Effect of preculturing conditions on growth of Lactobacillus rhamnosus on medium containing glucose and citrate

Lactobacillus rhamnosus can metabolize citrate through a citrate inducible transport system. The growth curves of L. rhamnosus on medium containing glucose and citrate was found to be highly dependent on preculturing conditions. It exhibited diauxic growth when precultured on glucose, but demonstrated simultaneous consumption when cultured on citrate. The maximum specific growth rate for cells growing on glucose + citrate was 0.38 h-1, which was higher than the growth rate on individual substrates (0.28 h-1). Simultaneous consumption also yielded higher net flavour compounds, diacetyl and acetoin. Flux analysis indicated that L. rhamnosus requires oxygen for balancing excess NADH through NADH oxidase. The flux analysis provided insights into the metabolic network of L. rhamnosus.     

Diacetyl production and growth of Lactobacillus rhamnosus on multiple substrates

Lactobacillus rhamnosus is a heterolactic acid bacterium, which can be used to produce flavour compounds like diacetyl and acetoin. Various startegies have been applied to improve the growth rate and diacetyl yield. The use of multiple substrates affected growth as well as the yield of diacetyl. Growth on a medium containing glucose demonstrated a diauxic growth profile, with the second phase of growth being on the product, lactic acid. L. rhamnosus also grew on a medium containing citrate. Growth on medium containing glucose+citrate demonstrated simultaneous utilization of carbon sources. L. rhamnosus did not grow in a medium containing acetate and also did not co-metabolize it with glucose. Maximum specific growth rate ( _max) was found to increase in the case of simultaneous utilization of glucose+citrate (0.38 h^–1) as compared to glucose as the sole carbon source (0.28 h^–1). The yields of diacetyl were also found to increase for glucose + pyruvate and glucose + citrate (0.10 and 0.05 g g^–1 of glucose, respectively) as compared to glucose alone (0.01 g g^–1 of glucose). The productivity of diacetyl on medium containing glucose and citrate was double that of a medium containing only citrate, although the yields were comparable.     

鼠李糖乳杆菌-格氏乳杆菌联用对阴道致病菌的抑制作用

目的本文通过提取细菌基因组进行16S rDNA PCR扩增和测序,分析菌株的进化树分支,鉴定一株乳酸菌菌株RD-0060并检测RD-0060与已有菌株RD-0046联用的抑菌能力和细胞粘附能力。方法结合现有菌株RD-0046(格氏乳杆菌,Lactobacillus gasseri),采用牛津杯法研究RD-0060单菌、RD-0060和RD-0046联用抑制致病菌的能力。通过共培养细菌和阴道上皮细胞VK2/E6E7,研究RD-0060单菌和RD-0060/RD-0046二联菌粘附能力。结果 RD-0060为鼠李糖乳杆菌(Lactobacillus rhamnosus),具有抑制阿托波菌、阴道加德纳菌和常见好氧型病菌的功能,对阴道上皮细胞也有较强的粘附能力;RD-0060和RD-0046二联菌的抑菌效果和细胞粘附能力比单菌株更强。结论鼠李糖乳杆菌和格氏乳杆菌联用能显著抑制阴道致病菌生长,并且能够大量粘附阴道细胞,而且两菌株联用有协同效果,具有良好的临床应用和开发前景。     

Survival of freeze-dried Lactobacillus plantarum and Lactobacillus rhamnosus during storage in the presence of protectants

No significant differences were observed in the viability of Lactobacillus plantarum and Lactobacillus rhamnosus cells during freeze-drying in the presence or absence of inositol, sorbitol, fructose, trehalose, monosodium glutamate and propyl gallate. However, survival was higher during storage when drying took place in the presence of these compounds. Sorbitol produced more significant effects than the other compounds toward maintaining viability of freeze-dried L. plantarum and L. rhamnosus.     

Effect of growth time on the surface and adhesion properties of Lactobacillus rhamnosus GG

Aims:  To investigate the changes in the surface properties of Lactobacillus rhamnosus GG during growth, and relate them with the ability of the Lactobacillus cells to adhere to Caco-2 cells.
Methods and Results:  Lactobacillus rhamnosus GG was grown in complex medium, and cell samples taken at four time points and freeze dried. Untreated and trypsin treated freeze dried samples were analysed for their composition using SDS-PAGE analysis and Fourier transform infrared spectroscopy (FTIR), hydrophobicity and zeta potential, and for their ability to adhere to Caco-2 cells. The results suggested that in the case of early exponential phase samples (4 and 8 h), the net surface properties, i.e. hydrophobicity and charge, were determined to a large extent by anionic hydrophilic components, whereas in the case of stationary phase samples (13 and 26 h), hydrophobic proteins seemed to play the biggest role. Considerable differences were also observed between the ability of the different samples to adhere to Caco-2 cells; maximum adhesion was observed for the early stationary phase sample (13 h). The results suggested that the adhesion to Caco-2 cells was influenced by both proteins and non-proteinaceous compounds present on the surface of the Lactobacillus cells.
Conclusion:  The surface properties of Lact. rhamnosus GG changed during growth, which in return affected the ability of the Lactobacillus cells to adhere to Caco-2 cells.
Significance and Impact of the Study:  The levels of adhesion of Lactobacillus cells to Caco-2 cells were influenced by the growth time and reflected changes on the bacterial surface. This study provides critical information on the physicochemical factors that influence bacterial adhesion to intestinal cells.     

