Angela Cassone

Synthesis of Angiotensin I-Converting Enzyme (ACE)-Inhibitory Peptides and γ-Aminobutyric Acid (GABA) during Sourdough Fermentation by Selected Lactic Acid Bacteria

This article aimed at investigating the synthesis of angiotensin I-converting enzyme (ACE)-inhibitory peptides and gamma-aminobutyric acid (GABA) during sourdough fermentation of white wheat, wholemeal wheat, and rye flours. Sourdough lactic acid bacteria, selected previously for proteinase and peptidase activities toward wheat proteins or for the capacity of synthesizing GABA, were used. The highest ACE-inhibitory activity was found by fermenting flour under semiliquid conditions (dough yield 330) and, especially, by using wholemeal wheat flour. Fourteen peptides, not previously reported as ACE-inhibitory, were identified from the water/salt-soluble extract of wholemeal wheat sourdough (IC 50 0.19-0.54 mg/mL). The major part of the identified peptides contained the well-known antihypertensive epitope VAP. The synthesis of GABA increased when the dough yield was decreased to 160. The highest synthesis of GABA (258.71 mg/kg) was found in wholemeal wheat sourdough.

Antifungal Activity of Wickerhamomyces anomalus and Lactobacillus plantarum during Sourdough Fermentation: Identification of Novel Compounds and Long-Term Effect during Storage of Wheat Bread

ABSTRACT This study aimed at investigating the antifungal activity of Wickerhamomyces anomalus and sourdough lactic acid bacteria to extend the shelf life of wheat flour bread. The antifungal activity was assayed by agar diffusion, growth rate inhibition, and conidial germination assays, using Penicillium roqueforti DPPMAF1 as the indicator fungus. Sourdough fermented by Lactobacillus plantarum 1A7 (S1A7) and dough fermented by W. anomalus LCF1695 (D1695) were selected and characterized. The water/salt-soluble extract of S1A7 was partially purified, and several novel antifungal peptides, encrypted into sequences of Oryza sativa proteins, were identified. The water/salt-soluble extract of D1695 contained ethanol and, especially, ethyl acetate as inhibitory compounds. As shown by growth inhibition assays, both water/salt-soluble extracts had a large inhibitory spectrum, with some differences, toward the most common fungi isolated from bakeries. Bread making at a pilot plant was carried out with S1A7, D1695, or a sourdough started with a combination of both strains (S1A7-1695). Slices of the bread manufactured with S1A7-1695 did not show contamination by fungi until 28 days of storage in polyethylene bags at room temperature, a level of protection comparable to that afforded by 0.3% (wt/wt) calcium propionate. The effect of sourdough fermentation with W. anomalus LCF1695 was also assessed based on rheology and sensory properties.

Antifungal activity of sourdough fermented wheat germ used as an ingredient for bread making

This study aimed at investigating the antifungal activity of sourdough fermented (Lactobacillus plantarum LB1 and Lactobacillus rossiae LB5) wheat germ (SFWG). Preliminarily, methanol and water/salt-soluble extracts from SFWG were assayed by agar diffusion towards Penicillium roqueforti DPPMAF1. As shown by hyphal radial growth rate, the water/salt-soluble extract showed the inhibition of various fungi isolated from bakeries. The antifungal activity was attributed to a mixture of organic acids and peptides which were synthesized during fermentation. Formic (24.7mM) acid showed the highest antifungal activity. Four peptides, having similarities with well known antifungal sequences, were identified and chemically synthesized. The minimal inhibitory concentration was 2.5-15.2mg/ml. Slices of bread made by addition of 4% (wt/wt) of freeze dried SFWG were packed in polyethylene bags and stored at room temperature. Slices did not show contamination by fungi until at least 28days of storage and behaved as the calcium propionate (0.3%, wt/wt).

Safety for Patients With Celiac Disease of Baked Goods Made of Wheat Flour Hydrolyzed During Food Processing

BACKGROUND & AIMS Celiac disease (CD) is characterized by an inflammatory response to wheat gluten, rye, and barley proteins. Fermentation of wheat flour with sourdough lactobacilli and fungal proteases decreases the concentration of gluten. We evaluated the safety of daily administration of baked goods made from this hydrolyzed form of wheat flour to patients with CD. METHODS Patients were randomly assigned to consumption of 200 g per day of natural flour baked goods (NFBG) (80,127 ppm gluten; n = 6), extensively hydrolyzed flour baked goods (S1BG) (2480 ppm residual gluten; n = 2), or fully hydrolyzed baked goods (S2BG) (8 ppm residual gluten; n = 5) for 60 days. RESULTS Two of the 6 patients who consumed NFBG discontinued the challenge because of symptoms; all had increased levels of anti-tissue transglutaminase (tTG) antibodies and small bowel deterioration. The 2 patients who ate the S1BG goods had no clinical complaints but developed subtotal atrophy. The 5 patients who ate the S2BG had no clinical complaints; their levels of anti-tTG antibodies did not increase, and their Marsh grades of small intestinal mucosa did not change. CONCLUSIONS A 60-day diet of baked goods made from hydrolyzed wheat flour, manufactured with sourdough lactobacilli and fungal proteases, was not toxic to patients with CD. A combined analysis of serologic, morphometric, and immunohistochemical parameters is the most accurate method to assess new therapies for this disorder.

