Lactobacillus strains isolated from Danbo cheese as adjunct cultures in a cheese model system

Isolates of Non-Starter Lactic Acid Bacteria (NSLAB) from six ripened Danbo cheeses of different ages and of different brands were examined. Special emphasis was on the genus Lactobacillus with the aim of investigating their role in cheese maturation. Thirty-three isolates were typed by the PCR-based method, Randomly Amplified Polymorphic DNA (RAPD). Ten RAPD types were found and 70% of the isolates were of RAPD types found in more than one cheese. The different RAPD types were identified to species level by Temporal Temperature Gradient Gel Electrophoresis (TTGE). Most of the isolates were identified as Lactobacillus paracasei (76%), but also Lactobacillus curvatus, Lactobacillus plantarum, Lactobacillus rhamnosus and some taxa originating from the starter culture were detected. In one cheese, no lactobacilli were found.One strain of the most frequent Lactobacillus RAPD type from each of the five cheeses with a Lactobacillus flora was used as adjunct cultures in a cheese model system. Four of the five adjuncts were re-isolated during ripening. Two adjunct containing model cheeses received higher flavour scores than the control while two other were associated with off-flavours. The two model cheeses with off-flavour had a similar microflora and both were after 13 weeks of ripening dominated by a strain identified as L. plantarum.     

Autoinducer-2-like activity in lactic acid bacteria isolated from minced beef packaged under modified atmospheres

Fifteen fingerprints (assigned to Leuconostoc spp., Leuconostoc mesenteroides, Weissella viridescens, Leuconostoc citreum, and Lactobacillus sakei) of 89 lactic acid bacteria (LAB) isolated from minced beef stored under modified atmospheres at various temperatures were screened for their ability to exhibit autoinducer-2 (AI-2)-like activity under certain growth conditions. Cellfree meat extracts (CFME) were collected at the same time as the LAB isolates and tested for the presence of AI-2-like molecules. All bioassays were conducted using the Vibrio harveyi BAA-1117 (sensor 1(-), sensor 2(+)) biosensor strain. The possible inhibitory effect of meat extracts on the activity of the biosensor strain was also evaluated. AI-2-like activity was observed for Leuconostoc spp. isolates, but none of the L. sakei strains produced detectable AI-2-like activity. The AI-2-like activity was evident mainly associated with the Leuconostoc sp. B 233 strain, which was the dominant isolate recovered from storage at 10 and 15°C and at the initial and middle stages of storage at chill temperatures (0 and 5°C). The tested CFME samples displayed low AI-2-like activity and inhibited AI-2 activity regardless of the indigenous bacterial populations. The LAB isolated during meat spoilage exhibited AI-2-like activity, whereas the LAB strains retrieved depended on storage time and temperature. The production of AI-2-like molecules may affect the dominance of different bacterial strains during storage. The results provide a basis for further research concerning the effect of storage temperature on the expression of genes encoding AI-2 activity and on the diversity of the ephemeral bacterial population.     

Listeria monocytogenes isolated from cold-smoked fish products in Osaka City, Japan

Listeria monocytogenes contamination of ready-to-eat seafood products commercially available in Osaka was examined between 1999 and 2000. L. monocytogenes was isolated from 12 (13%) of the 95 products tested. All positive samples were from cold-smoked fish with 9 being obtained during the summer. Thirteen isolates of L. monocytogenes were typed by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction (PCR)-based typing methods. Isolates of the same serotype originating from the same manufacturer gave similar DNA profiles, irrespective of the type of sample or date of isolation. The finding suggest that persistent strains in each manufacturing facility proliferate during the summer and contaminate products during manufacturing processes.     

Bioactive peptides released from Saccharomyces cerevisiae under accelerated autolysis in a wine model system

ABSTRACT:  The ACE inhibitory activity (IACE) and the oxygen radical absorbance capacity (ORAC-FL) values of yeast peptides isolated from a model wine during accelerated autolysis of Saccharomyces cerevisiae have been studied. Samples were taken at 6, 24, 48, 121, and 144 h of autolysis. Peptide concentration increased throughout autolysis process. Peptides were fractionated into 2 fractions: F1, constituted by hydrophilic peptides, and F2, containing hydrophobic peptides. Both IACE activity and ORAC-FL values increased during 121 h of autolysis, then decreased afterward. Peptide fraction F2 was the main fraction involved in IACE activity and ORAC-FL.     

