Functionality of exopolysaccharides produced by lactic acid bacteria in an in vitro gastric system

Aims:  To evaluate whether slime-exopolysaccharides (EPS) or capsular-polysaccharide (CPS) production could protect the polymer-producing strains Streptococcus thermophilus CRL 1190 and Lactobacillus casei CRL 87 against the harsh conditions of an in vitro gastric system (GS). EPS stability on the GS was studied.
Methods and Results:  An in vitro GS model containing human saliva and gastric juice was standardized. Polymer functionality on the cell viability and metabolic activity of the EPS-producing strains in the GS acidic conditions was evaluated. Two isogenic EPS/CPS deficient mutants were used for comparison. EPS or CPS conferred no significant protection on the cell viability of the studied strains after passage through the GS conditions. However, the phospho- and β-galactosidase activities of the EPS⁺ strains were higher than those of the EPS⁻. Cytoplasmic alterations in the wild-type and mutant strains and partial degradation of both EPS were detected.
Conclusions:  The presence of EPS/CPS protected the metabolic activity of the assayed LAB strains, but had no effect on survival at low pH.
Significance and Impact of the Study:  The presence of EPS/CPS as well as polymer resistance to the harsh conditions of the human GS could impact positively in probiotic strains to exert their properties in the host.     

Exocellular polysaccharides produced by lactic acid bacteria

Abstract The production of homopolysaccharides (dextrans, mutans) and heteropolysaccharides by lactic acid bacteria, their chemical composition, their structure and their synthesis are outlined. Mutans streptococci, which include Streptococcus mutans and S. sobrinus produce soluble and insoluble α-glucans. The latter may contain as much as 90%α-1–3 linkages and possess a marked ability to promote adherence to the smooth tooth surface causing dental plaque. Dextrans produced by Leuconostoc mesenteroides are high molecular weight α-glucans having 1–6, 1–4 and 1–3 linkages, varying from slightly to highly branched; 1–6 linkages are predominant. Emphasis is put on exopolysaccharide producing thermophilic and mesophilic lactic acid bacteria, which are important in the dairy industry. The produced polymers play a key role in the rheological behaviour and the texture of fermented milks. One of the main problems in this field is the transitory nature of the thickening trait. This instability is not yet completely understood. Controversial results exist on the sugar composition of the slime produced, but galactose and glucose have always been identified with galactose predominating in most cases.     

Vanillin production from simple phenols by wine-associated lactic acid bacteria

AIMS: The ability of lactic acid bacteria (LAB) to metabolize certain phenolic precursors to vanillin was investigated. METHODS AND RESULTS: Gas chromatography-mass spectrometry (GC-MS) or HPLC was used to evaluate the biosynthesis of vanillin from simple phenolic precursors. LAB were not able to form vanillin from eugenol, isoeugenol or vanillic acid. However Oenococcus oeni or Lactobacillus sp. could convert ferulic acid to vanillin, but in low yield. Only Lactobacillus sp. or Pediococcus sp. strains were able to produce significant quantities of 4-vinylguaiacol from ferulic acid. Moreover, LAB reduced vanillin to the corresponding vanillyl alcohol. CONCLUSIONS: The transformation of phenolic compounds tested by LAB could not explain the concentrations of vanillin observed during LAB growth in contact with wood. SIGNIFICANCE AND IMPACT OF THE STUDY: Important details of the role of LAB in the conversion of phenolic compounds to vanillin have been elucidated. These findings contribute to the understanding of malolactic fermentation in the production of aroma compounds.     

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.     

