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Combined effects of freezing rate, storage temperature and time on bread dough and baking properties
LWT - Food Science and Technology 42 (2009) 1474–1483
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Combined effects of freezing rate, storage temperature and time on bread dough and baking properties Jinhee Yi a, William L. Kerr b, * a b
Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011, USA Department Food Science and Technology, University of Georgia, Athens, GA 30602, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 October 2008 Received in revised form 13 May 2009 Accepted 15 May 2009
This study compares the effects of freezing temperature and rate as well as storage temperature and time on the quality of frozen dough. Yeasted bread dough was frozen using four freezing rates (19–69 C/h), then stored at 10, 20, 30, or 35 C for up to 180 days. Dough strength diminished with longer storage time and higher storage temperatures. Cryo-SEM showed that dough stored at 30 and 35 C had the least damaged gluten network. NMR studies showed that more rapidly frozen dough, and that stored at lower temperatures had lower transverse relaxation (T2) times (9–10 ms). However, dough stored at 20 C displayed the highest yeast activity among samples. Bread loaf volume decreased with storage time, and bread made from dough stored at 20 C showed the highest loaf volume. Breads produced from 30 and 35 C stored dough displayed less change in the texture profile during storage as well as less change in T2 values. Response surface analysis showed that optimal properties occurred at freezing rates of around 19–41 C/h and storage temperatures of 15 to 20 C. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Frozen dough Stickiness Extensibility Bread volume
1. Introduction Bread made from frozen dough has become an increasingly popular alternative to that made directly from unfrozen dough. Frozen dough can be manufactured in large quantities off-site, and then shipped to local restaurants or retail operations, saving on both equipment and labor costs. In recent years, the quality of these products has improved owing to advances in technology and formulation, but there is room for additional improvement. Problems associated with frozen dough include long proof time, low volume, poor texture, and variable performance (Kenny, Wehrle, Dennehy, & Arendt, 1999). Some of the poorer quality can be attributed to diminished yeast activity, the characteristics of the yeast and their survival after freezing (Baguena, Soriano, Martinezanaya, & Debarber, 1991; El-Hady, ElSamahy, Seibel, & Brummer, 1996; Hino, Takano, & Tanaka, 1987; Hsu, Hoseney, & Seib, 1979; Ribotta, Leon, & Anon, 2003; Wolt & D’Appolonia, 1984). In order to improve performance, frozen dough processors may add extra yeast, use short or no-time dough processing procedures, mix ingredients at relatively low temperatures, or incorporate new strains of freeze-tolerant yeasts.
* Corresponding author. Tel.: þ1 706 542 1085; fax: þ1 706 542 1050. E-mail address: [email protected] (W.L. Kerr). 0023-6438/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2009.05.017
During freezing, there are also deleterious effects on dough structure. Electron microscopy studies (Esselink, Aalst, Maliepaard, Henderson, et al., 2003) showed a breakdown of the reticular gluten network when subjected to freezing and thawing, and related these to changes in the rheological properties of the dough. Ice recrystallization and water migration in the dough also affects the dough structure (Bache & Donald, 1998; Rojas, Rosell, Benedito de Barber, Pe´rez-Munuera, & Lluch, 2000). Extended frozen storage alters the gluten protein matrix (Berglund, Shelton, & Freeman, 1991; Varrianomarston, Hsu, & Mahdi, 1980), results in dough weakening, and causes less gas retention (Autio & Sinda, 1992; Hsu et al., 1979; Inoue & Bushuk, 1991). In addition, injury to yeast membranes caused by freezing and thawing release cellular chemicals that have deleterious effects on the dough structure. However, researchers have different opinions on the effects of specific leachates, mainly reducing compounds such as glutathione, on the gluten network (Casey & Foy, 1995; Inoue & Bushuk, 1991; Wolt & D’Appolonia, 1984). While several researchers have studied the effects of storage time or temperature on yeast viability and dough structure (El-Hady et al., 1996; Havet, Mankai, & Le Bail, 2000; Mazur, 1961), less has been done on the influence of freezing rate. Especially in frozen dough preparations, where freezing and sometimes prolonged frozen storage intervene between dough formation and bread baking, several factors still have not been fully investigated (Giannou & Tzia, 2007). In the present study, we examined the
J. Yi, W.L. Kerr / LWT - Food Science and Technology 42 (2009) 1474–1483
combined effects of freezing rate, storage temperature and storage time on dough and bread quality. The dough properties were evaluated based on dough extensibility and adhesiveness, and the viability of yeast assayed through measurements of gas production. Both volume and firmness were measured in the finished breads. Direct examination of the dough in the frozen state was accomplished using cryogenic scanning electron microscopy (cryo-SEM), allowing visualization of the ultra-structure of gluten-starch association and the state of gluten strands in the network. We also used time-domain NMR to investigate changes in water dynamics (Esselink, Aalst, Maliepaard, & Duynhoven, 2003; Roman-Gutierrez, Guilbert, & Cuq, 2002; Ruan et al., 1999). 