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Научни трудове на Съюза на учените в България-Пловдив, серия Б. Естествени и хуманитарни науки, т. XV, 2013 г. Научна сесия „Техника и технологии, естествени и хуманитарни науки", 25-26 X 2012 Scientific researches of the Union of Scientists in Bulgaria-Plovdiv, series B. Natural Sciences and the Humanities, Vol. XV, ISSN 1311-9192, Technics, Technologies, Natural Sciences and Humanities Session, 25-26 oktober 2012.
THERMAL RESISTANCE OF WILD YEAST AND SACCHAROMYCES CARLSBERGENSIS IN "PEJA" PILSEN BEER
Xhemë Laj^i1*, Nushe Laj^i2, Blerim Baruti2, Dilaver Salihu2
xBeer Factory "Birra PEJA", Republic of Kosova 2Faculty of Mining and Metallurgy, University of Prishtina, Republic of Kosova
ABSTRACT
This study was performed to determine the thermal resistance of wild yeast and Saccharomyces carlsbergensis in Peja Pilsen beer (pH = 4.5 ± 0.2 and percentage of alcohol by volume = 4.7 ± 0.25 %). This investigation included determining Decimal reduction times (D-values) and Z-values. The corresponding D48°C, D50°C D52°C and D54°C values calculated from survivor plots for wild yeast were 4.60, 2.20, 0.515 and 0.348 minutes, respectively. D-values for Saccharomyces carlsbergensis at 47o, 48o, 49o, 50o, 51o and 52 °C were 3.86, 2.41, 1.43, 0.82, 0.59 and 0.32 minutes, respectively. The Z-value for wild yeast was 5.01 oC and for Saccharomyces carlsbergensis was 4.68 oC. These findings indicate that Saccharomyces carlsbergensis has greater temperature dependence than wild yeast. Relevant data, in the form of D and Z-values calculated in the various temperatures, potentially useful for the establishment of regimes of thermal control of wild/Saccharomyces carlsbergensis yeasts.
Key words: Beer, Wield yeast, Saccharomyces carlsbergensis, thermal resistance.
INTRODUCTION
Although beer is relatively resistant to spoilage, it is by no means entirely incapable of supporting the growth of microorganisms. For this reason, most beers are treated to eliminate any residual brewing yeast (Saccharomyces carlsbergensis) or infecting wild yeasts and bacteria before or during packaging. This can be achieved by pasteurization.
Saccharomyces carlsbergensis, the yeast most frequently used for the manufacture of beer, is the primary micro-organism present in the converted wort after fermentation and filtration, and prior to pasteurization [1, 2]. Beer spoilage due to the presence of wild yeast contaminants can take a number of forms and is influenced by the contaminant taxa [3]. Reported problems include the production of off-flavours, particularly phenolic off-flavours, which are formed by the carboxylation of ferulic and cinnamic acids, unacceptably high levels of turbidity and difficulties with clarification [3,4]
Most beers are pasteurized after filling to achieve microbiological stability and to inactivate molds and yeasts that might other-wise alter and deteriorate the product after processing. Although the thermal process applied in beer pasteurization is a low temperature process, it is desirable to apply the minimal thermal process in order to reduce undesirable organo-leptic changes in the final product. Knowledge of thermal resistance parameters of the target microorganism is essential for designing the minimal thermal process required for proper pasteurization. The
objective of this work was to determine the thermal resistance parameters D and Z for wild yeast and Saccharomyces carlsbergensis in Peja Pilsen beer.
MATERIALS AND METHODS
The study was conducted at microbiological laboratory of the beer factory "Birra Peja", Republic of Kosova. Thermal resistance parameters of wild yeast and Saccharomyces carlsbergensis in Peja Pilsen beer were investigated according to norms and standards: MEBAK-Methodensammlung der Mitteleuropäischen Brautechnischen Analysenkommission, (1997) and EBC-European Brewery Convention (2006) [5,6].
Product characterization
Physicochemical characteristics beer that could affect thermal resistance of the microorganism considered in this work were indicative of the following values (± standard deviation for 20 samples): pH = 4.5 ± 0.2; extract of original wort (EOW %) = 11.4 ± 0.25; percentage of alcohol by volume (%) = 4.7 ± 0.25; bitterness units (BU) = 22 ± 1.3; color (EBC) = 8.5 ± 1.5; carbonation of the beer (CO2, g/l )= 4.7 ± 0.5.
