Exiguobacterium sp. as a bioinoculant for plant-growth promotion and Selenium biofortification strategies in horticultural plants.

Plant growth-promoting rhizobacteria (PGPR) have a positive effect on plant development and being a promising way to enhance crop productivity and as substitution of chemical fertilizers. Selenium (Se) is an important trace element and its intake is usually lower than the daily minimum amount required for humans; hence, there is a demand on the design of Se biofortification strategies. Here, the genetic traits known to be associated with Plant-Growth Promotion (PGP) and Se biotransformation of Exiguobacterium sp. S17 were evaluated through genome analysis. Its growth-promoting capacity was tested through plant-growth promotion assays in laboratory and field conditions, using Brassica juncea (indian mustard), Beta vulgaris (chard), and Lactuca sativa (lettuce). Additionally, the Se biotransformation ability of Exiguobacterium sp. S17 was evaluated and the obtained selenized bacteria were tested in mustard plants. The sequenced bacteria genome revealed the presence of multiple genes involved in important functions regarding soil and plant colonization, PGP and Se biotransformation. Moreover, it was demonstrated that Exiguobacterium sp. S17 enhanced plant growth and could be useful to produce Se accumulation and biofortification in accumulator plants such as mustard. Thereby, Exiguobacterium sp. S17 might be used for developing new, sustainable, and environmentally friendly agro-technological strategies.

Biogenic selenium nanoparticles with antifungal activity against the wood-rotting fungus Oligoporus pelliculosus.

Selenium nanoparticles (SeNPs) have antimicrobial and antifungal activity. SeNPs using Se resistant bacteria is a low cost and eco-friendly technology. Fungal contamination of wood during drying is one of the main causes of economic losses in the wood industry. The bacterium Delftia sp. 5 resistance to Se and its ability to produce SeNPs able to inhibit the growth of the wood brown-rotting fungus Oligoporus pelliculosus was analyzed. The strain showed an optimal SeNPs production when selenite concentration was 160 mg L -1. The SeNPs were spherical with an average size 192.33 ± 8.6 nm and a zeta potential of -41.4 ± 1.3 nm. The SeNPs produced by Delftia sp. 5 (33.6 ± 0.1 mg L -1 Se) inhibited the growth of O. pelliculosus in agar plates and in Nothofagus pumilio (Lenga) wood samples. Delftia sp. 5 SeNPs could be used for embedding lenga wood prior to drying for preventing the growth of the deteriorating fungi O. pelliculosus.

Selenium stress response of the fruit origin strain Fructobacillus tropaeoli CRL 2034.

The fruit-origin strain Fructobacillus tropaeoli CRL 2034 can biotransform selenium into seleno-nanoparticles and selenocysteine. The proteomic analysis of F. tropaeoli CRL 2034 exposed to 5 and 100 ppm of Se showed a dose-dependent response since 19 and 77 proteins were deregulated, respectively. In the presence of 5 ppm of Se, the deregulated proteins mainly belonged to the categories of energy production and conversion or had unknown functions, while when cells were grown with 100 ppm of Se, most of the proteins were grouped into amino acid transport and metabolism, nucleotide transport and metabolism, or into unknown functions. However, under both Se conditions, glutathione reductases were overexpressed (1.8-3.1-fold), while mannitol 2-dehydrogenase was downregulated (0.54-0.19-fold), both enzymes related to oxidative stress functions. Mannitol 2-dehydrogenase was the only enzyme found that contained SeCys, and its activity was 1.27-fold increased after 5 ppm of Se exposure. Our results suggest that F. tropaeoli CRL 2034 counteracts Se stress by overexpressing proteins related to oxidative stress resistance and changing the membrane hydrophobicity, which may improve its survival under (food) storage and positively influence its adhesion to intestinal cells. Selenized cells of F. tropaeoli CRL 2034 could be used for producing Se-enriched fermented foods. KEY POINTS: • Selenized cells of F. tropaeoli showed enhanced resistance to oxidative stress. • SeCys was found in the Fructobacillus mannitol 2-dehydrogenase polypeptide chain. • F. tropaeoli mannitol 2-dehydrogenase activity was highest when exposed to selenium.

