RESEARCH Blackwell Oxford, International IDT Society 1364-0307 54 of UK Dairy Publishing Journal Technology of Ltd Dairy2006 Technology
O R I G I NAL R E SEARCH
Syneresis in set yogurt as affected by EPS starter cultures
and levels of solids THANUT AMATAYAKUL ,1 F R ANK S HE R KAT 2 and NAGE NDR A P S H A H 1 * 1
School of Molecular Sciences, Victoria University, Werribee Campus, Melbourne, Victoria, Australia and 2School of Applied Sciences-Food Science, RMIT University, City Campus, Melbourne, Victoria, Australia
This study compared the pattern of whey separation determined by siphon, drainage and centrifugation method in set yogurts as affected by solid contents (9% and 14%) and exopolysaccharide (EPS)producing starter cultures. The level of whey separation determined by all three methods decreased as the total solids increased. There was a further decrease in whey separation in yogurts made using EPSproducing starter cultures as determined by the siphon method at both solid contents as compared with those made with non-EPS starter cultures. There was no difference in the level of whey separation as determined by the drainage method in products made at the same solids content (both 9% and 14%). A higher level of whey separation was observed in the product made using EPS starter cultures as determined by centrifugation method than that with non-EPS starter cultures at 9% solids. At 14% solids level, the values were higher in the product made by non-EPS starter cultures than that made with EPS starter cultures. Keywords Exopolysaccahride, Set yogurts, Total solids, Whey separation.
I N T RO D U C T I O N
*Author for correspondence. E-mail: nagendra.shah@vu.edu.au 2006 Society of Dairy Technology
Syneresis or spontaneous whey separation on the
surface of set yogurt is regarded as a defect. This problem can be reduced or eliminated by increasing the level of milk solids to 15% (Tamime and Deeth 1980; Shah 2003). Other alternatives include the use of stabilizers (i.e. starch, gelatine and vegetable gum) or exopolysaccharide (EPS)-producing starter cultures. Due to consumer awareness of natural products, the use of stabilizers is restricted in some countries. Generally, EPS produced by yogurt starter cultures are heteropolysaccharides and exist in two forms: capsular (which is attached to the bacterial cell surface) and ropy (which is excreted in the environment) (Cerning 1990; De Vuyst and Degeest 1999; Broadbent et al. 2003). In some cases, bacteria can produce both forms of EPS. Both capsular and ropy EPS possess high water-binding ability. The use of EPS-producing starter cultures helps decrease the level of whey separation in set yogurt (Wacher-Rodarte et al. 1993; Hassan et al. 1996; Jaros et al. 2002) and increase the moisture content in mozzarella cheeses (Perry et al. 1997). To determine the best strategy for preventing syneresis in set yogurts, a correct method for the determination of whey syneresis is essential. The drainage and centrifugation methods are commonly used to determine whey syneresis (Harwalkar and
Kalab 1986; Guzmn-Gonzlez et al. 1999, 2000;
Bhullar et al. 2002; Jaros et al. 2002). For the drainage method, a certain quantity of set yogurt (disturbed or undisturbed gel) is placed on a sieve over a certain period at a fixed temperature and the whey is allowed to drain under gravitational force. The whey separation is calculated as the percent weight of the separated whey over the initial weight of the gel. The centrifugation method is generally used to study the water-holding capacity of a product. A certain quantity of set yogurt (disturbed or undisturbed gel) is centrifuged at a specified speed over a certain period at a fixed temperature. The whey separation is calculated as the percent weight of the separated whey over the initial weight of the gel. Although these methods give high-precision results, they do not represent the actual value of spontaneous whey separation in set yogurt. The breakage of the yogurt gel as well as the presence of EPS may influence the result. Lucey et al. (1998) developed a method for the measurement of spontaneous whey separation in set yogurt fermented in a volumetric flask. This study compared the syneresis as determined by three methods including drainage, centrifugation and siphon methods in set yogurts made at 9% and 14% total solids using non-EPS, capsular EPS or ropy EPS starter cultures. The siphon method was developed based on Lucey et al. (1998) with some modifications.
