Peptidomics-based identification of antihypertensive and antidiabetic peptides from sheep milk fermented using Limosilactobacillus fermentum KGL4 MTCC 25515 with anti-inflammatory activity: in silico, in vitro, and molecular docking studies.

This study investigated the synthesis of bioactive peptides from sheep milk through fermentation with Limosilactobacillus fermentum KGL4 MTCC 25515 strain and assessed lipase inhibition, ACE inhibition, α-glucosidase inhibition, and α-amylase inhibition activities during the fermentation process. The study observed the highest activities, reaching 74.82%, 70.02%, 72.19%, and 67.08% (lipase inhibition, ACE inhibition, α-glucosidase inhibition, and α-amylase inhibition) after 48 h at 37°C, respectively. Growth optimization experiments revealed that a 2.5% inoculation rate after 48 h of fermentation time resulted in the highest proteolytic activity at 9.88 mg/mL. Additionally, fractions with less than 3 kDa of molecular weight exhibited superior ACE-inhibition and anti-diabetic activities compared to other fractions. Fermentation of sheep milk with KGL4 led to a significant reduction in the excessive production of NO, TNF-α, IL-6, and IL-1ß produced in RAW 267.4 cells upon treatment with LPS. Peptides were purified utilizing SDS-PAGE and electrophoresis on 2D gels, identifying a maximum number of proteins bands ranging 10-70 kDa. Peptide sequences were cross-referenced with AHTPDB and BIOPEP databases, confirming potential antihypertensive and antidiabetic properties. Notably, the peptide (GPFPILV) exhibited the highest HPEPDOCK score against both α-amylase and ACE.

Probiotic potential of gluten degrading Bacillus tequilensis AJG23 isolated from Indian traditional cereal-fermented foods as determined by Multiple Attribute Decision-Making analysis.

The present study reported the characterization of gluten hydrolyzing strains of Bacillus sp. from fermented cereal dough. The strains were characterized for probiotic as well as technological attributes. A total of 45 presumptive gluten degrading isolates were obtained on gliadin agar plate assay. Based on hemolytic and antibiotic susceptibility pattern, only six isolates were considered safe which also indicated gliadinase activity on zymography. All the six strains were able to resist the pH 2.0, 0.25% bile and also possessed ability to adhere to the organic solvents and mucin. The cell free supernatant of five strains exhibited antimicrobial activities against Gram-positive and Gram-negative pathogens. A more than 50% survival of the isolated strains was obtained at a salt concentration of 2%, phenol concentration of 0.1% and temperature upto 45 °C. All the strains exhibited antioxidant activities and biofilm forming ability. Furthermore, the ranking of strains based on probiotic as well as other functional attributes was determined using multidimensional Technique for Order of Preference by Similarity to Ideal Solution (TOPSIS). A matrix of multidimensional indicators was prepared using alternatives and criteria, the analysis indicated the strain Bacillus tequilensis AJG23 as the potential probiotic candidate based on all screening criteria. Further work still needs to be done about the protective role of the potential strain against gluten sensitivity using in vitro models.

Nano-Conjugated Food-Derived Antimicrobial Peptides As Natural Biopreservatives: A Review of Technology and Applications.

In recent years, microbial food safety has garnered a lot of attention due to worldwide expansion of the food industry and processed food products. This has driven the development of novel preservation methods over traditional ones. Food-derived antimicrobial peptides (F-AMPs), produced by the proteolytic degradation of food proteins, are emerging as pragmatic alternatives for extension of the shelf-life of food products. The main benefits of F-AMPs are their wide spectrum antimicrobial efficacy and low propensity for the development of antibiotic resistance. However, direct application of F-AMPs in food limits its efficacy during storage. Therefore, the development of nanocarriers for the conjugation and distribution of potential AMPs may hold great potential to increase their bioactivity. This review highlights the significance of F-AMPs as a feasible and sustainable alternative to conventional food preservatives. The most recent developments in production, characterization, and mode of action of these AMPs against planktonic and biofilm forming pathogens are thoroughly discussed in this work. Moreover, nano-conjugation of F-AMPs with different nano-carriers and potential future application in food packaging are emphasized. This review may aid in comprehending the nano-conjugation of F-AMPs and offer insightful recommendations for further exploration and potential uses in the food processing industry.

