Jorge ReinheimerNational Scientific and Technical Research Council | conicet · INLAIN Instituto de Lactología Industrial
· Dr. en Quimica
INLAIN Instituto de Lactología Industrial
Head of Department
Probiotic microorganisms are mainly bacteria from the genera Lactobacillus and Bifidobacterium that exert a health benefit on the host when consumed on a regular basis as dietary supplements or in foods. The fermented dairy industry was the first in successfully marketing specific strains of probiotic bacteria in products such as yogurts and fresh cheeses. Factors such as growing vegetarianism including veganism among consumers, lactose intolerance, allergy to milk proteins, cholesterol content of dairy products, and religious or dietary habits have limited the growth of the market of probiotics in dairy products and impaired their consumption by people interested in a healthier diet. Plant material such as cereal, legumes, fruits, and vegetables, and their combinations, offer a plethora of possibilities for the development of probiotic products, fermented or not. Much of the knowledge learned in the last 30 years about probiotics in dairy products is paving the way for the development of new nondairy foods and drinks.
The aim of this study is to evaluate the influence of the technological processing on the functionality of the human breast milk probiotic strain Bifidobacterium lactis INL1. In vitro antagonistic activity of B. lactis INL1 was detected for Gram-positive and Gram-negative pathogens. B. lactis INL1 was administered to mice as fresh (F), frozen (Z), spray-dried (S), or lyophilized (L) culture. Immune parameters (IgA, IL-10, and IFN-γ) were determined and histological analysis was performed to assess functionality and protection capacity against Salmonella. In BALB/c mice, F and S cultures induced an increase in the number of IgA-producing cells in the small intestine and IL-10 levels were increased for L culture in the large intestine. In Swiss mice, B. lactis INL1 increased secretory-IgA levels in the small intestine before and after Salmonella infection, both as F or dehydrated culture. Also, an attenuation of damage in the intestinal epithelium and less inflammatory infiltrates were observed in animals that received F and S cultures, whereas in liver only F showed some effect. The anti-inflammatory effect was confirmed in both tissues by myeloperoxidase activity and by IFN-γ levels in the intestinal content. B. lactis INL1 showed inhibitory activity against pathogens and confirmed its probiotic potential in animal models. Technological processing of the probiotic strain affected its functionality.
Aims: To evaluate the effects of spray-drying of Lactobacillus rhamnosus 64 on its capacity to modulate the gut immune response and on the attenuation of TNBS-induced colitis in mice. Methods and results: L. rhamnosus 64 was spray-dried in cheese whey-starch solution and administered to mice for three, six or ten consecutive days. Peritoneal macrophage phagocytic activity, secretory-IgA (S-IgA) levels in the small intestinal fluid and TNFα, IFNγ, IL-10, IL-6 and IL-2 levels in homogenates of the small and large intestine were determined. The effects of spray drying were also evaluated in an acute model of TNBS-induced colitis. A shift in the regulation of immune parameters, particularly the cytokine profile, was observed for mice treated with the spray dried culture, compared to the profile observed in animals that received the strain as fresh culture. The spray dried culture of L. rhamnosus 64 showed antiinflammatory properties in murine model of TNBS-induced colitis. Conclusion: The spray-drying process of L. rhamnosus 64 in whey-starch modified its immunomodulating capacity in healthy animals and conferred enhanced protection in an in vivo model of inflammation. Significance and impact of the study: Probiotic capacity can be affected by spray-drying in relation to the properties observed for the strain as an overnight fresh culture. This fact should be taken into account when producing the culture for its application in the industry. This article is protected by copyright. All rights reserved.
This study was aimed to evaluate the potential of high pressure homogenization for the microencapsulation of two probiotic lactic acid bacteria, Lactobacillus paracasei A13 and Lactobacillus salivarius subsp. salivarius CET 4063 to produce functional fermented milks. Microcapsules of the considered functional microorganisms were obtained by HPH treatments at 50 MPa in the presence of sodium alginate and vegetable oil. The microencapsulated microorganisms were then inoculated as adjuncts to produce fermented milks. As controls were used fermented milks in which the two probiotic lactobacilli were inoculated without encapsulation. The viability of the strains was monitored during almost 2 months of refrigerated storage. The survival of lactic acid bacteria after the gastric-duodenal simulated test was determined. Fermented milk texture parameters, the presence of exo-polysaccharides and the production of volatile molecules were also evaluated over storage. The microcapsules, for both the considered probiotic strains, were homogeneous and with a size < 100 μM and therefore did not adversely affect the sensory properties of the fermented milks. The encapsulation decreased the hyperacidity phenomena generally related to the inclusion of probiotic microorganisms in fermented milks. The lower acidity of the products due to the microencapsulation was fundamental for the improvement of the viability of the starter culture and the sensory characteristics of the products. The microencapsulation conditions increased the resistance to the simulated digestion processes, although the strain Lb. paracasei A13 generally showed a higher resistance to the gastric barrier respect to Lb. salivarius CECT 4063. By contrast, the data obtained showed a reduction of EPS production by the microencapsulation. The volatile profiles showed specific profiles in relation to the probiotic strain used and microencapsulation process. In conclusion, the results of this study underlined the applicative potential of HPH microencapsulation of probiotic microorganisms to produce fermented milk with improved functionality and with enhanced sensory properties.
The aim of this study was to adjust technological parameters: acidification of the curd (pH 5.25) and time (2, 5, 10 and 20 min) and stretching temperature (58, 62.5 and 68 °C) in order to make a pasta filata cheese carrying a probiotic bacterium at levels higher than 10⁷ CFU/g. A control and a probiotic cheese were produced. Lactobacillus rhamnosus GG was used and its resistance to simulated gastrointestinal digestion (SGD) was evaluated. Gross composition and pH, microbiological analysis, proteolysis, physicochemical and sensory characteristics, volatile compounds, organic acids and sugar profiles were also determined. The probiotic remained above 3 × 10⁷ CFU/g during its shelf life and exhibited high resistance to SGD (matrix protection of about 60%). The addition of the probiotic increased secondary proteolysis (about 30% for SN fraction in trichloracetic and phosphotungstic acids) and the production of diacetyl, acetoin, lactic and acetic acids. Sensory characteristics (smell, astringency, acid taste and residual flavor) were also modified. The development of a probiotic Fior di Latte cheese that might contribute to disease prevention and generate improvements in sensory characteristics compared to traditional products would allow expanding the market of functional foods.
Gut microbiota dysbiosis plays a central role in the development and perpetuation of chronic inflammation in inflammatory bowel disease (IBD) and therefore is key target for interventions with high quality and functional probiotics. The local production of stable probiotic formulations at limited cost is considered an advantage as it reduces transportation cost and time, thereby increasing the effective period at the consumer side. In the present study, we compared the anti-inflammatory capacities of the Bifidobacterium animalis subsp. lactis (B. lactis) INL1, a probiotic strain isolated in Argentina from human breast milk, with the commercial strain B. animalis subsp. lactis BB12. The impact of spray-drying, a low-cost alternative of bacterial dehydration, on the functionality of both bifidobacteria was also investigated. We showed for both bacteria that the spray-drying process did not impact on bacterial survival nor on their protective capacities against acute and chronic colitis in mice, opening future perspectives for the use of strain INL1 in populations with IBD.
Lactobacillus fermentum Lf2, an autochthonous strain isolated as a non starter culture in Cremoso cheese, produces high EPS levels (~ 1 g/L) in optimized conditions (SDM broth, pH 6.0, 30 °C, 72 h). Technological (texture profile and rheological analysis) and sensory properties of non-fat yogurts with 300 and 600 mg EPS/L were studied at 3 and 25 days after manufacture. Yogurts with different EPS concentrations showed higher hardness values than the control group at both periods of time, being the only significant difference that remained stable during time. The consistency index was also higher for the treated samples at both times evaluated, being significantly different for samples with 300 mg/L of EPS extract, while the flow behavior index was lower for EPS-added yogurts. The thixotropic index was lower (P < 0.05) for samples with the highest EPS extract concentration at the end of the storage time. Regarding the sensory analysis, those yogurts with 600 mg/L of EPS extract presented the highest values of consistency at 3 days of storage. No considerable differences for defects (milk powder, acid, bitter and cooked milk flavors) were perceived between treated and control samples at both times evaluated. Syneresis was also studied and samples with 600 mg/L of EPS extract presented the lowest syneresis values at 25 days of storage, which considerably decreased with the time of storage.
Lactobacillus fermentum Lf2 is a strain which is able to produce high levels (approximately 1 g/l) of crude exopolysaccharide (EPS) when it is grown in optimised conditions. The aim of this work was to characterize the functional aspects of this EPS extract, focusing on its application as a dairy food additive. Our findings are consistent with an EPS extract that acts as moderate immunomodulator, modifying s-IgA and IL-6 levels in the small intestine when added to yogurt and milk, respectively. Furthermore, this EPS extract, in a dose feasible to use as a food additive, provides protection against Salmonella infection in a murine model, thus representing a mode of action to elicit positive health benefits. Besides, it contributes to the rheological characteristics of yogurt, and could function as a food additive with both technological and functional roles, making possible the elaboration of a new functional yogurt with improved texture.
Background Bacteriophages constitute a great threat to the activity of lactic acid bacteria used in industrial processes. Several factors can influence the infection cycle of bacteriophages. That is the case of the physiological state of host cells, which could produce inhibition or delay of the phage infection process. In the present work, the influence of Lactobacillus plantarum host cell starvation on phage B1 adsorption and propagation was investigated. Result First, cell growth kinetics of L. plantarum ATCC 8014 were determined in MRS, limiting carbon (S-N), limiting nitrogen (S-C) and limiting carbon/nitrogen (S) broth. L. plantarum ATCC 8014 strain showed reduced growth rate under starvation conditions in comparison to the one obtained in MRS broth. Adsorption efficiencies of > 99 % were observed on the starved L. plantarum ATCC 8014 cells. Finally, the influence of cell starvation conditions in phage propagation was investigated through one-step growth curves. In this regard, production of phage progeny was studied when phage infection began before or after cell starvation. When bacterial cells were starved after phage infection, phage B1 was able to propagate in L. plantarum ATCC 8014 strain in a medium devoid of carbon source (S-N) but not when nitrogen (S-C broth) or nitrogen/carbon (S broth) sources were removed. However, addition of nitrogen and carbon/nitrogen compounds to starved infected cells caused the restoration of phage production. When bacterial cells were starved before phage infection, phage B1 propagated in either nitrogen or nitrogen/carbon starved cells only when the favorable conditions of culture (MRS) were used as a propagation medium. Regarding carbon starved cells, phage propagation in either MRS or S-N broth was evidenced. Conclusions These results demonstrated that phage B1 could propagate in host cells even in unfavorable culture conditions, becoming a hazardous source of phages that could disseminate to industrial environments.
Functional properties of two Lactobacillus plantarum strains (Lp 813 and Lp 998) were evaluated (in vitro) before and after heat adaptation and shock. The stress conditions were selected considering a previous work: 55 C e 15 min (heat shock) and 45 C e 30 min (thermal adaptation), both performed in MRS broth. The functional properties evaluated were: a-survival to simulated gastrointestinal tract (GIT: saliva þ bovine pepsina solution brought to pH 2.3 in 90 min, bile solution 1% w/v pH 8e10 min and bile þ pancreatin solution pH 8e30 min), b-co-aggregation with Saccharomyces cerevisiae to simulate intestinal epithelia adhesion, c-auto-and co-aggregation with enteric pathogens (Escherichia coli and Salmonella enteritidis) and d-use of prebiotic compounds (inulin, soluble corn fiber, lactulose and raffi-nose) evaluated for kinetics of growth. Heat treatments (generally) affected the studied functional properties as follows: a-diminished the resistance to acidic step and improved survival undergoing bile-pancreatin step in GIT simulation, b-mean values of auto-aggregation and co-aggregation with yeast or pathogens were not significantly modified, despite an increase in variability and c-prebiotic use diminished in the case of strains subjected tothermal treatments except for raffinose, which showed to have been better employed in this last case. These results could be very useful when it comes to the selection of microorganisms to be used and conserved in processes and methodologies that involve high temperatures as a stress factor (spray drying or cheese elaboration).
In this study, we aimed at optimizing the growth of Lactobacillus rhamnosus 64 in whey permeate supplemented with different ingredients and we also studied the effects of mild stresses on the response to heat and oxidative challenge. Supplementation of diluted whey permeate with yeast extract, tryptone, Tween 80 and Mn–Mg produced ca. 1010 CFU/mL of L. rhamnosus 64 under pH controlled fermentation in anaerobiosis. Cells were exposed to different mild stresses (aerobic: 1 h air 0.3 vol vol−1 min, pH 6, 37 °C); heat (1 h, pH 6, 45 °C, anaerobiosis) and acid (1 h, pH 5, 37 °C, anaerobiosis), followed by heat (55 °C) or oxidative (0.4 mmol/L H2O2) challenge (15 min). Oxidative challenge was more detrimental compared to heat challenge. Anaerobic incubation, cell harvesting at the stationary phase and mild heat stress were more effective for reducing cell death towards heat challenge, whereas cheese whey-starch was effective towards cell protection to spray drying and storage. No effects on cell survival to spray drying were observed when mild stresses were used to increase resistance to spray drying.
