The Interior Surfaces of Wooden Barrels Are an Additional Microbial Inoculation Source for Lambic Beer Production.

Traditional lambic beer production takes place through wort inoculation with environmental air and fermentation and maturation in wooden barrels. These wooden casks or foeders are possible additional inoculation sources of microorganisms for lambic worts. To date, however, these lambic barrels have been examined only with culture-dependent techniques, thereby missing a portion of the microorganisms present. Moreover, the effects of the cleaning procedures (involving high-pressure water and/or fumigation) and the barrel type on the microbial community structures of the interior surfaces of wooden lambic barrels were unclear. The culture-dependent plating and culture-independent amplicon sequencing of swab samples obtained from the interior surfaces of different wooden casks and foeders used for traditional lambic beer production in Belgium revealed that the microbial compositions of these surfaces differed statistically throughout the barrel-cleaning procedures applied. At the end of the cleaning procedures, amplicon sequencing still detected fermentation- and maturation-related microorganisms, although only a few colonies were still detectable using culture-dependent methods. It is possible that some of the surviving microorganisms were missed due to the presence of many of these cells in a viable but not culturable state and/or engrained deeper in the wood. These surviving microorganisms could act as an additional inoculation source, besides brewery air and brewery equipment, thereby helping to establish a stable microbial community in the wort to diminish batch-to-batch variations in fermentation profiles. Furthermore, the microbial compositions of the interior barrel surfaces differed statistically based on the barrel type, possibly reflecting different characteristics of the lambic barrels in terms of age, wood thickness, and wood porosity.IMPORTANCE Although the coolship step is generally regarded as the main contributor to the spontaneous inoculation by environmental air of fresh worts for lambic beer production, it is known that microorganisms often associate with specific surfaces present in a brewery. However, knowledge about the association of microorganisms with the interior surfaces of wooden lambic barrels is limited. To clarify the role of casks and foeders as additional microbial inoculation sources, it was important to determine the influence of the barrel characteristics and the cleaning procedures on the microbial communities of the interior barrel surfaces. Moreover, this helped to elucidate the complex spontaneous lambic beer fermentation and maturation process. It will allow further optimization of the lambic beer production process, as well as the wooden-barrel-cleaning procedures applied.

Temporal and Spatial Distribution of the Acetic Acid Bacterium Communities throughout the Wooden Casks Used for the Fermentation and Maturation of Lambic Beer Underlines Their Functional Role.

Few data have been published on the occurrence and functional role of acetic acid bacteria (AAB) in lambic beer production processes, mainly due to their difficult recovery and possibly unknown role. Therefore, a novel aseptic sampling method, spanning both the spatial and temporal distributions of the AAB and their substrates and metabolites, was combined with a highly selective medium and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a high-throughput dereplication method followed by comparative gene sequencing for their isolation and identification, respectively. The AAB (Acetobacter species more than Gluconobacter species) proliferated during two phases of the lambic beer production process, represented by Acetobacter orientalis during a few days in the beginning of the fermentation and Acetobacter pasteurianus from 7 weeks until 24 months of maturation. Competitive exclusion tests combined with comparative genomic analysis of all genomes of strains of both species available disclosed possible reasons for this successive dominance. The spatial analysis revealed that significantly higher concentrations of acetic acid (from ethanol) and acetoin (from lactic acid) were produced at the tops of the casks, due to higher AAB counts and a higher metabolic activity of the AAB species at the air/liquid interface during the first 6 months of lambic beer production. In contrast, no differences in AAB species diversity occurred throughout the casks.IMPORTANCE Lambic beer is an acidic beer that is the result of a spontaneous fermentation and maturation process. Acidic beers are currently attracting attention worldwide. Part of the acidity of these beers is caused by acetic acid bacteria (AAB). However, due to their difficult recovery, they were never investigated extensively regarding their occurrence, species diversity, and functional role in lambic beer production. In the present study, a framework was developed for their isolation and identification using a novel aseptic sampling method in combination with matrix-assisted laser desorption ionization-time of flight mass spectrometry as a high-throughput dereplication technique followed by accurate molecular identification. The sampling method applied enabled us to take spatial differences into account regarding both enumerations and metabolite production. In this way, it was shown that more AAB were present and more acetic acid was produced at the air/liquid interface during a major part of the lambic beer production process. Also, two different AAB species were encountered, namely, Acetobacter orientalis at the beginning and Acetobacter pasteurianus in a later stage of the production process. This developed framework could also be applied for other fermentation processes.

