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Effect of carrot (Daucus carota) antifreeze proteins on the fermentation capacity of frozen dough
Food Research International 40 (2007) 763–769 www.elsevier.com/locate/foodres
Effect of carrot (Daucus carota) antifreeze proteins on the fermentation capacity of frozen dough Chao Zhang, Hui Zhang *, Li Wang School of Food Science and Technology, Southern Yangtze University, 1800 Lihu Avenue, Wuxi 214122, China Received 27 September 2006; accepted 7 January 2007
Abstract Carrot (Daucus carota) antifreeze proteins have strong anti-recrystallization ability and potential uses in food industry. In this study, the effects of carrot concentrated protein supplementation containing 18.3% carrot antifreeze protein on the fermentation capacity of frozen dough were evaluated. Adding carrot concentrated protein to frozen dough increased the retention capacity of the dough, decreased mortality of the yeast, caused a thermal-hysteresis phenomenon during freezing–thawing cycle, and retarded the crystal forming. The application of carrot concentrated protein in frozen dough was proved to be a promising improver for the frozen dough fermentation. 2007 Elsevier Ltd. All rights reserved. Keywords: Antifreeze protein; Frozen dough; Fermentation capacity; Carrot
1. Introduction Frozen dough technique has been of great interest since the 1960s, to deal with problems of short shelf-life of conventional dough (Ra¨sa¨nen, Blanshard, Mitchell, Derbyshire, & Autio, 1998). However, this technique results in several other problems that should be carefully considered to improve the quality of bread (Giannou, Kessoglou, & Tzia, 2003). It was reported that the freezing process weakens the dough structure and decreases the retention capacity of CO2. These situations can be improved by using strong wheat flour or freeze-tolerant yeasts (Panadero, Randez-Gil, & Prieto, 2005; Zounis, Quail, Wootton, & Dickson, 2002). In addition, the frozen dough technique prolongs fermentation time and deteriorates the texture of end products (Casey & Foy, 1995; Inoue, Sapirstein, Takayanagi, & Bushuk, 1994). These phenomena can be minimized by adding some additives (Bhattacharya, Langstaff, & Berzonsky, 2003). *
Corresponding author. Tel.: +86 510 85869382; fax: +86 510 85874723. E-mail address: [email protected] (H. Zhang). 0963-9969/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2007.01.006
Antifreeze proteins (AFPs) can lower freezing temperature non-equilibriumly, and strongly retard the recrystallization. AFPs are also incorporated within ice when frozen (Knight, Cheng, & DeVries, 1991). Even in the frozen state, AFPs inhibit the Ostwald ripening, when ice approaches the melting point and becomes more fluid (Knight, DeVries, & Oolman, 1984). AFPs in freezing and thawing process of cellular materials (e.g. tissues, red blood cells and sperms) have shown promising and beneficial effects. It was reported that the survival of oocytes, spermatozoa, and adenocarcinoma cells is raised at the presence of AFPs (Arav, Rubinsky, Fletcher, & Seren, 1993; Koushafar & Rubinsky, 1997; Payne, Oliver, & Upreti, 1996). Adding AFPs also increases the quality of ice cream and ice slurries (Grandum, Yabe, Tanaka, Takemura, & Nakagomi, 1997; Payne & Young, 1995; Warren, Mueller, & McKown, 1992). Carrot (Daucus carota) AFP (DcAFP) is a leucine-rich repeat protein with the molecular weight of 36.8 kDa (Meyer, Keil, & Naldrett, 1999) and has reportedly strong anti-recrystallization ability (Zhang, Liu, Feng, He, & Wang, 2004). However, few studies about application of DcAFP can be found so far. Therefore, we evaluated the
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effects of carrot concentrated protein supplementation containing DcAFP on the fermentation capacity of frozen dough in this study. 2. Materials and methods 2.1. Preparation of carrot concentrated protein (CCP) CCP was concentrated from cold acclimated carrot tap roots purchased from local supermarket. Tap roots (500 g) were cut into cubes, washed with deionized water and homogenized with 1 l 50 mM Tris/HCl buffer (pH 7.4) by kitchen blender (HR 2860, Philips, China). The mixture was centrifuged at 3500g and 4 C for 30 min. The supernatant was precipitated at the isoelectric point pH 4.5, and the precipitate was collected by centrifugation at 3500g and 4 C for 30 min, followed by re-dissolved in 50 mM Tris/HCl buffer (pH 7.4). Then the solution was dialyzed against deionized water at 4 C overnight. The dialyzed solution was concentrated to about 5% (v/v) of the original volume by polyethyleneglycol 20,000 and lyophilized. The protein content of the CCP powder was determined by Kjeltec Auto Analyzer Unit (Foss Tecator AB, Sweden). Moisture, ash and fat of the CCP powder were determined according to the standard AACC methods (2000). 2.2. Electrophoresis The molecular weight distribution of CCP was studied by SDS–PAGE. Laemmlis discontinuous high resolution SDS–PAGE system (1970), as modified by Fullington, Cole, and Kasarda (1983), was used to fractionate total proteins in 4% (w/v) concentrated polyacrylamide gels and 10% (w/v) isolated polyacrylamide gels. Electrophoresis was done at 80 mA and 100 mA constant current in the concentration and isolation gel, respectively. Low molecular weight marker (97.4, 66.2, 43.0, 31.0, 20.1, and 14.4 kDa) was purchased from Sino-American Biotechnology Company (Low molecular weight marker, Shanghai, China). Gels were stained for 2 h with 0.1% (w/v) Comassie Brilliant Blue R-250 in methanol/acetic-acid/water (25:10:65, v/v). 2.3. Stock dough A stock dough was prepared according to the following formula: 1000 g wheat flour, 20 g instant yeast, 40 g sucrose, 15 g NaCl, 620 g water, 50 g butter and 6.2 g protein (including, respectively, bovine serum albumin (BSA), soy protein isolated (SPI) or CCP). The control group had the same formula except for the 6.2 g protein. Yeast, sucrose and protein were dispersed fully in water before addition to the dough. The other ingredients were added as solid when the dough was nearly formed. This formula is currently applied in China for the preparation of bread and its simplicity allows a clear observation of changes during the processing of frozen dough. Ingredients were mixed in a Philips HR 1495 mixer (Philips, Argentina) for 7 min