Selenium bio-enrichment of Mediterranean fruit juices through lactic acid fermentation.

This work was carried out to elaborate selenium (Se) bio-enriched fermented Mediterranean fruit juices. To this purpose, pomegranate and table red grape juices were added with sodium selenite (Na2SeO3) and fermented by Levilactobacillus brevis CRL 2051 and Fructobacillus tropaeoli CRL 2034 individually or combined. To better evaluate the effect of selenite addition and starter strain inoculums on the total bacterial community of the fruit juices, fermentation trials were performed with raw and pasteurized fruit juices. No statistical significant differences were observed for total mesophilic microorganisms (TMM) and rod-shaped lactic acid bacteria (LAB) levels among raw and pasteurized juices inoculated with the starter strains, while significant differences between those juices with and without selenite were registered. LAB cocci, Pseudomonadaceae and yeasts were detected only for the raw juice preparations. The dominance of L. brevis CRL 2051 and F. tropaeoli CRL 2034 was confirmed by randomly amplified polymorphic DNA (RAPD)-PCR analysis. After fermentation, pH dropped for all inoculated trials and control raw juices. The soluble solid content (SSC) levels of the raw juices were higher than the corresponding pasteurized trials. The thermal treatment affected consistently yellowness of grape juice trials and redness of pomegranate juices. No microbial Se accumulation was registered for pomegranate juices, while F. tropaeoli CRL 2034 accumulated the highest amount of Se (65.5 µg/L) in the grape juice. For this reason, only trials carried out with raw grape juices were investigated by metagenomics analysis by Illumina MiSeq technology. Non-inoculated grape juices were massively fermented by acetic acid bacteria while Fructobacillus and Lactobacillus (previous genus name of Levilactobacillus) represented the highest operational taxonomy units (OTUs) relative abundance % of the trials inoculated with the starter strains as confirmed by this technique.

Fruits and fruit by-products as sources of bioactive compounds. Benefits and trends of lactic acid fermentation in the development of novel fruit-based functional beverages.

Current awareness about the benefits of a balanced diet supports ongoing trends in humans towards a healthier diet. This review provides an overview of fruits and fruit-by products as sources of bioactive compounds and their extraction techniques, and the use of lactic acid fermentation of fruit juices to increase their functionality. Fruit matrices emerge as a technological alternative to be fermented by autochthonous or allochthonous lactic acid bacteria (LAB such as Lactiplantibacillus plantarum, Lacticaseibacillus rhamnosus, and other Lactobacillus species), and also as probiotic vehicles. During fermentation, microbial enzymes act on several fruit phytochemicals producing new derived compounds with impact on the aroma and the functionality of the fermented drinks. Moreover, fermentation significantly reduces the sugar content improving their nutritional value and extending the shelf-life of fruit-based beverages. The generation of new probiotic beverages as alternatives to consumers with intolerance to lactose or with vegan or vegetarian diets is promising for the worldwide functional food market. An updated overview on the current knowledge of the use of fruit matrices to be fermented by LAB and the interaction between strains and the fruit phytochemical compounds to generate new functional foods as well as their future perspectives in association with the application of nanotechnology techniques are presented in this review.

Hydrolysis of raw fish proteins extracts by Carnobacterium maltaromaticum strains isolated from Argentinean freshwater fish.

