Edmund A. Zottola

Microbial biofilms in the food processing industry—Should they be a concern?

Biofilm formation will occur on solid surfaces in contact with a liquid. Organic and inorganic material in the liquid sediment onto the solid material. Subsequently, biologically active microorganisms will be attracted to this conditioned surface and adhere to it. The microbial cells will initiate growth, form an attachment matrix and develop into a complex community forming a microbial biofilm. Such microbial biofilms are common on solid surfaces in contact with many different kinds of liquids, fresh water, sea water, oil, milk and so on. These biofilms may be of benefit or be detrimental to the environment where they form. The goal of this review has been to summarize the literature on the development of microbial biofilms in these different environments with particular emphasis on what occurs in the environment of a food processing plant. Methods to control adherent microorganisms and subsequent biofilms in the food processing plant are discussed. It is apparent from the data that has been reviewed that the potential for the development of microbial biofilms in the environment of the food processing plant exists. However, the cleaning and sanitizing practices carried out in the food industry have been shown to control biofilm formation on food contact surfaces. Microbial attachment has been shown to occur on non-food contact surfaces and these attached microbes, if left undisturbed, will form biofilms. The potential for contamination of food with undesirable spoilage and pathogenic bacteria from attached microbes and biofilms exists in these food processing systems. Biofilm formation on non-food contact surfaces needs to be studied further and methods developed to prevent and control these biofilms.

Adherence to stainless steel by foodborne microorganisms during growth in model food systems

Biofilm formation on stainless steel by Salmonella typhimurium, Listeria monocytogenes, Escherichia coli O157:H7, Pseudomonas fragi and Pseudomonas fluorescens during growth in model food systems was studied. Test growth media included tryptic soy broth (TSB), diluted TSB (dTSB), 1% reconstituted skim milk (RSM) and diluted meat juice (DMJ). Adherent cells were stained with acridine orange and enumerated using epifluorescent microscopy and computerized image analysis. Cells were observed on the stainless steel surface after 1 h in all of the media. However, the increases in the number of adherent cells over time was seen only with S. typhimurium in DMJ, E. coli O157:H7 in TSB, dTSB and DMJ, P. fragi in RSM and P. fluorescens in RSM. The medium which produced the highest observed level of adherent cells was different for each microorganism.

Biofilms in food processing

Abstract Microbial colonization of surfaces (biofilms) have been documented in many environments. Recently, researchers have suggested that biofilms may be a source of contamination in food processing environments. This review will discuss some historical aspects of biofilms, possible mechanisms for the adherence of bacteria to surfaces, methods for studying biofilms and problems adherent microorganisms may cause in food processing.

Biofilm formation by Listeria monocytogenes utilizes a primary colonizing microorganism in flowing systems

Listeria monocytogenes serotype 3a and Pseudomonas fragi ATCC 4973 were examined for attachment capability and biofilm development on glass coverslips under flowing systems. Tryptic soy broth supplemented with yeast extract was the growth medium. A continuous flow slide chamber was developed for in situ observations using phase-contrast microscopy. Glass coverslips were examined by epifluorescent and scanning electron microscopy for biofilm formation. The ultrastructure of attached test organisms was examined for the presence of exopolymers using transmission electron microscopy. In pure cultures, attachment of L. monocytogenes to glass coverslips was sparse, while P. fragi accumulated on glass coverslips as a confluent layer of cells. When L. monocytogenes was grown in mixed culture with P. fragi , an exopolymer-producing microorganism, attachment and microcolony formation by L. monocytogenes was enhanced. Results suggest that under flowing conditions the presence of an exopolymer-producing microorganism may be more important than hydrophobicity, surface charge, or flagellar movement in attachment of L. monocytogenes to inert surfaces.

Scanning electron microscopy of microbial attachment to milk contact surfaces

Milk contact surfaces were observed by scanning electron microscopy (SEM) techniques for possible microbial attachment. Cultures of Pseudomonas fragi 4973, Staphylococcus aureus JAL, Streptococcus lactis C2, Streptococcus cremoris and Lactobacillus bulgaricus RR inoculated onto glass coverslips or stainless steel chips were examined. Stainless steel surfaces displayed many possible harborages for microbial colonization. SEM examination of P. fragi 4973 showed development of fibrous material, with numerous stick-like projections extending from the cell to the glass or stainless steel surface. These apparent attachment appendages became more pronounced as contact time increased. S. aureus , S. lactis , S. cremoris and L. bulgaricus did not display such fibrous material.

Growth media and surface conditioning influence the adherence of Pseudomonas fragi, Salmonella typhimurium, and Listeria monocytogenes cells to stainless steel

Microorganisms have been shown to adhere to food-contact surfaces and may provide a route for the contamination of processed food. To better understand this phenomenon, the effects of growth media and surface conditioning on the adherence of Pseudomonas fragi , Salmonella typhimurium and Listeria monocytogenes cells to stainless steel were studied. The microorganisms were grown in tryptic soy broth (TSB), 1% reconstituted skim milk (RSM) and RSM with 1% sucrose (RSM + S). Stainless-steel surfaces were conditioned by immersion in growth media for 1 h and then were rinsed in phosphate-buffered saline (PBS) prior to the adherence assay. After growing in each medium, cells were harvested, resuspended in PBS, and then allowed to contact the stainless steel for 30 min. Adherence was quantified by acridine orange-staining the cells and viewing under epifluorescence microscopy. Growth media had little influence on adherence to stainless steel that had not been preconditioned. P. fragi and L. monocytogenes cells adhered in the highest numbers when grown in RSM plus sucrose. S. typhimurium cells showed the highest level of adherence when grown in TSB. Analysis of variance yielded P values of less than 0.01, indicating that both growth media and surface conditioning were significant in the level of adherence observed.

