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starter culture cell concentrations are required. As-

suming that 1% inocula is ordinarily used, 10 kg (or

10 l) of starter culture would be sufficient to inoculate

1000 kg (or 1000 l) of substrate. Such an inoculum

could easily be produced from a colony or culture

slant, simply by successive transfers through inter-

mediate cultures (i.e., 1 ml into 100 ml, and 100 ml

into 10 l). Indeed, when pure cultures, rather than

backslopping techniques, were introduced nearly a

century ago in the cheese industry, the propagation

of a ‘mother’ culture and intermediate cultures was

the normal practice. However, as the size of the

cheese industry increased, such that starter culture

volumes of over 10 000 kg could be required for a

single day’s production, it no longer became feasible

for cheese manufacturers to prepare cultures in this

manner.

0015 Currently, there are two general types of cultures

that are manufactured and sold to the fermented food

and beverage industries. The first type, often referred

to as ‘bulk cultures,’ is used to inoculate a bulk tank,

which is then used to inoculate the production vat

(Figure 2). The bulk culture essentially represents the

intermediate cultures that have traditionally been re-

quired. After a suitable incubation period in the ap-

propriate culture medium, the fully grown bulk

culture is used to inoculate the raw material. Provided

that the starter culture organisms are protected

against acid damage, oxygen, hydrogen peroxide,

or other inhibitory end products, bulk cultures will

remain viable for many hours. Thus, in the cheese

industry, bulk cultures can be used to inoculate mul-

tiple cheese vats throughout a manufacturing day.

Maintenance of culture viability is an important

issue, however, and methods to insure that cultures

remain active will be discussed below.

0016 The second type of starter is designed to be inocu-

lated directly into the food material, thus eliminating

the need to grow bulk cultures. In the cheese industry,

these are often referred to as ‘direct-to-vat set’ or DVS

cultures. Of course, such cultures are also widely used

in the sausage, baking, and other industries, where

product volumes are usually small, compared to

cheese manufacture. These cultures have the obvious

advantage of eliminating the labor, hardware, and

accessory problems that accompany the preparation

and maintenance of bulk starter cultures. In order to

provide a sufficient inoculum into large volumes of

raw material, however, DVS cultures must be highly

concentrated, especially those used for cheese manu-

facture. The added concentration steps may reduce

culture viability, causing slow-starting fermentations.

Direct-to-vat cultures are also more expensive to pur-

chase than bulk cultures, and may not be economical

for large-scale operations.

Mixed versus Defined Cultures

0017Mixed cultures contain historically tested blends of

starter culture organisms. They can include several

genera, species, or even strains of organisms. Often

the actual identities of the organisms present in a

mixed culture are not known, and the individual

species may not have been characterized microbio-

logically or biochemically. Moreover, the proportion

of different organisms in a mixed culture may not be

constant from one product lot to another. Yet, mixed

cultures containing undefined strains are still used as

starter cultures for many applications because they

have a proven history of successful use. When used in

the manufacture of cheese, strains that are sensitive to

a specific bacteriophage may quickly die out. How-

ever, other strains present in the mixed culture may be

resistant to that particular phage and can then com-

plete the fermentation. Frequent exposure to different

bacteriophages, therefore, provides an effective

mechanism for insuring that phage-resistant strains

will be present in repeatedly propagated mixed cul-

tures. These so-called P starter cultures are commonly

used in the Netherlands for the manufacture of

Gouda, Edam, and related cheese varieties.

0018The main disadvantage of mixed cultures is that

they may yield finished products of inconsistent qual-

ity. In addition, fermentation rates may vary from day

to day, affecting production schedules. For applica-

tions where time is flexible, and product quality vari-

ations are more tolerable, such as in small-scale

cheese factories, mixed cultures are still widely used.

However, in large production facilities where precise

schedules are critical and consistent product quality is

expected, mixed cultures have become less common.

Instead, defined starter cultures have become preva-

lent.

0019Defined cultures simply refer to cultures that con-

tain microbiologically characterized strains, which

Production

vat

Bulk

tank

Lyophilized or

frozen cultures

Lyophilized or

frozen cultures

Bulk

DVS

fig0002 Figure 2 Bulk versus direct-to-vat set (DVS) culture prepar-

ation.

STARTER CULTURES 5587

are used individually or as blends. Most defined

strains have been isolated from wild or mixed cul-

tures, and characterized for metabolic properties,

phage-resistance, and other desirable traits. When

assembling a culture blend (containing multiple

defined strains), all strains must be compatible. That

is, one strain must not dominate over the others or

produce inhibitory agents that would affect the

growth of other organisms. Blends are often preferred

over single or paired strains for their predictability

and consistency. They are formulated to contain a

broad spectrum of strains having resistance to differ-

ent phage types, so that if one strain is affected, the

other strains can complete the fermentation.

