Chitosan from Syncephalastrum racemosum Using Sugar Cane Substrates as Inexpensive Carbon Sources

A strain of Syncephalastrum racemosum isolated from herbivores feces was studied for chitosan production using sugar cane substrates, such as juice or molasses, from Northeast Brazil. S. racemosus was batch grown in shake flasks over 5 days using sugar cane juice or molasses as carbon source. The effect of supplementary nitrogen source was studied by supplying medium with yeast extract or ammonium sulphate. The highest yield of the biopolymer (30 mg chitosan. g^?1 sucrose) was obtained using sugar cane juice (21 g sucrose. L^?1) plus 0.3% yeast extract. Batch fermentation in a 5-L bioreactor using the same medium, increased the yield to 50 mg chitosan. g^?1 sucrose. Chitosan extracted from S. racemosus cultured in sugar cane juice medium showed a low degree of acetylation with a high D-glucosamine content.     

Extracellular α-Amylase Production by Bacillus brevis MTCC 7521

Alpha amylases have various applications in food processing industries, for example, baking, brewing and distillery industries. Studies of the Ca²⁺ independent α-amylase production were carried out by a strain of Bacillus brevis MTCC 7521 isolated from a brick kiln soil. The optimum temperature, pH and incubation period for amylase production were 50°C, 6.0 and 36 h, respectively. The enzyme secretion was at par in the presence of any of the carbon sources (soluble starch, cassava starch and cassava flour). B. brevis produced more amylase in presence of beef extract as nitrogen source in comparison to other organic nitrogen sources (peptone, yeast extract and casein) and asparagine, potassium nitrate, ammonium sulphate, ammonium nitrate and urea reduced the enzyme activity. The addition of Ca²⁺ (10–40 mM) or surfactants (Tween 20, Tween 40, Tween 60, Tween 80, and sodium lauryl sulphate at 0.02% concentration) in culture medium did not result in further improvement in the enzyme production. The purified enzyme had a molecular mass of 205 kDa in native SDS-PAGE.     

Extracellular Pectinase Production and Purification from a Newly Isolated Bacillus subtilis Strain

Pectinase is widely used in the fruit juice industry with various aspects, especially to reduce blur in fruit juice. To produce pectinase, Bacillus subtilis were isolated from Kilis soil contaminated by agricultural crop wastes. The extracellular pectinase was purified by using three different technics (cell-free supernatant, ethanol, and ammonium sulphate precipitation) and enzyme activity was measured with spectrophotometric analysis. The maximum specific activity was observed in enzyme preparate purified by ethanol precipitation, 217.44 U/mg. The degradation products of reaction–glucose, saccharose, and fructose–were identified by Fourier transform infrared spectroscopy and thin layer chromatography analysis. Two protein bands belong to pectinase of B. subtilis were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Molecular weights of bands were approximately calculated as 60 and 64 kDa.     

COMBINED EFFECTS OF ENZYME DOSAGE AND REACTION TIME ON PAPAYA JUICE EXTRACTION WITH THE AID OF PECTIC ENZYMES – A PRELIMINARY REPORT

Pectic enzymes are widely used in the food industry for fruit juice extraction as well as in the clarification of cloudy juices. Our laboratory has been using pectic enzymes produced from a strain of Saccharomyces cerevisiae (ATCC 52712) in different applications including fruit juice extraction. The enzyme was produced in the laboratory by culturing the yeast in papaya juice supplemented with 1% pectin for 6 days. Known amounts of enzyme preparation (0–40 mg protein) were added to a measured weight of papaya mash for varying reaction periods (30–90 min) and the amount of free‐run juice obtained in each treatment compared with a control sample. Treatment of 200 g of papaya mash with different dosages of the pectic enzyme extract resulted in rapid increases in flow rate of free‐run juice. Mash treated with 32 mg of total protein extract with a 30‐min reaction time was the optimum for a maximum rate of juice flow (25 mL/min) when initial rates were measured. There was no significant difference (P > 0.05) between the red‐ and yellow‐fleshed varieties of the papaya used. When juice flow was monitored over 6 min, the treated samples gave a flow rate that was more than twice those of the untreated samples. This biotechnological approach could be adopted to enhance papaya‐juice production by local fruit juice processors when parameters for scale‐up processes are established.     

