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Effect of heat treatment and casein to whey protein ratio of skim milk on graininess and roughness of stirred yoghurt
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Food Research International 41 (2008) 165–171 www.elsevier.com/locate/foodres
Effect of heat treatment and casein to whey protein ratio of skim milk on graininess and roughness of stirred yoghurt A. Ku¨cu¨kcetin * Department of Food Engineering, Faculty of Agriculture, Akdeniz University, 07059 Antalya, Turkey Received 6 July 2007; accepted 4 November 2007
Abstract The aim of this work was to study how heat treatment and casein (CN) to whey protein (WP) ratio of skim milk affect physical characteristics of stirred yoghurt. Different heat treatments (95 °C/256 s, 110 °C/180 s and 130 °C/80 s) were applied to the yoghurt milk with the CN to WP ratios of 1.5:1, 2:1, 3:1 and 4:1. Physical properties, including graininess and roughness, of stirred yoghurt were determined during storage at 4 °C for 15 days. Visual roughness, number of grains, perimeter of grains, storage modulus, and yield stress decreased, when heating temperature or CN to WP ratio increased. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Stirred yoghurt; Physical characteristics; Heat treatment; Casein to whey protein ratio
1. Introduction Yoghurt represents a very significant dairy product around the world (Chandan, 2006). Since the 1950s, per capita consumption of fermented milks including yoghurt in most countries in the world has increased dramatically over the past three decades mainly due to the nutritional value and healthy aspects associated with these products (Tamime, 2004). Although there is great interest in the healthy-promoting properties of yoghurt, texture of stirred yoghurt plays an important quality and consumer acceptance (Lee & Lucey, 2004; Lucey, 2004). The texture of stirred yoghurt is influenced by milk composition, dry matter, heating, homogenization, type of starter culture, incubation temperature, cooling, storage time, etc. (Beal, Skokanova, Latrille, Martin, & Corrieu, 1999; Kessler, 2002; Martin, Skokanova, Latrille, Beal, & Corrieu, 1997; Tamime & Robinson, 1999; van Marle, 1998). Textural defects of stirred yoghurt like graininess (particle) or surface roughness (irregularities in the yoghurt *
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matrix) are undesirable as consumers expect smooth, uniform and fine-bodied products. Use of a high incubation temperature, excessive whey protein to casein ratio, certain types of starter culture and the use of excessive amounts of starter culture are associated with these types of defects (Lucey, 2004; Lucey & Singh, 1997). Although several works have been published determining the effects of technological process steps on physical properties of yoghurt (Beal et al., 1999; Krasaekoopt, Bhandari, & Deeth, 2004; Lee & Lucey, 2004; Sodini, Lucas, Oliveira, Remeuf, & Corrieu, 2002), interactions of the technological conditions regarding texture, have not been studied in detail. The objective of this research was to study the combined effects of heat treatment, casein/whey protein ratio, and storage time on the physical properties, including graininess and roughness, of stirred yoghurt. 2. Material and methods 2.1. Milk processing and yoghurt preparation Low heat skim milk powder, 36.1% (w/w) total protein, (BY 409 EG, Bayerische Milchindustrie eG, Landshut,
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Germany) and whey protein isolate, 93.5% (w/w) total protein (DSE 5627, NZMP, Wellington, New Zealand) were blended at suitable quantities in order to vary CN to WP ratios 4:1, 3:1, 2:1 and 1.5:1. Dry ingredients were blended to 11% total solids (w/w) and hydrated with distilled water overnight. The mean total protein content of milk blends was 4.5% (range, 4.3–4.8%). The standardized milk was heated at 95 °C for 256 s, at 110 °C for 180 s or at 130 °C for 80 s (at natural pH of milk blends) to denature the whey proteins to about 99%, and then subsequently cooled to 42 °C in the tubular heating equipment (200 L h1) of the Dairy for Research and Training of the University of Hohenheim (ASEPTO-Therm UHT-Pilotanlage, Asepto GmbH, Dinkelscherben, Germany). The temperature–time combinations for heat treatment were calculated based on kinetic data according to the Kessler (2002). After cooling, 0.1 g L1 of frozen pellets (starter culture Yo-Mix 621, Danisco A/S, Denmark) was added according to manufacture’s reference and the milk was incubated at 42 °C until pH decreased to 4.40. Fermentation was stopped by rapidly cooling to 4 °C in an ice-water bath. At the beginning of the cooling in an ice-water bath the yoghurt was manually stirred with a stainless-steel bored disk by up and down movements for almost 60 s. After setting the stirred product into 100 mL cups, the stirred yoghurt samples were stored at 4 °C for 15 days. The physical characteristics of the samples were analysed at days 1, and 15 of storage. 2.2. Physical properties measurements The total solids was determined according to method described in AOAC (1995). The protein content was determined using an FP-528 nitrogen/protein analyser (Leco Corporation, St. Joseph, MI, USA). The pH was measured by a Knick 765 pH meter (Knick Elektronische Messgerate GmbH & Co., Berlin, Germany). Syneresis in yoghurt samples was measured using a centrifugation method (Bhullar, Uddin, & Shah, 2002). At day 1 of the storage, yoghurt samples (m0 = 25 g) were poured into pre-weighed centrifuged tubes and centrifuged at 25 °C for 25 min at centrifugal force of 5031g. The supernatant (separated whey) was removed and weighed (m). The (w/w)% syneresis was calculated as m Syneresis ¼ 100 ð1Þ m0 2.3. Graininess The number and mean perimeter (diameter calculated from the measured boundary length) of grains as a measure for graininess were measured by image analysis. A glass plate (140 250 5 mm) was placed into a PVC frame and two metal bars with a height of 0.60 mm were fixed aside the plate. The yoghurt sample was poured onto the glass plate and spread evenly with a hand operated PVC plate to form a yoghurt layer of 0.60 mm thickness defined
