Prebiotic effect of different cereal bran (Sorghum, Barely and Millet) on growth of bifidobacterium longum BB536 during fermentation of goat milk

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This study was carried out to explore prebiotic effect of different cereal bran on Bifidobacterium longum BB536 for developing functional. All the bran (sorghum, barely and millet) were ground finely. Fermentation mediums were formulated from goat milk supplemented with 10% inulin and different cereal bran (sorghum, barely and millet). Probiotic strain B. longum BB 536 was used for fermentation.

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Nội dung Text: Prebiotic effect of different cereal bran (Sorghum, Barely and Millet) on growth of bifidobacterium longum BB536 during fermentation of goat milk

Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 625-634 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 10 (2020) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2020.910.074 Prebiotic Effect of Different Cereal Bran (Sorghum, Barely and Millet) on Growth of Bifidobacterium longum BB536 during Fermentation of Goat Milk Limia Hashim Mohamed, Barka Mohammed Kabeir*, Salma Elghali Mustafa and Salma Elzen Ibraheem *Department of Food Science and Technology, College of Agricultural Studies Sudan University of Science and Technology, Khartoum, Sudan *Corresponding author ABSTRACT This study was carried out to explore prebiotic effect of different cereal bran on Bifidobacterium longum BB536 for developing functional. All the bran (sorghum, barely and millet) were ground finely. Fermentation mediums were formulated from goat milk Keywords supplemented with 10% inulin and different cereal bran (sorghum, barely and millet). Probiotic strain B. longum BB 536 was used for fermentation. Different analyses including Cereal bran, proximate, mineral, strain BB536 viable count, physichochemical analysis were Prebiotic, conducted. Inulin was an excellent source of fiber, carbohydrate and minerals (Ca, K, Mg Bifidobacterium, and Na). Among cereal bran barley contained the highest level of fiber, followed by Goat milk, sorghum and then millet barn. Moreover, cereal bran are good source of protein, fat and Fermentation ash. During fermentation, the maximum growth of the strain BB 536 in all type of Article Info fermented goat milk supplemented with different fiber was attained at 12 h incubation. These high viable count of strain BB536 (7.53±0.16 b -8.43±0.03cLog CFU/ml) in all Accepted: fermented goat milk products exceed the minimum number (6 log CFU/ml products) 07 September 2020 required to presence in probiotic food. Further, rates of strain BB 536 increases induced by Available Online: different cereal bran are comparable to that of the commercial prebiotic inulin. Moreover, 10 October 2020 fermentation process was also accompanied with significant changes in physicochemical properties of fermented goat milk. Therefore, different types of cereal bran have prebiotic effect on strain BB 536 when supplemented to goat milk for development of functional food. Same time the fermented products can provide other essential nutrients such as protein, fat, minerals and fiber. Introduction normal foods and parts of the daily diet, but they contain a component that benefits some The growing interest in health and diet has particular physiological function and reduce recently produced the concept of functional the risk of diseases (Salovaaro, 1999). foods. By definition, functional foods are Nowadays, the wide applications of 625
Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 625-634 functional food are in form containing Dietary carbohydrates and soluble fiber that probiotics and non-digestible carbohydrate are able to stimulate, specifically the growth known as prebiotics (Fuller and Gibson, of potentially beneficial bacteria, e.g., 1997). bifidobacteria at the expense of the more harmful pathogenic microorganisms, are Reorganization of probiotic effect dates back called prebiotics. Presence of prebiotics in the to the 19th century when the French scientist colon helps to modify the microflora in such a Louis Pasteur (1822 – 1895) postulated the way that the health-promoting bacteria like importance of microorganisms in human life, bifidobacteria and lactobacilli become this was further reinforced by work done by predominant in numbers and may be 1908 Nobel Prize-winner Elie Metchnikoff, accompanied by elimination of pathogenic which led to the concept of probiotics. Strain bacteria (Kouane et al., 2005). of Bifidobacterium, lactobacillus and nonpathogenic yeast such as Saccharomyces Dietary fibers are often characterized by high boulardii are principally used individually or nutritional quality, as they are able to