Measurement and prediction of the susceptibility of lager beer to spoilage by lactic acid bacteria

J.L. FERNANDEZ AND W.J SIMPSON. 1995. The ability of 14 strains of hop-resistant lactic acid bacteria ( Lactobacillus spp., Pediococcus spp.) to grow in 17 different lager beers was assessed using a biological challenge test. All beers were spoiled by at least one strain: all strains could grow in at least one beer. The sensitivity of different beers to spoilage by lactic acid bacteria varied. Spoilage potential could be described in the form of a 'Resistance to Spoilage Value' (RSV) based on the ability of the organisms to grow in each beer. Parameters which correlated to RSV included pH, beer colour and the content of free amino nitrogen, total soluble nitrogen, a range of individual amino acids, maltotriose and the undissociated forms of SO₂ and hop bitter acids. Predictive models based on the technique of partial least squares regression and constructed using values for undissociated SO₂ content, undissociated hop bitter acids content, polyphenol content, maltotriose content, free amino nitrogen content and a colour intensity component allowed RSV to be predicted.     

Analysis of hopanoids in bacteria involved in food technology and food contamination

Hopanoids are pentacyclic triterpenoids which are believed to act as reinforcers of membranes in certain prokaryotic microorganisms. A rapid and sensitive method for their screening in bacteria was elaborated, involving extraction of non-saponifiable lipids and the analysis by gas chromatography-mass spectrometry, selectively monitoring the ion of m/z=191. Using the method, hopanoids were detected in strains of Acetobacter pasteurianus, but were found to be absent in lactic acid bacteria (Lactobacillus spp., Lactococcus spp.) and in food-contaminating bacteria (Salmonella spp., Listeria spp., Yersinia spp. and others).     

The use of multiplex PCR reactions to characterize populations of lactic acid bacteria associated with meat spoilage

A rapid, systematic and reliable approach for identifying lactic acid bacteria associated with meat was developed, allowing for detection of Carnobacterium spp., Lactobacillus curvatus, Lact. sakei and Leuconostoc spp. Polymerase chain reaction primers specific for Carnobacterium and Leuconostoc were created from 16S rRNA oligonucleotide probes and used in combination with species-specific primers for the 16S/23S rRNA spacer region of Lact. curvatus and Lact. sakei in multiplex PCR reactions. The method was used successfully to characterize lactic acid bacteria isolated from a vacuum-packaged pork loin stored at 2 degrees C. Seventy isolates were selected for identification and 52 were determined to be Lact. sakei, while the remaining 18 isolates were identified as Leuconostoc spp.     

FtsZ蛋白同源性分析在乳酸菌系统学研究中的应用

FtsZ是一种广泛存在于细菌和古菌中的结构保守的蛋白质,在细胞分裂的过程中起关键的作用。通过PCR扩增FtsZ基因的一段800bp的核苷酸,构建了干酪乳杆菌-片球菌及相关乳酸菌的FtsZ蛋白系统发育树。将此系统树和16SrDNA系统树比较发现二者的拓扑结构非常相似。在两个基因系统树中,片球菌与乳杆菌的种均显示了较近的亲缘关系,而与其它球状的乳酸菌,如链球菌和肠球菌的亲缘关系较远。研究还表明FtsZ蛋白序列的分辨率高于16SrDNA,更适用于乳酸菌种间的系统分类研究。     

Ecological studies on the occurrence of bacteria utilizing lactic acid at pH values below 4.5

Sites within two cheese factories, a silage tower and silage pit and two pet food factories were investigated for lactic acid utilizing bacteria. The bacteria isolated, together with known lactic acid utilizing and aciduric bacteria, were tested for their ability to 'alkalinize' a fermented meat product containing more than 20 g/1 lactic acid at pH > 4.3 a ten-fold dilution of this product and synthetic media, both at pH 4.5. Strains able to alkalinize the diluted product were 13 aerobic isolates (all Acetobacter spp.), nine anaerobic cultures, one strain of Megasphaera elsdenii and two strains of Clostridium tyrobutyricum. Eight of the aerobes could alkalinize undiluted product containing < 1 g/1 residual potassium sorbate, but had no effect on product containing > 3 g/1 sorbate; none of the anaerobic cultures was able to alkalinize undiluted product. Acetobacter spp., therefore, threatened the microbial stability of a fermented product containing < 3 g/1 potassium sorbate. The cultures able to alkalinize diluted product were all from sites contaminated with whey, silage or fermented product for at least 2 d, suggesting substantial accumulation of lactic acid utilizing bacteria at these sites.     

