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Yogurt: Technology and Biochemistry

Article in Journal of food protection · December 1980

DOI: 10.4315/0362-028X-43.12.939

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 939
Journal o_fFaod Protection Vol. 43, No.12. Pages 939-977 (December, 1980)
Copyright © 1980 International Association of Milk, Food, and Environmental Sanitarians

Yogurt: Technology and Biochemistry 1

A. Y. TAMIME2* and H. C. DEETH3
Department ofDairy Technology, West of Scotland Agricultural College, A uchincruive, A yr. KA6 5HW. Scotland and Otto Madsen Dairy Research
Laboratory, Queensland Department ofPrimary Industries, Hamilton, Queensland 4007, Australia

(Received for publication October 12, 1979)

ABSTRACT
which this knowledge is deficient. Some of these areas
were discussed in a recent book by Rasic and Kurmann
This review considers the physical and chemical changes (1978). This review concentrates on the biochemical
which occur during yogurt manufacture as a result of
changes occurring during the production of yogurt.
processing and microbial fermentation. Changes produced
during processes, such as heat treatment and physical TABLE 1. Yogurt and yogurt-like products known world-
manipulations, are reviewed. Microbial fermentation is wide.a
discussed in terms of the characteristics of the yogurt
organisms, Lactobacillus bulgaricus and Streptococcus ther- Traditional name Country
mophilus, and the biochemical reactions involved in carbohy- Jugurt/Eyran Turkey
drate metabolism, flavor production, proteolysis and lipolysis. Busa Turkestan
Kissel Mleka Balkans
Urgotnic Balkan Mountains
Acidification of milk by fermentation is one of the Leben/Leban Lebanon & Some Arab Countries
oldest methods of preserving milk and imparting to it Zabady Egypt & Sudan
special favorable organoleptic qualities. There are many Mast/Dough Iran & Afganistan
different methods of carrying out this fermentation in Rob a Iraq
various parts of the world and these give rise to a range of Dahi/Dadhi/Dahee India
Mazun/Matzoo Armenia
fermented milk proqucts, including kumiss, kefir,
Katyk Transcaucasia
acidophilus milk and yogurt. These products vary Greece
Tiaourti
considerably in composition, flavor and texture, accord- Cieddu Italy
ing to the nature of fermenting organisms, the type of Mezzoradu Sicily
milk and the manufacturing process used (Chandan et Gioddu Sardinia
al., 1969; Kosikowski, 1977; Berlin, 1962). Tarho Hungary
The word "yogurt" is derived from the Turkish word Fiili Finland
'jugurt' and Table 1 shows the variety of names by which Filmjolk/Fillbunke/
this product is known in different countries. Yogurt is a Filbunk/Surmelk
traditional food and beverage in the Balkans and the Taettemjolklfettemelk Scandinavia
Middle East. However, its popularity has now spread to Skyr Iceland
Yoghurt/Yogurt/Yaort/
Europe and to many other parts of the world, and, as
Yourt/Yaourti/
Table 2 shows, consumption has increased significantly Yahourth/Yogur I
in all countries in recent years. From these data it is Yaghourt Rest ofthe world
evident that yogurt plays an important role in the diets of ('Y' is replaced by 'I'
some European communities, particularly in Bulgaria, in some instances
and is making an increasingly significant contribution to aAfter: Orla-Jensen (1931), Hammer & Babel (1957), David-
the diets of many other countries. Consequently it is son & Passmore (1969), Pederson (1971), Nilson (1973); Kon
timely to review the state of knowledge of this product in (1972); El-Sadek et aL (1972a & b).
terms of its processing and manufacture; its microbiol-
ogy and biochemistry and its organoleptic, nutritive and Although some controversy still exists regarding the
therapeutic qualities, and in doing so to identify areas in exact definition of yogurt in terms of its chemical
composition and types of starter organisms, it is defined
1A second paper dealing with nutritional and therapeutic properties of
for the purpose of this review as the product resulting
yogurt will appear in a future issue ofthisjouma/. Since a large number
from milk by fermentation with a mixed starter culture
o_l references. appear in both papers. they are cited in style d(fferent
from that normally used in this journal. consisting only of Streptococcus thermophilus and
2
West ofScotland Agricultural College. Lactobacullus bulgaricus.
'Otto Madsen Dairy Research Laboratory. The justification for using such an approach has been

JOURNAL OF FOOD PROTECTION. VOL43, DECEMBER1980

940 TAMIME AND DEETH

TABLE 2. Per capita annual consumption ofyogurt (kg/head)a.

Country 1970 1971 1972 1973 1974 1975 1976 1977

Australia 1.0 1.1 1.2
Australiab 1.0 1.4
Austria 1.9 2.2 2.4 2.9 3.2 3.4 3.7 4.1
Belgiumc 2.9 4.0 4.7 3.8 3.7 3.9 3.8 3.6
Belgiumb 3.5 4.7 5.1 5.0 4.2
Brazil 0.3 0.02 0.6 0.6 0.6
Bulgaria 27.0 31.5
Canada 0.3 0.4 0.5 0.6 0.6 0.7 0.9 1.2
Czechoslovakia 1.2 1.3 1.4 1.6
Denmarkd 7.5 9.6 10.3 11.6 13.0 13.4 14.2
Denmarkb 1.7 2.3 3.5 5.1 5.9 6.6 7.6
Francec 6.2 6.5 7.0 7.2 7.4 7.8 7.9 8.0
Finland 2.7 5.2 6.9 7.8 6.5 6.3 6.4 6.7
Germany 3.8 4.5 8.4d 4.7 4.5 4.6 5.3 5.7
Ireland 7.7d 0.2 0.5 1.1 1.4 1.5
Israel 0.9 1.3 2.0 3.0 2.6 3.4 4.6 4.6
Japan 0.3 1.1 1.1 0.9 0.8 0.6 0.7
Luxembourg 4.1 4.2 4.2 4.7 5.1 6.2 6.0
Luxembourgb 2.3 2.2 2.5 3.2 3.3 3.9 4.4
Malta 0.2 0.2
13.6 13.4 13.1 13.4 13.2 14.1 14.7 14.9
Netherlands
New Zealand 1.5
0.2 0.4 0.6 0.9 1.2 1.2 1.4 1.7
Norway
2.0 2.6 2.8 3.2 3.2 3.2 2.9 2.7
Poland
2.4 3.0 2.9 3.2 3.2 3.4 3.4 4.3d
Spain
Sweden 0.7 1.2 1.7 1.7 1.7 2.3 2.5 2.9
Switzerland 7.5 8.1 8.9 9.8 10.4 10.9 12.0 12.2
0.7 0.8 1.0 1.5 1.7 1.6 1.7 1.8
UK
USA 0.1 0.5 0.6 0.7 0.7 0.9 1.1 1.2
0.04 0.02 0.1 0.1 0.1 7.2d 6.8d 6.8d
USSR
aAfter:- IDF (1974, 1975, 1976, 1977, 1978 & 1979), EEC (1975, 1977 & 1978), Anon. (1975a), Anon. (1975b), Australian Bureau of
Statistics (1978).
biDF figures.
coata of yogurt represents production/head.
doata of yogurt represents other fermented dairy products.

discussed elsewhere (Robinson and Tamime, 1975)
although a similar definition has been used in
formulating existing or proposed standards for yogurt in
many countries (Dehove, 1960; Netherland Standards,
1967; Danish Standards, 1968; Slump, 1971; Swiss Food
Act, 1972; FAO/WHO. 1973; Pien, 1973; New Zealand
Food & Drug Act, 1973; Bulgarian Standards, 1974 &
1976; South African Standards, 1975; UK Food
Standards Committee, 1975; Alary et al., 1976; Spanish
Standards, 1977).
PROCESSING AND MANUFACTURE
Traditionally, yogurt has been manufactured from
boiled milk which was concentrated to two thirds of the
original volume (Cronshaw, 1947; Elliker, 1949). The
traditional procedure, which is illustrated in Fig. 1, has
several drawbacks. First, successive inoculations of the
starter cultures tend to upset the balance between the S.
thermophilus and L. bulgaricus; second, the low
incubation temperature (ambient as compared with the
optimum temperature of 40 - 45 C) results in slow
acidification of the milk and can promote undesirable
side effects, e.g. whey syneresis, and adversely affect the
quality of the yogurt, and third, such a method provides
no control over the level of lactic acid produced during
manufacture. With improved methods of yogurt
production (see Fig. 2) these problems do not arise. In
such system, the starter, which is obtained from
commercial starter manufacturers or starter banks, is
propagated in rotation and inoculated into the milk
under aseptic conditions, the temperature is accurately
controlled and the yogurt is cooled very quickly when the
desired acidity is reached. Hence, the process can be well
controlled and the quality of yogurt standardized.
The more recent work on the manufacture of yogurt
has been reviewed by several authors (Humphreys and
Plunkett. 1969; Robinson and Tamime, 1975; Puhan,
1976a; Dalhuisen, 1972; Rasic and Obradovic, 1975;
Bottazzi, 1977; Klupsch, 1974) while the general
concepts of quality assessment of yogurt have been
recently discussed by Robinson and Tamime (1976),
Stocklin (1969), Kurmann (1976) and Kroger (1973,
1976a). In addition, Mann (1967, 1971, 1973a, b, c and
1976) has compiled several successively updated inter-
national digests on yogurt.

JOURNAL OF FOOD PROTECTION, VOL. 43, DECEMBER 1980

 r
YOGURT: BIOCHEMISTRY
Boil nilk cause parHal concentration
Cool to 1ncubation terrperature~ i.e. blDOd tef1)erature
' ' ""' ""' '" '"~""' 1
Inoculate with starter
1
lrcubate in bulk until flri"!; coagulurr is produced
or ir.cubate overnight at
room te~peraturc
1
Cool
j,.
Dispatch
Figure L Production of yogurt as traditionally made in the
Middle East.
TYPES OF YOGURT
The industrial manufacture of the different types of
yogurt is shown schematically in Fig. 3. The various types
differ according to their chemical composition, their
method of production, their t1avor and the nature of
post-incubation processing. The legal or proposed
standards for the chemical composition of yogurt in
various countries are based on three possible types of
yogurt classified according to fat content (full, medium
or low) of the product (FAO/WHO, 1973). Such a
classification is used in compositional standards to
facilitate standardization of product and to protect the
consumer.
However, there are two main types of yogurt, set and
stirred, based on the method of production and on the
physical structure of the coagulum. Set yogurt is the
product formed when fermentation/coagulation of milk
is carried out in the retail container, and the yogurt
produced is in a continuous semi-solid mass. By contrast,
stirred yogurt results when the coagulum is produced in
bulk a~d the ge_J structure is broken before cooling and
packagmg. Flutd yogurt may be considered as stirred
yogurt of low viscosity. Since viscosity is mainly governed
by the level of solids in the basic mix, fluid yogurt can be
produced by making a stirred yogurt from a mix with a
low level of total solids, e.g. 11 o/o (Rousseau, 1974).
H?":'ever, traditionally, fluid yogurt is manufactured by
m1xtng equal quantities of yogurt and water (see Fig. 3)
and one of the main characteristics of such a product is
~he separation of the solid and the whey phases. Hence, it
IS customary to shake the product before consumption.
Flavoring of yogurt is another method often used to
differentiate various types of yogurt. Flavored yogurts are
bas.ically divided into three categories (plain or natural,
frUit and t1avored yogurt). The first type, plain or natural
is the traditional yogurt with its typical sharp 'nutty'
t1avor. Sometimes the sharp acidic taste of the natural
product is masked by addition of sugar. Fruit yogurts are
made by addition of fruit, usually in the form of fruit
preserves, puree or jam, to yogurt. Flavored yogurts are
prepared by adding sugar or other sweetening agents and
JOURNAL OF FOOD PROTECTION. VOL. 43, DECEMBER 1980
941
synthetic flavorings and colorings to plain or natural
yogurt. Fruit and/or flavoring, and coloring material can
be easily incorporated and homogenously distributed in
stirred yogurt, but in set yogurt the fruit settles to the
bottom of the container and has to be mixed in by the
customer before eating (e.g. Swiss-type yogurt).
The post-incubation processing of yogurt may lead to
many different types of yogurt. The emergence of new
yogurt products has been largely due to recent
developments in this field. Typical examples of these are
pasteurized/UHT yogurt, concentrated yogurt, frozen
yogurt and dried yogurt. These products may vary
considerably in chemical composition, physical charac-
teristics and organoleptic qualities. Pasteurized/UHT
yogurt is heat-treated after incubation, a process which
leads to destruction of the yogurt starter bacteria and a
reduction in the level of volatile compounds which are
associated with the flavor of yogurt (see section on Recent
Developments).
Partial separation of the liquid phase from yogurt
leads to the production of concentrated/condensed
yogurt of around 24% total solids (Tamime, 1978a;
Tamime and Robinson, 1978; Robinson, 1977) with
rheological properties and characteristics considerably
different from those normally associated with yogurt.
Frozen yogurt, which can be either soft or hard, is a
product whose physical state resembles ice-cream rather
than yogurt, but which is similar to yogurt in chemical
composition and manufacturing detail up to the freezing
stage. However, in frozen yogurt, somewhat high
quantities of sugar and stabilizers are required to
maintain the air bubble structure during the freezing
process.
Dried yogurt can be produced by sun-drying,
spray-drying or freeze-drying. Sun-dried yogurt is
produced in many rural areas in the Middle East where
the surplus of summer milk is made into yogurt and
conserved by this method for winter consumption.
Industrial production of dried yogurt is acheived by
either spray-drying or freeze-drying (Robinson and
Tamime, 1975). The drying process transforms the
junket into powder. This causes a loss of some flavor
compounds are the destruction of starter culture.
Another type of yogurt which may find favor with the
diet-conscious consumer is low-calorie yogurt. Plain
yogurt normally contains about 250 - 335 KJ/100 g
(about 420 KJ/100 g for fruit yogurt). This can be
considerably reduced if stabilizers and thickening agents
are used to boost the viscosity. A typical low-calorie
yogurt contains ca. 9% milk solids-not-fat, 1 o/o fat and
0.5 · 1.0% stabilizer, e.g. carrageenan - gelatine (1:1)
(Grabs, 1979). It has a calorific value of about
170 KJ/100 g.
A recent development is the use of the enzyme,
/3-D-galactosidase, in yogurt manufacture to produce
another yogurt type, low-lactose yogurt. In this process,
hydrolysis of the lactose leads to a sweeter product
without addition of sugar (see later section).
942 TAMIME AND DEETH

(X ALFA·LAVAL
Yoghurt line

-
Product

Starter
''
-
- Flavouring
Bypass lor settype

Figure 2. Simplifedflowchartfor production of set and stirred-type yogurt. I. Balance tank, 2. Plate heat exchanger, 3. Vacuum
Chamber, 4. Homogenizer, 5. Holding tube, 6. Bulk starter tanks, 7. Incubation tanks, 8. Plate cooler, 9. Intermediate tanks, 10.
Fruit addition, 11. Packaging machine. Courtesy ofA/fa-Laval AlB, Lund, Sweden.
P::::-e lir;;ina!'y treatment of milk ( standardizati on/fortifi cati ::n/ lact.cse
}
Homogenization
~
Heat treatrmnt

Starter ~l:"'cpug:stion Cool tc incubatior, te:;,pe:-ctu::·e

retai
~
In cub ate
t
Concentrate sq ua l

~
qcaotity

Cocl rae,, Dry

t t ,[.
sn OispEJtch Dispatc'l ~oc~
YOGHURT
f'ASTEURIZED SOFT OR
t
;:lack
JR UHT QEEP -1-
YOGHURT FROZEi' Jis;:.:a-::c~

YJGHLJRT

Figure 3. Industrial production of various types of yogurt. (Flavoring, Sweeteners and colorants can be added to any of the
products.).
It is evident that there are many different types of inactivated starter culture), biochemical characteristics
yogurt products which may differ in chemical composi- or organoleptic properties. Thus it is extremely difficult
tion, (e.g. moisture content- 84% in yogurt and as low as to find a common definition for yogurt which adequately
3% in dried yogurt), microbiological status (viable or covers all of these types. It is therefore considered

JOURNAL OF FOOD PROTECTION, VOL. 43, DECEMBER 1980

 YOGURT: BIOCHEMISTRY 943
desirable to categorize these products according to some excess of 25 o/o adversely affected the availability of
basic property. The following scheme provided a moisture and hindered starter activity.
convenient classification based on the physical state of The level of total solids also affects the titratable
the product. acidity of the milk mix due to the buffering action of the
Physical state proteins, phosphates, citrates, lactates and other
Product type
I. Liquid/viscous miscellaneous milk constituents (Jenness and Patton,
Yogurt
II. Semi-Solid Concentrated/condensed yogurt 1959). An increase in total solids results in an increase in
III. Solid the titratable acidity (Humphreys and Plunkett, Joe. cit.)
Soft/hard frozen yogurt
IV. Powder and a reduction in the coagulation time. According to
Dried yogurt
Davis (1973), a doubling of the solids-not-fat content
The manner in which these products are interrelated l.s results in a doubling of the titratable acidity. An increase
shown schematically in Fig. 4. The present review will be in the total solids in milk can be achieved by many
mainly concerned with yogurt of Type 1. However, it is methods, some of which are discussed below.
evident from Fig. 4 that parts of the review will apply to Milk powder addition
Types II- IV also.
Milk powder (full cream, skim or buttermilk) is widely