Lactic acid bacteria (LAB) isolated from freshwater fish (hatcheries and captures) from Paraná river (Argentina) were analyzed by using culture-dependent approaches. The species belonging to Carnobacterium (C.) divergens, C. inhibens, C. maltaromaticum, C. viridans and Vagococcus (V.) salmoninarum were identify as predominant by RAPD-PCR and 16 s rRNA gene sequencing. C. maltaromaticum (H-17, S-30, B-42 and S-44) grew in raw fish extract and slightly reduced the medium pH (5.81-5.91). These strains exhibited moderate fish sarcoplasmic protein degradation (≤ 73 %) releasing small peptides and free amino acids, being alanine, glycine, asparagine and arginine concentrations increased in a higher extent (17.84, 1.47, 1.26 and 0.47 mg/100 mL, respectively) by S-44 strain at 96 h incubation. Interestingly C. maltaromaticum H-17 was able to inhibit Listeria monocytogenes. Results suggest that these strains would contribute to the development of new safe and healthy fishery products with improved nutritional and sensory characteristics.

Biotransformation of Selenium by Lactic Acid Bacteria: Formation of Seleno-Nanoparticles and Seleno-Amino Acids.

Selenium (Se) is an essential micronutrient for the majority of living organisms, and it has been identified as selenocysteine in the active site of several selenoproteins such as glutathione peroxidase, thioredoxin reductase, and deiodinases. Se deficiency in humans is associated with viral infections, thyroid dysfunction, different types of cancer, and aging. In several European countries as well as in Argentina, Se intake is below the recommended dietary Intake (RDI). Some lactic acid bacteria (LAB) can accumulate and bio-transform selenite (toxic) into Se-nanoparticles (SeNPs) and Se-amino acids (non-toxic). The microbial growth, Se metabolite distribution, and the glutathione reductase (involved in selenite reduction) activity of Se-enriched LAB were studied in this work. The ninety-six assayed strains, belonging to the genera Lactococcus, Weissella, Leuconostoc, Lactobacillus, Enterococcus, and Fructobacillus could grow in the presence of 5 ppm sodium selenite. From the total, eight strains could remove more than 80% of the added Se from the culture medium. These bacteria accumulated intracellularly between 1.2 and 2.5 ppm of the added Se, from which F. tropaeoli CRL 2034 contained the highest intracellular amount. These strains produced only the seleno-amino acid SeCys as observed by LC-ICP-MS and confirmed by LC-ESI-MS/MS. The intracellular SeCys concentrations were between 0.015 and 0.880 ppm; Lb. brevis CRL 2051 (0.873 ppm), Lb. plantarum CRL 2030 (0.867 ppm), and F. tropaeoli CRL 2034 (0.625 ppm) were the strains that showed the highest concentrations. Glutathione reductase activity values were higher when the strains were grown in the presence of Se except for the F. tropaeoli CRL 2034 strain, which showed an opposite behavior. The cellular morphology of the strains was not affected by the presence of Se in the culture medium; interestingly, all the strains were able to form spherical SeNPs as determined by transmission electron microscopy (TEM). Only two Enterococcus strains produced the volatile Se compounds dimethyl-diselenide identified by GC-MS. Our results show that Lb. brevis CRL 2051, Lb. plantarum CRL 2030, and F. tropaeoli CRL 2034 could be used for the development of nutraceuticals or as starter cultures for the bio-enrichment of fermented fruit beverages with SeCys and SeNPs.

Survival of selenium-enriched lactic acid bacteria in a fermented drink under storage and simulated gastro-intestinal digestion.

