Vitamin B12 biofortification of soymilk through optimized fermentation with extracellular B12 producing Lactobacillus isolates of human fecal origin.

The present study was designed to bio-fortify the soymilk (per se a B12-free plant food matrix). The PCR-based screening characterized the human fecal samples (4 out of 15 tested) and correspondingly identified novel lactobacilli isolates (n = 4) for their B12 production potential and rest (n = 62) as negative for this attribute. Further, 3 out of the 4 selected strains showed ability for extracellular vitamin production. The most prolific strain, Lactobacillus reuteri F2, secreted B12 (132.2 ± 1.9 µg/L) in cobalamin-free-medium with the highest ratio ever reported (0.97:1.00; extra-: intra-cellular). In next stage, the soymilk was biofortified in situ with B12 during un-optimized (2.8 ± 0.3 µg/L) and optimized (156.2 ± 3.6 µg/L) fermentations with a ∼54-fold increase at Artificial Neuro Fuzzy Inference System based R value of >0.99. The added-nutrients, temperature and initial-pH were observed to be the most important fermentation variables for maximal B12 biofortification. We report Lactobacillus rhamnosus F5 as the first B12 producing (101.7 ± 3.4 µg/L) strain from this species. The cyanocobalamin was extracted, purified and separated on UFLC as nutritionally-relevant B12. Besides, the vitamin was bioavailable in an auxotrophic-mutant. The lactobacilli fermentation is suggested, therefore, as an effective approach for B12 biofortification of soymilk.

Effect of different fat and protein levels in calf ration on performance of Sahiwal calves.

OBJECTIVE: The current study was carried out to examine the response of different levels of fat and protein in calf starter on nutrient utilisation, nitrogen metabolism, weight gain, blood parameters and immunity level in pre-ruminant calves. METHODS: Twenty four calves (5 days old) were divided into six groups in a 2x3 factorial design, with two levels of fat (10% and 14 %) and three levels of protein (18%, 21% and 24%). The calves were kept in individual pens for 120 days and fed with whole milk (1/10th of body weight) and calf starter ad-libitum. Daily dry matter intake (DMI) was recorded; whereas body weight was taken on fortnightly basis to calculate average daily gain (ADG). During the growth trial blood samples were collected at 30 days interval to estimate blood glucose, albumin, total protein, total leucocyte count, total immunoglobulin and IgG levels. A metabolic trial of seven days was carried out to find out the digestibility of different nutrients. RESULTS: The DMI was reduced (p<0.05) with higher fat and protein levels whereas feed conversion efficiency (FCE) was improved (p<0.05) with higher protein level. Different levels of fat and protein in calf ration did not affect average daily gain in calves.The dry matter (DM), organic matter (OM) and crude protein (CP) digestibility were significantly (p<0.01) higher with increased level of protein. The nitrogen retention was also significantly higher (p<0.05) at 24 % protein level, similarly the total immunoglobulin was significantly (p< 0.05) high in higher protein fed groups, showed better immunity. CONCLUSION: The present finding suggested that 10 % fat and 18 % protein level of calf starter could be used in Sahiwal calves for optimum performance in terms of weight gain and immunity.

Glutathione biosynthesis and activity of dependent enzymes in food-grade lactic acid bacteria harbouring multidomain bifunctional fusion gene (gshF).

AIMS: To assess glutathione (GSH) biosynthesis ability and activity of dependent enzymes in food-grade lactic acid bacteria (LAB) and correlating with genomic information on GSH system in LAB. METHODS AND RESULTS: Whole-genome sequences of 26 food-grade LAB were screened for the presence/absence of a set of genes involved in de novo GSH system. Multiple strains of Streptococcus thermophilus (37), Lactobacillus casei (37), Lactobacillus rhamnosus (4), Lactobacillus paracasei (8) Lactobacillus plantarum (23) and Lactobacillus fermentum (22) were screened for biochemical evidence of the GSH system. Multiple sequence analysis of GshF sequences was carried out for comparing the genomic signatures between GSH-producing and nonproducing species. CONCLUSIONS: Streptococcus thermophilus was found to have de novo GSH biosynthesis as well as import ability. Lactobacillus sp. were negative for GSH synthesis but could import it from the medium. All the species exhibited prolific GSH reductase and peroxidase activity. Sequence analysis revealed the absence of key amino acid residues as well as a truncated N-terminal region in lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides a comprehensive view on the status of an important antioxidative system (the GSH system) in LAB and is expected to serve as a primer for future work on the mechanistic role of GSH in the group.

