New method for exopolysaccharide determination in culture broth using stirred ultrafiltration cells

A new method to remove simple carbohydrates from culture broth prior to the quantification of exopolysaccharides (EPS) was developed and validated for the EPS-producing strain, Lactobacillus rhamnosus RW-9595M. This method uses ultrafiltration (UF) in stirred cells followed by polysaccharide detection in the retentate by the phenol-sulfuric acid method. The UF method was compared with a conventional method based on ethanol extraction, dialysis, protein removal by trichloroacetic acid (TCA) and freeze-drying. EPS production during pH-controlled batch fermentations in basal minimum medium, whey permeate (WP). and whey permeate supplemented with yeast extract, minerals and Tween-80 (SWP) was determined by the new UF and conventional methods. EPS recovery by the new method ranged from 83% to 104% for EPS added in the concentration range 40-1,500 mg/l in 0.1 M NaCl solution or culture medium. The UF method was rapid (8 h), accurate and simple, and required only a small sample volume (1-5 ml). A very high maximum EPS production was measured in SWP by both the UF and conventional methods (1,718 and 1,755 mg/l).     

Exopolysaccharide production during batch cultures with free and immobilized Lactobacillus rhamnosus RW-9595M

AIMS: Exopolysaccharides (EPS) were produced by Lactobacillus rhamnosus RW-9595M during pH-controlled batch cultures with free cells and repeated-batch cultures with cells immobilized on solid porous supports (ImmobaSil). METHODS AND RESULTS: Cultures were conducted in supplemented whey permeate (SWP) medium containing 5 or 8% (w/w) whey permeate. For free-cell batch cultures in 8% SWP medium, very high maximum cell counts (1.3 x 10(10) CFU ml(-1)) and EPS production (2350 mg l(-1)) were measured. A high EPS production (1750 mg l(-1)) was measured after four cycles for a short incubation period of only 7 h. Several methods for immobilized biomass determination based on analysis of biomass components (proteins, ATP and DNA) were tested. The DNA analysis method proved to be the most appropriate under these circumstances. This method revealed a high maximum immobilized biomass of 8.5 x 10(11) CFU ml(-1) support during repeated immobilized cell cultures in 5% SWP. The high immobilized biomass increased maximum EPS volumetric productivity (250 mg l(-1) h(-1) after 7 h culture) compared with free-cell batch cultures (110 mg l(-1) h(-1) after 18 h culture). CONCLUSIONS: High EPS productions were achieved during batch cultures of Lact. rhamnosus RW-9595M in SWP medium, exceeding 1.7 g EPS per litre. Repeated-batch cultures with immobilized cells resulted in increased EPS productivity compared with traditional free-cell cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The study clearly shows the high potential of the strain Lact. rhamnosus RW-9595M and immobilized cell technology for production of EPS as a functional food ingredient.     

Growth and exopolysaccharide production during free and immobilized cell chemostat culture of Lactobacillus rhamnosus RW-9595M

AIMS: Biomass and exopolysaccharide (EPS) production were studied during chemostat cultures in whey permeate medium with Lactobacillus rhamnosus RW-9595M-free cells and cells immobilized on solid porous supports (ImmobaSil). METHODS AND RESULTS: A continuous culture with free cells was conducted for 9 days at dilution rates (D) between 0.3 and 0.8 h(-1) in yeast extract (YE)/mineral supplemented whey permeate. Maximum EPS production (1808 mg l(-1)) and volumetric productivity (542.6 mg l(-1) h(-1)) were obtained for a low D of 0.3 h(-1). A continuous fermentation in a two-stage bioreactor system, composed of a first stage with immobilized cells and a second stage inoculated with free cells produced in the first reactor, was carried out for 32 days. The influence of YE concentration, temperature and dilution rate, and their interactions on biomass, EPS and lactic acid production was investigated. A statistically significant model was found only for lactic acid production. Marked cell morphological and physiological changes led to the formation of very large cell-containing aggregates and a low mean soluble EPS production (138 mg l(-1)). Aggregate volumetric productivity of the two-stage system varied between 5.7 and 49.5 g l(-1) h(-1) for different fermentation conditions and times. Aggregates contained a very high biomass concentration, estimated at 74% of aggregate dry weight by nitrogen analysis and 4.3 x 10(12) CFU g(-1) by a DNA extraction method and a high nonsoluble polysaccharide content (14.2%). At age 24 days, insoluble EPS concentration and volumetric productivity were 1250 mg l(-1) and 2240 mg l(-1) h(-1) respectively. The physiological changes were shown to be reversible when cells were incubated during three successive batch cultures. CONCLUSIONS: EPS production and volumetric productivity during continuous free-cell chemostat cultures with L. rhamnosus RW-9595M are among the highest values reported for lactobacilli in literature. Immobilization and continuous culture resulted in low soluble EPS production and large morphological and physiological changes of L. rhamnosus RW-9595M, with formation of macroscopical aggregates mainly composed of biomass and nonsoluble EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on continuous EPS production by immobilized LAB. Immobilization and culture time-induced cell aggregation and could be used to produce new synbiotic products with very high viable cell and EPS concentrations.     

