natural fermentation of lentils: influence of time, concentration and temperature on the kinetics of...

J Sci Food Agric lY96,71, 367--315

Natural Fermentation of Lentils : Influence of Time, Concentration and Temperature on the Kinetics of Hydrolysis of Inositol Phosphates Halina Kozlowska: Joanna Honke, Jadwiga Sadowska Centre for Agrotechnology a n d Veterinary Sciences, Polish Academy of Sciences, 10-7 18 Olsztyn 5, Poland

Juana Frias and Concepcion Vidal-Valverde Instituto de Fermentaciones Industriales (CSIC), Juan de la Cierva 3,28006 Madrid, Spain (Received 3 July 1995; revised version received 17 November 1995; accepted 8 February 1996)

Abstract: Lentil (Lens culinaris var oulgaris) flour was naturally fermented for 96 h at various conditions of concentration (79, 150 and 221 g litre-') and temperature (28"C, 35°C and 42°C). The content of total inositol phosphates (IP- total) and individual inositol phosphates (hexa- (IP,), penta- (IP,), tetra- (IP,) and tri- (IP?) phosphates) were analysed to establish the changes of these compounds during natural fermentation of lentils. The preparation of the lentil suspension brought about 16-27% reduction of the total inositol phosphates. At the end of 96 h of natural fermentation maximum IP loss (70-75%) was achieved for an experiment carried out at minimum concentration. For IP,, the largest decrease was achieved at the highest temperature, the fermentation condition that also brought about the highest IP, content.

Key words: natural fermentation, lentil, inositol phosphates, phytate, phytic acid.

INTRODUCTION (1994) compared phytate contents of basic food products of plant origin and found the greatest in cereals

Lentils have the ability to grow in high water stress and legume seeds. These compounds evoke high interest conditions and the nutritionally advantageous composi- in nutritionists due to their extensively documented tion of the stored seed is the main attribute which make negative properties as well as recently appearing indica- lentils an important crop, particularly in hot dry cli- tions of their beneficial influence on the human mates. Lentils have made a significant contribution to organism. Phytic acid (myoinositol 1,2,3,4,5,6-hexakis the human diet since the ancient times. Their high dihydrogen phosphate), is considered to be the major content in protein (250 g kg- '), carbohydrates factor causing impaired absorption of several essential (560 g kg-') and low content in lipids (10 g kg-') make minerals, such as Ca2+, Zn2+, Mg2+ and Fe2+ them an attractive and cheap source of nutrients in the (Thomson 1993; Reddy and Pierson 1994) turning them world, where there is an increasing demand for protein into insoluble forms which are unavailable to the (Adsule et a1 1989). organism. When diet is based on vegetable products (eg

Although lentils are considered to be one of the most vegetarian diets), deficiency in these valuable elements nutritious pulses, they contain several antinutritional may arise (Ellis et a1 1982). There are also known dis- factors which could limit their consumption (Savage advantageous properties of phytic acid related to 1988). Inositol phosphates commonly occur in food of protein complexing (Carnovale et al 1988) with proteo- plant origin. They have been demonstrated to be the lytic (Sarriano er a1 1985) and amylolytic enzymes major storage medium for phosphorus in seeds and (Desphande and Salunkhe 1982), which lower digest- grains constituting 60-90% of organically bound phos- ibility of proteins and starch. phorus found in seeds (Billington 1993). Khokhar et a1 Among the positive properties of phytate are their

ability to inhibit cell proliferation and hence to reduce * To whom correspondence should be addressed. the risk of colonic cancer (Graf and Eaton 1990), as well

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368 H Kozlowska et a1

as to bind the excess of iron, and thus to prevent dis- advantageous oxidation processes (McCord 1994). Phytate is also known to complex heavy metals which contaminate food (Inglemann et a1 1993). For this reason phytic acid is added to foodstuffs in Japan for the preservation of soybean oil, meat, fish, pastas, colouring agents and other foods (Six 1994).

