Bioprospecting of the probiotic potential of yeasts isolated from a wine environment.

Autochthonous yeasts of oenological origin are adapted to highly stressful and selective environments, which makes them potential candidates for probiotics. The objective of the present study was to explore the probiotic potential of 96 native yeasts of oenological origin, their biosafety, resistance to gastrointestinal tract conditions and adhesion properties. Regarding biosafety, 66 isolates shown negative hemolytic activity, negative urease activity and susceptibility to 3 or more antifungals. After the gastrointestinal resistance test, 15 isolates were selected that showed growth at different temperatures, tolerance to low pH and the presence of bile salts in in vitro tests. In general, survival after simulated conditions of the gastrointestinal tract was high and more restrictive was the duodenal. The results of the adhesion properties showed highly variable hydrophobicity and a high percentage of autoaggregation at 24 h. The maximum production of biofilm was detected in the Pichia strains. Of a total of 96 yeast strains, 15 non-Saccharomyces yeasts presented suitable properties as probiotic candidates. The native winemaking strains performed better than the reference probiotic strain, Saccharomyces cerevisiae var. boulardii CNCM I-745, which reaffirms that these strains are promising probiotic candidates and further studies are necessary to confirm their probiosis.

Synergistic synbiotic containing fructooligosaccharides and Lactobacillus delbrueckii CIDCA 133 alleviates chemotherapy-induced intestinal mucositis in mice.

Intestinal mucositis is a commonly reported side effect in oncology patients undergoing chemotherapy and radiotherapy. Probiotics, prebiotics, and synbiotics have been investigated as alternative therapeutic approaches against intestinal mucositis due to their well-known anti-inflammatory properties and health benefits to the host. Previous studies showed that the potential probiotic Lactobacillus delbrueckii CIDCA 133 and the prebiotic Fructooligosaccharides (FOS) alleviated the 5-Fluorouracil (5-FU) chemotherapy-induced intestinal mucosa damage. Based on these previous beneficial effects, this work evaluated the anti-inflammatory property of the synbiotic formulation containing L. delbrueckii CIDCA 133 and FOS in mice intestinal mucosa inflammation induced by 5-FU. This work showed that the synbiotic formulation was able to modulate inflammatory parameters, including reduction of cellular inflammatory infiltration, gene expression downregulation of Tlr2, Nfkb1, and Tnf, and upregulation of the immunoregulatory Il10 cytokine, thus protecting the intestinal mucosa from epithelial damage caused by the 5-FU. The synbiotic also improved the epithelial barrier function by upregulating mRNA transcript levels of the short chain fatty acid (SCFA)-associated GPR43 receptor and the occludin tight junction protein, with the subsequent reduction of paracellular intestinal permeability. The data obtained showed that this synbiotic formulation could be a promising adjuvant treatment to be explored against inflammatory damage caused by 5-FU chemotherapy.

Staphylococcus epidermidis role in the skin microenvironment.

Wound healing is a complex dynamic physiological process in response to cutaneous destructive stimuli that aims to restore the cutaneous' barrier role. Deciphering the underlying mechanistic details that contribute to wound healing will create novel therapeutic strategies for skin repair. Recently, by using state-of-the-art technologies, it was revealed that the cutaneous microbiota interact with skin immune cells. Strikingly, commensal Staphylococcus epidermidis-induced CD8+ T cells induce re-epithelization of the skin after injury, accelerating wound closure. From a drug development perspective, the microbiota may provide new therapeutic candidate molecules to accelerate skin healing. Here, we summarize and evaluate recent advances in the understanding of the microbiota in the skin microenvironment.

Development of a new DNA vaccine based on mycobacterial ESAT-6 antigen delivered by recombinant invasive Lactococcus lactis FnBPA+.

