In vitro and in vivo probiotic assessment of Leuconostoc mesenteroides P45 isolated from pulque, a Mexican traditional alcoholic beverage

Giles‑Gómez et al. SpringerPlus (2016) 5:708 DOI 10.1186/s40064-016-2370-7 Open Access RESEARCH In vitro and in vivo probiotic assessment of Leuconostoc mesenteroides P45 isolated from pulque, a Mexican traditional alcoholic beverage Martha Giles‑Gómez2, Jorge Giovanni Sandoval García2, Violeta Matus1, Itzia Campos Quintana2, Francisco Bolívar1 and Adelfo Escalante1* Abstract Pulque is a Mexican traditional alcoholic, non-distilled, fermented beverage produced by the fermentation of the sap, known as aguamiel, extracted from several maguey (Agave) species. Pulque has traditionally been considered a healthy beverage due to its nutrient content and also a traditional medicine for the treatment of gastrointestinal dis‑ orders and intestinal infections. During pulque fermentation, the development of acidity, alcohol and viscosity define its final sensorial properties, developing an enriched environment where dominant lactic acid bacteria (LAB), includ‑ ing diverse Leuconostoc species, are present. Because traditional pulque is consumed directly from the fermentation vessel, the naturally associated LAB are ingested and reach the human small intestine alive. Here, we report the in vitro and in vivo probiotic assessment of Leuconostoc mesenteroides strain P45 isolated from pulque. This isolated LAB spe‑ cies exhibited lysozyme, acid (pH 3.5) and bile salts (0.1 and 0.3 % oxgall) resistance. Antibacterial activity against the pathogens Listeria monocytogenes, enteropathogenic Escherichia coli, Salmonella enterica serovar Typhi and S. enterica serovar Typhimurium were observed in assays involving cell-to-cell contact, cell-free 2× concentrated supernatants and cell-to-cell contact under exopolysaccharide-producing conditions. The in vivo probiotic assessment showed an anti-infective activity of L. mesenteroides P45 against S. enterica serovar Typhimurium in challenged male and female BALB/c mice. Analysis of the available genome sequence of strain P45 allowed identified a pre-bacteriocin coding gene and six peptidoglycan hydrolase enzymes, probably involved in the antimicrobial activity of this strain. The results presented in this study support some potential microbial mechanisms associated with the beneficial effects on human health of this LAB involved in the fermentation of pulque. Keywords: Pulque, Leuconostoc mesenteroides, Probiotic, Antimicrobial activity, Bacteriocin, Glucan-hydrolase Background Pulque is a traditional alcoholic, non-distilled, fermented beverage produced by the fermentation of the sap known as aguamiel, extracted from several magueys (Agave) species such as A. salmiana, A. atrovirens and A. mapisaga. For its production, freshly collected aguamiel *Correspondence: 1 Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Col. Chamilpa, 62210 Cuernavaca, Morelos, México Full list of author information is available at the end of the article is transported to large open barrels where the fermentation takes place. The process is accelerated by the addition of a portion of previously produced pulque, and the fermentation time varies from a few hours to overnight, depending on whether the sap is collected at daybreak or dusk. The development of viscosity due to exopolysaccharide (EPS) synthesis, slight acidity and alcohol content are the main parameters used to determine the degree of fermentation. The process is static and performed under non-aseptic conditions; therefore, the populations of microorganisms involved in the fermentation are those © 2016 The Author(s). This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Giles‑Gómez et al. SpringerPlus (2016) 5:708 naturally occurring during sap accumulation in maguey plants and those incorporated during collection, transport, inoculation and manipulation (Escalante et al. 2004, 2008, 2012; Lappe-Oliveras et al. 2008; Sanchez-Marroquin and Hope 1953). Aguamiel and pulque have traditionally been considered as healthy beverages due to their nutrient content. They are a water substitute or alternative carbohydrate and protein source in places where drinking water is not available, of poor quality or where animal or vegetal proteins are scarce. Based on regular daily consumption, pulque is considered an important source of energy, vitamins and essential amino acids, such as lysine and tryptophan, which are deficient in the Mexican maize-based diet (Escalante et al. 2012; Lappe-Oliveras et al. 2008; Ortiz-Basurto et al. 2008; Sanchez-Marroquin and Hope 1953). Several of the health-promoting properties associated with the regular consumption of modest quantities of aguamiel or pulque are seen in rural populations (reviewed in Escalante et al. 2012). Scientific evidence supporting the relationship between the microbial diversity present in aguamiel and pulque has shown the antimicrobial effects of both the sap used as substrate and the final fermented product against pathogenic bacteria such as Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Listeria monocytogenes, Shigella flexneri and S. sonnei (Gómez-Aldapa et al. 2011). Although the results demonstrated antimicrobial activity during pulque fermentation, preventing the potential risk to consumers of contracting foodborne diseases by tested pathogens, the bactericidal effect was associated with the final alcohol content (~6 % v/v) and final pH (~4) but not with any specific bacterial activity. Aguamiel produced by maguey species for pulque production contains large quantities of carbohydrates (~75 % of dry weight in Agave masipaga plants) and among them, 10 % by wt were identified as fructooligosaccharides, suggesting a potential prebiotic effect (Ortiz-Basurto et al. 2008). Moreover, the role of hydrocolloids as thermoprotector prebiotics (including fructooligosaccharides) in aguamiel was shown to enhance the growth of Bifidobacterium bifidum (Rodríguez-Huezo et al. 2007). Pulque consumption for the treatment of gastrointestinal disorders and intestinal infections can also be explained by the possible probiotic activities associated with the presence of diverse lactic acid bacteria (LAB) such as Leuconostoc citreum, L. kimchi. L. mesenteroides and Lactobacillus acidophilus detected in fresh sap and during pulque fermentation (Escalante et al. 2004, 2008). To gain further evidence of the potential beneficial effects associated with LAB present in aguamiel and fermented pulque, we examined the probiotic properties of an LAB identified as L. mesenteroides strain P45 isolated from Page 2 of 10 this traditional Mexican be (...truncated)