batch fermentation of whey ultrafiltrate by lactobacillus helveticus for lactic acid production

Appl Microbiol Biotechnol (1986) 24:206--213 Applied o.d Microbiology Biotechnology © Springer-Verlag 1986

Batch fermentation of whey ultrafiltrate by Lactobacillus helveticus for lactic acid production

Denis Roy 1,2, Jacques Goulet 1, and Anh LeDuy 3

Department of Food Science and Technology, Laval University, Sainte-Foy, Qu6bec, Canada G1K 7P4 2 St-Hyacinthe Food Research Center, Agriculture Canada, St. Hyacinthe, Qu6bec, Canada J2S 4Z4 3 Department of Chemical Enegineering, Laval University, Sainte-Foy, Qu6bec, Canada G1K 7P4

Summary. Cheese whey ultrafiltrate (WU) was used as the carbon source for the production of lactic acid by batch fermentation with Lactobacil- lus helveticus strain milano. The fermentation was conducted in a 400 ml fermentor at an agitation rate of 200 rpm and under conditions of controlled temperature (42 ° C) and pH. In the whey ultrafiltrate-corn steep liquor (WU-CSL) medium, the optimal pH for fermentation was 5.9. Inocu- lum propagated in skim milk (SM) medium or in lactose synthetic (LS) medium resulted in the best performance in fermentation (in terms of growth, lactic acid production, lactic acid yield and maximum productivity of lactic acid), as compared to that propagated in glucose synthetic (GS) medium. The yeast extract ultrafiltrate (YEU) used as the ni trogen/growth factor source in the WU medium at 1.5% (w/v) gave the highest maximum productivity of lactic acid of 2.70 g/l-h, as compared to the CSL and the tryptone ultrafiltrate (TU). L. helveticus is more advantageous than Streptococcus thermophilus and Lactobacillus delbrueckii for the production of lactic acid from WU. The L. helveticus process will provide an alternative solution to the phage contamination in dairy industries using Lactobacillus bulgaricus.

Introduction

Fifteen years ago membrane processes were intro- duced in the dairy industry (De Boer and Hiddink 1980). Using ultrafiltration, cheese whey can be fractionated into a protein concentrate and a de-

Offprint requests to: Denis Roy (at address 1)

proteinized whey permeate fraction. This deproteinized ultrafiltrate fraction, namely the whey ultrafiltrate (WU), contains 4.0 to 4.5% lactose, which has to be removed before the deproteinized fraction is disposed of. Since lactose is the major component of WU solids, it is of interest to use it as carbon source for fermentation processes.

Previous studies in our laboratory (Dion et al. 1978; Champagne 1982) have demonstrated that lactose in WU could be integrated in the fermentation process for the production of baker's yeast. Since baker's yeast does not metabolize lactose, WU was transformed into a nitrogen enriched- wort which in turn was to be used as culture medium for growing baker's yeast. This nitrogen-enriched wort was prepared by fermenting lactose into lactic acid with Lactobacillus bulgaricus or Streptococcus thermophilus, and then by trans- forming lactic acid into ammonium lactate through neutralization with ammonia.

Studies on the lactic acid fermentation carried out elsewhere have been conducted as a batch process (Campbell 1953; Hanson and Tsao 1972; Marshall and Earle 1975; Reddy et al. 1976), as a continuous process (Hanson and Tsao 1972; Keller and Gerhardt 1975; Cox and MacBean 1977; Stieber and Gerhard 1979a; Major and Bull 1985), as a dialysis continuous process (Friedman and Gaden 1970; Stieber et al. 1977; Stieber and Gerhardt 1979b; Stieber and Gerhardt 1981), or in an immobilized cell bioreactor (Vick Roy et al. 1982; Stenroos et al. 1982). Among these, most of the work was done on whole cheese whey or on the synthetic media by using either Lactobacillus delbrueckii or L. bulgaricus. To this point, no in- formation had been available on the fermentation of WU by Lactobacillus helveticus, a thermophilic homofermentative lactic acid bacterium, for the production of lactic acid.