Manufacture of Starter Cultures

0020 Bacterial starter cultures, including those produced

for the dairy industry, are mass-produced in modern

fermenters, under aseptic conditions, not unlike those

used in the pharmaceutical industry for the produc-

tion of biomedical products. Although the size of

the fermentors may vary from as little as 10 l to over

300 l, the basic operational parameters and control

features are consistent. For production of dairy starter

cultures, milk- or whey-based media are most often

used; however, molasses or corn syrup can also be used

as the basal medium. Nutrients are usually added to

the medium. Vitamins, especially water-soluble

B-vitamins, are required for optimum growth of lac-

tococci and lactobacilli, and some species of Strepto-

coccus, Leuconostoc,andLactobacillus also require

specific amino acids. In addition, stability of some

lactobacilli species during frozen storage may be en-

hanced when the growth medium contains Tween 80,

a surfactant thought to stabilize cell membranes.

Acids produced during culture growth can reduce

final cell densities and decrease cell viability; thus

the pH is controlled by the addition of alkali, usually

gaseous NH

,NH

OH, Na

, or KOH. Catalase

is often added to degrade hydrogen peroxide that is

generated by growth of some LAB, and oxygen in-

corporation is minimized. Cells are typically grown to

achieve cell densities of 10

to 10

cells per ml, and

then packaged in a liquid form, concentrated or ly-

ophilized (freeze-dried). LAB are ideally harvested

either at late log phase or early stationary phase;

however, the optimum harvest time depends on the

specific organism. Liquid cultures are packaged in

cans having volumes ranging from 100 to 500 ml,

then rapidly frozen in liquid nitrogen (< 196



C).

It is important that the cultures remain frozen

(< 40



C) throughout storage, as well as during

transportation, since freezing and thawing lead to

the formation of ice crystals, which can puncture

and kill cells. Thawing can occur even when the

cells are held below 0



C, and so it is important to

put frozen cultures promptly into a 40



Cor

70



C freezer as soon as they arrive at the plant.

0021To produce frozen direct-to-vat cultures, cells

are prepared via high-cell-density fermentations (as

above), and then must be concentrated prior to freez-

ing. Either continuous centrifugation or cross-flow

membrane filtration is used. A washing step may be

included to remove the spent medium. Cell densities of

concentrated cultures can be as high as 10

to 10

cells per gram. Typically, a single can (usually 360 ml)

can be used to inoculate 2000 kg of milk.

0022For lyophilization, cells are grown and harvested,

as above, then freeze-dried under vacuum. Freeze

drying is simply a process whereby water is removed

from the frozen material by sublimation (See Freeze-

drying: The Basic Process; Structural and Flavor (Fla-

vour) Changes). It provides a means not only for

concentrating cultures, but also preserves them due

to the low water activity that is achieved. Freeze

drying is a far gentler process compared to other

drying technologies, such as spray drying. Freeze-

dried cultures can contain from 10

to 10

cells per

gram. The cells are usually packaged in foil pouches

or other oxygen-impermeable material. Lyophilized

cells are best maintained at 20



C, but are some-

what stable even at room temperature. Although ly-

ophilized cultures have become popular as DVS

starter cultures for cheese, yogurt, and cultured milk

products, cells may be sluggish after inoculation, with

a longer lag phase. To maintain cell viability for both

frozen and lyophilized cells, cryoprotectant agents are

usually added. Common cryoprotectant agents in-

clude glycerol, lactose, sucrose, trehalose, ascorbate,

and glutamate. It is important to note that freezing

and lyophilization parameters vary for different lactic

acid starter bacteria. For example, L. lactis subsp.

lactis is far more tolerant to freezing and lyophiliza-

tion than L. lactis subsp. cremoris.

Criteria for Evaluating Culture

Performance

0023In general, the job of most starter cultures is to carry

out the desired fermentation, promptly and consist-

ently, and to produce products having the appropriate

level of fermentation end products and desired flavor

and texture. The particular requirements for a given

strain, however, depend entirely on the application

for that culture (Table 3). For example, LAB used as

dairy starter cultures are selected based not only on

lactic acid production rates, but also on flavor- and

texture-producing properties, salt-sensitivity, phage

resistance, and durability during production and

5588 STARTER CULTURES

storage. The strains of Streptococcus thermophilus

and Lactobacillus delbrueckii subsp. bulgaricus used

as yogurt starter cultures, for example, are selected

based, in part, on acid development rates, acetalde-

hyde production, exopolysaccharide synthesis, and

lack of postfermentation acidity. However, different

strains of these bacteria may be used for Mozzarella

and Swiss cheese manufacture, since the latter prod-

ucts have their own requirements.

0024 To assess functional properties of dairy cultures,

several tests are routinely performed. Acid-production

rates are easily determined by inoculating heat-

treated milk with a suitable (and standardized) inocu-

lum (e.g., 0.1–3.0%), incubating the milk at a given

temperature, and then either measuring the decrease

in pH as a function of time or simply by determining

the pH or titratable acidity after a specific incubation

period. Lag times can also be estimated and are useful

in determining the effects of frozen or lyophilized

storage on culture activity. Another critical test is

determination of bacteriophage resistance, especially

under conditions that simulate cheese making. In

the Heap–Lawrence challenge test, cells are inocu-

lated into culture tubes of milk, representative or

factory phage mixtures are added, and the tubes are

incubated according to a cheese production time–

temperature profile. After incubation, the entire pro-

cess is repeated up to six times. An increase in pH (less

acid produced) in successive cycles indicates that the

culture has become sensitive to bacteriophage and

should be removed from the rotation.

