Comparative sequence analysis of plasmids from Lactobacillus delbrueckii and construction of a shuttle cloning vector

While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated with Lactobacillus delbrueckii, with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain of Lactobacillus delbrueckii subsp. bulgaricus was determined. It consisted of a circular DNA molecule of 6,220 bp with a G+C content of 44.6% and a characteristic ori and encoded six open reading frames (ORFs), of which functions could be predicted for three-a mobilization (Mob) protein, a transposase, and a fused primase-helicase replication protein. Comparative analysis of pDOJ1 and the other available L. delbrueckii plasmids (pLBB1, pJBL2, pN42, and pLL1212) revealed a very similar organization and amino acid identities between 85 and 98% for the putative proteins of all six predicted ORFs from pDOJ1, reflecting a common origin for L. delbrueckii plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids from Streptococcus thermophilus. This observation and the ability of the replicon to function in S. thermophilus support the idea that the origin of plasmids in L. delbrueckii was likely from S. thermophilus. This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons from L. delbrueckii has previously been constructed, an Escherichia coli-L. delbrueckii shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15A ori, the chloramphenicol resistance gene of pCI372, and the lacZ polylinker from pUC18. This cloning vector was successfully introduced into E. coli, L. delbrueckii subsp. bulgaricus, S. thermophilus, and Lactococcus lactis. This shuttle cloning vector provides a new tool for molecular analysis of Lactobacillus delbrueckii and other lactic acid bacteria.     

Survival of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in the Terminal Ileum of Fistulated Göttingen Minipigs

The ability of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus administered in yogurt to survive the passage through the upper gastrointestinal tract was investigated with Göttingen minipigs that were fitted with ileum T-cannulas. After ingestion of yogurt containing viable microorganisms, ileostomy samples were collected nearly every hour beginning 3 h after food uptake. Living L. delbrueckii subsp. bulgaricus and S. thermophilus were detected in the magnitude of 10⁶ to 10⁷ per gram of intestinal contents (wet weight) in all animals under investigation. A calculation of the minimum amount of surviving bacteria that had been administered is presented. Total DNA extracted from ileostomy samples was subjected to PCR, which was species specific for L. delbrueckii and S. thermophilus and subspecies specific for L. delbrueckii subsp. bulgaricus. All three bacterial groups could be detected by PCR after yogurt uptake but not after uptake of a semisynthetic diet. One pig apparently had developed an endogenous L. delbrueckii flora. When heat-treated yogurt was administered, L. delbrueckii was detected in all animals. S. thermophilus or L. delbrueckii subsp. bulgaricus was not detected, indicating that heat-inactivated cells and their DNAs had already been digested and their own L. delbrueckii flora had been stimulated for growth.     

Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids

We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 10⁴ transformants per μg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii.     

Genetically Engineered Immunomodulatory Streptococcus thermophilus Strains Producing Antioxidant Enzymes Exhibit Enhanced Anti-Inflammatory Activities

The aims of this study were to develop strains of lactic acid bacteria (LAB) having both immunomodulatory and antioxidant properties and to evaluate their anti-inflammatory effects both in vitro, in different cellular models, and in vivo, in a mouse model of colitis. Different Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were cocultured with primary cultures of mononuclear cells. Analysis of the pro- and anti-inflammatory cytokines secreted by these cells after coincubation with candidate bacteria revealed that L. delbrueckii subsp. bulgaricus CRL 864 and S. thermophilus CRL 807 display the highest anti-inflammatory profiles in vitro. Moreover, these results were confirmed in vivo by the determination of the cytokine profiles in large intestine samples of mice fed with these strains. S. thermophilus CRL 807 was then transformed with two different plasmids harboring the genes encoding catalase (CAT) or superoxide dismutase (SOD) antioxidant enzymes, and the anti-inflammatory effects of recombinant streptococci were evaluated in a mouse model of colitis induced by trinitrobenzenesulfonic acid (TNBS). Our results showed a decrease in weight loss, lower liver microbial translocation, lower macroscopic and microscopic damage scores, and modulation of the cytokine production in the large intestines of mice treated with either CAT- or SOD-producing streptococci compared to those in mice treated with the wild-type strain or control mice without any treatment. Furthermore, the greatest anti-inflammatory activity was observed in mice receiving a mixture of both CAT- and SOD-producing streptococci. The addition of L. delbrueckii subsp. bulgaricus CRL 864 to this mixture did not improve their beneficial effects. These findings show that genetically engineering a candidate bacterium (e.g., S. thermophilus CRL 807) with intrinsic immunomodulatory properties by introducing a gene expressing an antioxidant enzyme enhances its anti-inflammatory activities.     

