SHARMA/&POOR: Effect of fermentation on digestibility of pearl millet

Effect of various types of fermentation on in vifro protein and starch digestibility of differently processed pearl millet

A. Sharma and A. C. Kapoor

Summary tobacillus acidophilus, Candida ufilis and natural fermentation using freshly ground pearl millet flour as inoculum. All the processing treatments except coarse grinding improved the protein and starch digestibility. Autoclaving enhanced the digestibilities of processed samples which was further im- proved by different types of fermentation, the maximum being in case of germinated and naturally fermented pearl millet. A combination of Lactoba- cilli and yeast was more effective in increasing the protein as well ab starch digestibility as compared to pure culture fermentation.

Pearl millet (Pennisetum fyphoideum) grains were fermented with Lacto- bacilli and yeast alone, in combination and with natural flora at 30°C for 48 h after giving various processing treatments viz, fine and coarse grinding, soaking, debranning, dry heat treatment, germination and autoclaving after adding of water. Fermentation was carried out with Lactobacillus acidophi- [us and Rhodororulu isolated from naturally fermented pearl millet and Lac-

Introduction

Fermented foods have made important contributions to human diets for thousands of years and continue to do so. Certainly, the most significant role of fermentation in hu- man nutrition has been to make nutrients naturally present, more palatable and more widely available [l]. Other than preservation and providing variety to diet, there are further important consequences of fermentation like, production of acids and alcohols which are inhibitory to common patho- genic micro-organisms [2].

On the other hand, we are still witness to decreased availability of food supplies and lack of subsistence food stuffs for millions of human beings especially in less devel- oped areas of the world who suffer from hunger andlor malnutrition due to a number of reasons e.g. population explosion disturbed ecology etc. In such a situation, pearl millet (Pennisetum typhoideum) which is very efficient in utilization of moisture, has short growing season and ap- pear to have higher level of heat tolerance than other ce- reals and millets, can prove to be a good resort if utilized properly, so that its potentials are fully exploited. Pearl millet is no doubt superior to cereals with respect to some of the nutrients especially average protein, minerals and fat [3], but due to presence of various antinutrients, poor di- gestibility of protein and carbohydrates and low palatabil- ity, their maximum utilization is being affected. However, various processing treatments are known to affect the chemical composition of food, improve its digestibility and nutritive value. Dehulling, soaking, cooking, heat treat- ments, autoclaving, roasting, sprouting and natural fermen- tation have been reported to increase the protein digestibil- ity by a number of workers on different crops including millets [4, 5, 6, 7, 8, 9, 101.

Considering the nutritional value, ease to culture, low investment and proportionately high returns of millets, the present study was carried out to develop techniques for im- proving the protein and starch digestibility of pearl millet by giving various processing treatments and by carrying out pure culture as well as natural fermentation of pro- cessed samples.

Department of Foods and Nutrition, CCS Haryana Agricultural University, Hisar, India

Materials and methods

Materials

Pearl millet grains were procured from the Directorate of Farms, CCS Haryana Agricultural University, Hisar, India. They were cleaned of dust and other extraneous material prior to use. L. acidophilus (La) was obtained from National Dairy Research Insti- tute, Karnal, India and Candida urilis (Cu) from National Chemi- cal Laboratory, Pune, India. The other two microbes isolated from naturally fermented pearl millet as most dominating yeast and Lac- tobacillus, identified and used as inoculum were Rhodotorulu (R) and L. plantarurn (Lp), respectively.

Processing treatments

Grinding

Grains were ground to fine and coarse particles passing through 0.5 and 1.5 mm sieve, respectively.

Soaking

Pearl millet grains were soaked in water for 12 h at 30 "C with a grain to water ratio of 1 to 10 (w/v). After soaking, excess water was drained off and grains dried at 60 "C and powdered.

Debranning

Cleaned grains were tempered with to attain 16% moisture level and hand pounded to separate the bran. Debranned grains and bran were powdered after drying at 60°C in hot air oven till constant weight.

Dly heat treatment

Finely ground pearl millet flour was taken in a conical flask, plugged and autoclaved at 1.05 kg/cm2 pressure for 10 min.

Germination

Grains after 12 h soaking were germinated at 30 "C for 48 h by keeping them in trays lined with wet filter paper. Ungerminated seeds were separated and germinated ones dried at 60 "C and powdered.