Nonconventional starter cultures of coagulase-negative staphylococci to produce animal-derived fermented foods, a SWOT analysis.

Coagulase-negative staphylococci (CNS) are ubiquitous micro-organisms that are commonly present on animal skin and animal-derived foods. They are members of the beneficial microbial consortia of several fermented food products where they contribute to quality. Currently, only a few CNS species are included in commercial starter cultures, although many other ones with promising properties have been isolated from diverse food ecosystems. In the present study, a Strengths-Weaknesses-Opportunities-Threats (SWOT) analysis of the potential use of unconventional CNS starter cultures for the fermentation of animal-derived foods is carried out. An overview of both their desirable and worrisome metabolic traits is given. In general, the application of innovative CNS-based starter cultures offers opportunities to modulate flavour, improve the safety and health aspects and develop novel colour development strategies for clean label products. Yet, their implementation is often not straightforward as nontrivial obstacles or threats are encountered, which relate to technological, food safety and legal concerns. As most of the desirable and undesirable characteristics of CNS species are strain dependent, a case-by-case evaluation is needed when evaluating specific strains for their potential use as novel starter cultures.

The water kefir grain inoculum determines the characteristics of the resulting water kefir fermentation process.

AIMS: To investigate the influence of the water kefir grain inoculum on the characteristics of the water kefir fermentation process. METHODS AND RESULTS: Three water kefir fermentation processes were started with different water kefir grain inocula and followed as a function of time regarding microbial species diversity, community dynamics, substrate consumption profile and metabolite production course. The inoculum determined the water kefir grain growth, the viable counts on the grains, the time until total carbohydrate exhaustion, the final metabolite concentrations and the microbial species diversity. There were always 2-10 lactic acid bacterial cells for every yeast cell and the majority of these micro-organisms was always present on the grains. Lactobacillus paracasei, Lactobacillus hilgardii, Lactobacillus nagelii and Saccharomyces cerevisiae were always present and may be the key micro-organisms during water kefir fermentation. Low water kefir grain growth was associated with small grains with high viable counts of micro-organisms, fast fermentation and low pH values, and was not caused by the absence of exopolysaccharide-producing lactic acid bacteria. CONCLUSIONS: The water kefir grain inoculum influences the microbial species diversity and characteristics of the fermentation process. A select group of key micro-organisms was always present during fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study allows a rational selection of a water kefir grain inoculum.

The cocoa bean fermentation process: from ecosystem analysis to starter culture development.

Cocoa bean fermentation is still a spontaneous curing process to facilitate drying of nongerminating cocoa beans by pulp removal as well as to stimulate colour and flavour development of fermented dry cocoa beans. As it is carried out on farm, cocoa bean fermentation is subjected to various agricultural and operational practices and hence fermented dry cocoa beans of variable quality are obtained. Spontaneous cocoa bean fermentations carried out with care for approximate four days are characterized by a succession of particular microbial activities of three groups of micro-organisms, namely yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB), which results in well-fermented fully brown cocoa beans. This has been shown through a plethora of studies, often using a multiphasic experimental approach. Selected strains of several of the prevailing microbial species have been tested in appropriate cocoa pulp simulation media to unravel their functional roles and interactions as well as in small plastic vessels containing fresh cocoa pulp-bean mass to evaluate their capacity to dominate the cocoa bean fermentation process. Various starter cultures have been proposed for successful fermentation, encompassing both cocoa-derived and cocoa nonspecific strains of (hybrid) yeasts, LAB and AAB, some of which have been implemented on farms successfully.

