum 266

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rate of galactose consumption as it was in the period measured and assuming steady-267

state conditions similar in presence ( 105 plusmn 006 mmol l-1 h-1) or in absence (095 plusmn 268

005 mmol l-1 h-1) of the flavanol Growth was improved by the presence of catechin in 269

the medium (Fig 2) confirming the above results obtained in cultures carried out on 270

microtitre plates (Fig 1) (please note that as mentioned in the Material and Methods 271

section the optical densities of experiments displayed in Fig 1 and Fig 2 are different) 272

273

The fermentation performance of Lactobacillus plantarum RM71 in wine-resembling 274

medium and basal fermentation medium is improved by catechin 275

276

To test the effects of catechin in media relevant to practical fermentation 277

processes firstly a wine-resembling medium set up at an initial pH of 38 was 278

fermented with Lact plantarum RM71 strain This fermentation was monitored for two 279

days time needed for this bacterium to achieve its maximum log increase in this 280

medium Sugar consumption malic acid degradation and lactic acid production were 281

assayed daily in catechin-lacking or catechin-supplemented (100 mg l-1) media Lact 282

plantarum RM71 metabolized glucose and fructose simultaneously in both media but 283

the total sugar consumption was higher in the medium supplemented with catechin 284

compared to control (1877 vs 2149 mmol l-1 See Table 1) 285

The presence of catechin clearly accelerated sugar consumption at the initial 286

stage of fermentation (0-24 h) as it could be seen for glucose and fructose (Fig 3) 287

which proceeded about ten and six times faster respectively than their controls (Table 288

1) The mean consumption rates of glucose and fructose per day during the entire 289

fermentation period were of 545 mmol l-1 d -1 for glucose (529 for fructose) upon 290

catechin instead of 45 mmol l-1 d -1 for glucose (48 for fructose) in absence of the 291

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flavanol The fermentation of malic acid was faster than that of sugars and completed in 292

only one day A sample taken at ten hours from the onset of fermentation showed that 293

catechin clearly sped up the consumption of malic acid (Fig 3) Thus at this time (10 294

h) 613 of the total malic acid added (715 mmol l-1) was already consumed by Lact 295

plantarum RM71 in presence of catechin in contrast with the significantly lower 293 296

(335 mmol l-1) malic acid consumption by the control culture 297

Compared to control the presence of catechin gave rise to an increase of lactic 298

acid production as it is reflected by the daily and total amounts produced (Table 1) The 299

difference in lactic acid production was specially marked at the onset of fermentation 300

which could be expected because of the accelerated malic acid and sugar consumption 301

in the catechin supplemented cultures 302

To test if catechin exerted similar effects on Lact plantarum plant-derived 303

fermentations different from wine conditions a medium named BFM was prepared The 304

medium was devoid of wine characteristic components ie tartaric acid ethanol and 305

acetic acid as these were not used by Lact plantarum RM71 in the fermentation of wine 306

resembling medium (not shown) Furthermore glucose and fructose were replaced by 307

galactose (1) to investigate if catechin affected the fermentation of other sugars 308

Galactose is present in the diet and is usually present in dietary fibre from vegetables 309

and fruits such as the arabinogalactan which has been shown to increase the 310

Lactobacillus spp population in the human intestine (Robinson Feirtag amp Slavin 311

(2001) 312

As it can be observed in Table 2 the presence of catechin stimulated the growth 313

of Lact plantarum RM71 in BFM The patterns of malic acid degradation and galactose 314

utilization are shown in Fig 4 The fermentation of malic acid was completed in two 315

days regardless catechin supplementation however the presence of this flavanol clearly 316

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sped up the consumption of this acid Thus during the first day of fermentation 598 317

of the total malic acid added was consumed by Lact plantarum RM71 in presence of 318

catechin in contrast with the significantly lower 137 by the control culture 319

We observed that the extent of sugar utilization by Lact plantarum RM71 was 320

increased upon catechin (176 vs 2914 mmol l-1) In addition catechin promoted a faster 321

galactose consumption respect to controls The mean consumption rate of galactose per 322

day during the entire fermentation period was of 971 mmol l-1 d -1 upon catechin 323

instead of 586 mmol l-1 d -1 in absence of the flavanol Examination of Table 2 revealed 324

that the presence of catechin promoted earlier galactose utilization Thus at the initial 325

stage of fermentation (0-24 h) sugar consumption proceeded at a diminished rate of 326

084 mmol l-1 d-1 in the control cultures compared to a much faster rate of 435 mmol l-1 327

d-1 upon catechin Lactic acid production was also monitored as indicator of the 328

fermentation performance of Lact plantarum RM71 As it occurred in the wine-329

resembling medium in BFM daily and total lactic acid productions (as well as lactic 330

acid yields) were higher upon catechin (Table 2) being these increases more 331

pronounced at the onset of fermentation (0-24h) 332

333

Catechin is not metabolized by Lactobacillus plantarum RM71 334

335

Spectrometric analysis was carried out to search for potential catechin 336

degradationmetabolism resulting from the growth of Lact plantarum RM71 337

Supernatants from stationary phase cultures of Lact plantarum RM71 grown in pH 55 338

CDM wine- resembling medium or BFM upon 100 mg l-1 of catechin were compared 339

with cell-free negative controls incubated under the same conditions Experiments 340

performed in CDM appeared in Fig 5 which displayed practically no differences in the 341

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catechin peak eluting at 1817 min among HPLC chromatograms of cell-free control 342

and culture supernatants after 40 h incubation in CDM 343

344

DISCUSSION 345

346

The effects of phenolics compounds on lactic acid bacteria have been reported to 347

be rather variable (For a recent review see Rodriacuteguez et al 2009) This variability 348

depends on the compound (Vivas et al 1997 Campos et al 2003 Figueiredo et al 349

2008) the strain under study (Figueiredo et al 2008) or the medium used for growth 350

(Alberto et al 2001) Up to date some authors have associated the positive effects of 351

food relevant phenolic compounds on growth of lactic acid bacteria to the degradation 352

of the compound Thus the growth stimulating effects of gallate on O oeni (Vivas et al 353

1997) or Lact hilgardii (Alberto et al 2001) were reported to be dose-dependent and 354

suggested to be linked to gallate degradation In the case of anthocyanins it was 355

suggested that glucose moiety was used as energy source to justify the positive effect on 356

the growth of O oeni (Vivas et al 1997) 357

In this study it was observed that most of the catechin remained in the media 358

after fermentation with Lact plantarum RM71 This result was interpreted as this 359

bacterium did not degrade the largest catechin bulk otherwise the catechin peak would 360

have been disappeared or greatly diminished and new compounds would have been 361

appeared Consequently the metabolic and growth stimulations promoted by catechin 362

can not be ascribed to catechin metabolism by Lact plantarum RM 71 These results 363

are in agreement with those previously reported for O oeni by Reguant et al (2000) 364

who also uncoupled the stimulation of growth triggered by catechin from the 365

metabolism of this flavanol Since O oeni grows better in absence of oxygen these 366

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authors suggested that the oxygen scavenging ability of flavonoids tested including 367

catechin favoured the growth stimulating effects observed Contrarily other authors 368

could not apply the same reasoning as they found that catechin or epicatechin showed 369

neutral effects on the growth of O oeni and Lact hilgardii (Figueiredo et al 2008) It 370

has been recently reported that the radical scavenging capacity of catechins increased 371

with the pH of the medium (Muzolf et al 2008) however in this work the positive 372

effects of catechin on the growth of Lact plantarum were more pronounced at the lower 373

pH and not dependent on the dose of catechin used Therefore the positive influence of 374

catechin on growth found here can hardly be ascribed to its radical scavenging capacity 375

By time course of sugar consumption data tracking experiments it is shown that 376

at least in the case of Lact plantarum RM71 catechin stimulates growth by promoting 377

quicker sugar consumption increasing the extension of sugar utilization and stimulating 378

malic acid decarboxylation Clearly this is a different mode of action of catechin to that 379

suggested by this flavanol on Lact hilgardii where the growth stimulating effects were 380

linked to catechin degradation (Alberto et al 2001 Figueiredo et al 2008) or to that 381

suggested by Reguant et al (2000) for O oeni who justified the stimulating effects on 382

the antioxidant properties of catechin 383

The beneficial effects on growth were observed regardless of the catechin 384

concentration examined which support again the hypotheses that growth stimulation is 385

uncoupled from catechin degradation The lack of dose dependent effects is in 386

agreement with the weak bactericidal activity (Nakayama et al 1998) resulting from the 387

shallow location close to the phospholipidwater interface previously reported for 388

catechin (Caturla et al 2003) Otherwise an increased bactericidal effect would be 389

expected at increased catechin concentrations as it occurs with galloylated catechins 390

which possess a higher insertion capacity in the membrane (Ikagai et al 1993 Caturla 391

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et al 2003) Although the growth advantages were observed regardless the initial pH 392

values they were more pronounced in the pH 55 CDM which could indicate a stronger 393

interaction of catechin with membrane phospholipids at this lower pH than at pH 65 394

According to previous reports (Kajiya et al 2001 Caturla et al 2003) catechin 395

does not enter the cell so it is probable that the effects observed on growth and 396

metabolism were caused by alteration of the biophysical properties of the membrane of 397

Lact plantarum RM71 It is possible that catechin after had caused its biological effect 398

could had left the membrane by passive diffusion along time a process that has been 399

previously described for other flavonoids such as quercetin (Movileanu et al 2000) 400

Thus catechin once located in the membrane could alter the function of proteins 401

associated with the bilayer and affect the transport of materials across it Sugar and 402

malate transporters seem to be affected proteins accordingly to the changes in sugar and 403

malic acid consumption observed here upon catechin The citrate transport system could 404

be affected as well since catechin triggered variations in the growth patterns displayed 405

by Lact plantarum RM71 on citrate supplemented CDM Experiments are currently 406

under way to determine if catechin alters some biophysical properties of the membrane 407

of Lact plantarum and affects these transport systems 408

Citrate prolonged lag phase and induced sequential growth behaviour in CDM 409

The scope of this study was not to investigate this behaviour however the long lag 410

phase observed upon citrate indicates that there might be some limitations on the 411

substrate utilization apparatus at the onset of cultures which is usual when growing in 412

mixed-substrate environments (Daae et al 1998) The biphasic growth could be due to a 413

sequential metabolism of carbon sources since this behaviour was only observed at an 414

initial pH of 55 which is close to the optimum pH range for citrate metabolism in Lact 415

plantarum (Palles et al 1998) This type of sequential metabolism is not unprecedent 416

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18

for Lactobacillus ssp Thus diauxic growth has been described during growth also 417

under sugar limitation on a mixture of sugars and fructooligosaccharides (FOS) (Goh et 418

al 2007) and glucose and citrate (de Figueroa et al 1996) Catechin shorthened the lag 419

phase so it is probable that this flavanol relieved limitations in substrate utilization 420

Moreover in complete pH 55 CDM catechin replaced the biphasic growth by a diauxic 421

growth where a clear diauxic lag was evident This growth pattern is usually associated 422

to a better control of substrate assimilation (Daae et al 1998) so if catechin as it is 423

suspected inferred a more efficient substrate uptake a diauxic lag would be necessary 424

to fit the enzyme synthesis for the utilization of the second substrate 425

The results obtained in wine resembling medium or BFM revealed that catechin 426

sped-up malolactic fermentation stimulated the consumption of several sugars as well 427

as the lactic acid formation thus providing growth advantages to Lact plantarum 428

RM71 in these media Growth stimulation linked to malolactic fermentation is known to 429

be caused by its coupling to ATP synthesis by either the entry through F(H+)-ATPase 430

of environmental protons generated in the malate decarboxilation process and the 431

malatelactate antiport coupled also to F(H+)-ATPase Accordingly the growth 432

improvement of Lact plantarum RM71 upon catechin should not be surprising in view 433

of the accelerated malolactic fermentation and lactic acid production provided by the 434

flavanol Similar effects have been also described for the growth of O oeni CECT 435

4100T where this flavanol was found to stimulate the malolactic fermentation 436

development (Reguant et al 2000) Although malolactic fermentation is classically 437

associated to winemaking the importance of the growth benefit obtained by Lact 438

plantarum from the utilization of malic acid should not be restricted to the wine 439

environment but extended to other fermented food plants as recently reported (Plumed-440

Ferrer et al 2008) These authors found a significantly higher growth of Lact 441

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19

plantarum in cucumber juice compared to the rich MRS medium and highlighted the 442

crucial importance of the higher decarboxylation of malic acid in this stimulation 443

In summary the results presented here show that catechin improved malic acid 444

degradation fermentation of several sugars and therefore Lact plantarum RM71 445

growth which are desirable traits for a successful performance of this species as starter 446

in several food plant fermentations These stimulations are not linked to catechin 447

degradation 448

449

Acknowledgements 450

451

This work was supported by grants AGL2005-000470 and AGL2008-001052 (CICYT) 452

and 200670M058 (CAM) J A Curiel is a recipient of a fellowship from Ministerio de 453

Ciencia e Innovacioacuten (MICINN) 454

455

REFERENCES 456

457

Adlerberth I Ahrne S Johansson ML Molin G Hanson LA and Wold AE 458

(1996) A mannose specific adherence mechanism in Lactobacillus plantarum 459

conferring binding to the human colonic cell line HT-29 Appl Environ 460

Microbiol 62 2244-2251 461

462

Alberto MR Fariacuteas ME and Manca de Nadra MC (2001) Effect of gallic acid and 463

catechin on Lactobacillus hilgardii 5w growth and metabolism of organics 464

compounds J Agr Food Chem 49 4359-4363 465

466

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20

Amarowicz R Carle R Dongowski G Durazzo A Galensa R Kammerer D 467

Maiani G and Piskula MK (2009) Influence of postharvest processing and 468

storage on the content of phenolic acids and flavonoids in foods Mol Nutr Food 469

Res doi 101002mnfr200700486 470

471

Campos FM Couto JA and Hogg TA (2003) Influence of phenolics acids on 472

growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii J Appl 473

Microbiol 94 167-174 474

475

Caturla N Vera-Samper E Villalaiacuten J Reyes Mateo C and Micol V (2003) The 476

relationship between the antioxidant and the antibacterial properties of 477

galloylated catechins and the structure of phospholipids membranes Free Rad 478

Biol Med 34 648-662 479

480

Crespy V and Williamson G (2004) A review of the health effects of green tea 481

catechins in in vivo animal models J Nutr 134 3431-3440 482

483

Daae EB and Ison AP (1998) A simple structured model describing the growth of 484

Streptomyces lividans Biotechnol Bioeng 58 263-266 485

486

de Figueroa R M Benito de Caacuterdenas I L Sesma F Alvarez F de Ruiz Holgado 487

A P and Oliver G (1996) Inducible transport of citrate in Lactobacillus 488

rhamnosus ATCC 7469 J Appl Microbiol 81 348-354 489

490

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For Peer Review

21

de Man J C Rogosa M and Sharpe M E (1960) A medium for the cultivation of 491

lactobacilli J Appl Microbiol 23 130-135 492

493

Figueiredo AR Campos F de Freitas V Hogg T and Couto JA (2008) Effect of 494

phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus 495

oeni and Lactobacillus hilgardii Food Microbiol 25 105-12 496

497

Goh Y J Lee J H and Hutkins R W (2007) Functional analysis of the 498

fructooligosaccharide utilization operon in Lactobacillus paracasei 1195 Appl 499

Environ Microbiol 73 5716-5724 500

501

Hussein L Medina A Barrionuevo A Lammuela-Raventos RM and Andres-502

Lacueva C (2009) Normal distribution of urinary polyphenol excretion among 503

Egyptian males 7-14 years old and changes following nutritional intervention 504

with tomato juice (Lycopersicon esculentum) Int J Food Sci Nutr 60 302 ndash 311 505

506

Ikagai H Nakae T Hara Y and Shimamura T (1993) Bactericidal catechins 507

damage the lipid bilayer Biochim Biophys Acta 1147 132-136 508

509

Kajiya K S Kumazawa and Nakayama T (2001) Steric effects on the interaction of 510

tea catechins with lipid bilayers Biosci Biotechnol Biochem 65 2638-2643 511

512

Kostinek M Ban-Koffi L Ottah-Atikpo M Teniola D Schillinger U Holzapfel 513

WH and Franz C (2008) Diversity of predominant lactic acid bacteria 514

associated with cocoa fermentation in Nigeria Curr Microbiol 56 306-314 515

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10

by addition or omission of one or both of these acids in the formulation The 217

dependence of growth patterns on pH was relevant to subsequently interpret if catechin 218

effects were also pH-dependent Therefore we evaluated this effect by adjusting the 219

initial medium pH at 55 or 65 in absence of catechin 220

The growth patterns of Lact plantarum RM71 in the four CDM variants at pH 221

55 or 65 are shown in Fig 1 Lact plantarum RM71 grew well in absence of both 222

citrate and acetate (panels A and E) The presence of these acids in the medium 223

prolonged the lag phase (panels B and F) and elicited a biphasic growth pattern that did 224

only occur in the pH 55 CDM (panels B and C) It appeared that citrate was the only 225

acid leading to this growth profile because the lag phase prolongation (panels B C F 226

and G) as well as was biphasic growth (panels B and C) were observed only in citrate-227

supplemented CDM regardless of acetate inclusion in the medium 228

229

Catechin modifies growth and metabolism of Lactobacillus plantarum RM71 to improve 230

its fermentation performance in CDM 231

232

Results show that the presence of catechin provided advantages to Lact 233

plantarum RM71 for growing in CDM The two positive effects induced by this 234

flavanol were shorthening of the lag phase and higher final cell densities both occurring 235

regardless of the initial pH set-up (Fig1) 236

Examination of Fig 1 revealed that Lact plantarum RM71 achieved higher cell 237

densities in the catechin-supplemented CDM compared to control This growth 238

advantage was more pronounced in the pH 55 than in the pH 65 CDM The increase in 239

cell density reached in the pH 65 CDM upon catechin was of less magnitude than at pH 240

55 and it became equal to the cell density of the control soon after entering in the 241

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stationary phase (panels E to H) Contrarily in cultures grown at initial pH 55 the 242

higher cell density reached upon catechin was prolonged for a longer time during the 243

stationary phase (panel A) or maintained steadily higher than the control (panels B to 244

D) The increase in cell densitiy upon catechin was not a dose-dependent event 245

Compared to controls growth of Lact plantarum RM71 in catechin-containing 246

medium was quicker upon inoculation in the pH 55 CDM (panels A to E) than in the 247

pH 65 medium (panels E to F) Thus cultures carried out ie in complete pH 55 248

CDM supplemented with 100 mg l-1 of catechin shortened the duration of lag phase by 249

8 h (plusmn 087) compared to control (panel B) while a 45 h (plusmn 054) shorthening was 250

observed at an initial pH 65 (panel F) This quicker growth upon inoculation induced 251

by catechin was however not dose-dependent as the lengths of the lag phases were 252

similar at the two concentrations tested (Fig 1) We also observed that catechin induced 253

a shift in the growth pattern of Lact plantarum RM71 in complete pH 55 CDM This 254

pattern changed from a biphasic growth displayed in absence of catechin to a diauxic 255

pattern in presence of the flavanol (panel B) 256

Two key metabolic traits sugar consumption and lactic acid production were 257

monitored to evaluate the effect of catechin on Lact plantarum RM71 performance in 258

complete pH 55 CDM The time course of galactose consumption as well as of lactic 259

acid formation were compared between cultures grown upon 100 mg l-1 of catechin and 260

control cultures lacking the flavanol (Fig 2) The presence of catechin in CDM gave 261

rise to an increased galactose consumption respect to controls (increase of 39 when 262

compared to the control lacking catechin) A quicker galactose consumption and lactic 263

acid production were also observed upon catechin Thus the galactose consumption rate 264

in the first 48 h was of 555 mmol l-1 d-1 for the catechin supplemented cultures in 265

contrast with 344 mmol l-1 d-1 of the control Catechin had little effect on the maximum 266

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rate of galactose consumption as it was in the period measured and assuming steady-267

state conditions similar in presence ( 105 plusmn 006 mmol l-1 h-1) or in absence (095 plusmn 268

005 mmol l-1 h-1) of the flavanol Growth was improved by the presence of catechin in 269

the medium (Fig 2) confirming the above results obtained in cultures carried out on 270

microtitre plates (Fig 1) (please note that as mentioned in the Material and Methods 271

section the optical densities of experiments displayed in Fig 1 and Fig 2 are different) 272