2. Materials and methods 2.1. Dough preparation and freezing Bread dough was prepared from enriched, bleached hard winter wheat flour (Organic Select Artisan Flour, King Arthur Company, Norwich, Vermont) with 11.5 g/100 g protein content; Baker’s dry yeast (Fleischmann’s Yeast, Quebec, Canada); Dominion pure cane sugar (Dixie Crystal, Savannah, GA); non-iodized salt (Morton International, Inc., Chicago, IL); and soybean oil (Wesson Vegetable Oil, ConAgra Foods, Omaha, Nebraska). The basic formulation was (per 100 g flour weight): water (60 g/100 g), sugar (5 g/100 g), oil (5 g/100 g), yeast (3 g/100 g), and salt (2 g/100 g). The yeast was prehydrated with the water, and then all of the dough ingredients were placed in a 6-quart, 575 W mixer (Kitchen Aid Professional 600 Series, St. Joseph, MI) and mixed for 2 min at 120 rpm with a paddle, and for 8 min at 178 rmp with a dough hook. Once the dough was formed, it was separated into samples of 50 g, and shaped by hand into approximately 50 mm diameter circular slabs. Dough pieces were frozen in a built-in forced-air blast freezer located at the UGA Department of Food Science and Technology in Athens, GA. As part of the experimental design, the freezer was adjusted to give four different freezing rate schemes, as shown by the temperature-time plots in Fig. 1. Specific freezing times and rates depend on the conditions and final temperature. The average rate (and time) to reach 30 C were: Rate 1: 19 C/h (2.53 h); Rate 2: 41 C/h (1.18 h); Rate 3: 55 C/h (0.88 h); and Rate 4: 72 C/h (0.67 h). In terms of the time to go from the initial freezing point to 30 C, these times were: Rate 1: 2.27 h; Rate 2: 0.95 h; Rate 3: 0.62 h; and Rate 4: 0.45 h. After freezing to the desired temperature, dough samples were vacuum-packaged in plastic bags and placed at four different frozen storage temperatures (10, 20, 30 or 35 C), and stored for 30,
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60, 90, and 180 days. At each sampling period, 3 replications of the 16 treatments were withdrawn from the freezers and placed in an environmental chamber (Model HEC10R, HotPack, Warminster, PA) at 8 C and 35% RH in order to thaw. Thawed dough pieces were removed from the package, then placed in a chamber at 36 C and 85% RH for proofing. Dough samples were baked in a 5-rack gas convection oven (Blodgett BLD-DFG100, Burlington, Vermont) at 180 C for 15 min. After baking, bread samples were allowed to cool for approximately 1 h prior to subsequent measurements. 2.2. Dough extensibility and adhesiveness Extensibility of the dough was measured using a texture analyzer (TA-XT2i, Texture Technologies Corp., Scarsdale, NY) with a modified Kieffer extensibility rig and 5 kg load cell. Approximately 50 g dough samples were molded into strips approximately 7 mm in diameter and 60 mm in length. All samples were left to rest on a grooved plate at 8 C for 20 min and 90% RH prior to testing (Anderssen, Bekes, Gras, Nikolov, & Wood, 2004). The dough was pulled at a crosshead speed of 3.3 mm/s. The resistance to extension (maximum force) and extensibility (distance to break) were calculated from the force–deformation curves. One advantage of the Kieffer dough extensibility rig is that it uses a micro-extension method involving a very small sample size. It correlates highly with methods such as the extensigraph as indicated by baking performance (Kieffer, Wieser, Henderson, & Graveland, 1998; Sharadanant & Khan, 2003; Suchy, Lukow, & Ingelin, 2000). Adhesiveness was measured using the texture analyzer with a modified Chen–Hoseney stickiness rig, with conditions as described by Chen and Hoseney (1995). The sample was placed in a cylindrical cell on the base of the texture analyzer, which was then enclosed by a lid with a perforated hole. A small amount of dough was extruded through the hole. The upper cylindrical probe was brought in contact with the exposed dough to adhere to it, and the probe was pulled away from the base at a speed of 1.7 mm/s. Both the maximum force and the area under the force–deformation curve required to separate the probe from the test sample were used as measures of adhesiveness. 2.3. Cryo scanning electron microscopy A Jeol JSM-5410 scanning electron microscope with a CT-500C cryo-unit (Oxford Instruments, Oxfordshire, UK) was used to investigate the microstructure of the frozen bread dough. Each frozen dough sample was placed on the cryo-specimen holder, placed in liquid nitrogen, and then transferred to the cryo-unit in the frozen state. The dough specimens were fractured, sublimated (10 min at 70 C) and sputter coated with gold (4 min at 0.2 kPa). The prepared dough specimen was transferred to the microscope where it was observed at 15 kV and 120 C. Micrographs were taken at 500, 1000 and 2000 magnification. 2.4. NMR measurements of dough
Fig. 1. Freezing protocols for frozen bread dough. Average rate (and time) to reach 30 C. Rate 1: 19 C/h (2.53 h); Rate 2: 41 C/h (1.18 h); Rate 3: 55 C/h (0.88 h); and Rate 4: 72 C/h (0.67 h).
Proton relaxation measurements were made using a 20 MHz Proton (1H) NMR spectrometer (Resonance Instruments, Whitney, UK). Approximately 5 g dough samples were taken from the center of the thawed dough. Three replicates were taken from each piece of dough. Each sample was placed in a 10 mm diameter glass tube then covered with parafilm. The glass tube was placed in a 10 C bath for 20 min. After 20 min, the glass tubes with dough specimens were placed in 18 mm diameter NMR tubes. Transverse (T2) relaxation curves were developed using the CPMG pulse sequence: 90x(s-180y s-echo)n. Acquisition parameters were set to a 90 pulse of 4.2 ms and a recycle delay of 2 s. A pulse spacing (s) of
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100 ms was chosen to exclude the fast decaying solid-like signal. All measurements were made at 25 C 1 C. Relaxation curves were analyzed using the WinFIT multi-exponential routine (Resonance Instruments, Whitney, UK). 2.5. Yeast activity Yeast activity was measured using AACC method 89-01 (2000) with slight modification. Dough pieces (10 g) and 100 g water were placed into a 250 ml glass reaction vessel, capped, and placed in a water bath at 36 1 C. A hose was connected to the vessel, leading to a volumetric manometer. Gassing power was determined from the volume of carbon dioxide gas trapped after 180 min. Each 10 g sample contained 0.163 g dry yeast.