Industrially interesting lactic acid bacteria (LAB) for dairy starter cultures may be selected by searching for required phenotypic traits at the genotypic level using high-throughput screening approaches. Besides new strains to be used as primary starter cultures, an increasing attention is being given to the non-starter LAB (NSLAB) microbiota, which composes the so-called starter adjuncts (also defined as “subsidiary” or “complementary” cultures). Starter adjuncts can be used as ripening, protective, probiotic, and/or EPS-producing cultures. Food-grade genetic strategies should ensure novel solutions for starter culture improvement to produce highly desirable and innovative fermented dairy products, with functional properties, new sensory characteristics, and increased nutritional and health-promoting properties. The aptitude of phages to survive thermal treatments (including pasteurization) and their resistance to sanitation measures have forced the industry to rely on bacteria with naturally occurring phage resistance systems for the formulation of starter culture.
Applying sub-lethal levels of high-pressure homogenization (HPH) to lactic acid bacteria has been proposed as a method of enhancing some of their functional properties. Because the principal targets of HPH are the cell-surface structures, the aim of this study was to examine the effect of sub-lethal HPH treatment on the outermost cellular structures and the proteomic profiles of two known probiotic bacterial strains. Moreover, the effect of HPH treatment on the metabolism of probiotic cells within a dairy product during its refrigerated storage was investigated using SPME-GC-MS. Transmission electron microscopy was used to examine the microstructural changes in the outermost cellular structures due to HPH treatment. These alterations may be involved in the changes in some of the technological and functional properties of the strains that were observed after pressure treatment. Moreover, the proteomic profiles of the probiotic strains treated with HPH and incubated at 37°C for various periods showed different peptide patterns compared with those of the untreated cells. In addition, there were differences in the peaks that were observed in the low-mass spectral region (2000-3000 Da) of the spectral profiles of the control and treated samples. Due to pressure treatment, the volatile-molecule profiles of buttermilk inoculated with treated or control cells and stored at 4°C for 30 days exhibited overall changes in the aroma profile and in the production of molecules that improved its sensory profile, although the two different species imparted specific fingerprints to the product. The results of this study will contribute to understanding the changes that occur in the outermost cellular structures and the metabolism of LAB in response to HPH treatment. The findings of this investigation may contribute to elucidating the relationships between these changes and the alterations of the technological and functional properties of LAB induced by pressure treatment.
The survival of three Lactobacillus plantarum strains (Lp 790, Lp 813 and Lp 998) with functional properties was studied taking into account their resistance to thermal, osmotic and oxidative stress factors. Stress treatments applied were: 52 °C-15 min (Phosphate Buffer pH 7, thermal shock), H2O2 0.1% (p/v) - 30 min (oxidative shock) and NaCl aqueous solution at 17, 25 and 30% (p/v) (room temperature - 1 h, osmotic shock). The osmotic stress was also evaluated on cell growth in MRS broth added of 2, 4, 6, 8 and 10% (p/v) of NaCl, during 20 h at 30 °C. The cell thermal adaptation was performed in MRS broth, selecting 45 °C for 30 min as final conditions for all strains. Two strains (Lp 813 and Lp 998) showed, in general, similar behaviour against the three stress factors, being clearly more resistant than Lp 790. An evident difference in growth kinetics in presence of NaCl was observed between Lp 998 and Lp 813, Lp998 showing a higher optical density (OD570nm) than Lp 813 at the end of the assay. Selected thermal adaptation improved by 2 log orders the thermal resistance of both strains, but cell growth in presence of NaCl was enhanced only in Lp 813. Oxidative resistance was not affected with this thermal pre-treatment. These results demonstrate the relevance of cell technological resistance when selecting presumptive "probiotic" cultures, since different stress factors might considerably affect viability or/and performance of the strains. The incidence of stress conditions on functional properties of the strains used in this work are currently under research in our group. Copyright © 2014 Elsevier Ltd. All rights reserved.
Caseinomacropeptide (CMP) is the 64 C-terminal amino acid residue of κ-casein, formed by chymosin cleavage during cheese manufacture. This study examined the effects of oral administration of a CMP-enriched extract (CMPEE), obtained from a local dairy plant, on the Ca content of mouse femurs. Animals received low (0.1%, w/v), normal (0.5%, w/v) or high (1.2%, w/v) Ca diet for 3 or 8 weeks and CMPEE diluted (1:10) in their drinking water. No significant differences in Ca content were observed in faeces, kidney, urine or blood serum compared with control animals. The oral administration of CMP to mice significantly enhanced the Ca content in femur under a low-Ca diet model, especially during the period of full body development (3 weeks), in which case a significant 12% Ca increase was observed. These findings pave the way for further studies aimed at supplementing infant food with industrially-obtained CMP-enriched extract for enhanced bone health.
The probiotic Lactobacillus paracasei A13, treated at 50 MPa sub-lethal high pressure homogenisation (HPH), was used as adjunct for producing Caciotta cheese. The cell HPH treatment was used because it has been proven to increase the in vitro strain functionality. The starters and Lb. paracasei A13 viability, the cheese hydrolytic patterns and organoleptic profiles were monitored. After cheesemaking and during ripening, the Lb. paracasei A13 gastric acid resistance in cheese and the ability of the cheese, containing HPH-treated or untreated cells, to modulate the gut mucosal immune system in mice were evaluated. Traditional Caciotta was used as controls. The HPH-treated probiotic strain maintained high viability for 14 days whilst the physico-chemical analyses on Caciotta cheese containing HPH-treated cells showed a faster ripening, compared to other cheeses. For functional properties, the 50 MPa treatment increased the Lb. paracasei gastric resistance in Caciotta, maintaining high strain viability, but IL-10 producing capacity was lost by HPH-treatment whilst IgA production was not modified.
Phages infecting Leuconostoc mesenteroides strains can be overlooked during milk fermentation because they do not slowdown the acidification process. However, they can negatively impact the flavor profile of the final product. Yet, the information about these phages is still scarce. In this work, we investigated diverse factors influencing the adsorption of seven virulent Ln. mesenteroides phages, isolated from blue cheese manufacture in Argentina, to their host cells. The addition of calcium ions was generally necessary to observe complete cell lysis and plaque formation for four of the seven phages, but adsorption was very high even in the absence of this cation for all phages. The temperature barely influenced the adsorption process as it was high within the temperature range tested (0 to 50°C). Moreover, the kinetics of adsorption were similar on viable and non-viable cells, revealing that phage adsorption does not depend on physiological state of the bacterial cells. The adsorption rates were also high at pH values from 4 to 9 for all Ln. mesenteroides phages. We also analyzed the complete genome sequences of two of these phages. Complete nucleotide analysis of phages Ln-8 and Ln-9 showed dsDNA genomes with sizes of 28.5 and 28.9kb, and the presence of 45 and 48 open reading frames (ORFs), respectively. These genomes were highly similar to those of previously characterized Φ1-A4 (USA, sauerkraut, fermentation) and ΦLN25 (England, whey), both virulent Ln. mesenteroides phages. A detailed understanding of these phages will lead to better control strategies. Copyright © 2015 Elsevier B.V. All rights reserved.
Four spontaneous phage-resistant mutants, previously isolated from Lactobacillus plantarum ATCC 8014 using a phage cocktail (ATCC 8014-B1 and ATCC 8014-B2), were characterised with regard to their probiotic potential. Phage-resistant mutants exhibited, in general, the same properties as those found for Lb. plantarum ATCC 8014 strain. However, mutant M1 evidenced a remarkably high resistance to gastrointestinal passage. Low values of either β-galactosidase activity or hydrophobicity were observed. Antimicrobial activity against Gram-negative and Gram-positive bacteria was evidenced and lactulose was the most fermented carbohydrate. Strains were sensitive to gentamicin, erythromycin, ampicillin and chloramphenicol, whereas production of biogenic amines was not observed. Finally, a selected phage-resistant mutant (M1) produced the same immunological response as the sensitive strain in mice fed for 10 consecutive days. These natural mutants, with similar or improved potentially probiotic characteristics regarding their sensitive strain, could be used during fermented food manufacture to minimise failure due to phage.
The aim of this study was to evaluate the influence of microbiological and technological factors on the viability and functionality of probiotic Lactobacillus strains. In particular, the influence of harvesting time, food matrix, refrigerated storage, time of inoculation and refrigerated storage in fermented milk on the resistance to simulated gastric digestion (RSGD) was evaluated. Moreover, strain resistance to simulated gastric digestion was compared to human gastric fluid. Results showed that the variables studied affected, in a different way and in a strain‐dependent manner, the RSGD. No direct relation was observed between cell viability and RSGD.
Technological, biological and functional attributes of two groups of spontaneous bacteriophage-resistant mutants were investigated to verify features similar to those of their respective phage-sensitive parent probiotic strains (Lactobacillus casei/paracasei). A derivative from each group was challenged with phages in simulated probiotic product fermentation. Technological performance, compared with the original strains, was demonstrated. Minimal variations were detected in acidifying and enzymatic profiles. No β-glucuronidase activity was observed, but very high α-glucosidase and β-galactosidase activities were recorded. Low proteolytic activities and inability to ferment prebiotics were observed. Within one group, significant differences throughout simulated gastrointestinal-transit were observed. Neither of the strains deconjugated bile salts, but all showed inhibitory activity against four pathogens. Both mutants increased IgA producing cell numbers in vivo in a manner similar to that of the parent strains. Two derivatives demonstrated improved phage-resistance phenotype compared with the parental strains, resulting in alternatives for industrial rotation schemes.
The mouse has been largely used for the study of the protective capacity of probiotics against intestinal infections caused by Salmonella. In this work we aimed at comparing the mortality and translocation assay for the study of the protective capacity of the human breast milk-derived strain Bifidobacterium animalis subsp. lactis INL1 on a model of gut infection by Salmonella enterica subsp. enterica serovar Typhimurium. Different doses of S. Typhimurium FUNED and B. animalis subsp. lactis INL 1 were administered to Balb/c mice in a mortality or a translocation assay. The survival of the control group in the mortality assay resulted to be variable along experiments, and then we preferred to use a translocation assay where the preventive administration of 109 cfu of bifidobacteria/mouse for 10 consecutive days significantly reduced the number of infected animals and the levels of translocation to liver and spleen, with enhanced secretory immunoglobulin A and interleukin 10 production in the small and large intestine, respectively. Ten days of B. animalis subsp. lactis strain INL1 administration to mice significantly reduced both the incidence and the severity of Salmonella infection in a mouse model of translocation. This work provided the first evidence that a translocation assay, compared to a mortality assay, could be more useful to study the protective capacity of probiotics against Salmonella infection, as more information can be obtained from mice and less suffering is conferred to animals due to the fact that the mortality assay is shorter than the latter. These facts are in line with the guidelines of animal research recently established by the National Centre for the Replacement, Refinement & Reduction of Animals in Research.
The double use of cheese whey (culture medium and thermoprotectant for spray drying of lactobacilli) was explored in this study for adding value to this wastewater. In-house formulated broth (similar to MRS) and dairy media (cheese and ricotta whey and whey permeate) were assessed for their capacity to produce biomass of Lactobacillus paracasei JP1, Lb. rhamnosus 64 and Lb. gasseri 37. Simultaneously, spray drying of cheese whey-starch solution (without lactobacilli cells) was optimised using surface response methodology. Cell suspensions of the lactobacilli, produced in in house-formulated broth, were spray-dried in cheese whey-starch solution and viability monitored throughout the storage of powders for 2 months. Lb. rhamnosus 64 was able to grow satisfactorily in at least two of the in-house formulated culture media and in the dairy media assessed. It also performed well in spray drying. The performance of the other strains was less satisfactory. The growth capacity, the resistance to spray drying in cheese whey-starch solution and the negligible lost in viability during the storage (2 months), makes Lb. rhamnosus 64 a promising candidate for further technological studies for developing a probiotic dehydrated culture for foods, utilising wastewaters of the dairy industry (as growth substrate and protectant) and spray drying (a low-cost widely-available technology).
Nine Leuconostoc mesenteroides phages were isolated during blue cheese manufacture yielding faulty products with reduced eye formation. Their morphologies, restriction profiles, host ranges and long-term survival rates (25°C, 8°C, -20°C and -80°C) were analysed. Based on restriction analysis, six of them were further examined regarding resistance to physical (heat and high pressure homogenization, HPH) and chemical treatments (ethanol, sodium hypochlorite, peracetic acid, biocides A, C, E and F). According to their morphology, L. mesenteroides phages studied in the present work belonged to the Caudovirales order and Siphoviridae family. Six distinct restriction patterns were obtained with EcoRV, HindIII, ClaI and XhoI enzymes, revealing interesting phage diversity in the dairy environment. No significant reductions in phage counts were observed after ten months of storage at -20°C and -80°C, while slightly and moderate decrease in phage numbers were noticed at 8°C and 25°C, respectively. The phages subjected to heat treatments generally showed high resistance at 63°C and moderate resistance at 72°C. However, 80°C for 30min and 90°C for 2min led to complete inactivation of viral particles. In general, the best ethanol concentration tested was 75%, as complete inactivation for most Leuconostoc phages within 30min of incubation was achieved. Peracetic acid, and biocides A, C, E and F were highly effective when used at the same or at a moderately lower concentration as recommended by the producer. Usually, moderate or high concentrations (600-1600ppm) of sodium hypochlorite were necessary to completely inactivate phage particles. Leuconostoc phages were partially inactivated by HPH treatments as remaining viral particles were found even after 8 passes at 100MPa. This is the first report of L. mesenteroides phages isolated from an Argentinean dairy cheese plant. The results of this work could be useful for establishing the most effective physical and chemical treatments for inactivating phages in industrial plants and laboratory environments.
