The effect of reduced sodium chloride content on the microbiological and biochemical properties of a soft surface-ripened cheese.

Many health authorities have targeted salt reduction in food products as a means to reduce dietary sodium intake due to the harmful effects associated with its excessive consumption. In the present work, we evaluated the effect of reducing sodium chloride (NaCl) content on the microbiological and biochemical characteristics of an experimental surface-ripened cheese. A control cheese (1.8% NaCl) and a cheese with a reduced NaCl content (1.3% NaCl) were sampled weekly over a period of 27d. Reducing NaCl content induced microbial perturbations such as the lesser development of the yeast Debaryomyces hansenii and the greater development of the gram-negative bacterium Hafnia alvei. This was accompanied by changes in proteolytic kinetics and in profiles of volatile aroma compounds and biogenic amine production. Finally, the development of the spoilage microorganism Pseudomonas fragi was significantly higher in the cheese with a reduced salt content.

Behavior of Escherichia coli O26:H11 in the presence of Hafnia alvei in a model cheese ecosystem.

This study was designed to evaluate the capacity of three Hafnia strains to inhibit the growth of an E. coli strain O26:H11 in an uncooked pressed model cheese, in the presence or absence of a microbial consortium added to mimic a cheese microbial community. Inoculated at 2 log CFU/ml into pasteurized milk without Hafnia, the E. coli O26:H11 strain reached 5 log CFU/g during cheese-making and survived at levels of 4 to 5 log CFU/g beyond 40 days. Inoculated into milk at 6 log CFU/ml, all three tested Hafnia strains (H. alvei B16 and HA, H. paralvei 920) reached values close to 8 log CFU/g and reduced E. coli O26:H11 counts in cheese on day 1 by 0.8 to 1.4 log CFU/g compared to cheeses inoculated with E. coli O26:H11 and the microbial consortium only. The Hafnia strains slightly reduced counts of Enterococcus faecalis (~-0.5 log from day 1) and promoted Lactobacillus plantarum growth (+0.2 to 0.5 log from day 8) in cheese. They produced small amounts of putrescine (~1.3 mmol/kg) and cadaverine (~0.9 mmol/kg) in cheese after 28 days, and did not affect levels of volatile aroma compounds. Further work on H. alvei strain B16 showed that E. coli O26:H11, inoculated at 2 log CFU/ml, was inhibited by H. alvei B16 inoculated at 6 log CFU/ml and not at 4.5 log CFU/ml. The inhibition was associated neither with lower pH values in cheese after 6 or 24h, nor with higher concentrations of lactic acid. Enhanced concentrations of acetic acid on day 1 in cheese inoculated with H. alvei B16 (4 to 11 mmol/kg) could not fully explain the reduction in E. coli O26:H11 growth. A synergistic interaction between H. alvei B16 and the microbial consortium, resulting in an additional 0.7-log reduction in E. coli O26:H11 counts, was observed from day 8 in model cheeses made from pasteurized milk. However, E. coli O26:H11 survived better during ripening in model cheeses made from raw milk than in those made from pasteurized milk, but this was not associated with an increase in pH values. In vitro approaches are required to investigate the mechanisms and causative agents of this interaction. H. alvei B16 appears to be a promising strain for reducing E. coli O26:H11 growth in cheese, as part of a multi-hurdle approach.

Effects of Proteus vulgaris growth on the establishment of a cheese microbial community and on the production of volatile aroma compounds in a model cheese.

AIMS: To investigate the impact of Proteus vulgaris growth on a multispecies ecosystem and on volatile aroma compound production during cheese ripening. METHODS AND RESULTS: The microbial community dynamics and the production of volatile aroma compounds of a nine-species cheese ecosystem were compared with or without the presence of P. vulgaris in the initial inoculum. Proteus vulgaris was able to colonize the cheese surface and it was one of the dominant species, representing 37% of total isolates at the end of ripening with counts of 9.2 log(10) CFU g(-1). In the presence of P. vulgaris, counts of Arthrobacter arilaitensis, Brevibacterium aurantiacum and Hafnia alvei significantly decreased. Proteus vulgaris influenced the production of total volatile aroma compounds with branched-chain aldehydes and their corresponding alcohols being most abundant. CONCLUSIONS: Proteus vulgaris was able to successfully implant itself in a complex cheese ecosystem and significantly contributed to the organoleptic properties of cheese during ripening. This bacterium also interacted negatively with other bacteria in the ecosystem studied. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that the impact of a Gram-negative bacterium on cheese microbial ecology and functionality has been described.

