Vol. 1(3), pp.36-44, May 2013 Available online at http://www.accessinternationaljournals.org/arbs Copyright ©2013 Access International Journals
Full Length Research Paper
Isolation, characterization and identification of lactic acid bacteria and yeast involved in fermentation of Teff (EragrostisTef) Batter Askal Desiye1
and Kebede Abegaz2
1
Department of Biology, Hawassa University (HU), P.O.Box 05, Awassa, Ethiopia. Institute of Nutrition, Food Science and Technology, Hawassa University (HU), P.O.Box 5, Awassa, Ethiopia.
2
Corresponding author. Email:
[email protected]; Tel +251 916 869030. Accepted 15 April, 2013
Enjera, an indigenous Ethiopian pancake is one of the staple foods of Ethiopians. There is little information available concerning the succession and activities of microflora of its fermentation. Therefore this study was carried out to isolate, characterize and identify lactic acid bacteria and yeast. A total of 34 samples from enjera batter were collected during 96 h fermentation at 6 h intervals. The Teff sample was bought from Hawassa open market. A total of 107 lactic acid bacteria (LAB) and 68 yeast strains were isolated and identified. The LAB strains were identified as Pediococcus pentosaceus (49.53%), Lactobacillus fermentum (28.04%), Lactococcus piscium (5.61%), Lactococcus plantarum (4.67%), Pediococcus acidilactici (3.74%), Leuconostoc mesenteriodes subsp. mesenteriodes (2.80%), Lactococcus raffinolactis (2.80%), L. mesenteriodes subsp. dextranicum (1.87%), Enterococcus cassiiflavus (0.93%), and the yeast strains comprised Saccharomyces cerevisiae (48.53%), Candida humilis (22.06%), Candida tropicalis (17.65%), Saccharomyces exiguus (7.35%) and Pichia norvegensis (4.4%). The teff enjera batter contained both LAB and yeast species, which have great potential to impart the particular characteristics in the final product of enjera. Key words: Enjera, Lactic acid bacteria, Starter cultures, Teff batter, Yeast.
INTRODUCTION Fermentation is one of the most economical methods of producing and preserving foods. It provides a way to preserve food products, to improve organoleptic
properties by producing different flavors of foods, to improve the nutritive value and to reduce toxic substances (Damiani et al., 1996; Corsetti et al., 1998;
Desiye and Abegaz
Thiele et al., 2002). Fermentation is relatively a low-energy requiring preservation technology that promotes shelf life of food products. It is simple and easily adaptable to local household practices in traditional communities (Cooke et al., 1987). Food preparation is predominantly a household phenomenon in Ethiopia. Every household appears to process food starting from raw ingredients to the final products. In cases where fermentation is important to obtain a certain food, the microorganisms present on the raw ingredients or in the containers spontaneously take care of the process. The outcome and quality of spontaneous food fermentation may not be predictable. According to a review by Achi (2005), this is a major problem in African fermented foods. Generally, food fermentation relies on chance inoculation (that is either natural contamination or backslopping) which results in a product of inconsistent quality (Kimaryo et al., 2000). In order to maintain and sustain African indigenous fermented foods and beverages, controlled fermentation (Sadeghi et al., 2009; Agarry et al., 2010) and product quality characteristics (Kabeir et al., 2004; Theodore et al., 2007 and Taiwo, 2009) are strongly recommended. Controlled fermentation includes the use of pure or mixed starter cultures with appropriate technology (Glover et al., 2009). The ability to isolate strains of microorganisms with desirable physiological and metabolic characteristics for use as inocula in industrial fermentations resulted in a high degree of fermentation process (Dunga et al., 2005). Lactic acid bacteria and yeast are appreciated as starter culture and for their health benefits (Tamime et al., 2002). A wide variety of fermented foods and beverages are consumed in Ethiopia. These include: Enjera, kocho, tella, awaze, borde and tejj. Enjera is one of the fermented foods that is made from different cereals, including sorghum, teff, corn, wheat, barley, or a combination of some of these cereals. Enjera from teff (Eragrostis tef) is much more relished, by most Ethiopians, than that from any other source. Enjera is a pancake-like food usually obtained after the flour of cereals has been subjected to 24 to 96 h of traditional fermentation depending on the ambient temperature. In order to make teff enjera a starter (Ersho) saved from the previous fermented batter is used to initiate new batches of fermentation (back slopping). Housewives bake enjera every 2 - 3 days, and know the usefulness of this starter. However, occasional failures do occur in the fermentation that leads to inconsistencies in quality of enjera.
