Food fermentations: microorganisms with technological beneficial use.

Microbial food cultures have directly or indirectly come under various regulatory frameworks in the course of the last decades. Several of those regulatory frameworks put emphasis on "the history of use", "traditional food", or "general recognition of safety". Authoritative lists of microorganisms with a documented use in food have therefore come into high demand. One such list was published in 2002 as a result of a joint project between the International Dairy Federation (IDF) and the European Food and Feed Cultures Association (EFFCA). The "2002 IDF inventory" has become a de facto reference for food cultures in practical use. However, as the focus mainly was on commercially available dairy cultures, there was an unmet need for a list with a wider scope. We present an updated inventory of microorganisms used in food fermentations covering a wide range of food matrices (dairy, meat, fish, vegetables, legumes, cereals, beverages, and vinegar). We have also reviewed and updated the taxonomy of the microorganisms used in food fermentations in order to bring the taxonomy in agreement with the current standing in nomenclature.

Metabolism of nitrate in fermented meats: the characteristic feature of a specific group of fermented foods.

Within the universe of food fermentation processes the multi-purpose use of nitrate and/or nitrite is a unique characteristic of meat fermentations. These curing agents play a decisive role in obtaining the specific sensory properties, stability and hygienic safety of products such as fermented sausages, ham and, more recently, emulsion type of sausages. The use of nitrate is the traditional method in curing processes and requires its reduction to reactive nitrite. Thus, nitrate reduction is the key event that is exclusively performed by microorganisms. Under controlled fermentation conditions starter cultures are used that contain staphylococci and/or Kocuria varians, which in addition to strongly affecting sensory properties exhibit efficient nitrate reductase activity. To obtain clean label products some plant sources of nitrate have been in use. When producing thermally treated sausages (e.g. of emulsion type), starter cultures are used that form nitrite before cooking takes place. Staphylococci reduce nitrite to ammonia after nitrate has been consumed. K. varians is devoid of nitrite reductase activity. Nitrate and nitrite reductases are also present in certain strains of lactobacilli. It was shown that their application as starter cultures warrants efficient activity in sausages made with either nitrate or nitrite. NO is formed from nitrite in numerous chemical reactions among which disproportionation and reaction with reductants either added or endogenous in meat are of practical importance. Numerous nitrosation and nitrosylation reactions take place in the meat matrix among which the formation of nitrosomyoglobin is of major sensory importance. Safety considerations in meat fermentation relate to the safe nature of the starter organisms and to the use of nitrate/nitrite. Staphylococci ("micrococci") in fermented meat have a long tradition in food use but have not received the QPS status from the EFSA. They require, therefore, thorough assessment with regard to toxigenicity and pathogenicity determinants as well as presence of transferable antibiotic resistance. Nitrate and nitrite are still considered basically undesired in food. The main objections are based on their potential to form nitrosamines with carcinogenic potential. In view of new results from intensive research of NO, potential risks are opposed by positive effects on human health.

Microbial food cultures--opinion of the Senate Commission on Food Safety (SKLM) of the German Research Foundation (DFG).

In the EU, there are no specific legal regulations regarding microbial food cultures. However, at European and national level, there are regulations that require microbial cultures to be checked in terms of their compliance with legal requirements. Due to the lack of definitions for microbial food cultures with various applications, there are uncertainties regarding how they are to be assessed. The increased elaboration of microbial ecology and modern taxonomy has allowed the description of numerous new species that are attractive for use in food cultures or are already in use, on which, however, only limited experience is available. In view of these developments, the SKLM has prepared this statement, focusing on definitions, gaps in knowledge and further research needs. It aims to support the producers and users of microbial cultures as well as authorities responsible for consumer health protection with respect to safety assessment and to contribute to consumer information. The scientific status concerning these cultures in food technology, the traditional roots of their application and their potential for sustaining and/or furthering food variety and quality have not been adequately described up to now. This is the subject of the present SKLM statement. In addition, definitions are proposed for cultures used in food technology that may also be useful for the assessment in a legal context. The opinion was released in German on 29 March 2010, the English version was agreed on 15 November 2010.

