Production, statistical optimization, and functional characterization of alkali stable pectate lyase of Paenibacillus lactis PKC5 for use in juice clarification.

Pectate lyase is a hydrolytic enzyme used by diverse industries to clarify food. The enzyme occupies a 25% share of the total enzyme used in food industries, and their demand is increasing gradually. Most of the enzymes in the market belong to the fungal origin and take more time to produce with high viscosity in the fermentation medium, limiting its use. The bacteria belonging to the genus Bacillus have vast potential to produce diverse metabolites of industrial importance. The present experiment aimed to isolate pectate lyase-producing bacteria that can tolerate an alkaline environment at moderate temperatures. Bacillus subtilis PKC2, Bacillus licheniformis PKC4, Paenibacillus lactis PKC5, and Bacillus sonorensis ADCN produced pectate lyase. The Paenibacillus lactis PKC5 gave the highest protein at 48 h of incubation that was partially purified using 80% acetone and ammonium sulphate. Purification with 80% acetone resulted in a good enzyme yield with higher activity. SDS-PAGE revealed the presence of 44 kDa molecular weight of purified enzyme. The purified enzyme exhibits stability at diverse temperature and pH ranges, the maximum at 50 °C and 8.0 pH. The metal ions such as Mg2+, Zn2+, Fe2+, and Co2+ significantly positively affect enzyme activity, while increasing the metal ion concentration to 5 mM showed detrimental effects on the enzyme activity. The organic solvents such as methanol and chloroform at 25% final concentration improved the enzyme activity. On the other hand, detergent showed inhibitory effects at 0.05% and 1% concentration. Pectate lyase from Paenibacillus lactis PKC5 had Km and Vmax values as 8.90 mg/ml and 4.578 µmol/ml/min. The Plackett-Burman and CCD designs were used to identify the significant process parameters, and optimum concentrations were found to be pectin (5 gm%) and ammonium sulphate (0.3 gm%). During incubation with pectate lyase, the clarity percentage of the grape juice, apple juice, and orange juice was 60.37%, 59.36%, and 49.91%, respectively.

Antibacterial properties of Apis dorsata honey against some bacterial pathogens.

Now-a-days, different bioproducts are being used extensively for the welfare of mankind. However, for proper utility of any bioproduct, the exact biotechnological potential of that product should be explored. Honey is produced in almost every country on the planet. It has long been used as a medicinal agent in addition to its broader use as a popular food throughout the human history. It can be used to treat various diseases without causing any negative side effects. In the present study, the antibacterial potential of honey produced by A. dorsata was investigated at its variable concentrations (25, 50, 75 and 100 %) against four pathogenic bacterial species. The highest antimicrobial action was seen against E. coli at 100 % concentration of the honey while showing zone of inhibition of 37.5 ±â€¯3.5 mm. However, the lowest antibacterial action was observed against E. faecalis. The overall order of growth inhibition by the honey at its 100 % concentration for the implicated bacterial species appeared as: E. coli ˃ P. aeruginosa ˃ S. aureus ˃ E. faecalis. The honey couldn't show antibacterial action at its 25 % concentration. Our findings of the present study will be helpful for utility of the honey as an alternative medicine for curing different complications caused by microbial pathogens.

Production of Plant Beneficial and Antioxidants Metabolites by Klebsiellavariicola under Salinity Stress.

Bacteria that surround plant roots and exert beneficial effects on plant growth are known as plant growth-promoting rhizobacteria (PGPR). In addition to the plant growth-promotion, PGPR also imparts resistance against salinity and oxidative stress and needs to be studied. Such PGPR can function as dynamic bioinoculants under salinity conditions. The present study reports the isolation of phytase positive multifarious Klebsiella variicola SURYA6 isolated from wheat rhizosphere in Kolhapur, India. The isolate produced various plant growth-promoting (PGP), salinity ameliorating, and antioxidant traits. It produced organic acid, yielded a higher phosphorous solubilization index (9.3), maximum phytase activity (376.67 ± 2.77 U/mL), and copious amounts of siderophore (79.0%). The isolate also produced salt ameliorating traits such as indole acetic acid (78.45 ± 1.9 µg/mL), 1 aminocyclopropane-1-carboxylate deaminase (0.991 M/mg/h), and exopolysaccharides (32.2 ± 1.2 g/L). In addition to these, the isolate also produced higher activities of antioxidant enzymes like superoxide dismutase (13.86 IU/mg protein), catalase (0.053 IU/mg protein), and glutathione oxidase (22.12 µg/mg protein) at various salt levels. The isolate exhibited optimum growth and maximum secretion of these metabolites during the log-phase growth. It exhibited sensitivity to a wide range of antibiotics and did not produce hemolysis on blood agar, indicative of its non-pathogenic nature. The potential of K. variicola to produce copious amounts of various PGP, salt ameliorating, and antioxidant metabolites make it a potential bioinoculant for salinity stress management.