Phenolic Profile and Antioxidant Activity of Extracts Prepared from Fermented Heat‐Stabilized Defatted Rice Bran

Heat‐stabilized, defatted rice bran (HDRB) serves as a potential source of phenolic compounds which have numerous purported health benefits. An estimated 70% of phenolics present in rice bran are esterified to the arabinoxylan residues of the cell walls. Release of such compounds could provide a value‐added application for HDRB. The objective of this study was to extract and quantify phenolics from HDRB using fermentation technology. Out of 8 organisms selected for rice bran fermentation, Bacillus subtilis subspecies subtilis had the maximum phenolic release of 26.8 mg ferulic acid equivalents (FAE) per gram HDRB. Response surface methodology was used to further optimize the release of rice bran phenolics. An optimum of 28.6 mg FAE/g rice bran was predicted at 168 h, 0.01% inoculation level, and 100 mg HDRB/mL. Fermentation of HDRB for 96 h with B. subtilis subspecies subtilis resulted in a significant increase in phenolic yield, phenolic concentration, and radical scavenging capacity. Fermented rice bran had 4.86 mg gentistic acid, 1.38 mg caffeic acid, 6.03 mg syringic acid, 19.02 mg (‐)‐epicatechin, 4.08 mg p‐courmaric acid, 4.64 mg ferulic acid, 10.04 mg sinapic acid, and 17.59 mg benzoic acid per 100 g fermented extract compared to 0.65 mg p‐courmaric acid and 0.36 mg ferulic acid per 100 g nonfermented extract. The high phenolic content and antioxidant activity of fermented HDRB extract indicates that rice bran fermentation under optimized condition is a potential means of meeting the demand for an effective and affordable antioxidant.     

Enhanced Antioxidative Activity of Chinese Traditionally Fermented Okara (Meitauza) Prepared with Various Microorganism

In order to investigate the antioxidative activity of Meitauza koji (Meitauza, a Chinese traditionally fermented okara), the okara was fermented with Aspergillus oryzae 3.951, Rhizopus oligosporus 3.912, Actinomucor elegans 3.118 and Bacillus subtilis B2 as kojis. All the methanol extract yields and peptide content of the kojis were higher than that of non-fermented okara. Moreover, the Meitauza kojis displayed enhanced antioxidative activities in comparison with the non-fermented okara. Among the four kinds of kojis tested, those fermented with B. subtilis B2 exhibited the highest levels of 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals scavenging activities and reducing power. The ABTS and DPPH radicals scavenging activities of Meitauza koji fermented with B. subtilis B2 were ca. 12.53 and 7.92 folds that of the non-fermented okara. Analysis of the dose-response effect also revealed that before reaching a threshold point, there is a linear relationship between increases in antioxidative activity and increases in the concentration of koji extract. In addition, the peptide content of the Meitauza koji prepared with B. subtilis B2 increased from 8.6 mg to 34 mg per 100 mg extract. These results suggest the potential for developing traditional functional food with okara fermented by B. Subtilis B2.     

Bio-scouring process optimization of wool fiber and wastewater utilization

This paper is aimed at developing a method to optimize the wool‐scouring process with bio‐enzymes of Bacillus subtilis and Candida lipolytica. Good experimental conditions, first determined by single factor test, were the ratio of B. subtilis to C. lipolytica 1:4, temperature 40°C, pH 7.0, enzyme consumption 6%, bath ratio 1:35, and time 16 h. Based on these conditions, the Plackett–Burman design (P–B design) was used to determine the main influencing factors such as bath ratio, temperature, and time for enzymatic scouring. Then, a Response Surface Method (RSM) was used to optimize these factors, and the optimum parameters were solved with the quadratic regression equation: bath ratio 1:33.28, temperature 40.44°C, and time 18.11 h. Under these optimum conditions, the residual fat content in bio‐scoured wool was 0.75%. Furthermore, the wool surface was smoother and cleaner than the non‐optimized one. In addition, the lanolin was reclaimed as a valuable material in industry by centrifugation from wastewater, and it could reduce the content of organic substance to decrease the environmental pollution. Moreover, the residual lanolin, perspiration, dung, chaffy, short wool, and silt were utilized to make ecological organic fertilizer by compost treating. It could achieve the complex utilization of bio‐scouring wastewater.     

