Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine ␣ S1 -casein (␣ S1 -CN) 24-47 fragment (f24-47), f169-193, and ␤-CN f58-76; ovine ␣ S1 -CN f1-6 and ␣ S2 -CN f182-185 and f186-188; caprine ␤-CN f58-65 and ␣ S2 -CN f182-187; buffalo ␤-CN f58-66; and a mixture of three tripeptides originating from human ␤-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 g/ml (100 mol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC 50 ) of some of the crude peptide fractions was very low (16 to 100 g/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC 50 s were confirmed. An antibacterial peptide corresponding to ␤-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 g/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin.

The study of the microbiotas of 19 Italian sourdoughs used for the manufacture of traditional/typical breads allowed the identification, through a culture-dependent approach, of 20 and 4 species of lactic acid bacteria (LAB) and yeasts, respectively. Numerically, the most frequent LAB isolates were Lactobacillus sanfranciscensis (ca. 28% of the total LAB isolates), Lactobacillus plantarum (ca. 16%), and Lactobacillus paralimentarius (ca. 14%). Saccharomyces cerevisiae was identified in 16 sourdoughs. Candida humilis, Kazachstania barnettii, and Kazachstania exigua were also identified. As shown by principal component analysis (PCA), a correlation was found between the ingredients, especially the type of flour, the microbial community, and the biochemical features of sourdoughs. Triticum durum flours were characterized by the high level of maltose, glucose, fructose, and free amino acids (FAA) correlated with the sole or main presence of obligately heterofermentative LAB, the lowest number of facultatively heterofermentative strains, and the low cell density of yeasts in the mature sourdoughs. This study highlighted, through a comprehensive and comparative approach, the dominant microbiotas of 19 Italian sourdoughs, which determined some of the peculiarities of the resulting traditional/typical Italian breads. During the last decades, European and worldwide consumers have come ever more to appreciate traditional and typical foods. Traditional is the definition used for foods that historically are part of the cultural heritage of people living in a certain geographical area (29a). Typical is the attribute used for a food produced using one or more ingredients having characteristics strictly depending on the geographical area it comes from (9). Mainly due to the long history of regional political division, about 200 different types of bread are manufactured throughout Italy with large differences of recipes and traditions (24). Some breads have already received the Protected Designation of Origin (PDO) (Pane di Altamura and Pagnotta del Dittaino) or the Protected Geographical Indication (PGI) (Pane di Matera, Pane Casareccio di Genzano, and Coppia Ferrarese). In spite of the differences, almost all traditional/typical Italian breads use sourdough as the natural starter. Sourdough represents a very complex biological ecosystem (19) where yeasts and, especially, lactic acid bacteria (LAB) largely determine the sensory, technology, nutritional, and functional features of the resulting baked goods (20). In mature sourdoughs, LAB occur in numbers Ͼ10 8 CFU g Ϫ1 , whereas the number of yeasts is at least one order of magnitude lower (19). The microbial composition of the sourdough was subjected to numerous studies which have revealed a large species diversity (for reviews, see references 15, 16, 22, and 43). Overall, Lactobacillus brevis, Lactobacillus paralimentarius, Lactobacillus plantarum, Lactobacillus rossiae, and Lactobacillus sanfranciscensis dominate sourdough processes that are characterized by low incubation t...

The bacterial ecology during rye and wheat sourdough preparation was described by 16S rRNA gene pyrosequencing. Viable plate counts of presumptive lactic acid bacteria, the ratio between lactic acid bacteria and yeasts, the rate of acidification, a permutation analysis based on biochemical and microbial features, the number of operational taxonomic units (OTUs), and diversity indices all together demonstrated the maturity of the sourdoughs during 5 to 7 days of propagation. Flours were mainly contaminated by metabolically active genera (Acinetobacter, Pantoea, Pseudomonas, Comamonas, Enterobacter, Erwinia, and Sphingomonas) belonging to the phylum Proteobacteria or Bacteroidetes (genus Chryseobacterium). Their relative abundances varied with the flour. Soon after 1 day of propagation, this population was almost completely inhibited except for the Enterobacteriaceae. Although members of the phylum Firmicutes were present at very low or intermediate relative abundances in the flours, they became dominant soon after 1 day of propagation. Lactic acid bacteria were almost exclusively representative of the Firmicutes by this time. Weissella spp. were already dominant in rye flour and stably persisted, though they were later flanked by the Lactobacillus sakei group. There was a succession of species during 10 days of propagation of wheat sourdoughs. The fluctuation between dominating and subdominating populations of L. sakei group, Leuconostoc spp., Weissella spp., and Lactococcus lactis was demonstrated. Other subdominant species such as Lactobacillus plantarum were detectable throughout propagation. As shown by PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis, Saccharomyces cerevisiae dominated throughout the sourdough propagation. Notwithstanding variations due to environmental and technology determinants, the results of this study represent a clear example of how the microbial ecology evolves during sourdough preparation.