Use of alginate and cryo-protective sugars to improve the viability of lactic acid bacteria after freezing and freeze-drying

Summary In the present paper, the effect of cryo-protective sugars on the survival rate of different strains of Lactic Acid Bacteria (LAB, Lactobacillus acidophilus, Lactobacillus delbrueckii subspbulgaricus, Streptococcus salivarius subsp.thermophilus), after freezing or freeze-drying procedures, was compared. The cells were incubated at 4 °C in 32% final concentration sugar solutions (trehalose, maltose, sucrose, glucose and lactose), and viability was evaluated by the enumeration of colony-forming units. All sugars tested showed a protective effect on cell viability as compared to isotonic solution, especially after freeze-drying procedures (log c.f.u./ml ranging between 1.16 and 2.08, P < 0.001). Furthermore, the resistance to different stress agents (lysozyme, pepsin, bile salts) was estimated. Trehalose was the most effective sugar in preserving bacterial viability [% (log c.f.u. trehalose/log c.f.u. isotonic solution) ranging between 124 and 175, P < 0.001] although each strain showed a different sensitivity. Finally, the protective effect of immobilization of LAB in Ca-alginate beads was compared to that exercised by trehalose. The immobilization induced a good survival rate but lower as compared to the trehalose effect, mainly after freeze-drying in the presence of the selective agents [% (log c.f.u. alginate/log c.f.u. trehalose ranging between 81.1 and 94.5, P < 0.0001]. The protective effect of trehalose was evident in particular for Lactobacillus delbrueckii subsp. bulgaricus in presence of lysozyme. Therefore, because of its chemical inertness and low cost, trehalose could be easily utilized as excellent bacterial preservative, both to improve the viability of starter cultures and to obtain probiotic formulations more resistant to a variety of stressful conditions.     

Biogenic amine production by lactic acid bacteria isolated from cider

AIMS: To study the occurrence of histidine, tyrosine and ornithine decarboxylase activity in lactic acid bacteria (LAB) isolated from natural ciders and to examine their potential to produce detrimental levels of biogenic amines. METHODS AND RESULTS: The presence of biogenic amines in a decarboxylase synthetic broth and in cider was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). Among the 54 LAB strains tested, six (five lactobacilli and one oenococci) were biogenic amine producers in both media. Histamine and tyramine were the amines formed by the LAB strains investigated. Lactobacillus diolivorans were the most intensive histamine producers. This species together with Lactobacillus collinoides and Oenococcus oeni also seemed to produce tyramine. No ability to form histamine, tyramine or putrescine by Pediococus parvulus was observed, although it is a known biogenic amine producer in wines and beers. CONCLUSIONS: This study demonstrated that LAB microbiota growing in ciders had the ability to produce biogenic amines, particularly histamine and tyramine, and suggests that this capability might be strain-dependent rather than being related to a particular bacterial species. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of biogenic amines by food micro-organisms has continued to be the focus of intensive study because of their potential toxicity. The main goal was to identify the microbial species capable of producing these compounds in order to control their presence and metabolic activity in foods.     

Purification and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airag

AIMS: The aim of this study was to isolate and identify bacteriocin-producing lactic acid bacteria (LAB) issued from Mongolian airag (traditional fermented mare's milk), and to purify and characterize bacteriocins produced by these LAB. METHODS AND RESULTS: Identification of the bacteria (Enterococcus durans) was carried out on the basis of its morphological, biochemical characteristics and carbohydrate fermentation profile and by API50CH kit and 16S rDNA analyses. The pH-neutral cell-free supernatant of this bacterium inhibited the growth of several Lactobacillus spp. and food-borne pathogens including Escherichia coli, Staphylococcus aureus and Listeria innocua. The antimicrobial agent (enterocin A5-11) was heat stable and was not sensitive to acid and alkaline conditions (pH 2-10), but was sensitive to several proteolytic enzymes. Its inhibitory activity was completely eliminated after treatment with proteinase K and alpha-chymotrypsin. The activity was however not completely inactivated by other proteases including trypsin and pepsin. Three-step purification procedure with high recovery yields was developed to separate two bacteriocins. The applied procedure allowed the recovery of 16% and 64% of enterocins A5-11A and A5-11B, respectively, present in the culture supernatant with purity higher than 99%. SDS-PAGE analyses revealed that enterocin A5-11 has a molecular mass of 5000 Da and mass spectrometry analyses demonstrates molecular masses of 5206 and 5218 Da for fractions A and B, respectively. Amino acid analyses of both enterocins indicated significant quantitative difference in their contents in threonine, alanine, isoleucine and leucine. Their N-termini were blocked hampering straightforward Edman degradation. CONCLUSIONS: Bacteriocins A5-11A and B from Ent. durans belong to the class II of bacteriocins. SIGNIFICANCE AND IMPACT OF THE STUDY: Judging from molecular masses, amino acid composition and spectrum of activities, bacteriocins A5-11A and B from Ent. durans show high degree of similarity with enterocins L50A and L50B isolated from Enterococcus faecium (Cintas et al. 1998, 2000) and with enterocin I produced by Ent. faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation (Floriano et al. 1998).     

