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Item CHARACTERIZATION OF β-GALACTOSIDASE BY LACTIC ACID BACTERIA FROM MILK AND TRADITIONALLY FERMENTED MILK PRODUCTS FROM IBADAN(2014-01) PARKHA, O. A.Lactose intolerance (a condition in which man elucidates an immune reaction towards the presence of lactose due to inability to produce enzyme lactase) is a major nutritional deficiency among some adult consumers of milk and other dairy products worldwide. β–galactosidase hydrolysis of milk is one of the promising enzymatic applications in dairy industries for reducing lactose intolerance of milk products. However, plant and animal sources cannot meet the high demand of the enzyme in food industries. Hence, the aim of this study was to characterize β–galactosidase production by Lactic Acid Bacteria (LAB) isolated from locally fermented milk products. Raw milk from Sokoto Gudali was collected from Fulani settlement in Ojoo, Ibadan along with some fermented milk products (‘’Nono’’ and ‘’Wara’’). LABs were isolated from them and identified using conventional methods. The ability of the isolates to hydrolyze 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) was used to screen for β–galactosidase production. Isolates with the best β-galactosidase production were selected. The enzyme was extracted and optimization of growth conditions (temperature, pH, nitrogen, carbon sources, inoculums size and inoculums age) for β-galactosidase production was carried out using o-nitrophenyl-β-D-galactopyranoside (ONPG). The enzyme produced was characterized using pH, temperature, metal and non-metal ions, and inhibitors. Purification of the enzyme was carried out using dialysis and chromatographic methods. Hydrolytic effects of the purified β–galactosidase were determined in different concentrations of lactose using standard method. Data were analyzed using descriptive statistics. The isolated bacteria were identified as Lactobacillus plantarum (G11, E13 and E36), L. brevis, L. casei, L. lactis, Leuconostoc lactis, Streptococcus sp, and Bacillus subtilis. Lactobacillus plantarum (G11) had optimum growth value of 4.2 at 20°C and pH 7.0 with maximum enzyme value of 6.2U/mL at 30 hrs. The optimal β-galactosidase production occurred at neutral pH and 6% inoculum size. The best inoculums age varied between 18 hrs and 36 hrs. The best carbon source for enzyme production was raffinose with maximum value of 0.3U/mL while minimum activity was found in fructose with 0.2U/mL. The best nitrogen source was NH4NO3 with maximum value of 0.5U/mL and yeast extract had minimum value of 0.1U/mL. β–galactosidase activity increased with increase in molar concentration of the mono-valent chloride ions in which the highest was recorded in KCl at maximum value of 0.08U/mL while the minimum value of 0.001U/mL was obtained by NaCl at a concentration of 0.2 mmol respectively. The best sulfate ion was CuSO4 with maximum activity value of 0.2 U/mL at 0.2 mmol concentration and minimum value of 0.007 U/mL at 0.1 mmol by ZnSO4. The best enzyme inhibitor was KCN with maximum activity of 0.2U/mL at 0.2 mmol. The specific activity of β-galactosidase was 292.5 U/mg, 104.2 U/mg and 585.46 U/mg for G11, E13 and E36 respectively. The hydrolytic effects of the purified β-galactosidase showed a maximum yield of 35.8% glucose, 19.3% galactose and 35.3% glucose and 18.5% galactose at 80% and 60% lactose concentration respectively. β-galactosidase produced by Lactobacillus plantarum strain achieved lactose hydrolysis and could be of potential application for production of low lactose dairy products for consumption by lactose intolerant people.Item SELECTION, PRODUCT CHARACTERISTICS AND METABOLITE SPECTRUM OF A COMMON STARTER CULTURE FOR FUFU AND USI PRODUCTION(2015-07) OYINLOLA, K. A.Fermented cassava products like fufu and usi (edible starch) are important staple foods in many African homes. Natural fermentation time is usually long resulting in slower acidification process and inconsistent nutritional composition of products which could be overcome with the use of starter culture. However, most available starter cultures are used for single food fermentation and are uneconomical. This necessitates the development of a starter culture for multiple related food products to reduce cost. Hence this study was designed to produce a common starter culture for the production of fufu and usi. Cassava varieties TME 30572, TME 4(2)1425 and TME 50395 were obtained from the