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SHARMAKAPOOR: Effect of fermentation on digestibility of pearl millet

Table 1. Effect of fermentation on in vitro protein digestibility of processed and autoclaved pearl millet [% on dry matter basis]

Type of fermen- Finely Coarsely Soaked Debranned Dry heated Germinated SE CD tation ground ground (m) ( P S 0.05)

Processed 52.60 f 0.40 51.70 f 0.67 59.67 f 1.20 63.00 f 0.88 52.60 f 0.40 78.13 f 0.77 0.41 1.37 (unfermented) Processed and auto- 71.09 f 0.45 71.92 f 1.38 75.92 f 0.64 78.79 f 0.52 54.62 f 0.82 85.97 f 0.72 0.41 1.22 claved control (unfermented) La 76.11 f 0.49 73.88 f 0.91 80.17 f 0.74 80.88 f 1.28 65.57 f 0.84 88.97 f 0.30 0.41 1.22 c u 77.77f0.77 75.89f1.13 79.77f0.42 79.11f1.03 67.22f1.42 88.01f0.24 0.46 1.38 LP 76.91 f 0.42 77.03 f 1.55 80.95 f 0.26 80.85 f 0.43 67.87 f 0.92 88.99 f 0.61 0.41 1.22 R 77.18 f 0.80 76.96 f 0.55 80.04 f 0.61 82.64 f 0.88 67.06 f 1.29 88.93 f 0.49 0.41 1.23 La + Cu 77.92 f 0.80 76.15 f 0.59 80.55 f 0.62 82.43 f 0.80 69.00 f 0.41 88.95 f 0.93 0.72 2.14 Lp + R 76.80 f 0.90 76.97 f 1.20 81.84 f 0.64 82.85 f 1.05 67.96 f 0.26 87.92 f 0.62 0.52 1.55 NF 79.25 f 0.41 78.00 f 1.13 83.39 f 0.92 86.19 f 1.07 71.88 f 0.51 89.93 f 1.57 0.50 1.51

0.33 0.55 0.69 0.46 0.44 0.37 - - CD (P 5 0.05) 0.98 1.62 2.03 1.35 1.30 1.09 - - SE (m)

Values are mean f SD of four replications La = Lactobacillus acidophilus R = Rhodotorula NF = Natural fermentation

Interaction SE (m) = 0.49, CD = 1.36 Cu = Candida utilis Lp = Lactobacillus plantarum

Autoclaving

All the processed grains were autoclaved after addition of water sufficient to make a slurry fit for fermentation (1 : 9, w/v). Slurry, thus obtained was used for fermentation after cooling to room tem- perature.

Fermentation

One hundred gram of processed and autoclaved samples were inoculated with lo5 cells under laminar flow after cooling to room temperature and incubated at 30 "C for 48 h. For natural fermenta- tion 5 g freshly ground pearl millet was used as inoculum. The following pure culture and mixed culture fermentation were worked out:

La, Cu, Lp, R, La + Cu, Lp + R and Natural (NF) .

The fermented slurry was dried at 60 "C in hot air oven, ground to fine powder and packed in air tight containers for further analysis.

Chemical analysis

Protein digestibility (in vitro) was assessed by the method of AKESON and STAHMANN [ I l l as modified by SINGH and JAMBU- NATHAN [12] using pepsin and pancreatin. The nitrogen contents (digestible) were determined by Microkjeldahl method [13]. The digested protein of the samples was determined by multiplying the N content with the factor 6.25. Protein digestibility was calculated using the following formula:

Digested protein Total protein

Protein digestibility (%) = x 100.

Table 2. Effect of fermentation on in vitro starch digestibility of processed and autoclaved pearl millet [mg maltose released/g, on dry matter basis]

~ ~~~

Type of fermen- Finely Coarsely Soaked Debranned Dry heated Germinated SE CD tation ground ground (m) ( P S 0.05)

