122 research outputs found

    Inhibitory Activities of a Probiotic Bacterium (Bifidobacterium Pseudocatanulatum) on a Common Diarrheagrnic Pathogen (Salmonella Enterica) in Human

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    Sixteen strains of Salmonella were isolated from clinically diagnosed diarrhea patients. They were tested against a range of antimicrobial agents, and typed by serological test and RAPD fingerprinting. All the strains have the similar pattern of antimicrobial susceptibility. The serological test has typed them into 3 serovars but the RAPD fingerprinting has classed them into 2 major clusters. Three strains of bifidobacteria were analyzed for their survival rate in human stomach condition. It showed that the ability of bifidobacteria to survive was strains dependant. Bifidobacterium pseudocatanulatum F117 and Bifidobacterium infantis can survive at pH value of human stomach after exposure for 90 minutes but not Bifidobacterium pseudocatanulatum G48. The survival of bifidobacteria was higher in the pH after meal compared to the pH before meal (fasted state). The dose effect study demonstrated, that the initial concentration of bifidobacteria would affect the duration of inhibitory activity against Salmonella. Lower initial concentration exhibit greater inhibitory activity. The inhibition of Salmonella was due to the production of acetate and lactate by bifidobacteria and the effectiveness was higher at low pH. Acetate and lactate production was excessive when the initial concentration of bifidobacteria was low due to the high growth rates, metabolism, and competition of energy sources

    Otimização das condições de cultivo para a produção de enzimas degradadoras de fitato por Enterobacter sakazakii ASUIA279 isoladas da raiz de milho da Malásia

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    The production of extracellular phytase by Enterobacter sakazakii ASUIA279 was optimized using response surface methodology with full-factorial faced centred central composite design. Two sets of experiments were carried out to optimize the five most profound factors of the cultivation conditions in order to maximize phytase production. Incubation temperature, initial pH of the media and percentage of rice bran supplemented into the media were optimized in Erlenmeyer flasks while agitation and aeration effect were controlled in a bioreactor. This design reduced the number of actual experiment performed to optimize phytase production and allowed the study of possible interactions among the factors. In the first set of experiments linear and quadratic effect of initial pH was determined to be the most significant factor affecting phytase production. In the bioreactor both linear effects of agitation and aeration, were identified to be highly significant (> 99 %) in respect to phytase yields. Optimal phytase production was observed at a incubation temperature of 39.7 ºC, an initial pH of 7.1, supplementation with 13.6 % rice bran , 320 rpm of agitation and 0 vvm of aeration.  A produção de fitase extracelular por Enterobacter sakazakii ASUIA279 foi otimizada usando a metodologia de superfície de resposta com projeto de compósito central centralizado em face de fatorial completo. Dois conjuntos de experimentos foram realizados para otimizar os cinco fatores mais profundos das condições de cultivo, a fim de maximizar a produção de fitase. A temperatura de incubação, o pH inicial do meio e a porcentagem de farelo de arroz suplementado no meio foram otimizados em frascos de Erlenmeyer, enquanto o efeito de agitação e aeração foi controlado em um biorreator. Este projeto reduziu o número de experimentos reais realizados para otimizar a produção de fitase e permitiu o estudo de possíveis interações entre os fatores. No primeiro conjunto de experiências, o efeito linear e quadrático do pH inicial foi determinado como o fator mais significativo que afeta a produção de fitase. No biorreator, ambos os efeitos lineares da agitação e aeração foram identificados como altamente significativos (> 99%) em relação aos rendimentos da fitase. A produção ótima de fitase foi observada na temperatura de incubação de 39,7 ºC, pH inicial de 7,1, suplementação com 13,6% de farelo de arroz, 320 rpm de agitação e 0 vvm de aeração