0025Yeast cultures used for wine, beer, and bread should

ferment rapidly and have good sensory properties and

good storage stability. In addition, wine yeasts and

beer yeasts should sediment or flocculate to enhance

their separation and to reduce autolysis. Flocculation

is a heritable trait, requiring several genes that encode

for proteins involved in cell aggregation. Flocculated

cells sediment much faster relative to the free cells.

Other performance characteristics will depend on the

specific product. Many wine yeasts, for example, are

selected based on growth at low temperatures (10–



C), resistance to sulfiting agents, and high ethanol

tolerance. Beer yeasts should also be able to grow at

low temperature, produce flavorful end products, and

deplete fermentable carbohydrates (good attenu-

ation). Bread yeasts should withstand freezing (so

that frozen bread doughs will rise after thawing).

Using Starter Cultures

0026As described above, many starter culture prepar-

ations, whether in a frozen or lyophilized form, are

simply added directly into the food material. Frozen

canned cultures are routinely thawed in cold chlorin-

ated water immediately prior to use. Lyophilized cul-

tures may require additional stirring or mixing in the

vat to facilitate hydration. For bulk cultures, add-

itional steps are required to insure that the organisms

have reached sufficient levels and viability. In the

dairy industry, specialized culture media are almost

always used for bulk starter preparation, not only to

provide optimum growth conditions, but also to pro-

tect the cells from acid damage and from infection by

lytic bacteriophage. The bulk culture media is not

much different from the media used by the culture

supplier to mass-produce starter culture cells. A basal

medium containing a fermentable carbohydrate (lac-

tose, glucose, or sucrose) is supplemented with add-

itional sources of nitrogen, vitamins, minerals, and

other nutrients. Commercially available yeast extract

is an excellent source of many of these materials.

Phosphate salts are frequently added to provide buf-

fering as well as chelating properties.

0027Controlling pH during bulk culture preparation is

especially important since most LAB grow best above

pH 6.0, and can lose viability if held at pH values

below 5.0. The use of acid-injured starter cultures in

cheese manufacture will result in a long lag phase and

tbl0003 Table 3 Desirable properties of starter cultures

Culture Property

Dairy cultures Lactic acid production rates

Lag phase times

Phage resistance

Ease of manufacture

Stability and consistency

Produce desired flavor and texture

Preservation tolerance

Produce

Lack of off-flavors

Meat cultures Fast acidification

Produce desired flavor

Antimicrobial activity

Beer cultures Rapid fermentation

Produce desired flavor

Preservation tolerance and stability

Flocculation

Lack of off-flavors

Proper attentuation

Growth at wide temperature range

Tolerant to osmotic, temperature, and

handling stresses

Wine cultures Osmotolerant

Ethanol-tolerant

Flocculation, sedimentation

Growth at low temperature

Produce consistent flavor

Malolactic fermentation

Bread cultures Freeze-tolerant

Produce desired flavor

Produce adequate leavening

STARTER CULTURES 5589

prolonged fermentation times. Several notable ap-

proaches have been developed to minimize or prevent

acid damage to lactic starter cultures. The first ap-

proach, known in the industry as ‘external pH con-

trol,’ relies on the addition of alkaline solutions, such

as ammonium hydroxide, or ammonia gas, into the

bulk culture tank during fermentative growth.

The tank is fitted with a pH electrode, and a pH-

monitoring device that sends a signal to a pump

when the pH has decreased below a critical threshold.

Alkali is then added until an upper pH limit is

reached. Although the range at which the pH is con-

trolled depends on the specific organisms, most exter-

nal control systems maintain a pH between 5.8 and

6.2. Thus, as cells grow and produce acid, the pH

never falls below the set threshold. Such systems yield

starter cultures having high cell densities (as much as

10 times more cells) with enhanced viability. Lactate

salts that accumulate as a byproduct result in higher

media osmolality and ionic strength, but these effects

do not appear to have any significant negative effects

on cell viability. Due to the hardware expenses (pH

electrodes, pumps, pH-monitoring devices) inherent

in external pH control systems, however, a less ex-

pensive alternative is simply to manually neutralize

the bulk culture tank. This is done by a one- or two-

time addition of neutralizing agent (sodium or potas-

sium hydroxide) that raises the pH from around 5.0

to near 7.0.

0028 Although most culture media ordinarily contain

phosphate or citrate salts that provide some buffering,

it is not possible to add enough of these salts to main-

tain a near-neutral pH during growth by LAB. This is

because high phosphate concentrations are inhibitory

to some LAB used as starter cultures. Media can, how-

ever, still be formulated to contain buffer agents pro-

vided that the buffers are either in an insoluble or

encapsulated form. These so-called internal pH con-

trol systems typically contain carbonate or phosphate

salts (e.g., sodium carbonate or trimagnesium phos-

phate) that are released into the medium as a function

of low pH, such that the pH of the culture medium is

maintained above 5.0. Internal pH control systems

have the added advantage of not requiring the expen-

sive external pH control apparatus. Agitation is re-

quired, however, to prevent the buffer agents from

settling out during bulk culture growth.