and rested for 15 min in a fermentation cabinet at 30 C and 70% relative humidity followed by kneading and proofing, among other necessary operations. The resulting dough (120 g) was molded, covered with polymer film and immediately stored at 30 C. The molded dough was thawed in a fermentation cabinet at 38 C and 90% relative humidity for 160–180 min prior to baking in an oven (180 C top temperature and 210 C bottom temperature). 2.4. Sensory evaluation A panel consisted of nine members selected randomly from local staff members. They were trained and instructed to score the total volume, texture structure, flatness, flexibility and plasticity, loaf shape, crust texture, crust color, crumb color, and mouth feel of the bread according to the Chinese National Standard of bread sensory evaluation (National Standard: GB14611-93, China). The Chinese National Standard of bread sensory evaluation includes eight criteria that evaluate bread quality completely. The criterion is more rigorous and general than methods described by Monisha, Tami, and William (1991) and Rouille, Le Bail, and Courcoux (2000). The range of eight criteria was from 5 to 35 points and the sum of the eight criteria is 100 points. Bread was sliced into l cm thick and evaluated, 3 h after baking. From each sample group, one slice of bread was offered to every panelist at the same time for evaluation in an open area without special lighting. Water was provided for rinsing purposes. 2.5. Determination of yeast mortality Instant yeast (0.2410 g) was suspended in 1000 ml 0.9% (w/v) physiological saline solution. Then the protein content of the suspended yeast solution was adjusted to 10 mg ml1 for BSA, SPI and CCP respectively. The control group was the same formula except for the adjusting of the protein content. The final solution (5 ml) was stored in a pyxis at 18 C and thawed at 15 C. The yeast mortality (r) was determined by haemacytometer. Yeasts were dyed with methylene blue for 3 min, and the number of dead cell and total cell was counted. The mortality rate (r) was calculated by the following formula: r¼
CD CA
where CD is the number of dead cell; CA is the number of total cell. 2.6. Rheological properties of dough The rheological properties of dough during fermentation were determined by a Rheofermentometer F3 (Tripette et Renaud, France). Stock dough (200 g) was stored at 18 C for 1, 2, 3, 4, 5 and 6 weeks, respectively, and thawed at 30 C and 90% relative humidity until the surface temperature reached to 28.5 C (about 25 min). Then the
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dough was loaded into an aluminum tank and sealed after the environmental temperature (28.5 C) was reached. Registered parameters were: T1: time at which dough attained the maximum height; Hm: maximum dough fermentation height; h: the final dough fermentation height; H 0m : maximum gas formation height; T 01 : time of maximum gas formation; Tx: dough permeability by time when gas started to escape from the dough; AT: total gas volume; A1: gas retention volume (volume of the gas retained in the dough at the end of the assay) (Erdogdu-Arnoczky, Czuchajowska, & Pomeranz, 1996; Rosell, Rojas, & Benedito, 2001). Gas retention ratio R was also calculated. Each sample was measured in triplicate and the average value was recorded R¼
A1 AT
2.7. Determination of the freezing and thawing rate The stock dough (120 g) about 27 C was stored in an aluminium column (U60 mm · 50 mm) and then placed at 30 C with a temperature sensor in the core of the dough. The temperature was recorded every minute until it reached 18 C. Then the frozen column was thawed in a fermentation cabinet at 38 C and 90% relative humidity. The temperature was recorded every minute until it reached 25 C. The freezing (thawing) rate was calculated, in C min1. 2.8. Statistical analysis Results were presented as mean values ± standard deviation (S.D.) and the mean values of listed data were average of triplicates or more. Statistical significance of the differences between values was assessed by multifactor analysis of variance followed by Scheffe’s multiple range tests. If P is less than 0.05, it is considered statistically significant (Statgraphics, Statistical graphics System, USA). 3. Results and discussion 3.1. Molecular weight distribution of CCP Table 1 shows the composition of the CCP powder. The protein was 84.2% (w/w) of the CCP powder when the nitrogen factor was 6.25. Fig. 1 shows SDS–PAGE photograph of the CCP. The 36.8 kDa proteins were 18.41%, 19.83% and 16.68% grey of the total CCP in lanes 1, 2 and 3, respectively, in SDS– PAGE gel (Statistic analysis done by Glyko Bandscan Version 5.0). Our result includes a single band of DcAFP, Table 1 Composition of CCP powder Composition CCP powder a
Not detected.
Protein 84.2 ± 5.4
Fig. 1. SDS–PAGE photograph of the CCP. Lanes 1–3 is CCP, Lane 4 is the low molecular weight marker including rabit phosphorglase B (97,400 Da), bovine serum albumin (66,200 Da), rabbit actin (43,000 Da), bovine carbonic anhydrase (31,000), trypsin inhibitor (20,100 Da) and hen egg white lyssozyme (14,400 Da). DcAFP is 18.41%, 19.83% and 16.68% grey of the total CCP in Lanes 1, 2 and 3, respectively.
and is similar to the results reported by Meyer et al. (1999). DcAFP constituted 18.3 ± 1.58% of the CCP. So the DcAFP possessed 1.54 mg ml1 water in the dough. 3.2. Bread quality of the four groups Table 2 shows the results of bread quality evaluation ranked by nine panelists. The score of total volume of the CCP group was 32.0 ± 1.2 points and showed significantly difference with that of the control group. The score of crumb color of the CCP group was 4.0 ± 0.7 points and showed significantly difference with that of the control group. The score of mouth feel of the BSA and CCP group was 3.7 ± 0.7 points and 4.1 ± 0.7 points respectively. The two scores showed significantly difference with that of the control group. And other scores were similar to that of the control group. The sum of the four groups ranged from 80.90 points to 84.70 points and the mean score was 83.31 ± 1.66 points. The sum of the CCP groups was 83.70 points, close to the mean score. So the four groups had similar score, and the CCP supplementation did not give the negative influence on bread quality. 3.3. Yeast mortality after freezing–thawing process
Moisture
Fat
9.5 ± 0.3
a
–
Ash 0.4 ± 0.1
Fig. 2 shows the effect of frozen time on the yeast mortality (r). The r of the CCP group was lower than that of all other groups all the time. The r of the CCP group was
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Table 2 Bread sensory evaluation ranked by nine panelists Item
Control
BSA
SPI
CCP
Total volume (35 points) Texture structure (25 points) Flatness (10 points) Flexibility and plasticity (10 points) Bread sharp and crust texture (5 points) Crust color (5 points) Crumb color (5 points) Mouth feel (5 points)
31.4 ± 1.4 20.9 ± 1.5 5.7 ± 1.2 8.8 ± 0.8 4.1 ± 0.7 3.1 ± 0.7 3.4 ± 0.8 3.5 ± 0.5
32.3 ± 0.8 21.9 ± 1.4 6.0 ± 1.2 8.4 ± 1.0 4.5 ± 0.5 3.8 ± 0.6 3.4 ± 0.5 3.7 ± 0.7*
32.3 ± 1.3 21.6 ± 1.4 6.4 ± 1.1 8.9 ± 0.7 4.4 ± 0.5 3.5 ± 0.7 3.8 ± 0.8 3.8 ± 0.7
32.0 ± 1.2* 21.9 ± 1.5 5.7 ± 1.3 8.7 ± 0.7 4.4 ± 0.7 2.9 ± 0.7 4.0 ± 0.7* 4.1 ± 0.7*
Sum
80.90
83.95
84.70
83.70
*
P < 0.05.
