Isolation,characterisation and identification of lactic acid bacteria from bushera: a Ugandan traditional fermented beverage

One hundred and thirteen strains of lactic acid bacteria (LAB) were selected from 351 isolates from 15 samples of traditionally fermented household bushera from Uganda and also from laboratory-prepared bushera. Isolates were phenotypically characterised by their ability to ferment 49 carbohydrates using API 50 CHL kits and additional biochemical tests. Coliforms, yeasts and LAB were enumerated in bushera. The pH, volatile organic compounds and organic acids were also determined.

The LAB counts in household bushera varied between 7.1 and 9.4 log cfu ml⁻¹. The coliform counts varied between <1 and 5.2 log cfu ml⁻¹. The pH of bushera ranged from 3.7 to 4.5. Ethanol (max, 0.27%) was the major volatile organic compound while lactic acid (max, 0.52%) was identified as the dominant organic acid in household bushera.

The initial numbers of LAB and coliforms in laboratory-fermented bushera were similar; however, the LAB numbers increased faster during the first 24 h. LAB counts increased from 5.5 to 9.0 log cfu ml⁻¹ during the laboratory fermentation. Coliform counts increased from 5.9 to 7.8 log cfu ml⁻¹ at 24 h, but after 48 h, counts were less 4 log cfu ml⁻¹. Yeasts increased from 4.3 to 7.7 log cfu ml⁻¹ at 48 h, but thereafter decreased slightly. The pH declined from 7.0 to around 4.0. Lactic acid and ethanol increased from zero to 0.75% and 0.20%, respectively.

Lactic acid bacteria isolated from household bushera belonged to Lactobacillus, Streptococcus and Enterococcus genera. Tentatively, Lactobacillus isolates were identified as Lactobacillus plantarum, L. paracasei subsp. paracasei, L. fermentum, L. brevis and L. delbrueckii subsp. delbrueckii. Streptococcus thermophilus strains were also identified in household bushera. LAB isolated from bushera produced in the laboratory belonged to five genera (Lactococcus, Leuconostoc, Lactobacillus, Weissella and Enterococcus. Eight isolates were able to produce acid from starch and were identified as Lactococcus lactis subsp. lactis (four strains), Leuconostoc mesenteroides subsp. mesenteroides (one strain), Leuconostoc mesenteroides subsp. dextranicum (one strain), Weissella confusa (one strain) and L. plantarum (one strain).     

Some characteristics of lactic acid bacteria present in commercial sucuk samples

A total of 10 sucuk samples, obtained from Denizli, Turkey were analysed for some physical, chemical and microbiological characteristics. In addition, lactic acid bacteria (LAB) strains producing bacteriocin-like metabolites were isolated and identified. The production of some typical metabolites of the cultures isolated was investigated. At the end of the research, the average values of the pH, water and fat content were 5.1, and 37.2% and 30.5%, respectively. Microbiological analyses results were determined: average 8.34 log CFU/g TAMB, 8.91 log CFU/g LAB (at the MRS agar) and average 8.25 log CFU/g LAB (at the Elliker's lactic agar). The average counts of yeast-mould, coliform and Enterobacteriaceae were found to be 5.0 log CFU/g, 3.28 log CFU/g and 3.27 log CFU/g, respectively. In this study, counts of yeast-mould in the two samples, coliform counts in the five samples, and Enterobacteriaceae counts in the three samples were < 1.0 log CFU/g. A total of 6 of 100 LAB isolates obtained from the sucuk samples were found as a strain producing bacteriocin-like metabolites. These 6 strains were identified as follows; 3 strains Lactobacillus plantarum and 3 strains Pediococcus pentosaceus. According to the findings, these strains have the potential to be used as a sucuk starter culture. Additionally, acid and flavour compounds, other undesirable metabolite-producing activities of the strains, were determined in the model system. From these results it was concluded, after the determination of the toxicological properties, that the 4 strains of LAB identified (L. plantarum 13 P. pentosaceus 15 P. pentosaceus 74 and P. pentosaceus 75) would be useful as the starter and protective culture in the processing of the sucuk and similar fermented products.     