Phylogeny of spore-forming lactic acid bacteria based on 16S rRNA gene sequences

Abstract The phylogeny of spore-forming lactic acid bacteria was investigated on the basis of 16S rRNA gene sequences. Sixteen strains were separated into three lines of descent; one consisted of 14 strains assigned to Sporolactobacillus spp. and Bacillus spp., and the other two each consisted of " Sporolactobacillus dextrus " and Bacillus coagulans . Strains of all the first lineage but one composed a cluster of similarity values of 97.2% and higher, and were represented by the type of S. inulinus . The cluster was further separated into five subclusters, four catalase negative and one positive. The definition of the genus Sporolactobacillus should be amended to accomodate catalase positive strains. Spore-forming lactic acid bacteria originated at different phylogenetic positions, and would have evolved convergently in the area of Bacillus .     

Vaginal lactic acid bacteria in the mare: evaluation of the probiotic potential of native Lactobacillus spp. and Enterococcus spp. strains

Lactic acid bacteria (LAB) are important members of the human vaginal microbiota and their presence is considered beneficial. However, little is known about native vaginal bacteria in other animal species such as the horse. The aim of this work was to quantify the vaginal lactic acid bacteria and lactobacilli of mares and to establish if selected equine vaginal lactic acid bacteria, particularly Lactobacillus and Enterococcus spp. strains, could exhibit potential as probiotics. The vaginal lactic acid bacteria and lactobacilli of 26 mares were quantified by plate counts. Five strains (three Lactobacillus spp. and two Enterococcus spp.) were characterised and adhesion to vaginal epithelial cells, antimicrobial activity and ability to form biofilms were evaluated. Lactic acid bacteria were recovered from the 26 samples and lactobacilli counts were detected in 18 out of 26 mares (69%). Probiotic properties tested in this study varied among the isolates and showed promising features for their use as equine probiotics.     

Effects of cultivation conditions on folate production by lactic acid bacteria

A variety of lactic acid bacteria were screened for their ability to produce folate intracellularly and/or extracellularly. Lactococcus lactis, Streptococcus thermophilus, and Leuconostoc spp. all produced folate, while most Lactobacillus spp., with the exception of Lactobacillus plantarum, were not able to produce folate. Folate production was further investigated in L. lactis as a model organism for metabolic engineering and in S. thermophilus for direct translation to (dairy) applications. For both these two lactic acid bacteria, an inverse relationship was observed between growth rate and folate production. When cultures were grown at inhibitory concentrations of antibiotics or salt or when the bacteria were subjected to low growth rates in chemostat cultures, folate levels in the cultures were increased relative to cell mass and (lactic) acid production. S. thermophilus excreted more folate than L. lactis, presumably as a result of differences in the number of glutamyl residues of the folate produced. In S. thermophilus 5,10-methenyl and 5-formyl tetrahydrofolate were detected as the major folate derivatives, both containing three glutamyl residues, while in L. lactis 5,10-methenyl and 10-formyl tetrahydrofolate were found, both with either four, five, or six glutamyl residues. Excretion of folate was stimulated at lower pH in S. thermophilus, but pH had no effect on folate excretion by L. lactis. Finally, several environmental parameters that influence folate production in these lactic acid bacteria were observed; high external pH increased folate production and the addition of p-aminobenzoic acid stimulated folate production, while high tyrosine concentrations led to decreased folate biosynthesis.     