*Author for correspondence. E-mail: nagendra.shah@vu.edu.au
216
Vol 59, No 3 August 2006 International Journal of Dairy Technology
Vol 59, No 3 August 2006
M AT E R I A L S A N D M E T H O D S
Micro-organisms Non-EPS-producing Streptococcus thermophilus ASCC 1342 and Lactobacillus delbrueckii ssp. bulgaricus ASCC 1466, capsular EPS-producing S. thermophilus ASCC 285, and ropy EPS-producing S. thermophilus ASCC 1275 were used in this study. These bacteria were previously obtained from the Australian Starter Culture Research Centre (Werribee, Australia) and characterized by Zisu and Shah (2003) for their EPS production. The stock cultures were maintained at 80C in 12% (w/w) sterile reconstituted skim milk (RSM) containing 20% (v/v) sterile glycerol. The microorganisms were activated by growing separately in 9% (w/w) or 14% (w/w) sterile RSM for 18 h. The process was repeated three times. Yogurt manufacture Low-heat skim milk powder (SMP; Murray Goulbourne Co-operative Co. Ltd, Brunswick, Australia) was used to prepare six batches of nonfat set yogurts at 9% and 14% (w/w) total solids with non-EPS, capsular EPS or ropy EPS starter cultures. The SMP was reconstituted and allowed to hydrate overnight at 4C, followed by heat treatment at 85C for 30 min, cooling to 42C and inoculation with 2% (v/v) of starter cultures. A combination of 1% (v/v) of L. delbrueckii ssp. bulgaricus ASCC 1466 with either 1% (v/v) of S. thermophilus ASCC 1342 or ASCC 285 or ASCC 1275 was used resulting in nonEPS, capsular EPS or ropy EPS yogurt, respectively. The inoculated mix was aseptically transferred into separate plastic containers at 100 mL quantities. The incubation was carried out at 42C until pH 4.7 was reached, then the products were transferred to a cold room (4C). Quantification of lactic acid The concentration of lactic acid in set yogurts was determined using a high-performance liquid chromatography (HPLC) unit (Varian, Varian Associates, Walnut Creek, CA, USA) comprising of a solvent delivery system (model 9100) connecting with an autosampler (model 9012), an ultraviolet (UV) light detector (model 9050) and an organic acid analysis column (Aminex HPX-87H, 300 7.8 mm, Bio-Rad Laboratory, Richmond, CA, USA). The method of Shin et al. (2000) was followed. A mobile phase used was 0.009 N H2SO4 with the flow rate of 0.6 mL/min and the temperature of the column was set at 65C. Lactic acid was detected at 220 nm. Five grams of the sample was mixed with 100 L of 15.8 N HNO3 and 5.9 mL of 0.009 N H2SO4 before centrifuging a 1.5-mL aliquot at 11 600 g for 15 min at room temperature. The supernatant was filtered through a 0.45-m membrane filter (Schleicher & Schvell, Dassel, Germany) and 2006 Society of Dairy Technology
50 L of the filtrate was injected into the HPLC
system. The lactic acid standard was purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA).