Characterization of lactic acid bacteria isolated from a Thai low-salt fermented fish product and the role of garlic as substrate for fermentation

Lactic acid bacteria (LAB) isolated from raw materials (fish, rice, garlic and banana leaves) and processed som-fak (a Thai low-salt fermented fish product) were characterized by API 50-CH and other phenotypic criteria. Lactococcus lactis subsp. lactis and Leuconostoc citreum were specifically associated with fish fillet and minced fish, Lactobacillus paracasei subsp. paracasei with boiled rice and Weisella confusa with garlic mix and banana leaves. In addition, Lactobacillus plantarum, Lactobacillus pentosus and Pediococcus pentosaceus were isolated from raw materials. A succession of aciduric, homofermentative lactobacillus species, dominated by Lb. plantarum/pentosus, was found during fermentation. In total, 9% of the strains fermented starch and 19% fermented garlic, the two main carbohydrate components in som-fak. The ability to ferment garlic was paralleled by a capacity to ferment inulin. An increased percentage of garlic fermenting strains was found during fermentation of som-fak, from 8% at day 1 to 40% at day 5. No starch fermenting strains were isolated during fermentation. Three mixed LAB cultures, composed of either starch fermenting Lc. lactis subsp. lactis and Lb. paracasei subsp. paracasei, or garlic fermenting Lb. plantarum and Pd. pentosaceus, or a combination of these strains were inoculated into laboratory prepared som-fak with or without garlic. In som-fak without garlic, pH was above 4.8 after three days, irrespective of addition of mixed LAB cultures. The starch fermenting LAB were unable to ferment som-fak and sensory spoilage occurred after three days. Fermentation with the combined mix of starch and garlic fermenting strains led to production of 2.5% acid and a decrease in pH to 4.5 in two days. The fermentation was slightly slower with the garlic fermenting strains alone. This is the first report describing the role of garlic as carbohydrate source for LAB in fermented fish products.     

Antilisterial activity of lactic acid bacteria isolated from "Alheiras" (traditional Portuguese fermented sausages): In situ assays

A total of 226 lactic acid bacteria (LAB) isolated from “Alheira”, a traditional Portuguese fermented sausage, were screened for antagonistic activity against some pathogenic microorganisms, including Listeria monocytogenes. The objective was to isolate LAB with antibacterial activity from “Alheiras” and to select strains that could be used in “Alheira” production. Isolates displaying antibacterial activity against Listeria innocua and L. monocytogenes were investigated for the nature of the antibacterial compounds active against these microorganisms. Results showed that two LAB cultures retained activity in the supernatants after neutralization and catalase treatment. These two strains were both identified as Pediococcus pentosaceus. The final aim of this work was to test the antilisterial activity of these two strains during storage of “Alheira mass” (sterilized), at 4 °C. The growth of L. innocua population was significantly suppressed in the paste of “Alheira” when the samples were co-inoculated with the LAB strains, in comparison with the paste only inoculated with L. innocua or co-inoculated with a bacteriocin negative strain of Ped. pentosaceus (ca. 1 × 10⁷ CFU/g after 28 days of incubation).     