International Institute of Tropical Agriculture, Ibadan and landraces from Bodija market. Fresh, peeled, chipped and grated cassava tubers were spontaneously fermented in the laboratory. Lactic Acid Bacteria (LAB) were isolated from the fermenting mash and identified phenotypically. Genotypically identified starters were selected based on screening for starch hydrolysis, linamarase and pectinase enzyme production, antimicrobial compound production and rate of acidification using standard methods. The starters were utilised singly and randomly combined to initiate fermentation for production of fufu and usi. Un-inoculated fermentation mash served as control. Rate of production of organic acids, various sugars, metabolic enzyme assays, nutritional and anti-nutritional content of the resulting mashes were monitored using standard procedures. Best starter was applied in the final production of fufu and usi. Shelf-life of the products were evaluated and compared with the control. Data were subjected to descriptive statistics and ANOVA technique at p=0.05. Ninety-eight LABs were identified as Lactobacillus plantarum (50.0%), L. acidilactici (12.2%), L. brevis (11.3%), L. fermentum (10.3%), L. delbrueckii (8.2%), L. mesenteroides (6.0%), and L. lactis (2.0%). Screened isolates did not hydrolyse starch but produced pectinase, linamarase alongside hydrogen peroxide, diacetyl and lactate with a rapid decrease in medium pH (6.5 - 3.6). Selected potential starters were genotypically identified as L. pentosus F2A (A), L. plantarum subsp. argentolarensis F2B (B), L. plantarum F2C (C), L. plantarum U2A (G) and L. paraplantarum U2C (I). The best starter combination CGI gave significant reduction in fermentation pH (7.1 - 3.7) and lactic acid ranged between 0.04mg/ mL and 6.9mg/mL. Sugars produced include xylose (3.2µg/mL), arabinose (1.4µg/mL), fructose (26.2µg/mL), glucose (30.3µg/mL) and sucrose (99.7g/mL). Enzyme assay revealed peak amylase (10.1U/mL) and pectinase (4.4U/mL) activities at 24 hours as well as linamarase (0.8U/mL) at 48 hours in fufu, whereas, in usi, highest linamarase (0.7U/mL) and pectinase (1.0U/mL) activities were recorded at 72hours with no amylase activity. The CGI-produced fufu and usi had significant reduction in phytate (0.3-0.1mg/g and 0.3-0.27mg/g), tannin (35.4-34.0mg/g and 35.4-32.3mg/g), cyanide (0.1-0.05mg/g and 0.1-0.0mg/g), and moisture (7.3%-5.1% and 7.3%-5.4%) content while total protein content increased (1.0-1.3% and 1.0-1.8%) respectively. Starter fermented fufu and usi had shelf-life of five days while control had three days. The selected starter was able to ferment both fufu and usi to yield products with improved nutritional content, better shelf-life and reduced anti-nutritional composition. This could be employed in the production of indigenous fermented foods.Item NUTRITIONAL AND BIOCHEMICAL CHARACTERISTICS OF LACTIC ACID BACTERIA-CHALLENGED SORGHUM AND ITS FERMENTATION PRODUCT(2012-06) OJO, F. TSorghum is one of the major cereals malted for the brewing of beer. The conditions of transport and storage of this cereal predispose it to contamination by microbes thus affecting the quality of the end products. The contamination is usually controlled by treating the sorghum with chemicals, which however alter the chemical constituents of the sorghum. Therefore, the need to seek alternative functional methods of control of microbial contaminants becomes imperative. This study was aimed at investigating lactic acid bacteria as biocontrol agents against microbial pathogens of stored cereals for beer fermentation. Sorghum were obtained from Bodija market and from the Institute of Agricultural Research and Training, Ibadan. Lactic Acid Bacteria (LAB) were isolated from spontaneously-fermenting sorghum and identified using classical techniques. The abilities of the LAB strains to produce antimicrobials and their antagonistic activity against known cereal pathogens were used to select the best three strains for further work. The selected strains were applied singly and in combination at inoculum concentration of 2.3 x 104 cells/mL for five days to challenge sorghum seeds prior to malting and wort production. Sorghum wort was fermented for five days with Saccharomyces carlsbergensis. Physiological and nutritional characteristics of the unchallenged and challenged sorghum, and fermentative characteristics of the wort were determined using the European Brewery Convention methods. Data were analysed