0029 Although equipment and labor costs for external

pH control systems and for internal pH control media

are higher than those for conventional culture prepar-

ation systems, several advantages are realized. Since

higher cell densities are achieved, and cell viability is

enhanced, less culture is needed. In addition, because

the cells are maintained in a viable state, they can be

used for a longer period of time.

Bacteriophage

0030Bacteriophage are viruses that can infect and eventu-

ally lyse bacteria (Figure 3). They can multiply rap-

idly and decimate the starter culture. Bacteriophage

problems during bulk culture production are rela-

tively rare because culture media are heated to a

high temperature and aseptic conditions (i.e., closed,

sterilizable vats) are usually employed. However,

milk used for cheese manufacture receives only a

modest heat treatment, and cheesemaking, in general,

is an ‘open’ process, providing ready access by air- or

whey-borne phage. Thus, in most modern cheese pro-

duction factories, where vats are filled and refilled

during the production day, phage may have ample

opportunity to propagate and to reach high levels,

resulting in slow or arrested fermentations and poor-

quality product. Until recently, lactococcal bacterio-

phage were the main concern; however, increased

production of Mozzarella cheese, yogurt, and other

products that rely on thermophilic starter cultures has

led to the proliferation of phages against S. thermo-

philus and L. bulgaricus.

0031For nearly 50 years, problems caused by bacterio-

phage have been the driving force for much of the

Adsorption

Injection

DNA

replication

Phage

replication

Packaging

Lysis

Restriction/

modification

Abortive

infection

Adsorption

inhibition

Phage

Bacterial cell

fig0003Figure 3 Lytic cycle of lactic bacteriophage and steps at which

lactococcal phage resistance mechanisms operate.

5590 STARTER CULTURES

research on starter cultures. The dairy starter culture

industry and the cheese industry have adopted several

strategies to prevent or reduce the impact of bacterio-

phage problems. Strict sanitation and attention to

plant design are the first lines of defense against

phage. Ideally, rooms used to ‘step up’ starter cultures

to the concentrations needed for production are isol-

ated from the rest of the processing facility. Since

phage are commonly transmitted via air, these con-

tained areas should be held under positive pressure,

and filtered air should be used both for the intake into

the starter room, as well as into the starter tanks. The

production rooms should be designed such that

‘downstream’ product and waste flow do not contam-

inate the starter preparation room.

0032 Phage-inhibitory media have been embraced as an-

other means of phage control. Phage require calcium

ions to attach to, and subsequently invade, their

bacterial hosts. In 1956, the US Department of Agri-

culture introduced the idea of using phosphates in

culture media to bind calcium, thus making the cal-

cium ions unavailable for the phage. In addition to

their role in binding calcium, the phosphate salts in

phage-inhibitory media provide buffering capacity.

Citrate salts provide similar protection. Phage-

control strategies are summarized in Table 4.

0033 Most starter culture strains are only sensitive to a

specific bacteriophage, and some strains are naturally

resistant, due to one or more of several types of

phage-resistant mechanisms (Figure 3). In addition,

sensitive strains can become resistant by frequent

exposure to lytic phage (bacteriophage-insensitive

mutants or BIM) or by genetic manipulation. If the

phage-resistant variants still have good cheese-

making properties (which must be tested), then these

strains can be introduced as a starter culture, usually

as one of several other defined phage-resistant strains.

As described above, multiple defined strain starters

contain as many as five different strains, selected

largely on the basis of being phage-unrelated. These

cultures can be used on a continuous basis providing

consistent product quality. It is important, however,

to monitor phage levels in the cheese whey on a

regular daily basis, so that strains that become

phage-sensitive can be removed from the mixture

and replaced by a resistant strain.

0034Additional protection against phage can be

achieved by rotating cultures, meaning that a single

culture (containing as many as five or six different

strains) is used on a given day and then followed

by successive cultures containing strains whose

phage-sensitivity pattern is different from the preced-

ing culture. Thus, since phage proliferation requires

susceptible host strains, removing those strains from

the cheese production environment will reduce the

background level and accumulation of phages such

that normal fermentation rates can be achieved.

However, this practice is constrained by the limited

availability of phage-unrelated strains, as well as by

less consistent product quality. Many of the defined,

phage-resistant cultures now contain only two or

three strains.

New Developments in Starter Culture

Technology

0035The pioneering work of McKay and coworkers led

to the discovery that most of the phenotypic

traits necessary for lactic starter cultures, including

lactose fermentation, casein hydrolysis, and diacetyl

formation, were encoded by plasmid DNA. The

Klaenhammer group subsequently demonstrated

that phage-resistant determinants were also plasmid-

encoded. These discoveries, and the development of

gene-exchange and gene-transfer techniques, made it

possible to manipulate the genetic properties of lactic

starter cultures (See Biotechnology in Food Produc-

tion). However, because of regulatory and consumer

concerns regarding genetic modification of foods, the

applications of these technologies have generally been

restricted to noninvasive approaches. (See Genetically

Modified Foods.)