D. Roy et al.: Lactic acid from whey ultrafiltrate by L. helveticus 207

The species L. helveticus is of special interest because: (i) it produces almost twice concentration of lactic acid in milk as compared to other common lactic acid bacteria, including Streptococ- cus thermophilus and L. bulgaricus (Matteuzzi 1972; Jacques Goulet and Ren6e Roy, Internal Report, Centre de Recherche en Nutrition, Laval University, June 1980); and (ii) it produces an ra- cemic lactic acid (DL) as compared to only dex- trorotatory lactic acid (D) produced by other species, including Lactobacillus lactis, L. delbrueckii and L. bulgaricus (Bergey's manual 1974). L-lactic acid, being found naturally in the human metabolism, is absorbed more rapidly by human intes- tine (Alais 1984).

This paper will report the results of tests done to determine optimal conditions for the production of lactic acid by batch fermentation of WU. Our study is limited to the effects of pH, inoculum preparation and nitrogen/growth factor sources on the fermentation of WU by L. helveticus. The relevance of the choice of L. helveticus for lactic acid production from WU and the potential industrial application of such a process will be discussed.

Materials and methods

Organism and inoculum

The Lactobacillus helveticus strain milano used in this study was supplied by Institut Rosell, Montr6al, Qu6bec, Canada. Stocks cultures were maintained as suspensions in sterile skim milk (SM) medium and stored in liquid nitrogen.

Working cultures were propagated, by weekly transfer, in still cultures, on one of the following media: SM medium (at 10% v/v inoculum); whey ultrafiltrate-corn steep liquor (WU- CSL) medium (at 10% v/v inoculum): lactose synthetic (LS) medium (at 2% v/v inoculum); and glucose synthetic (GS) medium (at 2% v/v inoculum). Procedures for the preparation of these media will be described in the next section.

Active cultures for inoculating the fermentor (at 1% v/v) were prepared by growing the organism, from the working cultures, in still cultures at 42°C for 20 h. Cultures maintained in SM medium required only ~ hours of incubation time for inoculum preparation.

Culture media

The sterile SM medium was prepared by dissolving 12% (w/w) skim milk powder (low heat grade) in distilled water, and then by sterilizing the solution in an autoclave at 121°C for 10 min.

The GS medium used is that previously proposed by Friedman and Gaden 1970. It contained (in g/l): glucose (ACS standard grade) 50: yeast extract (Difco) 30; MgNO4 0.6; FeSO4 0.03; MnSO4 0.03; Na-acetate 1.0; KH2PO4 0.5 and K2HPO4 0.5. The LS medium was prepared by substituting lactose (ACS standard grade) for glucose.

Whey used in this study was prepared by dissolving 6.5% (w/v) spray-dried cheddar cheese whey powder (Kraffen, Kraft) in distilled water. The whey was then treated with an ultrafiltration system (Romicon HF2SSS system; with PM-50 hollow-fiber cartridges having molecular weight cut-off of 50,000). The resulting whey ultrafiltrate (WU) was stored at - 18°C and filter sterilized (Sartorius 0.2 gin) before use. The WU-CSL medium was prepared by adding 0.5% (w/v) corn steep liquor (CSL) to WU. The whey ultrafiltrate-yeast extract ultrafiltrate (WU-YEU) medium and the whey ultrafiltrate- tryptone ultrafiltrate (WU-TU) medium were prepared by adding the appropriate quantities of yeast extract (Difco, techni- cal grade) or tryptone (Difco) to the whey prior to the ultrafiltration step.

Experimental methods

All fermentation experiments were conducted on a 400 ml working volume fermentor (New Brunswick Scientific, Bio- Flo, Model C-30), at an agitation speed of 200 rpm, at controlled temperatures of 42°C (which is in the optimal range of 38 to 44°C for lactic acid production) and under aseptic conditions. The pH was maintained at the desirable values using a pH controller (Radiometer-pH meter, Copenhagen, Titrator II, Model 51) with the automatic addition of 3 N NH4OH.

