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Effect of transglutaminase-treated milk powders on the properties of skim milk yoghurt
International Dairy Journal 21 (2011) 628e635
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Effect of transglutaminase-treated milk powders on the properties of skim milk yoghurt C. Guyot*, U. Kulozik Chair for Food Process Engineering and Dairy Technology, Research Centre for Nutrition and Food Science (ZIEL) e Section Technology, Technische Universität München, Weihenstephaner Berg 1, 85354 Freising-Weihenstephan, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 June 2010 Received in revised form 22 September 2010 Accepted 5 October 2010
A novel method of transglutaminase (TGase) treatment for skim milk yoghurt production was investigated. In contrast to previous studies, TGase pre-treated skim milk powder (SMP) was used as protein fortification for yoghurt making, instead of treating the entire yoghurt milk. When the TGase concentration for powder production was increased from 0 to 10 U g1 protein, the viscosity of stirred skim milk yoghurt produced with addition of TGase-treated SMP increased from 247 to 453 mPas and the serum loss assessed using a centrifugation method decreased from 57.1% to 52.6%. Furthermore, by using enzyme-modified SMP, only half of the protein addition was required to obtain an equivalent viscosity compared to the control. The study showed that crosslinking the caseins by TGase only in the added SMP yields the desired positive effects while allowing for a complete elimination of the residual enzyme activity. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction The enzymatic crosslinking of proteins by means of microbial transglutaminase (TGase) strengthens protein-based food structures at the molecular level and positively affects properties such as serum holding capacity and gel firmness. The enzyme-catalysed reaction leads to the formation of intra- and inter-molecular crosslinks between the protein-bonded amino acids glutamine and lysine (Ikura, Yoshikawa, Sasaki, & Chiba, 1981; Kashiwagi et al., 2002; Nielsen, 1995; Seguro, Kumazawa, Kuraishi, Sakamoto, & Motoki, 1996a). Caseins, the major milk protein fraction, represent an excellent substrate for the TGase reaction (Christensen, Sorensen, Hojrup, Petersen, & Rasmussen, 1996; De Jong & Koppelman, 2002; Lorenzen, Schlimme, & Roos, 1998). In contrast, whey proteins in their native structure are hardly suitable for crosslinking due to their globular conformation and the limited accessibility of reactive amino acids (Faergemand, Otte, & Qvist, 1997; Han & Damodaran, 1996; Ju, Otte, Zakora, & Qvist, 1997; Sharma, Lorenzen, & Qvist, 2001). Therefore, TGase treatment is predominantly applied to casein-based milk products (Ikura, Goto, Yoshikawa, Sasaki, & Chiba, 1984) while whey proteins play only a minor role with regard to TGase-induced crosslinking in milk.
* Corresponding author. Tel.: þ49 816171 5317; fax: þ49 816171 4384. E-mail address: [email protected] (C. Guyot). 0958-6946/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2010.10.010
In many dairy products, the structure is mainly influenced by milk proteins. In the case of yoghurt, acid-induced gelation of caseins during fermentation leads to structure formation. An enzymatic crosslinking of caseins results in specific changes in yoghurt microstructure like reduced mesh size and a more homogeneous protein network (Lorenzen, Neve, Mautner, & Schlimme, 2002; Schorsch, Carrie, & Norton, 2000). In recent years, it was found for TGase-treated skim milk yoghurt that higher gel firmness, reduced syneresis and an improved mouth feel could be obtained (Faergemand & Qvist, 1997; Jaros, Heidig, & Rohm, 2007; Schorsch et al., 2000). Therefore, the application of TGase for yoghurt processing is of great interest to minimise frequent defects in yoghurt products, namely low viscosity and high level of syneresis (Amatayakul, Sherkat, & Shah, 2006). Generally, direct application of TGase treatment to yoghurt production can be accomplished by two different ways: incubation of the yoghurt milk prior to fermentation (Lauber, Henle, & Klostermeyer, 2000; Lorenzen et al., 2002; Ozer, Kirmaci, Oztekin, Hayaloglu, & Atamer, 2007) or simultaneous addition of TGase and the starter culture (Bönisch, Huss, Lauber, & Kulozik, 2007a; Yüksel & Erdem, 2010). However, both methods were shown to have undesirable side effects. The TGase reaction prior to fermentation requires additional processing time, but offers more flexibility regarding the incubation conditions independent of the fermentation conditions. Further, a thermal treatment to inactivate the enzyme after incubation can be applied prior to fermentation without affecting the fermentation of the lactic acid bacteria.
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In contrast, the incubation and simultaneous microbial acidification does not require additional processing time but a thermal enzyme inactivation is not possible. Due to the acidification by lactic acid bacteria the pH drops from initially pH 6.8 to 4.6, and the optimum pH range for TGase between pH 6 and 7 (Ando et al., 1989) is left soon after fermentation starts. The crosslinking effect may therefore be less pronounced. Overall, the simultaneous application of TGase and fermentation may cause a low remaining enzyme activity in the final product. Even at the low pH values from 4.2 to 4.6 after acidification outside the pH range optimal for the enzyme lead to negative structural changes during storage time. Lorenzen et al. (2002) described a strong increase in the gel strength during the first two weeks of storage of set-style yoghurt produced with simultaneous addition of TGase and the starter culture. Similar observations were made by Yüksel and Erdem (2010) who found an increase in gel hardness during storage for TGase-treated yoghurt samples without prior thermal TGase inactivation. For yoghurt samples with inactivated TGase, the hardness remained nearly constant during storage. According to standard practice, the protein content of yoghurt milk is increased from the initial 3.4% (w/w) to 4.0e5.0% (w/w) protein (Remeuf, Mohammed, Sodini, & Tissier, 2003; Sodini, Lucas, Oliveira, Remeuf, & Corrieu, 2002). Skim milk powder, whey protein isolates, whey protein concentrates or sodium caseinate can be used to fortify yoghurt milk. The kind and amount of protein used have a significant impact on the resulting yoghurt structure. As an alternative to the application of TGase to the liquid yoghurt milk, pre-treated skim milk powder (SMP) for yoghurt production has not been studied so far. By using enzymatically modified powder as the protein fortifier, additional processing time prior to fermentation is not required. Furthermore, it is possible to thermally inactivate the enzyme completely. Nevertheless, the influence of TGasetreated milk powder on yoghurt properties is barely investigated. To the best of our knowledge, no study has so far reported on the application of TGase pre-treated SMP for yoghurt processing. The key question is whether the TGase treatment of only a part of the proteins finally leads to the desired fortification effect compared to yoghurt produced by treating all proteins contained in yoghurt milk. Hence, the purpose of the present study was to investigate the impact of TGase-modified SMPs on the properties of fortified milk yoghurt. The objectives were to relate the key structural properties of yoghurt, namely flow behaviour and serum binding capacity, with respect to the powder used and, furthermore, to assess the optimal process parameters for the application of TGase-treated SMP to improve yoghurt quality.
(U) per g powder. One unit of enzyme was defined as the amount of enzyme that released 1 mmol of hydroxamic acid in 1 min at 37 C according to the hydroxamate test by Folk and Cole (1965). 2.2.2. Milk components and yoghurt starter culture Pasteurised (70 C, 30 s) skim milk with a total protein content of about 3.4% (w/w) was obtained from a local dairy. Spray-dried skim milk powder was purchased from Bayerische Milchindustrie eG (Landshut, Germany). The yoghurt starter culture ABT-21Ò was obtained from Chr. Hansen GmbH (Nienburg/Weser, Germany) in frozen form and is composed of Lactobacillus acidophilus, Streptococcus thermophilus and Bifidobacterium sp. According to the manufacturer’s data, the culture is suitable for the production of stirred yoghurt with mild flavour and low post-acidification. For the starter culture application to the yoghurt process, a pre-culture was prepared by mixing 10% (w/w) frozen starter culture with skim milk and stored for 24 h at 10 C. 2.2.3. Processing of TGase-treated milk protein powder TGase-treated SMP was produced according to the flow chart in Fig. 1. Skim milk concentrate with a total protein content of 8.0% (w/w) was obtained by mixing pasteurised skim milk with SMP. After rehydration of dissolved SMP for 24 h at 4 C, the skim milk concentrate was heat treated in a tubular heat exchanger at 85 C for 2 min to inactivate the indigenous TGase inhibitor contained in low heated milk (Bönisch, Tolkach, & Kulozik, 2006; De Jong, Wijngaards, & Koppelman, 2003). Then the milk concentrate was tempered to 40 C and the enzyme was added. TGase concentrations used were 0.5, 1.0, 3.0, 10.0 U g1 milk protein. In addition, a control without TGase was produced using the same conditions. After 2 h, the enzyme reaction was terminated by a thermal treatment (85 C, 2 min) for complete TGase inactivation (Seguro, Nio, & Motoki, 1996b). After cooling to 20 C, the TGase-incubated milk concentrate was spray dried using a Niro Atomizer (Copenhagen, Denmark). The
2. Material and methods 2.1. Chemicals CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate), sodium phosphate and hydrochloride acid (37%) were obtained from SigmaeAldrich (Steinheim, Germany); urea, 1,4-dithiothreitol (DTT), sodium chloride and sodium hydroxide from Merck (Darmstadt, Germany). All chemicals used were of analytical grade. 2.2. Preparation of materials 2.2.1. Transglutaminase The microbial transglutaminase (TGase, EC. 2.3.2.13) used in this study was the preparation ActivaÒ MP and was a gift from Ajinomoto Foods Deutschland GmbH (Hamburg, Germany). According to product specification the enzyme preparation consists of TGase, lactose and maltodextrin and has a declared activity of 100 Units
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Fig. 1. Processing of TGase-treated skim milk powder.
