Immune-Boosting, Antioxidant and Anti-inflammatory Food Supplements Targeting Pathogenesis of COVID-19.

The COVID-19 is an acute and contagious disease characterized by pneumonia and ARDS. The disease is caused by SARS-CoV-2, which belongs to the family of Coronaviridae along with MERS-CoV and SARS-CoV-1. The virus has the positive-sense RNA as its genome encoding for ~26 proteins that work together for the virus survival, replication, and spread in the host. The virus gets transmitted through the contact of aerosol droplets from infected persons. The pathogenesis of COVID-19 is highly complex and involves suppression of host antiviral and innate immune response, induction of oxidative stress followed by hyper inflammation described as the "cytokine storm," causing the acute lung injury, tissue fibrosis, and pneumonia. Currently, several vaccines and drugs are being evaluated for their efficacy, safety, and for determination of doses for COVID-19 and this requires considerable time for their validation. Therefore, exploring the repurposing of natural compounds may provide alternatives against COVID-19. Several nutraceuticals have a proven ability of immune-boosting, antiviral, antioxidant, anti-inflammatory effects. These include Zn, vitamin D, vitamin C, curcumin, cinnamaldehyde, probiotics, selenium, lactoferrin, quercetin, etc. Grouping some of these phytonutrients in the right combination in the form of a food supplement may help to boost the immune system, prevent virus spread, preclude the disease progression to severe stage, and further suppress the hyper inflammation providing both prophylactic and therapeutic support against COVID-19.

In vitro adhesion and anti-inflammatory properties of native Lactobacillus fermentum and Lactobacillus delbrueckii spp.

AIMS: This study aimed at characterizing the adhesion and immune-stimulatory properties of native probiotic Lactobacillus fermentum (MCC 2759 and MCC 2760) and Lactobacillus delbrueckii MCC 2775. METHODS AND RESULTS: Adhesion of the strains was assessed in Caco-2 and HT-29 cell lines. Expression of adhesion and immune markers were evaluated in Caco-2 cells by real-time qPCR. The cultures displayed >80% of adhesion to both cell lines and also induced the expression of mucin-binding protein (mub) gene in the presence of mucin, bile and pancreatin. Adhesion was mediated by carbohydrate and proteinaceous factors. The cultures stimulated the expression of inflammatory cytokines in Caco-2 cells. However, pro-inflammatory genes were down-regulated upon challenge with lipopolysaccharide and IL-10 was up-regulated by the cultures. Cell wall extract of L. fermentum MCC 2760 induced the expression of IL-6 by 5·47-fold, whereas crude culture filtrate enhanced the expression of IL-10 by 14·87-fold compared to LPS control. CONCLUSIONS: The bacterial cultures exhibited strong adhesion and anti-inflammatory properties. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to reveal the role of adhesion markers of L. fermentum and L. delbrueckii by qPCR. The strain-specific anti-inflammatory property of native cultures may be useful to alleviate inflammatory conditions and develop a target-based probiotic.

Conjugal transfer of aac(6')Ie-aph(2″)Ia gene from native species and mechanism of regulation and cross resistance in Enterococcus faecalis MCC3063 by real time-PCR.

