Use of encapsulated lactic acid bacteria as bioprotective cultures in fresh Brazilian cheese.

There is great interest for biopreservation of food products, and encapsulation may be a good strategy to extend the viability of protective cultures. In this study, Lactobacillus paraplantarum FT-259 and Lactococcus lactis QMF 11 were separately encapsulated in casein/pectin (C/P) microparticles, which were tested for antilisterial and anti-staphylococcal activity in fresh Minas cheese (FMC) stored at 8 °C. The encapsulation efficiency for both lactic acid bacteria (LAB) was 82.5%, with viability over 6.2 log CFU/g after storage of C/P microparticles for 90 days under refrigeration. Interestingly, free Lb. paraplantarum and free Lc. lactis grew significantly in refrigerated FMC, both in the presence and absence of pathogens, but only the first significatively grew when encapsulated. Encapsulation increased the antilisterial activity of Lb. paraplantarum in FMC. Moreover, Lc. lactis significantly inhibited listerial growth in FMC in both its free and encapsulated forms, whereas Staphylococcus aureus counts were only significantly reduced in the presence of free Lc. lactis. In conclusion, these results indicate that C/P microparticles are effective carriers of LAB in FMC, which can contribute for the assurance of the safety of this product.

Lactococcus lactis NZ9000 Prevents Asthmatic Airway Inflammation and Remodelling in Rats through the Improvement of Intestinal Barrier Function and Systemic TGF-ß Production.

INTRODUCTION: The use of probiotics has been broadly popularized due to positive effects in the attenuation of aberrant immune responses such as asthma. Allergic asthma is a chronic respiratory disease characterized by airway inflammation and remodelling. OBJECTIVE: This study was aimed to evaluate the effect of oral administration of Lactococcus lactis NZ9000 on asthmatic airway inflammation and lung tissue remodelling in rats and its relation to the maintenance of an adequate intestinal barrier. METHODS: Wistar rats were ovalbumin (OVA) sensitized and challenged and orally treated with L. lactis. Lung inflammatory infiltrates and cytokines were measured, and remodelling was evaluated. Serum OVA-specific immunoglobulin (Ig) E levels were assessed. We also evaluated changes on intestinal environment and on systemic immune response. RESULTS: L. lactis diminished the infiltration of proinflammatory leucocytes, mainly eosinophils, in the bronchoalveolar compartment, decreased lung IL-4 and IL-5 expression, and reduced the level of serum allergen-specific IgE. Furthermore, L. lactis prevented eosinophil influx, collagen deposition, and goblet cell hyperplasia in lung tissue. In the intestine, L. lactis-treated asthmatic rats increased Peyer's patch and goblet cell quantity and mRNA expression of IgA, MUC-2, and claudin. Additionally, intestinal morphological alterations were normalized by L. lactis administration. Splenocyte proliferative response to OVA was abolished, and serum levels of transforming growth factor (TGF)-ß were increased by L. lactis treatment. CONCLUSIONS: These findings suggest that L. lactis is a potential candidate for asthma prevention, and the effect is mediated by the improvement of intestinal barrier function and systemic TGF-ß production.

The influence of adaptive stresses on the survival of spray-dried Lactococcus lactis cells.

The research described in this technical research communication examines the hypothesis that sublethal stress conditions can improve the survival of Lactococcus lactis subsp. lactis during drying and subsequent storage. After drying, the L. lactis that had adapted to acid or osmotic stresses did not differ statistically in terms of cell viability loss compared to the control samples tested (~0.38 log cycles). However, the cells that had adapted to oxidative conditions demonstrated a cell viability loss of only 0.01 log cycles. After 45 d of storage at temperatures of 4 and 25 °C, the final L. lactis sample populations were shown to be higher (112.5%) when they had been submitted to sublethal conditions of oxidative stress. When the cell samples were exposed to acid stress conditions, they exhibited a viability loss (0.82 log cycles) that was statistically different from the control sample (0.58 log cycles) after 45 d. Osmotic stress conditions did not demonstrate any influence over cell survival rates. Thus, submitting cells to oxidative stress conditions prior to storage has been shown to be a potential strategy for producing dehydrated cultures of L. lactis strains that are less sensitive to oxygen exposure.