Analytical methods

The viable counts during fermentation were carried out by pour plate counts on Elliker agar (Difco) and were reported as logarithms of colony forming units (log CFU/ml). Plates were incubated at 42°C for 48 h before countings.

The lactic acid concentration was determined using the colorimetric procedure which measures total lactate concentration (Lawrence 1975). Appropriate dilutions of a stock solution containing lithium lactate solution were used to prepare a standard curve. Optical density was read at 570 nm with a spectophotometer (Bausch & Lomb, Model Spectronic 710).

The glucose and lactose concentrations were determined by the phenol-sulfuric acid method (Hanson and Phillips 1981).

Data from repetitive experiments were statistically treated by standard prodecures (Snedecor and Cochrane 1967).

Results and discussion

Effect o f pH on whey ultrafiltrate-corn steep liquor medium

Growth (viable counts) and lactic acid concentration in batch fermentations of WU-CSL medium at controlled pH varying from 4.7 to 6.3 are shown in Fig. 1. The inoculum used in this experiment was propagated in SM medium.

It was found that pH affects the growth curves of L. helveticus only after the exponential growth phase (after 8th hour, Fig. 1A). During this period, the death rate increases at higher pH values. The death phases of microorganism occurred in culture media where lactic acid concentration is

208 D. Roy et al. : Lactic acid from whey ultrafiltrate by L. helveticus

9 _.1

2 8 (,.3

.~ 5 0

50 .._J

LH/WU -CSL r T ~

I T I I I I L~ ®

I 1 L ] I I

40 0

3 o

g u .~ 20

o

-~ I0 g _J

O0 4 8 12 16 20 24 Time, h

Fig. 1. Batch fermentations in the WU-CSL medium at different pH values. All data are average values from 3 repetitive experiments

higher than 20 g/1. This observation was found for all medium pH values (Fig. 1). Therefore, the increase in death rate, as observed in Fig. 1, could be mainly due to the lactic acid toxicity.

Lactic acid production (= maximum concentration -- initial concentration), lactose utilization (= initial concentration -- minimum concentration), and maximum productivity of lactic acid (= maximum slope of the tangent of the product

5O 5 -J LH / WU - CSL , :

• .

7 2 • •

. . . - I -

o ~ iO - ,__.

0 4.5 5.0 5.5 6.0 6.5

pH

Fig. 2. Effect of pH on lactic acid production, lactose utilization and maximum productivity of lactic acid in batch fermentation of the WU-CSL medium. All data are average values from 3 repetitive experiments

concentration curve drawing from the point of initial product cencentration) are influenced by the pH of the culture medium. They increased linearly with an increase in pH over the range 4.7 to 6.3 (Fig. 2).

The pH of 5.9 obtained with the WU-CSL medium was then selected for subsequent experiments.

Effect of medium used for the propagation of inoculum

Inoculum prepared from different media does have an effect on growth and lactic acid production. Figure 3 shows growth and lactic acid concentration curves using inocula previously propagated on an SM medium and a WU-CSL medium respectively. The batch fermentation was done in a WU-CSL medium at a controlled pH of 5.9.

It was found that the specific growth rate Ix (slope of the exponential phase of the growth curves, Fig. 3) is the same for two different inocula. However, lactic acid production is much better with the inoculum propagated in an SM medium. Lactic acid production, lactic acid yield (= lactic acid product ion: lac tose utilization), and maximum productivity of lactic acid in the fermentation with the inoculum propagated in the SM medium are 32.5 g/l, 84% and 1.43 g/1-h. The corre- sponding values in the fermentation with inoculum propagated in the WU-CSL medium are 20.2 g/l, 74% and 0.75 g/1-h.

