Plant l~bods for Human Nutrition 40: 167-173, 1990. t67 © 1990 Kluwer Academic Publishers. Printed in the Netherlands.

Fermentation of pearl millet flour with yeasts and laetobaeilli: in vitro digestibility and utilisation of fermented flour for weaning mixtures

N. KHETARPAUL & B.M. CHAUHAN* Department of I'bods & Nutrition, Haryana Agricultural University, Hisar 125 004, India (*author for correspondence)

Received 20 November 1989; accepted in revised form 27 March 1990

Key words: pearl millet, yeasts, lactobacilli, starch digestibility, protein digestibility

Abstract, Single as well as mixed culture fermentations by yeasts (S. diastaticus; S. cerevistiae) and lactobacilli (L. brevis; L. fermentum) at 30 °C for 72 h improved the starch and protein digestibility (in vitro) of pearl millet flour significantly. The flour fermented by Saccharomyces diastaticus, a starch hydrolysing yeast, had the highest starch digestibility whereas fermenta- tion by Saccharomyces cerevisiae enhanced the in vitro protein digestibility of the flour significantly. Weaning mixtures prepared from the fermented flour were also found to be organoleptically acceptable.

Introduction

The carbohydrate as well as protein digestibility (in vitro) of pearl miUet (Penniseturn typhoideum), a staple food tbr a large segment of the population in Asian and African countries, is known to be low [4]. Pearl millet grains contain about 60% starch of which amylopectin is the major constituent [6]. The starch digestibility of pearl millet is low since it is relatively more resistant to attack by pancreatic amylase. Besides, phytate, known to inhibit the proteotytic and amylolytic enzymes [16, 21] is present in considerable amounts in pearl millet [4]. The formation of a ternary protein mineral phytate complex is believed to obstruct or inhibit the enzymatic degradation of protein and starch and results in their low digestibility. Fermentation has been known to improve the protein and starch digestibility of several food grains [3, 7]. This paper reports the effect of fermentation by pure cultures of yeasts and lactobacilli, the major microorganisms in naturally fermented pearl millet flour, on the starch and protein digestibility (in vitro) of pearl millet flour. Sensory evaluation of the products prepared from fermented flour for human consumption is also reported.

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Materials and methods

Materials

Pearl millet grains procured from the local market in a single lot, were cleaned of broken seeds, dust and other foreign material. The cultures of yeasts and lactobacilli namely, Saccharomyces diastaticus, Saccharomyces cerevisiae, Lactobacillus brevis and Lactobacillus fermentum were obtained from the Director, National Chemical Laboratory, Poona (India).

Fermentation

Pearl millet grains were coarsely ground by an electric grinder (Sumeet M/s E. Appliances, India) using 1.5 mm sieve. Coarsely ground pearl millet flour (100g) was mixed with distilled water (900ml), autoclaved (15psi for 15 min), cooled, inoculated with the above mentioned cultures of yeasts and lactobacilli and incubated at 30 °C for 72 h. For single culture fermentation, the culture of any one of the above mentioned yeasts or lactobacilli supplied initially 105cetls/ml in the fermenting mixture. For mixed fermentation, a combination of yeast (105cells/ml) and lactobacillus (105cells/ml) was deployed. Four different types of mixed fermentations included S. dias- taticus and L. brevis (SdLb), S. diastaticus and L. fermentum (SdLf), S. cerevisiae and L. brevis (ScLb) and S. cerevisiae and L. fermentum (ScLf) samples. The autoclaved unfermented pearl millet served as the control. The unfermented as well as fermented samples were oven dried for 48 h at 65 °C to a constant weight, finely ground using 0.5 mm sieve and used for further analysis,

Starch and protein digestibility (in vitro): Pancreatic amylase was em- ployed for assessing in vitro starch digestibility [17]. A suitable amount of defatted sample dispersed in 0.2 M phosphate buffer (pH 6.9) was acted upon by pancreatic amylase in a water bath at 37 °C for 2 h. The maltose so liberated was measured colorimetrically by using dinitrosalicylic acid reagent.