Invasive Lactococcus lactis producing mycobacterial Hsp65 ameliorates intestinal inflammation in acute TNBS-induced colitis in mice by increasing the levels of the cytokine IL-10 and secretory IgA.

AIMS: To investigate the anti-inflammatory activity of an invasive and Hp65-producing strain Lactococcus lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) in acute 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis in mice as an innovative therapeutic strategy against Crohn's disease (CD). METHODS AND RESULTS: The pXYCYT:Hsp65 plasmid was transformed into the L. lactis NCDO2118 FnBPA+ strain, resulting in the L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) strain. Then, the functionality of the strain was evaluated in vitro for Hsp65 production by Western blotting and for invasion into Caco-2 cells. The results demonstrated that the strain was able to produce Hsp65 and efficiently invade eukaryotic cells. Subsequently, in vivo, the anti-inflammatory capacity of the recombinant strain was evaluated in colitis induced with TNBS in BALB/c mice. Oral administration of the recombinant strain was able to attenuated the severity of colitis by mainly reducing IL-12 and IL-17 levels and increasing IL-10 and secretory immunoglobulin A levels. CONCLUSIONS: The L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) strain contributed to a reduction in inflammatory damage in experimental CD. SIGNIFICANCE AND IMPACT OF THE STUDY: This study, which used L. lactis for the production and delivery of Hsp65, has scientific relevance because it shows the efficacy of this new strategy based on therapeutic protein delivery into mammalian enterocytes.

Determination of ideal water activity and powder temperature after spray drying to reduce Lactococcus lactis cell viability loss.

Spray drying presents a promising technology for preserving bacteria despite a low survival rate of heat-sensitive cultures when subjected to the drying process. The aim of this study was to determine the ideal powder parameters [water activity (Aw) and temperature (T°Cpowder)] needed to produce dehydrated Lactococcus lactis ssp. lactis with a high viability after drying. Cell concentrates injected into a spray dryer using varying cell concentrate flow rates (Fconcentrate = 0.3 to 1.0 kg/h), inlet air temperatures (T°Cinlet air = 115 to 160°C), and outlet air temperatures (T°Coutlet air = 70 to 115°C) resulted in powders with different values of Aw and T°Cpowder, and levels of cell viability loss. Lower cell viability reduction (∼0.43 log cycles) was obtained in conditions of Aw = 0.198 and T°Cpowder = 52°C, which can be met by using T°Cinlet air ∼126°C and T°Coutlet air = 88.9°C regardless of Fconcentrate values. After 60 d of storage at room temperature, cell population varied from 7.0 × 105 to 1.1 × 108 cfu/g. The initial powder Aw had no influence on cell death rate, but T°Cpowder influence was observed. The approach adopted in this study can be applied to other bacteria or spray dryer equipment to determine optimal drying conditions.

In Vitro Probiotic and Antioxidant Potential of Lactococcus lactis subsp. cremoris LL95 and Its Effect in Mice Behaviour.

The composition of intestinal microbiota is widely believed to not only affect gut health but also influence behaviour. This study aimed to evaluate the probiotic characteristics, antioxidant activity, and antidepressant- and anxiolytic-like activities of Lactococcus lactis subsp. cremoris LL95. This strain showed probiotic properties such as resistance in a simulated gastric tract model and survival at different concentrations of NaCl and bile salts. Moreover, antioxidant activity of LL95 was demonstrated through DPPH radical scavenging activity, scavenging of ABTS•+ radical and ferric ion reducing antioxidant power (FRAP) assays. Female C57BL/6 mice received LL95 orally at a dose of 109 UFC/day for 28 days. LL95 improved depressive- and anxiety-like behaviour, demonstrated by decreased immobility time in the tail suspension test and forced swim test and increased per cent of time spent in the open arms on the elevated plus maze. These findings indicate the potential antioxidant activity of LL95 and its role in behaviour, suggesting that probiotic may have therapeutic applications.

Gut microbiome modulation during treatment of mucositis with the dairy bacterium Lactococcus lactis and recombinant strain secreting human antimicrobial PAP.