by the metal bars. The glass plate with the yoghurt layer was put into a dark chamber on an illuminated plate. An image of the transmitted sample was taken with an in-built CCD camera (TFP-M/WL, LTF-Labortechnik, Wasserburg, Germany) with a resolution of 768 576 pixel and 256 grey scale color depth, effectively corresponding to 74-pixel cm1. The image analysis was performed with Optimas 6.1 software (Media Cybergenetics, Silver Spring, Maryland, USA). The software was set for the evaluation of 3 mL sample. The number of grains indicating a perimeter greater than 1.0 mm per 3 mL of yoghurt and the mean perimeter of grains were evaluated. All measurements were performed in duplicate. 2.4. Visual roughness The yoghurt sample was prepared for the visual roughness according to the same procedure described above for the measurement of graininess. The visual roughness (Rvis) of the spreaded yoghurt sample was taken as an additional descriptive parameter of the digital image made for graininess. The mean absolute intensity deviation of each pixel from a median smoothed intensity line through the picture was defined as Rvis of the yoghurt sample. The intensity distributions of five lines (each 450 pixels) given as greyscale values were extracted from the digital image and were evaluated corresponding to Eqs. (2) and (3). In order to integrate the deviation of the background brightness the i ) was calculated as the median of mean greyscale value (A +/74 pixels around each pixel i (Eq. (2)). The Rvis was calculated using Eq. (3). The results are given as the mean of three individually prepared images (Fig. 1). J is the number of evaluated pixels in Eq. (3). i ¼ medianfAi74 ; Aiþ74 g A J 1 X ij jAi A Rvis ¼ J i¼1
ð2Þ ð3Þ
2.5. Storage modulus and yield stress Storage modulus (G0 ) and yield stress of yoghurt samples were determined using vane methodology described by Baravian, Lalante, and Parker (2002). The vane consisted of four blades arranged at equal angles around a thin vertical shaft and was mounted on an rheometer, type AR 2000 from TA Instruments (New Castle, DE, USA). For the measurements vane geometry was placed in the undisturbed sample with the top edge of the blades parallel to the sample surface. The blade radius was 14.0 mm and the height of the cylindrical part was 40.0 mm. The lower end was formed into a 20° cone with the lower edge of each blade being sharpened; the diameter of the 100 mL sample glass was 44.6 mm. Due to a distribution of shear stress and shear rate the vane geometry was calibrated to calculate shear stress from measured torque and shear rate from angular velocity. The shear stress factor at the inner effec-
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25
2
random line 1-2
greyscale value Ai
20 1
2
15
50 10
deviation I Ai - Ai I
25
0
Image with 768 x 576 Pixels and 256 greyscale values
median smoothed value Ai
75
5
0
100
200
300
400
Deviation value in %
1
Greyscale value in 100%
100
0 500
Pixel number j
Fig. 1. Steps to evaluate the visual roughness (Rvis). The intensity distribution given in grey scale values of a randomly chosen line is extracted from the digital image. The absolute deviation is calculated as the deviation between the intensity distribution and the median smoothed value.
tive cylinder and the shear rate factor were calculated to be 2.42 104 Pa N1 m1 and 1.834 1 rad1, respectively, according to Baravian et al. (2002). Storage modulus was determined at 1 Hz at a temperature of 7 °C using the above factors. Subsequently yield stress (s0 was determined by increasing the angular velocity of the rotor logarithmically from 5 104 rad s1 to 100 rad s1 in 4 min with the same sample. The yield stress was calculated at the break from linear stress increase in the strain–torque plot according to Steffe (1996). 2.6. Statistical analyses All statistical calculations were performed using SAS Statistical Software (release for Windows, SAS Institute Inc., Cary, NC, USA). A three-factor ANOVA with two interactions was performed to determine the effects of heat treatment, casein/whey protein ratio and storage time on graininess, visual roughness and rheological properties of stirred yoghurt. A two-factor ANOVA with interaction was carried out to analyse the effect of heat treatment and casein/whey protein ratio on syneresis. Duncan’s multiple range test was conducted to detect differences among the treatment means. 3. Results and discussion 3.1. Physical characteristics The pH at the end of the incubation period was similar for the different yoghurts regardless of the applied treatment with an average pH of 4.4. The pH values decreased significantly (p < 0.05) in each yoghurt sample during the storage at 4 °C for 15 days (results not shown). The pH values of the samples ranged from 3.88 to 3.99, by day 15. Effects of technological conditions on syneresis of yoghurt are presented in Fig. 2. The syneresis of the samples varied from 64.9% to 79.5%. As shown in Table 1, heat
treatment and ratio of CN to WP significantly (p 6 0.01) affected syneresis. Syneresis of yoghurt decreases progressively with an increase in the degree of whey protein denaturation, but above 90% denaturation this trend is less pronounced than in yoghurt obtained from milk containing 690% of heatdenatured whey protein. Furthermore, in yoghurt with the same degree of whey protein denaturation produced by heating, syneresis was lower in those made from heated at 85 °C rather than 130 °C (Kessler, 1997). Heating conditions (95 °C/256 s, 110 °C/180 s and 130 °C/80 s) chosen for our experiments ensure more than 90% whey protein denaturation in the yoghurt milk. Syneresis of yoghurt obtained from milk heated at 95 °C for 256 s was significantly (p < 0.05) lower than that from milk heated at 110 °C for 180 s or at 130 °C for 80 s (Table 1). The results of the current study agree with that from Kessler (1997). The levels of syneresis decreased as the ratios of CN to WP decreased (Table 1). This result was similar to that reported by Amatayakul, Halmos, Sherkat, and Shah (2006) and Puvanenthiran, Williams, and Augustin (2002). Additionally, the protein content (4.8%) of milk blend with low ratio of CN to WP (1.5:1) was higher than those with other ratios of CN to WP. Sodini, Remeuf, Haddad, and Corrieu (2004) underlined a clear positive effect of a high protein to total solids ratio of milk on yoghurt water-holding capacity. The visual roughness of the day 1 sample is shown in Fig. 2. The visual roughness was influenced by heat treatment, and ratio of CN to WP (p < 0.001). No effect of storage was detected (p > 0.05). The interaction between heat treatment and ratio of CN to WP was highly significant (p < 0.001). The visual roughness of yoghurt samples decreased significantly (p < 0.05) when heating temperature or ratio of CN to WP increased (Table 1). Moreover, the increase in visual roughness of yoghurt made with milk blend at a low CN to WP ratio (1.5:1) is due probably to the increase in the protein content of the milk blend.