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product inlet and outlet temperature was 180 C and 80 C, respectively. The TGase-treated milk powder was stored until usage at 10 C in closed plastic containers with a screw cap and a capacity of 50 L under dry conditions. 2.2.4. Yoghurt processing with TGase-treated milk powder Stirred skim milk yoghurt was prepared according to Bönisch et al. (2007a) with slight changes, as can be seen in Fig. 2. To determine the effect of protein fortification of skim milk with different amounts of TGase-treated milk powder, the treated powder was used to increase the original total protein content (w/w) of skim milk from initially 3.4% to 3.9%, 4.4%, 4.9% and 5.4%. For each yoghurt trial, 10 kg yoghurt milk was produced by adding 149.7 g, 299.4 g, 449.1 g or 598.8 g untreated or TGase-treated SMP to the corresponding amounts of pasteurised skim milk. Subsequently, the obtained yoghurt milk was stored for 12 h at 4 C for rehydration of all powder particles. Afterwards, the yoghurt milk was heat treated in a tubular heat exchanger at 95 C for 3 min. The chosen conditions were appropriate for low-fat yoghurt production (Dannenberg & Kessler, 1988). The yoghurt milk was tempered to 42 C in a water bath and inoculated with 0.2% (v/v) yoghurt pre-culture. The fermentation process was accomplished at 42 C in a laboratory heating cabinet and the decrease of pH as a function of time was monitored using an S40 SevenMulti pH meter (Mettler-Toledo GmbH, Giessen, Germany). After reaching pH 4.6, the yoghurt gel was pumped immediately with a flexible-tube pump (Verder 2005/1, Düsseldorf, Germany) with a flow rate of 80 L h1 through a fine sieve with a mesh size of 220 mm and a tubular cooler equipped with a static mixer (d ¼ 10 mm, l ¼ 2 m). The temperature at the end of the cooler was 20 1 C. The stirred yoghurt was collected and cooled to 4 C. After cooling, the stirred yoghurt samples were stored in 250 mL yoghurt jars at 4 C until analysis. For comparison of yoghurt made by protein fortification with TGase-treated SMP with yoghurt from directly incubated yoghurt milk, 10 kg of yoghurt with simultaneous addition of TGase and
starter culture was produced according to Bönisch et al. (2007a). Pasteurised skim milk was fortified with untreated SMP to a final protein content of 4.4% (w/w). Simultaneous with the yoghurt starter culture, 0.6 U TGase g1 protein was added to the tempered yoghurt milk. The enzymatic incubation and microbial fermentation occurred at the same time. All other process conditions are identical to the standard yoghurt production steps for yoghurt with application of enzyme-treated SMP (Fig. 2, optional proceeding). All yoghurt production processes were carried out in triplicate.
2.3. Analytical techniques 2.3.1. Size exclusion chromatography The degree of protein polymerisation was determined by using a size exclusion chromatography method (SEC) according to Lauber et al. (2000) and Bönisch, Lauber, and Kulozik (2004). An ÄKTA FPLC system provided with a P900 pump, a variable wavelength UV detector and a SuperdexÔ 200 10/300 GL gel filtration column (Amersham Biosciences, Freiburg, Germany) were used. The column was eluted at room temperature at a flow rate of 0.5 mL min1 with elution buffer composed of 6 M urea, 0.1 M sodium chloride, 0.1 M sodium phosphate and 0.1% (w/v) CHAPS. The TGase-treated and untreated SMP were dissolved at a protein concentration of 0.3% in elution buffer containing 1% (w/v) DTT as a reducing agent. To determine the resulting degree of protein polymerisation in the produced yoghurt samples, an aliquot of yoghurt was lyophilised by freeze-drying (DELTA 1-24, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany). The dried yoghurt was dissolved in the same way as the SMP. The samples were stored for 12 h at 4 C to afford a complete dissociation of casein aggregates and reduction of disulphide bonds. After membrane filtration (Chromafil Xtra RC-45/25, regenerated cellulose, MachereyeNagel GmbH & Co. KG, Düren, Germany), 50 mL of samples were injected and detection clocked to 280 nm. Data evaluation and peak integration were done by using the Unicorn 4.11 software (Amersham Biosciences, Freiburg, Germany). The degree of enzyme-induced protein polymerisation DDP was defined as the amount of crosslinked caseins related to the total amount of caseins in the sample (Bönisch et al., 2004). DDP was calculated according to equations (1) and (2):
Acrosslinked caseins 100% Atotal caseins Aoligomers;trimers;dimers ¼ 100% and Aoligomers;trimers;dimers;monomers
DP ¼
DDP ¼ DPwith TGase DPwithout TGase
(1)
(2)
where DP is the degree of polymerisation including thermal and enzyme-induced protein polymerisation and Ai is the area between the zero line and the absorption curve of fraction i.
Fig. 2. Production of stirred skim milk yoghurt using TGase-treated milk powder or milk powder without TGase treatment (control) as protein fortification (0.5e2.0% w/w).
2.3.2. Flow behaviour of yoghurt samples The rheological properties of stirred yoghurt samples were determined using an AR1000 N rheometer (TA instruments, New Castle, Delaware, USA) equipped with a coaxial cylinder geometry (radius 14 mm) and a sample cup (radius 15 mm). Before starting the measurement, both sample and sample cup were tempered to 10 C. A five-stage measuring profile consisting of pre-shearing (shear rate of 50 s1 for 30 s), equilibration step (2 min), upward ramp (shear rate from 0.1 to 100 s1 in 1 min), holding phase (shear rate of 100 s1 for 1 min) and downward ramp (shear rate from 100 to 0.1 s1 in 1 min) was applied and shear stress and apparent viscosity were detected.
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Stirred skim milk yoghurt exhibits a yield stress and shows a shear thinning flow behaviour during shear treatment that leads to irreversible structural changes. The shear rate versus shear stress curves of the downward ramp were adjusted to the HerscheleBulkley model as one of the most appropriate models to describe the steady-shear rheological behaviour of yoghurt (Fangary, Barigou, & Seville, 1999):
s g_ ¼ s0 þ Kg_ n
(3)
where s is the shear stress, s0 the yield stress, K the consistency index, g_ the shear rate and n the flow behaviour index. Using equation (3) the apparent viscosity happ is defined as
happ g_ ¼
s g_ s ¼ 0 þ K$g_ n1 g_ g_
(4)
For each trial, all rheological measurements were done in duplicate. 2.3.3. Serum binding capacity The serum binding capacity was assessed as the fraction of serum after centrifugation. Some 60 g yoghurt was filled in centrifugation glasses and centrifuged at 1500 g, 10 C for 20 min (Heraeus Multifuge 1 S-R, Thermo Fisher Scientific, Waltham, Massachusetts, USA). The amount of supernatant yoghurt serum was recorded and the relation between the amounts of serum mSerum and original yoghurt sample mYoghurt gives the serum loss SL in percent:
SL ¼
mSerum 100% mYoghurt
(5)
2.3.4. Water content The water content of SMP was determined using a gravimetric method according to IDF Standard 26A: 1993 (IDF, 1993). Some 3 g powder was dried in a T5028 oven (Heraeus GmbH, Hanau, Germany) at 102.0 C 1.0 C under atmospheric pressure for 2 h. To prove mass constancy, additional drying steps were performed until the differences in mass of powder sample were lower than 1.0 mg. The water content was calculated as the amount of weight loss after drying related to the initial amount of SMP in percent. All measurements were done in triplicate.
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production. Fig. 3 shows a representative chromatogram of SMP produced either without TGase treatment or with an enzyme addition of 1.0 and 10.0 U g1 protein, respectively. An increased TGase concentration led to an increasing amount of highly crosslinked caseins. The small oligomer peak of the control sample is known to result from the thermal treatment during heating and spray-drying of SMP (Singh, 2007) and was considered for calculation of the enzyme-induced crosslinking degree. The calculated DDPs are shown in Table 1. Depending on the enzyme concentration, the average degrees of protein polymerisation varied from 0 to 33.9%. The enzymatic crosslinking reaction leads to a molecular modification of milk proteins and variably high crosslinked milk powders can be obtained. Protein and water contents remained unaffected by the enzyme treatment (36.8% 0.3% protein, 3.8% 0.3% water) with no significant changes following enzyme treatment. Therefore, differences in the yoghurts produced by addition of varying TGase-modified powders can be linked to the respective degree of polymerisation.