Microbiological quality of wheat flour consumed in Morocco

Cereal products (soft and hard wheat) are a basic staple food in the Moroccan diet. A total of 60 samples of two types of wheat flours used for human consumption were collected; 30 samples among this collection were obtained from various households using Moroccan varieties of wheat produced in traditional flour mills. The rest of the samples were purchased from retail wheat flour sources in the Rabat and Sale city markets. Standard plate counts (SPC), total and faecal coliforms, Clostridium, Salmonella spp., Shigella spp., Staphylococcus aureus, Listeria monocytogenes, yeast, lactic acid bacteria, and molds, were carried out to assess the microbiological quality of wheat flour. Microbiological interpretation of the criteria was performed according to standards implemented by the Codex Alimentarius Commission. Most frequent counts, in traditional and industrial wheat flour, were total aerobic mesophilic bacteria with an average 4 × 104 and 2.5 × 104 cfu/g, respectively. The results showed higher coliform and fungi counts in house than in commercial samples. Pathogenic flora as Salmonella spp., Shigella spp., S. aureus, L. monocytogenes, and Clostridium were not detected in all investigated samples. Bacterial strains isolated from both flours belong to the following genera: Enterobacter spp., Serratia spp., Klebsiella spp., Pantoea spp., Leclercia spp., Proteus spp. The most frequent genus of the investigated isolates was Aspergillus (81 %). Microbial counts were lower than the limit laid down in the Codex Alimentarius, attributing to these flours a satisfactory microbiological quality.     

Cellular fatty acid composition of Leuconostoc oenos

The cellular fatty acid composition of 70 lactic acid bacteria was examined by capillary gas chromatography. Fifty-four Leuconostoc oenos strains, including three reference, type strains from the other Leuconostoc spp., nine Pediococcus spp. and two Lactobacillus spp. were studied. Eighteen fatty acids were determined, of which 10 were identified by gas chromatography-mass spectrometry. The relative percentages of the 18 fatty acids of the Leuconostoc strains were analyzed numerically and grouped using the unweighted pair-group method. Results show that four clusters could be defined at r = 0.920, with five strains unassigned. The major fatty acids of the Leuc. oenos strains were found to be palmitic acid (C16:0), palmitoleic acid (C16:1–9), oleic acid (C18: 1–9), vaccenic acid (C18: 1–11), dihyrosterculic acid (C19-cyclopropane-9) and lactobacillic acid (C19-cyclopropane-11). It was mainly on the basis of the amounts of oleic acid and the C19-cyclopropane fatty acids that the strains of Leuc. oenos could be distinguished from each other. This is the first report of the occurrence of dihydrosterculic acid in lactic acid bacteria. For the majority of Leuc. oenos strains, the result obtained with the cellular fatty acid analysis confirmed the phenotypic relationships.     

Gene expression systems for lactic acid bacteria.

Considerable advances have been made in the genetics and molecular biology of lactic acid bacteria, including Lactococcus, Lactobacillus, Leuconostoc, Pediococcus and Streptococcus spp. These have resulted in the construction of constitutive gene expression cassettes, inducible gene expression systems, and specific protein targeting systems for these bacteria. These developments are important in the food industry where lactic acid bacteria can be exploited as food-grade cell factories.     

Importance of lactic acid bacteria in Asian fermented foods

Lactic acid bacteria play important roles in various fermented foods in Asia. Besides being the main component in kimchi and other fermented foods, they are used to preserve edible food materials through fermentation of other raw-materials such as rice wine/beer, rice cakes, and fish by producing organic acids to control putrefactive microorganisms and pathogens. These bacteria also provide a selective environment favoring fermentative microorganisms and produce desirable flavors in various fermented foods. This paper discusses the role of lactic acid bacteria in various non-dairy fermented food products in Asia and their nutritional and physiological functions in the Asian diet.     