Histamine formation by Tetragenococcus muriaticus, a halophilic lactic acid bacterium isolated from fish sauce.

We examined histamine formation in cultures of Tetragenococcus muriaticus, a halophilic lactic acid bacterium isolated from fish sauce. T. muriaticus formed histamine in low acidity (pH 5.8), O2 limiting conditions with optimal NaCl and glucose concentrations of 5-7% (w/v) and above 1%, respectively. Histamine formation could not be prevented even at 20% (w/v) NaCl, indicating that NaCl could not prevent histamine formation by this bacterium. A conspicuous amount of histamine accumulated only during the late stationary phase regardless of the growth conditions. Studies of cell suspension experiments confirmed the results obtained from cultured cells.     

Suggestion of a protective function of proteinase inhibitors in potatoes: Inhibition of proteolytic activity of microorganisms isolated from spoiled potato tubers

Summary From infected potato tubers 108 microorganisms were isolated, of which 64 were strong proteolytes. Work on various isolates showed inhibition of growth and of proteolytec activity by inhibitors obtained from potatoes of the same variety.

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Hinweis auf eine Schutzfunktion der Proteinasen-Inhibitoren in Kartoffeln: Hemmung der proteolytischen Aktivität von Mikroorganismen, isoliert von verdorbenen Kartoffelknollen

Zusammenfassung Aus infizierten Kartoffelknollen wurden 108 Mikroorganismen isoliert, von denen 64 starke Proteolyten sind. Bei einer Reihe von Isolaten konnte eine Hemmung von Wachstum und proteolytischer Aktivität durch Inhibitoren der gleichen Kartoffelsorte nachgewiesen werden.

     

Evaluation of lactic acid bacteria,isolated from lightly preserved fish products,as starter cultures for new fish-based food products

Lactic acid bacteria suitable for fish fermentation were selected and their capacity as starter cultures in fish raw material was evaluated. Of 23 strains of lactic acid bacteria which were isolated from lightly preserved herring products and identified by the Biolog system mainly as Lactobacillus and Leuconostoc spp., three strains, which grew well at 5–10°C in the presence of food preservatives, were chosen for evaluation. Qualitative changes in minced fish, inoculated with lactic acid bacteria, and stored at 10°C for 7 weeks, were examined by organoleptic, bacteriological and chemical methods, and were found to be strongly strain specific. Inhibition of the competitive flora was more significant in the samples inoculated with Lactobacillus plantarum and Lactobacillus mesenteroides than in others. The best organoleptic and chemical results were obtained with Leuconostoc mesenteroides; this strain looks promising as a starter culture for fish fermentation and for the development of new fish products, which organoleptically resemble meat products.     

Production of biogenic amines by lactic acid bacteria and enterobacteria isolated from fresh pork sausages packaged in different atmospheres and kept under refrigeration

The occurrence of in vitro amino acid activity in bacterial strains associated with fresh pork sausages packaged in different atmospheres and kept in refrigeration was studied. The presence of biogenic amines in decarboxylase broth was confirmed by ion-exchange chromatography and by the presence of the corresponding decarboxylase genes by PCR. From the 93 lactic acid bacteria and 100 enterobacteria strains analysed, the decarboxylase medium underestimates the number of biogenic amine-producer strains. 28% of the lactic acid bacteria produced tyramine and presented the tdc gene. All the tyramine-producer strains were molecularly identified as Carnobacterium divergens. Differences on the relative abundance of C. divergens were observed among the different packaging atmospheres assayed. After 28 days of storage, the presence of argon seems to inhibit C. divergens growth, while packing under vacuum seems to favour it. Among enterobacteria, putrescine was the amine more frequently produced (87%), followed by cadaverine (85%); agmatine and tyramine were only produced by 13 and 1%, respectively, of the strains analysed. Packing under vacuum or in an atmosphere containing nitrogen seems to inhibit the growth of enterobacteria which produce simultaneously putrescine, cadaverine, and agmatine. Contrarily, over-wrapping or packing in an atmosphere containing argon seems to favour the growth of agmatine producer-enterobacteria. The production of putrescine and cadaverine was associated with the presence of the corresponding amino acid decarboxylase genes. The biogenic amine-producer strains were included in a wide range of enterobacterial species, including Kluyvera intermedia, Enterobacter aerogenes, Yersinia kristensenii, Serratia grimesii, Serratia ficaria, Yersinia rodhei, Providencia vermicola and Obesumbacterium proteus.     

