Safety properties and molecular strain typing of lactic acid bacteria from slightly fermented sausages

AIM: To evaluate the biodiversity of lactobacilli from slightly fermented sausages (chorizo, fuet and salchichon) by molecular typing, while considering their safety aspects. METHODS AND RESULTS: Species-specific PCR, plasmid profiling and randomly amplified polymorphic DNA (RAPD)-PCR were used to characterize 250 lactic acid bacteria (LAB) isolated from 21 low acid Spanish fermented sausages. Lactobacillus sakei was the predominant species (74%) followed by Lactobacillus curvatus (21.2%) and Leuconostoc mesenteroides (4.8%). By plasmid profiling and RAPD-PCR 144 different strains could be differentiated, 112 belonging to Lact. sakei, 23 to Lact. curvatus and 9 to Leuc. mesenteroides. Ion-pair high performance liquid chromatography was used to detect biogenic amine production. Tyramine and phenylethylamine were produced by 14.4 and 12.4% of the isolates, respectively, all belonging to the species Lact. curvatus. The production of tyramine was stronger than that of phenylethylamine. Partial sequencing of the tyrosine decarboxylase gene from Lact. curvatus was achieved. A specific PCR assay to detect the Lact. curvatus tyramine-producers was designed. The disc diffusion test was used to detect antibiotic resistance among the isolates. Most isolates displayed resistance to vancomycin and gentamicin. Only four strains were resistant to most of the antibiotics tested. None of the isolates were resistant to erythromycin. CONCLUSIONS: Lactobacillus sakei would be the species of choice for further use as starter culture in fermented sausage production. Strain typing and characterization of biogenic amine production together with antibiotic susceptibility testing for the selection of starter cultures could help to increase the quality and safety of the products. SIGNIFICANCE AND IMPACT OF THE STUDY: Species-specific PCR, RAPD and plasmid profiling proved to be efficient at typing LAB at species and strain level. Information on biogenic amine production and transferable antibiotic resistance is important in order to avoid selection of strains with undesirable properties as starter cultures.     

Heteropolysaccharides from lactic acid bacteria

Microbial exopolysaccharides are biothickeners that can be added to a wide variety of food products, where they serve as viscosifying, stabilizing, emulsifying or gelling agents. Numerous exopolysaccharides with different composition, size and structure are synthesized by lactic acid bacteria. The heteropolysaccharides from both mesophilic and thermophilic lactic acid bacteria have received renewed interest recently. Structural analysis combined with rheological studies revealed that there is considerable variation among the different exopolysaccharides; some of them exhibit remarkable thickening and shear-thinning properties and display high intrinsic viscosities. Hence, several slime-producing lactic acid bacterium strains and their biopolymers have interesting functional and technological properties, which may be exploited towards different products, in particular, natural fermented milks. However, information on the biosynthesis, molecular organization and fermentation conditions is rather scarce, and the kinetics of exopolysaccharide formation are poorly described. Moreover, the production of exopolysaccharides is low and often unstable, and their downstream processing is difficult. This review particularly deals with microbiological, biochemical and technological aspects of heteropolysaccharides from, and their production by, lactic acid bacteria. The chemical composition and structure, the biosynthesis, genetics and molecular organization, the nutritional and physiological aspects, the process technology, and both food additive and in situ applications (in particular in yogurt) of heterotype exopolysaccharides from lactic acid bacteria are described. Where appropriate, suggestions are made for strain improvement, enhanced productivities and advanced modification and production processes (involving enzyme and/or fermentation technology) that may contribute to the economic soundness of applications with this promising group of biomolecules.     

Development of RAPD protocol for typing of strains of lactic acid bacteria and enterococci

P.S. COCCONCELLI, D. PORRO, S. GALANDINI AND L. SENINI. 1995. A protocol for typing strains of lactic acid bacteria and enterococci based on randomly amplified polymorphic DNA (RAPD) fragments has been developed. Using a single 10-mer primer, fingerprints were achieved without the need to isolate genomic DNA. Different conditions of DNA release and amplification were investigated in order to obtain reproducible results and high discrimination among strains. This RAPD protocol was successfully applied for the typing of strains belonging to the species Lactobacillus acidophilus, Lact. helveticus, Lact. casei, Lact. reuteri, Lact. plantarum, Enterococcus faecalis, Ent. faecium and Streptococcus thermophilus.     