273

The fermentation performance of Lactobacillus plantarum RM71 in wine-resembling 274

medium and basal fermentation medium is improved by catechin 275

276

To test the effects of catechin in media relevant to practical fermentation 277

processes firstly a wine-resembling medium set up at an initial pH of 38 was 278

fermented with Lact plantarum RM71 strain This fermentation was monitored for two 279

days time needed for this bacterium to achieve its maximum log increase in this 280

medium Sugar consumption malic acid degradation and lactic acid production were 281

assayed daily in catechin-lacking or catechin-supplemented (100 mg l-1) media Lact 282

plantarum RM71 metabolized glucose and fructose simultaneously in both media but 283

the total sugar consumption was higher in the medium supplemented with catechin 284

compared to control (1877 vs 2149 mmol l-1 See Table 1) 285

The presence of catechin clearly accelerated sugar consumption at the initial 286

stage of fermentation (0-24 h) as it could be seen for glucose and fructose (Fig 3) 287

which proceeded about ten and six times faster respectively than their controls (Table 288

1) The mean consumption rates of glucose and fructose per day during the entire 289

fermentation period were of 545 mmol l-1 d -1 for glucose (529 for fructose) upon 290

catechin instead of 45 mmol l-1 d -1 for glucose (48 for fructose) in absence of the 291

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flavanol The fermentation of malic acid was faster than that of sugars and completed in 292

only one day A sample taken at ten hours from the onset of fermentation showed that 293

catechin clearly sped up the consumption of malic acid (Fig 3) Thus at this time (10 294

h) 613 of the total malic acid added (715 mmol l-1) was already consumed by Lact 295

plantarum RM71 in presence of catechin in contrast with the significantly lower 293 296

(335 mmol l-1) malic acid consumption by the control culture 297

Compared to control the presence of catechin gave rise to an increase of lactic 298

acid production as it is reflected by the daily and total amounts produced (Table 1) The 299

difference in lactic acid production was specially marked at the onset of fermentation 300

which could be expected because of the accelerated malic acid and sugar consumption 301

in the catechin supplemented cultures 302

To test if catechin exerted similar effects on Lact plantarum plant-derived 303

fermentations different from wine conditions a medium named BFM was prepared The 304

medium was devoid of wine characteristic components ie tartaric acid ethanol and 305

acetic acid as these were not used by Lact plantarum RM71 in the fermentation of wine 306

resembling medium (not shown) Furthermore glucose and fructose were replaced by 307

galactose (1) to investigate if catechin affected the fermentation of other sugars 308

Galactose is present in the diet and is usually present in dietary fibre from vegetables 309

and fruits such as the arabinogalactan which has been shown to increase the 310

Lactobacillus spp population in the human intestine (Robinson Feirtag amp Slavin 311

(2001) 312

As it can be observed in Table 2 the presence of catechin stimulated the growth 313

of Lact plantarum RM71 in BFM The patterns of malic acid degradation and galactose 314

utilization are shown in Fig 4 The fermentation of malic acid was completed in two 315

days regardless catechin supplementation however the presence of this flavanol clearly 316

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sped up the consumption of this acid Thus during the first day of fermentation 598 317

of the total malic acid added was consumed by Lact plantarum RM71 in presence of 318

catechin in contrast with the significantly lower 137 by the control culture 319

We observed that the extent of sugar utilization by Lact plantarum RM71 was 320

increased upon catechin (176 vs 2914 mmol l-1) In addition catechin promoted a faster 321

galactose consumption respect to controls The mean consumption rate of galactose per 322

day during the entire fermentation period was of 971 mmol l-1 d -1 upon catechin 323

instead of 586 mmol l-1 d -1 in absence of the flavanol Examination of Table 2 revealed 324

that the presence of catechin promoted earlier galactose utilization Thus at the initial 325

stage of fermentation (0-24 h) sugar consumption proceeded at a diminished rate of 326

084 mmol l-1 d-1 in the control cultures compared to a much faster rate of 435 mmol l-1 327

d-1 upon catechin Lactic acid production was also monitored as indicator of the 328

fermentation performance of Lact plantarum RM71 As it occurred in the wine-329

resembling medium in BFM daily and total lactic acid productions (as well as lactic 330

acid yields) were higher upon catechin (Table 2) being these increases more 331

pronounced at the onset of fermentation (0-24h) 332

333

Catechin is not metabolized by Lactobacillus plantarum RM71 334

335

Spectrometric analysis was carried out to search for potential catechin 336

degradationmetabolism resulting from the growth of Lact plantarum RM71 337

Supernatants from stationary phase cultures of Lact plantarum RM71 grown in pH 55 338

CDM wine- resembling medium or BFM upon 100 mg l-1 of catechin were compared 339

with cell-free negative controls incubated under the same conditions Experiments 340

performed in CDM appeared in Fig 5 which displayed practically no differences in the 341

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catechin peak eluting at 1817 min among HPLC chromatograms of cell-free control 342

and culture supernatants after 40 h incubation in CDM 343

344

DISCUSSION 345

346

The effects of phenolics compounds on lactic acid bacteria have been reported to 347

be rather variable (For a recent review see Rodriacuteguez et al 2009) This variability 348

depends on the compound (Vivas et al 1997 Campos et al 2003 Figueiredo et al 349

2008) the strain under study (Figueiredo et al 2008) or the medium used for growth 350

(Alberto et al 2001) Up to date some authors have associated the positive effects of 351

food relevant phenolic compounds on growth of lactic acid bacteria to the degradation 352

of the compound Thus the growth stimulating effects of gallate on O oeni (Vivas et al 353

1997) or Lact hilgardii (Alberto et al 2001) were reported to be dose-dependent and 354

suggested to be linked to gallate degradation In the case of anthocyanins it was 355

suggested that glucose moiety was used as energy source to justify the positive effect on 356

the growth of O oeni (Vivas et al 1997) 357

In this study it was observed that most of the catechin remained in the media 358

after fermentation with Lact plantarum RM71 This result was interpreted as this 359

bacterium did not degrade the largest catechin bulk otherwise the catechin peak would 360

have been disappeared or greatly diminished and new compounds would have been 361

appeared Consequently the metabolic and growth stimulations promoted by catechin 362

can not be ascribed to catechin metabolism by Lact plantarum RM 71 These results 363

are in agreement with those previously reported for O oeni by Reguant et al (2000) 364

who also uncoupled the stimulation of growth triggered by catechin from the 365

metabolism of this flavanol Since O oeni grows better in absence of oxygen these 366

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authors suggested that the oxygen scavenging ability of flavonoids tested including 367

catechin favoured the growth stimulating effects observed Contrarily other authors 368

could not apply the same reasoning as they found that catechin or epicatechin showed 369

neutral effects on the growth of O oeni and Lact hilgardii (Figueiredo et al 2008) It 370

has been recently reported that the radical scavenging capacity of catechins increased 371

with the pH of the medium (Muzolf et al 2008) however in this work the positive 372

effects of catechin on the growth of Lact plantarum were more pronounced at the lower 373

pH and not dependent on the dose of catechin used Therefore the positive influence of 374

catechin on growth found here can hardly be ascribed to its radical scavenging capacity 375

By time course of sugar consumption data tracking experiments it is shown that 376

at least in the case of Lact plantarum RM71 catechin stimulates growth by promoting 377

quicker sugar consumption increasing the extension of sugar utilization and stimulating 378

malic acid decarboxylation Clearly this is a different mode of action of catechin to that 379

suggested by this flavanol on Lact hilgardii where the growth stimulating effects were 380

linked to catechin degradation (Alberto et al 2001 Figueiredo et al 2008) or to that 381

suggested by Reguant et al (2000) for O oeni who justified the stimulating effects on 382

the antioxidant properties of catechin 383

The beneficial effects on growth were observed regardless of the catechin 384

concentration examined which support again the hypotheses that growth stimulation is 385

uncoupled from catechin degradation The lack of dose dependent effects is in 386

agreement with the weak bactericidal activity (Nakayama et al 1998) resulting from the 387

shallow location close to the phospholipidwater interface previously reported for 388

catechin (Caturla et al 2003) Otherwise an increased bactericidal effect would be 389

expected at increased catechin concentrations as it occurs with galloylated catechins 390

which possess a higher insertion capacity in the membrane (Ikagai et al 1993 Caturla 391

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et al 2003) Although the growth advantages were observed regardless the initial pH 392

values they were more pronounced in the pH 55 CDM which could indicate a stronger 393

interaction of catechin with membrane phospholipids at this lower pH than at pH 65 394

According to previous reports (Kajiya et al 2001 Caturla et al 2003) catechin 395

does not enter the cell so it is probable that the effects observed on growth and 396

metabolism were caused by alteration of the biophysical properties of the membrane of 397

Lact plantarum RM71 It is possible that catechin after had caused its biological effect 398

could had left the membrane by passive diffusion along time a process that has been 399

previously described for other flavonoids such as quercetin (Movileanu et al 2000) 400

Thus catechin once located in the membrane could alter the function of proteins 401

associated with the bilayer and affect the transport of materials across it Sugar and 402

malate transporters seem to be affected proteins accordingly to the changes in sugar and 403

malic acid consumption observed here upon catechin The citrate transport system could 404

be affected as well since catechin triggered variations in the growth patterns displayed 405

by Lact plantarum RM71 on citrate supplemented CDM Experiments are currently 406

under way to determine if catechin alters some biophysical properties of the membrane 407

of Lact plantarum and affects these transport systems 408

Citrate prolonged lag phase and induced sequential growth behaviour in CDM 409

The scope of this study was not to investigate this behaviour however the long lag 410

phase observed upon citrate indicates that there might be some limitations on the 411

substrate utilization apparatus at the onset of cultures which is usual when growing in 412

mixed-substrate environments (Daae et al 1998) The biphasic growth could be due to a 413

sequential metabolism of carbon sources since this behaviour was only observed at an 414

initial pH of 55 which is close to the optimum pH range for citrate metabolism in Lact 415

plantarum (Palles et al 1998) This type of sequential metabolism is not unprecedent 416

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18

for Lactobacillus ssp Thus diauxic growth has been described during growth also 417

under sugar limitation on a mixture of sugars and fructooligosaccharides (FOS) (Goh et 418

al 2007) and glucose and citrate (de Figueroa et al 1996) Catechin shorthened the lag 419

phase so it is probable that this flavanol relieved limitations in substrate utilization 420

Moreover in complete pH 55 CDM catechin replaced the biphasic growth by a diauxic 421

growth where a clear diauxic lag was evident This growth pattern is usually associated 422

to a better control of substrate assimilation (Daae et al 1998) so if catechin as it is 423

suspected inferred a more efficient substrate uptake a diauxic lag would be necessary 424

to fit the enzyme synthesis for the utilization of the second substrate 425

The results obtained in wine resembling medium or BFM revealed that catechin 426

sped-up malolactic fermentation stimulated the consumption of several sugars as well 427

as the lactic acid formation thus providing growth advantages to Lact plantarum 428

RM71 in these media Growth stimulation linked to malolactic fermentation is known to 429

be caused by its coupling to ATP synthesis by either the entry through F(H+)-ATPase 430

of environmental protons generated in the malate decarboxilation process and the 431

malatelactate antiport coupled also to F(H+)-ATPase Accordingly the growth 432

improvement of Lact plantarum RM71 upon catechin should not be surprising in view 433

of the accelerated malolactic fermentation and lactic acid production provided by the 434

flavanol Similar effects have been also described for the growth of O oeni CECT 435

4100T where this flavanol was found to stimulate the malolactic fermentation 436

development (Reguant et al 2000) Although malolactic fermentation is classically 437

associated to winemaking the importance of the growth benefit obtained by Lact 438

plantarum from the utilization of malic acid should not be restricted to the wine 439

environment but extended to other fermented food plants as recently reported (Plumed-440

Ferrer et al 2008) These authors found a significantly higher growth of Lact 441

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19

plantarum in cucumber juice compared to the rich MRS medium and highlighted the 442

crucial importance of the higher decarboxylation of malic acid in this stimulation 443

In summary the results presented here show that catechin improved malic acid 444

degradation fermentation of several sugars and therefore Lact plantarum RM71 445

growth which are desirable traits for a successful performance of this species as starter 446

in several food plant fermentations These stimulations are not linked to catechin 447

degradation 448

449

Acknowledgements 450

451

This work was supported by grants AGL2005-000470 and AGL2008-001052 (CICYT) 452

and 200670M058 (CAM) J A Curiel is a recipient of a fellowship from Ministerio de 453

Ciencia e Innovacioacuten (MICINN) 454

455

REFERENCES 456

457

Adlerberth I Ahrne S Johansson ML Molin G Hanson LA and Wold AE 458

(1996) A mannose specific adherence mechanism in Lactobacillus plantarum 459

conferring binding to the human colonic cell line HT-29 Appl Environ 460

Microbiol 62 2244-2251 461

462

Alberto MR Fariacuteas ME and Manca de Nadra MC (2001) Effect of gallic acid and 463

catechin on Lactobacillus hilgardii 5w growth and metabolism of organics 464

compounds J Agr Food Chem 49 4359-4363 465

466

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20

Amarowicz R Carle R Dongowski G Durazzo A Galensa R Kammerer D 467

Maiani G and Piskula MK (2009) Influence of postharvest processing and 468

storage on the content of phenolic acids and flavonoids in foods Mol Nutr Food 469

Res doi 101002mnfr200700486 470

471

Campos FM Couto JA and Hogg TA (2003) Influence of phenolics acids on 472

growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii J Appl 473

Microbiol 94 167-174 474

475

Caturla N Vera-Samper E Villalaiacuten J Reyes Mateo C and Micol V (2003) The 476

relationship between the antioxidant and the antibacterial properties of 477

galloylated catechins and the structure of phospholipids membranes Free Rad 478

Biol Med 34 648-662 479

480

Crespy V and Williamson G (2004) A review of the health effects of green tea 481

catechins in in vivo animal models J Nutr 134 3431-3440 482

483

Daae EB and Ison AP (1998) A simple structured model describing the growth of 484

Streptomyces lividans Biotechnol Bioeng 58 263-266 485

486

de Figueroa R M Benito de Caacuterdenas I L Sesma F Alvarez F de Ruiz Holgado 487

A P and Oliver G (1996) Inducible transport of citrate in Lactobacillus 488

rhamnosus ATCC 7469 J Appl Microbiol 81 348-354 489

490

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de Man J C Rogosa M and Sharpe M E (1960) A medium for the cultivation of 491

lactobacilli J Appl Microbiol 23 130-135 492

493

Figueiredo AR Campos F de Freitas V Hogg T and Couto JA (2008) Effect of 494

phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus 495

oeni and Lactobacillus hilgardii Food Microbiol 25 105-12 496

497

Goh Y J Lee J H and Hutkins R W (2007) Functional analysis of the 498

fructooligosaccharide utilization operon in Lactobacillus paracasei 1195 Appl 499

Environ Microbiol 73 5716-5724 500

501

Hussein L Medina A Barrionuevo A Lammuela-Raventos RM and Andres-502

Lacueva C (2009) Normal distribution of urinary polyphenol excretion among 503

Egyptian males 7-14 years old and changes following nutritional intervention 504

with tomato juice (Lycopersicon esculentum) Int J Food Sci Nutr 60 302 ndash 311 505

506

Ikagai H Nakae T Hara Y and Shimamura T (1993) Bactericidal catechins 507

damage the lipid bilayer Biochim Biophys Acta 1147 132-136 508

509

Kajiya K S Kumazawa and Nakayama T (2001) Steric effects on the interaction of 510

tea catechins with lipid bilayers Biosci Biotechnol Biochem 65 2638-2643 511

512

Kostinek M Ban-Koffi L Ottah-Atikpo M Teniola D Schillinger U Holzapfel 513

WH and Franz C (2008) Diversity of predominant lactic acid bacteria 514

associated with cocoa fermentation in Nigeria Curr Microbiol 56 306-314 515

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stationary phase (panels E to H) Contrarily in cultures grown at initial pH 55 the 242

higher cell density reached upon catechin was prolonged for a longer time during the 243

stationary phase (panel A) or maintained steadily higher than the control (panels B to 244

D) The increase in cell densitiy upon catechin was not a dose-dependent event 245

Compared to controls growth of Lact plantarum RM71 in catechin-containing 246

medium was quicker upon inoculation in the pH 55 CDM (panels A to E) than in the 247

pH 65 medium (panels E to F) Thus cultures carried out ie in complete pH 55 248

CDM supplemented with 100 mg l-1 of catechin shortened the duration of lag phase by 249

8 h (plusmn 087) compared to control (panel B) while a 45 h (plusmn 054) shorthening was 250

observed at an initial pH 65 (panel F) This quicker growth upon inoculation induced 251

by catechin was however not dose-dependent as the lengths of the lag phases were 252

similar at the two concentrations tested (Fig 1) We also observed that catechin induced 253

a shift in the growth pattern of Lact plantarum RM71 in complete pH 55 CDM This 254

pattern changed from a biphasic growth displayed in absence of catechin to a diauxic 255

pattern in presence of the flavanol (panel B) 256

Two key metabolic traits sugar consumption and lactic acid production were 257

monitored to evaluate the effect of catechin on Lact plantarum RM71 performance in 258

complete pH 55 CDM The time course of galactose consumption as well as of lactic 259

acid formation were compared between cultures grown upon 100 mg l-1 of catechin and 260

control cultures lacking the flavanol (Fig 2) The presence of catechin in CDM gave 261

rise to an increased galactose consumption respect to controls (increase of 39 when 262

compared to the control lacking catechin) A quicker galactose consumption and lactic 263

acid production were also observed upon catechin Thus the galactose consumption rate 264

in the first 48 h was of 555 mmol l-1 d-1 for the catechin supplemented cultures in 265

contrast with 344 mmol l-1 d-1 of the control Catechin had little effect on the maximum 266

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rate of galactose consumption as it was in the period measured and assuming steady-267

state conditions similar in presence ( 105 plusmn 006 mmol l-1 h-1) or in absence (095 plusmn 268

005 mmol l-1 h-1) of the flavanol Growth was improved by the presence of catechin in 269

the medium (Fig 2) confirming the above results obtained in cultures carried out on 270

microtitre plates (Fig 1) (please note that as mentioned in the Material and Methods 271

section the optical densities of experiments displayed in Fig 1 and Fig 2 are different) 272

273

The fermentation performance of Lactobacillus plantarum RM71 in wine-resembling 274

medium and basal fermentation medium is improved by catechin 275

276

To test the effects of catechin in media relevant to practical fermentation 277

processes firstly a wine-resembling medium set up at an initial pH of 38 was 278

fermented with Lact plantarum RM71 strain This fermentation was monitored for two 279

days time needed for this bacterium to achieve its maximum log increase in this 280

medium Sugar consumption malic acid degradation and lactic acid production were 281

assayed daily in catechin-lacking or catechin-supplemented (100 mg l-1) media Lact 282

plantarum RM71 metabolized glucose and fructose simultaneously in both media but 283

the total sugar consumption was higher in the medium supplemented with catechin 284

compared to control (1877 vs 2149 mmol l-1 See Table 1) 285

The presence of catechin clearly accelerated sugar consumption at the initial 286

stage of fermentation (0-24 h) as it could be seen for glucose and fructose (Fig 3) 287

which proceeded about ten and six times faster respectively than their controls (Table 288

1) The mean consumption rates of glucose and fructose per day during the entire 289

fermentation period were of 545 mmol l-1 d -1 for glucose (529 for fructose) upon 290

catechin instead of 45 mmol l-1 d -1 for glucose (48 for fructose) in absence of the 291

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flavanol The fermentation of malic acid was faster than that of sugars and completed in 292

only one day A sample taken at ten hours from the onset of fermentation showed that 293

catechin clearly sped up the consumption of malic acid (Fig 3) Thus at this time (10 294

h) 613 of the total malic acid added (715 mmol l-1) was already consumed by Lact 295

plantarum RM71 in presence of catechin in contrast with the significantly lower 293 296

(335 mmol l-1) malic acid consumption by the control culture 297

Compared to control the presence of catechin gave rise to an increase of lactic 298

acid production as it is reflected by the daily and total amounts produced (Table 1) The 299

difference in lactic acid production was specially marked at the onset of fermentation 300

which could be expected because of the accelerated malic acid and sugar consumption 301

in the catechin supplemented cultures 302

To test if catechin exerted similar effects on Lact plantarum plant-derived 303

fermentations different from wine conditions a medium named BFM was prepared The 304

medium was devoid of wine characteristic components ie tartaric acid ethanol and 305

acetic acid as these were not used by Lact plantarum RM71 in the fermentation of wine 306

resembling medium (not shown) Furthermore glucose and fructose were replaced by 307

galactose (1) to investigate if catechin affected the fermentation of other sugars 308

Galactose is present in the diet and is usually present in dietary fibre from vegetables 309

and fruits such as the arabinogalactan which has been shown to increase the 310

Lactobacillus spp population in the human intestine (Robinson Feirtag amp Slavin 311