Protein digestibility (in vitro) was determined by employing pepsin and pancreatin [1]. A suitable amount of defatted sample was mixed with pepsin solution and incubated at 37°C for 16h in a water bath shaker. After incubation, the pH was adjusted to 7.0 with 0.2N NaOH and again in- cubated at 37 °C in a water bath shaker for 24h after putting pancreatin solution. The digestible proteins were precipitated by 10% trichloroacetic acid. The supernatants were pooled and nitrogen content of the sample and of undigestible residue was determined by the microkjeldahl method [2].

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Protein digestibility was calculated using the following formula:

Protein digestibility (%) Digested protein

= x 100. Total protein

Development and utilization of the fermented product

Various types of weaning mixtures for children were prepared from the mixed cultures fermented flour. Weaning mixture A contained fermented pearl millet flour (55 g), green gram dal (15 g), peanut (10 g), sesame (5 g) and sugar (30 g) whereas the ingredients of weaning mixture B included ferment- ed flour (50 g), roasted bengal gram (15 g), whole wheat flour (15 g), peanut (15 g), sesame (19 g) and jaggery (35 g). Weaning mixture C was similar to weaning mixture B in composition except that it contained sugar instead of jaggery. All the ingredients except sugar or jaggery were first roasted, mixed and then sugar/jaggery was added.

Organoleptic evaluation

The weaning mixtures prepared as above were evaluated for colour, flavour, taste, texture and appearance by a panel of judges deploying 9-point hedonic scale and average of scores of all the above characteristics was expressed in terms of overall acceptability.

Stat~tkal analys~

The data were subjected to analysis of variance and correlation coefficients were derived in a completely randomised design [14].

R e s u l t s a n d d i s c u s s i o n

Starch digestibility

Mixed as well as single culture fermentations except L. brevis improved the starch digestibility of pearl millet flour significantly (Table 1). As compared to single culture fermentation by lactobacilli, both the yeasts deployed for fermentation resulted in higher starch digestibility; S. diastaticus fermented flour had significantly (P < 0.05) increased starch digestibility than the flour fermented by S. cerevisiae.

All the mixed culture fermentation combinations also enhanced the starch

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Table l. Effect of pure culture fermentation on in vitro starch digestibility (mg maltose released per g flour) and in vitro protein digestibility (%) of pearl millet flour (on dry matter basis)

Fermentation Starch digestibility Protein digestibility

Single fermentation Sd 38.3 ± 0.7 67.6 ± 0.1 Sc 35.6 ___ 0.8 81.6 _ 5.2 Lb 19.6 ± 0.6 68.4 ± 0.3 Lf 20.5 ± 0.8 68.7 _ 0.1

Mixed fermentation SdLb 35.6 4- 0.3 72.2 ± 0.1 SdLf 29.7 + 0.7 72.4 _ 3.5 ScLb 30.1 ___ 0.2 75.3 ___ 3.7 ScLf 28.5 ± 0.5 75.5 __+ 0.2

Control Raw pearl millet 17.8 ± 1.0 51.0 +__ 0.0 Unfermented autoclaved control 18.7 _+ 0.2 59.2 + 0.0 SE(m) b ± 0.4 ± 2.0 CD(P < 0.05) c 1.1 6.2

~Values are means ± SD of four replicates bStandard error of mean ~Critical difference determined by ANOVA (P < 0.05). Differences of two means within/ between the fermentation treatments exceeding this value are significant.

digestibility of pearl millet; all the four combinations studied had significant- ly (P < 0.05) different starch digestibility values. Among all the single and mixed culture fermentations, S. diastaticus fermented flour had the highest digestibility. The flour fermented by S. cerevisiae alone and SdLb combina- tion had the similar digestibility values. The improvement in starch diges- tibility of mixed culture fermentation combinations was significantly higher than that of single culture fermentation by lactobacilli.