~
used in the industry to fortify liquid milk for production
I of thick, smooth yogurt. The level of addition varies from
LIQUID/
VISCOUS
Yoghurt
Concentrated
Yoghurt
as little as 1 o/o (Stocklin, Joe. cit.; Wilcox, 1971;
Grozdova, 1971) to as much as 6 o/o (Hammond, 1972).
However, the generally recommended level of fortifica-
tion is around 3 - 4 o/o (Becker, 1971; Stadhouders and
Hassing, 1974; Davis, 1967,1971, 1973).
Concentration
Concentration of the basic mix is widely practiced in
the industry (Anon., 1976a, 1977a; Lundstedt, 1974;
Ill
SOLID Nielsen, 1974; Baustian, 1970), the process being carried
Soft /Hard j
\ :.,~oh•:,~ } out before homogenization and heat treatment. The
~ process usually takes place by evaporation, and, with the
exception of minor losses of volatile components in the
Figure 4. Proposed scheme for classification o.f' various types condensate, all the milk constituents are retained and
of yogurt. • This includes special therapeutic products and concentuted. The average amount of water removed is
yogurt fortified with vitamins.
between 10 and 25% (Anon., 1970; Dordevic et al., loc.
cit; Egli and Egli, 1976, 1977a, b; Malitz and
PREPARATION OF mE BASIC MIX Dubenkropp, 1971), and according to Wranghede (1973)
Total solids level the total solids is increased by 2 - 4o/o corresponding to
The level of total solids in the milk is significant for the recommended fortification with milk powder.
One advantage claimed for this process is in the
both the consistency and aroma of the manufactured
production of goat's milk yogurt. Evaporation improves
yogurt. In general, an increase in total solids will enhance
the consistency of and reduces the 'goaty' flavor of the
these properties (Galesloot, 1958; Ashton, 1963;
product (Hadland and Hoffmann, 1974). Vacuum
Siegenthaler and Ritter, 1964; Emmons and Tuckey,
evaporation also reduces the entrapped air in the basic
196 7). The percentage of total solids is largely dictated by
milk and this in turn reduces syneresis and improves the
the legal standards of the country concerned. Table 3
stability of fermented milk upon storage (Gradhage and
sets out the standards laid down by some countries.
Thurell, 1978).
The total solids level in milk for yogurt manufacture
can vary from as low as 9 o/o in skim yogurt to over 20 o/o in Whey powder addition
other types of yogurt. The review by Robinson and
Use of whey powder in the manufacture of yogurt has
Tamime (1975) shows that the recommended range is 14
been reported by Jelen and Horbal (1974), Prodanski
- 18 o/o. Kozhev et al. (1972) concluded that the best (1970), Nielsen (1976) and Korner (1973). The recom-
yogurt is made from milk containing 15.5 - 16.0% total mended level of fortification of the basic mix is 1 - 2o/o
solids. Although the composition of commercial yogurts (Spurgeon, 1976; Hartman, 1975). Higher levels impart
varies considerably, most low-fat yogurts fall within the
an undesirable whey flavor to the yogurt. Todoric and
range of 14 - 15 o/o.
Savadinovic (1973) reported that whey powder added at a
Total solids as high as 30o/o have been suggested by rate of 0.3o/o to the basic mix improved the viscosity of
some workers (Dordevic et al., 1973; Zmarlicki et al.,
yogurt and accelerated the rate of acid development.
1974). However, in a study by Pulay and Krasz (1974) on
the effect of the level of total solids on starter activity in Ultrafiltration (UF) and reverse osmosis (RO)
yogurt manufacture, it was concluded that levels in Concentration of milk by UP and RO is carried out at

JOURNAL OF FOOD PROTECTION, VOL. 43, DECEMBER 1980

944 T AMIME AND DEETH

TABLE 3. Existing or proposed standards for the chemical composition ofyogurt. a

Country of _ _ _ _T_:.y~p_es_o_f.:;_yo_:.g::_u_rt_b_a_se_d_o_n_"il_of_at_ _ _ _ Solids not fat/

Balkan Normal Medium Low Total solids
Argentina 2.8 0.5 0.5
Australia 3.0 1.5. 0.5 0.5
Denmark 3.8 1.8 1.5 0.3
FAO/WHO 3.0 3.0. 0.5 0.5 8.2SNF
Finland 2.5
France 3.0 1.0
Israel 3.0 1.5 0.5. 0.2
Italy 3.0 1.0
Lebanon 3.0 8.2SNF
Netherlands 4.5 3.2 0.3
Netherlands 0.5 13.3 TS
New Zealand 3.25 2.0 0.5 8.5 SNF
Switzerland 3.0 2.5. 1.5 0.5
Switzerland 3.5 2.0 0.5
Sweden 3.0 0.5
South Africa 3.3 2.5. 1.5 0.5 0.1 8.3-8.6 SNF
South Africa 3.3 2.2- 1.2 0.5. 0.0 6.0 6.4SNF
UK 3.5 2.0- 1.0 0.3 8.5 SNF
USA 2.0 2.0. 0.5 0.5
USA 3.8. 3.0 2.8. 2.0 1.0-0.5 8.25- 8.30 SNF
USSR 6.0 16.0 TS
West Germany 10.0 3.5 1.8. 1.5 0.5
West 3.5 1.8. 1.5 0.3
a After: Robinson & Tamime (1976). (Reproduced with permission ofJ. Soc. Dairy Techno!.)

ambient temperatures and hence avoids chemical
damage of milk constituents caused by heating.
Although the application of these methods is principally
in whey processing, their application for production of
various cultured dairy products has been reported
(Horton, 1974; Loo, 1975; Nielsen, 1975; Jepsen, 1977).
Milk concentrated by UF to 18 - 20~o total solids has
been reported to produce a good quality yogurt (smooth,
creamy and with typical acid flavor) without the need for
homogenization (Chapman et al., 1974; Glover et al.,
1974). However, Granier (1975) and Emaldi et al., (1975)
found that yogurt made from UF concentrated skim milk
with the lactose adjusted to 2% was superior to ordinary
commercial yogurt. It was milder in flavor and the level
of lactic acid did not increase during storage. The
concentration of milk to 13 or 15 ~o total solids by UF or
RO for the production of yogurt has been suggested by
Bundgaard et al. (1972) and Donnelly et al. (1974).
Davies et al. (1977) concluded that there was no marked
difference in the qualities of yogurt produced from milk
concentrated by RO (12.5 & 15 ~o total solids) and from
milk fortified with skim milk powder.
Comparison offortification methods
All the methods of fortification discussed can
successfully be used to increase the viscosity of the
manufactured yogurt to the desired level. The choice of
the fortification method in a particular situation is
usually determined on the bases of availability and cost
of the raw materials. However, there are some other
considerations which should be borne in mind.
The common commercial practice of fortification with
milk powder (skim or whole) can have its disadvantages,
e.g. causing excessive acid production and some taste
deviations (Gennip, 1973). The high acid production has
been attributed to the high lactose level and also to the
increased concentration of other bacterial nutrients. The
lactose concentration can be reduced by enzymic
hydrolysis using fJ-D-galactosidase (see Section Lactose
hydrolysed yogurt).
The viscosity of yogurt is almost wholly dependent on
the protein content of the milk. An illustration of this
relationship is given in Fig. 5 where the protein content
variation throughout the year shows a similar trend to
the consistency of the corresponding yogurt. Hence a
high protein concentration is essential for production of
a viscous yogurt. The fortification methods discussed
above raise the protein level in the mix but most of them
also raise the lactose level. UF concentrates, however,
can be used to raise the protein levels without
substantially raising the lactose levels. A comparison of
the chemical composition of UF and RO concentrates is
given in Table 4 and of the dried products in Table 5.
Another way of increasing protein levels but not
lactose levels is to use caseinate powders for fortification.
This has an additional advantage since the added
caseinate enhances the hydrophilic nature of the protein
in the mix and acts as a stabilizer. In so doing, it
enhances the viscosity and decreases the problem of
syneresis. (Gennip, Joe. cit.). The efficacy of sodium
caseinate vis a vis skim milk powder in enhancing
viscosity is shown in Fig. 6.

JOURNAL OF FOOD PROTECTION. VOL. 43, DECEMBER 1980

 YOGURT: BIOCHEMISTRY 945

HOMOGENIZATION Anon., 1969; Stanisic and Petricic, 1976; Christiansen

Homogenization is an integral part of the yogurt and Samuelsson, 1976; Girginov and Andreev, 1970;
manufacturing process. It is usually carried out before Wranghede, loc. cit.; Baustian, loc. cit.; Hrabova and
the heat treatment (Storgards, 1964; Martens, 1972; Hylmar, 1974; Malitz and Dubenkropp, Joe. cit.;
Hadland and Hoffmann, loc. cit) but in some cases may
take place after the heat treatment (Girginov, 1971;
1y 3. 78
Brink, 1971; Nikolov and Chernev, 196 7; Cernev, 1973).
""
"
:0
~
~
13
. }\ 3. 65
""..,.
0
It is carried out chiefly to effect a homogenous dispersion
of the milk mix constituents and to increase the viscosity
~
.c
"
({_"
12

11 \ ·--/1 ./ .
. 3. 52

3. 39
.,
~
~
and coagulum stability ofthe yogurt. It also improves the
'mouth-feel' of the product and thus increases the
organoleptic quality. Some of the physico-chemical

." I ·-·-
17
10 •....._/ 3.26 effects of homogenization of milk and their relevance in
~
0 yogurt manufacture are given in Table 6.
3. 13
~
>
............. Homogenization is particularly important in manu-
3. DO
facturing yogurt from milk containing fat (either low 1 -
2 D!o or full cream milk). The homogenization process
A M A splits the fat globules into smaller globules which become
coated with a new membrane comprised largely of casein
Figure 5. Relation between the protein content of milk and submicelles (Mulder and Walstra, 1974). This process
the consistency of yogurt during the year. • • effectively increases the density of the fat globules and
Consistency of yogurt; ~ ~ Protein content. After: reduces their tendency to agglutinate. In this way, the fat
Gennip (foe. cit.).
TABLE 4. Chemical analyses offractions and membrane retention levels of milk constituents of UF and RO concentrated whole
milk, skim milk and whey. a, b
WHOLE MILK SKIM MILK WHEY
Membrane Membrane Membrane
Process Milk Permeate retentionc Skim Permeate retentionc Whey Permeate retentionc

Total solids 11.8 5.4 78.4 9.4 3.3 87.3 6.6 5.4 31.1
Fat 2.9 100.0
Nitrogend 0.5 0.05 94.6 0.58 0.03 %.4 0.16 0.08 6.71
Lactose 4.9 4.6 57.0 4.7 3.1 76.1 4.5 4.2 38.1
Ash 0.7 0.5 68.4 0.8 0.3 85.7 0.6 0.5 69.1
Reverse osmosis
Total solids 11.7 0.08 96.6 8.8 0.33 98.0 6.8 0.11 99.1
Fat 3.2 100.0
Nitrogend 0.48 100.0 0.49 100.0 0.13 100.0
Lactose 4.3 100.0 4.7 100.0 4.4 100.0
Ash 0.7 0.03 97.8 0.7 0.2 84.6 0.6 0.09 90.3
a After: Glover (1971).
UF RO
bconcentration factors- Milk x 2.0 x 2.0
Skim x 1.6 x 2.2
Whey x 2.9 x 2.7
cMembrane retention stated as amount in concentrate expressed as 'l"o of the component in the original sample.
dNitrogen from protein and non-protein nitrogenous compounds.
TABLE 5. Comparison of the composition of raw materials used for the manufacture ofyogurt. a
--~~--~~~------------------------
Liquid Dried
Whey Whey Casein
Whole Skim Whole Skim
Component milk milk Cheddar Cottage milk milk Cheddar Cottage Commercial Co-precipitate

Moisture 87.4 90.5 93.5 94.8 2.0 3.0 4.5 3.2 7.0 4.0
Protein 3.5 3.6 0.8 0.6 26.4 35.9 12.9 13.0 88.5 83.0
Fat 3.5 0.1 0.04 27.5 0.8 0.9 0.2 1.5
Lactose 4.8 5.1 4.9 4.3 38.2 52.3 73.5 66.5 1.0
Ash 0.7 0.7 0.56 0.46 5.9 8.0 8.0 10.2 3.8 10.5
Calcium 0.1 0.12 0.9 1.3 0.64 1.4 2.5
Phosphorour 0.09 0.09 0.7 1.0 0.58 1.1
aAfter: Hargrove & Alford (1974), Harper & Seiberling (1976).

JOURNAL OF FOOD PROTECTION. VOL. 43, DECEMBER 1980

946 TAMIME AND DEETH

TABLE 6. Physical-chemical changes caused by homogenization of milk used for yogurt manufacture. a

Effect of Homogenization Changes Related to Yoghurt

A. Increase
Viscosity Reduction in fat globule size and increased adsorption on the
casein micelle which increases the effective total volume of 'sus-
pended' matter.
Xanthine Oxidase activitY. Due to disruption of the fat globule membrane which contains
about half of the enzyme present in milk.
Color (Whiter) Increase in number of fat globules which effects light reflectance
and scattering.
Lipolysis Increase in total fat surface area available to lipases. Destruction
of fat globule membrane which may enhance lypolysis by the
starter culture.
Proper mixing Especially if milk is fortified with dried milk powder.
Phospholipids in skim As a result of the physical action, more fat globule membrane
material is transferred to skim milk.
Foaming As a result of increased phospholipids in the skim milk phase.
Pumping of yoghurt milk can cause foaming in the incubation
tanks.
B. Decrease
Fat globule size Prevents 'cream line' formation in yogurt, especially during
incubation.
Oxidized flavour Due to migration of phospholipids to skim milk phase and forma-
tion of sulphydryl compounds which act as antioxidants, possibly
through denaturation of whey proteins causing exposure of hidden
SHgroups.
Protein stability Changes in protein-protein interaction as a result of some denatura-
tion and shift in salt balance.
Agglutination and effective buoyancy Decrease in fat clustering due to adsorption of casein micelles and
submicelles to fat globules.
Casein in skim phase Partial transfer to casein from skim milk to form a new membrane
around newly formed small fat globules.
Syneresis Increase in hydrophilicity and water binding capacity due to casein
fat globule membrane interaction and other protein-protein interac-
tions.
a After: Brunner (1974), Mulder & Walstra Ooc. cit.), Harper (1976).
4000 the liquid and does not separate out during incubation in
yogurt manufacture. A schematic representation of the
3600
,/ effect of homogenization is shown in Fig. 7.
Homogenization is an esential step when milk powders
3200 /
/ are used for fortification of yogurt milks. Homogeniza-
/ tion disrupts powder particles and even casein micelle
2800 /
": ;'
'-' Membrane·
c: 2400 ;' " 2-3'/o casein
NORMAL HOMOGENIZED
/
....>- FAT GLOBULE, CASEIN,
2000 25.1' OF MEft4JBRANE

"'u0 PROTEIN
(0.1-3 u dia}
1600
"'
> 10-20X FAT INTERACTS
Homogenization
1200
@ WITH CASEIN TO GIVE
HIGH DENSITY
COMPLEX
Boo
' PAATIALL~

~1 HVDROPHOBIZED FAT
GLOBULE, RARElY lN

0.2 0.6 1.0 1.8

Partia~ransfer J '\:;;;:1) "'ILK
WELL HOMOGENIZED
of phos holipids Pa ial
t nsfer of
of Protein Added
casein
Figure 6. Effect of increase of protein content on viscosity of
~~
~~
yogurt. - - - - Protein supplied by sodium caseinate; _ _ __
Protein supplied by skimmed milk powder. After: Gennip (lac.
cit.).
Figure 7. Schematic representation of the homogenization
becomes evenly and permanently dispersed throughout effect on fat globule size. After: Harper (lac. cit.).

JOURNAL OF FOOD PROTECTION, VOL. 43, DECEMBER 1980

 YOGURT: BIOCHEMISTRY
aggregates. Normally individual casein micelles are not
affected. High pressure homogenization is required to
disrupt normal-sized casein micelles.
The increase in viscosity caused by homogenization is
related to the change in the water-holding capacity of the
milk proteins. This also tends to reduce syneresis
(Grigorov, 1966a). The water-holding capacity may also
be improved by the increased amount of milk fat globule
membrane material (phospholipids and proteins) in the
skim phase (Samuelsson and Christiansen, 1978).
Storgards Ooc. cit.) and Fjaervoll (1971) reported that
yogurt viscosity was dependent on both the temperature
and the pressure of homogenization, with the best results
being achieved at 100 - 200 kg/cm 2 and at 50-60 C.
Galesloot Ooc. cit.) studied the effect of homogenization
and heat treatment on the firmness and viscosity of
yogurt and concluded that homogenization is essential
during the manufacture of wgurt (see Table 7).
TABLE 7. Effect of homogenization on the firmness and
viscosity ofyogurt made from milk heated at 86 C for 30 min. a
Firmness Viscosity
Process (sinking in em) (st:conds)
Homogenized 1.2 17
Not Homogenized 6.0 8.5
acompiled from Galesloot Ooc. cit.)
HEAT TREATMENT
In commercial practice, milk for yogurt manufacture
is pre-heated at 85 C for 30 min or 90 - 95 C for 5 -
10 min. This type of heat treatment in dairy processing is
unique to yogurt manufacture and it is therefore of
interest to examine the effects of this treatment on the
physical and chemical properties of the milk and the
manufactured yogurt.
A summary of the effects of heat treatment of milk and
the relevance of these in yogurt manufacture is given in
Table 8. From this table it is apparent that yogurt milk
undergoes several changes during its heat treatment but
that this heat treatment is insufficient to bring about
some of the more marked changes observed with more
intense heat treatments such as sterilization. The most
important changes in relation to yogurt manufacture are
changes in the physico-chemical structure of the
proteins, the lowering of the pH and the effect on the
nutritive properties both for bacterial starter growth and
human health.
Changes in the protein fraction involve both whey
proteins and casein and are very important in
determining the stability of the yogurt gel. A vast
literature exists on the heat denaturation of whey
proteins and the heat induced interactions between whey
proteins and caseins (e.g. Larson and Rolleri, 1955;
Sawyer, 1969) and it is beyond the scope of this review to
consider these topics in detail. However, Fig. 8
summarizes some of the changes which are relevant here.
It has long been recognized that /]-lactoglobulin (jJ-Lg)
interacts with K-casein but recent reports (Elfagm and
JOURNAL OF FOOD PROTECTION. VOL. 43, DECEMBER 1980
947
Wheelock, 1977, 1978a, b) indicate that ex-lactalbumin
(a:-La) is involved. In yogurt manufacture, most of the
whey proteins are denatured as shown in Table 9,
compiled from the data of Larson and Rolleri Ooc. cit.).
In particular, {3-Lg is almost completely denatured, a fact
which many authors have noted (e.g. Parry, 1974). The
interaction between the denatured /3-Lg and the casein is
very important as this increases the hydrophilic
properties of the casein (Grigorov, 1966b, c) and
facilitates the formation of a stable coagulum. It has
been noted that the hydration effect of the proteins is
maximal when milk is heated at 85 C but decreases as
the temperature is raised above 85 C (Prodanski, 1967;
Iyengar et al., 1967) and this adversely affects the quality
of the yogurt (Galesloot and Hassing, 1969). It therefore
appears that the usual heat treatment of yogurt milk
results in minimal syneresis and maximal firmness of the
yogurt coagulum. Some authors have noted that the size
of the casein micelles is markedly increased on heating
(Carrol et al .. 1971; Kalab and Harwalker, 1973; Morr,
1965). This can be explained by the interaction between
the whey proteins and the casein and by some interchain
cross-linking of caseins. A further effect of heating which
is relevant here is the sensitization of the whey proteins to
calcium ions, an effect which enhances coagulation
(Harper, loc. cit.).
Davies et al. (1978) reported the results of an ultra-
structural investigation of the changes occurring in milk
subjected to the heat treatment used in yogurt
manufacture (95 C/10 min). They observed formation of
filamentous appendages attached to casein micelles
which appeared to consist of denatured /]-lactoglobulin.
The interaction between them and the casein appeared to
involve disulfide linkages, probably to K-casein. This was
the first reported ultrastructural observation of this
interaction in heated milk, and it provides an interesting
structural explanation for the reported rheological
effects of heat treatment used in yogurt manufacture.
The appendages observed Davies et al. (1978) would tend
to inhibit micellar contact and fusion and therefore
decrease syneresis and increase gel firmness, the effects
noted by Grigorov (1966b) and Kalab et al. (1976) in
milks heated to at least 85 C.
It should be noted here that the amounts of denatured
whey protein and casein-whey protein complexes will be
largely dependent on the composition of the yogurt mix.
The remarks above apply only to milk per se. If milk is
fortified by evaporation, an increase in viscosity due to
interactions between proteins occurs during concentra-
tion (Beeby et al., 1971). Milk powder, which is
commonly used for fortification, has already undergone
heat-induced changes during the different stages of
forewarming, concentration and drying (Beeby et al., loc.
cit.). Use ofUF and RO concentrates presents a different
picture again since in these all the whey proteins are
retained in an undenatured condition and in RO
concentrates there is a high concentration of salts.
Therefore the mixes obtained by these different
TABLE B. Chemical and physical effects of heat treatment of milk and their relevance in yogurt manufacture.