Mucositis is an inflammatory condition of the gut, caused by an adverse effect of chemotherapy drugs, such as 5-fluorouracil (5-FU). In an attempt to develop alternative treatments for the disease, several research groups have proposed the use of probiotics, in particular, Lactic Acid Bacteria (LAB). In this context, the use of recombinant LAB, for delivering anti-inflammatory compounds has also been explored. In previous work, we demonstrated that either Lactococcus lactis NZ9000 or a recombinant strain expressing an antimicrobial peptide involved in human gut homeostasis, the Pancreatitis-associated Protein (PAP), could ameliorate 5-FU-induced mucositis in mice. However, the impact of these strains on the gut microbiota still needs to be elucidated. Therefore, in the present study, we aimed to characterize the effects of both Lactococci strains in the gut microbiome of mice through a 16 S rRNA gene sequencing metagenomic approach. Our data show 5-FU caused a significant decrease in protective bacteria and increase of several bacteria associated with pro-inflammatory traits. The Lactococci strains were shown to reduce several potential opportunistic microbes, while PAP delivery was able to suppress the growth of Enterobacteriaceae during inflammation. We conclude the strain secreting antimicrobial PAP was more effective in the control of 5-FU-dysbiosis.

Effect of sodium reduction and flavor enhancer addition on probiotic Prato cheese processing.

The effect of partial substitution of NaCl with KCl and the flavor enhancers addition (arginine, yeast extract and oregano extract) on Probiotic Prato cheese processing with (L. casei 01, 7logCFU/mL) was investigated. Microbiological (lactic acid bacteria and probiotic counts), physicochemical (proximate composition, pH, proteolysis), bioactivity (antioxidant and angiotensin I-converting enzyme inhibitory activity), rheological (uniaxial compression and creep tests), water mobility (time domain low field magnetic resonance), microstructure (scanning electron microscopy) and sensory evaluation (consumer test) were performed. Sodium reduction and flavor enhancers addition did not constitute an obstacle to the survival of lactic and probiotic bacteria. Proximate composition, antioxidant and angiotensin I-converting enzyme inhibitory activity, and the rheological parameters were affected by the addition of flavor enhancer. No change in the fatty acid profile of cheeses was observed while good performance in the consumer test was obtained by the addition of yeast extract and oregano extract. Prato cheese can be an adequate carrier of probiotics and the addition of different flavor enhancers can contribute developing this functional product in the cheese industry.

Secretion of biologically active pancreatitis-associated protein I (PAP) by genetically modified dairy Lactococcus lactis NZ9000 in the prevention of intestinal mucositis.

BACKGROUND: Mucositis is one of the most relevant gastrointestinal inflammatory conditions in humans, generated by the use of chemotherapy drugs, such as 5-fluoracil (5-FU). 5-FU-induced mucositis affects 80% of patients undergoing oncological treatment causing mucosal gut dysfunctions and great discomfort. As current therapy drugs presents limitations in alleviating mucositis symptoms, alternative strategies are being pursued. Recent studies have shown that the antimicrobial pancreatitis-associated protein (PAP) has a protective role in intestinal inflammatory processes. Indeed, it was demonstrated that a recombinant strain of Lactococcus lactis expressing human PAP (LL-PAP) could prevent and improve murine DNBS-induced colitis, an inflammatory bowel disease (IBD) that causes severe inflammation of the colon. Hence, in this study we sought to evaluate the protective effects of LL-PAP on 5-FU-induced experimental mucositis in BALB/c mice as a novel approach to treat the disease. RESULTS: Our results show that non-recombinant L. lactis NZ9000 have antagonistic activity, in vitro, against the enteroinvasive gastrointestinal pathogen L. monocytogenes and confirmed PAP inhibitory effect against Opportunistic E. faecalis. Moreover, L. lactis was able to prevent histological damage, reduce neutrophil and eosinophil infiltration and secretory Immunoglobulin-A in mice injected with 5-FU. Recombinant lactococci carrying antimicrobial PAP did not improve those markers of inflammation, although its expression was associated with villous architecture preservation and increased secretory granules density inside Paneth cells in response to 5-FU inflammation. CONCLUSIONS: We have demonstrated for the first time that L. lactis NZ9000 by itself, is able to prevent 5-FU-induced intestinal inflammation in BALB/c mice. Moreover, PAP delivered by recombinant L. lactis strain showed additional protective effects in mice epithelium, revealing to be a promising strategy to treat intestinal mucositis.