Effect of frozen time on the yeast mortality
Mortality (%)
50% 40% Control BSA SPI CCP
30% 20% 10% 0% 0
3
6
9
12 15 18 21 24 27 30 Time (days)
Fig. 2. Effect of frozen storage on the yeast mortality (r).
29.5% after frozen at 18 C for 28 days, while that of the control, BSA and SPI groups was 47.4%, 39.9% and 36.7%, respectively. The results showed that CCP supplementation to frozen dough protected the yeast during frozen storage. This result had also been proved on oocytes or spermatozoa at the present of AFP (Arav et al., 1993; Payne et al., 1996). 3.4. Changes of rheological properties of dough during frozen storage Table 3 shows the changes of rheological properties of dough during frozen storage, and this table can be divided into two parts: dough development part and gas release part. In dough development part, most dough reached the maximum height after three hours’ fermentation. Therefore, Hm was same in most of the dough, especially in the three-week frozen storage. T1, Hm and h decreased with frozen storage period went on. After the first storage week, Hm of the control, BSA, SPI and CCP groups was 28.7, 31.5, 28.5 and 29.4 mm, respectively. After a six-week frozen storage, Hm of the control, BSA, SPI and CCP groups was 24.3, 27.3, 26.8 and 27.6 mm, respectively. Changes of Hm of the SPI and the CCP group were smaller than those of the control and the BSA group during a sixweek storage. Therefore, the fermentation capacity of the SPI and the CCP group was stronger than that of the control and the BSA group. For gas release part, the values of T 01 and Tx mainly depended on the properties of the dough and thereby were
distinctly different in different groups. The results also revealed that the values of T 01 and Tx fluctuated within a six-week storage in each group. H 0m decreased with the storage time and did not display any difference in the four groups. Retention coefficient R is an important target and will be close to 100% in excellent dough (Rosell et al., 2001). In our experiment, R of the CCP group ranged from 89.9% to 95.2%, and that of the control, BSA and SPI group was from 85.3% to 90.4%, from 82.2% to 91.2% and from 82.0% to 89.0%, respectively. The value of R decreased when frozen storage went on. Remarkably, the value of R of the CCP group was 89.9% after a six-week storage and was higher than that of the control (85.3%), BSA (82.2%) and SPI (82.0%) group. The decrease of R was resulted from the decrease of retention capacity and the decrease of survival rate of yeast. Specifically, the retention capacity was dropped as the sharp ice crystal pierced the protein matrix in frozen process. The survival rate of yeast depends mainly on the frozen condition, species of yeast, and the damage from the dough. Here the damage from the dough would become an important factor if the frozen condition and species of yeast were fixed. It can be concluded that the CCP supplementation raised yeast survival rate and value of R, because the DcAFP can modify the ice morphology described by Meyer et al. (1999) avoiding the damage to the protein matrix and the yeast. Therefore, the fermentation capacity of the frozen dough was improved by using CCP as an additive. It is true that the fermentation capacity can be improved by other methods. For example, certain yeast strain with anti-frozen resistance is developed (Oda & Ouchi, 1990; Van Dijck et al., 2000). Although CCP supplementation might not the best way yet to improve fermentation capacity, but it proves to be a useful additive to improve frozen dough fermentation capacity, and further optimization may be achieved in the future. 3.5. Thermal hysteresis of the dough during the freezing– thawing process Fig. 3a shows the relation between time and central temperature of the dough in the freezing process. The CCP group took 166 min to reach 18 C from 25 C, while
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Table 3 Rheological properties of the dough during the storage Group
Storage time (week)
Dough development
Retention volume
T1 (h)
Hm (mm)
h (mm)
H 0m ðmmÞ
T 01 ðhÞ
Tx (h)
AT (ml)
A1 (ml)
R
Control
1 2 3 4 5 6
3:00:00 3:00:00 2:57:00 2:53:30 2:49:30 2:48:00
28.7 27.4 26.7 25.6 24.5 24.3
28.7 27.4 26.3 24.9 24.1 21.1
73.3 71.4 64.9 59.3 54.7 50.5
0:49:30 0:51:00 0:49:30 0:46:30 0:49:30 0:55:00
1:06:00 1:07:30 1:06:00 1:03:00 1:06:00 1:02:30
1396 1335 1234 1170 1103 1023
1262 1213 1114 1021 959 873
90.4% 90.9% 90.3% 87.3% 86.9% 85.3%
BSA
1 2 3 4 5 6
3:00:00 3:00:00 2:57:00 2:52:30 2:49:30 2:45:00
31.5 29.6 28.7 28.1 27.3 27.3
31.5 29.6 28.1 27.2 24.8 24.3
78.9 76.2 70.6 64.9 58.2 52.4
0:54:00 0:57:00 0:55:30 0:51:00 0:54:00 0:55:30
1:00:00 1:03:00 1:01:30 0:57:00 1:00:00 1:01:30
1388 1275 1206 1131 1067 989
1266 1126 1034 974 896 813
91.2% 88.3% 85.7% 86.1% 84.0% 82.2%
SPI
1 2 3 4 5 6
2:57:00 2:57:00 2:54:00 2:52:30 2:52:30 2:49:30
28.5 28.4 27.8 27.3 26.9 26.8
27.7 27.5 26.6 26.2 24.7 23.4
79.4 76.9 70.5 64.3 56.9 50.6
0:31:30 0:28:30 0:27:00 0:31:30 0:31:30 0:33:00
0:48:00 0:45:00 0:43:30 0:48:00 0:48:00 0:49:30
1330 1248 1159 1078 1007 937
1184 1059 968 894 822 768
89.0% 84.9% 83.5% 82.9% 81.6% 82.0%
CCP
1 2 3 4 5 6
2:58:30 2:58:30 2:58:30 2:55:30 2:54:00 2:51:00
29.4 29.3 29.3 28.7 28.4 27.6
29.1 29.0 29.1 27.6 26.7 26.2
69.7 68.6 63.7 57.3 51.9 48.6
0:33:00 0:33:00 0:30:00 0:33:00 0:36:00 0:34:30
1:21:00 1:21:00 1:18:00 1:21:00 1:24:00 1:22:30
1211 1153 1102 1041 975 894
1153 1084 1024 938 869 804
95.2% 94.0% 92.9% 90.1% 89.1% 89.9%
The definition of the items: T1: time at which dough attained the maximum height; Hm: maximum dough fermentation height; h: the final dough fermentation height; H 0m : maximum gas formation height; T 01 : time of maximum gas formation; Tx: dough permeability by time when gas started to escape from the dough; AT: total gas volume; A1: gas retention volume (volume of the gas retained in the dough at the end of the assay); R: gas retention ratio, described in Section 2.