Inhibition of growth of Shiga toxin-producing Escherichia coli by nonpathogenic Escherichia coli.

During routine quality control testing of diagnostic methods for Shiga toxin-producing Escherichia coli (STEC) using stool samples spiked with STEC, it was observed that the Shiga toxin could not be detected in 32 out of 82 samples tested. Strains of E. coli isolated from such stool samples were shown to be responsible for this inhibition. One particular isolate, named E. coli 1307, was intensively studied because of its highly effective inhibitory effect; this strain significantly reduced growth and Shiga toxin levels in coculture of several STEC strains regardless of serovar or Shiga toxin type. The probiotic E. coli Nissle 1917 inhibited growth and reduced Shiga toxin levels in STEC cultures to an extent similar to E. coli 1307, but commensal E. coli strains and several other known probiotic bacteria (enterococci, Bacillus sp., Lactobacillus acidophilus) showed no, or only small, inhibitory effects. Escherichia coli 1307 lacks obvious fitness factors, such as aerobactin, yersiniabactin, microcins and a polysaccharide capsule, that are considered to promote the growth of pathogenic bacteria. We therefore propose strain E. coli 1307 as a candidate probiotic for use in the prevention and treatment of infections caused by STEC.

Comparison of the compositional, microbiological, biochemical and volatile profile characteristics of three Italian PDO fermented sausages.

Three Italian PDO fermented sausages, Varzi, Brianza and Piacentino, were compared for compositional, microbiological, biochemical and volatile profile characteristics. Mean values for the gross composition varied especially due to moisture, fat, total protein and nitrate concentration which reflected differences in the ingredients and some technological parameters. Cell numbers of the major microbial groups were almost similar among sausages. The major differences were found for Brochothrix thermosphacta, enterococci and moulds. Apart from their use as starters, Lactobacillus sakei and Lactobacillus curvatus were the dominant lactic acid bacteria and, as well as Staphylococcus xylosus, dominated the population of coagulase-negative staphylococci. Sausages differed for the hydrolysis of myofibrillar proteins and secondary proteolysis. Varzi, the sausage subjected to prolonged fermentation at 23-25°C for 10 days before ripening, showed the highest degree of secondary proteolysis. Varzi and Brianza, the two fermented sausages manufactured by using microbial starters, showed the highest concentration and similar profiles of free amino acids. The peptidase activities contained in the aqueous extracts agreed with the above findings. A total of 52 volatile components, mainly alcohols, aldehydes and terpenes, were identified by solid-phase micro-extraction coupled with gas chromatography-mass spectrometry analysis. The volatile profiles of the three Italian PDO fermented sausages differed in part and, except for terpenes, the highest levels of the other chemical classes were found in Varzi and Brianza sausages. The composition of free fatty acids of the three Italian PDO sausages was rather similar. Monounsaturated fatty acids (MUFA) were found at the highest relative percentage followed by saturated (SFA) and polyunsaturated (PUFA) fatty acids. Oleic, palmitic, linoleic and stearic were the main free fatty acids found in all fermented sausages.

Characterization of the cultivable microbiota of sprouts and their potential for application as protective cultures.

The microbiota of ten seeds and ready-to-eat sprouts produced thereof was characterized by bacteriological culture and denaturing gradient gel electrophoresis (DGGE) of amplified DNA fragments of the 16S rRNA gene. The predominant bacterial biota of hydroponically grown sprouts mainly consisted of enterobacteria, pseudomonades and lactic acid bacteria (LAB). For adzuki, alfalfa, mung bean, radish, sesame and wheat, the ratio of these bacterial groups changed strongly in the course of germination, whereas for broccoli, red cabbage, rye and green pea the ratio remained unchanged. Within the pseudomonades, Pseudomonas gesardii and Pseudomonas putida have been isolated and strains of the potentially pathogenic species Enterobacter cancerogenes and Pantoea agglomerans were found as part of the main microbiota on hydroponically grown sprouts. In addition to the microbiota of the whole seedlings, the microbiota of root, hypocotyl and seed leafs were examined for alfalfa, radish and mung bean sprouts. The highest and lowest total counts for aerobic bacteria were found on seed leafs and hypocotyls, respectively. On the other hand, the highest numbers for LAB on sprouts were found on the hypocotyl. When sprouting occurred under the agricultural conditions, e.g. in soil, the dominating microbiota changed from enterobacteria to pseudomonades for mung beans and alfalfa sprouts. No pathogenic enterobacteria have been isolated from these sprout types. Within the pseudomonades group, Pseudomonas jessenii and Pseudomonas brassicacearum were found as dominating species on all seedling parts from soil samples. In practical experiments, a strain of P. jessenii was found to exhibit a potential for use as protective culture, as it suppresses the growth of pathogenic enterobacteria on ready-to-eat sprouts.