Table 1 Maximum resistance to extension of frozen bread dough (force, g) after thawing. Values for unfrozen dough: 69.98 g. Storage day
Freezing rate
10 C
20 C
30 C
35 C
30
Rate Rate Rate Rate
1 2 3 4
49.68op 51.19pq 52.19r 52.75s
53.28st 54.01t 55.60vw 56.05wx
54.33tu 54.19t 56.99y 56.41xy
54.73uv 55.25v 55.74w 56.02wx
60
Rate Rate Rate Rate
1 2 3 4
47.01kl 47.62m 48.89no 48.70n
49.62o 50.38p 52.62rs 53.38st
51.77q 51.86qr 52.65rs 52.74s
51.11pq 52.25rs 51.45q 52.22rs
90
Rate Rate Rate Rate
1 2 3 4
43.78h 44.84hi 45.11i 45.71j
45.45ij 46.34jk 47.31lm 47.30lm
45.33i 47.50m 47.73 46.64k
45.38 47.73m 47.02kl 47.32lm
180
Rate Rate Rate Rate
1 2 3 4
29.17a 29.54ab 29.49a 29.71ab
30.15bc 33.68fg 32.64def 32.39de
31.99cd 33.19fg 32.82ef 32.67ef
32.01d 33.06f 33.12fg 32.13de
2.6. Bread volume Loaf volume and weight were determined after baking and cooling for 1 h at ambient temperature (AACC 10-05, 2000). A container was filled with a known volume of rapeseed. The amount of seed displaced when the loaf was introduced was measured in a graduated cylinder, and measured the loaf volume. 2.7. Bread crumb firmness The firmness of the bread samples was determined using a texture analyzer (TA-XT2i, Texture Technologies Corp., Scarsdale, NY) with a 36 mm radius cylinder probe according to AACC Method 74-10A (2000). Three center slices of each loaf were measured at 25 C. The compression rate was set at 1.7 mm/s. Firmness was the force required to compress the slices (20 mm thick) by 40% strain. 2.8. Statistical analysis ANOVA was used to analyze all data, based on a three-way mixed design. Fisher’s LSD was used to determine significant differences between samples. A p-value of less than 0.05 was considered significant. A response surface methodology was used to examine regions of minimal or maximal response, particularly to see if there were similar regions of optimal response for the various response variables. Models surfaces were fit using linear, squared, and crossed terms using the JMP 7 software (SAS, Cary, NC). Contour plots were developed to where the response reached 90% of its minimum or maximum value. 3. Results and discussion 3.1. Dough extensibility and adhesiveness
Storage temperature
Values followed by different superscript letters are significantly different at p < 0.05.
of frozen dough. Freezing rate 2 produced dough with the highest extensibility, while those from freezing rate 1 had the lowest extensibility. Dough from freezing rate 1 also had lower MRE than those from other freezing rates. Frozen storage temperature also had some effect on dough extensibility. With lower frozen storage temperature (closer to 35 C), the extensibility increased but there was no significant difference between rates 3 and 4 (the fastest rates). The interaction between the freezing rate and frozen storage temperature determined extensibility and MRE. For the first 30 days, dough prepared at freezing rate 4 (the fastest freezing) and stored at 35 C had the highest extensibility (81.0 mm); dough prepared at freezing rate 1 and stored at 10 C had the lowest extensibility (74.45 mm). After 180 days, dough prepared at freezing rate 2 and stored at 35 C had the highest extensibility (71.39 mm); those produced at freezing rate 1 and stored at 10 C had the lowest extensibility (49.94 mm). Decreases in maximum resistance, and in extensibility may indicate deterioration in the gluten network. Extensibility has been attributed to the gluten structure, and particularly to high molecular weight glutenins (Belton, 1999; Bushuk & Macritchie, 1989; Table 2 Extensibility of frozen bread dough (distance to peak, mm) after thawing. Values for unfrozen dough: 84.99 mm. Storage day
Freezing rate
Storage temperature 10 C
Tables 1 and 2 show the maximum resistance to extension (MRE, in g force) and extensibility (in mm) of dough subject to different freezing and storage conditions. For the control (a freshly made, unfrozen dough), MRE was 69.98 g with an extensibility of 84.99 mm. All dough samples subject to freezing had lower resistance and extensibility than the control. Both the MRE and the extensibility decreased with time from 0 to 180 days of frozen storage. For example, maximum resistance ranged from 49.68 to 56.99 g at 30 days, and from 29.17 to 33.68 g at 180 days. Similarly, extensibility ranged from 74.45 to 81.00 mm at 30 days, and from 49.94 to 71.39 mm at 180 days. Storage time had a greater influence on extensibility than temperature. For samples prepared at freezing rate 1 (the slowest rate), and held for 30 days, extensibility varied from 74.45 mm (at 10 C) to 76.84 mm (at 35 C). After 180 days of storage, extensibility ranged from 49.94 mm (at 10 C) to 70.04 mm (at 35 C). Freezing rate also affected the extensibility
qr
20 C vw
30 C wx
35 C
30
Rate Rate Rate Rate
1 2 3 4
74.45 76.12uv 75.86tuv 75.22rs
76.23 78.10AB 77.42xy 77.69yzA
77.19 79.58BC 80.18CD 79.95CD
76.84vw 80.92D 80.99D 81.00D
60
Rate Rate Rate Rate
1 2 3 4
69.81hi 72.90no 71.64lmn 70.96kl
73.37nop 76.09uv 75.23rs 75.30rs
75.28rs 77.45xy 76.93w 77.30wx
73.46op 78.81AB 77.49xy 77.93zA
90
Rate Rate Rate Rate
1 2 3 4
63.94c 67.10e 66.66de 65.75d
72.59mn 73.58op 73.74pq 74.63qr
73.94pq 75.76tu 74.93rs 75.63st
74.71r 75.81tuv 75.15rs 75.42st
180
Rate Rate Rate Rate
1 2 3 4
49.94a 52.43b 52.00b 52.42b
67.59ef 69.24g 68.38f 69.66gh
69.19g 71.07kl 70.02hij 70.78jkl
70.04ij 71.39lmn 70.30ij 70.48jk
Values followed by different superscript letters are significantly different at p < 0.05.