Temperature (°C)
a
Dough freezing process
Control BSA SPI CCP
30 20 10 0
-10 -20 -30 0
50
100
150
200
Time (min)
b
Dough thawing process
Temperature (°C)
30 20 10 0 -10 -20 -30 0
20
40
60
Control BSA SPI CCP
80
Time (min)
Fig. 3. Dough freeze-thaw cycle. (a) Dough freezing process; (b) Dough thaw process. : ice forming stage (from 3 C to 8 C). Blank: non-ice forming stage (from 25 C to 3 C and 8 C to 18 C).
the control, BSA and SPI groups took 124 min, 106 min and 146 min to reach the same temperature, respectively. The average time (from 25 C to 18 C) was 135.5 ± 26.10 min for the four groups. Remarkably, during the ice forming stage (from 3 C to 8 C), the CCP group showed different thermal phenomenon that was the thermal hysteresis. The CCP group took 81 min to reach 8 C from 3 C, while the control, BSA and SPI groups took 50 min, 33 min and 54 min, respectively. The average time of the ice forming stage was 54.5 ± 19.87 min of the four groups. The non-ice forming stage (from 25 C to 3 C and from 8 C to 18 C) for the control, BSA, SPI and CCP groups was 74 min, 73 min, 92 min and 85 min, respectively. The average time of the non-ice forming stage was 81 ± 9.13 min for the four groups. Therefore, the freezing process of the four groups was similar in the non-ice forming stage and different in the ice forming stage. In the ice forming stage, the CCP group expended longer time than other groups, because the DcAFP retarded the formation of ice crystal. Fig. 3b shows the relation between time and central temperature of the dough in the thawing process. The CCP group needed 69 min to reach 25 C from 18 C, while the control, BSA and SPI groups reached the same temperature after 65, 57 and 73 min, respectively. Fig. 4a shows the freezing rate of the four groups. The freezing rate was lower than 0.5 C min1 in the ice forming
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Fig. 4. Freezing rate and thawing rate of the dough. (a) Freezing rate of the four groups; (b) Thawing rate of the four groups.
stage. In the non-ice forming stage, the range of freezing rate was from 0.867 C min1 to 1.167 C min1. The range of freezing rate of the CCP group was from 0 C min1 to 0.167 C min1 in the ice forming stage and from 0.167 C min1 to 0.80 C min1 in the non-ice forming stage, while that of the control, BSA and SPI groups was from 0.033 C min1 to 0.233 C min1 and from 0.20 C min1 to 0.933 C min1, from 0 C min1 to 0.167 C min1 and from 0.067 C min1 to 1.167 C min1, and from 0.033 C min1 to 0.167 C min1 and from 0.167 C min1 to 0.867 C min1, respectively. The freezing rate of the CCP group was lower than that of other groups, especially during the ice forming stage. The four groups had the similar valley-shaped curve. The difference only occurred in the extending valley-shaped curve of the CCP group. This difference was another proof that the CCP group needed longer time to be forzen than other groups, because the DcAFP retarded the formation of ice crystal. Fig. 4b shows the thawing rate of the four groups. In thawing process, the thawing rate of the four groups did not show much different phenomena. In ice forming stage, the thawing rates of all samples were decreased within the range from 0.2 C min1 to 0.467 C min1. In this experiment, the CCP group showed strong thermal hysteresis ability by retarding the freezing process and
lowering the freezing rate. Therefore, the characteristic of CCP was proved to belong to the AFPs category. 4. Conclusions DcAFP constituted about 18.3 ± 1.58% of the CCP supplementation. The fermentation capacity of the frozen dough was improved by the CCP supplementation through raising the yeast survival and retarding the ice crystal forming. Rheological properties of the CCP group showed stronger fermentation capacity than those of other groups. Retention capacity of the CCP group was also higher than other groups. The bread quality of the CCP group was similar to that of other groups according to the Chinese National Standard of bread sensory evaluation. CCP could be used as a beneficial additive to frozen dough. References AACC (2000). Approved methods of the AACC (10th ed.). St. Paul, MN: American Association of Cereal Chemists, Methods 08-01, 30-25, 4415A, 46-10, 54-10, 54-21. Arav, A., Rubinsky, B., Fletcher, G., & Seren, E. (1993). Cryogenic protection of oocytes with antifreeze proteins. Molecular Reproduction and Development, 36(4), 488–493.
C. Zhang et al. / Food Research International 40 (2007) 763–769 Bhattacharya, M., Langstaff, T. M., & Berzonsky, W. A. (2003). Effect of frozen storage and freeze–thaw cycles on the rheological and baking properties of frozen doughs. Food Research International, 36(4), 365–372. Casey, G., & Foy, J. (1995). Yeast performance in frozen doughs and Strategies for improvement. In K. Kulp, K. Lorenz, & J. Brummer (Eds.), Frozen and refrigerated doughs and batter (pp. 19–51). USA: American Association of Cereal Chemists, Inc. Erdogdu-Arnoczky, N., Czuchajowska, Z., & Pomeranz, Y. (1996). Functionality of whey and casein in fermentation and in breadbaking by fixed and optimized procedures. Cereal Chemistry, 73(3), 309–316. Fullington, J. G., Cole, E. W., & Kasarda, D. D. (1983). Quantitative sodium dodecyl sulfate–polyacrylamide gel electrophoresis of total proteins extracted from different wheat varieties: Effect of protein content. Cereal Chemistry, 60, 65–71. Giannou, V., Kessoglou, V., & Tzia, C. (2003). Quality and safety characteristics of bread made from frozen dough. Trends in Food Science and Technology, 14(3), 99–108. Grandum, S., Yabe, A., Tanaka, M., Takemura, F., & Nakagomi, K. (1997). Characteristics of ice slurry containing antifreeze protein for ice storage applications. Journal of Thermophysics and Heat Transfer, 11(3), 461–466. Inoue, Y., Sapirstein, H. D., Takayanagi, S., & Bushuk, W. (1994). Studies on frozen doughs III: Some factors involved in dough weakening during frozen storage and thaw–freeze cycles. Cereal Chemistry, 71(2), 118–121. Knight, C. A., Cheng, C. C., & DeVries, A. L. (1991). Adsorption of ahelical antifreeze peptides on specific ice crystal surface planes. Biophys Journal, 59, 409–418. Knight, C. A., DeVries, A. L., & Oolman, L. D. (1984). Fish antifreeze protein and the freezing and recrystallization of ice. Nature, 308, 295–296. Koushafar, H., & Rubinsky, B. (1997). Effect of antifreeze proteins on frozen primary prostatic adenocarcinoma cells. Urology, 49(3), 421–425. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685. Meyer, K., Keil, M., & Naldrett, M. J. (1999). A leucine-rich repeat protein of carrot that exhibits antifreeze activity. FEBS Letters, 447(2–3), 171–178.