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.     

一株能降解胆固醇的乳酸菌的选育

利用选择性培养基从成年人的粪便中分离出1株能降解胆固醇的乳酸菌,该菌能以胆固醇作为生长的唯一能源。在10％牛奶的发酵试验中,该菌能使牛奶发酵并凝固。在液体发酵中,胆固醇的降解率为34．6％。经初步鉴定,该菌为双歧杆菌（Bifidoacterium sp.)。     

一株产共轭亚油酸乳酸菌的鉴定及其特性

从酸菜汁中分离筛选到一株产共轭亚油酸(CLA)能力较高的乳酸菌。经鉴定,确定为植物乳杆菌Lactobacliius plantarum。微氧条件可提高CLA的产量,催化亚油酸(LA)生成CLA的酶受着LA的诱导。37℃对细胞生长和CLA生成最为有利。对数生长期为6～12h,18h后进入稳定期。在14～22h,CLA生成量快速增加,24h时达到最高值。该菌的培养物经萃取、甲酯化后,进行了气相色谱分离,生成的CLA产物为c9/t9,c11-CLA和t10,c12-CLA异构体的混合物。     

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.     

Survival of silage lactic acid bacteria in the goat gastrointestinal tract as determined by denaturing gradient gel electrophoresis

Aims: To determine the survival rate of silage lactic acid bacteria (LAB) in the ruminant gastrointestinal tract. Methods and Results: Wilted Italian ryegrass (Lolium multiflorum Lam.) silage (containing 1·9 × 10⁶ CFU LAB g^?1) was fed ad libitum to three goats equipped with rumen cannulae. Silage was given alone or with concentrates at a 1 : 1 ratio on a dry matter basis. Rumen fluid was then obtained 2, 4 and 8 h after the morning feeding. Denaturing gradient gel electrophoresis was performed to compare LAB communities in silage, rumen fluid and faeces. The LAB detected in the wilted silage included Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus murinus and Lactobacillus sakei. Bands indicative of Lact. murinus were detected in either the rumen fluid or faeces, whereas the bands indicative of Lact. plantarum, Lact. brevis and Lact. sakei were not. Although the rumen fluid LAB counts and volatile fatty acid concentrations were higher in goats fed silage plus concentrates compared with those fed silage alone, the LAB communities themselves remained unaffected. Sampling times and goat‐to‐goat variations did not affect the LAB communities found in the rumen fluid. Conclusion: LAB communities found in the gut are not remarkably affected by the consumption of silage LAB, even when the silage is accompanied by concentrates that facilitate gut fermentation. Significance and Impact of the Study: Although silage can improve probiotic function, it may be difficult for silage LAB to survive the digestive process in the ruminant gastrointestinal tract.     

Removal of patulin from apple juice using inactivated lactic acid bacteria

Aims: Apples and apple products are the most notably commodities contaminated with patulin (PAT), which cause detrimental effects on human health and economic problems. The primary objective of this study was to investigate the removal of PAT contamination from apple juice using 10 different inactivated lactic acid bacteria (LAB) strains. Methods and Results: Significant quantities of PAT ranging from 47 to 80% were bound to all tested bacterial strains, whereas Lactobacillus rhamnosus 6224 and Enterococcus faecium 21605 caused a decrease of PAT by 80·4 and 64·5%, respectively. The results showed that the binding of PAT depends on the initial concentration of toxin and the adsorption temperature, also the differences in biomass existed among the 10 bacterial strains. IR analysis was performed to identify potential functional groups and the possible binding sites related to PAT adsorption. Conclusions: The removal of PAT was observed to be strain specific. The results indicated that the biosorption process did not affect the quality of juice. FTIR analysis showed that the cell wall plays a key role in PAT adsorption. Significance and Impact of the Study: Our results proof that inactivated LAB have the potential as a novel and promising adsorbent to bind PAT effectively.     

