Effects of transglutaminase treatment on functional properties and probiotic culture survivability of goat milk yogurt

Small Ruminant Research 65 (2006) 113–121

Effects of transglutaminase treatment on functional properties and probiotic culture survivability of goat milk yogurt J.P. Farnsworth a , J. Li a , G.M. Hendricks b , M.R. Guo a,∗ a

Department of Nutrition and Food Sciences, University of Vermont, 215 Carrigan Hall, 536 Main Street, Burlington, VT 05405, USA b Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA Received 17 December 2004; received in revised form 26 April 2005; accepted 17 May 2005 Available online 18 July 2005

Abstract The objectives of this study were to investigate the effects of enzymatic cross-linking of milk proteins on the functional properties and probiotic culture survivability of goat milk yogurt. After pre-incubation (50 ◦ C, 1 h) of the milk with microbial transglutaminase [MTGase, 0–4 units (U)/g protein] and subsequent heat inactivation (75 ◦ C, 5 min), yogurt was prepared by inoculating the milk with a commercial probiotic yogurt starter containing probiotic cultures (Lactobacillus acidophilus, Bifidobacteria, and Lactobacillus subsp. casei) and incubating the inoculated milk at 43 ◦ C for 5 h. Compared to the untreated control trial, pretreatment of the milk with MTGase (2 and 4 U/g protein) increased (P < 0.05) the viscosity and decreased the syneresis of yogurt samples. The effect of enzyme treatment was more significant in improving the viscosity of yogurt compared to the method of increasing total solids (TS) in the milk. No significant differences in basic nutrient contents between control and enzyme-treated samples were observed. The probiotic cultures were relatively stable in the goat milk yogurt and their populations remained above 106 CFU/g during the 8-week storage period at 4 ◦ C. No significant differences in rate of change of population of all the three probiotic cultures were observed between the control and the MTGase treated groups. Microbiological analysis shows that the enzymatic cross-linking of proteins seems to have a positive role in the survivability of the probiotic cultures, which requires further investigation. Scanning electron microscopic studies revealed that the microstructure of the enzyme-treated samples appeared denser than the control, suggesting that pretreatment of milk by MTGase could be used to improve the microstructure of the yogurt gel. Results of this study indicate that enzymatic cross-linking of milk proteins by MTGase appears to be an effective means for improving the functional properties of goat milk yogurt. MTGase treatment may be a useful method in the production of probiotic goat milk yogurt products. © 2005 Elsevier B.V. All rights reserved. Keywords: Goat milk yogurt; Microbial transglutaminase; Cross-linking; Viscosity; Probiotic culture survivability

1. Introduction ∗ Corresponding author. Tel.: +1 802 656 8168; fax: +1 802 656 0001. E-mail address: [email protected] (M.R. Guo).

0921-4488/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2005.05.036

Goat milk products, such as yogurt and cheese, are becoming increasingly popular in the United States as

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specialty products and as substitutes for bovine milk products for those who have allergies against bovine milk (Haenlein, 1996; Park, 1994a). Unfortunately, it is difficult to produce goat milk yogurt with consistency comparable to bovine milk yogurt (Abrahamsen and Rysstad, 1991), mainly due to the impact of naturally low ␣sl -casein content and seasonally changing composition on the coagulation properties of the milk (Guo, 2003). ␣sl -Casein, one of major caseins in bovine milk, is a structural component of the casein micelle, and plays a major role in milk coagulation (Walstra et al., 1984). Depending on breeds, ␣sl -casein is found in relatively low or undetectable amounts in goat milk (Guo, 2003). Traditional methods used commercially to improve the texture of yogurt include increasing the total solids in the milk and in the case of stirred yogurt adding stabilizers, e.g. pectin and gelatin (Lucey and Singh, 1998). Emerging approaches to modifying the texture of cultured dairy products include: novel stabilizers, various types of milk-derived ingredients, use of various types of membrane concentrate or fractions, specific cultures (e.g. producing a specific exopolysaccharide type and content), enzymatic cross-linking of milk proteins, e.g. transglutaminase, use of high hydrostatic pressure (e.g. >200 MPa) to the milk to cause denaturation of whey proteins or of the yogurt to prevent post acidification, and very high pressure homogenization (Lucey, 2004). It has been shown recently that the yogurt microstructure can be improved by treatment of the milk with transglutaminase (TGase) (Faergemand et al., 1999; Lauber et al., 2000; Lorenzen et al., 2002). TGase (EC 2.3.2.13) catalyzes an acyl transfer reaction between ␥-carboxyamide groups of peptidebound glutamine residues and the ␧-amino groups of lysine residues, leading to the formation of intraand intermolecular isopeptide bonds (Motoki and Seguro, 1998). The enzyme reaction results in the formation of covalently cross-linked protein polymers. TGase is now widely used in prepared foods such as seafood, surimi products, meat products, noodles/pasta, dairy products, baked goods, etc. to improve their functional properties (Kuraishi et al., 2001). Many food proteins are good substrates for TGase and among the milk proteins the caseins in particular are excellent substrates for TGase (Lauber et al., 2000; Schorsch et al., 2000a,b). Due to the cost-

