Antioxidant and immunomodulatory potency of Lacticaseibacillus rhamnosus NCDC24 fermented milk-derived peptides: A computationally guided in-vitro and ex-vivo investigation.

Infections of microbial and non-microbial origins have been associated with significant immunological manifestations, thereby underscoring the need for a thorough understanding and investigation of novel immunomodulatory and antioxidant molecules that could prevent these incidences. To this end, we herein aim to identify fermented milk peptides with antioxidant and immunomodulatory properties that could be exploited for specific future applications. Our computational prediction models indicate that these peptides are non-toxic and possess considerable hydrophobicity (19.82-38.96 %) and functionality. Further analyses reveal that two of the four peptides, i.e., Pep 1 (AGWNIPM) and Pep 4 (YLGYLEQLLR), possess higher in-vitro antioxidant activity. The immunomodulatory potential of these two peptides (Pep 1 and Pep 4) is further demonstrated by using a combination of molecular simulation trajectory and ex-vivo approaches. Both peptides demonstrate ability to control the production of pro- inflammatory (TNF-α, IL-1, and IL-6) and anti-inflammatory (IL-10) cytokines as well as nitric oxide release in LPS-stimulated murine peritoneal macrophages. Similarly, peptide interferences also lead to significant (P < 0.05) improvement in macrophage phagocytic capacity. Taken together, these findings highlight the antioxidant and immunomodulatory properties of fermented milk peptides (Pep 1 and Pep 4) within the cellular environment and should facilitate prospective studies exploring such bioactive peptides and related functional molecules mediating the benefits of fermented milk products on human health.

Anaerobic Fungi: Past, Present, and Future.

Anaerobic fungi (AF) play an essential role in feed conversion due to their potent fiber degrading enzymes and invasive growth. Much has been learned about this unusual fungal phylum since the paradigm shifting work of Colin Orpin in the 1970s, when he characterized the first AF. Molecular approaches targeting specific phylogenetic marker genes have facilitated taxonomic classification of AF, which had been previously been complicated by the complex life cycles and associated morphologies. Although we now have a much better understanding of their diversity, it is believed that there are still numerous genera of AF that remain to be described in gut ecosystems. Recent marker-gene based studies have shown that fungal diversity in the herbivore gut is much like the bacterial population, driven by host phylogeny, host genetics and diet. Since AF are major contributors to the degradation of plant material ingested by the host animal, it is understandable that there has been great interest in exploring the enzymatic repertoire of these microorganisms in order to establish a better understanding of how AF, and their enzymes, can be used to improve host health and performance, while simultaneously reducing the ecological footprint of the livestock industry. A detailed understanding of AF and their interaction with other gut microbes as well as the host animal is essential, especially when production of affordable high-quality protein and other animal-based products needs to meet the demands of an increasing human population. Such a mechanistic understanding, leading to more sustainable livestock practices, will be possible with recently developed -omics technologies that have already provided first insights into the different contributions of the fungal and bacterial population in the rumen during plant cell wall hydrolysis.

Conjugated linoleic acid enriched skim milk prepared with Lactobacillus fermentum DDHI27 endorsed antiobesity in mice.

AIM: This study evaluated the antiobesity effect of skim milk prepared with conjugated linoleic acid producing probiotic Lactobacillus fermentum DDHI27 (PCLA). MATERIALS & METHODS: C57BL/6 J mice were divided into five groups, and different obesity-associated parameters were studied. RESULTS: PCLA supplementation alleviated body weight, epididymal and mesenteric fats and improves lipid profiles. Significant ameliorations in leptin, blood glucose, hepatic steatosis and reduction in adipocytes size were also observed. Additionally, feeding also led to positive alterations in the adipogenesis transcription factors and key lipogenesis genes. Improvement in the gut microbiota dysbiosis was also revealed. CONCLUSION: Results inferred that PCLA exerted an antiobesity effect in diet-induced obese mice and may be further developed in the functional foods for the management of obesity.

PCR and Omics Based Techniques to Study the Diversity, Ecology and Biology of Anaerobic Fungi: Insights, Challenges and Opportunities.