自然发酵中式香肠加工成熟过程中乳酸菌菌群研究

研究自然发酵川味、广味香肠加工成熟过程中乳酸菌菌群,旨在探明乳酸菌在香肠加工成熟过程中菌群分布及其变化规律。结果表明:(1)发酵初期两种香肠中乳酸菌数量均在104cfu/g水平,2d后不断增加,广味香肠中乳酸菌数量增加更快,第12天两者趋于一致,达到106cfu/g,第21天数量均跃升至107cfu/g,第30天达最大值,接近108cfu/g,40d后数量开始下降。(2)香肠中水分含量和pH值在加工成熟过程中总体呈下降的趋势,其乳酸菌数量变化与pH值和水分含量变化具有一定的相关性。(3)分离出的115株菌株经初步鉴定,杆菌80株,球菌35株,均为G ;从3个不同时期选出14株有代表性的菌株进行生理生化鉴定,拟确定S1-4、S3-2为清酒乳杆菌(Lactobacillus sakei),G21-10、C50-6、G50-4为戊糖乳杆菌(L.Pentosus),C21-6、C50-8、G50-9为植物乳杆菌(L.plantarum),其他的仅能鉴定到属,有乳杆菌、乳球菌。     

Antilisterial activity of lactic acid bacteria inoculated on cooked ham

This study was conducted to evaluate the ability of Lactobacillus sakei 1, a bacteriocin-producing (bac⁺) lactic acid bacterium (LAB), isolated from Brazilian fresh pork sausage to inhibit two Listeria monocytogenes strains (serotypes 4b and 1/2a) on cooked, sliced vacuum-packaged ham. L. sakei ATCC 15521 was used as a non-bacteriocin producer (bac⁻). L. monocytogenes (ca. 2 log CFU/mL) and LAB (ca. 6 log CFU/ml) were inoculated on the sterilized ham, vacuum-sealed and incubated at 8 °C for 10 days. A treatment with the bacteriocin Chrisin (UI/ml) was included. Both L. monocytogenes strains were significantly inhibited in the presence of either bac⁺ and bac⁻ LAB in comparison to the control (L. monocytogenes alone). Using a bacteriocinogenic strain of LAB did not offer an additional barrier to listerial growth in the studied meat system. The application of Chrisin did not affect at all the growth of L. monocytogenes.     

Enhanced lactic acid fermentation of silage by the addition of green tea waste

The effects of green tea waste (GTW) addition on the ensiling of forage were investigated. Wet and dried GTW added at 10, 50, 100 and 200 g kg^?1 of fresh matter (FM) and at 2, 10 and 20 g kg^?1 FM, respectively, decreased pH and increased lactic acid concentration of the silages, whereas the butyric acid concentration and ammonia nitrogen content, as a proportion of a total nitrogen, were lowered, compared with silage without additives (control). To investigate the effect of GTW‐associated LAB on silage fermentation, wet GTW was sterilized by autoclaving or gamma irradiation and added at 50 g kg^?1 FM. The silages made with sterilized GTW showed higher lactic acid concentrations, and lower pH and butyric acid concentrations than controls. The counts of lactic acid bacteria (LAB) were higher in silages made with sterilized GTW than control until 10 days after ensiling. The enhanced lactic acid fermentation was not found when green tea polyphenols (GTP) were added. These data suggested that GTW could enhance LAB growth and lactic acid production of silage, particularly when added at 50 g kg^?1 FM in a wet form or at the equivalent in a dry form. Although neither GTW‐associated LAB nor GTP accounted for the enhancement of lactic acid fermentation, GTW would possibly supply some nutrients which are heat‐stable and effective for LAB growth during silage fermentation. Copyright © 2004 Society of Chemical Industry     

ASSESSMENT OF POTENTIAL PROBIOTIC PROPERTIES OF LACTIC ACID BACTERIA AND YEAST STRAINS

Four lactic acid bacteria (LAB) and three yeast strains isolated from a traditional Bulgarian cereal-based fermented beverage were assessed for potential probiotic properties. Acid and bile resistance, antipathogenic activity and antibiotic resistance of the strains were evaluated. Tolerance to low pH values (2.0-3.0) and high bile concentrations (0.2-2.0%) of the LAB and yeast strains varied, but all strains kept viable throughout the experiments. Antagonistic activity towards most of the eight test-pathogens was observed for one LAB (Lactobacillus plantarum B28) and two yeast strains (Candida rugosa Y28 and Candida lambica Y30). Antibiotic resistance (39 antibiotics) of the LAB strains was variable, but showed their potential for therapeutic application.     

