Culture conditions affect Lactobacillus reuteri DSM 17938 ability to perform glycerol bioconversion into 3-hydroxypropionic acid.

The platform molecule 3-hydroxypropionic acid (3-HP) can be produced using Lactobacillus reuteri through a two-step bioprocess that involves a growth phase followed by a bioconversion phase. The bioproduction is performed by resting cells that convert glycerol into 3-HP and 1,3-propanediol in fed-batch mode. This work aimed at studying the effect of the growth conditions of L. reuteri DSM 17938 during the first step, on the glycerol bioconversion into 3-HP during the second step. A Plackett and Burman design was carried out to test, in controlled bioreactors, the effect of 11 growth conditions simultaneously, at fixed bioconversion conditions. The supplementation of the growth medium with vitamin B12 and cysteine displayed a negative effect on the 3-HP bioproduction. The addition of glucose, phytone peptone, Tween 80, 1,2-propanediol and betaine in the growth medium, together with a low temperature and an optimal pH of 6.0 during the growth phase increased the bioconversion duration from 56 h to 89 h at a glycerol feeding rate of 0.5 g·h-1. A validating experiment displayed that the 3-HP titer, 3-HP production yield and 3-HP specific production rate were significantly improved by 25 %, 150 % and 61 %, respectively.

Effect of dairy matrices on the survival of Streptococcus thermophilus, Brevibacterium aurantiacum and Hafnia alvei during digestion.

This study evaluated the ability of dairy matrices, different in composition (with and without fat) and structure (liquid and gel), to enhance microorganisms survival through digestion. The viability of three dairy microorganisms Streptococcus thermophilus, Brevibacterium aurantiacum and Hafnia alvei was measured during in vitro and in vivo digestion. S. thermophilus was highly sensitive to gastric stress, and was not found in the duodenal compartment. B. auranticum was moderately sensitive to gastric stress but resistant to duodenal stress. H. alvei was highly resistant to both stresses. LIVE/DEAD confocal microscopy's images, probed the effect of low pH on microorganisms survival. However, in vivo analyses (16S rRNA gene metabarcoding) failed to confirm in vitro observations since tested microorganisms were not detected. Despite of the different evolutions during digestion on buffer capacity, lipolysis, and rheological characteristics, we did not observe any protective effect of the dairy matrices on microorganisms survival.

Starvation induces physiological changes that act on the cryotolerance of Lactobacillus acidophilus RD758.

The relationship between lactose starvation and cryotolerance was investigated in Lactobacillus acidophilus RD758. Cryotolerance was measured from the acidification activity of cells recovered after 18-h lactose starvation. It was compared to that of nonstarved cells, both of them in a stationary phase and in the same medium. This measurement allowed quantifying the initial acidification activity before freezing, as well as the loss of acidification activity during freezing and the rate of loss during frozen storage. Even if initial acidification activity was similar for nonstarved and starved bacteria, the latter displayed a significantly better resistance to freezing and frozen storage at -20°C. To investigate the mechanisms that triggered these cryotolerance phenomena, the membrane fatty acid composition was determined by gas chromatography, and the proteome was established by 2-D electrophoresis, for starved and nonstarved cells. The main outcome was that the improved cryotolerance of starved cells was ascribed to two types of physiological responses as a result of starvation. The first one corresponded to an increased synthesis of unsaturated, cyclic, and branched fatty acids, to the detriment of saturated fatty acids, thus corresponding to enhanced membrane fluidity. The second response concerned the upregulation of proteins involved in carbohydrate and energy metabolisms and in pH homeostasis, allowing the cells to be better prepared for counteracting the stress they encountered during subsequent cold stress. These two phenomena led to a cross-protection phenomenon, which allowed better cryotolerance of Lb. acidophilus RD758, following cellular adaptation by starvation.

A proteomic and transcriptomic view of amino acids catabolism in the yeast Yarrowia lipolytica.

