Evaluation of the effect of malt, wheat and barley extracts on the viability of potentially probiotic lactic acid bacteria under acidic conditions

Abstract

In this work, the effect of cereal extracts, used as delivery vehicles for potentially probiotic lactic acid bacteria (LAB), on the acid tolerance of the cells was evaluated under conditions that simulate the gastric tract. More specifically, the effect of malt, barley and wheat extracts on the viability of Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus reuteri during exposure for 4 h in a phosphate buffer acidified at pH 2.5 was investigated. In the absence of cereal extracts all strains demonstrated a significant reduction in their cell population, particularly L. plantarum. The viability of L. plantarum was improved by approximately 4 log₁₀ cycles in the presence of malt and 3 log₁₀ cycles in the presence of wheat and barley. The survival of L. acidophilus and L. reuteri was increased by more than 1.5 and 0.7 log₁₀ cycle, respectively, upon addition of cereal extracts. In order to evaluate the contribution of the cereal constituents on cell survival, the individual effect of glucose, maltose and free amino nitrogen (FAN), which were added at concentrations that correlated to the reducing sugar and FAN content of the cereal extracts, was examined. The viability of L. plantarum was progressively improved as the maltose or glucose concentration increased; an increase by approximately 2 log₁₀ cycles was observed in the presence of 8.33 g/l sugar. The survival of L. acidophilus increased by more than 1 log₁₀ cycle, even at very low concentrations of maltose and glucose (e.g., 0.67 g/l), while L. reuteri stability was enhanced in the presence of maltose but no appreciable effect was demonstrated in the presence of glucose. Sugar analysis indicated that glycolysis was inhibited in all cases. Addition of tryptone and yeast extract, used as sources of FAN, enhanced L. acidophilus acid tolerance, but did not affect L. reuteri and L. plantarum. The results presented in this study indicate that malt, wheat and barley extracts exhibit a significant protective effect on the viability of L. plantarum, L. acidophilus and L. reuteri under acidic conditions, which could be mainly attributed to the amount of sugar present in the cereal extracts.

Introduction

The inclusion of potentially probiotic microorganisms in the diet has been established in the global food market leading to the development and commercialization of numerous probiotic products. In the dairy industry, a large variety of milk formulations have been used as delivery vehicles of probiotic lactic acid bacteria (Scheinbach, 1998). Cereals are also suitable substrates for lactic acid bacteria (LAB) growth, which has led to the commercialization of cereal-based probiotic products (Salovaara, 1996). Strains of the Lactobacillus genus, such as Lactobacillus acidophilus, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus casei, Lactobacillus rhamnosus and Lactobacillus gasseri constitute a significant proportion of cultures used in probiotic products (Shortt, 1999).

An important criterion when selecting a probiotic strain is its ability to survive the acidic conditions of the gastric tract. The stomach has a pH that may fall to as low as 1.5 and a salt content not less than 0.5% w/v Hill, 1990, Kailasapathy and Chin, 2000. The intrinsic tolerance of lactobacilli to low pH values, ranging between 1 and 4, has been examined in vitro using solutions such as acidified broths, buffers, complex media consisting of pancreatic enzymes, or fresh human gastric fluids Conway et al., 1987, Charteris et al., 1998, Gardiner et al., 1999, Vinderola et al., 2000. Lactobacillus species are considered intrinsically resistant to acid although differences exist between species and specific strains, however they generally demonstrate increased sensitivity at pH values below 3 Hood and Zottola, 1988, Jin et al., 1998, Kailasapathy and Chin, 2000. The acid tolerance of lactobacilli is attributed to their ability to maintain a constant pH gradient between the pH of the medium and their cytoplasmic pH. When the internal pH reaches a threshold value cellular functions are inhibited and the cell dies (Kashket, 1987).

Survival of the probiotic strains during gastric transit is also influenced by the physicochemical properties of the food carrier used for delivery. The buffering capacity and the pH of the carrier medium are significant factors, since food formulations with pH ranging from 3.5 to 4.5 and high buffering capacity, such as yoghurt, cheese, and skim milk, would increase the pH of the gastric tract and thus enhance the stability of the probiotic strain Gardiner et al., 1998, Kailasapathy and Chin, 2000, Zárate et al., 2000. In this regard, Charteris et al. (1998) reported improved gastric tolerance of several probiotic lactobacillus species upon addition of milk proteins. However, Gardiner et al. (1999) suggested that besides the buffering capacity, there are other factors, possibly including properties such as the presence of protective extracellular polysaccharides, the fat content and the dense matrix of cheese, which may enhance the survival of the probiotic strain.

In the present study, the effect of malt, wheat and barley extracts and several dietary constituents individually (reducing sugars, free amino nitrogen) on the viability of potentially probiotic L. plantarum, L. reuteri and L. acidophilus strains during exposure for 4 h in a phosphate buffer acidified at pH 2.5 was investigated. The overall aim was to identify the physicochemical factors that can generally improve the viability of a probiotic strain and select the appropriate cereal substrate as probiotic carrier, in relation to its composition. The strains were selected as representatives of the major LAB species present in the human gut (Collins et al., 1998). In addition, they are intrinsically stable to pH 1 (data not shown), are able to tolerate bile concentrations ranging from 2% to 4% (data not shown) and grow well in cereal substrates (Charalampopoulos et al., 2002). The media were prepared by adding the cereal suspensions to the phosphate buffer and subsequently adjusting the pH to 2.5, before adding the cell culture. Using the above preparation procedure, the effect of buffering capacity on cell viability was minimized.