Influence of Bacillus spp. Enzymes on Ultra High Temperature-treated Milk Proteins

Jan‰tová, B. , J . Luká‰ová, M. Draãková, L. Vorlová: Influence of Bacillus spp. Enzymes on Ultra High Temperature-treated Milk Proteins. Acta Vet. Brno 2004, 73: 393-400. A model case of long-life half-fat milk contamination with spores of 15 strains of B. licheniformis, B. subtilis a B. cereus isolated from farm environment and from raw milk was used to determine proteolysis by measuring the changes in milk protein contents. The methods of infrared spectroscopy, free tyrosine using Lowry’s method according to Juffs and setting of decrease in casein fractions using the SDS-PAGE were employed. Under storage temperature of 4 oC no proteolysis was recorded. However, under storage temperature of 24 °C the following changes in comparison with initial values were recorded after 3 weeks: a decrease in protein contents from 34.60 g⋅1-1 to 29.46 32.86 g⋅l-1 and increased free tyrosine contents from 0.65 mg⋅ml-1 to 2.13 1.59 mg⋅ml-1 depending on Bacillus spp. type. It was detected that milk heating at a temperature of 100 oC for 10 min and at 135 oC for 5 s, respectively, the spores of resistant strains Bacillus licheniformis may survive and show proteolytic activity. When monitoring the reduction of casein fractions it was found that the κ-casein was the most quickly broken down to 7.43%. The values of β-casein dropped to 27.53% and α-casein to 43.95%. Under storage temperature of 4 °C the reduction of all casein fractions was lower. A reduction to 79.42% for κ-casein, 78.30% for β-casein and 85.71% for α-casein was recorded. The intensity of changes was dependent on species and strain of Bacillus spp. used, on storage temperature and heat inactivation of spores. The initial spore concentration made itself felt, too. Proteolysis, Bacillus spores, Bacillus licheniformis, casein Aerobic and facultative anaerobic, sporulating gram-positive bacteria Bacillus spp. represent important contaminants of raw milk, mainly from the hygienic and technological point of view, some of them even from the point of view of human health. Spores of Bacillus spp. appear regularly in stable environment and they usually represent a secondary contamination of milk during milking process. Many representatives of the Bacillus strain create a part of the psychrotrophic microflora of milk. B. licheniformis and B. cereus are the most frequently isolated types of the Bacillus from raw milk (Criel ly et al. 1994; Phi l l ips and Griff i ths 1986). Páãová et al. (1996) found the most frequent occurrence of the type B. licheniformis. The occurrence of other types is lower (Luká‰ová et al. 2001; Vyletûlová et al. 2001). The post-pasteurization contamination is by spores from raw milk that either passed pasteurizing or come from the plant environment. Spores of thermostable bacteria may be introduced into the product even in the course of the technologic process. Microorganisms Bacillus spp. may also appear in UHT milk, as stated e.g. by Bahout (2000), who found spores in 18.3% of examined samples in the quantity of up to 2.6 × 102·ml-1. Vyletûlová (2001) monitored – on the basis of ribo-typing – the occurrence of B. licheniformis in milk up to the final product, the UHT milk, and in consideration of its frequency she supposes its penetration from the initial stage of production, while the origin of B. cereus in UHT milk ACTA VET. BRNO 2004, 73: 393–400 Address for correspondence: MVDr. Bohumíra Jan‰tová, Ph.D. Department of Milk Hygiene and Technology University of Veterinary and Pharmaceutical Sciences Palackého 1-3, 612 42 Brno, Czech Republic Phone: +420 541 562 712 Fax: +420 541 562 711 E-mail: janstovab@vfu.cz http://www.vfu.cz/acta-vet/actavet.htm cannot be clearly explained – it may come from the initial stage of production or the milk was re-contaminated in the course of processing. Bacillus spp. is a very difficult part of raw milk microflora, taking into consideration the problematic removal of its spores, related to their thermoresistance. The spores may also be partially damaged by pasteurization temperature, but they usually do not survive sterilization and the UHT process. An important characteristics of Bacillus spp. is the ability of vegetative cells to produce – after multiplication – thermostable extracellular enzymes (Meer et al. 1991; Ipsen et al. 2000), that – due to their proteolytic and lipolytic activity. These affect the nutritional and sensorial characteristics of products even when viable bacteria are absent (Boor et al. 1998). According to Brown (2000) Bacillus spp. are the microorganisms causing significant economic losses. Proteolysis of casein substances is one of the main features in proteolysis of milk proteins. For casein affection there 104 108 bacterial cells in l ml of milk are needed (Marth and Steele 1998). Kappa-casein is subject to the fastest hydrolysis of milk proteins. In the course of 7 days and under storage temperature of 20 oC it is completely lost, while para κ-casein is formed. The reduction of β-casein reaches 70%. Alfa-casein remains stable for the longest time and its loss is minimal (Dalgleish 1990). In comparison with fractions of κ-casein, α-lactalbumin and β-lactoglobulin are resistant (Swaisgood 1993; Madsen and Quist 1997). The effects of UHT milk treatment methods were monitored by Kel ly and Foley (1997), who recorded a more efficient inactivation of bacterial proteinase when using an indirect UHT method. García-Risco et al. (1999) recorded – in the course of storage at a temperature of 25 oC – more significant proteolysis in UHT skimmed milk in comparison with full milk. They recommend for this fact to be taken into consideration when setting the parameters of the UHT process depending on product type. The influences of proteolytic enzymes cause many defects of milk and dairy products, such as sweet curdling, colour and odour defects. A specifically monitored defect is gelformation by UHT milk (Dat ta and Deeth 2001; Dalgleish 1990; McMahon 1996; Kavanah et al. 2000). The defects are detected at the moment of reaching the concentration of microorganisms of 5.105 107 CFU·ml-1 (Vyletûlová et al. 2000; Marth and Steele 1998). Materials and Methods Collection strains of the Institute of Milk Hygiene and Technology, Faculty of Veterinary Hygiene and Ecology Brno, were used for the tests. Fifteen strains of Bacillus spp. were used isolated in the farm environment and from raw milk. Each test was performed with 5 strains of B. licheniformis, 5 strains of B. subtilis and 5 strains of B. cereus. Identification of isolated strains was performed by Luká‰ová et al. (2001), phenotype characteristics were tested on the basis of conventional growth tests. The long-life semi-full milk from the market was used as a medium for contamination with spores of Bacillus spp. Acrylamide, bisacrylamide, ammonium persulfate, TEMED, bromophenol blue, glycine, Tris (hydroxymethyl)aminomethane, 2-mercapthoethanol, Coomasie brilliant blue R-250 were supplied by the company Bio-Rad Laboratories (Richmond, CA). All chemical substances used were of p. a. grade. Inoculation of tested milk was performed using spore suspensions Bacillus spp. without thermo-inactivation, inoculated into the UHT milk in such a quantity so as to reach the concentration of spores of 102, 101, < 101 in l ml of milk. Then, the spore suspensions were heated to a temperature of 100 °C with exposure for 10 min and to a temperature of 135 °C with exposure for 5 seconds and inoculated to milk in identical quantity. Samples of milk were stored in closed sterile glass sampling bottles at a temperature of 24 oC in thermostat and at temperature of 4 °C in refrigerator. In order to be able to compare the growth dynamics of microorganisms growth and changes in values of monitored features, the microbiological examination of milk samples was performed – setting the total number of sporulating microorganisms. The Plate Count Agar (HiMedia, India) was used. The protein contents settings were performed at one-week intervals plus free tyrosine contents was set daily in the course of the first week. The samples were stored for three months. The protein contents was measured on the basis of the infra-red spectroscopy method MIR using the MilkoScan 104 device with multi-detection application (A/S N. Foss Electric, Denmark), using the wavelength of 645 nm. Before examination, the samples were conserved by potassium bichromate (K2Cr2O7) in 0.6 g⋅l -1 of milk. As 394

Aerobic and facultative anaerobic, sporulating gram-positive bacteria Bacillus spp.represent important contaminants of raw milk, mainly from the hygienic and technological point of view, some of them even from the point of view of human health.