Selenium (Se), which is present as SeCys in seleno-proteins, is involved in cancer prevention, thyroid functioning, and pathogen inhibition. Se is incorporated in the diet through Se-containing foods. Some lactic acid bacteria (LAB) can biotransform selenite (toxic) into Se-nanoparticles (SeNPs) and Se-amino acids. To exert their beneficial properties in the host, bacteria should survive the harsh conditions of the gastrointestinal tract and during food storage. We evaluated whether selenization of LAB influenced bacterial growth and survival during gastrointestinal digestion and after storage when present in a fermented fruit juice-milk (FJM) beverage. Lactobacillus brevis CRL 2051 and Fructobacillus tropaeoli CRL 2034 were grown in MRS with and without selenite, and used to inoculate the FJM matrix. Selenization had no effect on LAB growth (9.54-9.9 log CFU/mL) in the FJM drink. The presence of SeNPs was confirmed for both selenized strains in the FJM beverage; however, the highest Se concentration (100 µg/L) was detected for the fermented beverage with selenized L. brevis. Under storage conditions 1.1 log CFU/ml decrease in cell count of selenized cells of L. brevis was observed, while no effect on cell viability was detected for non-selenized L. brevis or both selenized and control cells of F. tropaeoli. Resistance of L. brevis during digestion of the fermented FJM beverage was not affected by selenization. Contrarily, an increase (1 log CFU/mL) in the resistance of F. tropaeoli was observed when cells were selenized. After digestion, Se was detected in the soluble fraction of the beverage fermented by both strains, being higher for L. brevis (23.6 µg/L). Although selenization did not exert a drastic effect on strains´ survival during storage and digestion, microbial selenization previous to food fermentation could be an interesting tool for Se enrichment avoiding thus the addition of toxic Se salts.

Silac-based quantitative proteomic analysis of Lactobacillus reuteri CRL 1101 response to the presence of selenite and selenium nanoparticles.

Stable isotope labeling in cell culture (SILAC) was applied for the first time on a lactic acid bacterium strain (L. reuteri CRL1101) for analyzing differential protein expression associated to selenite(Na2SeO3) and selenium nanoparticles (SeNPs) exposure. 57 and 47 proteins were found de-regulated by >1,5 fold in presence of selenite and SeNPs, respectively. Only 16 out of 104 proteins differentially expressed were commonly altered by selenite and SeNPs. The use of a clustered heat map allows us to visualize relations between the de-regulated proteins and exposure conditions. We identified a number of proteins involved in diverse functions and biological processes such as metabolism of carbohydrates, selenium and lipids; folding, sorting and degradation; environmental information and processing. In presence of both, selenite and SeNPs, proteins related to selenium metabolism such as cystathione beta-lyase and oxidoreductases (thioredoxine reductase and NAD/FAD oxidoreductase) were over expressed. Interestingly, the over expression of thioredoxin reductase could protect the host from oxidizing compounds. An over expression of phage proteins and chaperones with selenite was observed; this result and the fact that a lower cell count was detected when selenite was added could indicate that this latter Se species has a more deleterious effect than the nanoparticles.

Determination of size and mass-and number-based concentration of biogenic SeNPs synthesized by lactic acid bacteria by using a multimethod approach.

Selenium nanoparticles (SeNPs) were synthesized by a green technology using lactic acid bacteria (LAB, Lactobacillus acidophilus, L. delbrueckii subsp. bulgaricus and L. reuteri). The exposure of aqueous sodium selenite to LAB led to the synthesis of SeNPs. Characterization of SeNPs by transmission electron microscopy with energy dispersive X-ray spectrum (EDXS) analysis revealed the presence of stable, predominantly monodispersed and spherical SeNPs of an average size of 146 ± 71 nm. Additionally, SeNPs hydrodynamic size was determined by dispersive light scattering (DLS) and nanoparticle tracking analysis (NTA). For this purpose, a methodology based on the use of surfactants in basic medium was developed for isolating SeNPs from the bacterial pellet. The hydrodynamic size values provided by DLS and NTA were 258 ± 4 and 187 ± 56 nm, respectively. NTA measurements of number-based concentration reported values of (4.67±0.30)x109 SeNPs mL-1 with a relative standard deviation lower than 5% (n = 3). The quantitative results obtained by NTA were supported by theoretical calculations. Asymmetrical flow field flow fractionation (AF4) on line coupled to the inductively couple plasma mass spectrometry (ICP-MS) and off-line coupled to DLS was further employed to characterize biogenic SeNPs. The distribution of the particle size for the Se-containing peak provide an average size of (247 ± 14) nm. The data obtained by independent techniques were in good agreement and the developed methodology could be implemented for characterizing NPs in complex matrices such as biogenic nanoparticles embedded inside microbial material.