EPS purification and quantification
The method of separation and quantification of EPS in set yogurts was adapted from Zisu and Shah (2003). The proteins in 50 mL of diluted yogurt sample (1:1 yogurt : Mili-Q water; Millipore Corp, Bedford, MA, USA) were precipitated with 4 mL of 20% (w/v) trichloroacetic acid (SigmaAldrich Co., St. Louis, MO, USA) and separated by centrifugation (Sorvall RT 7, Kendro Instruments Australia Pty. Ltd, NSW, Australia) at 3313 g for 30 min (4C). The pH of the supernatant was adjusted to 6.8 with 40% (w/v) NaOH followed by boiling in a sealed container at 100C for 30 min to denature the whey proteins, which were separated by centrifugation (3313 g, 30 min, 4C). An equal volume of cold absolute ethanol was mixed with the supernatant and kept overnight at 4C to precipitate the carbohydrates, which were then separated by centrifugation (3313 g, 30 min, 4C). The resultant carbohydrate pellet was re-suspended in 10 mL of Milli-Q water and the suspension was sonicated for 1 h at room temperature using a sonication bath (FX 14PH sonication bath; Unisonics Pty Ltd, Sydney, Australia). The suspension was dialysed in a dialysis membrane tube of molecular weight cut-off value of 13 000 Da (Carolina Biological Supply Company, NC, USA) against tap water at 4C over 2 weeks. The water was changed twice a day. The EPS concentration in the suspension after dialysis was quantified using the phenol-sulphuric method of Dubois et al. (1956) and was expressed as glucose equivalent. Determination of spontaneous syneresis by the siphon method The level of spontaneous whey separation in undisturbed set yogurt was determined using a siphon method. The method was adapted from Lucey et al. (1998) in which the amount of spontaneous whey separated from milk gels fermented in a volumetric flask was measured. In our study, the method was modified in order to counteract the effects of the containers geometry and the difference in heat transfer between the volumetric flask and the yogurt cup. In our study, a cup of set yogurt was taken from the cold room (4C), weighed and kept at an angle of approximately 45 to allow whey collection at the side of the cup. A needle connected to a syringe was used to siphon the whey from the surface of the sample, and the cup of yogurt was weighed again. The siphon was carried out within 10 s to prevent further leakage of the whey from the gel. The syneresis was expressed as the percent weight of the whey over the initial weight of the yogurt sample. 217
Vol 59, No 3 August 2006
Table 1 The concentration of lactic acid in set yogurts made at 9% and 14% (w/w) total solids using non-EPS, capsular EPS and ropy EPS starter culture Total solids (%, w/w) 9 14
Lactic acid concentration (% w/w)c,d
Non-EPS 0.93 0.03 1.25 0.06b a
R E S U LT S
Capsular EPS
Ropy EPS
0.85 0.03 1.20 0.07a,b
0.89 0.02a,b 1.32 0.06a
Means of three replicate trials standard deviations.
Values in the same row do not share the same superscripts differ (P < 0.05, one-way ANOVA).
Table 2 The concentration of EPS in set yogurt made at 9% and 14% (w/w) total solids using non-EPS, capsular EPS and ropy EPS starter culture Total solids (%, w/w) 9 14
Lactic acid concentration (% w/w)c,d
Non-EPS
Capsular EPS
Ropy EPS
9.10 2.11b 20.77 3.05b
32.41 0.73a 114.63 10.34a
41.57 8.19a 109.80 9.25a
Mean of three replicate trials standard deviations.
Values in the same row do not share the same superscripts differ (P < 0.05, one-way ANOVA).
Determination of syneresis by the drainage
method A cup of set yogurt was taken out from the cold room and the surface whey was siphoned as in the siphon method. Approximately 30 g of the gel was cut in a single action by using a stainless steel ladle and the gel was weighed and drained on a 1 mm2 pore size sieve for 15 min at room temperature (20C). The whey was weighed and the syneresis was expressed as the percent weight of the whey separated from the gel over the initial weight of the gel. Determination of syneresis by the centrifugation method A cup of set yogurt removed from the cold room was stirred 20 times clockwise and anticlockwise with a glass rod. Approximately 30 g of the stirred yogurt was transferred into a 50-mL Blue max polypropylene conical tube (Becton Dickinson Laboratory, NJ, USA) using a 5-mL pipette and left at 4C for 2 h for stabilization. The stirred samples were then centrifuged (Sorvall RT 7) at 3313 g for 15 min at 10C. The separated whey was weighed. The syneresis was expressed as the percentage weight of the whey separated from the gel over the initial weight of the gel. Statistical analysis One-way analysis of variance (ANOVA) was used to analyse the data using Tukeys test for the multiple comparison test at 95% confidence level. The data 218
2006 Society of Dairy Technology
were analysed using spss version 10 for Windows
(SPSS Inc., Chicago, IL, USA).