Spores of Bacillus spp.appear regularly in stable environment and they usually represent a secondary contamination of milk during milking process.Many representatives of the Bacillus strain create a part of the psychrotrophic microflora of milk.B. licheniformis and B. cereus are the most frequently isolated types of the Bacillus from raw milk (Crielly et al. 1994;Phillips and Griffiths 1986).Páãová et al. (1996) found the most frequent occurrence of the type B. licheniformis.The occurrence of other types is lower (Luká‰ová et al. 2001;Vyletûlová et al. 2001).
The post-pasteurization contamination is by spores from raw milk that either passed pasteurizing or come from the plant environment.Spores of thermostable bacteria may be introduced into the product even in the course of the technologic process.Microorganisms Bacillus spp.may also appear in UHT milk, as stated e.g. by Bahout (2000), who found spores in 18.3% of examined samples in the quantity of up to 2.6 × 102•ml -1 .Vyletûlová (2001) monitored -on the basis of ribo-typing -the occurrence of B. licheniformis in milk up to the final product, the UHT milk, and in consideration of its frequency she supposes its penetration from the initial stage of production, while the origin of B. cereus in UHT milk cannot be clearly explained -it may come from the initial stage of production or the milk was re-contaminated in the course of processing.
Bacillus spp. is a very difficult part of raw milk microflora, taking into consideration the problematic removal of its spores, related to their thermoresistance.The spores may also be partially damaged by pasteurization temperature, but they usually do not survive sterilization and the UHT process.An important characteristics of Bacillus spp. is the ability of vegetative cells to produce -after multiplication -thermostable extracellular enzymes (Meer et al. 1991;Ipsen et al. 2000), that -due to their proteolytic and lipolytic activity.These affect the nutritional and sensorial characteristics of products even when viable bacteria are absent (Boor et al. 1998).According to Brown (2000) Bacillus spp.are the microorganisms causing significant economic losses.
Proteolysis of casein substances is one of the main features in proteolysis of milk proteins.For casein affection there 10 4 -10 8 bacterial cells in l ml of milk are needed (Marth and Steele 1998).Kappa-casein is subject to the fastest hydrolysis of milk proteins.In the course of 7 days and under storage temperature of 20 o C it is completely lost, while para κ-casein is formed.The reduction of β-casein reaches 70%.Alfa-casein remains stable for the longest time and its loss is minimal (Dalgleish 1990).In comparison with fractions of κ-casein, α-lactalbumin and β-lactoglobulin are resistant (Swaisgood 1993;Madsen and Quist 1997).
The effects of UHT milk treatment methods were monitored by Kelly and Foley (1997), who recorded a more efficient inactivation of bacterial proteinase when using an indirect UHT method.García-Risco et al. (1999) recorded -in the course of storage at a temperature of 25 o C -more significant proteolysis in UHT skimmed milk in comparison with full milk.They recommend for this fact to be taken into consideration when setting the parameters of the UHT process depending on product type.
The influences of proteolytic enzymes cause many defects of milk and dairy products, such as sweet curdling, colour and odour defects.A specifically monitored defect is gelformation by UHT milk (Datta and Deeth 2001;Dalgleish 1990;McMahon 1996;Kavanah et al. 2000).The defects are detected at the moment of reaching the concentration of microorganisms of 5.10 5 -10 7 CFU•ml -1 (Vyletûlová et al. 2000;Marth and Steele 1998).

Materials and Methods
Collection strains of the Institute of Milk Hygiene and Technology, Faculty of Veterinary Hygiene and Ecology Brno, were used for the tests.Fifteen strains of Bacillus spp.were used isolated in the farm environment and from raw milk.Each test was performed with 5 strains of B. licheniformis, 5 strains of B. subtilis and 5 strains of B. cereus.Identification of isolated strains was performed by Luká‰ová et al. (2001), phenotype characteristics were tested on the basis of conventional growth tests.The long-life semi-full milk from the market was used as a medium for contamination with spores of Bacillus spp.