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50
30 50 20
Rvis (unit of AIDm)
Syneresis (%) (w/w)
40
10
1.5:1
2:1
3:1
4:1
1.5:1
2:1
3:1
4:1
1.5:1
2:1
3:1
4:1
0
0 95°C / 256 s
110°C / 180 s
130°C / 80 s
Fig. 2. Syneresis (%) (w/w) ( ) and visual roughness (Rvis) (unit of AIDm) (h) of yoghurts obtained from different heat-treated milk with altered casein to whey protein ratios after 1 day of storage.
Table 1 Effect of the heat treatment (HT), the casein to whey protein ratio (CN/WP) and the storage time (ST) on the characteristics of stirred yoghurt Syneresis (%) (w/w)
RvisA (unit of AIDm)
Number of grains 3 mL1
PG (mm)
G0 (Pa)
Heat treatment 95 °C/256 s 110 °C/180 s 130 °C/80 s
71.9 ± 6.1bB 73.3 ± 5.3a 74.3 ± 5.1a
16.0 ± 18.0a 13.7 ± 15.0b 1.4 ± 0.2c
207 ± 180a 136 ± 130b 8 ± 13c
3.6 ± 1.2a 2.5 ± 0.8b 1.3 ± 0.9c
349.9 ± 157.0a 272.3 ± 122.4b 133.8 ± 62.4c
65.4 ± 48.0a 55.4 ± 43.7b 20.2 ± 11.0c
CN/WP 1.5:1 2:1 3:1 4:1
67.2 ± 1.6d 70.4 ± 1.9c 75.9 ± 1.0b 79.3 ± 0.5a
24.5 ± 19.7a 12.1 ± 8.5b 1.5 ± 0.1c 1.4 ± 0.1c
274 ± 185a 146 ± 121b 40 ± 30c 8 ± 7d
3.7 ± 1.4a 2.8 ± 0.9b 2.0 ± 0.9c 1.3 ± 0.9d
392.6 ± 127.1a 308.0 ± 147.4b 190.9 ± 77.5c 116.5 ± 34.4d
101.8 ± 49.6a 41.3 ± 18.6b 26.4 ± 9.8c 18.5 ± 6.1d
Storage time (days) 1 – 15 –
10.8 ± 15.4a 9.9 ± 14.2a
115 ± 150a 119 ± 154a
2.4 ± 1.3a 2.5 ± 1.4a
238.8 ± 142.9b 265.1 ± 155.7a
44.4 ± 39.4b 49.6 ± 45.4a
ANOVA HT CN/WP ST HT CN/WP HT ST CN/WP ST
*** *** n.s. *** n.s. n.s.
*** *** n.s. *** n.s. n.s.
*** *** n.s. ** n.s. n.s.
*** *** *** *** ** n.s.
A B C D E
**C *** n.d.D n.s.E n.d. n.d.
Yield stress (Pa)
*** *** ** *** n.s. n.s.
Rvis, visual roughness; AIDm, mean absolute intensity deviation; PG, mean perimeter of grains. Values are expressed mean ± standard deviation, different letters after values indicate significant differences; Duncan0 s multiple range test (P < 0.05). *, **, ***, significantly different at p < 0.05, p < 0.01, and p < 0.001, respectively. n.d., not determined. n.s., not significant.
The number and the mean perimeter of grains of the day 1 yoghurt varied from 0 to 442 per 3 mL of the sample and from 0.0 to 5.2 mm, respectively, according to heat treatment and ratio of CN to WP (Fig. 3). The number of grains was influenced strongly by the heat treatment and ratio of
CN to WP (p < 0.001) (Table 1). The effect of storage was not significant (p > 0.05). There were significant differences in the number and the mean perimeter of grains among yoghurts obtained from selected heat treatments. Furthermore, the number and
A. Ku¨cu¨kcetin / Food Research International 41 (2008) 165–171
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500
10
450 8
350 300
6
250 200
4
150 100
Mean perimeter of grains (mm)
Numbers of grains 3mL-1
400
2
50 1.5:1
2:1
3:1
4:1
1.5:1
2:1
3:1
4:1
1.5:1
2:1
3:1
0
4:1 0
95°C / 256 s
110°C / 180 s
130°C / 80 s
Fig. 3. Number of grains 3 mL1 ( ) and mean perimeter of grains (mm) (h) of yoghurts obtained from different heat-treated milk with altered casein to whey protein ratios after 1 day of storage.