effective production of TGase by microorganisms, especially by strains of Streptoverticillium (e.g. S. griseocameum, S. morbaraense), applications of the enzyme in industrial food production are possible (Zhu et al., 1995). Benefits of using microbial transglutaminase (MTGase) are lower costs for extraction and purification and their Ca2+ -independent catalytic action. Yogurt that contains probiotic bacteria such as L. acidophilus and Bifidobacteria is becoming popular due to the health promoting properties of the probiotics (Ravula and Shah, 1998). It is generally agreed that to be effective, yogurt should contain at least 106 live probiotic bacteria per ml (Ravula and Shah, 1998; Tamime and Robinson, 1999). The viability of probiotic bacteria in yogurt is affected by several factors, including acidity, pH, hydrogen peroxide, oxygen content, concentrations of lactic and acetic acids, temperature of storage, etc. during manufacture and storage of yogurt (Lankaputhra and Shah, 1995; Lankaputhra et al., 1996; Shah, 2000). Therefore, there is a growing industry interest in developing techniques to ensure adequate numbers of yogurt bacteria, particularly probiotic bacteria, throughout the shelf-life of yogurts. Extensive research has been conducted to study the effects of MTGase treatment on the resulting properties of yogurts made from bovine milk (Faergemand et al., 1999; Kuraishi et al., 1996, 2001; Lauber et al., 2000; Lorenzen et al., 1999, 2002; Lorenzen and Schlimme, 1997). However, this type of research on yogurt from goat milk is more limited (Farnsworth et al., 2002, 2003). On the other hand, the enzymatic cross-linking of milk proteins by MTGase introduces new covalent bonds into the yogurt gel. In contrast, in conventional yogurt, the milk gel is mainly stabilized by non-covalent physical cross-links (i.e., electrostatic interaction, hydrogen bonding and hydrophobic bonds) (Neve et al., 2001). Hence, these different protein networks may affect the growth behavior and viability of the yogurt starter bacteria and some alternative probiotic cultures (e.g. L. acidophilus, Bifidobacteria), which are fastidious microorganisms (Neve et al., 2001). The objectives of this study were (1) to investigate the effects of transglutaminase treatment on the functional properties of probiotic goat milk yogurt, and (2) to examine the survivability of the probiotic bacteria in yogurt during storage at low temperature.

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2. Materials and methods

2.3. Viscosity measurement

2.1. Preparation of materials

Viscosity measurements on unstirred and stirred yogurt samples were carried out under room temperature (22 ± 2 ◦ C) using a Brookfield Programmable DV-II+ Viscometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA), equipped with a No. 3 spindle running at different speeds ranging from 0.5 to 20 rpm. For relative comparison between treatments, viscosity reading was taken at the point of 30th second and torque was maintained at all times between 10 and 100%. All viscosity measurements were performed in triplicate.

