Evaluation of nutrient profile, biochemical composition and anti-gastric ulcer potentialities of khambir, a leavened flat bread.

Khambir is a leavened staple food among the native highlanders of Western Himalaya. It is prepared by sourdough fermentation of wheat flour with yeast (YAK) or buttermilk (BAK). Both types of bread were rich in carbohydrate, protein, dietary fiber, containing less fat and gluten, and enriched with lactic acid, vitamins, and minerals. The in vitro digestibility test showed a slow glucose-controlled release potential of khambir that reflected improved content of rapidly digestible starch, slowly digestible starch, resistant starch, and predicted glycemic index. The changes of crystallinity to amorphous structures of starch, content of protein and fatty acid, and accumulation of 17 major metabolites were evaluated through FTIR and GC-MS. The extracts of khambir alleviated cold-induced gastric ulcers in the animal model as it exhibited histoprotective and anti-inflammatory activities. This study demonstrated that the traditional leavened bread khambir is nutritious and can alleviate gastric lesions related to acute mountain sickness.

Elucidation of structural and functional integration of a novel antimicrobial peptide from Antheraea mylitta.

We report here the amino acid sequence of an antimicrobial peptide of Antheraea mylitta (peptide fraction II) effectively killed urinary tract associated MDR E. coli (Dutta et al., 2016), as Gly-Gly-Gly-Gly-Gly-Gly-His-Leu-Val-Ala. The physicochemical and biological properties of this peptide were evaluated by computational analysis and its isoelectric point, grand average of hydropathicity and Boman index values were found to be 6.74, 0.42 and -1.17kcal/mol, respectively. One valid model of peptide fraction II was constructed, that contains two antiparallel ß sheets with a hairpin and appeared as 'U' shaped structure. The glycine rich composition (Gly1, Gly5, Gly6 and Ala10) facilitates mostly for its flexibility or dynamicity, and in its other wing, aggregation prone residues (Leu8, Val9, Ala10) triggered its auto-aggregations when contacted only with the microbial membrane. We employed simulation of peptide binding on the membrane, showed stable and deep insertion of peptide fraction II into the membrane through its hydrophobic tail (up to 3.3±1.46Å). Molecular docking study with Patchdock server revealed that this peptide could interact with the lipid aliphatic chain of 1-palmitoyl-2-oleoyl-phosphoethanolamine (POPE) bilayer and may linked to membrane distortion as we have reported earlier. Further, the studied peptide has been predicted not to exhibit any antigenicity and non-responsive to RBC membrane. These data for the first time provide new insights of an antimicrobial peptide from silkworm A. mylitta and it may serve as the template for the design of novel peptide antibiotics from this group of insect against MDR Gram-negative bacteria.

Enzymatic hydrolyzed feather peptide, a welcoming drug for multiple-antibiotic-resistant Staphylococcus aureus: structural analysis and characterization.

This study aimed to explore the bactericidal activity of a feather-degraded active peptide against multiple-antibiotic-resistant (MAR) Staphylococcus aureus. An antibacterial peptide (ABP) was isolated from the chicken feathers containing fermented media of Paenibacillus woosongensis TKB2, a keratinolytic soil isolate. It was purified by HPLC, and its mass was found to be 4666.87 Da using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) spectroscopy. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of this peptide were 22.5 and 90 µg/ml, respectively. SEM study revealed the distorted cell wall of the test strain along with pore formation. The possible reason for bactericidal activity of the peptide is due to generation of reactive oxygen species (ROS), resulting in membrane damage and leakage of intracellular protein. Complete sequence of the peptide was predicted and retrieved from the sequence database of chicken feather keratin after in silico trypsin digestion using ExPASy tools. Further, net charge, hydrophobicity (77.7 %) and molecular modelling of the peptide were evaluated for better understanding of its mode of action. The hydrophobic region (17 to 27) of the peptide may facilitate for initial attachment on the bacterial membrane. The ABP exhibited no adverse effects on RBC membrane and HT-29 human cell line. This cytosafe peptide can be exploited as an effective therapeutic agent to combat Staphylococcal infections.

Role of probiotic Lactobacillus fermentum KKL1 in the preparation of a rice based fermented beverage.

