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an internal fragment of the target gene. With increas-

ing cycles of PCR amplification, the fluorescent

signal increases proportionately. A 5

nuclease assay

targeting the Yst gene has been shown to be highly

specific in detecting virulent Y. enterocolitica. This

assay has allowed a sensitive detection of 10

CFU

1

in pure culture and 10 CFU g

1

in spiked

ground pork. The 5

nuclease assay developed for

detecting virulent strains has also been shown to

yield a better estimation of contamination of pork

and other processed meat and a better detection level

than the ‘gold standard’ recommended by the US FDA

in the Bacteriological Analytical Manual for the detec-

tion of pathogenic Y. enterocolitica. The assay is rapid

and quantitative, can be performed for a number of

samples at a time, and is suitable for application as an

automated high-throughput detection system.

0021 Conventional PCR-based assays fail to discrimin-

ate between viable and nonviable cells. However, a

reverse-transcriptase 5

nuclease assay targeting a spe-

cific gene transcript can be potentially exploited for

the specific detection of viable Y. enterocolitica. PCR–

ELISA is another alternative to circumvent electro-

phoretic analysis of PCR products and reduce the

post-PCR analysis time. Essentially, a biotinylated

capture probe is employed to immobilize the labeled

PCR products on to microtiter plates. This colorimet-

ric assay performed in the microtiter format is amic-

able to automation, can be applied to a large number

of samples simultaneously, and allows the PCR prod-

uct to be quantified directly. A PCR–ELISA adopted

for detecting virulent strains has demonstrated a high

specificity and sensitivity comparable to gel electro-

phoresis in detecting 6 CFU of the bacterium per PCR

reaction. For the detection of Y. enterocolitica, the

BAC Screen and BAC Ident kits are available com-

mercially for conventional PCR and agarose gel

detection and the LightCycler and ABI Prism PCR

systems, respectively.

0022 Random-amplified polymorphic DNA (RAPD)

analysis is the amplification of random DNA

segments with single primers of arbitrary nucleotide

sequence. The difference in banding pattern allows

strains belonging to the same species to be identified.

Arbitrary primers used in RAPD have been shown

to differentiate pathogenic and nonpathogenic

strains and their serotypes. RAPD can also reveal

coinfection with more than one strain of Y. entero-

colitica, indicating the potential of this technique to

detect strain variations in epidemiological studies.

Treatment of

Yersinia enterocolitica

0023 Yersinia enterocolitica is significantly implicated in

foodborne illness leading to yersiniosis, arthritis, or

even septicemia in children. In the majority of cases,

patients recover without any treatment. Uncompli-

cated enterocolitis is a self-limiting disease. However,

in older children, certain complications like pseudo-

appendicular syndrome, mesentric adenitis, ileitis,

and extramesentric complications can be prevented

by administering antibiotics, like cotrimoxazole. Un-

complicated gastroenteritis is often treated with nor-

floxacin, ciprofloxacin, or ciprofloxacin/doxycycline.

0024Regardless of the age, any patient with culture-

proven septicemia should be treated. Treatment may

shorten both the duration of the disease and the

shedding of the bacterium. Aminoglycosides, in

combination with third-generation cephalosporins,

quinolones, or rifampin and trimethoprim-sulfa-

methoxazole, are effective against septicemia. No con-

trolled clinical trials have demonstrated the efficacy of

treatment, although septicemic patients should receive

ampicillin and tetracycline becausesevere infectionhas

a higher mortality (around 50%) rate.

0025Systemic infections, extraintestinal fecal infections,

and diseases in immunocompromised children have

been treated with cotrimoxazole (bactrim, septra,

sulfatrim), cefotaxime (cloforan), and for a long

time gentamycin (garamycin), which was later found

as a failed drug. For cotrimoxazole, the recommended

dosage is 160 mg day

1

PO bid in adults weighing

> 40 kg. The pediatric prescribed dosage is

8–10 mg kg

1

day

1

PO divided bid. Dose-related

complications include renal or hepatic impairment,

crystaluria, stone formation, and possible hemolysis

in glucose-6-phosphate dehydrogenase deficiency.

Cefotaxime (claforan) is recommended in an adult

dosage of 1–2 g day

1

divided q

6–8h

intravenously/

intramuscularly (IV/IM). Children are injected IV/

IM with 150–180 mg kg

1

day

1

divided q

6–8h

.In

patients with meningitis, 300 mg kg

1

day

1

IV/IM

divided q

6–8h

is recommended. Hypersensitivity to

cephalosporin antibiotics has been documented. A

dosage of 100 mg kg

1

day

1

ciprofloxacin started

early in the infection is an effective antibiotic treat-

ment recommended in Yersinia-triggered reactive

arthritis. However, antibiotic treatment has no effect

on fully developed arthritis.

0026The agents traditionally used to treat human infec-

tions including cotrimoxazole, doxycycline, and chlo-

ramphenicol may remain the drugs of first choice.

Very high failure rates of b-lactams like amoxycillin

with or without clavulanate, benzylpenicillin,

erythromycin, clindamycin, and first-generation

cephalosporins have been observed. Ninety-nine per

cent of all Yersinia strains have been found to be

resistant to amoxycillin. Variations in minimum

inhibitory concentrations (MICs) of different biovars

are shown in Table 3.

