Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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.0058 63



C for 10 min: typically used for shandies

0059 70



C for 10 min: typically used for squashes

0060 75



C for 15 min: typically used for susceptible

products such as high-blackcurrant-juice products.

0061 Tunnel pasteurization may be considered as a

continuous version of in-pack pasteurization.

0062 After the product has been filled, packed, and

seamed, the packages are transported through a

tunnel made up of different heat zones by means of

water of different temperatures being sprayed over

the packages, which gradually increase in tempera-

ture to the selected pasteurization level where they are

then held for the desired time (holding time) before

being cooled slowly to ambient temperature. Process-

ing time is approximately 40–50 min, resulting in

very effective pasteurization at relatively high cost

(it is energy-intensive and filling has to proceed at

the speed of the pasteurizer).

0063Flash pasteurization

Nonaseptic conditions This system may be used for

carton, polyvinylchloride, polyethylenetetraphthalate

(PET), and all other nonglass/metal drinks packaging

materials, and is suitable for both still and carbonated

beverages. The heat exchanger is connected in line

with the product mixing tank in the case of still prod-

ucts and the intermediate syrup tank in the case of

carbonated products. The syrup or finished product is

heated very quickly to the specified pasteurization

temperature, held for the specified time (usually

quoted as seconds), and cooled rapidly to the filling

temperature. The final drink may also be passed

through the heat exchanger in this way en route to

the carbonator if required.

0064The advantage of this type of pasteurization is that

the total heat input to the product is only a fraction of

that required for in-pack pasteurization and results

in considerably reduced flavor, color, and shelf-life

Ingredient blending

(quality check)

Intermediate

ingredient blending

(quality check)

Concentrates

Carbonates

Ingredient blending

Ready-to-drink still

Pasteurize

(90−95 8C for

10−45 s)

Pasteurize syrup

(90−95 8C for

10−45 s)

(where necessary)

Pasteurize

(90−978 C for

10−45 s)

Homogenize

(2000−3000 psi)

Dilute and carbonate

Fill: glass and

plastic bottles*

Case/palletize

Distribute

Case/palletize

Distribute Distribute

Fill: can, glass and

plastic bottles*

Either

1. Aseptic conditions

Pasteurized liquid is

filled into part-formed

containers which are

then sealed and finished

before proceeding to

the packing line

2. Hot-fill conditions

Hot pasteurized liquid

is filled into finished

packs which are sealed,

inverted for 1 min, and

cooled

3. Unpasteurized liquid is

filled into finished pack*

Case/palletize

Note: These processes may operate via an intermediate syrup or (partly or

throughout) on a ready diluted basis

* In-pack pasteurization performed where necessary with temperatures up to 70 8C for up to 20 min.

fig0003 Figure 3 Key stages in the processing of soft drinks.

SOFT DRINKS/Production 5357

effects. The disadvantage is that contamination may

occur during filling and/or capping and rigorous stand-

ards of hygiene and microbiological control must be

adhered to by fully trained and committed staff. It may

even be necessary to fill some products/packs in

enclosed areas with overpressure sterile air rooms.

0065 Aseptic conditions Before a discussion on the types

and methods of aseptic flash pasteurization, it is

necessary to consider exactly what this means in real

terms.

0066 Asepticity is sometimes referred to as commercial

sterility, but it is necessary to recognize the important

microbiological differences between the two types of

processing employed.

0067 With sterility it is realistic to expect that all meas-

urable organisms have been killed by the process,

which is almost always carried out at temperature

conditions well in excess of 100



C in hermetically

sealed containers, thus insuring the microbiological

integrity of the whole pack (product plus packaging).

0068 In aseptic packing the product is heat-treated separ-

ately from the container and maintained in positive air

pressure conditions (thus excluding any potentially

contaminating organisms) until filled and sealed into

the selected pack, which has been separately treated to

reduce any microbiological contamination on its sur-

face. Examples of aseptic conditions are those which

apply to the Tetra Pak process where the product is

flash-pasteurized at 96



C for 13 s and filled into card-

board previously treated with hydrogen peroxide; this

is formed into a tube and the individual pack seams

are carried out through the product.

0069 With aseptic processing/packaging it is generally

recognized that, independently of any pack integrity

problems, the normal failure rate will be of the order

of one pack in every 10 000 produced. A better rate

than this is rarely achieved and aseptic conditions are

sometimes quoted with failure rates as high as one in

3000, as in the case of carbonated products packed in

PET containers.

See also: Pasteurization: Principles; Preservation of

Food; Quality Assurance and Quality Control; Storage

Stability: Parameters Affecting Storage Stability; Water

Supplies: Water Treatment

Further Reading

Cullen KW, Ash DM, Warneke C and de Moor C (2002)

Intake of soft drinks, fruit-flavored beverages, and fruits

and vegetables by children in grades 4 through 6. Ameri-

can Journal of Public Health 92(9): 1475–1478.