Meyenberg instant powdered goat milk (whole milk, pasteurized and spray-dried) from JacksonMitchell, Inc. (Turlock, CA) was used for preparation of reconstituted goat milk (RGM). Commercially reduced fat (2%) homogenized and pasteurized goat milk (RFM) was purchased from a local store. A commercial enzyme product (ACTIVA TG-TI, 100 U/g transglutaminase activity), comprising of 1% Ca2+ -independent transglutaminase derived from the microorganism S. morbaraense and 99% maltodextrin, was gifted by Ajinomoto, USA, Inc. (Ames, IA). One commercial yogurt starter (Yo-Fast 10) containing probiotic cultures (L. acidophilus, Bifidobacteria, and Lactobacillus subsp. casei) was obtained from Chr. Hansen (Milwaukee, WI). All media and reagents were obtained from Fisher Scientific (Pittsburgh, PA) or Sigma Chemical Co. (St Louis, MO). 2.2. Yogurt preparation RGM (12.2% total solids, TS) or RFM was heated to 50 ◦ C in a water bath. Microbial transglutaminase (MTGase) solution (10%) was prepared and added to the milk at levels from 0 (control) to 4 U/g protein. The MTGase/milk solutions were incubated at 50 ◦ C for 1 h, and then heated to 75 ◦ C for 5 min to stop the enzymatic reaction. The samples were cooled to 43 ◦ C and inoculated with 0.04% probiotic yogurt starter culture. Set yogurt was made by incubating the inoculated milk at 43 ◦ C for 5 h followed by refrigeration. For making stirred yogurt, inoculated milk was incubated at 43 ◦ C for 5 h, cooled in an ice-water bath with overhead stirring (50 rpm) for 10 min, and then placed at refrigerator. Upon refrigeration at 4 ◦ C overnight, the yogurt samples from RGM were examined for physical properties including viscosity and syneresis, while the yogurt samples from RFM were subjected to chemical analysis for gross composition, titratable acidity (TA), pH, scanning electron microscopy (SEM) for microstructural characterization, and microbiological analysis for probiotic culture survivability. Meanwhile, RGM yogurt with various total solids (12.2% as a control, 13.2, 15.2, and 17.2%) were prepared for viscosity comparison.

2.4. Syneresis measurement Syneresis of the yogurt was determined by the centrifugation procedure of Keogh and O’Kennedy (1998) with modifications. Yogurt (200 g) was prepared in centrifuge cups and centrifuged at 2500 rpm (average 640 × g) for 10 min at 4 ◦ C. The clear supernatant was collected, weighed and syneresis was calculated according to the following equation (Keogh and O’Kennedy, 1998): syneresis (%) =

weight of supernatant (g) × 100% weight of yogurt sample (g)

2.5. Chemical analysis Effects of MTGase addition (2 U/g protein) on the chemical properties of the goat milk yogurt were compared to the control. Total solids (TS) of the yogurt samples were measured by the forced-draft oven method, while the protein and fat contents of the samples were determined by the Kjeldahl and Babcock Methods (AOAC, 2002). Ash content and titratable acidity (TA) were measured according to AOAC (2002) and lactose content was determined using the Chloramine-T method (James, 1999). The pH values of the yogurt samples were measured with a pH meter (model 240, IQ Scientific Instrument, Inc., San Diego, CA). For determination of mineral concentrations, yogurt samples (10 g) were dry-ashed in porcelain crucibles at 550 ◦ C for 6 h, solubilized with 10 ml of 6 M HC1, quantitatively transferred into 25 ml volumetric flasks, and diluted to volume with double-deionized water. Calcium (Ca), phosphorus (P), sodium (Na),

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magnesium (Mg), and trace mineral contents were determined by inductively coupled Argon Plasma Emission Spectrometry according to the procedures of Park (1994b) and Guo et al. (1997).

micrographs were digitized using a video camera connected to a personal computer. An imaging software (SigmaScan Pro) was used to determine the area of structure versus the area of pores.

2.6. Microbiological analysis

2.8. Statistical analysis

Control and MTGase treated (1, 2, and 4 U/g protein) goat milk yogurts were stored at 4 ◦ C, and sampled (5 g for each bacterial culture) weekly for 8 weeks. Survival cell count of the probiotic bacteria (L. acidophilus, Bifidobacteria, and Lactobacillus subsp. casei) was performed according to the procedures of the culture manufacturer (Chr. Hansen, Milwaukee, WI). L. acidophilus was counted with MRS-IM agar with maltose using a pour plate method, after 72 h incubation at 37 ◦ C under aerobic conditions. Bifidobacteria were enumerated with MRS-IM agar and glucose containing 0.05% dichloxallin, 0.1% lithium chloride, and 0.05% cysteine hydrochloride, after 72 h incubation at 37 ◦ C under anaerobic conditions. L. casei was determined with MRS-IM agar and glucose using the spread plate method, after 144 h incubation at 20 ◦ C under aerobic conditions. Cell counts, performed in triplicate, were calculated from the colonies on agar plates, and thus expressed as colony forming units per gram (CFU/g). 2.7. Scanning electron microscopy SEM was performed for control and MTGase treated (2 and 4 U/g protein) goat milk yogurts. The sampling was carried out by embedding the yogurt samples into agar as described by Schellhaass and Morris (1985). The agar cubes were fixed in glutaraldehyde (25 g/l) in 0.2 M sodium cacodylate buffer (pH 7.0) and post fixed in osmium tetroxide (10 g/l) followed by three rinses in diluted cacodylate buffer (cacodylate:water, 1:1). After staining in uranyl acetate (10 g/l) and dehydration using an acetone dehydration series, the samples were embedded in a mixture of Embed 812/Araladite 502 epoxy resin and sectioned before examination in a JEOL 100CX II TEMSCAN electron microscope operated at 80 kV. Micrographs were taken at two magnifications (×1000 and ×10,000). For unbiased results the microscopic fields for photography were selected by moving the microscope table to a random position before looking into the microscope. The