The use of the food-grade bacterium Lactococcus lactis as a vehicle for the oral delivery of DNA vaccine plasmids constitutes a promising strategy for vaccination. The delivery of DNA plasmids into eukaryotic cells is of critical importance for subsequent DNA expression and effectiveness of the vaccine. In this context, the use of the recombinant invasive L. lactis FnBPA+ (fibronectin-binding protein A) strain for the oral delivery of the eukaryotic expression vector vaccination using lactic acid bacteria (pValac), coding for the 6-kDa early secreted antigenic target (ESAT-6) gene of Mycobacterium tuberculosis, could represent a new DNA vaccine strategy against tuberculosis. To this end, the ESAT-6 sequence was cloned into the pValac vector; the L. lactis fibronectin-binding protein A (FnBPA)+ (pValac:ESAT-6) strain was obtained, and its immunological profile was checked in BALB/c mice. This strain was able to significantly increase interferon gamma (IFN-γ) production in spleen cells, showing a systemic T helper 1 (Th1) cell response. The mice also showed a significant increase in specific secretory immunoglobulin A (sIgA) production in colon tissue and fecal extracts. Thus, this is the first time that L. lactis has been used to deliver a plasmid DNA harboring a gene that encodes an antigen against tuberculosis through mucous membranes.

Cell-free supernatant of probiotic bacteria exerted antibiofilm and antibacterial activities against Pseudomonas aeruginosa: A novel biotic therapy.

Aim: This study aims to verify the antibacterial and antibiofilm action of cell-free spent medium (CFSM) from four lactic acid bacteria with potential probiotic characteristics (Lactiplantibacillus plantarum, Lactobacillus acidophilus, Lactobacillus johnsonii, and Lactobacillus delbrueckii) against two Pseudomonas aeruginosa strains. Main methods: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the CFSM, antibacterial activity by analysing the formation of inhibition zones, and inhibition of planktonic cultures were determined. Whether an increase in the concentration of CFSM influenced the growth of pathogenic strains and the anti-adhesive activity of the CFSM in biofilm formation (crystal violet and MTT assays) were determined, which were all corroborated by using scanning electron microscopy. Key findings: The relationship between the MIC and MBC values showed a bactericidal or bacteriostatic effect for all the cell-free spent media (CFSMs) tested for P. aeruginosa 9027™ and 27853™ strains. The CFSM supplemental doses of 18 or 22%, 20 or 22%, 46 or 48%, and 50 or 54% of L. acidophilus, L. delbrueckii, L. plantarum, and L. johnsonii, respectively, could completely inhibit the growth of both pathogen strains. The antibiofilm activity of the CFSM in three biofilm conditions (pre-coated, co-incubated, and preformed) demonstrated values ranging between 40% and 80% for biofilm inhibition, and similar results were observed for cell viability. Significance: This work provides strong evidence that the postbiotic derived from different Lactobacilli could be practical as an adjuvant therapy for reducing the use of antibiotics, being a good candidate to overcome the growing challenge of hospital infections due to this pathogen.

Paraprobiotic Lacticaseibacillus rhamnosus Protects Intestinal Damage in an Experimental Murine Model of Mucositis.

Intestinal mucositis (IM) is a common side effect resulting from cancer treatment. However, the management so far has not been very effective. In the last years, the role of the gut microbiota in the development and severity of mucositis has been studied. Therefore, the use of probiotics and paraprobiotics could have a potential therapeutic effect on IM. The aim of our study was to investigate the impact of the administration of Lacticaseibacillus rhamnosus (L. rhamnosus) CGMCC1.3724 and the paraprobiotic on IM in mice. For 13 days, male Balb/c mice were divided into six groups: control (CTL) and mucositis (MUC)/0.1 mL of saline; CTL LrV and MUC LrV/0.1 mL of 108 CFU of viable Lr; CTL LrI and MUC LrI/0.1 mL of 108 CFU of inactivated Lr. On the 10th day, mice from the MUC, MUC LrV, and MUC LrI groups received an intraperitoneal injection (300 mg/kg) of 5-fluorouracil to induce mucositis. The results showed that the administration of the chemotherapeutic agent increased the weight loss and intestinal permeability of the animals in the MUC and MUC LrV groups. However, administration of paraprobiotic reduced weight loss and maintained PI at physiological levels. The paraprobiotic also preserved the villi and intestinal crypts, reduced the inflammatory infiltrate, and increased the mucus secretion, Muc2 gene expression, and Treg cells frequency.

Diets Supplemented with Probiotics Improve the Performance of Broilers Exposed to Heat Stress from 15 Days of Age.

