EXOPOLYSACCHARIDE PRODUCTION BY SELECTED LACTIC ACID BACTERIA ISOLATED FROM FERMENTED VEGETABLES

Abstract Lactic acid bacteria (LAB) play a key role in the food fermentation process since they contribute to the texture, flavor, quality and conservation of the fermented products. Several EPS presence in the culture supernatant was detected through gel permeation chromatography (GPC). The EPS material was isolated from these strains by acetone precipitation, then dialysed, dried and weighted. The molecular mass was estimated by the same GPC method, while the monomer composition was determined by automated thin layer chromatography (TLC), after hydrolysis with 8N HCl. One of the positive strain, Leuconostoc mesenteroides/pseudomesenteroides 406 has been shown to produce large amounts of EPS, of about 15 g/L and two strains, Leuconostoc citreum/lacti

In Vitro Evaluation of Commercial Probiotic Products Containing <i>Streptococcus salivarius</i> K12 by Assessment of Probiotic Viability and Inhibitory Potency against Respiratory Pathogens

Upper respiratory infections (URI) are the most frequent illnesses, especially in children. The majority of those infections are prescribed broad-spectrum antibiotics, which are associated with various side effects and with the increase in multi-drug-resistant strains. A promising alternative approach is the administration of the probiotic strain Streptococcus salivarius K12 (SSK12) that colonizes the upper respiratory tract (URT) and produces the salivaricins A2 and B, which strongly antagonize the growth of key respiratory pathogens. However, since for food supplements no quality controls of the active probiotic ingredient are mandatory, the efficacy of commercial products containing SSK12 may vary. This study aimed to investigate the in vitro efficacy of several commercial SSK12-containing probiotics, positioned for the prevention of respiratory infections. The parameters evaluated to determine the in vitro efficacy included the viability of the probiotic bacterial strain and the minimum inhibitory dilution (MID) of the probiotic, determined by the agar spot method, against the pathogenic/potential pathogenic bacterial strains Streptococcus pyogenes FF22 and Micrococcus luteus T18. All tests were carried out both 12 and 24 months after manufacturing (AM) for each commercial product. The viability ranged from 9 × 108 to 4.4 × 109 CFU/serving at 12 months AM and from 8.5 × 107 to 2.8 × 109 CFU/serving at 24 months AM. The MID was, in general, positively correlated with the probiotic bacterium viability and varied between the commercial products, ranging from 10−5 to 10−7 at 12 months AM and from 10−4 to 10−7 at 24 months AM. Moreover, the inhibition zones related to the two indicator strains were variable in diameter for different products. The high variation of the in vitro efficacy of commercial products containing SSK12 may explain the different results reported in the literature regarding the clinical benefits of these preparations, and the determination of this parameter may be useful to evaluate the quality of probiotic products containing this bacterial strain

Biodiversity of Exopolysaccharides Produced by Streptococcus thermophilus Strains Is Reflected in Their Production and Their Molecular and Functional Characteristics

Twenty-six lactic acid bacterium strains isolated from European dairy products were identified as Streptococcus thermophilus and characterized by bacterial growth and exopolysaccharide (EPS)-producing capacity in milk and enriched milk medium. In addition, the acidification rates of the different strains were compared with their milk clotting behaviors. The majority of the strains grew better when yeast extract and peptone were added to the milk medium, although the presence of interfering glucomannans was shown, making this medium unsuitable for EPS screening. EPS production was found to be strain dependent, with the majority of the strains producing between 20 and 100 mg of polymer dry mass per liter of fermented milk medium. Furthermore, no straightforward relationship between the apparent viscosity and EPS production could be detected in fermented milk medium. An analysis of the molecular masses of the isolated EPS by gel permeation chromatography revealed a large variety, ranging from 10 to >2,000 kDa. A distinction could be made between high-molecular-mass EPS (>1,000 kDa) and low-molecular-mass EPS (<1,000 kDa). Based on the molecular size of the EPS, three groups of EPS-producing strains were distinguished. Monomer analysis of the EPS by high-performance anion-exchange chromatography with amperometric detection was demonstrated to be a fast and simple method. All of the EPS from the S. thermophilus strains tested were classified into six groups according to their monomer compositions. Apart from galactose and glucose, other monomers, such as (N-acetyl)galactosamine, (N-acetyl)glucosamine, and rhamnose, were also found as repeating unit constituents. Three strains were found to produce EPS containing (N-acetyl)glucosamine, which to our knowledge was never found before in an EPS from S. thermophilus. Furthermore, within each group, differences in monomer ratios were observed, indicating possible novel EPS structures. Finally, large differences between the consistencies of EPS solutions from five different strains were assigned to differences in their molecular masses and structures

Exopolysaccharide-producing Streptococcus thermophilus strains as functional starter cultures in the production of fermented milks