6250

YERSINIA ENTEROCOLITICA

/Detection and Treatment

0027 This implies a complex regulation of b-lactamases in

Y. enterocolitica. Some b-lactamases are prevalent and

significantly expressed in specific biovars. Biovar 1A,

1B 2,3, and 4and serotypes O:3; O:5,27; O:6, 30;O:7,

8; O:8, and O:9 are all found to be susceptible to most

of the cephalosporins, aminoglycosides, quinolones,

cotrimoxazole, doxycycline, piperacillin, tazobactam,

and imipenem and highly resistant to cefazolins,

amoxycillin, coamoxyclov, and macrolides. In clinical

trials, cotrimoxazole and doxycycline have been found

to be effective to an extent of 70–75%, whereas cefur-

ozine, ceftazidine, cefaparazone, piperacillin, and gen-

tamycin failed in seven of the eight courses.

0028 Hygienic practices avoiding close contact with

infected persons can prevent transmission of yersinio-

sis, thus obviating the need for drugs. Extreme care

needs to be taken while handling natural warm-

blooded reservoirs like pigs. Infection of Y. enteroco-

litica starts with the spread of the bacterium from the

intestinal contents of the host carrier to the carcass

and body parts. Virulent Y. enterocolitica serogroup

O:3 has been isolated from various surfaces in slaugh-

ter houses. Stringent measures must be taken to

prevent Y. enterocolitica contamination during

slaughter with potential growth during subsequent

storage and refrigeration. To prevent the initial col-

onization of Y. enterocolitica in reservoirs, applica-

tion of vaccines or even feeding with an avirulent

biotype 1A Y. enterocolitica capable of producing an

in-vivo antagonistic substance (bacteriocin) against

virulent Y. enterocolitica may be employed.

0029 Research has focused on the development of

vaccines and has shown a positive application when

tested in mice. Intranasal immunization with Y. entero-

colitica O:8 extracellular extracts have been shown to

induce local and systemic immunity and subsequent

protection against nasal infection. Efforts have also

been made to design a rational live vaccine by mutat-

ing virulence-associated genes like Yersinia adhesion

A(Yad A), Mn-cofactored superoxide dismutase. It

has been found that the mutants are able to colonize

Peyer’s patches for at least 12 days, resulting in the

induction of significant antibody titers against Yersi-

nia outer-membrane proteins and in priming of

Yersinia antigen specific CD4

Th1 cell isolates

from spleen. A high level of attenuation does not

diminish the immunogenic properties of the mutant

strains.

0030In many experimental cases, oral vaccination is less

successful as the colonization of Y. enterocolitica is

inhibited in the gut. Vaccination has been shown to be

more efficient by parenteral immunization than by

oral route. Research has also paved the way for

designing subunit vaccines using microbial heat-

shock protein as a candidate and also interleukin-12

as an efficient alternative adjuvant to immunostimu-

lating complexes for the induction of protective

CD4

Th1-cell-dependent immune responses against

bacterial pathogens.

Concluding Remarks

0031Yersinia enterocolitica remains in the limelight as a

prototype to study bacterial invasion and associated

pathogenesis. Armed with an array of temperature-

inducible, plasmid, and chromosomal-encoded viru-

lence factors,and capable of being transmitted through

food, water, and blood transfusions, Y. enterocolitica

has instigated significant public health concerns. Con-

sequently, extensive research is being carried out to

detect its presence in food products and blood meant

for human transfusion. Elegant detection techniques

have been devised, embracing inputs from immuno-

logical and nucleic acid-based techniques. Antibiotic

therapy has been adopted for effective treatment in

patients with active infection and immunocomprom-

ised individuals.

See also: Yersinia enterocolitica: Properties and

Occurrence

Further Reading

Aulisio CCG, Mehlman IJ and Snaders AC (1980)

Alkali method for rapid recovery of Yersinia entero-

colitica and Yersinia pseudotuberculosis from foods.

Applied and Environmental Microbiology 39: 135–140.

tbl0003 Table 3 Antibiogram of Yersinia enterocolitica

Type Response Antibiotic MIC (mgl

1

)

Biovar 1A R/IS Ticarcillin 16–64

Fosfomycin

Amoxycillin/clavulanate 4–32

S Chloramphenicol 4.0

Trimethoprim 1.0

Ciprofloxacin 0.016

Biovar 1B S Amoxycillin/clavulanate 0.5–2.0

Biovar 2 S Fosfomycin 4–16

Biovar 3 R Amoxycillin/clavulanate

Ampicillin

Cefoxitin

S Ticarcillin 4–32

Fosfomycin

Biovar 4 R/IS Ticarcillin

Amoxycillin/clavulanate

S Fosfomycin 1–4

Biovar 5 IS Amoxycillin/clavulanate

S Fosfomycin 1–32

IS, intermediate sensitivity; R, resistant; S, sensitive.

YERSINIA ENTEROCOLITICA

/Detection and Treatment 6251

Bhaduri S, Turner-Jones C, Taylor MM and Lachica VR

(1990) Simple assay of calcium dependency for virulent

plasmid-bearing clones of Yersinia enterocolitica. Ap-

plied and Environmental Microbiology 28: 798–800.

Bottone EJ (1999) Yersinia enterocolitica: overview and

epidemiological correlates. Microbes and Infection 1:

323–333.

Cleary TG (2000) Yersinia. In: Behrman RE, Kliegman RM

and Jenson HB (eds) Nelson Textbook of Pediatrics,

16th edn, pp. 857–859. London: W.B. Saunders, Har-

court Brace Jovanovich.