    Phytase: application in food industry

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    Phytates have been considered as a threat in human diet due to its antinutrients behaviour which known as strong chelators of divalent minerals such as Ca²+, Mg²+, Zn²+ and Fe²+. Phytic acid has a potential for binding positively charged proteins, amino acids, and/or multivalent cations or minerals in foods. The resulting complexes are insoluble, difficult for humans to hydrolyze during digestion, and thus, typically are nutritionally less available for absorption. The reduction of this phytates can be achieved through both enzymatic and non-enzymatic removal. Enzymatic degradation includes addition of either isolated form of wild-type or recombinant exogenous phytate-degrading enzymes microorganisms in the food matrix. Non-enzymatic hydrolysis of phytate occurred in the final food during food processing or physical separation of phytate-rich parts of the plants seed. The application of phytase with respect to breadmaking process, probiotics, animal feed supplement and transgenic crops are emphasised in this paper

    Valorisation of virgin coconut oil application in mayonnaise production as functional Ingredient

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    Mayonnaise is favourable by large number of consumers due to the pleasant taste that can enhance the appetite. However, several commercial mayonnaises are produced by using high concentrations of oil that may cause high threats of developing several diseases due to the link to health issues. On the other hand, oils known for health promoting properties such as virgin coconut oil are underutilized even though having high potential as plant-based alternative healthy oil that can be used in mayonnaise production. In this study, virgin coconut oil was used to partially and/or fully replace soybean oil that is commonly used in mayonnaise production. Three mayonnaise samples were prepared by using three oil combinations including 100% virgin coconut oil, 50%:50% soybean oil:virgin coconut oil and 100% soybean oil. The antioxidant activity, physiochemical properties, lipid profile and sensory evaluation of the mayonnaise samples were determined by using standard methods. The results showed significantly higher antioxidant activity for the mayonnaise sample prepared with 100% virgin coconut oil in comparison to other two samples. No significant differences were observed for the physical properties including pH and colour. However, 100% virgin coconut oil mayonnaise sample had the largest droplet size, lowest texture firmness and lowest viscosity among the samples. The sensory evaluation demonstrated higher preference to the 100% soybean oil mayonnaise that scored total of 7.050, while the 100% virgin coconut oil mayonnaise scored 5.57 for overall acceptability. The findings of this study demonstrated high potential for using virgin coconut oil in mayonnaise production to improve the quality and the biological functions of the product while maintaining the organoleptic properties

    Application of betel leaves (Piper betle L.) extract for preservation of homemade chili bo

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    This study is conducted to investigate the effect of different concentrations of betel leaves extract on color, pH and microbiological in homemade chili bo. The homemade chili bo with different concentrations (0 mg/ml, 0.75 mg/ml, 1.25 mg/ml and 1.75 mg/ml) of betel leaves extract were prepared for analysis. The results showed that the color of chili bo became darker as the concentration of betel leaves extract increased. The extract showed significant in the pH of chili bo after 7 days in which the highest concentration of extract showed the highest value of pH 4.31. The aerobic microbial count was decreased as the concentration of betel leaves extract increased in chili bo. After 7 days of storage, the highest concentration of betel leaves extract showed the highest percentage of reduction (6%), while the control sample showed 2.41% of aerobic reduction. The study also found that the extract contain lesser yeast and mold count (5.22 log CFU/ml) in homemade chili bo compared to the control sample (5.31 log CFU/ ml) after 7 days. Betel leaves extract can be considered as natural food preservatives in chili bo to reduce the growth of spoilage microorganism and thus enhance the shelf life of chili bo

    Efficacy of a locally produced microbial phytase from Enterobacter sakazakii ASUIA273 on body weight and hemato-biochemical constituents in broiler chickens