the perimeter of grains in yoghurt obtained from milk heated at 95 °C for 256 s was significantly (p < 0.05) higher than that from milk heated at 110 °C for 180 s or at 130 °C for 80 s (Table 1). At higher heat intensities, the precipitation of a-lactalbumin onto the micelle fills the gap formed by the b-lactoglobulin filaments, reduces the surface hydrophobicity, and causes a smoother micellar surface (Sodini et al., 2004). The number and the mean perimeter of grains of the day 1 yoghurt decreased significantly (p < 0.05) when ratio of CN to WP increased (Table 1). According to these results, graininess seemed to be linked to the amount of denatured whey protein and the ratio casein-to-denatured whey protein in the milk base. A similar trend was already reported by Rasmussen, Janhoj, and Ipsen (2007) and Remeuf, Mohammed, Sodini, and Tissier (2003). Lucey (2004) and Lucey and Singh (1997) reported that one of common reasons for occurrence of graininess was excessive whey protein to casein ratio. However, yoghurt with a high level protein, which made with milk blend at a low CN to WP ratio (1.5:1), was grainier than others. The graininess of the stirred yoghurt has also been found to depend on the level of protein in milk blends, which is consistent with results reported by Rasmussen et al. (2007). 3.2. Rheological properties The storage modulus values of the day 1 and day 15 stirred yoghurt samples are presented in Fig. 4. As shown in Table 1, G0 was significantly influenced by the heat treatment, ratio of CN to WP and storage time (p < 0.001). The interactions between heat treatment and ratio of CN
to WP and between heat treatment and storage time, were significant (p 6 0.01), whereas the interaction between ratio of CN to WP and storage time was not significant (p > 0.05). The G0 value increased significantly (p < 0.05) when CN to WP ratio was decreased (Table 1); a similar trend was reported by Lucey, Munro, and Singh (1999). The G0 value was higher in yoghurt made with the milk blend at a low CN to WP ratio (1.5:1) probably because of a higher level of protein (4.8%) in the milk blend with low ratio of CN to WP than in other milk blends. The G0 value decreased significantly (p < 0.05) when heating temperature was increased. During storage at 4 °C, G0 values increased for all yoghurt samples. These results are agreements with those obtained by Gustaw, Glibowski, and Mleko (2006). The yield stress of the day 1 and day 15 samples varied from 135.2 to 11.5 Pa and from 146.7 to 12.1 Pa, respectively, according to the technological conditions (Fig. 5). The yield stress was significantly affected by the heat treatment, ratio of CN to WP and storage time (p 6 0.01) (Table 1). The effect of the heat treatment, ratio of CN to WP and storage time on the yield stress in yoghurts was almost similar to that on G0 . The interaction between heat treatment and ratio of CN to WP was highly significant (p < 0.001), whereas the interactions between heat treatment and storage time and between ratio of CN to WP and storage time were not significant (p > 0.05). The yield stress significantly (p < 0.05) increased as heating temperature decreased, ratio of CN to WP decreased, or storage time increased (Table 1). The difference in the yield stress values among the yoghurts made from the milk
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A. Ku¨cu¨kcetin / Food Research International 41 (2008) 165–171 700
600
500
G' (Pa)
400
300
200
100
1.5:1
2:1
3:1
4:1
2:1
1.5:1
3:1
4:1
1.5:1
2:1
3:1
4:1
0 95˚C / 256 s
110˚C / 180 s
130˚C / 80 s
Fig. 4. G0 (Pa) of yoghurts obtained from different heat-treated milk with altered casein to whey protein ratios after 1 (h) and 15 ( ) days of storage.
200
Yield stress (Pa)
150
100
50
1.5:1
2:1
3:1
4:1
2:1
1.5:1
3:1
4:1
1.5:1
2:1
3:1
4:1
0 95°C / 256 s
110°C / 180 s
130°C / 80 s
Fig. 5. Yield stress (Pa) of yoghurts obtained from different heat-treated milk with altered casein to whey protein ratios after 1 (h) and 15 ( ) days of storage.
blends with different ratios of CN to WP, as observed in this study, may be due to difference in the protein contents of the milk blends. 4. Conclusions This study has shown that heat treatment and ratio of CN to WP of skim milk affect the physical properties of
yoghurt. As heating temperature or CN to WP ratio increased, the number of grains, perimeter of grains, visual roughness, G0 and yield stress decreased. For practical applications, heat treatment and ratio of CN to WP of skim milk can be optimized to improve quality or modified to create fermented milk products with different physical properties. Further work is addressed to analyse the composition of the grains and understand the mechanism of formation.
A. Ku¨cu¨kcetin / Food Research International 41 (2008) 165–171
Acknowledgements The author would like to thank Prof. Dr. Jo¨rg Hinrichs and Mr. Konrad Weidendorfer (Department of Animal Foodstuff Technology, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany), Alexander von Humboldt Stiftung (Germany), and The Scientific Research Projects Administration Unit of Akdeniz University (Turkey). References Amatayakul, T., Halmos, A. L., Sherkat, F., & Shah, N. P. (2006). Physical characteristics of yoghurts made using exopolysaccharideproducing starter cultures and varying casein to whey protein ratios. International Dairy Journal, 16, 40–51. AOAC (1995). Official methods of analysis of AOAC international (16th ed.). Arling, VA: AOAC International. Baravian, C., Lalante, A., & Parker, A. (2002). Vane rheometry with a large, finite gap. Applied Rheology, 12, 81–87. Beal, C., Skokanova, J., Latrille, E., Martin, N., & Corrieu, G. (1999). Combined effects of culture conditions and storage time on acidification and viscosity of stirred yogurt. Journal of Dairy Science, 82, 673–681. Bhullar, Y. S., Uddin, M. A., & Shah, N. P. (2002). Effects of ingredients supplementation on textural characteristics and microstructure of yoghurt. Milchwissenschaft, 57, 328–332. Chandan, R. C. (2006). History and consumption trends. In R. C. Chandan, C. H. White, A. Kilara, & Y. H. Hui (Eds.), Manufacturing Yogurt and Fermented Milks (pp. 3–15). Oxford: Blackwell Science Ltd. Gustaw, W., Glibowski, P., & Mleko, S. (2006). The rheological properties of yoghurt with incorporated whey protein aggregates/polymers. Milchwissenschaft, 61, 415–419. Kessler, H. G. (1997). The structure of fermented milk products as influenced by technology and composition. In Texture of fermented milk products and dairy desserts, International dairy federation symposium, Vicenza, Italy, 5–6 May 1997 (pp. 93–105). Brussels. Kessler, H. G. (2002). Food and bio process engineering–dairy technology (5th ed.). Mu¨nchen, Germany: Verlag A. Kessler, p. 694. Krasaekoopt, W., Bhandari, B., & Deeth, H. (2004). Comparison of texture of yogurt made from conventionally treated milk and UHT
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milk fortified with low-heat skim milk powder. Journal of Food Science, 69, 276–280. Lee, W. J., & Lucey, J. A. (2004). Structure and physical properties of yoghurt gels: Effect of inoculation rate and incubation temperature. Journal of Dairy Science, 87, 3153–3164. Lucey, J. A. (2004). Cultured dairy products: an overview of their gelation and texture properties. International Journal of Dairy Technology, 57, 77–84. Lucey, J. A., & Singh, H. (1997). Formation and physical properties of acid milk gels: a review. Food Research International, 30, 529–542. Lucey, J. A., Munro, P. A., & Singh, H. (1999). Effects of heat treatment and whey protein addition on the rheological properties and structure of acid skim milk gels. International Dairy Journal, 9, 275–279. Martin, N., Skokanova, J., Latrille, E., Beal, C., & Corrieu, G. (1997). Sensory and instrumental characterization of the texture of stirred yoghurt. In Texture of fermented milk products and dairy desserts, International dairy federation symposium, Vicenza, Italy, 5–6 May 1997 (pp. 24–33). Brussels. Puvanenthiran, A., Williams, R. P. W., & Augustin, M. A. (2002). Structure and visco-elastic properties of set yoghurt with altered casein to whey protein ratios. International Dairy Journal, 12, 383–391. Rasmussen, M. A., Janhoj, Th., & Ipsen, R. (2007). Effect of fat, protein and shear on graininess, viscosity and syneresis in low-fat stirred yoghurt. Milchwissenschaft, 62, 54–58. 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, 773–782. 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, 2479–2488. Sodini, I., Remeuf, F., Haddad, S., & Corrieu, G. (2004). The relative effect of milk base, starter, and process on yogurt texture: A review. Critical Reviews in Food Sciences and Nutrition, 44, 113–137. Steffe, J. F. (1996). Rheological methods in food process engineering, East Lansing. USA: Freeman Press. Tamime, A. Y. (2004). Yoghurt-latest developments and bridging the link with industry. In International dairy symposium, ‘‘Recent developments in dairy science and technology”, 24–28 May, Isparta, Turkey (1 pp.). Tamime, A. Y., & Robinson, R. K. (1999). Yoghurt science and technology (2nd ed.). Woolhead Publishing Limited: Cambridge, England, p. 619. van Marle, M. E. (1998). Structure and rheological properties of yoghurt gels and stirred yoghurts. Enschede, The Netherlands. PhD thesis, University of Twente, p. 153.