3.2. Influence of TGase pre-treated skim milk powders as protein fortification on the properties of stirred yoghurt 3.2.1. Effect of protein fortification with differently high crosslinked skim milk powders In the present study, it was found that yoghurt with addition of pre-treated SMP showed all the improved properties of yoghurt known for TGase-incubated milk. The viscosity and serum loss of the produced stirred skim milk yoghurts are depicted in Fig. 4. While the control sample with untreated SMP (DDP ¼ 0%) had a low viscosity of 247 mPas and a high amount of centrifugally forced serum loss of 57.1%, significantly higher viscosities and reduced serum loss were obtained by using TGase-treated SMP as protein fortifier. Especially the addition of high enzyme-treated SMP obtained by using 3.0 or 10.0 U TGase g1 protein for powder production (DDP is 23.6% or 33.9%, respectively) caused a strong increase in yoghurt viscosity to 376 or 453 mPas and a decrease in serum loss to 54.0% or 52.6%. In contrast to yoghurt produced with direct incubation of yoghurt milk, in these yoghurt samples only a small fraction of milk proteins is enzymatically modified. Nevertheless, the addition of
2.3.5. Total protein content The total protein content of TGase-treated milk powder and yoghurt samples was determined by the nitrogen content using a Leco FP 528 system (Leco Instrumente GmbH, Moenchengladbach, Germany) based on the Dumas method (Wiles, Gray, & Kissling, 1998). Total protein content was calculated by multiplying the total nitrogen content by 6.38. All measurements are carried out at least in duplicate. 2.3.6. Statistical analysis Statistical analysis was carried out using ANOVA. Differences were considered significant at P < 0.05. All statistic analyses were performed using Statgraphics software (Statistical Graphics Corporation, Rockville, MD, USA). 3. Results and discussion 3.1. Influence of enzymatic protein crosslinking on the properties of skim milk powder To investigate the influence of enzyme-catalysed protein polymerisation on the properties of SMP, different enzyme concentrations (0e10.0 U g1 protein) were used for powder
Fig. 3. Chromatographic pattern of skim milk powders produced without enzyme addition (eeee) and with TGase incubation at 1.0 (e e e) or 10.0 (e$$e$$) U g1 protein at 40 C for 2 h.
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Table 1 TGase-induced degrees of protein polymerisation DDP of differently TGasetreated skim milk powders.
DDP (%)a
TGase treatment
Without TGase, 40 C, 2 h (control) 0.5 U g1 protein, 40 C, 2 h 1.0 U g1 protein, 40 C, 2 h 3.0 U g1 protein, 40 C, 2 h 10.0 U g1 protein, 40 C, 2 h
0.0 6.7 12.5 23.6 33.9
1.4 1.3 1.7 1.7 4.6
a b c d e
a Results are given as average confidence interval (a ¼ 0.05); different lower case letters indicate significant differences with a 95% confidence level.
TGase pre-treated SMP leads to a significant structural enhancement of the yoghurt sample. In Fig. 5 the viscosity of stirred skim milk yoghurt with addition of TGase-treated SMP and direct enzyme incubation of yoghurt milk dependent on the resulting DDP in the yoghurt sample is shown. For skim milk yoghurt produced with direct implementation of TGase treatment, higher enzyme concentrations generally lead to firmer gel networks, improved viscosities and reduced amounts of serum loss during storage time (Bönisch, Huss, Weitl, & Kulozik, 2007c). The breaking strength of set-style yoghurt from TGase-incubated milk was increased with longer incubation times (Lauber et al., 2000). Similar observations were made for stirred skim milk yoghurt fortified with TGase-treated SMP. Bönisch et al. (2007a) found that for simultaneous addition of TGase and starter culture a DDP value
Fig. 4. Apparent viscosity (a) and serum loss (b) of stirred skim milk yoghurt produced with a protein fortification of þ1% (w/w) by means of differently high TGase-treated skim milk powders. The graphs show means confidence intervals (a ¼ 0.05, n ¼ 6).
Fig. 5. Viscosity of stirred skim milk yoghurt dependent on the resulting degree of polymerisation in the yoghurt sample for yoghurt produced with direct incubation of yoghurt milk (-) or addition of TGase-treated SMP (A). The graph shows means confidence intervals (a ¼ 0.05, n ¼ 6).
of about 15% is necessary to obtain a structure enhancement of skim milk yoghurt. Due to the direct incorporation of the enzyme in the yoghurt milk, all proteins contained in the yoghurt milk are available for the crosslinking reaction. Therefore, the resulting degree of polymerisation in the yoghurt sample has a high value of about 13.5%. For the addition of TGase pre-treated SMP, the major part of milk proteins from the pasteurised milk remains enzymatically untreated and only the proteins used for protein fortification are modified. Thus, the resulting degrees of protein polymerisation of the yoghurt samples depend on the fraction and kind of TGase pretreated SMP. For yoghurt with a protein fortification of 1.0% (w/w), the resulting degrees of polymerisation in the yoghurt samples were 1.5%, 2.8%, 5.4% or 7.7% for an enzyme concentration of 0.5, 1.0, 3.0 or 10.0 U g1 protein, respectively. Although the resulting degree of polymerisation in yoghurt is much lower for the addition of TGase-treated SMP with a high enzyme concentration of 10.0 U g1 protein compared to the yoghurt produced with direct TGase incubation, the viscosities of the yoghurt samples were similar, as can be seen in Fig. 5. The stronger impact of the resulting degree of polymerisation on the viscosity can be explained by the different protein compositions of yoghurt produced either by addition of TGase-treated SMP or with direct incubation. Fig. 6 shows the relative amounts of protein monomers, dimers, trimers and oligomers of yoghurt samples with direct incorporation of TGase to yoghurt milk and with protein fortification by TGase pre-treated SMP (3.0 U g1 protein). It can be noted that the direct TGase incubation leads to the formation of high amounts of casein dimers and trimers, but low amounts of oligomers. In the case of addition of high enzyme-treated SMP, the protein composition was different. Yoghurt produced by addition of TGase-treated SMP contained only small fractions of dimers and trimers but higher amounts of protein oligomers were obtained. Since the addition of intensively crosslinked milk proteins to the yoghurt milk significantly increases the oligomer fraction, the amounts of monomers, dimers and trimers remain close to the value for untreated yoghurt. It can be assumed that the higher fraction of casein oligomers strongly affects especially the yoghurt viscosity. Therefore, comparable rheological properties can be obtained with a lower value of the resulting degree of
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Fig. 6. Size exclusion chromatography pattern of yoghurt produced with direct incubation of yoghurt milk (e e e) or addition of 1.0% protein by TGase-treated SMP (eee; 3.0 U g1 protein).
polymerisation in the yoghurt sample by using TGase-treated SMP for protein fortification. 3.2.2. Influence of amount of protein fortification One of the main advantages of direct TGase treatment of yoghurt milk is the possibility to produce skim milk yoghurt with a reduced dry matter content but unchanged rheological and sensory properties (Bönisch, Lauber, & Kulozik, 2007b). Therefore, the influence of different amounts of powder addition on yoghurt structure was investigated. To fortify the protein content with 0.5e2.0% protein, TGase-treated and the untreated SMP were used. As TGase-treated powder, SMP with an enzyme treatment of 3.0 U g1 protein (DDP ¼ 23.6%) was chosen. According to Bönisch et al. (2007a), for direct TGase incorporation an enzyme concentration of 0.6 U g1 protein which leads to a DDP of 22% after fermentation is suitable for crosslinking all the proteins contained in yoghurt milk. Since for application of TGase-treated milk powder only a small fraction of proteins in the yoghurt milk is modified, the total amount of TGase preparation used is comparable with the amount applied for direct enzyme incorporation. For TGase treatment with 0.6 U g1 protein of the whole yoghurt milk containing 4.4% protein, an enzyme addition of 26.4 U kg1 yoghurt milk is necessary. In contrast to that, the relative amounts of TGase used for yoghurt produced with addition of TGase-treated SMP depend on the protein fortification. For the yoghurt fortified with 3.0 U TGase g1 protein treated SMP, the calculated enzyme concentrations required for crosslinking of the SMP added to yoghurt milk is 30.0 U kg1 yoghurt milk for a protein fortification of 1.0%. Due to the similarity, the TGase-treated SMP produced by the use of an enzyme concentration of 3.0 U g1 protein with an average DDP value of 23.6% was used for subsequent investigations. Fig. 7 shows the influence of the two different powders on yoghurt flow behaviour. For each level of protein fortification, the viscosities of yoghurt with addition of enzyme-treated milk powder was higher compared to yoghurt fortified with untreated SMP. Further, it was demonstrated that with the use of TGasetreated SMP, the same level of viscosity was achieved with less SMP addition to the yoghurt milk. While a protein fortification of about 1.6% is normally necessary for traditionally produced skim milk yoghurt without TGase treatment (Sodini et al., 2002), a protein fortification of only 1.0% is sufficient for skim milk yoghurt with
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Fig. 7. Viscosity of stirred skim milk yoghurt produced without fortification (;) or with addition of different amounts of TGase pre-treated (:; 3.0 U g1 protein, DDP of 23.6%) or untreated (A) skim milk powder to the yoghurt milk after 1-week storage at 4 C. The graphs show means confidence intervals (a ¼ 0.05, n ¼ 6).