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Monisha, B., Tami, M. L., & William, A. B. (1991). Effect of frozen storage and freeze–thaw cycles. Cereal Chemistry, 68, 105–107. Oda, Y., & Ouchi, K. (1990). Hybridization of Bakers’ yeast by the raremating method to improve leavening ability in dough. Enzyme and Microbial Technology, 12(12), 989–993. Panadero, J., Randez-Gil, F., & Prieto, J. A. (2005). Heterologous expression of type I antifreeze peptide GS-5 in baker’s yeast increases freeze tolerance and provides enhanced gas production in frozen dough. Journal of Agricultural and Food Chemistry, 53(26), 9966–9970. Payne, S. R., Oliver, J. E., & Upreti, G. C. (1996). Effect of antifreeze proteins on the motility of ram spermatozoa. Cryobiology, 31, 180–184. Payne, S. R., & Young, O. A. (1995). Effects of pre-slaughter administration of antifreeze proteins on frozen meat quality. Meat Science, 41, 147–155. Ra¨sa¨nen, J., Blanshard, J. M. V., Mitchell, J. R., Derbyshire, W., & Autio, K. (1998). Properties of frozen wheat doughs at subzero temperatures. Journal of Cereal Science, 28(1), 1–14. Rosell, C. M., Rojas, J. A., & Benedito, B. C. (2001). Influence of hydrocolloids on dough rheology and bread quality. Food Hydrocolloids, 15(1), 75–81. Rouille, J., Le Bail, A., & Courcoux, P. (2000). Influence of formulation and mixing conditions on bread making qualities of French frozen dough. Journal of Food Engineering, 43(4), 197–203. Van Dijck, P., Gorwa, M. F., Lemaire, K., Teunissen, A., Versele, M., Colombo, S., et al. (2000). Characterization of a new set of mutants deficient in fermentation-induced loss of stress resistance for use in frozen dough applications. International Journal of Food Microbiology, 55(1–3), 187–192. Warren, C. J., Mueller, G. M., & McKown, R. L. (1992). Ice crystal growth suppression polypeptides and methods of preparation. US Patent, 5 118, 792. Zhang, D. Q., Liu, B., Feng, D. R., He, Y. M., & Wang, J. F. (2004). Expression, purification, and antifreeze activity of carrot antifreeze protein and its mutants. Protein Expression and Purification, 35, 257–263. Zounis, S., Quail, K. J., Wootton, M., & Dickson, M. R. (2002). Studying frozen dough structure using low-temperature scanning electron microscopy. Journal of Cereal Science, 35(2), 135–147.
Effect of carrot (Daucus carota) antifreeze proteins on the fermentation capacity of frozen dough Chao Zhang, Hui Zhang *, Li Wang School of Food Science and Technology, Southern Yangtze University, 1800 Lihu Avenue, Wuxi 214122, China Received 27 September 2006; accepted 7 January 2007
Abstract Carrot (Daucus carota) antifreeze proteins have strong anti-recrystallization ability and potential uses in food industry. In this study, the effects of carrot concentrated protein supplementation containing 18.3% carrot antifreeze protein on the fermentation capacity of frozen dough were evaluated. Adding carrot concentrated protein to frozen dough increased the retention capacity of the dough, decreased mortality of the yeast, caused a thermal-hysteresis phenomenon during freezing–thawing cycle, and retarded the crystal forming. The application of carrot concentrated protein in frozen dough was proved to be a promising improver for the frozen dough fermentation. 2007 Elsevier Ltd. All rights reserved. Keywords: Antifreeze protein; Frozen dough; Fermentation capacity; Carrot
1. Introduction Frozen dough technique has been of great interest since the 1960s, to deal with problems of short shelf-life of conventional dough (Ra¨sa¨nen, Blanshard, Mitchell, Derbyshire, & Autio, 1998). However, this technique results in several other problems that should be carefully considered to improve the quality of bread (Giannou, Kessoglou, & Tzia, 2003). It was reported that the freezing process weakens the dough structure and decreases the retention capacity of CO2. These situations can be improved by using strong wheat flour or freeze-tolerant yeasts (Panadero, Randez-Gil, & Prieto, 2005; Zounis, Quail, Wootton, & Dickson, 2002). In addition, the frozen dough technique prolongs fermentation time and deteriorates the texture of end products (Casey & Foy, 1995; Inoue, Sapirstein, Takayanagi, & Bushuk, 1994). These phenomena can be minimized by adding some additives (Bhattacharya, Langstaff, & Berzonsky, 2003). *
Corresponding author. Tel.: +86 510 85869382; fax: +86 510 85874723. E-mail address: [email protected] (H. Zhang). 0963-9969/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2007.01.006
Antifreeze proteins (AFPs) can lower freezing temperature non-equilibriumly, and strongly retard the recrystallization. AFPs are also incorporated within ice when frozen (Knight, Cheng, & DeVries, 1991). Even in the frozen state, AFPs inhibit the Ostwald ripening, when ice approaches the melting point and becomes more fluid (Knight, DeVries, & Oolman, 1984). AFPs in freezing and thawing process of cellular materials (e.g. tissues, red blood cells and sperms) have shown promising and beneficial effects. It was reported that the survival of oocytes, spermatozoa, and adenocarcinoma cells is raised at the presence of AFPs (Arav, Rubinsky, Fletcher, & Seren, 1993; Koushafar & Rubinsky, 1997; Payne, Oliver, & Upreti, 1996). Adding AFPs also increases the quality of ice cream and ice slurries (Grandum, Yabe, Tanaka, Takemura, & Nakagomi, 1997; Payne & Young, 1995; Warren, Mueller, & McKown, 1992). Carrot (Daucus carota) AFP (DcAFP) is a leucine-rich repeat protein with the molecular weight of 36.8 kDa (Meyer, Keil, & Naldrett, 1999) and has reportedly strong anti-recrystallization ability (Zhang, Liu, Feng, He, & Wang, 2004). However, few studies about application of DcAFP can be found so far. Therefore, we evaluated the