Cell-free supernatant from Leuconostoc citreum MB1 revealed specific antilisterial activity. Preliminary studies demonstrated the proteinaceous, heat-stable, bacteriocin-like trait of the antimicrobial components present in the supernatant. Determination of the genes encoding bacteriocins by PCR and DNA sequencing led to amplification products highly homologous with leucocin A (found in diverse Leuconostoc species) and UviB (found in Leuc. citreum KM20) sequences. Additionally, antimicrobial activity of cell-free supernatant from Leuc. citreum MB1 was revealed by an inhibition halo of the SDS-PAGE gel subjected to a direct detection using Listeria monocytogenes as indicator strain. Different assays were carried out to assess the capacity of Leuc.citreum MB1 to control List. monocytogenes growth: (i) inactivation kinetics of the pathogen by antilisterial compounds present in concentrated cell-free supernatant from Leuc. citreum MB1, (ii) evaluation of optimal Leuc. citreum MB1 initial concentration to obtain maximum List. monocytogenes ATCC 15313 inhibition, and (iii) biocontrol of List. monocytogenes ATCC 15313 with Leuc. citreum MB1 during growth in milk at refrigeration temperature. According to our results, it is unquestionable that at least one bacteriocin is active in Leuc. citreum MB1, since important antilisterial activity was verified either in its cell-free supernatant or in co-culture experiments. Co-culture tests showed that ∼107 CFU/ml Leuc. citreum MB1 was the optimal initial concentration to obtain maximum pathogen inhibition. Moreover, Leuc. citreum MB1 was able to delay List. monocytogenes growth at refrigerated temperature.
Some strains of the genus Bifidobacterium are considered probiotics and can be added as adjunct cultures to functional dairy products. Lactobacillus delbrueckii subsp. lactis is used as a starter in food fermentations. The influence of co-culturing strains Bifidobacterium longum NCIMB 8809 and L. delbrueckii subsp. lactis 193 on the physiology and heat tolerance of these microorganisms was studied. 2DE coupled to MS protein analysis allowed the identification of several proteins from each bacterium whose expression changed when cells were grown in compartmentalized co-cultures compared to mono-cultures. Remarkably, production of stress response chaperones was enhanced in both strains and was related with increased survival to heat shock, a technologically suitable property in the manufacture of some dairy products. This study provides the first insight in understanding communication between B. longum and L. delbrueckii subsp. lactis.
The aim of this work was to assess if a sub-lethal high pressure homogenization (HPH) treatment could modulate in vitro functional and biological properties of probiotic bacteria. Lactobacillus paracasei A13, Lactobacillus acidophilus 08 and Dru, Lactobacillus delbrueckii subsp. lactis 200 and bile-resistant derivatives L. acidophilus Dru+ and L. delbrueckii subsp. lactis 200+ were suspended in phosphate buffered saline solution and treated at 50 MPa. Data obtained showed that HPH can modulate hydrophobicity and auto-aggregation without modification of viability and decarboxylase activity. Resistance to simulated gastric conditions resulted strain-dependent. High resistance was observed for treated L. paracasei A13, L. acidophilus Dru and 08 and L. delbrueckii subsp. lactis 200. The HPH-treatment reduced the resistance to simulated stomach duodenum-passage of L. acidophilus Dru while increased it for L. paracasei A13.Strain viability and resistance to simulated gastric conditions were evaluated treating at 50 MPa cells suspended in acidified buttermilk (pH 4.6) and stored at 4 °C for 30 days. The highest cell viability loss, after 30 d of refrigerated storage, was observed for L. acidophilus Dru, independently of the application of HPH. However, after 30 days of storage, the resistance of L. paracasei A13 to simulated gastric digestion significantly increased in HPH treated cells.
Unlabelled: High-pressure homogenization (HPH) has been proposed to be applied directly to lactic acid bacterial cells at sublethal levels to enhance some functional properties. As the principal target of HPH are the cell surface envelope structures, the aim of this work was to study the effect of a HPH treatment, applied at 50 MPa, on cell membrane stress responses of already-known functional strains, isolated from Argentinean products. Specifically, the membrane fatty acid composition of cells before and after the sublethal treatment was investigated, and the results showed that plasma membranes, their level of unsaturation and their composition are involved in response mechanisms adopted by microbial cells when subjected to a sublethal HPH stress. In fact, the data obtained demonstrated that the treatment was able to modify the fatty acid profile of the different strains, although a uniform response was not observed. Further studies are necessary both to elucidate the role of each fatty acid in the cell response mechanisms and to clarify the changes in membrane compositions induced by HPH treatment also in relation to the applicative potential of this technique. Significance and impact of the study: This study contributed to understand the response mechanisms activated in cells exposed to pressure stress. It has been demonstrated that high-pressure homogenization (HPH) treatments, conducted at sublethal levels, could increase some important functional and technological characteristics of nonintestinal probiotic strains. The findings of this paper can contribute to elucidate the mechanisms through which these treatments can modify these strain probiotic properties that are related to outermost cell structures, also principal target of HPH.
Buttermilk is a suitable substrate for fermentation with proteolytic strains of Lactobacillus in order to release peptide fractions able to enhance the gut mucosal immune system. We aimed to determine the influence of the degree of proteolysis of buttermilk proteins on their functionality. Animals received for seven consecutive days the cell-free fraction of 10 or 20% (w/v) buttermilk fermented with Lactobacillus delbrueckii subsp. lactis 210 at pH 6. The pH was controlled either with NaOH or Ca(OH)2. No significant differences in the number of IgA-producing cells in the small intestine of mice were found. The functional capacity of the product under study was not affected by the technological variables considered.
Specific strains should only be regarded as probiotics if they fulfill certain safety, technological and functional criteria. The aim of this work was to study, from a comprehensive point of view (in vitro and in vivo tests), three Lactobacillus strains (Lactobacillus paracasei JP1, Lactobacillus rhamnosus 64 and Lactobacillus gasseri 37) isolated from feces of local newborns, determining some parameters of technological, biological and functional relevance. All strains were able to adequately grow in different economic culture media (cheese whey, buttermilk and milk), which were also suitable as cryoprotectants. As selective media, LP-MRS was more effective than B-MRS for the enumeration of all strains. The strains were resistant to different technological (frozen storage, high salt content) and biological (simulated gastrointestinal digestion after refrigerated storage in acidified milk, bile exposure) challenges. L. rhamnosus 64 and L. gasseri 37, in particular, were sensible to chloramphenicol, erythromycin, streptomycin, tetracycline and vancomycin, increased the phagocytic activity of peritoneal macrophage and induced the proliferation of IgA producing cells in small intestine when administered to mice. Even when clinical trails are still needed, both strains fulfilled the main criteria proposed by FAO/WHO to consider them as potential probiotics for the formulation of new foods.
Three probiotic lactobacilli strains were spray-dried in 20% (w/v) skim milk and submitted to a simulated gastrointestinal digestion. Fresh or spray-dried cultures were administered to mice for 5 and 10 days, and Immunoglobulin A (IgA)-producing cells were enumerated in the small intestine by immunohistochemistry. Spray-drying significantly enhanced the resistance of Lactobacillus para-casei A13 and Lactobacillus casei Nad to a simulated gastrointestinal digestion (0.96 and 1.95 log orders, respectively), compared with fresh cultures. Also, a significant higher number of IgA-pro-ducing cells were induced by spray-dried cultures compared with fresh cultures. Spray-drying is a suitable, but strain-dependent, technological process for the development of probiotic cultures in skim milk with increased functionality.
Aims: The aim of this work was to identify 20 yeasts isolated from autochthonal cheese starters and evaluate their technological and functional properties. Methods and results: The capacities of the yeasts to grow at different temperatures, pH, NaCl and lactic acid concentrations as well as the proteolytic and lipolytic activities were studied. Moreover, survival to simulated gastrointestinal digestion, hydrophobicity, antimicrobial activity against pathogens and auto- and co-aggregation abilities were evaluated. The sequentiation of a fragment from the 26S rDNA gene indicated that Kluyveromyces marxianus was the predominant species, followed by Saccharomyces cerevisiae, Clavispora lusitaniae, Kluyveromyces lactis and Galactomyces geotrichum. RAPD with primer M13 allowed a good differentiation among strains from the same species. All strains normally grew at pH 4.7-5.5 and temperatures between 15 and 35°C. Most of them tolerated 10% NaCl and 3% lactic acid. Some strains showed proteolytic (eight isolates) and/or lipolytic (four isolates) capacities. All strains evidenced high gastrointestinal resistance, moderate hydrophobicity, intermediate auto-aggregation and variable co-aggregation abilities. No strains inhibited the growth of the pathogens assayed. Conclusions: Some strains from dairy sources showed interesting functional and technological properties. Significance and impact of the study: This study has been the first contribution to the identification and characterization of yeasts isolated from autochthonal cheese starters in Argentina. Many strains could be proposed as potential candidates to be used as probiotics and/or as co-starters in cheese productions.
Low levels of High Pressure of Homogenization (HPH) can be applied directly to lactic acid bacteria cells in order to enhance some functional properties. In a previous work we observed that a 50 MPa HPH treatment increased Lactobacillus paracasei A13 hydrophobicity and resistance to simulated gastric digestion. The aim of this work was to assess the in vivo effects of HPH treatment applied to probiotic lactobacilli on their interaction capacity with the gut and on their ability to induce IgA cell proliferation in mice intestine. BALB/c mice received FITC-labelled cultures of strains, previously treated or not (control) at 50 MPa. Fluorescently labelled cells were studied in the intestine of animals sacrificed 10 and 30 min after intragastric intubation. HPH-treated and control cultures of each strain were orally administered to mice for 2, 5 or 7 consecutive days. The number of IgA-producing cells in the gut was studied by immunohistochemistry. HPH treated probiotic lactobacilli modified their interaction with the small intestine. HPH-treated cells induced a higher IgA response compared to untreated ones, in a strain- and feeding period-dependent way. HPH treatment could increase some in vivo functional characteristics of probiotic strains, highlighting the potential of this technique for the development of probiotic cultures.
The effect of high pressure homogenization (HPH) with respect to a traditional heat treatment on the inactivation, growth at 8°C after treatments, and volatile profile of adventitious Leuconostoc strains isolated from Cremoso Argentino spoiled cheeses and ingredients used for their manufacture was evaluated. Most Leuconostoc strains revealed elevated resistance to HPH (eight passes, 100 MPa), especially when resuspended in skim milk. Heat treatment was more efficient than HPH in inactivating Leuconostoc cells at the three initial levels tested. The levels of alcohols and sulfur compounds increased during incubation at 8°C in HPH-treated samples, while the highest amounts of aldehydes and ketones characterized were in heated samples. Leuconostoc cells resuspended in skim milk and subjected to one single-pass HPH treatment using an industrial-scale machine showed remarkable reductions in viable cell counts only when 300 and 400 MPa were applied. However, the cell counts of treated samples rose rapidly after only 5 days of storage at 8°C. The Leuconostoc strains tested in this work were highly resistant to the inactivation treatments applied. Neither HPH nor heat treatment assured their total destruction, even though they were more sensitive to the thermal treatment. To enhance the inhibitory effect on Leuconostoc cells, HPH should be combined with a mild heat treatment, which in addition to efficient microbial inactivation, could allow maximal retention of the physicochemical properties of the product.
A novel treatment using polyphosphates to protect cheeses against superficial mould growth was assayed. The treatments were: control commercial paint with natamycin (I), commercial paint based on polyvinyl–water (II), immersion in a saturated solution of polyphosphate (III), immersion and commercial paint (IV), commercial paint of polyphosphate solutions (V) and immersion and commercial paint of a saturated solution of polyphosphates (VI). The cheeses were ripened for 6 months. Superficial mould growth was inhibited in groups IV and VI when compared to cheeses from the control group (I). Statistical sensory analysis made between cheeses from groups IV and I (control) showed no significant differences.
Commercial probiotic bacteria are delivered mainly as frozen or freeze-dried cultures. However, spray drying is a lower cost technology that could be used for the production of probiotic cultures. In this work we aimed at screening among lactobacilli strains for candidates able to survive to spray drying and to study the effects of a preliminary mild heat treatment and different food matrices on post-drying survival and simulated gas-tric acid resistance. Heat resistance (survival to exposure at 60 °C for 5 min) in MRS broth or in 10% (wt/vol) skim milk was assessed in 22 strains of Lactobacillus casei, Lactobacillus paracasei, Lactobacillus acidophilus and Lactobacillus plantarum. Five strains (L. casei Nad, L. plantarum com, L. paracasei A13, L. plantarum 8329 and L. acidophilus A9) were selected for spray drying in 20% (wt/vol) skim milk and storage at 5, 25 or 37 °C for 75 days. For L.p. A13, L.p. com and L.a. A9 no differences in cell viability were observed due to spray drying. However, for L.c. Nad and L.p. 8329 cell death due to spray drying was 0.16 and 0.49 log orders CFU ml −1 when a mild heat treatment (52 °C for 15 min) was applied and 0.85 and 0.95 log cycles, respectively, without preliminary mild heat treatment, showing that heat treatment enhanced survival to spray drying. The application of a heat treatment was effective for enhancing survival during storage of L.p. 8329, irrespective of the storage temperature and period. No significant cell loss at 5 and 25 °C was observed for L.c. Nad. For this strain, at 37 °C no cell counts of lactobacilli were observed after 30 days of storage. For L.a. A9, L.p. com and L.p. A13 a reduction in cell viability was observed along storage as temperature in-creased. Resistance to simulated gastrointestinal digestion was enhanced by spray drying. The application of a mild heat treatment before spray drying may enhance cell survival during storage and the resistance to gastro-intestinal digestion. Spray drying might be used for enhancing cell functionality in a strain-dependant way.
