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Jordan Journal of Agricultural Sciences, Volume 8, No.3 2012

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* Ass. Prof., Dept. of Food Sci., College of Agric. and Forestry, Mosul Univ., Iraq. Tel: +9647701667076 E-mail: [email protected] ** Dept. of Food Tech., College of Agric. Salahaddin Univ.,Iraq.Tel: +9647504451952 E-mail: [email protected] Received on 15/6/2011 and Accepted for Publication on 5/3/2012.

2012 DAR Publishers/University of Jordan. All Rights Reserved.

Improvement of Yogurt Properties by Microbial Transglutaminase

Waleed Ahmed Mahmood* and Nawal Hurmiz Sebo**

ABSTRACT

Yogurt was manufactured from cow's milk which was treated with different concentrations of free and immobilized Streptoverticillium mobaraense transglutaminase. The highest pH and lowest titratable acidity during three weeks of storage were detected in the sample treated with 75 U/l of free transglutaminase before starter addition. The concomitant MTGase treatment with starter addition resulted in significant increase in yogurt gel strength and viscosity and decreased water separation. This effect was continued during storage at 4 C. The effect of enzymatic treatment with 45 U/l almost resembled that of adding 3% of skimmed milk powder. Immobilized MTGase treatment had less effects on yogurt properties. Sensory evaluation of yogurt showed that treated samples were superior to untreated sample in all sensory aspects through storage periods. To reveal the occurrence of cross-linking among protein molecules, SDS-PAG electrophoresis was performed for yogurt proteins. New high molecular weight protein bands had occurred accompanied by decreasing in casein and whey proteins bands density.

Keywords: Transglutaminase, Yogurt, Streptoverticillium mobaraense.

INTRODUCTION

Texture and reological properties of some dairy

products, such as soft cheeses, ice cream and yogurt, are affected mainly by the balance among the milk components. Increasing protein level leads to an increase in gel strength. Yogurt and cheese usually suffer water separation during storage due to syneresis. The separated water contains substantial amounts of proteins. Moreover, consumers generally find such water separation objectionable (Han et al., 2003). Therefore, there remains a need for improved methods to produce

soft cheeses and yogurt wherein syneresis is reduced. Among the conventional methods of improving yogurt texture, is the addition of non fat dry milk (NFDM) and stabilizers to the milk (Guzman-Gonzalez et al., 1999; Shah, 2003), but since the addition of stabilizers is not permitted in many countries, alternative methods to achieve texture improvement have been of interest in recent years.

Transglutaminase (TGase) is an enzyme that catalyzes the cross linking of proteins through the formation of covalent bonds using two amino acids, glutamine and lysine, enabling the proteins to behave like larger molecules. The cross-links introduced by this enzyme change the protein structure and improve its functional properties, like texture, without decomposing the nutritional quality of the lysine residue (Seguro et al., 1996). Since microbial TGase (MTGase) is available in bulk from fermentation processes, the enzyme has

Improvement of Yogurt Waleed Ahmed Mahmood and Nawal Hurmiz Sebo

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been approved by food industries to improve quality of many foods such as meat, fish, soy products, yogurt, cheese, and ice cream (Zhu et al., 1995; Motoki and Seguro, 1998; Kuraishi et al., 2001; Jaros et al., 2007).

The present paper describes the effect of incorporation of the free and immobilized MTGase on the properties of yogurt as a function of the added enzyme concentration and storage period .

MATERIALS AND METHODS Bacterial cultures and growth conditions: A

lyophilized culture of Streptoverticillium mobaraense (DSM-40847) was obtained from DSMZ, Germany. The bacteria were propagated through submerged cultivation ( 30 C, 100 rpm) in a medium contained 2% starch, 2% peptone, 0.2% yeast extract, 0.2% dipotassium hydrogen phosphate, 0.1% magnesium sulfate and 0.05% glycerol (Ando et al., 1989). Yogurt starter culture consisted of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbueckii subsp. bulgaricus was from Rhodia Food.

Enzyme isolation and precipitation: At the end of incubation period (3 days), the Streptoverticillium mobaraense culture was filtered off. The enzyme was precipitated from the supernatant by adding two volumes of cold acetone. The precipitate was dissolved in Tris-acetate buffer (0.1M, pH 6) and lyophilized.