Fried EJ and Nestle M (2002) The growing political move-

ment against soft drinks in schools. Journal of the

American Medical Association 288(17): 2181.

Heath HB (1981) Source Book of Flavours. Westport: AVI.

Hicks D (1990) Non-Carbonated Fruit Juices and Fruit

Beverages. Glasgow: Blackie.

Matthews AC (1991) Food Flavourings. Glasgow: Blackie.

O’Donnell K (1990) Formulation and Production of

Carbonated Soft Drinks. Glasgow: Blackie.

Microbiology

M Stratford and C J Capell, Unilever Research,

Sharnbrook, UK

Background

0001‘Soft drinks’ is a term often used to describe all bever-

ages with the exception of alcoholic or hot beverages.

This includes a vast array of products ranging in

composition from mineral waters, sports drinks, diet

formulations, colas, mixers, and tonics to fruit juices.

Some soft drinks are sparkling (carbonated), whereas

others are still. Some are manufactured with a desig-

nated shelf-life greater than a year, whereas others are

sold for immediate consumption (freshly squeezed

juices). As a consequence of this variation in com-

position and packaging, the microbiology of soft

drinks also shows considerable variation. Some

soft drinks form an environment so hostile, or so

deficient in nutrients, that the microflora is nearly

nonexistent, and spoilage is consequently rare.

Other soft drinks, such as fruit juices of a higher pH

(Table 1) form an environment ideally suited to rapid

proliferation of yeasts, molds, and bacteria. For the

purposes of this article, the average soft drink will

be considered to have a pH between 2.5 and 3.5

(Table 1), sugar at 5–10%, 1–3gl

1

citric acid (or

titratable acidity equivalent), and fruit juices

or vegetable extracts sufficient to comprise a source

of minerals and vitamins for microbial growth.

0002In theory, the presence of microbes in a food prod-

uct can have any one of three possible effects. It can

be a positive attribute, growth of specific microbes

creating desirable organoleptic changes, as in

tbl0001Table 1 pH range and acids prevalent in various soft drinks

and fruit juices

Beverage pHrange Acids

Apple juice 2.9–4.5 Malic, citric

Orange juice 2.6–4.3 Citric, malic

Blackcurrant juice 3.0–3.8 Citric

Grapefruit juice 2.9–3.6 Citric

Lemonade 2.8–3.2 Citric

Cola 2.5–2.8 Phosphoric

5358 SOFT DRINKS/Microbiology

fermented foods or alcoholic beverages. It can be a

negative attribute, causing illness in the consumer or

spoilage of the food, or it can be entirely neutral, the

presence of microbes having no effect whatsoever on

the food or on the consumer. In soft drinks, microbes

are almost never beneficial, a possible exception being

the oriental acidic tea, Kombucha, fermented by

yeasts, molds and acetic acid bacteria. In the great

majority of instances, the presence of microbes in soft

drinks is entirely neutral. Most bacteria are sensitive

to the acidity of soft drinks, whereas the majority of

yeasts and molds may be suppressed by a lack of

oxygen within the soft drinks packaging (Table 2).

Any such organisms tend to arrive in bottles of soft

drinks in low numbers and have no impact whatso-

ever on the beverage. Most will die in this environ-

ment within a few hours to a few weeks, depending

on the organism.

0003 A small minority of microbes, however, find the

soft drink environment suitable for proliferation and

growth to substantial populations. These are the soft-

drinks spoilage flora (Table 2). Although the occa-

sional microbe per liter of beverage has little impact,

growth to 10

cells per milliliter results in visible

spoilage, and 10

–10

cells per milliliter are sufficient

to cause very considerable changes to the chemistry

of the beverage. Such changes are almost always

organoleptically unacceptable and in themselves

constitute spoilage.

Manufacturing Process of Soft Drinks

Process

0004 Fruit-juice manufacture typically occurs close to the

area of production. Fruit is processed to remove

leaves, stems, etc., washed, milled, and crushed. The

juice is usually extracted from the crushed pulp by

pressure. Single-strength juice is bulky and is often

concentrated at the site of production. Juices are

heated to near 100



C, a vacuum is applied, and

water is removed by evaporation. Volatile compon-

ents are recovered and added back after cooling. To

ensure microbiological stability, concentrates may

then be frozen, preserved, or aseptically filled into

drums.

0005Soft-drinks manufacture (Figure 1) essentially in-

volves dissolving weighed amounts of flavors, juices,

acids, antioxidants, and sugars into water. This is

then dispensed into the appropriate packaging.

Sugar enters the syrup room of the factory either as

granules, which are dissolved in hot water, typically



C/30 s or as 67



Brix syrup, known as ‘simple

sugar syrup.’ Other components of the soft drink,

acids, flavors, fruit juices, vegetable extracts, antioxi-

dants, etc., are weighed out and dissolved in water.