Processed 13.0 f 0.5 12.5 f 0.6 18.9 f 1.1 18.3 f 1.1 13.0 f 0.5 35.4 f 1.9 0.6 2.1 (unfermented) Processed and auto- 24.3 f 1.3 23.7 f 0.8 31.3 f 1.3 33.7 f 1.2 13.7 f 1.5 53.8 f 1.8 0.7 2.1 claved control (unfermented) La 35.3 f 0.6 34.4 f 0.4 43.4 f 0.4 45.1 f 0.5 20.8 f 1.3 86.0 f 1.6 0.5 1.4 cu 36.7 f 0.2 36.2 f 1.3 44.9 f 0.6 44.2 f 0.2 21.2 f 1.3 89.6 f 1.9 0.6 1.7 LP 36.2 f 0.5 35.7 f 0.6 43.9 f 0.6 43.9 f 1.9 22.4 f 2.7 87.6 f 0.7 0.7 2.3 R 34.7 f 0.8 33.7 f 1.6 39.8 f 1.3 41.0 f 0.4 20.0 f 0.9 80.8 f 1.4 0.6 1.8 La + Cu 38.9 f 1.9 36.3 f 1.4 45.5 f 0.6 46.7 f 0.8 22.4 f 0.8 90.8f 1.1 1.1 3.2 Lp + R 38.2 f 1.8 38.6 f 0.4 45.9 f 1.6 45.8 f 0.5 21.2 f 0.6 88.4 f 0 . 9 0.6 1.7 NF 40.0 f 1.6 40.3 i 0.5 44.7 f 1.3 46.7 f 0.8 22 .0f 1.3 109.6 f 1.0 0.6 1.8

0.6 0.5 0.5 0.5 0.8 1 .o CD (P 5 0.05) 1.9 1.5 1.6 1.4 2.3 3.0 - - SE (m) - -

Values are mean f SD of four replications La = Lactobacillus acidophilus R = Rhodotorula NF = Natural fermentation

Interaction SE (m) = 0.6, CD = 1.9 Cu = Candida utilis Lp = Lactobacillus plantarum

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SHARMA~KAPOOR : Effect of fermentation on digestibility of pearl millet

144 Nahrung 40 (1996) Nr. 3, S . 142-145

Starch digestibility (in vitro) was assessed by employing pan- creatic amylase and then measuring maltose liberated using di-ni- trosalicylic acid reagent [ 141.

Statistical analysis

Standard method for analysis of variance in a two factorial com- pletely randomized design was used [15].

Results and discussion

Protein digestibility

The protein digestibility of raw, finely ground pearl mil- let was 52.60 + 0.40% and it varied from 51.70 * 0.67 (coarsely ground) to 78.13 5 0.77% (germinated) in the processed samples (Table 1). In vitro protein digestibility in processed and autoclaved unfermented controls varied from 54.62% in dry autoclaved to 85.97% in germinated sam- ples.

Fermentation by La, Cu, Lp, R, their combinations and natural fermentation improved the protein digestibility over controls. Natural fermentation was most effective in im- proving the protein digestibility in all the processed sam- ples. Mixed culture fermentation had a greater effect than individual culture fermentation. The highest protein digest- ibility (89.93%) was observed in germinated and naturally fermented pearl millet.

Enhanced proteolytic activity during fermentation is gen- erally associated with improved protein digestibility. In- crease in amino nitrogen to signify a partial breakdown of proteins to peptides and amino acids has been reported [16]. A combination of S. cerevisiue + S. diastuticus and L. brevis + L. fermentum were more effective as compared to individual fermentation [8, 171. Sprouted pearl millet when fermented with S. cerevisiae and L. fermentum in- creased the protein digestibility up to 91%. The increase in protein digestibility in the present study may also be due to decrease in phytate content as phytate forms protein-phy- tate-mineral complex which is resistant to proteolytic en- zymes [18, 191.

Starch digestibility

The starch digestibility of raw finely ground pearl millet was 13.0 5 0.5 mg maltose/g and it ranged from 12.5 to 35.4 mg maltose/g in processed pearl millet (Table 2).

The starch digestibility in processed and autoclaved un- fermented samples i.e. controls varied from 13.7 in dry heated samples to 53.8 mg maltose released/g in germi- nated samples. After fermentation digestibility increased to a minimum level of 20.0 mg maltose released/g in dry heated pearl millet fermented with R and to a maximum up to 109.6mg maltose released/g in case of germinated and naturally fermented samples which was almost double from control.

Fermentation by all the micro-organisms increased the starch digestibility significantly from that of control. Com- bination of Lactobacilli and yeasts improved it to a greater

extent than individual culture fermentation in all types of processed pearl millet. Fermentation with R, though in- creased the starch digestibility, was least effective among all the microbes used. Natural fermentation was most effec- tive in increasing the starch digestibility. Finely ground pearl millet had better digestibility than coarsely ground on fermentation.