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Despite the fact that enjera is a favorite staple food, there is little information available concerning the succession and activities of microflora during enjera batter fermentation. Since enjera batter fermentation is based on natural inoculants, it is necessary to study the identity of the natural flora involved in its fermentation. In order to upgrade the quality of enjera and bring desirable changes, this study was aimed at isolating, characterizing and identifying lactic acid bacteria and yeast during fermentation of teff (Eragrostis Tef) batter.
MATERIALS AND METHODS Teff sampling Teff sample was bought from Hawassa open market. It was cleaned of dust, other seeds and foreign matter. On the day of fermentation, the teff was grounded into fine powder in a local flour mill before the start of batter preparation and fermentation.
Preparation and fermentation of Teff Batter Fermented teff batter was prepared under laboratory conditions. The fermentation was performed according to the traditional fermentation in households. It was initiated by sifting the flour and mixing 1 kg flour with 2 L water in fermenting plastic jar in a 1:2 ratio. Previously fermented dough (10%) was then added to the mixture of flour and water to act as a starter and mixing thoroughly until a homogenous slurry was obtained. The batter was allowed to ferment for 96 hours at room temperature.
Isolation and characterization of lactic acid bacteria Representative colonies were randomly selected based on their morphology, such as colour, shape and size from countable plates and purified by repeated plating. For characterization of LAB, twentyfour hours cultures of each isolate in MRS broth (Oxoid, Basingstoke, Hampshire, England) were used. All isolates were initially subjected to preliminary tests including Gram reaction, motility test, catalase reaction, cell morphology and cell arrangement (Kimaryo et al., 2000). The isolates were grouped as homofermentative or heterofermentative groups by their ability to produce gas from glucose fermentation. The isolates were
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inoculated into sterilized phenol red broth base supplemented with 1% glucose and containing inverted Durham tubes (Harrigan, 1998). The tolerance of isolates to different temperatures was observed by inoculating the pure culture in MRS broth and incubating for 5 days at 15, 37 and 45°C, and for 12 days at 4 and 10°C. Growth of LAB isolates in the presence of 0, 2, 4, 6, 8 and 10% (w/v) NaCl was recorded by inoculating the isolates in MRS broth for 5 days. The tolerance of isolates against high acid concentrations was recorded by inoculating the strains in MRS broth with pH 3.9 adjusted with HCl (Kimaryo et al., 2000). Isolates were tested for their ability to produce dextran from sucrose by streak plating onto sterilized MRS agar(Oxoid, Basingstoke, Hampshire, England) supplemented with 10% sucrose and incubated for 48 h at 25°C. Synthesis of dextran was indicated by the development of mucoid character (Damelin et al., 1995). Production of acetylmethylcarbinol from glucose was determined using Voges-Proskauer test (Harrigan, 1998). The ability of the isolates to hydrolyze starch was determined on starch agar and incubated at 30°C for 2-13 days. The plates were flooded with 5-10 ml of Gram’s iodine solution. Formation of blue colour was recorded as negative reaction while the development of clear zones was taken as positive reaction (Harrigan, 1998). The LAB isolates were tested for fermentation of the following sugars: arabinose, galactose, maltose, raffinose, trehalose, sorbitol, xylose, mannose, esculin, fructose, lactose, sucrose, melibiose, cellobiose, ribose and mannitol (Holt et al., 1994). All LAB strains were characterized and tentatively identified to species level according to Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994).