The aim of this work was to investigate how production and freeze-drying conditions of Bifidobacterium animalis subsp. lactis INL1, a probiotic strain isolated from breast milk, affected its survival and resistance to simulated gastric digestion during storage in food matrices. The determination of the resistance of bifidobacteria to simulated gastric digestion was useful for unveiling differences in cell sensitivity to varying conditions during biomass production, freeze-drying and incorporation of the strain into food products. These findings show that bifidobacteria can become sensitive to technological variables (biomass production, freeze-drying and the food matrix) without this fact being evidenced by plate counts.
To investigate the cell viability of Bifidobacterium longum 5(1A) in fermented milks and to study its immunostimulating and protective capacity against Salmonella enterica ssp. enterica serovar Typhimurium infection in mice. Bifidobacterium longum 5(1A) was added to milk fermented with different yoghurt starter cultures, before or after fermentation, and viability was monitored during storage (5°C, 28 days). Resistance to simulated gastric acid digestion was assessed. Fermented milks were orally administered to mice for 10 days followed by oral infection with Salmonella Typhimurium. The number of IgA+ cells in the small and large intestine was determined before infection. Survival to infection was monitored for 20 days. Bifidobacterium longum 5(1A) lost viability during storage, but the product containing it was effective for the induction of IgA+ cells proliferation in the gut and for the protection of mice against Salm. Typhimurium infection. Cell viability of Bif. longum 5(1A) in fermented milks along storage did not condition the capacity of the strain to enhance the number of IgA+ cells in the gut and to protect mice against Salmonella infection. The uncoupling of cell viability and functionality demonstrated that, in certain cases, nonviable cells can also exert positive effects.
Temperate bacteriophages ф iLp84 and ф iLp1308, previously isolated from mitomycin C-induction of Lactobacillus paracasei strains 84 and CNRZ1308, respectively, were tested for their resistance to several physical and chemical treatments applied in dairy industry. Long-term survival at 4 °C, -20 °C and -80 °C, resistance to either thermal treatments of 63 °C, 72 °C and 90 °C, high pressure homogenization (HPH, 100 MPa) or classic (ethanol, sodium hypochlorite and peracetic acid) and new commercial sanitizers, namely A (quaternary ammonium chloride), B (hydrogen peroxide, peracetic acid and peroctanoic acid), C (alkaline chloride foam), D (p-toluensulfonchloroamide, sodium salt) and E (ethoxylated nonylphenol and phosphoric acid), were determined. Phages were almost completely inactivated after eight months of storage at 25 °C, but viability was not affected at 4 °C, -20 °C or -80 °C. Both phages tolerated well HPH treatments. Phage iLp1308 showed higher thermal resistance than ф iLp84, but neither resisted 90 °C for 2 min. Best chemical inactivation was accomplished using peracetic acid or biocides A, C and E, whereas biocides B and D were completely ineffective. These results help to improve selection of chemical agents and physical treatments to effectively fight against phage infections in dairy plants.
A total of 81 spontaneous phage-resistant mutants were isolated from two sensitive Lactobacillus plantarum strains (ATCC 8014 and PLN) using two lytic bacteriophages (ATCC 8014-B1 and ATCC 8014-B2), either separately or mixed in a phage cocktail. Phenotypic characteristics related to their phage resistance abilities, as well as their technological properties, were determined. Random amplification of polymorphic DNA (RAPD-PCR) was used to determine genetic diversity among the selected isolates and their respective parent strains. Most of the mutants isolated were completely or partially unable to adsorb phage particles. Stability, high level of resistance and adequate technological properties were exhibited by the isolates of Lb. plantarum ATCC 8014. A selected variant, MC4, was able to resist the mixture of both lytic phages during fermented milk manufacture and the subsequent refrigerated storage. This phage-resistant derivative might be used in culture rotation programs when commercial strains become sensitive to phages present in industrial environments.
Six isolates of Bifidobacterium animalis subsp. lactis were obtained out of 16 samples of breast milk and were present in levels ranging from 1.4 to 2.8 log cfu mL−1. Isolates were stable in frozen storage (−20 °C and −70 °C, 6 months) and to refrigerated storage (5 °C, 4 weeks) in lactic acid-acidified milk (pH 4.5). Isolates were also able to grow in skim milk with yeast extract and they were stable to spray-drying in 20% (w/v) skim milk. Spray-dried cultures maintained a high number of viable cells when stored at 5 °C for 6 months and were stable to rehydration in water up to 50 °C. The oral administration of B. animalis subsp. lactis INL1 to mice increased the number of IgA+ cells in the small and large intestine. B. animalis subsp. lactis INL1 showed technological and probiotic potential for its incorporation to dairy products, whether fermented or not.
In this paper, the photocatalytic inactivation efficiency of phages infecting lactic acid bacteria (LAB) was evaluated. Ten bacteriophages (ATCC 15807-B1, 832-B1, 0241, 204, 342, MLC-A, J-1, OBJ, CHD and QF9) dispersed on TiO2-coated plates were exposed to UV-A radiation during different periods of time. Two kinds of efficiencies were defined and assessed: (i) the apparent photonic yield and (ii) the quantum yield of inactivation. Considering all phages studied in this work, dissimilar values of inactivation efficiencies were found; the results differ in more than one order of magnitude. The highest inactivation efficiency was found for the bacteriophage J-1, whereas the lowest one was obtained for the phage 342. The data presented in this work can provide useful information to design innovative and effective treatments to be applied in laboratories and industrial plants which handle LAB strains.
Ninety-eight Lactobacillus plantarum strains isolated from Italian and Argentinean cheeses were evaluated for probiotic potential. After a preliminary subtractive screening based on the presence of msa and bsh genes, 27 strains were characterized. In general, the selected strains showed high resistance to lysozyme, good adaptation to simulated gastric juice, and a moderate to low bile tolerance. The capacity to agglutinate yeast cells in a mannose-specific manner, as well as the cell surface hydrophobicity was found to be variable among strains. Very high β-galactosidase activity was shown by a considerable number of the tested strains, whereas variable prebiotic utilization ability was observed. Only tetracycline resistance was observed in two highly resistant strains which harbored the tetM gene, whereas none of the strains showed β-glucuronidase activity or was capable of inhibiting pathogens. Three strains (Lp790, Lp813, and Lp998) were tested by in vivo trials. A considerable heterogeneity was found among a number of L. plantarum strains screened in this study, leading to the design of multiple cultures to cooperatively link strains showing the widest range of useful traits. Among the selected strains, Lp790, Lp813, and Lp998 showed the best probiotic potential and would be promising candidates for inclusion as starter cultures for the manufacture of probiotic fermented foods.
The capacity of lactic acid bacteria to produce exopolysaccharides (EPS) conferring microorganisms a ropy phenotype could be an interesting feature from a technological point of view. Progressive adaptation to bile salts might render some lactobacilli able to overcome physiological gut barriers but could also modify functional properties of the strain, including the production of EPS. In this work some technological properties and the survival ability in simulated gastrointestinal conditions of Lactobacillus delbrueckii subsp. lactis 193, and Lb. delbrueckii subsp. lactis 193+, a strain with stable bile-resistant phenotype derived thereof, were characterized in milk in order to know whether the acquisition of resistance to bile could modify some characteristics of the microorganism. Both strains were able to grow and acidify milk similarly; however the production of ethanol increased at the expense of the aroma compound acetaldehyde in milk fermented by the strain 193+, with respect to milk fermented by the strain 193. Both microorganisms produced a heteropolysaccharide composed of glucose and galactose, and were able to increase the viscosity of fermented milks. In spite of the higher production yield of EPS by the bile-resistant strain 193+, it displayed a lower ability to increase viscosity than Lb. delbrueckii subsp. lactis 193. Milk increased survival in simulated gastric juice; the presence of bile improved adhesion to the intestinal cell line HT29-MTX in both strains. However, the acquisition of a stable resistance phenotype did not improve survival in simulated gastric and intestinal conditions or the adhesion to the intestinal cell line HT29-MTX. Thus, Lb. delbrueckii subsp. lactis 193 presents suitable technological properties for the manufacture of fermented dairy products; the acquisition of a stable bile-resistant phenotype modified some properties of the microorganism. This suggests that the possible use of bile-resistant derivative strains should be carefully evaluated in each specific application considering the influence that the acquisition of a stable bile-resistant phenotype could have in survival ability in gastric and intestinal conditions and in technological properties.
In a previous work, bile-salt-resistant derivatives were obtained from non-intestinal lactobacilli. The aim of this work was to investigate the impact of bile adaptation of Lactobacillus delbrueckii subsp. lactis 200 on morphology, surface properties, in vivo interaction capacity with the gut and ability to activate the gut immune response. Electron microscopy studies, growth kinetics in the presence of bovine and porcine bile, the capacity to deconjugate bile acids, hydrophobicity, autoaggregation and co-aggregation capacities were studied for the parental strain and its bile-resistant derivative in vitro. Additionally, survival in intestinal fluid, the interaction with the gut and the immunomodulating capacities were studied in mice. Bile salt adaptation conferred upon the adapted strain a higher capacity to withstand physiological concentrations of bile salts and greater survival capacity in intestinal fluid. However, bile salt exposure reduced cell hydrophobicity, autoaggregation and adhesion capacities, resulting in reduced persistence in the intestinal lumen and delayed capacity to activate the gut immune response. Insight into the effects of bile salts upon the interaction and immunomodulating capacity of lactobacilli with the gut is provided, relating in vitro and in vivo results.
Some species of Leuconostoc are very important for fermented dairy products, as they contribute to the organoleptic characteristics of butter and cream, and also contribute to the formation of openings in some soft, semi-hard (Edam and Gouda cheeses), many artisanal or in blue-veined cheeses, such as Roquefort. In this study, 14 Leuconostoc strains isolated from cheese and cheese-related products were characterized by genotypic and phenotypic methods, and their technological performance assessed for their potential use as dairy adjunct starters. Phenotypic characterization allowed these strains to be classified to genus level, and genotypic studies (RAPD-PCR and 16S rRNA gene sequencing) identified them to species/ subspecies level. Five Leuconostoc strains grew well and acidified milk, and most of them grew even at 8 °C. They showed moderate resistance to heat treatments (30 min t 63 °C) and grew well in the presence of 3% and 4% NaCl, and were significantly inhibited at pH ≤ 5. All strains showed resistance against the bacteriophages tested. In general, the antibacterial properties observed were slight and due to acid production, with the exception of Leuconostoc citreum MB1, which strongly inhibited Listeria monocytogenes ATCC 15313 by the production of a bacteriocin-like compound. All Leuconostoc strains studied were susceptible to gentamicin, tetracycline, erythromycin and ampicillin. Some strains also showed interesting technological and antimicrobial properties, thus being potentially appropriate as adjunct starters in fermented dairy products. This study highlights that adventitious lactic acid bacteria can be a great source of novel strains with interesting technological features that could be used for fermented dairy foods.
Aims: Characterization of four virulent Lactococcus lactis phages (CHD, QF9, QF12 and QP4) isolated from whey samples obtained from Argentinean cheese plants.Methods and Results: Phages were characterized by means of electron microscopy, host range and DNA studies. The influence of Ca2+, physiological cell state, pH and temperature on cell adsorption was also investigated. The double-stranded DNA genomes of these lactococcal phages showed distinctive restriction patterns. Using a multiplex PCR, phage QP4 was classified as a member of the P335 polythetic species while the three others belong to the 936 group. Ca2+ was not needed for phage adsorption but indispensable to complete cell lysis by phage QF9. The lactococci phages adsorbed normally between pH 5 and pH 8, and from 0°C to 40°C, with the exception of phage QF12 which had an adsorption rate significantly lower at pH 8 and 0°C.Conclusions: Lactococcal phages from Argentina belong to the same predominant groups of phages found in other countries and they have the same general characteristics.Significance and Impact of the Study: This work is the first study to characterize Argentinean L. lactis bacteriophages.