The yeast Yarrowia lipolytica has to develop dynamic metabolic adaptation mechanisms to survive within the cheese habitat. The availability of amino acids (AAs) is of major importance for microbial development and/or aroma production during cheese ripening. Using 2-D protein gel electrophoresis, we analyzed the adaptation mechanisms of Y. lipolytica for AAs limitation or supplementation in a batch culture containing lactate as a carbon source. Proteome analyses allow the identification of 34 differentially expressed proteins between the culture conditions. These analyses demonstrated that prior to the AAs addition, mainly proteins involved in the oxidative stress of the yeast were induced. Following the AAs addition, yeast cells reorganize their metabolism toward AAs catabolism and also generate a higher induction of proteins related to carbon metabolism and proteins biosynthesis. Using real-time reverse transcription PCR, we re-evaluated the expression of genes encoding proteins involved in these processes. The expression levels of the genes were in accordance with the proteomic results, with the up-regulation of genes encoding a branched-chain amino transferase BAT2, a pyruvate decarboxylase PDC6 and an Hsp70 protein SSZ1 involved in protein biosynthesis. A volatile compound analysis was also performed, and increased production of dimethyldisulfide from methionine and 3-methyl-butanal from leucine was observed in media supplemented with AAs.

Influence of cooling temperature and duration on cold adaptation of Lactobacillus acidophilus RD758.

The effect of different cooling temperatures and durations on resistance to freezing and to frozen storage at -20 degrees C in Lactobacillus acidophilus RD758 was studied, by using a central composite rotatable design. A cold adaptation was observed when the cells were maintained at moderate temperature (26 degrees C) for a long time (8h) before being cooled to the final temperature of 15 degrees C. These conditions led to a low rate of loss in acidification activity during frozen storage (0.64 minday(-1)) and a high residual acidification activity after 180 days of frozen storage (1011 min). The experimental design allowed us to determine optimal cooling conditions, which were established at 28 degrees C during 8h. Adaptation to cold temperatures was related to an increase in the unsaturated to saturated fatty acid ratio and in the relative cycC19:0 fatty acid concentration. Moreover, an increased synthesis of four specific proteins was observed as an adaptive response to the optimal cooling conditions. They included the stress protein ATP-dependent ClpP and two cold induced proteins: pyruvate kinase and a putative glycoprotein endopeptidase.

Arthrobacter bergerei sp. nov. and Arthrobacter arilaitensis sp. nov., novel coryneform species isolated from the surfaces of cheeses.

Fourteen isolates of two different bacterial species isolated from the surface of smear-ripened cheeses were found to exhibit many characteristics of the genus Arthrobacter. The isolates were aerobic, Gram-positive, catalase-positive, non-spore-forming and non-motile. The cell-wall peptidoglycan contained lysine, alanine and glutamic acid. rrs sequence analysis indicated that the new isolates Re117T and Ca106T are closely related to the Arthrobacter nicotianae group and showed highest sequence similarity (>98 %) to Arthrobacter nicotianae and Arthrobacter protophormiae. However, DNA-DNA hybridization studies indicated that the strains represented two novel genomic species within the genus Arthrobacter and did not belong to A. nicotianae or A. protophormiae (<43 % DNA-DNA relatedness). On the basis of the phylogenetic and phenotypic distinctiveness of the new isolates, these bacteria should be classified as two novel Arthrobacter species, for which the names Arthrobacter bergerei sp. nov. and Arthrobacter arilaitensis sp. nov. are proposed. Type strains have been deposited in culture collections as Arthrobacter bergerei Ca106T (=CIP 108036T=DSM 16367T) and Arthrobacter arilaitensis Re117T (=CIP 108037T=DSM 16368T).

Identification and functional analysis of the gene encoding methionine-gamma-lyase in Brevibacterium linens.

The enzymatic degradation of L-methionine and subsequent formation of volatile sulfur compounds (VSCs) is believed to be essential for flavor development in cheese. L-methionine-gamma-lyase (MGL) can convert L-methionine to methanethiol (MTL), alpha-ketobutyrate, and ammonia. The mgl gene encoding MGL was cloned from the type strain Brevibacterium linens ATCC 9175 known to produce copious amounts of MTL and related VSCs. The disruption of the mgl gene, achieved in strain ATCC 9175, resulted in a 62% decrease in thiol-producing activity and a 97% decrease in total VSC production in the knockout strain. Our work shows that L-methionine degradation via gamma-elimination is a key step in the formation of VSCs in B. linens.