By activation of the naturally occurring phytases of plant foods, phytic acid is hydrolyzed to myo-inositol and inorganic phosphate via intermediate myo-inositol phosphates (penta- to monophosphates). When the phosphate groups are removed from phytate, the mineral-binding strength decreases (Sandstrom and Sandberg 1992). Investigations performed on humans by Sandberg et a1 (1993) indicated an inhibiting effect of IP, and IP, on iron, zinc and calcium absorption, with no significant effect of IP, or IP, at levels found in human diets.

Phytate degradation in the digestive tract takes place when phytates are accompanied by an active phytase. Before consumption, dry legume seeds and another plant materials are most often thermally processed, which could inactivate endogenous enzymes (Larsson and Sandberg 1995), preventing phytate degradation. Honke et a1 (1994) reported significant reduction of phytate during treatment of legume seeds. Jany et a1 (1993), however, demonstrated that thermal treatment is not as effective as enzymatic hydrolysis. In order to affect the strong chelating properties of phytate new methods to carry out their extensive degradation are being sought. Fermentation has been suggested as an effective treatment for lowering phytate content (Turk and Sandberg 1992). Due to the production of lactic acid and other organic acids, the pH is lowered and, therefore, the phytase activated. Lactic fermentation of maize, soya bean and sorghum reduced the phytate content (Sudarmadji and Markakis 1977; Lopez et a1 1983; Svanberg and Sandberg 1987). During breadmaking the phytate content was reduced (by about 90%) as a result of increased endogenous phytase activity in response to and many factors such as pH, water content or amount of yeast present (Reddy and Pierson 1994). During scalding and sour-dough fermentation of bread the acidity of the dough seems to be of great importance for phytate degradation (Bartnik and Flory- siak 1988; Larsson and Sandberg 1991). The most marked phytate reduction of 96-97% occurred in bread made with 100 g kg-' sourdough (pH 4.6) or in bread in which the pH was adjusted with lactic acid to between 4.4 and 5.1 (Sandberg 1994).

Several experiments have demonstrated that fermentation of legumes enhances their nutritive value (Zamora and Fields 1979; Akpapunam and Achinewhu 1985), reduces some antinutritional endogenous compounds (Bressani 1983; Vidal-Valverde et a1 1993) and improves consumer acceptability (Ragaee et a1 1986a). In lentils, Vidal-Valverde et a1 (1993) reported that fer-

mentation for 96 h produced an increase in riboflavin content and in the ratio of available starch to total starch. Recently, Tabera et a1 (1994) showed that various conditions such as initial lentil concentration and temperature affected trypsin inhibitor activity and phenolic compounds differently. The objective of the present study was to investigate the changes in phytic acid (IP,) and its degraded forms (IP,, IP, and IP,) during natural fermentation carried out at various lentil-flour concentrations and temperatures. These studies encouraged us to conduct further studies on inositol phosphates.

MATERIAL AND METHODS

Fermentation

Lentil seeds (Lens culinaris var vulgaris, cultivar Magda- 20, from Albacete, Spain) were harvested in 1991. They were fine ground in a ball mill, sieved and the 50- 250 pm fraction collected. Suspensions of lentil flour in sterilised tap water were aseptically prepared at concentrations and temperatures giving the experimental design set out in Table 1. They were allowed to ferment naturally (with only the microorganisms borne on the seeds) for 4 days without aeration in a stirred fermentor (Infors ISF-100, Infors AG, Switzerland). The 0 time of fermentation was assigned when the complete suspension was under stirring and the controlled temperature had been obtained (10-40 min after placing in the fermentor). Samples were collected daily and freeze dried.

Determination of inositol phosphates

Inositol phosphates were determined according to Sandberg and Ahderinne (1986). From the ground raw and fermented samples the inositol phosphates were extracted with 0.5 M HCl acid at 20°C for 4 h. The extract was centrifuged and the supernatant was frozen overnight followed by thawing, centrifugation, evapo-

TABLE 1 Temperature and flour concentration of fer-

mented batches

Batches Temperature Flour ("C) concentration

(g litre - ')

BH 1 28 79 BH2 42 79 BH3 28 22 1 BH4 42 22 1 BH5 35 150

Natural fermentation of lentils 369

ration to dryness and dissolving in 0.025 M hydro- chloric acid. Samples were transferred to mini-columns filled with resin (Dowex 1 x 8), from which inositol phosphates were washed using 2 M HCI. The solvent was evaporated to dryness and the residue was dis- solved in the mobile phase and analysed by HPLC.