Relationships between exopolysaccharide (EPS) production (amount, molecular mass and sugar composition of the EPS) by different Streptococcus thermophilus strains as a functional starter culture, and textural characteristics (viscosity) of fermented milk and yoghurt have been studied. Five interesting heteropolysaccharide-producing strains have been tested. Both S. thermophilus LY03 and S. thermophilus CH101 produced the highest amounts of EPS and also displayed the highest apparent viscosities in fermented milk. S. thermophilus ST 111 and S. thermophilus STD differed considerably in EPS yields, but not in apparent viscosities of fermented milk. In addition, S. thermophilus ST 111 displayed a high variability in EPS amounts when cultivated in milk. In milk medium, S. thermophilus LY03 produced two heteropolysaccharides, a high-molecular-mass (HMM) EPS and a low-molecular-mass (LMM) EPS of the same composition (Gal/Glu/GalNAc=3.4:1.4:1.0). S. thermophilus ST 111 produced only a HMM-EPS (Gal/Rha=2.5:1.0), while S. thermophilus CH 101 (Gal/Glu=1.0:1.0), S. thermophilus ST 113 (Gal/Glu/Rha/GalNAc=1.7:3.9:1.5:1.0) and S. thermophilus STD (Gal/Glu/Rha/GalNAc=3.5:6.2:1.2:1.0) produced only LMM-EPS. Both HMM-EPS and LMM-EPS solutions (S. thermophilus LY03) demonstrated a pseudoplastic character; HMM-EPS solutions of 0.2% (m/v) displayed a high consistency as well. Although its production of high EPS amounts, S. thermophilus LY03 resulted in relatively thin yoghurts, so that texture values did not directly correlate with EPS production capacity. Once structure/function relationships are known, one can determine the molecular properties of the isolated and purified EPS (molecular size, structural characteristics) from candidate strains to predict their potential in texture formation. For a final selection of interesting EPS-producing starter strains one should test the EPS production under yoghurt manufacturing conditions

Streptococcus thermophilus ST 111 produces a stable high-molecular-mass exopolysaccharide in milk-based medium

Streptococcus thermophilus ST 111, grown in milk medium supplemented with whey protein hydrolysate, produced an exopolysaccharide (EPS) composed of galactose and rhamnose in a molar ratio of 2.5:1, and with a molecular mass of more than 5000 kDa. Fermentations in milk medium supplemented with whey protein hydrolysate further demonstrated the stability of this high-molecular-mass EPS, as its molecular mass was affected neither by the pH of the medium nor the fermentation time, indicating no enzymatic degradation. On the other hand, drying of the isolated EPS resulted in a decrease of the molecular mass. The stability of an EPS produced in milk-based media is an important asset for the production of fermented milk and yoghurt products

Non-Dairy Fermented Beverages Produced with Functional Lactic Acid Bacteria

At present, there is an increasing interest in beverages of non-dairy origin, as alternatives to those based on milk, but having similar health-promoting properties. Fermentation with specific bacteria or consortia may enhance the functionality of these products. In our study, selected lactic acid bacteria, that have been previously shown to possess functional properties (antimicrobial activity, probiotic potential), were used for the fermentation of wheat bran combined with root vegetables. Strains were investigated for their safety, while the obtained beverages were characterized in terms of microbial content, physical, chemical, nutritional, and functional properties. None of the strains harbors virulence genes, but all of them possess genes for survival at low pH, starch metabolism, and vitamin biosynthesis. Three strains (Lactiplantibacillus plantarum BR9, L. plantarum P35, and Lactobacillus acidophilus IBB801) and two substrates (5% wheat bran with 10% red beetroot/carrots) were selected based on a preliminary assessment of the beverage’s sensory acceptability. These strains showed good growth and stability over time in the stored beverages. No enterobacteria were detected at the end of fermentations, while the final pH was, in most cases, below 3.5. Free phenolics, flavonoids, and DPPH scavenging effect increased during fermentation in all drinks, reaching 24h values that were much higher than in the unfermented substrates. Most of the obtained drinks were able to prevent the growth of certain pathogens, including Listeria monocytogenes ATCC 19111, Salmonella enterica ATCC 14028, Staphylococcus aureus ATCC 25923, and Escherichia coli ATCC 25922. The obtained beverages would combine the nutritiveness of the raw ingredients with the beneficial effect of fermentation (increasing shelf life, health-promoting effect, pleasant flavor, etc.). They would also fill a gap in the non-dairy probiotics sector, which is constantly increasing due to the increasing number of vegan people or people that cannot consume dairy products

Screening of lactic acid bacteria isolates from dairy and cereal products for exopolysaccharide production and genes involved