Relevance in yogurt
Milk constituent Heat-induced changes manufacture Consequences for yogurt References

Nitrogenous
Whey proteins denaturation & aggregation,
inactivation of immunoglu-
bulins
active SH group production
a-La-fJ-Lg interaction
/3-Lg-K-Casein interaction
Casein partial hydrolysis, release of
glycopeptide from K-casein
dephosphorylation
aggregation, disaggregation,
interchain cross linking, e.g.
by isopeptide bonding
Enzymes inactivation
Other de com position of amino acids significant effect
to flavour compounds
amino acid lactose interac-
tion, Maillard reaction,
Schiff's base form'n.,
reduction in available lysine
almost complete
maximal at 90 C/10 min
occurs before interaction
with K-casein
very significant
of limited significance
very little
occurs especially with smaller
micelles
destruction of lipases & pro-
teases from milk & from bac-
teria
occurs to only small degree e.g. slight decrease in nutritive
lysine loss c. 0.3 o/o
destruction oflactenins, reduc- Davies & White Ooc. cit.),
tion in creaming ability
cooked flavour, lowering of
Eh, formation oflipid
antioxidant properties
contributes to gel stability
minimizes syneresis, increases Sawyer (1 %9), Grigorov (1966
micelle size, stabilizes gel
slight increase in free amino
acids and peptides
slight redistribution of phos-
phorus
increase in micelle size and
formation of protein network
minimizes rancid & bitter off
flavours
contributes to flavour
value, significant where yog-
urt fortified with high-heat
powders and concentrates
Auclair (1954), Larson &
Rolleri Ooc. cit.)
Burki & Blanc (1978)
Elfagrn & Wheelock (1978,
a & b)
a, b & c), Davies et al. (197.8)
Greene & Jezeski (1957 a, b &
c), Alais et al. (1 %7)
Belec & Jenness (1 %2)
Kalab & Harwalker Ooc. cit.)
Morr Ooc. cit.), Carrol et al.
Ooc. cit.)
Viani & Horman (1976)
Schalitschev et al. (1971),
Reimerdes et al. Ooc. cit.)

amino acid - amino acid inter- occurs to a limited degree minimal Klostermeyer (1977), Haagsma
action, e.g. formation oflysino- & Slump (1978)
alanine
Carbohydrates
Lactose decomposition to form occurs to small extent reduces pH & Eh, produces Morr et al. (1957), Greene~
organic acids, furfural & formic acid and affects growth Jezeski (1957b), Linklater
hydrox:ymethylfurfural of starter cultures, contributes (1965), Ramos (Joe. cit.)
reaction with amino acids (see to yogurt flavor
above)
 YOGURT: BIOCHEMISTRY 949

s-Lg

.-::
Type 1 or l 1° Aggregation (involves -SH groups)

0 A (Small aggregate of S-Lg)

1
0

=
0 Type 2 or J 2° Aggragation (does not involve -SH groups)

A2 [Large aggregate (S
20
= 295) of S-Lgl
+ rr.-la

1 + K-casein- (casein micelle)

~I
{(oo-La) - A J- K-casein- (caseir. micelle)
2
Figure 8. Heat-induced interaction between whey proteins
and casein. After: Elfagm and Wheelock (1977; 1978a, b),
~ McKenzie (1971) and Sawyer (1968).
>
o:s
Q

--0

"Ill'
; :!
TABLE 9. Effect of heat treatment on whey proteins a.

o/o Destruction at

-
.:J Protein 80 C/30 min 90 C/30 min
·;::::
.:
0
u
Bovine 100 100
Immunoglobulins 100 100
/1-Lactoglobulin >90 100
cc...Lactalbumin 60 90-100
Protease peptone c. 0 c. 0
acompiled from Larson & Rolleri Ooc. cit.).

procedures may react quite differently during the heat

treatment. There is evidence that the whey proteins are
protected from denaturation in the presence of high
solids levels (Nielsen, et al., 1973). Of course optimum
viscosity can be reached in high-solids mixes without
complete denaturation of the whey proteins.
In addition to the solids content, the type of milk also
influences the amount of denaturation which occurs
during heating. Ramos (1978) reported the effects on the
nitrogenous fractions of heating cow's, goat's and sheep's
milk at 80 C for 10 min. From the data, which are
reproduced in part in Table 10, it appears that sheep
milk is much more susceptible to heat-induced changes
than that of the other two species. The heat treatment
also shortens the coagulation time (Kalab et al., 1976).
This is partly due to the decrease in pH noted in milk
after such a heat treatment (Davies and White, 1959) and
partly to the whey protein-casein complex which is
formed. Table 11, adapted from Grigorov, illustrates the
decrease in coagulation time and the higher pH and
lower lactic acid concentration at coagulation in milks
heated for 30 min compared with those heated to the
same temperature and then cooled immediately. Kalab
et al., (1976), who obtained similar results, found that the
rate of acidity development was similar but the heated
milk gelled faster.
In an investigation of the effect of heat treatment of
milk on the performance of several single strain starters
Dutta et al., (1973) found little improvement in milks
heated at 85 C/10 min compared with those heated at 63
C/30 min (Table 12). In fact, there was a significant
reduction in proteolytic activity of the two organisms

JOURNAL OF FOOD PROTECTION, VOL. 43, DECEMBER 1980

950 TAMIMEANDDEETH

grown in milk subjected to the highest heat treatment. reduced to some extent, mainly through the interaction
Others workers, however, have noted stimulation of of w-NH 2 of lysine with lactose, though this appears to
growth of starter organisms partly due to destruction occur to only a limited degree (Reimerdes et al., 1978;
of the bactericidal lactenins (Storgards, loc. cit.; Kalab, et al., 1972). Some vitamins are also destroyed by
the heat treatment. Those most affected are ascorbic acid
TABLE 10. Percentage change in nitrogenous fractions of and some ofthe B group vitamins (B 1, B 6, Bw folic acid).
milk from cows, goats and sheep after heating at 80 C/10 The amount each of these is destroyed is largely
min. a dependent on the oxygen content of the milk. In the
absence of oxygen, the loss of vitamins is very low
N Fraction Cow Goat (Hartman and Dryden, 1974).
Casein +15.00 +24.4 +26.4
Non-casein -45.6 -35.9 -68.1 MECHANICAL HANDLING OF YOGURT
Soluble protein -62.4 -58.1 -79.4
Use of mechanical devices for handling yogurt is
fJ· Lactoglobulin -59.6 -74.3
Non
inevitable as the scale of production increases. Though
0.0 + 4.8 +15.0
these facilitate large-scale production, they can have
a Data selected from Ramos Ooc. cit.). adverse effects on the consistency of yogurt. A review on
Humphreys and Plunkett, loc. cit.) and to formation of the effect of mechanization on the quality of yogurt has
growth stimulants such as peptides, amino acids and recently been published by Tamime and Greig (1979).
formic acid (Miller et al., 1964; refer to section Structural damage to the coagulum of stirred yogurt
Symbiosis/stimulatory factors). may occur at several points between the incubation tank
As with almost all heat processes employed in food and the packaging machine (i.e. between stages 7 - 11,
processing, the primary aim of the heating procedure in see Fig. 2). The equipment units which may be involved
yogurt manufacture is destruction of microorganisms include paddles (agitators), pumps, tubular or plate
which may be pathogenic or which may adversely affect coolers, pipes, fittings, constrictions, extractors, fruit
the quality of the product. Almost all organisms with the metering/mixing machines and packaging units.
exception of the spore formers in the vegetative form are The agitator in the fermentation tank is required for
destroyed during yogurt manufacture. Unfortunately, either mixing the milk with the starter before the
some of the lipases and proteases elaborated by incubation period or breaking the coagulum after
contaminating psychrotrophs are not destroyed and may fermentation before cooling or pumping of the product.
cause flavor problems in yogurt on storage (Cousin, The speed of agitation is critical. For example, in the
1977). This, however, has not been identified as a Paasch and Silkeborg tank, two different paddles are
significant problem in yogurt as it has in other dairy used. The first type is a scraped-surface agitator that
products such as UHT milk, cheese and butter (Cogan, rotates at a speed of 7 R.P.M., and the second is a
1977). centrally mounted helical paddle rotating at 15 R.P.M.
Some reduction in the nutritive value of milk is caused Such low speeds of agitation are used to maximize the
by heat treatment. The nutritive value of the protein is efficiency of mixing and minimize the rate of shear

TABLE 11. Effect of heat treatment on the coagulation process during manufacture a,{yogurt. a

Heat treatment of milk

85 c 85 c 90C 90C 95 c 95 c
for for for
Item JOmin 30 30min
Coagulation time (hour) 2.43 2.01 2.34 2.04 2.29 2.04
Acidity (o/o La.) at coagulation 0.63 0.49 0.63 0.50 0.63 0.50
pH at coagulation 4.70 5.16 4.78 5.12 4.80 5.08
aAdapted from Grigorov (1 %6 b).

TABLE 12. Effect of heat treatment of milk on acid and.flavor production by S. thermophilus and L. bulgaricus.a

S. thennophilus L. bu lgaricus
Value 63 C/30 min 85 C/10 min 63 C/30 min 85 C/10 min

Acidity (o/o 1.a.) 1.0 0.85 1.6 1.7

Vol. acidity
(mg .01 N NaOH/50 g curd 9.0 9.0 40.0 34.5
Diacetyl (ppm) 13.0 12.0 12.0 13.0
Proteolytic activity
curd)' 0.34 0.25 0.25 0.18
acompiled from Dutta eta!. (1973).