Anaerobic fungi (phylum Neocallimastigomycota) are common inhabitants of the digestive tract of mammalian herbivores, and in the rumen, can account for up to 20% of the microbial biomass. Anaerobic fungi play a primary role in the degradation of lignocellulosic plant material. They also have a syntrophic interaction with methanogenic archaea, which increases their fiber degradation activity. To date, nine anaerobic fungal genera have been described, with further novel taxonomic groupings known to exist based on culture-independent molecular surveys. However, the true extent of their diversity may be even more extensively underestimated as anaerobic fungi continue being discovered in yet unexplored gut and non-gut environments. Additionally many studies are now known to have used primers that provide incomplete coverage of the Neocallimastigomycota. For ecological studies the internal transcribed spacer 1 region (ITS1) has been the taxonomic marker of choice, but due to various limitations the large subunit rRNA (LSU) is now being increasingly used. How the continued expansion of our knowledge regarding anaerobic fungal diversity will impact on our understanding of their biology and ecological role remains unclear; particularly as it is becoming apparent that anaerobic fungi display niche differentiation. As a consequence, there is a need to move beyond the broad generalization of anaerobic fungi as fiber-degraders, and explore the fundamental differences that underpin their ability to exist in distinct ecological niches. Application of genomics, transcriptomics, proteomics and metabolomics to their study in pure/mixed cultures and environmental samples will be invaluable in this process. To date the genomes and transcriptomes of several characterized anaerobic fungal isolates have been successfully generated. In contrast, the application of proteomics and metabolomics to anaerobic fungal analysis is still in its infancy. A central problem for all analyses, however, is the limited functional annotation of anaerobic fungal sequence data. There is therefore an urgent need to expand information held within publicly available reference databases. Once this challenge is overcome, along with improved sample collection and extraction, the application of these techniques will be key in furthering our understanding of the ecological role and impact of anaerobic fungi in the wide range of environments they inhabit.

Gut Microbiota Modulation and Its Relationship with Obesity Using Prebiotic Fibers and Probiotics: A Review.

In the present world scenario, obesity has almost attained the level of a pandemic and is progressing at a rapid rate. This disease is the mother of all other metabolic disorders, which apart from placing an added financial burden on the concerned patient also has a negative impact on his/her well-being and health in the society. Among the various plausible factors for the development of obesity, the role of gut microbiota is very crucial. In general, the gut of an individual is inhabited by trillions of microbes that play a significant role in host energy homeostasis by their symbiotic interactions. Dysbiosis in gut microbiota causes disequilibrium in energy homeostasis that ultimately leads to obesity. Numerous mechanisms have been reported by which gut microbiota induces obesity in experimental models. However, which microbial community is directly linked to obesity is still unknown due to the complex nature of gut microbiota. Prebiotics and probiotics are the safer and effective dietary substances available, which can therapeutically alter the gut microbiota of the host. In this review, an effort was made to discuss the current mechanisms through which gut microbiota interacts with host energy metabolism in the context of obesity. Further, the therapeutic approaches (prebiotics/probiotics) that helped in positively altering the gut microbiota were discussed by taking experimental evidence from animal and human studies. In the closing statement, the challenges and future tasks within the field were discussed.

Probiotic Properties of Lactobacillus plantarum RYPR1 from an Indigenous Fermented Beverage Raabadi.

Present study documents the potential probiotic Lactobacillus isolated from indigenous fermented beverage Raabadi, consumed during summers in Haryana and Rajasthan regions of India. A total of five Raabadi samples were collected aseptically and 54 isolates were purified using MRS medium. All the isolates were assessed for tolerance to low pH and bile salts. It was observed that out of 54 only 24 isolates could survive the simulated gastric conditions. These isolates were further evaluated in vitro for cell surface hydrophobicity, cell surface hydrophobicity, hypocholesteramic activity, anti-oxidative potential, BSH activity, antagonistic activity, and antibiotic resistance profile. In addition, the confirmation of phenol resistance was also done. On the basis of results obtained, the survival rate of isolates was noted and six isolates were finally selected for further studies. Among them Lactobacillus plantarum RYPR1 and RYPC7 showed good survival at pH 2 which shows good acid tolerance. Moreover, L. plantarum RYPR1 showed the highest hydrophobicity (79.13%) and represented the deconjugation of bile salts, which help in their adhesion to epithelial cells and colonization. Furthermore, RYPR1 also exhibited highest cholesterol reduction (59%) and subsequent analysis of results revealed that the above mentioned isolates further exhibit a good hypocholesterolemic effect and could be possibly used to prevent hypercholesterolemia. The present study divulges that L. plantarum RYPR1 has an excellent probiotic potential.

Catalytic Interactions and Molecular Docking of Bile Salt Hydrolase (BSH) from L. plantarum RYPR1 and Its Prebiotic Utilization.