Engineering Phage Resistance

0036Of particular importance has been the construction of

genetically modified, phage-resistant lactococcal

strains. In the early 1980s, the Klaenhammer group

identified a 46-kb plasmid in Lactococcus lactis

subsp. lactis that encoded for a heat-sensitive abortive

infection mechanism and that conferred resistance

to specific bacteriophage. This self-transmissible

tbl0004 Table 4 Phage-control strategies

Method Purpose

Sanitation Kill and remove phage in plant environment

Plant design Keep phage out of production area, prevent cross-contamination

Phage-inhibitory media Prevent phage from attaching to and infecting culture cells

Phage-resistant cultures Design starter cultures cells that will grow and perform well even in presence of phage

Culture rotation Prevent proliferation of phage by limiting access to suitable host

STARTER CULTURES 5591

plasmid, named p TR2030, was later found to con-

tain other regions, including genes coding for a re-

striction and modification system, that also conferred

bacteriophage resistance. Transfer of the plasmid into

a phage-sensitive, cheesemaking strain via conjugal

mating resulted in fast lactose-fermenting transconju-

gants having resistance to a broad range of lytic indus-

trial phages. Although this approach was successful

for a time, phages eventually appeared that could

overcome the host resistance. In fact, due to the dy-

namic nature of phage–host interactions and the non-

sterile manner in which cheese manufacturing occurs,

the propensity of lytic phages to adapt to and ultim-

ately appear against formerly resistant strains is nearly

inevitable.

0037 Several recent strategies have since been described

to provide more long-term phage protection. One

approach is simply to introduce or ‘stack’ two plas-

mids, each carrying phage resistance mechanisms

against different phage types, into a single strain. In

contrast, different phage-resistant genes can be intro-

duced individually into a single strain generating sev-

eral isogenic phage-resistant derivatives that can then

be used in a rotation scheme. Since each strain

harbors genes effective against different phage types,

the same parental strain can be used on a continuous

basis. Another system, called Per, relies on the inte-

gration of a phage DNA fragment (containing the

origin of replication region or ori site) into the

genome of the host strain. Ordinarily, when a bacter-

ium is infected by bacteriophage, phage DNA repli-

cation begins at the ori site. However, in the Per

strains, phage development is stalled, since the

chromosomal copy of the ori site acts as a decoy for

the phage replication machinery. Antisense RNA

strategies function in a similar manner, in that the

modified cell is directed to transcribe an antisense

messenger RNA that is complementary and capable

of hybridizing to the normally transcribed RNA. Re-

cently, a triggered-suicide system was described that,

when activated by the presence of a lytic phage,

causes the infected cells to lyse and die before phage

replication has occurred.

Strain Identification

0038 Advances in nucleic acid-based bacterial fingerprint-

ing methods have led to new identification tools and

renewed interest in bacterial taxonomy. The ability to

distinguish between strains of the same species is

important not only for identification purposes, but

also because it provides a basis for understanding

the phylogenetic and evolutionary relationships be-

tween LAB. Although morphological, biochemical,

and other phenotypic characteristics remain useful

for genus and species identification, molecular ap-

proaches that rely on nucleotide sequences have

proven to be more reliable, more reproducible, and

more robust. Several techniques, in particular, are

widely used, including pulsed-field gel electrophoresis

(PFGE), restriction fragment length polymorphism

(RFLP), ribotyping, 16S ribosomal RNA sequence

analysis, and various polymerase chain reaction-

based protocols. More precise methods of strain iden-

tification have also become important as proprietary

organisms are developed with unique characteristics

and production capabilities.

Encapsulated and Immobilized Cells

0039Another manner in which starter cultures can be pro-

duced and used is to encapsulate or surround cells in

inert materials, such as alginate beads. Alternatively,

starter culture cells can be adsorbed to a carrier sur-

face or covalently attached to inert support materials.

In theory, encapsulated cells may be less sensitive to

phage infections, be more stable during storage, and

be recovered after fermentation for reuse in subse-

quent fermentations. However, immobilization pro-

cedures can be costly and may alter starter culture

metabolism. For these reasons, immobilized culture

technology has not yet been adopted in the dairy

industry.

0040It is worth noting that a natural form of encapsu-

lated starter culture has long existed in the form

of kefir grains. Kefir, a fluid-fermented product

widely consumed in Russia and Eastern Europe, is

traditionally made by inoculating milk with kefir

grains. Kefir grains are irregularly shaped, polysac-

charide-containing particles up to 15 mm in diameter

that harbor a complex microflora, including homo-

and heterofermentative LAB and yeasts. Kefir grains

can be removed from the completed fermentation by

filtration or sieving, washed, and reused in subse-

quent fermentations. Pure cultures, usually contain-

ing strains of Lactobacillus spp. and Lactococcus

spp., are now available commercially, and are com-

monly used in the USA for kefir manufacture.

