Technological variables influencing the growth and stability of a silage inoculant based on spray-dried lactic acid bacteria.

AIMS: To investigate the influence of different culture media and fermentation conditions on growth performance and viability of three lactic acid bacteria (LAB) strains with potential as silage inoculants, and to optimize spray-drying in order to enhance survival to dehydration and storage stability. METHODS AND RESULTS: In house-formulated MRS was a suitable low-cost culture medium for Lactobacillus plantarum Hv75, Pediococcus acidilactici 3903 and L. buchneri B463. Uncontrolled pH biomass production conferred enhanced stability during storage at 4°C after spray-drying. The use of whey protein concentrate 35 (WPC)-maltodextrin (M) as matrix, inlet temperature of 145-150°C and air flow rate of 601 l h-1 was adequate for the production of dehydrated LAB. According to the desirability function, at this optimized condition, moisture content, yield and solubility were predicted to be 3·96, 73·68 and 90·36% respectively. Those conditions also showed a decrease of 0·855 log CFU per gram after drying, no loss in viability at 4°C for 6 months and 1 log CFU per gram reduction at 25°C. CONCLUSIONS: Stable and economically feasible dehydrated LAB cultures were obtained using alternative culture media, fermentation under uncontrolled pH and optimizing spray-drying process conditions through the desirability function method. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results can be utilized for efficient production and commercialization of several dry LAB.

Postbiotics produced at laboratory and industrial level as potential functional food ingredients with the capacity to protect mice against Salmonella infection.

AIM: To determine the protective capacity against Salmonella infection in mice of the cell-free fraction (postbiotic) of fermented milk, produced at laboratory and industrial level. METHODS AND RESULTS: The proteolytic activity (PA) of 5 commercial cultures and 11 autochthonous Lactobacillus strains was evaluated. The DSM-100H culture displayed the highest PA and it was selected for further studies. The capacity of the postbiotics produced by pH-controlled fermentation to stimulate the production of secretory IgA in faeces and to protect mice against Salmonella infection was evaluated. A significant increase in secretory IgA in faeces of mice fed 14 days the postbiotic obtained at the laboratory (F36) was detected compared to control animals. A significantly higher survival was observed in mice fed the F36 and the FiSD (industrial product) compared to controls. CONCLUSION: The postbiotics obtained showed immunomodulatory and protective capacity against Salmonella infection in mice. SIGNIFICANCE AND IMPACT OF THE STUDY: The pH-controlled milk fermentation by the proteolytic DSM-100H culture could be a suitable strategy to obtain a food ingredient to be added to a given food matrix, not adequate to host viable cells of probiotics, to confer it enhanced functionality and thus expand the functional food market.

Study of the effects of spray drying in whey-starch on the probiotic capacity of Lactobacillus rhamnosus 64 in the gut of mice.

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: Lactobacillus rhamnosus 64 was spray dried in cheese whey-starch solution and administered to mice for 3, 6 or 10 consecutive days. Peritoneal macrophage phagocytic activity, secretory 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 Trinitrobenzenesulfonic acid (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 (FC). The spray-dried culture of L. rhamnosus 64 showed anti-inflammatory properties in murine model of TNBS-induced colitis. CONCLUSIONS: 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 FC. This fact should be taken into account when producing the culture for its application in the industry.

Effect of a sublethal high-pressure homogenization treatment on the fatty acid membrane composition of probiotic lactobacilli.

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.

A comprehensive approach to determine the probiotic potential of human-derived Lactobacillus for industrial use.

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.

Cell viability and immunostimulating and protective capacities of Bifidobacterium longum 51A are differentially affected by technological variables in fermented milks.

AIM: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: The uncoupling of cell viability and functionality demonstrated that, in certain cases, nonviable cells can also exert positive effects.

Preservation of functionality of Bifidobacterium animalis subsp. lactis INL1 after incorporation of freeze-dried cells into different food matrices.

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.

Narrowing down the number of potential plant-based probiotic candidates by successive in vitro, technological and in vivo assays.

The interest on plant-based fermented food is in raise in Western countries. The aim of this study was to select interleukin (IL)-10 inducing strains for the development of potential probiotic plant-based fermented foods. Departing from a collection of 52 lactic acid bacteria (LAB) strains derived from plant material, in vitro co-culture with murine macrophages allowed us to narrow down the number of candidates to 21 strains able to induce IL-10 secretion. 14 of these strains were able to promote the production of tumour necrosis factor-α too. The capacity to induce IL-6 was used to further reduce the number of strains to 4, from which Lactiplantibacillus plantarum subsp. plantarum LpAv was selected to ferment oat and carrots. L. plantarum LpAv was able to ferment oat and carrots until reaching counts of ca. 108 and 109 cfu/ml. Fermented oat and carrots were orally administered to mice for 10 consecutive days and challenged with a single infective dose of Salmonella enterica serovar. Typhimurium. Counts of L. plantarum LpAv in fermented carrots were 9.23±0.05 cfu/ml and 9.27±0.01 cfu/ml, at day 1 and 10 of the feeding period. Fermented carrots were able to confer enhanced protection (80% of survival) against infection, when compared to control mice (less than 25% of survival). However, L. plantarum LpAv administered as pure culture was not able to confer protection against Salmonella infection. L. plantarum LpAv was selected among 52 plant-derived LAB and it was able to ferment oat and carrots, being only fermented carrots able to confer enhanced protection against Salmonella infection. A succession of in vitro to in vivo tests is proposed as a tool to narrow down the number of candidates when searching for potential novel probiotics from a collection of autochthonous strains.