Prebiotics are the non-digestible carbohydrate, which passes through the small intestine into unmetabolized form, reaches the large intestine and undergoes fermentation by the colonic bacteria thus; prebiotics stimulate the growth of probiotic bacteria. Further, bile salt hydrolase (BSH) is an enzyme that catalyses the deconjugation of bile salt, so it has enormous potential toward utilizing such capability of Lactobacillus plantarum RYPR1 toward detoxifying through BSH enzyme activity. In the present study, six isolates of Lactobacillus were evaluated for the co-aggregation assay and the isolate Lactobacillus plantarum RYPR1 was further selected for studies of prebiotic utilization, catalytic interactions and molecular docking. The prebiotic utilization ability was assessed by using commercially available prebiotics lactulose, inulin, xylitol, raffinose, and oligofructose P95. The results obtained revealed that RYPR1 is able to utilize these probiotics, maximum with lactulose by showing an increase in viable cell count (7.33 ± 0.02 to 8.18 ± 0.08). In addition, the molecular docking of BSH from Lactobacillus plantarum RYPR1 was performed which revealed the binding energy -4.42 and 7.03 KJ/mol. This proves a considerably good interactions among BSH and its substrates like Taurocholic acid (-4.42 KJ/mol) and Glycocholic acid (-7.03 KJ/mol). These results from this study establishes that Lactobacillus plantarum RYPR1 possesses good probiotic effects so it could be used for such applications. Further, molecular dynamics simulations were used to analyze the dynamic stability of the of modeled protein to stabilize it for further protein ligand docking and it was observed that residues Asn12, Ile8, and Leu6 were interacting among BSH and its substrates, i.e., Taurocholic acid and Lys88 and Asp126 were interacting with Glycocholic acid. These residues were interacting when the docking was carried out with stabilized BSH protein structure, thus, these residues may have a vital role in stabilizing the binding of the ligands with the protein.

A new anaerobic fungus (Oontomyces anksri gen. nov., sp. nov.) from the digestive tract of the Indian camel (Camelus dromedarius).

Two cultures of anaerobic fungi were isolated from the forestomach of an Indian camel (Camelus dromedarius). Phylogenetic analysis using both the internal transcribed spacer (ITS) and large-subunit (LSU) regions of the rRNA locus demonstrated that these isolates were identical and formed a distinct clade within the anaerobic fungi (phylum Neocallimastigomycota). Morphological examination showed that these fungi formed monocentric thalli with filamentous rhizoids and uniflagellate zoospores, broadly similar to members of the genus Piromyces. However, distinctive morphological features were observed, notably the pinching of the cytoplasm in the sporangiophore and the formation of intercalary rhizoidal swellings. Since genetic analyses demonstrated this fungus was only distantly related to Piromyces spp. and closer to the polycentric Anaeromyces clade, we have assigned it to a new genus and species Oontomyces anksri gen. nov., sp. nov. Interrogation of the GenBank database identified several closely related ITS sequences, which were all environmental sequences obtained from camels, raising the possibility that this fungus may be specific to camelids.

Anaerobic fungi (phylum Neocallimastigomycota): advances in understanding their taxonomy, life cycle, ecology, role and biotechnological potential.

Anaerobic fungi (phylum Neocallimastigomycota) inhabit the gastrointestinal tract of mammalian herbivores, where they play an important role in the degradation of plant material. The Neocallimastigomycota represent the earliest diverging lineage of the zoosporic fungi; however, understanding of the relationships of the different taxa (both genera and species) within this phylum is in need of revision. Issues exist with the current approaches used for their identification and classification, and recent evidence suggests the presence of several novel taxa (potential candidate genera) that remain to be characterised. The life cycle and role of anaerobic fungi has been well characterised in the rumen, but not elsewhere in the ruminant alimentary tract. Greater understanding of the 'resistant' phase(s) of their life cycle is needed, as is study of their role and significance in other herbivores. Biotechnological application of anaerobic fungi, and their highly active cellulolytic and hemi-cellulolytic enzymes, has been a rapidly increasing area of research and development in the last decade. The move towards understanding of anaerobic fungi using -omics based (genomic, transcriptomic and proteomic) approaches is starting to yield valuable insights into the unique cellular processes, evolutionary history, metabolic capabilities and adaptations that exist within the Neocallimastigomycota.

D1/D2 domain of large-subunit ribosomal DNA for differentiation of Orpinomyces spp.