Anti-cancer effect of lactic acid bacteria expressing antioxidant enzymes or IL-10 in a colorectal cancer mouse model.

The association between inflammatory bowel diseases and colorectal cancer is well documented. The genetic modification of lactic acid bacteria as a tool to increase the anti-inflammatory potential of these microorganisms has also been demonstrated. Thus the aim of the present work was to evaluate the anti-cancer potential of different genetically modified lactic acid bacteria (GM-LAB) producing antioxidant enzymes (catalase or superoxide dismutase) or the anti-inflammatory cytokine IL-10 (protein or DNA delivery) using a chemical induced colon cancer murine model. Dimethilhydrazine was used to induce colorectal cancer in mice. The animals received GM-LAB producing anti-oxidant enzymes, IL-10 or a mixture of different GM-LAB. Intestinal damage, enzyme activities and cytokines were evaluated and compared to the results obtained from mice that received the wild type strains from which derived the GM-LAB. All the GM-LAB assayed showed beneficial effects against colon cancer even though they exerted different mechanisms of action. The importance to select LAB with innate beneficial properties as the progenitor strain was demonstrated with the GM-LAB producing anti-oxidant enzymes. In addition, the best effects for the mixtures GM-LAB that combine different anti-inflammatory mechanism. Results indicate that mixtures of selected LAB and GM-LAB could be used as an adjunct treatment to decrease the inflammatory harmful environment associated to colorectal cancer, especially for patients with chronic intestinal inflammation who have an increased risk to develop colorectal cancer.

Heterologous expression of Streptococcus mutans Cnm in Lactococcus lactis promotes intracellular invasion, adhesion to human cardiac tissues and virulence.

In S. mutans, the expression of the surface glycoprotein Cnm mediates binding to extracellular matrix proteins, endothelial cell invasion and virulence in the Galleria mellonella invertebrate model. To further characterize Cnm as a virulence factor, the cnm gene from S. mutans strain OMZ175 was expressed in the non-pathogenic Lactococcus lactis NZ9800 using a nisin-inducible system. Despite the absence of the machinery necessary for Cnm glycosylation, Western blot and immunofluorescence microscopy analyses demonstrated that Cnm was effectively expressed and translocated to the cell wall of L. lactis. Similar to S. mutans, expression of Cnm in L. lactis enabled robust binding to collagen and laminin, invasion of human coronary artery endothelial cells and increased virulence in G. mellonella. Using an ex vivo human heart tissue colonization model, we showed that Cnm-positive strains of either S. mutans or L. lactis outcompete their Cnm-negative counterparts for tissue colonization. Finally, Cnm expression facilitated L. lactis adhesion and colonization in a rabbit model of infective endocarditis. Collectively, our results provide unequivocal evidence that binding to extracellular matrices mediated by Cnm is an important virulence attribute of S. mutans and confirm the usefulness of the L. lactis heterologous system for further characterization of bacterial virulence factors.

Recombinant invasive Lactococcus lactis can transfer DNA vaccines either directly to dendritic cells or across an epithelial cell monolayer.

Lactococcus lactis (L. lactis), a generally regarded as safe (GRAS) bacterium has recently been investigated as a mucosal delivery vehicle for DNA vaccines. Because of its GRAS status, L. lactis represents an attractive alternative to attenuated pathogens. Previous studies showed that eukaryotic expression plasmids could be delivered into intestinal epithelial cells (IECs) by L. lactis, or recombinant invasive strains of L. lactis, leading to heterologous protein expression. Although expression of antigens in IECs might lead to vaccine responses, it would be of interest to know whether uptake of L. lactis DNA vaccines by dendritic cells (DCs) could lead to antigen expression as they are unique in their ability to induce antigen-specific T cell responses. To test this, we incubated mouse bone marrow-derived DCs (BMDCs) with invasive L. lactis strains expressing either Staphylococcus aureus Fibronectin Binding Protein A (LL-FnBPA+), or Listeria monocytogenes mutated Internalin A (LL-mInlA+), both strains carrying a plasmid DNA vaccine (pValac) encoding for the cow milk allergen ß-lactoglobulin (BLG). We demonstrated that they can transfect BMDCs, inducing the secretion of the pro-inflammatory cytokine IL-12. We also measured the capacity of strains to invade a polarized monolayer of IECs, mimicking the situation encountered in the gastrointestinal tract. Gentamycin survival assay in these cells showed that LL-mInlA+ is 100 times more invasive than L. lactis. The cross-talk between differentiated IECs, BMDCs and bacteria was also evaluated using an in vitro transwell co-culture model. Co-incubation of strains in this model showed that DCs incubated with LL-mInlA+ containing pValac:BLG could express significant levels of BLG. These results suggest that DCs could sample bacteria containing the DNA vaccine across the epithelial barrier and express the antigen.