A high-molecular-mass surface protein (Lsp) and methionine sulfoxide reductase B (MsrB) contribute to the ecological performance of Lactobacillus reuteri in the murine gut.

Members of the genus Lactobacillus are common inhabitants of the gut, yet little is known about the traits that contribute to their ecological performance in gastrointestinal ecosystems. Lactobacillus reuteri 100-23 persists in the gut of the reconstituted Lactobacillus-free mouse after a single oral inoculation. Recently, three genes of this strain that were specifically induced (in vivo induced) in the murine gut were identified (38). We report here the detection of a gene of L. reuteri 100-23 that encodes a high-molecular-mass surface protein (Lsp) that shows homology to proteins involved in the adherence of other bacteria to epithelial cells and in biofilm formation. The three in vivo-induced genes and lsp of L. reuteri 100-23 were inactivated by insertional mutagenesis in order to study their biological importance in the murine gastrointestinal tract. Competition experiments showed that mutation of lsp and a gene encoding methionine sulfoxide reductase (MsrB) reduced ecological performance. Mutation of lsp impaired the adherence of the bacteria to the epithelium of the mouse forestomach and altered colonization dynamics. Homologues of lsp and msrB are present in the genomes of several strains of Lactobacillus and may play an important role in the maintenance of these bacteria in gut ecosystems.

Characterization of Lactobacillus coryniformis DSM 20001T surface protein Cpf mediating coaggregation with and aggregation among pathogens.

Phenotypic characterization of aggregation phenotypes of Lactobacillus coryniformis revealed that strain DSM 20001T coaggregated with Escherichia coli K88, Campylobacter coli, and Campylobacter jejuni but not with other human pathogens. In addition, cells of these pathogens aggregated in the presence of the spent culture supernatant (SCS) of strain DSM 20001T. Cells of E. coli K88 remained viable in the coaggregates and aggregates for up to 24 h. Both coaggregation and aggregation (co/aggregation) occurred at pH 3.5 to 7.5 and was sensitive to heat (85 degrees C for 15 min) and proteinase K. The co/aggregation-promoting factor (Cpf) was purified, and the gene was identified by PCR with degenerate primers derived from internal amino acid sequences. The cpf gene encoded a 19.9-kDa preprotein with a sec-dependent leader and an isoelectric point of 4.4. The amino acid sequence had no significant similarity to proteins with known functions. Northern analysis revealed not only major transcription from the promoter of cpf but also major transcription from the promoter of the preceding insertion element, ISLco1 belonging to the IS3 family. Recombinant Cpf produced in E. coli mediated aggregation of pathogens comparable to the aggregation obtained with purified Cpf or SCS of strain DSM 20001T. Cpf could be removed from cells of strain DSM 20001T by treatment with 5 M LiCl and could be subsequently reattached to the cell surface by using SCS or recombinant Cpf, which resulted in restoration of the co/aggregation property. These results together with those of the amino acid sequence analysis suggest that Cpf is a novel surface protein of L. coryniformis that mediates co/aggregation of some pathogens.

Characterisation of the microbiota of rice sourdoughs and description of Lactobacillus spicheri sp. nov.

