Molecular characterization of Lactobacillus species isolated from naturally fermented sausages produced in Greece,Hungary and Italy

The ecology of the lactic acid bacteria (LAB) in naturally fermented sausages produced in three European countries was investigated with the purpose of defining the geographic distribution of specific populations responsible for the fermentation and maturation of the sausages. Three-hundred and fifty eight strains belonging to the LAB group were isolated and identified by PCR-DGGE and sequencing. Three species were common to all three countries studied and were the most numerous. Lactobacillus curvatus, L. plantarum and L. sakei strains were subjected to RAPD-PCR followed by cluster analysis to obtain a genetic characterization. The distributions reflected in almost all cases the provenience of the strains, thereby distinguishing Italian, Greek and Hungarian LAB populations. The application of molecular methods for the identification and characterization of bacterial populations isolated from naturally fermented products allows a better understanding of the ecology. As a consequence measures can be taken to protect the biodiversity characterizing a specific system.     

Effects of post-fermentation processing on the stabilisation of naturally fermented green table olives (cv Nocellara etnea)

The effects of thermal treatment on green table olives were evaluated as a method to control enzymatic browning, to minimise the microorganism presence and to extend their shelf-life. However this treatment is often responsible for colour alterations, development of off-flavours and unfavourable texture changes. Moreover, the effect of different re-use of the natural brine of fermentation with or without different treatments was investigated. Calcium treatment was suggested to maintain firmness. Firming effects obtained from heat treatment combined with calcium treatment have been attributed to heat-activated pectin methylesterase and/or to increased calcium diffusion into tissues at higher temperatures. The results derived from this study will help in designing new processes which can be applied in table olive industry.     

Characterization of Lactobacillus isolates from fermented olives and their bacteriocin gene profiles

Near one hundred isolates of Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus plantarum from table olives were studied. Strains were genotyped by rep-PCR. Although the technique failed to differentiate some isolates at the species level, it proved a robust and easy procedure that could be useful for distinguishing between related strains of L. paraplantarum, L. pentosus and L. plantarum from a large pool of unrelated strains of these species. A PCR-based screening revealed the presence of the plantaricin encoding genes plnA, plnB, plnC, plnD, plnE/F, plnF, plnI, plnJ, plnK, plnG and plnN in most isolates of the three species. Sequences of bacteriocin genes present in L. paraplantarum and L. pentosus were homologous to L. plantarum genes. Through a discriminating analysis of the bacteriocin gene profiles, it was possible to establish a relationship between the origin of isolation and the LAB isolates, regardless of species.     

Potato juice fermented with Lactobacillus casei as a probiotic functional beverage

This study was conducted to evaluate probiotic potato juice as a potential substrate for the production of Lactobacillus casei, and the change in the functionality of potato juice was monitored during fermentation. L. casei grew well in potato juice without nutrient supplementation, and lactic acid bacteria of fermented ‘Haryoung’ juice reached 1.7×10⁹ CFU/mL after a 48 h fermentation. DPPH radical scavenging activities of the potato juices decreased after a 72 h fermentation, but fermented colored potato juice still maintained >50% radical scavenging activity. The survival rate of L. casei fermented in’ Haryoung’ juice was 89.0% after exposure to an acidic condition, and L. casei in all fermented potato juice samples showed the ability (50–85%) to survive in the presence of bile. These results suggest that fermented potato juice might serve as a probiotic functional beverage for vegetarians or consumers who are allergic to dairy products.     

