APTEFF, 38, 1-190 (2007) DOI:10.2298/APT0738045M UDC: 637.146.3:637.047/.05 BIBLID: 1450-7188 (2007) 38, 45-52 Original scientific paper PHYSICO-CHEMICAL PROPERTIES OF PROBIOTIC YOGHURT PRODUCED WITH TRANSGLUTAMINASE Spasenija D Milanoviü, Marijana Ĉ Cariü, Mirjana S uriỹ, Mirela D Iliỵiỹ and Katarina G Durakoviỹ 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 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 production and during days of storage Addition of TGase to milk (direct or after activation) 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 Ȗ-carboxyamide groups of peptide-bound glutamine residues and the İ-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 Milanoviü, Prof., Dr Marijana Ĉ Cariü, Prof., Dr Mirjana S uriỹ, Prof., Mirela D Iliỵiỹ, M.Sc., Katarina G Durakoviü, 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 systems (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 caseins 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 systems, in fish, and in plants Due to the cost-effective production of TGase by microorganisms, 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 substrates to transfer an adequate number of probiotic bacteria to the consumer (8) Probiotics 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, anticarcinogenic 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 Plan of the experiment 46 No Sample KY 0.02WAY 0.06 WAY 0.12 WAY 0.02AY 0.06AY 0.12AY TGase (g/100g) Without activation 0.02 0.06 0.12 With activation 0.02 0.06 0.12 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 acidophilus-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 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 solids, 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 h of filtration of 50 g sample at room temperature (14) Water-holding capacity of yoghurt was determined using a procedure by GuzmanGonzalez, Morais, Ramos and Amigo (15) 20g of yoghurt (Y) was centrifugated for 30 at 1250ug and 20°C (h = 4.8 cm) The whey expelled (WE) was removed and weighed The water-holding capacity (WHC) was determined as: WHC = 100 u Y WE Y Sensory quality of yoghurt samples was evaluated by experts judging: appearance (max point), colour (max points), consistency (max points), odour (max 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 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 fermentation with probiotic starter was shorter than in other yoghurt samples, and lasted about 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 KY 0.06AY 0.02WAY 0.12WAY 6.5 0.02AY 0.12AY 0.06WAY pH value 5.5 4.5 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 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 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 produced yoghurt samples was in the range from the 2.87% (sample 0.06 WAY) to the max 3.09% (sample 0.12 WAY) 10 Fat Total proteins Lactose Ash Total solids 0.02WAY 0.06WAY Components (g/100g) Milk KY 0.02AY 0.06AY 0.12AY 0.12WAY Probiotic yoghurt samples Fig 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 