Comparative effects of exopolysaccharides from lactic acid bacteria and fructo-oligosaccharides on infant gut microbiota tested in an in vitro colonic model with immobilized cells

The aim of this study was to compare the effects of purified exopolysaccharides from Lactobacillus rhamnosus RW-9595M with those of a well-known prebiotic (short-chain fructo-oligosaccharides) on infant colonic microbiota using a new three-stage chemostat model with immobilized infant faecal microbiota. Two continuous cultures with different faecal inocula were tested with different compositions of carbohydrate media. During the first fermentation (F1), fructo-oligosaccharides tested at a concentration of 9.8 g L(-1) increased the number of lactobacilli and decreased coliforms both in gel beads and in effluent from all three reactors, in agreement with data from the literature. During the second fermentation (F2), the effect of fructo-oligosaccharides tested at a lower concentration (7.5 g L(-1)) was reduced compared with F1. Fructo-oligosaccharides also increased total organic acid concentration and decreased ammonia production. Results obtained for exopolysaccharide tested at 1.5 g L(-1) indicate that exopolysaccharides from L. rhamnosus RW-9595M was not metabolized by infant microbiota and lacked any prebiotic effect.     

Consensus-degenerate hybrid oligonucleotide primers for amplification of priming glycosyltransferase genes of the exopolysaccharide locus in strains of the Lactobacillus casei group

A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3' degenerate core based on four highly conserved amino acids and a longer 5' consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L. casei, Lactobacillus zeae, and Streptococcus thermophilus. The priming GT gene was detected in the genome of both non-EPS-producing (EPS(-)) and EPS-producing (EPS(+)) strains of L. rhamnosus. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS(+) bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS(+) strains of L. rhamnosus. Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.     

Quantification by real-time PCR of Lactococcus lactis subsp. cremoris in milk fermented by a mixed culture

During cheese making, interactions between different strains of lactic acid bacteria play an important role. However, few methods are available to specifically determine each bacterial population in mixed cultures, in particular for strains of the same species. The aim of this study was to develop a real-time PCR quantification method to monitor the population of Lactococcus cremoris ATCC 19257 in mixed culture with Lactobacillus rhamnosus RW-9595M and the bacteriocin-producing microorganism Lc. diacetylactis UL719. The specificity of the two primers 68FCa33 and 16SR308 used to amplify a 240-bp fragment of DNA from Lc. cremoris was demonstrated by conventional PCR. Using these primers for real-time PCR, the detection limit was 2 cfu/reaction or 200 cfu of Lc. cremoris ATCC 19257 per millilitre of mixed culture in milk. In pure culture batch fermentation, good correlation was obtained between real-time PCR and the conventional plating method for monitoring Lc. cremoris growth. In mixed culture batch fermentation, Lb. rhamnosus and Lc. cremoris decreased due to nisin Z production by Lc. diacetylactis. The decrease of the Lc. cremoris cell population detected by real-time PCR was not possible to observe by the plate count method in the presence of a Lc. diacetylactis population that was 1 log higher.     

