Nanoencapsulation of casein-derived peptides within electrospun nanofibres.

BACKGROUND: Bioactive peptides derived from milk proteins are recognized as functional foods, but their consumption is limited by undesirable or bitter flavour, poor stability, and low bioavailability. Electrospinning is a versatile process for encapsulation of various bioactive compounds in the form of nanosized fibres, which can circumvent these disadvantages. This study was aimed at the preparation of casein-derived peptides-loaded nanofibres through electrospinning and characterizing them for fortification of milk. RESULTS: Pullulan at 100, 120, and 140 g kg-1 concentrations was used for electrospinning of peptides. Scanning electron and atomic force micrographs revealed the formation of clean bead-free peptides-loaded pullulan nanofibres at 120 and 140 g kg-1 concentrations with mean diameter of 60.45-133.05 nm and encapsulation efficiency of 72.95-86.04%. Fourier transform infrared spectra and X-ray diffractograms revealed the absence of interactions between the functional groups of pullulan and peptides during electrospinning. The zeta potential of the peptides-loaded nanofibres ranged from -15.6 to -24.6 mV, and the hydrodynamic diameter varied from 118.7 to 256.2 nm. The peptides from electrospun nanofibres showed sustained release to the extent of 75.3% after 8 h in gastrointestinal pH conditions. The release kinetics of peptides from nanofibres was best fitted to a Peppas-Sahlin model (R2  = 0.987), and through diffusion and erosion mechanisms. The antioxidant activity of pure peptides and those from nanofibres was comparable. The physico-chemical qualities of milk fortified with encapsulated peptides did not show noticeable difference either. CONCLUSIONS: From the morphological, ultrastructural, particle size, encapsulation efficiency, release kinetics, and antioxidant activity data, it was inferred that electrospinning could be an effective technique for nanoencapsulation of casein-derived bioactive peptides. These peptides-loaded nanofibres could be used for fortification of milk and milk products. © 2021 Society of Chemical Industry.

Evaluation of vacuum impregnation as a novel approach for soaking of fried Gulabjamun balls.

Vacuum impregnation of sugar syrup into sub-baric fried Gulabjamun was evaluated as a technological approach to prepare a product of most acceptable quality. Sugar syrup concentrations (40, 50 and 60 °Brix) in combination with process time (2, 4 and 6 min) were analyzed for their effect on product quality in terms of its overall acceptability, expansion ratio, hardness, juiciness and sugar content. The effect of the process conditions on the five listed responses during the vacuum impregnation process was evaluated using response surface methodology and modelled using a second order polynomial equation. The optimum combination was obtained as soaking in a syrup of 55 °Brix for 5 min and was experimentally validated for its real time adequacy. The experimental values of the quality parameters thus obtained were found to be in close agreement with the predicted values.

Development of mathematical model for prediction of adulteration levels of cow ghee with vegetable fat using image analysis.

The present study was undertaken to develop a protocol for acquisition and analysis of images of ghee samples to derive mathematical parameters related to adulteration of cow ghee with vegetable fat and to develop a model to predict the adulteration levels. The images acquired using a flatbed scanner were quantified in terms of their pixel intensity, colour, morphological, textural and skeleton parameters using ImageJ software. The selected parameters were measured for images of pure cow ghee and compared with that obtained for ghee adulterated with 5%, 10%, 15% and 20% vegetable fat. The parameters were assessed for their ability to detect the fixed adulteration levels on a discrete scale was assessed using discriminant analysis and the adulteration levels of the samples were correctly classified to the extent of 92.2%. An equation for predicting adulteration levels on a continuous scale using regression analysis (adjusted R 2 value 0.94) was developed, tested and further validated using a fresh data set including a commercially popular market sample of ghee giving a good fit (R 2 value of 0.85).

Effect of enzymatic hydrolysis of starch on pasting, rheological and viscoelastic properties of milk-barnyard millet (Echinochloa frumentacea) blends meant for spray drying.

The influence of enzymatic hydrolysis of starch on the pasting properties of barnyard millet was studied using a rheometer. The effects of blending hydrolyzed barnyard millet wort with milk at different ratios (0:1, 1:1, 1:1.5 and 1:2) on flow and viscoelastic behavior were investigated. From the pasting curves, it was evident that enzymatically-hydrolyzed starch did not exhibit typical pasting characteristics expected of normal starch. The Herschel-Bulkley model fitted well to the flow behaviour data, with coefficient of determination (R(2)) ranging from 0.942 to 0.988. All milk-wort blends demonstrated varying degree of shear thinning with flow behavior index (n) ranging from 0.252 to 0.647. Stress-strain data revealed that 1:1 blend of milk to wort had the highest storage modulus (7.09-20.06Pa) and an elastically-dominant behavior (phase angle <45°) over the tested frequency range. The crossover point of G' and G" shifted to higher frequencies with increasing wort content. From the flow and viscoelastic behavior, it was concluded that the 1:1 blend of milk to wort would have least phase separation and better flowability during spray drying.