HPLC analyses were carried out using a Shimadzu chromatograph (pump LC-10 AD, CR 4A Chromato- pac, a RID-6A detector, a CTO 6A column oven), a C,, pNovapak column, 35°C oven temperature, methanol/ 0.05 M formic acid (51 : 49, v/v) and 1.5 ml per 100 ml of tetrabutylammonium hydroxide as mobile phase. Identification of the inositol phosphates was achieved by comparison with the mixture of standards obtained during hydrolysis of phytic acid sodium salt (Sandberg et a1 1989) while ii quantitative analysis was conducted using an external standard (sodium phytate).

Statistical analysis

One-way ANOVA with Duncan’s multiple range test and multiple regression analyses according to Volk (1973) were applied. Calculations were done using SPSS for Windows 5.0.1.

RESULTS

The contents of particular forms of inositol phosphate (from IP, to IP,) in raw and fermented lentils are shown in Tables 2 to 6. The HPLC method resolved inositol phosphate forms from IP, to IP, but not the lower phosphate esters which might have been present in the fermented material. Figure 1 shows a representa- tive series of analytical results for sample BH4. It can also be assumed that myo-inositol and lower phosphate esters formed during fermentation could have been metabolised by the microflora. The presence of IP, , IP,, IP, and IP, was observed in raw lentil seeds.

Figure 2 shows the changes in total inositol phosphates throughout the natural fermentation of lentils carried out a t various initial lentil concentrations and temperatures.

The results show that the preparation of the sample brought about significant changes in the total inositol phosphate content and 16-27% reduction was usually obtained at zero time, the time required to complete the lentil suspension, and always within 40 min. During the fermentation procedure (from 0 to 96 h) higher reductions were achieved, 70-75% for BHl and BH2, 47% for BH3 and BH4 and 67% for BH5 (Table 2).

TABLE 2 Changes in total inositol phosphate (IP) content during natural fermentation of lentils (pmol ’ g DM)”

Batches Raw lentil 12.71 f 0.05

O h 24 h 48 h 72 h 96 h

BH 1 9.28 +_ 0.04ab 6.15 f 0.05a 3.81 f 0.03a 3.48 f 0-01a 3.22 f 0.03a BH2 9.30 f 0.02a 7.10 f 0.02b 6.69 f 0.02~ 5.04 f 0.07b 3.69 f 0.03b BH3 10.33 f 0.03bc 8.33 f 0 . 0 3 ~ 8.17 f 0.01d 7.97 f 0 .05~ 6.68 f 0.03d BH4 10.61 044~ 8.60 f 0.07~ 8.72 f 0.07d 7.94 f 0 .03~ 6.66 f 0.05d BH5 10.61 f 0*29b 6.52 f 0.62ab 5.46 f 0.72b 4.89 f 0 . 4 9 ~ 4.18 f 0 .39~

Values are means of three determinations from the same fermentation standard deviation. * The same following letters in the column denote the Duncan’s homogenous subsets.

TABLE 3 Changes in inositol hexaphosphate (IP,) content during natural fermentation of lentils (pmol- * g

DM)”


O h 24 h 48 h 72 h 96 h

BHl 8.82 f 0.05bb 4.75 f 0.04ab 3.31 f 0.01a 3.03 f 0.01a 2.90 f 0.02b BH2 8.1 1 f 044a 4.39 f 0.03a 3.45 +_ 0.04a 3.21 f 044a 2.42 f 0.04a BH3 9.13 f 0.03d 6.68 f 0.01d 5.08 f 0.01b 4.60 f 0.06b 3.96 0.03d BH4 8.72 f 0 .05~ 5.14 f 0 . 0 7 ~ 4.88 f 0.03b 4.48 f 0.01b 3.44 f 0 .05~ BH5 9.30 f 0.07d 5.17 f 0.52b 4.60 f 0.53b 440 f 0.56b 4.04 f 0.46d

‘ Values are means of three determinations from the same fermentation f standard deviation. The same following letters in the column denote the Duncan’s homogenous subsets.