addition of TGase-treated SMP (3.0 U g1 protein) to achieve equivalent flow behaviour, provided that a target viscosity of 200e300 mPas would be aimed at. Thus, yoghurt with comparable properties in respect of structural behaviour can be produced with a reduced amount of milk powder added to the yoghurt milk. 3.2.3. Comparison of structure stability of skim milk yoghurt with different TGase treatments during storage In the present study, it was clearly demonstrated that a protein fortification by means of TGase pre-treated SMP has a positive impact on the resulting yoghurt properties. By using high TGasetreated SMP, similar effects like increased viscosity, reduced syneresis and the option to reduce the protein and dry matter content without negative structural changes compared to the direct TGase incubation of yoghurt milk, were observed. Nevertheless, an important aim was to realise storage stability, i.e., avoiding structural defects showing up on the course of shelf life, by using the TGase-treated SMP in contrast to the direct incorporation of the enzyme in yoghurt production. Storage stability regarding structural properties is essential for yoghurt products. For direct implementation of TGase treatment in the process of yoghurt production, negative changes in yoghurt structure such as coarseness or lumpiness were often observed (Bönisch et al., 2007a; Demirkaya & Ceylan, 2009; Lorenzen et al., 2002; Ozer et al., 2007; Yüksel & Erdem, 2010). Generally, rebodying and rearrangements of casein clusters can lead to structural changes of conventionally produced yoghurt during storage time (Renan et al., 2008, 2009). However, the change of rheological properties due to rearrangement of caseins is rather low for conventional yoghurt products. Although rebodying is not completely understood so far, it was clearly demonstrated that active TGase in yoghurt affects the restructuring behaviour, and the resulting viscosity significantly increases during storage time. Hence, the aim is to combine the possibility of reducing the dry matter content of yoghurt by the TGase treatment without loss of structural properties with a constant viscosity during storage. Therefore, it was of major interest whether stirred skim milk yoghurt produced with addition of enzyme-treated SMP shows high storage stability. In Fig. 8 both the viscosity and the resulting
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no alteration of DDPYoghurt was found for the application of TGase pre-treated SMP (Fig. 8b). The comparison between the different enzyme treatments clearly indicates that the application of TGase-treated SMP is well suited for the protein fortification of yoghurt milk. By using the enzymatically modified powder the benefits of the TGase treatment can be combined with high structure stability during the whole storage time. 4. Conclusions Stirred skim milk yoghurts fortified with enzyme-modified SMP have higher viscosities and lower serum loss compared to yoghurt samples produced with addition of untreated SMP. Furthermore, due to complete enzyme inactivation by means of heat treatment and the spray-drying process, the viscosity of the yoghurt samples remained constant during storage time. In particular with regard to skim milk yoghurt, the application of TGase pre-treated SMP seems to be a promising alternative to previous TGase treatments. Improved properties without undesired structural changes during storage can be realised by addition of TGase pre-treated SMP as protein fortifier. Furthermore, with the use of TGase-treated SMP the same level of viscosity was reached with less milk powder addition to the yoghurt milk. Thus, yoghurt with comparable properties in respect of structural behaviour was produced with reduced amount of SMP. In further studies, the influence of different casein to whey protein ratios in the milk powders on the resulting yoghurt properties shall be investigated. Acknowledgement
Fig. 8. Apparent viscosity (a) and degree of polymerisation (b) during storage at 4 C of stirred skim milk yoghurts with different kinds of TGase application of TGase treatment, i. e., without enzyme treatment (A), with 0.6 U TGase g1 protein simultaneous with starter culture (-), and addition of 1% TGase pre-treated skim milk powder (;; 3.0 U g1 protein). The graph shows means confidence intervals (a ¼ 0.05, n ¼ 6).
DDP of stirred skim milk yoghurt with different TGase treatments as a function of storage time are depicted. Yoghurt without enzyme treatment always has a lower viscosity than the yoghurt samples either with direct enzyme incorporation or addition of pre-treated SMP. Although the resulting viscosity of yoghurt produced with addition of crosslinked SMP (3.0 U g1 protein) is lower compared to yoghurt samples with simultaneous addition of enzyme and starter culture. However, the positive effect of the enzyme treatment is still obvious and the addition of TGase-treated SMP led to an increased viscosity of 376 mPas and a reduced serum loss of 54.0%. The lower influence in this case can be explained by the use of SMP with 3.0 U g1 protein to fortify the protein content. Furthermore, it can be seen that for yoghurt produced with direct incubation of yoghurt milk, the viscosity increased significantly from 396 mPas to 456 mPas during storage time. In contrast to that, the viscosity of stirred skim milk yoghurt with TGasetreated SMP remained nearly constant during the whole storage time and no significant change was found. The absence of developing structural defects can be explained by the complete enzyme inactivation due to the heat treatment prior to applying the treated SMP. Since for direct TGase implementation the DDPYoghurt slightly increases from initially 12.8% to 14.8% within three weeks at 4 C,
The authors wish to thank Carina Gerhard and Michael Reitmaier for technical assistance as well as Dr.-Ing. Thomas Heidebach for helpful discussions and critical comments during the preparation of the manuscript. References Amatayakul, T., Sherkat, F., & Shah, N. P. (2006). Physical characteristics of set yoghurt made with altered casein to whey protein ratios and EPS-producing starter cultures at 9 and 14% total solids. Food Hydrocolloids, 20, 314e324. Ando, H., Adachi, M., Umeda, K., Matsuura, A., Nonaka, M., Uchio, R., et al. (1989). Purification and characteristics of a novel transglutaminase derived from microorganisms. Agricultural and Biological Chemistry, 53, 2613e2617. Bönisch, M. P., Huss, M., Lauber, S., & Kulozik, U. (2007a). Yoghurt gel formation by means of enzymatic protein cross-linking during microbial fermentation. Food Hydrocolloids, 21, 585e595. Bönisch, M. P., Lauber, S., & Kulozik, U. (2004). Effect of ultra-high temperature treatment on the enzymatic cross-linking of micellar casein and sodium caseinate by transglutaminase. Journal of Food Science, 69, E398eE404. Bönisch, M. P., Lauber, S., & Kulozik, U. (2007b). Improvement of enzymatic crosslinking of casein micelles with transglutaminase by glutathione addition. International Dairy Journal, 17, 3e11. Bönisch, M. P., Tolkach, A., & Kulozik, U. (2006). Inactivation of an indigenous transglutaminase inhibitor in milk serum by means of UHT-treatment and membrane separation techniques. International Dairy Journal, 16, 669e678. Bönisch, M. P., Huss, M., Weitl, K., & Kulozik, U. (2007c). Transglutaminase crosslinking of milk proteins and impact on yoghurt gel properties. International Dairy Journal, 17, 1360e1371. Christensen, B. M., Sorensen, E., Hojrup, P., Petersen, T. E., & Rasmussen, L. K. (1996). Localization of potential transglutaminase cross-linking sites in bovine caseins. Journal of Agricultural and Food Chemistry, 44, 1943e1947. Dannenberg, F., & Kessler, H. G. (1988). Effect of denaturation of beta-lactoglobulin on texture properties of set-style nonfat yogurt. 2. Firmness and flow properties. Milchwissenschaft e Milk Science International, 43, 700e704. De Jong, G. A. H., & Koppelman, S. J. (2002). Transglutaminase catalyzed reactions: impact on food applications. Journal of Food Science, 67, 2798e2806. De Jong, G. A. H., Wijngaards, G., & Koppelman, S. J. (2003). Transglutaminase inhibitor from milk. Journal of Food Science, 68, 820e825. Demirkaya, A. K., & Ceylan, Z. G. (2009). The effect of microbial transglutaminase on microbiological, chemical, textural and sensory properties of yogurt. Australian Journal of Dairy Technology, 64, 171e176.
C. Guyot, U. Kulozik / International Dairy Journal 21 (2011) 628e635 Faergemand, M., Otte, J., & Qvist, K. B. (1997). Enzymatic cross-linking of whey proteins by a Ca2þ-independent microbial transglutaminase from Streptomyces lydicus. Food Hydrocolloids, 11, 19e25. Faergemand, M., & Qvist, K. B. (1997). Transglutaminase: effect on rheological properties, microstructure and permeability of set style acid skim milk gel. Food Hydrocolloids, 11, 287e292. Fangary, Y. S., Barigou, M., & Seville, J. P. K. (1999). Simulation of yoghurt flow and prediction of its end-of-process properties using rheological measurements. Food and Bioproducts Processing, 77, 33e39. Folk, J. E., & Cole, P. W. (1965). Structural requirements of specific substrates for guinea pig liver transglutaminase. Journal of Biological Chemistry, 240, 2951e2960. Han, X. Q., & Damodaran, S. (1996). Thermodynamic compatibility of substrate proteins affects their cross-linking by transglutaminase. Journal of Agricultural and Food Chemistry, 44, 1211e1217. IDF. (1993). Dried milk and cream e Determination of water content. Brussels, Belgium: International Dairy Federation. Standard 26A. Ikura, K., Goto, M., Yoshikawa, M., Sasaki, R., & Chiba, H. (1984). Use of transglutaminase e reversible blocking of amino-groups in substrate proteins for a high-yield of specific products. Agricultural and Biological Chemistry, 48, 2347e2354. Ikura, K., Yoshikawa, M., Sasaki, R., & Chiba, H. (1981). Incorporation of amino-acids into food proteins by transglutaminase. Agricultural and Biological Chemistry, 45, 2587e2592. Jaros, D., Heidig, C., & Rohm, H. (2007). Enzymatic modification through microbial transglutaminase enhances the viscosity of stirred yogurt. Journal of Texture Studies, 38, 179e198. Ju, Z. Y., Otte, J., Zakora, M., & Qvist, K. B. (1997). Enzyme-induced gelation of whey proteins: effect of protein denaturation. International Dairy Journal, 7, 71e78. Kashiwagi, T., Yokoyama, K., Ishikawa, K., Ono, K., Ejima, D., Matsui, H., et al. (2002). Crystal structure of microbial transglutaminase from Streptoverticillium mobaraense. Journal of Biological Chemistry, 277, 44252e44260. Lauber, S., Henle, T., & Klostermeyer, H. (2000). Relationship between the crosslinking of caseins by transglutaminase and the gel strength of yoghurt. European Food Research and Technology, 210, 305e309. Lorenzen, P. C., Neve, H., Mautner, A., & Schlimme, E. (2002). Effect of enzymatic cross-linking of milk proteins on functional properties of set-style yoghurt. International Journal of Dairy Technology, 55, 152e157. Lorenzen, P. C., Schlimme, E., & Roos, N. (1998). Crosslinking of sodium caseinate by a microbial transglutaminase. NahrungeFood, 42, 151e154.