Data were analyzed by a general linear model procedure of the Fisher’s protected-least-significantdifference (PLSD) test using SAS (SAS Institute, Inc., Cary, NC). This test combines ANOVA with comparison of differences between the means of the treatments at the significance level of P < 0.05. 3. Results and discussion 3.1. Viscosity of goat milk yogurt Effects of total solids and MTGase treatment on the viscosity of unstirred (set-style) goat milk yogurt were compared (Figs. 1 and 2). Increasing total solids of the milk was shown to improve yogurt viscosity, but a significant increase (by about 70%) of viscosity was observed only at 5% added total solids. In contrast, pretreatment (50 ◦ C, 1 h) of the milk with MTGase increased (P < 0.05) the viscosity of the resulting goat milk yogurt samples at all levels of enzyme dosage. Enzyme treatment at the level of 0.25 U/g protein increased the viscosity of yogurt by 75% in comparison to the control, which is comparable to the increase

Fig. 1. Effect of increasing total solids in the milk on the viscosity of unstirred goat milk yogurt. The bar with different letters are different (P < 0.05) based on Fisher’s PLSD multiple comparisons.

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in viscosity achieved by adding a 5% goat milk powder. On the other hand, viscosity of unstirred yogurt might be associated with the strength of the gel resistant to breaking. As shown in Fig. 2, the viscosity of unstirred yogurt increased with increasing MTGase level, suggesting that the breaking strength of goat milk yogurt increases as a result of enzyme-induced cross-linking (Kuraishi et al., 2001). Similar results have also been reported on the gel strength of yogurt samples from transglutaminase-treated bovine milk (Faergemand et al., 1999; Lorenzen et al., 2002). According to Lauber et al. (2000), transglutaminase treatment can improve the gel-forming properties of caseins by intermolecular cross-linking and only a small amount of casein oligomerization is necessary for a significant enhancement of yogurt breaking strength. In the case of stirred yogurt, the viscosity is dramatically decreased in comparison with undisturbed samples presumably due to the destruction of the threedimensional network of the acidic yogurt gel by the stirring process. The viscosity of stirred yogurt increased with the amount of MTGase added (Fig. 3). Pretreatment of the milk with MTGase at the level of 2 U/g protein has increased the viscosity of the stirred yogurt more than 3.5 times compared to the untreated sample. However, due to a high standard deviation, significant increases (P < 0.05) in viscosity were observed only in the samples with MTGase at levels of 3 and 4 U/g protein. Our results are in agreement with Kuraishi et

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Fig. 3. Effect of MTGase treatment on the viscosity of stirred goat milk yogurt. The bars with different letters are different (P < 0.05) based on Fisher’s PLSD multiple comparisons.

al. (2001) where cross-linking catalyzed by MTGase increased the viscosity of stirred yogurt prepared from bovine milk as a result of improved water-holding properties. Various strategies exist to improve the gel stability of set-style yogurts, including increase in total solids of milk by evaporation or addition of milk powder, increasing protein content by ultrafiltration or addition of milk proteins, etc. (Abrahamsen and Holmes, 1981). On the other hand, stabilizers are commonly added to control textural defects and increase consistency in stirred-type yogurt (Lucey and Singh, 1998). Results of the present study show that MTGase treatment could be used as a substitute method for solids fortification or addition of stabilizers in the production of goat milk yogurt. Therefore, a cost reduction by a reduction in the non-fat solid content especially for non-fat goat milk powder is possible due to the increased viscosity with transglutaminase (Kuraishi et al., 2001). 3.2. Syneresis of goat milk yogurt

Fig. 2. Effect of MTGase treatment on the viscosity of unstirred goat milk yogurt. The bars with different letters are different (P < 0.05) based on Fisher’s PLSD multiple comparisons.

