Insights into lactic acid bacteria cryoresistance using FTIR microspectroscopy.

Freezing is widely used for bacterial cell preservation. However, resistance to freezing can greatly vary depending on bacterial species or growth conditions. Our study aims at identifying cellular markers of cryoresistance based on the comparison of three lactic acid bacteria (LAB) exhibiting different tolerance to freezing: Carnobacterium maltaromaticum CNCM I-3298, Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842, and Lactobacillus delbrueckii subsp. bulgaricus CFL1. A thorough characterization of their cytoplasmic membrane properties was carried out by measuring their fatty acid composition, membrane fluidity, and lipid phase transition upon cooling from 50 to -50 °C. Vitrification temperatures of the intra- and extra-cellular compartments were also quantified by differential scanning calorimetry. Additionally, the cell biochemical characterization was carried out using a recently developed Fourier transform infrared (FTIR) micro-spectroscopic approach allowing the analysis of live bacteria in an aqueous environment. The multivariate analysis of the FTIR spectra of fresh and thawed cells enabled the discrimination of the three bacteria according to their lipid, protein, and cell wall peptidoglycan components. It also revealed freezing-induced modifications of these three cellular components and an increase in bacteria heterogeneity for the two strains of L. bulgaricus, the freeze-sensitive bacteria. No cellular damage was observed for C. maltaromaticum, the freeze-resistant bacteria. Comparison of the results obtained from the different analytical methods confirmed previously reported cryoresistance markers and suggested new ones, such as changes in the absorbance of specific infrared spectral bands. FTIR microspectroscopy could be used as a rapid and non-invasive technique to evaluate the freeze-sensitivity of LAB.

Diversity of Lactic Acid Bacteria Involved in the Fermentation of Awa-bancha.

The lactic acid bacteria involved in fermentation and components in the tea leaves of Awa-bancha, a post-fermented tea produced in Naka, Kamikatsu, and Miyoshi, Tokushima, were investigated in the present study. Lactic acid bacteria were isolated from tea leaves after anaerobic fermentation and identified by multiplex PCR targeting of the recA gene and 16S ribosomal RNA gene homology. Lactiplantibacillus pentosus was the most frequently isolated species in Naka and Kamikatsu and Lactiplantibacillus plantarum in Miyoshi. In the phylogenetic tree based on the dnaK gene, L. pentosus isolated from Awa-bancha was roughly grouped by the production area and producer. The bacterial flora after anaerobic fermentation was dominated by Lactiplantibacillus spp. for most producers, and the compositions of samples from each producer varied. Organic acids, free amino acids, and catechins were analyzed as components related to the flavor of Awa-bancha. These components were unique to each producer. The present results revealed diversity in the lactic acid bacteria and flavor of Awa-bancha that depended on the producer.

Freeze-Drying of Lactic Acid Bacteria: A Stepwise Approach for Developing a Freeze-Drying Protocol Based on Physical Properties.

Freeze-drying or lyophilization has become a reference process for preserving lactic acid bacteria. The development of stable freeze-dried lactic acid bacteria (LAB) requires maintaining the biological activity of the cells and the macroscopic porous structure while increasing the efficiency of the manufacturing process. Physical properties of protective solutions, such as glass transition and collapse temperatures, are key elements not only for process optimization but also for the stability of freeze-dried LAB. This chapter provides a stepwise approach for developing a protective formulation for the long-term preservation of LAB and an efficient freeze-drying process. Methods for determining glass transition and collapse temperatures of protective solutions and cell suspensions, as well as water activity and water content of freeze-dried products, are described.

Pediococcus acidilactici strains as silage inoculants for improving the fermentation quality, nutritive value and in vitro ruminal digestibility in different forages.