Fermentation conditions affecting the bacterial growth and exopolysaccharide production by Streptococcus thermophilus ST 111 in milk-based medium

AIMS: To study the effect of different fermentation conditions and to model the effect of temperature and pH on different biokinetic parameters of bacterial growth and exopolysaccharides (EPS) production of Streptococcus thermophilus ST 111 in milk-based medium. METHODS AND RESULTS: The influence of temperature and pH was studied through fermentation and modelling. Fermentations under non-pH controlled conditions with S. thermophilus ST 111 indicated that the EPS production was low in milk medium, even if additional nitrogen sources were supplemented. Under pH-controlled conditions, addition of whey protein hydrolysate to the milk medium resulted in a fivefold increase of the EPS production. This medium did not contain polysaccharides interfering with EPS isolation. Primary and secondary modelling of different fermentations revealed an optimum temperature and pH of 40 degrees C and constant pH 6.2, respectively, for growth in milk medium supplemented with whey protein hydrolysate. Maximum EPS production was observed in the range of 32-42 degrees C and constant pH 5.5-6.6. Whereas growth and maximum EPS production were clearly influenced by temperature and pH, the specific EPS production was only affected by stress conditions (T = 49 degrees C). CONCLUSIONS: Addition of whey protein hydrolysate to milk medium resulted in an increased growth and EPS production of S. thermophilus ST 111 under pH-controlled conditions. A modelling approach allowed studying the influence of temperature and pH on the kinetics of both growth and EPS production. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of an appropriate milk-based medium and a combined model of temperature and pH can be of practical importance for the production of yoghurt or other fermented milks as well as for process optimization of the large-scale production of starter strains to be used for their EPS production.     

Identification and characterization of a conserved nuclease secreted by strains of the Lactobacillus casei group

AIMS: Nuclease secretion was evaluated for five species of Lactobacillus and the activity was characterized in terms of thermal resistance, molecular weight and mode of action on plasmid DNA. METHODS AND RESULTS: Assays of nuclease from L. rhamnosus ATCC 9595 on DNA of different origins indicates a broad activity spectrum. Secreted nuclease from this strain resists a thermal treatment of 20 min at 100 degrees C, is not sensitive to a treatment for disruption of disulphide bonds nor to EDTA treatment under 10 mM l(-1). Nuclease production is not growth linked and seems to be constitutive. Extracellular nuclease of L. rhamnosus ATCC 9595 introduces a single-stranded nick in supercoiled DNA, thus potentially reducing the transformability of plasmid DNA. In seven of eight tested strains, SDS-PAGE revealed a major protein with a molecular weight of ca 35 kDa. Minor degradation products also showed nuclease activity. CONCLUSIONS: A comparative analysis of the extracellular fractions of 14 different Lactobacillus strains indicate that nuclease secretion seems to be a widely distributed function among species of milk-related lactobacilli. The production of secreted nuclease may contribute to the low ability of Lactobacillus spp. to be transformed and maintain exogenous DNA. SIGNIFICANCE AND IMPACT OF THE STUDY: Determination of the characteristics and distribution of nuclease activity contribute to developing strategies to overcome this barrier to efficient transformation of milk lactobacilli.     

Factors influencing the production of water-soluble endopolysaccharides and exopolysaccharides from Lentinus lepideus and their effects on immune cytokine production

An efficient method to produce water-soluble polysaccharides from Lentinus lepideus is described. The productivity of both endopolysaccharides (PPS) and exopolysaccharides (EPS) was compared under various culture conditions. The effect of treating their own PPS and EPS on immune cytokine production was also studied in relation to culture factors. High yield production of EPS required moderate culture temperature (25 degrees ) as well as long culture period (16-20 days). In contrast, PPS production required high culture temperature (30 degrees ) and short culture period ( days). Most of the carbon sources did not affect polysaccharides and mycelial production except for sucrose. Immune cytokine levels in the EPS treatment varied among carbon sources or culture periods. PPS did not appear to affect much on the production of cytokines, regardless of the culturing factors, except for the culture period. These results suggest that the optimal culture conditions for L. lepideus vary according to culture purposes, and different culture conditions should be used for different targets including mycelial biomass, EPS, and PPS. Whereas the immunomodulating activity of EPS appeared to be affected by culture conditions in L. lepideus, that of PPS did not.     