Whey-derived valuable products obtained by microbial fermentation.

Whey, the main by-product of the cheese industry, is considered as an important pollutant due to its high chemical and biological oxygen demand. Whey, often considered as waste, has high nutritional value and can be used to obtain value-added products, although some of them need expensive enzymatic synthesis. An economical alternative to transform whey into valuable products is through bacterial or yeast fermentations and by accumulation during algae growth. Fermentative processes can be applied either to produce individual compounds or to formulate new foods and beverages. In the first case, a considerable amount of research has been directed to obtain biofuels able to replace those derived from petrol. In addition, the possibility of replacing petrol-derived plastics by biodegradable polymers synthesized during bacterial fermentation of whey has been sought. Further, the ability of different organisms to produce metabolites commonly used in the food and pharmaceutical industries (i.e., lactic acid, lactobionic acid, polysaccharides, etc.) using whey as growth substrate has been studied. On the other hand, new low-cost functional whey-based foods and beverages leveraging the high nutritional quality of whey have been formulated, highlighting the health-promoting effects of fermented whey-derived products. This review aims to gather the multiple uses of whey as sustainable raw material for the production of individual compounds, foods, and beverages by microbial fermentation. This is the first work to give an overview on the microbial transformation of whey as raw material into a large repertoire of industrially relevant foods and products.

Optimization of lactic ferment with quinoa flour as bio-preservative alternative for packed bread.

The consumers' demand for food with high nutritional quality and free of chemical additives increases the need to look for new products and preservation strategies. Quinoa (Chenopodium quinoa) is an Andean pseudocereal highly appreciated because of its nutritional properties. Moreover, it is an optimal substrate for growing and production of improved amounts of antifungal compounds by Lactobacillus plantarum CRL 778. The aim of this work was to optimize a lactic ferment for packaged breads with improved nutritional value and prolonged shelf life by applying a statistical experimental design model. The addition of 30 % quinoa to the wheat semiliquid ferment (QWF) could highly improve the amino acids release (4.3 g/L) during fermentation. Moreover, this quinoa proportion was sufficient to obtain the same concentration of the antifungal compounds, phenyllactic and hydroxiphenyllactic acids (PLA and OH-PLA) as with 100 % quinoa (ca. 36 and 51 mg/L, respectively). Statistical model analysis showed that citrate and skimmed milk enhanced significantly all evaluated parameters specially PLA (ca. 71 mg/L), HO-PLA (ca. 75 mg/L), and lactate (27 g/L) with a p value <0.005. The synergic effects of higher antifungal compounds production, acid release, and pH decrease allowed lowering the amount (about 50 %) of the chemical preservative calcium propionate commonly added to bread. Moreover, these breads show increased shelf life.

Lactobacillus delbrueckii subsp. bulgaricus CRL 454 cleaves allergenic peptides of ß-lactoglobulin.

Whey, a cheese by-product used as a food additive, is produced worldwide at 40.7 million tons per year. ß-Lactoglobulin (BLG), the main whey protein, is poorly digested and is highly allergenic. We aimed to study the contribution of Lactobacillus delbrueckii subsp. bulgaricus CRL 454 to BLG digestion and to analyse its ability to degrade the main allergenic sequences of this protein. Pre-hydrolysis of BLG by L. delbrueckii subsp. bulgaricus CRL 454 increases digestion of BLG assayed by an in vitro simulated gastrointestinal system. Moreover, peptides from hydrolysis of the allergenic sequences V41-K60, Y102-R124, C121-L140 and L149-I162 were found when BLG was hydrolysed by this strain. Interestingly, peptides possessing antioxidant, ACE inhibitory, antimicrobial and immuno-modulating properties were found in BLG degraded by both the Lactobacillus strain and digestive enzymes. To conclude, pre-hydrolysis of BLG by L. delbrueckii subsp. bulgaricus CRL 454 has a positive effect on BLG digestion and could diminish allergenic reactions.