The effect of cell surface components on adhesion ability of Lactobacillus rhamnosus

The aim of this study was to analyze the cell envelope components and surface properties of two phenotypes of Lactobacillus rhamnosus isolated from the human gastrointestinal tract. The ability of the bacteria to adhere to human intestinal cells and to aggregate with other bacteria was determined. L. rhamnosus strains E/N and PEN differed with regard to the presence of exopolysaccharides (EPS) and specific surface proteins. Transmission electron microscopy showed differences in the structure of the outer cell surface of the strains tested. Bacterial surface properties were analyzed by Fourier transform infrared spectroscopy, fatty acid methyl esters and hydrophobicity assays. Aggregation capacity and adhesion of the tested strains to the human colon adenocarcinoma cell line HT29 was determined. The results indicated a high adhesion and aggregation ability of L. rhamnosus PEN, which possessed specific surface proteins, had a unique fatty acid content, and did not synthesize EPS. Adherence of L. rhamnosus was dependent on specific interactions and was promoted by surface proteins (42–114 kDa) and specific fatty acids. Polysaccharides likely hindered bacterial adhesion and aggregation by masking protein receptors. This study provides information on the cell envelope constituents of lactobacilli that influence bacterial aggregation and adhesion to intestinal cells. This knowledge will help to understand better their specific contribution in commensal–host interactions and adaptation to this ecological niche.     

Structural analysis of the Lactobacillus rhamnosus strain KL37C exopolysaccharide

The exopolysaccharide from the lactic acid bacterium Lactobacillus rhamnosus strain KL37C isolated from human intestinal flora was prepared by sonication of bacterial cell mass suspended in water followed by centrifugation and cold ethanol precipitation of the supernatant. The polysaccharide material was purified by gel permeation chromatography on an TSK HW-50 column and characterised using chemical and enzymatic methods. On the basis of sugar and methylation analysis and 1H, 13C, 1D and 2D NMR spectroscopy the exopolysaccharide was shown to be composed of the following pentasaccharide repeating unit:-->3)-alpha-D-Glcp-(1-->2)-beta-D-Galf-(1-->6)-alpha-D-Galp-(1-->6)-alpha-D-Glcp-(1-->3)-beta-D-Galf-(1-->     

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%.     

Impact of spray-drying on the pili of Lactobacillus rhamnosus GG

The preservation of the viability of microorganisms in probiotic formulations is the most important parameter ensuring the adequate concentration of live microorganisms at the time of administration. The formulation and processing techniques used to produce these probiotic formulations can influence the preservation of the microbial viability. However, it is also required that the bacteria maintain their key probiotic capacities during processing, formulation and shelf life. In this study, we investigated the impact of spray-drying on different cell wall properties of the model probiotic strain Lactobacillus rhamnosus GG, including its adherence to intestinal epithelial cells. The dltD gene knock-out mutant, L. rhamnosus GG CMPG5540, displaying modified cell wall lipoteichoic acids, showed significantly increased colony-forming units after spray-drying and subsequent storage under standard conditions compared to wild-type L. rhamnosus GG. In contrast, disruption of the biosynthesis of exopolysaccharides or pili expression did not impact survival. However, spray-drying did significantly affect the adherence capacity of L. rhamnosus GG. Scanning electron microscopy confirmed that the pili, key surface factors for adherence to intestinal cells and mucus, were sheared off during the spray-drying process. These data thus highlight that both the functionality and viability of probiotics should be assessed during the spray-drying process and subsequent storage.     

Raman spectroscopic analysis of Lactobacillus rhamnosus GG in response to dehydration reveals DNA conformation changes

Dehydration of bacterial cells elicits cellular stress responses in bacteria. Microencapsulation has been used to protect cells against the environmental stress. In this study, Confocal Raman Spectroscopy was used to examine DNA changes in the chemical composition of non‐encapsulated and microencapsulated Lactobacillus rhamnosus GG and the reversibility of these changes upon freeze drying and rehydration. The viability of cells upon freeze drying was also enumerated using culture methods and membrane integrity was measured using BacLight Live/Dead staining. Raman analyses show changes in the spectral features associated with various biochemical compounds, which are interpreted as the result of detrimental freeze drying effects on the bacterial cells. Specifically, analyses based on Principal Components Analysis (PCA) of Raman spectra, confirm that microencapsulation protects cells from environmental stress. The results also reveal a B‐ to A‐like DNA conformation change in dormant cells that provided insights into the extent of reversibility of this transition upon rehydration. The extent of this reversibility is less in non‐encapsulated than in microencapsulated cells. These findings indicate the potential application of Raman spectroscopy in rapid sensing of microbial dehydration stress responses.