Effect of developed acidity and neutralization of milk on sensory, microstructural and textural changes in khoa prepared from cow and buffalo milk.

Khoa is a heat desiccated milk product which serves as base material for traditional dairy based Indian sweets. Sensory, textural and microstructural changes in khoa were studied to assess the effect of developed acidity and neutralization of milk. Noticeable changes were observed in sensory, textural and micro structural quality of khoa as affected by quality of milk. Lower hardness, springiness, cohesiveness and accordingly, gumminess and chewiness were observed in khoa manufactured using neutralized milk due to higher moisture retention as compared to control and acidic milk khoa. It was also evident from the Scanning Electron Microscopy that higher moisture retention in neutralized milk khoa resulted in elongation of native microstructure of khoa by suppressing the grain formation leading to smooth and plain surface while, developed acidity in milk resulted in greater granule formation in the resulting khoa causing shrinkage of native microstructure and acidic flavour in khoa. Similarly, acidification and subsequent neutralization of milk adversely affected sensory attributes of resulting khoa.

Techno-functional differentiation of two vitamin B12 producing Lactobacillus plantarum strains: an elucidation for diverse future use.

An appropriate selection of Lactobacillus strain (probiotic/starter/functional) on the basis of its techno-functional characteristics is required before developing a novel fermented functional food. We compared vitamin B12 (B12, cobalamin) producing Lactobacillus plantarum isolates, BHM10 and BCF20, for functional (vitamin over-production, genomic insight to B12 structural genes, and probiotic attributes) and technological [milks (skim and soy) fermentation and B12 bio-fortification] characteristics. Addition of B12 precursors (5-amonolevulinate and dimethylbenzimidazole) to cobalamin-free fermentation medium increased vitamin production in BHM10, BCF20, and DSM20016 (a positive standard) by 3.4-, 4.4-, and 3.86-folds, respectively. Three important B12 structural genes were detected in L. plantarum species (strains BHM10 and BCF20) by PCR for the first time. The gene sequences were submitted to NCBI GenBank and found phylogenetically closer to respective sequences in B12 producing Lactobacillus reuteri strains. During comparative probiotic testing, BCF20 showed significantly higher (p < 0.05 to p < 0.001) gastrointestinal tolerance and cell surface hydrophobicity (p < 0.05) than BHM10. Moreover, only BCF20 was found positive for BSH activity and also exhibited comparatively better antagonistic potential against potent pathogens. Conversely, high acid and bile susceptible strain BHM10 displayed significantly higher soy milk fermentation and resultant B12 bio-fortification abilities during technological testing. Two B12 quantification techniques, UFLC and competitive immunoassay, confirmed the in vitro and in situ bio-production of bio-available form of B12 after BHM10 fermentation. Conclusively, techno-functional differentiation of two B12 producing strains elucidates their diverse future use; BCF20 either for B12 over-production (in vitro) or as a probiotic candidate, while BHM10 for cobalamin bio-fortification (in situ) in soy milk.

Rayleigh-type waves in nonlocal micropolar solid half-space.

Propagation of Rayleigh type surface waves in nonlocal micropolar elastic solid half-space has been investigated. Two modes of Rayleigh-type waves are found to propagate under certain approximations. Frequency equations of these Rayleigh type modes and their conditions of existence have been derived. These frequency equations are found to be dispersive in character due to the presence of micropolarity and nonlocality parameters in the medium. One of the frequency equations is a counterpart of the classical Rayleigh waves and the other is new and has appeared due to micropolarity of the medium. Phase speeds of these waves are computed numerically for Magnesium crystal and their variation against wavenumber are presented graphically. Comparisons have been made between the phase speeds of Rayleigh type waves through nonlocal micropolar, local micropolar and elastic solid half-spaces.