Antiproliferative Effects of Homogenates Derived from Five Strains of Candidate Probiotic Bacteria

Unheated and heat-treated homogenates were separately prepared from candidate probiotic bacteria, including Lactobacillus rhamnosus GG, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Streptococcus thermophilus. We compared the phytohemagglutinin-induced proliferation of mononuclear cells in the presence of homogenates and in the presence of a control containing no homogenate by assessing thymidine incorporation in cell cultures. All homogenates suppressed proliferation, whether the enzymatic activity was inactivated or not inactivated by heating. When the proliferation assays were repeated with cytoplasmic and cell wall extracts derived from the homogenate of L. rhamnosus GG, the cytoplasmic extract but not the cell wall extract was suppressive. These findings indicate that candidate probiotic bacteria possess a heat-stable antiproliferative component(s). These bacteria may be used to generate microbiologically nonviable yet immunologically active probiotic food products that are easier to store and have a longer shelf life.     

Physiological Study of Lactobacillus delbrueckii subsp. bulgaricus Strains in a Novel Chemically Defined Medium

We developed a chemically defined medium called milieu proche du lait (MPL), in which 22 Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) strains exhibited growth rates ranging from 0.55 to 1 h⁻¹. MPL can also be used for cultivation of other lactobacilli and Streptococcus thermophilus. The growth characteristics of L. bulgaricus in MPL containing different carbon sources were determined, including an initial characterization of the phosphotransferase system transporters involved. For the 22 tested strains, growth on lactose was faster than on glucose, mannose, and fructose. Lactose concentrations below 0.4% were limiting for growth. We isolated 2-deoxyglucose-resistant mutants from strains CNRZ397 and ATCC 11842. CNRZ397-derived mutants were all deficient for glucose, fructose, and mannose utilization, indicating that these three sugars are probably transported via a unique mannose-specific-enzyme-II-like transporter. In contrast, mutants of ATCC 11842 exhibited diverse phenotypes, suggesting that multiple transporters may exist in that strain. We also developed a protein labeling method and verified that exopolysaccharide production and phage infection can occur in MPL. The MPL medium should thus be useful in conducting physiological studies of L. bulgaricus and other lactic acid bacteria under well controlled nutritional conditions.     

Use of PCR-Based Methods for Rapid Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412^T, which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.     

Survival of Yogurt Bacteria in the Human Gut

Whether Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus can be recovered after passage through the human gut was tested by feeding 20 healthy volunteers commercial yogurt. Yogurt bacteria were found in human feces, suggesting that they can survive transit in the gastrointestinal tract.     

In Silico Prediction of Horizontal Gene Transfer Events in Lactobacillus bulgaricus and Streptococcus thermophilus Reveals Protocooperation in Yogurt Manufacturing