Utilization of cheddar cheese containing nisin as an antimicrobial agent in other foods

Cheddar cheese made with nisin-producing lactococci contained between 400 and 1200 IU of nisin per gram of cheese. Cultures used were Lactococcus lactis ssp. cremoris JS102, a nisin-producing transconjugant developed in the laboratories of Dr. L.L. McKay and Lactococcus lactis ssp. lactis NCDO 1404 obtained from the National Collection of Food Bacteria, Reading, England. Pasteurized process cheese spreads with 53% and 60% moisture and 0, 301 and 387 IU nisin/g were manufactured and inoculated with 2000 spores of Clostridium sporogenes PA 3679 during manufacture. The heat process did not reduce nisin activity in the cheese spreads. The spreads were incubated at 22 degrees and 37 degrees C for 90 days. Spoilage was detected by the presence of gas and/or odor in the packages. The shelf-life of the nisin-containing cheese spreads was significantly greater than that of the control cheese spreads at the lower temperature at both moisture levels, whereas the keeping quality of the higher moisture cheeses at the higher temperature was not significantly different. Club cheese or cold pack cheese spreads with moisture levels of 44% and 60% and 0, 100 and 300 IU nisin/g were made. These cold processed cheese spreads were inoculated with 1000 cfu per g of Listeria monocytogenes V7, Staphylococcus aureus 196E and spores of C. sporogenes PA 3679. Heat shocked spores of PA 3769 at the same number were added to separate lots of the cheese spread. The cold pack cheese spreads were incubated at 23 degrees and 37 degrees C for up to 8 weeks. Samples were taken weekly and analyzed for surviving organisms. Significant reductions in numbers of the non-sporeforming test microbes were noted at both temperatures, at both moisture levels and both levels of nisin. Heat shocking the spores was needed to show reduction in numbers during the storage of the cold pack cheese spreads. The data obtained in this study suggest that the use of nisin-containing cheese as an ingredient in pasteurized process cheese or cold pack cheese spreads could be an effective method of controlling the growth of undesirable microorganisms in these processed foods.

Isolation and identification of adherent gram-negative microorganisms from four meat-processing facilities

Biofilms are described as a matrix of microorganisms which have adhered to and colonized a surface. Once formed, biofilms are difficult to remove and may be a source of contamination in food-processing environments. In this study, stainless-steel chips were fixed to surfaces adjacent to food-contact surfaces and cast-iron chips were suspended in the floor drains of four meat-processing plants. Biofilm formation was quantified by staining the attached cells and viewing them under epifluorescence microscopy. The stainless-steel and cast-iron chips removed from the plant environment showed some attached microorganisms. Floor drains appeared to provide an excellent environment for the formation of biofilms. Pseudomonas , Klebsiella , Aeromonas , and Hafnia species were identified as gram-negative microorganisms associated with the test surfaces.

Bacteriocidal activity of sanitizers against Enterococcus faecium attached to stainless steel as determined by plate count and impedance methods.

Enterococcus faecium attached to stainless steel chips (100 mm2) was treated with the following sanitizers: sodium hypochlorite, peracetic acid (PA), peracetic acid plus an organic acid (PAS), quaternary ammonium, organic acid, and anionic acid. The effectiveness of sanitizer solutions on planktonic cells (not attached) was evaluated by the Association of Official Analytical Chemists (AOAC) suspension test. The number of attached cells was determined by impedance measurement and plate count method after vortexing. The decimal reduction (DR) in numbers of the E. faecium population was determined for the three methods and was analyzed by analysis of variance (P < 0.05) using Statview software. The adhered cells were more resistant (P < 0.05) than nonadherent cells. The DR averages for all of the sanitizers for 30 s of exposure were 6.4, 2.2, and 2.5 for the AOAC suspension test, plate count method after vortexing, and impedance measurement, respectively. Plate count and impedance methods showed a difference (P < 0.05) after 30 s of sanitizer exposure but not after 2 min. The impedance measurement was the best method to measure adherent cells. Impedance measurement required the development of a quadratic regression. The equation developed from 82 samples is as follows: log CFU/chip = 0.2385T2-0.96T + 9.35, r2 = 0.92, P < 0.05, T = impedance detection time in hours. This method showed that the sanitizers PAS and PA were more effective against E. faecium than the other sanitizers. At 30 s, the impedance method recovered about 25 times more cells than the plate count method after vortexing. These data suggest that impedance measurement is the method of choice when evaluating the number of bacterial cells adhered to a surface.

Scanning electron microscopic examination of Yersinia enterocolitica attached to stainless steel at selected temperatures and pH values

Attachment of Yersinia enterocolitica to stainless steel surfaces at 35, 21, and 10°C was investigated using scanning electron microscopy (SEM). Cells adhered at all three temperatures, but, in general, the greatest number of adhered cells were observed at pH 8 and 21°C. Multi-flagellated cells were noted under these growth conditions. When grown at pH 9.5 and 21°C, fibrils were observed between cells and extending to the stainless steel surface. Fewer cells with flagella were seen at this pH. Adherence may be related to the flagella and any exopolymer surrounding the cells.