Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests

Abstract

The present study aims to evaluate the probiotic potential of lactic acid bacteria (LAB) isolated from naturally fermented olives and select candidates to be used as probiotic starters for the improvement of the traditional fermentation process and the production of newly added value functional foods. Seventy one (71) lactic acid bacterial strains (17 Leuconostoc mesenteroides, 1 Ln. pseudomesenteroides, 13 Lactobacillus plantarum, 37 Lb. pentosus, 1 Lb. paraplantarum, and 2 Lb. paracasei subsp. paracasei) isolated from table olives were screened for their probiotic potential. Lb. rhamnosus GG and Lb. casei Shirota were used as reference strains. The in vitro tests included survival in simulated gastrointestinal tract conditions, antimicrobial activity (against Listeria monocytogenes, Salmonella Enteritidis, Escherichia coli O157:H7), Caco-2 surface adhesion, resistance to 9 antibiotics and haemolytic activity. Three (3) Lb. pentosus, 4 Lb. plantarum and 2 Lb. paracasei subsp. paracasei strains demonstrated the highest final population (>8 log cfu/ml) after 3 h of exposure at low pH. The majority of the tested strains were resistant to bile salts even after 4 h of exposure, while 5 Lb. plantarum and 7 Lb. pentosus strains exhibited partial bile salt hydrolase activity. None of the strains inhibited the growth of the pathogens tested. Variable efficiency to adhere to Caco-2 cells was observed. This was the same regarding strains' susceptibility towards different antibiotics. None of the strains exhibited β-haemolytic activity. As a whole, 4 strains of Lb. pentosus, 3 strains of Lb. plantarum and 2 strains of Lb. paracasei subsp. paracasei were found to possess desirable in vitro probiotic properties similar to or even better than the reference probiotic strains Lb. casei Shirota and Lb. rhamnosus GG. These strains are good candidates for further investigation both with in vivo studies to elucidate their potential health benefits and in olive fermentation processes to assess their technological performance as novel probiotic starters.

Introduction

The term probiotic, literally meaning “for life”, was first addressed by Lilly and Stillwell (1965) and was used to describe substances produced by protozoa to stimulate the growth of other organisms. Nowadays, the term refers to viable, nonpathogenic microorganisms (bacteria or yeasts) that, when ingested, are able to reach the intestines in sufficient numbers to confer health benefits to the host (De Vrese and Schrezenmeir, 2008). Commonly used bacterial probiotics include various species of Lactobacillus, Bifidobacterium, and Streptococcus, as well as Lactococcus lactis and some Enterococcus species. Currently, the only probiotic yeast used is the nonpathogenic Saccharomyces boulardii (Morrow et al., 2012).

It is well established that probiotics confer a number of beneficial health effects to humans and animals. Intake of probiotics stimulates the growth of beneficial microorganisms and reduces the amount of pathogens improving thus the intestinal microbial balance of the host and lowering the risk of gastro-intestinal diseases (Fuller, 1989; Cross, 2002; Chiang and Pan, 2012). Their benefits include also the alleviation of certain intolerances (such as lactose intolerance), the enhancement of nutrients bioavailability, and prevention or reduction of the prevalence of allergies in susceptible individuals (Isolauri, 2001; Chiang and Pan, 2012). Probiotics are reported to have also antimutagenic, anticarcinogenic, hypocholesterolemic, antihypertensive, anti-osteoporosis, and immunomodulatory effects (Chiang and Pan, 2012). They relieve the symptoms of inflammatory bowel diseases, irritable bowel syndrome, colitis, alcoholic liver disease, constipation and reduce the risk for colon, liver and breast cancers (Prado et al., 2008).

Probiotic foods receive market interest as health-promoting, functional foods. Probiotic food products according to FAO/WHO (2002) are in general fermented foods containing an amount of viable and active microorganisms large enough to reach the intestine and exert an equilibrating action on the intestinal microflora. To deliver the health benefits, probiotic foods need to contain an adequate amount of live bacteria (at least 10⁶–10⁷ cfu/g) (Oliveira et al., 2001; Boylston et al., 2004), although there are recent convincing data on beneficial immunological effects derived from dead cells (Vinderola and Reinheimer, 2003; Mottet and Michetti, 2005).

Most probiotic bacteria are lactic acid bacteria and, among them, lactobacilli represent one of the fundamental microbial groups. They have been introduced in a wide range of food products. Many studies have reported that the best matrices to deliver probiotics are dairy fermented products, such as fermented milks and yogurt. On the other hand, nowadays there is a need for novel and non-dairy probiotics and it has been found that traditional fermented foods may constitute a good working base for the development of probiotic-type functional foods (De Vuyst et al., 2008; Ruiz-Moyano et al., 2008, 2011). A window of opportunity for the development of non-dairy probiotic products has arisen from the increasing number of lactose intolerance cases occurring in the world population, coupled with the unfavourable effect of cholesterol contained in fermented dairy products (Granato et al., 2010).

Among the traditional fermented foods, table olives could be a promising probiotic food through the use of functional probiotic starter cultures. Functional starter cultures contribute to microbial safety and offer organoleptic, technological, nutritional or health advantages. In contrast to well-adapted industrial starters, wild-type strains that naturally dominate traditional fermentations tend to have higher metabolic capacities, which can beneficially affect product quality, for instance with regard to aroma formation and/or food safety. Natural selection is likely to have forced such strains to be more competitive by endowing them with ecological advantages (Ayad et al., 2002). The information provided from traditional fermented foods and scientific research could help develop new probiotic products for the food industry (Rivera-Espinoza and Gallardo-Navarro, 2010).

Most of the studies published today about physiological properties of strains intended to be used as probiotics are performed on strains from human or animal internal cavities, considering that strains of these origins would be better adapted and colonize the human/animal gastrointestinal tract (Johansson et al., 1993; Prasad et al., 1998; Xanthopoulos et al., 2000; Ouwehand et al., 2002; Ruiz-Moyano et al., 2009; Zacarías et al., 2011). On the other hand, research has started to increase on probiotic functions of lactic acid bacteria isolated from foods like dairy products (Maragkoudakis et al., 2006; Bao et al., 2010; Monteagudo-Mera et al., 2012; Espeche et al., 2012), dry sausages (Papamanoli et al., 2003; Pennacchia et al., 2004; De Vuyst et al., 2008), foods of plant origin (Husmaini et al., 2011), fruits, cereals, meat or fish (Rivera-Espinoza and Gallardo-Navarro, 2010). Traditional fermented foods are a plentiful source of microorganisms and some of them show probiotic characteristics, although the research of these matrices as raw material for probiotic microorganisms is still scarce compared with their dairy counterpart (Rivera-Espinoza and Gallardo-Navarro, 2010).

The aim of this work was to perform established in vitro tests to evaluate the probiotic potential and safety of 71 LAB strains originating from olive microbiota and especially from naturally fermented olives. The candidate probiotic strains that fulfil the established criteria could therefore be potentially used as novel probiotic strains by the table olive industry and food industry in general.