Effect of extended shelf life milk processing on the bacterial composition associated with the nozzles of filling machines

Abstract

One of the possible reasons for post-contamination of extended shelf life (ESL) milk could be attachment and formation of biofilms on stainless steel pipe surfaces. In South Africa, ESL milk processors are still facing challenges extending the shelf life beyond 14 days. It is hypothesized that post-contamination along the milk processing line is responsible of reducing the shelf life of ESL milk. This assumption was investigated by assessing the microorganisms associated with the nozzles of aseptic filling machines post CIP process and this study was designed and divided into two phases. Phase one involved isolation and characterisation of the bacteria associated with the nozzles of aseptic filling machines after CIP process with the aim of determining the diversity of microorganisms attached to the nozzles of aseptic filling machines. Swab samples were collected from a plant processing ESL milk. Twenty swabs were taken from ten different nozzles of aseptic filling machines during 4 visits (n=80). The swab samples were plated on the day of sampling. A total bacterial count ranging from 1.75 - 1.95 log CFU/cm3 with an average of 1.81 log CFU/cm3 (n=80). MALDI-TOF revealed a high percentage of Gram-positive rods (69%), followed by Gram-positive cocci (20%) and then Gram-negative rods (7%). The Gram-positive rods belonging to genus Bacillus were identified as Bacillus cereus, followed by B. pumilus, B. subtilis and Paenibacillus spp. The Gram-positive cocci included S. hominis, S. epidermidis, Micrococcus luteus and Anaerococcus spp. The Gram-negative rods were identified as Acinetobacter junii. The prevalence of Bacillus spp. noted in the nozzles of ESL aseptic filling machines is attributed to their ability to resist heat treatment during CIP process and their ability to attach to stainless steel surfaces. There was a degree of similarity in terms of MALDI-TOF MS profiles for the strains of B. cereus, Staphylococcus spp. and Paenibacillus spp. originating from the nozzles of aseptic filling machines and the packaged ESL milk product. Furthermore, these isolates show close relatedness. These bacteria are likely to originate from the nozzles, dispensed into the final ESL milk during filling process. Phase two aimed at determining cell surface hydrophobicity of the isolates originating from the nozzles of aseptic filling machines. Co-currently the study further determined the ability of B. cereus, S. epidermidis, M. luteus and Paenibacillus spp. to attach and form biofilms on stainless surfaces. The bacterial strains were isolated from the nozzles of aseptic filling machines. The degree of hydrophobicity of the spore formers ranged from 8-91% while non-spore formers ranged from 6-67%. Hydrophobicity of S. hominis, S. epidermidis, Acinetobacter junii and Arthrobacter castelli differed significantly (p?0.05). Hydrophobicity of B. pumilus, Paenibacillus spp. and B. cereus (p?0.05) differed significantly. Spore formers showed the highest hydrophobicity to solvents, this can be contributed to their hydrophobic nature and their ability to attach to stainless surfaces. A continuous flow reactor system was used to grow biofilm of the isolates in skim milk. The skim milk was inoculated with spore suspension of B. cereus, Paenibacillus spp. and bacterial suspension of M. luteus and S. epidermidis. The bacterial suspensions were run separately over a period of 20h at 37°C. Stainless steel strips were submitted to Scanning Electron Microscopy (SEM) after 22h. The results suggested that spores of B. cereus and Paenibacillus spp. can only attach whilst M. luteus and S. epidermidis can attach and form biofilms on stainless steel. The ability of these isolates to form biofilms on stainless steel strips could be the main cause of contamination of ESL milk. Strains of Bacillus spp. can form biofilm on stainless steel and limit the shelf life of milk and milk products. However, toxins produced by some of these strains of B. cereus might be contagious to humans. The results confirmed that one of the reasons of contamination of ESL milk could be the ability of B. cereus to attach to stainless surfaces and M. luteus to form biofilms. Over and above the fact that the spores of B. cereus can lead to spoilage of milk and milk products, a concern is that the toxins produced by some of the strains of B. cereus are detrimental to human health.