Beverages

Carbonated Soft Drinks

Carbonated soft drinks are nonalcoholic beverages that contain water, carbon dioxide (2.0 to 4.0   volumes), nutritive or nonnutritive sweeteners, acidification agents (citric or phosphoric acids), colors, foaming and emulsifying agents, natural or synthetic flavors, and preservatives (benzoic and sorbic acids and their salts). Some drinks may also contain fruit juices, tea powder and be fortified with various vitamins (Compendium of Methods for the Microbiological Examination of Foods, 2001). They have a pH range from 2.5 to 4.0. Colas and ginger ales generally have the lowest pH (between 2.5 and 3.1) while root beers and cream sodas have the highest pH. Most of them are carbonated. In the United States, they are usually not pasteurized.

Due to the low pH, most bacteria, including pathogenic bacteria, die off rapidly in this type of beverages. Only aciduric bacteria, yeasts and molds are of significance that are rarely a public health issue (Compendium of Methods for the Microbiological Examination of Foods, 2001, 2015). Yeasts are the most important spoilage organisms since they can grow at acidic pH and under anaerobic conditions. Zygosaccharomyces bailii can tolerate high concentrations of preservatives as well as moderately high carbonation. Other yeasts isolated from spoiled products are Saccharomyces, Torulopsis, Brettanomyces (Dekkera), Candida, Kloeckera, Hansenula, and Pichia. Certain lactic acid bacteria (Lactobacillus and Leuconostoc) can also grow under these conditions when sufficient nutrients are present such as in the soft drinks containing fruit juices. These bacteria are also resistant to benzoic and sorbic acids.  Growth results in the production of haze, sediment, off-flavors, and gas. When yeasts cause spoilage, sufficient carbon dioxide is produced to burst the bottle or can. Some Lactobacillus and Leuconostoc spp. can cause ropiness due to the production of dextrans. The slime balls range in size from barely detectable to that of a large pea. Acetic acid bacteria and molds (Aspergillus, Penicillium, Mucor, and Fusarium) can grow only when dissolved oxygen is present as is in the case of noncarbonated soft drinks. Molds grow as delicate, fluffy, cottony white masses suspended in the liquid. From lack of oxygen, fruiting bodies cannot form. The molds grow slowly in bottled beverages and often are detected only after the beverages are in the marketplace. Mold spoilage, frequently traced to improperly sanitized equipment or environmental contamination, often is restricted to the products of a single bottling plant and can result in recall of hundreds of cases.

In the case of carbonated soft drinks, the flavor concentrates, water and other ingredients are rarely the source of yeasts and lactic acid bacteria (Compendium of Methods for the Microbiological Examination of Foods, 2001, 2015). Soft drinks that contain fruit juices, tea, etc. are more susceptible to spoilage than colas. Buildup of microorganisms in the plant environment is the major cause of spoilage. Good sanitation practices are critical for the preservation of soft drinks.

In our experience, liquid sweeteners can be a significant source of yeasts and molds and should be monitored. Packaging materials such as cans and bottles can also be a source of contamination if not adequately rinsed before filling.

Recommended tests

Test water, sugars, beverage syrups, and finished products for acid-producing bacteria, yeast and molds, preservative-resistant yeasts (PRY) (in preserved products), Brettanomyces/Dekkera (in carbonated products) and preservative-resistant molds (PRM) (in products not carbonated and preserved).

Water tested for heterotrophic count and total coliforms.

Swabs from key locations as well as rinse water tested for aciduric bacteria, yeasts and molds.

Air sampling to check air quality.

Preserved Drinks

Drinks containing fruit juices, tea, etc. with preservatives are usually not pasteurized even though some manufactures pasteurize them. Only aciduric bacteria, yeasts and molds  are of significance. Yeasts are the most important spoilage organisms since they can grow at acidic pH and under anaerobic conditions. Zygosaccharomyces bailii can tolerate high concentrations of preservatives as well as moderately high carbonation. Other yeasts isolated from spoiled products are Saccharomyces, Torulopsis, Brettanomyces, Candida, Kloeckera, Hansenula, and   Pichia. Certain lactic acid bacteria (Lactobacillus and Leuconostoc) can also grow under these conditions when sufficient nutrients are present such as in the soft drinks containing fruit juices. These bacteria are also resistant to benzoic and sorbic acids. Growth results in the production of haze, sediment,   off-flavors, and gas. When yeasts cause spoilage, sufficient carbon dioxide is produced to burst bottle or can. Some Lactobacillus and Leuconostoc spp.  can cause ropiness due to the production of dextrans. The slime balls range  in size from barely detectable to that of a large pea. Molds grow as   delicate, fluffy, cottony white masses suspended in the liquid. From lack of   oxygen, fruiting bodies cannot form. The molds grow slowly in bottled beverages and often are detected only after the beverages are in the marketplace. Mold spoilage, frequently traced to improperly sanitized equipment, often is restricted to the products of a single bottling plant and can result in recall of hundreds of cases.

Recommended tests

Monitor ingredients for aciduric bacteria, yeasts and molds, preservative-resistant yeasts (PRY) and preservative-resistant molds (PRM).

Swabs from key locations as well as rinse water and finished product tested for aciduric bacteria, yeasts and molds, preservative-resistant yeasts (PRY) and preservative-resistant molds (PRM).

Air sampling to check air quality for yeast and molds as well as preservative-resistant yeasts (PRY) and preservative-resistant molds (PRM). For recommended media, contact BCN Labs.