Kefir-isolated Lactococcus lactis subsp. lactis inhibits the cytotoxic effect of Clostridium difficile in vitro.

Kefir is a dairy product obtained by fermentation of milk with a complex microbial population and several health-promoting properties have been attributed to its consumption. In this work, we tested the ability of different kefir-isolated bacterial and yeast strains (Lactobacillus kefir, Lb. plantarum, Lactococcus lactis subps. lactis, Saccharomyces cerevisiae and Kluyveromyces marxianus) or a mixture of them (MM) to antagonise the cytopathic effect of toxins from Clostridium difficile (TcdA and TcdB). Cell detachment assays and F-actin network staining using Vero cell line were performed. Although incubation with microbial cells did not reduce the damage induced by C. difficile spent culture supernatant (SCS), Lc. lactis CIDCA 8221 and MM supernatants were able to inhibit the cytotoxicity of SCS to Vero cells. Fraction of Lc. lactis CIDCA 8221 supernatant containing components higher than 10 kDa were responsible for the inhibitory activity and heating of this fraction for 15 min at 100 °C completely abrogated this ability. By dot-blot assay with anti-TcdA or anti-TcdB antibodies, concentration of both toxins seems to be reduced in SCS treated with Lc. lactis CIDCA 8221 supernatant. However, protective effect was not affected by treatment with proteases or proteases-inhibitors tested. In conclusion, we demonstrated that kefir-isolated Lc. lactis CIDCA 8221 secreted heat-sensitive products able to protect eukaryotic cells from cytopathic effect of C. difficile toxins in vitro. Our findings provide new insights into the probiotic action of microorganisms isolated from kefir against virulence factors from intestinal pathogens.

In vitro inhibition of Citrobacter freundii, a red-leg syndrome associated pathogen in raniculture, by indigenous Lactococcus lactis CRL 1584.

Red-leg syndrome (RLS) is one of the main infectious diseases that cause economic losses in Lithobates catesbeianus hatcheries, Citrobacter freundii being an etiological agent. Treatment or prevention with therapeutics or chemicals results in modifications of the indigenous microbiota, development of antibiotic resistance, presence of their residues in food and enhancement of production costs. Thus, probiotics could be used as an alternative therapy. Lactic acid bacteria are part of the indigenous microbiota of healthy frogs and can prevent pathogen colonization by different mechanisms, including the production of antagonistic substances. In this work, the evaluation and characterization of the inhibition of C. freundii CFb by Lactococcus lactis subsp. lactis CRL 1584, a potentially probiotic candidate, were carried out. This strain produced lactic acid, H(2)O(2) and bacteriocin in static and shaken conditions and inhibited pathogen growth in associative cultures, with an earlier inhibition under agitated conditions. The elimination of each of the antimicrobial metabolites partially abolished the inhibition of the pathogen, suggesting that the inhibitory effect could be attributed to a combined action of the three antagonistic molecules. Electron microphotographs revealed the damage caused by L. lactis CRL 1584 supernatants to C. freundii cells. The addition of pure lactic acid, H(2)O(2) and bacteriocin to the culture media showed that each metabolite caused different morphological modifications in C. freundii, in agreement with the effect on viable cell counts. The results support the possibility that L. lactis CRL 1584 might be considered as a probiotic to be used in the prevention of RLS in raniculture.

Effect of freeze-drying on viability and in vitro probiotic properties of a mixture of lactic acid bacteria and yeasts isolated from kefir.

