Fermented foods of the Indian Himalaya are unexplored functional resources with high nutritional potential. Chhurpi cheese, fermented by defined native proteolytic lactic acid bacteria of Sikkim was assessed for ACE inhibitory, HOCl reducing, and MPO inhibitory, activity across varying stages of gastrointestinal (GI) digestion. The enhanced bioactivity of Lactobacillus delbrueckii WS4 chhurpi was associated with the generation of bioactive and multifunctional peptides during fermentation and GI digestion. Qualitative and quantitative in silico tools were employed for prediction of ACE inhibitory activity of novel chhurpi peptides. Selected peptides, with highest predictive ACE inhibitory potential were synthesized and in vitro validation revealed the ACE inhibitory potential of peptides HPHPHLSFM and LKPTPEGDL. LKPTPEGDL showed the most potent ACE inhibitory activity (IC50 of 25.82 ± 0.26 µmol) which slightly decreased upon GI digestion. The peptides demonstrated a non-competitive type mixed ACE inhibition modality. Furthermore, the two peptides exerted observable HOCl reducing and MPO inhibitory activity, demonstrating their antioxidative potential. HPHPHLSFM exhibited superior HOCl reduction (EC50 of 0.29 ± 0.01 mmol), while LKPTPEGDL demonstrated higher MPO (IC50 of 0.29 ± 0.01 mmol) inhibition. Molecular docking of the two peptides with MPO revealed proline and aspartate near peptidyl C-terminus to bind with enzyme catalytic residues. This study presents the first peptidome analysis of chhurpi produced through controlled fermentation and identifies novel peptides with MPO and ACE inhibitory activity. Furthermore, it marks the first synthesis and in vitro bioactivity validation of bioactive peptides from chhurpi cheese, highlighting its multifunctional potential.

The considerable value of whey is evident from its significant potential applications and contributions to the functional food and nutraceutical market.

Throughout the world, there are various extreme regions unhabitable for most living organisms which are inhabited by various extremophilic microorganisms. These extremophiles have adopted several strategies to thrive in extremely harsh environments such as by producing enzymes, proteins, metabolites, and exopolysaccharides that can withstand extreme conditions. The exopolysaccharides produced by the extremophiles help the microorganisms to thrive in the harsh environment by providing better cell-to-cell adhesion, cell-to-substratum adhesion, concentrating nutrients, preventing dehydration, and retaining the moisture level. Exopolysaccharides from extremophiles have unique compositions such as over-sulfation, which makes them an interesting candidate for industrial application. In this chapter, we summarize the production of exopolysaccharides from different extremophiles and their potential application in health and bioremediation of the environment.

With the growing demand for functional foods having better nutraceutical properties, lactic acid bacteria (LAB) has become an important industrial microorganism. LAB play a significant role in the functional food industry by exhibiting probiotic properties and has the ability to produce various biologically active metabolites such as γ-aminobutyric acid (GABA), exopolysaccharides (EPSs), conjugated linoleic acid (CLA), bacteriocins, reuterin and reutericyclin, which provides enhanced nutraceutical properties to the final food products. LAB are also known to produce several specific enzymes essential for producing substrate-derived bioactive compounds, such as polyphenols, bioactive peptides, inulin-type fructans and β-glucans, fatty acids, and polyols. These compounds exhibit many health benefits, including better mineral absorption, oxidative stress protection, blood glucose and cholesterol-lowering properties, prevention of gastrointestinal tract infections and improved cardiovascular function. Further, metabolically engineered LAB have been widely used for the nutritive enhancement of different food products and the application of CRISPR-Cas9 holds tremendous potential for the engineering of food cultures. This review provides an overview of the use of LAB as probiotics, its application in producing fermented foods and nutraceutical products, and its health benefits on the host.

