Fabio Minervini

Sourdough Bread Made from Wheat and Nontoxic Flours and Started with Selected Lactobacilli Is Tolerated in Celiac Sprue Patients

ABSTRACT 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 peptic-tryptic 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 bakers yeast. Two types of bread, containing ca. 2 g of gluten, were produced with bakers 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 bakers 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 bakers 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.

Angiotensin I-converting-enzyme-inhibitory and antibacterial peptides from Lactobacillus helveticus PR4 proteinase-hydrolyzed caseins of milk from six species.

ABSTRACT 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 αS1-casein (αS1-CN) 24-47 fragment (f24-47), f169-193, and β-CN f58-76; ovine αS1-CN f1-6 and αS2-CN f182-185 and f186-188; caprine β-CN f58-65 and αS2-CN f182-187; buffalo β-CN f58-66; and a mixture of three tripeptides originating from human β-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 μg/ml (100 μmol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC50) of some of the crude peptide fractions was very low (16 to 100 μg/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC50s were confirmed. An antibacterial peptide corresponding to β-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 μg/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin.

Glucan and fructan production by sourdough Weissella cibaria and Lactobacillus plantarum.

After a large screening on sourdough lactic acid bacteria, exopolysaccharide (EPS)-forming strains of Weissella cibaria, Lactobacillus plantarum, and Pediococcus pentosaceus were selected. After 6 days of incubation at 30 degrees C, the synthesis of EPS in MRS-based broth ranged from 5.54 to 7.88 mg mL-1. EPS had an apparent molecular mass of ca. 104 Da. As shown by carbohydrate consumption, the synthesis of EPS was found from sucrose only. Two types of homopolysaccharides were synthesized: glucans simultaneously with growth and fructans after 1 day of incubation. Two protein bands of ca. 180-200 kDa were in situ detected on SDS-PAGE gels incubated with sucrose. PCR products of ca. 220 bp were found for L. plantarum PL9 (100% of identity to putative priming glycosyltransferase of L. plantarum WCFS1) and W. cibaria WC4 (80% of identity to putative glycosyltransferase, epsD, of Bacillus cereus G9241) by using hybrid primers for the priming gtf genes. Degenerated primers DexreuR and DexreuV showed a unique PCR product, and the predicted amino acid sequences were identical for W. cibaria WC4 and L. plantarum PL9. The sequence had similarity with polysaccharide biosynthesis glycosyltransferases. W. cibaria WC4 or L. plantarum LP9 synthesized ca. 2.5 g kg-1 EPS during sourdough fermentation with sucrose added. Compared to the sourdough started with an EPS-negative strain, the sourdough started with W. cibaria WC4 or L. plantarum LP9 increased the viscosity, and the resulting bread had higher specific volume and lower firmness. The synthesis of EPS by selected sourdough lactic acid bacteria could be considered as a useful tool to replace the additives for improving the textural properties of baked goods.

Selection and use of autochthonous mixed starter for lactic acid fermentation of carrots, French beans or marrows.

Strains of Leuconostoc mesenteroides, Lactobacillus plantarum, Weissella soli/Weissella koreensis, Enterococcus faecalis, Pediococcus pentosaceus and Lactobacillus fermentum were identified from raw carrots, French beans and marrows by partial 16S rRNA gene sequence. L. plantarum M1, Leuc. mesenteroides C1 and P. pentosaceus F4 were selected based on the rates of growth and acidification in vegetable juice media, and used as the autochthonous mixed starter for the fermentation of carrots, French beans or marrows. An allochthonous starter, consisting of the same species, was also used for fermentation. A two-step fermentation process (1 day at 25 degrees C and 7 days at 15 degrees C) in brine (1% w/v) followed by storage at room temperature in olive oil until 40 days was set up. Unstarted vegetables subjected to the same treatments were used as the controls. Cell numbers of lactic acid bacteria in the started vegetables were ca. 10,000 (autochthonous starter) and 1000 (allochthonous starter) times higher than unstarted samples throughout the process. When fermented with the autochthonous starter, carrots, French beans or marrows were characterized by the rapid decrease of pH (<4.5), marked consumption of fermentable carbohydrates, and inhibition of Enterobacteriaceae and yeasts. Fermentation with the allochthonous starter did not acidify and inhibit bacteria and yeasts so rapidly. After 40 days, carrots, French beans and marrows fermented with the autochthonous starter had significantly (P<0.05) higher total concentration of vitamin C (ascorbate+dehydroascorbate) with respect to those fermented with the allochthonous starter and, especially unstarted vegetables. The same was found for the indexes of color. Firmness of both started vegetables was higher than unstarted vegetables. Sensory analysis differentiated started vegetables. Carrots and French beans fermented with the autochthonous starter were, especially, appreciated for fragrance. Appearance was the sensory attribute that mainly distinguished marrows fermented with the autochthonous starter.