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    An experiment was carried out in broiler chicks fed different doses of locally produced microbial phytase supplementation to observe their growth performance as health status, and to investigate the changes of hematological and biochemical values. A total of 144 chicks (Cobb) at one-day old were allocated to 4 treatment (T) groups with 12 cages comprising 3 replicates, each cage containing to 12 birds. Experimental formulating diets arranged with 4 levels of 0, 500, 1000 and 1500 phytase enzyme unit (FTU/kg-I) as considered as Th T2, T3, T4 respectively. They were maintained formulating diet on these dietary treatments from 1 to 42 d of age with feed and water made available for ad libitum consumption. At 1 week interval 2 birds from each treatment were weight through out experimental period for assessing the growth performance. For determining the changes of hematological (RBC, Hb, PCV, MCV, MCHC, WBC, Heterophil, Eosinophil, Basophil, Lymphocyte, Monocyte, Thrombocyte and Icterus Index) and biochemical (Albumin, Total Protein, ALT, ALP, AST, GGT, LDH, Cholesterol, Triglyceride, Glucose, Ca, P, Na, K, Cl, Urea, Creatine and Uric acid) values at the age of 6 weeks randomly selected 2 birds were slaughtered and blood were collected. Data were subjected to analysis of variance (ANOVA) using the least significant difference (LSD) by PC-SAS software (SAS Institute, 2009). Data showed that body weight was not affected at periods of 1si and 2nd weeks of age among different treatment groups. But, at ages from 3rd to 6th weeks, weight gains at four treatment gIZ.Qupwsere increased almost sequentially and consistantly, and had been showed more different and significant (p:s 0.05) increased at 4th and 5th weeks of age from the control. No significant and constant treatments effects were observed on blood and biochemical parameters except eosinophil: Accordingly, it can be recommended to use an uncentrifuged microbial phytase in broiler diet during the period from 4th - 5th weeks of age, to achieve increased weight gain without changing hemato-biochemical parameters

    Bacillus Multi-strain from Malaysian Fish Sauces Demonstrating Proteolytic, Lipolytic, Esterolytic and Glutamic-acid Production Activities