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effects of carrot concentrated protein supplementation containing DcAFP on the fermentation capacity of frozen dough in this study. 2. Materials and methods 2.1. Preparation of carrot concentrated protein (CCP) CCP was concentrated from cold acclimated carrot tap roots purchased from local supermarket. Tap roots (500 g) were cut into cubes, washed with deionized water and homogenized with 1 l 50 mM Tris/HCl buffer (pH 7.4) by kitchen blender (HR 2860, Philips, China). The mixture was centrifuged at 3500g and 4 C for 30 min. The supernatant was precipitated at the isoelectric point pH 4.5, and the precipitate was collected by centrifugation at 3500g and 4 C for 30 min, followed by re-dissolved in 50 mM Tris/HCl buffer (pH 7.4). Then the solution was dialyzed against deionized water at 4 C overnight. The dialyzed solution was concentrated to about 5% (v/v) of the original volume by polyethyleneglycol 20,000 and lyophilized. The protein content of the CCP powder was determined by Kjeltec Auto Analyzer Unit (Foss Tecator AB, Sweden). Moisture, ash and fat of the CCP powder were determined according to the standard AACC methods (2000). 2.2. Electrophoresis The molecular weight distribution of CCP was studied by SDS–PAGE. Laemmlis discontinuous high resolution SDS–PAGE system (1970), as modified by Fullington, Cole, and Kasarda (1983), was used to fractionate total proteins in 4% (w/v) concentrated polyacrylamide gels and 10% (w/v) isolated polyacrylamide gels. Electrophoresis was done at 80 mA and 100 mA constant current in the concentration and isolation gel, respectively. Low molecular weight marker (97.4, 66.2, 43.0, 31.0, 20.1, and 14.4 kDa) was purchased from Sino-American Biotechnology Company (Low molecular weight marker, Shanghai, China). Gels were stained for 2 h with 0.1% (w/v) Comassie Brilliant Blue R-250 in methanol/acetic-acid/water (25:10:65, v/v). 2.3. Stock dough A stock dough was prepared according to the following formula: 1000 g wheat flour, 20 g instant yeast, 40 g sucrose, 15 g NaCl, 620 g water, 50 g butter and 6.2 g protein (including, respectively, bovine serum albumin (BSA), soy protein isolated (SPI) or CCP). The control group had the same formula except for the 6.2 g protein. Yeast, sucrose and protein were dispersed fully in water before addition to the dough. The other ingredients were added as solid when the dough was nearly formed. This formula is currently applied in China for the preparation of bread and its simplicity allows a clear observation of changes during the processing of frozen dough. Ingredients were mixed in a Philips HR 1495 mixer (Philips, Argentina) for 7 min
and rested for 15 min in a fermentation cabinet at 30 C and 70% relative humidity followed by kneading and proofing, among other necessary operations. The resulting dough (120 g) was molded, covered with polymer film and immediately stored at 30 C. The molded dough was thawed in a fermentation cabinet at 38 C and 90% relative humidity for 160–180 min prior to baking in an oven (180 C top temperature and 210 C bottom temperature). 2.4. Sensory evaluation A panel consisted of nine members selected randomly from local staff members. They were trained and instructed to score the total volume, texture structure, flatness, flexibility and plasticity, loaf shape, crust texture, crust color, crumb color, and mouth feel of the bread according to the Chinese National Standard of bread sensory evaluation (National Standard: GB14611-93, China). The Chinese National Standard of bread sensory evaluation includes eight criteria that evaluate bread quality completely. The criterion is more rigorous and general than methods described by Monisha, Tami, and William (1991) and Rouille, Le Bail, and Courcoux (2000). The range of eight criteria was from 5 to 35 points and the sum of the eight criteria is 100 points. Bread was sliced into l cm thick and evaluated, 3 h after baking. From each sample group, one slice of bread was offered to every panelist at the same time for evaluation in an open area without special lighting. Water was provided for rinsing purposes. 2.5. Determination of yeast mortality Instant yeast (0.2410 g) was suspended in 1000 ml 0.9% (w/v) physiological saline solution. Then the protein content of the suspended yeast solution was adjusted to 10 mg ml1 for BSA, SPI and CCP respectively. The control group was the same formula except for the adjusting of the protein content. The final solution (5 ml) was stored in a pyxis at 18 C and thawed at 15 C. The yeast mortality (r) was determined by haemacytometer. Yeasts were dyed with methylene blue for 3 min, and the number of dead cell and total cell was counted. The mortality rate (r) was calculated by the following formula: r¼
CD CA
where CD is the number of dead cell; CA is the number of total cell. 2.6. Rheological properties of dough The rheological properties of dough during fermentation were determined by a Rheofermentometer F3 (Tripette et Renaud, France). Stock dough (200 g) was stored at 18 C for 1, 2, 3, 4, 5 and 6 weeks, respectively, and thawed at 30 C and 90% relative humidity until the surface temperature reached to 28.5 C (about 25 min). Then the
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dough was loaded into an aluminum tank and sealed after the environmental temperature (28.5 C) was reached. Registered parameters were: T1: time at which dough attained the maximum height; Hm: maximum dough fermentation height; h: the final dough fermentation height; H 0m : maximum gas formation height; T 01 : time of maximum gas formation; Tx: dough permeability by time when gas started to escape from the dough; AT: total gas volume; A1: gas retention volume (volume of the gas retained in the dough at the end of the assay) (Erdogdu-Arnoczky, Czuchajowska, & Pomeranz, 1996; Rosell, Rojas, & Benedito, 2001). Gas retention ratio R was also calculated. Each sample was measured in triplicate and the average value was recorded R¼
A1 AT
2.7. Determination of the freezing and thawing rate The stock dough (120 g) about 27 C was stored in an aluminium column (U60 mm · 50 mm) and then placed at 30 C with a temperature sensor in the core of the dough. The temperature was recorded every minute until it reached 18 C. Then the frozen column was thawed in a fermentation cabinet at 38 C and 90% relative humidity. The temperature was recorded every minute until it reached 25 C. The freezing (thawing) rate was calculated, in C min1. 2.8. Statistical analysis Results were presented as mean values ± standard deviation (S.D.) and the mean values of listed data were average of triplicates or more. Statistical significance of the differences between values was assessed by multifactor analysis of variance followed by Scheffe’s multiple range tests. If P is less than 0.05, it is considered statistically significant (Statgraphics, Statistical graphics System, USA). 3. Results and discussion 3.1. Molecular weight distribution of CCP Table 1 shows the composition of the CCP powder. The protein was 84.2% (w/w) of the CCP powder when the nitrogen factor was 6.25. Fig. 1 shows SDS–PAGE photograph of the CCP. The 36.8 kDa proteins were 18.41%, 19.83% and 16.68% grey of the total CCP in lanes 1, 2 and 3, respectively, in SDS– PAGE gel (Statistic analysis done by Glyko Bandscan Version 5.0). Our result includes a single band of DcAFP, Table 1 Composition of CCP powder Composition CCP powder a
Not detected.