Fig. 4 shows that syneresis of the goat milk yogurt samples treated with MTGase (2 and 4 U/g protein) was reduced (P < 0.05) by more than 40% compared to the untreated sample. No significant differences in syneresis were observed between the two MTGase treated samples. Less syneresis has also been reported for yogurt products from MTGase-treated bovine milk

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Stabilizers are often used during the manufacture of dairy products to enhance and maintain certain characteristics. However, excessive use of stabilizers can negatively impact the sensory properties by providing an unnatural flavor attribute or an over-stabilized (gellike) texture and mouth-feel (Lucey, 2004). Our results confirm that transglutaminase may be used to replace the use of stabilizers (Kuraishi et al., 2001). 3.3. Chemical composition and probiotic culture survivability of goat milk yogurt

Fig. 4. Effect of MTGase treatment on the syneresis of goat milk yogurt. The bars with different letters are different (P < 0.05) based on Fisher’s PLSD multiple comparisons.

(Kuraishi et al., 2001; Lorenzen et al., 2002; Schorsch et al., 2000b). Whey separation can be defined as the appearance of whey (serum) on the gel surface (e.g. of a set yogurt). Syneresis is the shrinkage of the gel, which then leads to whey separation. Common reasons for the occurrence of syneresis include the use of a high incubation temperature, excessive whey protein to casein ratio, low solids content and physical mishandling of the product during storage and retail distribution (Lucey, 2004). Yogurt, which is an acid milk gel formed by gradual acidification with a lactic starter, has some problems of whey separation, or syneresis, with a change of temperature or physical impact. Enrichment of dry matter and/or of protein content as well as addition of hydrocolloids like gelatin and starch are common means of avoiding whey syneresis. Cross-linking of protein chains to stabilize the three-dimensional network of the acidic yogurt gel may have a comparable effect (Lorenzen et al., 2002). As a gel formed with ␧-(␥-Glu)Lys bonds has improved water-holding capacities, the set-type yogurt which is made from transglutaminase-treated milk has a greater capacity for holding water, and whey separation is prevented (Kuraishi et al., 2001). On the other hand, reduction of syneresis may be caused by the effect of transglutaminase on the pore size of the milk gels. As pore size reduces, the protein network will result in smaller syneresis (Lorenzen et al., 2002).

Gross composition and mineral contents of goat milk yogurt prepared with and without MTGase are listed in Table 1. As expected, no differences (P > 0.05) in chemical composition between control and MTGase treated samples were observed. Overall mean percentage of total solids, protein, fat, and lactose for both control and MTGase treated yogurt samples were similar (11.0, 3.0, 3.0, and 3.7, respectively). The goat milk yogurt in this study had similar total solids, but lower protein and lactose, and higher fat contents when compared to commercial US goat milk yogurts (Park, 1994b). On the other hand, mineral contents of the samples in this study were comparable to those of commercial products despite some variations (Park, 1994b). Chemical composition of yogurt varies depending on the type of raw materials used, type of yogurt manufactured, and fortification methods, etc. As expected, the

Table 1 Chemical composition, titratable acidity (TA), and pH of goat milk yogurt samples Control Protein (%) Fat (%) Lactose (%) TS (%) Ash (%) Calcium (ppm) Phosphorus (ppm) Potassium (ppm) Magnesium (ppm) Sodium (ppm) Zinc (ppm) TA (%) pH

3.07 3.02 4.25 10.98 0.70 1047.90 894.70 1918.75 136.10 326.28 3.59 0.78 4.45

Values are mean ± S.D.

Treated with MTGase (2 U/g protein) ± ± ± ± ± ± ± ± ± ± ± ± ±

0.03 0.07 0.12 0.04 0.08 8.61 7.64 20.11 1.91 3.68 0.21 0.01 0.01

3.09 3.01 4.27 10.97 0.71 1114.91 956.00 2041.49 143.20 345.57 3.79 0.79 4.38

± ± ± ± ± ± ± ± ± ± ± ± ±

0.09 0.04 0.05 0.04 0.04 6.57 3.01 15.22 0.37 2.03 0.25 0.01 0.06

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Fig. 5. Changes of viable counts of L. acidophilus, Bifidobacteria, and L. casei in goat milk yogurt made from reduced fat (2%) homogenized and pasteurized goat milk (RFM) treated with MTGase (2 U/g protein) and from untreated RFM during cold storage. The cell numbers at day 0 represent viable counts at the end of the fermentation. Data are mean values of three experiments.