High level aminoglycoside resistance (HLAR) in the lactic acid bacteria (LAB) derived from food animals is detrimental. The aim of this study was to investigate the localization and conjugal transfer of aminoglycoside resistance genes, aac(6')Ie-aph(2″)Ia and aph(3')IIIa in different Enterococcus species. The cross resistance patterns in Enterococcus faecalis MCC3063 to clinically important aminoglycosides by real time PCR were also studied. Southern hybridization experiments revealed the presence of aac(6')Ie-aph(2″)Ia and aph(3')IIIa genes conferring HLAR in high molecular weight plasmids except in Lactobacillus plantarum. The plasmid encoded bifunctional aac(6')Ie-aph(2″)Ia gene was transferable from Enterococcus avium (n = 2), E. cecorum (n = 1), E. faecalis (n = 1) and Pediococcus lolii (n = 1) species into the recipient strain; E. faecalis JH2-2 by filter mating experiments thus indicating the possible risks of gene transfer into pathogenic strains. Molecular analysis of cross resistance patterns in native isolate of E. faecalis MCC3063 carrying aac(6')Ie-aph(2″)Ia and aph(3')IIIa gene was displayed by quantification of the mRNA levels in this study. For this, the culture was induced with increasing concentrations of gentamicin, kanamycin and streptomycin (2048, 4096, 8192, 16384 µg/mL) individually. The increasing concentrations of gentamicin and kanamycin induced the expression of the aac(6')Ie-aph(2″)Ia and aph(3')IIIa resistance genes, respectively. Interestingly, it was observed that induction with streptomycin triggered a significant fold increase in the expression of the aph(3')IIIa gene which otherwise was not known to modify the aminoglycoside. This is noteworthy as streptomycin was found to confer cross resistance to structurally unrelated kanamycin. Also, expression of the aph(3')IIIa gene when induced with streptomycin, revealed that bacteria harbouring this gene will be able to overcome streptomycin bactericidal action at specific concentrations. HLAR in E. faecalis MCC3063 may be due to the combined expression of both the aac(6')Ie-aph(2″)Ia and aph(3')IIIa genes which could be therapeutically challenging. A combined expression of both the genes in E. faecalis MCC3063 may yield HLAR which could be therapeutically challenging. The study highlights the significant alterations in the mRNA expression levels of aac(6')Ie-aph(2″)Ia and aph(3')IIIa in resistant pathogens, upon exposure to clinically vital aminoglycosides.

Emerging resistance to aminoglycosides in lactic acid bacteria of food origin-an impending menace.

Aminoglycosides are the most preferred choice of therapy against serious infections in humans. Therefore, its use in animal husbandry has been strictly regulated in the EU, UK, and USA to avoid the hazards of aminoglycoside resistance in gut microflora. Nevertheless, aminoglycosides are recommended for prophylaxis and therapeutics in food animals and agriculture owing to its bactericidal nature. In the recent past, the global surge in aminoglycoside-resistant lactic acid bacteria (LAB) from food sources has been noticed that might question its continued use in animal husbandry. Upon antibiotic administration, a selective pressure is created in the gut environment; in such instances, LAB could act as reservoirs of antibiotic resistance which may facilitate their transfer to pathogenic organisms contradicting its probiotic and industrial significance. This may be a risk to human health as the presence of one aminoglycoside resistance gene renders the bacteria tolerant to almost all antibiotics of the same class, thereby challenging its therapeutic efficacy. Low doses of aminoglycosides are recommended in farm animals due to its toxic nature and insolubility in blood. However, recent investigations indicate that use of aminoglycosides in sub-lethal concentrations can trigger the selection and conjugal transfer of aminoglycoside resistance in probiotic LAB. Resistance to erythromycin, tetracyclines, and fluoroquinolones in LAB were reported earlier to which immediate regulatory measures were adopted by some countries. Paradoxically, lack of regulations on antibiotic use in farms in most developing countries makes them a potential source of antibiotic resistance and its uncontrolled spread around the globe. The prevalence of aminoglycoside resistance was observed in enterococci from food origin earlier; however, its emergence in lactobacilli and pediococci suggests its spread in probiotic cultures which prompts immediate precautionary methods. This review highlights the emergence and hazards of aminoglycoside-resistant LAB which is in prime commercial demand both for preparing fermented food and also pharma-based therapeutics. It further focuses on the mode of aminoglycoside resistance and its occurrence in food-grade LAB, thus relating to its role in worldwide transfer via the food chain in spite of its limited use as compared to other antibiotics.

Extracellular chitin deacetylase production in solid state fermentation by native soil isolates of Penicillium monoverticillium and Fusarium oxysporum.

