Microorganisms that Thrive in Dairy Processing Environments

Microorganisms are organisms that are too small to see with the human eye and can be found on all surfaces (Goff, n.d.). In food production facilities, microorganisms can be the cause of food spoilage. They can also prevent spoilage through fermentation, or they can cause sickness in humans. Intrinsic factors that affect the growth and survival of microorganisms in food include “nutrient content, moisture content, pH, available oxygen, biological structures and antimicrobial constituents” (Goff, n.d.). The intent of this research is to review literature published on the prevalence of bacterial foodborne pathogens found in milk and dairy environments.  

The manifestation of psychrotropic bacteria in raw milk is studied all over the world because of the challenges linked with controlling the growth of bacteria during cold storage and the subsequent effects on milk and dairy products. In the midst of psychrotropic bacteria, the genus Pseudomonas has been the major cause of several defects in dairy products (Oliveira, 2015). A study was conducted by Cleto et al., 2012 sampling areas that were hard to reach when using sanitizing products in which bacteria can survive and establish biofilms. Sampling sites included the holding cell, cold storage tank, pasteurizer and storage tank- transfer pump junction. The bacteria found following the sanitation procedure were mainly Pseudomonas spp., Serratia spp., Staphylococcus sciuri and Stenotrophomonas maltophilia. Phenotypic characteristics were evaluated to understand how the bacteria were able to survive in a mixed species environment (Cleto, 2012). It was determined that the Pseudomonas spp. Isolates are able to “produce proteases or lecithinases at high levels” (Cleto, 2012). Production of protease indicated an “inverse correlation with siderophore production” (Cleto, 2012). Additionally, Serratia spp. Isolated were “strong biofilm formers and spoilage enzyme producers.” The organisms found were not ordinary contaminants of milk, but they were also producers of proteins with the ability to reduce the “quality and shelf-life of milk” (Cleto, 2012). There was also a considerable amount of the Serratia and Pseudomonas spp. isolated from the pasteurizer that were “capable of secreting compounds with antimicrobial properties” (Cleto, 2012).

A study by Schoder et al., 2011 was conducted to understand the route of transmission of Listeria spp. in dairy farms manufacturing fresh cheese prepared from ovine and caprine raw milk. This test was also to assess the impact of Listeria monocytogenes mastitis on raw milk contamination. A total of 5,799 samples were used including 230 milk and milk product samples, 835 environmental samples, and 4,734 aseptic half-udder foremilk samples. Samples were collected from 53 dairy farms in the dairy intensive area of Lower Austria. The farms were picked for the study because raw milk processed to cheese was being distributed straight to consumers. There were 153 samples that tested positive for Listeria spp. which yielded a frequency of 2.6%, L. monocytogenes was discovered in 0.9% of the samples (Schoder et al. 2011). Samples that were negative for Listeria spp. include bulk tank milk, cheese, and half-udder samples. Seeing that udder samples tested negative for L. monocytogenes mastitis, it was omitted as a source of raw milk contamination. L. monocytogenes was prevalent at 30.2% of all the inspected farms (Schoder et al. 2011). Swab samples collected from working boots and fecal samples had a higher occurrence of L. monocytogenes (15.7 and 30.0%) when compared to swab samples collected from the milk processing environment (7.9%) (Schoder et al. 2011). There was an association found between the occurrence of L. monocytogenes in the silage feeding practices, the milk processing environment, and the animals. Sequestration of L. monocytogenes was “three to seven times more likely” from farms where silage was served to the animals during the course of the year compared to farms that did not feed silage to animals that year (Schoder, 2011).

A study by Aktağ et al., 2019 was conducted in order to understand the contents of Maillard reaction products in various UHT-treated milk products and to determine how formation of these products and lysine blockage is altered by the composition. To understand this, several commercial UHT milks including follow-on infant milks, whole milk, semi-skimmed and skimmed, protein fortified, and, lactose-hydrolyzed milks were examined. Among the Millard reaction products, “dicarbonyl compounds, 5-hydroxymethylfurfural, furosine,  N-ε-carboxymethyllysine and N-ε-carboxyethyllysine” were observed (Aktağ, 2019). Results indicate that fortification of UHT milks with protein and carbohydrate and the hydrolysis of lactose promoted to Maillard reaction. Within the dicarbonyl compounds, 3-deoxygoucosone formation turned out to be the dominant dicarbonyl compound in milk (Aktağ, 2019). It varies between 3.12-12.67 mg/L, 13.45-21.98 mg/L and 4.59-40.38 mg/L in “lactose hydrolyzed, lactose-hydrolyzed protein-fortifies and follow on infant milks whereas it was 0.22-0.40 mg/L in milks” (Aktağ, 2019). Correspondingly, 5-hydroxymethylfurfural were not able to be detected in milks, where mean 5-hydroxymethylfurfural concentrations were discovered to be 56.3 mg/L and 31.5 mg/L in protein fortified milks and lactose hydrolyzed protein-fortified milks. Respectively amounts of “N-ε-carboxymethyllysine and N-ε-carboxyethyllysine” of different UHT milks were higher than in milks (Aktağ, 2019).