J. Yi, W.L. Kerr / LWT - Food Science and Technology 42 (2009) 1474–1483
Lindsay & Skerritt, 1999). While disulfide bonds help to maintain the gluten network and provide resistance to stretching, several theories have been developed to explain dough elasticity (Belton, 1999; MacRitchie & Lafiandra, 1997). Our results indicate that relatively rapid freezing and low storage temperatures result in the least changes to the gluten structure. Most deleterious were slow freezing rates, high storage temperature, and prolonged storage times. Rapid freezing and low storage temperatures promote the creation of a greater number of small ice crystals, due to enhanced nucleation and crystal growth rates. Low freezing rate promotes the growth of relatively large crystals. It has been suggested that ice crystals physically disrupt and damage the gluten network, and dehydrate the gluten so as to cause irreversible structural changes. Extensibility determines the ability of the dough to extend during gas production by yeast during proofing. Excessively high extensibility results in weak and slack dough, which collapses during the proofing stage or while baking in the oven (Sharadanant & Khan, 2003). MRE measures the ability of the dough to retain gas and subsequently to form springy bread. A very low resistance to extension results in poor gas retention and lower loaf volume. A very high resistance to extension also results in a lower loaf volume because the tough dough is not capable of proofing to an optimum height with the gas produced by the yeast. Contrary to our findings, Inoue and Bushuk (1991) reported that extensibility increased with storage time for one week but no clear trend was observed. However, Sharadanant and Khan (2003) reported that extensibility increased with extended storage time and maximum resistance to extension decreased with storage time. Table 3 shows the adhesiveness of dough samples prepared at different freezing rates, storage temperatures and storage times. Adhesiveness of unfrozen dough was 69.08 g. Adhesiveness of all samples from frozen dough were higher than that of control, indicating more sticky dough. In general, dough adhesiveness increased with storage time. For example, at 30 days adhesiveness ranged from 74.77 to 80.77 g, while at 180 days values ranged from 93.25 to 115.63 g. Storage temperature was not a significant factor for samples stored at 30 and 60 days, but was for samples stored at 90 and 180 days. At 90 and 180 days, samples stored at lower temperatures were less adhesive. Freezing rate was also a significant factor for adhesiveness, except for samples stored at 30 days. Samples prepared at freezing rate 2 were less adhesive; those at freezing rate 1 had the highest adhesiveness. In general, adhesiveness values followed a similar Table 3 Adhesiveness of frozen bread dough (tension force, g) after thawing. Values for unfrozen dough: 69.08 g. Storage day
Freezing rate
Storage temperature 10 C fg
20 C cd
30 C
35 C
ef
30
Rate Rate Rate Rate
1 2 3 4
79.47 75.04b 77.49c 78.73def
78.03 77.20c 81.00hi 80.77ghi
79.30 78.22def 80.10gh 79.28ef
78.07cd 75.12b 74.77a 77.82cd
60
Rate Rate Rate Rate
1 2 3 4
82.78klm 77.88cd 82.21jk 82.01jk
88.39mn 78.81ef 83.37lm 82.37jkl
87.70mn 78.16de 80.30gh 82.00jk
83.15lm 81.63ij 82.41kl 80.93hi
90
Rate Rate Rate Rate
1 2 3 4
108.45BC 105.86zA 107.84B 106.24A
97.48x 91.99p 92.36qr 92.37qr
94.37tu 90.83o 91.97p 92.00pq
93.15rs 91.63p 92.41qr 93.43rs
180
Rate Rate Rate Rate
1 2 3 4
115.63E 114.47DE 115.52E 113.74D
109.60C 98.83y 108.69BC 105.32z
97.23x 93.25rs 94.44uv 94.25tu
95.19vw 94.34tu 94.02st 93.46rst
Values followed by different superscript letters are significantly different at p < 0.05.
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dependence as extensibility. Samples stored at 35 C for short times (30 days) were more extensible (with higher force) and less adhesive. Samples stored for long time at low temperature (10 C) were less extensible (with lower maximum force) and were more adhesive. Adhesiveness is related to the force required to separate a material from a dissimilar surface. As discussed previously, freezing of dough and frozen storage may cause separation of water from the gluten network, particularly as water is redistributed due to ice recrystallization. This water may be more feely assessable at surfaces, leading to increased adhesiveness. Samples prepared at freezing rate 2 were less adhesive than those at other freezing rates. Several researchers have reported that dead yeast cells produce reducing substances that cause disruption of molecular bonds in the gluten network. It will be shown in a subsequent section that highest yeast activity incurred for samples prepared at freezing rate 2. One possibility is that fewer reducing substances are produced at freezing rate 2, as there are more viable yeast cells, and consequently less deterioration of the gluten network. This in turn results in less migration of water after freezing and thawing, and thus less adhesiveness. 3.2. Microstructure of frozen bread dough Cryo-SEM was used to visualize the microstructure of frozen dough (in the frozen state). The frozen dough structure at 90 days showed the typical structure of starch granules embedded in a gluten network (Figs. 2–5). All frozen dough samples had voids among the gluten network and starch granules. The gluten strands composing the network appeared to be quite damaged after 90 days. Dough produced at freezing rate 1 and 2 (the slowest rates) were less uniform and had thinner strands. Dough produced at freezing rates 3 and 4 (more rapid freezing) showed less disrupted structure. Storage temperature had a more notable effect on the gluten network in the dough. With lower storage temperature (closer to 35 C), the dough was more intact and had a more uniform gluten network. Regardless of freezing rate, dough stored at 35 C had less disrupted gluten strands than that stored at 10 C. Microstructural observations help explain the observed rheological properties of dough. As noted, dough stored at higher temperatures and for longer times was less extensible, and more adhesive, than dough stored at low temperatures for shorter times. Lower extensibility can be attributed to the thinner, more disrupted bonding in the gluten. As previously discussed, this disrupted structure is also less able to hold water, and thus more adhesive. Dough microstructure has been investigated by several researchers. Ribotta, Perez, Leon, and Anon (2004) studied the effect of emulsifiers on frozen bread dough and described the effects of extended frozen storage on the protein matrices. After long storage time, the gluten matrix was quite damaged. Rojas et al. (2000) described dough as a continuous gluten matrix, with starch granules scattered among the protein network. Discontinuities were observed in the lax matrix surrounding the starch granules under greater magnification. Berglund et al. (1991) found that after 24 weeks frozen storage, the gluten matrix had thinner strands, seemed more disrupted, and was separated from the starch granules. Nicolas et al. (2003) observed that during freezing, ice crystals appear to compress the gluten, leading to a significant phase separation between the gluten and ice. 3.3. NMR relaxometry Pulsed 1H NMR was used to investigate the relaxation characteristics of dough systems subject to different freezing rate and storage conditions. Table 4 shows the transverse relaxation times
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Fig. 2. Cryo scanning electron microscopy dough pieces stored for 90 days at 10 C and frozen at (a) Rate 1, (b) Rate 2, (c) Rate (3) and (d) Rate 4.
for the thawed dough samples. The T2 relaxation time increased somewhat with increased frozen storage time at all storage temperatures. At 30 days, T2 values ranged from 15.39 to 16.45 ms, while at 180 days T2 values ranged from 17.56 to 18.71 ms. In most cases, the T2 times were smallest for samples at freezing rate 2. Frozen storage temperature also had a significant, but small, effect on transverse relaxation times. Samples stored at lower
temperature had somewhat shorter T2 values. For example, at 30 days samples held at 10 C had values ranging from 16.28 to 16.42, while at 35 C values ranged from 15.39 to 15.93 ms. The amount and state of water play an important role in the preparation and properties of wheat flour dough and their products. Ruan et al. (1999) used several methods for presentation and analysis of relaxation time measurement of protons in dough, and
Fig. 3. Cryo scanning electron microscopy dough pieces stored for 90 days at 20 C and frozen at (a) Rate 1, (b) Rate 2, (c) Rate (3) and (d) Rate 4.