Lactobacillus bulgaricus and Streptococcus thermophilus, used in yogurt starter cultures, are well known for their stability and protocooperation during their coexistence in milk. In this study, we show that a close interaction between the two species also takes place at the genetic level. We performed an in silico analysis, combining gene composition and gene transfer mechanism-associated features, and predicted horizontally transferred genes in both L. bulgaricus and S. thermophilus. Putative horizontal gene transfer (HGT) events that have occurred between the two bacterial species include the transfer of exopolysaccharide (EPS) biosynthesis genes, transferred from S. thermophilus to L. bulgaricus, and the gene cluster cbs-cblB(cglB)-cysE for the metabolism of sulfur-containing amino acids, transferred from L. bulgaricus or Lactobacillus helveticus to S. thermophilus. The HGT event for the cbs-cblB(cglB)-cysE gene cluster was analyzed in detail, with respect to both evolutionary and functional aspects. It can be concluded that during the coexistence of both yogurt starter species in a milk environment, agonistic coevolution at the genetic level has probably been involved in the optimization of their combined growth and interactions.Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus) and Streptococcus thermophilus have been used in starter cultures for yogurt manufacturing for thousands of years. Both species are known to stably coexist in a milk environment and interact beneficially. This so-called protocooperation, previously defined as biochemical mutualism, involves the exchange of metabolites and/or stimulatory factors (38). Examples of biochemical protocooperation between L. bulgaricus and S. thermophilus include the action of cell wall-bound proteases, produced by L. bulgaricus strains, and formate, required for growth of L. bulgaricus and supplied by S. thermophilus (6, 7). An overview of the interactions between the two yogurt bacteria, including the exchange of CO₂, pyruvate, folate, etc., can be found in a recently published review by Sieuwerts et al. (43). Putative genetic mechanisms underlying protocooperation, however, so far have not been studied in detail.The genomes of two L. bulgaricus strains and three S. thermophilus strains, all used in yogurt manufacturing, have been fully sequenced (3, 32, 33, 34, 39, 44, 46). The available genomic information could provide new insights into the genetic aspects of protocooperation between L. bulgaricus and S. thermophilus through the identification of putative horizontal gene transfer (HGT) events at the genome scale. HGT can be defined as the exchange of genetic material between phylogenetically unrelated organisms (23). It is considered to be a major factor in the process of environmental adaptation, for both individual species and entire microbial populations. Especially HGT events between two species existing in the same niche can reflect their interrelated activities and dependencies (13, 17). Nicolas et al. (36) predicted HGT events between Lactobacillus acidophilus and Lactobacillus johnsonii by analyzing 401 phylogenetic trees, also including the genes of L. bulgaricus. Several HGT events have been predicted in the S. thermophilus strains CNRZ1066 and LMG 18311 (3, 10, 18) as well as in L. bulgaricus ATCC 11842 (46). Moreover, a core genome of S. thermophilus and possibly acquired genes were identified by a comparative genome hybridization study of 47 strains (40).In this study, we describe an in-depth bioinformatics analysis in which we combined gene composition (GC content and dinucleotide composition) and gene transfer mechanism-associated features. Thus, we predicted horizontally transferred genes and gene clusters in the five sequenced L. bulgaricus and S. thermophilus genomes, with a focus on niche-specific genes and genes required for bacterial growth. Identification of HGT events led to a list of putative transferred genes, some of which could be important for bacterial protocooperation and the adaptation to their environment. The evolution and function of the transferred gene cluster cbs-cblB(cglB)-cysE (originally called cysM2-metB2-cysE2 in S. thermophilus), involved in the metabolism of sulfur-containing amino acids, were analyzed in detail. On the basis of our analysis, it can be concluded that both species probably agonistically coevolved at the genetic level to optimize their combined growth in a milk environment and that protocooperation thus includes both biochemical and genetic aspects.     

Synthesis of γ-Aminobutyric Acid by Lactic Acid Bacteria Isolated from a Variety of Italian Cheeses

The concentrations of γ-aminobutyric acid (GABA) in 22 Italian cheese varieties that differ in several technological traits markedly varied from 0.26 to 391 mg kg⁻¹. Presumptive lactic acid bacteria were isolated from each cheese variety (total of 440 isolates) and screened for the capacity to synthesize GABA. Only 61 isolates showed this activity and were identified by partial sequencing of the 16S rRNA gene. Twelve species were found. Lactobacillus paracasei PF6, Lactobacillus delbrueckii subsp. bulgaricus PR1, Lactococcus lactis PU1, Lactobacillus plantarum C48, and Lactobacillus brevis PM17 were the best GABA-producing strains during fermentation of reconstituted skimmed milk. Except for L. plantarum C48, all these strains were isolated from cheeses with the highest concentrations of GABA. A core fragment of glutamate decarboxylase (GAD) DNA was isolated from L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48 by using primers based on two highly conserved regions of GAD. A PCR product of ca. 540 bp was found for all the strains. The amino acid sequences deduced from nucleotide sequence analysis showed 98, 99, 90, and 85% identity to GadB of L. plantarum WCFS1 for L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48, respectively. Except for L. lactis PU1, the three lactobacillus strains survived and synthesized GABA under simulated gastrointestinal conditions. The findings of this study provide a potential basis for exploiting selected cheese-related lactobacilli to develop health-promoting dairy products enriched in GABA.     

