Caracterização microbiológica, físico-química, proteômica e bioativa de queijos de coalho artesanal produzidos na Região Agreste do Estado de Pernambuco-Brasil

Abstract

The Artisanal “Coalho” cheese is a product typically northeastern and very popular, widely consumed by local population, representing an important socioeconomic activity for this region. The aim of this study was to obtain the microbiological, physicochemical, proteomics and bioactive characterizations of the Artisanal “Coalho” cheeses produced in the Agreste region of Pernambuco State - Brazil. The milks used as raw material in the manufacture of cheese and the cheeses were obtained from factures located in six Towns: Arcoverde, Cachoeirinha, Capoeiras, Correntes, São Bento do Una and Venturosa. The milk samples were analyzed for somatic cell counts (CCS) by flow cytometry in Somacount300®, physico-chemical parameters (total solids, total protein, total fat and lactose) by absorbance from infrared absorption equipment Bentley2000®. The cheeses were evaluated about physico-chemical parameters (proteins, lipids, moisture and lactose) according to Instruction n°68 of 12/12/2006 - MAPA, microbiological testing for the presence of total [AOAC/2002 (991.14)] and termotolerants [AOAC/2002 (986.33)] coliforms. Lactic microbiota was analyzed by polymerase chain reaction (PCR) using species-specific primers in spite of Lactobacillus genus, and the following: species Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecalis and Enterococcus faecium. The profiles of proteins of the aqueous extracts from cheeses were analyzed by SDS-PAGE electrophoresis and peptides by mass spectrometry (MALDI-ToF). Water-soluble peptides (WSP) from “Coalho” cheeses were tested for following activities: antioxidant (ABTS method); zinc-biding (AOAC method, 2000) and antimicrobial (NCCLS method, 2003). The results obtained for the physico-chemical and CCS parameters of milks used as raw material were within the limits established by Brazilian law, showing these results: proteins (2.99 – 3.48%); fats (3.24 – 4.0%); total dried extract (10.79 – 12.49%); lactose (4.26 – 4.61%) and CCS (9.2 x 104 – 5.28 x 105 cells/mL). The cheeses also had values for the physico-chemical and microbiological parameters within the standards established by law, such as: moisture (52.76 – 58.37%); proteins (17.04 – 21.59%); fats (21.25- 27%) and lactose (3.54 – 5.01%). It was observed that the “coalho” cheeses can be classified as high to very high moisture and non-fat to semi-fat cheese. All the cheese samples had total and thermotolerant coliforms according to the sanitarian standards of quality necessary for human consumers. The profile of lactic microbiota of cheeses evaluated showed heterogeneous comprising all genus (lactobacillus, lactococcus, estreptococcus e enterococcus) and e species (Enterococcus faecalis, Enterococcus faecium, Streptococcus thermophilus e Lactococcus lactis) tested. In spite of the proteic profile using aqueous extracts, it was possible to detect the presence of lactoferrin, lactoglobulin, lactoglobulin, serum albumin, casein, casein, casein and para casein and more 12 different proteins compared to the protein standard. The peptide profiles of the WSP samples showed high degree of proteolysis resulting in an average of 66 peptides containing 32 known peptides by literature obtained as product from casein hydrolysis (11 S1-Casein, 3 S2-Casein, 15 -casein and 3 -casein) and 7 caseinphosphopeptides. Among non-identified peptides, three of them showed peak at high intensity in all “Coalho” cheeses studied, with a molecular weight of 1597, 1725/1726, 2778/2779 Da, suggesting that these peptides may be a possible molecular marker for the “Coalho” cheese of Agreste Pernambuco State-Brazil. Regarding to bioactivities using WSP samples, the results showed the following activities: antioxidant (91.1 ± 0.43 -75.92 ± 0.7% inhibition); zinc-binding (75.47 ± 0.5 - 61.67 ± 0.65% of bind) and antimicrobial for bacteria Enterococcus faecalis, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. Finally, this work showed the great biotechnological potential of this cheese that lead to the high aggregation value for regional “Coalho” cheeses collaborating to socioeconomic development of the productive sector in the Pernambuco State.