Debaryomyces Hansenii Soft Drinks

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and fruit juices, oxygen levels are usually low, and CO2 levels either low or very high (in carbonated soft drinks). Spoilage is therefore due to facultative anaerobes, organisms that can grow with or without oxygen. In carbonated drinks, mould and bacterial growth is very unlikely as they are very sensitive to CO2.

The recent trend towards polyethylene terephthalate (PET) packaging presents its own problems. Typically PET containers cannot be hot filled; around 50°C is the upper temperature, although some recent types can be heated to 85°C. They are also permeable to oxygen (Rodriguez et al., 1992), which allows the growth of aerobic spoilage agents.

11.3 Background microbiology - spoilage

Many micro-organisms are found in soft drinks as environmental or raw material contaminants, but relatively few can grow within the acidic and low-oxygen environment. Yeasts are the most significant group of micro-organisms associated with spoilage of soft drinks and fruit juices. Spoilage will be seen as the growth and production of metabolic byproducts, for example, CO2, acid, and tainting compounds. As noted above, most spoilage is therefore by yeasts and mould species, with yeasts most important, and some spoilage is by acid-tolerant bacteria (Hocking & Jensen, 2001; Jay & Anderson, 2001).

11.3.1 Sources

Fruit and fruit juices are commonly contaminated with yeasts and moulds, often from insect damage. Fallen fruit should thus be avoided where possible, for all of the risks outlined below. Sugars and sugar concentrates are commonly contaminated with osmophilic yeasts, for example Z.rouxii. Growth is slow in concentrated solutions, but one cell per container of diluted stock is enough to cause spoilage (Davenport, 1996). Flavourings, water and other chemicals are all potential sources of microbial contamination. Process machinery and filling lines are particularly problematic and strict hygiene is essential.

11.3.2 Yeasts

There are over 800 species of yeasts currently described (Barnett et al., 2000), but only about 10 are commonly associated with spoilage of foods prepared in factories operating good standards of hygiene and using correctly applied chemical preservatives (Pitt & Hocking, 1997). Others are found if something goes wrong during manufacture; for example, incorrect preservative level, poor hygiene or poor-quality raw ingredients.

Davenport (1996, 1997, 1998) describes a simple classification scheme for yeasts causing spoilage in the soft drinks industry, which, for investigative purposes, is used in Sections 11.6 and 11.7 and Appendices 11.1-11.4 in this chapter. He found that the yeasts isolated could be divided into four categories: Groups 1-4. Group 1 yeasts are described as spoilage organisms that are well adapted to growth in soft drinks, able to cause spoilage from very low cell numbers (as few as one cell per container). The characteristics of Group 1 yeasts are osmotolerance, aggressive fermentation, resistance to preservatives (particularly weak organic acids) and a requirement for vitamins. Z.bailii is a typical example of this group, and this group corresponds closely with Pitt and Hocking's (1997) 10 key spoilage yeasts. Davenport describes Group 2 organisms as spoilage/hygiene types, able to cause spoilage of soft drinks, but only if something goes wrong during manufacturing, for example, too low a level (or absence) of preservative, ingress of oxygen, failure of pasteurisation or poor standards of hygiene. Group 2 organisms are common contaminants in factories, but can be severely restricted if good manufacturing practices (GMP) are adhered to strictly. Group 3 organisms are indicators of poor hygiene standards. These yeasts will not grow in soft drinks, even if present in high numbers, and are typical of the yeasts found in many factories; the higher the count, the worse the hygienic state of the factory. Group 4 yeasts, called 'aliens' by Davenport, are those out of their normal environment. An example would be Kluyveromyces lactis, a dairy spoilage yeast. Table 11.2 details the most common organisms in each group.