Bioprocess Optimization for Exopolysaccharides Production by Ganoderma lucidum in Semi-industrial Scale.

BACKGROUND: For many years, Ganoderma was highly considered as biofactory for the production of different types of bioactive metabolites. Of these bioactive compounds, polysaccharides gained much attention based on their high biotherapeutic properties. Therefore, special attention has been paid during the last years for the production of mushrooms bioactive compounds in a closed cultivation system to shorten the cultivation time and increase the product yield. OBJECTIVES: This work focuses on the development of a simple cultivation strategy for exopolysaccharides (EPS) production using Ganoderma lucidum and submerged cultivation system. METHODS: At first, the best medium supporting EPS production was chosen experimentally from the current published data. Second, like many EPS production processes, carbon and nitrogen concentrations were optimized to support the highest production of polysaccharides in the shake flask level. Furthermore, the process was scaled up in 16-L stirred tank bioreactor. RESULTS: The results clearly demonstrated that the best cultivation strategy was cultivation under controlled pH conditions (pH 5.5). Under this condition, the maximal volumetric and specific yield of EPS production were, 5.0 g/L and 0.42 g/g, respectively. CONCLUSION: The current results clearly demonstrate the high potential use of submerged cultivation system as an alternative to conventional solid-state fermentation for EPS production by G. lucidum. Furthermore, the optimization of both carbon and nitrogen sources concentration and scaling up of the process showed a significant increase in both volumetric and specific EPS production.

Scaling up of levan yield in Bacillus subtilis M and cytotoxicity study on levan and its derivatives.

This study focused on kinetics of levan yield by Bacillus subtilis M, in a 150 L stirred tank bioreactor under controlled pH conditions. The optimized production medium was composed of (g/L): commercial sucrose 100.0, yeast extract 2.0, K2HPO4 3.0 and MgSO4⋅7H2O 0.2; an increase in both carbohydrates consumption and cell growth depended on increasing the size of the stirred tank bioreactor from 16 L to 150 L. The highest levansucrase production (63.4 U/mL) and levan yield of 47 g/L was obtained after 24 h. Also, the specific levan yield (Yp/x) which reflects the cell productivity increased with the size increase of the stirred tank bioreactor and reached its maximum value of about 29.4 g/g cells. These results suggested that B. subtilis M could play an important role in levan yield on a large scale in the future. Chemical modifications of B. subtilis M crude levan (CL) into sulfated (SL), phosphorylated (PL), and carboxymethylated levans (CML) were done. The difference in CL structure and its derivatives was detected by FT-IR transmission spectrum. The cytotoxicity of CL and its derivatives were evaluated by HepGII, Mcf-7 and CaCo-2. In general most tested levans forms had no significant cytotoxicity effect. In fact, the carboxymethylated and phosphrylated forms had a lower anti-cancer effect than CL. On the other hand, SL had the highest cytotoxicity showing SL had a significant anti-cancer effect. The results of cytotoxicity and cell viability were statistically analyzed using three-way ANOVA.

Bioprocess optimization for pectinase production using Aspergillus niger in a submerged cultivation system.