The specific productivity of lactic acid ( = productivity:init ial inoculum concentration) is 0.26 g/h-109 and 0.60 g/h-109 inoculated cells in fermentations with inoculum propagated in SM and WU-CSL respectively. It is shown that the cells propagated in the WU-CSL medium are more active (productive) than those propagated in the SM medium. Despite this fact, the SM medium is pre- ferred for the propagation of cultures and for the preparation of inoculum in our work for the three following reasons:

(i) When the cultures had to be propagated in still cultures and under uncontrolled pH (as was done in this project), the maximum cell concentration obtained in the WU-CSL medium was always inferior to that obtained in the SM medium. Successive transfers further reduced the cell concentration in WU-CSL as compared to that in the SM medium. This resulted in the marked difference between the initial concentrations of viable cells found in the fermentation media seeded with two different inocula (Fig. 3);

D. Roy et al.: Lactic acid from whey ultrafiltrate by L. helvetieus 209

d 9 i= LH / WU - CSL

u_ 8

o

> j 4 0 I I I I ) I @ - . ®

30 I" from SM ] /

c

"6 I I I I I I -9 O0 4 B 12 16 20 24 2B

Time, h

Fig. 3. Batch fermentation in the WU-CSL medium with inocula propagated in different media: (V) SM medium; (m) WU-CSL medium. All data are average values from 20 repetitive experiments

(ii) In still cultures and under uncontrolled pH, the cell growth in the WU-CSL medium was much slower than that in the SM medium. In or- der to reach the maximum cell concentration in inoculum prior to seeding the fermentor, the inoculum on WU-CSL was incubated for 20 h as compared to only 4 h of incubation with the inoculum on the SM medium;

(iii) Many difficulties were experienced in our laboratory in terms of culture stability, on the WU-CSL medium, especially when the level of transfer inoculum was low. Indeed, when the working cultures propagated on the WU-CSL medium by weekly transfer at 1% (v/v) were used to prepare the inoculum for the fermentation experiments, cell growth in the fermentor seemed to fol- low a normal growth curve, but lactic acid production was inconsistent and non-reproducible. Such behavior was not observed with cultures propagated on the SM medium.

The beneficial effects of SM in stimulating growth and lactic acid synthesis is quite well- known. Indeed, SM is being used in the preparation of starting-cultures for cheese manufacturing and lactic acid microflora for the food industry. Champagne 1982 observed that the addition of SM to the WU-CSL medium increased the lactic acid production rate of S. thermophilus and L. bulgaricus. This observation also suggested that CSL is inadequate as a nitrogen/growth factor source

for the lactic acid fermentation of WU. This is because CSL, unlike SM, does not contain stimulatory peptides and /o r amino acids required for growth and lactic acid production.

Effect of nitrogen/growth factor sources on lactic acid production

CSL, yeast extract ultrafiltrate (YEU) and tryptone ultrafiltrate (TU) have been used as nitrogen/growth factors for the formation of the WU fermentation medium. Their effect on the maximum productivity of lactic acid is shown in Fig. 4. The maximum productivity of lactic acid is linearly correlated with the concentration of the ni- t rogen/growth factor over the range examined.

It was found that at a concentration level of 0.5% (w/v) in the culture medium, YEU gave slightly higher maximum productivity than the same concentration level of CSL. However, the maximum productivity of lactic acid is doubled (to 2.70 g/l-h) when YEU is used at a concentration of 1.5% (w/v) in the WU medium. The CSL was not tested at concentrations higher than 0.5% because lactic acid fermentation is inhibited under such conditions (Campbell 1953; Reddy et al. 1976). In all concentrations tested in this study, the TU always gave low maximum productivity.

This beneficial effect of YEU suggested that most, if not all, of the active compounds responsi- ble for stimulating effects on growth and on lactic acid synthesis were of low molecular weights (< 5o,ooo).