Studies by Nam et al., 2005 have been conducted to detect the presence of Campylobacter jejuni and Salmonella spp. using SYBR Green real-time polymerase chain reaction in dairy farm environmental samples. In the study to determine the presence of Campylobacter jejuni, eighty-two dairy farm samples were tested including “fecal slurry, feed/silage, lagoon water, drinking water, bulk tank milk, farm soil, and bedding material” (Nam, 2005). Results from the real-time PCR assay detected C. jejuni in 25 out of the 82 samples, with 17 of the samples being culture positive for C. jejuni. All the samples that tested positive by standard culture methods also tested positive by the real-time PCR method. The SYBR Green real-time PCR assay ensures a specific, reproducible, and simple method for detecting C. jejuni in dairy farm environmental samples. For the detection of Salmonella spp., type strains and 116 non-Salmonella strains were evaluated. All of the Salmonella tested were “invA-positive and non-Salmonella strains yielded no amplification products” (Nam, 2005). To confirm the real-time PCR assay, an experiment was performed using both “spiked and naturally contaminated samples” (Nam, 2005). Samples were obtained from lagoon water, feed/silage, bedding soil, and bulk tank milk from dairy farms. Samples were spiked with 10(0) to 10(5) CFU/ml of Salmonella enteritidis and sensitivities for detecting Salmonella in these samples were 10(3) to 10(4) CFU/ml of inoculums in broth without the inclusion of enrichment. Ninety-three environmental samples with the inclusion of “fecal slurry, feed/silage, lagoon water, drinking water, bulk tank milk, farm soil, and bedding soil” were examined for the existence of Salmonella by real-time PCR (Nam, 2005). These results were compared with results from conventional culture methods. Results indicate that all samples evaluated tested negative for Salmonella by “both real-time PCR and standard culture method” (Nam, 2005). There were no false positive or false negative results found. 

A study by Gurung et al., 2013 was conducted to test the prevalence and antimicrobial susceptibility of Acinetobacter spp. from raw bulk tank milk samples from different locations in Korea. There were 2,287 bulk tank milk samples tested. From the samples tested, Acinetobacter spp. were isolated from 176 bulk tank milk samples. From the 176 Acinetobacter spp., 57 isolates were found to be Acinetobacter baumannii. These isolates indicated non-resistant to “cefepime, imipenem, meropenem, ciprofloxacin, levofloxacin, or colistin” (Gurung, 2013). However, isolates showed resistance to “amikacin, gentamicin, piperacillin, and cefotaxime” with values of 2.3, 7.4, 2.3, and 4.0% (Gurung, 2013). It was determined that Acinetobacter spp. were least susceptible to tetracycline (17.6%), ceftazidime (10.8%), trimethoprim-sulfamethoxazole (15.9%), and ampicillin-sulbactam (10.2%). A. baumannii strains were susceptible to the majority of the antimicrobial agents that were tested compared to other Acinetobacter spp. The Acinetobacter isolates presented 17 different patterns of antimicrobial resistance. Among the 17, the most frequent detected was ampicillin-sulbactam (n = 13), followed by tetracycline (n = 9), ceftazidime-tetracycline (n = 8), and trimethoprimsulfamethoxazole-tetracycline (n = 8) (Gurung, 2013). These results indicate that Acinetobacter, as well as A. baumannii strains exist in bulk tank milk and thus revealed the importance of inspecting bulk tank milk for foodborne pathogens and Acinetobacter spp. that could potentially be a concern to the public health.

Milk and other dairy products carry a “natural microbial flora” as well as other microorganisms based on the many products accessible in the French market (Brisabois, 1997). How a pathogenic bacteria contaminates a product depends on the type of product and the mode of production and processing. The root cause of contamination in milk can be of endogenous or exogenous origin. The cause could be after the milk has been excreted from the udder of an infected animal, through contact with infected crowds, or through the environment (Brisabois, 1997). Treatment or processing of milk can either prevent the growth of micro-organisms or it can cause it to replicate. Most bacteria involved in dairy products include “mycobacteria, Brucella spp., Listeria monocytogenes, Staphylococcus aureus and enterobacteria (including toxigenic Escherichia coli and Salmonella)” (Brisabois, 1997). Presently, systems of testing and surveillance are required to monitor the control of pathogenic bacteria in milk and dairy products. Preventative measures should be taken into account concerning the microbiological influence of pathogenic bacteria on milk as well as dairy products.