Cloak OM, Duffy G, Sheridan JJ, Blair IS and McDowell

DA (1999) Isolation and detection of Listeria spp,

Salmonella spp and Yersinia spp using a simultaneous

enrichment step followed by a surface adhesion immu-

nofluorescent technique. Journal of Microbiological

Methods 39: 33–43.

Feng P (1992) Identification of invasive Yersinia species

using oligonucleotide probes. Molecular and Cellular

Probes 6: 291–297.

Gray JT, WaKabongo M, Campos FE et al. (2001) Recog-

nition of Yersinia enterocolitca multiple strain infection

in twin infants using PCR-based DNA fingerprinting.

Journal of Applied Microbiology 90: 358–364.

Kampfer P (1999) Yersinia. Introduction. In: Robinson RK,

Batt CA and Patel PD (eds) Encyclopedia of Food

Microbiology, vol. III, pp. 2342–2350. London: Aca-

demic Press.

Kapperaud G, Varadund T, Skjerve E, Hornes E and

Michaelsen TE (1993) Detection of pathogenic Yersinia

enterocolitica in foods and water by immunomagnetic

separation, nested PCR and colorimetric detection of

amplified DNA. Applied and Environmental Micro-

biology 59: 2938–2944.

Khare SS, Kamat AS, Doctor TR and Nair PM (1996)

Incidence of Yersinia enterocolitica and related species

in some fish, meat and meat products in India. Journal of

Science Food and Agriculture 72: 187–195.

Manafi M and Holzhammer E (1994) Comparison of the

vitek, API 20E and Gene-trak systems for the identifica-

tion of Yersinia enterocolitica. Letters in Applied Micro-

biology 18: 90–92.

Nagaraju NR, Isloor S, Rao MS and Rajasekhar M (2001)

Prevalence of yersinia antibodies in serodiagnosis of

bovine brucellosis. Journal of Veterinary Research 5:

197–204.

Schiemann DA and Toma S (1978) Isolation of Yersinia

enterocolitica from raw milk. Applied and Environmen-

tal Microbiology 35: 54–58.

Stock I and Wiedemann B (1999) An in vitro study of the

antimicrobial susceptibilities of Yersinia enterocolitica

and the definition of a database. Journal of Antimicro-

bial and Chemotherapy 43: 37–45.

Vishnubhatla A, Fung DYC, Oberst RD et al. (2000) Rapid

nuclease (TaqMan) assay for detection of virulent

strains of Yersinia enterocolitica. Applied and Environ-

mental Microbiology 66: 4131–4135.

Wauters G (1981) Antigens of Yersinia enterocolitica. In:

Bottone EJ (ed.) Yersinia enterocolitica, pp. 41–53. Boca

Raton, FL: CRC Press.

YOGURT

Contents

The Product and its Manufacture

Yogurt-based Products

Dietary Importance

The Product and its

Manufacture

N Shah, Victoria University of Technology, Victoria,

Australia

Introduction

0001 At the beginning of this century, Nobel Laureate, Elie

Metchnikoff, at the Pasteur Institute, linked health

and longevity to the ingestion of bacteria present in

fermented foods such as yogurt. This observation

provided a major boost to the manufacture and

consumption of yogurt and other fermented milk

products.

0002Although no records are available to trace the

origin of yogurt, it is believed that fermentation

was the first technique employed by humans to

preserve foods. The word ‘yogurt’ was derived from

the Turkish word ‘Jugurt.’ Yogurt is defined as ‘a

product resulting from milk by fermentation with a

mixed starter culture consisting of Streptococcus

6252 YOGURT/The Product and its Manufacture

thermophilus and Lactobacillus delbrueckii ssp. bul-

garicus.’ However, in some countries, including Aus-

tralia, other suitable lactic acid bacteria are permitted

for use as starter cultures. As a result, some yogurt

manufacturers use Lactobacillus helveticus and Lacto-

bacillus jugurti for yogurt manufacture. The first US

Federal Standards of Identity for yogurt were pub-

lished in 1981. The standard does not permit the use

of culture organisms other than Lb. delbrueckii ssp.

bulgaricus and Sc. thermophilus for making yogurt,

and the titratable acidity must be at least 0.9%, ex-

pressed as lactic acid.

0003 Yogurts differ according to their chemical compos-

ition, method of production, flavour used and the

nature of postincubation processing. Based on the

fat content, there are three main types of yogurt:

full-fat yogurt, reduced-fat yogurt, and low-fat

yogurt (Table 1).

0004 On the basis of the method of production and the

physical structure of the coagulum, yogurts are clas-

sified as either set or stirred. Set yogurt is the product

formed when the fermentation of milk is carried out

in a retail container, and the yogurt produced is in a

continuous semisolid mass. In contrast, stirred yogurt

results when the coagulum is produced from milk,

and the gel structure is broken before cooling and

packaging. Fluid yogurt can be considered as stirred

yogurt of low viscosity.

0005 On the basis of flavorings, yogurts are divided into

three categories. Plain or natural yogurt is the trad-

itional product, which has a typical sharp ‘nutty’

flavor. Fruit yogurts are made by addition of fruits,

usually in the form of fruit preserves, puree or jam.

Flavored yogurts are prepared from plain or natural

yogurt by adding sugar and/or other sweetening

agents, flavorings and colorings.

