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LALPROBIOME heat-treated bacterial strains support gut barrier function

The Lactobacillus genus contains the most widely used and well-documented strains of probiotics, but there are several practical limitations to their commercial application in animal feeds. The nutraceutical potential of heat-treated microbials (‘postbiotics’) processed from specific strains of bacteria is rapidly emerging. Heat-treated probiotic cells are recognized to contribute to immunomodulation, protection against enteropathogens and the maintenance of intestinal barrier integrity. At Lallemand, we implement a specific gentle heat treatment process inactivating microbial cells, which results in a shelf-stable bacterial cell still able to support pet health.

Lallemand Animal Nutrition recently conducted a study in collaboration with the University of Plymouth to compare the beneficial effect of dietary supplementation with Lactobacillus-based products, either live (probiotic) or LALPROBIOME heat-treated (postbiotic), in supporting gut barrier integrity using zebrafish as a vertebrate model.

Why Zebrafish?

Since 1960’s, the 3R’s principle (Replacement, Reduction, and Refinement) has been recognised as a scientific standard for the ethical use of animals and is required in the legislation framework of animal experimentation worldwide. Animal models remain an essential measure in helping researchers to identify and understand biological mechanisms and disease progression, as well as develop new therapeutic possibilities. Mammalian models are replaced by animals considered as less sentient and the use of zebrafish (Danio rerio) has exponentially increased among the scientific community over recent decades. Zebrafish genetic, anatomical and physiological homology to mammals provides an alternative model to rodents and provides superior information to in vitro modelling. Due to the considerable benefits, Lallemand are using zebrafish in many of their research projects.

Gut barrier function: why is it important?

Pathogens have two main pathways for entry into the body: the skin and mucosal surfaces. The gastro-intestinal tract is constantly challenged by multiple stimuli, required to discriminate against pathogens and food antigens, and encounters more antigens than any other bodily site. Gut barrier function and integrity is crucial for homeostasis and immune functioning. The microbiota, mucus and epithelial cells, all act as physical and chemical barriers to protect immuno-competent cells within the gut and the internal body itself.

Goblet cells (GCs) are specialized epithelial cells that line the mucosal surface and are integral for the secretion of mucus. Mucus forms part of the biochemical barrier found within the gut and acts as a sieve-like structure to allow passage of small and selected molecules, while separating and protecting epithelial cells from the microbiota and any potential pathogens. Beyond barrier maintenance, GCs also play a role in innate immunity, acting as antigen sensors. A higher density and coverage of GCs indicates the maintenance of a functional mucus barrier. After the mucus barrier, intestinal epithelial cells form a physical barrier to restrict and control the paracellular diffusion of essential ions, nutrients, microorganisms and toxins. In humans, the intestinal epithelium spans between 200-400m2 and consists of a single layer of cells. Between cells, multi-protein complexes dynamically regulate barrier integrity and are the rate-limiting factor for permeability. Zonula-Occludens (ZO) is an essential protein for the formation and maintenance of these junctions and is ubiquitously expressed in epithelial cells.

Interestingly, groups supplemented with LALPROBIOME heat-treated Lactobacillus helveticus HA122 (LHPost) and Lactiplantibacillus plantarum HA119 (LPPost) showed a significant increase in GC coverage and density at gut level compared to the Control group and the live LALPROBIOME Lactobacillus helveticus HA122 (LHPro) fed group. A higher density and coverage of GCs is indicative of the ability of LALPROBIOME heat-treated strains to enhance the functional mucus barrier. The gene expression of ZO (2a) was significantly upregulated in all treatment groups, with a 1.6 to 2-fold increase in comparison to the Control group.

The gut mucosal system: a unique network

Approximately 70% of the immune cells are located within the gut, acting as an immunological barrier. The intestinal immune system is a complex network dedicated to the defence of the body against pathogens while maintaining tolerance to beneficial microbes and food antigens. Several specialised cells (lymphocytes, dendritic cells and macrophages) are involved in this system, including, intra-epithelial lymphocytes (IELs). IELs are found within the epithelial layer of the intestinal mucosa and play a crucial role in immune surveillance, maintaining gut homeostasis, and responding to pathogens or other challenges. CD4+ and CD8+ are two subsets of IELs:

CD4+ T cells can include various subsets, which can help maintain tolerance to dietary antigens and commensal bacteria, participate in immune responses against pathogens, or assist in inflammatory processes.

CD8+ T cells are often cytotoxic T cells, capable of directly killing infected or aberrant (abnormal) host cells.

The trial showed higher mucosal IEL numbers in all supplemented groups as well as an elevation in lymphocyte populations for all groups. There was a significant increase in CD8 cells when zebrafish were fed LALPROBIOME Lactiplantibacillus plantarum HA 119 (LPPost). This demonstrates the impact of LALPROBIOME postbiotic bacterial strains on gut-associated lymphoid tissue (GALT).

The gut immune response: mucosal markers

Delving deeper into the immune system and cell functioning, we can also explore specific markers for certain aspects of gut immune responses, such as antimicrobial defence (lysozyme) and cellular processes involved in immune regulation, and tissue remodelling (cathepsin). Lysozymes are antimicrobial enzymes secreted into the mucus layer by epithelial cells, IELs and paneth cells in the small intestine. They are part of the innate immune system and can attack the cell walls of bacteria, especially gram-positive bacteria and are a natural form of protection against pathogens such as streptococcus. Cathepsins are involved in the regulation of tight junction proteins such as occludin and claudins. These two specific markers were significantly upregulated in both postbiotic groups during the trial. These results confirm the capacity of LALPROBIOME postbiotic strains to stimulate specific immune processes in the gut micro-environment.

Conclusion

Using the zebrafish model as a recognised mammalian model of research, this study demonstrates the potential of selected LALPROBIOME probiotic and postbiotic bacterial strains to support gut barrier integrity and natural defences which are crucial factors in supporting pet gastrointestinal homeostasis and immunity.

LALPROBIOME Heat-treated Bacteria