Probiotics and Antimicrobials

0041Although starter cultures usually consist of those or-

ganisms required to perform a specific fermentation,

the same technology, for the most part, is used to

produce bifidobacteria and other probiotic cultures,

even though they are used differently (See Bifidobac-

teria in Foods; Probiotics). Additionally, it has long

been known that some strains of LAB produce com-

pounds that inhibit other microorganisms. One

particular class of inhibitory compounds are the

5592 STARTER CULTURES

bacteriocins, proteinaceous materials produced by an

organism that inhibits other closely related organ-

isms. Nisin, a bacteriocin produced by strains of Lac-

tococcus lactis, is approved for use in processed

cheese and other foods as a direct food additive (See

Nisin). LAB that produce nisin and other bacteriocins

can be used as part of a lactic acid-producing starter

culture or as an adjunct in dairy, meat, and other

foods to inhibit pathogens as well as spoilage organ-

isms. In some cases, the organism produces bacterio-

cin in the food, but does not produce acids or other

fermentation end products that would alter the sens-

ory characteristics of the product. Alternatively, the

organism can be grown in a dairy- or nondairy-based

medium, and the fermented medium containing or-

ganic acids and bacteriocin is subsequently pasteur-

ized and concentrated. Viable cells are absent, but the

material, when added to foods, would still contribute

antimicrobial activity. These ‘bioprotective’ products

have been marketed as shelf-life extenders, although

pathogens may also be inhibited. Most of these prod-

ucts are effective only against Gram-positive bacteria;

however, some products also inhibit Gram-negative

spoilage bacteria, including psychrotrophs.

See also: Beers: History and Types; Raw Materials; Wort

Production; Biochemistry of Fermentation; Chemistry of

Brewing; Bifidobacteria in Foods; Biotechnology in

Food Production; Bread: Dough Mixing and Testing

Operations; Dough Fermentation; Cheeses: Types of

Cheese; Genetically Modified Foods; Lactic Acid

Bacteria; Probiotics; Vinegar; Wines: Types of Table

Wine

Further Reading

Allison GE and Klaenhammer TR (1998) Phage resistance

mechanisms in lactic acid bacteria. International Dairy

Journal 8: 207–226.

Caplice E and Fitzgerald GF (1999) Food fermentations:

role of microorganisms in food production and preser-

vation. International Journal of Food Microbiology 50:

131–149.

Champagne CP, Lacroix C and Sodini-Gallot I (1994)

Immobilized cell technologies for the dairy industry.

Critical Reviews in Biotechnology 14: 109–134.

Ford A and Fitzgerald GF (1999) Bacteriophage defence

systems in lactic acid bacteria. Antonie van Leeuwen-

hoek 76: 89–113.

Hammes WP and Hertel C (1998) New developments

in meat starter cultures. Meat Science 49(suppl.):

S125–S138.

Hutkins RW (2001) Metabolism of starter cultures. In:

Marth EH and Steele JL (eds) Applied Dairy Micro-

biology, pp. 209–241. New York: Marcel Dekker.

McKay LL and Baldwin KA (1990) Applications for bio-

technology: present and future improvements in lactic

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production of lactic acid bacteria. In: Salminen S and

von Wright A (eds) Lactic Acid Bacteria, pp. 73–103.

New York: Marcel Dekker.

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(2000) Novel cultures for cheese improvement. Trends

in Food Science and Technology 11: 96–104.

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Starvation See Famine, Starvation, and Fasting

STERILIZATION OF FOODS

M N Ramesh, Central Food Technological Research

Institute, Mysore, India

Background

0001 Sterilization is the complete destruction or elimin-

ation of all viable organisms in/on an object being

sterilized. Sterilization destroys yeasts, molds, vegeta-

tive bacteria, and spore formers, and allows the food

processor to store and distribute the products at am-

bient temperatures, thus extending the shelf-life. Ster-

ilization procedures involve the use of heat, radiation,

or chemicals, or physical removal of cells. The steril-

ization process involves four distinct stages. First, the

product must be heated at 110–125



C to ensure

sterilization. After this, the product requires a few

minutes to equilibrate, since the surface will be hotter,

and the central portion of the container will still be

cool. The equilibration stage allows a reduction in the

temperature gradient. Next, the product must be held

STERILIZATION OF FOODS 5593

at this temperature for a certain period of time to

ensure a predetermined sterilization value designated

by the F

value. Finally, the product has to be cooled

down mainly to arrest further heat treatment and to

avoid bursting of the container at hot conditions.

0002 The basic principles of sterilization technology as

applied to food processing are as follows:

0003 The processed product must be free from micro-

organisms capable of producing food poisoning

toxins and those microorganisms that cause food

spoilage during the product’s shelf-life, until it is

consumed.

0004 Clostridium botulinum spores are capable of grow-

ing in low acid (pH > 4.6) products during storage

and hence must be heat-treated to the equivalent of

at least 121.1



C for 3 min (an F

value of 3) to

achieve a 12-decimal reduction in the numbers of

the microorganism.

0005 The processing conditions should be applied to the

slowest-heating point, referred to as the ‘cold

point.’ This facilitates the assumption that, when

the slowest heating part is sterilized, by exposing it

to the required time–temperature profile, the rest of

the product will be sterilized.

Practically, complete sterilization leads to a deterior-

ation in product quality and nutrient retention

(Figure 1). Hence, in practice, commercial sterility is

defined as a product that has been processed so that,

under normal conditions, the product will neither

spoil nor endanger the health of the consumer. The

pH of the product is an important factor in determin-

ing the severity of the sterilization process.