Use of selected sourdough strains of Lactobacillus for removing gluten and enhancing the nutritional properties of gluten-free bread.

Forty-six strains of sourdough lactic acid bacteria were screened for proteolytic activity and acidification rate in gluten-free (GF) flours. The sourdough cultures consisted of Lactobacillus sanfranciscensis LS40 and LS41 and Lactobacillus plantarum CF1 and were selected and used for the manufacture of GF bread. Fermentation occurred in two steps: (i) long-time fermentation (16 h) and (ii) fast fermentation (1.5 h) using the previous fermented sourdough as inoculum (ca. 43%, wt/wt) with Saccharomyces cerevisiae (bakers yeast). GF bread started with bakers yeast alone was used as the control. Gluten was added to ingredients before fermentation to simulate contamination. Initial gluten concentration of 400 ppm was degraded to below 20 ppm only in the sourdough GF bread. Before baking, sourdough GF bread showed phytase activity ca. sixfold higher than that of GF bread started with bakers yeast alone. Atomic absorption spectrophotometric analysis revealed that the higher phytase activity resulted in an increased availability of free Ca2+, Zn2+, and Mg2+. The concentration of free amino acids also was the highest in sourdough GF bread. Sourdough GF bread had a higher specific volume and was less firm than GF bread started with bakers yeast alone. This study highlighted the use of selected sourdough cultures to eliminate risks of contamination by gluten and to enhance the nutritional properties of GF bread.

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.

Proteomic Analysis by Two-Dimensional Gel Electrophoresis and Starch Characterization of Triticum turgidum L. var. durum Cultivars for Pasta Making

Criteria for durum wheat quality are continuously evolving in response to market pressure and consumers preference. Specific attributes of durum wheat for different end products require more rapid and objective means to grade and classify wheat parcels based on processing potential. A total of 10 durum wheat cultivars were compared for compositional, protein, and starch characteristics. Mean values for the gross composition differed for total protein, gluten, and starch. Two-dimensional electrophoresis (2DE) analysis showed the proteome diversity among the cultivars. As shown by the principal component analysis (PCA) applied to 2DE data of gliadin and glutenin fractions, cultivars differed mainly from the number of proteins and levels of protein expression. As determined by the rapid viscoanalyzer (RVA), swelling power, starch damage, amylose content, and starch pasting properties of 10 cultivars differed significantly. 2DE fingerprinting and amylose content seemed to distinguish specific cultivars being useful tools for selecting suitable durum wheat cultivars for pasta making.

Use of selected enterococci and Rhizopus oryzae proteases to hydrolyse wheat proteins responsible for celiac disease

Aims:  This work aimed at using a pool of selected enterococci and fungal proteases to hydrolyse wheat gluten during long‐time fermentation.

Comparison of HPLC-RI, LC/MS-MS and enzymatic assays for the analysis of residual lactose in lactose-free milk

Lactose intolerance is the decreased ability to digest lactose, and the population involved is rapidly increasing all over the world. Different procedures have been reported in the literature to quantify lactose in dairy products, but the official method of analysis is based on enzymatic assay. In this paper, the effectiveness of two enzymatic kits in detecting residual lactose in lactose-free milk was investigated, and a comparison with two alternative chromatographic methods was done. The investigation used several samples of UHT milk containing different levels of lactose, and the results highlighted the inadequacy of the enzymatic assays and of the HPLC-RI method to analyse lactose-free milk. An LC-MS/MS method using the formate adduct was developed, and it allowed quantitation of lactose and lactulose in all samples at a high level of precision and repeatability.

Short communication: Chemical and sensory characteristics of Canestrato di Moliterno cheese manufactured in spring

Canestrato di Moliterno is an Italian Protected Geographical Indication hard cheese, made in winter and spring from a mixture of ewe and goat milks, that has been poorly investigated. The present study was aimed at characterizing the cheese made in the warm season. Two series of samples, ripened in traditional rooms called fondaco as indicated in the official protocol of production, were taken from the main certified producers. The cheeses were analyzed for gross composition; proteolysis and lipolysis; volatile fraction; and organoleptic features. Gross composition was not completely homogeneous among the samples, but primary proteolysis and lipolysis were quite uniform. We observed variations in secondary proteolysis, likely caused by fluctuations in environmental conditions in the fondaco. The sensory profiles of the samples were homogeneous: the cheese was soluble, greasy, and adhesive, with a sheepfold and buttery odor. The main taste attributes were fermented, pungent, and bitter. Overall, the results of this study provide an initial contribution to the characterization of Canestrato di Moliterno, and could be used to improve marketing strategies.