3 70 H Kozlowska et a1

TABLE 4 Changes in inositol pentaphosphate (IP,) content during natural fermentation of lentils

(pmol- ' g DM)"


O h 24 h 48 h 7 2 h 96 h

BHl 0.15 f O.O1ab 0.18 f 0.01a 0.08 f 0.01a 0.11 f 0.01a 0.06 f 0.01a

BH3 0.40 f 0.01b 0.22 f 0.01a 0.21 f 0.01b 0.13 f 0.01a 0.11 f O-OOb BH4 0.98 f 0.06d 0.93 f 0.06~ 0-98 & 0.05d 0.92 f 0.07d 0.92 f 0.03e

BH2 0.64 f 0.04~ 0.96 f 0 . 0 3 ~ 0.88 f 0 4 4 ~ 0.68 _+ 0 . 0 1 ~ 0.51 f 0.01d

BH5 0.41 f 0.08b 0.37 f 0.04b 0.25 f 0.04b 0.25 f 0.02b 0.25 f 0 . 0 3 ~

Values are means of three determinations from the same fermentation f standard deviation. The same following letters in the column denote the Duncan's homogenous subsets.

These results show that initial concentration of the lentil suspension has a strong influence on the decrease of the total content of inositol phosphates, reaching larger reductions at lower initial concentration, whilst temperature has less influence.

For raw lentils (Tables 3-6) the inositol hexaphosphate (IP,) was the inositol form present in the largest amount in the seeds (10.8 pmol g - I ) whilst ino-

sitol pentaphosphate (IPS), inositol tetraphosphate (IP,) and inositol triphosphate (IP,) were found in minor amounts (1.1, 0.5 and 0.4 pmol g-', respectively). In the preparation of the sample for fermentation, the time required for completing the lentil suspension caused significant changes in IP, (Table 3) and 14-29'0 reductions were achieved. After 96 h of fermentation a sharp decrease of IP, content was observed, which was

TABLE 5 Changes in inositol tetraphosphates (IP,) content during natural fermentation of lentils

(pmol- * g DM)"


O h 24 h 48 h 72 h 96 h

BH1 0.25 f 0.02ab 0.33 f 0.01b 0.08 f 0.01a 0.03 f 0.01a 0.02 f 0.01a

BH3 0.45 f 0.02~ 0.75 f 0 . 0 1 ~ 1.45 & 0.01e 1.44 f 0.01e 1.10 f 0.05e

BH5 0.35 f 0.03b 0.18 f 0.03a 0.14 f 0.01b 0.12 f 0.02b 0.10 f 0.02b

BH2 0.42 f 0.02~ 0.74 & 0 .02~ 1.01 f 0.01d 0.45 f 0 . 0 1 ~ 0.29 f 0 .02~

BH4 0.53 f 0.02d 1.22 f 0.06d 0.85 & 0 . 0 1 ~ 0.56 f 0.02d 0.39 f 0.01d

Values are means of three determinations from the same fermentation f standard deviation. * The same following letters in the column denote the Duncan's homogenous subsets.