Extracellular chitin deacetylase production by native soil isolates of Penicillium monoverticillium CFR 2 and Fusarium oxysporum CFR 8 in solid state fermentation (SSF) using commercial wheat bran (CWB) and shrimp processing by-products (SPP) as solid substrate has been studied. P. monoverticillium produced maximum chitin deacetylase activity of 547.7 ± 45 and 390.2 ± 31 units/g initial dry substrate (U/g IDS) at 96 h of incubation in CWB and SPP media, respectively. While, F. oxysporum produced maximum chitin deacetylase activity of 306.4 ± 22 U/g IDS at 72 h of incubation in CWB medium and 220.1 ± 20 U/g IDS at 120 h of incubation in SPP medium. Along with chitin deacetylase, P. monoverticillium and F. oxysporum produced other chitin degrading enzymes such as endo-chitinase and ß-N-acetylhexosaminidase. P. monoverticillium produced maximum activity (U/g IDS) of endo-chitinase 4.6 ± 0.20 at 120 h incubation and ß-N-acetylhexosaminidase 82.6 ± 03 at 120 h incubation in CWB medium. While, F. oxysporum produced maximum activity (U/g IDS) of endo-chitinase 7.8 ± 0.20 at 144 h incubation and ß-N-acetylhexosaminidase 38.3 ± 02 at 120 h incubation in CWB medium. Production of extracellular chitin deacetylase by P. monoverticillium CFR 2 and F. oxysporum CFR 8 in SSF is being reported for the first time.

Immunogenicity of Staphylococcus aureus LukM/F'-PV recombinant subunits: validation of diagnostic potential and evaluation of protective efficacy in vitro.

Leukotoxin M/F'-Panton Valentine (LukM/F'-PV), a beta pore-forming toxin secreted by Staphylococcus aureus, is a major virulence factor involved in the pathogenesis of bovine mastitis. The present study was aimed to determine immunogenicity of two recombinant subunits of LukM/F'-PV, rLukM (MW 38 kDa) and rLukF (MW 39 kDa), develop and validate an indirect enzyme linked immunosorbent assay (ELISA) using polyclonal antibodies raised in rabbits, and evaluate applicability of the assay to diagnose clinical and subclinical bovine mastitis. Additionally, in vitro assays were conducted to determine abilities of antibodies to neutralize cytotoxicity of the native leukotoxin. A total of 87 bovine milk samples (healthy, subclinical and clinical mastitis) were evaluated for the presence of toxin determinants. Receiver-operator characteristic curve for the experimental ELISA values statistically interpreted a cut-off score of >0.109 OD405, with an assay specificity of 100% and sensitivity in the range of 80-87.5%. In addition, area under curve of 0.93-0.98 revealed the test was accurate in categorizing samples from infected and non-infected bovine. The rLukF IgG-ELISA was more sensitive than rLukM IgG-ELISA. Furthermore, it was evident from MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium) dye reduction, indirect immunofluorescence and lactate dehydrogenase assays that anti-rLukM/rLukF antibodies, with high neutralizing titers, inhibited in vitro leukotoxic activity and protected bovine neutrophil membrane integrity from cytotoxicity of native leukotoxin. The findings demonstrated that antibodies produced from recombinant subunits contribute to specific and sensitive immunodiagnosis and may also have the potential to provide passive therapeutic benefit in the management of bovine mastitis.

Antioxidant and anticoagulant activity of polyphenol and polysaccharides from fermented Sargassum sp.