(2001) 312

As it can be observed in Table 2 the presence of catechin stimulated the growth 313

of Lact plantarum RM71 in BFM The patterns of malic acid degradation and galactose 314

utilization are shown in Fig 4 The fermentation of malic acid was completed in two 315

days regardless catechin supplementation however the presence of this flavanol clearly 316

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14

sped up the consumption of this acid Thus during the first day of fermentation 598 317

of the total malic acid added was consumed by Lact plantarum RM71 in presence of 318

catechin in contrast with the significantly lower 137 by the control culture 319

We observed that the extent of sugar utilization by Lact plantarum RM71 was 320

increased upon catechin (176 vs 2914 mmol l-1) In addition catechin promoted a faster 321

galactose consumption respect to controls The mean consumption rate of galactose per 322

day during the entire fermentation period was of 971 mmol l-1 d -1 upon catechin 323

instead of 586 mmol l-1 d -1 in absence of the flavanol Examination of Table 2 revealed 324

that the presence of catechin promoted earlier galactose utilization Thus at the initial 325

stage of fermentation (0-24 h) sugar consumption proceeded at a diminished rate of 326

084 mmol l-1 d-1 in the control cultures compared to a much faster rate of 435 mmol l-1 327

d-1 upon catechin Lactic acid production was also monitored as indicator of the 328

fermentation performance of Lact plantarum RM71 As it occurred in the wine-329

resembling medium in BFM daily and total lactic acid productions (as well as lactic 330

acid yields) were higher upon catechin (Table 2) being these increases more 331

pronounced at the onset of fermentation (0-24h) 332

333

Catechin is not metabolized by Lactobacillus plantarum RM71 334

335

Spectrometric analysis was carried out to search for potential catechin 336

degradationmetabolism resulting from the growth of Lact plantarum RM71 337

Supernatants from stationary phase cultures of Lact plantarum RM71 grown in pH 55 338

CDM wine- resembling medium or BFM upon 100 mg l-1 of catechin were compared 339

with cell-free negative controls incubated under the same conditions Experiments 340

performed in CDM appeared in Fig 5 which displayed practically no differences in the 341

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15

catechin peak eluting at 1817 min among HPLC chromatograms of cell-free control 342

and culture supernatants after 40 h incubation in CDM 343

344

DISCUSSION 345

346

The effects of phenolics compounds on lactic acid bacteria have been reported to 347

be rather variable (For a recent review see Rodriacuteguez et al 2009) This variability 348

depends on the compound (Vivas et al 1997 Campos et al 2003 Figueiredo et al 349

2008) the strain under study (Figueiredo et al 2008) or the medium used for growth 350

(Alberto et al 2001) Up to date some authors have associated the positive effects of 351

food relevant phenolic compounds on growth of lactic acid bacteria to the degradation 352

of the compound Thus the growth stimulating effects of gallate on O oeni (Vivas et al 353

1997) or Lact hilgardii (Alberto et al 2001) were reported to be dose-dependent and 354

suggested to be linked to gallate degradation In the case of anthocyanins it was 355

suggested that glucose moiety was used as energy source to justify the positive effect on 356

the growth of O oeni (Vivas et al 1997) 357

In this study it was observed that most of the catechin remained in the media 358

after fermentation with Lact plantarum RM71 This result was interpreted as this 359

bacterium did not degrade the largest catechin bulk otherwise the catechin peak would 360

have been disappeared or greatly diminished and new compounds would have been 361

appeared Consequently the metabolic and growth stimulations promoted by catechin 362

can not be ascribed to catechin metabolism by Lact plantarum RM 71 These results 363

are in agreement with those previously reported for O oeni by Reguant et al (2000) 364

who also uncoupled the stimulation of growth triggered by catechin from the 365

metabolism of this flavanol Since O oeni grows better in absence of oxygen these 366

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16

authors suggested that the oxygen scavenging ability of flavonoids tested including 367

catechin favoured the growth stimulating effects observed Contrarily other authors 368

could not apply the same reasoning as they found that catechin or epicatechin showed 369

neutral effects on the growth of O oeni and Lact hilgardii (Figueiredo et al 2008) It 370

has been recently reported that the radical scavenging capacity of catechins increased 371

with the pH of the medium (Muzolf et al 2008) however in this work the positive 372

effects of catechin on the growth of Lact plantarum were more pronounced at the lower 373

pH and not dependent on the dose of catechin used Therefore the positive influence of 374

catechin on growth found here can hardly be ascribed to its radical scavenging capacity 375

By time course of sugar consumption data tracking experiments it is shown that 376

at least in the case of Lact plantarum RM71 catechin stimulates growth by promoting 377

quicker sugar consumption increasing the extension of sugar utilization and stimulating 378

malic acid decarboxylation Clearly this is a different mode of action of catechin to that 379

suggested by this flavanol on Lact hilgardii where the growth stimulating effects were 380

linked to catechin degradation (Alberto et al 2001 Figueiredo et al 2008) or to that 381

suggested by Reguant et al (2000) for O oeni who justified the stimulating effects on 382

the antioxidant properties of catechin 383

The beneficial effects on growth were observed regardless of the catechin 384

concentration examined which support again the hypotheses that growth stimulation is 385

uncoupled from catechin degradation The lack of dose dependent effects is in 386

agreement with the weak bactericidal activity (Nakayama et al 1998) resulting from the 387

shallow location close to the phospholipidwater interface previously reported for 388

catechin (Caturla et al 2003) Otherwise an increased bactericidal effect would be 389

expected at increased catechin concentrations as it occurs with galloylated catechins 390

which possess a higher insertion capacity in the membrane (Ikagai et al 1993 Caturla 391

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17

et al 2003) Although the growth advantages were observed regardless the initial pH 392

values they were more pronounced in the pH 55 CDM which could indicate a stronger 393

interaction of catechin with membrane phospholipids at this lower pH than at pH 65 394

According to previous reports (Kajiya et al 2001 Caturla et al 2003) catechin 395

does not enter the cell so it is probable that the effects observed on growth and 396

metabolism were caused by alteration of the biophysical properties of the membrane of 397

Lact plantarum RM71 It is possible that catechin after had caused its biological effect 398

could had left the membrane by passive diffusion along time a process that has been 399

previously described for other flavonoids such as quercetin (Movileanu et al 2000) 400

Thus catechin once located in the membrane could alter the function of proteins 401

associated with the bilayer and affect the transport of materials across it Sugar and 402

malate transporters seem to be affected proteins accordingly to the changes in sugar and 403

malic acid consumption observed here upon catechin The citrate transport system could 404

be affected as well since catechin triggered variations in the growth patterns displayed 405

by Lact plantarum RM71 on citrate supplemented CDM Experiments are currently 406

under way to determine if catechin alters some biophysical properties of the membrane 407

of Lact plantarum and affects these transport systems 408

Citrate prolonged lag phase and induced sequential growth behaviour in CDM 409

The scope of this study was not to investigate this behaviour however the long lag 410

phase observed upon citrate indicates that there might be some limitations on the 411

substrate utilization apparatus at the onset of cultures which is usual when growing in 412

mixed-substrate environments (Daae et al 1998) The biphasic growth could be due to a 413

sequential metabolism of carbon sources since this behaviour was only observed at an 414

initial pH of 55 which is close to the optimum pH range for citrate metabolism in Lact 415

plantarum (Palles et al 1998) This type of sequential metabolism is not unprecedent 416

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for Lactobacillus ssp Thus diauxic growth has been described during growth also 417

under sugar limitation on a mixture of sugars and fructooligosaccharides (FOS) (Goh et 418

al 2007) and glucose and citrate (de Figueroa et al 1996) Catechin shorthened the lag 419

phase so it is probable that this flavanol relieved limitations in substrate utilization 420

Moreover in complete pH 55 CDM catechin replaced the biphasic growth by a diauxic 421

growth where a clear diauxic lag was evident This growth pattern is usually associated 422

to a better control of substrate assimilation (Daae et al 1998) so if catechin as it is 423

suspected inferred a more efficient substrate uptake a diauxic lag would be necessary 424

to fit the enzyme synthesis for the utilization of the second substrate 425

The results obtained in wine resembling medium or BFM revealed that catechin 426

sped-up malolactic fermentation stimulated the consumption of several sugars as well 427

as the lactic acid formation thus providing growth advantages to Lact plantarum 428

RM71 in these media Growth stimulation linked to malolactic fermentation is known to 429

be caused by its coupling to ATP synthesis by either the entry through F(H+)-ATPase 430

of environmental protons generated in the malate decarboxilation process and the 431

malatelactate antiport coupled also to F(H+)-ATPase Accordingly the growth 432

improvement of Lact plantarum RM71 upon catechin should not be surprising in view 433

of the accelerated malolactic fermentation and lactic acid production provided by the 434

flavanol Similar effects have been also described for the growth of O oeni CECT 435

4100T where this flavanol was found to stimulate the malolactic fermentation 436

development (Reguant et al 2000) Although malolactic fermentation is classically 437

associated to winemaking the importance of the growth benefit obtained by Lact 438

plantarum from the utilization of malic acid should not be restricted to the wine 439

environment but extended to other fermented food plants as recently reported (Plumed-440

Ferrer et al 2008) These authors found a significantly higher growth of Lact 441

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19

plantarum in cucumber juice compared to the rich MRS medium and highlighted the 442

crucial importance of the higher decarboxylation of malic acid in this stimulation 443

In summary the results presented here show that catechin improved malic acid 444

degradation fermentation of several sugars and therefore Lact plantarum RM71 445

growth which are desirable traits for a successful performance of this species as starter 446

in several food plant fermentations These stimulations are not linked to catechin 447

degradation 448

449

Acknowledgements 450

451

This work was supported by grants AGL2005-000470 and AGL2008-001052 (CICYT) 452

and 200670M058 (CAM) J A Curiel is a recipient of a fellowship from Ministerio de 453

Ciencia e Innovacioacuten (MICINN) 454

455

REFERENCES 456

457

Adlerberth I Ahrne S Johansson ML Molin G Hanson LA and Wold AE 458

(1996) A mannose specific adherence mechanism in Lactobacillus plantarum 459

conferring binding to the human colonic cell line HT-29 Appl Environ 460

Microbiol 62 2244-2251 461

462

Alberto MR Fariacuteas ME and Manca de Nadra MC (2001) Effect of gallic acid and 463

catechin on Lactobacillus hilgardii 5w growth and metabolism of organics 464

compounds J Agr Food Chem 49 4359-4363 465

466

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For Peer Review

20

Amarowicz R Carle R Dongowski G Durazzo A Galensa R Kammerer D 467

Maiani G and Piskula MK (2009) Influence of postharvest processing and 468

storage on the content of phenolic acids and flavonoids in foods Mol Nutr Food 469

Res doi 101002mnfr200700486 470

471

Campos FM Couto JA and Hogg TA (2003) Influence of phenolics acids on 472

growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii J Appl 473

Microbiol 94 167-174 474

475

Caturla N Vera-Samper E Villalaiacuten J Reyes Mateo C and Micol V (2003) The 476

relationship between the antioxidant and the antibacterial properties of 477

galloylated catechins and the structure of phospholipids membranes Free Rad 478

Biol Med 34 648-662 479

480

Crespy V and Williamson G (2004) A review of the health effects of green tea 481

catechins in in vivo animal models J Nutr 134 3431-3440 482

483

Daae EB and Ison AP (1998) A simple structured model describing the growth of 484

Streptomyces lividans Biotechnol Bioeng 58 263-266 485

486

de Figueroa R M Benito de Caacuterdenas I L Sesma F Alvarez F de Ruiz Holgado 487

A P and Oliver G (1996) Inducible transport of citrate in Lactobacillus 488

rhamnosus ATCC 7469 J Appl Microbiol 81 348-354 489

490

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For Peer Review

21

de Man J C Rogosa M and Sharpe M E (1960) A medium for the cultivation of 491

lactobacilli J Appl Microbiol 23 130-135 492

493

Figueiredo AR Campos F de Freitas V Hogg T and Couto JA (2008) Effect of 494

phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus 495

oeni and Lactobacillus hilgardii Food Microbiol 25 105-12 496

497

Goh Y J Lee J H and Hutkins R W (2007) Functional analysis of the 498

fructooligosaccharide utilization operon in Lactobacillus paracasei 1195 Appl 499

Environ Microbiol 73 5716-5724 500

501

Hussein L Medina A Barrionuevo A Lammuela-Raventos RM and Andres-502

Lacueva C (2009) Normal distribution of urinary polyphenol excretion among 503

Egyptian males 7-14 years old and changes following nutritional intervention 504

with tomato juice (Lycopersicon esculentum) Int J Food Sci Nutr 60 302 ndash 311 505

506

Ikagai H Nakae T Hara Y and Shimamura T (1993) Bactericidal catechins 507

damage the lipid bilayer Biochim Biophys Acta 1147 132-136 508

509

Kajiya K S Kumazawa and Nakayama T (2001) Steric effects on the interaction of 510

tea catechins with lipid bilayers Biosci Biotechnol Biochem 65 2638-2643 511

512

Kostinek M Ban-Koffi L Ottah-Atikpo M Teniola D Schillinger U Holzapfel 513

WH and Franz C (2008) Diversity of predominant lactic acid bacteria 514

associated with cocoa fermentation in Nigeria Curr Microbiol 56 306-314 515

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12

rate of galactose consumption as it was in the period measured and assuming steady-267

state conditions similar in presence ( 105 plusmn 006 mmol l-1 h-1) or in absence (095 plusmn 268

005 mmol l-1 h-1) of the flavanol Growth was improved by the presence of catechin in 269

the medium (Fig 2) confirming the above results obtained in cultures carried out on 270

microtitre plates (Fig 1) (please note that as mentioned in the Material and Methods 271

section the optical densities of experiments displayed in Fig 1 and Fig 2 are different) 272

273

The fermentation performance of Lactobacillus plantarum RM71 in wine-resembling 274

medium and basal fermentation medium is improved by catechin 275

276

To test the effects of catechin in media relevant to practical fermentation 277

processes firstly a wine-resembling medium set up at an initial pH of 38 was 278

fermented with Lact plantarum RM71 strain This fermentation was monitored for two 279

days time needed for this bacterium to achieve its maximum log increase in this 280

medium Sugar consumption malic acid degradation and lactic acid production were 281

assayed daily in catechin-lacking or catechin-supplemented (100 mg l-1) media Lact 282

plantarum RM71 metabolized glucose and fructose simultaneously in both media but 283

the total sugar consumption was higher in the medium supplemented with catechin 284

compared to control (1877 vs 2149 mmol l-1 See Table 1) 285

The presence of catechin clearly accelerated sugar consumption at the initial 286

stage of fermentation (0-24 h) as it could be seen for glucose and fructose (Fig 3) 287

which proceeded about ten and six times faster respectively than their controls (Table 288

1) The mean consumption rates of glucose and fructose per day during the entire 289

fermentation period were of 545 mmol l-1 d -1 for glucose (529 for fructose) upon 290

catechin instead of 45 mmol l-1 d -1 for glucose (48 for fructose) in absence of the 291

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flavanol The fermentation of malic acid was faster than that of sugars and completed in 292

only one day A sample taken at ten hours from the onset of fermentation showed that 293

catechin clearly sped up the consumption of malic acid (Fig 3) Thus at this time (10 294

h) 613 of the total malic acid added (715 mmol l-1) was already consumed by Lact 295

plantarum RM71 in presence of catechin in contrast with the significantly lower 293 296

(335 mmol l-1) malic acid consumption by the control culture 297

Compared to control the presence of catechin gave rise to an increase of lactic 298

acid production as it is reflected by the daily and total amounts produced (Table 1) The 299

difference in lactic acid production was specially marked at the onset of fermentation 300

which could be expected because of the accelerated malic acid and sugar consumption 301

in the catechin supplemented cultures 302

To test if catechin exerted similar effects on Lact plantarum plant-derived 303

fermentations different from wine conditions a medium named BFM was prepared The 304

medium was devoid of wine characteristic components ie tartaric acid ethanol and 305

acetic acid as these were not used by Lact plantarum RM71 in the fermentation of wine 306

resembling medium (not shown) Furthermore glucose and fructose were replaced by 307

galactose (1) to investigate if catechin affected the fermentation of other sugars 308

Galactose is present in the diet and is usually present in dietary fibre from vegetables 309

and fruits such as the arabinogalactan which has been shown to increase the 310

Lactobacillus spp population in the human intestine (Robinson Feirtag amp Slavin 311

(2001) 312

As it can be observed in Table 2 the presence of catechin stimulated the growth 313

of Lact plantarum RM71 in BFM The patterns of malic acid degradation and galactose 314

utilization are shown in Fig 4 The fermentation of malic acid was completed in two 315

days regardless catechin supplementation however the presence of this flavanol clearly 316

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14

sped up the consumption of this acid Thus during the first day of fermentation 598 317

of the total malic acid added was consumed by Lact plantarum RM71 in presence of 318

catechin in contrast with the significantly lower 137 by the control culture 319

We observed that the extent of sugar utilization by Lact plantarum RM71 was 320

increased upon catechin (176 vs 2914 mmol l-1) In addition catechin promoted a faster 321

galactose consumption respect to controls The mean consumption rate of galactose per 322

day during the entire fermentation period was of 971 mmol l-1 d -1 upon catechin 323

instead of 586 mmol l-1 d -1 in absence of the flavanol Examination of Table 2 revealed 324

that the presence of catechin promoted earlier galactose utilization Thus at the initial 325

stage of fermentation (0-24 h) sugar consumption proceeded at a diminished rate of 326

084 mmol l-1 d-1 in the control cultures compared to a much faster rate of 435 mmol l-1 327

d-1 upon catechin Lactic acid production was also monitored as indicator of the 328

fermentation performance of Lact plantarum RM71 As it occurred in the wine-329

resembling medium in BFM daily and total lactic acid productions (as well as lactic 330

acid yields) were higher upon catechin (Table 2) being these increases more 331

pronounced at the onset of fermentation (0-24h) 332

333

Catechin is not metabolized by Lactobacillus plantarum RM71 334

335

Spectrometric analysis was carried out to search for potential catechin 336

degradationmetabolism resulting from the growth of Lact plantarum RM71 337

Supernatants from stationary phase cultures of Lact plantarum RM71 grown in pH 55 338

CDM wine- resembling medium or BFM upon 100 mg l-1 of catechin were compared 339

with cell-free negative controls incubated under the same conditions Experiments 340

performed in CDM appeared in Fig 5 which displayed practically no differences in the 341

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15

catechin peak eluting at 1817 min among HPLC chromatograms of cell-free control 342

and culture supernatants after 40 h incubation in CDM 343

344

DISCUSSION 345

346

The effects of phenolics compounds on lactic acid bacteria have been reported to 347

be rather variable (For a recent review see Rodriacuteguez et al 2009) This variability 348

depends on the compound (Vivas et al 1997 Campos et al 2003 Figueiredo et al 349

2008) the strain under study (Figueiredo et al 2008) or the medium used for growth 350

(Alberto et al 2001) Up to date some authors have associated the positive effects of 351

food relevant phenolic compounds on growth of lactic acid bacteria to the degradation 352

of the compound Thus the growth stimulating effects of gallate on O oeni (Vivas et al 353

1997) or Lact hilgardii (Alberto et al 2001) were reported to be dose-dependent and 354

suggested to be linked to gallate degradation In the case of anthocyanins it was 355

suggested that glucose moiety was used as energy source to justify the positive effect on 356

the growth of O oeni (Vivas et al 1997) 357

In this study it was observed that most of the catechin remained in the media 358

after fermentation with Lact plantarum RM71 This result was interpreted as this 359

bacterium did not degrade the largest catechin bulk otherwise the catechin peak would 360

have been disappeared or greatly diminished and new compounds would have been 361

appeared Consequently the metabolic and growth stimulations promoted by catechin 362

can not be ascribed to catechin metabolism by Lact plantarum RM 71 These results 363

are in agreement with those previously reported for O oeni by Reguant et al (2000) 364

who also uncoupled the stimulation of growth triggered by catechin from the 365

metabolism of this flavanol Since O oeni grows better in absence of oxygen these 366

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16

authors suggested that the oxygen scavenging ability of flavonoids tested including 367

catechin favoured the growth stimulating effects observed Contrarily other authors 368

could not apply the same reasoning as they found that catechin or epicatechin showed 369

neutral effects on the growth of O oeni and Lact hilgardii (Figueiredo et al 2008) It 370

has been recently reported that the radical scavenging capacity of catechins increased 371

with the pH of the medium (Muzolf et al 2008) however in this work the positive 372

effects of catechin on the growth of Lact plantarum were more pronounced at the lower 373

pH and not dependent on the dose of catechin used Therefore the positive influence of 374

catechin on growth found here can hardly be ascribed to its radical scavenging capacity 375