Preparation and characterization of milk protein films and their application for packaging of Cheddar cheese.

Casein and whey protein concentrate (WPC) films, plasticized with glycerol and sorbitol independently, were prepared by casting. The film thickness, water vapour and oxygen permeation and tensile and moisture sorption properties of the films were determined. The tensile strength (TS), tensile strain (TE) and elastic modulus (EM) of the films ranged from 0.71 to 4.58 MPa, 19.22 to 66.63 % and 2.05 to 6.93 MPa, respectively. The film properties were influenced by the type of biopolymer (casein and whey protein concentrate), plasticizer and its concentration. Increasing the plasticizer concentration, increased the film thickness, TE and water vapour permeability (WVP), but decreased the TS and EM. As the concentration of plasticizer increased to the highest level, the film thickness increased from 0.168 to 0.305 mm for glycerol-plasticized films and from 0.251 to 0.326 mm for sorbitol-plasticized films. The film thickness increased because the amount of plasticizer in the film network increased and the amount of biopolymer remained same. Casein films showed superior tensile properties as compared to WPC films. The WVP of both casein and WPC films lied between 3.87 and 13.97 g.mm./(m(2).h.kPa). The moisture sorption isotherms of both films were typical of high-protein material, and were adequately described by the GAB model. The oxygen permeability of casein films was relatively lower than that of WPC films, regardless of the plasticizer used. The sensory data revealed that the organoleptic quality of Cheddar cheese was unaffected by milk-protein film packaging.

Extended shelf life flavoured dairy drink using dissolved carbon dioxide.

Cardamom flavoured dairy beverage prepared using standardized method was carbonated in glass bottles. Carbonation at 50 psi pressure for 30 s was recommended. The pasteurized flavoured drink, carbonated or otherwise was evaluated for sensory, chemical and microbial quality during its refrigerated storage. The uncarbonated control samples were found to be sensorily acceptable up to 14 days, while the carbonated beverage remained acceptable up to 30 days. Carbonation of drink significantly affected the pH and acidity of product without reducing its acceptability. Carbonation resulted in inhibition of microbes, the effect was pronounced on psychrotrophic count. There was a linear but marginal increase in the pH of the carbonated samples till the 17(th) day of storage; the values diminished thereafter. The carbonated samples also had significantly reduced contents of FFA and soluble nitrogen compared to that of uncarbonated control samples as storage progressed beyond 10 days and this was attributed to inhibited microbial growth.

Carbonated fermented dairy drink - effect on quality and shelf life.

Processing conditions were standardized for a carbonated sweetened fermented dairy beverage. The optimum level of carbonation for the beverage filled in 200 ml glass bottles was found to be at 50 psi pressure for 30 seconds. The beverage samples were stored under refrigerated conditions (7 °C) and evaluated at weekly intervals for their sensory, chemical and microbial quality. The uncarbonated control samples were found to keep well till 5 weeks of storage while the carbonated beverage was acceptable up to 12 weeks of storage. Carbonation did not significantly alter the pH of the beverage, while a marginal increase in titratable acidity was recorded for the carbonated samples. Carbonation was found to arrest the development of lipolysis and proteolysis in the beverage during storage. Microbiological investigations established the inhibition of yeast and mold growth due to dissolved CO2.

Molecular characterization and phylogenetic relationships among microsporidian isolates infecting silkworm, Bombyx mori using small subunit rRNA (SSU-rRNA) gene sequence analysis.