The effect of freeze-drying on viability and probiotic properties of a microbial mixture containing selected bacterial and yeast strains isolated from kefir grains (Lactobacillus kefir, Lactobacillus plantarum, Lactococcus lactis, Saccharomyces cerevisiae and Kluyveromyces marxianus) was studied. The microorganisms were selected according to their potentially probiotic properties in vitro already reported. Two types of formulations were performed, a microbial mixture (MM) suspended in milk and a milk product fermented with MM (FMM). To test the effect of storage on viability of microorganisms, MM and FMM were freeze-dried and maintained at 4°C for six months. After 180 days of storage at 4°C, freeze-dried MM showed better survival rates for each strain than freeze-dried FMM. The addition of sugars (trehalose or sucrose) did not improve the survival rates of any of the microorganisms after freeze-drying. Freeze-drying did not affect the capacity of MM to inhibit growth of Shigella sonnei in vitro, since the co-incubation of this pathogen with freeze-dried MM produced a decrease of 2 log in Shigella viability. The safety of freeze-dried MM was tested in mice and non-translocation of microorganisms to liver or spleen was observed in BALB/c mice feed ad libitum during 7 or 20 days. To our knowledge, this is the first report about the effect of freeze-drying on viability, in vitro probiotic properties and microbial translocation of a mixture containing different strains of both bacteria and yeasts isolated from kefir.

Listeria monocytogenes and Salmonella spp. in raw milk produced in Brazil: occurrence and interference of indigenous microbiota in their isolation and development.

This study aimed to verify the occurrence of Listeria monocytogenes and Salmonella spp. in raw milk produced in Brazil. On account of the poor microbiological quality of this product, possible interference from the indigenous microbiota in these pathogens was also evaluated. Two-hundred and ten raw milk samples were collected in four important milk-producing areas in Brazil, tested for L. monocytogenes and Salmonella spp. presence, and for enumeration of indicator microorganisms: mesophilic aerobes, total coliforms and Escherichia coli. The interference of the indigenous microbiota in the isolation procedures was also tested, as well the frequency of naturally occurring raw milk strains with antagonistic activity against both pathogens. The pathogens were not isolated in any raw milk sample, but poor microbiological quality was confirmed by the high levels of indicator microorganisms. When present at high levels, the indigenous microbiota generated an evident interference in the methodologies of L. monocytogenes and Salmonella spp. isolation, mainly when the pathogens appeared at low levels. Three-hundred and sixty raw milk strains were tested for antagonistic activity against both pathogens, and 91 (25.3%) showed inhibitory activity against L. monocytogenes and 33 (9.2%) against Salmonella spp. The majority of the antagonistic strains were identified as Lactic Acid Bacteria species, mainly Lactococcus lactis subsp. lactis and Enterococcus faecium, known by antimicrobial substance production.

Oral administration of a catalase-producing Lactococcus lactis can prevent a chemically induced colon cancer in mice.

Reactive oxygen species, such as hydrogen peroxide (H2O2), are involved in various aspects of tumour development. Decreasing their levels can therefore be a promising approach for colon cancer prevention. The objective of this study was to evaluate the effect of catalase-producing Lactococcus lactis on the prevention of an experimental murine 1,2-dimethylhydrazine (DMH)-induced colon cancer. DMH-treated BALB/c mice received either a catalase-producing L. lactis strain or the isogenic non-catalase-producing strain as a control, whereas other untreated mice did not receive bacterial supplementation. Catalase activity and H2O2 levels in intestinal fluids and blood samples were measured, and changes in the histology of the large intestines during tumour progression were evaluated. The catalase-producing L. lactis strain used in this study was able to slightly increase catalase activities in DMH-treated mice (1.19+/-0.08 U ml(-1)) and reduce H2O2 levels (3.4+/-1.1 microM) compared to (i) animals that received the non-catalase-producing strain (1.00+/-0.09 U ml(-1), 9.0+/-0.8 microM), and (ii) those that did not receive bacterial supplementation (1.06+/-0.07 U ml(-1), 10.0+/-1.1 microM). Using the histopathological grading scale of chemically induced colorectal cancer, animals that received the catalase-producing L. lactis had a significantly lesser extent of colonic damage and inflammation (2.0+/-0.4) compared to animals that received the non-catalase-producing L. lactis (4.0+/-0.3) or those that did not receive bacterial supplementation (4.7+/-0.5). The catalase-producing L. lactis strain used in this study was able to prevent tumour appearance in an experimental DMH-induced colon cancer model.