Summer Meeting 2013: growth and physiology of bifidobacteria.

Bifidobacteria are a minor fraction of the human colon microbiota with interesting properties for carbohydrate degradation. Monosaccharides such as glucose and fructose are degraded through the bifid shunt, a dedicated pathway involving phosphoketolase activity. Its stoechiometry learns that three moles of acetate and two moles of lactate are produced per two moles of glucose or fructose that are degraded. However, deviations from this 3 : 2 ratio occur, depending on the rate of substrate consumption. Slower growth rates favour the production of acetate and pyruvate catabolites (such as formate) at the cost of lactate. Interestingly, bifidobacteria are capable to degrade inulin-type fructans (ITF) (oligofructose and inulin) and arabinoxylan-oligosaccharides (AXOS). Beta-fructofuranosidase activity enables bifidobacteria to degrade ITF. However, this property is strain-dependent. Some strains consume both fructose and oligofructose, with different preferences and degradation rates. Small oligosaccharides (degree of polymerization or DP of 2-7) are taken up, in a sequential order, indicating intracellular degradation and as such giving these bacteria a competitive advantage towards other inulin-type fructan degraders such as lactobacilli, bacteroides and roseburias. Other strains consume long fractions of oligofructose and inulin. Exceptionally, oligosaccharides with a DP of up to 20 (long-chain inulin) are consumed by specific strains. Also, the degradation of AXOS by α-arabinofuranosidase and ß-xylosidase is strain-dependent. Particular strains consume the arabinose substituents, whether or not together with a consumption of the xylose backbones of AXOS, either up to xylotetraose or higher and either extra- or intracellularly. The production of high amounts of acetate that accompanies inulin-type fructan degradation by bifidobacteria cross-feeds other colon bacteria involved in the production of butyrate. However, bifidobacterial strain-dependent differences in prebiotic degradation indicate the existence of niche-specific adaptations and hence mechanisms to avoid competition among each other and to favour coexistence with other colon bacteria.

Antibacterial activities of coagulase-negative staphylococci from bovine teat apex skin and their inhibitory effect on mastitis-related pathogens.

AIMS: To explore antibacterial activities of coagulase-negative staphylococci (CoNS) from teat apices of dairy cows towards mastitis-causing pathogens. METHODS AND RESULTS: Of 254 CoNS, 38 displayed bacteriocin-like activity after a first screening. Seven of these strains displayed activity against at least one mastitis-related pathogen (Streptococcus uberis, Streptococcus dysgalactiae and Staphylococcus aureus). Staphylococcus chromogenes L217 displayed the strongest inhibitory effect, being active against all tested mastitis-related pathogens and most tested CoNS. Based on cation exchange and reversed-phase chromatography, in addition to N-terminal Edman degradation and PCR, the antibacterial peptide was identified as a nukacin-type bacteriocin and named nukacin L217. Although staphylococcal bacteriocins are generally found in the cell-free supernatants of liquid cultures, Staph. chromogenes L217 only led to detectable activity when grown on agar medium. CONCLUSIONS: Bacteriocin-like activities are not uncommon among CoNS from teat apices and may inhibit mastitis-causing pathogens, as found for nukacin L217 production by Staph. chromogenes L217. SIGNIFICANCE AND IMPACT OF THE STUDY: Nukacin L217 is the first identified bacteriocin of the species Staph. chromogenes and displays unusual production kinetics, that is, requiring surface growth of its producer. The fact that nukacins are produced by different CoNS species suggests a role in the teat skin ecosystem.

The use of nucleosides and arginine as alternative energy sources by coagulase-negative staphylococci in view of meat fermentation.