Fungi associated with post-harvest rot of sweet orange (Citrus sinensis) and aflatoxin B1 production by isolates of Aspergillus flavus on plain and supplemented orange juice

Samples of rotting sweet orange (Citrus sinensis) were obtained from the depots, sales counters and waste baskets. Fungi associated with rotting fruits were isolated and identified. Out of 12 species of fungi isolated, 8 are known to be producers of toxins. The 7 isolates of Aspergillus flavus obtained were screened for aflatoxin production in a nutrient solution, and 4 were found to be aflatoxigenic, producing primarily aflatoxin B₁. Aflatoxin B₁ production of the toxigenic isolates were further studied on plain juice and juice separately supplemented with 2.0% yeast extract and 2.0% sucrose. The highest yield of aflatoxin B₁ was produced on juice supplemented with yeast extract by the 4 toxigenic A. flavus isolates, followed by sucrose supplementation while the lowest amount of aflatoxin B₁ was detected on plain juice. Optimum temperature for aflatoxin B₁ production by A. flavus isolate (IBA-O1) was 25 °C to 30 °C, for an incubation period of 7–11 days on plain and supplemented juice media.     

A Maltooligosaccharides Producing α-Amylase from Bacillus subtilis SDP1 Isolated from Rhizosphere of Acacia cyanophylla Lindley

Maltooligosaccharides producing amylases are required in the food industry, especially in breadmaking. The Bacillus subtilis strain SDP1 amylase hydrolyses starch to produce maltotriose and maltotetraose along with maltose after prolonged reactions of 5 h. Bacillus subtilis strain SDP1 was isolated from the rhizosphere of Acacia cyanophylla Lindley from the Çukurova region of Turkey. The highest enzyme production was achieved with soluble starch as the carbon and yeast extract as the nitrogen source and at pH 7.0 and 37°C. Under optimized culture conditions, 68.49 U/mL activity was obtained. SDP1 α-amylase had molecular weight of 61 kD. The optimum pH of the enzyme was 7.0 and was highly active at pH ranging from 5.0 to 9.0. The optimum temperature of the crude enzyme was 60°C, and it retained 83% and 74% of its initial activity after 1 h and 2 h incubation periods, respectively, at 50°C. While, Mn⁺² has a stimulatory effect on the activity, Ca⁺², Mg⁺², Na⁺ did not effect the enzyme activity. Fe⁺³, Ni⁺², Cu⁺² and Co⁺² had an inhibitory effect on SDP1 amylase activity.     

Media optimisation for production of lactic acid by lactobacillus plantarum NCIM 2084 using response surface methodology

Abstract

For the production of lactic acid by Lactobacillus plantarum NCIM 2084, response surface methodology (RSM) using central composite rotatable design (CCRD) was adopted for optimising the media composition with respect to liquefied starch, wheat bran extract, ammonium nitrate and sodium acetate. A set of 28 experiments were carried out and cannonical analysis was employed. Significant (p ≤ 0.25) effects were observed for the first three variables. Quadratic and interaction effects of the variables were more prominent than the linear effects. The respective optimum concentrations obtained were 9.99, 0.05, 2.48 and 0.08% giving a substrate conversion of 92% and a lactic acid concentration of 72.9 g/L in 24 h. It has been possible to replace the expensive yeast extract by. a combination of wheat bran extract and ammonium nitrate.     

Effect of an additionally introduced degQ gene on di-d-fructofuranosyl 2,6′:2′,6 anhydride (DFA IV) production by recombinant Bacillus subtilis in a single culture production system