Protein 84.2 ± 5.4
Fig. 1. SDS–PAGE photograph of the CCP. Lanes 1–3 is CCP, Lane 4 is the low molecular weight marker including rabit phosphorglase B (97,400 Da), bovine serum albumin (66,200 Da), rabbit actin (43,000 Da), bovine carbonic anhydrase (31,000), trypsin inhibitor (20,100 Da) and hen egg white lyssozyme (14,400 Da). DcAFP is 18.41%, 19.83% and 16.68% grey of the total CCP in Lanes 1, 2 and 3, respectively.
and is similar to the results reported by Meyer et al. (1999). DcAFP constituted 18.3 ± 1.58% of the CCP. So the DcAFP possessed 1.54 mg ml1 water in the dough. 3.2. Bread quality of the four groups Table 2 shows the results of bread quality evaluation ranked by nine panelists. The score of total volume of the CCP group was 32.0 ± 1.2 points and showed significantly difference with that of the control group. The score of crumb color of the CCP group was 4.0 ± 0.7 points and showed significantly difference with that of the control group. The score of mouth feel of the BSA and CCP group was 3.7 ± 0.7 points and 4.1 ± 0.7 points respectively. The two scores showed significantly difference with that of the control group. And other scores were similar to that of the control group. The sum of the four groups ranged from 80.90 points to 84.70 points and the mean score was 83.31 ± 1.66 points. The sum of the CCP groups was 83.70 points, close to the mean score. So the four groups had similar score, and the CCP supplementation did not give the negative influence on bread quality. 3.3. Yeast mortality after freezing–thawing process
Moisture
Fat
9.5 ± 0.3
a
–
Ash 0.4 ± 0.1
Fig. 2 shows the effect of frozen time on the yeast mortality (r). The r of the CCP group was lower than that of all other groups all the time. The r of the CCP group was
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Table 2 Bread sensory evaluation ranked by nine panelists Item
Control
BSA
SPI
CCP
Total volume (35 points) Texture structure (25 points) Flatness (10 points) Flexibility and plasticity (10 points) Bread sharp and crust texture (5 points) Crust color (5 points) Crumb color (5 points) Mouth feel (5 points)
31.4 ± 1.4 20.9 ± 1.5 5.7 ± 1.2 8.8 ± 0.8 4.1 ± 0.7 3.1 ± 0.7 3.4 ± 0.8 3.5 ± 0.5
32.3 ± 0.8 21.9 ± 1.4 6.0 ± 1.2 8.4 ± 1.0 4.5 ± 0.5 3.8 ± 0.6 3.4 ± 0.5 3.7 ± 0.7*
32.3 ± 1.3 21.6 ± 1.4 6.4 ± 1.1 8.9 ± 0.7 4.4 ± 0.5 3.5 ± 0.7 3.8 ± 0.8 3.8 ± 0.7
32.0 ± 1.2* 21.9 ± 1.5 5.7 ± 1.3 8.7 ± 0.7 4.4 ± 0.7 2.9 ± 0.7 4.0 ± 0.7* 4.1 ± 0.7*
Sum
80.90
83.95
84.70
83.70
*
P < 0.05.
Effect of frozen time on the yeast mortality
Mortality (%)
50% 40% Control BSA SPI CCP
30% 20% 10% 0% 0
3
6
9
12 15 18 21 24 27 30 Time (days)
Fig. 2. Effect of frozen storage on the yeast mortality (r).
29.5% after frozen at 18 C for 28 days, while that of the control, BSA and SPI groups was 47.4%, 39.9% and 36.7%, respectively. The results showed that CCP supplementation to frozen dough protected the yeast during frozen storage. This result had also been proved on oocytes or spermatozoa at the present of AFP (Arav et al., 1993; Payne et al., 1996). 3.4. Changes of rheological properties of dough during frozen storage Table 3 shows the changes of rheological properties of dough during frozen storage, and this table can be divided into two parts: dough development part and gas release part. In dough development part, most dough reached the maximum height after three hours’ fermentation. Therefore, Hm was same in most of the dough, especially in the three-week frozen storage. T1, Hm and h decreased with frozen storage period went on. After the first storage week, Hm of the control, BSA, SPI and CCP groups was 28.7, 31.5, 28.5 and 29.4 mm, respectively. After a six-week frozen storage, Hm of the control, BSA, SPI and CCP groups was 24.3, 27.3, 26.8 and 27.6 mm, respectively. Changes of Hm of the SPI and the CCP group were smaller than those of the control and the BSA group during a sixweek storage. Therefore, the fermentation capacity of the SPI and the CCP group was stronger than that of the control and the BSA group. For gas release part, the values of T 01 and Tx mainly depended on the properties of the dough and thereby were
distinctly different in different groups. The results also revealed that the values of T 01 and Tx fluctuated within a six-week storage in each group. H 0m decreased with the storage time and did not display any difference in the four groups. Retention coefficient R is an important target and will be close to 100% in excellent dough (Rosell et al., 2001). In our experiment, R of the CCP group ranged from 89.9% to 95.2%, and that of the control, BSA and SPI group was from 85.3% to 90.4%, from 82.2% to 91.2% and from 82.0% to 89.0%, respectively. The value of R decreased when frozen storage went on. Remarkably, the value of R of the CCP group was 89.9% after a six-week storage and was higher than that of the control (85.3%), BSA (82.2%) and SPI (82.0%) group. The decrease of R was resulted from the decrease of retention capacity and the decrease of survival rate of yeast. Specifically, the retention capacity was dropped as the sharp ice crystal pierced the protein matrix in frozen process. The survival rate of yeast depends mainly on the frozen condition, species of yeast, and the damage from the dough. Here the damage from the dough would become an important factor if the frozen condition and species of yeast were fixed. It can be concluded that the CCP supplementation raised yeast survival rate and value of R, because the DcAFP can modify the ice morphology described by Meyer et al. (1999) avoiding the damage to the protein matrix and the yeast. Therefore, the fermentation capacity of the frozen dough was improved by using CCP as an additive. It is true that the fermentation capacity can be improved by other methods. For example, certain yeast strain with anti-frozen resistance is developed (Oda & Ouchi, 1990; Van Dijck et al., 2000). Although CCP supplementation might not the best way yet to improve fermentation capacity, but it proves to be a useful additive to improve frozen dough fermentation capacity, and further optimization may be achieved in the future. 3.5. Thermal hysteresis of the dough during the freezing– thawing process Fig. 3a shows the relation between time and central temperature of the dough in the freezing process. The CCP group took 166 min to reach 18 C from 25 C, while
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Table 3 Rheological properties of the dough during the storage Group