By time course of sugar consumption data tracking experiments it is shown that 376

at least in the case of Lact plantarum RM71 catechin stimulates growth by promoting 377

quicker sugar consumption increasing the extension of sugar utilization and stimulating 378

malic acid decarboxylation Clearly this is a different mode of action of catechin to that 379

suggested by this flavanol on Lact hilgardii where the growth stimulating effects were 380

linked to catechin degradation (Alberto et al 2001 Figueiredo et al 2008) or to that 381

suggested by Reguant et al (2000) for O oeni who justified the stimulating effects on 382

the antioxidant properties of catechin 383

The beneficial effects on growth were observed regardless of the catechin 384

concentration examined which support again the hypotheses that growth stimulation is 385

uncoupled from catechin degradation The lack of dose dependent effects is in 386

agreement with the weak bactericidal activity (Nakayama et al 1998) resulting from the 387

shallow location close to the phospholipidwater interface previously reported for 388

catechin (Caturla et al 2003) Otherwise an increased bactericidal effect would be 389

expected at increased catechin concentrations as it occurs with galloylated catechins 390

which possess a higher insertion capacity in the membrane (Ikagai et al 1993 Caturla 391

Page 16 of 33

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17

et al 2003) Although the growth advantages were observed regardless the initial pH 392

values they were more pronounced in the pH 55 CDM which could indicate a stronger 393

interaction of catechin with membrane phospholipids at this lower pH than at pH 65 394

According to previous reports (Kajiya et al 2001 Caturla et al 2003) catechin 395

does not enter the cell so it is probable that the effects observed on growth and 396

metabolism were caused by alteration of the biophysical properties of the membrane of 397

Lact plantarum RM71 It is possible that catechin after had caused its biological effect 398

could had left the membrane by passive diffusion along time a process that has been 399

previously described for other flavonoids such as quercetin (Movileanu et al 2000) 400

Thus catechin once located in the membrane could alter the function of proteins 401

associated with the bilayer and affect the transport of materials across it Sugar and 402

malate transporters seem to be affected proteins accordingly to the changes in sugar and 403

malic acid consumption observed here upon catechin The citrate transport system could 404

be affected as well since catechin triggered variations in the growth patterns displayed 405

by Lact plantarum RM71 on citrate supplemented CDM Experiments are currently 406

under way to determine if catechin alters some biophysical properties of the membrane 407

of Lact plantarum and affects these transport systems 408

Citrate prolonged lag phase and induced sequential growth behaviour in CDM 409

The scope of this study was not to investigate this behaviour however the long lag 410

phase observed upon citrate indicates that there might be some limitations on the 411

substrate utilization apparatus at the onset of cultures which is usual when growing in 412

mixed-substrate environments (Daae et al 1998) The biphasic growth could be due to a 413

sequential metabolism of carbon sources since this behaviour was only observed at an 414

initial pH of 55 which is close to the optimum pH range for citrate metabolism in Lact 415

plantarum (Palles et al 1998) This type of sequential metabolism is not unprecedent 416

Page 17 of 33

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18

for Lactobacillus ssp Thus diauxic growth has been described during growth also 417

under sugar limitation on a mixture of sugars and fructooligosaccharides (FOS) (Goh et 418

al 2007) and glucose and citrate (de Figueroa et al 1996) Catechin shorthened the lag 419

phase so it is probable that this flavanol relieved limitations in substrate utilization 420

Moreover in complete pH 55 CDM catechin replaced the biphasic growth by a diauxic 421

growth where a clear diauxic lag was evident This growth pattern is usually associated 422

to a better control of substrate assimilation (Daae et al 1998) so if catechin as it is 423

suspected inferred a more efficient substrate uptake a diauxic lag would be necessary 424

to fit the enzyme synthesis for the utilization of the second substrate 425

The results obtained in wine resembling medium or BFM revealed that catechin 426

sped-up malolactic fermentation stimulated the consumption of several sugars as well 427

as the lactic acid formation thus providing growth advantages to Lact plantarum 428

RM71 in these media Growth stimulation linked to malolactic fermentation is known to 429

be caused by its coupling to ATP synthesis by either the entry through F(H+)-ATPase 430

of environmental protons generated in the malate decarboxilation process and the 431

malatelactate antiport coupled also to F(H+)-ATPase Accordingly the growth 432

improvement of Lact plantarum RM71 upon catechin should not be surprising in view 433

of the accelerated malolactic fermentation and lactic acid production provided by the 434

flavanol Similar effects have been also described for the growth of O oeni CECT 435

4100T where this flavanol was found to stimulate the malolactic fermentation 436

development (Reguant et al 2000) Although malolactic fermentation is classically 437

associated to winemaking the importance of the growth benefit obtained by Lact 438

plantarum from the utilization of malic acid should not be restricted to the wine 439

environment but extended to other fermented food plants as recently reported (Plumed-440

Ferrer et al 2008) These authors found a significantly higher growth of Lact 441

Page 18 of 33

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19

plantarum in cucumber juice compared to the rich MRS medium and highlighted the 442

crucial importance of the higher decarboxylation of malic acid in this stimulation 443

In summary the results presented here show that catechin improved malic acid 444

degradation fermentation of several sugars and therefore Lact plantarum RM71 445

growth which are desirable traits for a successful performance of this species as starter 446

in several food plant fermentations These stimulations are not linked to catechin 447

degradation 448

449

Acknowledgements 450

451

This work was supported by grants AGL2005-000470 and AGL2008-001052 (CICYT) 452

and 200670M058 (CAM) J A Curiel is a recipient of a fellowship from Ministerio de 453

Ciencia e Innovacioacuten (MICINN) 454

455

REFERENCES 456

457

Adlerberth I Ahrne S Johansson ML Molin G Hanson LA and Wold AE 458

(1996) A mannose specific adherence mechanism in Lactobacillus plantarum 459

conferring binding to the human colonic cell line HT-29 Appl Environ 460

Microbiol 62 2244-2251 461

462

Alberto MR Fariacuteas ME and Manca de Nadra MC (2001) Effect of gallic acid and 463

catechin on Lactobacillus hilgardii 5w growth and metabolism of organics 464

compounds J Agr Food Chem 49 4359-4363 465

466

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20

Amarowicz R Carle R Dongowski G Durazzo A Galensa R Kammerer D 467

Maiani G and Piskula MK (2009) Influence of postharvest processing and 468

storage on the content of phenolic acids and flavonoids in foods Mol Nutr Food 469

Res doi 101002mnfr200700486 470

471

Campos FM Couto JA and Hogg TA (2003) Influence of phenolics acids on 472

growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii J Appl 473

Microbiol 94 167-174 474

475

Caturla N Vera-Samper E Villalaiacuten J Reyes Mateo C and Micol V (2003) The 476

relationship between the antioxidant and the antibacterial properties of 477

galloylated catechins and the structure of phospholipids membranes Free Rad 478

Biol Med 34 648-662 479

480

Crespy V and Williamson G (2004) A review of the health effects of green tea 481

catechins in in vivo animal models J Nutr 134 3431-3440 482

483

Daae EB and Ison AP (1998) A simple structured model describing the growth of 484

Streptomyces lividans Biotechnol Bioeng 58 263-266 485

486

de Figueroa R M Benito de Caacuterdenas I L Sesma F Alvarez F de Ruiz Holgado 487

A P and Oliver G (1996) Inducible transport of citrate in Lactobacillus 488

rhamnosus ATCC 7469 J Appl Microbiol 81 348-354 489

490

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21

de Man J C Rogosa M and Sharpe M E (1960) A medium for the cultivation of 491

lactobacilli J Appl Microbiol 23 130-135 492

493

Figueiredo AR Campos F de Freitas V Hogg T and Couto JA (2008) Effect of 494

phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus 495

oeni and Lactobacillus hilgardii Food Microbiol 25 105-12 496

497

Goh Y J Lee J H and Hutkins R W (2007) Functional analysis of the 498

fructooligosaccharide utilization operon in Lactobacillus paracasei 1195 Appl 499

Environ Microbiol 73 5716-5724 500

501

Hussein L Medina A Barrionuevo A Lammuela-Raventos RM and Andres-502

Lacueva C (2009) Normal distribution of urinary polyphenol excretion among 503

Egyptian males 7-14 years old and changes following nutritional intervention 504

with tomato juice (Lycopersicon esculentum) Int J Food Sci Nutr 60 302 ndash 311 505

506

Ikagai H Nakae T Hara Y and Shimamura T (1993) Bactericidal catechins 507

damage the lipid bilayer Biochim Biophys Acta 1147 132-136 508

509

Kajiya K S Kumazawa and Nakayama T (2001) Steric effects on the interaction of 510

tea catechins with lipid bilayers Biosci Biotechnol Biochem 65 2638-2643 511

512

Kostinek M Ban-Koffi L Ottah-Atikpo M Teniola D Schillinger U Holzapfel 513

WH and Franz C (2008) Diversity of predominant lactic acid bacteria 514

associated with cocoa fermentation in Nigeria Curr Microbiol 56 306-314 515

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flavanol The fermentation of malic acid was faster than that of sugars and completed in 292

only one day A sample taken at ten hours from the onset of fermentation showed that 293

catechin clearly sped up the consumption of malic acid (Fig 3) Thus at this time (10 294

h) 613 of the total malic acid added (715 mmol l-1) was already consumed by Lact 295

plantarum RM71 in presence of catechin in contrast with the significantly lower 293 296

(335 mmol l-1) malic acid consumption by the control culture 297

Compared to control the presence of catechin gave rise to an increase of lactic 298

acid production as it is reflected by the daily and total amounts produced (Table 1) The 299

difference in lactic acid production was specially marked at the onset of fermentation 300

which could be expected because of the accelerated malic acid and sugar consumption 301

in the catechin supplemented cultures 302

To test if catechin exerted similar effects on Lact plantarum plant-derived 303

fermentations different from wine conditions a medium named BFM was prepared The 304

medium was devoid of wine characteristic components ie tartaric acid ethanol and 305

acetic acid as these were not used by Lact plantarum RM71 in the fermentation of wine 306

resembling medium (not shown) Furthermore glucose and fructose were replaced by 307

galactose (1) to investigate if catechin affected the fermentation of other sugars 308

Galactose is present in the diet and is usually present in dietary fibre from vegetables 309

and fruits such as the arabinogalactan which has been shown to increase the 310

Lactobacillus spp population in the human intestine (Robinson Feirtag amp Slavin 311

(2001) 312

As it can be observed in Table 2 the presence of catechin stimulated the growth 313

of Lact plantarum RM71 in BFM The patterns of malic acid degradation and galactose 314

utilization are shown in Fig 4 The fermentation of malic acid was completed in two 315

days regardless catechin supplementation however the presence of this flavanol clearly 316

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14

sped up the consumption of this acid Thus during the first day of fermentation 598 317

of the total malic acid added was consumed by Lact plantarum RM71 in presence of 318

catechin in contrast with the significantly lower 137 by the control culture 319

We observed that the extent of sugar utilization by Lact plantarum RM71 was 320

increased upon catechin (176 vs 2914 mmol l-1) In addition catechin promoted a faster 321

galactose consumption respect to controls The mean consumption rate of galactose per 322

day during the entire fermentation period was of 971 mmol l-1 d -1 upon catechin 323

instead of 586 mmol l-1 d -1 in absence of the flavanol Examination of Table 2 revealed 324

that the presence of catechin promoted earlier galactose utilization Thus at the initial 325

stage of fermentation (0-24 h) sugar consumption proceeded at a diminished rate of 326

084 mmol l-1 d-1 in the control cultures compared to a much faster rate of 435 mmol l-1 327

d-1 upon catechin Lactic acid production was also monitored as indicator of the 328

fermentation performance of Lact plantarum RM71 As it occurred in the wine-329

resembling medium in BFM daily and total lactic acid productions (as well as lactic 330

acid yields) were higher upon catechin (Table 2) being these increases more 331

pronounced at the onset of fermentation (0-24h) 332

333

Catechin is not metabolized by Lactobacillus plantarum RM71 334

335

Spectrometric analysis was carried out to search for potential catechin 336

degradationmetabolism resulting from the growth of Lact plantarum RM71 337

Supernatants from stationary phase cultures of Lact plantarum RM71 grown in pH 55 338

CDM wine- resembling medium or BFM upon 100 mg l-1 of catechin were compared 339

with cell-free negative controls incubated under the same conditions Experiments 340

performed in CDM appeared in Fig 5 which displayed practically no differences in the 341

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15

catechin peak eluting at 1817 min among HPLC chromatograms of cell-free control 342

and culture supernatants after 40 h incubation in CDM 343

344

DISCUSSION 345

346

The effects of phenolics compounds on lactic acid bacteria have been reported to 347

be rather variable (For a recent review see Rodriacuteguez et al 2009) This variability 348

depends on the compound (Vivas et al 1997 Campos et al 2003 Figueiredo et al 349

2008) the strain under study (Figueiredo et al 2008) or the medium used for growth 350

(Alberto et al 2001) Up to date some authors have associated the positive effects of 351

food relevant phenolic compounds on growth of lactic acid bacteria to the degradation 352

of the compound Thus the growth stimulating effects of gallate on O oeni (Vivas et al 353

1997) or Lact hilgardii (Alberto et al 2001) were reported to be dose-dependent and 354

suggested to be linked to gallate degradation In the case of anthocyanins it was 355

suggested that glucose moiety was used as energy source to justify the positive effect on 356

the growth of O oeni (Vivas et al 1997) 357

In this study it was observed that most of the catechin remained in the media 358

after fermentation with Lact plantarum RM71 This result was interpreted as this 359

bacterium did not degrade the largest catechin bulk otherwise the catechin peak would 360

have been disappeared or greatly diminished and new compounds would have been 361

appeared Consequently the metabolic and growth stimulations promoted by catechin 362

can not be ascribed to catechin metabolism by Lact plantarum RM 71 These results 363

are in agreement with those previously reported for O oeni by Reguant et al (2000) 364

who also uncoupled the stimulation of growth triggered by catechin from the 365

metabolism of this flavanol Since O oeni grows better in absence of oxygen these 366

Page 15 of 33

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16

authors suggested that the oxygen scavenging ability of flavonoids tested including 367

catechin favoured the growth stimulating effects observed Contrarily other authors 368

could not apply the same reasoning as they found that catechin or epicatechin showed 369

neutral effects on the growth of O oeni and Lact hilgardii (Figueiredo et al 2008) It 370

has been recently reported that the radical scavenging capacity of catechins increased 371

with the pH of the medium (Muzolf et al 2008) however in this work the positive 372

effects of catechin on the growth of Lact plantarum were more pronounced at the lower 373

pH and not dependent on the dose of catechin used Therefore the positive influence of 374

catechin on growth found here can hardly be ascribed to its radical scavenging capacity 375

By time course of sugar consumption data tracking experiments it is shown that 376

at least in the case of Lact plantarum RM71 catechin stimulates growth by promoting 377

quicker sugar consumption increasing the extension of sugar utilization and stimulating 378

malic acid decarboxylation Clearly this is a different mode of action of catechin to that 379

suggested by this flavanol on Lact hilgardii where the growth stimulating effects were 380

linked to catechin degradation (Alberto et al 2001 Figueiredo et al 2008) or to that 381

suggested by Reguant et al (2000) for O oeni who justified the stimulating effects on 382

the antioxidant properties of catechin 383

The beneficial effects on growth were observed regardless of the catechin 384

concentration examined which support again the hypotheses that growth stimulation is 385

uncoupled from catechin degradation The lack of dose dependent effects is in 386

agreement with the weak bactericidal activity (Nakayama et al 1998) resulting from the 387

shallow location close to the phospholipidwater interface previously reported for 388

catechin (Caturla et al 2003) Otherwise an increased bactericidal effect would be 389

expected at increased catechin concentrations as it occurs with galloylated catechins 390

which possess a higher insertion capacity in the membrane (Ikagai et al 1993 Caturla 391

Page 16 of 33

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17

et al 2003) Although the growth advantages were observed regardless the initial pH 392

values they were more pronounced in the pH 55 CDM which could indicate a stronger 393

interaction of catechin with membrane phospholipids at this lower pH than at pH 65 394

According to previous reports (Kajiya et al 2001 Caturla et al 2003) catechin 395

does not enter the cell so it is probable that the effects observed on growth and 396

metabolism were caused by alteration of the biophysical properties of the membrane of 397

Lact plantarum RM71 It is possible that catechin after had caused its biological effect 398

could had left the membrane by passive diffusion along time a process that has been 399

previously described for other flavonoids such as quercetin (Movileanu et al 2000) 400

Thus catechin once located in the membrane could alter the function of proteins 401

associated with the bilayer and affect the transport of materials across it Sugar and 402

malate transporters seem to be affected proteins accordingly to the changes in sugar and 403

malic acid consumption observed here upon catechin The citrate transport system could 404

be affected as well since catechin triggered variations in the growth patterns displayed 405

by Lact plantarum RM71 on citrate supplemented CDM Experiments are currently 406

under way to determine if catechin alters some biophysical properties of the membrane 407

of Lact plantarum and affects these transport systems 408

Citrate prolonged lag phase and induced sequential growth behaviour in CDM 409

The scope of this study was not to investigate this behaviour however the long lag 410

phase observed upon citrate indicates that there might be some limitations on the 411

substrate utilization apparatus at the onset of cultures which is usual when growing in 412

mixed-substrate environments (Daae et al 1998) The biphasic growth could be due to a 413

sequential metabolism of carbon sources since this behaviour was only observed at an 414

initial pH of 55 which is close to the optimum pH range for citrate metabolism in Lact 415

plantarum (Palles et al 1998) This type of sequential metabolism is not unprecedent 416

Page 17 of 33

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18

for Lactobacillus ssp Thus diauxic growth has been described during growth also 417

under sugar limitation on a mixture of sugars and fructooligosaccharides (FOS) (Goh et 418

al 2007) and glucose and citrate (de Figueroa et al 1996) Catechin shorthened the lag 419

phase so it is probable that this flavanol relieved limitations in substrate utilization 420

Moreover in complete pH 55 CDM catechin replaced the biphasic growth by a diauxic 421

growth where a clear diauxic lag was evident This growth pattern is usually associated 422

to a better control of substrate assimilation (Daae et al 1998) so if catechin as it is 423

suspected inferred a more efficient substrate uptake a diauxic lag would be necessary 424

to fit the enzyme synthesis for the utilization of the second substrate 425

The results obtained in wine resembling medium or BFM revealed that catechin 426

sped-up malolactic fermentation stimulated the consumption of several sugars as well 427

as the lactic acid formation thus providing growth advantages to Lact plantarum 428

RM71 in these media Growth stimulation linked to malolactic fermentation is known to 429

be caused by its coupling to ATP synthesis by either the entry through F(H+)-ATPase 430

of environmental protons generated in the malate decarboxilation process and the 431

malatelactate antiport coupled also to F(H+)-ATPase Accordingly the growth 432

improvement of Lact plantarum RM71 upon catechin should not be surprising in view 433

of the accelerated malolactic fermentation and lactic acid production provided by the 434

flavanol Similar effects have been also described for the growth of O oeni CECT 435

4100T where this flavanol was found to stimulate the malolactic fermentation 436

development (Reguant et al 2000) Although malolactic fermentation is classically 437

associated to winemaking the importance of the growth benefit obtained by Lact 438

plantarum from the utilization of malic acid should not be restricted to the wine 439

environment but extended to other fermented food plants as recently reported (Plumed-440

Ferrer et al 2008) These authors found a significantly higher growth of Lact 441

Page 18 of 33

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19

plantarum in cucumber juice compared to the rich MRS medium and highlighted the 442

crucial importance of the higher decarboxylation of malic acid in this stimulation 443

In summary the results presented here show that catechin improved malic acid 444

degradation fermentation of several sugars and therefore Lact plantarum RM71 445

growth which are desirable traits for a successful performance of this species as starter 446

in several food plant fermentations These stimulations are not linked to catechin 447

degradation 448

449

Acknowledgements 450

451

This work was supported by grants AGL2005-000470 and AGL2008-001052 (CICYT) 452

and 200670M058 (CAM) J A Curiel is a recipient of a fellowship from Ministerio de 453