JOURNAL OF FOOD PROTECTION, VOL.43, DECEMBER 1980

 YOGURT: BIOCHEMISTRY
(Fuller, 1979). Figure 9 (from Norling, 1979) illustrates
how the consistency of yogurt is improved after cooling if
mechanical treatment, i.e. shearing stress is minimized
during production. Agitation of yogurt in the fermenta-
tion tank can be avoided completely by using the
Alfa-Laval system.
..
>
11'1
0 '
1,) ---
11'1
,_--
> Diameter of pipe (em)
-1
1-1------ ·-f-.-----_,_
cold store days
I COOLING I FILLING Time
PUMPING PUMPING
Figure 9. Viscosity curve of yogurt in relation to mechanical
treatment (shearing stress). Viscosity of yogurt in an
A/fa-Laval processing plant (the coagulum is extracted by
means of a positive displacement pump without agitation in the
fermentation tank). - - - - Viscosity curve of yogurt as result of
severe mechanical treatment. * Before the cooler, the yogurt
passes through an in-line "structurizing strainer". After:
Norling (1979).
The action of pumping can be detrimental to the
consistency of yogurt. Centrifugal and positive displace-
ment pumps are used to pump yogurt, but least damage
to the coagulum is caused by the latter type. Storgards
Ooc. cit.) reported the successful use of a centrifugal
pump, including the Imopump (Alfa-Laval; incidentally
this pump is not recommended any more by the
manufacturer) for pumping sour milk if sufficient care is
taken to eliminate incorporation of air by proper
adjustment of counter pressure in the pipeline.
Steenbergen (1971a) investigated various pumps (Prist-
ram FK 40, Waukesha 25-DO, Mono SH-22-R8,
Moineau LBF 6/RI46 and Jabsco) and concluded that
the Mono pump had the least effect on the consistency of
yogurt. He also found that the higher the RPM, the
greater the structural damage, e.g. 16-28% reduction in
viscosity of yogurt at 400 RPM as compared to 8.3-11.7%
at 100 RPM, depending on the type of pump. Hence he
concluded that a large-stroke volume pump must be used
for larger throughput rather than a higher pump speed.
However, Hauge et a!. (1974) recommended use of a
positive displacement pump (in particular the Mono and
the capsular type) in preference to centrifugal pumps for
the pumping of fermented milk products.
The flow of yogurt through pipelines, fittings and
constrictions can cause damage to the yogurt structure
and decrease its viscosity. Steenbergen (1971b) studied
the effect of pipeline dimensions, length and diameter,
on viscosity of yogurt being pumped through pipes.
Table 13 summarizes the results of this study. It can be
observed that: (a) if the velocity and the diameter of the
JOURNAL OF FOOD PROTECTION, VOL. 43, DECEMBER 1980
951
pipe are constant, the viscosity of yogurt is decreased by
increasing the length of the pipe and (b) if the velocity
and length of the pipe are constant, the least structural
damage occurs in a larger diameter pipe.
TABLE 13. Reduction in viscosity of yogurt as a result of
transport through pipes (temperature 13 C and flow rate 3600
1/h).a
Initial viscosity (sec): 30 40
Length of pipe (rn): 10 20 30 10 20 30
3.81 10 14 17 17 22 24
5.08 6 9 12 11 16 20
6.35 3 5 8 6 10 14
7.62 1 4 6 2 8 11
aAfter: Steenbergen (1971b).
Constrictions in the pipe line can reduce the viscosity
of yogurt by as much as 67% (Steenbergen, 1971b).
Steenbergen (1971c) noted a similar effect with filling
machines, but concluded that the reduction in viscosity
could be minimized by careful plant design.
The shearing effect of pumping and pipeline flow can
also be put to advantage in yogurt production. Nielsen
(1972) reported that the problem of nodulation can be
overcome by pumping with a centrifugal pump or by
pumping through a 30-mesh sieve fitted in the pipeline.
Homogenization can also be used for this purpose.
COOLING
Cooling is a critical step in yogurt production. It is
carried out directly after product reaches the desired
acidity. The objective is to reduce the metabolic activity
of the starter culture and hence to control the acidity of
the yogurt.
The cooling process can take place either in the
incubation vessel (in-tank cooling) or in tubular or plate
heat exchanger. The former system requires a longer
period of time. For example, in the Wincanton tank 4 h
are needed to cool yogurt from 42 to 5 C (Jay, 1979)
compared with 20- 30 min for the plate/tubular coolers
to cool yogurt to 15 C and further cooling to 5 C takes
place in the cold store. However, the rate of cooling can
be improved by utilizing all the surface area in contact
with yogurt, including the inner cylinder as happens in
the Diessel and Terlet type tanks (see Tamime and Greig,
Joe. cit.). In an investigation of agitation methods during
'in-tank cooling' of yogurt, Solms (1973) concluded that
the propeller type was most efficient, and produced a far
superior yogurt to the jet agitator type.
The plate and tubular coolers, which are widely used
in large-scale yogurt production, are usually of
conventional design and are quicker and more efficient
for cooling yogurt. However, to reduce the shearing effect
on yogurt, a wider plate assembly through which the
yogurt travels only once in each direction has been
recommended. Piersma and Steenbergen (1973) found
the Triplex plate cooler (Alfa-Laval) suitable for yogurt
952 TAMIMEANDDEETH
but suggested that throughput could be increased by
connecting units in parallel rather than in series as the
latter configuration could contribute to damage of the
yogurt structure. Steengergan (1971d) reported that a
tubular cooler was effective and caused little damage to
the coagulum as compared with the plate heat
exchanger.
There is a question about the final temperature to
which yogurt must be cooled before packaging. From a
microbiological point of view, the yogurt starters (S.
thermophilus and L. bulgan'cus) have limited growth at
around 10-15 C. However, in most industrial installa-
tions, yogurt is cooled to less than 20C, mixed with fruits,
packaged and finally cooled at 5 C (or less) in the
refrigerated store.
To retain the desired physical characteristics of stirred
yogurt, the Danish Dairy Research Institute (Anon.,
1977b) has recommended the following cooling proce-
dure: (a) stir in incubation tank until mixture is
homogeneous, (b) pre-cool to 24 C and package, (c) final
cooling, 5 - 6 in the cold store in an air temperature of 7
- 10 C and then at an air temperature of 1 - 2 C for the
remainder of the cooling period.
RECENT DEVELOPMENTS
Post production heat treatment
Yogurt is a perishable fermented dairy product which
has a shelf life of up to 3 weeks under refrigeration,
depending on the standard of hygiene during manufac-
ture and packaging. The keeping quality of yogurt can be
improved by various methods such as gas flushing, use of
preservatives and application of heat after incubation.
The latter method of preservation is now being used in
some instances and the product resulting after the heat
treatment is referred to as pasteurized or UHT yogurt
(Schulz, 1%6; Schulz and Voss, 1970; Voss, 1973;
Klupsch, 1970; Baustian and Burk, 1971).
Pasteurization conditions similar to those used for
milk are suitable for yogurt. However, it has been
reported that a shorter time of treatment may be
required because of the lower pH of yogurt as compared
to milk (Gavin, 1966). Rakshy (1966) reported a 100%
reduction in bacterial count in yogurt heated at 65-70 C
while Neirinckx (1972) heated yogurt at 60-65 C and
achieved a keeping quality of 6-8 weeks at 12 C. Hot
packaging of the product has been shown to extend the
shelf life at ambient temperature to 1 month (Mulcahy,
1972). In the industrial production of pasteurized yogurt
using, say, an Aseptjomatic (Alfa-Laval), the yogurt is
subjected to flash treatment at 60-70 C after incubation
(Bake, 1971; Siegenthaler et al., 1969; Klupsch, 1972;
Puhan, 1976b).
The main problems associated with pasteurized yogurt
are loss of flavor Uess significant in fruit-flavored yogurt)
and whey syneresis. Incorporation of stabilizers can
overcome the latter problem, but if set-yogurt is kept
perfectly still during pasteurization, there is no need for
addition of stabilizers (Stadhouders and Dijk, 1974).
Another recommended method to overcome whey
JOURNAL OF FOOD PROTECTION, VOL. 43, DECEMBER 1980
syneresis is to heat yogurt at 70 C for 30-40 sec and pack
at 55-60 C (Dellaglio, 1977; Luck and Mostert, 1971).
Alternatively, yogurt can be autoclaved at 60-85 C at 2
atmospheres after packing (Egli and Egli, 1976, 1977a
and b).
Although post-production heat treatment of yogurt
prolongs the shelf life of the product, it raises the
question of the definition of yogurt (FAO/WHO, 1977)
since most existing standards stipulate that yogurt must
contain an abundant viable count of S. thermophilus and
L. bulgan'cus (Davis, 1976; Kroger, 1975 and 1976b;
Kroger and Fram, 1975; Speck, 1977). However, it
appears reasonable to reserve the term "yogurt" for the
traditional product and to designate the new product
"pasteurized" or "UHT" yogurt.
Continuous manufacture ofyogurt
Yogurt production at the present time is carried out in
batches. Such a system can lead to a lack of uniformity
between different batches. It also sets a limit on the level
of production at a given time. The present trend is to
mechanize the system for continuous production to
obviate the limitations of the batch production.
According to Rasic (1975), the various continuous
methods can be divided into two types, those involving
continuous incubation and cooling (Ueno et al., 1%6;
Jahnert and Herberle, 1972; Hylmar et al., 1975; Anon.,
1976b; Pien, 1976; Hansen, 1977) and those involving
two-stage fermentation, i.e. pre-incubation and in-flow
inoculation (Girginov, 1965; Anon., 1975c; Stanisic and
Petricic, loc. cit.; Anon., 1976c; Ramaunuska and
Urbene,1976a and b; Macbean et al., 1978).
Although continuous fermentation in the yogurt
industry is still in its infancy, its feasibility has been
established (Lelieveld, 1976). Driessin et al. (1977a and
b), whose work has lead to development of a prototype
continuous fermenter, claimed the following advantages
for such a process: space saving, reduction in size of
equipment, reduction in yogurt losses in tanks and pipe
lines, reduction in capacity of cooling and filling sections,
better flexibility in total amount produced, lack of need
for all milk to be in stock at start of production,
uniformity of product quality and characteristics, better
control on acid development and the lack of necessity to
rush the cooling and packaging operations. On the other
hand, Macbean (1976) has pointed out some of the
problems associated with use of a continuous system
incorporating a pH stat fermenter fitted with a plug flow
meter.
Carbonated yogurt
A process for manufacture of a carbonated yogurt
beverage was reported by Duitschaever and Ketcheson
(1974). They found that carbonation of the finished
yogurt beverage improved its thirst-quenching quality
and enhanced its refreshing character. The yogurt
beverage was made by a process involving post-produc-
tion homogenization at a pressure of 175 Kg/cm 2 •
 YOGURT: BIOCHEMISTRY
Preservatives and gas-flushing
Preservatives are widely used in the food industry and
in particular for preservation of fruit. However, it is
reasonably safe to assume that use of preservatives is
prohibited in the yogurt industry although use of fruit to
flavor yogurt may carry some preservatives into it.
FAO/WHO (1976) has suggested that preservatives like
sorbic acid and its salts (potassium, sodium and
calcium), sulfur dioxide and benzoic acid can be used at
levels of 50 mg/Kg (singly in the fruit or in combination)
in the final product, if they come exclusively from the
fruit flavoring substances. Such a proposal is only
applicable to fruit-flavored yogurt and not the natural
flavor.
The presence of preservatives (e.g. sodium sorbate,
potassium sorbate or sodium dehydroacetate at levels as
low as 0.1 o/o (w /v) in the yogurt can affect growth of the
starter culture and cause the acetaldehyde level to
decrease more rapidly during storage (Hamdan et al.,
1971a; Nakae et a1.,1973).
An alternative method for prolonging the keeping
quality of yogurt, in particular the fruit-flavored types, is
the use of gas-flushing (carbon dioxide or nitrogen). The
purpose of such a process is to restrict the growth of
yeasts and molds in the product. Fltickiger and Walser
(1973) concluded that carbon dioxide flushing was not
suitable if yogurt was packed in PVC containers sealed
with an aluminium laminate because such containers are
permeable to carbon dioxide and oxygen. Furthermore,
they found that nitrogen flushing was effective in
suppressing Oidium spp. but ineffective against faculta-
tive anaerobic yeasts. However, Braun (1973) claimed
that carbon dioxide improved the shelf life of yogurt for
several months, but there was no indication of the type of
packaging material used. The combination of post
incubation heat treatment and gas-flushing with carbon
dioxide improved the keeping quality of yogurt for more
than 4 weeks at 15 C (Klupsh, 1970; 1971a and b),
Although application of gas-flushing can improve the
keeping quality of yogurt, the feasibility of using such a
process in the yogurt industry is mainly governed by
economic factors and depends on use of packaging
materials which are impermeable to gases used.
FERME~TATION AND MICROBIOLOGICAL ASPECTS
Class(fication of starter bacteria
The classification of the lactic acid bacteria by
Orla-Jensen (loc. cit.) is still recognized as the standard
method for classifying these organisms. In the 7th edition
of Bergey's Manual (1957) all these bacteria were
grouped in the family Lactobacillaceae, which was
subdivided into two tribes, the Streptococceae (coccal
shaped) and the Lactobacilleae (rod shaped). However,
in the 8th edition of Bergey's Manual (1974), lactic acid
bacteria are reclassified into two families, the Strepto-
coccaceae and Lactobacillaceae.
The yogurt starter bacteria, S. thermophilus and L.
bulgaricus are thermoduric, homofermentative lactic
10 URNAL OF FOOD PROTECTION, VOL 43, DECEMBER 1980
953
acid bacteria. Their general characteristics are shown in
Table 14. S. thermophilus is distinguished from other
members of the genus Streptococcus by its growth at
45 C and failure to grow at 10 C. Unlike most other
streptococci, this species lacks a recognizable group
antigen for serological identification and has to be
indentified by physiological procedures (Bergey's Man-
ual, 1974). Classification of S. thermophilus is well
documented and clear cut.
In contrast, the classification of L. bulgaricus and its
relationship to other Lactobacillus species have been the
subjects of some controversy. The organism appears to
be basically the same as that isolated from Bulgarian
yogurt by Metchnikoff and coworkers in the 1900's
(Davis, 1975a and b) and to be identical with
Orla-Jensen's Thermobacterium bulgaricum (Orla-
Jensen, Joe. cit.), according to Bergey's Manual (1974).
However, in editions of Bergey's Manual before the 8th
edition, (1974), descriptions of L. bulgaricus were
composites of what are now believed to beL. bulgaricus
and L. jugurti. The present classification, which has been
based on results of modern taxonomic methods, such as
guanosine plus cytosine (G and C) content of DNA, DNA
hybridization studies and enzyme homology studies,
clearly distinguishes species which have hitherto been
considered to be closely related. A summary of the data
for four such species is shown in Table 15. It is now
evident that L. helveticus and its maltose-negative
variant,L. jugurti, which share 84-100% DNA homology
(London, 1976), are readily distinguished from L.
bulgaricus and L. lactis. The latter two species are
considered to be very closely related, although L.
bulgaricus ferments fewer sugars than does L. lactis.
These two species together with L. delblilckii and L.
leichmanii have recently been grouped together because
of their similarities. Another species, L. jensenii, which
Bergey's Manual (1974) includes with these four species
has been shown to be clearly differentiated by its G and
C content (London, Joe. cit.).
Ratio of cocci: rods
The yogurt starter cultures are mixed strain starters of
S. thermophilus and L. bulgaricus which are normally
propagated together at around 42 C. Successive subcul-
turing may lead to mutation or to one organism
overgrowing the other. Hence, the starter strains are
either monitored microscopically (Breed smear) or
examined after culturing on a solid medium. Whatever
method is used, the objective is to monitor the ratio ofthe
cocci to rods. The "ratio" as it appears in the literature,
is somewhat confusing, since it might refer to the
colony : colony; clump : clump; chain : chain; or cell :
cell r::ttio of Streptococcus: Lactobacillus.
Enumeration of yogurt starter bacteria on solid media
is based on the morphology of the colony and/or
selectivity of the medium. Table 16 lists various media
which have been used. Selective media can be prepared
by incorporating antibiotics. However, with yogurt
954 TAMIME AND DEETH

TABLE 14. Selected characteristics starter culture. a, h

Characteristic S. thermophilus L. bulgaricus

DNA G + C (o/o) 50.3

Acid from glucose + +
Gas from glucose
Gas from gluconate
Aldolase +
Growth in media containing 2% NaCl
Lactic acid configuration L(+) D(-)
%Acid in milk 1.7
Ammonia from arginine
Growth at 15 C
Growth at 45 C + +
Serological group X E
Requirement for vitamins
Thiamine
Riboflavine +
Pyridoxal
Folic Acid
Thymidine
Vitamin B12
Carbohydrate utilization
Aesculin
Amygdalin
Arabinose ±
Cellobiose
Fructose + :t
Galactose ±
Gelatin
Lactose + +
Hippurate
Maltose
Mannitol
Mannose ±
Melezitose
Melibiose
Raffinose ±
Rhamnose
Ribose
Salicin
Starch +
Sorbitol
Sucrose +
Trehalose
±
aAfter: Rogosa & Hansen (1971), Sharpe et al. (1968), Rososa (1974), Deibel & Seeley (1974).
bKey +Positive reaction by 90% or more strains, - Negative reaction by 90% or more strains, ±Variable, slow or weak reaction, X
Ungrouped.

TABLE 15. Classification dataforsome Lactobacillus species. a

'7o Reassociation
DNA With
Lactic acid o/o Lactic acid G+C L. bulgaricus
Species configuration produced in milk (Moles o/o) DNA
L. bulgaricus D(-) 1.7 49.5 98
L. lactis D(-) 1.7 50.3 86
L.jugurti DL 2.7 39.0 0
L. helveticus DL 2.7 38.8 3-7
aAfter: Simonds et al. (1971), Gasser & Mandel (1968).

JOURNAL OF FOOD PRO TECTlON. VOL. 43, DECEMBER 1980

 YOGURT: BIOCHEMISTRY
955
organisms this has proved difficult because of their high
and strain-variable sensitivity to these substances (refer
to Table 21). The Breed smear can easily be adapted for Average number Number of fields
examining yogurt starters. The historic development of of organisms/field to be counted
this method was described by Wilson (1935). The 0-3 64
mechanical preparation of the smear and the possibility 4-6 32
of sampling errors make the technique prone to 7- 12 16
inaccuracy (Meynell and Meynell, 1970). The procedure 13-25 8
involves spreading 0.01 ml of the sample over an area ~-~ 4
1 cm 2 on glass microscopic slide and drying quickly to 51- 100 2
over 100 1
overcome bacterial multiplication. The highest concen-
3 After: Wilson (lac. cit.).
tration of organisms tends to be in the center of the
square because drying starts at the periphery of the Tofte-Jespersen, 1975), a ratio of 1 : 1 means 5 - 10 cells
smear and progresses gradua1ly toward the center. For of S. thermophilus to 1 cell of L. bulgaricus, and the
enumeration, Wang (1941) proposed that 50 fields work ofTamime (1977a and b) supports this view.
should be counted to make the final figure ofbacteria/ml
meaningful, but for practical purposes the work load Symbiosis/stimulatory factors
involved is excessive. To overcome this, Abrahamsen and The observation of a symbiotic relationship between
Solberg (1974) proposed that 10 fields could be counted the S. thermophilus and L. bulgaricus in the yogurt
to compensate for the uneven spreading. Robinson and starter culture was first reported by Orla-Jensen (Joe.
Tamime (1976) proposed that 10 fields be counted on a cit.). Pette and Lolkema (1950a) observed more rapid
five by five cross pattern, giving reasonable estimates of acid development in mixed cultures than in the single
bacterial load/mi. For accurate estimations, Wilson (Joe. strain cultures due to the increase in number of
cit.) proposed a scheme in which a different number of streptococci. They also demonstrated active growth of S.
fields are counted for different concentrations of thermophi/us in milk containing L. bu/garicus milk
organisms. This scheme is set out in Table 17. filtrate, and concluded that L. hulgaricus provided
essential growth requirements for stimulation of S.
TABLE 16. Culture media for enumbration of starter thermophi/us. Furthermore, the findings of Pette and
bacteria Lolkema (1950b) suggest that milk obtained at different
Agar Medium References times of the year may be deficient in different amino
acids. For example, 6 amino acids (leucine, lysine,
TGV Galesloot et al. (1961)
cystine, aspartic acid, histidine and valine) were found to
LAB Davis (1970, 197Sa & b), Davis et al. (1971).
TYLAB Tramer (1973) be essential for the streptococci during the spring
Lee A Lee et al. (1974) months, and 11 amino acids (the above amino acids plus
LA Elliker et al. (1956) glycine, isoleucine, tyrosine, glutamic acid and methio-
L-S Oxoid (1977) nine) to be essential for the autumn season. Valine was
HYA Porubcan & Sellars (1973), Sanders (1975) shown to be most stimulatory.
BTJ Briggs (1953), Cox & Briggs (1954) Bautista et al. (loc. cit.) confirmed that L. bu/garicus
M1e• M17 Trezaghi & Sandine (1975), Lowrie & stimulates S. thermophilus and concluded that glycine
Pearce (1971), Shankar & Davies (1977), and histidine are the essential amino acids; they failed to
Accolas et a!. (1978) observe stimulation by valine. However, Accolas et al.
MRS De-Mann et a!. (1960)
(1971) observed stimulation of S. thermophilus by
MRS (modified) Walter et al. (1978)
various mixtures of valine, leucine, histidine, and
NAP Davidson & Cronin (1977)
TSA McLaughlin (1946), Bautista eta!. (1966) isoleucine. Braquart et al. (1978a and b) concluded that
RA Sharpe & Fryer (1965) omission of glutamic acid, valine, leucine, histidine and
TSBA Moon et al. (1974) tryptophane from the growth medium reduced the
EWA Lucca (1968) stimulation by SOo/o. Similar findings were reported by
EA Lusinani (1975) Shankar (1977), Shankar and Davies (1977) and Higashio
MDT Ottogalli & Galli (1978) et al. (1977a). Galesloot et al. (1968) investigated the
PC&RC Johns et al. (1978a & b) opposite side of the relationship, and reached the
conclusion that S. thermophilus, under anaerobic
Another problem associated with microscopic count- conditions, produces a stimulatory substance for L.
ing of the starter is the high bacterial load, as it is bulgaricus that is equal to, or can be replaced by, formic
impossible to count only cells, clumps or chains in an acid. Furthermore, the same workers looked at the effect
undiluted sample. However, upon dilution the chains are of various heat treatments of milk and found that, in
broken, particularly those of the Streptococcus. There- intensively heated milk, i.e. autoclaved or UHT, this
fore the estimates of chain : chain ratio is not reliable stimulation was masked on account of a compound
and the ratio is usually based on cell numbers. According which could be replaced by formic acid (Auclair and
to the starter manufacturers/suppliers (Sellars, 1975; Portman, 1957). However, after the normal heat
JOURNAL OF FOOD PROTECTION, VOL 43, DECEMBER 1980
956 TAMIMEAND DEETH
treatment of milk used for yogurt, e.g. 85 - 90 C, L.
bulgaricus definitely needs the stimulatory factor
produced by S. thermophilus. The normal presence of
such a stimulatory factor in autoclaved milk appears to
have been over-looked by both Pette and Lolkema
(1950b) and Bautista et al. (toe. cit.).
Veringa et al. (1968) confirmed that the stimulatory
factor reported by Gales loot et al. (1968) was formic acid,
and that it was produced by S. thermophilus. This
ftnding was supported by Bottazzi et al. (1971) who
demonstrated the influence of UHT and autoclaved milk
on the cocci - rod ratio of yogurt cultures. They found
that the ratio moves in favor of the rods when formic acid
is present in milk, the effect of being most evident when
the formic acid concentration changes from 30- 50 y/ml
of milk. Accolas et al. (1971) observed the same
phenomona, while Higashio et al. (1977b) and Higashio
et al. (1978) concluded that L. bulgaricus is stimulated by
formic and pyruvic acids produced by S. thermophilus.
Other compounds found to be stimulatory to S.
thermophilus are peptides containing lysine resulting
from hydrolysis and casein by Micrococcus caseolyticus
(Desmazeaud and Hermier, 1972) or hepta- and
pentapeptides containing histidine obtained by hydroly-
sis of glucagon by papain (Dezmazeaud and Hermier,
1973). Lower peptides and free non-aromatic amino
acids resulting from enzymic digestion of casein or
trypsin-hydrolysed casein were also stimulatory to S.
thermophilus (Hayashi et al., 1974). L. bulgaricus can be
stimulated by purine, adenine, guanine, uracil and
adenosine (Weinmann et al., 1964; Cogan et al., 1968).
Psychrotrophic bacteria in raw milk are capable of
breaking down the casein but not the whey proteins
(DeBeukelar et at., 1977), and can provide nitrogenous
substances which are stimulatory to S. thermophilus and
L. bulgaricus (Cousin and Marth, 1977a). Yogurts made
from milk precultured with psychrotrophic bacteria
require shorter incubation times, but their organoleptic
quality can be slightly inferior (acidic, bitter, unclean
and fruity flavor) compared with control yogurts (Cousin
and Marth, 1977b).
It can be seen from the above findings that the
symbiotic behavior of the yogurt starter culture is
important. At some stage during the incubation period
the L. bulgaricus provides the essential amino acids
required by S. thermophilus and the latter provides
formic acid-like compounds stimulatory toL. bulgaricus.
It is unlikely thatL. bulgaricus is stimulated by peptides,
purines or pyrimidines during yogurt production
(Deutsch and Mattson, 1959).
Moon and Reinbold (1976) have demonstrated that S.
thermophilus can inhibit L. bulgaricus in the exponential
and stationary phase due to limited nutrient in the
medium, because the former organism is a better
competitor thanL. bulgaricus. However, this observation
is not reported by other workers.
Other factors that can influence the amount of
stimulation of the starter organisms are the level of total
solids in the yogurt milk and heat treatment of the milk.
JOURNAL OF FOOD PROTECTION, VOL. 43, DECEMBER 1980
Sellars and Babel (1978) noted variations in culture
activity were observed in different milks (reconstituted or
fresh) used for propagation of starter. The percentage of
solids-not-fat, presence or absence of fat and concentra-
tions of mineral salts in milk can all influence the rate of
acid production by the starter culture, and flavor and
aroma development. Their work is summarized in
Table 18, which shows that with an increase in solids, the
titratable acidity and viable bacterial count increased but
the pH values were higher. However, above 21 o/o total
solids the yogurt starter organisms, particularly L.
bulgaricus, are inhibited (framer, 1973; Pulay and
Krasz, loc. cit.).
TABLE 18. Effect of solids content of reconstituted skim-
milk on acid development and growth oflactic cultures. a,h
Titratable
acidity Viable count
____7£_S~F (o/o La.) pH (million/ml)
8 0.72 4.48 683.5
12 0.97 4.59 942.1
16 0.19 4.74 1302.5
a After: Sellars & Babel Ooc. cit.).
bMilk heated at 115 C for 15 min, cooled to 21 C, inoculated
with 1 o/o starter and incubated for 15 hat 20 C.
These changes are influenced by the buffering action
of the solids in the milk, which has an effect on the death
rate of cells, i.e. cells remain viable for longer periods at
higher pH values. The same workers recommend that the
incubation period should be shorter in low solids milk
than when a higher solids content is used, otherwise a
loss in activity is observed in subsequent propagations.
Although differences have been noted in different
batches of skim milk powder Greene and Jezeski, 1957a;
Sellars and Babel, loc. cit.; Barber, 1962), it is
advantageous to use reconstituted powder for starter
propagation because the raw material can be pre-tested
for suitability.
The effect of heat treatment on the physico-chemical
properties of milk is discussed elsewhere (see Table 8).
However, heating of milk can either inhibit or stimulate
the activity of the lactic starter cultures, and the work of
Greene and Jezeski (1957b) summarizes the overall
events as: (a) stimulation of starter in milk heated
between 62 C for 30 min and 72 C for 40 min; (b)
inhibition of starter in milk heated between 72 C for
45 min and 82 C for 10-120 min, as well as 90 C for
1-45 min; (c) stimulation of starter in milk heated to 90 C
for 60-180 min or autoclaved (120 C for 15-30 min) and
(d) inhibition of starter in milk autoclaved at 120 C for
longer than 30 min. According to Greene and Jezeski
(1957b), the effect of heat treatment of milk on starter
behavior, i.e. the apparent stimulation/inhibition/stimu-
lation/inhibition cycle, (confirmed by Swartling and
Mukherji, 1966; Bracquart et al., 1974), could be
duplicated by addition of denatured serum protein of
cysteine hydrochloride. The transition from one phase of
the cycle to another in response to different heat
treatment exposures occurred as a result of the release of
 YOGURT: BIOCHEMISTRY 957