Di-d-fructofuranosyl 2,6′:2′,6 anhydride (DFA IV) was produced directly from sucrose using a single culture of recombinant Bacillus subtilis 168 carrying the levan fructotransferase (lft) gene. In this study, three plasmids carrying the degQ36 gene, which is a degQ allele of B. subtilis (degQ36) with a degQ36 mutation on its promoter, were constructed to overproduce intact DegQ in B. subtilis 168. The transformant B. subtilis/pHT-D36 (with the degQ36 gene) consumed sucrose and produced levan at a higher rate than B. subtilis/pHT43 (without the degQ36 gene). The transformant B. subtilis/pLFT-GD36, carrying the lft and degQ36 genes, also consumed sucrose at a higher rate and produced more DFA IV than B. subtilis/pLFT-G, carrying the lft but without the degQ36 gene. B. subtilis/pLFT-GD36 produced 43.5 g/l of DFA IV and consumed 240 g/l of sucrose (96% of added sucrose) by 72 h of cultivation, whereas B. subtilis/pLFT-G produced 23.4 g/l of DFA IV with 76.9 g/l of sucrose still remaining in the system. Sucrose-inducible expression vectors were also constructed, which made it possible to produce DFA IV without IPTG induction. Using these vectors, sucrose consumption rates were enhanced and DFA IV production was increased upon introduction of the degQ36 gene. From these results, it can be concluded that the additionally introduced regulatory gene, degQ, was able to stimulate sucrose conversion to levan, and therefore increased DFA IV production in this system.     

Orange Finisher Pulp as Substrate for Polygalacturonase Production by Rhizopus oryzae

Orange finisher pulp, a juice processing by-product, was investigated as a substrate for polygalacturonase (PG) production by Rhizopus oryzae (ATCC 24563) via solid state fermentation. PG activity was indicated by increase in liquefaction and uronic acid solubilization and subsequent decrease in pectin content of the solid fraction over a 5-day fermentation. Partial characterization of the enzyme extract indicated pH optimum 5.0 for PG. Viscosity and reducing sugar measurements suggested endohydrolytic activity. Little or no pectinesterase, pectin or pectate lyase activities were detected; therefore, potential for direct utilization of the PG exists in the citrus industry. Orange finisher pulp was a suitable substrate for fungal PG production.     

Production of L(+) lactic acid from sweet sorghum,date palm,and golden syrup as alternative carbon sources

Lactic acid production has been carried out using (a) Lactobacillus delbruckii, (b) optimal production medium comprised of 10% carbon source derived from sweet sorghum (SS) (Sorghum bicolor)/golden syrup (GS; molasses after glucose crystallization)/date palm (DP) juice (Phoenix dactylifera L.), 1% yeast extract, 0.6% sodium acetate, 0.5% KH₂PO₄, and 0.5% MgSO₄ · 7H₂O, (c) in batch mode, (d) at 45 ± 1°C, 150 rpm, anaerobic condition, and pH 5.5 ± 0.1, and (e) sterilized CaCO₃ powder at regular intervals for neutralization to promote optimal utilization of sugar. The lactic acid (LA) productivity was higher from the use of GS and less from SS as well as DP juice. Decolorization of carbon sources prior to use in fermentation gave LA, meeting food grade specifications.     

Comparison of Effect of Gear Juicer and Colloid Mill on Microstructure,Polyphenols Profile,and Bioactivities of Mulberry (<Emphasis Type="Italic">Morus indica L</Emphasis>.)

Mulberry (Morus indica L.) fruits are rich sources of polyphenols with multiple health benefits. The gear juicer and colloid mill were compared in mulberry juice production and further marc water extraction based on polyphenol yield, phytochemical profiles, bioactivities, as well as microstructure of residues. Higher content of phenolic compounds, stronger antioxidative, and α-Glucosidase inhibition activities were obtained in colloid mill-processed juice. The extraction kinetics of marc was investigated under three conditions (stirring, ultrasound, and combined stirring and ultrasound) at 25 °C for 2.5 h. Based on total polyphenol and monomeric anthocyanin yield, the optimum extraction condition for gear juicer marc was combination of ultrasound with stirring for half an hour. While ultrasound for 1 h was optimized for water extraction of colloid mill marc. Using the optimized extraction condition, total polyphenols and monomeric anthocyanin obtained from colloid mill-processed samples, including juice and marc extract, were approximately 46 and 68 % higher than those in gear juicer-processed samples. Compared to gear juicer, colloid mill processing generated smaller particle size and more disrupted microstructure, which contributed to higher content of phenolic compounds in colloid mill juice. Moreover, changes in the microstructure of mulberry marc were intensified by extraction conditions, which improved polyphenol extraction efficiency, especially for colloid mill marc. These results provided valuable information for comparing gear juicer and colloid mill in mulberry as well as other berries for juice production and marc water extraction, and emphasized the great potential of colloid mill in berry processing.     