Encapsulation of antioxidant peptide enriched casein hydrolysate using maltodextrin-gum arabic blend.

Antioxidant peptide enriched casein hydrolysate (AO-CH) are receiving increasing attention due to their potential as functional ingredient. Encapsulation of AO-CH using maltodextrin-gum arabic (MD/GA) as wall material could represent an attractive approach to overcome the problems related to their direct application. Encapsulation parameter were optimized using different ratio of core to coat and proportion of coating material (10:0, 8:2, 6:4) under varying pH (2-8) for encapsulation efficiency (EE).The preparation P3 resulted in maximum EE (87%) using core to coat ratio 1:20, at pH 6.0 with 8:2 MD/GA ratio. The encapsulated preparation showed reduced bitterness (p < 0.05) compared to the casein hydrolysate together with maximum retention of antioxidant activity (93%). Further, the narrow range of particle size, indicates their better stability and represents a promising food additive for incorporation in food.

Pathogen-induced secretory diarrhea and its prevention.

Secretory diarrhea is a historically known serious health implication around the world which primarily originates through pathogenic microorganisms rather than immunological or genetical disorders. This review highlights infective mechanisms of non-inflammatory secretory diarrhea causing pathogens, known therapeutics and their efficacy against them. These non-inflammatory diarrheal pathogens breach cell barriers, induce inflammation, disrupt fluid secretion across the epithelium by alteration in ion transport by faulting cystic fibrosis transmembrane conductance regulator (CFTR), calcium activated chloride channels and ion exchanger functions. Currently, a variety of prevention strategies have been used to treat these symptoms like use of antibacterial drugs, vaccines, fluid and nutritional therapy, probiotics and prebiotics as adjuncts. In progression of the need for a therapy having quick physiological effects, withdrawing the symptoms with a wide and safe therapeutic index, newer antisecretory agents like potent inhibitors, agonists and herbal remedies are some of the interventions which have come into light through greater understanding of the mechanisms and molecular targets involved in intestinal fluid secretion. Although these therapies have their own pros and cons inside the host, the quest for new antisecretory agents has been a successful elucidation to reduce burden of diarrheal disease.

Phenotypic and genotypic screening of human-originated lactobacilli for vitamin B12 production potential: process validation by micro-assay and UFLC.

Vitamin B12 (B12) production is a strain specific, rare and hidden functional attribute of lactobacilli and a cogent protocol for selection of such isolates from the herd of lactobacilli is required. The present study included isolation of lactobacilli from human samples (milk and fecal), screening them by a polyphasic (three-phase) methodology for probable B12 production potential and validating the screening protocol by exploring selected strains for in vitro vitamin production (two-phase fermentation) and quantification [micro-assay and ultra fast liquid chromatography (UFLC)]. Fifty-nine Lactobacillus strains were recovered from tested biological samples. Contrary to screening inapplicabilities of first [growth potential (GP) in B12-free medium] and second phases (GP in B12-free and cobalt chloride-supplemented conditions), third phase (cbiK gene detection on genomic DNA) alone was revealed as a validated strategy for selection of two probable B12-producing lactobacilli. Microbiological assay confirmed production and bioavailability of produced vitamin, while UFLC testing validated the results by precisely quantifying the cyanocobalamin (industrially produced bio-available form of B12) in cell extracts of both possible B12 producers [BHM10 (10.91 ± 1.55 µg/l) and BCF20 (23.90 ± 1.73 µg/l)] and positive standard [Lactobacillus reuteri DSM20016 (20.03 ± 4.17 µg/l)]. Moreover, this study generates a novel report for genomic detection, partial amplification and sequencing of cbiK gene in Lactobacillus plantarum species (both BHM10 and BCF20). In conclusion, contrary to first two phases, cbiK gene detection strategy successfully selects B12-producing strains from a group of human-originated lactobacilli and can be used in the future for similar screening studies.

