APTEFF, 38, 1-190 (2007) UDC: 637.146.3:637.047/.

05
DOI:10.2298/APT0738045M BIBLID: 1450-7188 (2007) 38, 45-52
Original scientific paper

PHYSICO-CHEMICAL PROPERTIES OF PROBIOTIC YOGHURT

PRODUCED WITH TRANSGLUTAMINASE

Spasenija D. Milanovi, Marijana . Cari, Mirjana S. uri,

Mirela D. Ilii and Katarina G. Durakovi

The effect of different concentration of transglutaminase - TG (0.02%, 0.06% and

0.12%) on physico-chemical properties of probiotic yoghurt was investigated. Two series
of yoghurt were manufactured on a laboratory scale from pasteurised skim milk (0.1 %
w/w fat). Series I was produced with TGase activation during a period of 2 h at 40°C,
while series II was produced without enzyme activation. Then, the adequate quantities of
TGase and probiotic starter culture ABT-4 (Chr. Hansen A/S Denmark) were added.
Chemical composition, physico-chemical properties (water holding capacity and whey
separation) and sensory characteristics of yoghurt samples were determined after pro-
duction and during 5 days of storage. Addition of TGase to milk (direct or after activa-
tion) for probiotic yoghurt manufacture improved its overall characteristics. Activation
of TG in yoghurt production increases water holding capacity as well as decreases
syneresis during the storage.

KEY WORDS: Yoghurt, probiotic culture, transglutaminase, physico-chemical properties

INTRODUCTION

In recent years, enzymatic cross-linking of milk proteins using the enzyme (TGase,
EC 2.3.2.13) has attracted considerable attention in dairy research. This enzyme catalyzes
the acyl transfer reaction between -carboxyamide groups of peptide-bound glutamine
residues and the -amino groups of lysine residues, leading to the formation of intra- and
intermolecular isopeptide bonds (1).
The enzyme reaction results in formation of covalently cross-linked protein polymers.
TGase is now widely used in food production such as seafood, surimi products, dairy
products, meat products, noodles/pasta, baked goods, etc., to improve their functional
properties (2).

Dr. Spasenija D. Milanovi, Prof., Dr. Marijana . Cari, Prof., Dr. Mirjana S. uri, Prof., Mirela D. Ilii,
M.Sc., Katarina G. Durakovi, B.Sc, University of Novi Sad, Faculty of Technology, 21000 Novi Sad, Bul.
Cara Lazara 1, Serbia, e-mail: senadm@uns.ns.ac.yu

45
 Cross-linking of food proteins by TGase modifies the hydration ability, the gelation,
rheological and emulsifying properties, and heat stability of food proteins in model sys-
tems (3), but the rate of TGase cross-linking depends on the macromolecular structure of
each protein substrate.
Caseins are good substrates for TGase and, in a mixed system such as milk, the ca-
seins are cross-linked preferentially over the native whey proteins. Whey proteins require
structural modification, e.g., heat-induced denaturation, to allow their participation in
cross-linking reactions, either with caseins or other whey proteins (4-6).
Transglutaminases are widespread in nature. They can be found in mammalian sys-
tems, in fish, and in plants. Due to the cost-effective production of TGase by microorga-
nisms, especially by the strains of Streptoverticillium, applications of this enzyme in the
industrial food production are possible. Benefits of microbial transglutaminase (MTGase)
use are the lower costs of extraction and purification and their Ca2+-independent catalytic
action (7).
Fermented dairy products are generally considered to be one of the most suitable sub-
strates to transfer an adequate number of probiotic bacteria to the consumer (8). Pro-
biotics are live microbial food supplements exhibit a beneficial effect on the health of
consumers by maintaining or improving their intestinal microbial balance (9). A number
of health benefits have been proposed including antimicrobial, antimutagenic, anticarci-
nogenic and antihypertensive action, and reduction in serum cholesterol, alleviation of
lactose intolerance, and reduction of allergic symptoms (10-12).

EXPERIMENTAL

Materials

Yoghurt manufacture. Yoghurt samples were produced on a laboratory scale from

skimmed milk with 0.1% fat. Milk was pasteurized at 71°C during 15 s and cooled to
8°C. Two series of probiotic yoghurt with transglutaminase were produced (Table 1).