Storage time (week)
Dough development
Retention volume
T1 (h)
Hm (mm)
h (mm)
H 0m ðmmÞ
T 01 ðhÞ
Tx (h)
AT (ml)
A1 (ml)
R
Control
1 2 3 4 5 6
3:00:00 3:00:00 2:57:00 2:53:30 2:49:30 2:48:00
28.7 27.4 26.7 25.6 24.5 24.3
28.7 27.4 26.3 24.9 24.1 21.1
73.3 71.4 64.9 59.3 54.7 50.5
0:49:30 0:51:00 0:49:30 0:46:30 0:49:30 0:55:00
1:06:00 1:07:30 1:06:00 1:03:00 1:06:00 1:02:30
1396 1335 1234 1170 1103 1023
1262 1213 1114 1021 959 873
90.4% 90.9% 90.3% 87.3% 86.9% 85.3%
BSA
1 2 3 4 5 6
3:00:00 3:00:00 2:57:00 2:52:30 2:49:30 2:45:00
31.5 29.6 28.7 28.1 27.3 27.3
31.5 29.6 28.1 27.2 24.8 24.3
78.9 76.2 70.6 64.9 58.2 52.4
0:54:00 0:57:00 0:55:30 0:51:00 0:54:00 0:55:30
1:00:00 1:03:00 1:01:30 0:57:00 1:00:00 1:01:30
1388 1275 1206 1131 1067 989
1266 1126 1034 974 896 813
91.2% 88.3% 85.7% 86.1% 84.0% 82.2%
SPI
1 2 3 4 5 6
2:57:00 2:57:00 2:54:00 2:52:30 2:52:30 2:49:30
28.5 28.4 27.8 27.3 26.9 26.8
27.7 27.5 26.6 26.2 24.7 23.4
79.4 76.9 70.5 64.3 56.9 50.6
0:31:30 0:28:30 0:27:00 0:31:30 0:31:30 0:33:00
0:48:00 0:45:00 0:43:30 0:48:00 0:48:00 0:49:30
1330 1248 1159 1078 1007 937
1184 1059 968 894 822 768
89.0% 84.9% 83.5% 82.9% 81.6% 82.0%
CCP
1 2 3 4 5 6
2:58:30 2:58:30 2:58:30 2:55:30 2:54:00 2:51:00
29.4 29.3 29.3 28.7 28.4 27.6
29.1 29.0 29.1 27.6 26.7 26.2
69.7 68.6 63.7 57.3 51.9 48.6
0:33:00 0:33:00 0:30:00 0:33:00 0:36:00 0:34:30
1:21:00 1:21:00 1:18:00 1:21:00 1:24:00 1:22:30
1211 1153 1102 1041 975 894
1153 1084 1024 938 869 804
95.2% 94.0% 92.9% 90.1% 89.1% 89.9%
The definition of the items: T1: time at which dough attained the maximum height; Hm: maximum dough fermentation height; h: the final dough fermentation height; H 0m : maximum gas formation height; T 01 : time of maximum gas formation; Tx: dough permeability by time when gas started to escape from the dough; AT: total gas volume; A1: gas retention volume (volume of the gas retained in the dough at the end of the assay); R: gas retention ratio, described in Section 2.
Temperature (°C)
a
Dough freezing process
Control BSA SPI CCP
30 20 10 0
-10 -20 -30 0
50
100
150
200
Time (min)
b
Dough thawing process
Temperature (°C)
30 20 10 0 -10 -20 -30 0
20
40
60
Control BSA SPI CCP
80
Time (min)
Fig. 3. Dough freeze-thaw cycle. (a) Dough freezing process; (b) Dough thaw process. : ice forming stage (from 3 C to 8 C). Blank: non-ice forming stage (from 25 C to 3 C and 8 C to 18 C).
the control, BSA and SPI groups took 124 min, 106 min and 146 min to reach the same temperature, respectively. The average time (from 25 C to 18 C) was 135.5 ± 26.10 min for the four groups. Remarkably, during the ice forming stage (from 3 C to 8 C), the CCP group showed different thermal phenomenon that was the thermal hysteresis. The CCP group took 81 min to reach 8 C from 3 C, while the control, BSA and SPI groups took 50 min, 33 min and 54 min, respectively. The average time of the ice forming stage was 54.5 ± 19.87 min of the four groups. The non-ice forming stage (from 25 C to 3 C and from 8 C to 18 C) for the control, BSA, SPI and CCP groups was 74 min, 73 min, 92 min and 85 min, respectively. The average time of the non-ice forming stage was 81 ± 9.13 min for the four groups. Therefore, the freezing process of the four groups was similar in the non-ice forming stage and different in the ice forming stage. In the ice forming stage, the CCP group expended longer time than other groups, because the DcAFP retarded the formation of ice crystal. Fig. 3b shows the relation between time and central temperature of the dough in the thawing process. The CCP group needed 69 min to reach 25 C from 18 C, while the control, BSA and SPI groups reached the same temperature after 65, 57 and 73 min, respectively. Fig. 4a shows the freezing rate of the four groups. The freezing rate was lower than 0.5 C min1 in the ice forming
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Fig. 4. Freezing rate and thawing rate of the dough. (a) Freezing rate of the four groups; (b) Thawing rate of the four groups.
stage. In the non-ice forming stage, the range of freezing rate was from 0.867 C min1 to 1.167 C min1. The range of freezing rate of the CCP group was from 0 C min1 to 0.167 C min1 in the ice forming stage and from 0.167 C min1 to 0.80 C min1 in the non-ice forming stage, while that of the control, BSA and SPI groups was from 0.033 C min1 to 0.233 C min1 and from 0.20 C min1 to 0.933 C min1, from 0 C min1 to 0.167 C min1 and from 0.067 C min1 to 1.167 C min1, and from 0.033 C min1 to 0.167 C min1 and from 0.167 C min1 to 0.867 C min1, respectively. The freezing rate of the CCP group was lower than that of other groups, especially during the ice forming stage. The four groups had the similar valley-shaped curve. The difference only occurred in the extending valley-shaped curve of the CCP group. This difference was another proof that the CCP group needed longer time to be forzen than other groups, because the DcAFP retarded the formation of ice crystal. Fig. 4b shows the thawing rate of the four groups. In thawing process, the thawing rate of the four groups did not show much different phenomena. In ice forming stage, the thawing rates of all samples were decreased within the range from 0.2 C min1 to 0.467 C min1. In this experiment, the CCP group showed strong thermal hysteresis ability by retarding the freezing process and
lowering the freezing rate. Therefore, the characteristic of CCP was proved to belong to the AFPs category. 4. Conclusions DcAFP constituted about 18.3 ± 1.58% of the CCP supplementation. The fermentation capacity of the frozen dough was improved by the CCP supplementation through raising the yeast survival and retarding the ice crystal forming. Rheological properties of the CCP group showed stronger fermentation capacity than those of other groups. Retention capacity of the CCP group was also higher than other groups. The bread quality of the CCP group was similar to that of other groups according to the Chinese National Standard of bread sensory evaluation. CCP could be used as a beneficial additive to frozen dough. References AACC (2000). Approved methods of the AACC (10th ed.). St. Paul, MN: American Association of Cereal Chemists, Methods 08-01, 30-25, 4415A, 46-10, 54-10, 54-21. Arav, A., Rubinsky, B., Fletcher, G., & Seren, E. (1993). Cryogenic protection of oocytes with antifreeze proteins. Molecular Reproduction and Development, 36(4), 488–493.
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