The current investigation was carried out with an objective of determining the structural characteristic of polysaccharides extracted from fermented Sargassum sp. to be used as potent natural heparin substitute anticoagulant compound. Sargassum sp. fermented with marine lactic acid bacteria was initially subjected to ethanol precipitation for the recovery of bioactive compounds. Antioxidant activity was maximum in the soluble fraction whereas anticoagulant activity was observed to be high in the precipitate which correlated with the increased polyphenols and total sugars respectively. The precipitate was purified by anion exchange chromatography and the fractions collected were analyzed for total sugars and anticoagulant activity. There was 2.6-3.9-folds increase in anticoagulant activity in the final purified fractions, with a maximum activity in case of sample fermented with Enterococcus faecium (6.7±0.22 IU/mg). Structural elucidation of potential anticoagulant polysaccharide by Fourier Transform Infrared Spectroscopy (FT-IR) and Nuclear Magnetic Resonance (NMR) analysis indicated the presence of alginate rich in mannuronic acid.

Phenotypic expression, molecular characterization and transferability of erythromycin resistance genes in Enterococcus spp. isolated from naturally fermented food.

AIMS: To evaluate phenotypic resistance to macrolides-lincosamides and streptogramin B (MLSB ) antibiotics and to determine their localization as well as transferability of erythromycin resistance genes in enterococcal isolates of naturally fermented food-Idli batter. METHODS AND RESULTS: Diverse MLSB phenotypes observed among the enterococcal spp. (n = 32) were analysed through double disc and triple disc test. Standard minimum inhibitory concentration tests along with induction studies displayed synergistic or cross-resistance among MLSB antibiotics. Plasmid profiling and Southern hybridization revealed that erm(B) and msr(C) genes were localized either on chromosome or on high molecular weight plasmids and showed co-localization of these genes with lnu(B), tet(L) and tet(W) in one of the isolate. In vitro conjugation experiments demonstrated plasmid-mediated transfer of erm(B) gene from three Enterococcus durans strains to Enterococcus faecalis JH2-2. CONCLUSIONS: The study illustrated diverse MLSB phenotypes, multiple resistance genes and transferable plasmids among enterococci isolated from naturally fermented foods. SIGNIFICANCE AND IMPACT OF THE STUDY: From a public health point of view, the study identified that naturally fermented foods could represent a source of antibiotic resistance enterococci that can spread through foods. These results also suggest that vigilance may be exercised with the use of combination or novel MLSB antibiotics in treating enterococcal infections.

Development and application of active films for food packaging using antibacterial peptide of Bacillus licheniformis Me1.

AIMS: An attempt was made to evaluate the effectiveness of partially purified antibacterial peptide (ppABP) produced by Bacillus licheniformis Me1 for food preservation by means of active packaging. METHODS AND RESULTS: The active packaging films containing ppABP were developed using two different packing materials [low-density polyethylene (LDPE) and cellulose films] by two different methods: soaking and spread coating. The activated films showed antibacterial activity against pathogens. The release study of ppABP from coated film showed that the LDPE films liberated ppABP as soon as it comes in contact with water, while gradual release of coated ppABP was observed in case of cellulose films. The activated films showed residual activity in different simulating conditions, such as pH of food and storage temperatures. The activated films demonstrated its biopreservative efficacy in controlling the growth of pathogens in cheese and paneer. CONCLUSIONS: The ppABP-activated films were found to be effective for biopreservation. The ppABP from active films got diffused into the food matrix and reduced the growth rate and maximum growth population of the target micro-organism. SIGNIFICANCE AND IMPACT OF THE STUDY: Both types of ppABP-activated films can be used as a packaging material to control spoilage and pathogenic organisms in food, thereby extending the shelf life of foods.

Molecular Characterization and Toxicity Confirmation of LukM/F'-PV Producing Staphylococcus aureus Isolated from Bovine Mastitis Samples in Mysore, India.