Theory of Sterilization

0006 Thermal treatment of food products to render them

free of pathogenic microorganisms has been practiced

for several years. However, a method to quantify the

microbial destruction that takes place during thermal

treatment has only been understood for the last 75

years. In order to determine the amount of microbial

destruction that a thermal treatment delivers to a

process, an understanding of the amount of heat de-

livered to every portion of the food product and the

destruction kinetics of the microorganisms of interest

is required. The amount of heat delivered by a food

process is dependent on the way in which the product

is heated and its physical nature. Process-dependent

factors can include: processing equipment design,

type of heating media, container, food size and

shape, product composition, and viscosity (conduc-

tion- or convection-heated). The thermal destruction

kinetics of microorganisms or their ability to be killed

within the food matrix is dependent on a number of

factors, including: pH of the product, levels and types

of preservatives, water activity, the previous growth

conditions of the microorganisms of concern, product

composition, and competitive microorganisms.

0007The two types of bacteria of concern in food pre-

servation are organisms of public health significance

and spoilage-causing bacteria. In low-acid foods with

a pH greater than 4.6, the organism of public health

significance is Clostridium botulinum. Canned foods

are processed, based on the survival probability for

C. botulinum of 10

12

, or one survivor in 10

cans.

The organism most frequently used to characterize

low-acid food spoilage by mesophilic spore-formers

is PA 3679, a strain of C. sporogenes. Most food

companies accept thermal inactivation of 10

5

for

mesophilic spore-formers and 10

2

for thermophilic

spore-formers. The processing time depends on the

bioburden of the most resistant bacteria in a particu-

lar food, the spoilage risk involved, and whether food

can support the growth of potential contaminating

bacteria. Although a large amount of research work

has been carried out on the influence of different

factors on the processing time and the corresponding

sterilization value, a number of uncertainties still

exist on the application of these factors to arrive

scientifically at the exact processing conditions. In

order to avoid any risk resulting from these uncertain-

ties, a safety factor is added to increase the processing

time to sterilize the food product completely, which

invariably reduces the nutrient content and the

increase in energy cost.

Decimal Reduction Time or

value

0008The decimal reduction time, or D value, is the time at

any temperature to destroy 90% of the spores or

vegetative cells of a given organism. It is equal to the

number of minutes for the survivor curve to traverse

Time (min)

Quality

fig0001 Figure 1 Effect of sterilization on quality. (a) Degree of steril-

ization; (b) degree of nutrient loss. From Ramesh MN (1995)

Optimum sterilization of foods by thermal processing – a review.

Food Science and Technology Today 9(4): 217–227 with permission.

5594 STERILIZATION OF FOODS

one log cycle and can be calculated from the

reciprocal of the slope of the survivors, assuming a

logarithmic death rate:

N ¼ N

kt

;

where N is the final number of survivors after heat

treatment, N

is the initial number of organisms, e is

the exponential function, k is the thermal death rate

coefficient (traditionally measured in s

1

), and t is the

processing time. Rearranging gives

log N ¼ log N

ð60kt=2:3Þ:

Plotting log N against t in minutes gives the slope

60k/2.3 min

1

. The inverse of the slope is

D ¼ 2:3=60k min:

Thermal Death Time Constant or

Value

0052 The D value may be calculated from the inverse of the

slope of the survivor curve by regression analysis of

the data points. If a presence/absence (growth/no

growth) method is used to estimate the number of

survivors, N

can be calculated using the equation

¼ 2:303 logðn=rÞ,

where n is the number of units heated, r is the number

of sterile units, and N

is the most probable number

of survivors. The graphical representation of the

D-value concept is indicated in Figure 2.

0009 The change in D value with temperature can be

obtained by plotting log D against temperature. The z

value is the number of degrees for the thermal resist-

ance curve to transverse one log cycle and is equal to

the reciprocal of the slope of the curve. The equation

applied to the thermal resistance curve is given by

log D

Ref

¼ log D

¼ 1=½zðT  T

Ref

Þ;

where D

Ref

is the D value at the reference tempera-

ture (T

Ref

of 121.1



C), D

is the D value at any other

temperature, T, and z is the thermal death time

constant.

0010A graphical representation of the D-value concept

is shown in Figure 3.

Heat Resistance of Microorganisms

0011The most important bacterial spore former with

respect to heat processing is Clostridium botulinum,

because of the potent neurotoxin that it produces.

The organism occurs in seven distinct stereotypes

A–G, which are further subdivided into proteolytic

and nonproteolytic strains. The most heat-resistant

spores are those produced by type A and the proteo-

lytic B strains. The D value at 121.1



C of the most

resistant strains is generally considered to be

0.21 min. Clostridium sporogenes, which is closely

related to the proteolytic strains, produces spores

that are more heat-resistant. The D value of the

spores at 121.1 C



can be up to 1.5 min. C. sporo-

genes has been reported to be the most common

mesophilic putrefactive anaerobe in the spoilage of

low-acid (pH  4.6) canned foods.

0012The butyric anaerobes (C. butyricum, C. beijer-

inckii, and C. pasteurianum) are usually associated

with spoilage of products with pH values between 3.9

and 4.6 (e.g., tomatoes and pears), producing blown

cans and a butyric odor. Another organism that causes

spoilage of products down to pH 4.2, Bacillus coagu-

lans, has been reported to have a D value at 98.9



Cof

3.1 min and a z value of 16.1C



. Other mesophilic

Bacillus species that produce heat-resistant spores

and have been implicated in food poisoning out-

breaks are B. subtilis and B. licheniformis. Spores of

Minutes

Bacteria /g

D value

100

1000

10 000

10 15 20 25 30

120 ⬚C

110 ⬚C

Log N/N 0 = time/D

D = 12−6 = 6 min@ 120 ⬚C

D = 24−12 = 12 min@ 110 ⬚C

fig00 02 Figure 2 D-valueofamicroorganism. Fromhttp://cour-

ses.che.umn.edu/00fscn1102-1s/pdf_files/topic_11_Past_and_-

Ster.pdfwithpermission.