These are added to the simple sugar syrup, forming

the final syrup at 40–50



Brix. The final syrup is piped

from the syrup room to the proportioning pump in

the filling hall (Figure 1). Here, it is proportioned

with water to form the product, typically, 7–10



Brix.

This is filled into bottles/cans/tetrapaks at the filler,

which are then sealed at the capper.

Sources of Infection

0006Microbes may enter soft drinks and their production

facilities from a number of sources. These may dir-

ectly infect products or may colonize various sites

within the factory and subsequently cause infection

from these sites. Entry to the factory is most common

on raw materials, returned bottles, via insects or aerial

vectors.

0007Aerial contamination obviously depends on the

microbial loading and composition in the air. Nearby

sources of microbes greatly increase the microbial

load, e.g., breweries, dust sources (harvesting or

building work), fruit storage, or processing of fruit

or vegetables. Fresh fruit are routinely heavily sur-

face-contaminated with yeasts and molds, 10

–10

per gram. Factories processing fruits will inevitably

become contaminated with the molds associated with

those fruits’ spoilage flora. Ascomycetous molds pro-

duce conidiospores for dispersal and ascospores for

survival. Conidiospores are often airborne; asco-

spores often are not, making the presence of the

heat-sensitive conidiospores generally more common

as airborne contaminants. Aerial contamination is

highly seasonal, with mold spores predominating,

tbl0002 Table 2 Summary of the growth, spoilage, and safety significance of microbes in soft drinks

Microbe Growth Inhibition Spoilage Safety

Spoilage yeasts þ Excess gas Exploding bottles

Other yeasts  Low O



Spoilage molds þ Mycelium Possible mycotoxins

Other molds  Low O



Vegetative bacterial pathogens, e.g. Salmonella spp.,

E. coli VTEC (enteropathogenic E. c oli )

 Low pH  Food poisoning, in fresh,

unpasteurized, fruit juices

Acid-tolerant bacteria + Low O

Taints, slime 

Most bacteria  Low pH 

SOFT DRINKS/Microbiology 5359

and is probably the most significant cause of mold

spoilage. (See Microbiology: Classification of Micro-

organisms.)

0008 Insects are becoming increasingly recognized as a

vector for yeasts. Many insects carry yeasts and acid-

tolerant bacteria between flowers, actively consume

yeasts as part of their diet, and benefit from yeast

colonization of their digestive systems. Insect frass,

notably from fruit flies (Drosophila spp.), is particu-

larly rich in soft-drink spoilage yeasts.

0009 Returned bottles, when brought into the factory,

form a potent source of microbial infection of soft

drinks. Returned bottles are frequently cap-less and

unwashed, often containing a residue of soft drink.

Storage at ambient temperature allows selective

growth of spoilage microbes in the residue up to

high levels and formation of stationary-phase sur-

vival mechanisms, such as heat-resistant ascospores

by yeasts and molds. Stringent bottle-washing re-

moves almost all residues, but fungal debris and

viable ascospores can persist through the whole

process. Fruit-flies, attracted to the sugary residues

in returned bottles, can also spread infection around

the factory.

0010 The microbial loading of raw materials is normally

low where materials have been selected from good

quality sources. Sugar granules, 67



Brix syrups, or

fruit-juice concentrates are a potential source of

contamination. Fruit juices are often heavily contam-

inated; citrus fruits are especially associated with mold

spoilage, as the fruit defense is most susceptible to

pectinolytic molds. Pasteurization of the single-

strength juice or the concentration process should

result in a near-sterile product, except for rare heat-

resistant molds. Byssochlamys spp. and Talaromyces

spp. have been the cause of spoilage in pasteurized

fruit juices. The ascospores of these molds are rela-

tively uncommon, but significant growth of the mold

on the raw material must have occurred for the pres-

ence of problematic numbers of ascospores. Similarly,

mold spores in small numbers may survive the sugar

refining process. Both fruit juice concentrates and

sugar syrups are more likely to acquire an osmophilic

flora through mishandling after cooling. Owing to the

low water activity, only osmophilic yeasts and, more

rarely, molds can grow, albeit very slowly in sugar

syrups. Other ingredients, such as water, flavors, acid-

ulants, colorings, antioxidants, and preservatives, are

also possible sources of spoilage microorganisms.