Food Research International 41 (2008) 165–171 www.elsevier.com/locate/foodres
Effect of heat treatment and casein to whey protein ratio of skim milk on graininess and roughness of stirred yoghurt A. Ku¨cu¨kcetin * Department of Food Engineering, Faculty of Agriculture, Akdeniz University, 07059 Antalya, Turkey Received 6 July 2007; accepted 4 November 2007
Abstract The aim of this work was to study how heat treatment and casein (CN) to whey protein (WP) ratio of skim milk affect physical characteristics of stirred yoghurt. Different heat treatments (95 °C/256 s, 110 °C/180 s and 130 °C/80 s) were applied to the yoghurt milk with the CN to WP ratios of 1.5:1, 2:1, 3:1 and 4:1. Physical properties, including graininess and roughness, of stirred yoghurt were determined during storage at 4 °C for 15 days. Visual roughness, number of grains, perimeter of grains, storage modulus, and yield stress decreased, when heating temperature or CN to WP ratio increased. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Stirred yoghurt; Physical characteristics; Heat treatment; Casein to whey protein ratio
1. Introduction Yoghurt represents a very significant dairy product around the world (Chandan, 2006). Since the 1950s, per capita consumption of fermented milks including yoghurt in most countries in the world has increased dramatically over the past three decades mainly due to the nutritional value and healthy aspects associated with these products (Tamime, 2004). Although there is great interest in the healthy-promoting properties of yoghurt, texture of stirred yoghurt plays an important quality and consumer acceptance (Lee & Lucey, 2004; Lucey, 2004). The texture of stirred yoghurt is influenced by milk composition, dry matter, heating, homogenization, type of starter culture, incubation temperature, cooling, storage time, etc. (Beal, Skokanova, Latrille, Martin, & Corrieu, 1999; Kessler, 2002; Martin, Skokanova, Latrille, Beal, & Corrieu, 1997; Tamime & Robinson, 1999; van Marle, 1998). Textural defects of stirred yoghurt like graininess (particle) or surface roughness (irregularities in the yoghurt *
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matrix) are undesirable as consumers expect smooth, uniform and fine-bodied products. Use of a high incubation temperature, excessive whey protein to casein ratio, certain types of starter culture and the use of excessive amounts of starter culture are associated with these types of defects (Lucey, 2004; Lucey & Singh, 1997). Although several works have been published determining the effects of technological process steps on physical properties of yoghurt (Beal et al., 1999; Krasaekoopt, Bhandari, & Deeth, 2004; Lee & Lucey, 2004; Sodini, Lucas, Oliveira, Remeuf, & Corrieu, 2002), interactions of the technological conditions regarding texture, have not been studied in detail. The objective of this research was to study the combined effects of heat treatment, casein/whey protein ratio, and storage time on the physical properties, including graininess and roughness, of stirred yoghurt. 2. Material and methods 2.1. Milk processing and yoghurt preparation Low heat skim milk powder, 36.1% (w/w) total protein, (BY 409 EG, Bayerische Milchindustrie eG, Landshut,
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Germany) and whey protein isolate, 93.5% (w/w) total protein (DSE 5627, NZMP, Wellington, New Zealand) were blended at suitable quantities in order to vary CN to WP ratios 4:1, 3:1, 2:1 and 1.5:1. Dry ingredients were blended to 11% total solids (w/w) and hydrated with distilled water overnight. The mean total protein content of milk blends was 4.5% (range, 4.3–4.8%). The standardized milk was heated at 95 °C for 256 s, at 110 °C for 180 s or at 130 °C for 80 s (at natural pH of milk blends) to denature the whey proteins to about 99%, and then subsequently cooled to 42 °C in the tubular heating equipment (200 L h1) of the Dairy for Research and Training of the University of Hohenheim (ASEPTO-Therm UHT-Pilotanlage, Asepto GmbH, Dinkelscherben, Germany). The temperature–time combinations for heat treatment were calculated based on kinetic data according to the Kessler (2002). After cooling, 0.1 g L1 of frozen pellets (starter culture Yo-Mix 621, Danisco A/S, Denmark) was added according to manufacture’s reference and the milk was incubated at 42 °C until pH decreased to 4.40. Fermentation was stopped by rapidly cooling to 4 °C in an ice-water bath. At the beginning of the cooling in an ice-water bath the yoghurt was manually stirred with a stainless-steel bored disk by up and down movements for almost 60 s. After setting the stirred product into 100 mL cups, the stirred yoghurt samples were stored at 4 °C for 15 days. The physical characteristics of the samples were analysed at days 1, and 15 of storage. 2.2. Physical properties measurements The total solids was determined according to method described in AOAC (1995). The protein content was determined using an FP-528 nitrogen/protein analyser (Leco Corporation, St. Joseph, MI, USA). The pH was measured by a Knick 765 pH meter (Knick Elektronische Messgerate GmbH & Co., Berlin, Germany). Syneresis in yoghurt samples was measured using a centrifugation method (Bhullar, Uddin, & Shah, 2002). At day 1 of the storage, yoghurt samples (m0 = 25 g) were poured into pre-weighed centrifuged tubes and centrifuged at 25 °C for 25 min at centrifugal force of 5031g. The supernatant (separated whey) was removed and weighed (m). The (w/w)% syneresis was calculated as m Syneresis ¼ 100 ð1Þ m0 2.3. Graininess The number and mean perimeter (diameter calculated from the measured boundary length) of grains as a measure for graininess were measured by image analysis. A glass plate (140 250 5 mm) was placed into a PVC frame and two metal bars with a height of 0.60 mm were fixed aside the plate. The yoghurt sample was poured onto the glass plate and spread evenly with a hand operated PVC plate to form a yoghurt layer of 0.60 mm thickness defined