Probiotic and functional characterization of bifidobacteria of Indian human origin.

AIMS: To identify and characterize the probiotic and functional attributes of bifidobacteria isolated from human sources by in vitro methods. METHODS AND RESULTS: Twelve isolates of bifidobacteria were obtained from different human sources viz; human milk, infant and adult faeces. The preliminary identification of isolates was done using genus-specific PCR followed by species level identification using 16S rRNA sequencing. All the isolates invariably showed potential probiotic characteristics. Finally, three most promising isolates were subjected to safety evaluation and were found to be safe. These were further evaluated for their potential functional characteristics like, antioxidative, antimutagenic and cholesterol assimilation. The isolates viz; NBIF-5 (73·55 ± 0·03%) and NBIF-7 (64·06 ± 0·03%) assimilated significantly higher cholesterol than NBIF-2. The maximum antioxidative activity was observed in NBIF-2 (56·56 ± 0·28% of radical inhibition). High percentage antimutagenicity scores of 52·41 ± 2·25% and 53·68 ± 1·98% against sodium azide (NaN3) were shown by NBIF-2 and NBIF-7 respectively. CONCLUSIONS: Three strains (NBIF-2, NBIF-5 and NBIF-7) were found to be endowed with appreciable probiotic and functional activities. SIGNIFICANCE AND IMPACT OF THE STUDY: With growing demands for probioitcs and bifidobacteria being prominent ones, new strains of diversified origin have the potential to be explored for commercial and functional applications and thus can be added to the existing commercially available strains of this genera.

Preparation of iron bound succinylated milk protein concentrate and evaluation of its stability.

Major problems associated with the fortification of soluble iron salts include chemical reactivity and incompatibility with other components. Milk protein concentrate (MPC) are able to bind significant amount of iron due to the presence of both casein and whey protein. MPC in its native state possess very poor solubility, therefore, succinylated derivatives of MPC (succ. MPC) were also used for the preparation of protein-iron complex. Preparation of the complex involved centrifugation (to remove insoluble iron), ultrafiltration (to remove unbound iron) and lyophilisation (to attain in dry form). Iron binding ability of MPC enhanced significantly (P<0.05) upon succinylation. Stability of bound iron from both varieties of complexes was monitored under different conditions encountered during processing. Higher stability (P<0.05) of bound iron was observed in succ. MPC-iron complex than native protein complex. This method could be adopted for the production of stable iron enriched protein, an organic iron source.

Rumen modulatory effect of thyme, clove and peppermint oils in vitro using buffalo rumen liquor.

AIM: The present study was conducted to examine the rumen modulatory effect of thyme, clove and peppermint oils on rumen fermentation pattern in vitro using roughage based diet. MATERIALS AND METHODS: Thyme, clove and peppermint oils were tested at concentration of 0, 30, 300 and 600 mg/l (ppm) of total culture fluid using in vitro gas production technique in wheat straw based diet (concentrate: Wheat straw 50:50). Different in vitro parameters e.g., total gas production, methane production, nutrient degradability, volatile fatty acid (VFA) production and ammonia nitrogen concentration were studied using buffalo rumen liquor. RESULTS: Thyme oil at higher dose level (600 ppm) reduced (p<0.05) total gas production, feed degradability and ammonia nitrogen (NH3-N) concentration whereas total VFA concentration was significantly lower (p>0.05) in 300 and 600 ppm dose levels. 600 ppm dose level of clove oil reduced (p<0.05) total gas production, feed degradability, total VFA and acetate to propionate ratio. Methane production was significantly reduced (p<0.05) in 300 and 600 ppm dose levels of clove and peppermint oil. CONCLUSION: Right combination of these essential oils may prove to enhance performance of animals by reducing methane production and inhibiting protein degradation in rumen.