To isolate and characterize bacterial strains derived from Lactobacillus casei and Lactobacillus paracasei strains and resistant to phage MLC-A. Two of nine assayed strains rendered resistant mutants with recovery efficiencies of 83% (Lact. paracasei ATCC 27092) and 100% (Lact. casei ATCC 27139). DNA similarity coefficients (RAPD-PCR) confirmed that no significant genetic changes occurred while obtaining resistant mutants. Neither parent nor mutant strains spontaneously released phages. Phage-resistant mutants were tested against phages PL-1, J-1, A2 and MLC-A8. Lactobacillus casei ATCC 27092 mutants showed, overall, lower phage resistance than Lact. paracasei ATCC 27092 ones, but still higher than that of the parent strain. Lactobacillus paracasei ATCC 27092 mutants moderately adsorbed phage MLC-A only in calcium presence, although their parent strain successfully did it with or without calcium. Adsorption rates for Lact. casei ATCC 27139 and its mutants were highly influenced by calcium. Again, phage adsorption was higher on the original strain. Several isolates derived from two Lact. casei and Lact. paracasei strains showed resistance to phage MLC-A but also to other Lact. casei and Lact. paracasei phages. This study highlights isolation of spontaneous bacteriophage-resistant mutants from Lact. casei and Lact. paracasei as a good choice for use in industrial rotation schemes.
Probiotic bacteria, according to the definition adopted by the World Health Organization in 2002, are live microorganisms, which when administered in adequate amounts confer a health benefit to the host. Recent studies show that the same probiotic strain produced and/or preserved under different storage conditions, may present different responses regarding their susceptibility to the adverse conditions of the gastrointestinal tract, its capacity to adhere to the intestinal epithelium, or its immunomodulating capacity, the functionality being affected without changes in cell viability. This could imply that the control of cell viability is not always enough to guarantee the functionality (probiotic capacity) of a strain. Therefore, a new challenge arises for food technologists and microbiologists when it comes to designing and monitoring probiotic food: to be able to monitor the functionality of a probiotic microorganism throughout all the stages the strain goes through from the moment it is produced and included in the food vehicle, until the moment of consumption. Conventional methodological tools or others still to be developed must be used. The application of cell membrane functionality markers, the use of tests of resistance to intestinal barriers, the study of surface properties and the application of in vivo models come together as complementary tools to assess the actual capacity of a probiotic organism in a specific food, to exert functional effects regardless of the number of viable cells present at the moment of consumption.
Aims: The aim of this work was to study the adsorption step of two new temperate bacteriophages (Cb1/204 and Cb1/342) of Lactobacillus delbrueckii and to isolate phage-resistant derivatives with interesting technological properties. Methods and Results: The effect of divalent cations, pH, temperature and cell viability on adsorption step was analysed. The Ca2+ presence was necessary for the phage Cb1/342 but not for the phage Cb1/204. Both phages showed to be stable at pH values between 3 and 8. Their adsorption rates decreased considerably at pH 8 but remained high at acid pH values. The optimum temperatures for the adsorption step were between 30 and 40°C. For the phage Cb1/342, nonviable cells adsorbed a lower quantity of phage particles in comparison with the viable ones, a fact that could be linked to disorganization of phage receptor sites and/or to the physiological cellular state. The isolation of phage-resistant derivatives with good technological properties from the sensitive strains and their relationship with the cell heterogeneity of the strains were also made. Conclusions: Characterization of the adsorption step for the first temperate Lact. delbrueckii phages isolated in Argentina was made, and phage-resistant derivatives of their host strains were obtained. Significance and Impact of the Study: Some phage-resistant derivatives isolated exhibited good technological properties with the prospective to be used at industrial level.
Lactic acid bacteria (LAB) are widely used in food fermentation processes. The LAB starters are primarily used because of their capability to produce lactic acid from lactose, thus carrying out the fermentation process. Also, the decrease of pH is a requirement for the safety of products. Moreover, LAB are involved in the production of aroma compounds contributing with the sensorial characteristics of the products. Amongst the great diversity of LAB genera and species, Lactobacillus plantarum has the advantage of being able to grow in a wide variety of food matrix, thus acting as starter in several fermented products. Also, probiotic properties have been reported for Lactobacillus plantarum strains. Therefore, it could be used as starter/probiotic culture in the production of functional foods. However, it activity could be severely affected by bacteriophage attacks. Phage infection of bacterial cells is the main cause of reduction in acid production or complete starter failure, which generates serious economic losses and might result in low quality and/or unsafe foods. The first step in the bacteriophage lytic cycle takes place when the phage particle adsorbs to the host cell surface. This interaction is highly specific and dependent on the presence of specific recognition sites called receptors. A successful attachment may then be followed by the injection of the phage nucleic acid into the cell, its intracellular replication and release of phage progeny. The study of the first step in the infection cycle of bacteriophages (adsorption) and, specifically, the characterization of phage receptors are very valuable to develop appropriate and alternative defence tools against their attacks. Whole bacterial cells or purified cell walls are used to carry out characterization of phage receptors. Enzymatic and chemical treatments can be performed on purified cell walls to determine the nature of receptor components. Moreover, if phage receptors are carbohydrates in nature, phage inhibition by several saccharides or desorption and competition assays can be applied to determine the compounds involved. In this chapter, several methodologies to determine the nature of phage receptors are reviewed. Particularly, data obtained on the strain Lactobacillus plantarum ATCC 8014 and using these methodologies are detailed and compared with those reported previously for other LAB.
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.
Prophages account for most of the genetic diversity among strains of a given bacterial species, and represent a latent source for the generation of virulent phages. In this work, a set of 30 commercial, collection and dairy-isolated Lactobacillus casei group strains were used. A species-specific PCR assay allowed a reclassification, mainly of strains previously considered Lactobacillus casei, into either Lactobacillus paracasei or Lactobacillus rhamnosus. All the strains were induced with mitomycin C, allowing direct recovering of phage DNA in 25 cases, which corroborates the widely occurrence of lysogeny on Lactobacillus genomes, including probiotic strains of Lactobacillus casei group. Ten out of 11 commercial strains studied contained prophages, evidencing the potential risks of their use at industrial scale. Strains were also induced by treatment with different concentrations of hydrogen peroxide but, however, this agent was not able to evidence a prophage release for any of the strains tested. According to a RAPD-PCR fingerprinting with M13, 1254 and G1 primers, most of the commercial strains presented a high degree of homology and, regarding BglII- and BamHI-restriction profiles of phage DNA, six of them harboured the same prophage. Surprisingly, both Lactobacillus paracasei ATCC 27092 and Lactobacillus paracasei ATCC 27139 shared a second prophage with both an INLAIN collection and a commercial Lactobacillus paracasei strains, whereas two collection strains shared a third one. On the other hand, mitomycin C-inducible prophages were detected only on about a half of the strains isolated from dairy products, which had (with only one exception) from moderate to high correlation coefficients according to RAPD-PCR fingerprinting. After induction, supernatants were filtered and tested against nine Lactobacillus strains of the set sensitive to previously assayed virulent phages, allowing isolation of two new virulent phages: ф iLp1308 and ф iLp84. Both phages were able to lyse all but one strains sensitive to previously assayed phage MLC-A.
Bacteriophages can cause great economic losses due to fermentation failure in dairy plants. Hence, physical and chemical treatments of raw material and/or equipment are mandatory to maintain phage levels as low as possible. Regarding thermal treatments used to kill pathogenic bacteria or achieve longer shelf-life of dairy products, neither low temperature long time nor high temperature short time pasteurization were able to inactivate most lactic acid bacteria (LAB) phages. Even though most phages did not survive 90°C for 2 min, there were some that resisted 90°C for more than 15 min (conditions suggested by the International Dairy Federation, for complete phage destruction). Among biocides tested, ethanol showed variable effectiveness in phage inactivation, since only phages infecting dairy cocci and Lactobacillus helveticus were reasonably inactivated by this alcohol, whereas isopropanol was in all cases highly ineffective. In turn, peracetic acid has consistently proved to be very fast and efficient to inactivate dairy phages, whereas efficiency of sodium hypochlorite was variable, even among different phages infecting the same LAB species. Both alkaline chloride foam and ethoxylated non-ylphenol with phosphoric acid were remarkably efficient, trait probably related to their highly alkaline or acidic pH values in solution, respectively. Photocatalysis using UV light and TiO(2) has been recently reported as a feasible option to industrially inactivate phages infecting diverse LAB species. Processes involving high pressure were barely used for phage inactivation, but until now most studied phages revealed high resistance to these treatments. To conclude, and given the great phage diversity found on dairies, it is always advisable to combine different anti-phage treatments (biocides, heat, high pressure, photocatalysis), rather than using them separately at extreme conditions.
At least 345 bacteriophages infecting Streptococcus thermophilus starter cultures have been isolated; general characteristics include high thermal resistance, short latent periods and large burst size. Phages with such characteristics are primed to thrive in the cheese making environment, lysing bacterial cultures and generating low-quality fermented products. All S. thermophilus phages isolated to date are members of the Siphoviridae family and the Caudovirales order and appear to constitute a polythetic phage species comprising two large groups, cos- and pac-types, based on the mode of DNA packaging. Comparative analyses have shown that S. thermophilus phage genomes are similarly organized into distinct modular regions and allow the detection of a core genome region. Several PCR-based techniques have been designed to detect them in cheese whey and milk samples. Similar S. thermophilus phages are globally distributed and endemic in specific dairy environments. The biogeography of S. thermophilus phages reinforces their current classification.
Progressive adaptation to bile might render some lactobacilli able to withstand physiological bile salt concentrations. In this work, the adaptation to bile was evaluated on previously isolated dairy strains of Lactobacillus delbrueckii subsp. lactis 200 and L. delbrueckii subsp. lactis 200+, a strain derived thereof with stable bile-resistant phenotype. The adaptation to bile was obtained by comparing cytosolic proteomes of both strains grown in the presence or absence of bile. Proteomics were complemented with physiological studies on both strains focusing on glycolytic end-products, the ability to adhere to the human intestinal epithelial cell line HT29-MTX and survival to simulated gastrointestinal conditions. Protein pattern comparison of strains grown with and without bile allowed us to identify 9 different proteins whose production was regulated by bile in both strains, and 17 proteins that showed differences in their levels between the parental and the bile-resistant derivative. These included general stress response chaperones, proteins involved in transcription and translation, in peptidoglycan/exopolysaccharide biosynthesis, in the lipid and nucleotide metabolism and several glycolytic and pyruvate catabolism enzymes. Differences in the level of metabolic end-products of the sugar catabolism were found between the strains 200 and 200+. A decrease in the adhesion of both strains to the intestinal cell line was detected in the presence of bile. In simulated gastric and intestinal juices, a protective effect was exerted by milk improving the survival of both microorganisms. These results indicate that bile tolerance in L. delbrueckii subsp. lactis involves several mechanisms responding to the deleterious impact of bile salts on bacterial physiology.
Lysogeny entails more economical and technological risks in probiotic Lactobacillus casei/paracasei bacteria than in other lactic acid bacteria, due to economic value. Lysogeny is widely spread among L. casei/paracasei strains. Siphophages CL1 and CL2, isolated from noninfected lysed-cultures of commercial L. paracasei A, are thermoresistant, have identical host spectrum, latent and burst times, whereas burst sizes are 148 and 85, respectively. Mitomycin C induction of L. paracasei A yielded prophage iA2, whose presence was confirmed on the strain genome and whose restriction patterns differed from phages CL1 and CL2. The latter shared several restriction fragments, probably indicating a common origin.
The aim of this work was to study the efficiency of diverse chemical and thermal treatments usually used in dairy industries to control the number of virulent and temperate Lactobacillus delbrueckii bacteriophages. Two temperate (Cb1/204 and Cb1/342) and three virulent (BYM, YAB and Ib3) phages were studied. The thermal treatments applied were: 63 degrees C for 30 min (low temperature--long time, LTLT), 72 degrees C for 15 s (high temperature--short time, HTST), 82 degrees C for 5 min (milk destined to yogurt elaboration) and 90 degrees C for 15 min (FIL-IDF). The chemical agents studied were: sodium hypochlorite, ethanol, isopropanol, peracetic acid, biocides A (quaternary ammonium chloride), B (hydrogen peroxide, peracetic acid and peroctanoic acid), C (alkaline chloride foam), D (p-toluensulfonchloroamide, sodium salt) and E (ethoxylated nonylphenol and phosphoric acid). The kinetics of inactivation were drew and T(99) (time necessary to eliminate the 99% of phage particles) calculated. Results obtained showed that temperate phages revealed lower resistance than the virulent ones to the treatment temperatures. Biocides A, C, E and peracetic acid showed a notable efficiency to inactivate high concentrations of temperate and virulent L. delbrueckii phages. Biocide B evidenced, in general, a good capacity to eliminate the phage particles. Particularly for this biocide virulent phage Ib3 showed the highest resistance in comparison to the rest of temperate and virulent ones. On the contrary, biocide D and isopropanol presented a very low capacity to inactivate all phages studied. The efficiency of ethanol and hypochlorite was variable depending to the phages considered. These results allow a better knowledge and give useful information to outline more effective treatments to reduce the phage infections in dairy plants.