Inoculation of tested milk was performed using spore suspensions Bacillus spp.without thermo-inactivation, inoculated into the UHT milk in such a quantity so as to reach the concentration of spores of 10 2 , 10 1 , < 10 1 in l ml of milk.Then, the spore suspensions were heated to a temperature of 100 °C with exposure for 10 min and to a temperature of 135 °C with exposure for 5 seconds and inoculated to milk in identical quantity.Samples of milk were stored in closed sterile glass sampling bottles at a temperature of 24 o C in thermostat and at temperature of 4 °C in refrigerator.In order to be able to compare the growth dynamics of microorganisms growth and changes in values of monitored features, the microbiological examination of milk samples was performed -setting the total number of sporulating microorganisms.The Plate Count Agar (HiMedia, India) was used.The protein contents settings were performed at one-week intervals plus free tyrosine contents was set daily in the course of the first week.The samples were stored for three months.
The protein contents was measured on the basis of the infra-red spectroscopy method MIR using the MilkoScan 104 device with multi-detection application (A/S N. Foss Electric, Denmark), using the wavelength of 645 nm.Before examination, the samples were conserved by potassium bichromate (K 2 Cr 2 O 7 ) in 0.6 g⋅l -1 of milk.As a second method of bacterial proteolysis free tyrosine was measured by the Lowry's method according to Juffs (1973) with Folin-Ciocalteu phenol reagent, showing blue colour when mixed with the free tyrosine.The blue colour was measured using the spectrum photometer Helios β (Unicam, England, UK).
The decrease in casein fractions was monitored using the spore suspension B. licheniformis at a concentration of 10 1 ⋅ml -1 .The samples were stored at the above temperatures for a period of 1-4 months.In order to separate casein from whey proteins, the samples were precipitated with 10% acetic acid to pH 4.6.The residual fat was removed from the casein precipitate by three-time repeated washing in the solution of methylene dichloride -water (1:1) and by separation at 4500 g of 15 min.The final casein precipitate was processed using the lyophilizator Lyovac GT 2 (Amsco/Finn-Aqua, Finland) (López-Fandiño et al. 1993).The lyophilized casein was dissolved in Tris-HCl (pH 8.8) and sample buffer was added (2.4 ml Tris-HCl pH 6.8, 2 ml 10 % SDS (w/v), along with 1 ml glycerol, 4.4 ml distilled water, 0.1 ml bromophenol blue) at the rate of 1:4.The samples were boiled for 2 min.The separation of casein fractions was performed using separation gels (15% T, 2.6% C) and concentrating gels (3% T, 2.6% C) (Laemli 1970) while using the Mini-Protean III Cell Electrophoresis apparatus (Bio-Rad Laboratories, Richmond, CA).Separation buffer (30.3 g Tris, 144 g glycine, 10 g SDS completed up to 1 litre with distilled water) was used for migration, diluted 1:9 with distilled water.Electrophoresis was performed at 110 V at room temperature until bromophenol blue reached the end of the gel.Then, the gels were coloured with Commassie Brilliant Blue R-250 (450 ml ethanol, 100 ml acetic acid, 450 ml distilled water, 0.5 g Coomasie Blue R-250).The samples were coloured overnight with consequent colour removal (250 ml ethanol, 100 ml acetic acid, 650 ml distilled water).Then, the samples were dried, scanned and assessed using the computer program Image Quant 5.0 (Molecular Dynamics, USA).Quantification was performed on the basis of pixel density of individual areas of casein fraction bands.The decrease of individual casein fractions was always assessed within the scope of one coloured gel with applied samples, stored at given temperature from all extractions in the course of the given time period.The samples without Bacillus licheniformis were applied first.The coloration intensity (pixel density) was considered to be 100%.Values of pixels, measured for the other samples, were always related to the given value and re-calculated to percent data.While assessing the trials, the average values from results established at 5 strains of each type of Bacillus spp.were calculated.Statistical significance of differences at the levels of p < 0.01 and p < 0.05 for individual indicators was performed by using the Statistic and Graphic System STAT Plus (Matou‰ková et al. 1992).Data assessment was performed by dispersion analysis and by methods of consequent testing -the Scheffe's method of contracts and Tukey's test of significance of differences were used.In order to reach correctness of the results, the Box -Cox transformation was applied by dispersion analysis to the data so as to meet the condition of basic classification normality.Before the dispersion analysis it was necessary to verify the homogeneity of dispersion of compared selections.Bartlett's test was used for this purpose.