TABLE 6

DM)" Changes in inositol triphosphates (IP,) content during natural fermentation of lentils (pmol- g


O h 24 h 48 h 72 h 96 h

BH1 0.06 f 0.01a 0.89 f 0.01b 0.34 f 0.01a 0.31 f 0.01a 0.24 f 0.02a

BH3 0.35 f 0.01d 0.68 f 0.02a 1.43 f 0 . 0 1 ~ 1.80 f 0.01d 1.51 f 0 . 0 1 ~ BH4 0.38 f 0.02d 1.31 f 0@4d 1.67 f 0.04d 1.98 f 0.04e 1.91 f 0.04d BH5 0.29 f 0 .03~ 0.94 f 044b 0.61 f 0.01b 0.22 f 0.01a 0.18 f 0.02a

BH2 0.14 f 0.01b 1.01 f 0.04~ 1.35 f 0 . 0 1 ~ 0.75 f 0 . 0 1 ~ 0.46 f 0.04b

Values are means of three determinations from the same fermentation f standard deviation. The same following letters in the column denote the Duncan's homogenous subsets.

Natural fermentation of lentils

n 371

0 2 4 6 8 10

Retention time (min)

Fig 1. HRLC chromatogram of BH4 inositol phosphates. Fermentation (h): (1) 0; (2) 24; (3) 48; (4) 72; (5) 96.

related to the conditions in which the fermentation was carried out. The maximum decrease was found at the highest temperature (42°C) and the lowest concentration of lentils (79 g litre-*) (BH2).

For inositol pentaphosphate (IP,) (Table 4), the preparation of the lentil-suspension caused a sharp decrease and IPS values dropped from 1.1 pmol g-' found in the raw material to 0.2-0.6 pmol g-' at hour-0 of fermentation, except for the experiment carried out at high concentration and temperature (BH4, 221 g litre-' and 42°C) where the content of IP, was similar to the raw lentil (0.98 pmol g - ').

Throughout the fermentation procedure a consider- able reduction of IP, content was observed, except for

sample BH4 (221 mg litre-', 42°C). The minimum IP, content was detected in experiments carried out at 28°C (BH1 and BH3, 0.06 and 0.1 pmol g-', respectively), whilst batches incubated at 42°C (BH2 and BH4) presented maximum IP, values (0.5 and 0.9 pmol g-', respectively). The level of lentil concentration affected the IP, content of fermented lentils in the same manner as of that the IP, content. However the IPS content increased at higher temperature, contrary to effects on the IP, content.

The IP, content (Table 5 ) decreased during the preparation of the lentil-flour suspension, except for the experiment carried out at 221 g litre-' and 42°C (BH4) where no change was observed. After 24 h of natural

Fig 2.

2ol 10 I O ! r I I

12 36 60 84 0 24 40 72 96

Raw

hours Percentage changes in the total content of inositol phosphates during preparation and fermentation.

BH3; 0, BH4; x , BH5. ., BH1; +, BH2; *,


TABLE 7 Variable coefficients determined by linear multiple regression"

lonositol Variable coeficients Determination phosphates coefficient

form Constants Sample concentration Fermentation Fermentation time

Value S E Value S E Value S E temperature

Value S E

Total 4.9567 0.7104 0.0183 0.0018 0.0473 0.0182 - 0.0493 0.0029 0.7962 IP, 7.5919 0.2703 0.0083 0.0007 - 0.0287 0.0070 - 0.0501 0.001 1 0.7052 IP, - 1.3583 0'0947 0.0012 0.0002 0.0475 0.0024 - 0.0014 0.0004 0.8021

Calculation was made with confidence level = 0.05.

fermentation, except for sample BH5 (150 g litre-', 35°C) the IP, content of lentils increased, which was more pronounced at high lentil concentration and temperature. After 48 h of fermentation, a decrease in IP, content was observed for all the batches except for BH2 (79 g litre-', 42°C) and BH3 (221 g litre-', 28°C) where an increase was produced. After this time the IP, contents of all samples decreased to the end of experiment. The values of IP, at 96 h of fermentation were lower than the raw lentil, with the exception of batches BH3.

The content of IP, (Table 6 ) for the batches BH1 and BH2 changed in a manner similar to that of the IP, content. In the case of samples with the highest concentration (BH3 and BH4), an increase in the IP, content was observed throughout the fermentation procedure, reaching a very high value (1.5 and 1.91 pmol g-') after 96 h of fermentation.