Prevention of gastrointestinal infection using immunobiological methods with milk fermented with Lactobacillus casei and Lactobacillus acidophilus

The protective effect of feeding milk fermented with a mixture of Lactobacillus casei sp. and Lb. acidophilus sp. against Salmonella typhimurium infection in mice was compared with that obtained feeding milks fermented with these microorganisms individually. The survival rate obtained after oral infection with Sal. typhimurium was 100% in mice pretreated by feeding during 8 d with the mixture of Lb. casei and Lb. acidophilus fermented milks. Similar treatments with the individual milks were ineffective. Moreover, mice became more susceptible to infection with Sal. typhimurium after such treatment. The colonization of liver and spleen with the pathogen was markedly inhibited by the pretreatment with the mixture of fermented milk, while such inhibition was not observed using the Lb. casei and Lb. acidophilus milks. The highest levels of anti-salmonellae antibodies in serum and in intestinal fluid were found in the group of mice fed with the mixture and with Lb. casei fermented milk respectively. However, this latter milk was not effective in protecting against Sal. typhimurium. When the mice were first infected with Sal. typhimurium and then fed with the mixture of fermented milks, pathogen colonization was not prevented. The results suggest that the augmentation of resistance to salmonellae caused by the treatment with Lb. casei- + Lb. acidophilus-fermented milk was due to the anti-salmonellae protective immunity mainly mediated by the mucosal tissue. Milk fermented with this mixture could be used as an immunobiological method to prevent gastrointestinal infection.     

干酪乳杆菌发酵8种食用豆提取物抗氧化活性研究

该试验分析了干酪乳杆菌（Lactobacillus casei）发酵8种食用豆总酚、总黄酮含量以及抗氧化活性的变化。结果表明,发酵前豇豆总酚含量最高,为（5.53±0.18） mg 没食子酸当量（GAE）/g干质量;红豆总黄酮含量最高,为（11.55±0.16） mg 芦丁当量（RE）/g干质量。发酵后绿豆总酚、总黄酮含量增加最高,分别为182.35%、27.84%;豇豆总酚含量、菜豆总黄酮含量下降最多,分别为43.40%、82.13%。发酵后豌豆提取物1,1-二苯基-2-三硝基苯肼（DPPH）自由基清除活性上升最显著,菜豆提取物下降最显著;绿豆提取物2,2'-联氮-双（3-乙基苯并噻唑啉-6-磺酸）二铵盐（ABTS）自由基清除活性上升最显著,菜豆提取物下降最显著;豌豆提取物铁离子还原能力上升最显著,菜豆提取物下降最显著。相关性分析结果显示,总酚、总黄酮含量与抗氧化活性均极显著相关（P＜0.01）。发酵对食用豆酚类物质含量和抗氧化活性影响显著,为开发不同食用豆功能食品提供理论基础。     

Mineral content and sensory characteristics of Gordal green table olives fermented in chloride salt mixtures

This work studies the effects of the initial brine concentrations of NaCl, KCl, and CaCl(2) on the mineral content and gustatory and kinaesthetic sensations of fermented green table olives, using a simplex centroid mixture design augmented with interior points. The sodium in the flesh was linearly related to the mixture concentrations while potassium and calcium were linked by quadratic and special cubic models respectively. Acidity, saltiness, hardness, fibrousness, and crunchiness were expressed as linear funtions of the NaCl, KCl, and CaCl(2) initial brine contents but bitterness required quadratic equations. The models can be used to produce table olives with specific mineral contents in the flesh and to predict their corresponding sensory characteristics. PRACTICAL APPLICATION: The study provides the industry with models to estimate the Na, K, and Ca mineral contents in the flesh of fermented Gordal green table olives as well as their sensory characteristics as a function of the NaCl, KCl, and CaCl(2) initial compositions in the brining solution. Therefore, the paper provides tools which are able to support the production of commercial presentations which not only satisfy consumer demand for low Na, but are also K and Ca fortified table olive presentations with specific sensory profiles.     

Changes in gastric resistance of Lactobacillus casei in flavoured commercial fermented milks during refrigerated storage

This study investigated the gastric acid resistance ofLactobacillus caseiin commercial fermented milks during refrigerated storage. Samples of fermented milks (natural, strawberry, fruits of the forest, vanilla and multifruits) were obtained from three different manufacturers. In vitro gastric acid resistance (GAR) was assessed in samples upon arrival to the laboratory and after 10 and 20 days of refrigerated storage at 5 °C and 12 °C. The GAR ofL. caseiin fermented milks may increase or decrease during storage in relation to the flavour and storage conditions. The occurrence of this phenomenon in commercial fermented milks containing probiotic bacteria may induce changes in their functionality after consumption.     