The widespread status of subclinical condition of bovine mastitis is often associated with the production of leukotoxin M/F'-PV producing Staphylococcus aureus. The present study aims for the profiling of such leukotoxin producers through conventional and molecular methods in parallel to their leukotoxicity. The incidence of this particular pathogen was assessed in mastitis infected Holstein-Friesian cattle, where eight isolates of staphylococci were found to be present in 20 % of collected samples. Being intermediately resistant to vancomycin, they showed characteristic double zone hemolysis on 7 % sheep blood agar and typical type II reaction for coagulase test indicating the pathogenic attributes. Further with RAPD-PCR and 16S rDNA-RFLP, epidemiological specificity and genotypic relatedness of isolates to S. aureus was confirmed. Subsequently, the presence of leukotoxin (lukM) gene in native isolates was detected by leukotoxin gene specific PCR. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) assay evaluated for secreted leukotoxin in cell free supernatant was estimated to be 223 toxic units which had an LD50 cytotoxic activity on bovine neutrophil. Thus, the data acquired during study can be of prime diagnostic method for timely and accurate analysis of subclinical mastitis samples which goes undetected at consumer level.

Potential of marine lactic acid bacteria to ferment Sargassum sp. for enhanced anticoagulant and antioxidant properties.

AIM: To evaluate the suitability of marine lactic acid bacteria (LAB) as starter cultures for Sargassum sp. fermentation to enhance its antioxidant and anticoagulation activity. METHODS AND RESULTS: LAB isolated from marine source were characterized for their ability to utilize seaweed as a sole carbon source and applied to Sargassum fermentation. Fermentation period was optimized by monitoring the fermented sample at regular interval for a period of 18 days. Results revealed that a fermentation period of 12 days was effective with maximum culture viability and other desirable characteristics such as pH, total titratable acidity, total and reducing sugars. Under optimum fermentation period, the sample fermented with P1-2CB-w1 (Enterococcus faecium) exhibited maximum anticoagulation activity and antioxidant activity. CONCLUSIONS: The study reveals a novel well-defined starter culture from marine origin intended for seaweed fermentation for recovery of bioactive molecules. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides information for the enhancement of bioactive molecules in an eco-friendly manner and also paves a way towards the development of wide range of seaweed functional foods.

Isolation and characterization of potential lactic acid bacteria (LAB) from freshwater fish processing wastes for application in fermentative utilisation of fish processing waste

Proteolytic and/or lipolytic lactic acid bacteria (LAB) were isolated from visceral wastes of different fresh water fishes. LAB count was found to be highest in case of visceral wastes of Mrigal (5.88 log cfu/g) and lowest in that of tilapia (4.22 log cfu/g). Morphological, biochemical and molecular characterization of the selected LAB isolates were carried out. Two isolates FJ1 (E. faecalis NCIM5367) and LP3 (P. acidilactici NCIM5368) showed both proteolytic and lipolytic properties. All the six native isolates selected for characterization showed antagonistic properties against several human pathogens. All the native isolates were sensitive to antibiotics cephalothin and clindamycin; and, resistant to cotrimoxazole and vancomycin. Considering individually, P. acidilactici FM37, P. acidilactici MW2 and E. faecalis FD3 were sensitive to erythromycin. The two strains FJ1 (E. faecalis NCIM 5367) and LP3 (P. acidilactici NCIM 5368) that had both proteolytic and lipolytic properties have the potential for application in fermentative recovery of lipids and proteins from fish processing wastes.

Phytate-degrading Pediococcus pentosaceus CFR R123 for application in functional foods.

The present work looks at the role of phytate-degrading Pediococcus pentosaceus CFR R123 application in functional foods to evaluate the fate of phytate and calcium solubility during fermentation. Under standard conditions, CFR R123 grown in modified MRS containing sodium phytate CFR R123 showed 43% degradation of sodium phytate in 15 minutes. Fermentation of malted finger millet seed coat (MFSC) and soya milk (SM) with CFR R123 for 12 h resulted in 5.6-12% phytate degradation and a notable increase in calcium availability (125%) was observed. The isolate CFR R123 was found to decrease the phytic acid levels resulting in increased levels of calcium during MFSC and soya milk fermentation. This study introduces phytate-degrading P. pentosaceus CFR R123 that can be employed as a starter culture as well as an ingredient of functional food to provide nutritive benefits to the consumer with a natural phenomenon.