Ciencia e Innovacioacuten (MICINN) 454

455

REFERENCES 456

457

Adlerberth I Ahrne S Johansson ML Molin G Hanson LA and Wold AE 458

(1996) A mannose specific adherence mechanism in Lactobacillus plantarum 459

conferring binding to the human colonic cell line HT-29 Appl Environ 460

Microbiol 62 2244-2251 461

462

Alberto MR Fariacuteas ME and Manca de Nadra MC (2001) Effect of gallic acid and 463

catechin on Lactobacillus hilgardii 5w growth and metabolism of organics 464

compounds J Agr Food Chem 49 4359-4363 465

466

Page 19 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

20

Amarowicz R Carle R Dongowski G Durazzo A Galensa R Kammerer D 467

Maiani G and Piskula MK (2009) Influence of postharvest processing and 468

storage on the content of phenolic acids and flavonoids in foods Mol Nutr Food 469

Res doi 101002mnfr200700486 470

471

Campos FM Couto JA and Hogg TA (2003) Influence of phenolics acids on 472

growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii J Appl 473

Microbiol 94 167-174 474

475

Caturla N Vera-Samper E Villalaiacuten J Reyes Mateo C and Micol V (2003) The 476

relationship between the antioxidant and the antibacterial properties of 477

galloylated catechins and the structure of phospholipids membranes Free Rad 478

Biol Med 34 648-662 479

480

Crespy V and Williamson G (2004) A review of the health effects of green tea 481

catechins in in vivo animal models J Nutr 134 3431-3440 482

483

Daae EB and Ison AP (1998) A simple structured model describing the growth of 484

Streptomyces lividans Biotechnol Bioeng 58 263-266 485

486

de Figueroa R M Benito de Caacuterdenas I L Sesma F Alvarez F de Ruiz Holgado 487

A P and Oliver G (1996) Inducible transport of citrate in Lactobacillus 488

rhamnosus ATCC 7469 J Appl Microbiol 81 348-354 489

490

Page 20 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

21

de Man J C Rogosa M and Sharpe M E (1960) A medium for the cultivation of 491

lactobacilli J Appl Microbiol 23 130-135 492

493

Figueiredo AR Campos F de Freitas V Hogg T and Couto JA (2008) Effect of 494

phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus 495

oeni and Lactobacillus hilgardii Food Microbiol 25 105-12 496

497

Goh Y J Lee J H and Hutkins R W (2007) Functional analysis of the 498

fructooligosaccharide utilization operon in Lactobacillus paracasei 1195 Appl 499

Environ Microbiol 73 5716-5724 500

501

Hussein L Medina A Barrionuevo A Lammuela-Raventos RM and Andres-502

Lacueva C (2009) Normal distribution of urinary polyphenol excretion among 503

Egyptian males 7-14 years old and changes following nutritional intervention 504

with tomato juice (Lycopersicon esculentum) Int J Food Sci Nutr 60 302 ndash 311 505

506

Ikagai H Nakae T Hara Y and Shimamura T (1993) Bactericidal catechins 507

damage the lipid bilayer Biochim Biophys Acta 1147 132-136 508

509

Kajiya K S Kumazawa and Nakayama T (2001) Steric effects on the interaction of 510

tea catechins with lipid bilayers Biosci Biotechnol Biochem 65 2638-2643 511

512

Kostinek M Ban-Koffi L Ottah-Atikpo M Teniola D Schillinger U Holzapfel 513

WH and Franz C (2008) Diversity of predominant lactic acid bacteria 514

associated with cocoa fermentation in Nigeria Curr Microbiol 56 306-314 515

Page 21 of 33

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14

sped up the consumption of this acid Thus during the first day of fermentation 598 317

of the total malic acid added was consumed by Lact plantarum RM71 in presence of 318

catechin in contrast with the significantly lower 137 by the control culture 319

We observed that the extent of sugar utilization by Lact plantarum RM71 was 320

increased upon catechin (176 vs 2914 mmol l-1) In addition catechin promoted a faster 321

galactose consumption respect to controls The mean consumption rate of galactose per 322

day during the entire fermentation period was of 971 mmol l-1 d -1 upon catechin 323

instead of 586 mmol l-1 d -1 in absence of the flavanol Examination of Table 2 revealed 324

that the presence of catechin promoted earlier galactose utilization Thus at the initial 325

stage of fermentation (0-24 h) sugar consumption proceeded at a diminished rate of 326

084 mmol l-1 d-1 in the control cultures compared to a much faster rate of 435 mmol l-1 327

d-1 upon catechin Lactic acid production was also monitored as indicator of the 328

fermentation performance of Lact plantarum RM71 As it occurred in the wine-329

resembling medium in BFM daily and total lactic acid productions (as well as lactic 330

acid yields) were higher upon catechin (Table 2) being these increases more 331

pronounced at the onset of fermentation (0-24h) 332

333

Catechin is not metabolized by Lactobacillus plantarum RM71 334

335

Spectrometric analysis was carried out to search for potential catechin 336

degradationmetabolism resulting from the growth of Lact plantarum RM71 337

Supernatants from stationary phase cultures of Lact plantarum RM71 grown in pH 55 338

CDM wine- resembling medium or BFM upon 100 mg l-1 of catechin were compared 339

with cell-free negative controls incubated under the same conditions Experiments 340

performed in CDM appeared in Fig 5 which displayed practically no differences in the 341

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15

catechin peak eluting at 1817 min among HPLC chromatograms of cell-free control 342

and culture supernatants after 40 h incubation in CDM 343

344

DISCUSSION 345

346

The effects of phenolics compounds on lactic acid bacteria have been reported to 347

be rather variable (For a recent review see Rodriacuteguez et al 2009) This variability 348

depends on the compound (Vivas et al 1997 Campos et al 2003 Figueiredo et al 349

2008) the strain under study (Figueiredo et al 2008) or the medium used for growth 350

(Alberto et al 2001) Up to date some authors have associated the positive effects of 351

food relevant phenolic compounds on growth of lactic acid bacteria to the degradation 352

of the compound Thus the growth stimulating effects of gallate on O oeni (Vivas et al 353

1997) or Lact hilgardii (Alberto et al 2001) were reported to be dose-dependent and 354

suggested to be linked to gallate degradation In the case of anthocyanins it was 355

suggested that glucose moiety was used as energy source to justify the positive effect on 356

the growth of O oeni (Vivas et al 1997) 357

In this study it was observed that most of the catechin remained in the media 358

after fermentation with Lact plantarum RM71 This result was interpreted as this 359

bacterium did not degrade the largest catechin bulk otherwise the catechin peak would 360

have been disappeared or greatly diminished and new compounds would have been 361

appeared Consequently the metabolic and growth stimulations promoted by catechin 362

can not be ascribed to catechin metabolism by Lact plantarum RM 71 These results 363

are in agreement with those previously reported for O oeni by Reguant et al (2000) 364

who also uncoupled the stimulation of growth triggered by catechin from the 365

metabolism of this flavanol Since O oeni grows better in absence of oxygen these 366

Page 15 of 33

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For Peer Review

16

authors suggested that the oxygen scavenging ability of flavonoids tested including 367

catechin favoured the growth stimulating effects observed Contrarily other authors 368

could not apply the same reasoning as they found that catechin or epicatechin showed 369

neutral effects on the growth of O oeni and Lact hilgardii (Figueiredo et al 2008) It 370

has been recently reported that the radical scavenging capacity of catechins increased 371

with the pH of the medium (Muzolf et al 2008) however in this work the positive 372

effects of catechin on the growth of Lact plantarum were more pronounced at the lower 373

pH and not dependent on the dose of catechin used Therefore the positive influence of 374

catechin on growth found here can hardly be ascribed to its radical scavenging capacity 375

By time course of sugar consumption data tracking experiments it is shown that 376

at least in the case of Lact plantarum RM71 catechin stimulates growth by promoting 377

quicker sugar consumption increasing the extension of sugar utilization and stimulating 378

malic acid decarboxylation Clearly this is a different mode of action of catechin to that 379

suggested by this flavanol on Lact hilgardii where the growth stimulating effects were 380

linked to catechin degradation (Alberto et al 2001 Figueiredo et al 2008) or to that 381

suggested by Reguant et al (2000) for O oeni who justified the stimulating effects on 382

the antioxidant properties of catechin 383

The beneficial effects on growth were observed regardless of the catechin 384

concentration examined which support again the hypotheses that growth stimulation is 385

uncoupled from catechin degradation The lack of dose dependent effects is in 386

agreement with the weak bactericidal activity (Nakayama et al 1998) resulting from the 387

shallow location close to the phospholipidwater interface previously reported for 388

catechin (Caturla et al 2003) Otherwise an increased bactericidal effect would be 389

expected at increased catechin concentrations as it occurs with galloylated catechins 390

which possess a higher insertion capacity in the membrane (Ikagai et al 1993 Caturla 391

Page 16 of 33

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17

et al 2003) Although the growth advantages were observed regardless the initial pH 392

values they were more pronounced in the pH 55 CDM which could indicate a stronger 393

interaction of catechin with membrane phospholipids at this lower pH than at pH 65 394

According to previous reports (Kajiya et al 2001 Caturla et al 2003) catechin 395

does not enter the cell so it is probable that the effects observed on growth and 396

metabolism were caused by alteration of the biophysical properties of the membrane of 397

Lact plantarum RM71 It is possible that catechin after had caused its biological effect 398

could had left the membrane by passive diffusion along time a process that has been 399

previously described for other flavonoids such as quercetin (Movileanu et al 2000) 400

Thus catechin once located in the membrane could alter the function of proteins 401

associated with the bilayer and affect the transport of materials across it Sugar and 402

malate transporters seem to be affected proteins accordingly to the changes in sugar and 403

malic acid consumption observed here upon catechin The citrate transport system could 404

be affected as well since catechin triggered variations in the growth patterns displayed 405

by Lact plantarum RM71 on citrate supplemented CDM Experiments are currently 406

under way to determine if catechin alters some biophysical properties of the membrane 407

of Lact plantarum and affects these transport systems 408

Citrate prolonged lag phase and induced sequential growth behaviour in CDM 409

The scope of this study was not to investigate this behaviour however the long lag 410

phase observed upon citrate indicates that there might be some limitations on the 411

substrate utilization apparatus at the onset of cultures which is usual when growing in 412

mixed-substrate environments (Daae et al 1998) The biphasic growth could be due to a 413

sequential metabolism of carbon sources since this behaviour was only observed at an 414

initial pH of 55 which is close to the optimum pH range for citrate metabolism in Lact 415

plantarum (Palles et al 1998) This type of sequential metabolism is not unprecedent 416

Page 17 of 33

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For Peer Review

18

for Lactobacillus ssp Thus diauxic growth has been described during growth also 417

under sugar limitation on a mixture of sugars and fructooligosaccharides (FOS) (Goh et 418

al 2007) and glucose and citrate (de Figueroa et al 1996) Catechin shorthened the lag 419

phase so it is probable that this flavanol relieved limitations in substrate utilization 420

Moreover in complete pH 55 CDM catechin replaced the biphasic growth by a diauxic 421

growth where a clear diauxic lag was evident This growth pattern is usually associated 422

to a better control of substrate assimilation (Daae et al 1998) so if catechin as it is 423

suspected inferred a more efficient substrate uptake a diauxic lag would be necessary 424

to fit the enzyme synthesis for the utilization of the second substrate 425

The results obtained in wine resembling medium or BFM revealed that catechin 426

sped-up malolactic fermentation stimulated the consumption of several sugars as well 427

as the lactic acid formation thus providing growth advantages to Lact plantarum 428

RM71 in these media Growth stimulation linked to malolactic fermentation is known to 429

be caused by its coupling to ATP synthesis by either the entry through F(H+)-ATPase 430

of environmental protons generated in the malate decarboxilation process and the 431

malatelactate antiport coupled also to F(H+)-ATPase Accordingly the growth 432

improvement of Lact plantarum RM71 upon catechin should not be surprising in view 433

of the accelerated malolactic fermentation and lactic acid production provided by the 434

flavanol Similar effects have been also described for the growth of O oeni CECT 435

4100T where this flavanol was found to stimulate the malolactic fermentation 436

development (Reguant et al 2000) Although malolactic fermentation is classically 437

associated to winemaking the importance of the growth benefit obtained by Lact 438

plantarum from the utilization of malic acid should not be restricted to the wine 439

environment but extended to other fermented food plants as recently reported (Plumed-440

Ferrer et al 2008) These authors found a significantly higher growth of Lact 441

Page 18 of 33

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For Peer Review

19

plantarum in cucumber juice compared to the rich MRS medium and highlighted the 442

crucial importance of the higher decarboxylation of malic acid in this stimulation 443

In summary the results presented here show that catechin improved malic acid 444

degradation fermentation of several sugars and therefore Lact plantarum RM71 445

growth which are desirable traits for a successful performance of this species as starter 446

in several food plant fermentations These stimulations are not linked to catechin 447

degradation 448

449

Acknowledgements 450

451

This work was supported by grants AGL2005-000470 and AGL2008-001052 (CICYT) 452

and 200670M058 (CAM) J A Curiel is a recipient of a fellowship from Ministerio de 453

Ciencia e Innovacioacuten (MICINN) 454

455

REFERENCES 456

457

Adlerberth I Ahrne S Johansson ML Molin G Hanson LA and Wold AE 458

(1996) A mannose specific adherence mechanism in Lactobacillus plantarum 459

conferring binding to the human colonic cell line HT-29 Appl Environ 460

Microbiol 62 2244-2251 461

462

Alberto MR Fariacuteas ME and Manca de Nadra MC (2001) Effect of gallic acid and 463

catechin on Lactobacillus hilgardii 5w growth and metabolism of organics 464

compounds J Agr Food Chem 49 4359-4363 465

466

Page 19 of 33

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For Peer Review

20

Amarowicz R Carle R Dongowski G Durazzo A Galensa R Kammerer D 467

Maiani G and Piskula MK (2009) Influence of postharvest processing and 468

storage on the content of phenolic acids and flavonoids in foods Mol Nutr Food 469

Res doi 101002mnfr200700486 470

471

Campos FM Couto JA and Hogg TA (2003) Influence of phenolics acids on 472

growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii J Appl 473

Microbiol 94 167-174 474

475

Caturla N Vera-Samper E Villalaiacuten J Reyes Mateo C and Micol V (2003) The 476

relationship between the antioxidant and the antibacterial properties of 477

galloylated catechins and the structure of phospholipids membranes Free Rad 478

Biol Med 34 648-662 479

480

Crespy V and Williamson G (2004) A review of the health effects of green tea 481

catechins in in vivo animal models J Nutr 134 3431-3440 482

483

Daae EB and Ison AP (1998) A simple structured model describing the growth of 484

Streptomyces lividans Biotechnol Bioeng 58 263-266 485

486

de Figueroa R M Benito de Caacuterdenas I L Sesma F Alvarez F de Ruiz Holgado 487

A P and Oliver G (1996) Inducible transport of citrate in Lactobacillus 488

rhamnosus ATCC 7469 J Appl Microbiol 81 348-354 489

490

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For Peer Review

21

de Man J C Rogosa M and Sharpe M E (1960) A medium for the cultivation of 491

lactobacilli J Appl Microbiol 23 130-135 492

493

Figueiredo AR Campos F de Freitas V Hogg T and Couto JA (2008) Effect of 494

phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus 495

oeni and Lactobacillus hilgardii Food Microbiol 25 105-12 496

497

Goh Y J Lee J H and Hutkins R W (2007) Functional analysis of the 498

fructooligosaccharide utilization operon in Lactobacillus paracasei 1195 Appl 499

Environ Microbiol 73 5716-5724 500

501

Hussein L Medina A Barrionuevo A Lammuela-Raventos RM and Andres-502

Lacueva C (2009) Normal distribution of urinary polyphenol excretion among 503

Egyptian males 7-14 years old and changes following nutritional intervention 504

with tomato juice (Lycopersicon esculentum) Int J Food Sci Nutr 60 302 ndash 311 505

506

Ikagai H Nakae T Hara Y and Shimamura T (1993) Bactericidal catechins 507

damage the lipid bilayer Biochim Biophys Acta 1147 132-136 508

509

Kajiya K S Kumazawa and Nakayama T (2001) Steric effects on the interaction of 510

tea catechins with lipid bilayers Biosci Biotechnol Biochem 65 2638-2643 511

512

Kostinek M Ban-Koffi L Ottah-Atikpo M Teniola D Schillinger U Holzapfel 513

WH and Franz C (2008) Diversity of predominant lactic acid bacteria 514

associated with cocoa fermentation in Nigeria Curr Microbiol 56 306-314 515

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For Peer Review

15

catechin peak eluting at 1817 min among HPLC chromatograms of cell-free control 342

and culture supernatants after 40 h incubation in CDM 343

344

DISCUSSION 345

346

The effects of phenolics compounds on lactic acid bacteria have been reported to 347

be rather variable (For a recent review see Rodriacuteguez et al 2009) This variability 348

depends on the compound (Vivas et al 1997 Campos et al 2003 Figueiredo et al 349

2008) the strain under study (Figueiredo et al 2008) or the medium used for growth 350

(Alberto et al 2001) Up to date some authors have associated the positive effects of 351

food relevant phenolic compounds on growth of lactic acid bacteria to the degradation 352

of the compound Thus the growth stimulating effects of gallate on O oeni (Vivas et al 353

1997) or Lact hilgardii (Alberto et al 2001) were reported to be dose-dependent and 354

suggested to be linked to gallate degradation In the case of anthocyanins it was 355

suggested that glucose moiety was used as energy source to justify the positive effect on 356

the growth of O oeni (Vivas et al 1997) 357

In this study it was observed that most of the catechin remained in the media 358

after fermentation with Lact plantarum RM71 This result was interpreted as this 359

bacterium did not degrade the largest catechin bulk otherwise the catechin peak would 360

have been disappeared or greatly diminished and new compounds would have been 361

appeared Consequently the metabolic and growth stimulations promoted by catechin 362

can not be ascribed to catechin metabolism by Lact plantarum RM 71 These results 363

are in agreement with those previously reported for O oeni by Reguant et al (2000) 364

who also uncoupled the stimulation of growth triggered by catechin from the 365

metabolism of this flavanol Since O oeni grows better in absence of oxygen these 366

Page 15 of 33

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For Peer Review

16

authors suggested that the oxygen scavenging ability of flavonoids tested including 367

catechin favoured the growth stimulating effects observed Contrarily other authors 368

could not apply the same reasoning as they found that catechin or epicatechin showed 369

neutral effects on the growth of O oeni and Lact hilgardii (Figueiredo et al 2008) It 370

has been recently reported that the radical scavenging capacity of catechins increased 371

with the pH of the medium (Muzolf et al 2008) however in this work the positive 372

effects of catechin on the growth of Lact plantarum were more pronounced at the lower 373

pH and not dependent on the dose of catechin used Therefore the positive influence of 374

catechin on growth found here can hardly be ascribed to its radical scavenging capacity 375

By time course of sugar consumption data tracking experiments it is shown that 376

at least in the case of Lact plantarum RM71 catechin stimulates growth by promoting 377

quicker sugar consumption increasing the extension of sugar utilization and stimulating 378

malic acid decarboxylation Clearly this is a different mode of action of catechin to that 379

suggested by this flavanol on Lact hilgardii where the growth stimulating effects were 380

linked to catechin degradation (Alberto et al 2001 Figueiredo et al 2008) or to that 381

suggested by Reguant et al (2000) for O oeni who justified the stimulating effects on 382

the antioxidant properties of catechin 383

The beneficial effects on growth were observed regardless of the catechin 384

concentration examined which support again the hypotheses that growth stimulation is 385

uncoupled from catechin degradation The lack of dose dependent effects is in 386

agreement with the weak bactericidal activity (Nakayama et al 1998) resulting from the 387

shallow location close to the phospholipidwater interface previously reported for 388

catechin (Caturla et al 2003) Otherwise an increased bactericidal effect would be 389

expected at increased catechin concentrations as it occurs with galloylated catechins 390

which possess a higher insertion capacity in the membrane (Ikagai et al 1993 Caturla 391

Page 16 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

17

et al 2003) Although the growth advantages were observed regardless the initial pH 392

values they were more pronounced in the pH 55 CDM which could indicate a stronger 393

interaction of catechin with membrane phospholipids at this lower pH than at pH 65 394

According to previous reports (Kajiya et al 2001 Caturla et al 2003) catechin 395

does not enter the cell so it is probable that the effects observed on growth and 396

metabolism were caused by alteration of the biophysical properties of the membrane of 397

Lact plantarum RM71 It is possible that catechin after had caused its biological effect 398

could had left the membrane by passive diffusion along time a process that has been 399

previously described for other flavonoids such as quercetin (Movileanu et al 2000) 400