Yeast extract was also found as a better ni- t rogen/growth factor source for growth and lactic

._]

7,4

~ 2 E= E 6 I

LH/WU-

- YEU

- CSL____~./TU I I I

O0 5 I0 15 20 Nitrogen/growth factor concentration, g/L

Fig. 4. Effect of different ni t rogen/growth factor sources on the maximum productivity of lactic acid in batch fermentation of WU medium. All data on YEU and TU are average values of 3 repetitive experiments; on CSL, 20 repetitive experiments

210 D. Roy et al. : Lactic acid from whey ultrafiltrate by L. helveticus

acid production from WU by L. bulgaricus (Cox and MacBean 1977) as compared to CSL. In some circumstances, it is economically advantageous to use YE instead of CSL as an ingredient for lactic acid fermentation. This is the case when lactic acid fermentation is an integral part of the manu- facture of baker's yeast.

Comparison with the synthetic media

So far, it has been demonstrated that the best maximum productivity of lactic acid is obtained by propagating L. helvetieus in an SM medium, then inoculating the WU fermentation medium, supplemented with 1.5% (w/v) of YEU (Fig. 4). The comparison of lactic acid fermentation using this procedure with those using the GS and LS media is shown in Fig. 5. For the synthetic media, inocula were propagated in media of the same composition as the fermentation media.

It was found that the LS medium supported better growth of L. helveticus than did the GS medium. Consequently, lactic acid production, lactic acid yield, maximum productivity of lactic acid were higher in the LS medium. These are 40.2 g/l, 94% and 1.63 g/l-h, respectively, in the LS me-

J:t L H /

U

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j 5 0 ' I l I I I I (~) "~ WU-YEU ~ ) = 40 from LS--~_ ~ , , ~ L S ._o WU-YEU g~" " - -"

8 2o ~ G S 8 Io u

8 o 1 0 4 8 12 16 20 24 28

Time, h

Fig. 5. Batch fermentation in different culture media: ( • ) GS medium using inoculum propagated on GS medium; (m) LS medium using inoculum propagated on LS medium; ( 0 ) WU- YEU medium using inoculum propagated on SM medium; (©) WU-YEU medium using inoculum propagated on LS medium. All data are average values from 3 repetitive experiments, except (©) data are values from single experiment

dium compared to the corresponded values of 17.9 g/l, 86% and 0.75 g/1-h in the GS medium. Another observation indicating the difficulty of metabolizing glucose as compared to lactose by L. helvetieus is that the residual sugars measured at the end of the fermentations shown in Fig. 5 were 33.5 g/1 glucose and 10.7 g/1 lactose. Thus, during the same fermentation period, only about half of the available glucose in the GS medium had been utilized, whereas about 80% of the lactose in the LS medium had been consumed.

The maximum specific productivity of lactic acid was 1.19 g/h-109 inoculated cells in the GS medium and 1.03 g/h-109 inoculated cells in the LS medium. This suggests that the maximum productivity of lactic acid strongly depends on the initial concentration of viable cells at the beginning on the fermentation. Lower values of maximum productivity of lactic acid obtained with the GS medium were due to the lower initial concentration of viable cells at time zero, as compared to that of the LS medium. This lower concentration of viable cells was in turn due to the fact that the inoculum had been previously prepared on the GS medium; and the GS medium supported less growth as compared to the LS medium (the specific growth rate ~t, which is the slope of the growth curves in Fig. 5A, in the LS medium is greater than that of the GS medium).

The WU-YEU medium with inoculum propagated on the SM medium shown in Fig. 5, gave the highest productivity (2.70 g/l-h) and highest maximum specific productivity (1.45 g/h-109 inoculated cells) of lactic acid.

When inoculum is propagated in the LS medium prior to seeding the WU-YEU fermentation medium, the maximum productivity of lactic acid obtained in batch fermentation (2.37 g/l-h) was very close to that using inoculum propagated on the SM medium (Fig. 5B). This led to an interest- ing alternative method for the preparation of inoculum for certain experiments where the presence of SM is undesirable. An example of such a case is fermentation which requires a non-turbid medium for direct optical density readings; inoculum has to be propagated on the LS medium.