Table 11.2 Examples of yeast species found in soft drink factory environments

Group 1 - Fermentation Group 2 - Spoilage and and preservative resistance hygiene

Group 3 - Hygiene Group 4 - Aliens

Dekkera anomala

D. bruxellensis D. naardenensis Saccharomyces cerevisiae

(atypical strains) S. exiguus

Schizosaccharomyces pombe Zygosaccharomyces bailii Z. bisporus Z. lentus Z. rouxii

Candida davenportii

C. parapsilopsis Debaryomyces hansenii Hanseniaspora uvarum

Issatchenkia orientalis Lodderomyces elongisporus Pichia anomala P. membranifaciens Saccharomyces bayanus S. cerevisiae

Aureobasidium pullulans

Candida sake C. solani C. tropicalis

Kluyveromyces marxianus (dairy yeast) K. lactis

(dairy yeast)

Clavispora lusitania Cryptococcus albidus

C. laurentii

Debaryomyces etchellsii Rhodotorula glutinis R. mucilaginosa

Sources: Based on Davenport (1996); Stratford & James (2003).

Table 11.3 Names of the most important yeasts within the soft drinks industry, with synonyms

Current Name

Synonym

D. bruxellensis

Brettanomyces intermedius/B.bruxellensis

D. naardenensis

B. naardenensis

Saccharomyces cerevisiae

Saccharomyces carlsbergensis

S. bayanus

S. uvarum/S.heterogenicus

S. exiguus

Candida/Torulopsis holmii

Zygosacchaaromyces bailii

Saccharomyces bailii

Z. bisporus

Saccharomyces bisporus

Z. lentus

S. lentus

Z. rouxii

S. rouxii

Torulaspora delbreuckii

S. delbreuckii/S. rosei /Candida colliculosa

Pichia anomala

Hansenula anomala

P. membranifaciens

Candida valida

Issatchenkia orientalis

C. krusei

Kluyveromyces marxianus

C. kefyr

K. lactis

C. sphaerica

Debaryomyces hansenii

C. famata

Pichia guilliermondii

C. guilliermondii

Hanseniaspora uvarum

Kloeckera apiculata

Metchnikowia pulcherrima

Candida pulcherrima

One of the most confusing aspects of mould and yeast taxonomy is the frequency with which names are changed. Many yeasts and moulds have sexual and asexual stages that may have different names. Sometimes, the association of a yeast with a particular genus is recognised as wrong, and a name change then occurs. The current trend is to name the organism by the sexual name, with the asexual stage described as a synonym. Thus, Candida famata (asexual) becomes Debaryomyces hansenii (sexual). It is important to be aware of this variation when conducting literature searches. Names do not always reflect the spoilage potential - Saccharomyces cerevisiae is an important yeast for the brewing and baking industries, but a disaster for the soft drinks and fruit juice industry. Table 11.3 details alternative names for important yeasts.

11.3.3 Bacteria

Bacteria that have been associated with spoilage in the soft drinks industry include Acetobacter, Alicyclobacillus, Bacillus, Clostridium, Gluconobacter, Lactobacillus, Leuconostoc, Saccharobacter, Zymobacter and Zymomonas (Vasavada, 2003). Gluconobacter is a common spoilage agent of fruit juices; it is a strict aerobe, requiring free oxygen (Stratford et al., 2000).

Alicyclobacillus spoilage is becoming an important issue in heat-treated fruit juices, and the bacterium is commonly found contaminating fruit in the field. Growth of the organism is associated with the production of antiseptic and 'smoky' taints within juice. The former is due to 2,6-dibromophenol (2,6-DBP), giving a 'TCP' flavour (Jensen & Whitfield, 2003); the latter is due to guaiacol (2-methoxyphenol) (Jensen, 1999). Heat shock, taint precursors, incubation temperature and oxygen are all important factors in the production of taints (Jensen, 1999).