Taxonomic structure and monitoring of the dominant population of lactic acid bacteria during wheat flour sourdough type I propagation using Lactobacillus sanfranciscensis starters.

ABSTRACT The structure and stability of the dominant lactic acid bacterium population were assessed during wheat flour sourdough type I propagation by using singly nine strains of Lactobacillus sanfranciscensis. Under back-slopping propagation with wheat flour type 0 F114, cell numbers of presumptive lactic acid bacteria varied slightly between and within starters. As determined by randomly amplified polymorphic DNA-PCR and restriction endonuclease analysis-pulsed-field gel electrophoresis analyses, only three (LS8, LS14, and LS44) starters dominated throughout 10 days of propagation. The others progressively decreased to less than 3 log CFU g−1. Partial sequence analysis of the 16S rRNA and recA genes and PCR-denaturating gradient gel electrophoresis analysis using the rpoB gene allowed identification of Weissella confusa, Lactobacillus sanfranciscensis, Lactobacillus plantarum, Lactobacillus rossiae, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Pediococcus pentosaceus, and Lactobacillus spp. as the dominant species of the raw wheat flour. At the end of propagation, one autochthonous strain of L. sanfranciscensis was found in all the sourdoughs. Except for L. brevis, strains of the above species were variously found in the mature sourdoughs. Persistent starters were found in association with other biotypes of L. sanfranciscensis and with W. confusa or L. plantarum. Sourdoughs were characterized for acidification, quotient of fermentation, free amino acids, and community-level catabolic profiles by USING Biolog 96-well Eco microplates. In particular, catabolic profiles of sourdoughs containing persistent starters behaved similarly and were clearly differentiated from the others. The three persistent starters were further used for the production of sourdoughs and propagated by using another wheat flour whose lactic acid bacterium population in part differed from the previous one. Also, in this case all three starter strains persisted during propagation.

Lactic Acid Bacterium and Yeast Microbiotas of 19 Sourdoughs Used for Traditional/Typical Italian Breads: Interactions between Ingredients and Microbial Species Diversity

ABSTRACT 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.

Robustness of Lactobacillus plantarum starters during daily propagation of wheat flour sourdough type I.

This study aimed at investigating the robustness of selected sourdough strains of Lactobacillus plantarum. Seven strains were singly used as sourdough type I starters under daily back-slopping propagation (ten days) using wheat flour. Cell numbers of presumptive lactic acid bacteria varied slightly (median values of 9.13-9.46 log cfu g(-1)) between and within started sourdoughs, as well as the acidifying activity (median values of 1.24-1.33). After three days also the control sourdough (unstarted) had the same values. As shown by RAPD-PCR analysis, five (DB200, 3DM, G10C3, 12H1 and LP20) out of seven strains maintained elevated cell numbers (ca. 9 log cfu g(-1)) throughout ten days. The other two strains progressively decreased to less than 5 log cfu g(-1). As identified by partial sequencing of 16S rRNA and recA genes, L. plantarum (11 isolates), pediococci (7), Lactobacillus casei (3) and Lactobacillus rossiae (2) dominated the flour microbiota. Monitoring of lactic acid bacteria during sourdough propagation was carried out by culture dependent approach and using PCR-DGGE (Denaturing Gradient Gel Electrophoresis). Except for the sourdough started with L. plantarum LP20, in all other sourdoughs at least one autochthonous strain of L. plantarum emerged. All emerging strains of L. plantarum showed different RAPD-PCR profiles. L. rossiae and Pediococcus pentosaceus were only found in the control and sourdough started with strain 12H1. The characterization of the catabolic profiles of sourdoughs (Biolog System) showed that sourdoughs containing persistent starters behaved similarly and their profiles were clearly differentiated from the others. One persistent strain (DB200) of L. plantarum and Lactobacillus sanfranciscensis LS44, previously shown to be persistent (Siragusa et al., 2009), were used as the mixed starter to produce a wheat flour sourdough. Both strains cohabited and dominated during ten days of propagation.