cure in combination as probiotics (Tomasik and many chronic diseases and improve texture, Tomasik, 2003). Bifidobacteria is the sensory characteristics, and shelf life of foods. predominant species of human colonic and The fast growing food industry will likely faces microbiota. It has been extensively generate an ever-growing amount of introduced in the food industry and byproducts including bran, husk, peel, pharmaceutical applications (Guarner and pomace, and other products that are rich in Malagelada, 2003). dietary fibers (Betoret et al., 2011). Therefore, finding optimal use of dietary Bifidobacterium longum is one of the fibers becomes increasingly imperative. In bifidobacteria species found mainly in human this respect, the exploration of prebiotic effect faeces and it may be considered as the most for different Sudanese cereal bran is lacking. common species of bifidobacteria, being Therefore, the objective of this study is to found both in infant and adult. Potential evaluate the growth of B. longum BB536 in benefits from consumption of B. longum goat milk supplemented with cereal bran and include: antagonistic action toward intestinal evaluate its related physiochemical changes pathogens, improved lactose utilization, during fermentation process. anticarcinogenic action and control of serum cholesterol levels. Scientific studies showed Materials and Methods the benefits offered by Bifidobacterium longum BB536 (Kojima et al., 1996; Namba Raw materials et al., 2003). Thus there is considerable interest in incorporating these healths Inulin was obtained from A natural Product promoting bifidobacteria into food. However, Company (London, England). Different cereal most human origin probiotics are fastidious bran (sorghum, barley and millet) were when used alone, they are characterized by purchased from a local crops market at central low growth capability in food mediums market in Bahri (Khartoum state, Sudan). including the dairy, the main a recommended Goat fresh milk was obtained from the animal carrier of probiotics to human (FAW/WHO, farm at Department of Animal Production, 2001). Thus, scarcity of animal milk in many College of Agricultural Studies, Sudan countries makes it difficult to provide a University of Science and Technology adequate bifidobacteria intake. (Khartoum, Sudan). 626
Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 625-634 Preparation of cereal bran unaerobically at 37 oC for 48 h. The growth was calculated as Colony Forming Unit per Different cereal bran were ground and sieved ml (CFU/ml). using appropriate mesh. The resulting powder stored in a dark polyethylene bag at freezer Determination of titrable acidity until used. The titrable acidity (TA) of the different Preparation of fermentation inoculums fermented products was determined according to AOAC method (2006). Ten ml of sample B. longum BB536 was obtained from the were weighted into a conical flask. A distilled stock culture of microbiology laboratory water was added until the volume in the flask (Department of Food Science Technology, was 150 ml. The sample was then vigorously Collage of Agriculture Studies (SUST). The agitated and filtered. Twenty five milliliters of strain was maintained at -20 oC in 20% the filtrate were pipetted into porcelain dish, glycerol solution. Stock culture was prepared five drops of phenolphthalein added, and the by activation of the strain in skim milk, sample was titrated against 0.1N NaOH till a incubation an aerobically at 37 oC for 24h. fain pink colour that lasted for at least 30 The obtained culture was reactivated again seconds was obtained; then acidity of under the same conditions to prepare enough different products was calculated. stock for the experiment. The working culture was prepared by twice successive Determination of total soluble solids (TSS) transformations of stock culture in 10% sterilized skim milk (121oC for 15 min) and Total soluble solids (TSS) of the fermented incubation at 37 oC for 24h. products were determined at room Growth medium and fermentation temperature using digital refractometer with conditions degree Brixº scale 0-100 according to AOAC (1990) method. Growth medium were formulated from goat milk supplemented with 1% inulin or Determination of pH value different cereal bran (sorghum, barely and millet). Formulated medium were sterilized The pH value of the different fermented (121˚C for 15 min) and inoculated with a 3% products was determined using a pH-meter active culture working of B. longum BB536 (model HI 8521 microprocessor bench followed by incubation at 37 oC for 24h. PH/MV/C meter, Romania). Two standard buffer solution of pH 4.00 and 7.00 were used Enumeration of viable B. longum BB536 for calibration of the pH meter at room cell temperature. The pH meter was allowed to stabilize for one minute and then the pH of MRS medium was used to enumerate B. the fermented products samples was directly longum BB536 of different fermented measured. products using the plate count technique. Fermented samples were drawn at initial and Chemical composition every 6h intervals during fermentation.1ml of fermentation broth was diluted in peptone Determination of moisture content water, followed by plating on Demann Rogosa agar (MRS) supplement with 0.05% Moisture was determined according to the L-cystiene. The plates were incubated modified method of AOAC (1990). Five 627
Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 625-634 grams of the sample was weight using in Determination of crude fiber sensitive balance, after weighting the empty dishes and then transferred to an oven (Kat- Two gram of a defatted sample was placed NR.2851, Electrohelios, Sweden) at 105 ± into a conical flask containing 200ml of 0.1˚C for 6 hours .Afterwards, the dish with H2SO4 (0.26N). The flask was fitted to a sample was transferred to dessicator and condenser and allowed to boil for 30 minutes. allow to cool to room temperature before At the end of the digestion period, the flask reweighting to calculated moisture. was removed and the digestate was filtered through a proclaim filter crucible (No.3). Determination of fat content After that, the precipitate was repeatedly rinsed with distilled boiled water followed by Fat content was determined according to the boiling in 200ml NaOH (0.23) solution for 30 official method of AOAC (1990). A sample min under reflux condenser and the of 5g was weighed in extraction thimble and precipitate was filtered . Rinsed with hot covered with cotton, and then extracted with distilled water, 20ml ethyl alcohol (96%) and hexane. The thimble containing the sample 20ml diethyl ether . Finally, the crucible was and a pre-dried weight sample and flask dried at 105 ̊C until a constant weight was containing about 100 ml hexane was attached obtained and the difference in weight was to the extraction unit. The extraction process considered a crude fiber. was conducted for 16h. At the end of the extraction period, the flask was disconnected Calculation of carbohydrates from the unit and the solvent was evaporated. Later, the flask with the remaining crude Carbohydrates were calculated by difference hexane extracted was put in an oven, cooled according to the following: to room temperature reweight and the dried extract was registered as fat content. Total carbohydrates = 100% - [Moisture (%) + Protein (%) +Fat (%) + fiber (%) and Ash Determination of protein content (%)]. Protein content of different fermented Determination of minerals products was determined by Kjeldhal method according to the AOAC (1990) method. Potassium (K) and calcium (Ca) were determined by flame photometer (Sherwood Ash content Flame Photometer i410, Sherwood Scientific Ltd. Cambridge, UK) according to procedure The ash content of the sample was determined of AOAC (1990). The knob of flame according to the AOAC (1990) method. Two photometer was adjusted to potassium and grams of the deferent fermented products calcium respectively and reading was set to were weighed into a clean dry porcelain zero using deionized water. Blank solution crucible and placed in muffle furnace (model was run and read again the set zero. Standard Tipoforon Z A No 18203 Get Ran 1002. solution of each mineral was run and recorded England) at 600˚C for 6hours. The Crucible the reading of flame photometer. The reading was transferred to a desiccator, cooled to of potassium and calcium in products sample room temperature and weighed to calculate was obtained by running the sample one by ash content. one. Standard solution was run after each product sample. The standard curves were 628
Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 625-634 obtained by plotting absorbance values of the P. Inulin contained the lowest P as standards against appropriate concentrations compared to its level in cereal bran. All cereal of elements. One gram of dried product bran contained small amount of Ca, K, Mg samples was subjected to wet digestion and Na (Table 2). method as described by Richards (1968). Then analysis was carried out Chemical composition of Bifidobacterium spectrophotometer absorption (Varian AA longum BB536 fermented goat milk 240, Victoria, Australia) to determine Mg and supplemented with 1% of different types of Na via a standard curve. To determine fiber* phosphorus content of products, colorimetric estimation method was used as described by There was no significant (p
Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 625-634 presence in probiotic food (Viderola and decreases in pH levels of all types of Reinheimer, 2000). formulated goat milk by extended fermentation period to 24h (Table 5). The The rate of strain BB536 increases in different decreases in pH are due to increased acids fermented goat milk were 2.57, 2.31, 1.93 and production during fermentation process as a 1.18 log CFU/ml in fermented products result of fermented sugar by strain BB536; supplemented with sorghum bran, inulin, which produces acetic and lactic acid (De millet bran and barely bran, respectively. Vries et al., 1967). Moreover, the These rates of increases induced by different accumulated acids produced by cereal bran are comparable to the Bifidobacterium strain, reported to have prebitication (support growth of strain BB antibacterial activity such as prevention of the 536) with the commercial prebiotic. proliferation of pathogens (Bullen et al., Therefore, tested cereal bran might have 1976). The rate of pH decreases at maximum prebiotic effect on strain BB536. On the hand, strain BB536 growth (12h incubation) were the variations in growth rate of strain BB536 1.82, 1.07, 0.67 and 0.49 pH in fermented could be attributed to variances in availability goat milk supplemented with barely bran, of nutrients required for growth in the inulin, millet bran and sorghum bran, different formulated products. In fact, goat respectively (Table 5). Level of acidity milk contains almost the essential nutrient for increased by extended fermentation period strain growth. Together with different fiber and thus caused reduction in pH. combination could complement the nutrient component demand for strain BB 536 growth TSS changes during strain BB536 in formulated goat milk medium. fermentation of goat milk supplemented with inulin and different cereal bran However, after maximum growth (12 h) the strain started to decline in all types of Table 6 showed the changes in total soluble fermented goat milk products (Table 4). The solid (TSS) during fermentation of different decline of the strain might be due to the formulated goat milk with strain BB 536. accumulation of acids or reduction of There were significant (P
Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 625-634 Table.1 Proximate composition of inulin and different cereal bran (sorghum, barley and millet)* Components (%) Types of fiber Inulin Sorghum bran Barley bran Millet bran Moisture 3.000 ± 0.00 68.48± 0.332 50.15 ±0.156 68.71 ± 0.290 Fat ND 3.125 ± 1.039 4.935 ±0.679 5.935 ± 0.0212 Proteins ND 12.60 ± 2.97 13.915±1.110 11.375 ± 1.237 Fiber 89.00 ± 0.00 6.490 ± 0.297 19.685±0.870 4.635 ± 0.346 Ash ND 2.425 ± 0.0212 8.660±0.0141 3.190 ± 0.283 Carbohydrates 8.000± 0.000 6.870 ± 0.354 2.640±0.156 6.140 ± 0.0849 *Values are mean ± SD for replicate independent analysis ND= Not determined Table.2 Mineral content of inulin and different cereal bran (sorghum, barley and millet)* Components Types of fiber (mg/100g) Inulin Sorghum bran Barley bran Millet bran Ca 1.62±0.03a 0.08±0.01 b 0.08±0.00b 0.08±0.00b K 0.76±0.01a 0.11±0.01b 0.10±0.00b 0.09±0.01b Mg 0.82±0.02a 0.03±0.00c 0.02±0.00c 0.05±0.21b Na 1.74±0.00a 0.11±0.13 b 0.02±0.06c 0.02±0.00c P 0.26±0.00b 1.16±0.01a 1.01±0.02a 1.06±0. 35a *Values are mean ± SD for replicate independent runs. **Values that bear different superscript letter in the same raw of are significantly different at p
Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 625-634 Table.4 The viable count of Bifidobacterium longum BB536 (log CFU/ml) during fermentation of goat milk supplemented with different types of fiber* Fermented goat milk supplemented with different types of fiber Fermented inulin Sorghum bran Barley bran Millet bran time(h) 0 5.52±0.32c 5.86 ±0.06d 6.07±0.03c 6.03±0.11d 6 7.33±0.01b 6.07±0.09c 6.99±0.01b 6.31±0.42c 12 7.53±0.16a 8.43±0.03a 7.65±0.06a 7.96±0.02a 18 7.39±0.01b 7.25±0.06b 7.84±0.08a 7.25±0.22b 24 7.30±0.06b 6.93±0.17ab 6.79±0.06b 6.83±0.15bc * Values are mean ± SD for replicate independent runs. ** Values that bear different superscript letter in the same column are significantly different at p
Int.J.Curr.Microbiol.App.Sci (2020) 9(10): 625-634 Table.7 Titrable acidity (%) during strain BB536 fermentation of goat milk supplemented with inulin and different cereal bran Fermented Fermented goat milk supplemented with different types of fiber time (h) Inulin Sorghum bran Barley bran Millet bran 0 0.19± 0.00d 0.24±0.07d 0.26±0.00e 0.44± 0.00c 6 0.22±0.01d 0.20±0.00e 0.75± 0.01d 0.95± 0.01b c c c 12 0.65±0.07 0.44±0.01 0.82± 0.00 0.93± 0.01b 18 0.76± 0.04b 0.62±0.01b 0.85±0.01b 0.94± 0.02b 24 0.81±0.00a 0.73±0.00a 0.92± 0.07a 1.02± 0.01a * Values are mean ± SD for replicate independent runs. ** Values that bear different superscript letter in the same column are significantly different at p
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