STUDIES ON SOME TECHNOLOGICAL PROPERTIES OF PREDOMINANT LACTIC ACID BACTERIA ISOLATED FROM NIGERIAN FERMENTED FOODS

In a programme to develop starter cultures for improving the safety and quality of traditional fermented foods in Africa, a study was conducted on lactic acid bacterial (LAB) strains isolated from the traditional selected Nigerian fermented foods kunun-zaki, wara, nono, iru. The LAB strains representing the dominating population of each product, were identified as Enterococcus faecalis (1), Pediococcus pentosaceus (4), and Lactobacillus fermentum (19). All the strains grew at 450°C, an observation which could be attributed to the tropical environment of the fermented foods. They produced a moderate spectrum of enzymes of relevance to food processing, and exhibited similar patterns of enzymatic activity between species, but generally showed weak esterase and lipase activities as compared with peptidases. While no proteinase activity was detected, most strains showed high galactosidase activity. Two strains showed ability to degrade phytic acid. None of the strains produced any detectable bacteriocins or biogenic amines under the test conditions used, and all were unable to hydrolyse bile salt. Eleven (45.8%) of the strains coagulated skim milk at 30°C within 24-36 h, and at 37°C within 12-20 h together with a moderate drop in pH. The results are discussed to highlight the relevance of technological features of starter cultures in food processing in the African environment.     

Production of potentially probiotic beverages using single and mixed cereal substrates fermented with lactic acid bacteria cultures

In the present work, single and mixed cereal substrates were fermented with lactic acid bacteria to study and compare the effect of the media formulation on fermentation parameters. Three cereal flours namely malt, barley and barley mixed with malt (barley–malt) were selected and fermented with two probiotic strains: Lactobacillus plantarum (NCIMB 8826) and Lactobacillus acidophilus (NCIMB 8821). The effect of the single and mixed cereal flour suspensions on the fermentation of these two strains of lactic acid bacteria (LAB) was studied at an incubation temperature of 30 °C for 28 h. It was found that the LAB growth was enhanced in media containing malt and significant amounts of lactic acid were produced (0.5–3.5 g/L). A cell concentration between 7.9 and 8.5 Log₁₀ CFU/mL and a pH below 4.0 was achieved within 6 h of fermentation. Though the cell populations in the mixed culture fermentations of mixed substrates were similar to the ones obtained with single cereal flours, significant differences in the production of lactic acid were observed. These results suggest that the functional and organoleptic properties of these cereal-based probiotic drinks could be considerably modified through changes in the substrate or inocula composition.     

Inhibition of spoilage lactic acid bacteria by lysozyme during wine alcoholic fermentation

The efficacy of lysozyme against indigenous lactic acid bacteria (LAB) and four inoculated spoilage LAB cultures was investigated in laboratory scale Chardonnay winemaking trials (at pH 3.8). These LAB cultures included Lactobacillus kunkeei, Lactobacillus brevis, Pediococcus parvulus , and Pediococcus damnosus . Three concentrations of lysozyme were used: 0, 125 and 250 mg/L. Alcoholic fermentation of the grape juice was carried out at 20±0.5°C using Saccharomyces cerevisiae . Lysozyme did not have any negative impact on yeast growth and sugar reduction. This enzyme was found to be very effective in inhibiting the growth of all four LAB cultures investigated. Under the given experimental conditions, as high as an 8 log cell reduction was obtained for some of the strains. The acetic acid production by L. brevis and L. kunkeei was significantly reduced in the treatments with 125 and 250 mg/L lysozyme added ( P < 0.01). The effect of lysozyme on the cells of the LAB cultures was examined under a scanning electron microscope. It is evident that lysozyme had a detrimental impact on the cells of these cultures. Based on these observations, it is concluded that lysozyme may be a useful tool for winemakers to control the growth of spoilage LAB and to reduce the production of volatile acids. The addition of lysozyme may also prevent the increase of volatile acidity during stuck/sluggish alcoholic fermentation. This tool is particularly useful in high pH wines where SO₂ is less effective.     

