Microbiological Quality Control. Dr. Elke Just Nutrient Pad Sets in the Soft Drink Industry

Transcription

1 Microbiological Quality Control Dr. Elke Just Nutrient Pad Sets in the Soft Drink Industry

2 Over the past years, various methods for quick detection of microorganisms have been introduced on the market. What they all have in common is that they can only be used if the bioburden is relatively high, i.e /ml. In the beverage industry, however, it is important that trace infections, sometimes consisting of only a few microorganisms, can be detected per ml or per bottled unit. For such applications, the only feasible methods are ones that concentrate low microbial counts using relatively large volumes. The quickest method of concentration consists of filtration through a membrane filter (a type of filter that retains particles and microbes on its surface based on a screening effect). The filter is incubated on a special culture medium, thereby causing the relevant microorganisms to form visible colonies that can be counted. This process can be greatly simplified and is much more economical if nutrient pad sets ( NKS after the German abbreviation) are used instead of self-poured agar culture media. Nutrient pads are sterile, dehydrated culture media. They consist of biologically neutral cellulose pads that is free of inhibitors and contains no chemicals that might affect microbial growth. The pad is impregnated with culture medium, sterilized and preplated in disposable petri dishes under sterile conditions. Unlike agar plates, which are perishable and susceptible to contamination, nutrient pads can be stored for months, and some types can retain their good growth properties for years. To use the nutrient pads, just prewet them with sterile water. Demineralized water is better than tap water, which is often contaminated by chemicals. The manufacturer recommends using ml of water which should be dosed so that not all the water is absorbed by the pad. Instead a few extra drops should be allowed to collect in the petri dish. During growth, the microorganisms can draw on these water reserves. Thanks to this advantage, colonies on nutrient pads can often be analyzed earlier than those grown on other agar media. For example, when detecting yeast on Wort Medium, you can save half a day s time when you use nutrient pad sets instead of agar plates. Membrane Filtration Nutrient pad sets are used with the membrane filter method. Each nutrient pad set comes with the corresponding type of membrane filter; both are presterilized and individually packaged. If required, the nutrient pad can also be used as a streak plate. However, the membrane filter must always be used on the pad, otherwise distinct colony growth is not possible. What are the advantages of nutrient pad sets over self-poured agar culture media? They are easy to handle. Nutrient pad sets can be used in laboratories that do not have extensive microbiological equipment. No autoclaves, drying ovens or water baths are required. The sterile water for wetting the nutrient pad can be drawn into a syringe. An 0.2 mm disposable filter holder is attached to this syringe. They are economical. The time-consuming procedures for preparing culture media and cleaning and sterilizing vessels and equipment are eliminated. Consistent quality. Every batch of each type of nutrient pad is tested to compare it with the corresponding agar medium. This guarantees reproducible results for the user. Trouble-free storage. When stored in a laboratory cabinet, protected from light and moisture, most types of nutrient pad sets have a shelf life that lasts well beyond the use-by date stated on the package. It is not necessary or recommended to store them in the refrigerator. Highly versatile. The nutrient pad sets can be further modified by using additives in the wetting solution: The growth of acetic acid bacteria is promoted by adding approx. 5% ethanol to Wort or Orange Serum nutrient pads. The detection of yeasts can be narrowed down to osmophilic yeasts by adding glucose to the wetting solution. The general advantages of the membrane filter method also add to the benefits of nutrient pad sets: In contrast to the pour plate, drop or spiral plate methods, the sample volume to be tested can be varied; large volumes are no problem. Inhibitors, such as essential oils or disinfectants, can be flushed from the membrane filter after filtration. The incubated membrane filter can be used as a permanent record of the test by pasting it onto a report sheet (use solvent-free glue!). Always be sure to disinfect it beforehand by drying at about 60 C in an incubator. Growth Conditions in Beverages Bacteria, yeasts and molds are the microorganisms that can spoil the quality of soft drinks. In general, yeasts, which are relatives of fungi, require carbohydrate-rich nutrients in order to grow. Fungi and bacteria metabolize virtually all organic compounds. While bacteria and fungi are ubiquitous and settle in every environment, yeasts are usually limited to sugar-containing substrates like fruit, liquid and solid fruit preparations and sweet products. Except for mineral water, most soft drinks contain abundant amounts of sugar and therefore provide yeasts with the ideal growth conditions.

