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Discipline: contamination; Keywords: biofilms, exopolysaccharides, disinfectants, enzyme treatment, dairy equipment.
Microbial contamination is generally associated with the presence of microbial biofilms attached to the inner surfaces of tanks, pipes and milk processing lines. Biofilms in the dairy industry can be very harmful because of impaired quality of the finished products due to secretion of undesirable enzymes (e.g., proteases, lipases) within the biofilm, increased fluid frictional resistance of surface and increased corrosion rate of equipment surfaces. They are resistant to antimicrobial agents and therefore their removal has become a major problem in the dairy industry, since their detection is not always possible. Furthermore, the increase in temperature during transportation of bulk raw milk promotes biofilm formation in the tank. Removal is difficult due to the accumulated exopolysaccharides and the heterogeneity of cells forming the biofilm matrix.
Considerable efforts have been made to optimise cleaning and disinfection of dairy processing devices and equipment, including the use of sodium hydroxide (NaOH) and nitric acid (HNO3) in the cleaning-in-place method. Other chemical agents, including potassium hydroxide and hydrochloric acid, are also used. However, there is pressure on manufacturers to reduce effluent disposal mainly when using chemical cleaners that cause a negative environmental impact. Therefore alternative methods need to be investigated which was the purpose of the study of Dr S. Mnif and colleagues. The main objectives of their work were (i) to isolate microorganisms that colonise the internal surfaces of raw milk tankers, (ii) to study their ability to form biofilms, and (iii) to evaluate the resistance of the biofilms to cleaning products commonly used in the dairy industry. The results of the study were published in the International Dairy Journal, Volume 100 of 2020, No 104560, the tile being: Enzyme-based strategy to eradicate monospecies Macrococcus caseolyticus biofilm contamination in dairy industries
In the study, 197 bacterial strains were isolated from the milk collection centre and neighbouring farms. Six strains were selected according to their ability to form a thick biofilm on polystyrene microplates and on stainless steel surfaces. The biofilm formed by Macrococcus caseolyticus was used as a model for the evaluation of eradication efficacy. Protease, lipase, cellulase, a-amylase and DNase enzymes were applied to treat the biofilms. The buffer composition was also improved to increase the diffusion of molecules inside the formed biofilm and to kill bacteria by surfactin addition.
Of the 197 bacterial strains isolated, 6 strains revealed strong and mature biofilm formation on polystyrene surfaces after incubation at 300C. The strains in the biofilm were closely related to various genera, including Enterococcus, Lactococcus, Serratia and Macrococcus. The majority of these strains were protease producers. The main findings of the study were the following. (a) NaOH and HNO3 which are normally used for removal of mono-species biofilm showed that the biofilms obtained with strains 6B1, 20B1 and 27B1 resisted treatments to a certain extent with HNO3 and NaOH both individually and combined. The effect of the combination of NaOH and HNO3 was also studied. Although the bacterial removal was not complete, the synergic effect between the alkaline and acid cleaning agents was remarkable. The synergy did not remove all cells and biofilm residues were still attached on the surface. The moderate removal of the biofilm by these agents may be due to the presence of a protective exopolymeric matrix. Furthermore, biofilms form a gel phase where micro-organisms live inside, and therefore the matrix acts as a barrier. This could increase the resistance of biofilm cells to detergents and biocides. (b) Biofilms obtained from the 6 selected strains were treated with the alkaline detergent BASO CTC (1%, v/v). BASO CTC was partially inefficient in achieving total biofilm removal. (c) The results obtained from treatment with enzymes showed that protease, lipase and DNase at high concentration provided significant biofilm elimination. (d) The comparison between biofilm elimination after enzyme treatment with the used appropriate buffer for each enzyme (PBS 1, TriseHCl 1 and 50 mM citrate) and with distilled water control was also tested. The results revealed a potential action of the three buffers against biofilm with slight efficacy of PBS compared with TriseHCl and the citrate buffer, respectively. (e) After testing the effect of each enzyme individually, enzyme mixtures were developed to test their synergetic effect in removing biofilm formed in a TriseHCl buffer containing surfactin. The combination of enzymes targeting the exopolymeric matrix in the presence of surfactin resulted in improved biofilm removal. This combination could be an alternative to efficient biofilm cleaning.
The main message from this study is that conventional cleaning methods to prevent biofilms in dairy equipment may no longer be adequate and that alternative methods such as enzyme treatment may be required.