Comparison of exopolysaccharide production by strains of Lactobacillus rhamnosus and Lactobacillus paracasei grown in chemically defined medium and milk

Exopolysaccharide (EPS) production was compared among three strains of lactobacilli. Lactobacillus rhamnosus strain 9595M can be classified among the highest EPS-producing strains of lactic acid bacteria reported to date with a maximum EPS production of 1275 mg L⁻¹. Under controlled pH, no significant differences in the quantity of EPS produced could be detected between carbon source (glucose or lactose) or fermentation temperature (32 or 37°C). In milk, strains ATCC 9595M and R produced more than 280 mg L⁻¹ EPS whereas strain Type V produced less than 80 mg L⁻¹ EPS. Journal of Industrial Microbiology & Biotechnology (2000) 24, 251–255. Received 10 September 1999/ Accepted in revised form 22 December 1999     

Effects of medium composition on MFα1 promoter-directed secretion of a small protease inhibitor in Saccharomyces cerevisiae batch fermentation

Summary Casaminoacid supplementation of yeast nitrogen base (YNB) medium is widely used in the culture of recombinant yeasts as it enables respiratory growth on YNB and selection for URA3- or TRP1-bearing plasmids. Despite these advantages, this supplementation to YNB medium resulted in marked decreases in cellular productivity for the secretion of a protease inhibitor product in batch fermentation.     

Physiological approach to extracellular polysaccharide production by Lactobacillus rhamnosus strain C83

Extracellular polysaccharide production was studied by growing strain C83 of Lactobacillus rhamnosus in a chemically defined medium containing various carbon sources (glucose, fructose, mannose or maltose) at different concentrations. Mannose or a combination of glucose + fructose were by far the most efficient carbon sources. An optimization of growth medium composition led to an improvement in both growth and EPS production. When the strain was cultured at a lower temperature the EPS production increased. A temperature shift, at the end of the exponential growth phase, did not enhance the EPS production. The EPS synthesis by strain C83 was growth-related. The isolated slime had a sugar composition, as shown by three different methods, of glucose and galactose with a ratio of 1 : 1.     

抑制黑色素合成的乳酸菌胞外多糖的筛选和性质研究

【目的】筛选可抑制黑色素合成的乳酸菌胞外多糖。【方法】通过观察凝乳拉丝外观筛选产胞外多糖的乳酸菌菌株,测量胞外多糖对B16黑色素瘤细胞黑色素合成和细胞活力的影响。对胞外多糖进行纯化,并通过PMP衍生-HPLC、红外光谱、抑制酪氨酸酶活性、抗氧化能力对其单糖组成和结构、作用机制进行研究。【结果】筛选到一株乳酸菌Lactobacillus rhamnosus HLAB122,发酵产生的胞外多糖在5 g/L浓度下可使B16细胞黑色素产量下降至空白对照的32.7%,且在96 h内对细胞活力无影响。纯化后的多糖由鼠李糖、葡萄糖、半乳糖构成,各单糖摩尔比为1?5.44?5.37。该胞外多糖不抑制酪氨酸酶活力且抗氧化性微弱。【结论】L.rhamnosus HLAB122产生的胞外多糖在个人护理产品中有潜在应用价值。     

Media and process parameters affecting the growth, strain ratios and specific acidifying activities of a mixed lactic starter containing aroma-producing and probiotic strains