Evolution and identification of lactic acid bacteria isolated during the ripening of Sardinian sausages

Lactic acid bacteria (LAB) were isolated during the production and the ripening of Sardinian sausage, a typical Italian dry fermented sausage. Samples were taken at different stages, and 112 strains were isolated. The isolates were characterized using the micromethod proposed by Font de Valdez et al. [Font de Valdez, G., Savoy de Giori, G., Oliver, G., & De Ruiz Holgado, A. P. (1993). Development and optimization of an expensive microsystem for the biochemical characterization of lactobacilli. Microbiologie Aliments Nutrition, 11, 215–219]. Schillinger and Lücke’s [Schillinger, U., & Lücke, F. K. (1987). Identification of lactobacilli from meat and meat products. Food Microbiology. (4), 199–208] scheme and the biochemical patterns given by Bergey’s Manual of Systematic Bacteriology [Bergey’s Manual of Systematic Bacteriology (1986). Baltimore: William and Wilkins] were used for preliminary identification. A PCR-based method was then used to confirm the results. LAB were the dominant flora during ripening. They consisted mainly of homofermentative mesophilic rods. Lactobacillus sakei (43,3%), Lactobacillus plantarum (16,6%) and Lactobacillus curvatus (13,3%) were the main isolates. The results of the biochemical identification methods agreed well with those of PCR-based identification (91% agreement).     

Effect of lactic acid bacteria inoculant and beet pulp addition on fermentation characteristics and in vitro ruminal digestion of vegetable residue silage

The objective of this study was to determine the effect of beet pulp (BP) and lactic acid bacteria (LAB) on silage fermentation quality and in vitro ruminal dry matter (DM) digestion of vegetable residues, including white cabbage, Chinese cabbage, red cabbage, and lettuce. Silage was prepared using a small-scale fermentation system, and treatments were designed as control silage without additive or with BP (30% fresh matter basis), LAB inoculant Chikuso-1 (Lactobacillus plantarum, 5 mg/kg, fresh matter basis), and BP + LAB. In vitro incubation was performed using rumen fluid mixed with McDougall's artificial saliva (at a ratio of 1:4, vol/vol) at 39°C for 6 h to determine the ruminal fermentability of the vegetable residue silages. These vegetable residues contained high levels of crude protein (20.6-22.8% of DM) and moderate levels of neutral detergent fiber (22.7-33.6% of DM). In all silages, the pH sharply decreased and lactic acid increased, and the growth of bacilli, coliform bacteria, molds, and yeasts was inhibited by the low pH at the early stage of ensiling. The silage treated with BP or LAB had a lower pH and a higher lactic acid content than the control silage. After 6 h of incubation, all silages had relatively high DM digestibility (38.6-44.9%); in particular, the LAB-inoculated silage had the highest DM digestibility and the lowest methane production. The vegetable residues had high nutritional content and high in vitro DM digestibility. Also, both the addition of a LAB inoculant and moisture adjustment with BP improved the fermentation quality of the vegetable residue silages. In addition, LAB increased DM digestibility and decreased ruminal methane production.     

Comparison of the microflora isolated from spoiled cold-smoked salmon from three smokehouses

The microflora on spoiled, sliced and vacuum packed, cold-smoked salmon from three smokehouses was quantified and characterized in two independent experiments. Large variations in the microflora were observed both within (i.e. among vacuum packs from the same batch) and among the smokehouses. Lactic acid bacteria dominated the microflora, which reached 10⁷ cfu g⁻¹. Total viable counts of microorganisms alone were not related to quality, though spoilage characteristics were typical for microbiological spoilage. Among the lactic acid bacteria, Lactobacillus curvatus (ca 52–55%) was the most common species in both experiments with Lactobacillus saké, Lactobacillus plantarum, Carnobacterium spp. and Leuconostoc spp. present in smaller numbers. In some cases, large numbers of Enterobacteriaceae were also present and identified species were Serratia liquefaciens, Enterobacter agglomerans and Hafnia alvei. The microflora on cold-smoked salmon appeared to be related to the source of contamination i.e. the raw material and/or the smokehouse rather than being specific for the product, thus rendering the identification of the specific spoilage organisms difficult.     