Plant-derived proteins as a sustainable source of bioactive peptides: recent research updates on emerging production methods, bioactivities, and potential application.

The development of novel protein sources to compensate for the expected future shortage of traditional animal proteins due to their high carbon footprint is a major contemporary challenge in the agri-food industry currently. Therefore, both industry and consumers are placing a greater emphasis on plant proteins as a sustainable source of protein to meet the growing nutritional demand of ever increasing population. In addition to being key alternatives, many plant-based foods have biological properties that make them potentially functional or health-promoting foods, particularly physiologically active peptides and proteins accounting for most of these properties. This review discusses the importance of plant-based protein as a viable and sustainable alternative to animal proteins. The current advances in plant protein isolation and production and characterization of bioactive hydrolysates and peptides from plant proteins are described comprehensively. Furthermore, the recent research on bioactivities and bioavailability of plant protein-derived bioactive peptides is reviewed briefly. The limitations of using bioactive peptides, regulatory criteria, and the possible future applications of plant protein-derived bioactive peptides are highlighted. This review may help understand plant proteins and their bioactive peptides and provide valuable suggestions for future research and applications in the food industry.

Multifunctional bioactive properties of hydrolysates from colocynth (Citrullus colocynthis) seeds derived proteins: Characterization and biological properties.

Citrullus colocynthis (Colocynth) has gained a great deal of interest in their applications as indigenous nutraceutical and as a functional food ingredient. The intact colocynth seed protein was enzymatically hydrolyzed using proteolytic enzymes (alcalase, bromelain, and chymotrypsin) at different time intervals of 3, 6, and 9 h. The highest degree of hydrolysis (87.82%) was observed in chymotrypsin derived colocynth seed protein hydrolysates (CSPH) for 9 h. The CSPHs was further investigated through in-vitro assay to explore its potential biological activity such as antioxidant, inhibition of enzymatic marker related to diabetes (DPP-IV, α-glucosidase and α-amylase) and hyperlipidaemia (cholesteryl esterase and pancreatic lipase). Chymotrypsin hydrolysate showed the strongest DPPH (65.7 mM TEAC) and ABTS (525.2 mM TEAC) radical scavenging activity after 6 h of hydrolysis. Moreover, chymotrypsin-treated CSPH for 6 h inhibited cholesteryl esterase (IC50 = 13.68 µg/mL) and pancreatic lipase (IC50 = 14.12 µg/mL) significantly when compared to native protein. Whereas, bromelain and alcalase treated hydrolysate for 6 h effectively inhibited α-glucosidase and α-amylase at an inhibitory concentration of IC50 = 13.27 µg/mL and of IC50 = 17 µg/mL. Overall, the findings indicated that protein hydrolysates exhibited superior biological activity than intact colocynth seed proteins isolate (CSPI) and could be a sustainable source of bioactive peptides.

Milk-Derived Antimicrobial Peptides: Overview, Applications, and Future Perspectives.

The growing consumer awareness towards healthy and safe food has reformed food processing strategies. Nowadays, food processors are aiming at natural, effective, safe, and low-cost substitutes for enhancing the shelf life of food products. Milk, besides being a rich source of nutrition for infants and adults, serves as a readily available source of precious functional peptides. Due to the existence of high genetic variability in milk proteins, there is a great possibility to get bioactive peptides with varied properties. Among other bioactive agents, milk-originated antimicrobial peptides (AMPs) are gaining interest as attractive and safe additive conferring extended shelf life to minimally processed foods. These peptides display broad-spectrum antagonistic activity against bacteria, fungi, viruses, and protozoans. Microbial proteolytic activity, extracellular peptidases, food-grade enzymes, and recombinant DNA technology application are among few strategies to tailor specific peptides from milk and enhance their production. These bioprotective agents have a promising future in addressing the global concern of food safety along with the possibility to be incorporated into the food matrix without compromising overall consumer acceptance. Additionally, in conformity to the current consumer demands, these AMPs also possess functional properties needed for value addition. This review attempts to present the basic properties, synthesis approaches, action mechanism, current status, and prospects of antimicrobial peptide application in food, dairy, and pharma industry along with their role in ensuring the safety and health of consumers.