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    Background and Objective: Aroma and taste of fish sauce are derived from complex metabolic reactions involving bacteria and enzymes that degrade proteins and lipids. A longer fermentation time is needed to fully develop the rich flavor. The objective of this study was to isolate halotolerant bacteria from Malaysian fermented fish sauce that demonstrated significant proteolytic, lipolytic, esterolytic and glutamic acid-accumulating activities. These bacteria might potentially be used as starting cultures to enhance development of flavors during fish sauce fermentation. Material and Methods: The initial screening on proteolytic activity was carried out on saline skim milk agar. Isolates with high proteolytic index and positive lipase/esterase activity were further assessed using casein-based protease activity assay. Protease and lipase activities under various conditions of strains, NaCl concentrations (20–30% w/v), temperatures (30–35 °C) and incubation times (0–120 h) were assessed and analyzed using four-way ANOVA. The p-nitrophenyl butyrate and colorimetric assays were used to determine esterase and glutamic acid accumulating activities, respectively. Results and Conclusion: Six strains isolated from budu demonstrated hydrolytic activities and identified as Bacillus spp. Proteolytic activities were averagely highest in B. licheniformis 12N3A at 25% NaCl, 30 °C and 96 h of incubation. The highest lipolytic activities were achieved with B. haikouensis 12M1F at 30% NaCl, 35 °C and 72 h of incubation. Bacillus sp. 3M3A showed the highest esterase activity of 155.48 U, while Bacillus sp. 6M2A had the highest glutamic acid accumulation at 1993.02 mol. l-1. The strains of this study have demonstrated appropriateness for use as a cocktail culture to accelerate the fermentation process and enhance the flavor profile of fish sauces. Halotolerant nature of the strains and enzymes supports their potentials for wider use in the food industry. Conflict of interest: The authors declare no conflict of interest. Introduction   Budu is a Malaysian traditional fish sauce made from anchovies fermented with salt for 6–12 m and officially recognized as a national culinary heritage. Flavor is addressed as the primary quality attribute of budu. Function prediction of budu microbial communities based on meta-genomic composition has demonstrated increased protein and lipid breakdown pathways during fermentation and these pathways are known to play critical roles in formation of flavor [1]. Through spontaneous fermentation of high-salt fish sauce, a wide range of taste and aroma-active compounds belonging to aldehydes, acids, esters, alcohols and pyrazines are gradually formed, including 2-methyl-butanal, 3-methylbutanoic acid, methyl 2-ethyldecanoate, 1-octen-3-ol and 2,6-dimethylpyrazine [1]. This is achieved through the combined activities of fish endogenous enzymes and metabolic activities of halophilic and halotolerant microbes [2]. Additionally, microbes are capable of produc-ing microbial volatile organic compounds, which may stimulate production of secondary metabolites, especially during co-cultures [3]. Fatty acids, peptides and amino acids that are released through the process of lipid and protein hydrolyses serve as precursors for aroma compounds. They can be transformed into volatile organic compounds, including aldehydes, esters, ketones, alcohols, furans, hydrocarbons, lactones, pyrazines, amines, phenols, indoles, acids and sulfur-containing compounds [4]. The medium and short-chain fatty acid esters are important volatile organic compounds that can impart pleasant fruity flavors and aromas [5] and their presence in fish sauce can cover unpleasant smells and off flavors contributed by protein-derived compounds such as dimethyl trisulfide and trimethylamines. Microorganisms involved in fish sauce fermentation may facilitate glutamic acid accumulation by converting ketoglutaric acid into L-glutamic acid under the function of glutamate synthetase or glutamic acid dehydro-genase, as well as hydrolysis of proteins into umami amino acids and peptides [6]. Figure 1 shows schematic functions of protease, lipase and esterase as well as formation of glutamic acid in fermentation of fish sauces. At the onset of fermentation, fermentation-assisting bacteria are not the prevailing microorganisms. They need longer times to adapt to high salinity, establish dominance in the microbial community and produce flavor compounds [7].  Consequently, a prolonged fermentation time is needed to produce rich, flavorful fish sauces. The extended fermentation time can lead to an increase in overall produc-tion time, which may not be desirable for the producers who try to meet market demand and generate revenue quickly. In response to this, Malaysian fish-sauce producers terminate their fermentation process after only three months even though the flavor has not fully developed. Accelerating the fermentation process is therefore important for the production efficiency and profitability of fish sauce industries. Use of starting cultures has been suggested as an effective method to enhance proteolysis during fish-sauce fermentation [8], while improving and maintaining the consistent quality of the fermented fish [9]. Addition of Marinococcus halotolerans SPQ isolated from salt crystals showed increased contents of aspartic and glutamic acids with several