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 days In all other probiotic yoghurt samples the syneresis was lower by mL, and in yoghurt sample 0.02 AY by mL The water holding capacity of control yoghurt and samples produced without activation significantly decreased during days of storage During storage for days, the acidity of produced probiotic yoghurt samples decreased, and the measured values were from 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, 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 (%) Physico-chemical characteristics 60 50 40 30 20 10 KY 0.02AY 0.06AY 0.12AY 0.02WAY 0.06WAY 0.12WAY Yoghurt samples Fig Physico-chemical characteristics of probiotic yoghurt with TGase Total sensory scores of probiotic yoghurt variants were in the range from 18.2 (sample 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 significantly the physico-chemical and sensory characteristics of final product Table Physico-chemical properties of the yoghurt with TGase after days of storage Parameters Syneresis (mL) WHC (%) Acidity (qSH) pH KY 31 37 35.6 4.40 0.02 AY 30 55.5 36.4 4.38 Probiotic yoghurt samples 0.06 0.12 0.02 0.06 AY AY WAY WAY 29 30 30 30 56.0 57.0 44 43.5 36.1 36.5 34.6 34.8 4.42 4.43 4.39 4.38 0.12 WAY 30 56.5 35.6 4.37 49 K 20 18 16 14 0,12BA 0,02A 12 10 0,06BA 0,06A 0,02BA 0,12A Appearance Odour Colour Taste Consistency Total points Fig Sensory analysis of the probiotic yoghurt with TGase CONCLUSION Probiotic yoghurt variants were produced from skimmed milk (0.1% fat) with the different 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 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, produced 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 decrease of the syneresis and improved water holding capacity during storage Also, it improved 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 REFERENCES 50 Motoki, M and K Seguro: Transglutaminase and its use for food processing Trends Food Sci Technol (1998) 204-210 Kuraishi, C., K Yamazaki and Y Susa: Transglutaminase: its utilization in the food industry Food Rev Int 17 (2001) 221-246 3 10 11 12 13 14 15 16 Dickinson, E.: Enzymic crosslinking as a tool for food colloid rheology control and interfacial stabilization Trends Food Sci Technol (1997) 334-339 Ikura, K., M Yoshikawa, R Sasaki and H Chiba: Use of transglutaminase Reversible blocking of amino groups in substrate proteins for a high yield of specific products J Agric Food Chem 48 (1984) 2347-2354 Nonaka, M., H Tanaka, A Okiyama, M Motoki , H Ando and K Umeda: Polymerisation of several proteins by Ca2+-independent transglutaminase derived from micro-organism Agric Biol Chem 53 (1989) 2619-2623 Traoré, F and J C Meunier: Cross-linking activity of placental FXIIIa on whey proteins and casein J Agric Food Chem 40 (1992) 399-402 Farnsworth J P., J Li, G M Hendricks and M R Guo: Effects of transglutaminase treatment on functional properties and probiotic culture survivability of goat milk yoghurt Small Rumin Res 65 (2006) 113-121 Milanoviü S., M Cariü, M Iliỵiỹ, K Durakoviỹ and A