Thus, the inoculum could be propagated on the SM medium or on the LS medium for the optimal fermentation of the WU-YEU medium. This established procedure for fermentation experiments is now being used in our laboratory for subsequent work. An example of this is the observation of the effect of pH on the WU-YEU medium. The results in Fig. 6 show that the maximum productivity of lactic acid is linearly corre-

D. Roy et al.: Lactic acid from whey ultrafiltrate by L. helveticus 211

5

-f, J 4 ,%. o',

'_ 3

LH/WU-YEU

E O

-4n.5 P ~ I 5.0 5.5 6.0 6.5 pH

Fig. 6. Effect of pH on the maximum productivity of lactic acid in batch fermentation of the WU-YEU medium. All data are values from single experiments

lated to an increase in medium pH. This correla- tion straight-line is parallel to that obtained with the WU-CSL medium (Fig. 2), except that the former is shifted to higher values. This indicates that pH has the same effect on both media (WU- CSL and WU-YEU), but WU-YEU gave higher maximum productivity of lactic acid in all pH values.

Choice o f microorganism and evaluation of lactic acid fermentation

Ther are several reasons which justify the choice of L. helveticus for the fermentative production of lactic acid from WU as compared to commonly used species of lactic acid bacteria such as S. thermophilus, L. delbrueckii and L. bulgaricus. Besides two advantageous reasons previously mentioned (in the Introduction), the following will support the appropriateness of the choice of L. helveticus for such purposes.

Streptococcus thermophilus has two drawbacks : First, very few strains of S. thermophilus are able to ferment galactose, a breakdown product during the fermentation of lactose (Somkuti and Stein- berg 1979). In the contrary, L. helveticus can ferment lactose and all of its breakdown products, glucose and galactose (Bergey's manual 1974). Secondly, S. thermophilus requires some stimulatory peptides and/or amino acids for growth and for the production of lactic acid in a milk-base medium. S. thermophilus cannot sufficiently provide such stimulatory compounds for itself due to its limited amount of active cell-bound proteinase (especially aminopeptidases). The aminopeptidases, when, present in sufficient quantities, re-

lease stimulatory peptides and/or amino acids from casein. It could be done either by co-cultur- ing thermophilic lactobacilli, such as L. helveticus, L. lactis or L. bulgaricus, containing more active cell-bound proteinase, with S. thermophilus, or by supplementing the milk-base medium with the soluble extracts of such thermophilic lactobacilli (Hemme et al. 1981). Another alternative is the supplementing of the milk-base medium with a mixture of valine, histidine, methionine, glutamic acid, leucine and tryptophan, or dipeptides containing these amino acids (Sharpe 1979). Thus, with respect to S. thermophilus, it is more advantageous to use L. helveticus for the preparation of culture starters from milk-base media, such as the SM medium in this work, and also for the production of lactic acid from lactose-base substrates, such as the LS medium and the WU-YEU medium in this work.

Among other species of thermophilic lactobacilli commonly used for lactic acid fermentation, L. delbrueckii is obviously not suitable for WU medium because it cannot ferment lactose (Ber- gey's manual 1974). Previous studies in our laboratory on the screening of thermophilic lactobacilli revealed that two available strains of L. bulgaricus consistently yielded very low concentrations of lactic acid in milk as compared to that obtained from two available strains of L. helveticus (Jacques Goulet and Ren+e Roy, Internal Re- port, Centre de Recherche en Nutrition, Laval University, June 1980). Thus L. helveticus is the logical choice.

Several values of maximal lactic acid productivity which were reported from various sources are compiled in Table 1. Care should be taken when attempting to compare one set of results to another because these date were not obtained under the same experimental conditions. For in- stance, L. delbrueckii NRRL-B445 did not give the same maximal lactic acid productivity in glucose- YE medium by continuous fermentation (reported as 4.18 g/1-d from one researcher and 8.9 g/1-d other; Table 1). This could be caused by the slight difference in temperature and pH conditions used by these researchers.