The statistical analysis began with a one-way ANOVA with post hoc Duncan's multiple range test for results grouped in the two different ways. First, the significant differences in the content of examined I P forms among the BH1, BH2, BH3, BH4 and BH5 samples at the same stage of fermentation (columns in the tables) were analysed to confirm or exclude the effect of temperature and concentration of lentil flour. Duncan's homogeneous subsets were marked by the same letter superscripts. In the second arrangement, changes in the content of I P forms in the particular samples during fermentation were determined (rows in the tables). It enabled us to evaluate the dynamics of the I P form changes with the time of fermentation.

One-way ANOVA confirmed statistically significant differences (at P I 0.05) in all analysed groups of the first arrangement. The changes of IPtotal content (Table 2) and the distribution of Duncan's homogeneous subsets were very regular. Samples BH3 and BH4 (with higher concentration of lentil flour) made a homogeneous subset in all stages of fermentation. BH1 and BH2 samples (with lower concentration of lentil flour) did not make a homogeneous subset except one subset at the initial stage of fermentation. It suggested that in the samples with higher concentration of lentil flour, the

influence of fermentation temperature of IP,,,,, content was small or practically did not exist. The analysis of the changes in IP, content (Table 3) and Duncan's homogeneous subsets allows one to assume that the influence of lentil flour concentration was high in all stages of fermentation. Despite statistically significant differences of most of the groups, similar values of IP, content (Table 4) and the tendency for grouping in Duncan's homogeneous subsets by the samples fermented at the same temperature should be noticed. It might prove the influence of fermentation temperature on IP, content. Distribution of rarely appearing homogeneous subgroups in the Table 5, describing the changes of IP, content, was also random. However, the changes of IP, content in different stages of fermentation were differently directed. The samples with low concentration of lentil flour and fermented in low temperature contained always less IP, . On the other hand, in the samples with high concentration of lentil flour, this content was lower in those fermented at high temperature. Only three random homogeneous subsets were obtained from the results in the Table 6. These results showed, that the concentration of lentil flour influenced the IP, content, which the BH3 and BH4 samples confirmed.

One-way ANOVA with post hoc Duncan's multiple range test carried out for the second arrangement of results confirmed that the values of I P forms changed statistically significantly (P < 0.05) during fermentation, irrespective of concentration of lentil flour and fermentation temperature. Regular changes of the analysed values can be described by the function IP, =f(z), where r equals time. The changes of IP, and IP, values may be visualised by a set of different curves having mostly the maximum. The linear relationship between the determined values and time of fermentation was observed for IP, and IP,,,,,. The values of IP, changed specifically; linear dependence of the samples BH3, BH4 and BH5, and the maxima after 24 h of fermentation for BH1 and BH2.

The above results enabled the evaluation of the influence of all three factors. Multi-factor regression analysis

Natural fermentarion of lentils 373

was applied resulting in the equations:

IPIotal = 4.957 + 0.018C + 0.047~ - 0.049~

IP, = 7.592 + 0.008C - 0.029~ - 0.050~

IP, = - 1.358 + 0.095C + 0447t - 0.0014~

where C is the concentration of fermentation medium (g dl- I) , t is the temperature of fermentation ("C) and t is the time of fermentation (h).

The standard errors of estimation of equation constants, regression coefficients and determination coefficients are presented in Table 7. These values confirm that the equations accurately fit the experimental data. The equations demonstrate dissimilarity of an influence of the variables applied. Prolongation of fermentation time is related to the decrease in the content of IP forms while the higher concentration of lentil flour in fermentation medium increases I P content. The influence of temperature is irregular; the higher fermentation temperature inhibits the decrease of IPtOI,, and IP, content, while the opposite tendency is observed for IP,.

DISCUSSION

Natural lentil fermentation carried out in this work resulted in a reduction of inositol hexaphosphate accu- mulated in seeds to lower phosphoric esters IP,, IP, and IP3 (Tables 3 6) and to IP, and IP, and presum- ably even to free myo-inositol, which could not be detected with the method applied. In the fermentation conditions studied, time of fermentation, lentil concentration and temperature, it was observed that the content of total inositol phosphates mostly decreased at the lowest concentration (Fig 2). For the individual forms of inositol phosphates only 1P6 and IP, corre- lated with the conditions in which natural fermentation was carried out. The content of IP, decreased more at the highest temperature, a condition that also brought about the highest IP, content. This means that IP, could be hydrolysed to IP, and lower inositol forms during fermentation.

