Growth and Activity of Starter and Adjunct Lactobacilli and Lactococci during Ripening of Two Types Bulgarian White Brine Cheese

Growth and Activity of Starter and Adjunct Lactobacilli and Lactococci during Ripening of Two Types Bulgarian White Brine Cheese

Abstract

The growth and activity of starter and adjunct lactobacilli and lactococci during ripening of two types Bulgarian white brine cheese were studied. The pasteurization of milk used was 72°C for 10 min and 85°C for 10 min. The temperature of cheese ripening was 12°C and 6°C respectively. The count of lactobacilli and lactococci and the changes in nitrogen fractions (NCN/TN pH 4.6 soluble nitrogen/total nitrogen and NPN/TN - 12% CCl₃COOH soluble nitrogen/total nitrogen) were determined at 10, 17, 24 and 31st day of the ripening of the cheeses. The results obtained showed difference in the growth of lactobacilli and lactococci. The higher temperature of pasteurization of the milk and the lower temperature of ripening of the cheeses had bigger negative influence on the growth of lactobacilli and contrariwise the lower temperature of pasteurization of the milk and the higher temperature of ripening of the cheeses resulted in lower number of lactococci. The NCN/TN and NPN/TN values increased during the ripening of the both types cheese and it was higher for Batch B at all studied stages of ripening.

Key words: growth, proteolytic activity, lactobacilli, lactococci, Bulgarian white brine

cheese (BWBC)

Introduction

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The biochemical changes in cheese components during ripening are due to the activity of enzymes produced by microorganisms (Norani and Elmer, 1990). That is why the growth of the microorganisms is essential for the proteolysis, lipolysis and metabolism of lactose, citrate and lactate (Fox et al., 1989). The products of proteolysis are very important for the organoleptic characteristic of produced cheese ( Fox, 1989). The microflora of the cheese is presented mainly by the starter lactic acid bacteria, which are inoculated into milk and non-starter lactic acid bacteria which contaminate milk after pasteurization (Martley and Crow, 1993). A small percentage of non-starter lactic acid bacteria may survive pasteurization (Turner et al., 1986). They are mainly mesophilic lactobacilli (McSweeney et al., 1993). The lactic starter strain used in cheese manufacture has influence to the rate of growth and final count of non-starter lactic acid bacteria during ripening (Thomas, 1987; Martley and Crow, 1993; Crow et al., 1995).

The aim of the present study was to establish the growth and proteolytic activity of starter and adjunct lactobacilli and lactococci during ripening of two types Bulgarian white brine cheese.

Materials and Methods

Cheese making

The two types white brine cheeses were manufactured in industrial conditions. The cow milk was pasteurized at two different temperatures - 75°C for 10 min (Batch A - Control cheese) and 85°C for 10 min (Batch B - Experimental cheese) and then cooled to 34°C. The milk was inoculated with a mesophilic starter culture, consisted of Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Lactobacillus casei. Standard amounts of CaCl₂ and commercial animal rennet were added. After 60 min (from the addition of the rennet), the curd was cut and leaved to heal for 10 min, then the whey was removed and the curd was pressed until the moisture content reached 64%. The cheeses were put in salt solution (20-22%) for 16 hours and after that the salt content of cheeses was approximately 2.5%. Then the Control and Experimental cheeses were ripen at 12°C and 6 °C respectively, in hermetically closed metal cans filled with brine (10% concentration). The microbiological and physicochemical analyses were conducted at four stages of the ripening process - after 10, 17 , 24 and 31 days from the beginning of the ripening.

Physicochemical analysis

The Kashkaval loaves were analyzed for moisture content (IDF Standart 4 A:1982), fat (Gerber method; BSI 1955), salt (Reddy and Marth, 1993). Total nitrogen, noncasein nitrogen and nonprotein nitrogen content were determined by the Vakaleris and Price method (1959) modified to suit the specific conditions of the analysis. The total nitrogen content was determined after 20g of cheese was extracted in 10% sodium citrate solution (extract A) and  10 cm³ of the prepared extraction was used for determination by the Kjeldahl method. For the noncasein nitrogen determination 1.4N HCl was added to 80 cm³ of extract A, until pH reached 4.4 -  4.6 and after 10 min was filtered and the filtrate was used for determination by the Kjeldahl method. For the nonprotein nitrogen determination 12% CCl³ of extract A and af₃COOH was added to 40 cm ter 30 min was filtered and the filtrate was used for determination by the Kjeldahl method. The Kjeldahl method was performed in duplicate using Kjeltec Auto 1030 Analyzer (Tecator Sweden) combined with the Digestion System 20.

Microbiological analysis

The lactococci and lactobacilli count in the cheeses after 10, 17, 24 and 31 days of ripening were determined by cultivations on synthetic culture media (M17 and MRS respectively) and 48 hours incubation at 30°C. The methodology described in IDF Standard 149A:1997 was followed. The samples preparation was according to IDF Standard 122C: 1996 - 10g of the cheese samples were put into container of peristaltic-type blender and 90 cm³ of 20% sodium citrate solution was added. The mixture was stir up until complete dispersion of the samples. The suitable dilutions were made and 1 cm³ of each dilution was put into Petri dishes and the molten media (44±1°C) was added (M17 for lactococci and MRS for lactobacilli determination). After the media became solid the dishes were inverted and incubated at 30±1°C for 48 hours. After incubation all colonies were counted.

Statistical analysis

All statistical analyses were performed using two-way multivariate analysis of variance (MANOVA) and multiple comparison tests were carried out to study the effect of both pasteurization and ripening temperature on the physicochemical characteristics of cheeses and the count of lactobacilli and lactococci (Box et al., 1978). Statistical analyses were carried out on the averages of five results. Differences in the averages and F tests were considered sig-

Table 1

nificant when the computed probabilities were less than 0.05. All statistical procedures were computed using the Microsoft Excel 2003 and Sigma Plot 2002 software. Results

The moisture content, pH, fat content, NaCl content, NCN/TN and NPN/TN of the cheeses are shown in Table 1. It can be seen that the moisture content of experimental cheese is higher in all aging stages because of the higher denaturation of serum proteins, provoked by the higher pasteurization temperature, although the differences were not significant (P<0.05). Despite of this the way of alteration was similar for both cheeses. PH initially decreased until the 24^-th day and after that increased to the 31^-st day of ripening. This increasing of pH is probably due to the accumulation of alkaline products from the proteolysis of the cheese proteins and partial neutralization of the lactic acid. The pH of Batch A was slightly lower in all aging stages, except the 10^-th day due to the higher ripening temperature. The NaCl content was slightly higher in the 


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