by the metal bars. The glass plate with the yoghurt layer was put into a dark chamber on an illuminated plate. An image of the transmitted sample was taken with an in-built CCD camera (TFP-M/WL, LTF-Labortechnik, Wasserburg, Germany) with a resolution of 768 576 pixel and 256 grey scale color depth, effectively corresponding to 74-pixel cm1. The image analysis was performed with Optimas 6.1 software (Media Cybergenetics, Silver Spring, Maryland, USA). The software was set for the evaluation of 3 mL sample. The number of grains indicating a perimeter greater than 1.0 mm per 3 mL of yoghurt and the mean perimeter of grains were evaluated. All measurements were performed in duplicate. 2.4. Visual roughness The yoghurt sample was prepared for the visual roughness according to the same procedure described above for the measurement of graininess. The visual roughness (Rvis) of the spreaded yoghurt sample was taken as an additional descriptive parameter of the digital image made for graininess. The mean absolute intensity deviation of each pixel from a median smoothed intensity line through the picture was defined as Rvis of the yoghurt sample. The intensity distributions of five lines (each 450 pixels) given as greyscale values were extracted from the digital image and were evaluated corresponding to Eqs. (2) and (3). In order to integrate the deviation of the background brightness the i ) was calculated as the median of mean greyscale value (A +/74 pixels around each pixel i (Eq. (2)). The Rvis was calculated using Eq. (3). The results are given as the mean of three individually prepared images (Fig. 1). J is the number of evaluated pixels in Eq. (3). i ¼ medianfAi74 ; Aiþ74 g A J 1 X ij jAi A Rvis ¼ J i¼1
ð2Þ ð3Þ
2.5. Storage modulus and yield stress Storage modulus (G0 ) and yield stress of yoghurt samples were determined using vane methodology described by Baravian, Lalante, and Parker (2002). The vane consisted of four blades arranged at equal angles around a thin vertical shaft and was mounted on an rheometer, type AR 2000 from TA Instruments (New Castle, DE, USA). For the measurements vane geometry was placed in the undisturbed sample with the top edge of the blades parallel to the sample surface. The blade radius was 14.0 mm and the height of the cylindrical part was 40.0 mm. The lower end was formed into a 20° cone with the lower edge of each blade being sharpened; the diameter of the 100 mL sample glass was 44.6 mm. Due to a distribution of shear stress and shear rate the vane geometry was calibrated to calculate shear stress from measured torque and shear rate from angular velocity. The shear stress factor at the inner effec-
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25
2
random line 1-2
greyscale value Ai
20 1
2
15
50 10
deviation I Ai - Ai I
25
0
Image with 768 x 576 Pixels and 256 greyscale values
median smoothed value Ai
75
5
0
100
200
300
400
Deviation value in %
1
Greyscale value in 100%
100
0 500
Pixel number j
Fig. 1. Steps to evaluate the visual roughness (Rvis). The intensity distribution given in grey scale values of a randomly chosen line is extracted from the digital image. The absolute deviation is calculated as the deviation between the intensity distribution and the median smoothed value.
tive cylinder and the shear rate factor were calculated to be 2.42 104 Pa N1 m1 and 1.834 1 rad1, respectively, according to Baravian et al. (2002). Storage modulus was determined at 1 Hz at a temperature of 7 °C using the above factors. Subsequently yield stress (s0 was determined by increasing the angular velocity of the rotor logarithmically from 5 104 rad s1 to 100 rad s1 in 4 min with the same sample. The yield stress was calculated at the break from linear stress increase in the strain–torque plot according to Steffe (1996). 2.6. Statistical analyses All statistical calculations were performed using SAS Statistical Software (release for Windows, SAS Institute Inc., Cary, NC, USA). A three-factor ANOVA with two interactions was performed to determine the effects of heat treatment, casein/whey protein ratio and storage time on graininess, visual roughness and rheological properties of stirred yoghurt. A two-factor ANOVA with interaction was carried out to analyse the effect of heat treatment and casein/whey protein ratio on syneresis. Duncan’s multiple range test was conducted to detect differences among the treatment means. 3. Results and discussion 3.1. Physical characteristics The pH at the end of the incubation period was similar for the different yoghurts regardless of the applied treatment with an average pH of 4.4. The pH values decreased significantly (p < 0.05) in each yoghurt sample during the storage at 4 °C for 15 days (results not shown). The pH values of the samples ranged from 3.88 to 3.99, by day 15. Effects of technological conditions on syneresis of yoghurt are presented in Fig. 2. The syneresis of the samples varied from 64.9% to 79.5%. As shown in Table 1, heat
treatment and ratio of CN to WP significantly (p 6 0.01) affected syneresis. Syneresis of yoghurt decreases progressively with an increase in the degree of whey protein denaturation, but above 90% denaturation this trend is less pronounced than in yoghurt obtained from milk containing 690% of heatdenatured whey protein. Furthermore, in yoghurt with the same degree of whey protein denaturation produced by heating, syneresis was lower in those made from heated at 85 °C rather than 130 °C (Kessler, 1997). Heating conditions (95 °C/256 s, 110 °C/180 s and 130 °C/80 s) chosen for our experiments ensure more than 90% whey protein denaturation in the yoghurt milk. Syneresis of yoghurt obtained from milk heated at 95 °C for 256 s was significantly (p < 0.05) lower than that from milk heated at 110 °C for 180 s or at 130 °C for 80 s (Table 1). The results of the current study agree with that from Kessler (1997). The levels of syneresis decreased as the ratios of CN to WP decreased (Table 1). This result was similar to that reported by Amatayakul, Halmos, Sherkat, and Shah (2006) and Puvanenthiran, Williams, and Augustin (2002). Additionally, the protein content (4.8%) of milk blend with low ratio of CN to WP (1.5:1) was higher than those with other ratios of CN to WP. Sodini, Remeuf, Haddad, and Corrieu (2004) underlined a clear positive effect of a high protein to total solids ratio of milk on yoghurt water-holding capacity. The visual roughness of the day 1 sample is shown in Fig. 2. The visual roughness was influenced by heat treatment, and ratio of CN to WP (p < 0.001). No effect of storage was detected (p > 0.05). The interaction between heat treatment and ratio of CN to WP was highly significant (p < 0.001). The visual roughness of yoghurt samples decreased significantly (p < 0.05) when heating temperature or ratio of CN to WP increased (Table 1). Moreover, the increase in visual roughness of yoghurt made with milk blend at a low CN to WP ratio (1.5:1) is due probably to the increase in the protein content of the milk blend.