Table 1. Plan of the experiment

TGase (g/100g)
No. Sample
Without activation
1 KY 0
2 0.02WAY 0.02
3 0.06 WAY 0.06
4 0.12 WAY 0.12
With activation
5 0.02AY 0.02
6 0.06AY 0.06
7 0.12AY 0.12

46
 In the first series milk was cooled to 43°C and the appropriate quantities of TGase
(0.02%. 0.06% and 0.12%) and probiotic starter culture ABT-4 (Lactobacillus acido-
philus-5. Bifidobacterium-12. S. thermophilus. Chr. Hansen A/S Denmark) were added.
In the second series TGase was activated in milk during a period of 2 h at 40°C, after that
the milk was heat treated at 80°C for 1min, cooled to 43°C and inoculated with the same
probiotic starter at 45°C.
In both trials the fermentation lasted for 4-7 hours, until the pH 4.5 was reached. The
fermented milk was cooled to 8°C and gently homogenized. Characteristics of all yoghurt
samples were determined, such as: macro-chemical composition (dry matter, fat, total
proteins, total nitrogen, ash content, and acidity) physico-chemical (pH, water holding
capacity, and whey separation) and sensory characteristics.

Methods

Chemical composition of probiotic yoghurt samples produced with TGase (total so-
lids, fat content, proteins content and ash content) was analyzed by standard methods
(13), pH value was measured on a pH meter PHSPEAR (Eutech Instruments Oakton).
Whey syneresis is expressed in mL of whey separated after 3 h of filtration of 50 g
sample at room temperature (14).
Water-holding capacity of yoghurt was determined using a procedure by Guzman-
Gonzalez, Morais, Ramos and Amigo (15). 20g of yoghurt (Y) was centrifugated for 30
min at 1250ug and 20°C (h = 4.8 cm). The whey expelled (WE) was removed and weig-
hed. The water-holding capacity (WHC) was determined as:

100 u Y  WE 
WHC = .
Y
Sensory quality of yoghurt samples was evaluated by experts judging: appearance
(max 1 point), colour (max 2 points), consistency (max 4 points), odour (max 3 points)
and taste (max 10 points).

RESULTS AND DISCUSSION

Probiotic yoghurt samples with TGase were produced from the pasteurized skimmed
milk (0.1% fat). The milk quality was in accordance with the actual Regulations of milk
and dairy products (16).
Changes of the pH during milk fermentation are presented in Fig. 1. The pH value of
samples decreased during fermentation, but it was different between control yoghurt,
yoghurt produced with TGase activation and yoghurt samples produced without TGase
activation.
Fermentation time of the yoghurt produced with enzyme activation prior to fermen-
tation with probiotic starter was shorter than in other yoghurt samples, and lasted about 6
h.
Fermentation of the control yoghurt was finished after 9.26 h, and of yoghurt samples
produced without activation, the fermentation time was 9.10 h (sample 0.02 WAY) and
8.5 h (sample 0.12 WAY).

47
 7

KY 0.02AY
0.06AY 0.12AY
6.5
0.02WAY 0.06WAY
0.12WAY

6
pH value

5.5

4.5

4
0 3.83 4.33 4.58 4.83 5.08 5.33 5.5 5.66 5.82 5.98 6.26 6.51 7.01 7.26

Fermentation time (h)

Fig 1. Changes of the pH value of the probiotic yoghurt with TGase

during milk fermentation
Chemical composition of the probiotic yoghurt produced with TGase is presented in
Figure 2. Since the fat content of all probiotic yoghurt samples with TGase is 0.1%, the
produced samples belong to the group of dietary yoghurts. The protein content in pro-
duced yoghurt samples was in the range from the min. 2.87% (sample 0.06 WAY) to the
max. 3.09% (sample 0.12 WAY).