The microbiota of two industrially processed rice sourdoughs was characterised by bacteriological culture in combination with PCR-denaturing gradient gel electrophoresis (DGGE) and 16S/28S rDNA sequence analysis. Rice sourdough I was continuously propagated for several years by back-slopping every week, whereas sourdough II was processed by using a commercial starter culture and back-slopping daily for three days. In rice sourdough II Candida krusei and Saccharomyces cerevisiae as well as Lactobacillus fermentum, Lactobacillus gallinarum, Lactobacillus kimchii, Lactobacillus plantarum, and Lactobacillus pontis dominated at the first day of fermentation. RAPD analysis of lactobacilli revealed identical profiles for each of the species except for L. fermentum and L. pontis indicating the presence of different strains. Fluctuations within the LAB community during fermentation were monitored by PCR-DGGE. L. pontis decreased in numbers over time and L. curvatus became dominant after 3 days of fermentation. Rice sourdough I contained S. cerevisiae, Lactobacillus paracasei (present with three different RAPD types), Lactobacillus paralimentarius, and a Lactobacillus strain which could not be allotted to any valid species. Phylogenetic analysis based on 16S rDNA sequences revealed Lactobacillus brevis as the closest relative (97.3% sequence similarity). Differences in some phenotypic characteristics and DNA-DNA relatedness indicated that the strain represents a new Lactobacillus species, for which the name Lactobacillus spicheri is proposed.

Effect of food components and processing parameters on DNA degradation in food.

The effect of food components on degradation of DNA by DNase I (EC 3.1.21.1) was monitored by electrotransformation of Escherichia coil, making it possible to determine the number of plasmid molecules capable of giving rise to transformed cells. The transformation frequency increased linearly with the plasmid number within the range of 2 x 10(6) to 2 x 10(10). DNA degradation was reduced by one order of magnitude in the presence of 0.05% (w.v(-1)) maltol or 1 mM putrescine. Complete inhibition of degradation was observed with > or = 0.2% (w.v(-1)) maltol, > or = 0.01% (w.v(-1)) octyl gallate or > or = 0.5 mM of spermine. To monitor degradation of plant DNA during food processing, a real-time PCR system was established. The ratio of copy numbers of a potato gbss DNA fragment of 325 bp and a nested 96 bp fragment was determined. The latter served as internal reference for normalization. The system made it possible to exclude process-dependent changes of DNA concentration in the food matrix. Processing of genetically modified potatoes to dried potato sticks, crisps or flakes was studied and drying steps were shown to exert the strongest effect on DNA degradation, resulting in a drop of the ratio from 0.73 to 0.16.

Effect of food processing on the fate of DNA with regard to degradation and transformation capability in Bacillus subtilis.

Soymilk, tofu, corn masa, and cooked potato were produced from transgenic raw materials and the effect of processing on the degradation of DNA was studied. Major degrading factors were for soymilk and tofu the mechanical treatment of soaked soybeans and for corn masa and cooked potatoes the thermal treatment. In the processed foods no DNA fragments > 1.1 kb were detected. We included in our studies the effect of the size of donor DNA and length of the homologous sequence on the marker rescue transformation of B. subtilis LTH 5466, which was monitored by restoration of deleted nptII. When DNA fragments (168, 414, 658, and 792 bp) of nptII and linearized plasmid DNA (pGEM-T-1, 3168 bp and pGEM-T-2, 3792 bp) containing the 168 bp or 792 bp fragments, respectively, were used as donor DNA, it was observed that the efficiency of marker rescue decreased with decreasing length of homologous sequence. The use of a larger plasmid (pMR2, 5786 bp) containing the 792 bp fragment revealed higher efficiency of marker rescue compared to pGEM-T-2. The nptII fragments resulted in lower efficiencies compared to plasmid DNA containing the same fragment. For the 792 bp fragment and the linearized plasmid pMR2 a first-order dependency of the frequency of marker rescue transformation on the DNA concentration was observed. Based on the acquired data, the hypothetical frequency of transformation of transgenic DNA to B. subtilis in cooked potatoes was calculated to be equal to 8.5 x 10(-19) and 1.2 x 10(-27) for homologous and illegitimate recombination, respectively. These data permit to roughly estimate the time after which a person (10(8) years) or the world population (15 days) is exposed to one transformant generated by homologous recombination event, when the daily consumption per person is 130 g of cooked potatoes.

Identification and population dynamics of yeasts in sourdough fermentation processes by PCR-denaturing gradient gel electrophoresis.