Natural fermentation of legumes is characterised by decreases in pH. Vidal-Valverde et a1 (1993) observed that the pH value during natural fermentation of lentils fell from 6.0 to 3.8 after 4 days. Ragaee et al (1985) reported that lactic acid bacteria play a major role in natural fermentation of lentil. The pH drop during lentil fermentation experiments performed in this work was from 6.4 to 4.6--3.8 (Frias et a1 1996). Considering that plant phytases have pH optima of 4-6 (Irving 1980). it can be assumed that conditions favourable for lentil phytase occurred in the process of fermentation. Additionally, an influence of enzymes of microbiological origin on degradation of phytates cannot be excluded (Jany 1993). Naturally occurring phytase is activated at low pH (Svanberg and Sandberg 1987) and the hydro-

lysis of higher inositol phosphates to phosphate and several lower phosphoric esters of myo-inositol, and in some cases also to free myo-inositol can take place.

Phytate present in raw materials and foods of plant origin are suggested to be a major factor responsible for lowering the availability of minerals and some proteins (Konietzny et al 1994). Mahajan and Chauhan (1988) noted that natural fermentation improved the HCl- extractability of minerals including calcium, magnesium and copper in pearl milled flour. Lonnerdal et a1 (1989) suggested that aqueous solutions of pure fractions of inositol phosphates with less than five phosphate groups produced via non-enzymatic hydrolysis, have no effect on zinc and calcium absorption in suckling rats. Sandstrom and Sandberg (1992) reported that adding IP, to white bread produced via non-enzymatic hydrolysis has no effect on zinc absorption in humans, while IP, and IP, depressed zinc absorption. Chichlowska et a1 (1994) reported that phytic acid modified the level of thyroid hormones and insulin, affecting the metabolism of carbohydrates, peptides and fats in rats after consumption of phytic acid in the diet.

Fermented legumes are an integral and significant part of the diet in developing countries (Salunkhe and Kadam 1989) and fermentation has been suggested as an economical method of processing and preserving foods. Legume-based fermented foods present desirable modifications including a partial or complete elimi- nation of antinutritional compounds (Reddy and Salunkhe 1989). Zamora and Fields (1979) found an increase in methionine, isoleucine, niacin, riboflavin and thiamin as a result of natural fermentation of chickpeas and cowpeas. Vidal-Valverde et a1 (1993) reported that natural fermentation of lentils totally eliminated ci- galactosides, compounds related to flatus production. These authors also noted an increase in riboflavin content and in the digestibility of starch. Fermentation of lentils for 96 h also brought about a decrease in trypsin inhibitor activity and a sharp drop in the ratio of tannin/catechin, a desirable feature from a nutritional point of view (Tabera et al 1994). Ragaee et a1 (1986b) observed an increment in total amino acids and in uitro protein digestibility in fermented lentils, which increased consumer acceptability (Ragaee et a1 1986a).

In conclusion, natural fermentation of lentils could be an acceptable process to enhance the nutritive value of lentils because of the increase of some nutrients such as vitamins, to remove some antinutritional factors such as a-galactosides and trypsin inhibitor activity and to degrade IP, to IP,, IP, and IP, forms which have a reduced effect on the bioavailability of minerals.

ACKNOWLEDGEMENT

This work has been supported by the Polish State Com- mittee for Scientific Research Grant 031 08, the Spanish Comision Interministerial de Ciencia y Technologia


ALI-91-1092-C02-01 and Copernicus Network EU (CIPA-CT92-4020). One of the authors (JF) acknow- ledges support from the European Union through an individual bursary (AIR3-BM93-1118). The authors are indebted to Dr Tabera for the preparation of fermented lentils.

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