In the present study, starch digestibility increased on fer- mentation with different microbes. It may be related to en- zymatic properties of microbes which ferment the substrate. The presence of alpha-amylase in the fermenting bacteria was indicated by BERNFELD [20]. The enzymes bring about cleavage of amylose and amylo-pectin to approximately six parts of maltose and five parts of glucose. Elimination of alpha-amylase inhibitors during processing treatments in- cluding germination, fermentation and heat treatments has been reported [14, 21, 22, 231. Natural fermentation was more effective than S. diastaticus, S. cerevisiae, L. brevis and L. fermentum individually or in combination in improv- ing the starch digestibility of pearl millet [8, 241.

In conclusion, it may be inferred that among various pro- cessing treatments and fermentation, germination and natu- ral fermentation proved to be the best. A combination of microbes was more effective in increasing the in vitro pro- tein and starch digestibility than individual culture fermen- tation.

References

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NIAN, Nutritive Value of Indian Foods. ICMR, Hyderabad, In- dia 1989.

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[7] CHOWDHURY, S., M. Sc. Thesis, CCSHAU, Hisar, India (1993).

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[lo] MAHAJAN, S., M. Sc. Thesis, HAU, Hisar, India (1986). [ I l l AKESON, W. E., and M. A. STAHMANN, J. Nutr. 83 (1964)

[12] SINGH, U., and R. JAMBUNATHAN, J. Food Sci. 46 (1981)

[13] A.O.A.C., Official Methods of Analysis of the Association of

[I41 SINGH, U., M. S. KHERKAR and R. JAMBUNATHAN, J. Food

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RANI/JOOD et al. : Cultivar differences of pigeon pea seeds

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(1990) 883. Received 20 November 1995 Revised manuscript received 5 March 1996

Cultivar differences and effect of pigeon pea seeds boiling on trypsin inhibitor activity and in vitro digestibility of protein and starch

S. Rani, S. Jood and S. Sehgai

Summary activity and improved protein and starch digestibility when compared to un- processed seeds. Cultivar differences were also significant. Boiling of soaked seeds proved most effective in improving nutritional quality. Soaking and ordinary boiling of soaked seeds of five cultivars of pigeon

pea (Cajunus cajan L.) significantly (P 5 0.05) lowered trypsin inhibitor

Introduction

Pigeon pea is one of the most important food legumes grown in tropical and sub-tropical areas [l]. Its consump- tion by human beings is limited by the presence of antinu- tritional factors. Trypsin inhibitor is the most prominent in disrupting digestive process due to undesirable physiologi- cal reactions [2, 3, 4, 51. Reduction or elimination of these undesirable attributes is necessary to improve the nutri- tional quality and acceptability of food legumes. Various methods of processing have been used for this purpose [6, 7, 8, 91. The objective of this study was to investigate the effect of soaking and/or boiling on trypsin inhibitor activity and protein and starch digestibility (in vitro) of five cultivars of pigeon pea.

Materials and Methods

Materials

The seeds of five highly yielding cultivars (H 89-5, H 82-1, H89-2, H86-1 and Manak) of pigeon pea grown under similar soil and agroclimatic conditions at research farms, CCS Haryana Agricultural University, were procured from the Department of Plant Breeding. Seeds were cleaned and soaked in water for 12 h (overnight) at 37 "C, rinsed twice with distilled water and dried to a constant weight at 55 "C. For boiling, overnight soaked seeds were boiled (water two times the weight of soaked seeds) in round

Department of Foods & Nutrition, CCS Haryana Agricultural Uni- versity, Hisar, India

mouthed beakers fitted with condensors connected to running water until they became soft. Unsoaked seeds were boiled in the same way using a seed to water ratio of 1 :5 (w/v). The boiled seeds were mashed and dried at 55 "C. The oven dried processed as well as unprocessed samples were ground and stored at room temperature for further analysis.

Following chemicals such as, Trypsin, T 8253, from bovine pan- creas; pepsin, P 6887, from porcine stomach; Pancreatin, P 1500, from porcine pancreas and pancreatin amylase, A 6255, from por- cine pancreas, were purchased from Sigma Chemical Company St. Louis, USA.

Chemical analysis

Trypsin inhibitor activity was determined by the modified meth- od [lo]. Protein digestibility (in vitro) was assessed by employing pepsin and pancreatin [ I l l . Starch digestibility (in vitro) was as- sessed by employing pancreatic amylase [12].

Statistical analysis

Data were statistically analysed for analysis of variance ~ 3 1 .

Results and discussion

Variation in trypsin inhibitor activity (741 -869 TIU/g) among cultivars was significant (Table 1). Trypsin inhibitor activity found maximum (869 TIU/g) in Man& and mini- mum (741 TIU/g) in H 89-2. Variation in activity was also

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