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50
30 50 20
Rvis (unit of AIDm)
Syneresis (%) (w/w)
40
10
1.5:1
2:1
3:1
4:1
1.5:1
2:1
3:1
4:1
1.5:1
2:1
3:1
4:1
0
0 95°C / 256 s
110°C / 180 s
130°C / 80 s
Fig. 2. Syneresis (%) (w/w) ( ) and visual roughness (Rvis) (unit of AIDm) (h) of yoghurts obtained from different heat-treated milk with altered casein to whey protein ratios after 1 day of storage.
Table 1 Effect of the heat treatment (HT), the casein to whey protein ratio (CN/WP) and the storage time (ST) on the characteristics of stirred yoghurt Syneresis (%) (w/w)
RvisA (unit of AIDm)
Number of grains 3 mL1
PG (mm)
G0 (Pa)
Heat treatment 95 °C/256 s 110 °C/180 s 130 °C/80 s
71.9 ± 6.1bB 73.3 ± 5.3a 74.3 ± 5.1a
16.0 ± 18.0a 13.7 ± 15.0b 1.4 ± 0.2c
207 ± 180a 136 ± 130b 8 ± 13c
3.6 ± 1.2a 2.5 ± 0.8b 1.3 ± 0.9c
349.9 ± 157.0a 272.3 ± 122.4b 133.8 ± 62.4c
65.4 ± 48.0a 55.4 ± 43.7b 20.2 ± 11.0c
CN/WP 1.5:1 2:1 3:1 4:1
67.2 ± 1.6d 70.4 ± 1.9c 75.9 ± 1.0b 79.3 ± 0.5a
24.5 ± 19.7a 12.1 ± 8.5b 1.5 ± 0.1c 1.4 ± 0.1c
274 ± 185a 146 ± 121b 40 ± 30c 8 ± 7d
3.7 ± 1.4a 2.8 ± 0.9b 2.0 ± 0.9c 1.3 ± 0.9d
392.6 ± 127.1a 308.0 ± 147.4b 190.9 ± 77.5c 116.5 ± 34.4d
101.8 ± 49.6a 41.3 ± 18.6b 26.4 ± 9.8c 18.5 ± 6.1d
Storage time (days) 1 – 15 –
10.8 ± 15.4a 9.9 ± 14.2a
115 ± 150a 119 ± 154a
2.4 ± 1.3a 2.5 ± 1.4a
238.8 ± 142.9b 265.1 ± 155.7a
44.4 ± 39.4b 49.6 ± 45.4a
ANOVA HT CN/WP ST HT CN/WP HT ST CN/WP ST
*** *** n.s. *** n.s. n.s.
*** *** n.s. *** n.s. n.s.
*** *** n.s. ** n.s. n.s.
*** *** *** *** ** n.s.
A B C D E
**C *** n.d.D n.s.E n.d. n.d.
Yield stress (Pa)
*** *** ** *** n.s. n.s.
Rvis, visual roughness; AIDm, mean absolute intensity deviation; PG, mean perimeter of grains. Values are expressed mean ± standard deviation, different letters after values indicate significant differences; Duncan0 s multiple range test (P < 0.05). *, **, ***, significantly different at p < 0.05, p < 0.01, and p < 0.001, respectively. n.d., not determined. n.s., not significant.
The number and the mean perimeter of grains of the day 1 yoghurt varied from 0 to 442 per 3 mL of the sample and from 0.0 to 5.2 mm, respectively, according to heat treatment and ratio of CN to WP (Fig. 3). The number of grains was influenced strongly by the heat treatment and ratio of
CN to WP (p < 0.001) (Table 1). The effect of storage was not significant (p > 0.05). There were significant differences in the number and the mean perimeter of grains among yoghurts obtained from selected heat treatments. Furthermore, the number and
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500
10
450 8
350 300
6
250 200
4
150 100
Mean perimeter of grains (mm)
Numbers of grains 3mL-1
400
2
50 1.5:1
2:1
3:1
4:1
1.5:1
2:1
3:1
4:1
1.5:1
2:1
3:1
0
4:1 0
95°C / 256 s
110°C / 180 s
130°C / 80 s
Fig. 3. Number of grains 3 mL1 ( ) and mean perimeter of grains (mm) (h) of yoghurts obtained from different heat-treated milk with altered casein to whey protein ratios after 1 day of storage.