aroma compounds belonged to alcohol group [10]. Use of protease-producing Tetragenococcus halophi-lus has decreased fermentation time to six months and improved quantity of desirable amino acids [11]. When selecting starter cultures for fish-sauce fermentation, ease of cultivation is primarily important. It is essential for the strains to possess pro-technological metabolic character-istics such as the ability to grow and produce high-activity hydrolases that are active at high salt concentrations (20–30% NaCl). Therefore, a candidate derived from fish sauce is an excellent choice due to its adaptability, resistance to osmotic stress and ability to thrive in high salt environments. This enables them to grow more efficiently and fulfil their roles within the microbial community. Fermentation of fish, which naturally contains high protein and low sugar contents, differs significantly from conventional carbohydrate fermentation. Rather than targeting sugars, this process focuses majorly on fish protein and lipid components [12]. Therefore, proteolytic and lipolytic activities are two key characteristics that should be addressed when selecting starter candidates for fish fermentation. Release of proteases and lipases by halophilic Staphylococcus simulans PMRS35 and Haloarcula sp. isolated from salted and fermented fish [13,14] as well as M. halotolerans SPQ and Chromohalobacter sp. from salt environments [10,15] has been documented in several investigations. It is suggested that fish sauce contains a diverse enzymes-producing microbiota that contribute to the formation of taste-active compounds, making it an ideal source for the selection of starter cultures. Nevertheless, a complete assessment of various strains from budu has not been carried out, specifically for assessing production of multiple enzymes that are likely to improve budu flavor. The aim of the current study was to isolate, identify and assess bacterial cultures from budu, displaying proteolytic, lipolytic, esterolytic and glutamic-acid accumulating activities. This study provides a novel contribution to the existing literature as it assessed the bacterial enzymes involved in the flavor development of Malaysian fish sauce, an area with limited attentions. Halotolerant strains thoroughly assessed for their major abilities to degrade fish proteins and lipids make promises for potential uses in the food industry. They can provide a viable solution for improving production and fermentation of high-salt fermented foods, particularly fish sauces. Materials and Methods 2.1. Isolation of halotolerant proteolytic bacteria Fish-sauce samples from 3, 6 and 12-m fermentation tanks were used for bacterial isolation. A modified isolation approach [8] was used to concurrently identify halotolerant proteolytic bacteria during first screening. To increase the likelihood of collecting indigenous proteolytic fish-sauce origin strains, media types and salt concentrations were modified. Twenty-five grams of fish sauce were diluted with 225 ml of sterile 0.85% (w/v) NaCl and stirred for 1 min. To recover further halophilic species, three decimal dilutions of the samples were plated on four media of marine agar (MA) with 20% NaCl for bacteria tolerant to 20% NaCl; De Man, Rogosa and Sharpe agar (MRSA) with 20% NaCl for 20% NaCl-tolerant lactic acid bacteria (LAB); mannitol salt agar (MSA) with 10% NaCl for salt-tolerant Staphylococci; and nutrient agar (NA) with 30% NaCl for bacteria tolerant to 30% NaCl. Each medium received 1% of skim milk for proteolytic activity screening. The agar medium was incubated at 30 °C for 3–14 d until colonies formed. Four-quadrant streaking pattern was used to pick and streak clear zone-forming bacterial colonies with diverse morphologies onto newly prepared agar plates for purification. 2.2. Screening of protease and lipase/esterase activities using agar plate assay Prior to the screening, one loop of every single isolate was transferred to marine broth (MB) containing 20% NaCl and incubated at 30 °C for 3 d, except for colonies recovered from MSA that were grown in nutrient broth supplemented with 10% NaCl. Cells were collected by centrifugation at 4000 rpm for 10 min at 4 °C following a 3-d incubation time. To prepare pellets for resuspension, pellets were first rinsed twice with 10 ml of pH-7.4 phosphate buffered saline (PBS). For each strain, absorbance of the suspended cells at 600 nm was adjusted to 0.85 before well-diffusion method was used. For the screening of bacteria with proteolytic activity, aliquots of the bacterial suspension (50 µl) were added to a 6-mm well on agar media consisted of NA supplemented with 10% NaCl and 1% skim milk for MSA-derived strains and MA supplemented with 1% skim milk for the rest of strains. Proteolytic indices were assessed after incubating plates at 30 °C for 2 d [9]. Agar spot techniques in three various media of phenol red, tributyrin and Tween 80 agar were used to screen bacteria that produced lipase or esterase. Each medium was prepared according to [16]. Five microliters of resuspended cells were dropped onto the surface of the phenol red, tributyrin and Tween 80 agar and allowed to absorb. Inoculated plates were incubated at 30 °C for 3–14 d until visible signs of lipolysis activity were observed. The color shift of phenol-red agar from red to orange indicated lipase activity, which was caused by a slight pH drop induced by fatty acid releases. A clear zone on tributyrin agar indicated the presence of extracellular esterase. The FFAs were precipitated by calcium (giving a white zone) around colonies showing lipase and esterase activity on Tween 80 agar. Isolates demonstrating a high proteolytic index and/or indicating lipolytic activity were selected for further growth and protease activity assessment using liquid assay. 