Effect of Culture Variables on Mycelial Arachidonic acid Production by Mortierella alpina

Effect of culture conditions on biomass, lipid, and arachidonic acid production was investigated in the oleaginous fungus Mortierella alpina CBS 528.72 under shake flask conditions. Several factors have been found to affect the biomass buildup and lipogenesis in this fungus, complicated by the fact that different strains demonstrate varying optimization conditions. Growth, lipid accumulation, and arachidonic acid production in the strain investigated were influenced by media, pH, temperature, carbon source, nitrogen source, etc. The results indicated that the most effective medium for growth and arachidonic acid production was glucose yeast extract medium. The optimum pH and temperature were found to be 6.5 and 28°C, respectively. On the same weight basis, glucose was the most efficient carbon source for biomass and lipid production in this fungal strain which yielded 6.8 g/L dry biomass and 40.2% (w/w) total lipid after 7 days of cultivation. Maximum arachidonic acid (ARA) production of 40.41% achieved in rhamnose-containing media was not concomitant with higher biomass and lipid yields. Efficacy of organic carbon sources, viz, yeast extract and peptone over inorganic sources like sodium nitrate, ammonium sulfate, ammonium chloride, etc, was established in the present study. M. alpina CBS 528.72 grown in peptone acquired the highest lipid content (42.0% (w/w)). However, the ARA content (28.74%) proved to be significantly less than that grown in yeast extract (35.28%). Furthermore, it was found that the biomass and ARA production declined drastically in a medium with vegetable oils as the sole carbon source but triggered the lipogenic pathway leading to higher accumulation of total lipids. Under the ideal conditions mentioned above, the maximum biomass, total lipid, and arachidonic acid production were 6.8 g/L, 41.6%, and 35.28% total fatty acid, respectively, in shake flask system.     

Safe preparation of beefy meaty peptide with Bacillus subtilis

Preparation of flavour peptides with microorganisms is an attractive choice for its large-scale production. However, beefy meaty peptide (BMP), as a research hotspot in the field of umami peptide, has been studied in few microorganisms, and furthermore, the safe preparation of BMP has not yet been reported. In this study, multi-copy BMP (8BMP) was successfully expressed and produced in the ‘generally recognized as safe’ Bacillus subtilis (B. subtilis). Firstly, 8BMP with the potentially intense umami was screened out based on molecular docking analysis. Then, it was successfully expressed in the B. subtilis 168 and verified through sensory evaluation. Finally, the scaling capacity of 8BMP was evaluated in a 5-L fermenter, with the highest yield (0.84 g L⁻¹) 6.4 times than that of shake flask, which is conductive for industrial production of BMP and other food peptides.     

Use of Bacillus subtilis to enrich isoflavone aglycones in fermented natto

BACKGROUND: Natto is a food made by fermenting cooked soybeans with Bacillus subtilis. Soybean isoflavones are reported to provide many health benefits, including oestrogenic effects. However, isoflavone aglycones may be absorbed faster and in higher amounts in the human intestine than their glucosides. This study aimed to investigate the content of isoflavone components in commercial natto products as well as the use of B. subtilis strains to ferment cooked soybeans to produce a high level of isoflavone aglycones in natto. RESULTS: The content and composition of isoflavones in commercial natto products were predominantly (>76%) isoflavone glucosides. Fermentation of cooked soybeans with B. subtilis BCRC 14718 at 37 °C for 48 h was more effective in converting glucosides to aglycones than with other strains of B. subtilis, increasing the proportion of isoflavone aglycones from 12 to 68% of the total isoflavones in the fermented natto. The proportions of the isoflavone aglycones daidzein and genistein in cooked soybeans fermented with B. subtilis BCRC 14718 for 48 h increased from 6 to 54% and from 5 to 13% respectively. CONCLUSION: Bacillus subtilis BCRC 14718 incubated with cooked soybeans produces higher levels of isoflavone aglycones, which may enhance health benefits over traditional fermented natto. Copyright © 2008 Society of Chemical Industry     

Evaluation of in vitro probiotic potential of phytase‐producing bacterial strain as a new probiotic candidate