Suitability of buttermilk for fermentation with Lactobacillus helveticus and production of a functional peptide-enriched powder by spray-drying.

AIM: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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. SIGNIFICANCE AND IMPACT OF THE STUDY: 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.

Breast-milk derived potential probiotics as strategy for the management of childhood obesity.

Obesity and overweight, and their concomitant metabolic diseases, emerge as one of the most severe health problems in the world. Prevention and management of obesity are proposed to begin early in childhood, when probiotics may have a role. The Simulator of the Human Intestinal Microbial Ecosystem (SHIME®), in a dynamic validated in vitro system able to simulate the different parts of the gastrointestinal tract, has proven to be useful in analyzing the human intestinal microbial community. L. plantarum 73a and B. animalis subsp. lactis INL1, two strains isolated from breast milk, were assayed in the SHIME® using the fecal microbiota of an obese child. L. plantarum 73a alone or in combination with B. animalis subsp. lactis INL1 demonstrated survival capacity in the SHIME® system. The administration of both strains increased the alpha diversity of the microbiota and reduced the levels of the phylum Proteobacteria. In particular, the genera Escherichia, Shigella, and Clostridium_sensu_stricto_1 were significantly reduced when both strains were administered. The increase of Proteobacteria phylum is generally associated with the microbiota of obese people. Escherichia and Shigellacan be involved in inflammation-dependent adiposity and insulin resistance. L. plantarum73a supplementation reduced ammonia production. L. plantarum 73a alone or in combination with B. animalis subsp. lactis INL1 are potential probiotic candidates for the management of infant obesity.

Occurrence of bacteria with technological and probiotic potential in Argentinian human breast-milk.

Breast milk can be a source of potential probiotic bacteria, but the technological capacity of isolates obtained from this source is not always guaranteed. We aimed at isolating lactobacilli from breast milk samples collected in Argentina, focusing on isolates with functional and technological potential as probiotics. Fourteen Lactobacillus and one Bifidobacterium isolates were obtained from 164 samples donated by 104 mothers. The isolates preliminarily identified by MALDI-TOF, and then the identity was confirmed by partial 16S rRNA gene sequencing. Hydrophobicity was determined (hexadecane and xylene partition). The strains were also co-cultured with murine RAW 264.7 macrophages for screening the capacity to induce the anti-inflammatory cytokine interleukin (IL)-10. Hydrophobicity ranged from 7.4 and 95.9%. The strains Lactobacillus gasseri (70a and 70c) and Lactobacillus plantarum (73a and 73b) were the strains with a higher capacity to induce IL-10 production by macrophages. The technological application was evaluated by freezing dried in 10% lactose or 10% polydextrose. The survival was assessed after accelerated (37 °C, 4 weeks) or long-term (5 and 25 °C, 12 months) storage. Except for Lactobacillus gallinarum 94d, strains lost less than 1 Log10 order cfu/g after long-term (12 months) storage at 5 °C in lactose and polydextrose as protectants. A low correlation between survival to accelerated and long-term storage tests was observed. L. gasseri (70a and 70c) and L. plantarum (73a and 73b) deserve further studies as potential probiotics due to their capacity to induce IL-10 from murine macrophages and their hydrophobicity. In special, L. plantarum 73a was able to confer enhanced protection against Salmonella infection by promoting the immunity of the small intestine.

Postbiotics: facts and open questions. A position paper on the need for a consensus definition.

The past definitions of probiotics and prebiotics have been reviewed and updated recently. According to these concepts, probiotics comprise live microorganisms that confer a health benefit on the host when administered in adequate amounts, whereas a prebiotic is a substrate that is selectively utilised by host microorganisms, conferring a health benefit. The words probiotics and prebiotics can be found on labels of many foods and supplements. Consumers have a growing awareness of these terms' meanings, and many countries are increasingly using them for regulation purposes. At the same time, there is increasing evidence on the health effects of non-viable microorganisms and the metabolites that they can produce by fermentation or by their action on food components. Different terms have been used in the literature to refer to these bioactive compounds, which do not fall under the known categories of probiotics, prebiotics or synbiotics. The tentative term postbiotics has been the most used one so far. However, no definition of the term has gained international consensus to date. This work aims to provide information on the facts and the open questions about the so-called postbiotics.

Probiotic Crescenza cheese containing Lactobacillus casei and Lactobacillus acidophilus manufactured with high-pressure homogenized milk.

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.

Nonstarter Lactobacillus strains as adjunct cultures for cheese making: in vitro characterization and performance in two model cheeses.

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.

Mortality and translocation assay to study the protective capacity of Bifidobacterium lactis INL1 against Salmonella Typhimurium infection in mice.

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.

Growth of Lactobacillus paracasei A13 in Argentinian probiotic cheese and its impact on the characteristics of the product.

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.