2.3. Growth assessment of the strains One loop of each selected strain was transferred to MB media and incubated at 30 °C for 48 h. After incubation, cells were centrifuged at 4000 rpm for 10 min at 4 °C. Collected pellets were washed and resuspended in PBS with an OD600 adjusted to 1.1-1.2. A 1-ml volume of the cell suspension was transferred to 40 ml of MB using 125-ml amber bottles. The mixture was incubated at 30 °C with 120-rpm agitation speed. Growth assessment was carried out by measuring aliquots of the growth media at 600 nm using Biomate 3 UV-vis spectrophotometer (Thermo-Fisher Scientific, Massachusetts, USA) at specified intervals for 216 h. Sterile MB medium was used as blank for each reading. 2.4. Extraction of the bacterial crude enzymes Cell-free supernatant (CFS) was prepared for the enzyme assays by centrifuging aliquots of the harvested growth media at 11,000 × g for 10 min at 4 °C. 2.5. Assessment of protease activity Protease activity of the isolates was determined based on the Cupp-Enyard and Aldrich method [17], where casein was used as substrate. Casein was diluted to 6.5 mg.ml-1 in 50 mM potassium phosphate buffer (pH 7.5). Temperature of the solution gradually increased to 80–85 °C with gentle stirring for nearly 10 min until a homogeneous dispersion was achieved. Casein solution was set at 37 °C prior to use. Moreover, CFS was added to 5 ml of casein solution and incubated at 37 °C for 10 min. Every tube was then treated with 5-ml solution of 110 mM trichloroacetic acid (TCA) following incubation to terminate the reaction. To verify that each tube had an identical end volume and account for the enzyme absorbance value, an equal quantity of the enzyme solution was added to the blank. Solutions were incubated at 37 °C for 30 min and then centrifuged at 4000 rpm for 5 min to remove insoluble substances. For colori-metric determination of tyrosine, 400 µl of the supernatant were immediately added with 1 ml of sodium carbonate and 0.2 ml of Folin reagent. The mixture was mixed gently and permitted to stabilize at 37 °C for 30 min. Absorbance measurement was carried out at 660 nm using spectrophoto-meter. Tyrosine concentration was determined using standard tyrosine curve. Bacterial protease unit (U) was defined as the quantity of enzyme that produced the equivalent of 1 μmol. L-1 of L-tyrosine per min in pH 7.5 and 37 °C. Isolates with significant protease activity were subjected to further analyses. The protease activity was determined using the Eq. 1   Eq.1 2.6. Effects of time, temperature and NaCl concentration on protease and lipase activities Using MB media, the selected strains were assessed for their protease and lipase activities under various temperatures (30 and 35 °C) and NaCl concentrations (20, 25 and 30%). In this study, a temperature range of 30–35 °C was selected as it represented the average temperature used in the industry. Salt concentration varied 20–30% during budu 12-m fermentation; therefore, this salt range was used to assess its effects on the strains’ enzyme activity. Protease assay was carried out using the method described in Section 2.5. To assess lipolytic activity, titration method [18] was used with emulsion of fish oil and polyvinyl alcohol at a ratio of 1:3 as the substrate for the assay. Briefly, 2.5 ml of phosphate buffer (pH 4.8) were added to 2.5 ml of the substrate, followed by adding 0.5 ml of CFS. A 10-min enzymatic reaction was carried out at 37 °C. Blank was prepared without addition of CFS. A final volume of 7.5 ml of 95% ethanol was added to terminate the reaction. Phenolphthalein indicator was used to titrate the released fatty acids against 0.1 M NaOH. The quantity of lipase that might release 1 µmol of fatty acids was measured in units of lipase activity. The mole of fatty acids was equal to the mole of the titrant and 1 mol NaOH was equal to 106 × µmol fatty acid equivalent. Lipase activity was determined using the Eqs. 2 and 3:   Eq. 2 Eq. 3 2.7. Esterase activity Colorimetric method was used to determine the esterase activity using modified method [19, 20]. Prior to esterase assessment, an overnight culture (24 h) of each strain was grown in modified Gibbon’s media containing 20% of NaCl, 2.5% of fish oil, 0.1% of MgSO4.7H2O, 0.3% of Na3C6H5O, 0.2% of KCl, 0.75% of tryptone and 0.1% of yeast extract for 3 d at 30 °C and 120 rpm. The CFS was used for the analysis. A mixture of 14 mM p-nitrophenyl butyrate in acetonitrile, 66.67 mM of Tris HCl (pH 8) and 3M of NaCl at a ratio of 1:8:1 was prepared as the substrate for the esterase activity assay. A volume of 20 µl of CFS and 200 µl of substrate were transferred into 96-well microplates and gently agitated. Double-distilled water was used as blank instead of CFS. Absorbance reading was carried out at 410 nm every 30 s at 37 °C over 5 min to measure the p-nitrophenol (p-NP) release using microplate reader (Powerwave X 340 microplate scanning spectro-photometer, BIO-TEK Instruments, Vermont, USA). Extinction coefficient (ε) for p-NP of this assay was 8222.1 M-1 cm-1, which was derived from the slope of the p-NP standard curve under similar detection conditions. For one unit (U) of esterase activity, enzyme quantity needed to release 1 µM of p-NP per minute was calculated based on Eq. 4 and Beer-Lambert Law as follows:                                                                                                                                                                                                                                Eq. 4 Where, ΔAbs showed changes in absorbance over time; ε denoted the molar extinction coefficient in M-1 cm-1; volume of assay represented the total volume of reaction mixture (ml); and Δt represented the incubation time (min). 