The poultry sector demands alternative additives to antibiotics that can be used as performance enhancers. Therefore, this experiment was conducted to evaluate the probiotics effects on performance, intestinal health, and redox status of 720 broilers exposed to heat stress from 15 days of age. Eight dietary treatments were evaluated: basal diet (BD) without antibiotic and probiotic (T1); BD supplemented with antibiotic zinc bacitracin (T2), BD supplemented with commercial probiotic of Bacillus subtilis DSM 17,299 (T3), BD supplemented with non-commercial probiotic of Lactococcus lactis NCDO 2118, Lactobacillus delbrueckii CNRZ 327, Escherichia coli CEC15, or Saccharomyces boulardii (T4 to T7), and BD simultaneously supplemented with the four non-commercial probiotics (T8). Feed intake, weight gain, and feed conversion were determined in the period from 1 to 42 days of age. Carcass and cuts yield, abdominal fat deposition, cloacal temperature, weight and length of intestine, activity of myeloperoxidase and eosinophilic peroxidase enzymes in the jejunum, jejunal histomorphometry, relative gene expression in the jejunum (occludin, zonulin, interleukin-8, cholecystokinin, ghrelin, and heat shock protein-70), and liver (heat shock protein-70), in addition to malondialdehyde level and superoxide dismutase activity in the intestine, liver, and blood, were measured in broilers at 42 days old. As main results, broilers fed T1 diet exhibited lower weight gain (3.222 kg) and worse feed conversion (1.70 kg/kg). However, diets containing non-commercial probiotics resulted in up to 3.584 kg of weight gain and improved feed conversion by up to 10%, similar to that observed for broilers of the T2 and T3 groups.

Growth differentiation factor 11 delivered by dairy Lactococcus lactis strains modulates inflammation and prevents mucosal damage in a mice model of intestinal mucositis.

Mucositis is an inflammation of the gastrointestinal mucosa that debilitate the quality of life of patients undergoing chemotherapy treatments. In this context, antineoplastic drugs, such as 5-fluorouracil, provokes ulcerations in the intestinal mucosa that lead to the secretion of pro-inflammatory cytokines by activating the NF-κB pathway. Alternative approaches to treat the disease using probiotic strains show promising results, and thereafter, treatments that target the site of inflammation could be further explored. Recently, studies reported that the protein GDF11 has an anti-inflammatory role in several diseases, including in vitro and in vivo results in different experimental models. Hence, this study evaluated the anti-inflammatory effect of GDF11 delivered by Lactococcus lactis strains NCDO2118 and MG1363 in a murine model of intestinal mucositis induced by 5-FU. Our results showed that mice treated with the recombinant lactococci strains presented improved histopathological scores of intestinal damage and a reduction of goblet cell degeneration in the mucosa. It was also observed a significant reduction of neutrophil infiltration in the tissue in comparison to positive control group. Moreover, we observed immunomodulation of inflammatory markers Nfkb1, Nlrp3, Tnf, and upregulation of Il10 in mRNA expression levels in groups treated with recombinant strains that help to partially explain the ameliorative effect in the mucosa. Therefore, the results found in this study suggest that the use of recombinant L. lactis (pExu:gdf11) could offer a potential gene therapy for intestinal mucositis induced by 5-FU.

Safety Evaluation of Lactobacillus delbrueckii subsp. lactis CIDCA 133: a Health-Promoting Bacteria.

Lactobacillus delbrueckii subsp. lactis CIDCA is a new potential probiotic strain whose molecular basis attributed to the host's benefit has been reported. This study investigated the safety aspects of Lactobacillus delbrueckii subsp. lactis CIDCA 133 based on whole-genome sequence and phenotypic analysis to avoid future questions about the harmful effects of this strain consumption. Genomic analysis showed that L. delbrueckii subsp. lactis CIDCA 133 harbors virulence, harmful metabolites, and antimicrobial resistance-associated genes. However, none of these genetic elements is flanked or located within prophage regions and plasmid sequence. At a phenotypic level, it was observed L. delbrueckii subsp. lactis CIDCA 133 antimicrobial resistance to aminoglycosides streptomycin and gentamicin antibiotics, but no hemolytic and mucin degradation activity was exhibited by strain. Furthermore, no adverse effects were observed regarding mice clinical and histopathological analysis after the strain consumption (5 × 107 CFU/mL). Overall, these findings reveal the safety of Lactobacillus delbrueckii subsp. lactis CIDCA 133 for consumption and future probiotic applications.