Determination of transglutaminase activity: The activity was measured by the colorimetric hydroxamate procedure with N-carbo-benzoxy-L-glycine as the substrate according to the method of Folk (1970). Reaction mixture containing 50 l of the enzyme, 350 l of Tris-acetate buffer (0.1 M, pH 6), 25 l of 2 M hydroxylamine and 75 l of 0.1 M CBZ-L-GLN-GLY, was incubated at 37 C for 10 minutes and then the reaction was stopped by adding 500 l of 15% trichloroacetic acid (TCA) containing 5% FeCl3. The

absorbance was recorded at 525 nm using CECIL 3021 spectrophotometer. One unit of transglutaminase activity was defined as the amount of enzyme which causes the formation of one micromole of hydroxamic acid per minute at 37C. A calibration curve was prepared using L-glutamic -monohydroxamate.

Immobilization of transglutaminase: The enzyme was entrapped in calcium alginate gel beads following the procedure of Fraser and Bickerstaff (1997). The enzyme solution was mixed with a same volume of 2% sodium alginate solution. The mixture was transferred to a 10-ml syringe and drop wise into a beaker containing 0.15 M calcium chloride solution with gentle stirring. Gel beads of calcium alginate were formed which left for 60 minutes for hardening, then they were collected, washed with distilled water, suspended in 0.1M, pH 6 Tris-acetate buffer and stored at 4 C. To determine the immobilization efficiency, the beads gel was dissolved by immersion in 0.05 M EDTA, pH 7 for 60 minutes and the activity of the obtained free enzyme was determined.

Milk treatment and yogurt manufacturing: Whole fresh cow's milk (containing 3.7% fat, 3.38% protein and 12.44% total solids) was heated to 90 C for 5 minutes, cooled down to 43 C and divided into 4 samples. To the first sample, the free MTGase was added at the same time of the starter addition. The second sample was treated with MTGase as above and incubated at 40 C for 60 minutes then the enzyme was inactivated by heating to 90 C for 5 minutes, then the yogurt starter was added at 43 C. To the third sample, the immobilized enzyme beads were added, incubated at 40 C for one hour, then the beads were removed by filtration through cheesecloth and the starter was added at 43 C. To the last sample, non-fat dry milk (NFDM) was added without MTGase addition. These samples were used to manufacture yogurt according to the method of Tamime and Robinson (1999). Yogurt


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samples were then cooled and kept at 4 C to the next day for analysis. Concerning the effect of the MTGase treatment on the stored yogurt properties, the concentration of the added enzyme in all cases was 75 unit/l. The set yogurt samples were stored at 4 C for 21 days and the samples were weekly analyzed for acidity, pH, physical and sensory properties.

Chemical analysis of milk and yogurt: pH of yogurt was measured by pH-meter (WTW-530). Titratable acidity of milk and yogurt was estimated according to IDF/ISO Standard (1991) by calculating SH value which is defined as the volume (ml) of 0.25 M NaOH that neutralize the acidity in 100 ml of milk. To detect the formation of cross-links, SDS-PAG electrophoresis was conducted for yogurt proteins by using vertical electrophoresis apparatus (Jookoh Co. LTD) following the procedure described by Laemmli (1970).

Physical tests of yogurt: Yogurt gel firmness was measured using texture analyzer (Stevens-LFRA with TA26 probe). To measure the quantity of separated water, the procedure described by Harwalker and Kalab (1983) was followed by centrifuging 25 gm of yogurt sample at 1400 g and 5 C for 15 minutes. Yogurt viscosity was estimated using Bostwick consistometer by measuring the distance which the stirred yogurt sample moves on a declined metallic surface in 10 seconds.

Sensory evaluation: Sensory evaluation of the produced yogurt was carried out for appearance, texture, flavor and taste after 1, 7, 14 and 21 days of storage according to Nelson and Trout (1964).

Statistical analysis: The data were analyzed by using the Complete Random Design (CRD) according to

SAS (2001). RESULTS AND DISCUSSION The effects of milk treatment with transglutaminase

or non-fat dry milk (NFDM) on the properties of the produced yogurt are summarized in Table (1).

Most MTGase treatments, with the different methods of application, caused non-significant increase (p0.05) in yogurt pH values as compared to the untreated yogurt and with the yogurt containing NFDM. The effect of enzymatic treatment on pH and acidity was found to be proportional to the concentration of the added enzyme. The highest pH (4.53) was detected in the sample treated with 75 U/l of free transglutaminase before the addition of the starter in which the increase in pH became significant upon storage at 4 C for three weeks and it was accompanied by significant decrease in titratable acidity. A significant drop in all samples pH was observed upon increasing the storage period (Table 2).