Four sourdoughs (A to D) were produced under practical conditions, using a starter obtained from a mixture of three commercially available sourdough starters and baker's yeast. The doughs were continuously propagated until the composition of the microbiota remained stable. A fungi-specific PCR-denaturing gradient gel electrophoresis (DGGE) system was established to monitor the development of the yeast biota. The analysis of the starter mixture revealed the presence of Candida humilis, Debaryomyces hansenii, Saccharomyces cerevisiae, and Saccharomyces uvarum. In sourdough A (traditional process with rye flour), C. humilis dominated under the prevailing fermentation conditions. In rye flour sourdoughs B and C, fermented at 30 and 40 degrees C, respectively, S. cerevisiae became predominant in sourdough B, whereas in sourdough C the yeast counts decreased within a few propagation steps below the detection limit. In sourdough D, which corresponded to sourdough C in temperature but was produced with rye bran, Candida krusei became dominant. Isolates identified as C. humilis and S. cerevisiae were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively. The yeast species isolated from the sourdoughs were also detected by PCR-DGGE. However, in the gel, additional bands were visible. Because sequencing of these PCR fragments from the gel failed, cloning experiments with 28S rRNA amplicons obtained from rye flour were performed, which revealed Cladosporium sp., Saccharomyces servazii, S. uvarum, an unculturable ascomycete, Dekkera bruxellensis, Epicoccum nigrum, and S. cerevisiae. The last four species were also detected in sourdoughs A, B, and C.

Marker rescue studies of the transfer of recombinant DNA to Streptococcus gordonii in vitro, in foods and gnotobiotic rats.

A plasmid marker rescue system based on restoration of the nptII gene was established in Streptococcus gordonii to study the transfer of bacterial and transgenic plant DNA by transformation. In vitro studies revealed that the marker rescue efficiency depends on the type of donor DNA. Plasmid and chromosomal DNA of bacteria as well as DNA of transgenic potatoes were transferred with efficiencies ranging from 8.1 x 10(-6) to 5.8 x 10(-7) transformants per nptII gene. Using a 792-bp amplification product of nptII the efficiency was strongly decreased (9.8 x 10(-9)). In blood sausage, marker rescue using plasmid DNA was detectable (7.9 x 10(-10)), whereas in milk heat-inactivated horse serum (HHS) had to be added to obtain an efficiency of 2.7 x 10(-11). No marker rescue was detected in extracts of transgenic potatoes despite addition of HHS. In vivo transformation of S. gordonii LTH 5597 was studied in monoassociated rats by using plasmid DNA. No marker rescue could be detected in vivo, although transformation was detected in the presence of saliva and fecal samples supplemented with HHS. It was also shown that plasmid DNA persists in rat saliva permitting transformation for up to 6 h of incubation. It is suggested that the lack of marker rescue is due to the absence of competence-stimulating factors such as serum proteins in rat saliva.

Identification of Lactobacillus reuteri genes specifically induced in the mouse gastrointestinal tract.

Lactobacilli are common inhabitants of the gastrointestinal tracts of mammals and have received considerable attention due to their putative health-promoting properties. Little is known about the traits that enhance the ability of these bacteria to inhabit the gastrointestinal tract. In this paper we describe the development and application of a strategy based on in vivo expression technology (IVET) that enables detection of Lactobacillus reuteri genes specifically induced in the murine gut. A plasmid-based system was constructed containing 'ermGT (which confers lincomycin resistance) as the primary reporter gene for selection of promoters active in the gastrointestinal tract of mice treated with lincomycin. A second reporter gene, 'bglM (beta-glucanase), allowed differentiation between constitutive and in vivo inducible promoters. The system was successfully tested in vitro and in vivo by using a constitutive promoter. Application of the IVET system with chromosomal DNA of L. reuteri 100-23 and reconstituted lactobacillus-free mice revealed three genes induced specifically during colonization. Two of the sequences showed homology to genes encoding xylose isomerase (xylA) and peptide methionine sulfoxide reductase (msrB), which are involved in nutrient acquisition and stress responses, respectively. The third locus showed homology to the gene encoding a protein whose function is not known. Our IVET system has the potential to identify genes of lactobacilli that have not previously been functionally characterized but which may be essential for growth of these bacteria in the gastrointestinal ecosystem.

