New types of antimicrobial compounds produced by Lactobacillus plantarum

Many factors contribute to a successful natural fermentation of carbohydrate-rich food and feed products. Metabolic activities of lactic acid bacteria (LAB) play a leading role. Their ability to rapidly produce copious amounts of acidic end products with a concomitant pH reduction is the major factor in these fermentations. Although their specific effects are difficult to quantitate, other LAB metabolic products such as hydrogen peroxide and diacetyl can also contribute to the overall antibiosis and preservative potential of these products. The contribution of bacteriocins is also difficult to evaluate. It is suggested that they may play a role in selecting the microflora which initiates the fermentation. Bacteriocins are believed to be important in the ability of LAB to compete in non-fermentative ecosystems such as the gastrointestinal tract. During the past few decades interest has arisen in the use of the varied antagonistic activities of LAB to extend the shelf-life of protein-rich products such as meats and fish. Recent findings indicate that the newly discovered Lactobacillus reuteri reuterin system may be used for this purpose.

Lactobacillus reuteri converts glycerol into a potent cell growth inhibitor. This substance, termed reuterin, inhibits the growth of gram-positive and gram-negative bacteria as well as yeasts, fungi, and protozoa. Semipreparative chromatography was used to purify reuterin, and Fourier transform infrared spectroscopy and liquid chromatography-mass spectrometry were used to establish the molecular weight as well as the molecular functionality of the reuterin molecule. Nuclear magnetic resonance studies of purified reuterin carried out with deuterium oxide confirmed the presence of two three-carbon compounds, beta-hydroxypropionaldehyde and the corresponding hydrated acetal, and a six-carbon cyclic dimer of the aldehyde. Further nuclear magnetic resonance studies with deuterated methanol revealed that in this solvent the compound existed as a three-carbon compound in a methoxy form. Trimethylsilyl derivatives of reuterin were analyzed by gas chromatography-mass spectrometry, and a molecule was identified which had a molecular weight corresponding to a disilylated dimeric structure. On the basis of the above information, reuterin was determined to be an equilibrium mixture of monomeric, hydrated monomeric, and cyclic dimeric forms of beta-hydroxypropionaldehyde. This was subsequently confirmed by chemical synthesis.

Lactic acid bacteria (LAB) play an essential role in the majority of food fermentations, and a wide variety of strains are routinely employed as starter cultures in the manufacture of dairy, meat, vegetable and bakery products. One of the most important contributions of these microorganisms is the extended shelf life of the fermented product by comparison to that of the raw substrate. Growth of spoilage and pathogenic bacteria in these foods is inhibited due to competition for nutrients and the presence of starter-derived inhibitors such as lactic acid, hydrogen peroxide and bacteriocins (Ray and Daeschel, 1992). Bacteriocins, are a heterogenous group of anti-bacterial proteins that vary in spectrum of activity, mode of action, molecular weight, genetic origin and biochemical properties. Currently, artificial chemical preservatives are employed to limit the number of microorganisms capable of growing within foods, but increasing consumer awareness of potential health risks associated with some of these substances has led researchers to examine the possibility of using bacteriocins produced by LAB as biopreservatives. The major classes of bacteriocins produced by LAB include: (I) lantibiotics, (II) small heat stable peptides, (III) large heat labile proteins, and (IV) complex proteins whose activity requires the association of carbohydrate or lipid moieties (Klaenhammer, 1993). Significantly however, the inhibitory activity of these substances is confined to Gram-positive bacteria and inhibition of Gram-negatives by these bacteriocins has not been demonstrated, an observation which can be explained by a detailed analysis and comparison of the composition of Gram-positive and Gram-negative bacterial cell walls (Fig. 1). In both types the cytoplasmic membrane which forms the border between the cytoplasm and the external environment, is surrounded by a layer of peptidoglycan which is significantly thinner in Gram-negative bacteria than in Gram-positive bacteria. Gram-negative bacteria possess an additional layer, the so-called outer membrane which is composed of phospholipids, proteins and lipopolysaccharides (LPS), and this membrane is impermeable to most molecules. Nevertheless, the presence of porins in this layer will allow the free diffusion of molecules with a molecular mass below 600 Da. The smallest bacteriocins produced by lactic acid bacteria are approximately 3 kDa and are thus too large to reach their target, the cytoplasmic membrane (Klaenhammer, 1993; Stiles and Hastings, 1991). However, Stevens et al. (1991) and Ray (1993) have demonstrated that Salmonella species and other Gram-negative bacteria become sensitive to nisin after exposure to treatments that change the permeability barrier properties of the outer membrane (see below). This review will focus on the mode of action of lantibiotics (class I) and class II LAB bacteriocins and their potentials in food preservation and control of food poisoning.