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This work was aimed at producing a sourdough bread that is tolerated by celiac sprue (CS) patients. Selected sourdough lactobacilli had specialized peptidases capable of hydrolyzing Pro-rich peptides, including the 33-mer peptide, the most potent inducer of gut-derived human T-cell lines in CS patients. This epitope, the most important in CS, was hydrolyzed completely after treatment with cells and their cytoplasmic extracts (CE). A sourdough made from a mixture of wheat (30%) and nontoxic oat, millet, and buckwheat flours was started with lactobacilli. After 24 h of fermentation, wheat gliadins and low-molecular-mass, alcohol-soluble polypeptides were hydrolyzed almost totally. Proteins were extracted from sourdough and used to produce a peptictryptic digest for in vitro agglutination tests on K 562(S) subclone cells of human origin. The minimal agglutinating activity was ca. 250 times higher than that of doughs chemically acidified or started with baker's yeast. Two types of bread, containing ca. 2 g of gluten, were produced with baker's yeast or lactobacilli and CE and used for an in vivo double-blind acute challenge of CS patients. Thirteen of the 17 patients showed a marked alteration of intestinal permeability after ingestion of baker's yeast bread. When fed the sourdough bread, the same 13 patients had values for excreted rhamnose and lactulose that did not differ significantly from the baseline values. The other 4 of the 17 CS patients did not respond to gluten after ingesting the baker's yeast or sourdough bread. These results showed that a bread biotechnology that uses selected lactobacilli, nontoxic flours, and a long fermentation time is a novel tool for decreasing the level of gluten intolerance in humans.

Bioactivities of peptides encrypted in major milk proteins are latent until they are released and activated by enzymatic proteolysis. After a brief description of the properties of some bioactive peptides, an overview of the angiotensin I‐converting‐enzyme (ACE)‐inhibitory and antimicrobial peptides is given. Emphasis is on the capacity of lactic acid bacteria to generate ACE‐inhibitory peptides and on the exploitation of the potential role of antimicrobial peptides derived from milk proteins.

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Seven mature type I sourdoughs were comparatively back-slopped (80 days) at artisan bakery and laboratory levels under constant technology parameters. The cell density of presumptive lactic acid bacteria and related biochemical features were not affected by the environment of propagation. On the contrary, the number of yeasts markedly decreased from artisan bakery to laboratory propagation. During late laboratory propagation, denaturing gradient gel electrophoresis (DGGE) showed that the DNA band corresponding to Saccharomyces cerevisiae was no longer detectable in several sourdoughs. Twelve species of lactic acid bacteria were variously identified through a culture-dependent approach. All sourdoughs harbored a certain number of species and strains, which were dominant throughout time and, in several cases, varied depending on the environment of propagation. As shown by statistical permutation analysis, the lactic acid bacterium populations differed among sourdoughs propagated at artisan bakery and laboratory levels. Lactobacillus plantarum, Lactobacillus sakei, and Weissella cibaria dominated in only some sourdoughs back-slopped at artisan bakeries, and Leuconostoc citreum seemed to be more persistent under laboratory conditions. Strains of Lactobacillus sanfranciscensis were indifferently found in some sourdoughs. Together with the other stable species and strains, other lactic acid bacteria temporarily contaminated the sourdoughs and largely differed between artisan bakery and laboratory levels. The environment of propagation has an undoubted influence on the composition of sourdough yeast and lactic acid bacterium microbiotas. Many studies have undoubtedly demonstrated the nutritional, sensory, texture, and shelf-life advantages of using sourdough for the manufacture of baked goods (14). Nowadays, sourdough is largely used for making wheat and rye breads, crackers, pizza, various sweet baked goods, and gluten-free products (2,14,20).Based on the biotechnology protocol, sourdough fermentations are usually classified into three types (types I, II, and III) (48). Type I, or traditional, sourdoughs are characterized by continuous (daily) propagation (back-slopping) of the dough at ambient temperatures (20 to 30°C). Typical and traditional Italian breads are mainly made using sourdoughs belonging to type I (27), and, in most cases, only one back-slopping per day is carried out (26,27). A microbial consortium mainly consisting of obligately and/or facultatively heterofermentative lactobacilli and yeasts usually dominates the mature sourdough (48). For sourdoughs made with various cereals, the stable population of lactic acid bacteria seemed to occur through a three-phase evolution within a few days of back-slopping (45). Nevertheless, the stability of the mature sourdough depends on a number of factors, which include the environmental microbiota (e.g., flour and other ingredients, house microbiota), its metabolic activity (e.g., amylase activity, cofactor regeneration capability, and energy synthesis from various ...