Suppressive effect of Tetragenococcus halophilus,isolated from fish-nukazuke,on histamine accumulation in salted and fermented fish

Occasionally, quantities higher than 1000 mg/kg of histamine (Hm) accumulate in salted and fermented fish products by the histidine decarboxylase of halophilic lactococci Tetragenococcus sp. In a total of 200 isolates from fish nukazuke (salted and fermented fish with rice bran), 13 strains produced Hm more than 200 μg/ml in 0.5% histidine containing broth, whereas 130 isolates produced absolutely no Hm. Among the strains, 22 strains suppressed the Hm production of the Hm-forming (HmF) strains. Both the HmF and Hm-suppressing (HmS) strains were identified as Tetragenococcus halophilus. To observe the Hm-suppressing effect, a specified quantity of live cells was needed. In the case of 10% NaCl salted sardine, inoculation with 3 log cells/g of a strain HmF-131 resulted in a significant Hm accumulation, 2800 μg/g in 30 days at 20 °C. The increase in Hm was clearly suppressed by 9 log live cells/g of strain HmS-129. These results suggest that HmS-129 can be used as a starter for salted and fermented fish products, enhancing food safety.     

Molecular typing to trace Listeria monocytogenes isolated from cold-smoked fish to a contamination source in a processing plant

In this study, Listeria monocytogenes contamination in a cold-smoked fish processing plant in Osaka, Japan, was examined from 2002 to 2004. A total of 430 samples were collected and divided into five categories: raw fish, materials during processing, processing equipment, environment, and finished products. A total of 59 finished products were examined throughout this study. L. monocytogenes was isolated from four of these samples during summer and autumn but was not found during winter or spring. During the warmer seasons, L. monocytogenes was more prevalent on processing equipment, especially slicing machines (8 of 54 samples in summer and autumn versus 1 of 50 samples in winter and spring). L. monocytogenes was not detected on whole skins removed from 23 frozen raw fish. L. monocytogenes strains isolated from 56 samples were characterized by serotyping, pulsed-field gel electrophoresis, and three PCR-based methods. Seventy-seven L. monocytogenes strains were recognized as contaminants of the samples: 2 distinguishable strains were identified in each of 13 samples, 3 strains were identified in 2 samples, 5 strains were identified in 1 sample, and the other 40 strains were identified in 40 samples. Combining the results from these techniques, 77 strains were classified into 13 different types. Three of these types prevailed throughout the plant, and two of the three were also isolated from final products. The DNA subtype found in the product was also found on the slicing machines. Our findings suggest that the slicing machines at this plant were the source of the product contamination. Implementing an appropriate cleaning regime for the slicing machines was effective in preventing contamination.     

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.     

Analysis of a 30 kbp plasmid encoding histidine decarboxylase gene in Tetragenococcus halophilus isolated from fish sauce

In order to analyze the genes related to the histamine production, a strain of histamine producing halophilic bacteria, referred to as strain H, was isolated using enrichment culture and dilution-to-extinction methods with histidine broth inoculated from the fish sauce mashes. The two Japanese fish sauce mashes used, accumulate over 1000 mg/l of histamine. Phenotypic and 16 S rRNA gene sequence analyses identified strain H as Tetragenococcus halophilus, the predominant histamine producing bacteria present during fish sauce fermentation. Genetic analyses (PCR and Southern blot) of the histamine producing strain confirmed that the strain harbored a 30 kbp plasmid (pHDC) encoding a single copy of the pyruvoyl dependent histidine decarboxylase gene (hdc). A comparison of hdcA that is a structural gene of histidine decarboxylase among strain H, Lactobacillus hilgardii 0006, L. sakei LTH2076, Oenococcus oeni 9204, T. halophilus and T. muriaticus JCM10006 (T) indicated >99% sequence similarity. The hdc gene cluster consisted of 4 ORFs, hdcP, hdcA, hdcB, and hdcRS, and were almost identical to that of L. hilgardii 0006 with 99% sequence similarity including the structural hdc spacer region. However, the approximately 500 bp regions upstream and downstream of the hdc gene were different between that of strain H and L. hilgardii 0006. The complete sequence of pHDC revealed 29,924 nucleotides including 28 ORFs, two pairs of IR (inverted repeat), similar sequence of plasmid conjugative elements, and a theta-type replicon. These results suggested that hdc could be encoded on transformable elements among lactic acid bacteria.     