Effect of varying levels of formaldehyde treatment of mustard oil cake on rumen fermentation, digestibility in wheat straw based total mixed diets in vitro.

AIM: The aim of the current study was to protect the protein in mustard cake by different levels of formaldehyde treatment with a view to optimize the level of formaldehyde. MATERIALS AND METHODS: Different levels of formaldehyde treatment (0, 1, 1.5 and 2% of crude protein) containing concentrate and roughages diet in 40:60 ratio were tested for their effect on nutrients digestibility, in vitro ammonia release, in vitro gas production and change in protein fractions. Non-significant (p≤0.05) effect on pH, microbial biomass, partitioning factor, total gas production (TGP), TGP per g dry matter and TGP per g digestible dry matter (ml/g) was observed in almost all the treatments. RESULTS: Total volatile fatty acids at 2% formaldehyde treatment level of mustard cake was lower (p<0.05) as compared to other groups, while in vitro dry matter digestibility and in vitro organic matter digestibility were reported to be low in 1% formaldehyde treated group. CONCLUSION: On a holistic view, it could be considered that formaldehyde treatment at 1.5% level was optimal for protection of mustard oil cake protein.

Multigrid methods for isogeometric discretization.

We present (geometric) multigrid methods for isogeometric discretization of scalar second order elliptic problems. The smoothing property of the relaxation method, and the approximation property of the intergrid transfer operators are analyzed. These properties, when used in the framework of classical multigrid theory, imply uniform convergence of two-grid and multigrid methods. Supporting numerical results are provided for the smoothing property, the approximation property, convergence factor and iterations count for V-, W- and F-cycles, and the linear dependence of V-cycle convergence on the smoothing steps. For two dimensions, numerical results include the problems with variable coefficients, simple multi-patch geometry, a quarter annulus, and the dependence of convergence behavior on refinement levels [Formula: see text], whereas for three dimensions, only the constant coefficient problem in a unit cube is considered. The numerical results are complete up to polynomial order [Formula: see text], and for [Formula: see text] and [Formula: see text] smoothness.

Performance of crossbred calves with dietary supplementation of garlic extract.

Twelve crossbred calves (Holstein cross) in their pre-ruminant stage were used to study the effect of garlic extract feeding on their performance and they were randomly allotted into treatment and control groups in equal number. Performance was evaluated by measuring average body weight (BW) gain, feed intake [dry matter (DM); total digestible nutrient (TDN) and crude protein (CP)], feed conversion efficiency (DM, TDN and CP), fecal score and fecal coliform count. Diets were same for both groups. In addition, treatment group received garlic extract supplementation at 250 mg/kg BW/day/calf. BW measured weekly, feed intake measured twice daily, proximate analysis of feeds and fodders analysed weekly, fecal scores monitored daily and fecal coliform count done weekly. There was a significant (p < 0.01) increase in mean BW gain and feed intake and a significant (p < 0.01) decrease in severity of scours as measured by fecal score in the treatment group compared to the control group. The results suggest that garlic extract can be supplemented to the calves for better performance.

Galactooligosaccharides: novel components of designer foods.

Since introduction of functional foods, commercialization of the traditionally used probiotics has ushered in more followers into the new fraternity of sophisticated, health-conscious consumers. In 1995, this was followed by the first introduction of prebiotics. Prebiotics are defined as "a non-digestible feed supplement, beneficially affecting the host by selectively stimulating growth and/or activity in one or a limited number of bacteria in the colon." The number of new product introductions with prebiotics has steeply increased over the last few years. Paradoxically, probiotics have limited applications as these cannot be used in wide range of food products because of their viability issue. Fortunately, prebiotics do not suffer from any such constraint and can be used in a wide range of food products. Probiotics do not have a long shelf life in their active form. In most cases, refrigeration is required to maintain the shelf life. While probiotics are predominantly used in fermented dairy products, the use of prebiotics has expanded into other food categories. Prebiotics have successfully been incorporated in a wide variety of human food products such as baked goods, sweeteners, yoghurts, nutrition bars, and meal replacement shakes. For instance, the introduction of galacto-oligosaccharides (GOS) into baby foods has been very successful. GOS, which are identical to the human milk oligosaccharides, has emerged with strong clinical support for both digestive and immune health. Various aspects related to GOS such as types and functions of functional food constituents with special reference to GOS, their role as prebiotics, and enhanced industrial production through microbial intervention are dealt in this review.

