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Bacillus stearothermophilus
B. stearothermophilus are often related to the contamination of dairy products, especially milk powder. They produce thermophilic spores that can withstand pasteurisation at 73 °C for 15s and can grow at 65 °C. They can cause spoilage in circumstances involving the reconstitution of milk powder. The growth of B. stearothermophilus during the manufacture of milk powder is considered to take place as a biofilm. Biofilms are defined as the growth of microorganisms and their extracellular polymeric material on a surface (Abraham, Debray, Candau & Piar, 1990). B. stearothermophilus have been found to enhance the acidic conditions in milk and lactose solution. Their growth effects protein stability during processing which results in accelerated aggregation of milk proteins (Yoo, Hardin & Chen, 2006).
Molecular identification of isolates
(a) Preparation of DNA for PCR The isolation of genomic DNA for PCR with agarose gel detection was conducted as described by Scheldeman et al. (2002), with some modifications. DNA templates were extracted from 3-day-old pure cultures from BHI agar plates which had been washed three times by centrifugation at 13 000 × g for 3 min followed by resuspension in distilled water. 100 μl solution of 0.1 M NaOH and 0·25% sodium lauryl sulphate (Merck, South Africa) was added to the washed pellets and heated for 17 min at 90 °C. The boiled samples were again centrifuged at 13 000 × g for 3 min and crude DNA was collected from the supernatants.
(b) PCR Procedure The PCR reactions were conducted using B. sporothermodurans specific primers of BSPO-F2 (5’-ACG GCT CAA CCG AG-3’) and BSPO-R2 (5’-GTA ACC TCG CGG TCT A-3’) as described by Schelderman et al. (2002). Primers prepared by Iqaba Biotechnical, Pretoria, South Africa were used to amplify fragments of about 664 base pairs from the 16S rDNA gene. For the PCR detection with agarose gel electrophoresis, 100 ng of genomic DNA was used as template for the PCR reaction in a total reaction volume of 25 μl, using 1.25 U Taq DNA polymerase, 200 nM of each primer, 50 mM KCl, 10mM Tris-HCl (pH 8.3), 1.5 mM MgCl2 and 0.2 mM of NTP mix (Fermentas, South Africa). The mixture was subjected to 30 cycles of amplification in a thermal cycler (iCycler Thermal Cycler, BIO-RAD, South Africa).
The first cycle was preceded by an initial denaturation step of 2 min at 94 °C.
Subsequently, each cycle consisted of a denaturation step of 20s at 94 °C, an annealing step of 45s at 45 °C and an elongation step of 60s at 72 °C. The last cycle included a final extension of 7 min at 72 °C. 10 μl of PCR products were analysed on a 1% agarose gel containing 0.5 μg/ml ethidium.
(c) Preparation of DNA for RT PCR DNA for real time PCR was extracted from 3-day-old pure cultures on BHI agar plates. About 5 or 6 colonies were dissolved in 300 μl of double distilled water and pure DNA was extracted and purified using the ZR Fungal/Bactrial DNA KitTM (Zymo Research Corporation, USA), following the manufacturer’s protocol. The concentration of extracted DNA was analysed using the DNA Nanodrop Spectrophotometer and samples were stored at -20 °C until they were used as PCR templates.
(d) RT PCR procedure For the real time PCR, 1 ng of pure genomic DNA was mixed with iQTM SYBR® Green Supermix (Bio-rad, South Africa) primers and sterile distilled water to a total of 25 μl reaction volume. The reaction was conducted using the DNA Engine® Peltier Thermal Cycler incorporated with a Chromo 4 real time PCR detector (Bio-rad, South Africa). Primer concentrations and reaction conditions were the same as that of the PCR detection with agarose gel electrophoresis.
Treatment regimes used on B. sporothermodurans isolates
(a) Preparation of raw and pasteurised milk isolates After plating and incubation of raw and pasteurised samples, about five individual colonies that displayed the morphology of a Bacillus colony were harvested and transferred into different eppendorf tubes containing BHI broth for the establishment of a pure culture.