The ability of coagulase-negative staphylococci (CNS) to use alternative energy sources in meat may partially explain their occurrence in fermented meats. Of 61 CNS strains tested, all metabolized adenosine and inosine in a meat simulation medium (MSM). The ability to catabolize arginine via the arginine deiminase (ADI) pathway varied between strains. All tested strains of Staphylococcus carnosus and Staphylococcus epidermidis possessed an arcA gene and showed ADI activity, whereas other species, such as Staphylococcus equorum and Staphylococcus succinus, did not. Arginine catabolic mobile elements (ACME), as in the positive control S. epidermidis ATCC 12228, were uncommon and only found in Staphylococcus xylosus 3PA6 (sausage isolate) and Staphylococcus chromogenes G222 (teat apex isolate). Monoculture experiments were performed in MSM with S. carnosus 833 and SS3-4, S. xylosus G211, and S. epidermidis ATCC 12228 and 2S7-4. At all pH values tested (5.3, 5.8, and 6.5), the strains of S. carnosus catabolized arginine faster than the strains of S. xylosus and S. epidermidis. Only at pH 6.5 could a low ADI activity be found for S. xylosus G211. Increased ADI activity occurred in the case of the ACME-positive S. epidermidis ATCC 12228, when compared to the ACME-negative S. epidermidis 2S7-4.

Microbial ecology of sourdough fermentations: diverse or uniform?

Sourdough is a specific and stressful ecosystem inhabited by yeasts and lactic acid bacteria (LAB), mainly heterofermentative lactobacilli. On the basis of their inocula, three types of sourdough fermentation processes can be distinguished, namely backslopped ones, those initiated with starter cultures, and those initiated with a starter culture followed by backslopping. Typical sourdough LAB species are Lactobacillus fermentum, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus sanfranciscensis. Typical sourdough yeast species are Candida humilis, Kazachstania exigua, and Saccharomyces cerevisiae. Whereas region specificity is claimed in the case of artisan backslopped sourdoughs, no clear-cut relationship between a typical sourdough and its associated microbiota can be found, as this is dependent on the sampling, isolation, and identification procedures. Both simple and very complex consortia may occur. Moreover, a series of intrinsic and extrinsic factors may influence the composition of the sourdough microbiota. For instance, an influence of the flour (type, quality status, etc.) and the process parameters (temperature, pH, dough yield, backslopping practices, etc.) occurs. In this way, the presence of Lb. sanfranciscensis during sourdough fermentation depends on specific environmental and technological factors. Also, Triticum durum seems to select for obligately heterofermentative LAB species. Finally, there are indications that the sourdough LAB are of intestinal origin.

Species diversity, community dynamics, and metabolite kinetics of spontaneous leek fermentations.

Leek (Allium ampeloprasum var. porrum) is one of Belgium's most important vegetables. All or part of the green leek parts are often left on the fields because of their limited cooking applications compared to the white leek parts. Therefore, the possibility to perform leek fermentations in view of product valorization and diversification was investigated. This study deals with the community dynamics, species diversity, and metabolite kinetics of spontaneous leek fermentations, thereby studying the influence of added NaCl concentration, harvesting season, and duration of the fermentation. The combination of a culture-dependent and culture-independent approach revealed the prevalence of lactic acid bacteria (LAB) from the third day of fermentation onwards, which was not influenced by the fermentation conditions applied. Enterobacteriaceae, Pseudomonadaceae, and yeasts disappeared after one week of fermentation. Leuconostoc mesenteroides, Lactobacillus sakei, and Lactobacillus plantarum, Lactobacillus brevis, and Lactobacillus parabrevis were the most frequently isolated LAB species. Both added NaCl concentrations were suitable to perform successful fermentations within three weeks. By that time, glucose and fructose, the main leek carbohydrates, were metabolized into mainly lactic acid, acetic acid, ethanol, and mannitol. A sensory analysis revealed that the fermented white leek parts were generally more appreciated than the fermented green leek parts.

Unraveling the microbiota of teat apices of clinically healthy lactating dairy cows, with special emphasis on coagulase-negative staphylococci.