Bioactive peptides in the management of lifestyle-related diseases: Current trends and future perspectives.

Lifestyle-related diseases constitute a major concern in the twenty-first century, with millions dying worldwide each year due to chosen lifestyles and associated complications such as obesity, type 2 diabetes, hypertension, and hypercholesterolemia. Although synthetic drugs have been shown to be quite effective in the treatment of these conditions, safety of these compounds remains a concern. Natural alternatives to drugs include food-derived peptides are now being explored for the prevention and treatment of lifestyle-related complications. Peptides are fragments nascent in the primary protein sequences and could impart health benefits beyond basic nutritional advantages. Evidence suggests that by controlling adipocyte differentiation and lipase activities, bioactive peptides may be able to prevent obesity. Bioactive peptides act as agents against type 2 diabetes because of their ability to inhibit enzymatic activities of DPP-IV, α-amylase, and α-glucosidase. Moreover, bioactive peptides can act as competitive inhibitors of angiotensin-converting enzyme, thus eliciting an antihypertensive effect. Bioactive peptides may have a hypocholesterolemic effect by inhibiting cholesterol metabolism pathways and cholesterol synthesis. This review addresses current knowledge of the impact of food-derived bioactive peptides on lifestyle diseases. In addition, future insights on the clinical trials, allergenicity, cytotoxicity, gastrointestinal stability, and regulatory approvals have also been considered.

Probiotic mediated NF-κB regulation for prospective management of type 2 diabetes.

Diabetes and other lifestyle disorders have been recognized as the leading cause of morbidity and mortality globally. Nuclear factor kappa B (NF-κB) is a major factor involved in the early pathobiology of diabetes and studies reveal that hyperglycemic conditions in body leads to NF-κB mediated activation of several cytokines, chemokines and inflammatory molecules. NF-κB family comprises of certain DNA-binding protein factors that elicit the transcription of pro-inflammatory molecules. Various studies have identified NF-κB as a promising target for diabetic management. Probiotics have been proposed as bio-therapeutic agents for treatment of inflammatory disorders and many other chronic clinical stages. The precise mechanisms by which probiotics acts is yet to be fully understood, however research findings have indicated their role in NF-κB modulation. The current review highlights NF-κB as a bio-therapeutic target for probable management of type 2 diabetes through probiotic intervention.

Impairment of Cronobacter sakazakii and Listeria monocytogenes biofilms by cell-free preparations of lactobacilli of goat milk origin.

Biofilm-associated bacterial infections represent one of the major threats to modern medical treatments. Bacteria encased in biofilm matrix are more resistant towards antimicrobials and thus the capability of microbes to persist and nurture in a biofilm seems to be the foremost aspect of pathogenesis and therapeutic failure. Therefore, there is a pressing demand for new drugs active against microbial biofilms. In the current study, anti-biofilm potential of Lactobacillus spp. cell-free supernatants (CFSs) against Cronobacter sakazakii and Listeria monocytogenes was characterized using crystal violet staining and MTT assay. CFSs of goat milk origin lactobacilli not only prevented biofilm formation but also disrupted preformed biofilms. Neutralized and heat-treated preparations of Lactobacillus CFSs also inhibited biofilm formation by test pathogens. The results were quantitatively confirmed by light and fluorescent microscopy observations. Biofilms developed under static conditions displayed typical compact microcolonies with uniform distribution over the surface, while upon CFS challenge, biofilms were disrupted with presence of dead cells. These findings highlight the anti-biofilm potency of Lactobacillus spp. strains of goat milk origin and their potential application in food industries.

Screening of cell surface properties of potential probiotic lactobacilli isolated from human milk.