3 Yeasts and molds on a Wort nutrient pad Mixed culture of beverage-spoiling yeasts from soda on a Schaufus-Pottinger nutrient pad Lactic-acid bacteria on a VLB-57-S nutrient pad Among the other microbial flora, only specialized variants are capable of using the conditions available in soft drinks to multiply because inhibitory factors also come into play: Low ph. Fruit juice, cola beverages, sodas and other soft drinks have a ph between 3.5 to 2.5 which greatly inhibits bacterial growth. Lactic acid and acetic acid bacteria are primarily acid-tolerant. Lack of oxygen. Especially in carbonated beverages, the lack of oxygen suppresses aerobic forms, such as fungi and acetic acid bacteria. Fastidious microbes that require great amounts of protein, vitamins and growth promoting nutrients will find the conditions in soft drinks to be everything but optimum. Therefore, proteolytes, for example, Bacillus, Pseudomonas and Enterobacteriaceae, are excluded. The essential oils present in fruits and essences provide a certain degree of natural protection against microbial spoilage. Only few microorganisms rank among the typical beverage-spoiling microbes that can cause changes in appearance, taste and aroma. Table 1 lists the particular spoilage microbes that can be expected in the different beverages. The first to be mentioned are yeasts, particularly Saccharomyces, with high fermentation activity, Pichia, Hansenula, Torulopsis, and the slow-growing form, Brettanomyces. Lactic acid bacteria of the genera Lactobacillus and Leuconostoc will also spoil beverages. Molds and acetic-acid bacteria (e.g., Gluconobacter) are spoilage microbes found in beverages that are low in carbonation or non-carbonated. Commonplace microbes such as water microbes, yeasts with oxidative metabolism, spores of Bacillus or micrococci can be detected in beverages even though their microbial counts may not be very high. Table 2 shows the types of nutrient pad to be used for specific tests. Testing of Beverage Types Water Typical representatives of Gram-negative water microbes are pseudomonads, flavobacteria, Gluconobacter and Alcaligenes, Grampositive micrococci, spirilla, vibrios and the photosynthetic Cyanobacteria. Commonly encountered microbes that contaminate or affect the taste of beverages are Enterobacteriaceae and enteric cocci. Spore-forming bacteria can also be present in nutrient-rich water. Carbonated waters in which strictly anaerobic conditions prevail are less susceptible to microbial spoilage than slightly carbonated and carbonated bottled water. In compliance with the European Drinking Water Directive, the aerobic mesophilic colony count must be determined by testing 1 ml of water according to the pour-plate method. These regulations do not apply when internal in-line controls are conducted or additional samples are taken. In such cases, you can use larger sample volumes and therefore the much more practical membrane filter method. This similarly applies to the microbiological testing of bottled water, where 250-ml samples have to be tested to rule out the presence of problem microbes (E. coli, coliforms, fecal streptococci and Pseudomonas aeruginosa).

4 Table 1 Beverage type Microbial group to be tested Soft Fruit juice Concentrate Liquid sugar. Potable Water Vegetable General drinks sugar water in closed juice hygiene (according to containers Südzucker) Yeasts Osmotoleront yeasts Molds Aerobic mesophilic beverage-spoiling microbes Lactic acid bacteria Leuconostoc oenos + Leuconostoc mesenteroides + + Aerobic mesophiles, totol CFU count Thermophilic Bacillus spores + + Mesophilic Clostridium spores + + E coli and coliforms Fecal streptococci + + Pseudomonas aeruginosa + + Table 2 Test for Nutrient pad type Additive to Incubation conditions wetting solution Yeasts and molds Wort, Malt extract 25 C 2 5 days aerobic Schaufus-Pottinger 28 C 30 C 2 3 days aerobic Osmotoleront yeasts Wort, Malt extract Sugar 25 C 28 C 3 5 days aerobic Schaufus-Pottinger Beverage-spoiling microbes Orange Serum 30 C 2 3 days aerobic (aerobic mesophiles) normal or ph 3.2 Lactic acid bacteria Orange Serum 25 C 28 C 2 3 days microaerophilic VLB-S7-S 25 C 28 C 3 5 days to anaerobic Acetic acid bacteria Orange Serum 3% 5% Ethanol 30 C 5 7 days aerobic Wort Leuconostoc oenos Tomato Juice 30 C 7 days microaerophilic ( Jus de Tomate ) Leuconostoc mesenteroides Weman 28 C 30 C 2 3 days aerobic Aerobic mesophiles, Standard 30 C 2 3 days aerobic total CFU count Standard TTC Thermophilic spore formers Glucose Tryptone 55 C 1 2 days aerobic (after pretreatment 10 at 80 C) Mesophilic Clostridia Standard 37 C 2 3 days strictly anaerobic (after 10 at 80 C) Caso E. coli and coliform microbes Endo and Tergitol 37 C 20 ±4 hours aerobic Fecal streptococci Azide 37 C 1 2 days aerobic Pseudomonas aeruginosa Cetrimide 37 C 2 days aerobic