Swab samples (n=72) obtained from the teat apex of lactating dairy cows without visual signs of inflammation (n=18) were gathered on 2 well-managed Flemish dairy herds (herds 1 and 2) during the same month to assess the bacterial diversity of teat apices before milking. A combination of both culture-dependent [plating and (GTG)(5)-PCR fingerprinting of the colonies] and culture-independent [denaturing gradient gel electrophoresis (PCR-DGGE)] techniques indicated that the teat apices contain a wide diversity of bacterial genera. Despite a low bacterial load, 20 bacterial genera of 3 phyla (Actinobacteria, Firmicutes, and Proteobacteria) were present. The most prevalent bacteria were the coagulase-negative staphylococci (CNS), encompassing a total of 15 species, which were identified to the species level using a combination of (GTG)(5)-PCR fingerprinting, gene sequencing (16S ribosomal RNA and rpoB genes), and a novel PCR-DGGE technique based on the tuf-PCR amplicon. Overall bacterial diversity did not differ significantly between the herds or between noninfected and subclinically infected quarters in herd 1. In herd 1, borderline significant lower CNS species diversity was found on teat apices of noninfected quarters compared with subclinically infected quarters. The most prevalent CNS species were Staphylococcus haemolyticus and Staphylococcus equorum in both herds and Staphylococcus carnosus in herd 2.

Expression of the arginine deiminase pathway genes in Lactobacillus sakei is strain dependent and is affected by the environmental pH.

The adaptation of Lactobacillus sakei to a meat environment is reflected in its metabolic potential. For instance, the ability to utilize arginine through the arginine deiminase (ADI) pathway, resulting in additional ATP, represents a competitive benefit. In L. sakei CTC 494, the arc operon (arcABCTDR) shows the same gene order and organization as that in L. sakei 23K, the genome sequence of which is known. However, differences in relative gene expression were found, and these seemed to be optimal in different growth phases, namely, the highest relative gene expression level was in the end exponential growth phase in the case of L. sakei CTC 494 and in the mid-exponential growth phase of L. sakei 23K. Also, the environmental pH influenced the relative expression level of the arc operon, as shown for L. sakei CTC 494, with the highest relative expression level occurring at the optimal pH for growth (pH 6.0). Deviations from this optimal pH (pH 5.0 and pH 7.0) resulted in an overall decline of the relative expression level of all genes of the arc operon. Furthermore, a differential relative expression of the individual genes of the arc operon was found, with the highest relative gene expression occurring for the first two genes of the arc operon (arcA and arcB). Finally, it was shown that some L. sakei strains were able to convert agmatine into putrescine, suggesting an operational agmatine deiminase pathway in these strains, a metabolic trait that is undesirable in meat fermentations. This study shows that this metabolic trait is most probably encoded by a previously erroneously annotated second putative arc operon.

Species diversity and metabolic impact of the microbiota are low in spontaneously acidified Belgian sausages with an added starter culture of Staphylococcus carnosus.

Quality of fermented sausages is affected by acidifying lactic acid bacteria (LAB) and colour- and flavour-promoting coagulase-negative staphylococci (CNS), whether or not used as starter culture. Artisan fermented sausages are often perceived as superior to industrial variants, partially because of the specific microbiota due to spontaneous acidification, which may be considered as an artisan characteristic. Therefore, two kinds of spontaneously acidified Belgian sausages were prepared (Belgian-type salami and Boulogne sausage), but with addition of a Staphylococcus carnosus culture. The Belgian-type salami was made from pork and beef, whereas the Boulogne sausage contained pork and horse meat. In all cases, Lactobacillus sakei was the dominant LAB species present on the raw materials and during fermentation, whereas enterococci remained present in the background. Enterobacteriaceae vanished after fermentation. The CNS species diversity on the raw materials was large and differed between the pork, beef, and horse meat. Nevertheless, this species diversity was annihilated during fermentation by the added S. carnosus culture. The volatiles fraction was mainly composed of aldehydes that originated from lipid oxidation and spices-derived compounds. Aromatic compounds that are typically associated to CNS activity, such as end-products from the metabolism of branched-chain amino acids, were not present in the Belgian-type salami and only marginally present in the Boulogne sausage. In conclusion, spontaneous acidification of Belgian-type fermented sausages leads to dominance of L. sakei and is no guarantee for bacterial contribution to the aroma profile when S. carnosus is added as a starter culture.