0011Build-up or colonization of a spoilage flora within

the factory is probably the most significant source of

infection. It has been estimated that 95% of yeast

infections of soft drinks are caused by poor plant

hygiene. Soft-drink manufacture is generally a very

high-volume throughput operation, with up to

36 000 l h

1

passing through each filling line. Little

time is therefore allowed for growth of microbes

within the bulk volume of liquid, before pasteuriza-

tion or addition of preservatives. Microbial build-up

is therefore most significant in dead spaces or on

Delivery

Granulated

sugar

Fruit juice

Water

Chemicals

flavours, acids,

antioxidants,

preservatives

Ingredient

preparation

Water

Final

syrup

Syrup room

Filling hall

Returned

bottles

Bottle washer

Filler Capper

Proportioner

Simple syrup

storage

Conveying

Secondary

packaging and

warehousing

fig0001 Figure 1 Schematic diagram of a soft-drinks production facility. In the syrup room, sugars are dissolved in water to form simple

syrup, and juices and flavors are added to form the final syrup. This is pumped into the filling hall, diluted with water by the

proportioning pump, and filled into bottles. Bottles are then capped, packaged, and distributed. Tunnel pasteurization is applied to

capped bottles or ‘in-line’ pasteurization between the proportioner and filler.

5360 SOFT DRINKS/Microbiology

surfaces proximate to the product stream. Areas most

at risk include the proportioning pump, filler and filler

heads, and capper. Other areas of concern include old,

dried splashes of fruit juice or soft drink, the syrup

room, particularly where ingredients have been spilled

or allowed to become damp, and drains into which

sugars are washed. (See Contamination of Food.)

Microbial Stability

0012 Microbial stability can be achieved by preventing

viable microbes entering the product, (aseptic filling),

sterilization (pasteurization), or by factors preventing

microbial growth in soft drinks (preservation). Pas-

teurization is the most effective method of eliminating

microbes in soft drinks (Table 3). Vegetative yeast and

mold cells are generally heat-sensitive, almost all being

killed in 10 min at 60



C. Pasteurization of fruit juices,

such as orange juice, which are typically heavily con-

taminated with yeasts and molds, range from 10 to

15 min at 70–75



C. Such pasteurization will not elim-

inate heat-resistant formsof molds, such as ascospores.

0013 The microbial stability of soft drinks can be

achieved most simply and effectively by ‘in-pack’ ster-

ilization, downstream from the capper using a tunnel

pasteurizer (Table 3). This insures absence of microbes

in the bottle and cap as well as the product. Alterna-

tively, products may be sterilized ‘in-line’ using a plate

pasteurizer and aseptically filled into sterile bottles, or

products may be given a flash in-line pasteurization to

lower the microbial loading and preservatives added

to prevent the growth of surviving microbes. Fresh-

pressed fruit juices that would normally spoil within 2

days may be preserved by chilled storage for a limited

period of time, e.g. 2–3 weeks.

0014 Some factors suppressing microbial growth are

intrinsic to the composition of soft drinks, such as

the phosphoric acid in cola or the low pH that inhibits

or kills the majority of bacteria. Pressurized CO

carbonated soft drinks has a pronounced antimicro-

bial effect, particularly against molds and bacteria.

Fermentative yeasts are more resistant, particularly

Brettanomyces spp., but growth can be inhibited at

high carbonation. The lack of nutrients in many soft

drinks, such as vitamins, phosphate, or a usable ni-

trogen source, is also an aid to preservation. It has

been noted that soft drinks improved by the addition

of real fruit juices often became increasingly prone to

microbial spoilage.

0015Preservatives are added to soft drinks to kill or

prevent the growth of microbes. The preservatives

commonly encountered in soft drinks are sorbic acid

and benzoic acid. Less frequently found are sulfites

(SO

), parabens, and dimethyldicarbonate (DMDC).

Preservative usage and permitted concentrations vary

greatly, depending on the legislation in force where

soft drinks are produced and consumed. Sorbic and

benzoic acids function far better as preservatives at

lower pH. Resistance to preservatives has been

reported to the greatest extent in Zygosaccharomyces

spoilage yeasts, but also occurs to a lesser extent in

certain molds and bacteria, such as Gluconobacter

(Acetomonas).

Characteristic of Soft-drinks Spoilage

0016Soft drinks may be spoiled by yeasts, molds, or

bacteria. Yeast and mold spoilage predominates,

being favored by the low pH and high sugar/assimil-

able nitrogen ratio. Spoilage of soft drinks may be

clear and easily detectable by a number of visual

or organoleptic symptoms. However, it is by no

means uncommon not to isolate viable microbes

from a spoiled product. It appears that spoilage

organisms often grow and cause the symptoms

of spoilage before dying. This may be due to a lack

of nutrients or oxygen, or to the formation of toxic

byproducts such as ethanol. Consequentially, it is not

always possible to identify the causative agent of

spoilage.

0017Yeast growth in excess of 10

cells per milliliter

results in a visible cloud or haze. Some spoilage yeasts

secrete proteolytic or pectolytic enzymes that can

destroy the natural hazes of fruit juices. Spoilage

yeasts may be flocculent, forming particulates,

whereas others form a surface skin or vellum, notably

Pichia membranaefaciens. The most significant spoil-

age yeasts are highly fermentative, producing CO

to high pressure, and consequently distorting and

bursting the packaging (Figure 2). Fermentation also

results in ethanol production, and yeast spoilage is

often associated with a sweet alcoholic taste.