Bacteriophage infection of lactic acid bacteria (LAB) constitutes one of the major problems in the dairy industry, causing economic losses and a constant risk of low quality and/or unsafe foods. The first step in the phage biology is the adsorption on the host cell surface. In a previous study, a remarkable thermal, chemical and photocatalytic resistance was demonstrated by four phages of Lactobacillus plantarum (ATCC 8014-B1, ATCC 8014-B2, FAGK1 and FAGK2). In the present work, these phages were used to characterize the adsorption process on L. plantarum ATCC 8014. Clearly, the characterization of this process could increase the possibilities of design useful strategies in order to prevent phage infections. The influence of Ca(2+), temperature, pH and physiological cell state on phage adsorption was investigated. Burst sizes of phages ATCC 8014-B1 and ATCC 8014-B2 were 60 and 83 PFU/infective centre, respectively. The four phages exhibited a high infectivity even at pH 4 and pH 11. Calcium or magnesium ions were not indispensable for cell lysis and plaque formation, and more than 99% of phage particles were adsorbed either in the presence or absence of Ca(2+), after 15 min at 37 degrees C. Phage adsorption was only partially affected at 50 degrees C, while reached its maximum between 30 and 42 degrees C. The highest adsorption values (99.9%) were observed from pH 5 to 7, after 30 min at 37 degrees C. Adsorption rates decreased after the thermal inactivation of cells, though, when 20 microg/ml of chloramphenicol was used, adsorption values were similar on treated and untreated cells. All these results showed that the adsorption process was only partially affected by a few conditions: thermally killed host cells, an incubation temperature of 50 degrees C and pH values of 9 and 10. Nevertheless, and unfortunately, those conditions are not commonly applied during fermented food manufacturing, thus restricting highly the application of strategies currently available to reduce phage infections in industrial environments. This work also contributes to increase the currently knowledge on the biological aspects of L. plantarum bacteriophages.
To ferment buttermilk, a low-cost by-product of the manufacture of butter, with a proteolytic strain of Lactobacillus helveticus, to enhance its value by the production of a functional peptide-enriched powder. Buttermilk was fermented with Lact. helveticus 209, a strain chosen for its high proteolytic activity. To enhance the release of peptidic fractions, during fermentation pH was kept at 6 by using NaOH, Ca(CO)(3) or Ca(OH)(2). Cell-free supernatant was recovered by centrifugation, supplemented or not with maltodextrin and spray-dried. The profile of peptidic fractions released was studied by RP-HPLC. The lactose, Na and Ca content was also determined. The powder obtained was administered to BALB/c mice for 5 or 7 consecutive days, resulting in the proliferation of IgA-producing cells in the small intestine mucosa of the animals. Buttermilk is a suitable substrate for the fermentation with Lact. helveticus 209 and the release of peptide fractions able to be spray-dried and to modulate the gut mucosa in vivo. A powder enriched with peptides released from buttermilk proteins, with potential applications as a functional food additive, was obtained by spray-drying. A novel use of buttermilk as substrate for lactic fermentation is reported.
Introduction General Aspects Types of Starter Cultures New Sources of Primary Starters Specialized Cultures of LAB Future Trends and Final Considerations References
Baby's intestine is (or was said to be) sterile at birth and gut microbiota developmentis a gradual process after delivery. Quantitative and qualitative differences inbifidobacterial and lactic acid bacteria levels and species composition have been shownbetween breastfed and formula-fed infants, bifidobacteria being the most dominantmicroorganisms in the former group. Establishment of the gut microbiota is a stepwiseprocess which provides the earliest and most massive source of microbial stimuli for thenormal maturation of the gut mucosal immune system, contributing to its development ininfancy and to the control of the gut-associated immunological homeostasis later in life.Probiotic intervention in the neonatal period has attracted scientific interest after recentdemonstrations showing that specific strains reduce the symptoms and risk of allergicand infectious diseases or improve feeding tolerance. However, no all early interventionsin children reported rendered positive results. The question of the right dose and thespecific pathologies that probiotic administration, to infants less than 6 month of age,could be helpful for is still under a vigorous debate. Breast milk contains several factors,including nutrients, antimicrobial agents, IgA antibodies and TGF-β, which contributebeneficially to the immunologic maturation and well-being of the infant as well as factorsthat promote the growth of bifidobacteria in the infant's intestine. Additionally, healthybreast milk contains significant numbers of bacteria. In 2003 it was reported the isolationof lactobacilli from breast milk as potential probiotics. Breast milk seems to be a natural source of probiotic bacteria for infants. In this context, supplementation of infantformulas with these kinds of probiotics might beneficially alter the composition of themicroflora of formula-fed infants in such a way that it resembles that of breast-fedinfants. However, to date there is no available information concerning the technologicalpotential of these strains for their industrialization (growth in milk, resistance to lacticacid, freezing or spray-drying, among others) if they are thought to be included in dairyproducts or in formulas for infants.
The growth capacity of probiotic Lactobacillus paracasei A13, Bifidobacterium bifidum A1 and L. acidophilus A3 in a probiotic fresh cheese commercialized in Argentina since 1999 was studied during its manufacture and refrigerated storage at 5 degrees C and 12 degrees C for 60 days. Additionally, viable cell counts for probiotic bacteria in the commercial product are reported for batch productions over the last 9 years. L. paracasei A13 grew a half log order at 43 degrees C during the manufacturing process of probiotic cheese and another half log order during the first 15 days of storage at 5 degrees C, without negative effects on sensorial properties of the product. However, a negative impact on sensorial characteristics was observed when cheeses were stored at 12 degrees C for 60 days. Colony counts in the commercial product showed variations from batch to batch over the last 9 years. However, colony counts for each probiotic bacterium were always above the minimum suggested. Growth capacity of L. paracasei A13 in cheese during manufacturing and storage, mainly at temperatures commonly found in retail display cabinets in supermarkets (12 degrees C or more), would make it necessary to re-evaluate its role as possible probiotic starter and the consequences on food sensorial characteristics if storage temperature during commercial shelf life is not tightly controlled.
Two greatly related Lactobacillus plantarum bacteriophages (named FAGK1 and FAGK2) were isolated from Kefir grains of different origins. Both phages belonged to the Siphoviridae family (morphotype B1) and showed similar dimensions for head and tail sizes. The host range of the two phages, using 36 strains as potential host strains, differed only in the phage reactivity against one of them. The phages showed latent periods of 30 min, burst periods of 80+/-10 min and burst size values of 11.0+/-1.0 PFU per infected cell as mean value. Identical DNA restriction patterns were obtained for both phages with PvuI, SalI, HindIII and MluI. The viral DNA apparently did not present extremes cos and the structural protein patterns presented four major bands (32.9, 35.7, 43.0 and 66.2 kDa). This study reports the first isolation of bacteriophages of Lb. plantarum from Kefir grains and adds further knowledge regarding the complex microbial community of this fermented milk.
Two Streptococcus thermophilus phages (ALQ13.2 and phiAbc2) were previously isolated from breakdowns of cheese manufacture in Argentina. Complete nucleotide sequence analysis indicated that both phages contained linear double-stranded DNA: 35,525 bp in length for the pac-type phage ALQ13.2 and 34,882 bp for the cos-type phage phiAbc2. Forty-four and 48 open reading frames (ORF) were identified for ALQ13.2 and phiAbc2, respectively. Comparative genomic analysis showed that these isolates shared many similarities with the eight previously studied cos- and pac-phages infecting different S. thermophilus strains. In particular, part of the phiAbc2 genome was highly similar to a region of phage 7201, which was thought to be unique to this latter phage. Protein analysis of the pac-phage ALQ13.2 using SDS polyacrylamide gel electrophoresis (SDS-PAGE) identified three major proteins and seven minor proteins. Parallel structural proteome analysis of phiAbc2 revealed seven protein bands, two of which were related to major structural proteins, as expected for a cos-type phage. Similarities to other S. thermophilus phages suggest that the streptococcal phage diversity is not extensive in worldwide dairy factories possibly because related high-performing bacterial strains are used in starter cultures.
Nineteen Streptococcus thermophilus phages isolated from an Argentinean cheese plant were studied regarding their host range, morphology, packaging mechanism and nucleotide sequence of the variable region VR2 (one of the genetic determinants of host specificity). According to their features, they were morphologically classified into Siphoviridae family, morphotype B1. Among the phages tested, 13 distinct restriction patterns were found, and only one phage showed a pac-type mechanism. The classification based on the VR2 sequence proved to be very useful, since phages with very different VR2 sequences showed clearly different host ranges.
The bacteriophages Cb1/204 and Cb1/342 were obtained by induction from the commercial strain Lactobacillus delbrueckii subsp. lactis Cb1, and propagated on Lactobacillus delbrueckii subsp. lactis 204 (Lb.l 204) and Lactobacillus delbrueckii subsp. bulgaricus 342 (Lb.b 342), respectively. By cross sensitivity, it was possible to detect a delay in the lysis of Lb.l 204 with Cb1/342 phage, while the adsorption rate was high (99.5%). Modified and unmodified phages were isolated using phage Cb1/342 and strain Lb.l 204. The EOP (Efficiency of Plaquing) values for the four phages (Cb1/204, Cb1/342, Cb1/342modified and Cb1/342unmodified) suggested that an R/M system modified the original temperate phage, and the BglII-DNA restriction patterns of these phages might point out the presence of a Type II R/M system. Also, the existence of a Type I R/M system was demonstrated by PCR and nucleotide sequence, being the percentages of alignment homology with Type I R/M systems reported previously higher than 95%. In this study it was possible to demonstrate that the native phage resistant mechanisms and the occurrence of prophages in commercial host strains, contribute strongly to diversify the phage population in a factory environment.
The growth of commercial strains of thermophilic lactic acid bacteria (LAB) was determined in reconstituted dried whey and buttermilk. Kinetic (μ, /h and g, h) and growth (N, cfu/mL; X, cell yield and IV , percentual variation) parameters were determined using Elliker (streptococci) and MRS (lactobacilli) broths, and a commercial medium as controls. Cheese whey was the least effective substrate for growing streptococci and lactobacilli strains. However, whey and buttermilk with added yeast extract gave the best performance. These media could be efficient and easily available alternatives for producing industrial biomass of thermophilic LAB.
To investigate the effect of high pressure homogenization on virus inactivation, phages specific for Lactobacillus delbrueckii, Lactobacillus helveticus, Streptococcus thermophilus, Lactococcus lactis, Lactobacillus paracasei and Lactobacillus plantarum were studied. The influence of pressure, number of passes, suspension medium and phage concentration were studied at 25 °C. Reductions in viability were proportional to pressure and number of passes, though the inactivation extent was phage-dependent. At 100 MPa, some bacteriophages were completely inactivated (6 log10 reduction) after 3 or 5 passes, while others remained infective after 8 passes. For all phages, treatment at 60 MPa was insufficient for complete inactivation, even after 8 passes. No clear influence of suspending medium was observed. Inactivation seems to depend on phage concentration; the higher the initial load, the bigger the reduction achieved. Although these results showed that several phages studied are resistant to high-pressure homogenization, this strategy could be combined with others to control their presence in raw milk.
To evaluate the phage diversity in the environment of a dairy industry which manufactures a product fermented with a probiotic strain of Lactobacillus paracasei. Twenty-two Lact. paracasei phages were isolated from an industrial plant that manufactures a probiotic dairy product. Among them, six phages were selected based on restriction profiles, and two phages because of their notable thermal resistance during sample processing. Their morphology, host range, calcium dependency and thermal resistance were investigated. All phages belonged to the Siphoviridae family (B1 morphotype), were specific for Lact. casei and paracasei strains showing identical host spectrum, and only one phage was independent of calcium for completing its lytic cycle. Some of the phages showed an extraordinary thermal resistance and were protected by a commercial medium and milk. Phage diversity in a probiotic product manufacture was generated to a similar or greater extent than during traditional yogurt or cheese making. This work emphasizes probiotic phage infections as a new ecological situation beyond yogurt or cheese manufactures, where the balanced coexistence between phages and strains should be directed toward a favourable state, thus achieving a successful fermentation.
The effect of several biocides, thermal treatments, and photocatalysis on the viability of four Lactobacillus plantarum phages was investigated. Times to achieve 99% inactivation (T99) of phages at 63, 72, and 90 degrees C were evaluated in four suspension media: deMan Rogosa Sharpe broth, reconstituted skim milk, a commercial EM-glucose medium, and Tris magnesium gelatin buffer. The four phages studied were highly resistant to 63 degrees C (T99 > 45 min); however, counts < 10 PFU/ml were achieved by heating at 90 degrees C for 5 min. Higher thermal resistance at 72 degrees C was observed when reconstituted skim milk and EM-glucose medium were assayed. Peracetic acid (0.15%, vol/vol) was an effective biocide for the complete inactivation of all phages studied within 5 min of exposure. Sodium hypochlorite (800 ppm) inactivated the phages completely within 30 min. Ethanol (100%) did not destroy phage particles even after 45 min. Isopropanol did not have any effect on phage viability. Phage counts < 50 PFU/ml were obtained within 180 min of photocatalytic treatment. The results obtained in this work are important for establishing adequate methods for inactivating phages in industrial plants and laboratory environments.
High pressure homogenization (HPH) is one of the most promising alternatives to traditional thermal treatment for food preservation and diversification. In order to evaluate its potential for the production of fermented milks carrying probiotic bacteria, four types of fermented milks were manufactured from HPH treated and heat treated (HT) milk with and without added probiotics. Microbiological, physicochemical and organoleptic analyses were carried out during the refrigerated period (35 d at 4 degrees C). HPH application to milk did not modify the viability of the probiotic cultures but did increase the cell loads of the starter cultures (ca. 1 log order) compared with traditional products. The coagula from HPH-milk was significantly more compacted (P<0.05) (higher firmness) than that obtained with HT-milk, and it had the highest values of consistency, cohesiveness and viscosity indexes compared with fermented milks produced without HPH treatment. All the samples received high sensory analysis scores for each descriptor considered. HPH treatment of milk can potentially diversify the market for probiotic fermented milks, especially in terms of texture parameters.