Isolation and characterization of yeast isolates Morphological, physiological, and biochemical tests were carried out for yeast characterization (Barnett et al., 2000). Colonies with distinct morphological differences, such as color, shape, and size were picked and purified by streaking on yeast malt glucose peptone agar (yeast extract, 3 g; malt extract, 3 g; dextrose, 10 g; peptone, 5 g; agar ,20 g; chloramphenicol, 0.1 g ;distilled water,1000 ml; pH 6.4). The tolerance of yeasts at high osmotic pressures was determined by inoculating actively growing cultures into a media containing high concentration of NaCl and glucose (Barnett et al., 2000). Growth in the presence of cycloheximide was performed in yeast
extract broth with filter sterilized 0.01 and 0.1% solutions of cycloheximide (Smith and Yarrow, 1995). Urea test was performed in the Urea broth and incubated at 37°C for 2 h. The test was taken to be positive when there was development of a deep red colour in the broth, which indicates urease activity (Barnett et al., 2000). Growth in the presence of acetic acid was tested by inoculating the pure yeast strains into sterilized yeast extract broth containing a 1% acetic acid (Smith and Yarrow, 1995). Utilization of ethanol as a sole carbon source was determined in ethanol broth (ammonium sulphate, 1.0 g; potassium dihydrogen phosphate, 1.0 g; magnesium sulphate, hydrated (MgSO4.7H2O), 0.5 g; distilled water, 1000 ml). After incubating for 3 to 7 days at 25°C the growth was recorded (Harrigan, 1998). The yeast isolates were tested for fermentation of the following carbohydrates: glucose, galactose, maltose, sucrose, trehalose, melibiose, lactose, cellobiose, melezitose, raffinose, inulin, starch and xylose. The yeast isolates were identified to species level according to the tests described by Barnett et al. (2000).
RESULTS Characterization of LAB A total of 107 lactic acid bacteria isolates were characterized and identified to species level. All isolates were Gram-positive, catalase negative and non-motile. In physiological characteristics all isolates also grew at 15°C. But the ability of growth at 50°C and in 8% and 10% NaCl were different among the isolates. In pattern of fermented carbohydrates all isolates showed positive for galactose, maltose, xylose, fructose and sucrose. Fermentation of Lactose, cellobiose and melobiose were important for differentiation the isolates. The results of the characterization using standard physiological and biochemical tests (Table 1) were identified as Pediococcus pentosaceus (49.53%), Lactobacillus fermentum (28.04%), Pediococcus acidilactici (3.74%), Leuconostoc mesenteriodes subsp. mesenteriodes (2.80%), L. mesenteriodes subsp. dextranicum (1.87%), Enterococcus cassiiflavus (0.93%), Lactococcus plantarum (4.67%), Lactococcus raffinolactis (2.80%) and Lactococcus piscium (5.61%). Cocci were the dominant LAB accounting for 71.96% whereas rods accounted for 28.04%. Among the 107 LAB isolates, 32.71% produced gas (CO2) from glucose and were referred to as heterofermentative, whereas 67.29% were
Desiye and Abegaz
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Table 1. Morphological, physiological and biochemical properties of LAB isolated during teff batter fermentation.
Number of strains with positive reaction Characteristics
Codes of the species
A
B
C
D
E
F
G
H
I
No. of strains Cell morphologya Gas production from glucose
53 C 0
30 R 30
6 C 0
5 C 0
4 C 0
3 C 3
3 C 0
2 C 2
1 C 0
Growth at temperature (°C) 4 10 15 37 45 50
0 0 53 53 53 0
30 30 30 30 30 0
6 6 6 0 0 0
5 5 5 5 0 0
0 0 4 4 4 4
0 0 3 3 0 0
3 3 3 3 0 0
2 2 2 2 2 0
1 1 1 1 1 0
Growth in NaCl (%) 2 4 6 8 10
53 53 53 0 0
30 30 30 0 0
6 0 0 0 0
5 0 0 0 0
4 4 4 0 0
3 3 3 3 0
0 0 0 0 0
2 2 0 0 0
1 1 1 0 0
Growth at pH 3.9 Hydrolysis of starch
53 0
30 21
6 0
0 0
4 0
3 0
3 0
2 0
0 0
Production of: Dextran from sucrose Acetion from glucose
0 0
0 0
0 6
5 0
0 0
3 0
0 3
2 2
0 0
a
R-rod shape, C- cocci shape , A- P. pentosaceus, B- Lb. fermentum, C- Lac. Piscium, D- Lac. Plantarum, E- P. acidilactici , F- Leu. mesenteriodes subsp. mesenteriodes, G- Lac. raffinolactis, H- Leu. mesenteriodes subsp. dextranicum, I- Ent. cassiiflavus
homofermentative (produced no gas). These heterofermentative cocci were tentatively identified as Leuconostoc spp (Table 2).