Isolation and cultivation of microorganisms
The strains used in this work wild yeast and Saccharomyces carlsbergensis were isolated and identified from non-pasteurized Peja Pilsen beer obtained from industrial process. The Isolation and cultivation of microorganisms were developed based on Koch method in selective terrene in five phases: sampling, dilution preparation, plating, incubation in aerobic condition and counting of Colony Formed Uunits (CFU's).
As microbial suspension were taken a sample of 100 ml beer in sterile condition at the end of beer fermentation with fermentation stage of 81 ± 3%, alcohol content 4.7± 0.25% and pH 4.5± 0.2. Sample of beer was stirred and 1 ml was withdrawn and transferred into tube 1 with 9 ml Ringov physiologic solution. The successive dilution was prepared in principle 1:10', 1:102, 1:103, 1:104 and 1:105. The selective plating was developed in selective culture medium: Crystal Violet Agar (Liofilchem s.r.l) for wild yeast and Malt Agar (Liofilchem s.r.l. Bakteriology Products) for Saccharomyces carlsbergensis yeast. Incubation was developed at optimal temperature 25° C on sterile Petri dishes for 48 hours in aerobic condition.Total aerobic counts were determined on Plate Count Agar. Experiment was replicated five times. Cell counts of wild yeast was 5.2x105 CFU ml-1 and for Saccharomyces carlsbergensis was 7.5x104 CFU ml-1.
Thermal resistance study
Test samples of beer using sealed glass tubes were submerged in a pre-heated water bath to various temperatures in the range 47o - 54 oC and continuously stirred. A high precision thermometer (Thermo-Schneider, ± 0.1°C, previously calibrated and certified) was inserted in water bath in order to measure the temperature in the water bath center.
Determination of Decimal reduction time (D-value) and Z-values
The rate of heat inactivation of a population is proportional to the number of cells, N. At constant temperature, the rate of decrease [7,8] in the number of viable microorganisms (-dN/dt) is proportional to the number of viable microorganisms present (N):
d) = kN
dt
where N is the number of microorganisms, k (s-1) is the first-order rate constant also called the death rate constant and t (min) is the heat treatment time.
The Decimal reduction time (D-value) is the time required to kill 90% of a cell population and can be calculated with the relationship:
(2) D = , 1
log No - log N
where No is the initial number of microorganisms.
Thermal resistance constant (Z-value) defined for the temperature increase that involves a decrease in the D value of 90%. The Z value can be determined with the equation:
T - T
Z 2 1 =-2-1-
l°gDT1 - logDT2
Thermal resistance constant (Z) were calculated from the negative inverse of the slope of log(D) versus temperature treatment.
RESULTS AND DISCUSSIONS
Heating remains the principal method of microbial inactivation. The classical method of D- and Z-values, developed by Stumbo, 1973, is widely accepted and practiced. This method assumes first-order kinetics as a model to describe inactivation of microorganisms. A mechanistic explanation for this is that death will be caused by inactivation of some critical enzyme and it is commonly held that enzyme inactivation is governed by first-order kinetics.
Determination of D- and Z-value for wild yeast
Number of survived wield yeast during the heat treatment at temperatures 48, 50, 52 and 54°C for determined time are shown in table 1. When heating was performed in beer, a slight effect on cell survival is observed at 48 oC and significant inactivation begins at 52 oC (fig.1).
Table 1. Number of survived wield yeast cells. 48°C 50°C 52°C 54°C
Time, min N, CFU/ml Time, min N, CFU/ml Time, min N, CFU/ml Time, min N, CFU/ml
0 520000 0 520000 0 520000 0 520000
1 362500 2 73720 0,25 193420 0,25 98950
5 36180 4 6718 0,5 46240 0,5 12104
10 4007 6 1120 0,75 20235 0,75 2710
15 242 8 102 1 4838 1 331
20 26 10 17 2 72 2 1
When the microbial populations as a function of time are presented on semi-logarithmic coordinates, a linear decrease in microbial population with time is observed as shown in figure 1. It is the survivor curve. The log numbers apparently decreasing in a linear manner with time. Reproducible semi-logarithmic curve were obtained without any apparent deviations associated with variable heat resistance within the cell population.
Figurel. The semi-logarithmic curve of survived wild yeast.
Decimal reduction times (D) were calculated from the negative inverse of the slope of the linear regression line of log number of microorganism versus time at a particular temperature (equation 2) and obtained results are shown in table 2.
Table 2. Decimal reduction times (D) and linear regression lines at temperature treatment.
