Lactic acid bacteria isolated from equid milk and their extracellular metabolites show great probiotic properties and anti-inflammatory potential

Abstract

Traditionally, equid milk has been used as an alternative to human milk for infants or children mainly because of its similarities in nutrient composition, hypoallergenicity, immune modulation and antimicrobial activity. As a highly nutritious substrate, it possesses great potential for probiotic bacteria isolation. The aim of this study was to assess the probiotic potential of Lactobacillus plantarum M2 and L. plantarum KO9 isolated from donkey and mare milk, respectively, and determine TNF-α suppression attributed to their extracellular metabolites in lipopolysaccharide- (LPS-)stimulated human peripheral blood mononuclear cells (PBMCs). Resistance to simulated gastric and intestinal conditions, antimicrobial activity against pathogens, autoaggregation, coaggregation, biofilm quantification, antioxidative potential and haemolytic activity were determined. The isolated strains demonstrated great probiotic potential and their extracellular metabolites with molecular mass smaller than 2000 Da supressed TNF-α production up to 67% in LPS-stimulated PBMCs exerting high anti-inflammatory activity. Extracellular metabolites did not show any cyto/genotoxic effects toward PBMCs.

Introduction

Since the classical era, the consumption of equid (mare and donkey) milk has had a large presence in Europe and Asia. It was generally thought that its consumption results in numerous health benefits. Recently, equid milk has been successfully used as an alternative for infants with food allergies and other health-promoting properties from its consumption have been reported (Salimei & Fantuz, 2012). Modulation of antimicrobial peptides level and anti-inflammatory properties of equid milk were also documented in animal models (Yvon et al., 2018).

Generally, milk as a highly nutritious substrate is a suitable environment for the growth of many microorganisms (Šarić et al., 2012) and a great potential source of probiotic bacteria as fermented products from mare and donkey milk also have documented positive effects to the consumer (Jacobsen et al., 1999; Shu et al., 1999; Uniacke-Lowe & Fox, 2011). Both types of milk contain a diverse lactic acid bacteria (LAB) composition (Andrighetto, Dalmasso, Lombardi, Civera, & Bottero, 2016; Pieszka et al., 2016). LAB are widely used in food industry and are generally recognised as safe (GRAS) (Liu & Pan, 2010).

LAB with documented health promoting effects are often classified as probiotics. Beneficial LAB used in functional foods and pharmaceutical preparations interact with the immune cells in the gut and affect their activation signals (Perdigon, Galdeano, Valdez, & Medici, 2002). Characteristics such as immune modulation and production of bioactive substances are restricted to some strains (Ferreira dos Santos, Alves Melo, Almeida, Passos Rezende, & Romano, 2016), whereas lactobacilli and bifidobacteria are the most common species used as probiotics in the food industry (Isolauri, Salminen, & Ouwehand, 2004).

Numerous studies of probiotics have been conducted in the last few years reporting in vivo evidence of IgA secretion stimulation (Frece et al., 2009), stimulation of peripheral immunoglobin production and inhibition of proinflammatory cytokine production have been reported (Azad, Kalam, Sarker, & Wan, 2018). Effects on cytokine production in an inflammation model study demonstrated that not only contact mechanisms were present but through the production of small metabolites, capable of intestinal transport, LAB can modulate cytokine production (Menard et al., 2004). Tumour necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine released by immune cells that triggers a wide range of responses including inflammation, cell apoptosis, and maturation of immune cells (Locksley, Killeen, & Lenardo, 2001). TNF-α is an immune mediator that has also been linked with inflammatory bowel disease, rheumatologic conditions, and even depression (Dowlati et al., 2010). Immune cell TNF-α production can be induced in the presence of lipopolysaccharides (LPS) which are a major component of the outer membrane of Gram-negative bacteria.

Despite the anti-inflammatory properties of equid milk consumption and reported isolation of LAB strains with bacteriogenic properties from equid milk (Murua et al., 2013; Sa, Krishnaa, Pavithrab, Hemalathab, & Ingalea, 2011) studies evaluating the probiotic potential of LAB strains naturally present in mare and donkey raw milk are scarce.

Therefore, the aim of this study was to isolate LAB strains naturally present in equid milk and select potential probiotic candidates. Furthermore, we determined in vitro if extracellular bacterial metabolites, with a molecular mass smaller than 2000 Da (Da) that can cross the epithelial barrier, supress TNF-α production in LPS stimulated peripheral blood lymphocytes. Since we evaluated the beneficial potential of newly isolated LAB strains, we wanted to exclude their possible cyto/genotoxic properties toward human circulating blood cells; thus, the cytotoxicity and genotoxicity of bacterial fractions on PBMC were also determined.