Pasteurized Drinks

Drinks containing fruit juices, teas, etc. with no preservatives added that are usually pasteurized at less than 200 -205 F have the same kind of spoilage that sports drinks have. Their main spoilage is due to the survival of the ascospores of the heat-resistant molds of the genera Paecilomyces (syn. Byssochlamys), Talaromyces, and Aspergillus species with neosartorya morph (syn. Neosartorya). Other molds that have been isolated from pasteurized fruit drinks are Byssochlamys spectabilis/Paecilomyces variotii and Penicillium arenicola.

In this case, the fruit concentrates such as the ones of white grape and strawberries, are one of the sources of heat-resistant mold spores as well as  Alicyclobacillus guaiacol producing (ACB) spores. In the case of the drinks that have additional sweeteners, the same considerations as in the case of the sport drinks should be made. Plant environment, air quality, and bottle cleanliness should be taken into consideration.

Recommended tests

Incoming fruit concentrates  and sugars tested for yeast and molds as well as heat-resistant  molds (HRM).

Air sampled for yeast, molds and heat-resistant molds (per at least 1000 L of air with heat-shock).

Swabs to check efficacy of sanitation procedures and environmental sponges for HRM.

Additional tests

Heat-resistant mold spores per 100 g sweetener or juice concentrate.

Alicyclobacillus guaiacol positive (ACB) if spoilage is suspected.

Sport Drinks

Sport drinks are nonalcoholic drinks that contain flavorings, colors, citric acid, electrolytes, and sweeteners (sucrose and/or high fructose corn syrup). They usually do not contain any preservatives or carbonation. The pH range of sport drinks is from 2.5 to 3.5. Citric acid is the most widely used  acidulant. The carbohydrate (sucrose and fructose) content ranges from 2 to   5%.

To prevent growth of potential spoilage microorganisms, sport drinks are usually  pasteurized at temperatures above 185oF and hot filled at above 170oF. These  temperatures are high enough to kill bacteria, yeasts and non-heat resistant mold spores (conidia). However, bacterial and heat-resistant mold spores (ascospores) can survive this treatment. Bacterial spores would not be able  to germinate and grow at the low pH of most sport drinks. The molds most frequently isolated belong to the genera Byssochlamys,Talaromyces, and Neosartorya. Another mold that has been isolated from pasteurized drinks Byssochlamys spectabilis/Paecilomyces variotii (Samson et al., 2003).

Heat-resistant  ascospores can be activated by this heat treatment thus allowing germination.   Heat-resistant molds such as Byssochlamys fulva and B. nivea possess the ability to grow at very low oxygen tensions. It has been reported that in the presence of very low levels of oxygen, these species appear to grow  anaerobically and produce C02. A small amount of oxygen contained in the headspace of a bottle or dissolved in the liquid can provide sufficient  oxygen for these fungi to grow. Molds grow as fluffy, cottony white masses  suspended in the liquid. It usually takes about two weeks for them to be noticeable..

Ingredients  such as the liquid sweeteners (sucrose and fructose) can be a source of  yeast, molds, heat-resistant molds and even Alicyclobacillus guaiacol positive (ACB) spores.  Osmophilic yeasts (yeasts that can grow at high concentrations of sugar) can grow in liquid sugar during storage in tanks, especially in areas where  condensate forms. At the same time, heat-resistant molds as well as non-heat  resistant species can grow on the walls of the sugar tanks. The higher the  microbial load of the sugar, the higher the microbial load in the product.  The microbial load of the product affects the effectiveness of the pasteurization step. Conidia of the heat-resistant molds can be isolated from the air quite frequently. These should be considered as an indication that ascospores may also be present. These ascospores can get inside the bottles before the  filler and cause spoilage if the filling temperature is maintained at 80 C.  These spores have been also isolated from empty bottles. Therefore, empty  bottles can be a source of contamination if not adequately rinsed before filling.

Environmental  buildup of microorganisms in the plant environment can be another cause of  beverage contamination. If the concentration of these molds in air reach to a certain point, the plant environment can be fogged using chlorine dioxide.

Recommended tests

Incoming sugars tested for yeast and molds as well as heat-resistant mold (per 10 g).

Air sampled for yeast, molds and heat-resistant molds (per at least 100 L).

Swabs to check efficacy of sanitation procedures.

Additional tests

Heat-resistant mold spores per 100 g sweetener.

Alicyclobacillus guaicol positive  (ACB) if spoilage is suspected.

Adapted from

DiGiacomo, R. and Gallagher, P. 2015. Soft Drinks, chapter 59, p. 569. In F. P. Downes and K. Ito   (eds.), Compendium of Methods for the Microbiological Examination of Foods. 4th ed., American Public Health Association, Washington, DC.

Hatcher, W. S., Jr., Parish, M. E., Weihe, J. L., Splittstoesser, D. F., and Woodward,   B. B. 2001. Fruit Beverages, p. 565. In F. P. Downes and K. Ito (eds.),   Compendium of Methods for the Microbiological Examination of Foods. American   Public Health Association, Washington, DC.

Hatcher, W. S., Jr., Parish, M. E., Weihe, J. L., Splittstoesser, D. F., and Woodward,   B. B. 2015. Fruit Beverages, chapter 58, p. 565. In F. P. Downes and K. Ito (eds.),   Compendium of Methods for the Microbiological Examination of Foods. 4th ed., American   Public Health Association, Washington, DC.