This study presents the suitability of D1/D2 domain of large-subunit (LSU) ribosomal DNA (rDNA) for differentiation of Orpinomyces joyonii and Orpinomyces intercalaris based on PCR-restriction fragment length polymorphism (RFLP). A variation of G/T in O. intercalaris created an additional restriction site for AluI, which was used as an RFLP marker. The results demonstrate adequate heterogeneity in the LSU rDNA for species-level differentiation.

Conjugated linoleic acid producing potential of lactobacilli isolated from the rumen of cattle.

Lactobacilli isolated from the rumen of cattle were subjected to morphological and biochemical characterizations followed by PCR-based identification. Among isolates, Lactobacillus brevis was found to be the most prevalent species in the rumen. For in vitro conjugated linoleic acid (CLA) production, the two isolates of L. brevis and one each of Lactobacillus viridescens and Lactobacillus lactis were selected. The sunflower oil (i.e., 0.25, 0.5 and 1.0%; a rich source of linoleic acid) was added to skim milk as a substrate for CLA production by isolates at 37 degrees C/12 h. L. brevis 02 was found to be the most potential CLA producer (10.53 mg CLA/g fat) at 0.25% concentration of sunflower oil followed by L. brevis 01 (8.27 mg CLA/g fat). However, at higher level of sunflower oil (i.e., 1.0%), L. lactis was the highest CLA producer (9.22 mg/g fat) when compared to L. brevis and L. viridescens. The results indicated that L. brevis and/or CLA production was inhibited with increasing concentration of sunflower oil in skim milk. In contrast, L. lactis and L. viridescens could tolerate the increasing concentrations of sunflower oil and produced higher CLA. Overall, L. brevis extends a possibility to be used as a direct-fed microbial for ruminants to increase the CLA content in milk, however, in vivo trials are needed for validation of results obtained.

Effect of administration of anaerobic fungi isolated from cattle and wild blue bull (Boselaphus tragocamelus) on growth rate and fibre utilization in buffalo calves.

Fifteen Murrah buffalo calves (age about 10 months, 163-176 kg BW) were divided into three groups. Group I (Control) was fed a complete feed mixture consisted of 50% wheat straw and 50% concentrate mixture (contained per kg: maize 330 g, groundnut cake 210 g, mustard cake 120 g, wheat bran 200 g, de-oiled rice bran 110 g, mineral mixture 20 g and common salt 10 g) along with 2 kg green oats per animal and day to meet the vitamin A requirements. Calves of Groups II and III were fed with the Control diet supplemented with Orpinomyces sp. C-14 and Piromyces sp. WNG-12 cultures, respectively. The digestibility of DM was significantly highest with Piromyces sp. WNG-12 in Group III (62.2%) followed by Orpinomyces sp. C-14 in Group II (60.3%), and Control (53.5%). A similar pattern of increase in digestibility of crude protein and cell-wall contents was observed in treatment groups. The digestible energy in terms of percent total digestible nutrients was also significantly enhanced in Groups II (56.6%) and III (59.9%) when compared to Control (49.2%). The rumen fermentation parameters such as pH and NH3-N were found to be lower, whereas total nitrogen, tricarboxylic acid precipitable-, nitrogen, total volatile fatty acids and zoospore counts per millilitre of rumen liquor were significantly higher in fungal administered groups. After administration of fungal cultures, improvements of animal growth rate (i.e. body weight gain) and feed efficiency were also observed.

In vitro degradation of wheat straw by anaerobic fungi from small ruminants.

Anaerobic ruminal fungi may play an active role in fibre degradation as evidenced by the production of different fibrolytic enzymes in culture filtrate. In the present study, 16 anaerobic fungal strains were isolated from ruminal and faecal samples of sheep and goats. Based on their morphological characteristics they were identified as species of Anaeromyces, Orpinomyces, Piromyces and Neocallimastix. Isolated Neocallimastix sp. from goat rumen showed a maximum activity of CMCase (47.9 mIU ml(-1)) and filter paper cellulase (48.3 mIU ml(-1)), while Anaeromyces sp. from sheep rumen showed a maximum xylanolytic activity (48.3 mIU ml(-1)). The cellobiase activity for all the isolates ranged from 178.0-182.7 mIU ml(-1). Based on the enzymatic activities, isolated Anaeromyces sp. from sheep rumen and Neocallimastix sp. from goat rumen were selected for their potential of in vitro fibre degradation. The highest in vitro digestibility of NDF (23.2%) and DM (34.4%) was shown for Neocallimastix sp. from goat rumen, as compared to the digestibility of NDF and DM in the control group of 17.5 and 25.0%, respectively.