The aim of this work was to study the relationship between the cell morphological heterogeneity and the phage-resistance in the commercial strain Lactobacillus delbrueckii subsp. lactis Ab1. Two morphological variants (named C and T) were isolated from this strain. Phage-resistant derivatives were isolated from them and the percentage of occurrence of confirmed phage-resistant cells was 0.001% of the total cellular population. Within these phage-resistant cell derivatives there were T (3 out of 4 total isolates) and C (1 out of 4 total isolates) variants. The study of some technological properties (e.g. proteolytic and acidifying activities) demonstrated that most of phage-resistant derivatives were not as good as the parental strain. However, for one derivative (a T variant), the technological properties were better than those of the parental strain. On the other hand, it was possible to determinate that the system of phage-resistance in the T variants was interference in adsorption step, with adsorption rates <15%. For the C variant derivative it was possible to demonstrate the presence of a restriction/modification system and, moreover, to determinate that this system could be Type I R/M.
A set of simple in vitro tests (identification by species-specific PCR, genetic diversity, phage sensitivity, growth and viability in milk, resistance to salts and flavor compounds, bacterial interactions, tolerance to simulated gastric juice and bile, bile salts deconjugation, hydrophobicity and β-galactosidase and antibacterial activities), that can be carried out in almost every laboratory of microbiology, mainly in developing countries where there is often limited access to sophisticated techniques, allowed us to identify, among 19 intestinal human isolates, a potential candidate for new probiotic dairy foods for the local market. Lactobacillus gasseri LgF37/1 performed well in the culture media used for the enumeration of probiotic bacteria in argentinian dairy products. This strain showed also high tolerance to the technological challenges assessed, bile salts resistance, the capacity to produce bacteriocin-like metabolites, to inhibit pathogenic bacteria, to deconjugate bile salts and high hydrophobicity. Further in vivo research should be carried out with this strain before claiming probiotic properties for it. However, the use of a set of simple in vitro techniques proved to be important to determine which strains should undergo future and more complex studies.
A total of 16 soft and semihard Argentinean cheeses, and 95 pasteurized cheese–milk samples, were analysed for microorganisms responsible for blowing. Lactic acid bacteria (starter microflora) resulted in high numbers (> 107 colony-forming units (cfu)/g). Coliform and yeast counts were lower than 104 cfu/g. Cremoso cheeses were blown by leuconostocs, lactobacilli and Bacillus polymyxa. Mozzarella was spoiled by B. polymyxa and Bacillus macerans. Semihard cheeses were affected by spore-forming (Clostridium and Bacillus), propionic and lactobacilli strains. Clostridia, Bacillus, leuconostocs and heterofermentative lactobacilli were detected in pasteurized milk. Bacillus strains were not previously associated with blowing in soft and semihard cheeses.
Frequency of lysogeny in Lactobacillus delbrueckii strains (from commercial and natural starters) and preliminary characterization of temperate bacteriophages isolated from them. Induction of strains (a total of 16) was made using mitomycin C (MC) (0.5 mug ml(-1)). For 37% of the MC-treated supernatants, it was possible to detect phage particles or presence of killing activity, but only two active bacteriophages were isolated. The two temperate phages isolated were prolate-headed phages which belonged to group c of Lact. delbrueckii bacteriophages classification. Different DNA restriction patterns were obtained for each phage, while the structural protein profiles and packaging sites were identical. Distinctive one-step growth curves were exhibited by each phage. An influence of calcium ions was observed for their lysis in broth but not on the adsorption levels. Our study showed that lysogeny is also present in Lact. delbrueckii strains, including commercial strains. Commercial strains could be lysogenic and this fact has a great practical importance since they could contribute to the dissemination of active-phage particles in industrial environments.
A PCR protocol for detection of Lactobacillus helveticus bacteriophages was optimized. PCR was designed taking into account the sequence of the lys gene of temperate bacteriophage Phi-0303 and optimized to obtain a fragment of 222 bp using different Lb. helveticus phages from our collection. PCR was applied to total phage DNA extracted from 53 natural whey starters used for the production of Grana cheese and all gave the expected fragment. The presence of actively growing phages in the cultures was verified by traditional tests. Several PCR products of the lys gene were sequenced and aligned. The resulting sequences showed variable heterogeneity between the phages.
Bacteriophage infections of starter lactic acid bacteria (LAB) pose a serious risk to the dairy industry. Nowadays, the expanding use of valuable Lactobacillus strains as probiotic starters determines an increase in the frequency of specific bacteriophage infections in dairy plants. This work describes a simple and rapid Polymerase Chain Reaction (PCR) method that detects and identifies bacteriophages infecting Lactobacillus casei/paracasei, the main bacterial species used as probiotic. Based on a highly conserved region of the NTP-binding genes belonging to the replication module of L. casei phages phiA2 and phiAT3 (the only two whose genomes are completely sequenced), a pair of primers was designed to generate a specific fragment. Furthermore, this PCR detection method proved to be a useful tool for monitoring and identifying L. casei/paracasei phages in industrial samples since specific PCR signals were obtained from phage contaminated milk (detection limit: 10(4) PFU/mL milk) and other commercial samples (fermented milks and cheese whey) that include L. casei/paracasei as probiotic starter (detection limit: 10(6) PFU/mL fermented milk). Since this method can detect the above phages in industrial samples and can be easily incorporated into dairy industry routines, it might be readily used to earmark contaminated milk for use in processes that do not involve susceptible starter organisms, or processes which involve phage-deactivating conditions.
The growth of six probiotic commercial strains of lactobacilli was assessed in reconstituted dried whey and buttermilk supplemented with yeast extract, meat peptone, soy peptone, tryptone or casein acid hydrolysate at 0.3%, 0.6% or 1%. The addition of 1% glucose was also tested. Growth and acidification kinetics were determined at 37°C using MRS broth and a commercial culture medium as references. The suitability of whey and buttermilk as cryoprotectants at –20°C and –70°C was also assessed. Whey and buttermilk with 0.3% yeast extract were chosen for the growth of probiotic lactobacilli, since no satisfactory growth was observed without an external nitrogen source, whereas glucose did not improve the growth of any of the strains assayed. In general, buttermilk performed as satisfactorily as the reference media. The effectiveness of these media as cryoprotectants was strain dependent: skimmed milk and whey were the most suitable ones, especially for long-term storage at –20°C. However, at –70°C, no significant differences were observed between the culture media assessed. The use of whey or buttermilk as culture media for the production of probiotic lactic acid bacteria and for their cryopreservation implies a novel use of these low-cost products, offering an alternative way of utilizing the by-products of the dairy industry, helping to minimize their negative impact on the environment.
Our aim was to obtain derivatives of non-intestinal lactobacilli—commonly used in the dairy industry—able to grow under physiological concentrations of bile salts. Six resistant derivatives (five from Lactobacillus delbrueckii subsp. lactis and one from L. acidophilus) growing in the presence of 0.5% bile salts were obtained from 24 strains of L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis and L. helveticus, and also one strain of L. acidophilus with low resistance to bile salts. Carbohydrate fermentation profiles remained unchanged but the rate of fermentation of several sugars was generally slower in bile-resistant derivatives. No significant differences with respect to the parent strains were observed for the cell-wall hydrophobicity, capacity to grow in milk and survival during frozen storage. Some bile-resistant derivatives were also resistant to low pH, but in other strains this capacity was acquired following progressive adaptation to low pH. Adaptation to bile salts and low pH might be valuable tools for increasing the survival under gastrointestinal conditions of non-intestinal lactobacilli used for the manufacture of dairy products.
The effects of microencapsulation of AB-type culture (Lactobacillus acidophilus LA-5 and Bifidobacterium lactis Bb-12) with calcium alginate on cell survival in Iranian yogurt drink (Doogh) during storage at 4°C for 42 days, as well as under simulated gastrointestinal conditions, were studied. The pH of the product at the beginning of storage was 4.53 and the final pH at the end of storage were 4.52 and 3.78 for the samples containing encapsulated and free cells, respectively. The acetic acid content in the encapsulated-cellcontaining Doogh increased by 0.01% (from 0.05 to 0.06%) during the storage period, whereas for free-cell-containing Doogh the increase was 0.04% (from 0.05 to 0.09%). At day 42, the viable counts of L. acidophilus and bifidobacteria in the samples containing encapsulated cells were 5.5 and 4.0 log cycles higher than those containing free cells, respectively. To evaluate the protective impact of encapsulation on cell survival in in vivo situations, the product was subjected to three simulated gastrointestinal conditions, including extreme conditions (pH 1.5, 90 min/2% bile, 90 min), intermediate conditions (pH 1.5, 90 min/1% bile, 90 min) and normal conditions, i.e. the situation in the gastrointestinal tract of a normal healthy person after the consumption of a probiotic-containing dairy drink, when the stomach has not been free for a relatively long time (pH 2.0, 30 min/0.6% bile, 60 min). The viability of the probiotic cells increased from 0.6% and 0.2% (L. acidophilus and bifidobacteria, respectively) as free cells to 18.0% and 9.5% under the extreme gastrointestinal conditions, after encapsulation. Under normal gastrointestinal conditions, the cell survival rates were 16.1% for L. acidophilus and 21% for bifidobacteria before encapsulation, and 26.3 and 34.0% (L. acidophilus and bifidobacteria, respectively) after encapsulation.
High-pressure homogenization (HPH) is one of the most promising alternatives to traditional thermal treatment of food preservation and diversification. Its effectiveness on the deactivation of pathogenic and spoilage microorganisms in model systems and real food is well documented. To evaluate the potential of milk treated by HPH for the production of Crescenza cheese with commercial probiotic lactobacilli added, 4 types of cheeses were made: HPH (from HPH-treated milk), P (from pasteurized milk), HPH-P (HPH-treated milk plus probiotics), and P-P (pasteurized milk plus probiotics) cheeses. A strain of Streptococcus thermophilus was used as starter culture for cheese production. Compositional, microbiological, physicochemical, and organoleptic analyses were carried out at 1, 5, 8, and 12 d of refrigerated storage (4 degrees C). According to results obtained, no significant differences among the 4 cheese types were observed for gross composition (protein, fat, moisture) and pH. Differently, the HPH treatment of milk increased the cheese yield about 1% and positively affected the viability during the refrigerated storage of the probiotic bacteria. In fact, after 12 d of storage, the Lactobacillus paracasei A13 cell loads were 8 log cfu/ g, whereas Lactobacillus acidophilus H5 exhibited, in P-P cheese, a cell load decrease of about 1 log cfu/g with respect to the HPH-P cheese. The hyperbaric treatment had a significant positive effect on free fatty acids release and cheese proteolysis. Also, probiotic cultures affected proteolytic and lipolytic cheese patterns. No significant differences were found for the sensory descriptors salty and creamy among HPH and P cheeses as well as for acid, piquant, sweet, milky, salty, creamy, and overall acceptance among HPH, HPH-P, and P-P Crescenza cheeses.
Characterization of four virulent Lactococcus lactis phages (CHD, QF9, QF12 and QP4) isolated from whey samples obtained from Argentinean cheese plants. Phages were characterized by means of electron microscopy, host range and DNA studies. The influence of Ca(2+), physiological cell state, pH and temperature on cell adsorption was also investigated. The double-stranded DNA genomes of these lactococcal phages showed distinctive restriction patterns. Using a multiplex PCR, phage QP4 was classified as a member of the P335 polythetic species while the three others belong to the 936 group. Ca(2+) was not needed for phage adsorption but indispensable to complete cell lysis by phage QF9. The lactococci phages adsorbed normally between pH 5 and pH 8, and from 0 degrees C to 40 degrees C, with the exception of phage QF12 which had an adsorption rate significantly lower at pH 8 and 0 degrees C. Lactococcal phages from Argentina belong to the same predominant groups of phages found in other countries and they have the same general characteristics. This work is the first study to characterize Argentinean L. lactis bacteriophages.
Microencapsulation of probiotic cells with hydrocolloid materials, as an efficient method of viability increase of probiotics, has been recently under special concern and investigation. In present study, the protective effect of microencapsulation with calcium alginate on survival of probiotic cells (Lactobacillus acidophilus La-5 and Bifidobacterium lactis Bb-12) incorporated in Iranian yogurt drink (Doogh) was studied after the product exposure to simulated gastrointestinal conditions. The 3 simulated gastrointestinal conditions were: reinforced/strict gastrointestinal condition (pH1.5-90 min/2% bile-90 min), semi-alleviated gastrointestinal condition (pH1.5-90min/1% bile-90min) and real-alleviated gastrointestinal condition (pH2.0-30 min/0.6% bile-60 min). The viability of free probiotic cells increased from 0.6 and 0.2% (L. acidophilus and bifidobacteria, respectively) to 18.0 and 9.5% at reinforced gastrointestinal conditions, after encapsulation. At real-alleviated conditions, the cell survival rates were 16.1% for L. acidophilus and 21% for bifidobacteria before encapsulation, whilst these amounts improved to 26.3 and 34.0% (L. acidophilus and bifidobacteria, respectively) after encapsulation.