Boza, a natural source of probiotic lactic acid bacteria

Aims: To evaluate the probiotic properties of strains isolated from boza, a traditional beverage produced from cereals. Methods and Results: The strains survived low pH conditions (pH 3·0), grew well at pH 9·0 and were not inhibited by the presence of 0·3% (w/v) oxbile. Cytotoxicity levels of the bacteriocins, expressed as CC₅₀, ranged from 38 to 3776 μg ml^?1. Bacteriocin bacST284BZ revealed high activity (EC₅₀ = 735 μg ml^?1) against herpes simplex virus type 1. Growth of Mycobacterium tuberculosis was 69% repressed after 5 days in the presence of bacST194BZ. Various levels of auto‐cell aggregation and co‐aggregation with Listeria innocua LMG 13568 were observed. Adhesion of the probiotic strains to HT‐29 cells ranged from 18 to 22%. Conclusions: Boza is a rich source of probiotic lactic acid bacteria. All strains survived conditions simulating the gastrointestinal tract and produced bacteriocins active against a number of pathogens. Adherence to HT‐29 and Caco‐2 cells was within the range reported for Lactobacillus rhamnosus GG, a well‐known probiotic. In addition, the high hydrophobicity readings recorded define the strains as good probiotics. Significance and Impact of the Study: Boza contains a number of different probiotic lactic acid bacteria and could be marketed as a functional food product.     

A method for the identification of proteins secreted by lactic acid bacteria grown in complex media

Lactic acid bacteria (LAB) are known for their special nutritional requirements, being usually cultured in complex media to achieve optimal growth. In this paper, a protocol based on trichloroacetic acid precipitation of peptides and proteins is presented. The method has been tested on four probiotic LAB strains grown in De Man Rogosa Sharpe (MRS) broth, a complex medium that is often used for the culture of such bacteria. This protocol allowed the detection of 19 proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 10 of them being successfully identified by tandem MS. Thereafter, the 10 were found to be secreted or surface associated by bioinformatic means. In conclusion, this work supplies a method for the identification of proteins secreted by LAB, allowing discrimination between the proteins present in the MRS and those produced by probiotic LAB.     

Pediocin production by recombinant lactic acid bacteria

Production of the anti-listerial bacteriocin, pediocin, by lactic acid bacteria (LAB) transformed with the cloning vector pPC418 (Ped⁺, 9.1 kb) was influenced by composition of media and incubation temperature. Maximum pediocin production, tested against Listeria innocua, by electrotransformants of Lactococcus lactis ssp. lactis was measured in tryptone/lactose/yeast extract medium after 24 h growth at 30 °C, while incubation at 40 °C was optimum for Ped⁺ transformants of Streptococcus thermophilus and Enterococcus faecalis. The amount of pediocin produced by S. thermophilus in skim milk and cheese whey supplemented with 0.5% yeast extract was estimated as 51000 units ml^–1 and 25000 units ml^–1, respectively. Pediocin production remained essentially unchanged in reconstituted skim milk or whey media diluted up to 10-fold. The results demonstrate the capacity of recombinant strains of LAB to produce pediocin in a variety of growth media including skim milk and inexpensive cheese whey-based media, requiring minimum nutritional supplementation.     