the perimeter of grains in yoghurt obtained from milk heated at 95 °C for 256 s was significantly (p < 0.05) higher than that from milk heated at 110 °C for 180 s or at 130 °C for 80 s (Table 1). At higher heat intensities, the precipitation of a-lactalbumin onto the micelle fills the gap formed by the b-lactoglobulin filaments, reduces the surface hydrophobicity, and causes a smoother micellar surface (Sodini et al., 2004). The number and the mean perimeter of grains of the day 1 yoghurt decreased significantly (p < 0.05) when ratio of CN to WP increased (Table 1). According to these results, graininess seemed to be linked to the amount of denatured whey protein and the ratio casein-to-denatured whey protein in the milk base. A similar trend was already reported by Rasmussen, Janhoj, and Ipsen (2007) and Remeuf, Mohammed, Sodini, and Tissier (2003). Lucey (2004) and Lucey and Singh (1997) reported that one of common reasons for occurrence of graininess was excessive whey protein to casein ratio. However, yoghurt with a high level protein, which made with milk blend at a low CN to WP ratio (1.5:1), was grainier than others. The graininess of the stirred yoghurt has also been found to depend on the level of protein in milk blends, which is consistent with results reported by Rasmussen et al. (2007). 3.2. Rheological properties The storage modulus values of the day 1 and day 15 stirred yoghurt samples are presented in Fig. 4. As shown in Table 1, G0 was significantly influenced by the heat treatment, ratio of CN to WP and storage time (p < 0.001). The interactions between heat treatment and ratio of CN
to WP and between heat treatment and storage time, were significant (p 6 0.01), whereas the interaction between ratio of CN to WP and storage time was not significant (p > 0.05). The G0 value increased significantly (p < 0.05) when CN to WP ratio was decreased (Table 1); a similar trend was reported by Lucey, Munro, and Singh (1999). The G0 value was higher in yoghurt made with the milk blend at a low CN to WP ratio (1.5:1) probably because of a higher level of protein (4.8%) in the milk blend with low ratio of CN to WP than in other milk blends. The G0 value decreased significantly (p < 0.05) when heating temperature was increased. During storage at 4 °C, G0 values increased for all yoghurt samples. These results are agreements with those obtained by Gustaw, Glibowski, and Mleko (2006). The yield stress of the day 1 and day 15 samples varied from 135.2 to 11.5 Pa and from 146.7 to 12.1 Pa, respectively, according to the technological conditions (Fig. 5). The yield stress was significantly affected by the heat treatment, ratio of CN to WP and storage time (p 6 0.01) (Table 1). The effect of the heat treatment, ratio of CN to WP and storage time on the yield stress in yoghurts was almost similar to that on G0 . The interaction between heat treatment and ratio of CN to WP was highly significant (p < 0.001), whereas the interactions between heat treatment and storage time and between ratio of CN to WP and storage time were not significant (p > 0.05). The yield stress significantly (p < 0.05) increased as heating temperature decreased, ratio of CN to WP decreased, or storage time increased (Table 1). The difference in the yield stress values among the yoghurts made from the milk
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600
500
G' (Pa)
400
300
200
100
1.5:1
2:1
3:1
4:1
2:1
1.5:1
3:1
4:1
1.5:1
2:1
3:1
4:1
0 95˚C / 256 s
110˚C / 180 s
130˚C / 80 s
Fig. 4. G0 (Pa) of yoghurts obtained from different heat-treated milk with altered casein to whey protein ratios after 1 (h) and 15 ( ) days of storage.
200
Yield stress (Pa)
150
100
50
1.5:1
2:1
3:1
4:1
2:1
1.5:1
3:1
4:1
1.5:1
2:1
3:1
4:1
0 95°C / 256 s
110°C / 180 s
130°C / 80 s
Fig. 5. Yield stress (Pa) of yoghurts obtained from different heat-treated milk with altered casein to whey protein ratios after 1 (h) and 15 ( ) days of storage.
blends with different ratios of CN to WP, as observed in this study, may be due to difference in the protein contents of the milk blends. 4. Conclusions This study has shown that heat treatment and ratio of CN to WP of skim milk affect the physical properties of
yoghurt. As heating temperature or CN to WP ratio increased, the number of grains, perimeter of grains, visual roughness, G0 and yield stress decreased. For practical applications, heat treatment and ratio of CN to WP of skim milk can be optimized to improve quality or modified to create fermented milk products with different physical properties. Further work is addressed to analyse the composition of the grains and understand the mechanism of formation.
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Acknowledgements The author would like to thank Prof. Dr. Jo¨rg Hinrichs and Mr. Konrad Weidendorfer (Department of Animal Foodstuff Technology, Institute of Food Science and Biotechnology, University of Hohenheim, Stuttgart, Germany), Alexander von Humboldt Stiftung (Germany), and The Scientific Research Projects Administration Unit of Akdeniz University (Turkey). References Amatayakul, T., Halmos, A. L., Sherkat, F., & Shah, N. P. (2006). Physical characteristics of yoghurts made using exopolysaccharideproducing starter cultures and varying casein to whey protein ratios. International Dairy Journal, 16, 40–51. AOAC (1995). Official methods of analysis of AOAC international (16th ed.). Arling, VA: AOAC International. Baravian, C., Lalante, A., & Parker, A. (2002). Vane rheometry with a large, finite gap. Applied Rheology, 12, 81–87. Beal, C., Skokanova, J., Latrille, E., Martin, N., & Corrieu, G. (1999). Combined effects of culture conditions and storage time on acidification and viscosity of stirred yogurt. Journal of Dairy Science, 82, 673–681. Bhullar, Y. S., Uddin, M. A., & Shah, N. P. (2002). Effects of ingredients supplementation on textural characteristics and microstructure of yoghurt. Milchwissenschaft, 57, 328–332. Chandan, R. C. (2006). History and consumption trends. In R. C. Chandan, C. H. White, A. Kilara, & Y. H. Hui (Eds.), Manufacturing Yogurt and Fermented Milks (pp. 3–15). Oxford: Blackwell Science Ltd. Gustaw, W., Glibowski, P., & Mleko, S. (2006). The rheological properties of yoghurt with incorporated whey protein aggregates/polymers. Milchwissenschaft, 61, 415–419. Kessler, H. G. (1997). The structure of fermented milk products as influenced by technology and composition. In Texture of fermented milk products and dairy desserts, International dairy federation symposium, Vicenza, Italy, 5–6 May 1997 (pp. 93–105). Brussels. Kessler, H. G. (2002). Food and bio process engineering–dairy technology (5th ed.). Mu¨nchen, Germany: Verlag A. Kessler, p. 694. Krasaekoopt, W., Bhandari, B., & Deeth, H. (2004). Comparison of texture of yogurt made from conventionally treated milk and UHT
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