Monitoring the bacterial population dynamics in sourdough fermentation processes by using PCR-denaturing gradient gel electrophoresis.

Four sourdoughs (A to D) were produced under practical conditions by using a starter mixture of three commercially available sourdough starters and a baker's yeast constitutively containing various species of lactic acid bacteria (LAB). The sourdoughs were continuously propagated until the composition of the LAB flora remained stable. Two LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) systems were established and used to monitor the development of the microflora. Depending on the prevailing ecological conditions in the different sourdough fermentations, only a few Lactobacillus species were found to be competitive and became dominant. In sourdough A (traditional process with rye flour), Lactobacillus sanfranciscensis and a new species, L. mindensis, were detected. In rye flour sourdoughs B and C, which differed in the process temperature, exclusively L. crispatus and L. pontis became the predominant species in sourdough B and L. crispatus, L. panis, and L. frumenti became the predominant species in sourdough C. On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran), L. johnsonii and L. reuteri were found. The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which L. fermentum was also detected. Isolates of the species L. sanfranciscensis and L. fermentum were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively.

IL-10 producing CD14low monocytes inhibit lymphocyte-dependent activation of intestinal epithelial cells by commensal bacteria.

Intestinal epithelial cell (IEC) activation by non-pathogenic, commensal bacteria was demonstrated to require the presence of immunocompetent cells. In this study, HT-29 and CaCO-2 transwell cultures, reconstituted with CD4+ and CD8+ T cells, CD19+ B cells and CD14high monocytes, were challenged with non-pathogenic Gram negative Escherichia coli and Gram positive lactobacilli. Cytokine expression was analysed by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assays (ELISA). Expression of tumour necrosis factor alpha (TNF-alpha) and interleukin (IL)-8 mRNA in E. coli or L. sakei challenged IEC was promoted by lymphocyte populations predominantly CD4+ T cells, while monocytes failed to mediate an inflammatory cytokine response. The monocyte phenotype and function were further characterised by flow cytometry and mixed lymphocyte reaction (MLR). During the co-culture with IEC and bacterial stimulated IEC, CD14high peripheral blood monocytes acquired a CD14low CD16low phenotype with reduced expression co-stimulatory (CD80, CD86, CD58) cell surface molecules. Immunosuppressive functions of IEC conditioned CD14low monocytes were demonstrated by the predominant secretion of IL-10 and IL-IRa and their reduced potential to trigger an allogeneic lymphocyte response. In conclusion, IEC contribute to the development of CD14low CD16low monocytes with immunosuppressive function and antagonised a lymphocyte-mediated activation of the intestinal epithelium in response to intestinal and food derived bacteria. These results strengthen the hypothesis that the gut epithelium constitutes an important functional element in the regulation of mucosal immune homeostasis towards commensal bacteria.

Construction of a marker rescue system in Bacillus subtilis for detection of horizontal gene transfer in food.

A marker rescue system based on the repair of the kanamycin resistance gene nptII was constructed for use in Gram-positive bacteria and established in Bacillus subtilis 168. Marker rescue was detected in vitro using different types of donor DNA containing intact nptII. The efficiency of marker rescue using chromosomal DNA of E. coli Sure as well as plasmids pMR2 or pSR8-30 ranged from 3.8 x 10(-8) to 1.5 x 10(-9) transformants per nptII gene. Low efficiencies of ca. 10(-12) were obtained with PCR fragments of 792 bp obtained from chromosomal DNA of E. coli Sure or DNA from a transgenic potato. B. subtilis developed competence during growth in milk and chocolate milk, and marker rescue transformation was detected with frequencies of ca. 10(-6) and 10(-8), respectively, using chromosomal DNA of E. coli Sure as donor DNA. Although the copy number of nptII genes of the plant DNA exceeded that of chromosomal E. coli DNA in the marker rescue experiments, a transfer of DNA from the transgenic plant to B. subtilis was detectable neither in vitro nor in situ.