Lactic acid concentration
The concentration of lactic acid in set yogurts made at 9% and 14% (w/w) solids using non-EPS, capsular EPS and ropy EPS starter cultures is shown in Table 1. In general, the lactic acid concentration was higher in the products made at 14% solids than that at 9%. The capsular EPS starter cultures produced the lowest amount of lactic acid in both 9% and 14% yogurts. EPS concentration The EPS concentration in set yogurts made at 9% and 14% total solids using non-EPS, capsular EPS and ropy EPS starter cultures is shown in Table 2. The 14% products had higher EPS concentration (100 mg/L) than those at 9% products (40 mg/ L). The level of EPS detected was at 9.10 and 20.77 mg/L in non-EPS yogurts made at 9% and 14% solids, respectively. At both 9% and 14% solids levels, the EPS concentration in products made by using capsular EPS and ropy EPS starter cultures was not significantly different (P < 0.05). Syneresis determined by the siphon method Figure 1 shows the levels of syneresis as determined by the siphon method in yogurts made at 9% and 14% solids using non-EPS, capsular EPS and ropy EPS starter cultures. At 9% solids, the yogurt made using EPS starter cultures showed a significantly lower level of syneresis (P < 0.05). The product made using capsular EPS starter cultures showed the lowest values. The level of whey separation in the product made at 14% solids using non-EPS starter cultures decreased significantly (P < 0.05) as compared to that at 9% solids. There was no significant difference (P < 0.05) in whey separation in the products made at 9% and 14% solids content with capsular and ropy starter cultures. Syneresis determined by the drainage method The level of syneresis determined by the drainage method in yogurts made at 9% and 14% solids using non-EPS, capsular EPS and ropy EPS starter cultures is shown in Figure 2. There was no difference in the level of syneresis among products made using nonEPS, capsular EPS or ropy EPS starter cultures. This was observed at both 9% and 14% solids levels. However, the level of syneresis decreased by approximately 25% when the total solids was increased to 14%. Syneresis determined by the centrifugation method The level of syneresis as determined by the centrifugation method in yogurts made at 9% and 14%
Vol 59, No 3 August 2006
made at 14% solids, the results showed the opposite
trend. The products made by using ropy EPS starter cultures had the lowest level of whey separation, whereas those made using non-EPS starter cultures had the highest level of whey separation. DISCUSSION
Figure 1 The level of spontaneous whey separation
determined by the siphon method in set yogurt made at 9% and 14% (w/w) total solids using non-EPS-, capsular EPS- or ropy EPS-producing starter cultures. n = 3; error bars represent standard deviation. Bars did not share the same letters differ (P < 0.05, one-way ANOVA).
Figure 2 The level of whey separation determined by the
drainage method in set yogurts made at 9% and 14% (w/w) total solids using non-EPS-, capsular EPS- or ropy EPSproducing starters. n = 3; error bars represent standard deviation. Bars did not share the same letters differ (P < 0.05, one-way ANOVA).
Figure 3 The level of whey separation determined by the
centrifugation method in set yogurts made at 9% and 14% (w/ w) total solids using non-EPS-, capsular EPS- or ropy EPSproducing starters. n = 3; error bars represent standard deviation. Bars did not share the same letters differ (P < 0.05, one-way ANOVA).