using ANOVA. One hundred and twenty seven strains of LAB were isolated and identified as Lactobacillus plantarum (32), Lactobacillus brevis (31), Lactobacillus fermentum (25), Lactobacillus delbrueckii (8), Lactobacillus casei (12) and Lactobacillus acidophilus (19). Lactobacillus plantarum, Lactobacillus fermentum and Lactobacillus casei produced high antimicrobial lactic acid (2.5±0.5g/L, 2.4±0.3g/L and 2.5±0.5g/L respectively) and had high inhibitory activities (17mm, 14mm and 17mm respectively). Lactobacillus brevis produced antimicrobial lactic acid with the highest mean concentration of 2.7±0.5g/L from local sorghum but was not used for further work because the inhibitory activity was low when tested against pathogenic organisms. All the LAB produced bacteriocin with antagonistic effects on all the pathogens tested, and Lactobacillus plantarum had the highest zone of inhibition (17mm) against Bacillus subtilis. All the LAB grew at temperature of 30oC, pH 5.0-5.5, high glucose and peptone concentration (1.5-2.0mg/ml). The malted untreated -sorghum had 13.2 % protein, 3.0 % crude fat, 1.9 % ash, 1.8 % crude fibre, 42.5 mg/g phytate, 36.0 mg/g tannin, 2.0 mg/g protein inhibitor and 16.0 IoBunits diastatic power. Fermentation of the unchallenged wort (pH 6.2) yielded ethanol content of 2.2 %. With LAB treatment, there was a reduction in protein (12.2 %), crude fat (2.1 %) and crude fibre (1.1%); and significant (p<0.05) reduction in antinutrients (phytate 32.7 mg/g, tannin 22.4 mg/g, protease inhibitor 0.0 mg/g and wort pH 4.2). There was also an increase in diastatic power (24.0 IoBunits). The subsequent fermentation produced 4.8 % ethanol. The microbial profiles of the challenged malted sorghum showed a steady decrease in Bacillus, Staphylococcus and Pseudomonas count compared with the unchallenged where they showed steady increase. Lactobacillus starter cultures reduced spoilage pathogens, antinutritional factors of sorghum during malting and improved the end products.Item PHYSIOLOGICAL, GENOMIC AND PRESERVATIVE PROPERTIES OF PEDIOCOCCUS ISOLATES FROM MEATS UNDER LOW TEMPERATURE STORAGE(2012-06) DUYILEMI, O. P.The need for meat preservation cannot be overemphasised. However, existing methods of meat preservation including the use of artificial preservatives have toxic side effects. There is a dearth of information on the use of Lactic Acid Bacteria (LAB), which is known as good preservatives for food, in the preservation of meat. Hence, the aim of this study was to examine the use of Pediococcus acidilactici and low temperature in improving the quality of beef, chicken and turkey meat samples. Samples of beef, chicken and turkey were obtained from open market retailers and stored for 28 days at 4oC, 2oC, -4oC and -15oC. From these, LAB were isolated and identified using conventional methods. Quantities of lactic acid and acetic acid were determined using high performance liquid chromatography, while diacetyl and hydrogen peroxide were determined by enzymatic methods. High production of lactic acid was used as a criterion for selecting five isolates of Pediococcus acidilactici and the 16S rDNA genes were amplified and sequenced. The isolates were checked for plasmid presence and tested for bacteriocin production using gel electrophoresis and agar well assay. Antimicrobials produced by the isolates were tested in vitro against known meat spoilage organisms: Staphylococcus aureus, Escherichia coli, Pseudomonas faecalis, Listeria monocytogenes, Salmonella typhimurium and Bacillus cereus. The isolates and their filtrates were applied to fresh meat at -4oC and -15oC. Microbial load, proximate and biochemical parameters of the meat samples were monitored at seven days interval for 28 days. Data were analysed using ANOVA at p = 0.05. One hundred and ten LAB isolates from beef [Lactobacillus (24), Pediococcus (6), Leuconostoc (9)], chicken [Lactobacillus (27), Pediococcus (4), Leuconostoc (2)] and turkey [Lactobacillus (31), Pediococcus (5), Leuconostoc 2] were identified. Fast freezing (-15oC) and freezing (-4oC) gave significantly lower LAB count (4.1 ± 0.03 - 5.1 ± 0.02 logcfu/mL) than those of chilling (2oC) and refrigeration (4oC) (4.2 ± 0.04 - 5.4 ± 0.02 logcfu/mL) in all the meat samples. Lactic acid and acetic acid production peaked at 30.7 g/L and 32.0 mg/mL respectively while diacetyl and hydrogen peroxide production peaked at 40.8 ng/L and 16.0 µg/L respectively. Sizes of the 16S rDNA from the five strains of P. acidilactici ranged from 145 bp to 161 bp. Band size of plasmid DNA ranged from 861 - 20643 bp. Bacteriocin inhibition zones ranged from 1.0 to 6.5 mm. The highest zone of inhibition of antimicrobial action was 12 mm against P. faecalis. Proximate and biochemical analyses gave lower values compared with control samples: pH (4.8 / 5.7), thiobarbituric acid (0.2 / 0.5 mg malonaldehyde/kg), free fatty acid (0. 