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Fig. 4. Cryo scanning electron microscopy dough pieces stored for 90 days at 30 C and frozen at (a) Rate 1, (b) Rate 2, (c) Rate (3) and (d) Rate 4.
suggested there is a continuous distribution of protons having different relaxation times in heterogeneous systems such as dough (Esselink, Aalst, Maliepaard, & Duynhoven, 2003; Esselink, Aalst, Maliepaard, Henderson, et al., 2003; Ruan et al., 1999). Timedomain NMR techniques have demonstrated that at high water contents, water is no longer bound in the physical sense. However,
the T2 relaxation times of this water population are still small due to hydrogen exchange with surface hydroxyl groups. In gluten, generally much longer T2 relaxation times are observed, and this has been attributed to a higher degree of water mobility (Cherian & Chinachoti, 1996; Esselink, Aalst, Maliepaard, & Duynhoven, 2003; Esselink, Aalst, Maliepaard, Henderson, et al., 2003).
Fig. 5. Cryo scanning electron microscopy dough pieces stored for 90 days at 35 C and frozen at (a) Rate 1, (b) Rate 2, (c) Rate (3) and (d) Rate 4.
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Table 4 Transverse relaxation time (T2, ms) of frozen bread dough after thawing. Storage day
Freezing rate
Storage temperature 10 C
20 C
30 C
35 C
30
Rate Rate Rate Rate
1 2 3 4
16.42 16.35ef 16.28d 16.35ef
16.32 16.01bc 16.25cd 16.45f
16.01 16.35ef 16.37ef 16.21cd
15.93 15.52a 15.66ab 15.39a
60
Rate Rate Rate Rate
1 2 3 4
16.91j 16.76h 16.82hi 16.89ij
16.98jk 16.59g 16.87ij 16.93j
16.84i 16.43f 16.79h 17.06kl
16.73h 16.42f 16.80h 16.62g
90
Rate Rate Rate Rate
1 2 3 4
17.84o 18.01pq 18.20rs 17.61m
17.96pq 17.65m 17.82o 17.67mn
17.50m 17.85o 17.15l 17.51m
17.80o 17.73n 17.60m 17.84o
180
Rate Rate Rate Rate
1 2 3 4
18.59tu 18.46t 18.71w 18.54t
17.56m 18.15r 18.30s 18.03q
18.65uv 18.31st 18.00pq 17.89op
18.00pq 17.78no 17.62m 17.91pq
Values followed by different superscript letters are significantly different at p < 0.05.
In general, longer relaxation times indicate the presence of more mobile water, or at least water that has less restricted rotational freedom. In our studies, T2 values were greatest at 180 days storage at 10 C, and least at 30 days storage at 35 C. At 180 days at 10 C, we also found the least dough extensibility and maximum dough stickiness, while at 30 days storage at 35 C we found maximum extensibility and the least stickiness. This suggests that changes in T2 values are most related to changes in the gluten and starch network. As discussed previously, greater changes in the gluten structure and separation of water occur at high storage temperatures and longer storage times. This greater mobility of water once removed from the vicinity of gluten could well explain the increased T2 values. 3.4. Yeast activity and gas production Table 5 presents the total gas production for thawed dough slurries after 180 min at 36 C. Yeast activity in frozen dough produced under all freezing and storage conditions was lower than that for an unfrozen control. For example, total gas was 112 ml/g
Table 5 Yeast activity (ml CO2 gas/g dry yeast) in frozen dough after thawing. Values for unfrozen dough: 147.0 ml/g dry yeast. Storage day
Freezing rate
Storage temperature 10 C qr
20 C s
30 C q
35 C
30
Rate Rate Rate Rate
1 2 3 4
75.6 84.0s 61.2mn 58.8lm
86.4 95.4t 64.8op 60.0no
74.4 74.4q 57.0lm 55.2k
73.2q 73.2q 55.2k 56.4klm
60
Rate Rate Rate Rate
1 2 3 4
64.8op 72.0q 54.0k 48.6gh
75.6qr 84.6s 56.4klm 51.6ij
63.0no 64.2nop 49.2hi 49.2hi
61.2mn 62.4n 46.8fg 48.0gh
90
Rate Rate Rate Rate
1 2 3 4
51.0ij 56.4klm 43.2ef 40.8d
55.8kl 63.0no 50.4hij 50.4hij
48.6gh 55.8kl 42.6ef 41.4de
47.4fgh 54.6k 41.4de 41.4de
180
Rate Rate Rate Rate
1 2 3 4
43.2ef 46.8fg 36.6bc 34.2a
47.4fgh 51.6ij 41.4de 39.6bcd
41.4de 47.4fgh 36.0ab 35.4a
40.8d 46.2f 34.8a 36.0ab
Values followed by different superscript letters are significantly different at p < 0.05.