A dominant lactic acid bacteria, Lactobacillus fermentum KKL1 was isolated from an Indian rice based fermented beverage and its fermentative behavior on rice was evaluated. The isolate grown well in rice and decreased the pH, with an increase of total titratable acidity on account of high yield in lactic acid and acetic acid. The production of α-amylase and glucoamylase by the strain reached plateau on 1st and 2nd day of fermentation respectively. The accumulation of malto-oligosaccharides of different degrees of polymerization was also found highest on 4th day. Besides, phytase activity along with accumulation of free minerals also unremittingly increased throughout the fermentation. The fermented materials showed free radical scavenging activity against DPPH radicals. In-vitro characteristics revealed the suitability of the isolate as probiotic organism. The above profiling revealed that probiotic L. fermentum KKL1 have the significant impact in preparation of rice beer and improves its functional characteristics.

Production of chitin and bioactive materials from Black tiger shrimp (Penaeus monodon) shell waste by the treatment of bacterial protease cocktail.

The main objective of this study was to obtain chitin in pure form from a new crustacean waste material for industrial applications. Black tiger shrimp shell wastes are a rich source of protein and valuable bioactive carbohydrate polymers such as chitin. After removal of carotenoid, Black tiger shrimp shell wastes (BTSHWs) were treated with chemicals and protease enzyme to extract chitin. Box-Behnken response surface methodology was applied to optimize the deproteinization process to obtain chitin. At optimal pH (8.82), temperature (50.05 °C), agitation speed (100.98 rpm), enzyme substrate ratio of 1:8 (wv-1) and 72 h of incubation with Paenibacillus woosongensis TKB2 crude protease cocktail, 80 % deproteinization was found along with 77.28 % recovery of chitin. The valuable oligopeptides were determined by MALDI-TOF analysis and analysis of adequate amount of free amino acids in protein hydrolysate from BTSHW, indicating a high nutritional value used for food, feed or as a nitrogen source in growth medium for microorganisms. The chitin obtained was compared with the commercial chitin using scanning electron microscopy, Fourier transform infrared spectrometer, X-ray diffraction and 13C CP/MAS-NMR. Chitin obtained from crude protease treatment showed comparable physicochemical and structural properties to those of the commercial chitin. The carotenoid obtained after treatment can be used for medicinal purpose.

Microbial, saccharifying and antioxidant properties of an Indian rice based fermented beverage.

Haria, a popular rice based ethnic fermented beverage, is consumed as a staple food and refreshing drink by the vast number of Indian tribal people. In this study, the composition of microbial consortia and the occurrence of some important nutraceuticals during haria preparation were investigated. The quantities of moulds and yeasts were highest at 2nd day, and then declined, but, on the contrary, the quantity of Lactic Acid Bacteria and Bifidobacterium sp. increased concurrently during the course of fermentation. Accumulation of starch hydrolytic enzymes along with different types of malto-oligosaccharides like maltotetrose (26.18µg/gm), maltotriose (28.16µg/gm), and maltose (26.94µg/gm) were also noted. Furthermore, GC-MS analysis indicated the occurrence of pyranose derivatives in the fermented products. The fermented materials showed higher free radicals scavenging activity (82.54%, 4th day) against DPPH radicals. These studies clearly demonstrated that the microbial interaction during fermentation of rice makes it more nutritious, and most likely more beneficial for health.

Production of cellulolytic enzymes by Aspergillus fumigatus ABK9 in wheat bran-rice straw mixed substrate and use of cocktail enzymes for deinking of waste office paper pulp.

Response surface methodology was employed to optimize mixed substrate solid state fermentation for the production of cellulases and xylanase by Aspergillus fumigatus ABK9. Among 11 different parameters, fermentation time (86-88 h), medium pH (6.1-6.2), substrate amount (10.0-10.5 g) and substrate ratio (wheat bran:rice straw) (1.1) had significantly influences on enzyme production. Under these conditions endoglucanase, ß-glucosidase, FPase (filter paper degrading activity) and xylanase activities of 826.2, 255.16, 102.5 and 1130.4 U/g, respectively were obtained. The enzyme cocktail extracted (solid to water ratio of 1:10) from the ferments increased brightness of waste office paper pulp by 82.8% ISO, Ink(D) value by 82.1%, removed chromophores (2.53 OD; A(237)nm) and hydrophobic compounds (1.15 OD; A(465)nm) and also decreased the kappa number to 13.5 from 16.8.