Selection of tropical lactic acid bacteria for enhancing the quality of maize silage

The objective of this study was to select lactic acid bacteria (LAB) strains isolated from silage and assess their effect on the quality of maize silage. The LAB strains were inoculated into aqueous extract obtained from maize to evaluate their production of metabolites and pH reduction. The ability to inhibit the pathogenic and silage-spoilage microorganisms’ growth was evaluated. Nine LAB strains that showed the best results were assessed in polyvinyl chloride experimental silos. The inoculation of the LAB strains influenced the concentration of lactic and acetic acids and the diversity of Listeria. The inoculation of silages with Lactobacillus buchneri (UFLA SLM11 and UFLA SLM103 strains) resulted in silages with greater LAB populations and improvements after aerobic exposure. The UFLA SLM11 and SLM103 strains identified as L. buchneri showed to be promising in the treatment of maize silage.     

Physiological properties of Lactobacillus paracasei, L. danicus and L. curvatus strains isolated from Estonian semi-hard cheese

Growth and survival of Lactobacillus paracasei (six strains), L. danicus sp. nov. (four isolates, two strains) and L. curvatus (two strains) from semi-hard Estonian cheeses were comparatively studied in different environmental conditions of relevance for their growth in cheese and survival in gastric environment. Maximum specific growth rates for L. paracasei strains varied between 0.40 and 0.57 h⁻¹, and all strains were tolerant to low water activities, heating at 60 °C for 30 min and pH 3. The newly discovered genetically distinct species L. danicus was characterized by low maximum growth rates (0.26–0.38 h⁻¹) and low temperature optimum (<30 °C). It was acid and heat sensitive and inhibited at salt concentrations from 4% and water activities below 0.93. L. curvatus was characterized by the highest growth rates (0.65–0.70 h⁻¹), tolerance to high NaCl concentrations, but sensitivity to heating, bile salts and low pH. The study showed that genetically different LAB species isolated from cheese could be distinguished by simple cultivation experiments.     

Effects of indigenous starter cultures on the microbial and physicochemical characteristics of Urutan, a Balinese fermented sausage

Urutan is a Balinese traditional dry fermented sausage prepared from lean pork and various kinds of spice. Urutan is different from the European sausages, because it is fermented under warm condition with fluctuating temperatures of approximately 25 degrees C at night to 50 degrees C during sun drying. In this study, two of the 71 strains of lactic acid bacteria (LAB) isolated from natural urutan fermentation were used as starter cultures: Lactobacillus plantarum U201, the dominant LAB, and Pediococcus acidilactici U318, a bacteriocin producer. A soft urutan with yellowish brown color was produced using these strains as multiple starters. The starter cultures grew in characteristic succession which reconstructed the natural fermentation process. Lactobacilli were dominant until 48 h fermentation and pediococci dominated at the later stage of fermentation. Proliferation of starter cultures produced lactic acid which resulted in the decrease in pH and coagulation of soluble protein in urutan. Both strains could eliminate the Enterobacteriaceae in urutan after 24 h fermentation, and could suppress and eliminate the occurrence of micrococci at 120 h fermentation. By using a single starter culture, no succession was observed to occur in urutan and the time of elimination of Enterobacteriaceae was delayed. Thus, the strains of L. plantarum U201 and P. acidilactici U318 have great potential for use as multiple starter cultures in urutan fermentation.     