The expected explanation for this relationship is that the amino acids and small peptides, which are needed for the growth of lactic acid bacteria, are cross linked by the transglutaminase treatment and became unavailable to the bacteria. This might led to the retardation in bacterial growth and decrease in acid production. The same observation was found by Lorenzen et al. (2002) who stated that the pre incubation of milk with MTGase followed by thermal inactivation of the enzyme caused the elongation of fermentation time while the concomitantly addition of the enzyme with the starter did not affect the acidity of the yogurt. On the other hand, Bonisch et al. (2007) detected that 88% of cross linking had taken place in the pH range of 5.7-6.6 and the enzyme was inhibited at pH lower than 4.5.


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Table (1): Effect of treatment with transglutaminase or non-fat dry milk (NFDM) on some properties of yogurt. Syneresis (ml/25gm)

Viscosity (cm/10 sec)

Gel strength (gm/cm2)

Acidity (SH)

pH MTGase conc.(U/l)

Treatment

10.00a 10.41a 8.66n 39.1cd 4.36a 0 Control

8.33c 9.75b 9.66mn 38.75d-f 4.37a 15

7.20ef 7.83gh 13.66gh 38.51e-g 4.40a 30

6.50gh 6.25k 15.33ef 38.40e-h 4.42a 45

6.40hi 5.23l 21.00c 38.26f-h 4.43a 60

6.10i 4.50m 29.00a 38.10g-i 4.45a 75

Addition of free MTGase with the starter

9.10b 9.83b 9.33mn 38.53e-g 4.42a 15

7.63d 8.63e 11.33k 38.16g-i 4.47a 30

7.06f 8.33ef 14.33fg 38.03h-j 4.49a 45

6.73g 7.45hi 16.00e 37.73ij 4.51a 60

6.10i 7.10ij 22.33b 37.60j 4.53a 75

Addition of free MTGase before the starter

9.80a 10.01ab 9.33mn 38.78de 4.40a 15

9.10b 9.16c 11.66i-k 38.61e-g 4.43a 30

8.56c 8.75de 14.00g 38.49f-h 4.44a 45

8.30c 8.10fg 14.66fg 38.35e-h 4.45a 60

7.60d 7.18ij 17.33d 38.21e-h 4.48a 75

Addition of immobilized MTGase before the starter

9.76a 9.63b 10.00lm 39.26gh 4.35a 1

9.16b 9.13cd 11.00kl 39.56bc 4.33a 1.5

8.36c 8.5ef 12.66hi 39.75ab 4.31ab 2

7.70d 7.83gh 12.33ij 39.80bc 4.30ab 2.5

7.40de 7.00j 12.66hi 40.00a 4.29b 3

Addition of NFDM (%)

Means within columns followed by different letters are significantly different at p 0.05.

Concerning the effects of MTGase treatment on physical properties of the product, a significant increase (p0.05) in yogurt gel strength was detected upon the treatment with all methods except with low enzyme concentration (15 U/l). This increase was proportional to the concentration of the added enzyme. The directaddition of 75 U/l of free MTGase along with the starter was significantly superior to all other treatments. The least effect was detected with immobilized enzyme treatment which could be due to the decrease in the

enzyme-substrate affinity especially with high molecular weight substrates where the mass transfer was reduced leading to the obstruction of the substrate to reach the active site of the enzyme (Schorch et al., 2000). The effect of adding 30 U/l of free enzyme almost resembled the effect of adding 3% of NFDM (Table 1). Lorenzen et al. (2002) found that the treatment of milk with MTGase had reduced water separation and improved the texture of set style yogurt. During the first week of storage, all free MTGase-treated samples (75 U/l) showed significant


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increase (p0.05) in gel strength but the highest strength was detected in the sample to which free enzyme was added concomitantly with the starter in which strength increase was continued during all storage period.

The addition of immobilized enzyme caused non-significant increase during storage (Table 2). Schorch et al. (2000) demonstrated that after a certain time period, the firmness of MTGase-induced gels reached nearly a plateau value, suggesting that most of the protein connections were already built up during this time period. They also stated that the basic function of TGase is to cross-link milk proteins covalently, which

results in a finer and stronger gel network in the yogurt as compared with acid-induced gels. This indicates that the concomitantly addition of MTGase with the starter stimulated the crosslinking of protein molecules which was continued during and after fermentation resulting in the formation of new covalent bonds which caused the development of gel stiffness during storage period (Ozer et al., 2007). The enzymatic crosslinking of proteins by TGase is considered as an important way for improving the texture of yogurt and other protein-containing foods (Tamime, 2006; Jaros et al., 2007).