Sixty‐three phytase‐producing bacterial strains were isolated from natural sources, and their probiotic potential was evaluated. Of these, only fifteen strains were selected on the basis of confirmatory plate assay. Among these, five phytase‐producing strains exhibiting potent probiotic properties were identified as Bacillus cereus P1, Bacillus subtilis P6, B. subtilis P7, Pseudomonas aeruginosa P12 and P. aeruginosa P15 on the basis of morphological, biochemical and molecular characteristics. Maximum phytase activity (2.74 EU mL^?1) with potential probiotic properties, that is more than 70% survivability at pH 2.0, up to 2.0% bile salt tolerance, sporulation efficiency of more than 80% and survival in anaerobic condition (94.31%), was revealed by B. subtilis P6 as compared to the well‐established commercial probiotic strains Lactobacillus sporogenes and Lactobacillus casei. Thus, phytase‐producing B. subtilis P6 with promising probiotic features can be used in food and animal feed applications for betterment of mankind after further validation.     

Enzymatic synthesis of glycosylated puerarin using maltogenic amylase from Bacillus stearothermophilus expressed in Bacillus subtilis

BACKGROUND: The maltogenic amylase from Bacillus stearothermophilus (BSMA) is a valuable biocatalyst that has been used to transglycosylate natural glycosides to improve solubility. To ensure safety, BSMA was produced in Bacillus subtilis, using new shuttle vector‐based expression vectors. The transglycosylation of puerarin was also conducted with crude BSMA and analyzed. RESULTS: Two expression systems, each containing one of the promoters from the genes encoding Bacillus licheniformis maltogenic amylase (BLMA) and an α‐amylase from B. subtilis NA64 (amyR2), were constructed. The amyR2 promoter system was chosen as the best system; it yielded 107 mg of pure BSMA from a 2 L culture. In the transglycosylation reactions of puerarin using crude BSMA, relative amounts for maltosyl‐α‐(1 → 6)‐puerarin, glucosyl‐α‐(1 → 6)‐puerarin, glucosyl‐α‐(1 → 3)‐puerarin, and puerarin were determined as 26:18:7:49. A two‐step purification process, including gel permeation chromatography, yielded 1.7 g of the transfer products from 3 g of puerarin. CONCLUSION: The crude BSMA produced from a host generally recognized as safe (B. subtilis) can be used to transglycosylate various functional compounds. The expression system developed in this study will be helpful for the production of other food‐grade enzymes by B. subtilis. Copyright © 2010 Society of Chemical Industry     

Incidence ofBacillus cereusandBacillus subtilisin foods in the Netherlands

The incidence ofB. cereusandB. subtilisin various food products in the Netherlands was investigated. In total, 229 samples (milk, yeast, flour, pasta products, Chinese meals, cocoa, chocolate, bakery products, meat products, herbs and spices) were analysed. Of these 109 (48%) containedB. cereus. The contamination level ranged from 10²–10⁶bacteria g⁻¹or ml⁻¹B. subtiliswas present in 18 of 72 (25%) samples examined, the levels varied from 10²–10⁵bacteria g⁻¹. From 12 of these 72 (17%) samples, bothB. cereusandB. subtiliscould be isolated. In total, 91 presumptiveB. cereuscolonies were isolated from the various samples. According to the ISO conformation tests and the carbohydrate patterns (API 50 CHB) 81 (89%) of these isolates were confirmed to beB. cereus. Of the 50 suspectB. subtiliscolonies, 10 isolates were shown to beB. pumilisbased on inability to ferment sorbitol and carbohydrate utilization pattern in the API 50 CHB. Of the remaining 40 isolates 30 (75%) were determined to beB. subtilisaccording to the confirmation tests and the carbohydrate patterns, four (10%) wereB. licheniformisand six (15%) were shown to beB. amyloliquefaciens. TheseBacillusspecies, belonging to the `B. subtilisgroup', are also suspected as food-poisoning agents. Numbers ofB. subtilisappeared to decrease more readily by processing and storage of food products than those ofB. cereus. This phenomenon, and the fact that direct isolation methods forB. subtilisare not available, indicate that the role ofB. subtilisin food spoilage and foodborne infections and intoxication may be underestimated.