dry yeast for unfrozen dough as compared to 95.4 ml/g for frozen dough produced under freezing rate 2 and stored at 20 C for 30 days. In general, the total gas production decreased with longer storage periods. The highest gas production at 30 days was obtained from frozen dough produced at freezing rate 2 and stored at 20 C storage temperature (95.4 ml/g), and the lowest from dough produced at freezing rates 3 and 4 (most rapid freezing) and stored at 30 or 35 C (55.2–57.0 ml/g). Dough frozen at rates 1 and 2 (relatively slow freezing) and stored at 10 and 20 C had higher gas production for all frozen dough samples. This same relative gas production was maintained for all storage periods. However, the total gas volume obtained was lower at longer storage periods at all freezing rates and storage temperatures. The highest gas volume for frozen dough was 84.6 ml/g at 60 days storage, 63.0 ml/g at 90 days, and 51.6 ml/g ml at 180 days; the lowest volume was 46.8 ml/g at 60 days, 40.8 ml/g at 90 days, and 34.2 ml/ g at 180 days. Greatest volumes were obtained from dough produced at freezing rate 2. In almost all cases, maximum gassing power was observed for dough samples held at 20 C, particularly at freezing rates 2 or 1 (41 C or 19 C/h). This temperature is often recommended for storage in industrial settings, although the reasons for this are somewhat empirical (Trevedi, Hauser, Nagodawithana, & Reed, 1989). It has been observed by several researchers that yeast activity is diminished by freezing and storage (Autio & Sinda, 1992; El-Hady et al., 1996; Neyreneuf & Delpuech, 1993; Wolt & D’Appolonia, 1984). Fluctuations in temperature have been found to be especially damaging (Berglund et al., 1991; El-Hady et al., 1996; Inoue & Bushuk, 1991). In addition, yeast may be less viable after freezing (Baguena et al., 1991). Several factors influence yeast activity in frozen dough, including the influence of temperature on metabolism, osmotic stress incurred as ice forms, the ability of yeast to form osmoregulating or other protective compounds, and the effects that freeze-damage of the gluten network may play in limiting diffusion of nutrients and byproducts to and from metabolizing yeast. Lower temperature per se should improve the survival of yeast. However, in the frozen state, temperature determines the amount of ice present in the system, and the rate of freezing determines how the freezing process ensues. Early work (Mazur, 1970; Mazur & Schmidt, 1968) showed that optimal survival of Saccharomyces cerevisiae occurs at cooling rates of 7 C/min. It was suggested that water in yeast cells remains supercooled down to 10 C, as the cell membrane prevents extracellular ice from propagating into the cell. At relatively slow freezing rates this allows time for water to diffuse from the cytoplasm to external spaces, due to the osmotic gradient. Concentration of intercellular solutes and electrolytes leads to dehydration and changes in ionic strength and pH that affect the metabolic functions of the cell and integrity of the cell membrane. At rates faster than 10 C/min, less time is allowed for cell dehydration, and intercellular freezing is reached more quickly. This also contributes to freeze concentration of solutes in the cell. Interestingly, much of the cell water is frozen at 20 C. One might expect that faster freezing rates and lower storage temperatures would be more beneficial to yeast survival. However, Mazur (1970) postulated that in these conditions, intercellular ice is subject to recrystallization during warming, and that this leads to direct damage of cell membranes. Alternately, Muldrew and McGann (1990) speculated that rapid cooling leads to greater osmotic pressure gradients across the membrane, rupturing the membrane and allowing ice to propagate into the cell. In any event, thawing is also a critical determinant of yeast survival. For example, survival in slowly cooled-slowly warmed systems is greater than in slowly cooled-rapidly warmed systems (Mazur & Schmidt, 1968).
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The direct effect of temperature on yeast cell membranes also cannot be discounted. Lower temperature can lead to phase transitions and loss of fluidity of the lipid bilayer, and alter proteinbilayer interactions (Morris & Clark, 1987). Thermotropic transitions have been measured in a variety of unicellular organisms and various ‘‘lesions’’ observed during freezing. The membrane can become leaky at low temperature. For example, liposomal models progressively released more glucose at temperatures between 0 and 25 C (Morris & McGrath, 1981). The amount released and response to temperature depended on the lipid composition of the membrane. A few other factors should also be considered in understanding survival and activity of yeast in frozen dough. First, most of the above studies are done in isolated systems. Yeast in dough will be subject to a different physical and osmotic environment than in compressed yeast (Ribotta et al., 2003). Optimal freezing rates in 2dough are typically below 2 C/min as compared to 7 C/min in isolated yeast cultures (Gelinas, Deaudelin, & Grenier, 1995). In our work, rates around 0.86–1.2 C/min provided optimal yeast activity. In addition, S. cerevisiae have developed mechanisms to combat stress conditions. The accumulation of trehalose in the cell seems to be particularly important in combating the osmotic stresses of freezing and drying, as well as stabilizing the cell membrane (Hino, Mihara, Nakashima, & Takano, 1990; Majara, Oconnor-Cox, & Axcell, 1996; Van Dijck, Colavizza, Smet, & Thevelein, 1995). Indeed, much work is ongoing in producing S. cerevisiae with elevated trehalose, as by disrupting the ATH1 gene that produces trehalose-hydrolyzing enzyme (Kim, Alizadeh, Harding, Hafner-Gravink, & Klionsky, 1996). In addition, membrane proteins such as aquaporin also help transport water across the yeast cell membrane, and it is thought that this controlled efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage (Tanghe et al., 2002). Rapid freezing may limit the ability of yeast to adapt to freezing stress. On the whole, relatively slow freezing rates are optimal for maintaining yeast activity after thawing (El-Hady et al., 1996; Havet et al., 2000; Lorenz, 1974). 3.5. Bread volume Table 6 shows the volume of bread made from the various frozen dough treatments. The volume of bread made from unfrozen control dough was 137.8 ml. All breads made from frozen dough
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had lower volumes. Typically, greatest loaf volumes were attained from dough frozen at rate 2. However, freezing rate was not a strong determinant for loaf volume. For example, after 10 C and 30 days, loaf volume varied only from 111.7 to 113.3 ml, while at 35 C it varied from 91.7 to 94.7 ml. Similarly, for dough stored for 180 days at 10 C loaf volume varied from 86.3 to 90.0 ml. Greatest overall loaf volume (119.0 ml) was seen with bread made from dough frozen at rate 2, and stored at 20 C for 30 days, the same conditions found for greatest yeast activity. Lowest volumes were found with bread made from dough frozen at rates 3 or 4 and stored at 30 or 35 C (83.7–84.0 ml at 180 days). There was no significant difference between freezing rate 3 and 4 for all storage times. Loaf volumes for all breads decreased with increased dough storage time. For example, at 10 C volumes decreased from 111.7 to 113.3 ml at 10 C and 30 days to 86.3–90.0 ml after 180 days. At storage temperatures of 30 and 35 C, the effect of storage time was less pronounced. After 180 days, bread volumes from samples frozen at rate 2, and held at 10 or 20 C, were slightly higher than the other treatments, but there were no significant differences amongst volumes for all other samples held for 180 days. Storage temperature also had a major influence on subsequent loaf volume. In general, samples held at 20 C produced bread with greatest loaf volume, although storage temperature effects interacted with storage time as described above. Again, this seems to reflect, at least in part, the effects of treatment on yeast activity.