Table (2): Effect of transglutaminase treatment (75 U/l) on some properties of stored yogurt stored at 4C for 21 days.

Treatment

Storage period (day)

pH Acidity (SH) Gel strength (gm/cm2)

Viscosity (cm/10sec.)

Syneresis (ml/25gm)

1 4.39de 39.10k 8.66k 9.75a 9.10a

7 4.25g 45.30f 10.66jk 8.75b 8.30b

14 4.17h-j 48.03c 10.66jk 8.45b 8.16b

Control

21 3.91l 49.16a 10.66jk 8.35b 8.06b

1 4.49ab 38.00m 24.33c 4.50f 5.76e-g

7 4.35ef 43.30i 38.33b 3.81g 5.23fg

14 4.24g 45.52f 42.00a 3.61g 5.13g


21 4.15ij 47.70d 42.33a 3.46g 5.03g

1 4.51a 37.60n 14.33hi 6.61c-e 7.03cd

7 4.40cd 42.65j 17.66ef 6.73c-e 6.36de

14 4.25g 45.33f 17.66ef 6.80c-e 6.30de


21 4.18hi 46.80e 17.00f 6.80c-e 6.16e

1 4.46bc 38.65l 12.62ij 6.88c-e 7.20c

7 4.34ef 43.80g 13.66hi 6.90cd 6.80cd

14 4.22gh 46.92e 14.00hi 6.96c 6.70cd

Addition of immobilized MTGase

21 4.12i 48.26c 13.66hi 6.63c-e 6.56cd

Means within columns followed by different letters are significantly different at p 0.05.

Cross-linked yogurt with all treatments showed a highly viscous structure especially at high added

MTGase concentrations as compared to untreated sample and that contained non-fat dry milk. The highest


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viscosity (4.5 cm/10 sec.) had occurred upon concomitantly addition of 75 U/l of free MTGase with the starter. All treated samples showed significant increase (p0.05) in viscosity except the treatment with low concentration of immobilized enzyme (Table 1). The viscosity had continued to increase during the first week of storage after which lower increase was noticed during the remaining storage period with some variations among the treatments (Table 2). This could be attributed to differences in samples pH which may alter the protein-protein interactions resulting in slow protein rearrengments in the acid casein gels during cold storage (Ozer et al., 1998). Kuraishi et al. (2001) observed increased yogurt viscosity upon increasing the added enzyme concentration. They mentioned to the benefit of this treatment in decreasing non fat solids content and reducing the production cost.

Syneresis, or whey separation, is an important factor that affects consumer acceptance of yogurt. In unstored yogurt, MTGase treatment with all methods and concentrations caused significant decrease (p0.05) in water separation except the treatment with 15 U/l of immobilized enzyme. The least quantity of separated water (6.1ml/25 gm) was detected upon treating with 75 U/l of free MTGase along with the starter as compared to the untreated sample (10 ml/25 gm). The addition of NFDM had also reduced water separation. The effect of adding 3% of NFDM (7.4 ml/25 gm) resembled the effect of the treatment with 45 U/l of free MTGase (Table 1). The treatment with 75 U/l of free MTGase along with the starter resulted in minimal separated water (5.03 ml/25 gm) at the end of storage period. On the contrary, the untreated sample had lost 9.1 ml/25 gm. at the end of the storage period (Table 2). This was due

to the decreased permeability which had occurred as a result of the formation of permanent cross-links among the milk proteins. The decreased permeability causes more compact and stable microstructure with smaller compartments in yogurt which allowed more free water to be entrapped in the yogurt gel network (Moon and Hong, 2003). Motoki and Seguro (1998) mentioned that syneresis could be reduced by MTGase treatment which improves the water holding capacity of yogurt gel network. On the other hand, the slow development of acidity had an important role in decreasing water separation (Lorenzen et al., 2002).

The addition of the free MTGase in all concentrations along with the starter was found to be the most effective way for improving all physical properties. Treatment with the immobilized enzyme caused less effect than the free form. This may be due to the physical obstruction caused by the alginate gel lattice against the movement of substrates and products throughout the gel resulting in retarding and lowering the enzyme reaction rate. On the other hand, the immobilized enzyme treatment is unable to be applied concomitantly with the starter and it is only applicable by adding it prior to the addition of the starter.