An evaluation of chelex-based DNA purification protocols for the typing of lactic acid bacteria

An easy and rapid protocol to extract DNA to be used as template for polymerase chain reaction (PCR) fingerprinting experiments from cultivable lactic acid bacteria (LAB) is proposed. Different procedures for rapid extraction of DNA by chelex (iminodiacetid acid) ionic resin were compared. Factors affecting the quality and reproducibility of PCR fingerprinting profiles were also investigated. Two out of three chelex-based protocols allowed to obtain DNA samples which, after PCR amplification, provided electrophoretic patterns comparable with those obtained by classical lysozyme and phenol-chloroform DNA extraction. A good level of reproducibility and consistency of the InstaGene procedure was verified. The procedure is fast, practical, and the DNA is of quality similar to that obtained by phenol-chloroform extraction. Although applied to a little number of LAB strains, chelex-based protocols are potentially applicable to a vast array of organisms and/or biological materials.     

Exploiting expolysaccharides from lactic acid bacteria

Microbial exopolysaccharides (EPSs) synthesized by lactic acid bacteria (LAB) play a major role in the manufacturing of fermented dairy products. EPS production is characterized by a large variety in terms of quantity, chemical composition, molecular size, charge, type of sidechains and rigidity of the molecules. Monosaccharide unit's composition, linkages, charge and size determine the EPS' intrinsic properties and their interactions with other milk constituents. EPSs contribute to texture, mouthfeel, taste perception and stability of the final product. Furthermore, it was reported that EPS from food grade organisms, particularly LAB, have potential as food additives and as functional food ingredients with both health and economic benefits. A better understanding of structure-function relationships of EPS in a dairy food matrix and of EPS biosynthesis remain two major challenges for further applications of EPS and the engineering of functional polysaccharides.     

Evaluation of lactic acid bacteria autolysate for the supplementation of lactic acid bacteria fermentation

At the end of culture in a carbon-limited medium, i.e. the best conditions for subsequent autolysis, lactic acid bacteria were harvested and autolysed at 50 °C for 24 h. The resulting supernatant was then successfully tested as a substitute for industrial yeast extract for the supplementation of whey permeate and its conversion into lactic acid: for almost equivalent total nitrogen amounts of both supplements, the same growth and production rates were recorded.     

Identification of industrial strains of lactic acid bacteria by methods of molecular genetic typing

Various methods currently used in microbiology for determining taxonomic state of bacteria are discussed. The main focus is aimed at identifying and gene typing of lactic acid bacteria, used as starter cultures for industrial process of production of sour milk products, meat products, and probiotics.     

Identification of industrial strains of lactic acid bacteria by methods of molecular genetic typing

Various methods currently used in microbiology for determining taxonomic state of bacteria are discussed. The main focus is aimed at identifying and gene typing of lactic acid bacteria, used as starter cultures for industrial process of production of sour milk products, meat products, and probiotics.     

乳酸菌发酵上清液成分分析

目的 乳酸菌发酵上清液为益生菌生产企业的废弃物,为了开发其在食品和饲料中的应用,对其进行初步的成分分析。 方法 采用本公司乳酸菌生产离心上清液为材料,用化学分析法测定其中的总固形物、总糖、总蛋白质和总酸等成分含量;采用抑菌圈法检测以大肠埃希菌为革兰阴性菌指示菌、金黄色葡萄球菌为革兰阳性菌指示菌的抑菌活性,并测定以大肠埃希菌为革兰阴性菌指示菌的抑菌效价;采用点样培养法测定菌种分泌淀粉酶、蛋白酶等消化酶的活力。 结果 乳酸菌发酵上清液分别含总固形物4.96%~7.04%、总糖0.11%~0.60%、总蛋白质1.67%~2.45%、总酸(以乳酸计)2.41~7.12 g/L;其对革兰阴性菌、革兰阳性菌都具有一定的抑制作用,抑菌效价范围为374.87~878.05 μ/g;测得菌种分泌淀粉酶活力(H/C)范围为4.33~8.67。 结论 初步揭示了乳酸菌发酵上清液中含有一定量的营养成分及抑菌活性、抑菌效价和淀粉酶活力,为开发其在食品和饲料中的应用提供了依据。     