5 Soft Drinks and Fruit Juices Water microbes usually pose no problems in the majority of soft drinks because they are suppressed by the inhibitory factors already mentioned. Only acid-tolerant microaerophilic microbes can multiply in beveragespoiling proportions. This propagation tends to start after long periods in which the microorganisms have had time to adapt to the living conditions of the substrate. For this reason, it is important to be able to detect trace infections, i.e. of only a few microbes per bottle, with reliable accuracy. This can be performed simply by passing the contents of an entire bottle through a membrane filter. Fruit juices, cola beverages and sodas are especially easy to filter in this manner. However, beverages clouded with fruit pulp, especially viscous fruit nectars, are difficult to filter. Depending on the size of the fruit pulp particles, the filterable amount can be increased in some cases by prefiltration. Despite this, the prefilter must also be incubated, at least for yeast detection, in order to include the count of relatively large yeast cells. Adequate sample volumes of beverages clouded with fruit pulp can be obtained when a membrane filter with a larger pore size is used as a microbe detection filter. Pore sizes of 1.2 mm to 3 mm, and sometimes even up to 8 mm, can be used to detect yeast infections and allow the fruit pulp to pass through the membrane. Fruit Juice Concentrates and Syrup Since these products are generally protected from microbial spoilage, precautions must be taken to prevent contamination by only a few special variants that are spoilage microbes. These mainly involve yeasts, particularly the osmotolerant forms, and certain slime-forming bacteria. Because microorganisms do not affect the concentrate or syrup itself, but are only capable of accelerated growth and increased fermentability when diluted to a final drinking concentration, microbiological control in concentrates is targeted more at establishing the microbial count than at determining product spoilage. Sugar This similarly applies to sugar, especially sugar in liquid form. In addition to the sporeformers which only multiply above a ph of 4.5, the slime-forming bacteria Leuconostoc mesenteroides poses the greatest spoilage risk to sugar. This bacterium is also called the frog s egg bacillus because it can produce gelatinous, slime-coated cell masses. Vegetable Juices Vegetable juices have a ph above 4.5 so that the presence of any spore-forming bacteria will cause spoilage problems. There is a particular risk when sterilization has been inadequate. Any remaining spores will survive for long periods, and months later can ultimately develop into viable cells. In anaerobic sporeformers, growth is accompanied by gas formation and swelling of the container; aerobic spore-formers can also cause flat-sour spoilage especially when the product is stored at high temperatures. General Hygiene Monitoring General monitoring of hygiene conditions is imperative for in-line control during beverage production. This involves testing the empty bottles, the bottle cleaning machine, the filling machine as well as all cleaning and disinfectant solutions and the rinse water. The rinsing method is the most suitable technique for bottle monitoring: fill the bottle with 50 ml of sterile rinsing solution, such as physiological saline solution or 0.1% peptone water, close and shake well to remove any microbes from the inner walls. Then pour the contents of the bottle through a membrane filter, rinse well and incubate on Standard or Standard TTC nutrient pad sets. Caps can also be shaken with peptone water or physiological saline solution. The cleaning disinfectant and rinse water are also membrane filtered. Always make sure that the filter is rinsed thoroughly, since traces of residual disinfectant can delay or suppress microbial growth. Fillers and capping machines are critical sites that can harbor secondary infections. Samples can be taken with sterile cotton swabs which are then rinsed. This liquid is subsequently tested using a membrane filter. Meticulous testing of the bottled product, all raw materials, every production step and hygienically critical sites is indispensable for producing beverages that have the microbiological stability required to withstand long storage periods and transport times without undergoing any changes in appearance, taste or aroma. References: Baumgart I., Mikrobiologische Untersuchung von Lebensmitteln, 1986, Behr. Schmidt-Lorenz W. Sammlung von Vorschriften zur mikrobiologischen Untersuchung von Lebensmitteln, 1980, Chemie. Pichhardt K. Lebensmittelmikrobiologie, 1980, Springer. Das Südzucker Handbuch, 2nd Edition [ Südzucker Manual ], Sartorius Nutrient Pad Sets and Culture Media, 1984.* * Updates of this literature are available in German only.

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