Charged casein-derived oligopeptides competitively inhibit the transport of a reporter oligopeptide by Lactococcus lactis.

AIM: To study the effect of casein-derived peptides, accumulated during growth of Lactococcus lactis in milk, on its oligopeptide transport (Opp) function. METHODS AND RESULTS: This effect was estimated by analysing the ability of casein-derived peptides to compete for the transport of a reporter peptide by whole L. lactis cells. The transport of the reported peptide was monitored by determining the intracellular concentrations of the corresponding amino acids by means of reverse-phase high-performance liquid chromatography (HPLC). Uptake of the reporter peptide was competitively inhibited by casein-derived peptides. The competition was only because of charged casein-derived peptides, including anionic peptides. The design of specific pure peptides made it possible to evidence for a positive (or negative) influence exerted by the positively (or negatively) charged side chain of the N-terminal amino acid on the competition. CONCLUSIONS: Charged casein-derived peptides impaired the oligopeptide transport function of L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate an inhibition of Opp when too many peptides are produced by the proteinase. Peptide transport by Opp therefore represents a bottleneck for increasing the growth rate of L. lactis in milk.

Sulfur compound production by Geotrichum candidum from L-methionine: importance of the transamination step.

L-methionine degradation products and catabolic enzymatic activities involved in methanethiol generation were investigated in Geotrichum candidum GcG. L-methionine was easily degraded by G. candidum and the transamination product, 4-methylthio-2-oxobutyric acid (KMBA), was found to transiently accumulate. In parallel, considerable L-methionine aminotransferase activity was found in this microorganism. L-methionine and KMBA demethiolating activities were also detected. The degradation of KMBA corresponded to an overall increase in the production of volatile sulfur compounds. These results show that the transamination pathway is of major importance in the initial breakdown of L-methionine by this cheese-ripening microorganism.

Enzymatic versus spontaneous S-methyl thioester synthesis in Geotrichum candidum.

The synthesis of short chain S-methyl thioesters was investigated in Geotrichum candidum strain GcG. The results indicated the involvement of an enzymatic reaction in this microorganism that led to the synthesis of S-methyl thioacetate (MTA) when methanethiol and acetyl-CoA were used as substrates. MTA was generated from these substrates by enzymatic or spontaneous reactions, whose relative importance depended largely on pH and temperature. For longer chain acyl-CoA compounds (C3 to C6), thioester synthesis was primarily spontaneous. Short chain fatty acid activation by a CoA residue probably is a prerequisite for the synthesis of S-methyl thioesters.

The contribution of caseins to the amino acid supply for Lactococcus lactis depends on the type of cell envelope proteinase.

The ability of caseins to fulfill the amino acid requirements of Lactococcus lactis for growth was studied as a function of the type of cell envelope proteinase (PI versus PIII type). Two genetically engineered strains of L. lactis that differed only in the type of proteinase were grown in chemically defined media containing alphas1-, beta-, and kappa-caseins (alone or in combination) as the sources of amino acids. Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase. Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation. This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for L. lactis in milk (S. Helinck, J. Richard, and V. Juillard, Appl. Environ. Microbiol. 63:2124-2130, 1997) as a function of the type of proteinase. Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined. The results were in agreement only in the case of the PI-type proteinase. Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a PIII-type proteinase.

The effects of adding lactococcal proteinase on the growth rate of Lactococcus lactis in milk depend on the type of enzyme.

Increasing the proteolytic activity of Lactococcus lactis cultures in milk by adding the corresponding proteinase resulted in a stimulation of the growth rate regardless of the strain and the type of proteinase, demonstrating that the rate of casein degradation was responsible for the growth rate limitation of L. lactis in milk. However, the stimulation was only transient, and the reduction in growth rate in the poststimulation phase depended on the type of cell envelope proteinase. When a PI-type proteinase was added, three causes were involved in the subsequent reduction in growth rate: degradation of the added proteinase, repression of the proteolytic activity expressed by the cells, and competition for peptide uptake. When a PIII-type proteinase was added, the cessation of stimulation was due to the autoproteolysis of the added enzyme only.