denatured serum protein nitrogen of concentrations of
0.15 - 0.20 mg/ml or cysteine (10 - 20 y /ml). Further-
more, the same workers found out that when cysteine was
added artificially, it augmented the sulfhydryl groups
made available by heating. Thus the cysteine became
stimulatory in raw and slightly heated milks, but it was
inhibitory in highly heated milks. These changes in the
amino-nitrogen levels are obviously only part of the
picture, and Greene and Jezeski (1957c) have dealt in
length with the possible interactions that could be
involved. Perhaps formation of formic acid may also be
important.
Inhibitory substances
Inhibitory substances in milk can originate from
various sources, including antibiotic therapy of the dairy
cow, disinfectant and detergent residues on equipment
and bacteriophages. Natural inhibitors may also be
present in the milk. The presence of inhibitory residues
in milk, especially antibiotics, is of concern to both dairy
technologists and health authorities.
Antibiotic residues. Antibiotic therapy, mainly intra-
mammary injection, is widely practiced for treatment of
mastitis in the dairy cow. Table 19 lists the various types
of antibiotics used in the United Kingdom. These
antibiotics are retained in the udder tissues and
gradually diffuse into the milk and contaminate it.
Hence, it is recommended that milk from treated cows be
with held for 72 h. The significance of antibiotics in
yogurt milk is two fold: first, small quantities can affect
the activity of the sta£ter culture, leading to a poor
quality food for the consumer and economic loss for the
manufacturer, and second, antibiotic residues in milk
and other dairy products are a potential public health
threat. To minimize the amount of antibiotics in milk for
yogurt manufacture it is advisable to reject contaminated
supplies. Unfortunately at present there is no quick test
which can be used on milk at reception to enable this to
be done. However, The Milk Marketing Boards in the
UK have adopted a payment scheme incorporating
penalties for milk containing more than 0.02 Interna-
tional Unit (I.U .) of antibiotics/mi. Although milk
containing up to 0.02 I.U. penicillin per ml does not
attract a penalty under the UK system, such levels are
sufficient to affect the acitivty of the yogurt starter
culture. Tramer (1964) claimed that milk containing 0.02
I.U. penicillin per ml causes 'slow yogurt' and 0.03 I.U.
per milk complete failure. The most popular methods
used to detect such residues in milk are the disc assay
and the 2,3,5-triphenyl-tetrazolium chloride (TTC)
method. In the latter method (Wright and Tramer,
1961), the test organism used isS. thennophilus because
of its high sensitivity to antibiotics (see Table 20 and 21).
L. bulgaricus is more sensitive to streptomycin than S.
thermophilus, and hence a milk which passes the TIC
test may contain enough of this antibiotic to cause
failure in yogurt manufacture.
Antibiotic residues in milk are a constant problem in
the yogurt industry since this can cause slow acid
TABLE 19. Use of various antibiotics in the UK for
intramammary treatment o.fmastitis in the dairy cow. a
Intramammary Dry cow
Antibiotics injection therapy o/o
Penicillin • •
Streptomycin • 35
Penicillin + Streptomycin • •
Neomycin • • 5
Chloramphenicol • rare
Tetracyclines • 5
Framycetinb • •
Penethemateb • 15
Er}ihromycin b •"' •
Nitrofurans • rare
Sulphonamides 5
Cloxacillin •"' • 35
Ampicillin •
Spiramycin •
aAfter: Baron (1976).
boften used in conjunction with other antibiotics.
production and lead to unpalatable and wheyed-off
yogurt. It is therefore desirable that measures be taken to
overcome this problem. Some possible measures are: (a)
testing milk used for yogurt production to ensure
freedom from antibiotics; (b) reduction of effective levels
by heat treatment; in some instances this can reduce the
potency of some antibiotics, e.g. commercial sterilization
inactivates SOo/o of penicillin, but streptomycin and
chloramphenicol are unaffected by heat (Tramer, 1964);
(c) the use of penicillinase-producing micrococci to
reduce penicillin contamination (Reiter et al., 1961;
Vazquez & Reiter, 1962); such an approach, however,
conflicts with the definition of yogurt adopted in this
review; (d) development of yogurt strains resistant to
antibiotics, such as those reported by Hargrove et al.
(1950) (resistant to 3 I. U. penicillin and 500 1-lg
streptomycin per ml) and by Solomon et al. (1%6)
(resistant to 70 - 120 1-lg chlortetracycline, 40 - SO 1-lg
chloramphenicol and 500 ~-tg streptomycin per ml).
Although these workers failed to report on the quality of
yogurt from the developed strains, Rasic and Kurmann
(loc. cit.) reported that the resistant S. thermophilus and
L. bu[garicus strains had a reduced rate of acid
development and flavor production.
Disinfectant and detergent residues. Disinfectants and
detergents are extensively used in the dairy industry for
cleansing and sanitation of dairy equipment on the farm
and in the creamery. These preparations are classified
into five groups: chlorine compounds, quaternary
ammonium compounds (QAC), iodophors, ampholytes
and alkalies. Residues of these compounds in milk are
indicative of bad management or negligence. Contami-
nation is more likely to occur on the farm. The presence
of disinfectants or detergents in milk is undesirable for

JOURNAL OF FOOD PROTECTION, VOL.43, DECEMBER 1980

958 TAMIME AND DEETH

TABLE 20.

Inhibitory level/ml
Antibiotics S, thermophilus L. bulgaricus References

Penicillin 0.004 0.01I.U. 0.020-0.100l.U. Neal & Calbert (1955), Bester & Lomb art (1974), Adams (1955);
Kosikowski & Mocquot (1958), Kosikowski (Joe. cit.), Grossklaus &
Levetzow (1966), Nikolov (1967), Dinchev (1967), Abo- Elnaga et al.
(1973), Mocquot & Hurel (1970), Becker (1966), Teply & Cerminova
(1974), Kondratenko et al. (1978), Berridge (1956), Tramer (1973)
0.007. O.Q15 j.lg 0.026. >0.150 Eg Cogan (1972)
Streptomycin 0.001 13.000 mg 0.001- 13.000 mg Bester & Lombard (Joe. cit.), Obiger (1966)
1.000 . 21.000 j.lg 0.100 - >8.000 ).lg Cogan (1972), Nikolov (1967)
4.200 y Neal & Calbert (Joe. cit.)
0.380- 1.000 LU. 0.380 1.000 I.U. Mocquot & Hurel (Joe. cit.), Teply & Cerminova (Joe. cit.)
Tetracycline 1.000 l.U. 1.000-2.000 l.U. Teply & Cerminova (Joe. cit.)
0.130 0.500 j.lg 0.340- 2.000 LU. Cogan (1972)
0.800- 6.900 mg 0.800- 6.900 mg Kondratenko et al. (Joe. cit.)
Chlortetracycline 0.060- 1.000 ).lg 0.060 . 1 .000 j.lg Mocquot & Hurel (Joe. cit.)
0.200- 0.300 y Neal & Calbert (Joe. cit.), Nikolov (1967)
> 0.1 LU. > 0.1 I.U. Teply & Cerminova (Joe. cit.)
Oxytetracycline 0.250- 1.000 y Neal & Calbert (loc. cit.)
0.310. 1.000 ).lg 0.310. 1.000 j.lg Nikolov (1% 7)
0.400 I.U. 0.700 l.U. Teply & Cerminova (Joe. cit.)
Cloxacillin 0.340-0.700 j.lg 0.260. >1.000 ).lg Cogan (1972)
Bacitracin 0.040-0.120 l.U. 0.040 · 0.1000 l.U. Teply & Cerminova (Joe. cit.)
Erythromycin 0.300 - 1.300 mg 0.070 1.300 mg Kondratenko et al. (Joe. cit.)
Oleandomycin 0.800 - 3 .300 mg 0.800- 3.300 mg Kondratenko et al. (Joe. cit.)
Chloramphenicol 0.800- 13.00 mg 0.800- 13.000 mg Kondratenko et al. (Joe. cit.)
0.500 LU. 0.500 LU. & Cerminova (Joe. cit.)

yogurt manufacture since the starter cultures are readily
inhibited by these compounds. Inhibitory levels are
shown in Table 22. Bester and Lombard (1974) reported
inhibition of S. thermophilus and L. bulgaricus by QAC,
chlorine and iodophor compounds at lower concentra-
tion than those for mixed cultures shown in Table 22.
These differences may be attributed to variation in the
commercial preparations used or to differences in the
yogurt strains.
In countries where glass bottles are still used for
packaging yogurt, the bottle wash liquid can be a source
of detergent residue. Nikolov (1975) concluded that
inhibition of the yogurt starter culture occurs if the milk
contains 2.5o/o bottle wash consisting of 1 o/o sodium
hydroxide and >100 mg active chlorine per litre.
Bacteriophages. The lactic starter cultures are
vulnerable to bacteriophage attack, which can result in
failure of lactic acid production. Pette and Kooy (1952)
concluded that on the basis of phage sensitivity, S.
thermophilus strains could be divided into three groups:
the phage insensitive, the phage carriers (which, when
infected, have slower growth rates and produce yogurt
with soft coagulum) and those that undergo complete
lysis by phage (see also Sozzi and Prella, 1969; Koroleva
et a!., 1978).
Phage is eliminated by heating at 85 C for 20 min
(Stolk, 1955) and hence the heat treatment of yogurt milk
ensures destruction of these viruses if the raw milk is
their origin. Although investigations of phage in yogurt
starters has mainly been concerned with S. thermophilus
(Stolk, Joe. cit.; Ciblis. 1966; Sozzi and Prella, Joe. cit.;
Sozzi, 1972; Reinbold and Reddy, 1973; Sarimo and
Maksunen, 1978; Koroleva et a!., Joe. cit.), failure in
yogurt production can be caused by phage attacking L.
bulgaricus (Peake and Stanley, 1978a and b). It is
important to note that phage-induced lysis of L.
bulgaricus retards growth of S. thermophilus, due to the
breakdown of the symbiotic relationship between the
two organisms. Furthermore, Sozzi et a!. (1978) reported
that the optimum temperature for phage attacking the
yogurt starter culture ranges from 39 - 43 C, which is the
same as the growth temperature of S. thermophilus and
L. bulgaricus. It is evident, then, that the same
precautionary measures practiced in the cheese industry
must be adopted in the yogurt industry. These include
aseptic techniques for propagation of the starter culture,
effective sterilization of air and equipment, and daily
rotation of phage-unrelated starter strains or use of
phage-resistant strains. Ciblis (Joe. cit.) reported that
chemical sterilization/sanitation can be effectively used
against S. thermophilus phage by using 0.1 o/o QAC,
70-90% ethanol, 0.5-l.Oo/o potassium permanganate or
50-100 mg of available chlorine per litre.
Miscellaneous Inhibitors. Milk contains natural
antibodies (lactenins/agglutinins) which inhibit growth
of organisms, including lactic starters (Auclair and
Hirsch, 1953; Auclair and Berridge, 1953). Lactenins (L 1
and L 2) are heat-sensitive (Auclair, Joe. cit.) and are
destroyed by heating yogurt milk at 85 C for 30 min
(Storgards, Joe. cit.). The mechanism of the inhibitory

JOURNAL OF FOOD PROTECTION. VOL. 43. DECEMBER 1980

 YOGURT: BIOCHEMISTRY 959
TABLE 21. Antibiotic and antimicrobial starter culture. a, h

Antibiotic or S. thennophilus L. bulgaricus

antimicrobial Cone en tra tion .... .... ....
agent per disc ::r:r:'r:r:
VlVlVlr.l'lVl
.... ....
c:oc:oc:oc:oc:o
,..J....l....l,...l....l

Penicillin G 2 units 33333 23333

Polymyxim B 50 units 00000 10000
Polymyxim B 300 units 11111 22000
Phenethicillin 3 units 33333 33332
Tetracycline Smcg 33333 00212
Tetracycline 30mcg 33333 33333
Vanocomycin Smcg 33323 33222
Chlortetracycline Smcg 33333 1123 3
Chlortetracycline 30mcg 33333 33333
Ampicillin 2mcg 33333 11 2 3 1
Ampicillin lOmcg 33333 33332
Bacitracin 2 units 33333 33333
Chloramphenicol Smcg 33322 22123
Chloramphenicol 30mcg 33333 33233
Cephalothin 30mcg 33333 33333
Erythromycin 2mcg 22222 12332
Erythromycin 15mcg 33333 33333
Kanamycin Smcg 00000 00212
Kanamycin 30mcg 11111 21323
Lincomycin 2mcg 33333 33333
Methacycline Smcg 33333 32333
Methici!lin Smcg 22222 00330
Nafcillin 1 meg 33333 00131
Colistin 10mcg 00000 11000
Cloxacillin 1 meg 22122 00230
Demethylchiortetracycline Smcg 33233 122 33
Demethylchlortetracycline 30mcg 33333 33333
Doxycycline Smcg 33331 22333
Doxycycline 30mcg 33333 33333
Dihydrostreptomycin 2mcg 3 1111 00112
Dihydrostreptomycin 10mcg 32232 33333
Novobiocin 5mcg 32233 11333
Novobiocin 30mcg 33333 33333
Neomycin Smcg 1 111 1 00 101
Neomycin 30mcg 22222 22222
Oleandomycin 2mcg 33333 10233
Oleandomycin 15mcg 33333 33333
Oxacillin 1 meg 33333 00 23 0
Oxytretacycline Smcg 32333 20233
Oxytretacycline 30mcg 33333 32333
Furadantin SOmcg 33333 33003
Furadantin 100mcg 33333 33 113
Mandelamine 1.0mg 11111 00000
Mandelamine 2.5mg 21222 00021
Nalidixoc Acid 30mcg 00000 00000
Triburon 1 33333 33333
""''""uuou from: Reinbold & Reddy (1974).
bo: No inhibition, 1 : Inhibitory zone .;;; 2 mm, 2 : Inhibitory zone > 2 mm but< 5 mm, 3 : Inhibitory zone ~ 5 mm.

action of lactenins on lactic starter cultures has not been
elucidated. Patel (1970) observed inhibition of S.
thennophilus in raw buffalo's milk for the first 2 h, but
resumption of growth afterwards. He attributed this to
either adaptation of the organism or destruction of
lactenins. Other inhibitors in milk can result from
feeding cows forage such as vetch blends, turnip foliage,
rape seed or moldy silage. In such milk, yogurt starter
cultures show a reduced rate of acid formation even
after the milk is heated at 90 C for 15 min (Zeitrag, 1973;
Popovic- Vranjes, 1979). This could be due to the
presence ofthiocyanates in milk (Rutkowski et al., 1973).
Lawrence (1970) reported that milk from late lactation
cows fed on ordinary pastures contained as much as
10 ppm thiocyanates. Natural inhibitors in milk affect
yogurt production during the spring months (30-80% of