2 / 0.5 KOH/g lipid), total volatile nitrogen (0.6 / 1.4 mgN/100) and crude fat (3.4 / 4.5 %); but increased crude protein (22.1 / 17.7 %). Lowest microbial load (total bacteria count 2.2 logcfu/ml, coliform count 1.4 logcfu/ml, fungal count 2.0 logcfu/ml) and highest LAB count (4.9 logcfu/ml) were observed on the 28th day. Pediococcus acidilactici with optimum physiological characteristics prolonged the keeping quality of meat under low temperature storage.Item RAFFINOSE METABOLISM AND UTILISATION BY L. PLANTARUM ISOLATED FROM INDIGENOUSLY FERMENTED CEREAL GRUELS FOR NUTRITIONAL IMPROVEMENT(2012-08) ADEYEMO, S. M.Most African foods used in weaning are usually cereal-based gruels fermented by Lactic Acid Bacteria (LAB). The food mainly supplies carbohydrate; excessive intake of which might cause malnutrition in growing children. Minimum dietary requirements of a child could be met through fortification with protein-rich supplements. Soybean is rich in dietary protein but contains some antinutritional factors and raffinose, an oligosaccharide responsible for gas formation, bloating and flatulence in weaning children. The use of microorganisms for hydrolysing raffinose has not been fully exploited in Nigeria. In this study, the use of LAB to hydrolyse raffinose, reduce antinutritional factors and improve nutritional composition of such food blends were investigated. Commercially- hawked “Ogi” (CO) samples, Local Varieties (LV) of sorghum and maize were obtained from Bodija market, Ibadan and Typed Varieties (TV)-Samsorg 40, Samsorg 41 and Ex-Kano from the Institute of Agricultural Research and Training, Ibadan. The LAB were isolated from spontaneously-fermenting cereal gruels and identified using standard methods. Nine strains of Lactobacillus plantarum were selected based on the abundant production of -galactosidase, and characterized by PCR amplification of 16SrDNA genes. Plasmid presence was determined using agarose gel electrophoresis and the effect of plasmid curing was monitored. The growth of the organisms and metabolites production in different carbon sources were monitored at 200C to 800C and pH of 3.0 to 9.6. Soyabean was pre-treated by milling, cooking and roasting while the relationship of the isolates to raffinose metabolism during fermentation was monitored daily for 5 days. Reducing sugar, residual oligosaccharides, nutritional, antinutritional factors and alpha-galactosidase were determined using UV-spectrophotometer following Association of Official Analytical Chemist procedures. Data were analysed using ANOVA at p=0.05. One hundred and twenty LAB isolates were obtained and identified as L. plantarum (35.8 %), L. fermentum (12.5 %), L. pentosus (7.5 %), L. acidophilus (15.8 %), L. casei (5.8 %), L. brevis (6.7 %), L. cellobiosus (6.7 %), L. jensenii (5.0 %) and L. reuterii (4.2 %). Analyses of the nine L. plantarum isolates revealed high sequence identities (97.0 %). These isolates exhibited significant differences in utilization of raffinose at varying concentrations of 0.2 -1.0 mg/mL, while isolates obtained from LV performed better than those from TV and CO. Fermentation reduced the oligosaccharide content of the soyabean by 74.6 % while the reducing sugars increased by 65.0 %. Fortification of the gruel with soyabeans using uncured L. plantarum strains improved the nutritional quality (protein: 8.4 to 17.8 %, fat: 3.6 to 12.9 %, ash: 2.0 to 3.8 %, Fe: 6.4 to 10.7 mg/100g and Ca: 156.7 to 211.0 mg/100g), and a significant reduction in antinutritional factors (Tannin: 1.9 to 0.1 mg/g, Phytate: 1.2 to 0.1 mg/g and Trypsin Inhibitor : 1.2 to 0.0 mg/g) was observed after fermentation . Oligosaccharide content, reducing sugar, nutritional and antinutritional composition and organoleptic attributes of the end product were significantly affected by plasmid curing. Utilisation of raffinose by Lactobacillus plantarum from local food sources reduced antinutritional factors and oligosaccharides in soybeans. Nutritional quality of cereal gruels were improved by inclusion of Lactobacillus plantarum.