Temperature treatment Linear regression Determinant coefficient (r2) D (min)
48°C y = -0.217-x + 5.457 0.9987 4.60
50°C y = -0.454-x + 5.72 0.9986 2.20
52°C y = -1.941-x + 5.71 0.9979 0,515
54°C y = -2.870-x + 5.61 0.9953 0,348
By increasing the heating temperature, the degree of inactivation of microorganisms grows whereas the D value decreases. The thermal survivor curves for the temperatures considered in this study indicated that thermal destruction followed the classical decimal reduction pattern corresponding to a first order kinetics. In all cases, high determinant coefficients were obtained in the adjustments (0.9953 < r2 < 0.9987).
Thermal resistance constant (Z) were calculated from the negative inverse of the slope of the linear regression line log (D) versus temperature as shown in figure 2.
Figure 2. The semi-logarithmic curve of decimal reduction time as a function of temperature treatment for the wild yeast.
From the negative inverse slope 1/k where k=0.1997 oC-1 the thermal resistance constant was calculate to be Z = 5.01 oC.
Determination of D- and Z-value for Saccharomyces carlsbergensis yeast
The number of survived Saccharomyces carlsbergensis cells during the heat treatment at temperatures: 47, 48, 49, 50 51 and 52°C for determined time are shown in table 3.
Table 3. Number of survived Saccharomyces carlsbergensis at different temperature treatment.
47°C 48°C 49°C 50°C 51°C 52°C
Time. N, CFU/ Time, N, CFU/ Time, N, CFU/ Time, N, CFU/ Time, N, CFU/ Time, N, CFU/
min ml min ml min ml min ml min ml min ml
0 75000 0 75000 0 75000 0 75000 0 75000 0 75000
5,0 4794 4,0 2240 2,0 4443 1,0 8062 1,0 1368 0,5 3048
10,0 172 8.0 24 4.0 95 2.0 312 2.0 9.0 1.0 45
15,0 11 12,0 1 6,0 6 3,0 20 3,0 1.0 1,5 2
The decimal reduction time was determined by one fold linear regression and obtained results are shown in figure 3. The semi-logarithmic curve resulting when log N is plotted vs. treatment time is frequently referred to as the survival curve. The log numbers apparently decreasing in a linear manner with time.
TreJ[mtrtLllme | minuets]
Figure 3. The semi-logarithmic curve of survived Saccharomyces carlsbergensis yeast.
Decimal reduction times (D) were calculated from the negative inverse of the slope of the linear regression line of log number of microorganism versus time at a particular temperature and obtained results are shown in table 4. In all cases, high determinant coefficients were obtained in the adjustments (0.97 < r2< 0.99).
Table 4. Decimal reduction time (D) and linear regression lines at temperature treatment.
Temperature treatment Linear regression Determinant coefficient (r2) D (min)
47°C y= -0.2589-x + 4.90 0.9985 3.86
48°C y= -0.4149-x + 4.89 0.9957 2.41
49°C y= -0.698-x + 4.914 0.9957 1.43
50°C y= -1.2134-x + 4.96 0.9953 0.82
51°C y= -1.6807-x + 4.76 0.9796 0.59
52°C y= -3.1106-x + 4.91 0.9965 0.32
Thermal resistance constant (Z) for Saccharomyces carlsbergensis yeast was determined from the negative inverse of the slope of the linear regression line log (D) versus temperature and obtained result are shown in figure 4.
Figure 4. The semi-logarithmic curve of decimal reduction time as a function of temperature treatment for Saccharomyces carlsbergensis yeast.
From the negative inverse slope 1/k where k= -0.2138oC-1 the thermal resistance constant was calculated to be Z = 4.68 oC.
CONCLUSIONS
The thermal resistance of wild yeast and Saccharomyces carlsbergensis isolated from Peja Pilsen beer gave the following results:
The corresponding D48°C, D50°C D52°C and D54°C values calculated from survivor plots for wild yeast were 4.60, 2.20, 0.515 and 0.348 minutes, respectively. D-values for Saccharomyces carlsbergensis for the corresponding temperatures were: D47°C= 3.86 min, D48°C= 2.41 min, D49°C = 1.43 min, D50°c= 0.82 min, D51°C= 0.59 min and D52°C= 0.32 min. The Z-value for wild yeast was 5.01 oC and for Saccharomyces carlsbergensis was 4.68 oC. These findings indicate that Saccharomyces carlsbergensis has greater temperature dependence than wild yeast. The D and Z-values calculated in the various temperatures, potentially useful for the establishment of regimes of thermal control of wild/Saccharomyces carlsbergensis yeasts.
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Рецензент: доц. Вяра Иванова