0006 The method of yogurt production has changed very

little over the years. However, some newer varieties

have been added including frozen-, concentrated-,

dried-, and pasteurized yogurt. Postincubation pro-

cessing of yogurt may lead to pasteurized/UHT

yogurt, concentrated yogurt, frozen yogurt, and

dried yogurt. Pasteurized/UHT yogurt is prepared by

heat- treating yogurt after incubation. Heat treatment

destroys yogurt starter bacteria and reduces the levels

of volatile compounds associated with the flavor of

yogurt. In some countries, such as Australia, heat

treatment after production to inactivate the starter

culture is not permitted. In other countries, such

as the USA and several countries in Europe, heat

treatment is permitted; however, auxiliary labeling

‘heat-treated after culturing’ is required if yogurts

are heat-treated after culturing. Frozen yogurt resem-

bles ice cream, and the chemical composition and

manufacturing details up to the freezing stage are

similar to those of yogurt. Frozen yogurt can be either

soft or hard. Dried yogurt can be produced by

sun-drying, spray-drying or freeze-drying of yogurt.

The drying process transforms the junket into

powder. Drying also causes loss of some flavor com-

pounds and destruction of the starter culture. An-

other type of yogurt, which may find favor among

diet-conscious consumers is low-calorie yogurt, in

which intense sweeteners are used, and the viscosity

of the product is improved by the addition of stabil-

izers and thickening agents such as carrageenan and/

or gelatin.

0007In the Middle East, concentrated yogurt is prepared

by adding wheat flour or parboiled wheat, and the

yogurt–wheat mixture is shaped into rolls and sun-

dried. The product is popularly known as ‘kishk.’ In

some countries, such as India, Nepal, and Bangla-

desh, yogurt is made in earthenware pots, and

because of evaporation of moisture through the

pores of the earthenware pots, the product becomes

concentrated.

Processing and Manufacture of Yogurt

Preparation of Mix

0008A flow diagram for making yogurt is shown in

Figure 1. Traditionally, yogurt is manufactured from

milk, which has been concentrated by boiling to in-

crease the viscosity of the product. Nowadays, milk is

fortified with nonfat dry milk for the same purpose.

The level of fortification varies from as little as 1% to

as much as 6%. However, the generally recom-

mended level of fortification is around 3–4%. This

increases the total solids level in yogurt mix to

15–17%.

0009Ultrafiltration and reverse osmosis may also be

used to achieve a higher solids content in order to

improve the viscosity of the product. Milk concen-

trated by ultrafiltration to 18–20% total solids has

been reported to produce good-quality yogurt. The

viscosity of yogurt is almost wholly dependent on the

protein content of milk. Hence, a high protein con-

centration is essential for the production of viscous

yogurt. Casein is the major contributor of viscosity

tbl0001 Table 1 Compositional standards for yogurt

Full-fat

yogurt

Reduced-fat

yogurt

Low-fat

yogurt

Fat (%) 3.0% 0.5–2 0.5

Milk solids not fat (%) 8.25 8.25 8.25

Titratable acidity (%) 0.9 >0.9 >0.9

pH 4.5 4.5 4.5

YOGURT/The Product and its Manufacture 6253

followed by fat and whey proteins. Stabilizers can

also influence the consistency of yogurt. In practice,

gelatin, starch, vegetable gums, carrageenan, and

pectin are used most widely as stabilizers for yogurt.

The best yogurt texture is achieved by using gelatin at

0.3–0.8%. Good yogurt can be made without the use

of added stabilizers, but yogurt without a stabilizer is

more vulnerable to a number of stress factors than

one that has not been stabilized. When properly

chosen and used, stabilizers play an important role

in improving the body, texture, mouth feel, and ap-

pearance of yogurt. The fat content may vary from

0.5 to 3.5%. Milk fat also tends to ‘mask’ the acid

flavor of yogurt. Obviously, when milk fat is incorp-

orated into the mix formulation, homogenization

becomes important for the overall texture quality of

yogurt.

0010 Sweeteners are added to plain yogurts, generally

with fruits. Addition of sugar is a method of cutting

the sharpness of yogurt flavor. An appropriate level of

sugar should be used to mask the full degree of acid-

ity, but enough sugar should remain in the product

after fermentation for a desirable acid–sugar blend.

Up to 10% sucrose is usually used when fruit is added

to yogurt. According to Ravula and Shah, a mix

containing 4% or more sucrose, may reduce acid

production and lower cell counts of both microbial

species when incorporated in the mix prior to fermen-

tation. Nonnutritive or intense sweeteners such as

aspartame (NutraSweet) may find favor among diet

conscious consumers. Since aspartame is approxi-

mately 200 times sweeter than sucrose, only a small

amount is required for desirable sweetness. Because

of this, yogurts containing intense sweeteners require

a bulking agent such as polydextrose.

0011 As yogurt mix is prepared, particular attention

should be given to blending, homogenization, and

heat treatment of the mix. The blending and

homogenization steps are important to the uniformity

of ingredient distribution. Homogenization is usually

carried out before heat treatment, but in some cases,

it may take place after the heat treatment.

Homogenization

0012Homogenization is the typical industrial process used

to effect stabilization of the lipid phase against separ-

ation by gravity. The specific gravity of milk fat is 0.9,

whereas that of skim milk is 1.036. Fat, being lighter,

tends to separate from the serum or skim milk if left

undisturbed. The diameter of the fat globules in milk

ranges from 1 to 15 mm, with an average of 3–4 mm.