Thus catechin once located in the membrane could alter the function of proteins 401

associated with the bilayer and affect the transport of materials across it Sugar and 402

malate transporters seem to be affected proteins accordingly to the changes in sugar and 403

malic acid consumption observed here upon catechin The citrate transport system could 404

be affected as well since catechin triggered variations in the growth patterns displayed 405

by Lact plantarum RM71 on citrate supplemented CDM Experiments are currently 406

under way to determine if catechin alters some biophysical properties of the membrane 407

of Lact plantarum and affects these transport systems 408

Citrate prolonged lag phase and induced sequential growth behaviour in CDM 409

The scope of this study was not to investigate this behaviour however the long lag 410

phase observed upon citrate indicates that there might be some limitations on the 411

substrate utilization apparatus at the onset of cultures which is usual when growing in 412

mixed-substrate environments (Daae et al 1998) The biphasic growth could be due to a 413

sequential metabolism of carbon sources since this behaviour was only observed at an 414

initial pH of 55 which is close to the optimum pH range for citrate metabolism in Lact 415

plantarum (Palles et al 1998) This type of sequential metabolism is not unprecedent 416

Page 17 of 33

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For Peer Review

18

for Lactobacillus ssp Thus diauxic growth has been described during growth also 417

under sugar limitation on a mixture of sugars and fructooligosaccharides (FOS) (Goh et 418

al 2007) and glucose and citrate (de Figueroa et al 1996) Catechin shorthened the lag 419

phase so it is probable that this flavanol relieved limitations in substrate utilization 420

Moreover in complete pH 55 CDM catechin replaced the biphasic growth by a diauxic 421

growth where a clear diauxic lag was evident This growth pattern is usually associated 422

to a better control of substrate assimilation (Daae et al 1998) so if catechin as it is 423

suspected inferred a more efficient substrate uptake a diauxic lag would be necessary 424

to fit the enzyme synthesis for the utilization of the second substrate 425

The results obtained in wine resembling medium or BFM revealed that catechin 426

sped-up malolactic fermentation stimulated the consumption of several sugars as well 427

as the lactic acid formation thus providing growth advantages to Lact plantarum 428

RM71 in these media Growth stimulation linked to malolactic fermentation is known to 429

be caused by its coupling to ATP synthesis by either the entry through F(H+)-ATPase 430

of environmental protons generated in the malate decarboxilation process and the 431

malatelactate antiport coupled also to F(H+)-ATPase Accordingly the growth 432

improvement of Lact plantarum RM71 upon catechin should not be surprising in view 433

of the accelerated malolactic fermentation and lactic acid production provided by the 434

flavanol Similar effects have been also described for the growth of O oeni CECT 435

4100T where this flavanol was found to stimulate the malolactic fermentation 436

development (Reguant et al 2000) Although malolactic fermentation is classically 437

associated to winemaking the importance of the growth benefit obtained by Lact 438

plantarum from the utilization of malic acid should not be restricted to the wine 439

environment but extended to other fermented food plants as recently reported (Plumed-440

Ferrer et al 2008) These authors found a significantly higher growth of Lact 441

Page 18 of 33

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For Peer Review

19

plantarum in cucumber juice compared to the rich MRS medium and highlighted the 442

crucial importance of the higher decarboxylation of malic acid in this stimulation 443

In summary the results presented here show that catechin improved malic acid 444

degradation fermentation of several sugars and therefore Lact plantarum RM71 445

growth which are desirable traits for a successful performance of this species as starter 446

in several food plant fermentations These stimulations are not linked to catechin 447

degradation 448

449

Acknowledgements 450

451

This work was supported by grants AGL2005-000470 and AGL2008-001052 (CICYT) 452

and 200670M058 (CAM) J A Curiel is a recipient of a fellowship from Ministerio de 453

Ciencia e Innovacioacuten (MICINN) 454

455

REFERENCES 456

457

Adlerberth I Ahrne S Johansson ML Molin G Hanson LA and Wold AE 458

(1996) A mannose specific adherence mechanism in Lactobacillus plantarum 459

conferring binding to the human colonic cell line HT-29 Appl Environ 460

Microbiol 62 2244-2251 461

462

Alberto MR Fariacuteas ME and Manca de Nadra MC (2001) Effect of gallic acid and 463

catechin on Lactobacillus hilgardii 5w growth and metabolism of organics 464

compounds J Agr Food Chem 49 4359-4363 465

466

Page 19 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

20

Amarowicz R Carle R Dongowski G Durazzo A Galensa R Kammerer D 467

Maiani G and Piskula MK (2009) Influence of postharvest processing and 468

storage on the content of phenolic acids and flavonoids in foods Mol Nutr Food 469

Res doi 101002mnfr200700486 470

471

Campos FM Couto JA and Hogg TA (2003) Influence of phenolics acids on 472

growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii J Appl 473

Microbiol 94 167-174 474

475

Caturla N Vera-Samper E Villalaiacuten J Reyes Mateo C and Micol V (2003) The 476

relationship between the antioxidant and the antibacterial properties of 477

galloylated catechins and the structure of phospholipids membranes Free Rad 478

Biol Med 34 648-662 479

480

Crespy V and Williamson G (2004) A review of the health effects of green tea 481

catechins in in vivo animal models J Nutr 134 3431-3440 482

483

Daae EB and Ison AP (1998) A simple structured model describing the growth of 484

Streptomyces lividans Biotechnol Bioeng 58 263-266 485

486

de Figueroa R M Benito de Caacuterdenas I L Sesma F Alvarez F de Ruiz Holgado 487

A P and Oliver G (1996) Inducible transport of citrate in Lactobacillus 488

rhamnosus ATCC 7469 J Appl Microbiol 81 348-354 489

490

Page 20 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

21

de Man J C Rogosa M and Sharpe M E (1960) A medium for the cultivation of 491

lactobacilli J Appl Microbiol 23 130-135 492

493

Figueiredo AR Campos F de Freitas V Hogg T and Couto JA (2008) Effect of 494

phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus 495

oeni and Lactobacillus hilgardii Food Microbiol 25 105-12 496

497

Goh Y J Lee J H and Hutkins R W (2007) Functional analysis of the 498

fructooligosaccharide utilization operon in Lactobacillus paracasei 1195 Appl 499

Environ Microbiol 73 5716-5724 500

501

Hussein L Medina A Barrionuevo A Lammuela-Raventos RM and Andres-502

Lacueva C (2009) Normal distribution of urinary polyphenol excretion among 503

Egyptian males 7-14 years old and changes following nutritional intervention 504

with tomato juice (Lycopersicon esculentum) Int J Food Sci Nutr 60 302 ndash 311 505

506

Ikagai H Nakae T Hara Y and Shimamura T (1993) Bactericidal catechins 507

damage the lipid bilayer Biochim Biophys Acta 1147 132-136 508

509

Kajiya K S Kumazawa and Nakayama T (2001) Steric effects on the interaction of 510

tea catechins with lipid bilayers Biosci Biotechnol Biochem 65 2638-2643 511

512

Kostinek M Ban-Koffi L Ottah-Atikpo M Teniola D Schillinger U Holzapfel 513

WH and Franz C (2008) Diversity of predominant lactic acid bacteria 514

associated with cocoa fermentation in Nigeria Curr Microbiol 56 306-314 515

Page 21 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

16

authors suggested that the oxygen scavenging ability of flavonoids tested including 367

catechin favoured the growth stimulating effects observed Contrarily other authors 368

could not apply the same reasoning as they found that catechin or epicatechin showed 369

neutral effects on the growth of O oeni and Lact hilgardii (Figueiredo et al 2008) It 370

has been recently reported that the radical scavenging capacity of catechins increased 371

with the pH of the medium (Muzolf et al 2008) however in this work the positive 372

effects of catechin on the growth of Lact plantarum were more pronounced at the lower 373

pH and not dependent on the dose of catechin used Therefore the positive influence of 374

catechin on growth found here can hardly be ascribed to its radical scavenging capacity 375

By time course of sugar consumption data tracking experiments it is shown that 376

at least in the case of Lact plantarum RM71 catechin stimulates growth by promoting 377

quicker sugar consumption increasing the extension of sugar utilization and stimulating 378

malic acid decarboxylation Clearly this is a different mode of action of catechin to that 379

suggested by this flavanol on Lact hilgardii where the growth stimulating effects were 380

linked to catechin degradation (Alberto et al 2001 Figueiredo et al 2008) or to that 381

suggested by Reguant et al (2000) for O oeni who justified the stimulating effects on 382

the antioxidant properties of catechin 383

The beneficial effects on growth were observed regardless of the catechin 384

concentration examined which support again the hypotheses that growth stimulation is 385

uncoupled from catechin degradation The lack of dose dependent effects is in 386

agreement with the weak bactericidal activity (Nakayama et al 1998) resulting from the 387

shallow location close to the phospholipidwater interface previously reported for 388

catechin (Caturla et al 2003) Otherwise an increased bactericidal effect would be 389

expected at increased catechin concentrations as it occurs with galloylated catechins 390

which possess a higher insertion capacity in the membrane (Ikagai et al 1993 Caturla 391

Page 16 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

17

et al 2003) Although the growth advantages were observed regardless the initial pH 392

values they were more pronounced in the pH 55 CDM which could indicate a stronger 393

interaction of catechin with membrane phospholipids at this lower pH than at pH 65 394

According to previous reports (Kajiya et al 2001 Caturla et al 2003) catechin 395

does not enter the cell so it is probable that the effects observed on growth and 396

metabolism were caused by alteration of the biophysical properties of the membrane of 397

Lact plantarum RM71 It is possible that catechin after had caused its biological effect 398

could had left the membrane by passive diffusion along time a process that has been 399

previously described for other flavonoids such as quercetin (Movileanu et al 2000) 400

Thus catechin once located in the membrane could alter the function of proteins 401

associated with the bilayer and affect the transport of materials across it Sugar and 402

malate transporters seem to be affected proteins accordingly to the changes in sugar and 403

malic acid consumption observed here upon catechin The citrate transport system could 404

be affected as well since catechin triggered variations in the growth patterns displayed 405

by Lact plantarum RM71 on citrate supplemented CDM Experiments are currently 406

under way to determine if catechin alters some biophysical properties of the membrane 407

of Lact plantarum and affects these transport systems 408

Citrate prolonged lag phase and induced sequential growth behaviour in CDM 409

The scope of this study was not to investigate this behaviour however the long lag 410

phase observed upon citrate indicates that there might be some limitations on the 411

substrate utilization apparatus at the onset of cultures which is usual when growing in 412

mixed-substrate environments (Daae et al 1998) The biphasic growth could be due to a 413

sequential metabolism of carbon sources since this behaviour was only observed at an 414

initial pH of 55 which is close to the optimum pH range for citrate metabolism in Lact 415

plantarum (Palles et al 1998) This type of sequential metabolism is not unprecedent 416

Page 17 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

18

for Lactobacillus ssp Thus diauxic growth has been described during growth also 417

under sugar limitation on a mixture of sugars and fructooligosaccharides (FOS) (Goh et 418

al 2007) and glucose and citrate (de Figueroa et al 1996) Catechin shorthened the lag 419

phase so it is probable that this flavanol relieved limitations in substrate utilization 420

Moreover in complete pH 55 CDM catechin replaced the biphasic growth by a diauxic 421

growth where a clear diauxic lag was evident This growth pattern is usually associated 422

to a better control of substrate assimilation (Daae et al 1998) so if catechin as it is 423

suspected inferred a more efficient substrate uptake a diauxic lag would be necessary 424

to fit the enzyme synthesis for the utilization of the second substrate 425

The results obtained in wine resembling medium or BFM revealed that catechin 426

sped-up malolactic fermentation stimulated the consumption of several sugars as well 427

as the lactic acid formation thus providing growth advantages to Lact plantarum 428

RM71 in these media Growth stimulation linked to malolactic fermentation is known to 429

be caused by its coupling to ATP synthesis by either the entry through F(H+)-ATPase 430

of environmental protons generated in the malate decarboxilation process and the 431

malatelactate antiport coupled also to F(H+)-ATPase Accordingly the growth 432

improvement of Lact plantarum RM71 upon catechin should not be surprising in view 433

of the accelerated malolactic fermentation and lactic acid production provided by the 434

flavanol Similar effects have been also described for the growth of O oeni CECT 435

4100T where this flavanol was found to stimulate the malolactic fermentation 436

development (Reguant et al 2000) Although malolactic fermentation is classically 437

associated to winemaking the importance of the growth benefit obtained by Lact 438

plantarum from the utilization of malic acid should not be restricted to the wine 439

environment but extended to other fermented food plants as recently reported (Plumed-440

Ferrer et al 2008) These authors found a significantly higher growth of Lact 441

Page 18 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

19

plantarum in cucumber juice compared to the rich MRS medium and highlighted the 442

crucial importance of the higher decarboxylation of malic acid in this stimulation 443

In summary the results presented here show that catechin improved malic acid 444

degradation fermentation of several sugars and therefore Lact plantarum RM71 445

growth which are desirable traits for a successful performance of this species as starter 446

in several food plant fermentations These stimulations are not linked to catechin 447

degradation 448

449

Acknowledgements 450

451

This work was supported by grants AGL2005-000470 and AGL2008-001052 (CICYT) 452

and 200670M058 (CAM) J A Curiel is a recipient of a fellowship from Ministerio de 453

Ciencia e Innovacioacuten (MICINN) 454

455

REFERENCES 456

457

Adlerberth I Ahrne S Johansson ML Molin G Hanson LA and Wold AE 458

(1996) A mannose specific adherence mechanism in Lactobacillus plantarum 459

conferring binding to the human colonic cell line HT-29 Appl Environ 460

Microbiol 62 2244-2251 461

462

Alberto MR Fariacuteas ME and Manca de Nadra MC (2001) Effect of gallic acid and 463

catechin on Lactobacillus hilgardii 5w growth and metabolism of organics 464

compounds J Agr Food Chem 49 4359-4363 465

466

Page 19 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

20

Amarowicz R Carle R Dongowski G Durazzo A Galensa R Kammerer D 467

Maiani G and Piskula MK (2009) Influence of postharvest processing and 468

storage on the content of phenolic acids and flavonoids in foods Mol Nutr Food 469

Res doi 101002mnfr200700486 470

471

Campos FM Couto JA and Hogg TA (2003) Influence of phenolics acids on 472

growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii J Appl 473

Microbiol 94 167-174 474

475

Caturla N Vera-Samper E Villalaiacuten J Reyes Mateo C and Micol V (2003) The 476

relationship between the antioxidant and the antibacterial properties of 477

galloylated catechins and the structure of phospholipids membranes Free Rad 478

Biol Med 34 648-662 479

480

Crespy V and Williamson G (2004) A review of the health effects of green tea 481

catechins in in vivo animal models J Nutr 134 3431-3440 482

483

Daae EB and Ison AP (1998) A simple structured model describing the growth of 484

Streptomyces lividans Biotechnol Bioeng 58 263-266 485

486

de Figueroa R M Benito de Caacuterdenas I L Sesma F Alvarez F de Ruiz Holgado 487

A P and Oliver G (1996) Inducible transport of citrate in Lactobacillus 488

rhamnosus ATCC 7469 J Appl Microbiol 81 348-354 489

490

Page 20 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

21

de Man J C Rogosa M and Sharpe M E (1960) A medium for the cultivation of 491

lactobacilli J Appl Microbiol 23 130-135 492

493

Figueiredo AR Campos F de Freitas V Hogg T and Couto JA (2008) Effect of 494

phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus 495

oeni and Lactobacillus hilgardii Food Microbiol 25 105-12 496

497

Goh Y J Lee J H and Hutkins R W (2007) Functional analysis of the 498

fructooligosaccharide utilization operon in Lactobacillus paracasei 1195 Appl 499

Environ Microbiol 73 5716-5724 500

501

Hussein L Medina A Barrionuevo A Lammuela-Raventos RM and Andres-502

Lacueva C (2009) Normal distribution of urinary polyphenol excretion among 503

Egyptian males 7-14 years old and changes following nutritional intervention 504

with tomato juice (Lycopersicon esculentum) Int J Food Sci Nutr 60 302 ndash 311 505

506

Ikagai H Nakae T Hara Y and Shimamura T (1993) Bactericidal catechins 507

damage the lipid bilayer Biochim Biophys Acta 1147 132-136 508

509

Kajiya K S Kumazawa and Nakayama T (2001) Steric effects on the interaction of 510

tea catechins with lipid bilayers Biosci Biotechnol Biochem 65 2638-2643 511

512

Kostinek M Ban-Koffi L Ottah-Atikpo M Teniola D Schillinger U Holzapfel 513

WH and Franz C (2008) Diversity of predominant lactic acid bacteria 514

associated with cocoa fermentation in Nigeria Curr Microbiol 56 306-314 515

Page 21 of 33

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17

et al 2003) Although the growth advantages were observed regardless the initial pH 392

values they were more pronounced in the pH 55 CDM which could indicate a stronger 393

interaction of catechin with membrane phospholipids at this lower pH than at pH 65 394

According to previous reports (Kajiya et al 2001 Caturla et al 2003) catechin 395

does not enter the cell so it is probable that the effects observed on growth and 396

metabolism were caused by alteration of the biophysical properties of the membrane of 397

Lact plantarum RM71 It is possible that catechin after had caused its biological effect 398

could had left the membrane by passive diffusion along time a process that has been 399

previously described for other flavonoids such as quercetin (Movileanu et al 2000) 400

Thus catechin once located in the membrane could alter the function of proteins 401

associated with the bilayer and affect the transport of materials across it Sugar and 402

malate transporters seem to be affected proteins accordingly to the changes in sugar and 403

malic acid consumption observed here upon catechin The citrate transport system could 404

be affected as well since catechin triggered variations in the growth patterns displayed 405

by Lact plantarum RM71 on citrate supplemented CDM Experiments are currently 406

under way to determine if catechin alters some biophysical properties of the membrane 407

of Lact plantarum and affects these transport systems 408

Citrate prolonged lag phase and induced sequential growth behaviour in CDM 409

The scope of this study was not to investigate this behaviour however the long lag 410

phase observed upon citrate indicates that there might be some limitations on the 411

substrate utilization apparatus at the onset of cultures which is usual when growing in 412

mixed-substrate environments (Daae et al 1998) The biphasic growth could be due to a 413

sequential metabolism of carbon sources since this behaviour was only observed at an 414

initial pH of 55 which is close to the optimum pH range for citrate metabolism in Lact 415

plantarum (Palles et al 1998) This type of sequential metabolism is not unprecedent 416

Page 17 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

18

for Lactobacillus ssp Thus diauxic growth has been described during growth also 417

under sugar limitation on a mixture of sugars and fructooligosaccharides (FOS) (Goh et 418

al 2007) and glucose and citrate (de Figueroa et al 1996) Catechin shorthened the lag 419

phase so it is probable that this flavanol relieved limitations in substrate utilization 420

Moreover in complete pH 55 CDM catechin replaced the biphasic growth by a diauxic 421

growth where a clear diauxic lag was evident This growth pattern is usually associated 422

to a better control of substrate assimilation (Daae et al 1998) so if catechin as it is 423

suspected inferred a more efficient substrate uptake a diauxic lag would be necessary 424

to fit the enzyme synthesis for the utilization of the second substrate 425

The results obtained in wine resembling medium or BFM revealed that catechin 426

sped-up malolactic fermentation stimulated the consumption of several sugars as well 427

as the lactic acid formation thus providing growth advantages to Lact plantarum 428

RM71 in these media Growth stimulation linked to malolactic fermentation is known to 429

be caused by its coupling to ATP synthesis by either the entry through F(H+)-ATPase 430

of environmental protons generated in the malate decarboxilation process and the 431

malatelactate antiport coupled also to F(H+)-ATPase Accordingly the growth 432

improvement of Lact plantarum RM71 upon catechin should not be surprising in view 433

of the accelerated malolactic fermentation and lactic acid production provided by the 434

flavanol Similar effects have been also described for the growth of O oeni CECT 435

4100T where this flavanol was found to stimulate the malolactic fermentation 436

development (Reguant et al 2000) Although malolactic fermentation is classically 437

associated to winemaking the importance of the growth benefit obtained by Lact 438

plantarum from the utilization of malic acid should not be restricted to the wine 439

environment but extended to other fermented food plants as recently reported (Plumed-440

Ferrer et al 2008) These authors found a significantly higher growth of Lact 441

Page 18 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

19

plantarum in cucumber juice compared to the rich MRS medium and highlighted the 442

crucial importance of the higher decarboxylation of malic acid in this stimulation 443

In summary the results presented here show that catechin improved malic acid 444

degradation fermentation of several sugars and therefore Lact plantarum RM71 445

growth which are desirable traits for a successful performance of this species as starter 446

in several food plant fermentations These stimulations are not linked to catechin 447

degradation 448

449

Acknowledgements 450

451

This work was supported by grants AGL2005-000470 and AGL2008-001052 (CICYT) 452

and 200670M058 (CAM) J A Curiel is a recipient of a fellowship from Ministerio de 453