Evaluation of eleven candidate probiotic Lactobacillus strains isolated from human milk showed that some of the strains were well endowed with desirable cell surface and attachment attributes. The cell surface properties (hydrophobicity, auto-aggregation, attachment to collagen and HT-29 monolayer) of probiotic Lactobacillus species of human milk origin were compared with reference probiotic/ non-probiotic species and pathogenic strains. The bacterial adhesion to hydrocarbons (BATH) was determined using three aliphatic (Chloroform, n-Hexane and n-Octane) and two aromatic (Toluene and Xylene) solvents. Maximum affinity of Lactobacillus strains towards chloroform and toluene indicated the presence of low electron acceptor/ acidic surface components on cell surface of most of the strains. The highest value of per cent hydrophobicity was recorded with chloroform in HM1 (L. casei) (97·10 ± 3·35%) and LGG (98·92 ± 1·24%). A moderate auto-aggregation attribute was observed in all of our Lactobacillus isolates. Only HM10, HM12 and HM13 exhibited comparatively enhanced precipitation rate after 7 h of incubation period. The adhesion potential to collagen matrix was highest in LGG (26·94 ± 5·83%), followed by HM1 (11·07 ± 3·54%) and HM9 (10·85 ± 1·74%) whereas, on HT-29 cells, HM8 (14·99 ± 3·61%), HM3 (13·73 ± 1·14%) and HM1 (11·21 ± 3·18%) could adhere effectively. In this manner, we noticed that although the cell surface properties and adhesion prospective of probiotic bacteria were strain dependent, five of our isolates viz. HM1, HM3, HM8, HM9 and HM10 exhibited promising cell surface properties, which could be further targeted as indigenous probiotic.

Antibiotic sensitivity pattern of indigenous lactobacilli isolated from curd and human milk samples.

The gut microbiota plays a vital role in host well-being and lactic acid bacteria (LAB) have gained an overwhelming attention as health promoter. This perception has evolved from traditional dairy products to a money-spinning market of probiotics. The safety of probiotics is coupled to their intended use and LAB may act as pool of antimicrobial resistance genes that could be transferred to pathogens, either in food matrix or in gastrointestinal tract, which could be detrimental to host. This study evaluated the antibiotic susceptibility patterns of LAB isolated from curd (20) and human milk (11) samples. Antibiotic susceptibility was determined against 26 common antibiotics, following reference disc diffusion assay. A varied response in terms of susceptibility and resistance towards antibiotics was recorded. Among curd isolates, D7 (Lactobacillus plantarum) was the most resistant followed by D4, D8, D10 and D25. Among human milk isolates, HM-1 (L. casei) showed the highest resistance profile. All LAB isolates displayed high susceptibility pattern towards imipenem and meropenem. In general, high resistivity was exhibited by human milk isolates. The present study showed that antibiotic resistance is widespread among different lactobacilli, which may pose a food safety concern. Therefore, antibiotic sensitivity should be considered as a vital tool for safety assessment of probiotics.

Antibacterial effects of Lactobacillus isolates of curd and human milk origin against food-borne and human pathogens.

This study was undertaken to assess the antibacterial efficacy of lactobacilli isolated from curd and human milk samples. Identities of thirty-one different lactobacilli (20 from curd and 11 from human milk) were confirmed by genus-specific PCR and 16S rRNA-based sequencing. These strains belonged to five species, Lactobacillus casei, L. delbrueckii, L. fermentum, L. plantarum, and L. pentosus. Antibacterial activities of cell-free supernatants (CFSs) of all the Lactobacillus isolates were estimated through standard agar-well diffusion assay, against commonly occurring food-borne and clinically important human pathogens. None of the lactobacilli cell-free supernatant (CFS) exhibited inhibitory activity against four pathogens, namely Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Klebsiella pneumoniae. Bacillus cereus, Salmonella enterica serovar Typhi, and Shigella flexneri were moderately inhibited by majority of CFSs, whereas, weak activity was observed against Pseudomonas aeruginosa and Proteus mirabilis. CFS of some of the curd isolates displayed antagonistic activity against Streptococcus mutans; however, human milk lactobacilli did not displayed any inhibitory activity against them. As expected, Nisin (Nisaplin®) showed inhibitory activity against Gram-positive, S. aureus, B. cereus, and L. monocytogenes. Interestingly, few of the examined CFSs exhibited inhibitory activities against both Gram-positive and Gram-negative pathogens. Findings from this study support the possibility to explore the tested lactobacilli and their CFSs as natural bio-preservatives, alone or in combination with approved bacteriocins in food and pharma formulations after validating their safety.