AIMS: The effects of medium-composition and fermentation parameters on the properties of mixed mesophilic starters were studied. The starter was composed of Lactococcus lactis ssp. lactis (L. lactis), Lactococcus lactis ssp. cremoris (L. cremoris), Lactobacillus rhamnosus (Lact. rhamnosus) and Leuconostoc mesenteroides ssp. cremoris (Leuc. cremoris). METHODS AND RESULTS: The media used were reconstituted skim milk (RSM), and whey-based media with either citrate or phosphate buffers. The fermentation parameters were incubation temperature (22 degrees C or 32 degrees C), no pH control, and pH control in pH zones of either pH 6.0-5.8 or pH 6.0-5.2. The starter properties were strain ratio, specific acidifying activity (SAA), total population, residual carbohydrates and organic acids produced. The growth of L. lactis was favoured under pH control in whey-based media. High concentrations of Lact. rhamnosus were favoured in whey-based media prepared at 32 degrees C. The highest contents of Leuc. cremoris were obtained in starters prepared in RSM at 22 degrees C without pH control. Starters prepared under pH control gave the highest populations and made it possible for significantly lower inoculation rates (IR) to be used to carry out subsequent milk fermentations. However, the SAA of starters prepared under pH control were lower than the SAA of starters grown without any pH control. CONCLUSIONS: None of the conditions enabled the strain ratio at inoculation to be maintained. The data show that it is possible to prepare a mesophilic starter that has a significant probiotic Lact. rhamnosus content; this starter could be used in the preparation of probiotic-containing cheeses or in Leuc. cremoris for aroma production in fermented milks. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data on what should be expected with respect to strain ratios and IR if cheesemakers decide to shift their aroma-producing starter production method from the traditional 'milk-based without pH control' method to whey-based media used with pH-zone control strategies.     

Production of exopolysaccharide by Lactobacillus rhamnosus R and analysis of its enzymatic degradation during prolonged fermentation

The potential of Lactobacillus rhamnosus R for producing exopolysaccharide (EPS) when grown on basal minimum medium supplemented with glucose or lactose was investigated. EPS production by L. rhamnosus R is partially growth associated and about 500 mg of EPS per liter was synthesized with both sugars. The product yield coefficient (Y(EPS/S)) was 3.15 (0.0315 g of EPS [g of lactose](-1)) and 2.88 (0.0288 g of EPS [g of glucose](-1)). It was clearly shown that the amount of EPS produced declined upon prolonged fermentation. Degradation of EPS in fermentation processes was also assessed by measuring its molecular weights and viscosities. As these reductions might have a negative effect on the yield and viscosifying properties of EPS, it was essential to examine possible causes related to this breakdown. The decrease in viscosities and molecular weights of EPS withdrawn at different cultivation times permitted us to suspect the presence of a depolymerizing enzyme in the fermentation medium. Our study on enzymatic production profiles showed a large spectrum of glycohydrolases (alpha-D-glucosidase, beta-D-glucosidase, alpha-D-galactosidase, beta-D-galactosidase, beta-D-glucuronidase, and some traces of alpha-L-rhamnosidase). These enzymes were localized, two of them (alpha-D-glucosidase and beta-D-glucuronidase) were partially purified and characterized. When incubated with EPS, these enzymes were capable of lowering the viscosity of the polymer as well as liberating some reducing sugars. Upon prolonged incubation (27 h), the loss of viscosity was increased up to 33%.     

Intermediate chains of exopolysaccharides from Lactobacillus rhamnosus RW‐9595M increase IL‐10 production by macrophages

Aims: To evaluate the immunosuppressive properties of the exopolysaccharide (EPS) from high‐EPS producer Lactobacillus rhamnosus RW‐9595M on inflammatory cytokines produced by macrophages. Methods and Results: The conditioned media (CM) were produced by macrophages treated with parental Lact. rhamnosus ATCC 9595 and its isogenic variant, the high‐EPS producer Lact. rhamnosus RW‐9595M, and the levels of TNF‐α, IL‐6, IL‐10 and IL‐12 were evaluated. Results revealed that CM from parental Lact. rhamnosus induced higher levels of TNF‐α, IL‐6 and IL‐12 but inhibited IL‐10 production, whereas its mucous variant induced low or no TNF‐α and IL‐6. Addition of purified EPS to macrophages treated with parental Lact. rhamnosus decreased the inflammatory cytokines and inhibited the metabolic activity of lymphocytes. The intermediate polysaccharide chains (16–30 units) produced by time‐controlled hydrolysis of EPS increased the IL‐10 produced by macrophages. Conclusions: Polysaccharide chains of EPS induced immunosuppression by the production of macrophagic anti‐inflammatory IL‐10. Significance and impact of the Study: These results indicate that the EPS from Lact. rhamnosus RW‐9595M may be useful as a new immunosuppressive product in dairy food.     