Ciencia e Innovacioacuten (MICINN) 454

455

REFERENCES 456

457

Adlerberth I Ahrne S Johansson ML Molin G Hanson LA and Wold AE 458

(1996) A mannose specific adherence mechanism in Lactobacillus plantarum 459

conferring binding to the human colonic cell line HT-29 Appl Environ 460

Microbiol 62 2244-2251 461

462

Alberto MR Fariacuteas ME and Manca de Nadra MC (2001) Effect of gallic acid and 463

catechin on Lactobacillus hilgardii 5w growth and metabolism of organics 464

compounds J Agr Food Chem 49 4359-4363 465

466

Page 19 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

20

Amarowicz R Carle R Dongowski G Durazzo A Galensa R Kammerer D 467

Maiani G and Piskula MK (2009) Influence of postharvest processing and 468

storage on the content of phenolic acids and flavonoids in foods Mol Nutr Food 469

Res doi 101002mnfr200700486 470

471

Campos FM Couto JA and Hogg TA (2003) Influence of phenolics acids on 472

growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii J Appl 473

Microbiol 94 167-174 474

475

Caturla N Vera-Samper E Villalaiacuten J Reyes Mateo C and Micol V (2003) The 476

relationship between the antioxidant and the antibacterial properties of 477

galloylated catechins and the structure of phospholipids membranes Free Rad 478

Biol Med 34 648-662 479

480

Crespy V and Williamson G (2004) A review of the health effects of green tea 481

catechins in in vivo animal models J Nutr 134 3431-3440 482

483

Daae EB and Ison AP (1998) A simple structured model describing the growth of 484

Streptomyces lividans Biotechnol Bioeng 58 263-266 485

486

de Figueroa R M Benito de Caacuterdenas I L Sesma F Alvarez F de Ruiz Holgado 487

A P and Oliver G (1996) Inducible transport of citrate in Lactobacillus 488

rhamnosus ATCC 7469 J Appl Microbiol 81 348-354 489

490

Page 20 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

21

de Man J C Rogosa M and Sharpe M E (1960) A medium for the cultivation of 491

lactobacilli J Appl Microbiol 23 130-135 492

493

Figueiredo AR Campos F de Freitas V Hogg T and Couto JA (2008) Effect of 494

phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus 495

oeni and Lactobacillus hilgardii Food Microbiol 25 105-12 496

497

Goh Y J Lee J H and Hutkins R W (2007) Functional analysis of the 498

fructooligosaccharide utilization operon in Lactobacillus paracasei 1195 Appl 499

Environ Microbiol 73 5716-5724 500

501

Hussein L Medina A Barrionuevo A Lammuela-Raventos RM and Andres-502

Lacueva C (2009) Normal distribution of urinary polyphenol excretion among 503

Egyptian males 7-14 years old and changes following nutritional intervention 504

with tomato juice (Lycopersicon esculentum) Int J Food Sci Nutr 60 302 ndash 311 505

506

Ikagai H Nakae T Hara Y and Shimamura T (1993) Bactericidal catechins 507

damage the lipid bilayer Biochim Biophys Acta 1147 132-136 508

509

Kajiya K S Kumazawa and Nakayama T (2001) Steric effects on the interaction of 510

tea catechins with lipid bilayers Biosci Biotechnol Biochem 65 2638-2643 511

512

Kostinek M Ban-Koffi L Ottah-Atikpo M Teniola D Schillinger U Holzapfel 513

WH and Franz C (2008) Diversity of predominant lactic acid bacteria 514

associated with cocoa fermentation in Nigeria Curr Microbiol 56 306-314 515

Page 21 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

18

for Lactobacillus ssp Thus diauxic growth has been described during growth also 417

under sugar limitation on a mixture of sugars and fructooligosaccharides (FOS) (Goh et 418

al 2007) and glucose and citrate (de Figueroa et al 1996) Catechin shorthened the lag 419

phase so it is probable that this flavanol relieved limitations in substrate utilization 420

Moreover in complete pH 55 CDM catechin replaced the biphasic growth by a diauxic 421

growth where a clear diauxic lag was evident This growth pattern is usually associated 422

to a better control of substrate assimilation (Daae et al 1998) so if catechin as it is 423

suspected inferred a more efficient substrate uptake a diauxic lag would be necessary 424

to fit the enzyme synthesis for the utilization of the second substrate 425

The results obtained in wine resembling medium or BFM revealed that catechin 426

sped-up malolactic fermentation stimulated the consumption of several sugars as well 427

as the lactic acid formation thus providing growth advantages to Lact plantarum 428

RM71 in these media Growth stimulation linked to malolactic fermentation is known to 429

be caused by its coupling to ATP synthesis by either the entry through F(H+)-ATPase 430

of environmental protons generated in the malate decarboxilation process and the 431

malatelactate antiport coupled also to F(H+)-ATPase Accordingly the growth 432

improvement of Lact plantarum RM71 upon catechin should not be surprising in view 433

of the accelerated malolactic fermentation and lactic acid production provided by the 434

flavanol Similar effects have been also described for the growth of O oeni CECT 435

4100T where this flavanol was found to stimulate the malolactic fermentation 436

development (Reguant et al 2000) Although malolactic fermentation is classically 437

associated to winemaking the importance of the growth benefit obtained by Lact 438

plantarum from the utilization of malic acid should not be restricted to the wine 439

environment but extended to other fermented food plants as recently reported (Plumed-440

Ferrer et al 2008) These authors found a significantly higher growth of Lact 441

Page 18 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

19

plantarum in cucumber juice compared to the rich MRS medium and highlighted the 442

crucial importance of the higher decarboxylation of malic acid in this stimulation 443

In summary the results presented here show that catechin improved malic acid 444

degradation fermentation of several sugars and therefore Lact plantarum RM71 445

growth which are desirable traits for a successful performance of this species as starter 446

in several food plant fermentations These stimulations are not linked to catechin 447

degradation 448

449

Acknowledgements 450

451

This work was supported by grants AGL2005-000470 and AGL2008-001052 (CICYT) 452

and 200670M058 (CAM) J A Curiel is a recipient of a fellowship from Ministerio de 453

Ciencia e Innovacioacuten (MICINN) 454

455

REFERENCES 456

457

Adlerberth I Ahrne S Johansson ML Molin G Hanson LA and Wold AE 458

(1996) A mannose specific adherence mechanism in Lactobacillus plantarum 459

conferring binding to the human colonic cell line HT-29 Appl Environ 460

Microbiol 62 2244-2251 461

462

Alberto MR Fariacuteas ME and Manca de Nadra MC (2001) Effect of gallic acid and 463

catechin on Lactobacillus hilgardii 5w growth and metabolism of organics 464

compounds J Agr Food Chem 49 4359-4363 465

466

Page 19 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

20

Amarowicz R Carle R Dongowski G Durazzo A Galensa R Kammerer D 467

Maiani G and Piskula MK (2009) Influence of postharvest processing and 468

storage on the content of phenolic acids and flavonoids in foods Mol Nutr Food 469

Res doi 101002mnfr200700486 470

471

Campos FM Couto JA and Hogg TA (2003) Influence of phenolics acids on 472

growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii J Appl 473

Microbiol 94 167-174 474

475

Caturla N Vera-Samper E Villalaiacuten J Reyes Mateo C and Micol V (2003) The 476

relationship between the antioxidant and the antibacterial properties of 477

galloylated catechins and the structure of phospholipids membranes Free Rad 478

Biol Med 34 648-662 479

480

Crespy V and Williamson G (2004) A review of the health effects of green tea 481

catechins in in vivo animal models J Nutr 134 3431-3440 482

483

Daae EB and Ison AP (1998) A simple structured model describing the growth of 484

Streptomyces lividans Biotechnol Bioeng 58 263-266 485

486

de Figueroa R M Benito de Caacuterdenas I L Sesma F Alvarez F de Ruiz Holgado 487

A P and Oliver G (1996) Inducible transport of citrate in Lactobacillus 488

rhamnosus ATCC 7469 J Appl Microbiol 81 348-354 489

490

Page 20 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

21

de Man J C Rogosa M and Sharpe M E (1960) A medium for the cultivation of 491

lactobacilli J Appl Microbiol 23 130-135 492

493

Figueiredo AR Campos F de Freitas V Hogg T and Couto JA (2008) Effect of 494

phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus 495

oeni and Lactobacillus hilgardii Food Microbiol 25 105-12 496

497

Goh Y J Lee J H and Hutkins R W (2007) Functional analysis of the 498

fructooligosaccharide utilization operon in Lactobacillus paracasei 1195 Appl 499

Environ Microbiol 73 5716-5724 500

501

Hussein L Medina A Barrionuevo A Lammuela-Raventos RM and Andres-502

Lacueva C (2009) Normal distribution of urinary polyphenol excretion among 503

Egyptian males 7-14 years old and changes following nutritional intervention 504

with tomato juice (Lycopersicon esculentum) Int J Food Sci Nutr 60 302 ndash 311 505

506

Ikagai H Nakae T Hara Y and Shimamura T (1993) Bactericidal catechins 507

damage the lipid bilayer Biochim Biophys Acta 1147 132-136 508

509

Kajiya K S Kumazawa and Nakayama T (2001) Steric effects on the interaction of 510

tea catechins with lipid bilayers Biosci Biotechnol Biochem 65 2638-2643 511

512

Kostinek M Ban-Koffi L Ottah-Atikpo M Teniola D Schillinger U Holzapfel 513

WH and Franz C (2008) Diversity of predominant lactic acid bacteria 514

associated with cocoa fermentation in Nigeria Curr Microbiol 56 306-314 515

Page 21 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

19

plantarum in cucumber juice compared to the rich MRS medium and highlighted the 442

crucial importance of the higher decarboxylation of malic acid in this stimulation 443

In summary the results presented here show that catechin improved malic acid 444

degradation fermentation of several sugars and therefore Lact plantarum RM71 445

growth which are desirable traits for a successful performance of this species as starter 446

in several food plant fermentations These stimulations are not linked to catechin 447

degradation 448

449

Acknowledgements 450

451

This work was supported by grants AGL2005-000470 and AGL2008-001052 (CICYT) 452

and 200670M058 (CAM) J A Curiel is a recipient of a fellowship from Ministerio de 453

Ciencia e Innovacioacuten (MICINN) 454

455

REFERENCES 456

457

Adlerberth I Ahrne S Johansson ML Molin G Hanson LA and Wold AE 458

(1996) A mannose specific adherence mechanism in Lactobacillus plantarum 459

conferring binding to the human colonic cell line HT-29 Appl Environ 460

Microbiol 62 2244-2251 461

462

Alberto MR Fariacuteas ME and Manca de Nadra MC (2001) Effect of gallic acid and 463

catechin on Lactobacillus hilgardii 5w growth and metabolism of organics 464

compounds J Agr Food Chem 49 4359-4363 465

466

Page 19 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

20

Amarowicz R Carle R Dongowski G Durazzo A Galensa R Kammerer D 467

Maiani G and Piskula MK (2009) Influence of postharvest processing and 468

storage on the content of phenolic acids and flavonoids in foods Mol Nutr Food 469

Res doi 101002mnfr200700486 470

471

Campos FM Couto JA and Hogg TA (2003) Influence of phenolics acids on 472

growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii J Appl 473

Microbiol 94 167-174 474

475

Caturla N Vera-Samper E Villalaiacuten J Reyes Mateo C and Micol V (2003) The 476

relationship between the antioxidant and the antibacterial properties of 477

galloylated catechins and the structure of phospholipids membranes Free Rad 478

Biol Med 34 648-662 479

480

Crespy V and Williamson G (2004) A review of the health effects of green tea 481

catechins in in vivo animal models J Nutr 134 3431-3440 482

483

Daae EB and Ison AP (1998) A simple structured model describing the growth of 484

Streptomyces lividans Biotechnol Bioeng 58 263-266 485

486

de Figueroa R M Benito de Caacuterdenas I L Sesma F Alvarez F de Ruiz Holgado 487

A P and Oliver G (1996) Inducible transport of citrate in Lactobacillus 488

rhamnosus ATCC 7469 J Appl Microbiol 81 348-354 489

490

Page 20 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

21

de Man J C Rogosa M and Sharpe M E (1960) A medium for the cultivation of 491

lactobacilli J Appl Microbiol 23 130-135 492

493

Figueiredo AR Campos F de Freitas V Hogg T and Couto JA (2008) Effect of 494

phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus 495

oeni and Lactobacillus hilgardii Food Microbiol 25 105-12 496

497

Goh Y J Lee J H and Hutkins R W (2007) Functional analysis of the 498

fructooligosaccharide utilization operon in Lactobacillus paracasei 1195 Appl 499

Environ Microbiol 73 5716-5724 500

501

Hussein L Medina A Barrionuevo A Lammuela-Raventos RM and Andres-502

Lacueva C (2009) Normal distribution of urinary polyphenol excretion among 503

Egyptian males 7-14 years old and changes following nutritional intervention 504

with tomato juice (Lycopersicon esculentum) Int J Food Sci Nutr 60 302 ndash 311 505

506

Ikagai H Nakae T Hara Y and Shimamura T (1993) Bactericidal catechins 507

damage the lipid bilayer Biochim Biophys Acta 1147 132-136 508

509

Kajiya K S Kumazawa and Nakayama T (2001) Steric effects on the interaction of 510

tea catechins with lipid bilayers Biosci Biotechnol Biochem 65 2638-2643 511

512

Kostinek M Ban-Koffi L Ottah-Atikpo M Teniola D Schillinger U Holzapfel 513

WH and Franz C (2008) Diversity of predominant lactic acid bacteria 514

associated with cocoa fermentation in Nigeria Curr Microbiol 56 306-314 515

Page 21 of 33

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Peer Review

20

Amarowicz R Carle R Dongowski G Durazzo A Galensa R Kammerer D 467

Maiani G and Piskula MK (2009) Influence of postharvest processing and 468

storage on the content of phenolic acids and flavonoids in foods Mol Nutr Food 469

Res doi 101002mnfr200700486 470

471

Campos FM Couto JA and Hogg TA (2003) Influence of phenolics acids on 472

growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii J Appl 473

Microbiol 94 167-174 474

475

Caturla N Vera-Samper E Villalaiacuten J Reyes Mateo C and Micol V (2003) The 476

relationship between the antioxidant and the antibacterial properties of 477

galloylated catechins and the structure of phospholipids membranes Free Rad 478

Biol Med 34 648-662 479

480

Crespy V and Williamson G (2004) A review of the health effects of green tea 481

catechins in in vivo animal models J Nutr 134 3431-3440 482

483

Daae EB and Ison AP (1998) A simple structured model describing the growth of 484

Streptomyces lividans Biotechnol Bioeng 58 263-266 485

486

de Figueroa R M Benito de Caacuterdenas I L Sesma F Alvarez F de Ruiz Holgado 487

A P and Oliver G (1996) Inducible transport of citrate in Lactobacillus 488

rhamnosus ATCC 7469 J Appl Microbiol 81 348-354 489

490

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21

de Man J C Rogosa M and Sharpe M E (1960) A medium for the cultivation of 491

lactobacilli J Appl Microbiol 23 130-135 492

493

Figueiredo AR Campos F de Freitas V Hogg T and Couto JA (2008) Effect of 494

phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus 495

oeni and Lactobacillus hilgardii Food Microbiol 25 105-12 496

497

Goh Y J Lee J H and Hutkins R W (2007) Functional analysis of the 498

fructooligosaccharide utilization operon in Lactobacillus paracasei 1195 Appl 499

Environ Microbiol 73 5716-5724 500

501

Hussein L Medina A Barrionuevo A Lammuela-Raventos RM and Andres-502

Lacueva C (2009) Normal distribution of urinary polyphenol excretion among 503

Egyptian males 7-14 years old and changes following nutritional intervention 504

with tomato juice (Lycopersicon esculentum) Int J Food Sci Nutr 60 302 ndash 311 505

506

Ikagai H Nakae T Hara Y and Shimamura T (1993) Bactericidal catechins 507

damage the lipid bilayer Biochim Biophys Acta 1147 132-136 508

509

Kajiya K S Kumazawa and Nakayama T (2001) Steric effects on the interaction of 510

tea catechins with lipid bilayers Biosci Biotechnol Biochem 65 2638-2643 511

512

Kostinek M Ban-Koffi L Ottah-Atikpo M Teniola D Schillinger U Holzapfel 513

WH and Franz C (2008) Diversity of predominant lactic acid bacteria 514

associated with cocoa fermentation in Nigeria Curr Microbiol 56 306-314 515

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21

de Man J C Rogosa M and Sharpe M E (1960) A medium for the cultivation of 491

lactobacilli J Appl Microbiol 23 130-135 492

493

Figueiredo AR Campos F de Freitas V Hogg T and Couto JA (2008) Effect of 494

phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus 495

oeni and Lactobacillus hilgardii Food Microbiol 25 105-12 496

497

Goh Y J Lee J H and Hutkins R W (2007) Functional analysis of the 498

fructooligosaccharide utilization operon in Lactobacillus paracasei 1195 Appl 499

Environ Microbiol 73 5716-5724 500

501

Hussein L Medina A Barrionuevo A Lammuela-Raventos RM and Andres-502

Lacueva C (2009) Normal distribution of urinary polyphenol excretion among 503

Egyptian males 7-14 years old and changes following nutritional intervention 504

with tomato juice (Lycopersicon esculentum) Int J Food Sci Nutr 60 302 ndash 311 505

506

Ikagai H Nakae T Hara Y and Shimamura T (1993) Bactericidal catechins 507

damage the lipid bilayer Biochim Biophys Acta 1147 132-136 508

509

Kajiya K S Kumazawa and Nakayama T (2001) Steric effects on the interaction of 510

tea catechins with lipid bilayers Biosci Biotechnol Biochem 65 2638-2643 511

512

Kostinek M Ban-Koffi L Ottah-Atikpo M Teniola D Schillinger U Holzapfel 513

WH and Franz C (2008) Diversity of predominant lactic acid bacteria 514

associated with cocoa fermentation in Nigeria Curr Microbiol 56 306-314 515

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22

516

Morales G Linares JF Beloso A Albar JP Martiacutenez JL and Rojo F (2004) 517

The Pseudomonas putida Crc global regulator controls the expression of genes 518

from several chromosomal catabolic pathways for aromatic compounds J 519

Bacteriol 186 1337-1344 520

521

Moreno-Arribas M V Polo M C Jorganes F and Muntildeoz R (2003) Screening of 522

biogenic amine production by lactic acid bacteria isolated from grape must and 523

wine Int J Food Microbiol 84 117ndash123 524

525

Movileanu L Neagoe I and Flonta M L (2000) Interaction of the antioxidant 526

flavonoid quercetin with planar bilayers Int J Pharm 205 135-146 527

528

Muzolf M Szymusiak H Gliszczynska-Swiglo A Rietjens IMCM Tyrakowska 529

B (2008) pH-dependent radical scavenging capacity of green tea catechins J 530

Agr Food Chem 13 816-823 531

532

Nakayama T Ono K and Hashimoto K (1998) Affinity of antioxidative polyphenols 533

for lipid bilayers evaluated with a liposome system Biosci Biotechnol Biochem 534

62 1005-1007 535

536

Okada S Takahashi N Ohara N Uchimura T and Kozaki M (1996a) 537

Microorganisms involving in fermentation of awa-bancha japanese fermented 538

tea leaves microorganisms involving in fermentation of japanese fermented tea 539

leaves Part I Nippon Shokuhin Kogyo Gakkai-Shi 43 12-20 540

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23

541 Okada S Takahashi N Ohara N Uchimura T and Kozaki M (1996b) 542

Microorganisms involving in fermentation of goishi-cha japanese fermented tea 543

leaves Microorganisms involving in fermentation of japanese fermented tea 544

leaves Part II Nippon Shokuhin Kogyo Gakkai-Shi 43 1019-1027 545

546

Palles T Beresford T Condon S and Cogan T M (1998) Citrate metabolism in 547

Lactobacillus casei and Lactobacillus plantarum J Appl Microbiol 85 147ndash154 548

549

Plumed-Ferrer C Koistinen KM Tolonen TL Lehesranta SJ Kaumlrenlampi SO 550

Elina Maumlkimattila E Joutsjoki V Virtanen V and von Wright A (2008) 551

Comparative study of sugar fermentation and protein expression patterns of two 552