2.8. Glutamic acid assessment Prior to assessment, the overnight culture of each strain was inoculated (1% v/v) into the glutamic acid production media (pH 7). This medium consisted of 5% of glucose, 0.8% of urea, 0.0002% of biotin, 0.1% of K2HPO4, 0.25% of MgSO4.7H2O, 0.01% of MnSO4.7H2O and 0.16% of CaCO3. Incubation was carried out at 30 °C for 7 d at 120 rpm. The crude glutamic acid source was the CFS that separated following centrifugation at 10,000 rpm for 10 min at 4 °C [21]. Glutamic acid was determined using glutamic acid colorimetric assay kit (Elabscience Biotechnology, Texas, USA). 2.9. Genome-based identification of the isolates Extraction of the bacterial DNA was carried out using Nucleospin microbial DNA extraction kit (Machery-Nagel, Duren, Germany). One loop of a single bacterial colony was pelleted and transferred to a microcentrifuge tube containing 40–400 μm of glass beads, added with buffer and proteinase and processed with FastPrep-24 system (MP Biomedicals, Ohio, USA). Lysed cells were centrifuged at 11,000× g for 30 s and DNA in the supernatant was purified using microbial DNA column based on the manufacturer’s protocol. Genome-based identification of the strains was carried out using Patriot Biotech, Selangor, Malaysia, based on the manufacturer’s standard protocol. Primers of 27F (TTTCTGTTGGTGCTGATATTGCAGRGTTYGATYMTGGCTCAG) and 1492R (ACTTGCCTGTCGCTCTAT-CTTCTACGGYTACCTTGTTACGACTT) with a Nano-pore partial adapter on the 5' end were used to amplify 16S rRNA full-length sequence of the strains. The PCR was carried out using WizBio HotStart 2× mastermix (Wizbiosolutions, Gyeonggi-do, Korea) with the following conditions of an initial denaturation step at 95 °C for 3 min; followed by 35 cycles of denaturation at 95 °C for 20 s, annealing at 50 °C for 20 s and extension at 72 °C for 120 s. The PCR products were visualized on agarose gels and purified using solid-phase reversible immobilization beads. Nanopore Flongle flow cell (Oxford Nanopore Technol-ogies, Oxford, UK) was used for the 24-h sequencing process. Sequences were compared to known sequences using BLAST online tool of the National Centre for Biotechnology (NCBI) website to verify the homology. The neighbor-joining trees were then constructed using MEGA 11 software. The rRNA sequence data of the selected strains were submitted to GenBank database. 2.10. Statistical analysis Minitab 19 statistical package (Minitab, Pennsylvania, USA) was used for the analysis. Results of the ANOVA analysis was reported statistically significant when p < 0.05. One-way ANOVA was carried out on variables to detect significant differences between the samples. Four-way ANOVA was used to assess effects of the multiple factors (time, temperature, NaCl concentration and strain) on the protease and lipase activities. To find out which groups of the samples were distinct from others, post hoc Tukey's honest significant difference (HSD) test was carried out. 2.11. Ethical considerations No experiments involving humans or animals were carried out in this study. Results and Discussion 3.1. Initial screening and isolation of proteolytic and lipolytic halotolerant bacteria For selecting bacteria for budu starter cultures, strains were initially assessed based on their ability to persist high-salt concentrations associated with budu fermentation. A total of 50 bacterial isolates were capable of growing on isolation media supplemented with 10–30% of NaCl (w/v) (data not shown). Twenty-nine isolates were originated from 12-m samples, eight from 6-m samples and 13 from 3-m samples. Only five isolates were recovered from NA containing 30% of NaCl, while 40 isolates were recovered from MA (20% NaCl). No colonies were observed in the MRSA containing 20% of NaCl, whereas, a few staphylococci were detected on MSA with 10% of NaCl. Decreased number of strains recovered from budu when grown on high-salt media could be due to the inactivation of key enzymes for microbial metabolism and growth. Additi-onally, the osmotic stress and metabolic adaptation forced bacteria to allocate their energy for adaptation rather than multiplication. Under further severe conditions, bacteria might enter viable but nonculturable (VBNC) states [22]. In a previous study, LAB and halophilic bacteria were isolated from Vietnamese fish sauces using salt concentrations ranging 5-18 and 18-25%, respectively. No growth of colonies was detected in high-salt media (approximately 29% NaCl) [10]. In the present study, strains capable of growing at 30% NaCl could be recovered; however, a longer incubation time of 14 d was needed for the colonies to become visible in the media, in contrast to the growth on MA that occurred within 72 h. Presence of LAB has been reported in Thai and