3.6. Bread crumb firmness Table 7 shows crumb firmness for bread made from frozen dough. Measured firmness was 4.03 N for bread made from unfrozen control dough. For that made from frozen/thawed dough, crumb firmness increased significantly with frozen storage time of the dough. For example, at 20 C firmness values ranged from 5.99 to 6.88 N at 30 days, and from 7.76 to 8.04 N at 180 days. In general, bread made from dough frozen at rates 1 and 2 was less firm than bread made from dough frozen at rates 3 and 4 for all storage times. The storage temperature also had a significant effect on bread firmness. All dough samples stored at 10 and 20 C produced bread that was less firm than other samples for all storage times. At 30 and 60 days, bread made from dough stored at 10 C was less firm than that from dough stored at 20 C.
Table 6 Volume of bread (ml) made from frozen dough. Values of bread from unfrozen dough: 137.8 ml.
Table 7 Firmness (force, N) of bread made from frozen bread dough. Values of bread from unfrozen dough: 4.03 N.
Storage day
storage day
Freezing rate
Storage temperature 10 C w
20 C
o
Freezing rate
35 C no
Storage temperature 10 C
tu
35 C
1 2 3 4
111.7 113.3xy 113.3xy 112.7wx
115.3 119.0A 113.3xy 113.0wxy
94.3 95.3pq 93.7mno 94.7op
94.0 94.7op 91.7jk 92.0jkl
30
Rate Rate Rate Rate
1 2 3 4
5.00 4.56a 5.78c 5.91c
6.12 5.99cd 6.47e 6.88fg
10.60 10.76tu 10.46stu 10.38rs
9.05no 8.92no 9.33p 9.52pqr
60
Rate Rate Rate Rate
1 2 3 4
108.7tu 109.3uv 109.0u 108.0t
113.3x 116.3z 111.7w 112.7wx
92.3kl 93.7mno 91.3ij 91.7jk
92.3kl 93.3mn 91.3ij 91.3ij
60
Rate Rate Rate Rate
1 2 3 4
5.93c 5.87c 6.47e 6.16de
6.80f 6.64f 6.90g 6.95gh
11.81wx 12.18yz 12.29yzA 11.74w
11.40v 11.68vw 11.42v 11.75w
90
Rate Rate Rate Rate
1 2 3 4
104.7s 107.3t 101.0r 102.0r
109.0u 112.0w 107.7t 107.7t
92.0jkl 92.7lm 88.7e 88.7e
90.3ghi 90.7hi 88.3de 88.7e
90
Rate Rate Rate Rate
1 2 3 4
8.00kl 7.71k 9.07op 8.86mno
7.28hij 7.12hi 6.96gh 7.35ij
12.26yz 12.36zA 12.04xy 12.40zAB
12.39zA 11.91wxy 11.92wxy 12.38zA
180
Rate Rate Rate Rate
1 2 3 4
88.7e 91.3ij 87.0c 87.7cd
88.0cde 89.3fg 85.3ab 84.0a
86.0b 89.0ef 84.0a 83.7a
180
Rate Rate Rate Rate
1 2 3 4
8.03kl 8.04kl 7.76k 8.04kl
12.48AB 12.73CD 12.34zA 12.94CD
12.91CD 12.77BCD 12.60BC 12.82CD
8.78mn 8.46lm 10.18rs 9.73qr
cd
30 C
Rate Rate Rate Rate
Values followed by different superscript letters are significantly different at p < 0.05.
ab
20 C
30
88.3de 90.0gh 86.3bc 87.7cd
z
30 C
Values followed by different superscript letters are significantly different at p < 0.05.
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optimize bread quality. Prolonged storage times lead to detrimental changes in both gluten structure and yeast viability.
References
Fig. 6. Contour plots for elasticity (E), yeast activity (Y), and bread volume (V) at 90% of their maximum value; and adhesiveness (A) and firmness (F) at 90% of their minimum value. Arrows show direction toward more optimal values.
One usually assumes that a less firm, tenderer bread is most desirable for consumers. As noted, firmness of bread from the control was lower than that from frozen dough. Crumb firmness arises from the integrity of the gluten network, the degree of crosslinking, the amount of gas incorporated, and from other structural components of the bread. Ribbotta, Perez, Leon & Anon (2004) reported similar results to the ones reported in this work with frozen storage temperature. With longer frozen storage time, the firmness increased, which may come from depolymerization of glutenin and higher retrogradation of amylopectin in bread made from frozen dough.
3.7. Response surface Fig. 6 shows a response surface for the various quality measurements as a function of freezing rate and frozen storage temperature. The overlapping contour lines show each variable at 90% of their optimal levels, with arrows pointing to more optimal regions. Both volume and firmness depended primarily on storage temperature, and storage temperatures below 20 C were most beneficial. Elasticity and adhesiveness depended on both storage temperature and freezing rate, and generally lower temperatures and higher freezing rates were beneficial. Yeast activity also depended on temperature and freezing rate, but in this case lower temperatures and slower freezing rates were more beneficial. An optimal region for all attributes (shaded in grey) occurs at temperatures approximately between 15 and 20 C and centered around freezing rate 2.