Effects of a Lactobacillus casei 393 fermented milk product on bone metabolism in ovariectomised rats

The effects of a Lactobacillus casei 393 fermented milk product (FMP) on bone metabolism were examined. FMP (>0.1 mg mL^?1) supplementation significantly increased osteoblastic MC3T3-E1 cells proliferation whereas skim milk powder supplementation did not show any positive effect up to 1 mg mL^?1. The FMP (1%) supplemented ovariectomised rats had increased bone weight, bone mineral density, and bone breaking force compared with control ovariectomised rat. In addition, the activity of tartrate resistant acid phosphatase, a biomarker of osteoclasts, was significantly reduced in the FMP group. Based on the results, the L. casei 393 FMP had a preventative effect on bone loss in ovariectomised rats.     

Characterization of the angiotensin-converting enzyme inhibitory activity of fermented milks produced with Lactobacillus casei

Our study assayed angiotensin-converting enzyme (ACE) inhibitory activity and fermentation characteristics of 41 food-originated Lactobacillus casei strains in fermented milk production. Twenty-two of the tested strains produced fermented milks with a high ACE inhibitory activity of over 60%. Two strains (IMAU10408 and IMAU20411) expressing the highest ACE inhibitory activity were selected for further characterization. The heat stability (pasteurization at 63°C for 30 min, 75°C for 25 s, and 85°C for 20 s) and resistance to gastrointestinal proteases (pepsin, trypsinase, and sequential pepsin/trypsinase treatments) of the ACE inhibitory activity in the fermented milks produced with IMAU10408 and IMAU20411 were determined. Interestingly, such activity increased significantly after the heat or protease treatment. Because of the shorter milk coagulation time of L. casei IMAU20411 (vs. IMAU10408), it was selected for optimization experiments for ACE inhibitory activity production. Our results show that fermentation temperature of 37°C, inoculum density of 1 × 10⁶ cfu/g, and fermentation time of 12 h were optimal for maximizing ACE inhibitory activity. Finally, the metabolite profiles of L. casei IMAU20411 after 2 and 42 h of milk fermentation were analyzed by ultra-HPLC electron spray ionization coupled with time-of-flight mass spectrometry. Nine differential abundant metabolites were identified, and 2 of them showed a strong and positive correlation with fermented milk ACE inhibitory activity. To conclude, we have identified a novel ACE inhibitory L. casei strain, which has potential for use as a probiotic in fermented milk production.     

Microbiological and physicochemical changes of naturally black olives fermented at different temperatures and NaCl levels in the brines

The effect of different temperatures (25°C, 18°C and ambient temperature) and NaCl levels in brines (4%, 6% and 8%) on the microbiological and physicochemical characteristics of naturally black olives of Conservolea variety was studied for up to 190 days. Fermentation was carried out according to the traditional anaerobic method. The initial microflora consisted of Gram-negative bacteria, lactic acid bacteria and yeasts. Inhibition of Gram-negative bacteria was evident in all fermentations. The prevailing micro-organisms were lactic acid bacteria and yeasts, the association of which was dependent on the conditions of fermentation. At 25°C and 18°C in brines containing 4% and 6% NaCl, the growth of lactic acid bacteria was favoured resulting in a lactic acid fermentation, as indicated by the high free acidity levels and low pH values in the brines. On the contrary, 8% NaCl concentration affected the growth of lactic acid bacteria and enhanced the activity of fermentative yeasts, producing a final product with lower free acidity and higher pH value. At ambient temperature, the counts of lactic acid bacteria followed the fluctuation of temperature regardless of salt concentration, while yeasts did not seem to be affected. The lactic acid bacteria identified belonged to the species Lactobacillus mesenteroides, Lactobacillus brevis, Lactobacillus plantarum and Lactobacillus pentosus. The best conditions for fermentation were at 25°C and 6% NaCl, developing free acidity of 142 mM (1·28% w/v) lactic acid and pH value of 3·8. After 5 months of brining, olives fermented at 25°C were judged by panelists as being debittered and ready to eat. No off-odour development was detected in any case due to anomalous fermentation. The HPLC analysis revealed that citric, malic, tartaric, succinic, lactic and acetic were among the end products of fermentation.     