From Table 1, the maximal lactic acid productivity of L. helveticus in WU-YEU medium in batch fermentation is 2.7 g/1-h which is lower than that reported by other researchers on L. bulgaricus 2217. However, the substrate conversion (= substrate consumed • initial substrate concentration) was highest with L. helveticus (93%). The lactic acid concentration in the culture medium with L. helveticus falls within the lower range re-

212 D. Roy et al.: Lactic acid from whey ultrafiltrate by L. helveticus

Table 1. Compiled data on maximal lactic acid productivity

Culture and Maximal At maximal lactic acid productivity References fermentation conditions lactic acid

productivity Lactic Fermen- Dilution Substrate (g/l-h) acid con- tation rate con-

centration time version (g/l) (h) (h - 1) (%)

L. helveticus strain milano Batch (0.4 1) WU-YEU medium 42 o C; pH 5.9 Lactose initial 37.2 g/1 (i) Inoculum from LS medium 2.4 (ii) Inoculum from SM medium 2.7

L. bulgaricus 2217 Batch (9.5 1) Whey-CSL medium 43°C; pH 5.5 Lactose initial 50 g/1 5

Continuous (3.6 1) WU-YE medium 44°C; pH 5.5 Feed lactose concentrat ion 62 g/1 5

Dialysis continuous (2--3.6 1) WU-YE medium 44°C; pH 5.5 Feed lactose concentrat ion 242 g/1 10.7

L. delbrueckii NRRL-B445 Batch ( < 7 1) Glucose-YE medium 45°C; pH 5.3 Glucose initial 20 g/1 2.38

Continuous ( < 7 1) Glucose-YE medium 45°C; pH 5.3 Feed glucose concentrat ion 20 g/1 4.18

Continuous (1.4 1) Glucose-YE medium 42°C: pH 6.0 Feed glucose concentrat ion 50 g/1 8.9

37 13 - - - - This work 35 12 - - 93 This work

60 10 - - 91 Reddy et al. 1976

30 - - 0.17 51 Stieber and Gerhardt 1979b

Stieber and 65 (fermenter) - - 0.14 44 Gerhardt 20 (dialyzer) 1979a

9.5 4 - - 63 Hanson and Tsao 1972

20.8 - - 0.20 62 Hanson and Tsao 1972

22.3 - - 0.35--0.40 60--67 Major and Bull 1985

ported for L. bulgaricus (from 30 to 85 g/l; Table 1).

The most significant feature of this work comes from the fact that it gives a potential solution to the phage contamination problem in industrial application. L. bulgaricus is a common species of thermopfiilic lactic acid bacteria used in the manufacturing of yogurt and cheese (Sharpe 1979). Virulent phages specific to L. bul- 9aricus, which upset the normal acidification process, resulting in faulty cheese or yogurt, have been isolated and investigated by Accolas and

Spillmann 1979. These researchers revealed that these phages belong to a different morphological group (Bradley's group B) than the phages active on L. helveticus (which belong to Bradley's group A). Thus, based on different host specificities of such phages, the use of L. helvetieus for lactic acid production from WU, when conducted in .the yogurt or cheese factory (using L. bulgaricus), could alleviated the phage propagations when phage contamination occurs. This application could be extended to the manufacturing of lactic acid as a sole product. L. helveticus could be very useful as

D, Roy et al.: Lactic acid from whey ultrafiltrate by L. helveticus 213

an alternate culture to L. bulgaricus when phage contamination occurs.

Acknowledgements. This work was supported by grants from Qu+bec CRESAQ (Conseil des recherches et services agricoles du Qu6bec). The statistical analyses of data by Pierre Prov- encher was appreciated. Issued as St-Hyacinthe Food Re- search Center No. 24.

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Received September 5, 1985/ Revised January 30, 1986

batch fermentation of whey ultrafiltrate by lactobacillus helveticus for lactic acid production

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