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Nielsen, P. M. (1995). Reactions and potential industrial applications of transglutaminase. Review of literature and patents. Food Biotechnology, 9, 119e156. Ozer, B., Kirmaci, H. A., Oztekin, S., Hayaloglu, A., & Atamer, M. (2007). Incorporation of microbial transglutaminase into non-fat yogurt production. International Dairy Journal, 17, 199e207. Remeuf, F., Mohammed, S., Sodini, I., & Tissier, J. P. (2003). Preliminary observations on the effects of milk fortification and heating on microstructure and physical properties of stirred yogurt. International Dairy Journal, 13, 773e782. Renan, M., Guyormarc’h, F., Arnoult-Delest, V., Paquet, D., Brule, G., & Famelart, M. H. (2008). The rebodying of stirred yoghurt: interactions between proteins. Journal of Dairy Research, 75, 450e456. Renan, M., Guyomarc’h, F., Arnoult-Delest, V., Paquet, D., Brule, G., & Famelart, M. H. (2009). Rheological properties of stirred yoghurt as affected by gel pH on stirring, storage temperature and pH changes after stirring. International Dairy Journal, 19, 142e148. Schorsch, C., Carrie, H., & Norton, I. T. (2000). Cross-linking casein micelles by a microbial transglutaminase: influence of cross-links in acid-induced gelation. International Dairy Journal, 10, 529e539. Seguro, K., Kumazawa, Y., Kuraishi, C., Sakamoto, H., & Motoki, M. (1996a). The epsilon-(gamma-glutamyl)lysine moiety in crosslinked casein is an available source of lysine for rats. Journal of Nutrition, 126, 2557e2562. Seguro, K., Nio, N., & Motoki, M. (1996b). Some characteristics of a microbial protein cross-linking enzyme: transglutaminase. In N. Parris, A. Kato, L. K. Creamer, & J. Pearce (Eds.), Macromolecular interactions in food technology. ACS symposium series, Vol. 650 (pp. 271e280). Washington, DC, USA: American Chemical Society. Sharma, R., Lorenzen, P. C., & Qvist, K. B. (2001). Influence of transglutaminase treatment of skim milk on the formation of epsilon-(gamma-glutamyl)lysine and the susceptibility of individual proteins towards crosslinking. International Dairy Journal, 11, 785e793. Singh, H. (2007). Interactions of milk proteins during the manufacture of milk powders. Lait, 87, 413e423. Sodini, I., Lucas, A., Oliveira, M. N., Remeuf, F., & Corrieu, G. (2002). Effect of milk base and starter culture on acidification, texture, and probiotic cell counts in fermented milk processing. Journal of Dairy Science, 85, 2479e2488. Wiles, P. G., Gray, I. K., & Kissling, R. C. (1998). Routine analysis of proteins by Kjeldahl and Dumas methods: review and interlaboratory study using dairy products. Journal of AOAC International, 81, 620e632. Yüksel, Z., & Erdem, Y. K. (2010). The influence of transglutaminase treatment on functional properties of set yoghurt. International Journal of Dairy Technology, 63, 86e97.
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Effect of transglutaminase-treated milk powders on the properties of skim milk yoghurt C. Guyot*, U. Kulozik Chair for Food Process Engineering and Dairy Technology, Research Centre for Nutrition and Food Science (ZIEL) e Section Technology, Technische Universität München, Weihenstephaner Berg 1, 85354 Freising-Weihenstephan, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 June 2010 Received in revised form 22 September 2010 Accepted 5 October 2010
A novel method of transglutaminase (TGase) treatment for skim milk yoghurt production was investigated. In contrast to previous studies, TGase pre-treated skim milk powder (SMP) was used as protein fortification for yoghurt making, instead of treating the entire yoghurt milk. When the TGase concentration for powder production was increased from 0 to 10 U g1 protein, the viscosity of stirred skim milk yoghurt produced with addition of TGase-treated SMP increased from 247 to 453 mPas and the serum loss assessed using a centrifugation method decreased from 57.1% to 52.6%. Furthermore, by using enzyme-modified SMP, only half of the protein addition was required to obtain an equivalent viscosity compared to the control. The study showed that crosslinking the caseins by TGase only in the added SMP yields the desired positive effects while allowing for a complete elimination of the residual enzyme activity. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction The enzymatic crosslinking of proteins by means of microbial transglutaminase (TGase) strengthens protein-based food structures at the molecular level and positively affects properties such as serum holding capacity and gel firmness. The enzyme-catalysed reaction leads to the formation of intra- and inter-molecular crosslinks between the protein-bonded amino acids glutamine and lysine (Ikura, Yoshikawa, Sasaki, & Chiba, 1981; Kashiwagi et al., 2002; Nielsen, 1995; Seguro, Kumazawa, Kuraishi, Sakamoto, & Motoki, 1996a). Caseins, the major milk protein fraction, represent an excellent substrate for the TGase reaction (Christensen, Sorensen, Hojrup, Petersen, & Rasmussen, 1996; De Jong & Koppelman, 2002; Lorenzen, Schlimme, & Roos, 1998). In contrast, whey proteins in their native structure are hardly suitable for crosslinking due to their globular conformation and the limited accessibility of reactive amino acids (Faergemand, Otte, & Qvist, 1997; Han & Damodaran, 1996; Ju, Otte, Zakora, & Qvist, 1997; Sharma, Lorenzen, & Qvist, 2001). Therefore, TGase treatment is predominantly applied to casein-based milk products (Ikura, Goto, Yoshikawa, Sasaki, & Chiba, 1984) while whey proteins play only a minor role with regard to TGase-induced crosslinking in milk.
* Corresponding author. Tel.: þ49 816171 5317; fax: þ49 816171 4384. E-mail address: [email protected] (C. Guyot). 0958-6946/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2010.10.010
In many dairy products, the structure is mainly influenced by milk proteins. In the case of yoghurt, acid-induced gelation of caseins during fermentation leads to structure formation. An enzymatic crosslinking of caseins results in specific changes in yoghurt microstructure like reduced mesh size and a more homogeneous protein network (Lorenzen, Neve, Mautner, & Schlimme, 2002; Schorsch, Carrie, & Norton, 2000). In recent years, it was found for TGase-treated skim milk yoghurt that higher gel firmness, reduced syneresis and an improved mouth feel could be obtained (Faergemand & Qvist, 1997; Jaros, Heidig, & Rohm, 2007; Schorsch et al., 2000). Therefore, the application of TGase for yoghurt processing is of great interest to minimise frequent defects in yoghurt products, namely low viscosity and high level of syneresis (Amatayakul, Sherkat, & Shah, 2006). Generally, direct application of TGase treatment to yoghurt production can be accomplished by two different ways: incubation of the yoghurt milk prior to fermentation (Lauber, Henle, & Klostermeyer, 2000; Lorenzen et al., 2002; Ozer, Kirmaci, Oztekin, Hayaloglu, & Atamer, 2007) or simultaneous addition of TGase and the starter culture (Bönisch, Huss, Lauber, & Kulozik, 2007a; Yüksel & Erdem, 2010). However, both methods were shown to have undesirable side effects. The TGase reaction prior to fermentation requires additional processing time, but offers more flexibility regarding the incubation conditions independent of the fermentation conditions. Further, a thermal treatment to inactivate the enzyme after incubation can be applied prior to fermentation without affecting the fermentation of the lactic acid bacteria.