Bacterial strains were isolated from undisturbed regions of Sikkim Himalaya for the production of highly active xylanase and xylooligosaccharides (XOS). Seven bacterial isolates showed xylanase production while fermenting beechwood xylan (BX) and the agrowastes corncob (CC), rice straw (RS), and sugarcane bagasse (SB). Bacillus altitudinis XYL17, isolated from soil at an elevation of 4083 m, produced the highest amount of xylanase. Maximum xylanase production was observed during BX fermentation after 24 h with the an observed activity of 6576.96 +/- 82.75 U/mg. CC was observed to be the best agrowaste substrate for xylanase production with an observed activity of 4150.25 +/- 94.64 U/mg. Xylopentaose was the most prominent XOS, with a highest observed content of 11.206 +/- 0.331 mg/mL in RS hydrolysate. Significant increase of total phenolic content was observed for hydrolysates of B. altitudinis XYL17 fermented agro-wastes as compared to the control, while there was no significant difference (p < 0.05) in phenolic content between the control and fermented BX. The biotechnological potential of the XOS-rich hydrolysate was demonstrated by the observed radical scavenging, HOCl reducing, and myeloperoxidase (MPO) inhibitory activity. MPO inhibitory IC50 of 6.88 mg/mL and 9.4 mg/mL was observed for hydrolysates of SB and CC, respectively. We conclude that B. altitudinis XYL17 can be used for the valorization of agricultural wastes for the simultaneous production of highly efficient xylanase along with XOS and other antioxidant compounds.

Fermented food products are consumed by about 30% of the world's population due to their high nutritional value and health properties. The use of LAB in the fermentation process has resulted in a variety of fermented food products derived from both plant and animal sources. LAB have been used as starter cultures for food fermentation both traditionally and industrially, having certain specific characteristics such as rapid growth, product yield, higher biomass and also unique organoleptic properties, and are employed in food fermentation. The advancement of highthroughput genome sequencing methods has resulted in a tremendous improvement in our understanding of LAB physiology and has become more essential in the field of food microbiology. The complete genome sequence of Lactococcus lactis in 2001 resulted in a better understanding of metabolic properties and industrial applications of LAB. Genes associated with β-galactosidase, antimicrobial agents, bile salt hydrolase, exopolysaccharide, and GABA producing LAB have received a lot of attention in recent years. Genome editing techniques are required for the development of strains for novel applications and products. They can also play an important part as a research method for acquiring mechanistic insights and identifying new properties. The genome editing of lactic acid bacterial strains has a lot of potential applications for developing functional foods with a favourable influence on the food industries.

Microbial enzymes have been applied for production of nutraceutical on hydrolysis and transformation of different types of biomolecules. Biomolecules that are transformed for the production of nutraceuticals include proteins, carbohydrate, polyphenols, and lipids. The health benefits exhibited by enzymatically produced bioactive molecules include antioxidant, prebiotic, antihypertensive, antimicrobial, anticancer, and immunomodulatory properties. Enzymatic hydrolysis can also result in breakdown of antinutritional factors present in foods. The type of bioactive compounds produced depends on the specificity of the biocatalyst and biochemical composition of the substrate. The present chapter describes the different types of nutraceutical produced on hydrolysis/transformation using microbial enzymes.

We live in an era where consumption of unhealthy processed foods has become a part of our daily life contributing to various life-threatening diseases and malnutrition globally. Awareness among the people has led to the demand for foods and nutraceuticals that not only provide nutrition but also provide health beneficial functional properties. Consumption of nutraceuticals and functional foods has increased worldwide due to the health beneficial properties they exert in combating diseases while providing nutrition. In order to meet the global demand of functional food and nutraceuticals, safe and eco-friendly biotechnological approaches are required for the mass production of these products. This chapter focuses on the biological properties and the recent developments of biological technologies for the production of biologically active compounds such as bioactive peptides, polyphenols, oligosaccharides, selenium, pigments, vitamins, gamma-aminobutyric acid, and polyunsaturated fatty acid. Biological processes currently in use or under development for the production of these biologically active compounds are through the use of native microorganisms, microbial enzyme technologies, engineered microbes, and renewable source of substrates.

The genomic analysis of industrially important bacteria can help in understanding their capability to withstand extreme environments and shed light on their metabolic capabilities. The whole genome of a previously reported broad temperature active lipase-producing Pseudomonas sp. HS6, isolated from snow-covered soil of the Sikkim Himalayan Region, was analyzed to understand the capability of the bacterium to withstand cold temperatures and study its lipolytic nature. Pseudomonas sp. HS6 was found to be psychrotolerant with an optimal growth temperature ranging between 25 and 30 °C, with the ability to grow at 5 °C. The genome harbours various cold-adaptation genes, such as cold-shock proteins, fatty acid alteration, and cold stress-tolerance genes, supporting the psychrotolerant nature of the organism. The comparative analysis of Pseudomonas sp. HS6 genome showed the presence of amino acid substitutions in genes that favor efficient functioning and flexibility at cold temperatures. Genome mining revealed the presence of four triacylglycerol lipases, among which the putative lipase 3 was highly similar to the broad temperature-active lipase purified and characterized in our previous study. In silico studies of putative lipase 3 revealed broad substrate specificity with partial and no inhibition of the enzyme activity in the presence of PMSF and orlistat. The presence of genes associated with cold adaptations and true lipases with activity at broad temperature and substrate specificity in the genome of Pseudomonas sp. HS6 makes this bacterium a suitable candidate for industrial applications.

