M. Faccia

Proteolysis by Sourdough Lactic Acid Bacteria: Effects on Wheat Flour Protein Fractions and Gliadin Peptides Involved in Human Cereal Intolerance

ABSTRACT Sourdough lactic acid bacteria were preliminarily screened for proteolytic activity by using a digest of albumin and globulin polypeptides as a substrate. Based on their hydrolysis profile patterns, Lactobacillus alimentarius 15M, Lactobacillus brevis 14G, Lactobacillus sanfranciscensis 7A, and Lactobacillus hilgardii 51B were selected and used in sourdough fermentation. A fractionated method of protein extraction and subsequent two-dimensional electrophoresis were used to estimate proteolysis in sourdoughs. Compared to a chemically acidified (pH 4.4) dough, 37 to 42 polypeptides, distributed over a wide range of pIs and molecular masses, were hydrolyzed by L. alimentarius 15M, L. brevis 14G, and L. sanfranciscensis 7A. Albumin, globulin, and gliadin fractions were hydrolyzed, while glutenins were not degraded. The concentrations of free amino acids, especially proline and glutamic and aspartic acids, also increased in sourdoughs. Compared to the chemically acidified dough, proteolysis by lactobacilli positively influenced the softening of the dough during fermentation, as determined by rheological analyses. Enzyme preparations of the selected lactobacilli which contained proteinase or peptidase enzymes showed hydrolysis of the 31-43 fragment of A-gliadin, a toxic peptide for celiac patients. A toxic peptic-tryptic (PT) digest of gliadins was used for in vitro agglutination tests on K 562 (S) subclone cells of human myelagenous leukemia origin. The lowest concentration of PT digest that agglutinated 100% of the total cells was 0.218 g/liter. Hydrolysis of the PT digest by proteolytic enzymes of L. alimentarius 15M and L. brevis 14G completely prevented agglutination of the K 562 (S) cells by the PT digest at a concentration of 0.875 g/liter. Considerable inhibitory effects by other strains and at higher concentrations of the PT digest were also found. The mixture of peptides produced by enzyme preparations of selected lactobacilli showed a decreased agglutination of K 562 (S) cells with respect to the whole 31-43 fragment of A-gliadin.

Selection and use of autochthonous multiple strain cultures for the manufacture of high-moisture traditional Mozzarella cheese.

Lactobacillus plantarum 18A, Lactobacillus helveticus 2B, Lactobacillus delbrueckii subsp. lactis 20F, Streptococcus thermophilus 22C, Enterococcus faecalis 32C and Enterococcus durans 16E were the most acidifying strains within 146 isolates for natural whey starters. The effect of media and temperature on 2 autochthonous multiple strain cultures (AMSI: 18A, 2B, 20F and 22C, 32C and 16E and AMSII: 18A, 2B, 20F and 22C) was studied. Genomic analysis showed a constant cell numbers for AMSII during 16 days of propagation in whey milk. Mozzarella cheese was made by using AMSII, commercial starter (CS) or citric acid (DA). Compared to other cheeses, the DA had a lower level of protein, ash, Ca, free amino acids and a higher level of moisture. Based on confocal laser scanning microscopy analysis, AMSII cheese showed the lowest microstructural variations during the period of storage compared to other cheeses. All the sensory attributes were scored highest for AMSII cheese. ASMII extend the shelf-life to ca. 12-15 days instead of the 5-7 days of traditional high-moisture Mozzarella cheese.

Manufacture of Fior di Latte cheese by incorporation of probiotic lactobacilli

This work aimed to select heat-resistant probiotic lactobacilli to be added to Fior di Latte (high-moisture cow milk Mozzarella) cheese. First, 18 probiotic strains belonging to Lactobacillus casei, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus reuteri were screened. Resistance to heating (65 or 55°C for 10 min) varied markedly between strains. Adaptation at 42°C for 10 min increased the heat resistance at 55°C for 10 min of all probiotic lactobacilli. Heat-adapted L. delbrueckii ssp. bulgaricus SP5 (decimal reduction time at 55°C of 227.4 min) and L. paracasei BGP1 (decimal reduction time at 55°C of 40.8 min) showed the highest survival under heat conditions that mimicked the stretching of the curd and were used for the manufacture of Fior di Latte cheese. Two technology options were chosen: chemical (addition of lactic acid to milk) or biological (Streptococcus thermophilus as starter culture) acidification with or without addition of probiotics. As determined by random amplified polymorphic DNA-PCR and 16S rRNA gene analyses, the cell density of L. delbrueckii ssp. bulgaricus SP5 and L. paracasei BGP1 in chemically or biologically acidified Fior di Latte cheese was approximately 8.0 log(10)cfu/g. Microbiological, compositional, biochemical, and sensory analyses (panel test by 30 untrained judges) showed that the use of L. delbrueckii ssp. bulgaricus SP5 and L. paracasei BGP1 enhanced flavor formation and shelf-life of Fior di Latte cheeses.

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.

Short communication: Chemical-sensory and volatile compound characterization of ricotta forte, a traditional fermented whey cheese

Ricotta forte is a traditional whey cheese, obtained through natural fermentation of fresh ricotta, that is getting increasing attention by food traders. In view of possible initiatives for its valorization, the chemical and sensory characteristics were investigated. Samples were obtained from 14 different manufacturer, and were subjected to chemical, biochemical, volatile organic compound, and sensory analyses. All samples presented low pH with high moisture (62-66%) and fat content (57-60% on dry matter). From a biochemical point of view, the electrophoretic patterns evidenced that β-lactoglobulin was the main protein present at all sample ages. Only intermediate levels of proteolysis (20.69% ripening index) took place during aging, whereas the main biochemical event in this dairy product was lipolysis (2.10 mEq/g of acid degree value). Accordingly, free fatty acids dominated the volatile organic compound profile and strongly influenced the sensory characteristics with flavor described as rancid, pungent, acrid, and smelly feet: all associated with short-chain fatty acids such as acetic, propionic, butyric, and caproic. Finally, the sample age did not influence chemical composition, whereas it had significant effect on lipolysis and flavor intensity.