Changes in the Microbiology of Vacuum-packaged Beef

The development of the microbial flora on meat stored in vacuum-bags at 0–2° for up to 9 weeks was studied. Although the proportion of lactic acid bacteria increased relative to the aerobic spoilage organisms, the numbers of the latter continued to increase throughout storage. The initial contamination of the meat before vacuum-packaging was important; meat with a very low initial number had lower numbers of bacteria throughout storage for up to 9 weeks and steaks cut from such meat which had been stored always had 1–2 days' additional aerobic shelf life at 4°. Spoilage of these steaks was due either to slime formation and off-odour associated with high counts of presumptive Pseudomonas spp., or by discoloration and souring (lactic acid bacteria). Extract release volume and pH measurements performed on the vacuum-packaged primal joints were only of value in determining the onset of aerobic spoilage when large numbers of Gram negative organisms were present, whereas the titrimetric method of spoilage evaluation of the vacuum-packaged meat showed a correlation with spoilage due to lactic organisms.     

Effect of Packaging under Carbon Dioxide, Nitrogen or Air on the Microbial Flora of Pork Stored at 4°C

Changes in the microbial flora of pork packed in laminated plastic bags and stored at 4 °C were studied in an initial atmosphere of carbon dioxide, nitrogen or air. The time needed for the total aerobic count at 28 °C to reach 5 × 10⁶ organisms/cm² was about 7 times longer in carbon dioxide than in air, whilst in nitrogen it was about twice as long.
The predominant organisms on fresh pork taken directly from the processing line were: Acinetobacter calcoaceticus , non-fluorescent Pseudomonas spp. and Flavobacterium spp. After storage in air for 7 d, more than 90% of the flora consisted of non-fluorescent Pseudomonas spp. After storage in nitrogen for 10 d, 70% of the flora consisted of non-fluorescent Pseudomonas spp. with lower levels of fluorescent Pseudomonas spp., Kurthia zopfii, Aeromonas hydrophila and Lactobacillus plantarum. The non-fluorescent Pseudomonas spp. could be divided into three different groups, on proteolytic and lipolytic ability; the distribution of the groups was markedly different between pork loins stored in air and nitrogen.
On pork stored in carbon dioxide for 21 d the flora consisted of L. plantarum together with lower levels of heterofermentative lactic acid bacteria. When the storage time in carbon doxide was prolonged to 35 d, the proportion of heterofermentative lactic acid bacteria increased to about 50% of the flora.     

Randomly-amplified microsatellite polymorphism for preliminary typing of lactic acid bacteria from Bryndza Cheese

A high-throughput, medium-discrimination method for preliminary typing and selecting non-identical isolates of lactic acid bacteria in cheeses was developed. RAMP, a PCR with one microsatellite-targeted and one random primer, was used for preliminary typing of 1119 isolates of lactic acid bacteria from Slovak Bryndza cheese. A total of 59 genotypes were identified based on RAMP profiles consisting of 12–23 DNA fragments of 150–3000 bp. For example, 18, 17, 13 and 7 different RAMP-types were identified in Lactobacillus brevis, L. plantarum, L. paracasei and L. fermentum, respectively. The method facilitated well reproducible, medium-discrimination typing of Lactobacillus spp. and Pediococcus spp. at a subspecies level and proved to be suitable for preliminary typing of lactic acid bacteria isolated from cheese.     

A note on the microbiology of retail packs of prepared salad vegetables

Retail packs of mixed, prepared salad vegetables from two different manufacturers were stored at 7°C until the end of storage-life (sell-by date plus 1 d), when the microbial flora was examined. The quality of the salads was acceptable at the end of storage-life. The oxygen concentrations in packs were lower, and the carbon dioxide concentrations were higher, than those in air. High numbers of bacteria were present, with Pseudomonas spp. and Enterobacter agglomerans predominating in packs of both salads, together with lactic acid bacteria in one of the salads. Significant numbers of pectolytic bacteria including Pseudomonas spp. and Erwinia carotovora were detected. Despite the presence of high numbers of coliform bacteria, Escherichia coli was not detected in batches of one salad, and was detected in relatively low numbers in batches of the other. Yersinia spp., predominantly environmental strains of Yersinia enterocolitica , were isolated by enrichment from all samples tested; Staphylococcus aureus and enterococci were not detected.     