Hydrolysis of Sequenced β-Casein Peptides Provides New Insight into Peptidase Activity from Thermophilic Lactic Acid Bacteria and Highlights Intrinsic Resistance of Phosphopeptides

The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening. To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified β-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the β-casein hydrolysate, as shown by tandem mass spectrometry. Cell extracts prepared from Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus delbrueckii subsp. lactis ITG LL14 and ITG LL51, L. delbrueckii subsp. bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized in protein. The peptidase activities were assessed with the β-casein hydrolysate as the substrate at pH 5.5 and 24°C (conditions of warm room ripening) by (i) free amino acid release, (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry. Regardless of strain, L. helveticus was the most efficient in hydrolyzing β-casein peptides. Interestingly, cell extracts of S. thermophilus were not able to release a significant level of free proline from the β-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline. With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species.     

A new methodology for rapid detection of Lactobacillus delbrueckii subsp. bulgaricus based on multiplex PCR

In this study we present a novel multiplex PCR assay for rapid and efficient detection of Lactobacillus delbrueckii subsp. bulgaricus. The accuracy of our method was confirmed by the successful identification of L. delbrueckii subsp. bulgaricus in commercial yoghurts and food supplements and it may be readily applied to the food industry.     

Ability of Thermophilic Lactic Acid Bacteria To Produce Aroma Compounds from Amino Acids

Although a large number of key odorants of Swiss-type cheese result from amino acid catabolism, the amino acid catabolic pathways in the bacteria present in these cheeses are not well known. In this study, we compared the in vitro abilities of Lactobacillus delbrueckii subsp. lactis, Lactobacillus helveticus, and Streptococcus thermophilus to produce aroma compounds from three amino acids, leucine, phenylalanine, and methionine, under mid-pH conditions of cheese ripening (pH 5.5), and we investigated the catabolic pathways used by these bacteria. In the three lactic acid bacterial species, amino acid catabolism was initiated by a transamination step, which requires the presence of an α-keto acid such as α-ketoglutarate (α-KG) as the amino group acceptor, and produced α-keto acids. Only S. thermophilus exhibited glutamate dehydrogenase activity, which produces α-KG from glutamate, and consequently only S. thermophilus was capable of catabolizing amino acids in the reaction medium without α-KG addition. In the presence of α-KG, lactobacilli produced much more varied aroma compounds such as acids, aldehydes, and alcohols than S. thermophilus, which mainly produced α-keto acids and a small amount of hydroxy acids and acids. L. helveticus mainly produced acids from phenylalanine and leucine, while L. delbrueckii subsp. lactis produced larger amounts of alcohols and/or aldehydes. Formation of aldehydes, alcohols, and acids from α-keto acids by L. delbrueckii subsp. lactis mainly results from the action of an α-keto acid decarboxylase, which produces aldehydes that are then oxidized or reduced to acids or alcohols. In contrast, the enzyme involved in the α-keto acid conversion to acids in L. helveticus and S. thermophilus is an α-keto acid dehydrogenase that produces acyl coenzymes A.     

Pediocin production in milk by Pediococcus acidilactici in co-culture with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus

The production of pediocin in milk by Pediococcus acidilactici was evaluated in co-culture with the dairy fermentation cultures Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. The cultures were tested singly and in different combinations in milk (0 or 2% fat content) during incubation at 40°C for up to 10 h. Cell-free milk samples taken every 60 min were tested for bacteriocin activity against Listeria monocytogenes. Pediocin activity was not detectable when P. acidilactici was inoculated into milk as a monoculture. When P. acidilactici was grown in combination with the yogurt starter cultures S. thermophilus and Lb. delbrueckii ssp. bulgaricus, pediocin concentration reached 3,200–6,400 units ml⁻¹ after 8 h of incubation. The results showed that pediocin producing pediococci may be useful adjunct components in mixed cultures of S. thermophilus and Lb. delbrueckii ssp. bulgaricus to amplify the bioprotective properties of fermented dairy foods against Listeria contamination.     