Efficient mechanical disruption of Lactobacillus helveticus, Lactococcus lactis and Propionibacterium freudenreichii by a new high-pressure homogenizer and recovery of intracellular aminotransferase activity.

Microbiological studies often involve bacterial cell fractionation, which is known to be difficult for Gram-positive as compared to Gram-negative bacteria. Our purpose was to test the breaking efficiency of a new high-pressure pilot homogenizer for three Gram-positive species involved in dairy technology and to assess the activity of an intracellular aminotransferase. Varied pressures (50, 100 and 200 MPa) were applied to concentrated bacterial suspensions (1.2 mg dry weight/ml) of Lactobacillus helveticus, Lactococcus lactis and Propionibacterium freudenreichii. Breaking efficiency was estimated by decreases in optical density at 650 nm, cellular dry weight and viability. The proteins released were quantified and the residual intracellular aminotransferase activity was estimated using leucine as substrate. One run at 50 MPa was sufficient to break 80% of lactobacilli cells whereas 200 MPa were required for the same efficiency for L. lactis and P. freudenreichii. Whatever the pressure, leucine aminotransferase activity was recovered in the supernatant after cell breaking. This new high-pressure pilot homogenizer can allow rapid (20 s/run), easy, continuous and highly efficient cell breaking for intracellular enzyme recovery or other purposes. As the species tested were not phylogenetically related, and had different morphologies and cell wall compositions, we conclude that most Gram-positive bacteria may be broken efficiently by this new device.

[Viability and activity of the lactic bacteria (Streptococcus salivarius ssp thermophilus y Lactobacillus delbrueckii ssp bulgaricus) del yogurt en Venezuela]. / Viabilidad y actividad de la flora láctica (Streptococcus salivarius ssp thermophilus y Lactobacillus delbrueckii ssp bulgaricus) del yogurt en Venezuela.

National and international legislations have agreed that the population of lactic bacteria in yogurt must be viable and not less than 10(6) ufc/g. In Venezuela, during last years, observations indicate that the number of viable cells in some commercial samples show high variations, as low levels. This research attempted to find the origin of this problem in the local industry. For this purpose 105 commercial samples were analyzed during their shelf life and 32 samples of yogurt prepared in the laboratory following the flow diagram of the local industry. The different conditions of freeze dried lactic culture, were also analyzed. These samples were evaluated for viable cell count of lactic bacteria and possible variations of pH and acidity. The absence or low number of lactic bacteria detected in some commercial samples is due to the use of inadequate working cultures that show imbalanced proportions of the two microorganisms, besides a low count below 106 ufc/g. The succesive propagation and storage time of mother culture, and the overacidification of the product, produce subletal injury to the microbial cells of the yogurt starter culture. The data indicate that manufacturing practices significantly affect the survival of the lactic flora.

[Trials of cheese-making using concentrated bacterial starters]. / Ensayos de elaboración de quesos con fermentos bacterianos concentrados.

Studies about cheese preparation with frozen and concentrated bacterial starters have been carried out. The Pategras cheeses were obtained from raw milk. The starters were prepared with a selected strain of Streptococcus lactis, concentrated until reaching a value of 3.10(9) colony forming units/ml and resuspended in milk previously supplemented with 8% of yeast extract. These concentrates were frozen at -40 degrees C and kept at -20 degrees C for 60 days. Three kinds of starters were tested: one thawed by placing the flask in a 40 degrees C water bath, another added to the cheese vat without previous thawing and a control sample prepared in steamed reconstituted milk. In order to evaluate the convenience of each technique several chemical and microbiological analysis were performed during the preparation (Table 1 and 2) and the ripening of the cheese (Table 3). The results have showed that the direct use of thawed frozen concentrates in the cheese vat allows the obtaining of high quality cheese. On the other hand, the technique based on thawing through a water bath did not lead to good results.