solids using non-EPS, capsular EPS and ropy EPS
starter cultures is shown in Figure 3. The yogurts made at 9% solids using non-EPS starter cultures had the lowest level of whey separation. The highest level of whey separation was detected in the product made using ropy EPS starter cultures. In yogurts 2006 Society of Dairy Technology
The increase in the concentration of EPS and lactic
acid in 14% yogurts compared with those at 9% suggested the influence of milk solids content on the activities of bacteria. Other workers have also shown that the increase in the concentration of available nutrients affected the EPS and lactic acid production by lactic acid bacteria (Amrane and Prigent 1998; Hassan et al. 2001; Zisu and Shah 2003). The difference in the patterns of syneresis detected from each method may suggest that these methods measured different data. The siphon method determines the level of spontaneous whey separated on the surface of gels. This method would represent the level of spontaneous syneresis in set yogurts. The drainage method measures the level of whey separated from cut gels under the influence of gravity. The centrifugation method measures the level of whey separated from the collapsed gels as a result of centrifugal force. Lucey and Singh (1997) suggested that the determination of whey collected from the drainage method is more relevant to measurement of whey separation in products such as cottage cheese than yogurt gels. The whey collected as a result of centrifugation would be influenced by other factors such as the rigidity and rheological properties of gels. In general, the level of whey separation in set yogurts as determined by the siphon method decreased due to an increase in solids content and the use of EPS starter cultures. This result is in agreement with that of others (Hassan et al. 1996; Jaros et al. 2002). The influence of EPS on syneresis disappeared as determined by the drainage method. This was in contrast with the results reported by others (Hassan et al. 1996; Jaros et al. 2002). They observed lower level of syneresis of unbroken yogurt gels made using EPS starter cultures as determined by the drainage method as compared with that made using non-EPS starter cultures. The difference between the results in our study and those of others (Hassan et al. 1996; Jaros et al. 2002) may be due to the leakage of whey at the cut surface. However, the level of syneresis decreased significantly (P < 0.05) in the products made at 14% solids. This was in agreement with the results of Jaros et al. (2002) and Harwalkar and Kalab (1986), who observed a reduction in whey separation of set yogurt when the total solids were increased. The level of syneresis as determined by the centrifugation method in the products made at 9% 219
Vol 59, No 3 August 2006
solids using EPS starter cultures was higher than
that made using non-EPS starter cultures. Hassan et al. (1995) observed larger pore sizes in the microstructure of set yogurts made using EPS starter cultures compared to those made using non-EPS starter cultures. In general, when mixing milk proteins and polysaccharides together at a certain concentration, the solution may show phase separation into milk protein-rich phase and polysaccharide-rich phase if they are incompatible. Hassan et al. (2002) observed that the EPS separated from the protein matrix after set yogurt was stirred and formed a large phase. In our study, the centrifugation method is likely to accelerate the phase separation between milk proteins and EPS in stirred yogurt made at 9% solids. This may explain the increase in whey separation in the products made using EPS starter cultures. Yogurts made at 14% solids with EPS starter cultures showed lower level of syneresis than those made with non-EPS starter cultures. Many factors may be responsible for those results including a decrease in the phase separation of milk protein EPS, an increase in EPS concentration causing an increase in viscosity as well as water adsorption as the total solids was elevated to 14% (Marshall and Rawson 1999). CONCLUSION The pattern of whey separation as determined by the siphon method, drainage method and centrifugation method was different. The level of syneresis could be influenced by method of determination as well as by the level of solids content and the type of starter culture. Therefore, it is important to select the right method for the determination of syneresis and to be cautious of other factors as well as the interpretation of the results. By comparing between the three methods, the siphon method would be more appropriate in the determination of the level of spontaneous whey separation on the surface of set yogurt. REFERENCES Amrane A and Prigent Y (1998) Lactic acid production rates during the different growth phases of Lactobacillus helveticus cultivated on whey supplemented with yeast extract. Biotechnology Letters 20 379383. Bhullar Y S, Uddin M A and Shah N P (2002) Effects of ingredients supplementation on textural characteristics and microstructure of yoghurt. Milchwissenschaft 57 328332. Broadbent J R, McMahon D J, Welker D L, Oberg C J and Moineau S (2003) Biochemistry, genetics, and applications of exopolysaccharide production in Streptococcus thermophilus: a review. Journal of Dairy Science 86 407423. Cerning J (1990) Exocellular polysaccharides produced by lactic acid bacteria. FEMS Microbiology Reviews 87 113 130.