The effect of MTGase treatments and storage periods on sensory evaluation of yogurt is indicated in Table (3). All MTGase-treated samples during all storage periods were superior to the untreated yogurt in all sensory properties. The treated samples were characterized by dry and soft surfaces which could be attributed to the formation of glutamyl-lysine bonds and their effect in improving the water-holding capacity of the yogurt gel (Motoki and Seguro, 1998). A similar observation was reported by Kuraishi et al. (2001).


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Table (3): Effect of transglutaminase treatment (75 U/l) on some sensory properties of stored yogurt stored at 4C for 21 days.

Treatment Storage period (day)

Appearance (10)

Texture (30)

Flavor (45)

Sour taste (15)

Total (100)

1 8.00cd 20.00f-h 38.75ab 13.00ab 79.75d-g

7 8.25b-d 23.75c-g 41.75ab 14.00a 87.75a-d

14 7.75cd 19.50i 38.50ab 10.50e 76.25e-g

Control

21 7.00d 19.25i 36.00b 10.50e 72.75g

1 9.50ab 26.75a-c 35.75b 10.75de 82.75b-f

7 10.00a 28.00ab 40.00ab 11.75b-e 89.75a-c

14 9.75ab 28.75a 41.50ab 12.75a-c 92.75a


21 9.25a-c 26.75a-d 38.50ab 13.25ab 87.75a-d

1 9.50ab 25.50a-e 36.00b 10.75de 81.75c-f

7 10.00a 28.25a 40.25ab 12.00b-e 90.50a-c

14 9.75ab 27.75a-c 41.75ab 12.75a-c 92.00ab


21 9.25a-c 26.25a-d 38.25ab 13.25ab 87.00a-d

1 9.25a-c 23.25d-h 37.25ab 11.25c-e 81.00c-g

7 10.00a 26.00a-d 40.00ab 12.25b-d 88.25a-d

14 9.25a-c 25.75a-e 40.75ab 12.50a-c 88.25a-d

Addition of immobilized MTGase

21 9.25a-c 25.00a-e 37.25ab 12.50a-c 84.00a-e

Means within columns followed by different letters are significantly different at p 0.05. Seven days were found to be a preferable period of

yogurt storage for all treatments to get the best appearance of the product. There was non-significant decrease in this property in all treatments upon increasing the storage time. The least degree was recorded for the untreated sample after storage for 21 days.

Concerning the sensory evaluation of yogurt texture, it was found that the MTGase-treated samples were superior to the untreated sample throughout all storage periods. The best texture was obtained by the concomitantly addition of free MTGase with the starter but the differences among the treatments were non-significant. This result was in accordance to instrumental

analysis of this property and was in agreement with Kuraishi et al. (2001) who stated that the sensory evaluated texture of MTGase-treated yogurt was highly superior to the untreated yogurt.

During the first week of storage, the flavor of the untreated sample was found to be superior to the MTGase-treated samples. This could be due to the effect of the enzymatic treatment on the obstruction of the growth of lactic acid bacteria and retarding the development of acidity and the production of flavor compounds such as acetaldehyde, acetoin and diacetyl through the first days of storage (Ozer et al., 2007). After two weeks of storage, the treated samples retained the preference in the flavor property. Treated yogurt


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samples were also characterized by a fatty flavor. This was in agreement with Metwally (2007) who observed that the treatment with MTGase produces fatty flavor which can replace some of added fat to the low-fat dairy products.

The effect of MTGase treatment on yogurt protein molecules of one day-old yogurt was revealed by SDS-PAG electrophoresis (Fig. 1). The treated yogurt was characterized by a decrease in the intensity of casein and whey protein bands accompanied by increasing the

intensity of high molecular weight polymers bands depending on the degree of cross-linking that occurred according to the treatment type. The highest cross-linking was observed upon the addition of 75 U/l of free enzyme along with the starter followed by the addition of the same concentration prior to the starter addition. A relatively lower cross-linking had occurred upon using the immobilized MTGase which could be due to the effect of the alginate gel lattice on lowering the enzymatic reaction rate.

Fig. 1: SDS-PAGE profile of MTGase-treated yogurt proteins. 1-Control, 2- Addition of 30 U/l of free MTGase with the starter, 3- Addition of 75 U/l of free MTGase with the starter, 4- Addition of 75 U/l of free

MTGase before the starter, 5- Addition of 75 U/l of immobilized MTGase before the starter.

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