Antimicrobial Resistance: Its Surveillance, Impact, and Alternative Management Strategies in Dairy Animals.

Antimicrobial resistance (AMR), one among the most common priority areas identified by both national and international agencies, is mushrooming as a silent pandemic. The advancement in public health care through introduction of antibiotics against infectious agents is now being threatened by global development of multidrug-resistant strains. These strains are product of both continuous evolution and un-checked antimicrobial usage (AMU). Though antibiotic application in livestock has largely contributed toward health and productivity, it has also played significant role in evolution of resistant strains. Although, a significant emphasis has been given to AMR in humans, trends in animals, on other hand, are not much emphasized. Dairy farming involves surplus use of antibiotics as prophylactic and growth promoting agents. This non-therapeutic application of antibiotics, their dosage, and withdrawal period needs to be re-evaluated and rationally defined. A dairy animal also poses a serious risk of transmission of resistant strains to humans and environment. Outlining the scope of the problem is necessary for formulating and monitoring an active response to AMR. Effective and commendably connected surveillance programs at multidisciplinary level can contribute to better understand and minimize the emergence of resistance. Besides, it requires a renewed emphasis on investments into research for finding alternate, safe, cost effective, and innovative strategies, parallel to discovery of new antibiotics. Nevertheless, numerous direct or indirect novel approaches based on host-microbial interaction and molecular mechanisms of pathogens are also being developed and corroborated by researchers to combat the threat of resistance. This review places a concerted effort to club the current outline of AMU and AMR in dairy animals; ongoing global surveillance and monitoring programs; its impact at animal human interface; and strategies for combating resistance with an extensive overview on possible alternates to current day antibiotics that could be implemented in livestock sector.

ß-Glucosidase activity and bioconversion of isoflavones during fermentation of soymilk.

BACKGROUND: Lactobacillus rhamnosus C6 strain showed higher ß-glucosidase activity as well as biotransformation of isoflavones from glycones (daidzin and genistin) to aglycones (daidzein and genistein) in soymilk. However, L. rhamnosus C2 and Lactobacillus casei NCDC297 also exhibited similar activity during soymilk fermentation. These three strains can be selected for the development of functional fermented soy foods enriched with aglycone forms of isoflavones, such as soy yoghurt, soy cheese, soy beverages and soy dahi. RESULTS: The study determined ß-glucosidase activity of probiotic Lactobacillus cultures for bioconversion of isoflavones to aglycones in fermenting soymilk medium. Soymilk was fermented with six strains (L. rhamnosus C6 and C2, L. rhamnosus NCDC19 and NCDC24 and L. casei NCDC17 and NCDC297) at 37 °C for 12 h. The highest ß-glucosidase activity and isoflavone bioconversion after 12 h occurred by L. rhamnosus C6 culture during fermentation in soymilk. Increased isoflavone aglycone content in fermented soymilk is likely to improve the biological functionality of soymilk (e.g. antioxidant activity, alleviation of hormonal disorders in postmenopausal women, etc.). CONCLUSION: Lactobacillus rhamnosus C6 culture can be used for the development of functional fermented soy-based products.

Functional significance of bioactive peptides derived from soybean.

Biologically active peptides play an important role in metabolic regulation and modulation. Several studies have shown that during gastrointestinal digestion, food processing and microbial proteolysis of various animals and plant proteins, small peptides can be released which possess biofunctional properties. These peptides are to prove potential health-enhancing nutraceutical for food and pharmaceutical applications. The beneficial health effects of bioactive peptides may be several like antihypertensive, antioxidative, antiobesity, immunomodulatory, antidiabetic, hypocholesterolemic and anticancer. Soybeans, one of the most abundant plant sources of dietary protein, contain 36-56% of protein. Recent studies showed that soy milk, an aqueous extract of soybean, and its fermented product have great biological properties and are a good source of bioactive peptides. This review focuses on bioactive peptides derived from soybean; we illustrate their production and biofunctional attributes.