0018Mold spoilage of soft drinks is usually visual and

obvious. Molds given more oxygen via loose seals,

large headspace, or oxygen-permeable packaging

tend to form surface pellicles and may sporulate.

Mold growth in suboptimum conditions, low oxygen

or preservatives, tends to form submerged mycelial

tbl0003 Table 3 Common methods of achieving microbial stability in

soft drinks

Method Comment

Tunnel pasteurization Sterilizes product and packaging

together

In line, plate pasteurization Sterilizes product only, then

requires aseptic or hot filling

Flash pasteurization þ

preservatives

Flash lowers the microbial load,

and preservatives inhibit

survivors

Frozen/chill storage Inhibition or delay of microbial

growth

SOFT DRINKS/Microbiology 5361

masses, often resembling small wispy balls of cotton

wool. Mold growth can also eliminate clouds in citrus

fruit juices. Taints and musty flavors may be produced.

0019 Bacterial spoilage is usually associated with fruit or

vegetable juices and the presence of oxygen. Spoilage

is often associated with loss of astringency, souring,

slime or ropiness in texture, turbidity, and off-flavors.

Spoilage Microorganisms of Soft Drinks

0020Currently, some 800 yeast species, thousands of

molds, and vast numbers of bacteria are recognized.

The ability of microorganisms to exchange DNA

often makes species identification of an organism

causing spoilage to be of little value. In the ‘forensic

approach’ to spoilage microbiology, Professor Daven-

port proposed a simplified taxonomy based on the

behavior of microbes, rather than on their species

names. Group 1 were the spoilage microoganisms,

well suited to proliferate in soft drinks with the

potential to cause spoilage from as few as one cell

per container. Group 1 spoilage microbes for soft

drinks were limited to a number of yeasts, listed in

Table 4. All are generally osmotolerant, highly fer-

mentative, vitamin-requiring and resistant to preser-

vatives, such as acetic, sorbic, or benzoic acids. These

yeasts are fortunately rare in soft-drink environments

and do not form any part of the flora of a well-run

soft-drink production facility.

0021Group 2 microbes, described as spoilage/hygiene,

are capable of causing spoilage of soft drinks but only

as a result of mistakes occurring during manufacture.

This group, examples of which are listed in Table 4,

includes yeast and mold species and acid-tolerant

bacteria. These can be regarded as opportunistic

spoilage organisms, ready to exploit errors such as

under-dosing of preservatives, failure of pasteuriza-

tion, hygiene failures, or ingress of oxygen. Such or-

ganisms are often present in soft-drink factories,

usually in low numbers and restricted by good

hygiene procedures.

0022Group 3 microbes are hygiene indicators and will

not cause spoilage. Most yeasts found in soft drinks

production and products are Group 3 organisms

(Table 4). Their numbers can be used as a measure

of the general hygiene of the facility, and their origin

may indicate the origin of the contamination. For

fig0002 Figure 2 Ruptured can of a soft drink caused by yeast spoilage.

The can was infected with Zygosaccharomycesbailii and incubated

for 2 weeks at 25



C. Cans were visibly swollen within 7 days.

tbl0004 Table 4 Yeasts associated with soft drinks and fruit juices

Group1 Group 2 Group 3

Zygosaccharomyces bailii Candida parapsilosis Aureobasidium pullulans

Zygosaccharomyces bisporus Debaryomyces hansenii Cryptococcus albidus

Zygosaccharomyces lentus Pichia membranaefaciens Cryptococcus laurentii

Zygosaccharomyces rouxii Saccharomyces exiguus Rhodotorula glutinis

Saccharomyces exiguus Saccharomyces cerevisiae Rhodotorula mucilaginosa

Saccharomyces cerevisiae Candida krusei Candida sake

Brettanomyces bruxellensis Pichia anomala Candida intermedia

Schizosaccharomyces pombe Kloeckera apiculata Candida zeylandoides

Torulaspora delbrueckii

Zygosaccharomyces fermentati

Zygosaccharomyces florentinus

Zygosaccharomyces microellipsoides

Group 1 are spoilage yeasts, Group 2 are opportunistic spoilage microbes, and Group 3 are hygiene indicators. The most significant species are in bold.

Saccharomyces exiguus and S. cerevisiae are listed in two groups in respect of their strain variability, abnormal strains causing spoilage.

5362 SOFT DRINKS/Microbiology

example, the yeast-like mold Aureobasidium pullu-

lans, or red-pigmented yeasts, Rhodotorula glutinis

or R. mucilaginosa, can be used to indicate airborne

dust contamination. The presence of such organisms

in beverages is relatively common and not a cause for

concern. Such organisms are usually present in very

low numbers, are harmless to the consumer, and are

unable to grow or cause spoilage. It has been esti-

mated that up to a third of all commercial fruit juices

contain a few viable yeasts.

