Changes in membrane fatty acids of Lactobacillus helveticus during vacuum drying with sorbitol

Aims:  To examine changes in membrane fatty acid profile attributed to the physiological adaptation of Lactobacillus helveticus during vacuum drying.
Methods and Results:  The viability and membrane integrity of the cells after vacuum drying were measured by plate counts and DNA fluorescence dyes. The physiological adaptation of cells dried in the presence of sorbitol was observed by determining changes in membrane fatty acid composition using gas chromatography. Results showed that viability and membrane integrity of Lact. helveticus cells increased when drying in the presence of sorbitol. The occurrence of the very low melting point polyunsaturated fatty acids linoleic and arachidonic acid was observed in cells dried in the presence of sorbitol.
Conclusions:  The physiological adaptation of cells occurred with cell membrane of Lact. helveticus during vacuum drying of cells in the presence of sorbitol.
Significance and Impact of the Study:  The study showed that physiological adaptation with membrane of the cells occurred during the drying process. The insight implies that instead of viability improvement of dried cells by the conventional stress induction during cultivation, the induction may be exercised thereafter without compromising growth of the cells.     

Damage of cell envelope of Lactobacillus helveticus during vacuum drying

AIMS: The aim of this study was to gain insight into the inactivation mechanisms of Lactobacillus helveticus during vacuum drying. METHODS AND RESULTS: Early stationary phase cells of L. helveticus were dried in a vacuum drier. Viability, cell integrity and metabolic activity of cells were assessed over time by plate counts on de Man Rogosa and Sharpe broth agar medium and cytological methods employing fluorescent reagents and nucleic acid stains. The cell envelope damage was visualized by atomic force microscopy (AFM). Fourier transform infrared spectroscopy (FT-IR) was used to indirectly observe changes in cell components during drying. Viability, metabolic activity and cell integrity decreased during vacuum drying, and different inactivation curves, characterized by the loss of ability to resume growth, and cell injuries were found. AFM images showed cracks on the surface of dried cells. Main changes in FT-IR spectra were attributed to the damage in cell envelope. CONCLUSION: The cell envelope was the main site of damage in L. helveticus during vacuum drying. SIGNIFICANCE AND IMPACT OF THE STUDY: Inactivation mechanisms of L. helveticus during vacuum drying were partly elucidated. This information is useful for the improvement of the viability of vacuum-dried starter cultures.     

Inactivation mechanisms of lactic acid starter cultures preserved by drying processes

The preservation of lactic acid starter cultures by drying are of increased interest. A further improvement of cell viability is, however, still needed, and the insight into inactivation mechanisms of the cells is a prerequisite. In this present work, we review the inactivation mechanisms of lactic acid starter cultures during drying which are not yet completely understood. Inactivation is not only induced by dehydration inactivation but also by thermal- and cryo-injuries depending on the drying processes employed. The cell membrane has been reported as a major site of damage during drying or rehydration where transitions of membrane phases occur. Some drying processes, such as freeze drying or spray drying, involve subzero or very high temperatures. These physical conditions pose additional stresses to cells during the drying processes. Injuries of cells subjected to freezing temperatures may be due to the high electrolyte concentration (solution effect) or intracellular ice formation, depending on the cooling rate. High temperatures affect most essential cellular components. It is difficult to identify a critical component, although ribosomal functionality is speculated as the primary reason. The activation during storage is mainly due to membrane lipid oxidation, while the storage conditions such as temperature moisture content of the dried starter cultures are important factors.     

l-glutamate transport in Lactobacillus helveticus

An energy source (glucose or lactose) was required for the transport of l-glutamic acid by Lactobacillus helveticus. Mg²⁺, K⁺ and Li⁺ increased its accumulation while Ca²⁺ and Na⁺ decreased it. It was inhibited by NaF, indicating that ATP may be involved in uptake. Optimum transport was at pH 7.3 and 45°C. l-Glutamic acid transport showed a high degree of stereospecificity, as neither d-glutamate nor d-aspartate were active. Proton-conducting uncouplers, like carbonyl cyanide-m-chlorophenylhydrazone, and ionophores (nigericin, monensin and gramicidin) were strongly inhibitory. These results indicate that a proton motive force may be involved in the transport of l-glutamic acid.The authors are with the Centro de Referencia para Lactobacilos, Chacabuco 145 4000 S.M. de Tucumán, Argentina and the Cátedra de Microbiologia Superior, Universidad Nacional de Tucumán, Argentina.     