Lactobacillus plantarum strains grown in three different media Appl Environ 553

Microbiol 74 5349-5358 554

555

Reguant C Bordons A Arola L Rozegraves N (2000) Influence of phenolic compounds 556

on the physiology of Oenococcus oeni from wine J Appl Microbiol 88 1065-71 557

558

Robinson RR Feirtag J and Slavin JL (2001) Effects of dietary arabinogalactan on 559

gastrointestinal and blood parameters in healthy human subjects J Am Coll Nutr 560

20 279-281 561

562

Rodas AM Ferrer Sand Pardo I (2005) Polyphasic study of wine Lactobacillus 563

strains taxonomic implications Int J Syst Evol Microbiol 55 197-207 564

565

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24

Rodriacuteguez H Landete J M de las Rivas B and Muntildeoz R (2008) Metabolism of 566

food phenolic acids by Lactobacillus plantarum CECT 748T Food Chem 107 567

1393-1398 568

569

Rodriacuteguez H Curiel JA Landete JM de las Rivas B Loacutepez de Felipe F 570

Goacutemez-Cordoveacutes C Manchentildeo JM Muntildeoz R (2009) Food phenolics and 571

lactic acid bacteria Int J Food Microbiol 132 79-90 572

573

Teusink B van Enckevort F Francke C Wiersma A Wegkamp A Smid E J and 574

Siezen R J (2005) In silico reconstruction of the metabolic pathways of 575

Lactobacillus plantarum Comparing predictions of nutrient requirements with 576

those from growth experiments Appl Environ Microbiol 71 7253-7262577

578

Vivas N Lonvaud-Funel A and Glories Y (1997) Effect of phenolic acids and 579

anthocyanins on growth viability of a lactic acid bacterium Food Microbiol 14 580

291-300 581

582

Ugliano M Genovese A and Moio L (2003) Hydrolysis of wine aroma precursors 583

during malolactic fermentation with four commercial starter cultures of 584

Oenococcus oeni J Agr Food Chem 51 5073-5078 585

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25

Legends to Figures 586

587

Fig 1 Catechin effects on the growth curves of Lactobacillus plantarum RM71 depend 588

on the initial pH and CDM formulation Selected growth curves of cells grown at 30ordmC 589

in pH 55 (panels A to D) or in pH 65 (panels E to H) CDM are shown Panels A to D 590

or E to H correspond consecutively to growth in CDM lacking both citrate and acetate 591

(A E) including citrate and acetate (B F) including citrate only (CG) and acetate only 592

(DH) Catechin was added at the following concentrations no catechin (controls) 593

100 mg l-1 200 mg l-1594

595

Fig 2 Time course of galactose consumption and lactic acid production by 596

Lactobacillus plantarum RM71 in the presence of catechin Cultures (15 ml) of Lact 597

plantarum RM71 were grown in pH 55 CDM supplemented with 05 galactose 598

Growth was carried out in presence () or in the absence () of 100 mg l-1 of catechin 599

Triangles represent the time course of galactose consumption in the presence () or 600

absence () of catechin Diamonds represent the time course of lactic acid production 601

in the presence () or absence () of catechin Values are the average of three 602

independent replicates with maximum standard deviation of lt7 603

604

Fig 3 Catechin accelerates sugar consumption malolactic fermentation and lactic acid 605

production during growth of Lactobacillus plantarum RM71 in wine resembling 606

medium The wine resembling medium was set-up at an initial pH of 38 and fermented 607

with Lact plantarum RM71 for two days at 25ordmC Monitoring of the fermentation 608

metabolic parameters was carried out in cultures grown in presence of catechin (100 mg 609

l-1) (solid symbols) or in its absence (open symbols) Symbols Fructose () 610

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26

Glucose () L-lactic acid () L-malic acid () Each point is the average 611

value of at least two independent determinations Standard deviations are represented by 612

vertical bars 613

614

Fig 4 Effects of catechin on fermentation metabolic traits of Lactobacillus plantarum 615

RM71 in basal fermentation medium (BFM) The medium was set-up at an initial pH of 616

38 and fermented with Lactplantarum RM71 for three days at 25ordmC Monitoring of the 617

fermentation metabolic parameters was carried out in presence of catechin (100 mg l-1) 618

(solid symbols) or in its absence (open symbols) Triangles represent L-malic acid 619

() squares galactose () diamonds L-lactic acid ()Each point is the 620

average value of at least two independent determinations Standard deviations are 621

represented by vertical bars 622

623

624

Fig 5 HPLC chromatograms of Lactobacillus plantarum strain RM71 supernatants 625

grown in the presence of catechin Supernatants of Lact plantarum RM71cultures and 626

cell-free control culture after 40 h incubation in CDM were analyzed by HPLC as 627

described The medium was set-up at an initial pH of 55 and 100 mg l-1 of catechin in 628

absence (A) or in presence of cells (B) Absorbance was recorded at 280 nm 629

Page 26 of 33

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27

Table 1 The fermentation performance of Lactobacillus plantarum RM71 in wine-resembling medium is improved by catechin

mmol l-1 consumption of mmol l-1 production of

Time (d) Glucose Fructose L-malic L-lactic Log cfu

Control 0-1 02 (22) 038 (4) dagger 1165 97 (108) Dagger 462 sect

1-2 89 (978) 929 (96) 00 2523 (07) 73

Total 91 967 115 3493 (078)

CAT 0-1 222 (204) 225 (212) 1141 1555 (09) 505 1-2 868 (796) 834 (787) 00 2845 (09) 77

Total 109 1059 1178 44 (086)

Consumption or production values are calculated using the average values represented in Fig 3 dagger Values in parentheses are percentages of

glucose fructose or L-malic acid consumed respect to total amount used by Lact plantarum RM71 in the fermentationsDagger Values in parentheses

represent the daily or total lactic yield by glucose fructose and L-malic acid consumed (theoretical value of the ratio lacticmalic acid =067)

CAT (cultures grown upon 100 mg l-1 of catechin) sect Consumption or production values and cfu counts are the average of at least two

independent fermentations Values do not differ significantly by using non parametric tests (Plt005)

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28

Table 2 The fermentation performance of Lactobacillus plantarum RM71 in basal fermentation medium (BFM) is improved by catechin

mmol l-1

Time (d) Galactose consumption L-malic consumption L-lactic production Log cfu

Control 0-1 084 (48) 159 (137) dagger 078 (028) Dagger 387 sect

1-2 1032 (586) 999 (863) 1507 (055) 604 2-3 644 (366) 0 (0) 761 (059) 848

Total 176 1158 2268 (053)

CAT 0-1 435 (149) 683 (598) 557 (042) 427 1-2 142 (487) 459 (402) 2637 (084) 67 2-3 1059 (363) 0 (0) 123 (058) 876

Total 2914 1142 3866 (058)

Consumption or production values are calculated using the average values represented in Fig 4 dagger Values in parentheses are percentages of

galactose or L-malic acid consumed respect to total amount used by Lact plantarum RM71 in the fermentationsDagger Values in parentheses

represent the daily or total lactic yield by galactose and L-malic acid consumed (theoretical value of the ratio lacticmalic acid =067) CAT

(cultures grown upon 100 mg l-1 of catechin) sect Consumption or production values and cfu counts are the average of at least two independent

fermentations Values do not differ significantly by using non parametric tests (Plt005)

Page 28 of 33

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Fig 1 Loacutepez de Felipe Curiel amp Muntildeoz

A

0

005

01

015

02

025

03

035

0 10 20 30 40 50

Time (h)

OD

60

0

G

0

01

02

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04

05

06

07

08

0 10 20 30 40 50

Time (h)

OD

60

0

H

0

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04

05

06

07

0 10 20 30 40 50

Time (h)

OD

60

0

E

0

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02

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04

05

06

07

0 10 20 30 40 50

Time (h)

OD

60

0F

0

01

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06

07

0 10 20 30 40 50

Time (h)

OD

60

0B

0

01

02

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04

05

0 10 20 30 40 50

Time (h)

OD

60

0

C

0

01

02

03

04

05

0 10 20 30 40 50

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OD

60

0

D

0

01

02

03

04

05

0 10 20 30 40 50

Time (h)

OD

600

Page 29 of 33

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For Peer Review

Fig 2 Loacutepez de Felipe Curiel amp Muntildeoz

0

02

04

06

08

1

12

14

16

0 5 10 15 20 25 30 35 40 45

Time (hr)

Gro

wth

(O

D600)

0

005

01

015

02

025

03

035

04

045

05

Gala

cto

se

L-L

acti

c a

cid

(

)

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For Peer ReviewFig 3 Loacutepez de Felipe Curiel amp Muntildeoz

0

2

4

6

8

10

12

0 05 1 15 2 25

Time (days)

mM

(G

luco

se

Fru

cto

se)

0

5

10

15

20

25

30

35

40

45

50

mM

(L

-lacti

c a

cid

L

-mali

c a

cid

)

Page 31 of 33

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For Peer ReviewFig 4 Loacutepez de Felipe Curiel amp Muntildeoz

0

10

20

30

40

50

60

0 05 1 15 2 25 3 35

Time (days)

mM

(G

ala

cto

se

L

-la

cti

c a

cid

)

0

2

4

6

8

10

12

14

16

18

20

mM

(L

-ma

lic

)

Page 32 of 33

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For Peer ReviewFig 5 Loacutepez de Felipe Curiel amp Muntildeoz

mA

U

0

100

200

300

Minutes

0 20 40 60 80

mA

U

0

100

200

Time (min)

A

B

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For Peer Review

26

Glucose () L-lactic acid () L-malic acid () Each point is the average 611

value of at least two independent determinations Standard deviations are represented by 612

vertical bars 613

614

Fig 4 Effects of catechin on fermentation metabolic traits of Lactobacillus plantarum 615

RM71 in basal fermentation medium (BFM) The medium was set-up at an initial pH of 616

38 and fermented with Lactplantarum RM71 for three days at 25ordmC Monitoring of the 617

fermentation metabolic parameters was carried out in presence of catechin (100 mg l-1) 618

(solid symbols) or in its absence (open symbols) Triangles represent L-malic acid 619

() squares galactose () diamonds L-lactic acid ()Each point is the 620

average value of at least two independent determinations Standard deviations are 621

represented by vertical bars 622

623

624

Fig 5 HPLC chromatograms of Lactobacillus plantarum strain RM71 supernatants 625

grown in the presence of catechin Supernatants of Lact plantarum RM71cultures and 626

cell-free control culture after 40 h incubation in CDM were analyzed by HPLC as 627

described The medium was set-up at an initial pH of 55 and 100 mg l-1 of catechin in 628

absence (A) or in presence of cells (B) Absorbance was recorded at 280 nm 629

Page 26 of 33

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27

Table 1 The fermentation performance of Lactobacillus plantarum RM71 in wine-resembling medium is improved by catechin

mmol l-1 consumption of mmol l-1 production of

Time (d) Glucose Fructose L-malic L-lactic Log cfu

Control 0-1 02 (22) 038 (4) dagger 1165 97 (108) Dagger 462 sect

1-2 89 (978) 929 (96) 00 2523 (07) 73

Total 91 967 115 3493 (078)

CAT 0-1 222 (204) 225 (212) 1141 1555 (09) 505 1-2 868 (796) 834 (787) 00 2845 (09) 77

Total 109 1059 1178 44 (086)

Consumption or production values are calculated using the average values represented in Fig 3 dagger Values in parentheses are percentages of

glucose fructose or L-malic acid consumed respect to total amount used by Lact plantarum RM71 in the fermentationsDagger Values in parentheses

represent the daily or total lactic yield by glucose fructose and L-malic acid consumed (theoretical value of the ratio lacticmalic acid =067)

CAT (cultures grown upon 100 mg l-1 of catechin) sect Consumption or production values and cfu counts are the average of at least two

independent fermentations Values do not differ significantly by using non parametric tests (Plt005)

Page 27 of 33

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28

Table 2 The fermentation performance of Lactobacillus plantarum RM71 in basal fermentation medium (BFM) is improved by catechin

mmol l-1

Time (d) Galactose consumption L-malic consumption L-lactic production Log cfu

Control 0-1 084 (48) 159 (137) dagger 078 (028) Dagger 387 sect

1-2 1032 (586) 999 (863) 1507 (055) 604 2-3 644 (366) 0 (0) 761 (059) 848

Total 176 1158 2268 (053)

CAT 0-1 435 (149) 683 (598) 557 (042) 427 1-2 142 (487) 459 (402) 2637 (084) 67 2-3 1059 (363) 0 (0) 123 (058) 876

Total 2914 1142 3866 (058)

Consumption or production values are calculated using the average values represented in Fig 4 dagger Values in parentheses are percentages of

galactose or L-malic acid consumed respect to total amount used by Lact plantarum RM71 in the fermentationsDagger Values in parentheses

represent the daily or total lactic yield by galactose and L-malic acid consumed (theoretical value of the ratio lacticmalic acid =067) CAT

(cultures grown upon 100 mg l-1 of catechin) sect Consumption or production values and cfu counts are the average of at least two independent

fermentations Values do not differ significantly by using non parametric tests (Plt005)

Page 28 of 33

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Fig 1 Loacutepez de Felipe Curiel amp Muntildeoz

A

0

005

01

015

02

025

03

035

0 10 20 30 40 50

Time (h)

OD

60

0

G

0

01

02

03

04

05

06

07

08

0 10 20 30 40 50

Time (h)

OD

60

0

H

0

01

02

03

04

05

06

07

0 10 20 30 40 50

Time (h)

OD

60

0

E

0

01

02

03

04

05

06

07

0 10 20 30 40 50

Time (h)

OD

60

0F

0

01

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07

0 10 20 30 40 50

Time (h)

OD

60

0B

0

01

02

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Time (h)

OD

60

0

C

0

01

02

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04

05

0 10 20 30 40 50

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OD

60

0

D

0

01

02

03

04

05

0 10 20 30 40 50

Time (h)

OD

600

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For Peer Review

Fig 2 Loacutepez de Felipe Curiel amp Muntildeoz

0

02

04

06

08

1

12

14

16

0 5 10 15 20 25 30 35 40 45

Time (hr)

Gro

wth

(O

D600)

0

005

01

015

02

025

03

035

04

045

05

Gala

cto

se

L-L

acti

c a

cid

(

)

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For Peer ReviewFig 3 Loacutepez de Felipe Curiel amp Muntildeoz

0

2

4

6

8

10

12

0 05 1 15 2 25

Time (days)

mM

(G

luco

se

Fru

cto

se)

0

5

10

15

20

25

30

35

40

45

50

mM

(L

-lacti

c a

cid

L

-mali

c a

cid

)

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For Peer ReviewFig 4 Loacutepez de Felipe Curiel amp Muntildeoz

0

10

20

30

40

50

60

0 05 1 15 2 25 3 35

Time (days)

mM

(G

ala

cto

se

L

-la

cti

c a

cid

)

0

2

4

6

8

10

12

14

16

18

20

mM

(L

-ma

lic

)

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For Peer ReviewFig 5 Loacutepez de Felipe Curiel amp Muntildeoz

mA

U

0

100

200

300

Minutes

0 20 40 60 80

mA

U

0

100

200

Time (min)

A

B

Page 33 of 33

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27

Table 1 The fermentation performance of Lactobacillus plantarum RM71 in wine-resembling medium is improved by catechin

mmol l-1 consumption of mmol l-1 production of

Time (d) Glucose Fructose L-malic L-lactic Log cfu

Control 0-1 02 (22) 038 (4) dagger 1165 97 (108) Dagger 462 sect

1-2 89 (978) 929 (96) 00 2523 (07) 73

Total 91 967 115 3493 (078)

CAT 0-1 222 (204) 225 (212) 1141 1555 (09) 505 1-2 868 (796) 834 (787) 00 2845 (09) 77

Total 109 1059 1178 44 (086)

Consumption or production values are calculated using the average values represented in Fig 3 dagger Values in parentheses are percentages of

glucose fructose or L-malic acid consumed respect to total amount used by Lact plantarum RM71 in the fermentationsDagger Values in parentheses

represent the daily or total lactic yield by glucose fructose and L-malic acid consumed (theoretical value of the ratio lacticmalic acid =067)

CAT (cultures grown upon 100 mg l-1 of catechin) sect Consumption or production values and cfu counts are the average of at least two

independent fermentations Values do not differ significantly by using non parametric tests (Plt005)

Page 27 of 33

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28

Table 2 The fermentation performance of Lactobacillus plantarum RM71 in basal fermentation medium (BFM) is improved by catechin

mmol l-1

Time (d) Galactose consumption L-malic consumption L-lactic production Log cfu

Control 0-1 084 (48) 159 (137) dagger 078 (028) Dagger 387 sect

1-2 1032 (586) 999 (863) 1507 (055) 604 2-3 644 (366) 0 (0) 761 (059) 848

Total 176 1158 2268 (053)

CAT 0-1 435 (149) 683 (598) 557 (042) 427 1-2 142 (487) 459 (402) 2637 (084) 67 2-3 1059 (363) 0 (0) 123 (058) 876

Total 2914 1142 3866 (058)

Consumption or production values are calculated using the average values represented in Fig 4 dagger Values in parentheses are percentages of

galactose or L-malic acid consumed respect to total amount used by Lact plantarum RM71 in the fermentationsDagger Values in parentheses

represent the daily or total lactic yield by galactose and L-malic acid consumed (theoretical value of the ratio lacticmalic acid =067) CAT

(cultures grown upon 100 mg l-1 of catechin) sect Consumption or production values and cfu counts are the average of at least two independent

fermentations Values do not differ significantly by using non parametric tests (Plt005)

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Fig 1 Loacutepez de Felipe Curiel amp Muntildeoz

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Fig 2 Loacutepez de Felipe Curiel amp Muntildeoz

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For Peer ReviewFig 3 Loacutepez de Felipe Curiel amp Muntildeoz

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For Peer ReviewFig 4 Loacutepez de Felipe Curiel amp Muntildeoz

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For Peer ReviewFig 5 Loacutepez de Felipe Curiel amp Muntildeoz

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28

Table 2 The fermentation performance of Lactobacillus plantarum RM71 in basal fermentation medium (BFM) is improved by catechin

mmol l-1

Time (d) Galactose consumption L-malic consumption L-lactic production Log cfu

Control 0-1 084 (48) 159 (137) dagger 078 (028) Dagger 387 sect

1-2 1032 (586) 999 (863) 1507 (055) 604 2-3 644 (366) 0 (0) 761 (059) 848

Total 176 1158 2268 (053)

CAT 0-1 435 (149) 683 (598) 557 (042) 427 1-2 142 (487) 459 (402) 2637 (084) 67 2-3 1059 (363) 0 (0) 123 (058) 876

Total 2914 1142 3866 (058)

Consumption or production values are calculated using the average values represented in Fig 4 dagger Values in parentheses are percentages of

galactose or L-malic acid consumed respect to total amount used by Lact plantarum RM71 in the fermentationsDagger Values in parentheses

represent the daily or total lactic yield by galactose and L-malic acid consumed (theoretical value of the ratio lacticmalic acid =067) CAT

(cultures grown upon 100 mg l-1 of catechin) sect Consumption or production values and cfu counts are the average of at least two independent

fermentations Values do not differ significantly by using non parametric tests (Plt005)

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Fig 1 Loacutepez de Felipe Curiel amp Muntildeoz

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Fig 2 Loacutepez de Felipe Curiel amp Muntildeoz

0

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For Peer ReviewFig 3 Loacutepez de Felipe Curiel amp Muntildeoz

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For Peer ReviewFig 4 Loacutepez de Felipe Curiel amp Muntildeoz

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For Peer ReviewFig 5 Loacutepez de Felipe Curiel amp Muntildeoz

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Fig 1 Loacutepez de Felipe Curiel amp Muntildeoz

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Fig 2 Loacutepez de Felipe Curiel amp Muntildeoz

0

02

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Gro

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For Peer ReviewFig 3 Loacutepez de Felipe Curiel amp Muntildeoz

0

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For Peer ReviewFig 4 Loacutepez de Felipe Curiel amp Muntildeoz

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For Peer ReviewFig 5 Loacutepez de Felipe Curiel amp Muntildeoz

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Fig 2 Loacutepez de Felipe Curiel amp Muntildeoz

0

02

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1

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Gro

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For Peer ReviewFig 3 Loacutepez de Felipe Curiel amp Muntildeoz

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For Peer ReviewFig 4 Loacutepez de Felipe Curiel amp Muntildeoz

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For Peer ReviewFig 5 Loacutepez de Felipe Curiel amp Muntildeoz

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For Peer ReviewFig 3 Loacutepez de Felipe Curiel amp Muntildeoz

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For Peer ReviewFig 4 Loacutepez de Felipe Curiel amp Muntildeoz

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For Peer ReviewFig 5 Loacutepez de Felipe Curiel amp Muntildeoz

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For Peer ReviewFig 5 Loacutepez de Felipe Curiel amp Muntildeoz

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