Alicyclobacillus acidoterrestris is a thermotolerant, spore-forming aerobic organism that can survive fruit juice heat treatments to grow in the juice. Spoilage can occur from inoculum levels as low as 1 spore per 10 ml (Pettipher & Osmundon, 2000; Walls & Chuyate, 2000a). Growth minima and maxima at temperatures of 26-50°C, minimum pH 2.0, D95 of 2.4 min. Isolation methods have been compared (Pacheco, 2002; Pettipher & Osmundson, 2000; Silva & Gibbs, 2001, Walls & Chuyate 2000). Effective control can be achieved by rapid chilling of juice to below 20°C after pasteurization, use of sorbate or benzoate, removal of oxygen or addition of ascorbic acid (Cerny et al., 2000) or by using an appropriate pasteurisation regime (Silva & Gibbs, 2001). Surface disinfection of fruit using chlorine or 4% peroxide has also proved effective (Orr & Beuchat, 2000).

11.3.4 Moulds

Mould problems can be divided into two types: growth of a variety of moulds due to poor hygiene within the factory or field environment, and growth of heat-resistant moulds within heat-processed juices. The former type can cause tainting, discolouration and other general problems associated with gross mould growth. The latter type can result in slow growth of the mould within the processed product. There is some overlap between the two groups. Xerophilic (highly sugar-tolerant) fungi are likely contaminants if hygiene is poor.

Heat-resistant moulds able to cause spoilage of fruit juices and soft drinks include Aspergillus ochraceus, Aspergillus tamarii, Aspergillus flavus, Byssochlamys nivea, Byssochlamys fulva, Paecilomyces variotii, Neosartorya fischeri, Eupenicillium brefeldianum, Phialophora mustea, Talaromyces flavus, Talaromyces trachyspermus and Thermoascus aurantiacum. Others include Penicillium notatum, Penicillium roquefortii and Cladosporum spp. (de Nijs et al., 2000; Pitt & Hocking, 1997; Stratford et al., 2000). Mould spoilage within the factory is associated with poor hygiene. Various types of heat-resistant spores can be produced: ascospores, chlamydospores and sclerotia.

Many of the above moulds are found on fruits pre- and post-harvest. Most moulds require oxygen to grow. Growth is exhibited as surface mats, sometimes producing copious spores. Some moulds produce extracellular degradative enzymes, such as pectinases. Detection of heat-tolerant moulds is usually carried out by plating out a sample of heat-shocked juice. Mould identification is carried out by reference to standard morphological texts, and is difficult unless carried out by experienced personnel. An excellent scheme that combines basic physiology with simple morphological attributes was developed by Pitt and Hocking (1997). It uses growth on three media at three temperatures to differentiate moulds: 5, 25 and 37°C, a high sugar content (xerophilic), a closely defined medium with nitrate as a nitrogen source and sucrose as hexose sugar, and a general medium. This technique is particularly useful for the differentiation of Penicillium species.

11.3.4.1 Mycotoxins

Mycotoxins are toxic secondary metabolites produced by fungi growing within or on foods. They can be a serious threat to human and animal health (Nagler et al., 2001). Table 11.4 details mycotoxins associated with soft drinks and fruit juice manufacture and raw materials. Patulin is the most common mycotoxin associated with fruit juice, particularly apple juice (Pitt & Hocking, 1997). It commonly occurs if juice is produced from stored apples. Mould growth in infected apples increases with time, raising levels of patulin. The use of windfall apples for juice is also a factor. Avoidance of windfall apples, filtration of juice and pressing quickly after harvest are all methods to reduce the incidence of patulin in juice. Patulin can be destroyed by fermentation to cider or by the addition of ascorbic acid (Marth, 1992). Within Europe, the European Union has set a limit of 50 ^g/kg for patulin in both apple juice and cider. A recent survey of apple products in Chile found that 28% of samples of juice and concentrate exceeded this limit (Canas & Aranda, 1996).

Table 11.4 Examples of toxin-producing moulds associated fruit with fruit raw materials and soft drinks products

Fruit(s)

Mould

Toxin(s)

Apple

Penicillium expansum

Patulin, citrinin,

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