Characterization of coagulase-negative staphylococcus species from cows' milk and environment based on bap, icaA, and mecA genes and phenotypic susceptibility to antimicrobials and teat dips.

The aim of this study was to investigate whether the main coagulase-negative staphylococci (CNS) species involved in bovine intramammary infections (IMI) possess specific characteristics that promote colonization of the udder. Virulence markers associated with biofilm formation, antimicrobial resistance, and biocide tolerance were compared between typically contagious CNS species (Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus simulans) and those rarely causing IMI (Staphylococcus sciuri, Staphylococcus equorum, and others) to find possible associations with pathogenicity. Coagulase-negative staphylococci isolates (n=366) belonging to 22 different species were analyzed by PCR for the presence of the biofilm-associated genes bap and icaA, and the methicillin resistance gene mecA. A selection of 82 isolates was additionally tested for their susceptibility to 5 antibiotics and 2 commercial teat dip products. Minimum inhibitory concentrations of antimicrobials were determined by Etest (AB bioMérieux, Marcy l'Etoile, France), and a microdilution method was optimized to determine minimum biocidal concentrations of teat dips. The bap, icaA, and mecA genes were detected significantly more in isolates from CNS species typically living in the cows' environment than in isolates from IMI-causing species. Antimicrobial resistance was mainly against erythromycin (23%) or oxacillin (16%), and was detected more often in the environmental species. The isolates least susceptible to the teat dips belonged to the IMI-causing species Staph. chromogenes and Staph. simulans. We concluded that carriage of biofilm genes and antimicrobial resistance were not associated with the ability to colonize the mammary gland because free-living CNS species constituted a more significant reservoir of biofilm and resistance determinants than did IMI-causing species. In contrast, increased tolerance to biocides may favor the establishment of bovine IMI by some CNS species.

The pentose moiety of adenosine and inosine is an important energy source for the fermented-meat starter culture Lactobacillus sakei CTC 494.

The genome sequence of Lactobacillus sakei 23K has revealed that the species L. sakei harbors several genes involved in the catabolism of energy sources other than glucose in meat, such as glycerol, arginine, and nucleosides. In this study, a screening of 15 L. sakei strains revealed that arginine, inosine, and adenosine could be used as energy sources by all strains. However, no glycerol catabolism occurred in any of the L. sakei strains tested. A detailed kinetic analysis of inosine and adenosine catabolism in the presence of arginine by L. sakei CTC 494, a fermented-meat starter culture, was performed. It showed that nucleoside catabolism occurred as a mixed-acid fermentation in a pH range (pH 5.0 to 6.5) relevant for sausage fermentation. This resulted in the production of a mixture of acetic acid, formic acid, and ethanol from ribose, while the nucleobase (hypoxanthine and adenine in the case of fermentations with inosine and adenosine, respectively) was excreted into the medium stoichiometrically. This indicates that adenosine deaminase activity did not take place. The ratios of the different fermentation end products did not vary with environmental pH, except for the fermentation with inosine at pH 5.0, where lactic acid was produced too. In all cases, no other carbon-containing metabolites were found; carbon dioxide was derived only from arginine catabolism. Arginine was cometabolized in all cases and resulted in the production of both citrulline and ornithine. Based on these results, a pathway for inosine and adenosine catabolism in L. sakei CTC 494 was presented, whereby both nucleosides are directly converted into their nucleobase and ribose, the latter entering the heterolactate pathway. The present study revealed that the pentose moiety (ribose) of the nucleosides inosine and adenosine is an effective fermentable substrate for L. sakei. Thus, the ability to use these energy sources offers a competitive advantage for this species in a meat environment.

Linoleate isomerase activity occurs in lactic acid bacteria strains and is affected by pH and temperature.

