Melanie Huch

Enterococci as probiotics and their implications in food safety.

Enterococci belong to the lactic acid bacteria (LAB) and they are of importance in foods due to their involvement in food spoilage and fermentations, as well as their utilisation as probiotics in humans and slaughter animals. However, they are also important nosocomial pathogens that cause bacteraemia, endocarditis and other infections. Some strains are resistant to many antibiotics and possess virulence factors such as adhesins, invasins, pili and haemolysin. The role of enterococci in disease has raised questions on their safety for use in foods or as probiotics. Studies on the incidence of virulence traits among enterococcal strains isolated from food showed that some can harbour virulence traits, but it is also thought that virulence is not the result of the presence of specific virulence determinants alone, but is rather a more intricate process. Specific genetic lineages of hospital-adapted strains have emerged, such as E. faecium clonal complex (CC) 17 and E. faecalis CC2, CC9, CC28 and CC40, which are high risk enterococcal clonal complexes. These are characterised by the presence of antibiotic resistance determinants and/or virulence factors, often located on pathogenicity islands or plasmids. Mobile genetic elements thus are considered to play a major role in the establishment of problematic lineages. Although enterococci occur in high numbers in certain types of fermented cheeses and sausages, they are not deliberately added as starter cultures. Some E. faecium and E. faecalis strains are used as probiotics and are ingested in high numbers, generally in the form of pharmaceutical preparations. Such probiotics are administered to treat diarrhoea, antibiotic-associated diarrhoea or irritable bowel syndrome, to lower cholesterol levels or to improve host immunity. In animals, enterococcal probiotics are mainly used to treat or prevent diarrhoea, for immune stimulation or to improve growth. From a food microbiological point of view, the safety of the bacteria used as probiotics must be assured, and data on the major strains in use so far indicate that they are safe. The advantage of use of probiotics in slaughter animals, from a food microbiological point of view, lies in the reduction of zoonotic pathogens in the gastrointestinal tract of animals which prevents the transmission of these pathogens via food. The use of enterococcal probiotics should, in view of the development of problematic lineages and the potential for gene transfer in the gastrointestinal tract of both humans and animals, be carefully monitored, and the advantages of using these and new strains should be considered in a well contemplated risk/benefit analysis.

Diversity of lactic acid bacteria from Hussuwa, a traditional African fermented sorghum food

The diversity of lactic acid bacteria associated with Hussuwa fermentation, a Sudanese fermented sorghum food, was studied using a polyphasic taxonomical approach. Predominant strains could be well characterised based on a combination of phenotypic tests and genotypic methods such as ARDRA, rep-PCR and RAPD-PCR, as well as 16S rRNA gene sequencing of representative strains. Thus, the majority (128 of 220, 58.3%) of strains exhibited phenotypic properties typical of heterofermentative lactobacilli and of these, 100 strains were characterised more closely using the genotyping methods. The majority (97/100) strains could be characterised as Lactobacillus fermentum strains. Seventy-two of 220 strains (32.7%) showed phenotypic properties that are characteristic of pediococci. Of 41 selected strains investigated by genotyping techniques, 38 (92.7%) could be characterised as Pediococcus acidilactici strains, while three (7.3%) could be characterised as Pediococcus pentosaceus strains. The Hussuwa fermentation thus appears to be dominated by L. fermentum strains and P. acidilactici strains. For this reason, we selected representative and predominant strains as potential starter cultures for Hussuwa fermentation. These strains, L. fermentum strains BFE 2442 and BFE 2282 and P. acidilactici strain BFE 2300, were shown on the basis of RAPD-PCR fingerprinting to predominate in a model fermentation when used as starter cultures inoculated at 1 x 10(6) CFU/g and to lower the pH of the fermentation to below pH 4.0 within 48 h. These cultures should be studied for further development as starter preparations in pilot scale studies in actual field fermentations.

Quantification of Slackia and Eggerthella spp. in Human Feces and Adhesion of Representatives Strains to Caco-2 Cells