Influence of yeast extract concentrationon batch cultures of Lactobacillus helveticus: growth and production coupling

Lactobacillus helveticus was cultivated batchwise on whey permeate supplemented with 2–30g yeast extract l^-1. For low supplementation levels, growth was slow, and a long maintenance phase in the absence of any cell autolysis was observed. When the yeast extract concentration was increased, growth time increased slightly; under these circumstances, a large increase of maximum biomass concentration was recorded, indicating a clear nitrogen source starvation at low supplement concentration. At the same time the length of the maintenance phase decreased; for a 30gl^-1 supplementation, it vanished and a death phase was observed instead: this behaviour highlighted a shift from nitrogen to carbon source starvation. For more and more supplemented media, the final lactic acid concentration was constant, while fermentation time decreased significantly; a growth-associated production phase was followed by a maintenance production phase. The part played by the former mecha nism was under tight control of yeast extract concentration: for 30gl^-1 supplementation, 83% of total lactic acid was provided by a growth-linked production process.     

1H NMR investigation on the role of sorbitol for the survival of Lactobacillus paracasei ssp. paracasei in vacuum‐dried preparations

Aims: The aim of this work was to study the effect of sorbitol as protective agent on the survival of the probiotic strain Lactobacillus paracasei ssp. paracasei (F19) after vacuum drying. Methods and Results: The survival was studied after different drying times and for various concentrations of sorbitol by plate count method. Furthermore, time domain ¹H NMR studies on dehydrated suspensions of Lact. paracasei ssp. paracasei were performed to study the proton mobility in the dried samples. From the obtained signal, T2 relaxation times of single components and fractions with different proton mobility were determined. It was found out that the survival is increased by the presence of a minimum amount of sorbitol that is dependent on drying time. Furthermore, it is shown that the protective effect can only be observed below a critical water content of c. 20%. Nuclear magnetic resonance (NMR) results indicate a transition of sorbitol from liquid to solid like behaviour during drying. The onset of the transition coincides with the critical water content found for a protective effect. Conclusions: The data suggest that sorbitol protons are incorporated into the dried cells of Lact. paracasei ssp. paracasei (F19) below the critical water content and therefore leading to an enhanced survival. Significance and Impact of the Study: The results help to better understand the underlying mechanism of protection of Lact. paracasei ssp. paracasei using sorbitol and to establish vacuum drying as potential alternative drying technique to standard freeze drying.     

Growth and exopolysaccharide production by Lactobacillus helveticus ATCC 15807 in an adenine-supplemented chemically defined medium

AIMS: To analyse the exopolysaccharide (EPS) production by Lactobacillus helveticus ATCC 15807 in a chemically defined medium (CDM) and the effect of nutrients and stress culture conditions on cell growth and EPS formation. METHODS AND RESULTS: Cultures were conducted in CDM: (i) containing essential and nonessential bases and vitamins; (ii) without nonessential bases and vitamins [Simplified CDM (SCDM)]; (iii) SCDM supplemented individually with vitamins and bases. The influence of carbohydrates, pH and osmotic culture conditions on growth and polymer formation was analysed. Adenine and lactose stimulated both growth and EPS production. Constant pH fermentations (4.5 and 6.2) did not improve EPS synthesis while NaCl and glycerol were detrimental for growth and polymer formation. In all media the EPS monomer composition was glucose and galactose (2.5 : 1). CONCLUSIONS: A SCDM containing adenine and lactose was optimal for cell growth and EPS formation by Lact. helveticus ATCC 15807. Controlled pH (6.2 and 4.5) and osmotic stress culture conditions did not improve polymer production. The EPS characteristics were identical in all media. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides a better knowledge on EPS synthesis by Lact. helveticus. A CDM to perform regulation studies on EPS production by Lact. helveticus species is now available.     

L-乳酸细菌培养条件的优化

实验通过对L-乳酸细菌(Lactobacillus casei FH-2)菌体生长、产酸速率、耐酸水平、高糖发酵性能的优化,实现了优化该菌生长和条件的目的。并对培养基中不同辅料的添加进行了研究。结果证明：共同加入麦根一麸皮浸泡液时,可以提高乳酸的产量,其最佳方案为50℃、4h、1％麸皮浸泡液和2％麦根。乳酸产量为40．5g/L。结论：实验通过优化L-乳酸细菌的发酵条件提高了乳酸的产量。     

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.     