AIMS: To investigate the ability of lactic acid bacteria (LAB) to convert linoleic acid (LA) and α-linolenic acid (α-LNA) to conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA), respectively. To assess pH and temperature influences on CLA and CLNA production by Lactobacillus sakei LMG 13558. METHODS AND RESULTS: A screening of 48 LAB yielded one Lactobacillus curvatus, five Lactobacillus plantarum and four Lact. sakei strains displaying linoleate isomerase (LAI) activity. CLNA conversion percentages varied largely (1-60%). CLA conversion, occurring in three strains, was lower (2-5%). The LAI gene sequences of the ten LAI-positive strains shared 75-99% identity with the LAI gene sequence of a Lact. plantarum AS1.555. At pH 6.2, CLA and CLNA production by Lact. sakei LMG 13558 was higher at 30°C than at 20 and 25°C. At pH 5.5 (30°C) or 37°C (pH 6.2), LA was not converted and α-LNA only slightly converted. CONCLUSIONS: LAB show strain-dependent LAI activity. Production of CLA and CLNA is affected by pH and temperature, as shown for Lact. sakei LMG 13558. SIGNIFICANCE AND IMPACT OF THE STUDY: Several LAB produce CLA and/or CLNA, as shown for Lact. sakei and Lact. curvatus for the first time. These findings offer potential for the manufacturing of fermented functional foods.

The kinetics of the arginine deiminase pathway in the meat starter culture Lactobacillus sakei CTC 494 are pH-dependent.

Lactobacillus sakei is frequently present as the dominant lactic acid bacterium in spontaneously fermented meat products, demonstrating its competitiveness in and adaptation to the meat environment. Since meat is generally low in carbohydrate content, the ability to utilize other energy sources to generate ATP, such as arginine via the arginine deiminase (ADI) pathway, represents a competitive benefit. In this study, the kinetics of growth and arginine conversion capabilities of Lb. sakei CTC 494 were analyzed, and a model was set up to describe the influence of pH on growth and arginine conversion. A series of in vitro batch fermentations using reconstituted MRS medium at different constant pH values (pH 4.50-pH 7.75) was performed. Arginine conversion through the ADI pathway, which was activated from the stationary growth phase on, resulted in the production of both citrulline and ornithine for all pH conditions tested. However, the pattern and the ratio of the end-products of the ADI pathway were influenced by pH. For certain pH values (between pH 5.0 and 6.5), a further conversion of citrulline into ornithine was found when all arginine was depleted. Characterization of responses of the ADI pathway in Lb. sakei CTC 494 to environmental conditions will allow a better understanding and control of this important starter culture in meat fermentations.

Sources other than unused sawdust can introduce Klebsiella pneumoniae into dairy herds.

A longitudinal study was carried out to detect intramammary infections caused by Klebsiella pneumoniae and to identify potential sources of this bacterial species in the environment of the cows. The study was performed in 6 well-managed Belgian dairy herds from May 2008 to May 2009. Monthly (n=13), unused and used sawdust bedding samples as well as individual quarter milk and feces samples were collected from 10 randomly selected cohort cows in each herd. Cases of clinical mastitis of all lactating cows in the 6 herds were also sampled (n=64). From the 3,518 collected samples, 153 K. pneumoniae isolates were obtained, of which 2 originated from milk (clinical mastitis cases). In feces (n=728), used bedding (n=73), and unused bedding (n=73), respectively, 125 (17.2%), 20 (27.4%), and 6 (8.2%) isolates were found. The isolates were fingerprinted by means of pulsed field gel electrophoresis. In total, 109 different pulsotypes were differentiated, indicating a high degree of genetic diversity within the isolates. All isolates from unused bedding belonged to pulsotypes other than those from the other sources, suggesting that sources other than unused sawdust may introduce K. pneumoniae into the herd. Only 2 pulsotypes contained isolates originating from different sources. Pulsotype 10 was found in milk and used bedding and pulsotype 21 was found in feces and used bedding. The 2 milk isolates originated from 2 cows in the same herd but they belonged to a different pulsotype. The results indicate that K. pneumoniae can be prevalent in the environment without causing significant mastitis problems. Most cows were shedding K. pneumoniae in feces, substantiating findings under very different conditions (i.e., American dairy herds). Contamination of used bedding in the cubicles with K. pneumoniae from feces was confirmed, whereas unused bedding was not an important source of K. pneumoniae for the environment of the cows.