Characterization of lactic acid bacteria isolated from seafood

Lactic acid bacteria were isolated from various samples of seafood: fresh pollock, brine shrimp, gravad fish, vacuum-packed seafood (surimi, smoked tuna, salted cod), and fish stored under 100% CO₂ at 5°C (smoked tuna, fresh and salted cod, salmon). Eighty-six independent isolates were obtained and were grouped according to cell morphology, presence or absence of diaminopimelic acid in the cell wall, and lactate configuration. Fifty-four isolates were identified as belonging to the genus Lactococcus and most of them exhibited DNA homologies with L. lactis subsp. lactis. Four strains were identified as Lactobacillus plantarum , eight strains as genus Leuconostoc and 16 belonged to the genus Carnobacterium. One facultative heterofermentative Lactobacillus and three other isolates were not identified. Of the strains 47% showed similar patterns of carbohydrate fermentations especially among strains belonging to the genera Lactococcus and Carnobacterium. Most of the strains (64%) grew at 5°C, in salted media and in fish extract medium without added sugar. Carnobacterium piscicola and Carn. divergens were the only reference strains able to grow in the same conditions as well as psychrotroph strains isolated from seafood. A numerical analysis could not be used because of the divergent properties of isolates of the same genus and strong similarities between different genera.     

Simple method for extracting plasmid DNA from lactic acid bacteria

Rapid screening and large-scale plasmid DNA isolation procedures are described for lactic acid bacteria, using glass beads to break cells. The rapid screening procedure allows one to obtain plasmid DNA pellets in less than 1 h. This method has been successfully tested on various bacteria from the genera Lactococcus, Leuconostoc, Lactobacillus, Pediococcus, Streptococcus, Enterococcus and Propionibacterium. This procedure yields plasmid DNA with minor chromosomal and plasmid DNA-degraded form contaminations.     

Health and nutritional benefits from lactic acid bacteria

There are several potential health or nutritional benefits possible from some species of lactic acid bacteria. Among these are: improved nutritional value of food, control of intestinal infections, improved digestion of lactose, control of some types of cancer, and control of serum cholesterol levels. Some potential benefits may result from growth and action of the bacteria during the manufacture of cultured foods. Some may result from growth and action of certain species of the lactic acid bacteria in the intestinal tract following ingestion of foods containing them. In selecting a culture to produce a specific benefit it is necessary to consider not only the wide variation among species of the lactic acid bacteria but also that among strains within a given species. With the possible exception of improving lactose utilization by persons who are lactose maldigestors, no specific health or nutritional claims can yet be made for the lactic acid bacteria.     

Health and nutritional benefits from lactic acid bacteria

Abstract There are several potential health or nutritional benefits possible from some species of lactic acid bacteria. Among these are: improved nutritional value of food, control of intestinal infections, improved digestion of lactose, control of some types of cancer, and control of serum cholesterol levels. Some potential benefits may result from growth and action of the bacteria during the manufacture of cultured foods. Some may result from growth and action of certain species of the lactic acid bacteria in the intestinal tract following ingestion of foods containing them. In selecting a culture to produce a specific benefit it is necessary to consider not only the wide variation among species of the lactic acid bacteria but also that among strains within a given species. With the possible exception of improving lactose utilization by persons who are lactose maldigestors, no specific health or nutritional claims can yet be made for the lactic acid bacteria.     

Comparative studies of lactic acid dehydrogenases in lactic acid bacteria

The stability, pH-dependence and kinetic properties of the Mn²⁺ and FDP-activated NAD-dependent lactic acid dehydrogenases from Lactobacillus casei ssp. casei (ATCC 393) and L. curvatus (DSM) 20010) were studied after the enzymes were purified to homogeneity by affinity chromatography. Both enzymes are virtually unidirectional, catalysing efficiently only the reduction of pyruvate. They are similar with respect to the effector requirement and pH-optimum. They differ, however, in their electrophoretic mobility, heat stability, pH-dependence of the Mn²⁺ requirement and several kinetic properties. It is suggested that most of these differences are caused by differences of the negative charges in the vicinity of the FDP-binding site or the site responsible for the interaction of the subunits of the enzymatically active oligomeres.Abbreviations l-LDH l-Lactic acid dehydrogenase - FDP Fructose-1,6-bisphosphate - DTE Dithioerythrol AddendumIn the case of the L. casei-LDH the shape of the NADH saturation curve is not changed by omitting the effectors FDP and Mn 2⁺. The K _M under these conditions is 3 fold higher (10.10 ^–5 M).     