Lactobacillus casei LcY decreases milk protein immunoreactivity of fermented buttermilk but also contains IgE-reactive proteins

This study evaluates Lactobacillus casei LcY protein immunactive properties and assesses its potential to decrease milk protein immunoreactivity in fermented buttermilk before and after simulated digestion. Competitive ELISA analysis of α-lactalbumin, β-lactoglobulin, α-casein, β-casein, κ-casein, bovine serum albumin and lactoferrin determined significant (P < 0.001) immunoreactivity reduction after fermentation, and even greater decrease after three-stage simulated digestion. Immunoblotting of fermented buttermilk with human allergic sera revealed that α-casein remained the most allergenic milk protein. The LcY cell fractionation and further separation by SDS-PAGE and 2DE, immunoblotting and MALDI-TOF MS/MS identification all highlighted that cyclopropane-fatty-acyl-phospholipid synthase and carboxylate-amine ligase in the cell wall/membrane protein fraction reacted with human IgE antibodies. In silico analysis of these proteins' allergenic potential indicated that these are new allergens rather than already known cross-reacting allergens.To our best knowledge, this is the first study providing evidence of human IgE reactive protein presence in lactic acid bacteria.     

New insights in antibiotic resistance of Lactobacillus species from fermented foods

Bacteria belonging to the genus Lactobacillus are used as starter cultures or that develop naturally as fermenting microbiota in the production of various foods. On the detrimental side, lactobacilli may act as reservoir of antibiotic resistance genes, which can spread to commensal bacteria in humans or animals, or to food-associated pathogens. In the last decade, advances in molecular biology and in genome sequencing have provided more information on antibiotic resistances in foodborne bacteria. The aim of this review was to consider and provide an up-to-date status on phenotypic and genotypic antibiotic resistance profiles in Lactobacillus species from fermented foods and also to highlight new information on the distribution of glycopeptide and chloramphenicol resistance genes in Lactobacillus genomes. In silico screening of vanZ (glycopeptide resistance) and cat (chloramphenicol resistance)-like sequences in Lactobacillus species isolated from fermented foods revealed for the first time the occurrence of vanZ and cat genes in Lactobacillus species being highly conserved genes in the chromosome of each species, presumably non-transferable. Further studies involving genome sequences of Lactobacillus isolated from fermented foods, especially those relying on spontaneous fermentation, is crucial to increase knowledge on the potential presence and spread of antibiotic resistance genes via the food route.     

The biological activity of fermented milk produced by Lactobacillus casei ATCC 393 during cold storage

Lactobacillus casei ATCC 393 is an important probiotic strain widely known in dairy technology. However, its capability to produce bioactive peptides from milk proteins has not been studied. The viability of the Lb. casei ATCC 393 strain and some physicochemical properties in fermented milk throughout storage for 21 days at 4 °C was evaluated; biological activity, i.e., antioxidant, angiotensin converting enzyme inhibitory and anticancer activities of water soluble extract and its filtrate (< 2 kDa; F1) were determined. Lb. casei counts remained over 9 log cfu g⁻¹ during the storage period in fermented milk. These bioactivities were increased significantly (P < 0.01) during storage. F1 of fermented milk after three weeks of storage showed the highest bioactivity impact. De novo sequencing assay for peptide identification was applied to the mass spectrum of F1. The promising capability of Lb. casei ATCC 393 to release bioactive peptides from milk proteins was demonstrated.     