Shelf-life of a chilled precooked chicken product stored in air and under modified atmospheres: microbiological, chemical, sensory attributes

This study evaluated the effect of modified atmosphere packaging on shelf-life extension of a precooked chicken meat product stored at 4 degrees C using microbiological, physico-chemical and sensory analyses. The following gas mixtures were used: M1: 30%/70% (CO2/N2), M2: 60%/40% (CO2/N2) and M3: 90%/10% (CO2/N2). Identical chicken samples were aerobically packaged and used as control samples. Sampling was carried out at predetermined time intervals namely: 0, 4, 8, 12, 16 and 20 days. Total viable counts (TVC), Lactic acid bacteria (LAB), Brochothrix thermosphacta, pseudomonads, yeasts and molds, and Enterobacteriaceae were monitored. TVC of precooked chicken product reached 7 log cfu/g, after days 12 and 16 of storage (air and M1 samples), respectively. The M2 and M3 gas mixture packaged samples did not reach this value throughout the 20 days storage period under refrigeration. LAB and to a lesser degree B. thermosphacta, constituted part of the natural microflora of precooked chicken samples stored in air and under MAP reaching 7.0-8.1 log cfu/g at the end of storage period. Of the remaining bacterial species monitored, both pseudomonads and yeasts/molds were significantly higher (P<0.05) for chicken samples stored in air than under MAP (M1, M2, M3) throughout the entire storage period under refrigeration. Finally, counts of Enterobacteriaceae were low (<2 log cfu/g) in all chicken samples irrespective of the packaging conditions throughout the entire storage period. Of the chemical indices determined, thiobarbituric (TBA) values in all cases remained low, equal or lower than 3.0 mg malonaldehyde (MA)/kg during the entire storage period. Results of the present work show that the limit of sensory acceptability was only reached for the aerobically stored and M1 gas mixture chicken samples somewhat before days 16 and 20 of storage, respectively. This limit coincided with high TVC and LAB populations (>6.8 log cfu/g), increased lipid oxidation (aerobic storage only) and apparent growth of yeasts/moulds on the surface of chicken samples. The use of MAP as shown in the present study, resulted in an extension of shelf-life of precooked chicken by ca. 4 days (M1 gas mixture), and by more than 6 days (M2 and M3 gas mixtures), respectively. Precooked chicken meat was better preserved under M2 and M3 mixtures maintaining desirable odor/taste attributes even on final day of storage tested.     

Flaniostatin, a new isoflavonoid glycoside isolated from the leaves of Cudrania tricuspidata as a tyrosinase inhibitor

In the course of screening program for skin whitening compounds, flaniostatin (FST) was isolated from the leaves of Cudrania tricuspidata as a novel inhibitor of tyrosinase. The structure of FST was determined by ESI-MS and NMR spectroscopic analyses as a new isoflavone glycoside. The FST was exhibited a tyrosinase inhibitory effect in a dose-dependent manner. This effect was higher than that of arbutin at the same concentrations. These results indicated that FST isolated from C. tricuspidata may be a positive tool for skin-whitening agent research.     

Changes in free amino acids during chilled storage of hake (Merluccius merluccius L.) in controlled atmospheres and their use as a quality control index

This paper examines the evolution of FAAs in hake stored in ice and kept in controlled atmospheres for 33 days with four different gas mixes (CO₂/O₂/N₂): M1 (60/15/25), M3 (40/40/20), M4 (60/40/0), and M5 (40/60/0). The most abundant FAAs in hake muscle are threonine, glutamic acid, glycine, alanine, #-alanine, tryptophan, 1-methylhistidine, and anserine. Anserine was the compound initially present in the highest concentration (61.65 mg/100 g of fish wet weight). As storage progressed, anserine concentrations fell, the smallest decrease being found in the lot kept in M1. At the end of storage the lot with the lowest level of anserine was M5C, which contained the highest O₂ levels. The levels in the other lots were very similar. This fall in anserine coincided with an increase in 1-methylhistidine and #-alanine, the two amino acids to which it becomes hydrolyzed. As well as the fall in anserine, there was a progressive increase in muscle tryptophan during storage of the hake. This increase became more pronounced from day 12 of storage and was greatest in the control and lots M1C and M3C, which relates to the results of the other biochemical indexes. Both anserine and tryptophan could be used as quality control indexes for chilled hake. In general, no clear relationship was found between the effect of the atmosphere and the behavior of the FAAs.     

