Influence of Proteolytic Microorganisms on Dairy Products Quality

Transcription

1 Bulletin UASVM Agricultue, 66 (2)/2009 Print ISSN ; Electronic ISSN Influence of Proteolytic Microorganisms on Dairy Products Quality Mihaela- Ancuţa ROTAR, Cristina SEMENIUC, Sorin APOSTU, Camelia GUŞ, Dan VODNAR, Livia ŢICREA 1) Department of Food Technology, University of Agricultural Sciences and Veterinary Medicine, Faculty of Agriculture, 3-5 Mănăştur str.,400372,cluj-napoca, România, Abstract: We aimed to study the proteolytic microorgamism activity, which are present in dairy products such as yogurt, buttermilk, sana and the influence of them about quality. Thus, conditions under which these microorganisms multiply, appear and measures to avoid the presence in yogurt, buttermilk and sana were analyzed. Monitorization of this conditions and measures lead to the achievement of quality products which meet the quality requirements of consumers. The viability of proteolytic microorganisms inoculated at different concentration in dairy products was investigated. The proteolytic microorganisms studied were coliforms bacteria and Escherichia coli, Bacillus subtilis, Clostridium sporogenes, Proteus vulgaris şi Pseudomonas aeruginosa, which are representative for this class of microorganisms, using specific medium. We compared the results with a control sample (called multi loops) for eatch proteolytic microorganism. media. Keywords: dairy products, proteolysis, proteolytic microorganisms, multi-loops, specific INTRODUCTION Proteolysis is one of the most complex biochemical events which occur during dairy ripening. Proteolysis is catalysed by proteinases and peptidases from several sources including indigenous enzyme from the milk, coagulant, starter lactic acid bacteria, nonstarter lactic acid bacteria and probiotic adjuncts. The activities of these enzymes hydrolyze caseins (αs1-, αs2-, β- and κ-casein) to smaller peptides and amino acids, which contribute to flavour and texture of the cheeses (Sasachi et al, 1995;Antonsson et al,2002 ). There are many microorganisms that act on proteins in milk that hydrolysis in peptone, polypeptides, and amino acids. These amino acids decompose into ammonia, indol, skatol, hydrogen sulphide, etc. (Yazici et al, 2004). These microorganisms act primarily on the casein, using enzymes. Usually, the simplification of the casein is rarely by the enzymes of the same bacterial species. Most enzymes that are involved in various phases of degradation of casein derived from various species,witch pollutes milk. Most of this group of microorganisms can produce glycosides and lipholitic enzymes. For this reason, most effects of proteolytic microorganisms in milk and milk products is not only proteolysis specific changes, but also through other glycosides and lipholitic processes. In this large group of microorganisms enter species in almost all taxonomic divisions: Gram-positive cocci, Gram-negative bacillus, Gram positive bacillus, aerobic or anaerobic (Bacillus and Clostridium), actinomycetes, yeasts, molds (Apostu, 2006; 2003). In our study we investigated the microbiological composition of three dairy products: yogurt, buttermilk and sana, in terms of quantitative assessment of five bacteria with 432

2 proteolytic activity: Escherichia coli, Bacillus subtilis, Clostridium sporogenes, Proteus vulgaris and Pseudomonas aeruginosa. We determinated also, the influence of these microorganisms on physical and chemical composition of the product. MATERIALS AND METHODS The methods used to determined the proteolytic bacteria of dairy products have been standardized using the average enrichment, isolation and confirmation characteristic of each microbial species. Were used isolation media: Medium Levine, Rambach - for Escherichia coli and Proteus vulgaris - Incubation 24 h at 37 C, with sulphite agar medium azide and sodium for Clostridium sporogenes - Incubation 3-5 days at 45 C, especially environmental for Bacillus subtilis - Incubation 24 h at 37 C - agar F, P agar, agar with citrimid - for Pseudomonas - Incubation 24 h at 37 C. The results were compared with control samples of bacteria, which were obtained from pure cultures of microorganisms, figured in the microbiological chances form. To check resistance proteolytic bacteria were sown in dairy products five microorganisms in pure culture from different dilution (from 10 1 to 10 6 ). After incubation period was checked the proteolytic microorganisms which can survive in dairy products. Studied concurrently with rapid Lactoscan Analyzer and the influence of proteolytic microorganisms on protein, fat and lactose from products. Lactoscan determination is based on intensity comparation of absorption of two rays of light from the same source of infrared rays (one standard and one design), which via a rotating mirror camera is transmitted through the sample of acid milk products (detector). With a special device, based on the length of each wave component analysis, expresses the percentage displayed on a screen or recorded on a PC connected to the device. RESULTS AND DISCUSSION Regarding microbiological load of dairy products studied was found that 60% of cases the presence of coliforms and Escherichia coli species and in 30% of these cases are recorded exceeded the reference values of Ord MS 975 / 98. The presence of coliforms and Echerichia coli (considered health indicator) Fig. 1 and Fig. 2 shows serious deficiencies in hygiene. This may come from water or from the staff manipulator, indicating recent faecal contamination. Embedded in the substrate protein and fat resistant and temperature pasteurization. Fig 1 E. coli pozitive -yogurt Fig 2. E. coli negative for. Buttermilk and pozitive for sana 433