A total of 174 lactic acid bacteria (LAB) strains isolated from dairy and cereal products were screened for the production of exopolysaccharides (EPS). Therefore, a rapid screening method was developed based on ultrafiltration and gel permeation chromatography. Furthermore, a screening through the polymerase chain reaction (PCR) was performed with primer pairs targeting different genes involved in EPS production. Nine isolates produced a homopolysaccharide of the glucan type, whereas only one strain produced a heteropolysaccharide. The production of a glucan by a strain of Lactococcus lactis and the production of a heteropolysaccharide by a strain of Lactobacillus curvatus are reported for the first time. The PCR screening revealed many positive strains. For three of the ten EPS-producing strains, no corresponding genes could be detected. Furthermore, a lot of strains possessed one or more eps genes but did not produce an EPS. Therefore, a screening on the molecular level should always be accompanied by another screening method that is able to distinguish true EPS producer strains from non-producing ones. Statistical analysis did not reveal any relationship between the type and origin of the strains, the presence or absence of a capsular polysaccharide or EPS, and the presence or absence of eps genes.Fil: Van der Meulen, Roel. Vrije Universiteit Brussel; BélgicaFil: Grosu Tudor, Silvia. Vrije Universiteit Brussel; BélgicaFil: Mozzi, Fernanda Beatriz. Vrije Universiteit Brussel; Bélgica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Vaningelgem, Frederik. Vrije Universiteit Brussel; BélgicaFil: Zamfir, Medana. Vrije Universiteit Brussel; Bélgica. Romanian Academy. Cell Biology Department. Institute of Biology; RumaniaFil: Font, Graciela Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; Argentina. Universidad Nacional de Tucumán. Facultad de Bioquímica, Química y Farmacia; ArgentinaFil: De Vuyst, Luc. Vrije Universiteit Brussel; Bélgic

<i>Lactobacillus</i> Strains for Vegetable Juice Fermentation—Quality and Health Aspects

Vegetable juices are new carrier variants for beneficial bacteria, representing an alternative to dairy-fermented products, especially for vegan, strict vegetarian, or allergic consumers. The aim of this study was to characterize several Romanian native lactic acid bacteria (LAB) strains to select valuable nutritional and probiotic strains for vegetable juice fermentation. Nineteen LAB strains were analyzed for antibiotic susceptibility (disc-diffusion method), the presence of antibiotic resistance genes, the presence of functional genes. and the production of organic acids by HPLC. Antibiotic resistant strains were observed only with ampicillin (Amp10) and kanamycin (K30), 79% and 32%, respectively, with results partially confirmed by molecular analysis. Multiplex PCR revealed the presence of LBA1272, dltD, folP, agl, α-amy, malL, and ribA genes, related to stress resistance, starch metabolism, and production of vitamins, except for folK. HPLC analyses were performed on beet roots (SF), tomato (TM), and a mixture of carrots, celery, and beet (MTS) juices. High values of lactic acid were recorded in all cases of LAB fermentation (5034–14,176 µg/mL). The maximum values recorded for acetic acid did not exceed 2.5 mg/mL having a positive influence on the product’s taste

S-layer production by Lactobacillus acidophilus IBB 801 under environmental stress conditions

The ability of microorganisms to synthesize S-layer, the outermost structure of the microbial cell envelope composed of non-covalently bound proteins, has been ascribed to help microorganisms to exert their probiotic properties in the host. In this work, formation of S-layer by the potentially probiotic strain Lactobacillus acidophilus IBB 801 under different stress culture conditions (high incubation temperatures, presence of bile salts or NaCl, and acidic pH) was assayed. A marked S-layer synthesis by L. acidophilus IBB 801 was detected when the strain was grown at 42 °C and in the presence of 0.05 % bile salts or 2.0 % NaCl. The presence of S-layer proteins was further confirmed by transmission electron microscopy and protein identification by MS/MS. The differential expression of the proteome of this strain at 42 °C, when a marked formation of S-layer was detected, revealed the overexpression of six proteins mainly related to general stress and protein biosynthesis and translation, while four proteins detected in lower amounts were involved in DNA repair and energy metabolism. As L. acidophilus IBB 801 produces both a bacteriocin and S-layer proteins, the strain could be of interest to be used in the formulation of functional food products with specific properties.Fil: Grosu Tudor, Silvia Simona. Academia Romana, Institutul de Biologie Bucuresti; RumaniaFil: Brown, Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Hebert, Elvira Maria. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Brezeanu, Aurelia. Academia Romana, Institutul de Biologie Bucuresti; RumaniaFil: Brinzan, Alexandru. Academia Romana, Institutul de Biologie Bucuresti; RumaniaFil: Fadda, Silvina G.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Mozzi, Fernanda Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucuman. Centro de Referencia Para Lactobacilos; ArgentinaFil: Zamfir, Medana. Academia Romana, Institutul de Biologie Bucuresti; Rumani