A novel soy chhurpi product was developed by fermentation of soymilk using proteolytic Lactobacillus delbrueckii strains isolated from traditional chhurpi production of Sikkim Himalaya. Soymilk fermentation by L. delbrueckii WS4 was associated with the hydrolysis of globulin proteins, with observed antioxidant, and ACE-inhibitory activity which further increased upon simulated in vitro gastrointestinal digestion. Peptidomics analysis of soy chhurpi and its gastrointestinal digest resulted in the identification of bioactive peptides with ACE-inhibitory and antioxidant properties. In silico antihypertensive property prediction followed by molecular docking study demonstrated strong binding affinity of selected peptides with ACE. The glycinin-derived peptide, SVIKPPTDE escaped gastrointestinal digestion and demonstrated strong non-bond interactions with ACE catalytic residues. QSAR models predicted an ACE-inhibitory IC50 of 21.29 µM for SVIKPPTDE. This is the first report on the production of novel functional soy chhurpi cheese using defined starter strains and the identification of bioactive peptides in undigested and gastrointestinal digested soy chhurpi.

Betacoronaviruses (β-CoVs) have caused major viral outbreaks in the last two decades in the world. The mutation and recombination abilities in β-CoVs resulted in zoonotic diseases in humans. Proteins responsible for viral attachment and replication are highly conserved in β-CoVs. These conserved proteins have been extensively studied as targets for preventing infection and the spread of β-CoVs. Peptides are among the most promising candidates for developing vaccines and therapeutics against viral pathogens. The immunostimulatory and viral inhibitory potential of natural and synthetic peptides has been extensively studied since the SARS-CoV outbreak. Food-derived peptides demonstrating high antiviral activity can be used to develop effective therapeutics against β-CoVs. Specificity, tolerability, and customizability of peptides can be explored to develop potent drugs against β-CoVs. However, the proteolytic susceptibility and low bioavailability of peptides pose challenges for the development of therapeutics. This review illustrates the potential role of peptides in eliciting an adaptive immune response and inhibiting different stages of the β-CoV life cycle. Further, the challenges and future directions associated with developing peptide-based therapeutics and vaccines against existing and future β-CoV pathogens have been discussed.

In this study, simulated in vitro GI digestion of the Himalayan hard chhurpi cheese resulted in the increase of hydrolyzed protein content, antioxidant and ACE-inhibitory activities. LC-MS/MS-based peptidomics revealed a total of 1473 peptides in the samples originating from different milk proteins, including α-S1-casein, α-S2-casein, β-casein, κ-casein, α-lactalbumin, and β-lactoglobulin, out of which 60 peptides have been reported for different functional properties. A total of 101 peptides were predicted to be antihypertensive using the bioactivity prediction web servers, AHTpin and mAHTPred. In silico molecular docking studies predicted 20 antihypertensive peptides, exhibiting non-bond interactions between hard chhurpi peptides and ACE catalytic residues. A peptide, SLVYPFPGPI, identified in GI digested cow hard chhurpi and undigested, and GI digested samples of yak hard chhurpi, showed a stronger binding affinity towards ACE. Identifying antioxidant and ACE inhibitory peptides in hard cheese products adds value to them as functional foods of the Himalayan region.