JOURNAL OF FOOD PROTECTION, VOL.43,DECEMBER 1980

960 TAMIME AND DEETH
TABLE 22. Inhibition ofyogurt starter cultures by disinfectant/detergent residues in milk. a
Mixed culture
Chlorine
Iosan CP ~2500
MetaK ~2500
Chloramine > so
Quaternary Ammonium Compounds
MeripoleM ~ 250
Ommisan ~ 250
Ampholyte
Tego 51 ~ 1000
Iodophor
Iosan S ~2000
Alkaline detergents
Recablue > 500-1000
Recablue (White) >500-1000
a After: Posthumus (l %8), Bester & Lombard (loc. cit.) Sotlar (1975), Sotlar & Arsov (1976).
bTest method TTC.
milk samples- Cerna et al., 1974). The same problem has
been reported by Davis (1973), Meiklejohn (1978) and
Cooper et a!. (1974). It could be speculated that
thiocyanates in milk may be responsible for some of the
inhibitor-related problems.
Furthermore, milk containing a high somatic ceil
count can cause a reduction in the activity of the yogurt
starter culture by up to 3So/o. However, heating the milk
at 90 C for 20 min inactivates the inhibitory substance(s)
(Gajdusek and Sebela, 1973).
Other compounds in milk that can cause inhibition of
the yogurt starter culture are formic, acetic, butyric and
decanoic acids in concentrations up to 100 ppm
(Kulshrestha and Marth, 1974), and insecticides such as
malathion and trichlorphon which can gain access to
milk through environmental pollution (Deane and
Patten, 1971; Gajduskova and Lat, 1972).
Recent developments
Recent developments in selection of appropriate
strains of S. thermophilus and L. bulgaricus for the
preparation of yogurt can be summarized as follows. (a)
Choice of compatible strains to ensure that the symbiotic
relationship exists between the yogurt organisms. (b) Use
of phage-unrelated starters to avoid failure or slow
acidification. (c) Use of frozen-concentrated starter
cultures. These are added directly to the vat, and hence
obviate the problems associated with bulk starter
preparation. (d) Use of mucogenic or slime-producing
yogurt starter cultures, e.g. 'RR' developed in The
Netherlands (Galesloot and Hassing, 1966). These
starters improve the viscosity of yogurt (Kosikowski et
a!.. 1979) and its resistance to mechanical damage (see
section on Carbohydrate Metabolism). (e) The develop-
ment of mutant strains of L. bulgaricus with increased
proteolytic activity. Such mutants can be produced by
using X-radiation (Dilanian et a!., 1970). UV radiation
(Krsev, 1976), gamma radiation (Singh and Rangana-
than, 1974a and 1977a and b, 1979; Singh eta!.. 1978) or
JOURNAL OF FOOD PROTECTION. VOL. 43, DECEMBER 1980
Inhibitory level rng/litre
S. thermophilusb L. bulgaricusb
100 100
100-500 50-100
60 60
chemical mutagens (Singh and Ranganathan, 1974b).
The improved proteolytic activity of L. bulgaricus may
prove useful in yogurt manufacture since the increased
release of amino acids can stimulateS. thermophil us and
enhance its metabolic activity. This could lead to
enhanced production of lactic acid and thus to a shorter
incubation period in yogurt production. Unfortunately,
little information is available on the effectiveness of L.
bulgaricus mutants in the yogurt field. It would appear
that there is considerable scope for further research in
this field to assess the significance of the effect of such
strains on the technology of yogurt manufacture and on
the organoleptic qualities of yogurt (see also section on
Proteolysis).
BIOCHEMICAL CHANGES
Carbohydrate metabolism
Pathways. Energy is required by micro-organisms to
maintain their life cycle. Such energy can be provided to
the bacterial cell via different systems, and as the lactic
acid bacteria do not possess the cytochrome system for
electron transport or enzymes to operate the anaplerotic
pathways and tricarboxylic acid cycle (Lawrence et a!.,
1976), the energy must be mainly supplied by
fermentation of carbohydrates. In milk, lactose is the
only available carbohydrate for this purpose. The initial
step by S. thermophilus and L. bulgaricus is the
transport of lactose through the cell membrane before
utilization by the intracellular enyzme systems. The
transport mechanism probably involves the phosphoe-
nolpyruvate (PEP) dependent phosphotransferase (PTS)
system which is present in Staphylococcus aureus and
Escherichia coli (Roseman, 1969 and 1972, Wilson et a!.,
1972; Saier, 1977) and in cheese starter cultures (McKay
et al.. 1969 and 1970; Molskness et al., 1973; Johnson
and McDonald. 1974; Thomas, 1976). However, this
system has apparently not been studied in the yogurt
organisms.
 YOGURT: BIOCHEMISTRY
Lactose is hydrolysed inside the bacterial cell by the
enzyme {3-D-galactosidase {{1-gal) to glucose and galact-
ose (Wierzbicki and Kosikowski, 1973; Blankenship and
Wells, 1974; Kilara and Shahani, 1976; Rao and Dutta,
1977 and 1978). A second enzyme {1-D-phosphogalactosi-
dase {{1-P gal) which yields glucose and galactose-6-phos-
phate from lactose phosphate has also been reported to
be present in L. bulgaricus (Permi et al., 1972) and in S.
thermophilus (Somkuti and Steinberg, 1978a and b).
Glucose is metabolised via the Embden Meyerhof
pathway (Stainer et al., 1971; Marth, 1974) to pyruvic
acid, which in turn is converted to lactic acid via the
action of lactate dehydrogenase present in both S.
thermophilus and L. bulgaricus (see section on Lactic
Acid Production). The mechanism of glucose metabolism
by the yogurt starter culture is now well understood, but
the mechanism of galactose metabolism is not yet clear.
Bissett and Anderson (1973, 1974a and b) reported that
galactose could be metabolised via glucose or via the
D-tagatose-6P and Leloir pathways by cheese starter
culture. In any event, galactose accumulates in yogurt
(Tamime, 1977a and b; Goodenough and Kleyn, 1976)
and is not catabolized to any great extent. This may be
because galactose is not metabolised in the presence of
the readily fermentable sugar, glucose, which may
suppress the Leloir and D-tagatose-6P pathways, or
because S. thermophilus and L. bulgaricus are devoid of
suitable metabolic pathways (for further discussion refer
to Lactose Hydrolysed Yogurt).
As mentioned above, some yogurt starter cultures
produce a polysaccharide (slime) during fermentation,
which enhances the viscosity of yogurt, e.g. culture RR
which was developed in The Netherlands (see Galesloot
and Hassing, 1966; Tamime and Robinson, Joe. cit.;
Tamime, 1978b and c; Luczynska et al., 1978). The
structure of this polymer is not known, but Sharpe et al.,
(1972) reported that the 'slime' produced by the
heterofermentative species of the genus Lactobacillus
was a glucan, probably a dextran, containing primariliy
oo-1, 6-glycosidic linkages. The work of Tamime, (1977a
and 1978c) suggested that the 'slime' produced by starter
culture RR was a glucan, after comparison with a sample
of {1-glucan. Acid hydrolysis yielded only glucose,
according to gas liquid chromatographie analysis.
Lactic acid production. Production of lactic acid is the
most important chemical process which occurs during
yogurt manufacture. The lactic acid helps to destabilize
the casein micelle and this leads to coagulation of the
milk protein and formation of the yogurt gel (Ca-
caseinate-phosphate complex + Lactic acid--->
Casein complex+ Ca-lactate + Ca-phosphate) (Dyatch-
enko, 1971). The lactic acid also gives the sharp, acid
taste to yogurt and contributes to the typical 'aromatic'
flavor.
Lactic acid bacteria possess the enzyme lactate
dehydrogenase (LDH) for the synthesis of lactic acid. The
configuration of lactic acid formed can be D(-), L(+) or
DL(±). Different bacterial species produce different
configurations, depending on whether D(-), L(+) or
961
both enzymes are present. S. thermophilus produces the
L(+) form of lactic acid, while the D(-) form is produced
by L. bulgaricus (Steffen et al., 1973; Garvie, 1978;
Lunder, 1972; Benner, 1976). The report that L.
bulgaricus produced DL lactic acid (Schober et al., 1964)
has not been confirmed by other workers, and such a
finding is not typical of this organism (see Table 15).
Some Lactobacillus species are, however, known to
produce D(-) lactate during the exponential phase and
some L(+) lactate during the later phases (London, loc.
cit.).
During manufacture of yogurt, L(+) lactate is produced
at a faster rate than D(-) lactate (Blumenthal and
Helbing, 1972). This phenomenom corresponds with the
growth patterns of S. thermophilus and L. bulgaricus
during yogurt fermentation (see Fig. 10). The reported
percentage of the two lactic acid isomers in yogurt may
be from 45-60o/o L(+) lactate to 40-55o/o D(-) lactate
(Benner, loc. cit.; Puhan et al., 1973a and b and 1974;
Wiesner et al., 1975; Vanderpoorten and Renterghem,
1974). The percentages are affected by the temperature
of incubation, starter culture inoculum rate, ratio
between cocci and rods, age of the yogurt and the level of
lactic acid produced. For example, the ratio of L(+) to
D(-) lactate was reported to range from 0.34 to 8.28 with
an average of 1 for 269 samples of commercial yogurt
(Puhan et al., 1973b and 1974). The level of D(-) lactate
in yogurt increases progressively during storage due to
the activity of L. bulgaricus, which is more tolerant than
S. thermophilus (Abrahamsen, 1978).
Figure 10. Production of lactic acid in milk by yogurt starter
cultures. • Mixed culture, .& L. bulgaricus, • S. thermophilus.
After: Pette andLolkema (1950 C).
Addition of glucose or sucrose does not affect the ratio
of L(+) and D(-) lactate produced in yogurt (Vander-
poorten and Renterghem, Joe. cit.), although Tramer
(1973) reported a slight reduction in total lactic acid
production when 6o/o sugar was added to the basic mix.
The fact may be important in commercial production of
yogurt where sugar is added before heating and
incubation or with the fruit before packaging. With the
former method, some reduction in starter culture activity

JOURNAL OF FOOD PROTECTION, VOL. 43, DECEMBER 1980

962 TAMIME AND DEETH
may be experienced.
The higher the level of D(-) and the lower the ratio of
L(+) to D(-) lactate, the sharper the flavor of yogurt. If
the L(+) : D(-) lactate is used to assess the quality
yogurt, then a good yogurt is considered to be one
having a ratio of 2 or less. A ratio above 2 is an indication
of faulty yogurt (Blumenthal and Helbling, 1974).
D(-) lactic acid is less readily metabolized in the
human body than the L(+) isomer, and in cases of
extremely unbalanced diets it might be expected to cause
some disturbances. However, Krusch (1978) commented
that findings in this respect have so far not been reported
and it is considered safe to consume up to a litre of
yogurt per day. If necessary, the percentage of D(-)
lactate in yogurt can be reduced by using a smaller
inoculum and incubating at a lower temperature
(V anderpoorten and Renterghem, Joe. cit.).
Lactose hydrolysed yogurt. During the manufacture of
yogurt not all the lactose is fermented (Shanley, 1973;
Goodenough, 1975; Goodenough and Kleyn, Joe. cit.;
Tamime, 1977b). Engel (1973) advocated that the excess
lactose could be utilized to sweeten the yogurt without
increasing its calorific value. Furthermore, reduction of
lactose level in dairy products could be desirable for
lactose-intolerant people (Gallagher et al., 1974). The
lactose can be readily hydrolysed by commercial
preparations of {3-D-galactosidase Oactase) which are
produced from Aspergillus niger, Saccharomyces lactis
and E. coli (Shukla, 1975). Theoretically, when the
enzyme hydrolyses lactose, glucose and galactose are
produced in equal amounts. However, in practice this
does not happen, because other sugars, oligosaccharides,
are formed in addition to glucose and galactose,
(Aronson, 1952; Pazur, 1954; Roberts and McFarren,
1953; Nickerson, 1974). Roberts and Pettinati (1957)
reported that the action of the 'lactase' from Saccharo-
myces fragilis on lactose in pure solution yielded 11
different oligosaccharides. The percentage conversion of
lactose to oligosaccharides increased as the concentra-
tion was increased. It reached a maximum when lactose
was 35o/o (w/v) and the percentage conversion to
oligosaccharides was then 44.6%. Tamime (1978c)
observed formation of oligosaccharides during the action
of a commercial {3-D-galactosidase preparation (Maxi-
lact) on lactose in milk (see Table 23.)
Although commercial production of yogurt from
lactose-hydrolyzed milk is not widely carried out,
research work in this field has been reported by many
workers (Engel, Joe. cit.; Thompson and Gyuricsek,
1974; Thompson and Gyuricsek, 1976; Gyuricsek and
Thompson, 1976; Rossi and Terence-Jemmet, 1978;
Thomasow and Klostermeyer, 1977; Antilla et al., 1978;
Tamime, 1977b, 1978b and c). They have unanimously
reported an enhanced rate of lactic acid development in
lactose-hydrolyzed milk, as compared with the controls,
and hence a reduction of the incubation period required
(Holsinger and Guy, 1974; Woychik et al., 1974;
Woychik and Holsinger, 1977). Stimulation of the yogurt
JOURNAL OF FOOD PROTECTION. VOL 43, DECEMBER 1980
TABLE 23. Determination of mono and disaccharides
present in milk as a result of lactose hydrolysis by Maxilact
(40mgl200 ml milk 16% T.S.) at 35 C. a
--~-----------------------
Duration of
hydrolysis Weight (g) of sugar present in milk
(Hours) Lactose Galactose Glucose Oligos"accharideb
0 6.73 0.04 0.00 0.00
1 2.31 1.23 1.78 1.35
2 0.79 1.73 2.48 1,77
3 0.32 1.98 2.75 1.72
4 0.19 2.14 2.86 1.58
aAfter: Tamime (1978c).
bcalculated by difference (assuming weight remains app.
constant).
starter culture could be attributed to free glucose in milk,
as reported by Gilliland et al. (1972) for lactic
streptococci. Hemme et al. (1978) concluded that
stimulation of S. thermophilus was not due to the
presence of glucose as result of lactose hydrolysis, but to
contaminating proteolytic activity in the {3-D-galactosi-
dase preparation.
Accumulation of galactose in yogurt has been reported
by Tamime (1977a and b), Mouillet et al. (1977), Brathen
(1977), O'Leary and Woychik (1976a and b). However,
the latter workers observed in one strain of yogurt starter
culture that galactose was utilized at a later stage during
fermentation. This suggests that some L. bulgaricus
strains they used are capable of catabolizing galactose
(see Table 14). Popov and Zakhariev (1976) reported that
galactose accumulated in fermented milk because S.
thermophilus showed a tendency to utilize only glucose
while L. bulgaricus metabolized both glucose and
galactose. It is evident, however, that further investiga-
tion is required on the subject of galactose metabolism in
yogurt. Another aspect of starter gro\\oih in lactose-
hydrolyzed milk which is still in dispute is the cell count
of the organisms. Rossi and Terence-Jemmet Ooc. cit.)
reported a low count of S. thermophilus in low-lactose
milk, O'Leary and Woychik (1976b) observed similar
patterns for S. thermophilus and L. bulgaricus counts
(enumerated on solid media) in low- and high-lactose
milk and Tamime (1977b) reported an increase in the cell
count (microscopic examination) of both organisms,
especially in 50% lactose-hydrolysed milk. The difference
in such observations may be related to different
bacteriological methods of analysis, to variation in
culture strains or to the purity of the enzyme lactase
used. There is a need to improve the agar media available
for enumeration of the yogurt starter culture.
Technologically, it is feasible to manufacture yogurt
from lactose-hydrolyzed milk; the process of hydrolysis
can take place during overnight storage of cold milk or
before heat treatment if the milk is tempered to the
optimum temperature of the enzyme, e.g. 35-37 C. In
either event, only slight disruption in factory routine
occurs. It is generally recognized that yogurt manufac-
tured from lactose-hydrolysed milk is milder and sweeter
due to the presence of monosaccharides and is
 YOGURT: BIOCHEMISTRY 963
TABLE 24. Production of carbonyl compounds (ppm) by yogurt starter culture.

Starter culture Acetaldehyde Acetone Acetoin Diacetyl References

S. thennophilus 1.7-2.3 1.2-5.2. 1.5-3.0 Trace Bottazzi & Vescovo (1969)
1.6-8.3 0.1-3.8 Bottazzi & Dellaglio (196 7)
3.0 Hamdan et al. (1971 b)
6.5 7.0 2.0 Mutai eta!. (Joe. cit.)
1.0-3.0 0.1-0.7 El-Sadek et al. (1972 a & b)
7.6 Baisya & Bose (Joe. cit.)
2.8-3.1 0.2-0.5 0.25 Yu & Nakanishi (1975 a)
6.0-13.0 Dutta et al. (1973)
L. bulgaricus 1.4-8.5 2.4-3.2. Trace Bottazzi & Vescovo (Joe. cit.}
10.0 Hamden et a!. (1971 b)
12.0 2.0 0.5 Mutai et a!. (Joe. cit.)
3.3-12.2 EI -Sadek et a!. (1972 a & b)
2.7-3.2 0.3-0.4 Yu & Nakanishi (1975 a)
12.0-13.0 Dutta et a!. (1973)
Mixed cultures 2.5 O'Leary & Woychik (1976 b)
10.0-17.0 4.0 Vescovo (1971)
10.4-37.5 Robinson et a!. (1977)
13.0-15.0 Vanderpoorten & Waes (1972)
13.4-13.9 2.2-5.7 0.4-0.9 Bottazzi et a!. (1971)
13.6-16.7 Veringa & Davelaar (1970)
15.0-22.9 Abrahamsen (Joe. cit.)
17.0 1.0 4.0 Vescovo & Bottazzi (1970)
18.7-34.7 1.3 Yu & Nakanishi (1975 d)
22.0-26.0 Hamdan eta!. (1971 b)
23.0-33.0 Gomer eta!. (1975 a & b)
23.0-41.0 Gomer et a!. (1968)
28.0-37.0 Bills et al.