Homogenization reduces the average diameter of fat

globules to 1 or 2 mm. As a result, the fat globules do

not cream during the incubation of yogurt. Because of

the size reduction, there is usually a four-to sixfold

increase in the surface area.

0013The fat-globule membrane protects the fat globules

from lipase. Upon homogenization, the fat-globule

membrane is destroyed, and the fat globule is vulner-

able to attack by lipase, which is naturally present in

milk. Because of this, the milk is pasteurized to inacti-

vate lipase before homogenization. If the milk is to be

homogenized before pasteurization, the milk must

be pasteurized immediately to prevent lipolysis.

0014Since the efficiency of homogenization is much

better at higher temperatures, the mix is warmed to

around 65



C to liquefy fat and then forced at high

pressure (1.8 10

kPa) through a small orifice to

reduce the size of the fat globules.

Heat Treatment

0015Yogurt mixes are heated to a high temperature, typic-

ally 85



C for 30 min. Such a high heat treatment has

many objectives: (1) to destroy all pathogenic bac-

teria, (2) to inactivate all the enzymes that may be

Preparation of mix (standardization, fortification with skim milk powder)

Homogenization (65

⬚C

,1.8 × 10

kPa)

Heat treatment (85

⬚C

/30 min)

Cool to incubation temperature (42

⬚C

)

Inoculation with starter (1% each of Sc. thermophilus and Lb. delbrueckii ssp. bulgaricus)

Pack in retail container (or inoculate in bulk for fruit yogurt)

Incubate (42

⬚C

for ~4 h)

Cool (4

⬚C

) (for fruit yogurt, mix with fruit when the product is cooled to 4

⬚C

)

fig0001 Figure 1 Flow diagram for yogurt manufacture.

6254 YOGURT/The Product and its Manufacture

present in milk, including lipase, (3) to destroy most

of the spoilage-causing bacteria, including thermo-

durics, and, most importantly, (4) to denature whey

proteins, b-lactoglobulin and a-lactalbumin. This

heat treatment denatures more than 90% of the

b-lactoglobulin compared with 60% of the a-lacto-

globulin. A complex is formed between k-casein and

denatured b-lactoglobulin, which increases the

hydrophilic properties of the casein, reduces the pro-

pensity of the gel to syneresis, and facilitates the

formation of a stable coagulum. The hydration effect

of the protein is maximal when milk is heated at



C but decreases as the temperature is raised

above 85



C. A higher heat treatment decreases the

hydrophilic properties of the b-lactoglobulin–k-

casein complex. As a result, syneresis occurs, and the

structure of the yogurt gel becomes weak and fragile.

0016 After heat treatment and complex formation be-

tween b-lactoglobulin and k-casein, the heated milk

forms a smooth gel-like structure when the pH drops

during fermentation to 4.6, which is the isoelectric

point of casein (the pH at which casein proteins carry

no electrical charge).

Inoculation and Incubation

0017 After pasteurization, the mix is cooled to 45



C and

inoculated at a level that varies from 0.5 to 5%.

The former level (0.5–1%) may be susceptible to

growth conditions and a longer incubation time.

The maximum amount recommended is 5%. This

level will cause very rapid acid production, but leads

to defects in aroma, and a large amount of culture

must be prepared. The optimum level is 2%, 1%

each of Lb. delbrueckii ssp. bulgaricus and Sc.

thermophilus.

0018 Lb. delbrueckii ssp. bulgaricus hydrolyzes milk

proteins, the caseins, thus releasing essential amino

acids, including valine, which stimulate the growth of

Sc. thermophilus. Initially, Sc. thermophilus grows

rapidly, reducing the pH to around 5.4, which stimu-

lates the growth of Lb. delbrueckii ssp. bulgaricus,

which is acid-tolerant and produces large amounts of

lactic acid, which reduces the pH. Sc. thermophilus

uses oxygen during its growth, which makes

oxidation–reduction potential more favorable for

Lb. delbrueckii ssp. bulgaricus; it also produces

formic acid, which stimulates the growth of the lacto-

bacillus.

0019 The starter bacterial cells are distributed in the

inoculated mix by stirring for 10–15 minutes after

the addition of the cultures. This is followed by dis-

pensing the mixes into consumer-sized containers and

incubating at 42



C for approximately 4 h until the

pH decreases to 4.5. For the manufacture of stirred

yogurt, the inoculated mix is incubated in bulk, and

when the pH reaches the desired value, the yogurt

is stirred, cooled, mixed with various flavorings, if

required, and filled into retail containers.

0020The incubation temperature of 42



C is optimal for

the associated growth; the optimum temperature of

the growth of Sc. thermophilus alone is 37



C, and

that for Lb. delbrueckii ssp. bulgaricus is 45



C. Incu-

bation temperatures above 42



C will promote the

growth of the Lactobacillus, whereas an incubation

temperature of less than 42



C will favor the growth

of Sc. thermophilus, and lack of flavor results due to

the poor growth of Lb. delbrueckii ssp. bulgaricus.

Deviation in either direction will lead to a disturbance

in the ratio of lactobacilli to streptococci. The ratio of

lactobacilli and streptococci in natural yogurt for

optimum flavor should be 1:1. By using monocul-

tures, this ratio of 1:1 can be maintained easily by

adjusting the inoculum level of both Lb. delbrueckii

ssp. bulgaricus and Sc. thermophilus.

0021When a 2% inoculum is used at an incubation

temperature of 42



C, the milk will coagulate, and a

firm gel will form in 3.5–4 h, and the pH will decrease

to 4.4–4.5. When frozen concentrated cultures are

used, an incubation period of 5–8 h may be required.