Characterization of yeasts A total of sixty eight yeast strains isolated during enjera fermentation were characterized and identified as belonging to 5 species. These were Saccharomyces cerevisiae (48.53%), Candida humilis (22.06%), Candida tropicalis (17.65%), Saccharomyces exiguous (7.35%) and Pichia norvegensis (4.4%) during fermentation of teff batter. Growth on 5 and 10% NaCl, 50% glucose, 0.01% cycloheximide and ethanol utilization were important for
differentiation. All isolates were positive for glucose fermentation. In pattern of fermented carbohydrates galactose, maltose, trehalose, melibiose, melezitose, raffinose and starch were important for differentiation. None of the isolate grew in 16% NaCl, 60% glucose, 0.1% cycloheximide and 1% acetic acid. None of those 68 strains fermented Inulin, lactose, cellobiose and xylose (Table 3).
Successions of LAB and Yeast Species during Traditional Fermentation of Teff Batter Pediococcus pentosaceus and L. fermentum were found from the beginning to the end of teff enjera batter
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Table 2. Carbohydrate fermentation of LAB isolated during teff batter fermentation. Number of strains with positive reaction Carbohydrates Arabinose Galactose Maltose Raffinose Trehalose Sorbitol Xylose Mannose Esculin Fructose Lactose Sucrose Mannitol Ribose Cellobiose Melibiose
Codes of species
A
B
C
D
E
F
G
H
I
No. of strains
53 53 53 53 0 53 0 53 53 0 53 53 53 0 53 53 0
30 30 30 30 30 30 0 30 30 0 30 30 30 0 30 0 30
6 0 6 6 6 0 6 6 6 0 6 6 6 6 0 0 0
5 5 5 5 0 5 5 5 5 0 5 0 5 5 5 0 0
4 4 4 4 0 4 0 4 4 0 4 4 4 0 4 4 0
3 3 3 3 3 3 0 3 3 0 3 3 3 0 3 0 0
3 3 3 3 0 3 0 3 0 3 3 0 3 3 0 0 0
2 0 2 2 2 2 0 2 2 0 2 2 2 2 0 0 0
1 1 1 1 1 1 0 1 1 0 1 1 1 0 1 0 0
A- P. pentosaceus, B- Lb. fermentum, C- Lac. Piscium, D- Lac. Plantarum, E- P. acidilactici , F- Leu. mesenteriodes subsp. mesenteriodes, G- Lac. raffinolactis, H- Leu. mesenteriodes subsp. dextranicum, I- Ent. cassiiflavus
fermentation. Enterococcus cassiiflavus was also present at early period of fermentation of teff batter due to the initial pH of the batter. L. piscium, L. mesenteriodes sub sp. mesenteriodes and sub sp. dextranicum were isolated after middle period of fermentation. C. humilis, S. cerevisiae, C. tropicalis and S. exiguus were found from the beginning to the end of teff enjera batter fermentation. P. norvegensis was also present at early period of fermentation of teff batter (Table 4).