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In contrast, the incubation and simultaneous microbial acidification does not require additional processing time but a thermal enzyme inactivation is not possible. Due to the acidification by lactic acid bacteria the pH drops from initially pH 6.8 to 4.6, and the optimum pH range for TGase between pH 6 and 7 (Ando et al., 1989) is left soon after fermentation starts. The crosslinking effect may therefore be less pronounced. Overall, the simultaneous application of TGase and fermentation may cause a low remaining enzyme activity in the final product. Even at the low pH values from 4.2 to 4.6 after acidification outside the pH range optimal for the enzyme lead to negative structural changes during storage time. Lorenzen et al. (2002) described a strong increase in the gel strength during the first two weeks of storage of set-style yoghurt produced with simultaneous addition of TGase and the starter culture. Similar observations were made by Yüksel and Erdem (2010) who found an increase in gel hardness during storage for TGase-treated yoghurt samples without prior thermal TGase inactivation. For yoghurt samples with inactivated TGase, the hardness remained nearly constant during storage. According to standard practice, the protein content of yoghurt milk is increased from the initial 3.4% (w/w) to 4.0e5.0% (w/w) protein (Remeuf, Mohammed, Sodini, & Tissier, 2003; Sodini, Lucas, Oliveira, Remeuf, & Corrieu, 2002). Skim milk powder, whey protein isolates, whey protein concentrates or sodium caseinate can be used to fortify yoghurt milk. The kind and amount of protein used have a significant impact on the resulting yoghurt structure. As an alternative to the application of TGase to the liquid yoghurt milk, pre-treated skim milk powder (SMP) for yoghurt production has not been studied so far. By using enzymatically modified powder as the protein fortifier, additional processing time prior to fermentation is not required. Furthermore, it is possible to thermally inactivate the enzyme completely. Nevertheless, the influence of TGasetreated milk powder on yoghurt properties is barely investigated. To the best of our knowledge, no study has so far reported on the application of TGase pre-treated SMP for yoghurt processing. The key question is whether the TGase treatment of only a part of the proteins finally leads to the desired fortification effect compared to yoghurt produced by treating all proteins contained in yoghurt milk. Hence, the purpose of the present study was to investigate the impact of TGase-modified SMPs on the properties of fortified milk yoghurt. The objectives were to relate the key structural properties of yoghurt, namely flow behaviour and serum binding capacity, with respect to the powder used and, furthermore, to assess the optimal process parameters for the application of TGase-treated SMP to improve yoghurt quality.
(U) per g powder. One unit of enzyme was defined as the amount of enzyme that released 1 mmol of hydroxamic acid in 1 min at 37 C according to the hydroxamate test by Folk and Cole (1965). 2.2.2. Milk components and yoghurt starter culture Pasteurised (70 C, 30 s) skim milk with a total protein content of about 3.4% (w/w) was obtained from a local dairy. Spray-dried skim milk powder was purchased from Bayerische Milchindustrie eG (Landshut, Germany). The yoghurt starter culture ABT-21Ò was obtained from Chr. Hansen GmbH (Nienburg/Weser, Germany) in frozen form and is composed of Lactobacillus acidophilus, Streptococcus thermophilus and Bifidobacterium sp. According to the manufacturer’s data, the culture is suitable for the production of stirred yoghurt with mild flavour and low post-acidification. For the starter culture application to the yoghurt process, a pre-culture was prepared by mixing 10% (w/w) frozen starter culture with skim milk and stored for 24 h at 10 C. 2.2.3. Processing of TGase-treated milk protein powder TGase-treated SMP was produced according to the flow chart in Fig. 1. Skim milk concentrate with a total protein content of 8.0% (w/w) was obtained by mixing pasteurised skim milk with SMP. After rehydration of dissolved SMP for 24 h at 4 C, the skim milk concentrate was heat treated in a tubular heat exchanger at 85 C for 2 min to inactivate the indigenous TGase inhibitor contained in low heated milk (Bönisch, Tolkach, & Kulozik, 2006; De Jong, Wijngaards, & Koppelman, 2003). Then the milk concentrate was tempered to 40 C and the enzyme was added. TGase concentrations used were 0.5, 1.0, 3.0, 10.0 U g1 milk protein. In addition, a control without TGase was produced using the same conditions. After 2 h, the enzyme reaction was terminated by a thermal treatment (85 C, 2 min) for complete TGase inactivation (Seguro, Nio, & Motoki, 1996b). After cooling to 20 C, the TGase-incubated milk concentrate was spray dried using a Niro Atomizer (Copenhagen, Denmark). The
2. Material and methods 2.1. Chemicals CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate), sodium phosphate and hydrochloride acid (37%) were obtained from SigmaeAldrich (Steinheim, Germany); urea, 1,4-dithiothreitol (DTT), sodium chloride and sodium hydroxide from Merck (Darmstadt, Germany). All chemicals used were of analytical grade. 2.2. Preparation of materials 2.2.1. Transglutaminase The microbial transglutaminase (TGase, EC. 2.3.2.13) used in this study was the preparation ActivaÒ MP and was a gift from Ajinomoto Foods Deutschland GmbH (Hamburg, Germany). According to product specification the enzyme preparation consists of TGase, lactose and maltodextrin and has a declared activity of 100 Units
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Fig. 1. Processing of TGase-treated skim milk powder.
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product inlet and outlet temperature was 180 C and 80 C, respectively. The TGase-treated milk powder was stored until usage at 10 C in closed plastic containers with a screw cap and a capacity of 50 L under dry conditions. 2.2.4. Yoghurt processing with TGase-treated milk powder Stirred skim milk yoghurt was prepared according to Bönisch et al. (2007a) with slight changes, as can be seen in Fig. 2. To determine the effect of protein fortification of skim milk with different amounts of TGase-treated milk powder, the treated powder was used to increase the original total protein content (w/w) of skim milk from initially 3.4% to 3.9%, 4.4%, 4.9% and 5.4%. For each yoghurt trial, 10 kg yoghurt milk was produced by adding 149.7 g, 299.4 g, 449.1 g or 598.8 g untreated or TGase-treated SMP to the corresponding amounts of pasteurised skim milk. Subsequently, the obtained yoghurt milk was stored for 12 h at 4 C for rehydration of all powder particles. Afterwards, the yoghurt milk was heat treated in a tubular heat exchanger at 95 C for 3 min. The chosen conditions were appropriate for low-fat yoghurt production (Dannenberg & Kessler, 1988). The yoghurt milk was tempered to 42 C in a water bath and inoculated with 0.2% (v/v) yoghurt pre-culture. The fermentation process was accomplished at 42 C in a laboratory heating cabinet and the decrease of pH as a function of time was monitored using an S40 SevenMulti pH meter (Mettler-Toledo GmbH, Giessen, Germany). After reaching pH 4.6, the yoghurt gel was pumped immediately with a flexible-tube pump (Verder 2005/1, Düsseldorf, Germany) with a flow rate of 80 L h1 through a fine sieve with a mesh size of 220 mm and a tubular cooler equipped with a static mixer (d ¼ 10 mm, l ¼ 2 m). The temperature at the end of the cooler was 20 1 C. The stirred yoghurt was collected and cooled to 4 C. After cooling, the stirred yoghurt samples were stored in 250 mL yoghurt jars at 4 C until analysis. For comparison of yoghurt made by protein fortification with TGase-treated SMP with yoghurt from directly incubated yoghurt milk, 10 kg of yoghurt with simultaneous addition of TGase and
starter culture was produced according to Bönisch et al. (2007a). Pasteurised skim milk was fortified with untreated SMP to a final protein content of 4.4% (w/w). Simultaneous with the yoghurt starter culture, 0.6 U TGase g1 protein was added to the tempered yoghurt milk. The enzymatic incubation and microbial fermentation occurred at the same time. All other process conditions are identical to the standard yoghurt production steps for yoghurt with application of enzyme-treated SMP (Fig. 2, optional proceeding). All yoghurt production processes were carried out in triplicate.
2.3. Analytical techniques 2.3.1. Size exclusion chromatography The degree of protein polymerisation was determined by using a size exclusion chromatography method (SEC) according to Lauber et al. (2000) and Bönisch, Lauber, and Kulozik (2004). An ÄKTA FPLC system provided with a P900 pump, a variable wavelength UV detector and a SuperdexÔ 200 10/300 GL gel filtration column (Amersham Biosciences, Freiburg, Germany) were used. The column was eluted at room temperature at a flow rate of 0.5 mL min1 with elution buffer composed of 6 M urea, 0.1 M sodium chloride, 0.1 M sodium phosphate and 0.1% (w/v) CHAPS. The TGase-treated and untreated SMP were dissolved at a protein concentration of 0.3% in elution buffer containing 1% (w/v) DTT as a reducing agent. To determine the resulting degree of protein polymerisation in the produced yoghurt samples, an aliquot of yoghurt was lyophilised by freeze-drying (DELTA 1-24, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany). The dried yoghurt was dissolved in the same way as the SMP. The samples were stored for 12 h at 4 C to afford a complete dissociation of casein aggregates and reduction of disulphide bonds. After membrane filtration (Chromafil Xtra RC-45/25, regenerated cellulose, MachereyeNagel GmbH & Co. KG, Düren, Germany), 50 mL of samples were injected and detection clocked to 280 nm. Data evaluation and peak integration were done by using the Unicorn 4.11 software (Amersham Biosciences, Freiburg, Germany). The degree of enzyme-induced protein polymerisation DDP was defined as the amount of crosslinked caseins related to the total amount of caseins in the sample (Bönisch et al., 2004). DDP was calculated according to equations (1) and (2):
Acrosslinked caseins 100% Atotal caseins Aoligomers;trimers;dimers ¼ 100% and Aoligomers;trimers;dimers;monomers
DP ¼
DDP ¼ DPwith TGase DPwithout TGase
(1)
(2)
where DP is the degree of polymerisation including thermal and enzyme-induced protein polymerisation and Ai is the area between the zero line and the absorption curve of fraction i.