Similarly, freeze-dried cultures may require longer

times than fresh and active cultures.

0022A new trend in yogurt making is to incubate the

yogurt mix at a significantly lower temperature than

normal for a longer period. One of the advantages is

that products require less starter cultures and shorter

cooling times. An incubation temperature of 30



with 0.25% starter culture may take 12–14 h. If the

incubation temperature is too low, the growth of

yogurt starter may be affected adversely.

0023The final acidity of yogurt is 0.9–1%. The US

standard requires a titratable acidity of 0.9% or

higher, whereas the Australian standard requires a

pH of 4.5 or lower. According to IDF, the minimum

acidity of yogurt should be 0.7%.

0024The yogurt cultures produce acetaldehyde, which

gives natural yogurt its typical flavor that resembles

that of green apples. Other volatile compounds

include acetic acid and diacetyl.

Cooling

0025Once the desired acidity has been reached, the prod-

uct is cooled to <10



C as quickly as possible. In the

case of stirred yogurt, in one-phase cooling, the prod-

uct is cooled from the incubation temperature to

<10



C prior to the addition of flavoring material

and filling. In two-phase cooling, the temperature of

the product is reduced to 15–20



C during the first

phase cooling before addition of flavoring materials

and filling of containers followed by the second stage

YOGURT/The Product and its Manufacture 6255

cooling to <10



C in a cold store. Yogurt quality may

be improved by packaging at 24



C followed by final

cooling of the product in the container. To maximize

the effect, the second stage of cooling should be

carried out as slowly as possible over a 12-h period.

0026 The viscosity of yogurt improves during storage for

1–2 days. During the first 24–48 h of cold storage, an

improvement in the physical characteristics of the

coagulum is observed, mainly hydration and/or sta-

bilization of casein micelles. Proper hydration is re-

quired to avoid syneresis. It is therefore important to

delay the sale or distribution of yogurt for 24–48 h.

0027 During cold storage, it is important to minimize

rough mechanical handling of the packaged yogurt,

and to maintain the temperature of <5



C. During

transport, especially in summer, shaking of yogurt

can lead to a reduction in viscosity and syneresis.

Flavored yogurts

0028 Flavored yogurts are prepared by adding flavorings to

plain yogurt. Sundae-style yogurt is prepared by

layering 15–18% of total weight of yogurt with fruit

pure

e or syrup on the bottom of the containers and

then filling the containers with warm inoculated mix,

followed by sealing the containers and incubation.

The fruit in the product may be mixed with the yogurt

gel by consumers before eating.

0029 Swiss or stirred-style yogurts are prepared by

blending fruit pure

e, sucrose, or glucose into bulk

prepared fresh plain yogurt. Since the coagulum is

broken during blending, plain yogurt is usually pre-

pared with a higher level of stabilizer (0.7%) than

normal (0.3%). The product after mixing with fruit is

chilled to 4



Frozen yogurt

0030 Frozen yogurt is a fermented milk or yogurt mix

containing a typical yogurt flavor and has been sub-

jected to fermentation by the two yogurt bacteria to a

pH of 4.5 or lower or a titratable acidity of 0.9% or

higher, expressed as lactic acid. Frozen yogurts,

frozen flavored yogurt desserts, yogurt frozen on a

stick, or frozen fermented dairy desserts are relatively

new products that were developed in the 1960s.

Frozen yogurts are prepared in a similar fashion as

icecream, and therefore are made most conveniently

in icecream factories.

Low-lactose Yogurt

0031 Lactose malabsorbers do not produce sufficient lac-

tase (b-d-galactosidase) and thus cannot hydrolyze

the ingested lactose completely. In addition to the

gastrointestinal discomfort, such as stomach upset

and diarrhea brought about by ingestion of milk by

these individuals, a general impairment in the normal

digestion process has been observed.

0032Low-lactose or lactose-free milk for lactose sensi-

tive individuals can be prepared either by the physical

removal of lactose by ultrafiltration or by hydrolysis

of lactose into the corresponding monosaccharides,

glucose, and galactose.

0033Lactose can be hydrolyzed using strong mineral

acid or enzymes. The enzyme b-galactosidase (b-

d-galactoside galactohydrolase, EC 3.2.1.23), com-

monly called lactase, catalyzes the hydrolysis of the

b-1 !4-galactosidic linkage present in lactose. Lac-

tase has been isolated commercially from the fungi,

Aspergillus niger, A. oryzae, and A. flavus, or the

yeast Saccharomyces lactis or from the bacterium

Escherichia coli. Yogurt bacteria (Lb. delbrueckii

ssp. bulgaricus and Sc. thermophilus) possess the

highest amount of b-galactosidase activity among

the lactic acid bacteria.

0034b-Galactosidase from Saccharomyces (known as

neutral lactase) has an optimum activity at pH 6.8–

7.0, is stable in the pH range of 6.0–8.5, and works

best at 35



C. It is suitable for treating milk (pH 6.6)

or sweet whey (pH 6.2), but the lack of stability

below pH 6.0 precludes its use for treating acid

whey (pH 4.5). A. niger lactase (also known as

acid lactase), with a pH optimum of 4.0–4.5, good

stability over a wide range of pH (3.0–7.0), and an

optimum temperature of 55



C, is suitable for the

modification of acid whey.

