Efficient Characterisation of Bacterial Contaminants in the Milk industry: A Case for Molecular Sequencing

Transcription

1 SA Society of Dairy Technology 2015 Dairy Symposium April, 2015 Cape St. Francis Resort, Cape St. Francis, Eastern Cape Efficient Characterisation of Bacterial Contaminants in the Milk industry: A Case for Molecular Sequencing Rodney Owusu-Darko PhD Food Science, University of Pretoria Supervisor: Prof. Elna Buys

2 Overview Bacterial contaminants in milk (Spore formers) Why molecular sequencing in the food industry What is molecular sequencing Case Study: Molecular characterisation of Bacillus sporothermodurans Summary Implications for milk processors 2

3 Bacterial Contaminants in Milk Sporeforming bacteria that are present in milk are important because the formation of the spore by the bacterium allows it to be resistant to heat, freezing, chemicals, and other adverse environments during processing Although the vegetative cell is killed by these conditions, the spores can survive and need harsher conditions to be inactivated (implications on sensory and nutritional quality) Some of the bacteria that are important belong to the genus Bacillus, these microbes can grow under mesophilic temperatures (15 C about 40 C) or some grow under thermophilic temperatures ( 45 C - about 80 C ) These Bacillus species can cause food spoilage and some cause food-borne illnesses 3

4 Why Molecular Sequencing in the Food Industry Thermal processing conditions of UHT milk and other milk products have been based on phenotypic (physical) & biochemical attributes and mostly neglecting genotypic (genetic) attributes Strains that arise within species which can be captured genotypically can differ highly in their heat resistance and other process characteristics Better sequencing techniques of differentiating between strains (closely related species); use of rpob as compared to the old 16S rdna 4

5 What is molecular sequencing Process used to determine the nucleotide sequence in an organism. 5

6 Case Study: Molecular characterisation of Bacillus sporothermodurans in UHT milk 6

7 Bacillus sporothermodurans Non pathogenic (hopefully no pathogenic strains out there), spore forming Highly heat resistant strains and type of environment may play role in spore structure (Cremona & Buys, 2014); strains have been isolated from silage, feed concentrate, raw milk, UHT milk and powdered milk Growth of up to 10 5 CFU/ml in stored UHT milk (spoilage causing OR presence just contrary to GMP) Use of H 2 O 2 induces higher heat resistance of laboratory grown strains (Scheldeman et al., 2006); implications on industrial cleaning processes 7

8 Methodology 8

9 Results Genetic diversity using GTG5 PCR Fingerprinting M B M: Invitrogen 1Kb DNA ladder B: Blank 1: B. sporothermodurans (UHT milk, South Africa) 2: B. sporothermodurans (UHT milk, Germany) 3: B. sporothermodurans (UHT milk, Germany) 4: B. sporothermodurans (Raw milk, Belgium) 5: B. sporothermodurans (UHT milk, Belgium) 6: B. sporothermodurans (Feed concentrate, Belgium) 7: B. sporothermodurans (Feed concentrate, Belgium) 8: B. sporothermodurans (Silage, Belgium) 9: B. sporothermodurans (Feed concentrate, Belgium) 10: Paenibacillus lactis (Food micro lab, UP, South Africa) 11: B. pumilus (Food mirco lab, UP, South Africa) 12: B. cereus (Food micro lab, UP, South Africa) Figure 1: Gel image of B. sporothermodurans strains and other Bacillus species from milk obtained by GTG5-PCR 9

10 Results 2 rpob gene Single copy in bacteria 16S rrna gene Multiple copies in bacteria 10

11 Summary Comparison between sequences can allow for discrimination and characterisation between spoilage organisms. Level of discrimination dependent on differences in nucleotides a. 16S rdna is good for genus identification but may not discriminate between strains b. rpob provides appropriate number of unique bases for discrimination between strains 11

12 Implications for Milk Processors Developing effective screening tools using sequencing technology can help identify process resistant organisms (e.g. heat resistant strains) and help formulate interventions, hence improving milk safety and quality We found out that rpob gene sequencing is more effective at differentiating sub species as compared to the 16S rdna sequencing Silage, feed concentrate, raw milk, storage & processing equipment etc 12

13 Thank You 13