particularly suitable for production of flavored yogurt
(Ihomasow and Klostermyer, loc. cit.; Tamime, 1977b;
Engel, loc. cit.). The crucial factor that can influence
commercial production of yogurt from lactose-hydro-
lysed milk is the cost of the enzyme compared with the
price of sugar. When the question of cost is of secondary
importance, e.g. in production of yogurt for therapeutic
purposes, this process may prove ideal for production of
sweet yogurt.
Flavor production. The two main roles of the starter
culture during manufacture of yogurt are production of
lactic acid and development of flavor in the product. The
major flavor components in yogurt are the carbonyl
compounds, acetaldehyde, acetone, acetoin and diacetyl.
Fette and Lolkema (19.50c) concluded that acetaldehyde
and other unidentifiable compounds are responsible for
the flavor in yogurt, while Schulz and Hingst (1954)
reported that flavor in natural yogurt need not include
high levels of acetaldehyde. However, the reports by
many workers suggest that the presence of acetaldehyde
is important for good yogurt flavor (Keenan and Bills,
1968; Sandine and Elliker, 1970; Sandine et al., 1972;
Dwivedi, 1973).
Reported levels of the acetaldehyde, acetone, acetoin
and diacetyl produced by single and mixed yogurt starter
cultures are collated in Table 24. From these data it
appears that: (a) the major flavor component is
acetaldehyde. Some high levels of diacetyl and acetoin
have been reported for cultures of the individual
organisms (e.g. Dutta et al., 1973; Mutai et al., 1972;
Baisya and Bose, 1975) but similar findings have not
been reported for mixed cultures. (b) the symbiotic
relationship which exists between S. thennophilus and L.
bulgaricus increases the level of acetaldehyde produced
in yogurt compared with cultures of the single strains. In
general, L. bulgaricus has been reported to be the main
flavor contributor in yogurt, (Hamdan et al., 1971b;
Bottazzi et al., 1973). However, Shankar and Davies
(1978) and Shankar (1977) have recently claimed that S.
thennophilus produces more acetaldehyde than L.
bulgaricus, provided that the growth stimulatory factors
are present in the milk medium. Flavor production varies
according to the strain of bacteria used and hence, strain
selection could be based on this criterion. Other
compounds associated with flavor enhancement in
yogurt are volatile fatty acids (Iurcic, et al., 1969;
Dumont and Adda, 1973) and amino acids (Groux,
1976). Yu and Nakanishi (1975a) reported that
production of n-pentaldehyde and 2-heptanone by L.
bulgaricus but not by S. thennophilus with slightly
higher levels produced in skim milk than in whole milk.
These compounds may s::ontr'bute to yogurt flavor (see
also Groux and Moinas, 1974). Viani and Horman Ooc.
cit.) identified the various flavor compounds associated
with typical yogurt aroma, and these are tabulated in
Table 25 together with their possible origins. It can be
seen that the flavor components can arise from fat,
protein or lactose, but the important ones are formed by
microbial fermentation of lactose. The significance of
those derived from thermal degradation will depend on

JOURNAL OF FOOD PROTECTION, VOL. 43, DECEMBER 1980

964 T AMIME AND DEETH

the amount of heat treatment used during manufacture. the presence of glucose. They also showed that S.
TABLE 25. Flavor compounds in yogurt. a thermophilus showed a reduced activity as the incuba-
tion temperature was increased from 30 to 37 C. Hence,
Flavor component Origin & precursor in yogurt production, the activity of this enzyme could be
Acetaldehyde, acetoin & Microbial fermentation of expected to mainly originate from L. bulgaricus.
diacetyl lactose-citrate cycle Another pathway that can lead to production of
Acetylpropionate, ~ Threonine cycle or thermal acetaldehyde is via the activity of the enzyme
2-Hydroxy-3-pentanone, degradation of fat deoxyriboaldose which degrades DNA and breaks down
3-Hydroxy-2-pentanone thymidine to acetaldehyde. Lees and I ago (1977) detected
Acetone, butanone, ~ Thermal degradation of fat this enzyme in some lactic acid bacteria. However, it was
3-pentene-2-one, from ketoacids
2-Hexanone
not present in L. bulgaricus and only present in one of
2-Heptanone, 2-nonanone, four strains of S. thennophilus. It is evident, therefore,
2-undecanone that more than one metabolic pathway for production of
y- Va.lerolactone, d-caprolac-} Thermal degradation of fat acetaldehyde may operate simultaneously in yogurt
tone. d-caprilactone, d-Tri- from hydroxyacids cultures. Figure 11 illustrates some ofthese mechanisms
decalactone (see also Lees and Iago, 1978a and b). Finally, the lactic
Pentane, methylcyclopentane acid produced by the starter culture during the
Benzaldehyde, benzyl alcohol, Thermal degradation of fat fermentation process contributes to the flavor of yogurt.
methyl benzoate and/or lactose It imparts an acidic and refreshing taste, while the
Furfural, furfurylalcohol. Thermal de grad at ion of lactose carbonyl compounds are associated with the aroma and
5-Methylfurfural,
flavor of yogurt.
FuryI meth ylketone,
2, 5-Dimethylfurane, Proteolysis
2-Furyl-3-propional, Although lactic acid bacteria are usually considered to
Furylethylketone, be only weakly proteolytic (Dutta et al., 1971), they do
2-Pentylfurane cause a significant degree of proteolysis in many
Dimethysulfide Thermal degradation of p~:otein fermented dairy products, including yogurt. This leads to
Dimethylsulfone from methionine changes in the physical structure of the product and
Isobutyraldehyde Thermal degradation of ptotein contributes to the production of flavor compounds
from valine
(Groux, Joe. cit.). The products of proteolysis, peptides of
Phenylacetaldehyde Thermal degradation of protein
various sizes and amino acids, seldom contribute to
from phenylalanine
flavor directly (Groux, Joe. cit.) but act as precursors for a
a After: Viani & Horman Ooc. cit.). variety of enzymic and chemical reactions which produce
The propensity of the yogurt starter culture to produce flavor compounds (Viani and Horman, Joe. cit.).
acetaldehyde is much lower in goat's milk than in cow's In yogurt proteolysis by starter bacteria increases the
milk (Garner et al., 1971). Abrahamsen et al. (1978) amount of soluble non-protein nitrogen by about SOo/o
reported the production of 17.1 ppm of acetaldehyde in (Breslaw and Kleyn, 1973; Blanc, 1973) to about 8% of
cow's milk and 4.7-5.5 ppm in goat's milk after 3 h of total nitrogen. Of the two yogurt bacteria, L. bulgaricus
incubation. The reason for this difference is presently not is the more proteolytic (Maret et al., 1974; Rapp, 1969;
understood. Luca, 1972; Yu and Nakanishi, 1975b) and has the
Acetaldehyde in yogurt has been reported to be ability to hydrolyse casein, whereas S. thennophilus has
produced from lactose, valine and acetyl phosphate only limited proteinase activity. However, the latter has
(Humphreys and Plunkett, loc. cit.), by decarboxylation peptidase activity and can hydrolyse the intermediate
of pyruvate (Keenan and Bills, loc. cit.) and by cleavage products of casein proteolysis by L. bulgaricus (Poznan-
of threonine to glycine and acetaldehyde (Sandine and ski et al., 1965; Tourneur, 1974; Carini and Lodi, 1974).
Elliker, Joe. cit.). Lees and I ago (1976a and b and 1977) As mentioned elsewhere in this review, this is an
have recently elucidated metabolic pathways for produc- important aspect of the synergistic relationship between
tion of acetaldehyde in lactic starters. They established these two organisms in yogurt (Moon and Reinbold, Joe.
that glucose was metabolized to acetaldehyde and cit.).
ethanol by the yogurt starter bacteria via the activity of Proteolytic enzymes. There is considerable evidence
aldehyde dehydrogenase (reduces acetyl - CoA to that S. thennophilus does produce proteinases and can
acetaldehyde) and alcohol dehydrogenase (reduces degrade casein under favorable conditions. Maret et al.
acetaldehyde to ethanol), respectively. The former Ooc. cit.) reported that although this organism initially
enzyme was only present in L. bulgaricus and the latter uses nitrogenous substances present in the medium, it
enzyme was present in high activity in two out of four shows definite proteolytic activity after 24 h of gto\\-ih in
strains of S. thennophilus (Lees and I ago, 1976a). reconstituted skim milk.
Lees and Iago (1976b) demonstrated threoine aldolase A summary of the results of several investigations into
activity in S. thermophilus and L. bulgaricus. The proteinase activity of S. thennophilus is given in
activity was higher in the latter organism, especially in Table 26. The most successful assays of this activity were

JOURNAL OF FOOD PROTECTION. VOL. 43. DECEMBER 1980

 YOGURT: BIOCHEMISTRY
Qlucose
catechol
I
•
l
4-hyc:lroxy-2-kela
-n-valerate
l
l
phenylpf'OCIIDII<IIe
ntlroelhane
Figure 11. Diagramatic representation of known reactions involving acetaldehyde. After: Lees andJago (1978a).
performed by Desmazeaud and Juge (1976) using
12 5J-casein as substrate. This sensitive, short-time assay
revealed a 10-fold difference in proteinase activity in the
seven strains tested. The proteinase activity appears to be
endocellular and mostly membrane-bound. (Formisano
et a1.,1973; Shankar and Davies,1978).
Many authors have made reference to the peptidase
activity of S. thermophilus, but the work of the French
group (Rabier and Desmazeaud, Joe. cit.; Desmazeaud,
1974; Deszmeaud and Juge,loc. cit.) and that of Shankar
and Davies (1978) has been the most comprehensive. A
summary of their results and those of Bottazzi (1967) is
given in Table 27.
The information available on proteinases elaborated
by L. bulgaricus is summarized in Table 26. Most
workers have detected proteinase activity in the strains
examined. There are however significant differences in
proteinase activity of different strains (Chebbi et al.,
1974) and some work has been reported from India on
highly proteolytic strains (Chebbi et al., 1977). Mutant
strains with increased proteolytic activity have been
produced with X-radiation (Dilanyan et al., 1971) and
mutagenic chemicals such as N-methyl-N-nitro-N-nitro-
guanadine (Singh and Ranganathan, 1974a and b).
L. bulgaricus also produces peptidases but generally
these are of lower activity and less importance than the
proteases (Sasaki and Nakae, 1960). Aminopeptidases
have been detected but no carboxy- or endopeptidases
have been reported. Only some strains have dipeptidase
activity {EI-Soda et al., 1978; Shankar and Davies, 1978).
The results are summarized in Table 27.
JOURNAL OFFOODPROTECTlON, VOL.43, DECEMBER 1980
965
0· pnoaplloryi~TIIanolal!line
acetylene
The literature varies considerably on the question of
which caseins are attacked most readily by the
proteinases of the yogurt bacteria. Table 28 gives a
summary of the relative rates of hydrolysis of caseins
given in several reports. In the paper by Desmazeaud and
Juge Ooc. cit.), in which they reported that (3 and K but
no a:s were attacked by S. thermophilus, they noted that
this appeared to be contrary to what had been found for
other lactic bacteria. From Table 28, however, it would
be difficult to make such a generalization. As with other
aspects of protein degradation by yogurt organisms and
other lactic acid bacteria, strain differences are probably
very significant. Unfortunately, data on casein break-
down in yogurt are scarce. One report, however, suggests
that a: casein undergoes change during extended storage
of yogurt (Ottogalli et al., 1974).
Conditions affecting proteolysis. The most intense
proteolysis occurs during the log phase of growth of the
yogurt organisms (Maret et al., Joe. cit.; Miller and
Kandler, 1967a; Argyle et al., Joe. cit.; Sato and
Nakashima, Joe. cit.). It then declines slowly after the
stationary phase is reached. Hence, most of the
proteolysis takes place in the first 24-48 h. Table 29,
taken from Formisano et al., (1974), shows an initial
burst of proteolysis followed by a decrease in the rate of
proteolysis during storage of cultures of the yogurt
organisms and ofyogurt.
In yogurt, the ratio of L. bulgaricus to S.
thermophilus influences the amount of proteolysis. Luca
(1974) found that more proteolysis, as judged by the
extent of amino acid liberation, occurred with a 1:1 ratio
966 TAMIMEANDDEETH

TABLE 26. Proteinases ofS. thennophilus and L. bulgaricus.

Results and comments References

Str. thermophilus
Casein (native and low activity, more in presence of Poznanski et al. (1965)
iso lectric) rennet
Sodium caseinate 12.3'l"o hydrolysed after 14 days at Shidlovskaya &
37 c D'yachanko (1968)
Casein 6/6 strains caused increase in TCA Carini & Resmini (1968)
soluble N but no change in electro-
phoresis pattern
Casein activity in sediment from lysozyme- Formisano et al. (1973)
treated cells, opt. pH 7
Casein activity in cell free extracts Rabier & Desmazeaud (1973)
125 1 - casein 10 fold difference in activity for Desmazeaud & Juge
7 strains, sensitive short-time- Ooc. cit.); Desmazeaud
assay used (1978)
N, N-dimethyi-[J- cell-bound, opt, pH 7 .5, opt. temp. Shankar & Davies (1978)
casein 40 C, activity 0.15 n moles min" 1
mg· 1 cell mass
L. bulgaricus
Casein intracellular, opt. pH 7 at 20-37 C, Sato & Nakashima (1965)
opt. temp. 37 Cat pH 6.7
Casein intracellular activity against native Poznanski et al. (1965)
casein > activity against isolectric
casein
Milk extracellular protease in cell-free Miller & Kandler (1967a)
filtrates
a:s and K-casein intracellular, sialic acid release from Ohmiya & Sato (1969)
o:s 2X sialic acid release from K
Casein activity at pH 7 >activity at pH 4,5 or Formisano et al. (1973)
6. Both cell-bound & cell-free activity
from lysozyme -treated cells
Casein and whey strain used produced a single proteinase, Argyle et al. (1976)
proteins both cell-bound & soluble, opt. pH 5.2-
5.8, opt. temp. 45-SQ C
Casein high proteinase strains, chemical and Dilanyan et al. (1971);
X-ray mutants Singh & Ranganathan
(1974b & 1977a); Chebbi
et al. (1977)
N, N-dimethyi-[J-casein cell-bound, opt. pH 6.0, opt. temp. Shankar & Davies (1978)
40 C, activity 4.0 n moles min" 1 ,
mg· 1 cell mass

(70 mg%) than with a 1:2 (41 mg%) or 2:1 (50 mgo/q)
mixture. Luca (1972) found that hydrolysis of whey
proteins in milk, as judged by amounts of non-protein
nitrogen, decreased as the ratio of L. bulgaricus to S.
thermophilus decreased. The spectrum of free amino
acids also produced changes with the ratio. Kapac-Park-
aceva et al. (1975) reported that in 1:1 yogurt, tyrosine,
phenylalanine and leucine accounted for 56 o/o free amino
acids, whereas in a 1:3 yogurt proline constituted 71 %of
free amino acids after 24 h.
The presence of high levels of free fatty acids in yogurt
milk may be detrimental to the amount of proteolysis by
the starter organisms and to yogurt quality. Capric acid
has been found to have an inhibitory effect on proteolysis
by L. bulgaricus alone or in combination with S.
thermophilus and to affect the texture of the coagulum.
Oleic acid however has little effect (Poznanski et al.,
1968; Trovarelli, 1974).
Use of commercial preparations of (3-D-glactosidase in
manufacturing low-lactose yogurt may also affect the
amount of proteolysis in yogurt. Use of this enzyme
preparation in cheese has resulted in enhanced
proteolysis and accelerated ripening (Marschke and
Dulley, 1978). and it now appears that this may be
partially due to the presence of proteases in the
yeast-derived (3-D-galactosidase preparations (Dulley,
1978; Hemme et al., loc. cit.).
Increased proteolysis may also occur in yogurt from
milk contaminated with proteolytic psychrotrophs.
Cousin and Marth (1977 a and b) found an increase in the
amount of non-protein nitrogen in such yogurts and
reported that the proteolysis was accompanied by
development of unacceptable flavors when milks were
pre-cultured with a Pseudomonas or a Flavobacterium
species.
An adverse effect of proteolysis in dairy products is

JOURNAL OF FOOD PROTECTION. VOL. 43. DECEMBER 1980

 YOGURT: BIOCHEMISTRY 967
TABLE 27. Peptidases of S. thennophilus and L. bulgaricus.

Enzyme Results and comments References

S. thermophilus
Peptidases strongest activity against di- & tri- Bottazzi (1 96 7)
peptides with N-terminalleucine,
N-terminal amino acid generally
released
a:-Aminoacylpeptidase opt. activity at pH 6.4 and at 35 C, Rabier & Desmazeaud
MW 62,000, metal ion requirement, Ooc. cit.)
N-terminal amino acid residues
released
Dipeptidase opt. activity at pH 7.5 and at SOC, Rabier & Desmazeaud
MW 50,000, metal ion requirement, Ooc. cit.)
specific for dipeptides with a polar,
bulky N-terminal amino acids
Endopeptidase present in all strains, Desmazeaud (1974, 1978)
active on glucagon & insulin-{3 chain,
split bond involving a:-NH2 of hydro-
phobic amino acids; metal ion (esp.
Mn*) requirement, intracellular
Arginineamino-peptidase present in some strains, inactive on Desmazeud & J uge
dipeptides Ooc. cit.)
Prolylidipeptidase present in all strains, active on Desmazeud & Juge
numerous dipeptides Ooc. cit.)
Leucineaminopeptidase present in all strains Desmazeud & J uge
active on dipeptides, Ooc. cit.)
Leucineaminopeptidase cell-bound, opt. pH 7.0, opt. temp. Shankar & Davies (1978)
42 C, activity 14.5 n mole min" 1
mg" 1 cell mass
Dipeptidase cell-bound, opt, pH 7.5 opt. temp. Shankar & Davies (1978)
42 C, activity 51.0 moles min" 1
mg" 1 protein
Carboxypeptidase not detected in 7 strains tested Desmazeaud (1978)
(Vs L-Leu-gly)
L. bulgaricus
Aminopeptidases detected in 6/6 strains, four-fold El-Soda et al.
(Vs leu-pNA) range of activity Ooc. cit.)
· Aminopeptidases cell-bound, opt. pH 6.0, opt. temp. Shankar & Davies (1978)
(Vs leu-pNA) 37 C, activity4.0 n mole min" 1
mg" 1 cell mass
Dipeptidase (Vs gly-tyr) detected in some strains El-Soda et al.
Ooc. cit.)
Dipeptidase not detected Shankar & Davies (1978)
(Vs leu-gly)
Carboxypeptidases not detected El-Soda et al., Ooc. cit.)
(Vs CBZ glu-arg)
& Endopeptidases
(Vs phe-pNA)

production of bitter peptides. In yogurt this has been
attributed to proteolysis by L. hulgaricus during storage
(Renz and Puhan, 1975). It was found that the conditions
of manufacture influenced the amount of bitterness,
yogurts produced at 44 C being less likely to be bitter
than those produced at 38 C.
Products of proteolysis. The amount of soluble
nitrogenous compounds increases when milk is cultured
with L. hulgaricus and S. thermophilus. Table 30,
adapted from Miller and Kandler (1967a), shows the
distribution of soluble nitrogen among the different
fractions when the two species are cultured separately in
milk. The range of values observed for each species
further indicates the large strain differences noted above.
The relatively low urea nitrogen and high ammonia
nitrogen observed for S. thermophilus compared with L.
hulgaricus is probably due to the ability of the former to
split urea (Miller and Kandler, 1967b).
The total amount and the profile offree amino acids in
yogurt is dependent on several variables such as strain of
starter bacteria and age of yogurt (Ogai et al., 1972),
ratio of lactobacilli to streptococci, type of milk and
method of manufacture (Rasic et al., 1971a and b;
Stojslavljevic et al., 1971). Regarding the strain of

JOURNAL OF FOOD PROTECTION, VOL. 43, DECEMBER 1980

968 TAMIME AND DEETH

TABLE 28. Proteolysis .of individual caseins by yogurt bacteria.