BACKGROUND: Pectinase enzymes present a high priced category of microbial enzymes with many potential applications in various food and oil industries and an estimated market share of $ 41.4 billion by 2020. RESULTS: The production medium was first optimized using a statistical optimization approach to increase pectinase production. A maximal enzyme concentration of 76.35 U/mL (a 2.8-fold increase compared with the initial medium) was produced in a medium composed of (g/L): pectin, 32.22; (NH4)2SO4, 4.33; K2HPO4, 1.36; MgSO4.5H2O, 0.05; KCl, 0.05; and FeSO4.5H2O, 0.10. The cultivations were then carried out in a 16-L stirred tank bioreactor in both batch and fed-batch modes to improve enzyme production, which is an important step for bioprocess industrialization. Controlling the pH at 5.5 during cultivation yielded a pectinase production of 109.63 U/mL, which was about 10% higher than the uncontrolled pH culture. Furthermore, fed-batch cultivation using sucrose as a feeding substrate with a rate of 2 g/L/h increased the enzyme production up to 450 U/mL after 126 h. CONCLUSIONS: Statistical medium optimization improved volumetric pectinase productivity by about 2.8 folds. Scaling-up the production process in 16-L semi-industrial stirred tank bioreactor under controlled pH further enhanced pectinase production by about 4-folds. Finally, bioreactor fed-batch cultivation using constant carbon source feeding increased maximal volumetric enzyme production by about 16.5-folds from the initial starting conditions.

Bioprocess development for kefiran production by Lactobacillus kefiranofaciens in semi industrial scale bioreactor.

Lactobacillus kefiranofaciens is non-pathogenic gram positive bacteria isolated from kefir grains and able to produce extracellular exopolysaccharides named kefiran. This polysaccharide contains approximately equal amounts of glucose and galactose. Kefiran has wide applications in pharmaceutical industries. Therefore, an approach has been extensively studied to increase kefiran production for pharmaceutical application in industrial scale. The present work aims to maximize kefiran production through the optimization of medium composition and production in semi industrial scale bioreactor. The composition of the optimal medium for kefiran production contained sucrose, yeast extract and K2HPO4 at 20.0, 6.0, 0.25 g L(-1), respectively. The optimized medium significantly increased both cell growth and kefiran production by about 170.56% and 58.02%, respectively, in comparison with the unoptimized medium. Furthermore, the kinetics of cell growth and kefiran production in batch culture of L. kefiranofaciens was investigated under un-controlled pH conditions in 16-L scale bioreactor. The maximal cell mass in bioreactor culture reached 2.76 g L(-1) concomitant with kefiran production of 1.91 g L(-1).

Anaerobic Probiotics: The Key Microbes for Human Health.

Human gastrointestinal microbiota (HGIM) incorporate a large number of microbes from different species. Anaerobic bacteria are the dominant organisms in this microbial consortium and play a crucial role in human health. In addition to their functional role as the main source of many essential metabolites for human health, they are considered as biotherapeutic agents in the regulation of different human metabolites. They are also important in the prevention and in the treatment of different physical and mental diseases. Bifidobacteria are the dominant anaerobic bacteria in HGIM and are widely used in the development of probiotic products for infants, children and adults. To develop bifidobacteria-based bioproducts, therefore, it is necessary to develop a large-scale biomass production platform based on a good understanding of the ideal medium and bioprocessing parameters for their growth and viability. In addition, high cell viability should be maintained during downstream processing and storage of probiotic cell powder or the final formulated product. In this work we review the latest information about the biology, therapeutic activities, cultivation and industrial production of bifidobacteria.

Scaling up, characterization of levan and its inhibitory role in carcinogenesis initiation stage'.

Honey isolate Bacillus subtilis M was cultivated in shake flasks and in 16-l bioreactor cultures to investigate cell growth, bio-metabolites production kinetics and bioprocess scalability. The respective maximal levan and levansucrase productions of 59.5 g/l and 74.1 U/ml were achieved in bioreactor cultures under pH controlled condition (pH=7.0) after only 24 h. Crude levan (levE) was isolated, characterized and fractionated into F1, F2, and F3 with different molecular weight (21.8, 13.118, 9.53 kDa). (1)H NMR and (13)C NMR spectroscopy proved that LevE and their fractions were mainly ß-(2, 6)-linked levan-type polysaccharide. The cancer chemo-preventive activity indicated that the levE and its fraction 3 were promising inhibitors of cytochrome P-450 1A activity, inducers of glutathione-S-transferase activity in Murine hepatomaHepa1c1c7cells and possessed highest radical scavenging affinity to both ROO and OH. They inhibited the induced-DNA fragmentation. None of the tested samples triggered apoptosis or necrosis in splenocytes, except F2.