DISCUSSION The association of LAB and yeasts are common in several traditional cereal based fermented foods and beverages (Berhanu, 1985; Gobbetti, 1998 and Kebede, 2002). These lactic acid bacteria and yeasts identified in fermented teff enjera batter have been isolated in other fermented foods. The results of the present study are in line with a review by Gobbetti (1998) that discussed Leuconostoc sp. and Enterococcus sp. S. cerevisiae, S. exiguus, P. norvegensis isolated from sourdough fermentation. L. fermentum, Pediococcus spp,
Leuconostoc spp, Enterococcus spp, C. humilis and S. exiguus have been isolated from sourdough (Vuyst and Vancanneyt, 2007). L. fermentum and L. mesenteriodes subsp. mesenteriodes have been isolated during cassava fermentation (Yao et al., 2009) and from Mawe fermentation (Nout, 2009). L. fermentum have been suggested to be the predominating microorganisms during fermentation of sorghum dough in Sudanese kisra (Asmahan and Muna, 2009). Halm et al. (1993) also reported the dominance of Candida spp. followed by Saccharomyces spp. in fermented maize dough. P. pentosaceus, L. fermentum C. humilis, S. cerevisiae, C. tropicalis and S. exiguus were isolated from the beginning to the end of enjera batter fermentation. They are the predominating microflora and remained until the end of fermentation by steam baking. E. cassiiflavus, Pichia norvegensis and Lactococcus piscium were also present at the early period of fermentation. L. mesenteriodes sub sp. mesenteriodes, sub sp. dextranicum and also Lactococcus piscium were identified after 48 h of fermentation. This succession could be because of utilization of substrate of teff and tolerance of the pH enjera batter. The results of the
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Table 3. Physiological and biochemical properties of yeast isolated during teff batter fermentation. Number of strains with positive reaction Characteristics
Code of species
No. of strains Growth in 5% NaCl 10% NaCl 16% NaCl 50% Glucose 60% Glucose 0.01% cycloheximide 0.1% cycloheximide 1% acetic acid Ethanol utilization Carbohydrate fermentation Glucose Galactose Maltose Sucrose Trehalose Melibiose Melezitose Raffinose Inulin Starch lactose cellobiose xylose
A
B
C
D
E
15
12
3
33
5
0 0 0 0 0 0 0 0 0
12 12 0 12 0 12 0 0 0
3 3 0 0 0 0 0 0 0
33 33 0 33 0 0 0 0 19
0 0 0 0 0 5 0 0 5
15 15 0 15 0 0 0 0 0 0 0 0 0
12 0 12 12 0 0 0 0 0 0 0 0 0
3 0 0 0 0 0 0 0 0 0 0 0 0
33 33 33 33 33 33 21 33 0 33 0 0 0
5 5 0 5 5 0 0 0 0 0 0 0 0
A- Candida humilis, B- Candida tropicalis, C-Pichia norvegensis D-Saccharomyces cerevisiae and E-Saccharomyces exiguous.
present findings are in line with Berhanu (1985), who isolated members of Enterobacteriaceae during the first 18 h of fermentation when pH of the dough was about 5.8. Enterobacteriaceae were then succeeded by L. mesenteriodes and S. faecalis because of the low pH. As the pH was further reduced to about 4.7 P. faecalis, L. brevis, L. plantarum and L. fermentum become the predominating flora and remain until the batter fermentation is stopped for steam baking (Berhanu, 1985).
yeast species, which have great potential to impart the particular characteristics in the final product of enjera. The results of this study also provide basic information on select promising starter cultures for the production of predictable quality of enjera with different sensory quality.
ACKNOWLEDGEMENT The authors wish to thank Mr. Wondwosen Tadesse for his guidance and assistance during the research work.
Conclusion REFERENCES In general, the spontaneously fermented teff enjera batter contained both homo and heterofermentative LAB and
Achi K (2005). The potential for upgrading traditional
Adv.Res.Biol.Sci.
42
Table 4. Distribution of identified LAB and yeast species during teff (Eragrostis tef) enjera batter fermentation.
Ferm. Time (h)
Isolate no.