Evaluation of beneficial attributes for phytate-degrading Pediococcus pentosaceus CFR R123.

In this study we have screened and selected potent phytate-degrading lactic acid bacteria (LAB), and evaluated their beneficial attributes. Around 60 LAB strains were isolated from several cereal- and pulse-based conventional fermented preparations. They were screened for their ability to degrade myo-inositol hexakisphosphate (IP6) by cobalt chloride qualitative staining method (plate assay). One of the cultures, Pediococcus pentosaceus CFR R123, was capable of degrading both calcium and sodium salts of phytic acid. Additionally, we have carried out an in vitro evaluation for the beneficial attributes of phytate degrading CFR R123. P. pentosaceus CFR R123 showed 53% survivability at pH 2 and 62% at pH 2.5, whereas cultures of Lactobacillus rhamnosus GG showed a survivability of 55% and 82%, respectively. CFR R123 could also withstand 0.3% ox-bile, whereas no growth was observed for GG. The strain CFR R123 exhibited 62.8% hydrophobicity to xylene whereas 59% was found for GG. Both the tested strains showed a good spectrum of antibacterial activity against food-borne pathogens like Escherichia coli, Listeria monocytogenes Scott A, etc. P. pentosaceus CFR R123 possessed ß-galactosidase activity and cholesterol reduction ability. In conclusion, LAB with phytate degrading ability and several beneficial attributes could potentially be used as a starter culture to improve the nutritional security of functional food.

Characterisation of the heat-stable bacteriocin-producing and vancomycin-sensitive Pediococcus pentosaceus CFR B19 isolated from beans.

A bacteriocin-producing lactic culture with antilisterial activity was isolated from beans and identified as Pediococcus pentosaceus CFR B19. It was able to grow and produce bacteriocin at 41 °C but not at 50 °C. This isolate was found to be sensitive to vancomycin and produced heat-stable (at 121 °C for 15 min) bacteriocin. Molecular weight of the purified bacteriocin was found to be ∼4.8 kDa. This isolate can be used as a starter culture or co-culture in fermented milk products and the bacteriocin can be used as a natural preservative in various food products.

Utilization of tannery fleshings: Optimization of conditions for fermenting delimed tannery fleshings using Enterococcus faecium HAB01 by response surface methodology.

Conditions for fermentation of delimed tannery fleshings--to obtain higher degree of protein hydrolysis and reasonably better antioxidant activity--using Enterococcus faecium HAB01 (GenBank #FJ418568) were optimized. Three independent variables--viz., inoculum level (X1), glucose level (X2) and fermentation time (X3)--were optimized using response surface method considering degree of hydrolysis (DH; %) and total titrable acidity (TTA) as response variables. The optimized conditions were found to be 12.5% (v/w) inoculum, 17.5% (w/w) glucose and 96h of fermentation at 37+/-1 degrees C to obtain a maximum DH%. The usefulness of the predicted model was further validated by considering random combinations of the independent factors. The chemical score of the hydrolysate revealed an excess amount of essential amino acids, viz., arginine and leucine compared to reference protein. The liquor portion had relatively high antioxidant activities, indicating its potential for use as a high value feed ingredient.

Lactobacillus farciminis MD, a newer strain with potential for bacteriocin and antibiotic assay.

A native isolate Lactobacillus farciminis MD isolated from fermenting mushroom exhibited a high degree of sensitivity to the majority of the bacteriocins produced by strains of lactobacilli, leuconostoc and pediococci. Also, the efficacy of Lact. farciminis MD as a sensitive strain for antibiotic assay was established against different antibiotics including ampicillin, cefazoline, chloramphenicol and nitrofurantoin at concentrations of 30 microg each, showing an inhibition zone of 30 mm diameter. The high degree of sensitivity towards bacteriocins and antibiotics provide potential for the exploitation of Lact. farciminis MD in establishing very well-defined bacteriocin producers.