Paraprobiotics and Postbiotics of Lactobacillus delbrueckii CIDCA 133 Mitigate 5-FU-Induced Intestinal Inflammation.

Intestinal mucositis is a commonly reported side effect in oncology practice. Probiotics are considered an excellent alternative therapeutic approach to this debilitating condition; however, there are safety questions regarding the viable consumption of probiotics in clinical practice due to the risks of systemic infections, especially in immune-compromised patients. The use of heat-killed or cell-free supernatants derived from probiotic strains has been evaluated to minimize these adverse effects. Thus, this work evaluated the anti-inflammatory properties of paraprobiotics (heat-killed) and postbiotics (cell-free supernatant) of the probiotic Lactobacillus delbrueckii CIDCA 133 strain in a mouse model of 5-Fluorouracil drug-induced mucositis. Administration of paraprobiotics and postbiotics reduced the neutrophil cells infiltrating into the small intestinal mucosa and ameliorated the intestinal epithelium architecture damaged by 5-FU. These ameliorative effects were associated with a downregulation of inflammatory markers (Tlr2, Nfkb1, Il12, Il17a, Il1b, Tnf), and upregulation of immunoregulatory Il10 cytokine and the epithelial barrier markers Ocln, Cldn1, 2, 5, Hp and Muc2. Thus, heat-killed L. delbrueckii CIDCA 133 and supernatants derived from this strain were shown to be effective in reducing 5-FU-induced inflammatory damage, demonstrating them to be an alternative approach to the problems arising from the use of live beneficial microorganisms in clinical practice.

Lactobacillus delbrueckii CIDCA 133 Ameliorates Chemotherapy-Induced Mucositis by Modulating Epithelial Barrier and TLR2/4/Myd88/NF-κB Signaling Pathway.

Intestinal mucositis promoted by the use of anticancer drugs is characterized by ulcerative inflammation of the intestinal mucosa, a debilitating side effect in cancer patients undergoing treatment. Probiotics are a potential therapeutic option to alleviate intestinal mucositis due to their effects on epithelial barrier integrity and anti-inflammatory modulation. This study investigated the health-promoting impact of Lactobacillus delbrueckii CIDCA 133 in modulating inflammatory and epithelial barrier markers to protect the intestinal mucosa from 5-fluorouracil-induced epithelial damage. L. delbrueckii CIDCA 133 consumption ameliorated small intestine shortening, inflammatory cell infiltration, intestinal permeability, villus atrophy, and goblet cell count, improving the intestinal mucosa architecture and its function in treated mice. Upregulation of Muc2, Cldn1, Hp, F11r, and Il10, and downregulation of markers involved in NF-κB signaling pathway activation (Tlr2, Tlr4, Nfkb1, Il6, and Il1b) were observed at the mRNA level. This work suggests a beneficial role of L. delbrueckii strain CIDCA 133 on intestinal damage induced by 5-FU chemotherapy through modulation of inflammatory pathways and improvement of epithelial barrier function.

Lactic Acid Bacteria as Delivery Vehicle for Therapeutics Applications.

Lactic acid bacteria comprise a large group of Gram-positive organisms capable of converting sugar into lactic acid. They have been studied due to their therapeutic potential on the mucosal surface. Among the species, Lactococcus lactis is considered the model bacterium and it has been explored as an important vehicle for providing therapeutic molecules and antigens in the mucosa. They can be genetically engineered to produce a variety of molecules as well as deliver heterologous DNA and protein. DNA vaccines consist of the administration of a bacterial plasmid under the control of a eukaryotic promoter encoding the antigen of interest. The resulting proteins are capable of stimulating the immune system, becoming a promising technique for immunization against a variety of tumors and infection diseases and having several advantages compared to conventional nucleic acid delivery methods (such as bioballistic delivery, electroporation, and intramuscular administration).

Main Features of DNA-Based Vectors for Use in Lactic Acid Bacteria and Update Protocols.

DNA vaccines have been used as a promising strategy for delivery of immunogenic and immunomodulatory molecules into the host cells. Although, there are some obstacles involving the capability of the plasmid vector to reach the cell nucleus in great number to promote the expected benefits. In order to improve the delivery and, consequently, increase the expression levels of the target proteins carried by DNA vaccines, alternative methodologies have been explored, including the use of non-pathogenic bacteria as delivery vectors to carry, deliver, and protect the DNA from degradation, enhancing plasmid expression.