experimental data from this study indicate that application of MTGase in the production of goat milk yogurt will not significantly affect the nutritional value of the products. Moreover, no significant differences in titratable acidity and pH were also noted between control and MTGase-treated yogurt samples (Table 1). The changes in the viable counts of L. acidophilus, Bifidobacteria, and L. casei in probiotic goat milk yogurts during refrigerated storage are presented in Fig. 5. The probiotic cultures were relatively stable in the yogurt and their populations remained above 106 CFU/g up to 8 weeks at 4 ◦ C, suggesting that goat milk yogurt may be an excellent carrier for the probiotics. No differences (P > 0.05) in rate of change of population of all the three probiotic cultures were observed between the control and the MTGase-treated

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samples. When MTGase is used in goat milk yogurt preparation, milk was pretreated for 1 h with enzyme, which was subsequently inactivated before addition of the probiotic yogurt starter. This setup mimics conditions considered to be suitable for MTGase application for industrial yogurt manufacture. However, the enzyme can also be added to milk at the same time as the starter culture. In this case, the enzyme reaction proceeds during fermentation. Neve et al. (2001) have shown that if the enzyme is supplied simultaneously with the yogurt starter bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) without subsequent inactivation, an increased viability of the L. bulgaricus culture was observed during cold storage of the yogurt from bovine milk. Hence, it will be interesting to also study the effect of enzymatic cross-linking of milk proteins without inactivation of enzyme on the probiotic culture survivability, as well as functional properties of probiotic goat milk yogurt. At present, probiotics are increasingly incorporated into fermented dairy products. Because some probiotic strains grow slowly in milk, the usual practice is to add yogurt starter bacteria to enhance the fermentation process for making probiotic yogurt (Samona and Robinson, 1994). Neve et al. (2001) found that pretreatment of bovine milk with MTGase induces a minor imbalance of the yogurt starter bacteria during fermentation, whereas a long-term application of MTGase (without enzyme inactivation) in bovine yogurt results in a stabilizing effect on the viability of L. bulgaricus. Since probiotic goat milk yogurt contains cultures of S. thermophilus and L. bulgaricus, it may be of interest now to study the effect of an MTGase treatment on the growth behavior and viability of these conventional yogurt cultures during the production and storage of probiotic goat milk yogurt products. 3.4. Microstructure of goat milk yogurt SEM micrographs (×10,000) of yogurt gels prepared with and without MTGase are shown in Fig. 6. In general, the microstructures of the yogurt samples were similar in protein matrices composed of casein micelle chains and clusters. However, SEM revealed that the protein matrices of the MTGase treated samples appeared to be relatively more compact (denser) than

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Fig. 6. Scanning electron micrographs (×10,000) of goat milk yogurt from MTGase-treated (2 and 4 U/g protein) or untreated milk (C).

the control (Fig. 6). The results of imaging analysis by SigmaScan Pro confirmed that there was less open area in the yogurt gels with MTGase. The effect of MTGase on the microstructure is quite clear: the aggregates are denser, and a finer-meshed network that leads to improved gel strength and less syneresis. Thus, crosslinking by transglutaminase clearly inhibits the local phase separation which is present in a normal acidinduced casein gel (Schorsch et al., 2000b). A more dense network was formed at a 4 U/g protein enzyme level (compared to 2 U/g protein), which explains the stronger gel. SEM microstructure analysis confirmed that the higher strength of yogurt gels from cross-linked milk is due to a well organized protein network with smaller pores in the product (Faergemand and Qvist, 1997; Lorenzen et al., 2002; Schorsch et al., 2000b). 4. Conclusions Enzymatic cross-linking of milk proteins by MTGase appears to be an effective means for improving the functional properties of goat milk yogurt. The consistency of the goat milk yogurt is significantly improved by applying MTGase. Furthermore, whey separation could be reduced significantly by enzymatic cross-linking. SEM micrographs demonstrate that pretreatment of milk by MTGase could be used to improve the microstructure of the yogurt gel. Microbiological analysis shows that the enzymatic cross-linking of proteins seems to have a positive role in the survivability of the probiotic cultures, which requires further investigation. Results of this study indicate that MTGase treatment may be a useful method in the production of probiotic goat milk yogurt.

Acknowledgement We would like to thank Dr. R. Akuzawa for sending some references.

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