0035Low-lactose yogurt is then produced by the use of

low-lactose milk during processing.

Heated or Pasteurized Yogurt

0036Even though natural yogurt is held at 4



C, the titrat-

able acidity increases due to residual activity of the

starter bacteria. This process is referred in the indus-

try as ‘post-acidification,’ as a result of which the

product becomes too tart. Without heat treatment,

the shelf-life of yogurt is 4–5 weeks at 4



C. Heating

destroys most of the starter bacteria and yeast and

molds, and if post-processing contamination is

avoided, the shelf-life of the product can be extended

to 8 weeks. No postacidification occurs in heated

yogurt. Heated or pasteurized yogurt can be prepared

by heat-treating yogurt in the package at about 55



for 30 min, followed by cooling. The main problems

associated with pasteurized yogurt are loss of flavor

and syneresis. Stabilizers can be used to overcome the

latter problem.

0037Controversies still exist regarding the heat treat-

ment of yogurt after fermentation as heat-treated

yogurt contains very few or no viable yogurt bacteria.

It also raises questions about the definition of yogurt,

6256 YOGURT/The Product and its Manufacture

which must contain an abundant viable count of Sc.

thermophilus and Lb. delbrueckii ssp. bulgaricus.

Yogurt is considered to be an excellent nutritional

food, and consumers ingest several million live cells

through a typical serving of yogurt.

Prebiotics, Probiotics, and Synbiotics

0038 The term ‘probiotic’ is derived from two Greek words

meaning ‘for life.’ Probiotic organisms and sub-

stances produced by these organisms contribute to

the microbial balance in the intestines. The generally

accepted definition of probiotics is that ‘they are live

microbial food or feed supplements that provide a

beneficial effect on hosts (human or animal) by

improving the microbial balance in the intestine.’

The intake of these bacteria is reported to help restore

the balance in the intestinal microflora, which may

have been lost due to stress, antibiotic use, or illness.

The major strains of bacteria used in probiotics, Lb.

acidophilus and Bifidobacterium spp., are dominant

organisms of human large intestines. These microor-

ganisms are claimed to inhibit the growth of patho-

genic organisms through the production of organic

acids and bacteriocins. Other benefits include reduc-

tion in lactose malabsorption, suppression of poten-

tially harmful enzymes, increased immune response

due to increased production of secretory immuno-

globulins A, reduction in serum cholesterol, and

antimutagenic effects.

0039 The beneficial effects of the presence of bifidobac-

teria in the gastrointestinal tract are dependent on

their viability and metabolic activity. The growth

of bifidobacteria is dependent on the presence of

complex carbohydrates such as oligosaccharides and

other substrates such as N-acetyl glucosamine

and lactulose. These carbohydrates that stimulate

the growth of bifidobacteria are known as ‘bifido-

genic factors.’ Some oligosaccharides, due to their

chemical structure, are resistant to digestive enzymes

and therefore pass into the large intestine, where they

become available for fermentation by bifidobacteria.

0040 Compounds that are either partially degraded or

not degraded by the host and are preferentially util-

ized by bifidobacteria as carbon and energy sources

are defined as ‘prebiotics.’ Some of the bifidogenic

factors that are of commercial significance include

fructo-oligosaccharides, lactose derivatives (such as

lactulose, galacto-oligosaccharides), isomalto-oligo-

saccharides, xylo-oligosaccharides, gluco-oligosac-

charides and soybean oligosaccharides. Resistant

starch and nonstarch polysaccharides are classified

as colonic foods but not as prebiotics because they

are not metabolized by certain beneficial bacteria.

Oligosaccharides have been recognized for their

health benefits in Japan, and many products con-

taining oligosaccharides were developed during the

1990s. Products that contain both prebiotics and

probiotics are referred to as ‘synbiotics.’ An example

of synbiotic includes SymBalance yogurt, which con-

tains inulin as prebiotic and Lb. reuteri, Lb. acido-

philus, and Lb. casei as probiotics.

0041It is not clear how probiotics work. Acidification of

the gut is claimed to be one of the mechanisms.

Breast-fed infants have a much higher percentage of

Bifidobacterium bifidum than formula-fed infants.

The intestinal microflora of the latter group is more

like that of adults; it is a mixed microflora, including

coliform bacteria. Bifidobacteria produce acetic acid,

butyric acid, lactic acid, and pyruvic acid. The lactic

acid and acetic acid account for more than 90% of

organic acids produced. It is widely accepted that

because of acid production by Lb. acidophilus and

bifidobacteria, the enteropathogenic bacteria are

unable to grow. The growth of clostridia and E. coli,

when cocultured with bifidobacteria, has been found

to be inhibited, even at a neutral pH, suggesting that

acid production may not be solely responsible for

inhibition. Metabolites produced by bifidobacteria

may be partly responsible for the inhibition of

pathogens.

Acidophilus and Bifidus Yogurt (AB Yogurt)

0042Lb. acidophilus and bifidobacteria are normal inhab-

itants of the intestine of many animals including man.

Lb. acidophilus is Gram positive and rod-shaped,

while bifidobacteria are Gram-positive rods of vari-

able morphology that show branching and pleo-

morphism. Bifidobacteria were first isolated by

Tissier at the Pasteur Institute, Paris, France, and

predominate the gut flora in breast-fed infants.