Xenografting of sheep testis tissue and isolated cells as a model for preservation of genetic material from endangered ungulates

Recovery of germ cells could be an option for preservation of the genetic pool of endangered animals. In immature males, xenografting of testis tissue provides the opportunity to recover sperm from these animals. In adult animals, xenografting has been less successful, but de novo morphogenesis of functional testis tissue from dissociated testis cells could be an alternative. To assess the potential use of these techniques in endangered bovid species, the domestic sheep was used as a model. Testes from 2-week-old lambs were grafted as tissue fragments or cell suspensions into nude mice. Grafts were recovered at 4, 8, 12 and 16 weeks post grafting. For isolated cells, two additional time points at 35 and 40 weeks after grafting were added. In addition, to analyse the possible effect of social stress among mice within a group on the development of the grafts, testis tissue grafts were recovered 13 weeks post grafting from mice housed individually and in groups. Complete spermatogenesis occurred in sheep testis xenografts at 12 weeks, similar to the situation in situ. Isolated sheep testis cells were able to reorganize and form functional testicular tissue de novo. Housing mice individually or in groups did not have any effect on the development of xenografts. Xenografting of testis tissue might be useful to obtain sperm from immature endangered ungulates that die prematurely. Testis tissue de novo morphogenesis from isolated cells could open interesting options to recover germ cells from mature males with impaired spermatogenesis.     

Presence of lactobacilli in the intestinal content of freshwater fish from a river and from a farm with a recirculation system

Lactobacilli are Gram-positive and catalase negative rods commonly found in lactic acid fermented foods and in the gastrointestinal tract of mammals and birds. Few studies have described lactobacilli in freshwater fish. We analysed the presence of lactobacilli in the intestines of young and adult freshwater fish inhabiting a river environment and from fish reared in an aquaculture unit with a water recirculation system. Various species of lactobacilli were present in relatively high number in the intestines of edible fresh water fish from the river, especially in the warm season but in low numbers in the cold season. Lactobacilli were scarcely found in the intestines of edible farmed fish reared in a recirculation system in warm water. Lactobacilli are reported for the first time from the intestines of wild European eel, perch, rudd, ruffe, bleak, silver bream, chub, somnul and farmed African catfish. The two first fishes, and the last one are highly valuable species for fisheries and aquaculture. Additionally, improved methods for storage and bacteriological analysis of fish intestinal content are described. The natural presence of lactic acid bacteria in fish may be of great interest in producing fermented fish products worldwide.     

Aerated whey protein gels as a controlled release system of creatine investigated in an artificial stomach

A controlled creatine-release system has been developed from whey protein-based gels. Their functionalization was carried out by aeration and sodium ions induced “cold gelation” processes. The effect of protein concentration in the aerated whey protein gels at pH 7.0 and 8.0 was analyzed. Physicochemical properties of the aerated gels were evaluated. It was possible to obtain the ions induced whey protein aerated gel with well distributed creatine and different microstructure as well as rheological properties. Different protein concentrations and pH enabled obtaining gels with different rheological properties, texture, air fraction, diameter of air bubbles, microstructure and surface roughness. An increase in the protein concentration enhanced the hardness of the samples, regardless of their pH. The mechanical strength of gels prepared at pH 8 were higher than those obtained at pH 7, as was manifested by the smaller storage modulus of the latter. The former gel exhibited a microstructure between particulate and fine-stranded. A stronger gel matrix produced smaller air bubbles. Aerated gels produced at pH 7.0 had higher roughness than those obtained at pH 8.0. Optimal conditions for inclusion of air bubbles into the gel matrix were: 9% protein concentration at pH 8.0 and this aerated gel was selected for digestion in the artificial stomach. There is a small conversion of creatine to creatinine in the artificial stomach digestion process (9.6% after 6 h). The diffusion of creatine crystals from the aerated gel matrix was the mechanism responsible for the release process. Aerated whey protein gels can be used as matrices for time extended releasing of creatine in the stomach.