Bioactive peptides are released during the production of fermented dairy, vegetables, fruits, legumes, fish, and meat products. The proteolytic specificity of lactic acid bacteria, Bacillus spp., yeasts, and mold, apart from their ability to synthesize bioactive peptides, plays an important role in the generation of specific bioactive peptides in traditional fermented foods. Controlled fermentation using defined starter strains has been explored for the development of bioactive peptides enriched novel fermented foods with potential functionality. Bioactive peptides enriched foods exert diverse health benefits, such as antioxidant, antihypertensive, antidiabetic, and immunomodulatory effects. Bioactive peptides can be used as alternatives to synthetic compounds due to negligible side effects and high valuation in the nutraceutical and functional food market. However, challenges associated with the identification, quantification, organoleptic properties, and bioavailability of bioactive peptides need to be addressed before exploiting the potential of bioactive peptides in the functional food industry. In the present review, we have discussed the production of bioactive peptides in diverse fermented foods. Structural and sequence specificity of peptides and their effect on the expression of distinct health beneficial effects have been described. The potential of utilizing these bioactive peptides for the development of novel functional fermented foods is discussed. Recent advances in peptide identification, quantification, debittering of peptides, and increasing peptide bioavailability have been explained.

A by-product of the dairy and non-dairy food industry, whey is generated in large quantities. It can become a major pollutant if disposed of in the environment under untreated conditions. Whey is a rich source of several nutrients that include carbohydrates, proteins, isoflavones, and micronutrients. Valorization of whey using different biotechnological approaches can help to manage the by-product, with the economic and efficient production of bioactive compounds and value-added products. Microbial and enzymatic bioprocesses can be exploited for the treatment of whey for the release of nutraceuticals such as bioactive peptides, prebiotics, exopolysaccharides, organic acids, bacteriocins, isoflavone aglycones, and for the production of industrially important enzymes including beta-galactosidase, protease, and amylase. Whey enriched with bioactive compounds can be utilized for the functional and nutritional enhancement of foods and the development of novel functional foods with health beneficial effects.

The human gastrointestinal tract is inhabited by a diversity of microbial population responsible for important metabolic systems that are necessary for nutrition uptake and digestion. The gut microbiota consists of a wide array of bacterial species that shape the biochemical profile of the diet and thus have a huge impact on the host health and disease. Various studies have focused on identification and characterisation of metabolic pathways undergone by specific microorganisms that result in metabolism of dietary components, which play a major role in human metabolism and nutrient absorption. Considered as a virtual organ of the human body, the gut microbiome refers to collective genomes of microorganisms residing in the human gut and consists of over three million genes. These genes encode for metabolic pathways that are responsible for fermentation of various substrates, including non-digestible dietary fibres, such as exopolysachharides (EPS) into short-chain fatty acids (SCFAs). Deconjugation reactions of polyphenolic compounds, such as isoflavones, lignans, flavones and flavanols result in generation of health-beneficial compounds, such as aglycone metabolites, phenolic acids and enterolignans which have multiple functional properties. The functional properties offered by metabolites generated during gut fermentation include anticancer, antihypertensive, antidiabetic, antiobesity and antioxidant activity. People suffering from diseases, such as arthritis, Crohn's disease, obesity, atopic eczema and diabetes have commonly showed decreased gut microbial diversity. Thus, high bacterial diversity in the gut is of prime importance for generation of health-beneficial metabolites with functional properties.

Cheese is a product of ancient biotechnological practices, which has been revolutionized as a functional food product in many parts of the world. Bioactive compounds, such as peptides, polysaccharides, and fatty acids, have been identified in traditional cheese products, which demonstrate functional properties such as antihypertensive, antioxidant, immunomodulation, antidiabetic, and anticancer activities. Besides, cheese-making probiotic lactic acid bacteria (LAB) exert a positive impact on gut health, aiding in digestion, and improved nutrient absorption. Advancement in biotechnological research revealed the potential of metabolite production with prebiotics and bioactive functions in several strains of LAB, yeast, and filamentous fungi. The application of specific biocatalyst producing microbial strains enhances nutraceutical value, resulting in designer cheese products with multifarious health beneficial effects. This review summarizes the biotechnological approaches applied in designing cheese products with improved functional properties.

Microorganisms striving in extreme environments and exhibiting optimal growth and reproduction at low temperatures, otherwise known as psychrophilic microorganisms, are potential sources of cold-active enzymes. Owing to higher stability and cold activity, these enzymes are gaining enormous attention in numerous industrial bioprocesses. Applications of several cold-active enzymes have been established in the food industry, e.g., β-galactosidase, pectinase, proteases, amylases, xylanases, pullulanases, lipases, and β-mannanases. The enzyme engineering approaches and the accumulating knowledge of protein structure and function have made it possible to improve the catalytic properties of interest and express the candidate enzyme in a heterologous host for a higher level of enzyme production. This review compiles the relevant and recent information on the potential uses of different cold-active enzymes in the food industry.