     

Proteomic analysis of lactose-starved Lactobacillus casei during stationary growth phase

Aims:  Starvation stress is a condition that nonstarter lactic acid bacteria (NSLAB) normally encounter. This study was aimed to investigate starvation-induced proteins in Lactobacillus casei during stationary growth phase.
Methods and Results:  The impact of carbohydrate starvation on L. casei GCRL163 was investigated using two different media (a modified de Man, Rogosa and Sharpe broth and a semi-defined medium). Cells were grown in the presence of excess lactose (1%) or starvation (0%) and differences in the patterns of one-dimensional sodum dodecyl sulfate–polyacrylamide gel electrophoresis and two-dimensional electrophoresis of the cytosolic protein fractions were investigated. Differentially regulated proteins were identified by MALDI-TOF/TOF mass spectrometry. Many differentially regulated proteins were enzymes of various metabolic pathways involved in carbohydrate metabolism to yield energy. Differences in protein expression were also observed in the two culture conditions tested in this experiment.
Conclusion:  Numerous glycolytic enzymes were differentially regulated under lactose starvation. The differential expression of these glycolytic enzymes suggests a potential survival strategy under harsh growth conditions (i.e. lactose starvation).
Significance and Impact of the Study:  This paper reports improved understanding of stress responses and survival mechanism of NSLAB under lactose-depleted cheese-ripening condition. This knowledge of how NSLAB bacteria adapt to lactose starvation could be applied to predict the performances of bacteria in other industrial applications.     

The effect of temperature and pH gradients on Lactobacillus rhamnosus gene expression of stress-related genes

In this study, Lactobacillus rhamnosus, a renowned probiotic, was cultivated in fluctuating environment. Base gradients caused by a pH control in an industrial process and temperature gradients caused by uneven heating were simulated with a scale-down method. A pH gradient was created in a plug flow reactor (PFR). Expression of pH stress-related genes (atpA, aldB, cfa, groEL, hrcA and pstS) were studied as a relative gene expression study using ldhD as a reference gene. Expression measurements were carried out with the TRAC method. The responses of groEL, hrcA and atpA genes to temperature and pH changes were observed. The expression of phosphate uptake system-related pstS gene was induced almost linearly in the chemostat cultivation experiments when the base gradient in the PFR was increased. Correlations between the results from gene expression studies and freeze stability or acid stress survival were studied. However, by measuring the expression of these genes, we were not able to predict eventual freeze stability or survival from the acid stress test.     

Functional analysis of D-alanylation of lipoteichoic acid in the probiotic strain Lactobacillus rhamnosus GG

Lipoteichoic acid (LTA) is a macroamphiphile molecule which performs several functions in gram-positive bacteria, such as maintenance of cell wall homeostasis. D-alanylation of LTA requires the proteins encoded by the dlt operon, and this process is directly related to the charge properties of this polymer strongly contributing to its function. The insertional inactivation of dltD of the probiotic strain Lactobacillus rhamnosus GG (ATCC 53103) resulted in the complete absence of D-alanyl esters in the LTA as confirmed by nuclear magnetic resonance analysis. This was reflected in modifications of the bacterial cell surface properties. The dltD strain showed 2.4-fold-increased cell length, a low survival capacity in response to gastric juice challenge, an increased sensitivity to human beta-defensin-2, an increased rate of autolysis, an increased capacity to initiate growth in the presence of an anionic detergent, and a decreased capacity to initiate growth in the presence of cationic peptides compared to wild-type results. However, in vitro experiments revealed no major differences for adhesion to human intestinal epithelial cells, biofilm formation, and immunomodulation. These properties are considered to be important for probiotics. The role of the dlt operon in lactobacilli is discussed in view of these results.     

Kinetic study of the conversion of different substrates to lactic acid using Lactobacillus bulgaricus

Lactic acid fermentation includes several reactions in association with the microorganism growth. A kinetic study was performed of the conversion of multiple substrates to lactic acid using Lactobacillus bulgaricus. Batch experiments were performed to study the effect of different substrates (lactose, glucose, and galactose) on the overall bioreaction rate. During the first hours of fermentation, glucose and galactose accumulated in the medium and the rate of hydrolysis of lactose to glucose and galactose was faster than the convesion of these substrates. Once the microorganism built the necessary enzymes for the substrate conversion to lactic acid, the conversion rate was higher for glucose than for galactose. The inoculum preparation was performed in such a way that healthy young cells were obtained. By using this inoculum, shorter fermentation times with very little lag phase were observed. The consumption patterns of the different substrates converted to lactic acid were studied to determine which substrate controls the overall reaction for lactic acid production. A mathematical model (unstructured Monod type) was developed to describe microorganism growth and lactic acid production. A good fit with a simple equation was obtained. It was found experimentally that the approximate ratio of cell to substrate was 1 to 10, the growth yield coefficient (Y(XS)) was 0.10 g cell/g substrate, the product yield (Y(PS)) was 0.90 g lactic acid/g substrate, and the alpha parameter in the Luedeking-Piret equation was 9. The Monod kinetic parameters were obtained. The saturation constant (K(S)) was 3.36 g/L, and the specific growth rate (microm ) was 1.14 l/h.