Probiogenomics of Lactobacillus delbrueckii subsp. lactis CIDCA 133: In Silico, In Vitro, and In Vivo Approaches.

Lactobacillus delbrueckii subsp. lactis CIDCA 133 (CIDCA 133) has been reported as a potential probiotic strain, presenting immunomodulatory properties. This study investigated the possible genes and molecular mechanism involved with a probiotic profile of CIDCA 133 through a genomic approach associated with in vitro and in vivo analysis. Genomic analysis corroborates the species identification carried out by the classical microbiological method. Phenotypic assays demonstrated that the CIDCA 133 strain could survive acidic, osmotic, and thermic stresses. In addition, this strain shows antibacterial activity against Salmonella Typhimurium and presents immunostimulatory properties capable of upregulating anti-inflammatory cytokines Il10 and Tgfb1 gene expression through inhibition of Nfkb1 gene expression. These reported effects can be associated with secreted, membrane/exposed to the surface and cytoplasmic proteins, and bacteriocins-encoding genes predicted in silico. Furthermore, our results showed the genes and the possible mechanisms used by CIDCA 133 to produce their beneficial host effects and highlight its use as a probiotic microorganism.

Intake of Lactobacillus delbrueckii (pExu:hsp65) Prevents the Inflammation and the Disorganization of the Intestinal Mucosa in a Mouse Model of Mucositis.

5-Fluorouracil (5-FU) is an antineoplastic drug that causes, as a side effect, intestinal mucositis, acute inflammation in the small bowel. The Heat Shock Protein (Hsp) are highly expressed in inflammatory conditions, developing an important role in immune modulation. Thus, they are potential candidates for the treatment of inflammatory diseases. In the mucositis mouse model, the present study aimed to evaluate the beneficial effect of oral administration of milk fermented by Lactobacillus delbrueckii CIDCA 133 (pExu:hsp65), a recombinant strain. This approach showed increased levels of sIgA in the intestinal fluid, reducing inflammatory infiltrate and intestinal permeability. Additionally, the histological score was improved. Protection was associated with a reduction in the gene expression of pro-inflammatory cytokines such as Tnf, Il6, Il12, and Il1b, and an increase in Il10, Muc2, and claudin 1 (Cldn1) and 2 (Cldn2) gene expression in ileum tissue. These findings are corroborated with the increased number of goblet cells, the electronic microscopy images, and the reduction of intestinal permeability. The administration of milk fermented by this recombinant probiotic strain was also able to reverse the high levels of gene expression of Tlrs caused by the 5-FU. Thus, the rCIDCA 133:Hsp65 strain was revealed to be a promising preventive strategy for small bowel inflammation.

Lactococcus lactis FNBPA+ (pValac:e6ag85a) Induces Cellular and Humoral Immune Responses After Oral Immunization of Mice.

The development of a new vaccine strategy against tuberculosis is urgently needed and has been greatly encouraged by the scientific community worldwide. In this work, we constructed a lactococcal DNA vaccine based on the fusion of two Mycobacterium tuberculosis antigens, ESAT-6 and Ag85A, and examined its immunogenicity. The coding sequences of the ESAT-6 and Ag85A genes were fused and cloned into the eukaryotic expression pValac vector, and the functionality of the vector was confirmed in vitro. Then, L. lactis FnBPA+ (pValac:e6ag85a) was obtained and used for oral immunization of mice. This strain induced significant increases in IFN-γ, TNF-α, and IL-17 cytokines in stimulated splenocyte cultures, and significant production of antigen-specific sIgA was observed in the colonic tissues of immunized mice. We demonstrated that L. lactis FnBPA+ (pValac:e6ag85a) generated a cellular and humoral immune response after oral immunization of mice. The strategy developed in this work may represent an interesting DNA mucosal vaccine candidate against tuberculosis, using the fusion of two highly immunogenic antigens delivered by safe lactic acid bacteria.

Novel Strategies for Efficient Production and Delivery of Live Biotherapeutics and Biotechnological Uses of Lactococcus lactis: The Lactic Acid Bacterium Model.