Distribution of coagulase-negative Staphylococcus species from milk and environment of dairy cows differs between herds.

In many parts of the world, coagulase-negative staphylococci (CNS) are the predominant pathogens causing intramammary infections (IMI) in dairy cows. The cows' environment is thought to be a possible source for CNS mastitis and this was investigated in the present paper. A longitudinal field study was carried out in 6 well-managed dairy herds to determine the distribution and epidemiology of various CNS species isolated from milk, causing IMI and living freely in the cows' environment, respectively. In each herd, quarter milk samples from a cohort of 10 lactating cows and environmental samples from stall air, slatted floor, sawdust from cubicles, and sawdust stock were collected monthly (n=13). Isolates from quarter milk samples (n=134) and the environment (n=637) were identified to species level using amplified fragment length polymorphism (AFLP) genotyping. Staphylococcus chromogenes, S. haemolyticus, S. epidermidis, and S. simulans accounted for 81.3% of all CNS milk isolates. Quarters were considered infected with CNS (positive IMI status) only when 2 out of 3 consecutive milk samples yielded the same CNS AFLP type. The species causing IMI were S. chromogenes (n=35 samples with positive IMI status), S. haemolyticus (n=29), S. simulans (n=14), and S. epidermidis (n=6). The observed persistent IMI cases (n=17) had a mean duration of 149.4 d (range 63.0 to 329.8 d). The CNS species predominating in the environment were S. equorum, S. sciuri, S. haemolyticus, and S. fleurettii. Herd-to-herd differences in distribution of CNS species were observed in both milk and the environment, suggesting that herd-level factors are involved in the establishment of particular species in a dairy herd. Primary reservoirs of the species causing IMI varied. Staphylococcus chromogenes and S. epidermidis were rarely found in the environment, indicating that other reservoirs were more important in their epidemiology. For S. haemolyticus and S. simulans, the environment was found as a reservoir, suggesting that IMI with these species were possibly environmental in origin.

Molecular source tracking of predominant lactic acid bacteria in traditional Belgian sourdoughs and their production environments.

AIMS: To investigate the circulation of predominant sourdough lactic acid bacteria (LAB) species in the production environment of two Belgian artisan sourdough bakeries. METHODS AND RESULTS: Isolates were collected from sourdoughs, flour, hands of the baker and air in the bakery setting and taxonomically characterized using repetitive element sequence-based PCR fingerprinting, pheS and/or 16S rRNA gene sequencing and amplified fragment length polymorphism (AFLP) analysis. In parallel, PCR-DGGE (denaturing gradient gel electrophoresis) analysis of V3-16S rDNA amplicons was applied to visualize the predominant bacterial population in the sourdoughs and the corresponding bakery environment (flour, hands of the baker, air and bakery equipment). Both approaches revealed that sourdoughs produced at D01 and D10 were mainly dominated by Lactobacillus spicheri and L. plantarum and by L. sanfranciscensis, respectively, and that these LAB species also circulated in the corresponding bakery environment. Furthermore, AFLP fingerprinting demonstrated that sourdough and bakery environment isolates of these species were genetically indistinguishable. For more sensitive source-tracking, SYBR Green-based real-time PCR assays were developed using species-specific primers targeting the pheS gene of L. plantarum and L. sanfranciscensis, detected in air samples from D01 and D10, respectively. CONCLUSIONS: The results obtained in this study indicate that specific strains of LAB persist in artisan doughs over years and circulate in the bakery environment. Furthermore, the importance of air as a potential carrier of LAB in artisan bakery environments was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: PheS-based real-time PCR can be used to detect, quantify and/or monitor specific LAB species (e.g. starter cultures) in sourdough and bakery environment samples.