Improvement in starch digestibility during fermentation may be due to breakdown of starch to oligosaccharides by fermenting microflora [5]. Reduction in the phytate content of pearl millet during fermentation [8, 10] may also account for improvement in its starch digestibility as a significant (P < 0.05) negative correlation (0.5610) was obtained between the phytic acid and starch digestibility (in vitro). An improvement in starch digestibility through fermentations has been reported earlier in the case of sorghum [20], soybean [31 and tef [15].

Protein digestibility (in vitro)

A significant (P < 0.05) increase in protein digestibility was noticed when the pearl millet flour was autoclaved at 15psi for 15min (Table 1).

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Table 2. Overall acceptability of weaning mixtures prepared from fermented pearl millet"

Fermentation Weaning mixture

A B C

S. diastaticus + L. brevis 7.32 _+ 0.42 6.94 -{- 0.79 6.90 + 0.52 S. diastaticus -t- L.fermentum 7.08 + 0.50 6.38 _+ 0.7t 7.00 +_ 0.59 S. cerevisiae + L. brevis 7.20 + 0.59 6.44 + 0.74 7.12 + 0.57 S. cerevisiae + L. fermentum 7.02 _+ 0.52 6.70 + 0.69 6.98 + 0.50 Se (m) u _+ 0.22 _+ 0.21 + 0.22 CD (P < 0.05) c 0.66 0.63 0.66

~Average score of seven characteristics (colour, appearance, flavour, texture, taste, bitterness and sourness) given by 10 judges on 9-point hedonic scale, i.e. Like extremely (9), Like very much (8), Like moderately (7), Like slightly (6) Neither like nor dislike (5), Dislike slightly (4), Dislike moderately (3), Dislike very much (2) and Dislike extremely (1). bStandard error of means. cCritical difference determined by ANOVA (P < 0.05). Differences of two means within/ between the fermentation treatments exceeding this value are significant.

Fermentat ions by yeasts and lactobacilli further significantly improved the protein digestibility of the autoclaved unfermented flour. There was not significant (P < 0.05) difference among the improvement in protein diges- tibility brought about by S. diastaticus, L. brevis and L. fermentum whereas S. cerevisiae fermented flour resulted in the highest amount of increase in protein digestibility.

Mixed culture fermentations seemed to be more effective for enhancing the protein digestibility than all the single culture fermentations except S. cerevisiae fermented group. Improvement brought about in in vitro protein digestibility by various mixed culture fermented groups, i.e. SdLb, SdLf, ScLb and ScLf did not vary significantly (P < 0.05) among themselves.

Microflora may produce some proteolytic enzymes [13, 18] during fer- mentat ion [9] which may be responsible for increased protein digestibility of pearl millet. Phytic acid known to inhibit the proteolytic enzymes [12] is considerably reduced during fermentat ion [8, 10] which may also contribute to the increase in protein digestibility. A significant (P < 0.05) negative correlation was obtained between the phytic acid and in vitro protein diges- tibility. Increased protein digestibility in various fermented products includ- ing tempeh and miso [18], germinated and fermented soybean [3] and rabadi [7] has been reported earlier.

Utilisation o f f ermented products

The pearl millet flour fermented by pure cultures of yeasts and lactobacilli was incorporated in weaning mixtures (Table 2). Weaning mixtures A and

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C were 'moderately liked' when various mixed fermented flour were incor- porated for their preparation. Weaning mixtures B prepared from SdLf and ScLb were slightly 'liked'. Among all the mixtures prepared, weaning mix- ture A containing fermented flour, roasted peanut, green gram dal, sesame and sugar had the higher overall acceptability. Taur, Pawar and Ingle [19] did sensory evaluation of the chips made from germinated-fermented sorg- hum and they were 'neither liked nor disliked' by the judging panel.

Overall, single as well as mixed culture fermentations enhanced the starch and protein digestibility (in vitro) of pearl millet flour significantly. The weaning mixtures prepared from the flour mixed fermented with yeasts and lactobacilli were slightly to moderately liked. Fermentation, therefore, seems to be a potential processing method for enhancing the digestibility of cereals like pearl millet. Yeast fermentation is very simple and less expensive technique and can be adopted at household level for better utilisation of the protein and starch of the pearl millet flour.

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