Spoilage Yeasts

0023 The most widely reported yeasts in soft drinks are the

Group 1 spoilage yeasts, which include Zygosacchar-

omyces bailii, Z. rouxii, Z. lentus, and Z. bisporus.

These are rarely found in soft drinks or production

facilities, but when they do occur, spoilage is usually

widespread and devastating. These yeasts share many

physiological characteristics, being osmotolerant,

preservative-resistant, and highly fermentative, and

most are taxonomically closely related.

0024 Zygosaccharomyces bailii (Figure 3) is the most

preservative-resistant organism known, able to resist

high concentrations of acetic acid and ethanol.

Strains of Z. bailii can tolerate sorbic and benzoic

acids at pH 4.0 at up to 1000 p.p.m. (Figure 4). The

European statutory limit for these preservatives is

currently 300 p.p.m.! Adaptation to preservatives by

prior exposure can increase tolerance by some 50–

100%. Z. bailii readily forms ascospores, although

these are not markedly heat-resistant. Z. bailii grows

well in sugar syrups and juice concentrates, contain-

ing 50–60% sugar, thus allowing easy infection of

soft drinks via the raw materials. The infective dose

of Z. bailii is low; this yeast is reputed to cause

spoilage from as little as one cell per container.

Z. bailii can cause spoilage of fruit juices, cordials

and concentrates, and carbonated and still soft

drinks. Z. bailii requires B-group vitamins and is

less likely to spoil products lacking fruit juices or

similar vitamin sources.

0025 Zygosaccharomyces bisporus shares most of the

characteristics of its close relative Z. bailii. Z. bisporus

is much less frequently encountered than Z. bailii.

Examination of the characteristics of several strains

of Z. bisporus show much diversity; some strains

being comparable with Z. bailii in terms of preserva-

tive resistance and osmotolerance, whereas other

strains are more sensitive. Overall, Z. bisporus strains

are less preservative-resistant than Z. bailii (Figure 4).

0026 Zygosaccharomyces lentus is a newly discovered

species, members of which had previously been mis-

takenly identified as Z. bailii. Z. lentus strains are

often more preservative-resistant than Z. bisporus.

All of the Z. lentus strains isolated to date are from

spoiled foods, predominantly from orange and

tomato products. Z. lentus strains are unusual in

growing very slowly and having a low temperature

range. Growth is poor above 25



C, but these yeasts

will grow at 4



C and cause spoilage of refrigerated

soft drinks and fruit juices.

0027Zygosaccharomyces rouxii is extremely osmotoler-

ant being able to tolerate water activity down to 0.62

in fructose. Consequently, this yeast is probably

the most significant spoilage organism in syrups

and fruit-juice cordials and concentrates, but also

spoils fruit juices and soft drinks. Like the other

Zygosaccharomyces spoilage yeasts, Z. rouxii is

highly fermentative, spoilage of concentrates often

being detectable by bulging containers. Z. rouxii is

also reported capable of growth from very low con-

tamination levels. Direct comparisons show that Z.

rouxii is significantly less preservative-resistant than

Z. bailii (Figure 4) and shows significantly less resist-

ance to pasteurization than most other yeasts. The

rarer honey spoilage yeast, Z. mellis, shows very

similar characteristics to Z. rouxii. Many Z. mellis

strains have been shown by 26S rDNA sequencing to

have been misidentified, in reality, Z. rouxii strains.

0028Saccharomyces exiguus, sometimes known as Can-

dida holmii or Torulopsis holmii, is also closely related

to the central Zygosaccharomyces spoilage family. S.

exiguus strains are vigorous-growing, moderately

osmotolerant, and highly fermentative, generally re-

sembling Saccharomyces cerevisiae. Some strains of S.

exiguus are capable of growth in extreme acid condi-

tions and high levels of preservatives.

fig0003Figure 3 Scanning electron micrograph of the spoilage yeast

Zygosaccharomyces bailii NCYC 1766. Cells are ovoid, 4 mmin

length, with prominent surface bud scars.

SOFT DRINKS/Microbiology 5363

0029 Brettanomyces, and its ascospore-forming teleo-

morph, Dekkera, is a genus containing species noted

for spoilage of low nutrient, carbonated beverages

such as soda water, tonic, cola, or clear lemon. The

most commonly encountered species are Dekkera

bruxellensis and D. naardenensis, causing sediments

and characteristic off-flavors and odors. These yeast

species are characteristically sensitive to preservatives

(Figure 4), high sugar, and heat, but are regarded as

spoilage Group 1 yeasts owing their its resistance to

and spoilage of carbonated beverages.

0030 Other spoilage yeast species are less commonly

encountered, including other near-relatives of the

Zygosaccharomyces spoilage family, Z. fermentati.