乳酸菌的系统分类概况

乳酸菌是重要的益生菌资源,在食品、农业、化工业、医学等领域具有广阔的应用前景。目前,人们熟知的乳酸菌主要集中在乳杆菌属、双歧杆菌属、链球菌属、肠球菌属、乳球菌属、片球菌属和明串珠菌属等少数属种。为了拓宽人们对乳酸菌的认知,本文就乳酸菌的系统分类学进行阐述。在系统分类学上,乳酸菌分别隶属于厚壁菌门4纲7目18科39属653种和放线菌门2纲2目3科12属88种。最后,对乳酸菌的益生作用、安全性与有效来源、益生潜能的体外评价指标等进行简要的讨论。     

Class II antimicrobial peptides from lactic acid bacteria

Strains of lactic acid bacteria (LAB) produce a wide variety of antibacterial peptides. More than fifty of these so-called peptide bacteriocins have been isolated in the last few years. They contain 20-60 amino acids, and are cationic and hydrophobic in nature. Several of these bacteriocins consist of two complementary peptides. The peptide bacteriocins of LAB are inhibitory at concentrations in the nanomolar range, and cause membrane permeabilization and leakage of intracellular components in sensitive cells. The inhibitory spectrum is limited to gram-positive bacteria, and in many cases to bacteria closely related to the producing strain. Among the target organisms are food spoilage bacteria and pathogens such as Listeria, so that many of these antimicrobial peptides could have a potential as food preservatives as well as in medical applications.     

Bacteriocins of lactic acid bacteria

Lactic acid bacteria produce a variety of antagonistic factors that include metabolic end products, antibiotic-like substances and bactericidal proteins, termed bacteriocins. The range of inhibitory activity by bacteriocins of lactic acid bacteria can be either narrow, inhibiting only those strains that are closely related to the producer organism, or wide, inhibiting a diverse group of Gram-positive microorganisms. The following review will discuss biochemical and genetic aspects of bacteriocins that have been identified and characterized from lactic acid bacteria.     

Genomics of lactic acid bacteria

Lactic acid bacteria (LAB) are found to occupy a variety of ecological niches including fermented foods as well as mucosal surfaces of humans and other vertebrates. This review is based on the genomic content of LAB that is responsible for the functional and ecological diversity of these bacteria. These genomes reveal an ongoing process of reductive evolution as the LAB have specialized to different nutritionally rich environments. Species-to-species variation in the number of pseudogenes as well as genes directing nutrient uptake and metabolism reflects the adaptation of LAB to food matrices and the gastrointestinal tract. Although a general trend of genome reduction was observed, certain niche-specific genes appear to be recently acquired and appear on plasmids or adjacent to prophages. Recent work has improved our understanding of the genomic content responsible for various phenotypes that continue to be discovered, as well as those that have been exploited by man for thousands of years.     

Identification of lactic acid bacteria and yeast from boza

Boza is a low-alcohol beverage produced from the fermentation of barley, oats, millet, maize, wheat or rice. The number of lactic acid bacteria isolated from three boza samples ranged from 9 × 10⁶ to 5 × 10⁷ CFU/mL. Carbohydrate fermentation reactions and PCR with species-specific primers classified the isolates as Lactobacillus paracasei subsp. paracasei, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus rhamnosus and Lactobacillus fermentum. No filamentous fungi were isolated. Yeasts were isolated from two of the three boza samples, with cell numbers ranging from 1.3 × 10² to 1.8 × 10³ CFU/mL. Results obtained from sequencing of the D1/D2 rDNA region identified the yeasts as Candida diversa, Candida inconspicua, Candida pararugosa, Issatchenkia orientalis, Pichia fermentans, Pichia guillliermondii, Pichia norvegensis, Rhodotorula mucilaginosa and Torulaspora delbrueckii. C. inconspicua has been isolated from human sputum and tongue and is an opportunistic pathogen. R. mucilaginosa is also an opportunistic pathogen implicated in fungaemia, endocarditis and meningitis. P. norvegensis has been associated with septicaemia in humans. Saccharomyces cerevisiae, commonly associated with fermented beverages, has not been detected in any of the boza samples, despite enrichment.