Modification of azo dyes by lactic acid bacteria

Aim:  The ability of Lactobacillus casei and Lactobacillus paracasei to modify the azo dye, tartrazine, was recently documented as the result of the investigation on red coloured spoilage in acidified cucumbers. Fourteen other lactic acid bacteria (LAB) were screened for their capability to modify the food colouring tartrazine and other azo dyes of relevance for the textile industry.
Methods and Results:  Most LAB modified tartrazine under anaerobic conditions, but not under aerobic conditions in modified chemically defined media. Microbial growth was not affected by the presence of the azo dyes in the culture medium. The product of the tartrazine modification by LAB was identified as a molecule 111 daltons larger than its precursor by liquid chromatography-mass spectrometry. This product had a purple colour under aerobic conditions and was colourless under anaerobic conditions. It absorbed light at 361 and 553 nm.
Conclusion:  LAB are capable of anabolizing azo dyes only under anaerobic conditions.
Impact and Significance of the Study:  Although micro-organisms capable of reducing the azo bond on multiple dyes have been known for decades, this is the first report of anabolism of azo dyes by food related micro-organisms, such as LAB.     

Presence of sourdough lactic acid bacteria in commercial total mixed ration silage as revealed by denaturing gradient gel electrophoresis analysis

Aims: To characterize the bacterial communities in commercial total mixed ration (TMR) silage, which is known to have a long bunk life after silo opening. Methods and Results: Samples were collected from four factories that produce TMR silage according to their own recipes. Three factories were sampled three times at 1‐month intervals during the summer to characterize the differences between factories; one factory was sampled 12 times, three samples each during the summer, autumn, winter and spring, to determine seasonal changes. Bacterial communities were determined by culture‐independent denaturing gradient gel electrophoresis. All silages contained lactic acid as the predominant acid, and the contents appeared stable regardless of factories and product seasons. Acetic acid and 1‐propanol contents were different between factories and indicated seasonal changes, with increases in warm seasons compared to cool seasons. Both differences and similarities existed among the bacterial communities from each factory and product season. Lactobacillus parabuchneri was found in the products from three of four factories. Various sourdough lactic acid bacteria (LAB) were identified in commercial TMR silage; Lactobacillus panis, Lactobacillus hammesii, Lactobacillus mindensis, Lactobacillus pontis, Lactobacillus frumenti and Lactobacillus farciminis were detected in many products. Moreover, changes owing to product season were distinctive, and Lact. pontis and Lact. frumenti became detectable in summer products. Conclusion: Sourdough LAB are involved in the ensiling of commercial TMR silage. Silage bacterial communities vary more by season than by factory. The LAB species Lact. parabuchneri was detected in the TMR silage but may not be essential to the product’s long bunk life after silo opening. Significance and Impact of the Study: Commercial TMR silage resembles sourdough with respect to bacterial communities and long shelf life. The roles of sourdough LAB in the ensiling process and aerobic stability are worth examining.     

Characterization of exopolysaccharides produced by three moderately halophilic bacteria belonging to the family Alteromonadaceae

Aims:  To study the exopolysaccharides (EPSs) produced by three novel moderately halophilic species belonging to the family Alteromonadaceae to optimize EPS yields, characterize their physical and chemical properties and evaluate possible biotechnological applications for these polymers.
Methods and Results:  EPSs synthesized by Idiomarina fontislapidosi F32^T, Idiomarina ramblicola R22^T and Alteromonas hispanica F23^T were collected and analysed under optimum conditions: MY medium supplemented with 7·5% (w/v) salts; 32°C; and 1% (w/v) glucose. Polymers were synthesized mainly during the early stationary growth phase with yields ranging from 1 to 1·5 g l⁻¹. The Idiomarina species each produced an anionic EPS composed mainly of glucose, mannose and galactose. A. hispanica synthesized an anionic EPS composed mainly of glucose, mannose and xylose. Solutions of all the polymers were low in viscosity and pseudoplastic in their behaviour. They showed emulsifying activity and the capacity to bind some metals.
Conclusions:  The Alteromonadaceae species studied in this work produced EPSs with physical and chemical properties different from those produced by other halophilic and nonhalophilic bacteria, suggesting that the wide diversity of micro-organisms being encountered nowadays in hypersaline environments offers enormous potential resources for biotechnological applications.
Significance and Impact of the Study:  We have optimized the EPS production and analysed new biopolymers produced by some recently described, moderately halophilic bacteria. These biopolymers are chemically and physically different from others already in use in biotechnology and offer hopes for new applications, especially in the case of A. hispanica , which may prove to be a viable source of xylo-oligosaccharides.