220
2006 Society of Dairy Technology
De Vuyst L and Degeest B (1999) Heteropolysaccharides
from lactic acid bacteria. FEMS Microbiology Reviews 23 153177. Dubois M, Gilles K A, Hamilton J K, Rebers P A and Smith F (1956) Colorimetric method for determination of sugars and related substances. Analytical Chemistry 38 350 356. Guzmn-Gonzlez M, Morais F, Ramos M and Amigo L (1999) Influence of skimmed milk concentrate replacement by dry dairy products in a low-fat set-type yoghurt model system. I: Use of whey protein concentrates, milk protein concentrates and skimmed milk powder. Journal of the Science of Food and Agriculture 79 11171122. Guzmn-Gonzlez M, Morais F and Amigo L (2000) Influence of skimmed milk concentrate replacement by dry dairy products in a low-fat set-type yoghurt model system. II: Use of caseinates, co-precipitate and blended dairy powders. Journal of the Science of Food and Agriculture 80 433 438. Harwalkar V R and Kalab M (1986) Relationship between microstructure and susceptibility to whey separation in yoghurt made from reconstituted nonfat dry milk. Food Microstructure 5 287294. Hassan A N, Corredig M and Frank J F (2002) Capsule formation by nonropy starter cultures affects the viscoelastic properties of yogurt during structure formation. Journal of Dairy Science 85 716720. Hassan A N, Frank J F, Farmer M A, Schmidt K A and Shalabi S I (1995) Formation of yogurt microstructure and threedimensional visualization as determined by confocal scanning laser microscopy. Journal of Dairy Science 78 26292636. Hassan A N, Frank J F, Schmid K A and Shalabi S I (1996) Textural properties of yogurt made with encapsulated nonropy lactic cultures. Journal of Dairy Science 79 20982103. Hassan A N, Frank J F and Shalabi S I (2001) Factors affecting capsule size and production by lactic acid bacteria used as dairy starter cultures. International Journal of Food Microbiology 64 199203. Jaros D, Rohm H, Haque A and Kneifel W (2002) Influence of the starter cultures on the relationship between dry matter content and physical properties of set-style yogurt. Milchwissenschaft 57 325326. Lucey J A, Munro P A and Singh H (1998) Whey separation in acid milk gels made with glucono-delta-lactone: effects of heat treatment and gelation temperature. Journal of Texture Studies 29 413426. Lucey J A and Singh H (1997) Formation and physical properties of acid milk gels. Food Research International 30 529542. Marshall V M and Rawson H L (1999) Effects of exopolysaccharide-producing strains of thermophilic lactic acid bacteria on the texture of stirred yoghurt. International Journal of Food Science and Technology 34 137143. Perry D B, McMahon D J and Oberg C J (1997) Effect of exopolysaccharide-producing cultures on moisture retention in low-fat mozzarella cheese. Journal of Dairy Science 80 799805. Shah N P (2003) Yogurt: The product and its manufacture. In Encyclopedia of Food Science and Nutrition, Vol. 10, 2nd edn. Caballero B, Trugo L C and Finglas P M, eds. London: Academic Press.
Vol 59, No 3 August 2006
Shin H S, Lee Jh, Pestka J J and Ustunol Z (2000) Growth and
viability of commercial Bifidobacterium spp. in skim milk containing oligosaccharides and inulin. Journal of Food Science 65 884887. Tamime A Y and Deeth H C (1980) Yogurt: technology and biochemistry. Journal of Food Protection 43 939977. Wacher-Rodarte C, Galvan M V, Farres A, Gallardo F, Marshall V M and Garcia-Garibay M (1993) Yogurt
2006 Society of Dairy Technology
production from reconstituted skim milk powders using
different polymer and non-polymer-forming starter cultures. Journal of Dairy Research 60 247254. Zisu B and Shah N P (2003) Effects of pH, temperature, supplementation with whey protein concentrate, and adjunct cultures on the production of exopolysaccharides by Streptococcus thermophilus 1275. Journal of Dairy Science 86 34053415.