Effect of exopolysaccharide-producing strains of Streptococcus thermophilus on technological attributes of fat-free lassi.

Sixty-four exopolysaccharide-producing thermophilic lactic acid bacteria (LAB) were isolated from traditionally made Indian fermented milk products. On the basis of morphological and biochemical tests, these isolates were identified as the species of Lactobacillus, Streptococcus, and Enterococcus genera. Initial screening for technological attributes revealed that Streptococcus thermophilus IG16 was a promising isolate, and produced both capsular and ropy polysaccharides at the concentration of 211 mg/L. Exopolysaccharide produced by IG16 was a heteropolysaccharide containing rhamnose and galactose in a ratio of 5.3:1 and had a molecular weight of 3.3 x 10(4) Da. Use of IG16 as a starter culture controlled whey separation and improved viscosity, flavor, consistency, and color and appearance of lassi. Use of IG16 resulted in lassi having optimal acidity, less syneresis, high viscosity, and better scores for flavor, consistency, and color and appearance.

Technological and functional applications of low-calorie sweeteners from lactic acid bacteria.

Lactic acid bacteria (LAB) have been extensively used for centuries as starter cultures to carry out food fermentations and are looked upon as burgeoning "cell factories" for production of host of functional biomolecules and food ingredients. Low-calorie sugars have been a recent addition and have attracted a great deal of interest of researchers, manufacturers, and consumers for varied reasons. These sweeteners also getting popularized as low-carb sugars have been granted generally recommended as safe (GRAS) status by the U.S. Federal Drug Administration (USFDA) and include both sugars and sugar alcohols (polyols) which in addition to their technological attributes (sugar replacer, bulking agent, texturiser, humectant, cryoprotectant) have been observed to exert a number of health benefits (low calories, low glycemic index, anticariogenic, osmotic diuretics, obesity control, prebiotic). Some of these sweeteners successfully produced by lactic acid bacteria include mannitol, sorbitol, tagatose, and trehalose and there is a potential to further enhance their production with the help of metabolic engineering. These safe sweeteners can be exploited as vital food ingredients for development of low-calorie foods with added functional values especially for children, diabetic patients, and weight watchers.

Folate: a functional food constituent.

Folate, a water-soluble vitamin, includes naturally occurring food folate and synthetic folic acid in supplements and fortified foods. Mammalian cells cannot synthesize folate and its deficiency has been implicated in a wide variety of disorders. A number of reviews have dwelt up on the health benefits associated with increased folate intakes and many countries possess mandatory folate enrichment programs. Lately, a number of studies have shown that high intakes of folic acid, the chemically synthesized form, but not natural folates, can cause adverse effects in some individuals such as the masking of the hematological manifestations of vitamin B(12) deficiency, leukemia, arthritis, bowel cancer, and ectopic pregnancies. As fermented milk products are reported to contain even higher amounts of folate produced by the food-grade bacteria, primarily lactic acid bacteria (LAB), the focus has primarily shifted toward the natural folate, that is, folate produced by LAB and levels of folate present in foods fermented by/or containing these valuable microorganisms. The proper selection and use of folate-producing microorganisms is an interesting strategy to increase "natural" folate levels in foods. An attempt has been made through this review to share information available in the literature on wide ranging aspects of folate, namely, bioavailability, analysis, deficiency, dietary requirements, and health effects of synthetic and natural folate, dairy and nondairy products as a potential source of folate, microorganisms with special reference to Streptococcus thermophilus as prolific folate producer, and recent insight on modulation of folate production levels in LAB by metabolic engineering.