Z. florentinus, Z. microellipsoides, Torulaspora del-

brueckii, and the fission yeast Schizosaccharomyces

pombe. These are all fermentative yeasts with moder-

ate osmotolerance. Their resistance to preservatives is

generally moderate to low. (See Yeasts.)

Spoilage Molds

0031 Molds are, by and large, aerobic multicellular hyphal

fungi, but a spectrum of phenotypes exist. Food spoil-

age molds are generally fast-growing opportunistic

organisms. Fungi have a greater tolerance of low

pH and low water availability than bacteria and are

therefore most closely associated with foods with

these characteristics. Their requirement for oxygen

and sensitivity to carbon dioxide mean that mold

spoilage may be effectively controlled by carbon-

ation. Some molds can produce heat-resistant stages,

including sclerotia, chlamydiospores, and ascospores,

and are potentially capable of surviving normal

pasteurization (Table 5). Although molds produce

hyphal growth, they are able to grow well in liquid

culture, either growing on the surface or completely

submerged. Although molds are generally regarded

as obligate aerobes, some do ferment sugars to

ethanol and carbon dioxide; those best studied, the

dimorphic yeast-like molds such as Mucor spp., are

also associated with the spoilage of soft drinks. As

these molds do not produce exceptionally heat-resist-

ant stages, they should be eliminated by pasteuriza-

tion, and such spoilage is an indication of hygiene

failure. Mold resistance to the common food preser-

vatives is generally reported to be less than spoilage

yeasts. However, many molds will degrade sorbic

acid to strong-smelling 1,3-pentadiene, making

mold spoilage of preserved beverages particularly

distinctive.

0032Many molds have the potential to cause spoilage of

soft drinks. Those most commonly encountered in-

clude Aspergillus niger, A. ochraceus, A. fischeri, and

A. tamarii, Byssoclamys nivea, B. fulva, Paecilomyces

variotii, Neosartorya fischeri, Eupenicillium brefel-

dianum, Phialophora mustea, Taleromyces flavus,

T. trachyspermus,andThermoascus aurantiacum.

Spoilage Bacteria

0033The ecological conditions of soft drinks limit the

growth of bacteria, with formulation pH having the

clearest effect on the development of potential spoil-

age organisms. Bacterial soft-drink spoilage organ-

isms fall into three main categories: spore-formers,

246810

Z. bailii

Z. lentus

Z. bisporus

Z. rouxii

S. exiguus

S. cerevisiae

C. parapsilosis

Dekkera spp.

R. glutinis

Benzoic acid (mM) Glucose(M)

Inhibitory concentration

fig0004 Figure 4 Bar chart showing the relative preservative resistance and osmotolerance of a number of spoilage yeasts (unpublished

data from H. Steels, with permission). Inhibitory concentrations were measured in YEPD (yeast extract, peptone, glucose) broth, pH

4.0, at 25



C. Data are the mean values of 28 strains of Z. bailli, 10 strains of Z. lentus, six Z. bisporus, nine Z. rouxii, five S. exiguus,11

S. cerevisiae, seven C. parapsilosis, five Dekkera/Brettanomyces spp., and five R. glutinis.

5364 SOFT DRINKS/Microbiology

lactic acid bacteria, and acetic acid bacteria. As most

soft drinks are pasteurized, acidoduric/philic spore-

forming bacterial species are of greatest threat to

fruit-juice manufacturers. High-pH fruit or vegetable

juices, tomato, apple, pear, or carrot juices have been

reported spoiled by Clostridium spp. or Bacillus spp.

including C. butyricum, C. pasteurianum, Bacillus

coagulans, B. licheniformis, B. macerans, B. subtilis,

and B. polymyxa, the thermophilic B. coagulans being

the causative agent of ‘flat-souring’ in tomato juices.

0034 Alicyclobacillus acidoterrestris is an obligately aer-

obic spore-forming thermophilic bacteria, associated

with soils and acidic fruit juices principally from

warm climates. Although not common at present,

this organism has the potential to become a major

cause of spoilage of fruit-containing soft drinks

(Tables 5 and 6). It will grow at pH 2.5, and its spores

have been reported to be very resistant or immune to

pasteurization (D



of 2–12 min; D



, time at

temperature leaving 10 % survivors in the popula-

tion). It may grow and produce tarry off-odors, in-

cluding guaiacol and 2,6-dibromophenol, through

the reduction of taint precursors. It does not produce

gas, and bacterial haze is often not an important

consideration as fruit juice may be naturally cloudy.

Potentially, removal of taint precursors, limitation of

storage temperature to < 20



C, for example, preven-

tion of ingress of oxygen or super-pasteurization will

eliminate spoilage, though practicalities may limit

these solutions.