Lactic acid bacteria (LAB) are traditionally used in fermentation and food preservation processes and are recognized as safe for consumption. Recently, they have attracted attention due to their health-promoting properties; many species are already widely used as probiotics for treatment or prevention of various medical conditions, including inflammatory bowel diseases, infections, and autoimmune disorders. Some LAB, especially Lactococcus lactis, have been engineered as live vehicles for delivery of DNA vaccines and for production of therapeutic biomolecules. Here, we summarize work on engineering of LAB, with emphasis on the model LAB, L. lactis. We review the various expression systems for the production of heterologous proteins in Lactococcus spp. and its use as a live delivery system of DNA vaccines and for expression of biotherapeutics using the eukaryotic cell machinery. We have included examples of molecules produced by these expression platforms and their application in clinical disorders. We also present the CRISPR-Cas approach as a novel methodology for the development and optimization of food-grade expression of useful substances, and detail methods to improve DNA delivery by LAB to the gastrointestinal tract. Finally, we discuss perspectives for the development of medical applications of recombinant LABs involving animal model studies and human clinical trials, and we touch on the main safety issues that need to be taken into account so that bioengineered versions of these generally recognized as safe organisms will be considered acceptable for medical use.

Probiotics, Prebiotics, Synbiotics, and Paraprobiotics as a Therapeutic Alternative for Intestinal Mucositis.

Intestinal mucositis, a cytotoxic side effect of the antineoplastic drug 5-fluorouracil (5-FU), is characterized by ulceration, inflammation, diarrhea, and intense abdominal pain, making it an important issue for clinical medicine. Given the seriousness of the problem, therapeutic alternatives have been sought as a means to ameliorate, prevent, and treat this condition. Among the alternatives available to address this side effect of treatment with 5-FU, the most promising has been the use of probiotics, prebiotics, synbiotics, and paraprobiotics. This review addresses the administration of these "biotics" as a therapeutic alternative for intestinal mucositis caused by 5-FU. It describes the effects and benefits related to their use as well as their potential for patient care.

Neuroprotective effects of p62(SQSTM1)-engineered lactic acid bacteria in Alzheimer's disease: a pre-clinical study.

Alzheimer's disease (AD) is a progressive neurodegeneration characterized by neuron death ending in memory and cognitive decline. A major concern in AD research is the identification of new therapeutics that could prevent or delay the onset of the disorder, with current treatments being effective only in reducing symptoms. In this perspective, the use of engineered probiotics as therapeutic tools for the delivery of molecules to eukaryotic cells is finding application in several disorders. This work introduces a new strategy for AD treatment based on the use of a Lactobacilluslactis strain carrying one plasmid (pExu) that contains a eukaryotic expression cassette encoding the human p62 protein. 3xTg-AD mice orally administered with these bacteria for two months showed an increased expression of endogenous p62 in the brain, with a protein delivery mechanism involving both lymphatic vessels and neural terminations, and positive effects on the major AD hallmarks. Mice showed ameliorated memory, modulation of the ubiquitin-proteasome system and autophagy, reduced levels of amyloid peptides, and diminished neuronal oxidative and inflammatory processes. Globally, we demonstrate that these extremely safe, non-pathogenic and non-invasive bacteria used as delivery vehicles for the p62 protein represent an innovative and realistic therapeutic approach in AD.

Vector Development Timeline for Mucosal Vaccination and Treatment of Disease Using Lactococcus lactis and Design Approaches of Next Generation Food Grade Plasmids.

Lactococcus lactis has been used historically in fermentation and food preservation processes as it is considered safe for human consumption (GRAS-Generally Recognized As Safe). Nowadays, in addition to its wide use in the food industry, L. lactis has been used as a bioreactor for the production of molecules of medical interest, as well as vectors for DNA delivery. These applications are possible due to the development of promising genetic tools over the past few decades, such as gene expression, protein targeting systems, and vaccine plasmids. Thus, this review presents some of these genetic tools and their evolution, which allow us to envision new biotechnological and therapeutic uses of L. lactis. Constitutive and inductive expression systems will be discussed, many of which have been used successfully for heterologous production of different proteins, tested on animal models. In addition, advances in the construction of new plasmids to be used as potential DNA vaccines, delivered by this microorganism, will also be viewed. Finally, we will focus on the scene of gene expression systems known as "food-grade systems" based on inducing compounds and safe selection markers, which eliminate the need for the use of compounds harmful to humans or animal health and potential future prospects for their applications.