0043Yogurt containing Lb. acidophilus and bifidobac-

teria has gained popularity in many countries, includ-

ing Japan, France, Germany, Canada, Australia, and

USA, and more than 70 products containing Lb. acid-

ophilus and bifidobacteria, including sour cream,

buttermilk, yogurt, milk powder, and frozen desserts,

are produced world-wide. It is estimated that about

11% of all yogurt sold in France now contains

Lb. acidophilus and Bifidobacterium spp. In 1978,

the Yakult Co. launched a Bifidus fluid yogurt named

Milmil

, which contains Bifidobacterium breve,

B. bifidum and Lb. acidophilus. More than 54 differ-

ent types of milk products containing Lb. acidophilus

and Bifidobacterium bifidum are marketed in Japan.

0044Lb. acidophilus and Bifidobacterium spp. are

difficult to propagate because of their specific nutri-

tional requirements. Bifidobacteria are not as acid-

tolerant as Lb. acidophilus, and the growth of

YOGURT/The Product and its Manufacture 6257

Bifidobacterium species is significantly retarded

below pH 4.0. Lb. acidophilus and Bifidobacterium

spp. are slow acid producers; the slow growth rate of

these organisms can be compensated by adding a

higher level of inoculum, such as 5 or 10%. Yogurt

bacteria are usually added to carry out fermentation.

If pure cultures of Lb. acidophilus and/or Bifidobac-

terium spp. are used, the time required to reduce the

pH of milk to 4.5 could be as long as 18–24 h at



C. When yogurt bacteria (Lb. delbruecckii ssp.

bulgaricus and Sc. thermophilus) and AB (Lb. acid-

ophilus and Bifidobacterium spp.) cultures are used,

the incubation time is about 4 h.

Recent Advances in Probiotic Yogurt

0045 The most commonly used species in commercial pro-

biotic products are Lb. acidophilus, Lb. casei, Lacto-

bacillus GG (a close relative of Lb. casei subgroup

rhamnosus, ATCC 53103), B. bifidum, B. longum,

B. breve, and B. infantis. Additional blends are also

being investigated, such as Lb. reuteri, Lb. plan-

tarum,andLb. casei.

0046 In Australia and Europe, yogurt containing

Lb. acidophilus and Bifidobacterium spp. is referred

to as AB yogurt. The recent trend is to incorporate

Lb. casei in addition to Lb. acidophilus and Bifido-

bacterium spp, and such products are known as ‘ABC

yogurt.’

0047 A wide variety of probiotic cultured products are

now available world-wide such as Nu-Trish a/B, USA

(acidophilus þbifidus), AB-yogurt, Denmark (acido-

philus þbifidobacteria þyogurt culture), Miru-Miru,

Japan (Lb. acidophilus þLb. casei þB. breve), Bifigh-

urt (B. longum þSc. thermophilus). Products that

contain both prebiotics and probiotics include Sym-

Balance yogurt (Lb. reuteri þLb. acidophilus þLb.

casei þbifidobacteria, inulin) produced in Switzer-

land and Fysiq (Lb. acidophilus þRaftiline brand

prebiotic).

0048 Several probiotic products contain both yogurt

bacteria and one or more types of probiotic bacteria.

Because of sensitivity to acid, Lb. acidophilus and

Bifidobacterium spp. in yogurt begin to die within a

few days after manufacture because of acid produced

during manufacture and storage. In order to provide

health benefits, the suggested level for probiotic

bacteria is 10

cfu per gram of a product. However,

studies have shown a low viability of probiotics in

market preparations. Many yogurt manufacturers use

a starter culture devoid of Lb. delbrueckii ssp. bulgar-

icus but a combination of Lb. acidophilus, bifido-

bacteria and Sc. thermophilus (known as ‘ABT’)as

starter cultures to overcome the postacidification

problem. However, the use of ABT starter culture

increases incubation time significantly as Sc. thermo-

philus is the main organism responsible for fermenta-

tion in ABT starter cultures, and this organism is less

proteolytic than Lb. delbrueckii ssp. bulgaricus. Bifi-

dobacteria are anaerobic and are fastidious organ-

isms, requiring specific growth factors and prefering

a low oxidation–reduction potential for growth.

0049Cysteine is usually incorporated in the media used

for selective enumeration of bifidobacteria. Cysteine

is an essential growth factor for bifidobacteria and

also reduces the oxidation–reduction potential for

optimum growth of anaerobic bifidobacteria. Dave

and Shah have shown a better viability of bifidobac-

teria, in yogurt made from ABT starter cultures, with

an additional source of nitrogen, such as acid casein

hydrolysate, than with lowering the oxidation–

reduction potential with cysteine. These authors

have also shown that incorporation of cysteine (at

> 250 mg l

1

) to lower the redox potential affected

the growth of Sc. thermophilus, as this organism is

considered aerobic. Since Sc. thermophilus is the sole

fermenting organism in ABT starter cultures, the

incubation time increased significantly.

0050The viability of bifidobacteria in yogurt made with

starter cultures containing both yogurt bacteria was

not affected as much, as Lb. delbrueckii ssp. bulgar-

icus is proteolytic and provides peptides and amino

acids for the growth of bifidobacteria.

See also: Bifidobacteria in Foods; Casein and

Caseinates: Uses in the Food Industry; Heat Treatment:

Chemical and Microbiological Changes;

Homogenization; Lactic Acid Bacteria; Pasteurization:

Principles; Prebiotics; Probiotics; Starter Cultures;

Sweeteners: Intensive; Others; Yogurt: Yogurt-based

Products; Dietary Importance