0
1, 2, 3, 61, 62 ,63
6
4, 5, 6, 4,65,66
P. pentosaceus, Lb. fermentum, L.piscium
3, 4,5,35,36,37
12
7, 8,9, 10 ,67, 68 ,69
P.acidilactici ,Lb. fermentum, P. pentosaceus
6,7,38,39
18
11, 12, 70, 71, 72
Lb. fermentum, P. pentosaceus
8, 9,10,40,41
C. humilis, S. cerevisiae, C. tropicalis, P. norvegensis
24
13, 14, 15,73, 74,75, 76
Lb. fermentum, P.acidilactici P. pentosaceus
11,12,13,42, 43,44
P. norvegensis, S. cerevisiae, C. tropicalis
30
16, 17, 7,78,79
14,45,46
S. cerevisiae, C. humilis
36
18,19, 80, 81, 82
15, 16, 47, 48,49
C. humilis, S. cerevisiae
42
20, 21, 22, 83, 84,85, 86
Lb. fermentum, subsp. mesenteriodes, L. plantarum, P. pentosaceus
17,50,51
S. cerevisiae, C. humilis
48
23, 87,88
Lb. fermentum, P. pentosaceus
18,19,52
C. humilis, S. cerevisiae, P. norvegensis
Name of LAB species P. pentosaceus, E. cassiiflavus, Lb. fermentum
P. pentosaceus, Lb. fermentum Lb. fermentum, P. pentosaceus, L. plantarum, subsp. mesenteriodes
fermented foods through biotechnology. Afr. J. Biotechnol. 4 (5): 375-380. Agarry O, Nkama I, Akoma O (2010). Production of Kununzaki (A Nigerian fermented cereal beverage) using starter culture. Inter.Res. J.Microbiol. 1(2):18-25. Asmahan A, Muna M (2009). Use of Starter Cultures of Lactic Acid Bacteria and Yeasts in the Preparation of Kisra, a Sudanese Fermented Food. Pak. J. Nutr. 8 (9): 1349-1353. Barnett J, Payne R, Yarrow D (2000). Yeasts: Characteristics and Identification. 3rd ed. Cambridge University Press. Cambridge. Berhanu G (1985). Involvement of Lactic acid bacteria in the fermentation of tef (Eragrostis tef), an Ethiopian fermented food. J.Food Sci. 50: 800-801. Cooke R, Twiddy D, Reilly P (1987). Lactic acid fermentation as a low-cost means of food preservation in tropical countries. FEMS Microbiol. Rev. 46: 369-379. Corsetti M, Gobbetti M, Balestrieri F, Paoletti L, Russi, Rossi J (1998). Sourdough Lactic Acid Bacteria Effects
Isolate no.
Name of yeast species
1,2,33,34
C. humilis ,S. cerevisiae C. tropicalis, S. cerevisiae, C. humilis, S. exiguus S. exiguus, S. cerevisiae
on Bread Firmness and Staling. J. Food Sci. 63(2): 347351. Damelin L, Dykes G, Von Holy A (1995). Biodiversity of lactic acid bacteria from food-related ecosystems. J. Microbios. 83: 13-22. Damiani P, Gobbetti M, Cossignani L, Corsetti A, Simonetti S, Rossi J (1996). The Sourdough Microflora. Characterization of Hetero-and Homofermentative Lactic Acid Bacteria, Yeasts and Their Interactions on the Basis of the Volatile Compounds Produced. Technol. 29: 63-70. Dunga N, Romboutsb M, Nout J (2005). Development of defined mixed-culture fungal fermentation starter granulate for controlled production of rice wine. J. Innov. Food Sci. Emerging Technol. 6: 429 - 441. Glover R, Sawadogo H, Diawara B, Jespersen L, Jakobsen M (2009). Utilization of Lactobacillus fermentum and Saccharomyces cerevisiae as starter cultures in the production of ‘dolo’.
Desiye and Abegaz
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Table 4 contd. Distribution of identified LAB and yeast species during teff (Eragrostis tef) enjera batter fermentation.