Microencapsulation of probiotic bacteria by using hydrocolloid materials provides one of the best possible cares to protect them against detrimental conditions of fermented milks and to extend the shelf life of probiotic fermented products. The most widely used encapsulating material is Ca-alginate. In this research, the effect of microencapsulation of AB-type culture (L. acidophilus LA-5 and Bifidobacterium lactis BB-12) with calcium alginate on cell survival over a 42- day refrigerated storage period (at 4°C) in Iranian yogurt drink (Doogh) was studied. The initial pH of product at the start of storage time was 4.522 and the final pHs at the end of storage period were 4.510 and 3.775 for the samples containing encapsulated and unencapsulated cells, respectively; indicating considerably higher metabolic activity of free cells compared with entrapped ones. At d 42, viable counts of L. acidophilus and bifidobacteria in the samples containing encapsulated cells were about 5.5 and 4.0 log cycles higher than those containing free cells, respectively.
Three commercial phage-sensitive strains of Lactobacillus delbrueckii (strains Ab(1), YSD V and Ib(3)) and four spontaneous phage-resistant mutants (strains A(7), A(17), V(2) and I(39)) isolated from them, all with a probiotic potential previously demonstrated were studied for their tolerance of bile salts (ox gall). Minimal Inhibitory Concentrations (MICs) ranged from 0.30% to 0.35% (w/v) of ox gall. These strains were exposed to gradually increasing concentrations of ox gall with the aim of isolating bile resistant derivatives. Stable derivatives able to tolerate up to 0.9% of ox gall were obtained from L. delbrueckii Ab(1), as well as from its spontaneous phage-resistant mutants A(7) and A(17). Random Amplified Polymorphic DNA (RAPD-PCR) analysis revealed a strong genetic homology between the ox gall-tolerant derivatives and their respective non-adapted original strains. These derivatives maintained, in general, the phage resistance phenotype of the non-adapted strains, with only one exception (phage-resistant mutant A(7)). After progressive ox gall adaptation, the phage-resistant mutant A(7) also exhibited progressive reversion of the phage resistance phenotype. The derivative with the highest ox gall-acquired tolerance (A(7)(0.9)) became sensitive to the phage, but derivatives with low (A(7)(0.3)) and intermediate (A(7)(0.6)) ox gall-acquired tolerance retained phage resistance. The technological properties of ox gall derivatives were comparable to those of their respective parent strains. However, the cells of the former were smaller than those of the original strains. Finally, the tolerant derivatives grew faster in the presence of ox gall than the parent strains. Our results demonstrated that it was possible to obtain, by a natural selection strategy, probiotic strains with acquired ox gall-tolerance from three (L. delbrueckii Ab(1) and their phage-resistant mutants A(7) and A(17)) of seven tested strains. Since such derivatives keep both phage resistance and other useful technological properties, they could be used for production of functional foods.
Nonstarter lactic acid bacteria are the main uncontrolled factor in today's industrial cheese making and may be the cause of quality inconsistencies and defects in cheeses. In this context, adjunct cultures of selected lactobacilli from nonstarter lactic acid bacteria origin appear as the best alternative to indirectly control cheese biota. The objective of the present work was to study the technological properties of Lactobacillus strains isolated from cheese by in vitro and in situ assays. Milk acidification kinetics and proteolytic and acidifying activities were assessed, and peptide mapping of trichloroacetic acid 8% soluble fraction of milk cultures was performed by liquid chromatography. In addition, the tolerance to salts (NaCl and KCl) and the phage-resistance were investigated. Four strains were selected for testing as adjunct cultures in cheese making experiments at pilot plant scale. In in vitro assays, most strains acidified milk slowly and showed weak to moderate proteolytic activity. Fast strains decreased milk pH to 4.5 in 8 h, and continued acidification to 3.5 in 12 h or more. This group consisted mostly of Lactobacillus plantarum and Lactobacillus rhamnosus strains. Approximately one-third of the slow strains, which comprised mainly Lactobacillus casei, Lactobacillus fermentum, and Lactobacillus curvatus, were capable to grow when milk was supplemented with glucose and casein hydrolysate. Peptide maps were similar to those of lactic acid bacteria considered to have a moderate proteolytic activity. Most strains showed salt tolerance and resistance to specific phages. The Lactobacillus strains selected as adjunct cultures for cheese making experiments reached 10(8) cfu/g in soft cheeses at 7 d of ripening, whereas they reached 10(9) cfu/g in semihard cheeses after 15 d of ripening. In both cheese varieties, the adjunct culture population remained at high counts during all ripening, in some cases overcoming or equaling primary starter. Overall, proximate composition of cheeses with and without added lactobacilli did not differ; however, some of the tested strains continued acidifying during ripening, which was mainly noticed in soft cheeses and affected overall quality of the products. The lactobacilli strains with low acidifying activity showed appropriate technological characteristics for their use as adjunct cultures in soft and semihard cheeses.
A total of 100 spontaneous phage-resistant mutants isolated from nine commercial Streptococcus thermophilus strains were characterized preliminarily by randomly amplified polymorphic DNA (RAPD) and the nature of their phage-resistance mechanisms was investigated. Only for mutants isolated from one strain, free phages were detected in their culture supernatants when these were titrated on the sensitive strain, suggesting that the mutants could have acquired the resistance phenotype by integrating the phage in their genomes (lysogeny). Adsorption interference was observed in the derivatives isolated from two strains. For mutants isolated from two other strains, restriction–modification (R–M) type systems were detected. In one of these cases, R–M was probably combined with another intracellular anti-phage system. In most cases, the molecular profiles (RAPD fingerprints) obtained with four arbitrary primers showed a high similarity among parent strains and their respective phage-resistant mutants. Some of these mutants were identified as potentially improved strains for industrial use.
Three commercial phage sensitive strains of Lactobacillus delbrueckii and spontaneous phage resistant mutants isolated from them were studied by focusing on their biological and probiotic features. After incubation in a simulated gastric solution (pH 2.0), viable cell counts decreased moderately, but a protective effect on strains was observed when milk was added. A limited resistance to bile was observed, while most of the strains tolerated lysozyme, and grew in the presence of bile salts and fermented prebiotics. Some strains showed high hydrophobicity values and β-galactosidase activity. The strong antibacterial activity displayed toward pathogens was due to the production of lactic acid. Sensitive strains and their phage resistant variants were able to adhere to Caco-2/TC-7 monolayers and significantly inhibited the invasion of Salmonella enterica serovar Enteritidis into Caco-2/TC-7 cells. All strains tested showed potential probiotic features, suggesting a potential probiotic role of L. delbrueckii.
The capacity of three phosphates to interrupt the lytic cycle of four specific autochthonal bacteriophages of lactic acid bacteria used as starters was assayed. The phosphates used (polyphosphates A and B and sodium tripolyphosphate-high solubility [TAS]) were selected on the basis of their capacity to sequester divalent cations, which are involved in the lytic cycle of certain bacteriophages. The assays were performed in culture media (deMan Rogosa Sharpe and Elliker broths) and reconstituted (10%, wt/vol) commercial skim milk to which phosphates had been added at concentrations of 0.1, 0.3, and 0.5% (wt/vol). Phosphate TAS was the most inhibitory one, since it was able to inhibit the lytic cycle of all bacteriophages studied, in both broths and milk. In broth, polyphosphates A and B inhibited the lytic cycle of only two bacteriophages at the maximal concentration used (0.5%), whereas in milk, they were not capable of maintaining the same inhibitory effect.
Three commercial phage sensitive Lactobacillus delbrueckii strains (identified as Ab(1), YSD V and Ib(3)), and four spontaneous phage-resistant mutants isolated from them were tested for their capacity to activate the gut mucosal immune response in mice, as indicated by the numbers of IgA-producing cells. Random Amplified Polymorphic DNA (RAPD) analysis revealed a strong genetic homology between the sensitive strains and their respective derivatives. The phage-resistant mutants exhibited high levels of phage resistance, elevated stability of this phenotype and technological properties comparable to those of their respective parent strains. The tolerance to acidic conditions, bile salts and lysozyme was strain dependent and total cell viability losses as a result of exposure to all three stresses ranged from 2.0 to 3.7 log units. All the strains were highly resistant to a simulated gastric solution of pH 3, while significant additional losses in cell viability were observed when acid treated cells were exposed to bile salts and lysozyme. BALB/c mice received pure cultures of Lb. delbrueckii sensitive and phage-resistant strains for 2, 5 or 7 consecutive days. The ability of the parent strains to activate the small intestine immune response was preserved or enhanced in phage-resistant mutants. The maximal proliferation of IgA(+) cells was observed at day 5 or 7, depending on the strain. Mutants isolated in this study using natural selection strategies had improved phage resistance, adequate technological properties and satisfactory gut mucosal immunostimulation ability, and so would be good candidates for industrial applications in functional foods.
The effect of refrigerated storage temperature was studied at 2, 5 and 8°C on the viability of probiotics in ABY (Lactobacillus acidophilus, Bifidobacterium lactis BB-12 and yogurt bacteria. Bulgaricus, i.e. Streptococcus thermophilus and Lactobacillus delbrueckii ssp. Bulgaricus) probiotic yogurt. The study was carried out during a 20-day refrigerated storage period to identify the best storage temperature(s). Also, the viability change of the probiotic micro-organisms was analysed at 5-day intervals throughout the refrigerated storage period. After 20 days, storage at 2°C resulted in the highest viability of L. acidophilus, whereas for Bifidobacterium lactis the highest viability was obtained when yogurt was stored at 8°C.
Spontaneous phage-resistant mutants were isolated from 12 commercial Streptococcus thermophilus strains by secondary-culture method. They were characterized by cell and colony morphology, carbohydrate fermentation patterns, phage-resistance stability, efficiency of plaquing (EOP), acidifying and proteolytic powers and milk acidification kinetics. Only 22.6% (n ¼ 100) of isolates proved to be true phage-resistant mutants. Phage resistance stability was a variable parameter among the mutants isolated, while EOP values were mostly very low (o10 À7). Acidifying and proteolytic powers did not differ (p40:05) between phage-sensitive strains and their respective phage-resistant derivatives. Groups of mutants were found showing a milk acidifying activity which was slower or faster than that observed for the parent strain. Some mutants exhibiting high phage resistance and good technological capacities could be used as improved strains for industrial processes.
To determine the presence, diffusion and variability of the integrase (int) gene in Lactobacillus delbrueckii ssp. lactis isolated from natural whey starters used for the production of Italian hard cheeses. A PCR-based protocol aimed to amplify an internal fragment of the int gene was optimized taking into account phage genome sequences available from public databases. Thirty-seven of the 39 strains tested showed the presence of the putative int gene. Southern blot hybridization experiments confirmed data obtained by PCR. The presence of the putative int gene was observed also in 20 of 23 Lact. delbrueckii ssp. lactis lytic phages isolated from the same starter cultures used to isolate strains. Phylogenetic analysis of partial int gene revealed a high similarity both within and between strain- and phage-derived sequences. Sixty per cent of the int-positive strains resulted inducible with mitomycin C, and two of them released active phage particles. Our preliminary findings seem to suggest that an important number of Lact. delbrueckii ssp. lactis strains associated with the whey starters are lysogenic. Further contribution to obtain a clearer picture of the complex relationship between thermophilic lactic acid bacteria phage and host in whey starters for Italian, hard-cooked cheeses.
This paper contains a study on the suitability of MRS-bile agar medium for enumeration of mixed probiotic cultures in cultured dairy products, beside an analysis of the morphological characteristics of probiotic colonies using SDM (stereo digital microscopy) method. Three probiotic cultures (Lactobacillus acidophilus, Lactobacillus casei and bifidobacteria) were enumerated in various mixed culture compositions, i.e. AY (L. acidophilus and yogurt bacteria), CY (L. casei and yogurt bacteria), BY (bifidobacteria and yogurt bacteria), ABY (L. acidophilus, bifidobacteria and yogurt bacteria), ACY (L. acidophilus, L. casei and yogurt bacteria) and BCY (bifidobacteria, L. casei and yogurt bacteria). MRS-bile agar showed a good suitability for differential enumeration of probiotics in ACY culture composition under both aerobic and anaerobic conditions, differential and selective enumeration of probiotics in BCY culture composition (anaerobiosis and aerobiosis, respectively) and selective enumeration in ABY culture composition only for L. acidophilus under aerobiosis. Furthermore, by using the subtractive enumeration method (SEM) proposed in this article based on the subtraction of the colonies growth under aerobic condition (only L. acidophilus) from the colonies growth under anaerobic condition (both L. acidophilus and bifidobacteria), the number of bifidobacteria in ABY culture composition could also be well deter-mined.
The effect of refrigerated storage temperature was studied at 2, 5 and 8 ° C on the viability of probiotics in ABY ( Lactobacillus acidophilus, Bifidobacterium lactis BB-12 and yogurt bacteria. Bulgaricus, i.e. Streptococcus thermophilus and Lactobacillus delbrueckii ssp. Bulgaricus) probiotic yogurt. The study was carried out during a 20-day refrigerated storage period to identify the best storage temperature(s). Also, the viability change of the probiotic micro-organisms was analysed at 5-day intervals throughout the refrigerated storage period. After 20 days, storage at 2 ° C resulted in the highest viability of L. acidophilus, whereas for Bifidobacterium lactis the highest viability was obtained when yogurt was stored at 8 ° C.