(b) Preparation of UHT milk isolates Similarly, four individual colonies were randomly picked from each BHI plate spread with samples from different UHT milk packs and transferred into separate eppendorf tubes containing BHI broth to establish pure cultures of UHT milk isolates.
(c) Preparation of control strains A reference strain of Bacillus sporothermodurans (DSM No 10599), a control being obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), Germany, and a local B. sporothermodurans isolate from UHT milk in South Africa, were used to determine the effect of UHT processing stresses on their survival. In order to prepare pure cultures, a single colony of each isolate was collected from BHI agar plates and transferred into 1.5 ml eppendorf tubes containing 1 ml of freshly prepared Brain Heart Infusion broth and then incubated for 24h at 37 °C. A ml of each culture was centrifuged at 10 000 × g for 3 min and pellets were subsequently diluted to approximately 0.5 McFarland Standard prepared by adding 0.5 ml of 0.048 M BaCl2 to 99.5 ml of 0.18 M H2SO4 (1% w/v) with physiological saline (8.5 g/L NaCl) as diluents (Andrews, 2005).
(d) Preparation of spore suspension of test isolates From an overnight pure culture of each strain, 1 ml of culture was collected and spread on BHI agar plates and incubated for 15 days at 37 °C. Growths on each plate were checked for sporulation using a light microscope until ca. 100% sporulation was attained. Spores were harvested by scraping the dense growth on the surface of agar plates with a sterile loop and transferred into a 1.5 ml eppendorf tube containing sterile physiological saline. Spores were washed with double distilled water, centrifuged at 10 000 × g for 3 min and pellets were diluted to ca. 108 spores per ml with sterile physiological saline using 0.5 McFarland Standard.
(e) UHT treatment process Heating was conducted with some modifications as described by Huemer et al. (1998). UHT treatment was conducted with sterile pressure tubes with threaded type
B plugs (Sigma Aldrich, Midrand, South Africa) with an outer diameter of 25 mm and wall thickness of 2 mm and an oil bath. The tubes were filled with 3 ml spore suspension and 1 ml of BHI broth. The tubes were closed with stoppers and were submerged in an oil bath for either 120 ± 1 °C for 4s or 140 ± 1 °C for 4s. The temperature equilibrium time was determined with the aid of a thermocouple mounted on a pressure tube.
CHAPTER 1: INTRODUCTION AND PROBLEM STATEMENT
1.1 Problem statement
CHAPTER 2: LITERATURE REVIEW
2.1 Current trends in UHT milk production and consumption
2.2 Quality and safety of raw milk
2.3 Bacterial composition of raw milk
2.4 Quality and age gelation of UHT milk
2.5 Rapid methods to determine the bacterial quality of milk
2.6 Milk contamination routes
2.7 Background to UHT milk contamination
2.8 Thermal processes and properties of treated milk
2.9 Bacillus sporothermodurans
2.10 B. sporothermodurans characterisation techniques
2.11 Bacillus sporothermodurans spores
2.12 Analysis B. sporothermodurans spore components
2.13 Thermal inactivation kinetics of Bacillus sporothermodurans
2.14 Stress response adaptation of Bacillus Species
2.15 Other Bacillus species related to thermally processed milk
CHAPTER 3: OBJECTIVES AND HYPOTHESES
3.1 Objectives
3.2 Hypotheses
CHAPTER 4: RESEARCH
4.1 Incidence and survival of Bacillus sporothermodurans during UHT processing
4.2 The effects of wet heat treatment on the structural and chemical components of Bacillus sporothermodurans spores
CHAPTER 5: GENERAL DISCUSSION
5.1 Methodological considerations
5.2 Incidence and survival of Bacillus sporothermodurans during UHT processing
5.3 Effects of wet heat treatment on the structural and chemical components of Bacillus sporothermodurans spores
5.4 Proposals for future research
CHAPTER 6: CONCLUSION AND RECOMMENDATIONS
CHAPTER 7: REFERENCES