Diversity in proteinase specificity of thermophilic lactobacilli as revealed by hydrolysis of dairy and vegetable proteins.

Ability of industrially relevant species of thermophilic lactobacilli strains to hydrolyze proteins from animal (caseins and ß-lactoglobulin) and vegetable (soybean and wheat) sources, as well as influence of peptide content of growth medium on cell envelope-associated proteinase (CEP) activity, was evaluated. Lactobacillus delbrueckii subsp. lactis (CRL 581 and 654), L. delbrueckii subsp. bulgaricus (CRL 454 and 656), Lactobacillus acidophilus (CRL 636 and 1063), and Lactobacillus helveticus (CRL 1062 and 1177) were grown in a chemically defined medium supplemented or not with 1 % Casitone. All strains hydrolyzed mainly ß-casein, while degradation of αs-caseins was strain dependent. Contrariwise, κ-Casein was poorly degraded by the studied lactobacilli. ß-Lactoglobulin was mainly hydrolyzed by CRL 656, CRL 636, and CRL 1062 strains. The L. delbrueckii subsp. lactis strains, L. delbrueckii subsp. bulgaricus CRL 656, and L. helveticus CRL 1177 degraded gliadins in high extent, while the L. acidophilus and L. helveticus strains highly hydrolyzed soy proteins. Proteinase production was inhibited by Casitone, the most affected being the L. delbrueckii subsp. lactis species. This study highlights the importance of proteolytic diversity of lactobacilli for rational strain selection when formulating hydrolyzed dairy or vegetable food products.

Fermentation of quinoa and wheat slurries by Lactobacillus plantarum CRL 778: proteolytic activity.

Quinoa fermentation by lactic acid bacteria (LAB) is an interesting alternative to produce new bakery products with high nutritional value; furthermore, they are suitable for celiac patients because this pseudo-cereal contains no gluten. Growth and lactic acid production during slurry fermentations by Lactobacillus plantarum CRL 778 were greater in quinoa (9.8 log cfu/mL, 23.1 g/L) than in wheat (8.9 log cfu/mL, 13.9 g/L). Lactic fermentation indirectly stimulated flour protein hydrolysis by endogenous proteases of both slurries. However, quinoa protein hydrolysis was faster, reaching 40-100% at 8 h of incubation, while wheat protein hydrolysis was only 0-20%. In addition, higher amounts of peptides (24) and free amino acids (5 g/L) were determined in quinoa compared to wheat. Consequently, greater concentrations (approx. 2.6-fold) of the antifungal compounds (phenyllactic and hydroxyphenyllactic acids) were synthesized from Phe and Tyr in quinoa by L. plantarum CRL 778, an antifungal strain. These promising results suggest that this LAB strain could be used in the formulation of quinoa sourdough to obtain baked goods with improved nutritional quality and shelf life, suitable for celiac patients.

Diversity in growth and protein degradation by dairy relevant lactic acid bacteria species in reconstituted whey.

The high nutritional value of whey makes it an interesting substrate for the development of fermented foods. The aim of this work was to evaluate the growth and proteolytic activity of sixty-four strains of lactic acid bacteria in whey to further formulate a starter culture for the development of fermented whey-based beverages. Fermentations were performed at 37 °C for 24 h in 10 and 16% (w/v) reconstituted whey powder. Cultivable populations, pH, and proteolytic activity (o-phthaldialdehyde test) were determined at 6 and 24 h incubation. Hydrolysis of whey proteins was analysed by Tricine SDS-PAGE. A principal component analysis (PCA) was applied to evaluate the behaviour of strains. Forty-six percent of the strains grew between 1 and 2 Δlog CFU/ml while 19% grew less than 0·9 Δlog CFU/ml in both reconstituted whey solutions. Regarding the proteolytic activity, most of the lactobacilli released amino acids and small peptides during the first 6 h incubation while streptococci consumed the amino acids initially present in whey to sustain growth. Whey proteins were degraded by the studied strains although to different extents. Special attention was paid to the main allergenic whey protein, ß-lactoglobulin, which was degraded the most by Lactobacillus acidophilus CRL 636 and Lb. delbrueckii subsp. bulgaricus CRL 656. The strain variability observed and the PCA applied in this study allowed selecting appropriate strains able to improve the nutritional characteristics (through amino group release and protein degradation) and storage (decrease in pH) of whey.