Todoroviỹ: Effect of transglutaminase and probiotic starter on yoghurt quality, International Congress on Bioprocesses in Food Industries, Univesity of Patras, Rio-Patras, Greece, 18-21 June 2006, Congress Proceedings, p 84 Menrad, K.: Market and marketing of functional food in Europe J Food Eng 56 (2003) 181-188 Lourens-Hattingh, A and C B Viljeon: Yoghurt as probiotic carrier food Int Dairy J 11 (2001) 1-17 Schrezenmeir, J and M de Vrese: Probiotics, prebiotics and synbiotics – Approaching a definition American J Clinical Nutr 73 (2001) 361S-364S Shah, N P.: Some beneficial effects of probiotic bacteria Biosci Microflora 19 (2000) 99-106 Cariü M Milanoviü S Vucelja D.: Standard , Methods of Analysing Milk and Dairy Products Faculty of Technology Novi Sad (2000) p.204 Atamer, M., Cariü, M., Milanoviü, S., Gavariü, D „Quality of the yoghurt produced from UF milk“, Zbornik Matice srpske za prirodne nauke, Matica srpska Novi Sad, No 91, (1996) 27-35 (in Serbian) Guzman-Gonzalez M Morais F Ramos M and Amigo L.: Influence of skimmed milk concentrate replacement by dry dairy products in a low fat set-type yoghurt model system: I: Use of whey protein concentrates Milk protein concentrates and skimmmed milk powder Journal of the Science of Food and Agriculture, 79, (1999) 1117-1122 Regulations on quality and other demand for milk and dairy products, composite dairy products and starter culture Yugoslav Official Register 26/2002 (in Serbian) ɎɂɁɂɑɄɈ-ɏȿɆɂȳɋɄȿ ɄȺɊȺɄɌȿɊɂɋɌɂɄȿ ɉɊɈȻɂɈɌɋɄɈȽ ȳɈȽɍɊɌȺ ɉɊɈɂɁȼȿȾȿɇɈȽ ɍɁ ɉɊɂɆȿɇɍ ɌɊȺɇɋȽɅɍɌȺɆɂɇȺɁȿ ɋɩɚɫɟɧɢʁɚ Ⱦ Ɇɢɥɚɧɨɜɢʄ, Ɇɚɪɢʁɚɧɚ ȭ ɐɚɪɢʄ, Ɇɢɪʁɚɧɚ ɋ ȭɭɪɢʄ, Ɇɢɪɟɥɚ Ⱦ ɂɥɢɱɢʄ ɢ Ʉɚɬɚɪɢɧɚ Ƚ Ⱦɭɪɚɤɨɜɢʄ ɍ ɪɚɞɭ ʁɟ ɢɫɩɢɬɚɧ ɭɬɢɰɚʁ ɪɚɡɥɢɱɢɬɢɯ ɤɨɧɰɟɧɬɪɚɰɢʁɚ ɬɪɚɧɫɝɥɭɬɚɦɢɧɚɡɟ (0,02%, 0,04% ɢ 0,08%) ɧɚ ɮɢɡɢɱɤɨ-ɯɟɦɢʁɫɤɟ ɨɫɨɛɢɧɟ ɩɪɨɛɢɨɬɫɤɨɝ ʁɨɝɭɪɬɚ ɩɪɨɢɡɜɟɞɟɧɨɝ ɭɡ 51 ɩɪɢɦɟɧɭ ɬɪɚɧɫɝɥɭɬɚɦɢɧɚɡɟ ɍ ɥɚɛɨɪɚɬɨɪɢʁɫɤɢɦ ɭɫɥɨɜɢɦɚ ɩɪɨɢɡɜɟɞɟɧɟ ɫɭ ɞɜɟ ɫɟɪɢʁɟ ɭɡɨɪɚɤɚ ʁɨɝɭɪɬɚ ɢɡ ɨɛɪɚɧɨɝ ɦɥɟɤɚ ɫɚ 0,1% ɦɥɟɱɧɟ ɦɚɫɬɢ ɉɪɜɚ ɫɟɪɢʁɚ ʁɟ ɩɪɨɢɡɜɟɞɟɧa ɭɡ ɚɤɬɢɜɚɰɢʁɭ ɬɪɚɧɫɝɥɭɬɚɦɢɧɚɡɟ ɧɚ 40°C, ɫɚɬɚ, ɚ ɞɪɭɝɚ ɫɟɪɢʁɚ ɩɪɨɛɢɨɬɫɤɨɝ ʁɨɝɭɪɬɚ ɩɪɨɢɡɜɟɞɟɧɚ ʁɟ ɛɟɡ ɩɪɟɬɯɨɞɧɟ ɚɤɬɢɜɚɰɢʁɟ ɟɧɡɢɦɚ Ɂɚ ɩɪɨɢɡɜɨɞʃɭ ɨɛɟ ɫɟɪɢʁɟ ɤɨɪɢɲʄɟɧɚ ʁɟ ɩɪɨɛɢɨɬɫɤɚ ɫɬɚɪɬɟɪ ɤɭɥɬɭɪɚ ABT-4 (Chr Hansen A/S, Ⱦɚɧɫɤɚ) ɏɟɦɢʁɫɤɢ ɫɚɫɬɚɜ, ɮɢɡɢɱɤɨ-ɯɟɦɢʁɫɤɚ ɫɜɨʁɫɬɜɚ (ɫɩɨɫɨɛɧɨɫɬ ɜɟɡɢɜɚʃɚ ɜɨɞɟ, ɫɢɧɟɪɟɡɢɫ ɢ pH ɜɪɟɞɧɨɫɬ) ɢ ɫɟɧɡɨɪɧɢ ɤɜɚɥɢɬɟɬ ɩɪɨɢɡɜɟɞɟɧɢɯ ɜɚɪɢʁɚɧɬɢ ɩɪɨɛɢɨɬɫɤɨɝ ʁɨɝɭɪɬɚ ɢɫɩɢɬɚɧɢ ɫɭ ɧɚɤɨɧ ɩɪɨɢɡɜɨɞʃɟ ɢ ɬɨɤɨɦ ɞɚɧɚ ɫɤɥɚɞɢɲɬɟʃɚ Ƚɟɧɟɪɚɥɧɨ ɩɨɫɦɚɬɪɚɧɨ, ɞɨɞɚɬɚɤ ɬɪɚɧɫɝɥɭɬɚɦɢɧɚɡɟ (ɞɢɪɟɤɧɨ ɢɥɢ ɭɡ ɩɪɢɦɟɧɭ ɚɤɬɢɜɚɰɢʁɟ) ɡɧɚɱɚʁɧɨ ɩɨɛɢʂɲɚɜɚ ɮɢɡɢɱɤɨ-ɯɟɦɢʁɫɤɟ ɤɚɪɚɤɬɟɪɢɫɬɢɤɟ ɩɪɨɛɢɨɬɫɤɨɝ ʁɨɝɭɪɬɚ Ⱥɤɬɢɜɚɰɢʁɚ ɬɪɚɧɫɝɥɭɬɚɦɢɧɚɡɟ ɭ ɩɪɨɢɡɜɨɞʃɢ ɩɪɨɛɢɨɬɫɤɨɝ ʁɨɝɭɪɬɚ ɞɨɩɪɢɧɨɫɢ ɛɨʂɨʁ ɫɩɨɫɨɛɧɨɫɬɢ ɜɟɡɢɜɚʃɚ ɜɨɞɟ ɧɚɤɨɧ ɩɪɨɢɡɜɨɞʃɟ ɢ ɬɨɤɨɦ ɫɤɥɚɞɢɲɬɟʃɚ Received 25 June 2007 Accepted September 2007 52 ... 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 Since the fat content of. .. the probiotic yoghurt production improved significantly the physico- chemical and sensory characteristics of final product Table Physico- chemical properties of the yoghurt with TGase after days of. .. 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