Fig. 2. Production of stirred skim milk yoghurt using TGase-treated milk powder or milk powder without TGase treatment (control) as protein fortification (0.5e2.0% w/w).
2.3.2. Flow behaviour of yoghurt samples The rheological properties of stirred yoghurt samples were determined using an AR1000 N rheometer (TA instruments, New Castle, Delaware, USA) equipped with a coaxial cylinder geometry (radius 14 mm) and a sample cup (radius 15 mm). Before starting the measurement, both sample and sample cup were tempered to 10 C. A five-stage measuring profile consisting of pre-shearing (shear rate of 50 s1 for 30 s), equilibration step (2 min), upward ramp (shear rate from 0.1 to 100 s1 in 1 min), holding phase (shear rate of 100 s1 for 1 min) and downward ramp (shear rate from 100 to 0.1 s1 in 1 min) was applied and shear stress and apparent viscosity were detected.
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Stirred skim milk yoghurt exhibits a yield stress and shows a shear thinning flow behaviour during shear treatment that leads to irreversible structural changes. The shear rate versus shear stress curves of the downward ramp were adjusted to the HerscheleBulkley model as one of the most appropriate models to describe the steady-shear rheological behaviour of yoghurt (Fangary, Barigou, & Seville, 1999):
s g_ ¼ s0 þ Kg_ n
(3)
where s is the shear stress, s0 the yield stress, K the consistency index, g_ the shear rate and n the flow behaviour index. Using equation (3) the apparent viscosity happ is defined as
happ g_ ¼
s g_ s ¼ 0 þ K$g_ n1 g_ g_
(4)
For each trial, all rheological measurements were done in duplicate. 2.3.3. Serum binding capacity The serum binding capacity was assessed as the fraction of serum after centrifugation. Some 60 g yoghurt was filled in centrifugation glasses and centrifuged at 1500 g, 10 C for 20 min (Heraeus Multifuge 1 S-R, Thermo Fisher Scientific, Waltham, Massachusetts, USA). The amount of supernatant yoghurt serum was recorded and the relation between the amounts of serum mSerum and original yoghurt sample mYoghurt gives the serum loss SL in percent:
SL ¼
mSerum 100% mYoghurt
(5)
2.3.4. Water content The water content of SMP was determined using a gravimetric method according to IDF Standard 26A: 1993 (IDF, 1993). Some 3 g powder was dried in a T5028 oven (Heraeus GmbH, Hanau, Germany) at 102.0 C 1.0 C under atmospheric pressure for 2 h. To prove mass constancy, additional drying steps were performed until the differences in mass of powder sample were lower than 1.0 mg. The water content was calculated as the amount of weight loss after drying related to the initial amount of SMP in percent. All measurements were done in triplicate.
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production. Fig. 3 shows a representative chromatogram of SMP produced either without TGase treatment or with an enzyme addition of 1.0 and 10.0 U g1 protein, respectively. An increased TGase concentration led to an increasing amount of highly crosslinked caseins. The small oligomer peak of the control sample is known to result from the thermal treatment during heating and spray-drying of SMP (Singh, 2007) and was considered for calculation of the enzyme-induced crosslinking degree. The calculated DDPs are shown in Table 1. Depending on the enzyme concentration, the average degrees of protein polymerisation varied from 0 to 33.9%. The enzymatic crosslinking reaction leads to a molecular modification of milk proteins and variably high crosslinked milk powders can be obtained. Protein and water contents remained unaffected by the enzyme treatment (36.8% 0.3% protein, 3.8% 0.3% water) with no significant changes following enzyme treatment. Therefore, differences in the yoghurts produced by addition of varying TGase-modified powders can be linked to the respective degree of polymerisation.
3.2. Influence of TGase pre-treated skim milk powders as protein fortification on the properties of stirred yoghurt 3.2.1. Effect of protein fortification with differently high crosslinked skim milk powders In the present study, it was found that yoghurt with addition of pre-treated SMP showed all the improved properties of yoghurt known for TGase-incubated milk. The viscosity and serum loss of the produced stirred skim milk yoghurts are depicted in Fig. 4. While the control sample with untreated SMP (DDP ¼ 0%) had a low viscosity of 247 mPas and a high amount of centrifugally forced serum loss of 57.1%, significantly higher viscosities and reduced serum loss were obtained by using TGase-treated SMP as protein fortifier. Especially the addition of high enzyme-treated SMP obtained by using 3.0 or 10.0 U TGase g1 protein for powder production (DDP is 23.6% or 33.9%, respectively) caused a strong increase in yoghurt viscosity to 376 or 453 mPas and a decrease in serum loss to 54.0% or 52.6%. In contrast to yoghurt produced with direct incubation of yoghurt milk, in these yoghurt samples only a small fraction of milk proteins is enzymatically modified. Nevertheless, the addition of
2.3.5. Total protein content The total protein content of TGase-treated milk powder and yoghurt samples was determined by the nitrogen content using a Leco FP 528 system (Leco Instrumente GmbH, Moenchengladbach, Germany) based on the Dumas method (Wiles, Gray, & Kissling, 1998). Total protein content was calculated by multiplying the total nitrogen content by 6.38. All measurements are carried out at least in duplicate. 2.3.6. Statistical analysis Statistical analysis was carried out using ANOVA. Differences were considered significant at P < 0.05. All statistic analyses were performed using Statgraphics software (Statistical Graphics Corporation, Rockville, MD, USA). 3. Results and discussion 3.1. Influence of enzymatic protein crosslinking on the properties of skim milk powder To investigate the influence of enzyme-catalysed protein polymerisation on the properties of SMP, different enzyme concentrations (0e10.0 U g1 protein) were used for powder
Fig. 3. Chromatographic pattern of skim milk powders produced without enzyme addition (eeee) and with TGase incubation at 1.0 (e e e) or 10.0 (e$$e$$) U g1 protein at 40 C for 2 h.
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Table 1 TGase-induced degrees of protein polymerisation DDP of differently TGasetreated skim milk powders.
DDP (%)a
TGase treatment
Without TGase, 40 C, 2 h (control) 0.5 U g1 protein, 40 C, 2 h 1.0 U g1 protein, 40 C, 2 h 3.0 U g1 protein, 40 C, 2 h 10.0 U g1 protein, 40 C, 2 h
0.0 6.7 12.5 23.6 33.9
1.4 1.3 1.7 1.7 4.6
a b c d e
a Results are given as average confidence interval (a ¼ 0.05); different lower case letters indicate significant differences with a 95% confidence level.
TGase pre-treated SMP leads to a significant structural enhancement of the yoghurt sample. In Fig. 5 the viscosity of stirred skim milk yoghurt with addition of TGase-treated SMP and direct enzyme incubation of yoghurt milk dependent on the resulting DDP in the yoghurt sample is shown. For skim milk yoghurt produced with direct implementation of TGase treatment, higher enzyme concentrations generally lead to firmer gel networks, improved viscosities and reduced amounts of serum loss during storage time (Bönisch, Huss, Weitl, & Kulozik, 2007c). The breaking strength of set-style yoghurt from TGase-incubated milk was increased with longer incubation times (Lauber et al., 2000). Similar observations were made for stirred skim milk yoghurt fortified with TGase-treated SMP. Bönisch et al. (2007a) found that for simultaneous addition of TGase and starter culture a DDP value
Fig. 4. Apparent viscosity (a) and serum loss (b) of stirred skim milk yoghurt produced with a protein fortification of þ1% (w/w) by means of differently high TGase-treated skim milk powders. The graphs show means confidence intervals (a ¼ 0.05, n ¼ 6).