Eggerthella and Slackia spp. are gut associated bacteria that have been suggested to play roles in host lipid and xenobiotic metabolism. A quantitative PCR method for the selective enumeration of bacteria belonging to either the genus Eggerthella or Slackia was developed in order to establish the numbers of these bacteria occurring in human feces. The primers developed for selective amplification of these genera were tested first in conventional PCR to test for their specificity. Representative species of Eggerthella and Slackia, as well as closely related genera of the Coriobacteriia, were included in the investigation. The selected primers were shown to be capable of specific amplification of species of the genera Eggerthella and Slackia, but not all species of the genera may be amplified by the respective primers. Their use in qPCR experiments to assess the levels of Slackia equolifaciens and Eggerthella lenta in the feces of 19 human volunteers showed they occurred at mean counts of 7 × 105 and 3.1 × 105 CFU/g for Eggerthella spp. and Slackia spp., respectively. Electron microscopy investigations showed that while E. lenta cells exhibited slender and very regular shaped rods, Slackia cells showed a remarkably pleomorphic phenotype. Both species did not appear to have fimbriae or pili. Some S. equolifaciens cells showed a characteristic “ribbon” of presumably extracellular material around the cells, particularly at the areas of cell division. The two species also differed markedly in their adhesion behavior to Caco-2 cells in cell culture, as E. lenta DSMZ 15644 showed a high adhesion capacity of 74.2% adherence of the bacterial cells added to Caco-2 cells, while S. equolifaciens DSM 24851T on the other hand showed only low adhesion capability, as 6.1% of bacterial cells remained bound. Speculatively, this may imply that the ecological compartments where these bacteria reside in the gut may be different, i.e., E. lenta may be associated more with the gut wall, while Slackia may be free living in the lumen.

Anti-listerial activity of bacteriocin-producing Lactobacillus curvatus CWBI-B28 and Lactobacillus sakei CWBI-B1365 on raw beef and poultry meat.

Aim:  The study aimed to evaluate the effect of the bacteriocins produced by Lactobacillus sakei CWBI‐B1365 and Lactobacillus curvatus CWBI‐B28 on the growth and survival of Listeria monocytogenes in raw beef and poultry meat.

Stability of Individual Maillard Reaction Products in the Presence of the Human Colonic Microbiota

Maillard reaction products (MRPs) are taken up in substantial amounts with the daily diet, but the majority are not transported across the intestinal epithelium. The aim of this study was to obtain first insights into the stability of dietary MRPs in the presence of the intestinal microbiota. Four individual MRPs, namely, N-ε-fructosyllysine (FL), N-ε-carboxymethyllysine (CML), pyrraline (PYR), and maltosine (MAL), were anaerobically incubated with fecal suspensions from eight human volunteers at 37 °C for up to 72 h. The stability of the MRPs was measured by HPLC with UV and MS/MS detections. The Amadori product FL could no longer be detected after 4 h of incubation. Marked interindividual differences were observed for CML metabolism: Depending on the individual, at least 40.7 ± 1.5% of CML was degraded after 24 h of incubation, and the subjects could thus be tentatively grouped into fast and slow metabolizers of this compound. PYR was degraded by 20.3 ± 4.4% during 24 h by all subjects. The concentration of MAL was not significantly lowered in the presence of fecal suspensions. In no case could metabolites be identified and quantified by different mass spectrometric techniques. This is the first study showing that the human colonic microbiota is able to degrade selected glycated amino acids and possibly use them as a source of energy, carbon, and/or nitrogen.

Use of Lactobacillus strains to start cassava fermentations for Gari production

Two Lactobacillus strains, Lactobacillus plantarum BFE 6710 and Lactobacillus fermentum BFE 6620, were used to start cassava fermentations in a pilot study under field production conditions in Kenya, to determine their potential to establish themselves as predominant lactobacilli during the fermentation. Predominant strains from three fermentations were isolated throughout the 48 h fermentation period. The use of these strains in high numbers clearly resulted in 1 to 2 log higher lactic acid bacteria (LAB) counts over the course of the fermentation when compared to the uninoculated control. 178 predominant LAB isolates were grouped based on their phenotypic characteristics, and were characterised to strain level by RAPD-PCR, followed by PFGE strain typing. Overall, L. plantarum strains represented the majority of the isolates, followed by Weissella confusa and Lactococcus garvieae strains. The results of RAPD-PCR and PFGE strain typing techniques indicated that L. plantarum BFE 6710 was successful in asserting itself as a predominant strain. In contrast, L. fermentum BFE 6620 failed to establish itself as a predominant organism in the fermentation. The success of the L. plantarum strains to predominate in the cassava fermentation demonstrates the potential for development of Lactobacillus starter cultures to industrialise the Gari production process.

Genetic analysis of the plantaricin EFI locus of Lactobacillus plantarum PCS20 reveals an unusual plantaricin E gene sequence as a result of mutation