Temperature

D-value

z value

0.1

1.0

10.0

100.0

1000.0

100 110

Z = 110 − 102 = 8 ⬚C

fig0003Figure 3 z-valueofamicroorganism. Fromhttp://cour-

ses.che.umn.edu/00fscn1102-1s/pdf_files/topic_11_Past_and_-

Ster.pdfwithpermission.

STERILIZATION OF FOODS 5595

the thermophilic organisms that have an optimum

growth temperature of 55



C are usually much

more resistant to wet heat than mesophilic organisms.

0013 The most resistant thermophile is Clostridium ther-

mosaccharolyticum, which produces blown cans and

a butyric or ‘cheesy’ odor, with D values as high as

68–195 min at 121.1



C. The spores of Bacillus stear-

othermophilus have often been used in process evalu-

ation studies because of their high heat resistance.

The organism can cause spoilage of products with

pH values > 5.3. Another unusual bacterial spore

former is Sporolactobacillus inulinus. This organism

has been isolated from a number of food and environ-

mental sources and has been reported to have a

D value at 90



C of 5.1 min and a z value of 13 C



Quality-control personnel should be aware, there-

fore, that not all isolates of Gram–positive spore-

forming rods belong to the genera Bacillus or

Clostridium. Typical D and z values for some of the

spore formers are listed in Table 1.

Lethal Rate

0014 Using the z value, the lethal rate, L, can be calculated

from

L ¼ log

1

½ððT  T

Ref

Þ=zÞ:

The lethal rate is a measure of the lethality of any

temperature, T, relative to the reference temperature

Ref

. For example, for a reference temperature of

121.1



Candaz of 10C



, the lethal rate at 111.1



will be

log

1

½ð111:1  121:1Þ=10Þ ¼ 0: 1:

Thus, 1 min at 111.1



C is worth 0.1 min at 121.1



in terms of lethality. Note that z is written C



and not



C, since z represents a change of 10 Centigrade

degrees in the above example and not a temperature

of 10 degrees Centigrade.

Value

0015The symbol F designates the equivalent in minutes at

121.1



C of the combined lethalities of all time–tem-

perature relationships at the point of slowest heating

for a product during heat processing. Thus, the F

value is a measure of the killing power of a heat

process. The term F

denotes the F value at the center

of a pack, F

denotes the equivalent F value in

minutes at 121.1



C, and F

denotes the integrated

lethality of heat received by all points in a container.

can be related to the D value by the equation

¼ D

Ref

ðlog N

 log NÞ;

where D

Ref

is the D value at 121.1



C, log N

is log of

the initial number of organisms, and log N is the log

of the final number of surviving organisms. In the

above equation, F

can be considered as equivalent

to F

or F

in rapid heating systems such as the

thermoresistometer. The F

value of a process can be

obtained in practice by summing the lethal rates at

1-min intervals from the heating and cooling curve of

a product during a heat process. This is a simplified

view, and process calculations in the industry involve

rather more complicated mathematics and usually do

not include the cooling curve contribution to the

process. The process time can be calculated using

the equation

tbl0001 Table 1 Typical heat resistances of selected spore formers

Organism Medium Temperature (



C) D value (min) z (C



)

Clostriduim sporogenes (PA 3679) Several substrates including pea pure

e 104.4–143.3 0.75–2.03 9.0–14.7

Phosphate buffer pH 7 112.8–148.9 1.06 9.3

Clostriduim botulinum Several substrates 104.0–132.2 0.051–0.58 8.2–10.4

Phosphate buffer pH 7 120.0–140.0 0.13 11.0

Other Clostridium sp. Several substrates 85.0–121.0 0.2–195 6.9–11

Bacillus sp. Several substrates including milk 100.0–121.0 0.3–16.0 4.1–7.7

Desulfotomaculum nigrificans 121 55.0 9.5

Escherichia coli Nutrient broth 70 0.006 4.9

Milk 70 0.04 6.5

Lactobacillus sp. Tomato juice 70 4.0–11.0 11.5–12.5

Listeria monocytogenes Beef, chicken, and carrot homogenates 70 0.14–0.27 5.98–7.39

Salmonella typhimurium Aqueous sucrose/glucose (a

0.995) 70 0.03–816 6.8–19.0

Milk chocolate (51% milk)

Staphylococcus aureus Milk 70 0.30 5.1

Streptococcus sp. Broth and ham 70 0.015–2.84 3.5–17.0

Microbacterium lacticum Skim milk 70 4.0

From Brown KL (1991) Principles of heat preservation. In: Rees JAG and Bettison J (eds) Processing and Packaging of Heat Preserved Foods, pp. 15–49.

New York: Van Nostrand Reinhold.

5596 STERILIZATION OF FOODS