AIMS: To examine the characteristics of three isolated Pediococcus acidilactici strains (LTG7, LOG9 and LH9) and evaluate their effects on silage quality, nutritive value and in vitro ruminal digestibility in a variety of forages. METHODS AND RESULTS: One commercial inoculant Lactobacillus plantarum MTD-1 (G) and three isolated lactic acid bacteria (LAB) strains were measured by morphological, physiological and biochemical tests. All the LAB strains were added to Italian ryegrass (Lolium multiflorum Lam.), tall fescue (Festuca arundinacea Schred.) and oat (Avena sativa L.) for ensiling 30 days in laboratory silos (1 l) respectively. Isolated strains could grow normally at 5-20°C, pH 3·5-7·0 and NaCl (3·0, 6·5%), and were identified as P. acidilactici by sequencing 16S rDNA. In Italian ryegrass and oat silages, all inoculants obviously (P < 0·05) increased lactic acid (LA) contents, LAB numbers and in vitro dry matter digestibility (IVDMD), and decreased pH, undesirable micro-organism numbers, butyric acid and ammonia nitrogen (NH3 -N) contents compared with the corresponding controls. LTG7, LOG9 and G silages in Italian ryegrass and oat had markedly (P < 0·05) higher LA content and IVDMD, and lower pH and NH3 -N contents than LH9 silages. In tall fescue silage, LAB inoculants had no obvious (P > 0·05) effect on fermentation quality, while markedly (P < 0·05) enhanced IVDMD. CONCLUSIONS: Based on our results, strains LTG7 and LOG9 had similar potential with the commercial inoculant G in silage making. SIGNIFICANCE AND IMPACT OF THE STUDY: Few studies involved inoculation of silage with P. acidilactici in different forage types. Analysis of effects of LAB strains with their physiological and biochemical characteristics help understand how LAB inoculants affect the digestibility.

Antifungal effect of organic acids from lactic acid bacteria on Penicillium nordicum.

The control of fungal contamination is particularly important to avoid both spoilage of food and feed products and the occurrence of toxic compounds, known as mycotoxins. Some lactic acid bacteria (LAB) strains have shown the capacity to inhibit fungal growth and the production of mycotoxins. In this work, cell-free supernatants (CFS) of Lactobacillus plantarum UM55 and Lactobacillus buchneri UTAD104 were tested against Penicillium nordicum radial growth and OTA production. When CFS of these strains were used, the radial growth of the fungus was inhibited by less than 20%, but the production of OTA was reduced by approx. 60%. These antifungal effects resulted from organic acids produced by LAB. The CFS of L. plantarum UM55 contained lactic acid, phenyllactic acid (PLA), hydroxyphenyllactic acid (OH-PLA) and indole lactic acid (ILA), while L. buchneri UTAD104 CFS contained acetic acid, lactic acid and PLA. These organic acids were further tested individually for their inhibitory capacity. Calculation of the inhibitory concentrations (ICs) showed that acetic acid, ILA and PLA were the most effective in inhibiting P. nordicum growth and OTA production. When the inhibitory activity of LAB cells incorporated into the culture medium was tested, L. buchneri UTAD104 inhibited the production of OTA entirely in all conditions tested, but fungal growth was only inhibited completely by the highest concentrations of cells. Acetic acid production was primarily responsible for this effect. In conclusion, the ability of LAB to inhibit mycotoxigenic fungi depends on strain capability to produce specific organic acids, and those acids may differ from strain to strain. Also, the use of LAB cells, especially from L. buchneri, in food products prone to contamination with P. nordicum (e.g. dry-cured meats and cheeses) may be an alternative solution to control fungal growth and OTA production.

Immuno-electron microscopy of primary cell cultures from genetically modified animals in liquid by atmospheric scanning electron microscopy.

High-throughput immuno-electron microscopy is required to capture the protein-protein interactions realizing physiological functions. Atmospheric scanning electron microscopy (ASEM) allows in situ correlative light and electron microscopy of samples in liquid in an open atmospheric environment. Cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required. Here, cells were imaged by optical or fluorescence microscopy, and at high resolution by gold-labeled immuno-ASEM, sometimes with additional metal staining. Axonal partitioning of neurons was correlated with specific cytoskeletal structures, including microtubules, using primary-culture neurons from wild type Drosophila, and the involvement of ankyrin in the formation of the intra-axonal segmentation boundary was studied using neurons from an ankyrin-deficient mutant. Rubella virus replication producing anti-double-stranded RNA was captured at the host cell's plasma membrane. Fas receptosome formation was associated with clathrin internalization near the surface of primitive endoderm cells. Positively charged Nanogold clearly revealed the cell outlines of primitive endoderm cells, and the cell division of lactic acid bacteria. Based on these experiments, ASEM promises to allow the study of protein interactions in various complexes in a natural environment of aqueous liquid in the near future.