Proteolytic action of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 reduces antigenic response to bovine ß-lactoglobulin.

The whey protein ß-lactoglobulin (BLG) is highly allergenic. Lactic acid bacteria can degrade milk proteins. The capacity of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 to hydrolyse the major BLG epitopes (V41-K60; Y102-R124; L149-I162) and decrease their recognition by IgE of allergic patients was evaluated. The intensity of BLG degradation was analysed by Tricine SDS-PAGE and RP-HPLC. Peptides released were identified by LC-MS/MS and the hydrolysates were tested for their capacity to inhibit IgE binding by ELISA test. L. delbrueckii subsp. bulgaricus CRL 656 degraded BLG (35%, 8h). The sequence analysis of the released peptides indicated that this strain degraded three main BLG epitopes. BLG-positive sera (3-5year old children) were used for testing IgE binding inhibition of BLG hydrolysates from the Lactobacillus strain. The hydrolysates were less immuno-reactive (32%) than the heated BLG. L. delbrueckii subsp. bulgaricus CRL 656 could be used for developing hypoallergenic dairy products.

Functional fermented whey-based beverage using lactic acid bacteria.

Whey protein concentrate (WPC) is employed as functional food ingredient because of its nutritional value and emulsifying properties. However, the major whey protein beta-lactoglobulin (BLG) is the main cause of milk allergy. The aim of this study was to formulate a fermented whey beverage using selected lactic acid bacteria and WPC35 (WPC containing 35% of proteins) to obtain a fermented product with low lactose and BLG contents and high essential amino acid concentration. Cell viability, lactose consumption, lactic acid production, proteolytic activity, amino acid release and BLG degradation by the selected strains Lactobacillus acidophilus CRL 636, Lactobacillus delbrueckii subsp. bulgaricus CRL 656 and Streptococcus thermophilus CRL 804, as single or mixed (SLaB) cultures were evaluated in WPC35 (10%, w/v) incubated at 37 degrees C for 24h. Then, the fermented WPC35 was mixed with peach juice and calcium lactate (2%, w/v) and stored at 10 degrees C for 28 days. During fermentation, single cultures grew 1.7-3.1 log CFU/ml and produced 25.1-95.0 mmol/l of lactic acid as consequence of lactose consumption (14.0-41.8 mmol/l) after 12h fermentation. L. delbrueckii subsp. bulgaricus CRL 656 was the most proteolytic strain (626 microg/ml Leu) and released the branched-chain essential amino acids Leu (16 microg/ml), Ile (27 microg/ml) and Val (43 microg/ml). All strains were able to degrade BLG in a range of 41-85% after 12h incubation. The starter culture SLaB grew 3.0 log CFU/ml, showed marked pH reduction, produced 122.0 mmol/l of lactic acid, displayed high proteolytic activity (484 microg/ml Leu) releasing Leu (13 microg/ml), Ile (18 microg/ml) and Val (35 microg/ml), and hydrolyzed 92% of BLG. The addition of calcium lactate to WPC35 maintained the drink pH stable during shelf life; no contamination was detected during this period. After 28 days, a decrease in cell viability of all strains was observed being more pronounced for L. delbrueckii subsp. bulgaricus CRL 656 and L. acidophilus CRL 636 (2.3 and 1.9 log CFU/ml, respectively). The results showed that WPC fermentation by rationally selected lactic acid bacteria might be used for developing functional beverages with improved characteristics such as reduced BLG content and increased branched-chain essential amino acids.