3 According to standards only Escherichia coli species has the obligation to be identify. However, the study was deemed necessary for these bacteria with proteolytic activity, because their presence in food can affect their quality. Other identified germs were Clostridium sporogenes (Fig. 3) and Pseudomonas aeruginosa (Fig. 4) in a percentage of 20% and 10%, respectively. Clostridium are earthly microorganism, reaching in milk during milking, handling, atmospheric dust, dirt from the animals, through contact with milking vessels or from operators hands. Being bacteria witch growth very easier, can resist to the thermal treatments applied to dairy products. Some species of Gram-negative bacillus, especially Pseudomonas, Proteus, often pollute water, which contaminated equipment, facilities and milk after pasteurization or sterilization. Fig. 3 Clostridium pozitive from yogurt and sana Fig. 4 Pseudomnas confirmed In can be noted that in samples of dairy products, bacteria of the species Bacillus subtilis (Fig. 6) and Proteus vulgaris (Fig. 5), witch are sensitive species to high temperature during pasteurization of milk, were absent. Fig. 5 Idntification of Proteus negative sample Fig. 6 Bacillus subtilis negative sample Proteolytic microorganisms produced important physico-chemical changes of the protein content through reducing -and in some cases, even slightly higher - until the end of shelf-life, caused by the decomposition of proteins due to proteolysis processes and the proteins environmental releases of microorganisms development. Because of this aspect, was considered necessary seeding dairy products with five microorganisms in pure culture, in different dilution to see the influence of physico-chemical composition of the products. After incubation and confirmation of colonies on selective media, characteristic of each microorganism in part, can be concluded that: Escherichia coli was present at all dilution 434

4 Pseudomonas aeruginosa was identified only in yoghurt to 10 6 Bacillus subtillus was not detected in any dilution Proteus vulgaris was present in yoghurt to sample 10 6 Clostridium sporogenes was present in all three samples of dairy products in eatch dilution. Dairy products sown with pure cultures of microorganisms were analyzed using Lactoscan Analyzer, quickly, achieved the following results present in Tab. 1. The samples were abbreviated as follows: yogurt-i sana-s and buttermilk- LB. Tab. 1 Lactoscan Lab values of dairy products sown with pure culture Samples Fat Lactose Protein I B.subtilis S B.subtilis 2,28 2,08 2,7 LB B.subtilis 2,2 2,08 2,67 I E.coli (4) 2,28 2,32 2,58 S E.coli (4) 3,54 2,28 2,78 LB E.coli (4) 3 2,12 2,72 I Clostridium (4) 2,84 2,18 2,52 S Clostridium (4) 3,54 2,2 2,78 LB Clostridium (4) 2,4 2,16 2,82 I Proteus (7)) 2,92 2,08 2,52 S Proteus (7) 3,76 2,18 2,9 LB Proteus (7) 2,42 2,08 2,72 I Proteus (6) 2,96 2,16 2,54 S Proteus (6) 2,96 2,3 2,92 LB Proteus (6) 2,46 2,14 2,8 I Pseudomonas (7) 2,98 2,1 2,54 S Pseudomonas (7) 3,62 2,22 2,84 LB Pseudomonas (7) 2,44 2,16 2,8 I Pseudomonas (6) 3,1 2,12 2,64 S Pseudomonas (6) 3,82 2,24 2,92 LB Pseudomonas (6) 2,56 2,08 2,84 The microorganisms witch were identified only at a certain dilution are given in parentheses (eg E.coli S (4) Sana sown with Escherichia coli at dilutions 10 4 ) as can be seen in Tab. 1. CONCLUSIONS Samples of dairy products sown with pure cultures of proteolytic microorganisms with a known concentration of germs, presents: Except Bacillus subtilis species, all other four organisms can survive in the dairy products, even if some of them only to contamination with high titres (Proteus vulgaris to 10 6 only). Lactoscan tests show that amount of protein decrease and lactose increase with one unit to the amount of protein of non-sown products. This proves that the proteolytic and lipolytic ativity of sown microorganisms in dairy products is present in germs that are not identified by microbiological analysis of dairy products in which they were sown. 435

5 These microorganisms cause changes in products witch can be observed organoleptic,also. Some proteolytic microorganisms have also lipolytic activity, recorded by decreasing of fat, compared to a significant amount of fat found in products. We appreciate that their presence (of proteolytic microorganisms) in dairy products, produce significant changes in the physico-chemical composition of the products, they develop, implicitly affecting their quality. REFERENCES 1. Antonsson, M., Y. Ardo, B. F. Nilsson, G. Molin (2002). Screening and selection of Lactobacillus strains for use as adjunct cultures in production of semi-hard cheese. Journal of Dairy Research, 69, Apostu S., Mihaela-Anca Rotar (2003). Lucrări practice de Microbiologie alimentară Ed. Risoprint Cluj-Napoca. 3. Apostu, S. (2006). Microbiologia produselor agroalimentare, Cluj- Napoca, Ed. Risoprint. 4. Sasaki M., B. W. Bosman, and P. S. T. Tan (1995). Comparison of proteolytic activities in various lactobacilli. Journal of Dairy Research, 62, STAS ISO 4831, 4832/ Yazici F., A. Akgun (2004). Effect of some protein based fat replacers on physical, chemical, textural, and sensory properties of strained yoghurt, Journal of Food Engineering 62,