Influence of carbohydrates on the isolation of lactic acid bacteria

Aims: To determine the influence of carbohydrates on enrichment isolation of lactic acid bacteria from different niches. Methods and Results: Lactic acid bacteria in three traditional fermented products in southern Africa (amasi, mahewu and tshwala) and in three fresh samples (two flowers and a fruit) were enrichment cultured in media supplemented with 13 different carbohydrates. Diversity of lactic acid bacteria was determined by PCR‐denaturing‐gradient gel electrophoresis. Carbohydrates used in enrichment media had a big impact on the isolation of lactic acid bacteria from fermented products. Depending on the carbohydrates tested, the number of species detected ranged from one to four in amasi, one to five in mahewu and one to three in tshwala. Fructose and mannitol selected for relatively higher numbers of lactic acid bacteria in fermented products. Specific relationships between substrates and lactic acid bacteria have been noted. On the other hand, small influences were found among carbohydrates tested in flowers and fruit. Conclusion: Carbohydrates have a big impact on the isolation of a variety of lactic acid bacteria in fermented food. Significance and Impact of the Study: This is the first study that reports the influence of carbohydrates on the enrichment of lactic acid bacteria.     

Effect of protective agents, freezing temperature, rehydration media on viability of malolactic bacteria subjected to freeze-drying

AIMS: The effects of protective agents, rehydration media and freezing temperature on the viabilities of Lactobacillus brevis and Oenococcus oeni H-2 when subjected to freeze-drying were investigated. METHODS AND RESULTS: Several protectants and rehydration media were tested to improve the survival after freeze-drying. The cells were also frozen at -65 and -20 degrees C to check the effect of freezing temperature on the viability. CONCLUSIONS: The best protectant and rehydration medium to obtain the highest viability after freeze-drying varied with the species of bacteria. Yeast extract (4.0%) and sodium glutamate (2.5% ) gave maximum viability of L. brevis and O. oeni (67.8% and 53.6% respectively). The highest survival of L. brevis and O. oeni were obtained when rehydrated with 10% sucrose and MGY medium respectively. When the bacterial cells were frozen quickly (-65 degrees C) than slowly (-20 degrees C), L. brevis and O. oeni both showed increased viability after freeze-drying. SIGNIFICANCE AND IMPACT OF THE STUDY: The viabilities of L. brevis and O. oeni after freeze-drying were shown to be strain specific and dependent on protective agents, rehydration media and freezing temperature.     

Improved batch culture of Lactobacillus helveticus with reused cells reactivated at acidic pH: the minimal volume of reused culture fraction

Three modes of batch culture of Lactobacillus helveticus were compared: the conventional culture inoculated with 11% (v/v) seed culture and cultures inoculated with 0.1 or 11% (v/v) of a volume fraction of the preceding culture, reactivated first at acidic pH. Culture reuse was useful with low nitrogen supplementation of culture medium (5 g yeast extract l^–1). Even when the volume of reused culture fraction was minimal (0.1% v/v), a 14% increase in the mean production rate was observed, compared to the conventional mode of batch culture.     

Influence of lyophilization, fluidized bed drying, addition of protectants, and storage on the viability of lactic acid bacteria

Aims:  The present study focuses on the impact of two different drying technologies and the influence of protectants on process survival and storage stability of the two lactic acid bacterial strains Enterococcus faecium and Lactobacillus plantarum .
Methods and Results:  After incubation with the protectants glucose, sucrose, trehalose, and maltodextrin the concentrated bacterial suspensions were subjected to fluidized bed drying and lyophilization and subsequently stored at 4, 22, and 35°C for half a year. Lactobacillus plantarum turned out to be more sensitive to both drying methods than Ent. faecium . Without the addition of a protectant cells of both strains suffered higher losses during fluidized bed drying. Elevated storage temperatures correlate with a higher decline of viable bacterial cells.
Conclusions:  Although survival rates varied between the strains, the nonreducing disaccharides revealed overall best protection for both investigated lactic acid bacteria during processing and storage. The addition of protective carbohydrates can prevent the decline in viability during fluidized bed drying.
Significance and Impact of the Study:  The influence of protectants proved to be species specific and therefore needs to be determined on a case-to-case basis. Survival rates, duration, and energy consumption appear to be the crucial parameters to evaluate the economy of production processes for industrial starter cultures.     