Lactobacillus plantarum strains produce a variety of chromosomally encoded bacteriocins and often multiple bacteriocins are encoded by a single strain. In this study, the genetic loci for bacteriocin production of L. plantarum strains BFE 5092 and PCS20 were studied. These strains were investigated for their possible application as protective cultures in food preservation. The bacteriocin locus of strain BFE 5092 showed remarkable similarity to the plantaricin loci previously described for L. plantarum strains C11 and WCFS1. However, the locus of the L. plantarum PCS20 strain was unusual in that it showed an interesting mutation as a result of deletions within the plnE gene. These deletions led to a hypothetically produced peptide which is 2 amino acids shorter than plantaricin E. Furthermore, it differs by 24 amino acids, while it shares 30 identical amino acids i.e., 15 at the amino end and 15 at the carboxyl end of the hypothetical peptide. As a consequence, the amino acid sequence is changed such that a double-glycine-type leader peptide would not be encoded. This raises the question whether a functional peptide is being produced, even though RT-PCR studies showed that the plnE gene is obviously expressed. Furthermore, a transposase gene was located upstream of the plnEFI gene cluster and was inserted into a bacteriocin regulatory gene, the histidine protein kinase gene. Taken together, these facts indicate a loss of plantaricin gene function in L. plantarum PCS20 as a result of transposition and mutation.

Diversity of bacillus species isolated from okpehe, a traditional fermented soup condiment from Nigeria.

The diversity of Bacillus species isolated from the fermented soup condiment okpehe in Nigeria was studied using a combination of phenotypic and genotypic methods. Fifty strains presumptively characterized as Bacillus spp. using the API 50 CHB test were further identified by PCR of randomly amplified polymorphic DNA (RAPD) and by amplified ribosomal DNA restriction analysis (ARDRA) genotyping methods. ARDRA fingerprinting with HhaI, HinfI, and Sau3AI restriction enzymes did not allow successful differentiation between the Bacillus species, except for distinguishing B. cereus from other Bacillus species. This problem was overcome with the combination of RAPD PCR and ARDRA genotypic fingerprinting techniques. Sequencing of 16S rRNA genes of selected strains representative of the major clusters revealed that the Bacillus strains associated with this fermentation were B. subtilis, B. amyloliquefaciens, B. cereus, and B. licheniformis (in decreasing order of incidence). The presence of enterotoxin genes in all B. cereus strains was demonstrated by multiplex PCR. The high incidence of detection (20%) of possibly pathogenic B. cereus strains that contained enterotoxin genes indicated that these fermented foods may constitute a potential health risk.

A genus‐specific PCR method for differentiation between Leuconostoc and Weissella and its application in identification of heterofermentative lactic acid bacteria from coffee fermentation

A genus-specific PCR analysis method was developed for a rapid and reliable differentiation between the two heterofermentative lactic acid bacteria genera Leuconostoc and Weissella. Primer sets specific for target regions of the 16S rRNA genes were designed and the specificity of the PCR was evaluated using the type strains of 13 species of Leuconostoc and 11 species of Weissella. In addition, the newly developed genus-specific PCR analysis was applied to characterize 72 lactic acid bacteria (LAB) strains isolated from coffee fermentation and which were presumptively classified as Leuconostoc or Weissella species. Additionally, a total of 34 LAB isolates from various other fermented foods were included. The investigations of these strains were conducted to test the effectiveness of correct characterization of field isolates using the genus-specific PCR approach. The correct assignment to one of these two genera by the application of the genus-specific primers was confirmed by further identifying the strains using repetitive extragenic palindromic-PCR and 16S rRNA gene sequencing.

Characterization and Technological Properties of Lactic Acid Bacteria in the Production of “Sorghurt,” a Cereal-Based Product

A total of 57 predominant LAB strains isolated from cassava and maize grains fermentation processes for fufu and ogi were identified using phenotypic and genomic fingerprinting methods such as rep-PCR and ARDRA. They were divided into facultatively heterofermentative rods (26.3%), obligately heterofermentative rods (31.6%), and tetrad forming homofermentative cocci (42.1%). Selected strains were further identified by sequencing the 16S rDNA gene. Technological studies such as acidification, hydrogen peroxide production, starch hydrolysis, enzymatic activities, degradation of oligosaccharides, and in vitro adherence properties were carried out. Lactobacillus plantarum strains demonstrated better and rapid acid production capability, followed by the Pediococcus strains, while L. fermentum strains exhibited slower acid production. Hydrogen peroxide production was observed among the LAB groups. The test strains utilized the indigestible sugars raffinose and stachyose. Only L. pentosus demonstrated high amylase activity comparable to that of L. amylovorus DSM 20531. Lactobacillus plantarum and L. fermentum strains showed high β-glucosidase activity. Six strains were selected as starter cultures. The strains were tolerant to acidic pH levels and bile salt. The yoghurt-like “sorghurt” produced using the selected starter cultures had a final pH of less than pH 4.0, and a viable count of less than 5.5 Log10cfu/mL at the end of a 24 h fermentation period. The samples were generally acceptable to the taste panelists. The starter organisms demonstrated varying degree of adherence to HT29 MTX cell line. Therefore, employing functionally defined LAB strains may be one practical approach for incorporating health-promoting features into appropriate food products suitable for targeted population.