Preparation of marine silage of swordfish, ray and shark visceral waste by lactic acid bacteria

The goal of the present work was to study the efficacy of several lactic acid bacteria (LAB) as bio-silage inoculants of swordfish, ray and shark viscera by-products. A sterilised medium was initially used as a model system for assessing the potential of these microorganisms in batch and fed-batch cultures with re-neutralisation. In all cases, batch cultivations without re-neutralisation led to the highest production and yields of the main metabolites of LAB fermentation (lactic and acetic acids). The dynamics of these metabolites followed a conversion pattern from lactic to acetic acid with a final joint concentration over 16 g/L and final pH lower than 4.5. Both productions were modelled by means of logistic modified equations. In addition, the capability of LAB to ferment the fish visceral wastes was always high and easily reproducible. Finally, the results obtained for non-sterilised fermentations with Lactobacillus casei CECT 4043 were similar to those obtained for sterilised media, and a stable material was obtained after 72 h of culture.     

Behavior of Escherichia coli O157:H7 during the manufacture and ripening of Fontina Protected Designation of Origin cheese

This study was conducted to describe the cheese-making procedure of Fontina Protected Designation of Origin (PDO) cheese and to evaluate the behavior of Escherichia coli O157:H7 during cheese manufacture and ripening. The study was divided into 2 phases: the production of Fontina PDO cheese was monitored at 3 different dairies in the Aosta Valley and an E. coli O157 challenge was conducted at a fourth dairy. The dairies employ different commercial starter cultures for cheese making. The growth of lactic acid bacilli (LAB) and the decrease in pH were slower in the first hours and the LAB concentrations were overall higher in dairy A than in the other 2 dairies. The pH remained substantially unchanged during ripening (range 5.2 to 5.4) in all dairies. Water activity remained constant at around 0.98 until d 21, when it decreased to around 0.97 until d 80 in dairies A and B and 0.95 in dairy E. Whole raw cow milk was used for making Fontina cheese according to the standard procedure. For the experimental production, the milk was inoculated with E. coli O157:H7 at a concentration of approximately 5 log₁₀ cfu/mL and commercial starter cultures were used according to the Fontina PDO regulation. An increase of 2.0 log₁₀ cfu/g in E. coli O157:H7 was observed during the first 9.5 h of cheese making, followed by a decrease at 46 h when pH decreased to 5.4 in all trials. Fresh cheeses were salted and held at 10°C for ripening for 80 d. Water activity was decreased to 0.952 at the end of the ripening stage. The LAB concentrations declined gradually; this trend was more marked for the lactobacilli than either the thermophilic or the mesophilic lactococci. The increase in LAB count and the decrease in pH in the first hours did not seem to affect E. coli O157 growth. Ripening was found to inhibit pathogen survival, however, as seen in the reduction of 3 log₁₀ from the maximum concentration measured during the earlier stages of production.     

Application of molecular methods for the classification and identification of lactic acid bacteria

Phylogenetic analysis has revealed that the typical lactic acid bacteria (LAB) belong to the Gram-positive bacteria with a low guanine plus cytosine DNA content. The genera Lactobacillus, Leuconostoc and Pediococcus can be traditionally differentiated on the basis of morphological and physiological properties but phylogentically they are intermixed. The former genus Streptococcus has been split up into the four genera Enterococcus, Lactococcus, Streptococcus and Vagococcus.

The traditional phenotypic identification of LAB is rather tedious and not always reliable. Nucleic acid probe technology may be an alternative for a faster and more reliable differentiation. 16S or 23S rRNA-targeted oligonucleotides have been used for the specific identification of LAB. It is even now possible to identify various LAB in fermented food, without any preceding enrichment or cultivation step, at the species level within one working day. Application of fluorescently labelled oligonucleotides also allowed the in situ detection and identification of whole cells of lactococci and enterococci. DNA restriction fragment analysis and ribotyping have been used to distinguish LAB at the strain level or groups of closely related strains. Specific hybridization probes are also useful tools for the identification of genetically modified nucleic acids and the corresponding organisms.