Effects of copper supplement on growth and viability of strains used as starters and adjunct cultures for Emmental cheese manufacture

Aims: To determine the effects of supplemented copper (Cu²⁺) on growth and viability of strains used as starters and adjunct cultures for Emmental cheese manufacture. Methods and Results: Thirteen strains belonging to Lactobacillus delbrueckii, Lactobacillus helveticus, Lactobacillus rhamnosus, Streptococcus thermophilus or Propionibacterium freudenreichii species were exposed to various copper concentrations in the proper growth medium at relevant growth temperatures, and the effects of supplemented copper on bacterial growth and cell viability were determined by optical density and pH measurements, also by platings. Among the species considered, L. delbrueckii was the most copper resistant and S. thermophilus the most sensitive to copper. Anaerobic conditions increased this sensitivity significantly. There was also a considerable amount of variation in copper resistance at strain level. Conclusions: Copper resistance is both a species- and strain-dependent property and may reflect variability in copper-binding capacities by cell wall components among species and strains. In addition, the chemical state of copper may be involved. Significance and Impact of the Study: This study revealed that copper resistance is a highly variable property among starter and adjunct strains, and this variability should be considered when strains are selected for Emmental cheese manufacture.     

植物乳杆菌多孔淀粉直接包埋工艺的研究

利用多孔淀粉的吸附特性,将植物乳杆菌包埋于多孔淀粉内,通过测定多孔淀粉对植物乳杆菌的包埋率,研究菌体浓度、多孔淀粉添加量、振荡转速、包埋温度、pH值、时间对包埋率的影响,确定最适包埋条件。结果表明:菌体浓度10~8cfu/mL,多孔淀粉添加量为2%,pH 6.0、20℃,200 r/min振荡处理40 min,在此条件下包埋率为79.5%,对包埋后的菌体进行喷雾干燥试验,其存活率较未包埋的从0.35%提高到29.5%。     

Random amplified polymorphic DNA-PCR based cloning of markers to identify the beer-spoilage strains of Lactobacillus brevis, Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis

AIMS: Beer-spoilage ability of lactic acid bacteria such as Lactobacillus brevis is a strain-dependent phenomenon in which the mechanism has not yet been completely clarified. In order to systematically identify genes that contribute to beer-spoilage, large-scale random amplified polymorphic DNA (RAPD)-based cloning methods was carried out. METHODS AND RESULTS: A systematic RAPD polymerase chain reaction (PCR) analysis using 600 primers was performed on beer-spoilage and on nonspoilage strains of L. brevis. Among 600 primers, three were found to amplify a single locus highly specific to beer-spoilage strains. DNA sequencing of this locus revealed a three-part operon encoding a putative glycosyl transferase, membrane protein and teichoic acid glycosylation protein. PCR analysis of typical beer-spoilage lactic acid bacteria suggested that this locus is highly specific to beer-spoilage strains. CONCLUSION: The cloned markers are highly specific to identify the beer-spoilage strains not only in L. brevis but also in Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper proves that RAPD-PCR is an efficient method for cloning the strain-specific genes from bacteria. The markers described here is one of the most useful tools to identify the beer-spoilage strains of lactic acid bacteria.     

Preliminary characterization of lactic acid bacteria isolated from Zlatar cheese

AIMS: Isolation, characterization and identification of lactic acid bacteria (LAB) from artisanal Zlatar cheese during the ripening process and selection of strains with good technological characteristics. METHODS AND RESULTS: Characterization of LAB was performed based on morphological, physiological and biochemical assays, as well as, by determining proteolytic activity and plasmid profile. rep-polymerase chain reaction (PCR) analysis and 16S rDNA sequencing were used for the identification of LAB. PCR analysis was performed with specific primers for detection of the gene encoding nisin production. Strains Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcus faecium and Enterococcus faecalis were the main groups present in the Zlatar cheese during ripening. CONCLUSIONS: Temporal changes in the species were observed during the Zlatar cheese ripening. Mesophilic lactobacilli are predominant microflora in Zlatar cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study we determined that Zlatar cheese up to 30 days old could be used as a source of strains for the preparation of potential starter cultures in the process of industrial cheese production. As the Serbian food market is adjusting to European Union regulations, the standardization of Zlatar cheese production by using starter culture(s) based on autochtonous well-characterized LAB will enable the industrial production of this popular cheese in the future.