The life cycle, spore morphology, pathogenicity, tissue specificity, mode of transmission and small subunit rRNA (SSU-rRNA) gene sequence analysis of the five new microsporidian isolates viz., NIWB-11bp, NIWB-12n, NIWB-13md, NIWB-14b and NIWB-15mb identified from the silkworm, Bombyx mori have been studied along with type species, NIK-1s_mys. The life cycle of the microsporidians identified exhibited the sequential developmental cycles that are similar to the general developmental cycle of the genus, Nosema. The spores showed considerable variations in their shape, length and width. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIWB-15mb was found to be more virulent than other isolates. All of the microsporidians were found to infect most of the tissues examined and showed gonadal infection and transovarial transmission in the infected silkworms. SSU-rRNA sequence based phylogenetic tree placed NIWB-14b, NIWB-12n and NIWB-11bp in a separate branch along with other Nosema species and Nosema bombycis; while NIWB-15mb and NIWB-13md together formed another cluster along with other Nosema species. NIK-1s_mys revealed a signature sequence similar to standard type species, N. bombycis, indicating that NIK-1s_mys is similar to N. bombycis. Based on phylogenetic relationships, branch length information based on genetic distance and nucleotide differences, we conclude that the microsporidian isolates identified are distinctly different from the other known species and belonging to the genus, Nosema. This SSU-rRNA gene sequence analysis method is found to be more useful approach in detecting different and closely related microsporidians of this economically important domestic insect.

Genetic diversity and phylogenetic relationships among microsporidia infecting the silkworm, Bombyx mori, using random amplification of polymorphic DNA: morphological and ultrastructural characterization.

Random amplification of polymorphic DNA polymerase chain reaction (RAPD-PCR) and pathological, morphological and ultrastructural characterization were used to differentiate seven new microsporidian isolates infecting the mulberry silkworm, Bombyx mori. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIK-4m was found to be more virulent than other isolates. However, all the isolates, except NIK-4m, showed heavy gonadal infection and vertical transmission in the infected silkworms. Differences in the spore shape ranging from oval to elongate were observed, and the polar filament has 8-16 coils arranged in one or two rows. Of the 80 decamer random primers tested, 50 generated reproducible RAPD profiles and yielded a total of 600 fragments, of which 594 were polymorphic (99%). Forty nine RAPD primers produced 179 unique genetic markers, whose presence or absence differed among the microsporidians, albeit with varied efficiency of polymorphism detection. The degree of band sharing was used to evaluate genetic distances between different microsporidian isolates and to construct a phylogenetic tree using Dice coefficients. Cluster analysis based on Dice coefficients resulted in the formation of one major cluster consisting of NIK-1s, NIAP-7g, NIK-2r and NIK-5d and NIK-4m in the other; while NIAP-6p was intermediate between these two. NIK-8b and NITN-9n were found to be entirely different from others. Reproducible RAPD patterns of all microsporidian isolates enabled us to differentiate the microsporidian isolates. The results demonstrate that besides ultrastructural studies, RAPD-PCR can be a useful and reliable tool to detect polymorphism, genetic relationships, and for the identification of the microsporidians. In addition, DNA fingerprints generated in this process have potential applications as diagnostic tools for identification of different microsporidia with considerable accuracy.

Characterization and phylogenetic relationships among microsporidia infecting silkworm, Bombyx mori, using inter simple sequence repeat (ISSR) and small subunit rRNA (SSU-rRNA) sequence analysis.

This study is the first report on the genetic characterization and relationships among different microsporidia infecting the silkworm, Bombyx mori, using inter simple sequence repeat PCR (ISSR-PCR) analysis. Six different microsporidians were distinguished through molecular DNA typing using ISSR-PCR. Thus, ISSR-PCR analysis can be a powerful tool to detect polymorphisms and identify microsporidians, which are difficult to study with microscopy because of their extremely small size. Of the 100 ISSR primers tested, only 28 primers had reproducibility and high polymorphism (93%). A total of 24 ISSR primers produced 55 unique genetic markers, which could be used to differentiate the microsporidians from each other. Among the 28 SSRs tested, the most abundant were (CA)n, (GA)n, and (GT)n repeats. The degree of band sharing was used to evaluate genetic similarity between different microsporidian isolates and to construct a phylogenetic tree using Jaccard's similarity coefficient. The results indicate that the DNA profiles based on ISSR markers can be used as diagnostic tools to identify different microsporidia with considerable accuracy. In addition, the small subunit ribosomal RNA (SSU-rRNA) sequence gene was amplified, cloned, and sequenced from each of the 6 microsporidian isolates. These sequences were compared with 20 other microsporidian SSU-rRNA sequences to develop a phylogenetic tree for the microsporidia isolated from the silkworms. This method was found to be useful in establishing the phylogenetic relationships among the different microsporidians isolated from silkworms. Of the 6 microsporidian isolates, NIK-1s revealed an SSU-rRNA gene sequence similar to Nosema bombycis, indicating that NIK-1s is similar to N. bombycis; the remaining 5 isolates, which differed from each other and from N. bombycis, were considered to be different variants belonging to the species N. bombycis.