Effect of Selected Types of Beer on Bacteria of the Genus Arcobacter

The genus Arcobacter is related to the well-known human pathogen, Campylobacter jejuni, and has been linked to human diseases. In this study, the survival of Arcobacter spp. in various concentrations of ethanol, in various samples of beers, and in a model stomach has been investigated. For most of these bacteria, a concentration of 10 % ethanol was determined to be the minimum inhibitory concentration. The fact that these organisms are able to survive under these conditions may have an impact in the food processing industry. We studied the activity of beer against arcobacters. These bacteria were killed in all samples of beer within 30 min. A model stomach, containing a food matrix and a synthetic gastric fluid, was used to deduce the effect of beer against Arcobacter spp. during food consumption. Complete inactivation of all monitored arcobacters was detected usually within 15 min. However, the presence of beer does not potentiate the effect of gastric fluid against these bacteria. This is apparently the first study focusing upon the effect of beer on Arcobacter spp.     

A note on the microbiology of retail packs of prepared salad vegetables

Retail packs of mixed, prepared salad vegetables from two different manufacturers were stored at 7 degrees C until the end of storage-life (sell-by date plus 1 d), when the microbial flora was examined. The quality of the salads was acceptable at the end of storage life. The oxygen concentrations in packs were lower, and the carbon dioxide concentrations were higher, than those in air. High numbers of bacteria were present, with Pseudomonas spp. and Enterobacter agglomerans predominating in packs of both salads, together with lactic acid bacteria in one of the salads. Significant numbers of pectolytic bacteria including Pseudomonas spp. and Erwinia carotovora were detected. Despite the presence of high numbers of coliform bacteria, Escherichia coli was not detected in batches of one salad, and was detected in relatively low numbers in batches of the other. Yersinia spp., predominantly environmental strains of Yersinia enterocolitica, were isolated by enrichment from all samples tested; Staphylococcus aureus and enterococci were not detected.     

Amino acid fermentation in non-starter Lactobacillus spp. isolated from cheddar cheese

Amino acid fermentation profiles of nine strains of Lactobacillus spp., initially isolated from a 3-year-old Cheddar cheese, were determined using the Biolog MT microplatetrade mark method. Eight of the isolates were able to ferment amino acids, but only when incubated in the presence of exogenously supplied alpha-ketoglutaric acid that served as an acceptor in the initial transamination step in the fermentative degradation. The range of amino acids catabolized was strain dependent. Amino acid catabolites were detected by gas chromatography mass spectrometry (GCMS) in culture supernatant fluids of a representative non-starter lactic acid bacteria isolate Lactobacillus paracasei CI6.     

rpoB gene: a target for identification of LAB cocci by PCR-DGGE and melting curves analyses in real time PCR

Lactic acid bacteria (LAB) are essential in the quality of many fermented beverages like beer, cider and wine. In the two later cases, they convert malic acid into lactic acid during the malolactic fermentation. After fermentation, microbial stabilization is needed to prevent the development of spoilage bacteria species. Among them, cocci lead to different alterations: Pediococcus sp., and some strains of Leuconostoc mesenteroides and Oenococcus oeni can produce exopolysaccharides which modify wine viscosity and lead to ropiness. They also can produce acetic acid, biogenic amine, ethyl carbamate and volatile phenols. Therefore detection and identification are crucial. Results of phenotypic tests and DNA-DNA probes are not accurate enough. 16S RNA gene which is currently used for bacterial species identification presents intraspecies heterogeneity. The rpoB gene is an alternative to this limitation. However previous PCR targeting partial sequence of rpoB gene could not delimit cocci species. Therefore we compared the rpoB gene sequence of the six main cocci species found in fermented beverages: P. damnosus, P. dextrinicus, P. parvulus, P. pentosaceus, L. mesenteroides and O. oeni. The most discriminating partial sequence of the rpoB gene was chosen for designing primers. By PCR-DGGE the reliability of these primers was verified. It was controlled in a mixture of several cocci and other lactic acid bacteria (Lactobacillus sp.). Then we adapted the primers and the PCR conditions in order to achieve the identification of cocci species by real time PCR program including the fluorescent dye SYBR Green I, which gives faster results. PCR melt curves were established and a specific T(m) was attributed to each species.