0035Unpasteurized fruit juices with pHs higher than

about 3.2 may be spoiled by the growth of lactic acid

bacteria. Lactobacillus or Leuconostoc spp. can cause

slime, ropiness, and off-flavors in fruit juices, particu-

larly where adapted strains have been allowed to build

up in production facilities. Lactic acid bacteria are

heat-sensitive and lose viability in chilled juices. (See

Bacillus: Occurrence; Lactic Acid Bacteria.)

0036Gluconobacter spp. (Acetomonas) is the most fre-

quently encountered cause of bacterial spoilage at low

pH. Spoilage changes flavor characteristics and may

lead to pack swelling and haze. Gluconobacter spp. are

resistant to preservatives such as sorbic acid, benzoic

acid, and DMDC, but are heat-sensitive and absolutely

dependent on the presence of free oxygen. Spoilage is a

problem only in gas-permeable packaging, such as still

soft drinks in plastic beakers (Table 6).

Safety and MicrobialHazards in Soft Drinks

0037The hazards posed by the presence of microorganisms

on soft drinks are much lower than for the majority of

foods. Owing to the low pH of soft drinks, bacterial

spores will not germinate, and vegetative cells pro-

gressively die off. Pathogenic bacteria are therefore of

little consequence in acidic soft drinks. Fruit juices,

notably apple juices prepared from ripe fruit, are

generally less acidic than the majority of soft drinks

and allow a longer survival of pathogens. Recent,

much-publicized cases of illnesses caused by Escher-

ichia coli 0157:H7 in unpasteurized apple juice

appeared to be caused by animal fecal contamination

of the fruit. Pathogens, such as E. coli or Salmonella

spp., die in acidic apple juices, but viability can be

maintained for several days and progressively lost

over 3–5 weeks, respectively. Occurrences of salmon-

ellosis in fruit juices are rare but finite. Pathogens in

fruit juices are easily inactivated by pasteurization or

by the addition of preservatives. Soft drinks may also

very rarely contain viruses or the parasitic protozoan,

tbl0005 Table 5 Heat resistance of yeasts, molds, mold ascospores,

and bacterial spores associated with soft drinks

Microbe Form D-values (min)

Byssochlamys fulva Ascospores D



300,



90–900

Byssochlamys nivea Ascospores D



45–48

Neosartorya fischeri Ascospores D



84,



72–450

Aspergillus flavus Conidiospores D



186

Ta l a r o m y c e s f l av u s Ascospores D



468,



60–420

Talaromyces macrosporus Ascospores D



420–1320,



132–360

Alicylobacillus acidoterrestris Spores D



960–1380

Saccharomyces cerevisiae Ascospores D



6360

Saccharomyces cerevisiae Vegetative cells D



Zygosaccharomyces bailii Ascospores D



600–2220

Zygosaccharomyces bailii Vegetative cells D



120–240

tbl0006 Table 6 Niche habitats formed by different varieties of soft drinks, and spoilage microbes associated with these habitats

Habitat Spoilage microbe Spoilage indications

High carbonation Brettanomyces/Dekkera spp. Clouds, off-flavors, odors

High preservatives Z. bailii, Z. bisporus, Z. lentus Excess gas, blown cans

Juices in thin plastic Gluconobacter (Acetomonas) Off-flavors, haze

Tomato juices Bacillus coagulans Flat souring

Syrups or concentrates Z. rouxii, Z. bailii Excess gas, blown cans

Refrigerated juices Z. lentus Clouds, off-flavors

Heat-treated juices Alicyclobacillus acidoterrestris Tarry taints, guaiacol

Heat-treated juices Ascospore-forming molds Mycelium, off-flavors

SOFT DRINKS/Microbiology 5365

Cryptosporidium parvum, infection occurring via

contaminated water supplies. Illnesses have been

reported, caused by small, round-structured viruses

in orange juice or Cryptosporidium parvum in apple

juices and flavored fruit drinks.

0038 The presence of fermentative yeasts in soft drinks

also poses a physical hazard to the consumer and is

caused by fermentation to high pressure within pack-

ages. Pressures comparable with that in Champagne

bottles will cause metal cans and kegs to split or

rupture (Figure 2), plastic bottles to distort and effer-

vesce violently when opened, and glass bottles to

shatter. Exploding bottles caused by Zygosaccharo-

myces. yeasts are a recognized cause of eye injuries.

Molds do not generate gas to the same extent but, in

higher pH products, could pose a threat from the

formation of mycotoxins. Some concern has been

raised regarding formation of patulin in stored apples

by Penicillium expansum. Apple juices prepared from

moldy fruit have been found to contain 1130 mgl

1

Consequently, European and FDA limits have been

set for patulin in apple juices.

Seealso: Acids:NaturalAcidsandAcidulants;

Pasteurization:PasteurizationofLiquid Prod ucts;

Preservatives:Classificationsand Properties;Food

Uses; Soft Drinks:ChemicalComposition; Production;

Spoilage:BacterialSpoilage;Molds inSpoilage;Yeasts

inSpoilage; Yeasts