Potentialities of newly isolated Bacillus subtilis and Lactobacillus sp for curd preparation and a comparative study of its physico-chemical parameters with other marketed curds.

Two Bacillus sp. were isolated from the local fermented milk and identified on the basis 16S rRNA sequence profile as Bacillus subtilis AKL1 and by biochemical process as Lactobacillus acidophilus AKL2. These isolates were used as fresh inoculums for curd preparation individually and in combinations. Different physico-chemical and therapeutic properties of the newly prepared curd were examined and compared with marketed local (sweet and sour) and branded (Mother Dairy and Thackar) curds. The total hydrolyzed peptides, free amino acids, lactic acid were significantly higher, whereas, total solid, ash content, syneresis and free reducing sugar were lower in the curd prepared by a mixture of AKL1 and AKL2 (0.5:0.5, v/v). The antioxidant activity against ABTS+, DPPH8, OH* and Fe3+ were also higher in the newly formulated curd. Polyphenols (85.5 microg/g), flavonoids (12.5 microg/g) and free aromatic amino acids contents were also higher in AKL1+AKL2. All these components prevent excess protein oxidation that was revealed by SDS-PAGE. The curd also exhibited potent antimicrobial activity against some entero-pathogens like Clostridium perfringens, Escherichia coli, Shigella dysentery, Vibrio cholerae and Staphylococcus aureus. It can be concluded that the combination of these Lactobacillus sp. will be a fruitful inoculum for the preparation of curd having better health promoting effects.

Low cost single-step purification of endoglucanase from Aspergillus fumigatus ABK-9.

Low cost agro-waste was used as adsorption support for single-step purification of endoglucanase from the culture filtrate of A. fumigatus ABK-9. Among various agro-waste substrates, 1% NaOH pretreated rice bran was proved to be the best for adsorbing about 74.8 and 71.1% of endoglucanase at 4 degrees C and 10 degrees C respectively. Langmuir type adsorption isotherm at 4 degrees C showed maximum adsorption of enzyme at pH 5.0, which was in the range of optimum pH of the enzyme. The rice bran column bound enzyme was maximally eluted by a mixture of acetate buffer (0.05 M, pH 5.5) and ethanol (40%, v/v) at a ratio of 3:2 and a flow rate of 1 mL/min. A 5.52-fold purification of the enzyme was achieved from culture supernatant. The specific activity and recovery yield after purification were 294.0 U/mg and 40.15%, respectively, which were comparable with other contemporary protocols. The homogeneity of the enzyme was tested through sodium dodecyl sulphate polyacrylamide gel electrophoresis and a single band of 56.3 kDa was observed. Zymogram analysis finally confirmed the occurrence of endoglucanase in the single band.

Xylanase isozymes from the newly isolated Bacillus sp. CKBx1D and optimization of its deinking potentiality.

Recycling of civic paper waste by enzyme-based technology is nowadays a point of much concern for pollution-less green environment. In this study, the deinking effectiveness of purified xylanase from a newly isolated bacterium was evaluated for recycling of laser jet paper waste. A potent xylanases-producing bacterium from the microbial consortia of termite gut was isolated, which was further identified on the basis of 16S rRNA sequence as Bacillus sp. CKBx1D. In submerged fermentation condition, the isolate produced the highest level of xylanase (480 U/ml) at 36 h of growth. The extracellular xylanase system comprises of three distinct isozymes (est. Mw 35.28, 28.63, 18.94 kDa). The deinking of laser printed paper waste was performed using the purified enzyme mixture. Whole operational parameters were optimized using the Response Surface Methodology; it was found that at pH 6.8 with 47.2 h of continuous shaking at constant temperature of 35 °C, enzymes showed best deinking activity. After enzyme treatment, the physical properties of the pulp like brightness and ERIC (effective residual ink content) values were enhanced, whereas the pulp opacity was more reduced than the control treatment. Hence, the bacterial isolate and its xylanolytic enzyme system could efficiently be used in recycling paper waste as deinking agent.