Results and Discussion
The decrease of protein contents due to influence of proteolytic enzymes was recorded using the MilcoScan device only at a storage temperature of 24 o C and it corresponded with the growth of the number of microorganisms.More significant proteolysis was found in samples with higher initial contents of inoculated thermally inactivated spores in milk.A smaller range was recorded in samples with sporadic spore presence.
When using the highest concentration of spores and storage for 3 weeks (see Table 1a), the examined strains of B. licheniformis caused a significant reduction of milk protein content depending on initial concentration used -from 34.60 g•1 -1 to 32.58 -31.40 -31.18 g•1 -1 of milk.The proteolytic effect of B. subtilis enzymes may be assessed in comparison with the other examined types as the least significant one, the protein contents founddepending on initial spores concentration used -the values of 33.22 -33.26 -32.86 g•1 -1 of milk.When monitoring the milk samples containing B. cereus spores a significant reduction in protein contents was recorded for all the examined strains.The values of 31.26 -29.79 -29.46 g•1 -1 of milk were found.Even with initial contents of spores in milk of < 10 1 •ml -1 the detected protein contents was very low (Table 1a).Significant (p < 0.05) or highly significant (p < 0.01) differences were recorded among the effects of enzymes of individual monitored types of Bacillus spp.
When storing the samples at temperature of 4 o C the reduction of proteins did not occur due to any of the monitored types of Bacillus spp.-in comparison with the initial protein contents.The numbers found are comparable with control samples.
The MilcoScan device was used for determination of proteolysis also by Kelly and Foley (1997), who monitored the influence of native proteolytic enzymes of milk.The questions of protein contents in milk caused by activities of lytic enzymes were also studied by Corzo et al. (1994), Recio et al. (2000), Zhao et al. (1998), Madsen and Qvist (1997).
Lower values of free tyrosine (Table 2a) were found in samples with sporadic occurrence of spores, while a significant increase was detected with higher spore contents.Changes were monitored as soon as from the second day of monitoring, when the average number of sporulating bacteria reached 10 4 -10 5 CFU⋅ml -1 during tests with all the monitored types of bacilli.In the course of storage of milk samples with spores of B. licheniformis -an increase of free tyrosine was detected after a period of 28 days depending on initial spore concentration up to 1.34 -1.59 -1.58 mg⋅ml -1 .The highest proteolytic activity was recorded for B. cereus, where free tyrosine values of 2.09 and 2.13 mg⋅ml -1 were detected at higher spore concentrations and 1.66 mg⋅ml -1 in case of < 10 1 ⋅ml -1 concentration.The lowest range of protein breakdown was detected in milk inoculated with B. subtilis spores, when the free tyrosine contents increased only to 1.06 -1.26 and 1.28 mg⋅ml -1 .When studying the proteolytic activities of psychrotrophic microorganisms in milk, Luká‰ová (1985) detected an increase of free tyrosine up to 1.21 mg⋅ml -1 at 10 8 CFU⋅ml -1 , which corresponds to values detected by us while reaching the same number of microorganisms ( Pfii skladovací teplotû 4 o C nebyla proteol˘za zaznamenána, zatímco pfii skladovací teplotû 24 °C byly zji‰tûny po 3 t˘dnech následující zmûny od pÛvodních hodnot: pokles bílkovin z 34.60 g⋅1 -1 mléka na hodnoty 29.46 -32.86 g⋅l -1 mléka a zv˘‰ení obsahu volného tyrosinu z 0.65 mg⋅ml -1 na hodnoty 2.13 -1.59 mg⋅ml -1 mléka v závislosti na druhu Bacillus spp.. Bylo zji‰tûno, Ïe teplotní a ãasov˘ parametr záhfievu na 100 o C 10 minut a 135 o C 5 sekund mohou spóry rezistentních kmenÛ Bacillus licheniformis pfieÏít a vykazovat proteolytickou aktivitu.

Fig. 2b .
Fig. 2b.SDS-PAGE pattern of casein fractions proteolysis by Bacillus licheniformis of storage UHT milk at 4 °C.The number at the lines means week of storage.
o C Fig. 1a.Proteolytic influence of Bacillus licheniformis on casein fractions in the UHT milk stored at 24 °C Fig. 2a.Proteolytic influence of