Organism Casein proteolysis Reference

S. thermophilus Shidlovskaya &
D'yachenko Ooc. cit.)
{J & Khyd. but not cx:s 1 Desmazeaud & Juge (loc. cit.)
L. bulgaricus ex: hyd. but not {J Ohmiya & Sato (1968)
o:5 > K (based on sialic acid release) Ohmiya & Sato (1969)
{J>cx: D'yachenko & Shidlovskaya
(1971), Ohmiay & Sato (1978)
K>cx:s&fJ Chebbi et al. (1974)
K & cx:s hyd. but not {J Chebbi et al. (1977)

~
{J > cx:s• K (with whole casein)
cx:5 > {J, K variable (with purified Singh & Ranganathan
casein) (1977a & b), Singh
cx:s&K>{J & Ranganathan (1979)
> > whole casein > K Shankar & Davies (1978)

TABLE 29. Effect of storage on proteolysis. a
After refrigeration
Organism (s) After 3 h (app. 18 h) 6 days 20 days
S. thermophilus .036b .083
L. bulgaricus .230 .550
Yogurt (S. & L.
60:40) .070 .283
aAfter:Formisano et al. (1974).
bproteolysis values expressed in O.D. units.
bacteria, Nachev (1970) made an intensive investigation
of 125 strains of L. bulgaricus and concluded that they
could be classified into three varieties on the basis of
sugar fermentation and that the three varieties were
characterized by the amino acids they released: the first
group (118 strains) by the presence of leucine, glutamic
acid, asparagine and proline and absence of aminobuty-
ric acid, J3-alanine and tryptophan, the second group (6
strains) by the absence of glutamic acid and the third
group (1 strain) by the presence of tryptophan. This may
help to explain some of the apparent discrepancies in the
free amino acid profiles which have been reported. A
selection of results from four different laboratories is
presented in Table 31. Glutamic acid and proline stand
out as the two most prominent free amino acids in yogurt
from these data. Whereas most other amino acids are
consumed to some extent during growth of the bacteria
in yogurt manufacture, proline and glutamic acid are not
utilized by the bacteria and hence accumulate in the
yogurt (Groux, Joe. cit.; Grudzinskaya and Koroleva,
1971). The final content of the other amino acids reflects
the balance between proteolysis and consumption.
The total amino acid content of both milk and yogurt
.Q99 .108 has been reported by several workers. For milk this
.658 .851 figure appears to be "10 mg/100 ml (Miller and
Kandler, 1967b; Rasic et al., 1971a; Miller et al., 1964)
.466 .562 and for yogurt the reported values vary from 23.6 to
70 mg/100 ml (Rasic et al., 1971a; Ottogalli et al., 1974;
Luca, 1974). Ottogalli et al. Ooc. cit.) showed that the
total amino acid content increased during storage at 20 C
and 4 C though the increase at 4 C was only
0.2 mg/100 ml over 60 days. Rasic et al. (1971a) found
that the method of manufacture influenced the final
content. Incubation of yogurt at 41-42 C for 2-3 h yielded
a higher amino acid content than incubation at 42 C for
1 h followed by S-6 h at 30-32 C (23.6 vs. 19.4 mg/
100 ml). This group has also compared the free amino
acids of cow's, goat's and sheep's milk and yogurts.
Table 32, compiled from their papers, shows that the
goat's milk and the corresponding yogurt had much
higher amino acid levels. The levels of threonine, serine,
glutamic acid and glycine in particular were much higher
in goat's milk and yogurt than in the corresponding cow
products.
There is ample evidence that peptides are produced in
yogurt. However, little work has been reported on
this aspect. In a recent paper by Taner and Zivkova
(1977), presence of several short-chain peptides which
change in number and structure during storage was

TABLE 30. Soluble nitrogenous fractions from milk and milk cultured with L. bulgaricus and S. thermophilus. a

Dialysable N AmmaniaN Peptide N

Sample mg/1 'Vo mg/1 % mg/1 o/o

Milk 249 4.7 30 0.6 20 0.4 62 1.2 137 2.6

Av (6) 490 9.3 73 1.4 166 3.1 96 1.8 155 2.9
L. bulgaricus Range 438-545 8.3-10.3 63-89 1.2-7.9 56-314 1-5.7 51-146 1-2.8 71-270 1.3-5.4
Av (5) 302 5.7 144 2.7 21 0.4 lO 0.6 127 2.4
S. thermophilus 222-4064.2-7.7 88-190 1.7-3.6 16-26 0.3-0.5 3-30 0.1-0.9117-197 2.0-3.7
a Adapted from Miller & Kandler (196 7 a).

JOURNAL OF FOOD PROTECTION, VOL. 43, DECEMBER 1980

 YOGURT: BIOCHEMISTRY 969
TABLE 31. Free amino acid content (mg/100 m{) in milk and yogurt.
Rasic et a!. (1971 a) Miller et a!. (loc. cit.) Groux (Joe. cit.) a Blanc (loc. cit.)
Amino acid Milk Yogurt Milkb Yogurtc Milkd Yogurt Milk Yogurt
Lysine 0.25 0.80 0.22 0.88 0.83 0.72 0.94 1.11
Histidine 0.11 0.80 0.11 1.09 -(e) 1.70
Arginine 0.16 0.70 0.96 1.39
Aspartic acid 0.52 0.70 0.23 1.20 0.51 0.75
Threonine 0.26 0.70 0.09 0.60 0.24 0.24 0.05 0.70
Serine 1.35 2.90 0.08 2.21 0.06 1.50
Glutamic acid 1.48 4.80 3.90 7.06 4.15 6.10
Proline 0.12 5.40 0.12 6.33 7.05
Glycine 0.30 0.45 0.53 0.38 0.13 0.28
Alanine 0.64 3.80 0.16 1.43 0.04 1.17
Valine 0.10 0.90 0.10 1.41 0.31 0.25 1.86
Methionine 0.05 0.20 0.05 0.05 0.08 0.05 0.19
Isoleucine 0.06 0.40 0.07 0.52 0.15 0.15 1.03
Leucine 0.26 0.70 0.06 0.92 0.58 0.22 1.82
Tyrosine 0.14 0.25 0.06 0.18 0.61 tr 0.25
Phenylalanine 0.13 0.45 0.05 0.17 0.11 tr 0.61
tr 0.20
a Estimated from figures by Groux Ooc. cit.) assuming 10 g dry matter/100 ml supertant fluid.
b Average of two samples.
cyogurt "D" in Miller et al. Ooc. cit.).
dchemically acidified milk.
esignifies no reported value.
TABLE 32. Free amino acid content (mg/100 m{) in cow's, abnormally high; it appears that an error was made in
and sheep's milk and yogurt. a the designation of units for these data. Nevertheless, the
relative increase in free fatty acids over 21 days (30%) is
Cow's Goat's Sheep's most significant. In some studies, no decrease in fat
Amino Acid Mille Yogurt Mille Yogurt Milk Yogurt content in yogurt compared with milk was observed. In
Lysine 0.25 0.80 0.60 2.35 0.19 0.72 fact, in another table in the paper by Formisano et al.
Histidine 0.11 0.80 0.45 1.28 0.10 0.50 (1974) the fat contents of milk and 20 days-old
Arginine 0.16 0.70 0.40 0.67 0.26 0.85 corresponding yogurt were 3. 75 and 3.738 OJo, respectively.
Aspartic acid 0.52 0.70 0.22 1.37 0.18 1.75
TABLE 33. Fat and free acid contents of yogurt during
Threonine 0.26 0.70 3.34 2.80 0.13 0.55
storage. a
Serine 1.35 2.90 3.05 3.51 0.20 2.00
Glutamic acid 1.48 4.80 3.54 3.78 1.08 4.10 Fat 48h 6 days 21 days
Proline 0.12 5.40 0.65 4.35 0.11 4.30
Glycine 0.30 0.45 5.91 6.06 0.15 0.25 Neutral fat ( %)
Alanine 0.64 3.80 1.33 3.83 0.56 1.30 (determined gravimetrically) 4.05 3.96 3.91
Valine 0.10 0.90 0.30 0.50 0.24 0.90 Neutral fat (total esters
Methionine 0.05 0.20 0.10 0.35 0.05 0.15 determined colormetrically) 4.24 4.14 3.96
Isoleucine 0.06 0.40 0.18 0.43 0.06 0.25 Free fatty acids
Leucine 0.26 0.70 0.21 1.25 0.23 0.45 NaOH/100 yogurt)b 200 220 260
Tyrosine 0.14 0.25 0.30 0.60 0.16 0.24 aAfter: Formisano et at. (1974).
0.13 0.45 0.11 0.35 0.08 0.15 bunits probably incorrect (see text).
acompiled from Rasic et al. (1971a & b); Stojslavljevic et al.
Ooc. cit.) The work ofTannous and Merat (1969), in which ghee
manufactured from yogurt showed a higher rate of
neatly demonstrated by peptide mapping. No data on
lipolysis than ghee made from milk after homogenization
composition or size were given.
with water, has been quoted as evidence for the presence
Lipolysis of lipolytic activity in yogurt. However, it is unlikely that
Although fat metabolism occurs to only a small degree this lipolysis could be attributed to starter enzymes since
in yogurt, it is thought to make a significant contribution these would be destroyed by the severe heat treatment
to flavor. Formisano et al. (1974) reported a decrease in used in ghee manufacture (up to 115 C). The lipolysis was
fat content of yogurt over 21 days of 3.4 OJo determined probably due to either heat-stable bacterial enzymes or
gravimetrically or 6.60Jo determined colorimetric ally. to bacterial contamination ofthe homogenate.
They also noted a corresponding increase in the free fatty Liploysis occurring in yogurt is attributed to the
acid content. Their data are reproduced in Table 33. The lipolytic enzymes ofthe starter bacteria, the natural milk
values reported by these authors for free fatty acids are lipase being completely inactivated by pasteurization

JOURNAL OF FOOD PROTECTION, VOL. 43, DECEMBER 1980

970 TAMIMEANDDEETH

TABLE 34. Lipase and esterase activities in yogurt starter bacteria.

Enzyme Activity (,ueq/min)

Organism Substrate Intact cells
S. thermophilus Lipase Tributyrin
Lipase Triolein
L. bulgaricus Lipase Tributyrin
Lipase Triolein
Lipase Tributyrin
Esterase Triacetin
aActivitv/mg dry matter.
bvalues in parenthesis are for cells grown in the absence offat, other values are for cells grown in the presence of fat.
cy /mg protein.
dsignifies not detected.
(Deeth and Fitz-Gerald, 1976). Many lactic acid bacteria
have been shown to produce lipases (Fryer, et al., 1967)
and esterases (Morichi et al., 1968; Otterholm et al.,
1968), but activity of these enzymes are usually relatively
low. In some strains, higher activities can be induced by
culturing in the presence of fat (Formisano et ai., 1974).
Lipases of lactic acid bacteria usually show a preference
for short-chain triglycerides, and have been reported to
show higher activity against partial glycerides than
against triglycerides (Stadhouders and Veringa, 1973).
Both organisms used in yogurt production have been
reported to have lipolytic ability although the reports
differ markedly in the amounts and specificity of the
lipase activity measured. This is presumably due to the
different strains examined and to the different assay
procedures employed. S. thermophilus has been shown to
exhibit tributyrinase activity (Formisano et al., 1972 &
1974; Angeles and Marth, 1971), trioleinase activity
(Angeles and Marth, loc. cit.; Umanskii et al., 1974) and
low activity against milkfat and Tween 40 and 60
(Umanskii et al., loc. cit.). Some authors have, however,
been unable to observe any lipolytic activity even against
tributyrin in yogurt organisms (Chander and Chebbi,
1972). L. bulgaricus has been shown to have tributyrinase
activity (Otterholm et al., loc. cit.) though to a lesser
degree than S. thermophilus (Formisano et al., 1974).
Esterases active against soluble substrates such as
ex-naphthyl acetate have been observed in both L.
bulgaricus and S. thermophilus (Morichi et al., loc. cit.;
Otterholm et al., loc. cit.). The former authors have
suggested that the esterase patterns shown on electro-
phoretograms may be species-specific and may be useful
for identification purposes.
The lipolytic enzymes in yogurt starters are intra-
cellular and mostly present in the cytoplasm with very
little activity being associated with the cell membranes
after cell disruption. This is demonstrated by the data in
Table 34.
Several authors have investigated the nature of the free
fatty acids in yogurt. Formisano et al., (1974) reported a
Cell free Cell
extracts
29.5c (23.37)
0.08 (0.6) 0.4 (0.35) Formisano
et al. (1974)
0.37c Otter holm
Traces et al. (loc. cit.)
fatty acids, both free and esterified, of yogurt made from
cow's, sheep's and goat's milk, Rasic and Vucurovic
(1973) reported extensive data on the changes which
occurred when milk was converted to yogurt. They
showed that some acids decreased whereas other
increased but from the data presented it is not possible to
observe a consistent change in any fatty acid during
yogurt production from the three milks. Further work is
required in this area as it seems unlikely that the yogurt
bacteria would behave very differently towards the
milkfat of these three ruminant species.
An increase in volatile fatty acids has been noted
during production and storage of yogurt (Formisano et
al., 1973 and 1974) and in mixed cultures of L.
bulgaricus and S. thermophilus (Dutta et al., 1971; Yu et
al., 1974; Yu and Nakanishi, 1975c). Volatile acidity data
reported by Dutta et al., (1971), Yu et al. Ooc. cit.) and
Yu and Nakanishi (1975c) for the two yogurt organisms
(Tables 35 and 36) indicate that L. bulgaricus produced
more volatile acids than does S. thermophilus. Yu and
Nakanishi (1975c) obtained a volatile acidity of 7.2 and
7.55 meq/kg when a 1: 1 mixture of the two yogurt
starters was cultured in skim and whole milk,
respectively. They observed more C2, C3 and C4 and less
C6. C8 and ClO acids from whole milk than from skim
milk. The highest volatile acid produced were n-C4,
n-C6, C8 and ClO. Earlier data reported by these authors
(Table 36) (Yu et al., loc. cit.) for cultures of S.
thermophilus and L. bulgaricus in whole and skim milk
suggested only marginal differences between the contents
of individual volatile acids produced in whole and skim
TABLE 35. Total and volatile acidity produced by yogurt
organisms. a
Period of Total Volatile
incubation acidity acidity
(h) (o/o lactic acid) (meg/kg)
S. thermophilus 24 0.67 0.82
48 0.84 1.00
.,., 0.86 0.88
I I

g.l.c. profile which showed a predominance of long-chain L. bulgaricus 24 1.41 3.34

fatty acids and which was similar to the fatty acid profile 48 2.31 6.18
of milkfat. They maintained that this pattern did not 77 2.52 8.26
change appreciably during storage. From a study of the a From Dutta et al. (1971).

10 URNAL OF FOOD PRO TECTTON, VOL. 43, DECEMBER 1980

 YOGURT: BIOCHEMISTRY
971
TABLE 36. Changes in volatile fatty acids in whole and skim milk fermented with L. bulgaricus and S. thermophilus. a

L. bulgaricus Str. thermophilus Mixed

Acids Milk Control 37 C/24 h 36 C/72 h 37 C/24 h 37 C/72 h 37 C/24 h 37 C/72 h

Total whole 3.20 6.05 6.26 4.90 4.19 6.88 7.55

VFA skim 2.97 5.89 6.32 4.88 3.79 6.80 7.20
(mg/100 g)
C2 whole 0.21 0.55 1.26 0.51 0.45 0.57 0.48
skim 0.20 1.95 1.36 0.45 0.37 0.12 0.20
C3 whole trace trace trace 0.05 O.o3 0.22 0.11
skim 0.05 0.05 O.o3 0,03 trace trace
i-C4 whole 0.03 0,03 0.05 0.05 0.04 0.13 0.14
skim 0.03 0.04 0.61 0.05 0.05 0.03 0.06
n-C4 whole 0.39 0.74 0.94 1.21 0.97 1.05 1.44
skim 0.38 0.50 0.96 1.20 0.90 0.66 1.08
i-C5 whole 0.05 0.21 0.21 0.14 0.10 0.15 0.06
skim 0,03 0.13 0.18 0.11 0.09 O.o7 0.17
nC5 whole _b -b
skim trace trace -b -
b
n-C6 whole 1.09 1.73 1.24 1.24 1.05 1.56 2.57
skim 1.13 1.72 1.35 1.25 1.07 2.40 2.04
C8 whole 0.97 1.44 0.99 0.74 0.53 1.78 1.64
skim 0.96 1.30 1.18 0.87 0.56 2.26 2.36
ClO whole 1.21 1.59 1.30 0.91 1.10 2.65 2.22
skim 1.10 1.81 1.74 1.06 0.68 3.11 2.92
aAfter: Yu et al. (1974), Yu & Nakanishi (1975c).
bTest was not determined.

milk. This is somewhat at variance with the conclusion
reached by Turcic et al., (loc, cit.) that, because the
relative levels of C4, C6, C8 and C10 acids in cultures of
L. bulgaricus resembled those in milkfat, hydrolysis of
milkfats probably occurred during metabolism of L.
bulgaricus. Formisano et al. (1973 and 1974) reported
increases in low molecular weight acids, including
butyric and four non-milkfat fatty acids during storage.
It appears then that although a small degree of
lipolysis may take place in yogurt, most of the volatile
acid content of yogurt is derived from non-fat
components. Probably the most important precursors of
these are amino acids (Nakae and Elliot, 1965). The
higher production of yolatile acids by L. bulgaricus is
probably related to its high proteolytic activity (see
section of proteolysis).
ACKNOWLEDGMENT
The authors gratefully acknowledge the technical drawings given by
Alfa-Laval AB, Lund, Sweden, and S. Crawford for preparation of
figures and graphs.
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