C. humilis, C. tropicalis, S. cerevisiae
54
24,25,89,90
L.piscium, P.acidilactici, P. pentosaceus
60
26,27,91,92
P. pentosaceus, Sub sp. dextranicum
21,22,56
S. cerevisiae, C. humilis
72
31,32,33, 34, 35,36,96,97,98
Lb. fermentum, P. pentosaceus Sub sp. mesenteriodes, L.piscium
25,59,60
S. cerevisiae, C. tropicalis, S. exiguus
78
37,38,39,40,99
Lb. fermentum, P. pentosaceus
84
41,42, 43,44, 45,46, 47,48,49,50, 51, 100, 101
90
52, 53, 102, 103, 104
P. pentosaceus, Lb. fermentum, L.piscium
29,65
96
54, 55, 56, 57, 58 , 59, 60, 105, 106, 107
P. pentosaceus, Lb. fermentum
30,31,32,66, 67,68
P. pentosaceus, L.piscium, L. raffinolactic, Lac. plantarum
J. Appl. Biosci. 22: 1312 –1319. Gobbetti M (1998). The sourdough microflora: Interactions of lactic acid bacteria and yeasts. Trends Food Sci. Technol. 9: 267-274. Halm M, Lillie A, Sorensen A, Jakobsen M(1993). Microbiological and aromatic characteristics of fermented maize dough for kenkey production in Ghana. Inter.J. Food Microbiol.19: 135-143. Harrigan W (1998). Laboratory methods in food microbiology. 3rd ed. Acadamic press. Holt J, Krieg R, Senath A, Staley T, Williams S (1994). Bergey’ s manual of determinative bacteriology. Hensly RW and Forlifer E L (eds). 9th ed. Williams and Wilkins. Baltirnore, USA. pp. 787. Kabeir B, Shuhaimi M, Muhammad K, Suraini A, Yazid A (2004). A Nutritious Medida (Sudanese Cereal Thin Porridge) Prepared by Fermenting Malted Brown Rice Flour with Bifidobacterium Longum BB 536. Mal. J. Nutr. 10(2): 183-193. Kebede A, Fekadu B, Langsrud T, Narvhus J (2002). Parameters of processing and microbial changes during fermentation of borde, a traditional Ethiopian beverage. J. Food Technol. Africa. 7(3): 85-92. Kimaryo V, Massawe G, Olasupo N, Holzapfel W (2000). The use of a starter culture in the fermentation of
20, 53,54,55
26,61 27,28,62,63, 64
S. cerevisiae S. cerevisiae, C. humilis, C. tropicalis S. cerevisiae, C. humilis C. tropicalis, S. exiguus C. humilis, S. cerevisiae
cassava for the production of “Kivunde”, a traditional Tanzanian food product. Inter. J. Food Microbiol. 56: 179190. Nout R (2009). Rich nutrition from the poorest - Cereal fermentations in Africa and Asia. J. Food Microbiol. 26: 685-692. Sadeghi A, Shahidi F, Mortazavi S, Mahallati M, Koocheki A, Rezaie R (2009). Sourdough effect on reduction of Barbari bread staling. J. Sci. Technol. Agric. Natur. Resour. 13(47): 47-54. Smith M, Yarrow (1995). Yeasts. In: Samson, R., and Hoekstra, S. (eds). Introduction to food borne fungi. 4th ed. Printed by Ponsens and Looyen, Wageningen, Netherlands. Taiwo O (2009).Physical and nutritive properties of fermented cereal foods. Afr. J. Food Sci. 3(2): 023-027. Tamime A (2002). Microbiology of starter cultures. Dairy Microbiology Handbook (ed. Robinson RK), 3rd edn, pp. 261-366, John Wiley & Sons Inc., New York. Theodore I, Ikenebomeh J, Ahonkhai I (2007). Nutritional Status of Maize Fermented Meal by Fortification with Bambara-Nut. Afr. J. Food Agric. Nutr. Dev. 7(2): 56-63. Thiele C, Ganzle G, Vogel R (2002). Contribution of sourdough lactobacilli, yeast, and cereal enzymes to the generation of amino acids in dough relevant for bread
Adv.Res.Biol.Sci.
44
flavour. Cereal Chem. 79:45-51. Vuysta L, Vancanneyt M (2007). Biodiversity and identification of sourdough lactic acid bacteria. J. Food Microbiol. 24: 120-127. Yao A, Dortu C, Egounlety M, Pinto C, Vinodh A, Melanie H, Charles M, Franz A, Holzapfel W, Mbugua S, Mengu M, Thonart P (2009). Production of freeze-dried lactic acid bacteria starter culture for cassava fermentation into gari. Afr. J. Biotechnol. 8 (19): 4996-5004.
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