Induced lactic acid fermentation of untreated green olives of the Conservolea cultivar by Lactobacillus pentosus

Inoculation of untreated green olives of the Conservolea cultivar (Olea europea media rotunda) with a commercial strain of Lactobacillus pentosus with/without glucose supplement was studied. Despite an initial loss in viability of 0.5 log cycles on average, due to lack of adaptation of the starter to the saline environment of the brine, cultures grew well and initiated an accelerated fermentation process. Inoculation reduced the survival period of Enterobacteriaceae, and consequently potential spoilage, and caused a quicker acidification of brines and decrease in pH compared with control uninoculated processes. The diffusion of phenolic compounds was slow, hindered by the epidermis of the fruits, and had no effect on the growth of the starter. HPLC analysis revealed that lactic and acetic acids were among the end‐products during the fermentation of green olives. Citric, tartaric and malic acids were also detected but at much lower concentrations. Results obtained from this work can be applied directly to industry for the effective use of starter cultures on natural fermentation processes of green olives without prior lye treatment. © 2003 Society of Chemical Industry     

Enumeration of Lactobacillus casei in the presence of L. acidophilus,bifidobacteria and lactic starter bacteria in fermented dairy products

The aim of this work was to select a set of culture media to perform the enumeration of L. casei when it appears together with L. acidophilus, bifidobacteria and bacteria of lactic acid starters, in fermented dairy products. A number of L. acidophilus, Bifidobacterium and L. casei strains (8 of each) were tested for their ability to grow in two selective/differential media (LP-MRS agar and B-MRS agar). The enumeration of these bacteria using the media mentioned above was also carried out in fermented dairy products available on the market. B-MRS agar and LP-MRS agar were able to inhibit the lactic acid bacteria from the starters. B-MRS agar could be useful for the selective colony count of L. acidophilus or L. casei, or for a differential enumeration when these bacteria appear together in a fermented dairy product. In the case of bifidobacteria, a selective colony count could be performed on LP-MRS agar, even when it appears with L. casei, because a differential cell enumeration between both organisms was possible. The three probiotic bacteria could be simultaneously enumerated from dairy products that contain them along with starter bacteria using media developed in this work.     

Stress Responses in Probiotic Lactobacillus casei

Survival in harsh environments is critical to both the industrial performance of lactic acid bacteria (LAB) and their competitiveness in complex microbial ecologies. Among the LAB, members of the Lactobacillus casei group have industrial applications as acid-producing starter cultures for milk fermentations and as specialty cultures for the intensification and acceleration of flavor development in certain bacterial-ripened cheese varieties. They are amongst the most common organisms in the gastrointestinal (GI) tract of humans and other animals, and have the potential to function as probiotics. Whether used in industrial or probiotic applications, environmental stresses will affect the physiological status and properties of cells, including altering their functionality and biochemistry. Understanding the mechanisms of how LAB cope with different environments is of great biotechnological importance, from both a fundamental and applied perspective: hence, interaction between these strains and their environment has gained increased interest in recent years. This paper presents an overview of the important features of stress responses in Lb. casei, and related proteomic or gene expression patterns that may improve their use as starter cultures and probiotics.     

Diffusion of sodium chloride in green olives

The diffusivity of sodium chloride in green olives was investigated in laboratory experiments. The olives were immersed in brines of limited volume and salt absorption was measured from changes in brine concentration. The salt diffusivity was determined by a numerical method of non-linear regression analysis. Low salt diffusivity values, ranging from 3·2 × 10⁻¹¹ to 4·3 × 10⁻¹¹ m² s⁻¹, with a mean value of 3·8 × 10⁻¹¹ m² s⁻¹, were obtained, due to the high resistance to mass transfer of the olive skin and flesh. Pretreatment of the olives in 1·8% sodium hydroxide solution, used to remove the bitter component oleuropein, has the added advantage of increasing the salt diffusivity almost 5-fold.