Improved production of live cells of Lactobacillus rhamnosus by continuous cultivation using glucose-yeast extract medium

In this study, the growth kinetics of Lactobacillus rhamnosus and lactic acid production in continuous culture were assessed at a range of dilution rates (0.05 h(-1) to 0.40 h(-1)) using a 2 L stirred tank fermenter with a working volume of 600 ml. Unstructured models, predicated on the Monod and Luedeking-Piret equations, were employed to simulate the growth of the bacterium, glucose consumption, and lactic acid production at different dilution rates in continuous cultures. The maximum specific growth rate of L. rhamnosus, mu-max, was estimated at 0.40 h(-1), and the Monod cell growth saturation constant, Ks, at approximately 0.25 g/L. Maximum cell viability (1.3 x 10(10) CFU/ml) was achieved in the dilution rate range of D = 0.28 h(-1) to 0.35 h(-1). Both maximum viable cell yield and productivity were achieved at D = 0.35 h(-1). The continuous cultivation of L. rhamnosus at D = 0.35 h(-1) resulted in substantial improvements in cell productivity, of 267% (viable cell count) that achieved via batch cultivation.     

Production of a Novel Extracellular Polysaccharide by Lactobacillus sake 0-1 and Characterization of the Polysaccharide

A novel exopolysaccharide (EPS) produced by Lactobacillus sake 0-1 (CBS 532.92) has been isolated and characterized. When the strain was grown on glucose, the produced EPS contained glucose and rhamnose in a molar ratio of 3:2 and the average molecular mass distribution (M(infm)) was determined at 6 x 10(sup6) Da. At a concentration of 1%, the 0-1 EPS had better viscosifying properties than xanthan gum when measured over a range of shear rates from 0 to 300 s(sup-1), while shear-thinning properties were comparable. Rheological data and anion-exchange chromatography suggested the presence of a negatively charged group in the EPS. Physiological parameters for optimal production of EPS were determined in batch fermentation experiments. Maximum EPS production was 1.40 g (middot) liter(sup-1), which was obtained when L. sake 0-1 had been grown anaerobically at 20(deg)C and pH 5.8. When cultured at lower temperatures, the EPS production per gram of biomass increased from 600 mg at 20(deg)C to 700 mg at 10(deg)C but the growth rate in the exponential phase decreased from 0.16 to 0.03 g (middot) liter(sup-1) (middot) h(sup-1). EPS production started in the early growth phase and stopped when the culture reached the stationary phase. Growing the 0-1 strain on different energy sources such as glucose, galactose, mannose, fructose, lactose, and sucrose did not alter the composition of the EPS produced.     

Influence of nutritional and environmental factors on polysaccharide production by Azotobacter vinelandii cultured on 4-hydroxybenzoic acid

The capacity of 4-hydroxybenzoic acid to support exopolysaccharide (EPS) biosynthesis was investigated. Carbon source concentration, nitrogen supplementation, and other nutritional and environmental factors were optimized to obtain maximal EPS recovery. Higher EPS yields were obtained in nitrogen-free media amended with 20–30 mM 4-hydroxybenzoic acid. In general, modifications in inorganic salt concentration did not alter EPS production, except in the case of magnesium ions. Increased levels of this cation were correlated to greater EPS yields. Production was strongly influenced by certain environmental factors. Optimal values of 34°C, 80 rpm and neutral or slightly basic conditions were selected. Under these conditions, more than 25% of the carbon source supplied was converted to EPS and the production was improved about 42% in comparison to that observed in the initial media. Journal of Industrial Microbiology & Biotechnology (2001) 27, 5–10. Received 05 November 2000/ Accepted in revised form 30 April 2001