In a study conducted by Quiley et al., 2012, in order to extract bacterial DNA from raw milk and raw milk cheese, a comparison of methods was used. The objective of this study was to compare seven different methods that have been altered to remove the total DNA from raw milk as well as raw cheese with a view to its potential use for the PCR of bacterial DNA. Food sources used in this experiment included fresh milk samples collected in triplicate from a milking parlor kept under aseptic conditions and instantly placed in “isothermic conditions” (Quigley, 2012). Once ready, samples were transported to the lab in order to extract DNA. A “commercial, soft, raw milk cheese manufactured from cows milk” with starter samples were sampled as well (Quigley, 2012). The samples were in triplicate, under aseptic conditions. The seven methods used were Chemagic Food Basic kit, Milk Bacterial DNA Isolation kit, Modified QIAamp DNA stool mini kit, Power Food Microbial DNA Isolation kit, the Guanidine Thiocyanate method and the Lytic method. Of the seven methods used, Power Food Microbial DNA Isolation kit was the most suitable for DNA extraction from raw milk and raw cheese due to its rapid generation of highly concentrated DNA (Quigley, 2012). It was very pure and served as a great template for PCR amplifications that may be done in the future. This kit also extracted DNA from Gram-positive and Gram-negative pathogens. This has been the first report of the Power Food Microbial DNA Isolation kit used to remove DNA from dairy products (Quigley, 2012). 

There are many important factors that contribute to the “ripening and flavor development” of cheeses, yogurts, and soured creams. These factors include growth, location, and distribution in the developing casein matrix of dairy foods (Hickey, 2015). For us to be able to see these bacterial colonies as well as the environment that surrounds them, we must use a microscope. Using several different microscopy methods permits the immediate identification, “enumeration, and distribution of starter, non-starter and pathogenic bacteria in dairy foods” (Hickey, 2015). Confocal laser scanning microscopy is mainly used for the identification of bacteria location through the use of florescent dyes. Extensive studies should be conducted in relation to the growth of micro-gradients and localized ripening parameters in dairy products because of the location of bacteria at the protein-fat interface (Hickey, 2015). The use of microscopy techniques and florescent dyes will aid in identifying onset spoilage/pathogenic bacteria in product manufacture. These techniques will benefit both financially and in the safety of the product. 

Works Cited

Aktağ, I. G., Hamzalıoğlu, A., & Gökmen, V. (2019). Lactose hydrolysis and protein fortification pose an increased risk for the formation of Maillard reaction products in UHT treated milk products. Journal of Food Composition and Analysis, 84, 103308. doi: 10.1016/j.jfca.2019.103308

Brisabois, A., V. Lafarge, A. Brouillaud, M. L. de Buyser, C. Collette, B. Garin-Bastuji, and M. F. Thorel. 1997. Pathogenic organisms in milk and milk products: The situation in France and in Europe. Rev. Sci. Tech. 16:452–471.

Cleto S, Matos S, Kluskens L, Vieira MJ (2012) Characterization of Contaminants from a Sanitized Milk Processing Plant. PLoS ONE 7(6): e40189. https://doi.org/10.1371/journal.pone.0040189 

Goff, D. (n.d.). The Dairy Science and Technology. Retrieved from https://www.uoguelph.ca/foodscience/book/export/html/1884.

Gurung, M., Nam, H., Tamang, M., Chae, M., Jang, G., Jung, S., & Lim, S. (2013). Prevalence and antimicrobial susceptibility of Acinetobacter from raw bulk tank milk in Korea. Journal of Dairy Science, 96(4), 1997–2002. doi: 10.3168/jds.2012-5965

Hickey, C. D., Sheehan, J. J., Wilkinson, M. G., & Auty, M. A. (2015). Growth and location of bacterial colonies within dairy foods using microscopy techniques: a review. Frontiers in microbiology, 6, 99. doi:10.3389/fmicb.2015.00099 

Nam, H.-M., Srinivasan, V., Gillespie, B. E., Murinda, S. E., & Oliver, S. P. (2005). Application of SYBR green real-time PCR assay for specific detection of Salmonella spp. in dairy farm environmental samples. International Journal of Food Microbiology, 102(2), 161–171. doi: 10.1016/j.ijfoodmicro.2004.12.020

Nam, H., Srinivasan, V., Murinda, S., & Oliver, S. (2005). Detection of Campylobacter jejuni in Dairy Farm Environmental Samples Using SYBR Green Real-Time Polymerase Chain Reaction. Foodborne Pathogens and Disease, 2(2), 160–168. doi: 10.1089/fpd.2005.2.160 

Oliveira, G. B. D., Favarin, L., Luchese, R. H., & Mcintosh, D. (2015). Psychrotrophic bacteria in milk: How much do we really know? Brazilian Journal of Microbiology, 46(2), 313–321. doi: 10.1590/s1517-838246220130963

Quigley, L., O’Sullivan, O., Beresford, T., Ross, R. P., Fitzgerald, G., & Cotter, P. (2012). A comparison of methods used to extract bacterial DNA from raw milk and raw milk cheese. Journal of Applied Microbiology, 113(1), 96–105. doi: 10.1111/j.1365-2672.2012.05294.x 

Schoder, D., Melzner, D., Schmalwieser, A., Zangana, A., Winter, P., & Wagner, M. (2011). Important Vectors for Listeria monocytogenes Transmission at Farm Dairies Manufacturing Fresh Sheep and Goat Cheese from Raw Milk. Journal of Food Protection, 74(6), 919–924. doi: 10.4315/0362-028x.jfp-10-534