Plasmid-encoded ropiness production inLactobacillus casei SSP.casei

Summary Genetic determinants of the Muc⁺ character were investigated in two ropy strains,Lactobacillus delbrueckii ssp.bulgaricus 201 andL. casei ssp.casei NCIB 4114, which secrete a large amount of slime in culture media. Plasmid DNA analysis revealed the presence of two plasmids (4.5 and 2.3 Mdal) inL. casei ssp.casei, whileL. delbrueckii ssp.bulgaricus was plasmid free, suggesting a chromosomal location of Muc⁺ character in this strain. Curing experiments carried out onL. casei ssp.casei NCIB 4114 indicated a correlation between the Muc⁺ phenotype and the 4.5 Mdal plasmid.     

Growth and Proteolytic Activity of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus in Reduced Sodium Kashkaval Cheese

Summary Reduced sodium Kashkaval cheese was produced from cow’s milk. Mixtures of NaCl, NaCl:KCl (1:1, 2:1) and NaCl:KH₂PO₄ (1:1, 2:1) were used for hot brining and salting of the cheddarized cheese curd. There were no significant differences (P < 0.05) in the count of Lb. delbrueckii ssp. bulgaricus after aging of Kashkaval samples. At the end of the ripening process the counts of Lb. delbrueckii ssp. bulgaricus reached 10⁶ c.f.u./g and the counts of Streptococcus thermophilus varied from 10⁴ to 10⁵ c.f.u./g. Proteolysis during ripening of reduced sodium Kashkaval cheese, initiated by the starter microorganisms Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, was studied through the changes in the levels of non-casein and non-protein nitrogen. It was observed that non-casein and non-protein nitrogen increased significantly (P < 0.05) during ripening. The amounts of non-casein and non-protein nitrogen accumulated in the studied Kashkaval samples were similar. That indicates that the partial replacement of NaCl with KCl or KH₂PO₄ does not cause significant changes in the course of proteolysis of Kashkaval cheese by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus.     

Comparison of growth,acidification and productivity of pure and mixed cultures of Streptococcus salivarius subsp. thermophilus 404 and Lactobacillus delbrueckii subsp. bulgaricus 398

Pure and mixed controlled-pH batch cultures of Streptococcus salivarius subsp. thermophilus 404 and Lactobacillus delbrueckii subsp. bulgaricus 398 have been conducted. The characteristics of growth and acidification and the productivity of the cultures were compared. During the mixed cultures, the growth characteristics revealed a pronounced stimulation of S. thermophilus whereas L. bulgaricus metabolism was not significantly improved. The final total population was 1.4 to 4.9 higher than in pure cultures. The acidification characteristics were not enhanced by the mixed culture conditions. The productivity of mixed cultures was 1.7 to 2.4 times higher as compared to an equivalent mixing of pure cultures.Correspondence to: C. Béal     

thyA as a Selection Marker in Construction of Food-Grade Host-Vector and Integration Systems for Streptococcus thermophilus

We constructed food-grade host-vector and integration systems for Streptococcus thermophilus by using a thymidylate synthase gene (thyA) as the selection marker. Two thyA genes, thyA_St and thyA_Lb, were cloned from S. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, respectively. Thymidine-requiring mutants of S. thermophilus were obtained after successive cultures in the presence of trimethoprim, and one of them, TM1-1, was used as the host. Food-grade vectors were constructed by using either thyA_St or thyA_Lb as the selection marker. Transformants of TM1-1 created by using these vectors were selected for thymidine autotrophy as efficiently as for erythromycin resistance. By using the host-vector system developed in this way, a foreign amylase gene (amyA) was expressed in TM1-1 and was also integrated into the chromosome by use of a temperature-sensitive integration vector constructed with thyA_Lb as the selection marker via a double-crossover event. The results obtained show that thyA is an efficient and safe selection marker for S. thermophilus that is suitable for food applications.     

Assimilation of cholesterol by some cultures of lactic acid bacteria and bifidobacteria

Summary Cultures ofStr. thermophilus assimilated less cholesterol than those ofLactobacillus delbrueckii subsp.bulgaricus. A significant difference was found between strains ofL. delbrueckii subsp.bulgaricus — LB1 and LB2 and LB3 (p<0.001).Bif. bifidum actively assimilated cholesterol, but no significant difference was observed between their two strains (p>0.05). Cultures ofL. asidophilus assimilated significantly more cholesterol than those ofStr. thermophilus and a commercial yoghurt culture.     

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 β-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 β-casein, while degradation of α_s-caseins was strain dependent. Contrariwise, κ-Casein was poorly degraded by the studied lactobacilli. β-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.