Microbial Ecology Dynamics during Rye and Wheat Sourdough Preparation

ABSTRACT 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.

Ecological parameters influencing microbial diversity and stability of traditional sourdough.

The quality of some leavened, sourdough baked goods is not always consistent, unless a well propagated sourdough starter culture is used for the dough fermentation. Among the different types of sourdough used, the traditional sourdough has attracted the interest of researchers, mainly because of its large microbial diversity, especially with respect to lactic acid bacteria. Variation in this diversity and the factors that cause it will impact on quality and is the subject of this review. Sourdough microbial diversity is mainly caused by the following factors: (i) sourdough is obtained through spontaneous, multi-step fermentation; (ii) it is propagated using flour, whose nutrient content may vary according to the batch and to the crop, and which is naturally contaminated by microorganisms; and (iii) it is propagated under peculiar technological parameters, which vary depending on the historical and cultural background and type of baked good. In the population dynamics leading from flour to mature sourdough, lactic acid bacteria (several species of Lactobacillus sp., Leuconostoc sp., and Weissella sp.) and yeasts (mainly Saccharomyces cerevisiae and Candida sp.) outcompete other microbial groups contaminating flour, and interact with each other at different levels. Ecological parameters qualitatively and quantitatively affecting the dominant sourdough microbiota may be classified into specific technological parameters (e.g., percentage of sourdough used as inoculum, time and temperature of fermentation) and parameters that are not fully controlled by those who manage the propagation of sourdough (e.g., chemical, enzyme and microbial composition of flour). Although some sourdoughs have been reported to harbour a persistent dominant microbiota, the stability of sourdough ecosystem during time is debated. Indeed, several factors may interfere with the persistence of species and strains associations that are typical of a given sourdough: metabolic adaptability to the stressing conditions of sourdough, nutritional and antagonistic interactions among microorganisms, intrinsic robustness of microorganisms, and existence of a stable house microbiota. Further studies have to be performed in order to highlight hidden mechanisms underlying the microbial structure and stability of sourdough. The comprehension of such mechanisms would be helpful to assess the most appropriate conditions that allow keeping a given traditional sourdough as a stable microbial ecosystem, thus preserving, during time, the typical traits of the resulting product.

Mechanism of degradation of immunogenic gluten epitopes from Triticum turgidum L. var. durum by sourdough lactobacilli and fungal proteases.

ABSTRACT As shown by R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay (ELISA), selected sourdough lactobacilli, in combination with fungal proteases, hydrolyzed gluten (72 h at 37°C) of various cultivars of Triticum turgidum L. var. durum to less than 20 ppm. Complementary electrophoretic, chromatography, and mass spectrometry techniques were used to characterize the gluten and epitope hydrolysis. Nine peptidases were partially purified from the pooled cytoplasmic extract of the sourdough lactobacilli and used to hydrolyze the 33-mer epitope, the most immunogenic peptide generated during digestion of Triticum species. At least three peptidases (general aminopeptidase type N [PepN], X-prolyl dipeptidyl aminopeptidase [PepX], and endopeptidase PepO) were necessary to detoxify the 33-mer without generation of related immunogenic epitopes. After 14 h of incubation, the combination of all or at least six different peptidases totally hydrolyzed the 33-mer (200 mM) into free amino acids. The same results were found for other immunogenic epitopes, such as fragments 57-68 of α9-gliadin, 62-75 of A-gliadin, and 134-153 of γ-gliadin. When peptidases were used for fermentation of durum wheat semolina, they caused the hydrolysis of gluten to ca. 2 ppm. The in vivo digestion was simulated, and proteins/peptides extracted from pepsin-trypsin (PT) digestion of durum wheat semolina fermented with selected sourdough lactobacilli induced the expression of gamma interferon and interleukin 2 at levels comparable to those of the negative control. Durum wheat semolina fermented with sourdough lactobacilli was freeze-dried and used for making Italian-type pasta. The scores for cooking and sensory properties for this pasta were higher that those of conventional gluten-free pasta.