    Candida sp. as a starter culture for cocoa (Theobroma cacao L.) beans fermentation

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    Two cocoa bean fermentation methods (spontaneous fermentation and the use of starter culture) for 7 days fermentation were compared in terms of safety and quality fermented beans. Candida sp. was used as a starter culture in this study. The safety of the fermented cocoa beans were measured by the growth colonies of pathogenic microorganisms namely Bacillus cereus, Escherichia coli, Salmonella sp., Staphylococcus aureus, and Pseudomonas sp., on Bacillus cereus agar, eosin-methylene blue (EMB) agar, xylose lysine deoxycholate (XLD) agar, Baird-Parker agar (BPA), and Pseudomonas agar, respectively. B. cereus, E. coli and Salmonella sp. were early present in both fermentations. Candida sp.-fermentation showed detection of B. cereus at 5.34 log10 CFU/g and absence after 24 hours of fermentation while in spontaneous-fermentation B. cereus was too few to count. Moreover, the log10 E. coli number in Candida sp.-fermentation and spontaneous-fermentation were reduced from 5.72 to 3.66 and from 7.15 to 4.46 on day 1 to day 3, respectively. There were no presences of pathogenic microorganisms on day 5 and day 7 for both fermentations. In term of quality, proximate analysis of spontaneous-fermentation resulted that the content of moisture, ash, fat, crude protein, crude fibre and carbohydrate was 56.47%, 2.32%, 3.17%, 7.02%, 28.14% and 2.88%, meanwhile for the Candida sp.-fermentation was 53.96%, 2.19%, 3.44%, 8.25%, 25.46% and 6.70%, respectively. This study showed that both fermentations are considered to be safe and there is no significant difference in proximate value in fermented cocoa beans from spontaneous-fermentation and Candida sp.-fermentation

    Cultivation conditions for phytase production from recombinant escherichia coli DH5α

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    Response surface methodology (RSM) was used to optimize the cultivation conditions for the production of phytase by recombinant Escherichia coli DH5α. The optimum predicted cultivation conditions for phytase production were at 3 hours seed age, a 2.5% inoculum level, an L-arabinose concentration of 0.20%, a cell concentration of 0.3 (as measured at 600 nm) and 17 hours post-induction time with a predicted phytase activity of 4194.45 U/mL. The model was validated and the results showed no significant difference between the experimental and the predicted phytase activity (P = 0.305). Under optimum cultivation conditions, the phytase activity of the recombinant E. coli DH5α was 364 times higher compared to the phytase activity of the wild-type producer, Enterobacter sakazakii ASUIA279. Hence, optimization of the cultivation conditions using RSM positively increased phytase production from recombinant E. coli DH5α
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