10
Fat Total proteins Lactose Ash Total solids
9

7
Components (g/100g)

0
Milk KY 0.02AY 0.06AY 0.12AY 0.02WAY 0.06WAY 0.12WAY

Probiotic yoghurt samples

Fig 2. Chemical composition of probiotic yoghurt with Tgase

The physico-chemical characteristics, water holding capacity in the first place, are
very different between the probiotic yoghurt variants with TGase in certain series. The
lowest water holding capacity was found in the control sample of probiotic yoghurt –
52.5%, and the highest in the yoghurt sample produced without activation with 0.12% of
TGase.
The water holding capacity of samples produced with TGase activation was lower
compared to the probiotic yoghurt samples produced without enzyme activation (Fig. 3).
The yoghurt samples syneresis was very uniform: from 29 mL (sample 0.12 AY) to 32
mL (sample 0.02 AY).

48
 The characteristics of probiotic yoghurt samples were analyzed after 5 days of storage
(Table 2). The syneresis of control yoghurt and samples produced without activation, and
using 0.06% and 0.12% of enzyme, was the same during 5 days. In all other probiotic
yoghurt samples the syneresis was lower by 1 mL, and in yoghurt sample 0.02 AY by 2
mL.
The water holding capacity of control yoghurt and samples produced without activa-
tion significantly decreased during 5 days of storage.
During storage for 5 days, the acidity of produced probiotic yoghurt samples decrea-
sed, and the measured values were from min 34.5°SH (sample 0.02 WA) to max.
36.4°SH (sample 0.02 WAY).
pH Value of probiotic yoghurt samples decreased during the storage, min. by 0.07 pH
units in sample 0.06 AY to max 0.13 pH units in yoghurt sample 0.12 WAY.

70
Syneresis (mL)
Water holding-capacity (%)
60
Physico-chemical characteristics

50

40

30

20

10

0
KY 0.02AY 0.06AY 0.12AY 0.02WAY 0.06WAY 0.12WAY
Yoghurt samples

Fig. 3. Physico-chemical characteristics of probiotic yoghurt with TGase

Total sensory scores of probiotic yoghurt variants were in the range from 18.2 (sam-
ple 0.06 BA) to max. 20 (samples produced with enzyme activation). The consistency of
probiotic yoghurt variants produced with TGase enzyme activation is homogenous,
whereas samples produced without enzyme activation have grained consistency.
Generally, enzyme activation in the probiotic yoghurt production improved signifi-
cantly the physico-chemical and sensory characteristics of final product.

Table 2. Physico-chemical properties of the yoghurt with TGase

after 5 days of storage

Probiotic yoghurt samples

Parameters 0.02 0.06 0.12 0.02 0.06 0.12
KY
AY AY AY WAY WAY WAY
Syneresis (mL) 31 30 29 30 30 30 30
WHC (%) 37 55.5 56.0 57.0 44 43.5 56.5
Acidity (qSH) 35.6 36.4 36.1 36.5 34.6 34.8 35.6
pH 4.40 4.38 4.42 4.43 4.39 4.38 4.37
49
 K
20
18
16
14
0,12BA 0,02A
12
10
8
6
4
2
0

0,06BA 0,06A

0,02BA 0,12A
Appearance Colour Consistency
Odour Taste Total points

Fig. 4. Sensory analysis of the probiotic yoghurt with TGase

CONCLUSION

Probiotic yoghurt variants were produced from skimmed milk (0.1% fat) with the dif-
ferent amounts of TGase added.
The fermentation time of control yoghurt sample was the longest – 7.26 h, and of
yoghurt samples with previous enzyme activation at 40°C for 2 h, was shortest – 6.26 h.
For samples produced without enzyme activation, fermentation time was from 5.98 h
(sample 0.06 WAY) to 7.01 h (sample 0.02 WAY).
The content of macrocomponents was uniform in all probiotic yoghurt samples, pro-
duced both with and without enzyme activation.
Syneresis of yoghurt samples with TGase was slightly (insignificantly) different after
the production.
TGase activation in the production of probiotic yoghurt results in a significant de-
crease of the syneresis and improved water holding capacity during storage. Also, it im-
proved the physico-chemical and sensory characteristics of the final product.

ACKNOWLEDGEMENT

This study as a part of Project Eureka, E! 3488 is financially supported by Ministry

of Science of the Republic Serbia.

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50
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Received 25 June 2007

Accepted 5 September 2007

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