Fig. 5. Viscosity of stirred skim milk yoghurt dependent on the resulting degree of polymerisation in the yoghurt sample for yoghurt produced with direct incubation of yoghurt milk (-) or addition of TGase-treated SMP (A). The graph shows means confidence intervals (a ¼ 0.05, n ¼ 6).
of about 15% is necessary to obtain a structure enhancement of skim milk yoghurt. Due to the direct incorporation of the enzyme in the yoghurt milk, all proteins contained in the yoghurt milk are available for the crosslinking reaction. Therefore, the resulting degree of polymerisation in the yoghurt sample has a high value of about 13.5%. For the addition of TGase pre-treated SMP, the major part of milk proteins from the pasteurised milk remains enzymatically untreated and only the proteins used for protein fortification are modified. Thus, the resulting degrees of protein polymerisation of the yoghurt samples depend on the fraction and kind of TGase pretreated SMP. For yoghurt with a protein fortification of 1.0% (w/w), the resulting degrees of polymerisation in the yoghurt samples were 1.5%, 2.8%, 5.4% or 7.7% for an enzyme concentration of 0.5, 1.0, 3.0 or 10.0 U g1 protein, respectively. Although the resulting degree of polymerisation in yoghurt is much lower for the addition of TGase-treated SMP with a high enzyme concentration of 10.0 U g1 protein compared to the yoghurt produced with direct TGase incubation, the viscosities of the yoghurt samples were similar, as can be seen in Fig. 5. The stronger impact of the resulting degree of polymerisation on the viscosity can be explained by the different protein compositions of yoghurt produced either by addition of TGase-treated SMP or with direct incubation. Fig. 6 shows the relative amounts of protein monomers, dimers, trimers and oligomers of yoghurt samples with direct incorporation of TGase to yoghurt milk and with protein fortification by TGase pre-treated SMP (3.0 U g1 protein). It can be noted that the direct TGase incubation leads to the formation of high amounts of casein dimers and trimers, but low amounts of oligomers. In the case of addition of high enzyme-treated SMP, the protein composition was different. Yoghurt produced by addition of TGase-treated SMP contained only small fractions of dimers and trimers but higher amounts of protein oligomers were obtained. Since the addition of intensively crosslinked milk proteins to the yoghurt milk significantly increases the oligomer fraction, the amounts of monomers, dimers and trimers remain close to the value for untreated yoghurt. It can be assumed that the higher fraction of casein oligomers strongly affects especially the yoghurt viscosity. Therefore, comparable rheological properties can be obtained with a lower value of the resulting degree of
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Fig. 6. Size exclusion chromatography pattern of yoghurt produced with direct incubation of yoghurt milk (e e e) or addition of 1.0% protein by TGase-treated SMP (eee; 3.0 U g1 protein).
polymerisation in the yoghurt sample by using TGase-treated SMP for protein fortification. 3.2.2. Influence of amount of protein fortification One of the main advantages of direct TGase treatment of yoghurt milk is the possibility to produce skim milk yoghurt with a reduced dry matter content but unchanged rheological and sensory properties (Bönisch, Lauber, & Kulozik, 2007b). Therefore, the influence of different amounts of powder addition on yoghurt structure was investigated. To fortify the protein content with 0.5e2.0% protein, TGase-treated and the untreated SMP were used. As TGase-treated powder, SMP with an enzyme treatment of 3.0 U g1 protein (DDP ¼ 23.6%) was chosen. According to Bönisch et al. (2007a), for direct TGase incorporation an enzyme concentration of 0.6 U g1 protein which leads to a DDP of 22% after fermentation is suitable for crosslinking all the proteins contained in yoghurt milk. Since for application of TGase-treated milk powder only a small fraction of proteins in the yoghurt milk is modified, the total amount of TGase preparation used is comparable with the amount applied for direct enzyme incorporation. For TGase treatment with 0.6 U g1 protein of the whole yoghurt milk containing 4.4% protein, an enzyme addition of 26.4 U kg1 yoghurt milk is necessary. In contrast to that, the relative amounts of TGase used for yoghurt produced with addition of TGase-treated SMP depend on the protein fortification. For the yoghurt fortified with 3.0 U TGase g1 protein treated SMP, the calculated enzyme concentrations required for crosslinking of the SMP added to yoghurt milk is 30.0 U kg1 yoghurt milk for a protein fortification of 1.0%. Due to the similarity, the TGase-treated SMP produced by the use of an enzyme concentration of 3.0 U g1 protein with an average DDP value of 23.6% was used for subsequent investigations. Fig. 7 shows the influence of the two different powders on yoghurt flow behaviour. For each level of protein fortification, the viscosities of yoghurt with addition of enzyme-treated milk powder was higher compared to yoghurt fortified with untreated SMP. Further, it was demonstrated that with the use of TGasetreated SMP, the same level of viscosity was achieved with less SMP addition to the yoghurt milk. While a protein fortification of about 1.6% is normally necessary for traditionally produced skim milk yoghurt without TGase treatment (Sodini et al., 2002), a protein fortification of only 1.0% is sufficient for skim milk yoghurt with
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Fig. 7. Viscosity of stirred skim milk yoghurt produced without fortification (;) or with addition of different amounts of TGase pre-treated (:; 3.0 U g1 protein, DDP of 23.6%) or untreated (A) skim milk powder to the yoghurt milk after 1-week storage at 4 C. The graphs show means confidence intervals (a ¼ 0.05, n ¼ 6).
addition of TGase-treated SMP (3.0 U g1 protein) to achieve equivalent flow behaviour, provided that a target viscosity of 200e300 mPas would be aimed at. Thus, yoghurt with comparable properties in respect of structural behaviour can be produced with a reduced amount of milk powder added to the yoghurt milk. 3.2.3. Comparison of structure stability of skim milk yoghurt with different TGase treatments during storage In the present study, it was clearly demonstrated that a protein fortification by means of TGase pre-treated SMP has a positive impact on the resulting yoghurt properties. By using high TGasetreated SMP, similar effects like increased viscosity, reduced syneresis and the option to reduce the protein and dry matter content without negative structural changes compared to the direct TGase incubation of yoghurt milk, were observed. Nevertheless, an important aim was to realise storage stability, i.e., avoiding structural defects showing up on the course of shelf life, by using the TGase-treated SMP in contrast to the direct incorporation of the enzyme in yoghurt production. Storage stability regarding structural properties is essential for yoghurt products. For direct implementation of TGase treatment in the process of yoghurt production, negative changes in yoghurt structure such as coarseness or lumpiness were often observed (Bönisch et al., 2007a; Demirkaya & Ceylan, 2009; Lorenzen et al., 2002; Ozer et al., 2007; Yüksel & Erdem, 2010). Generally, rebodying and rearrangements of casein clusters can lead to structural changes of conventionally produced yoghurt during storage time (Renan et al., 2008, 2009). However, the change of rheological properties due to rearrangement of caseins is rather low for conventional yoghurt products. Although rebodying is not completely understood so far, it was clearly demonstrated that active TGase in yoghurt affects the restructuring behaviour, and the resulting viscosity significantly increases during storage time. Hence, the aim is to combine the possibility of reducing the dry matter content of yoghurt by the TGase treatment without loss of structural properties with a constant viscosity during storage. Therefore, it was of major interest whether stirred skim milk yoghurt produced with addition of enzyme-treated SMP shows high storage stability. In Fig. 8 both the viscosity and the resulting
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no alteration of DDPYoghurt was found for the application of TGase pre-treated SMP (Fig. 8b). The comparison between the different enzyme treatments clearly indicates that the application of TGase-treated SMP is well suited for the protein fortification of yoghurt milk. By using the enzymatically modified powder the benefits of the TGase treatment can be combined with high structure stability during the whole storage time. 4. Conclusions Stirred skim milk yoghurts fortified with enzyme-modified SMP have higher viscosities and lower serum loss compared to yoghurt samples produced with addition of untreated SMP. Furthermore, due to complete enzyme inactivation by means of heat treatment and the spray-drying process, the viscosity of the yoghurt samples remained constant during storage time. In particular with regard to skim milk yoghurt, the application of TGase pre-treated SMP seems to be a promising alternative to previous TGase treatments. Improved properties without undesired structural changes during storage can be realised by addition of TGase pre-treated SMP as protein fortifier. Furthermore, with the use of TGase-treated SMP the same level of viscosity was reached with less milk powder addition to the yoghurt milk. Thus, yoghurt with comparable properties in respect of structural behaviour was produced with reduced amount of SMP. In further studies, the influence of different casein to whey protein ratios in the milk powders on the resulting yoghurt properties shall be investigated. Acknowledgement
Fig. 8. Apparent viscosity (a) and degree of polymerisation (b) during storage at 4 C of stirred skim milk yoghurts with different kinds of TGase application of TGase treatment, i. e., without enzyme treatment (A), with 0.6 U TGase g1 protein simultaneous with starter culture (-), and addition of 1% TGase pre-treated skim milk powder (;; 3.0 U g1 protein). The graph shows means confidence intervals (a ¼ 0.05, n ¼ 6).
DDP of stirred skim milk yoghurt with different TGase treatments as a function of storage time are depicted. Yoghurt without enzyme treatment always has a lower viscosity than the yoghurt samples either with direct enzyme incorporation or addition of pre-treated SMP. Although the resulting viscosity of yoghurt produced with addition of crosslinked SMP (3.0 U g1 protein) is lower compared to yoghurt samples with simultaneous addition of enzyme and starter culture. However, the positive effect of the enzyme treatment is still obvious and the addition of TGase-treated SMP led to an increased viscosity of 376 mPas and a reduced serum loss of 54.0%. The lower influence in this case can be explained by the use of SMP with 3.0 U g1 protein to fortify the protein content. Furthermore, it can be seen that for yoghurt produced with direct incubation of yoghurt milk, the viscosity increased significantly from 396 mPas to 456 mPas during storage time. In contrast to that, the viscosity of stirred skim milk yoghurt with TGasetreated SMP remained nearly constant during the whole storage time and no significant change was found. The absence of developing structural defects can be explained by the complete enzyme inactivation due to the heat treatment prior to applying the treated SMP. Since for direct TGase implementation the DDPYoghurt slightly increases from initially 12.8% to 14.8% within three weeks at 4 C,
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