Effect of exopolysaccharides on the hydrolysis of beta-lactoglobulin by Lactobacillus acidophilus CRL 636 in an in vitro gastric/pancreatic system.

An analysis of the peptides generated by hydrolysis of BLG by nonproliferating cells of the strain Lactobacillus acidophilus CRL 636 was carried out. The effect of polysaccharides (pectin, and two EPS synthesized by two Streptococcus thermophilus strains, EPS1190 and EPS804) on BLG digestibility using an in vitro gastric/pancreatic system was analyzed. Polysaccharides are commonly used in the dairy industry to improve food texture; these hydrocolloids may interact with proteins, affecting their digestibility. Nonproliferating cells of Lb. acidophilus CRL 636 were able to hydrolyze 52% of BLG. Twenty-six resulting peptides with molecular masses in the range 544-4119 Da were identified by LC-MS/MS. These peptides resulted mostly from the hydrolysis of the more accessible N-terminal part of BLG. Degradation of BLG by pepsin was poor (8%). When BLG was previously hydrolyzed by Lb. acidophilus CRL 636, peptic hydrolysis was of 54.8%, while when pectin and EPS1190 were added, hydrolysis was higher (58.2 and 57.2%, respectively). Peptides crossing 8 kDa dialysis membranes after trypsin/chymotrypsin hydrolysis were analyzed by HPSEC. The produced peptides were smaller when BLG was hydrolyzed previously by the Lb. acidophilus strain. Moreover, in the presence of pectin, the amount of the larger peptide (3.5 kDa) observed in the size exclusion chromatograms was considerably decreased. Our studies showed that prehydrolysis of BLG by Lb. acidophilus CRL 636 had a positive influence on BLG digestibility and that polysaccharides may change the peptide profile yielded by trypsin/chymotrypsin hydrolysis, releasing smaller size peptides, which are known to be less immune-reactive. Moreover, Lb. acidophilus CRL 636 was able to hydrolyze the main epitopes (41-60, 102-124, and 149-162) of BLG, reducing its allergenic content.

Whey fermentation by thermophilic lactic acid bacteria: evolution of carbohydrates and protein content.

Whey, a by-product of the cheese industry usually disposed as waste, is a source of biological and functional valuable proteins. The aim of this work was to evaluate the potentiality of three lactic acid bacteria strains to design a starter culture for developing functional whey-based drinks. Fermentations were performed at 37 and 42 degrees C for 24h in reconstituted whey powder (RW). Carbohydrates, organic acids and amino acids concentrations during fermentation were evaluated by RP-HPLC. Proteolytic activity was measured by the o-phthaldialdehyde test and hydrolysis of whey proteins was analyzed by Tricine SDS-PAGE. The studied strains grew well (2-3log cfu/ml) independently of the temperature used. Streptococcus thermophilus CRL 804 consumed 12% of the initial lactose concentration and produced the highest amount of lactic acid (45 mmol/l) at 24h. Lactobacillus delbrueckii subsp. bulgaricus CRL 454 was the most proteolytic (91 microg Leu/ml) strain and released the branched chain amino acids Leu and Val. In contrast, Lactobacillus acidophilus CRL 636 and S. thermophilus CRL 804 consumed most of the amino acids present in whey. The studied strains were able to degrade the major whey proteins, alpha-lactalbumin being degraded in a greater extent (2.2-3.4-fold) than beta-lactoglobulin. Two starter cultures were evaluated for their metabolic and proteolytic activities in RW. Both cultures acidified and reduced the lactose content in whey in a greater extent than the strains alone. The amino acid release was higher (86 microg/ml) for the starter SLb (strains CRL 804+CRL 454) than for SLa (strains CRL 804+CRL 636, 37 microg/ml). Regarding alpha-lactalbumin and beta-lactoglobulin degradation, no differences were observed as compared to the values obtained with the single cultures. The starter culture SLb showed high potential to be used for developing fermented whey-based beverages.