4. Conclusions The quality of bread made from frozen dough depends on the rate of freezing, storage temperature, and the length of time stored. Faster freezing and lower storage temperatures promote less damage to the gluten network, thus help retain elastic properties of the dough. However, relatively lower freezing rates and storage temperatures promote yeast viability and gassing power. As such, a compromise in freezing rate and storage temperature is needed to
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J. Yi, W.L. Kerr / LWT - Food Science and Technology 42 (2009) 1474–1483 Mazur, P. (1970). Cryobiology – freezing of biological systems. Science, 168(934), 939–949. Mazur, P., & Schmidt, J. J. (1968). Interactions of cooling velocity, temperature, and warming velocity on the survival of frozen and thawed yeast. Cryobiology, 5(1),1–17. Morri, G. J., & Clarke, A. (1987). Cells at low temperatures. In G. J. Morris, & B. W. W. Grout (Eds.), The Effects of Low Temperature on Biological Systems (pp. 71–129). Baltimore, MD: Edward Arnold. Morris, G. J., & McGrath, J. (1981). The response of multilamellar liposomes to freezing and thawing. Cryobiology, 18, 390–398. Muldrew, K., & McGann, L. E. (1990). Mechanisms of intracellular ice formation. Biophysical Journal, 57, 525–532. Neyreneuf, O., & Delpuech, B. (1993). Freezing experiments on yeasted dough slabs – effects of cryogenic temperatures on the baking performance. Cereal Chemistry, 70(1), 109–111. Nicolas, Y., Smit, R. J. M., Aalst, Hv., Esselink, F. J., Weegels, P. L., & Agterof, W. G. M. (2003). Effect of storage time and temperature on rheological and microstructural properties of gluten. Cereal Chemistry, 80(4), 371–377. Ribotta, P. D., Leon, A. E., & Anon, M. C. (2003). Effect of yeast freezing in frozen dough. Cereal Chemistry, 80(4), 454–458. Ribotta, P. D., Perez, G. T., Leon, A. E., & Anon, M. C. (2004). Effect of emulsifier and guar gum on micro structural, rheological and baking performance of frozen bread dough. Food Hydrocolloids, 18(2), 305–313. Rojas, J. A., Rosell, C. M., Benedito de Barber, C., Pe´rez-Munuera, I., & Lluch, M. A. (2000). The baking process of wheat rolls followed by cryo scanning electron microscopy. European Food Research Technology, 212, 57–63.
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Roman-Gutierrez, A. D., Guilbert, S., & Cuq, B. (2002). Frozen and unfrozen water contents of wheat flours and their components. Cereal Chemistry, 79(4), 471–475. Ruan, R. R., Wang, X. A., Chen, P. L., Fulcher, R. G., Pesheck, P., & Chakrabarti, S. (1999). Study of water in dough using nuclear magnetic resonance. Cereal Chemistry, 76(2), 231–235. Sharadanant, R., & Khan, K. (2003). Effect of hydrophilic gums on the quality of frozen dough: II. Bread characteristics. Cereal Chemistry, 80(6), 773–780. Suchy, J., Lukow, O. M., & Ingelin, M. E. (2000). Dough microextensibility method using a 2-g mixograph and a texture analyzer. Cereal Chemistry, 77(1), 39–43. Tanghe, A., Van Dijck, P., Dumortier, F., Teunissen, A., Hohmann, S., & Thevelein, J. A. (2002). Aquaporin expression correlates with freeze tolerance in baker’s yeast, and overexpression improves freeze tolerance in industrial strains. Applied and Environmental Microbiology, 68(12), 5981–5989. Trevedi, N., Hauser, J., Nagodawithana, T., & Reed, G. (1989). Update on baker’s yeast. AIB Technical Bulletin, XI(2). American Institute of Baking. Van Dijck, P., Colavizza, D., Smet, P., & Thevelein, J. M. (1995). Differential importance of trehalose in stress resistance in fermenting and nonfermenting Saccharomyces cerevisiae cells. Applied Environmental Microbiology, 61(1), 109–115. Varrianomarston, E., Hsu, K. H., & Mahdi, J. (1980). Rheological and structuralchanges in frozen dough. Bakers Digest, 54(1), 32–34. Wolt, M. J., & D’Appolonia, B. L. (1984). Factors involved in the stability of frozen dough. II. The effects of yeast type, flour type, and dough type additives on frozen-dough stability. Cereal Chemistry, 61(3), 213–221.
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LWT - Food Science and Technology journal homepage: www.elsevier.com/locate/lwt
Combined effects of freezing rate, storage temperature and time on bread dough and baking properties Jinhee Yi a, William L. Kerr b, * a b
Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011, USA Department Food Science and Technology, University of Georgia, Athens, GA 30602, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 October 2008 Received in revised form 13 May 2009 Accepted 15 May 2009
This study compares the effects of freezing temperature and rate as well as storage temperature and time on the quality of frozen dough. Yeasted bread dough was frozen using four freezing rates (19–69 C/h), then stored at 10, 20, 30, or 35 C for up to 180 days. Dough strength diminished with longer storage time and higher storage temperatures. Cryo-SEM showed that dough stored at 30 and 35 C had the least damaged gluten network. NMR studies showed that more rapidly frozen dough, and that stored at lower temperatures had lower transverse relaxation (T2) times (9–10 ms). However, dough stored at 20 C displayed the highest yeast activity among samples. Bread loaf volume decreased with storage time, and bread made from dough stored at 20 C showed the highest loaf volume. Breads produced from 30 and 35 C stored dough displayed less change in the texture profile during storage as well as less change in T2 values. Response surface analysis showed that optimal properties occurred at freezing rates of around 19–41 C/h and storage temperatures of 15 to 20 C. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Frozen dough Stickiness Extensibility Bread volume
1. Introduction Bread made from frozen dough has become an increasingly popular alternative to that made directly from unfrozen dough. Frozen dough can be manufactured in large quantities off-site, and then shipped to local restaurants or retail operations, saving on both equipment and labor costs. In recent years, the quality of these products has improved owing to advances in technology and formulation, but there is room for additional improvement. Problems associated with frozen dough include long proof time, low volume, poor texture, and variable performance (Kenny, Wehrle, Dennehy, & Arendt, 1999). Some of the poorer quality can be attributed to diminished yeast activity, the characteristics of the yeast and their survival after freezing (Baguena, Soriano, Martinezanaya, & Debarber, 1991; El-Hady, ElSamahy, Seibel, & Brummer, 1996; Hino, Takano, & Tanaka, 1987; Hsu, Hoseney, & Seib, 1979; Ribotta, Leon, & Anon, 2003; Wolt & D’Appolonia, 1984). In order to improve performance, frozen dough processors may add extra yeast, use short or no-time dough processing procedures, mix ingredients at relatively low temperatures, or incorporate new strains of freeze-tolerant yeasts.
* Corresponding author. Tel.: þ1 706 542 1085; fax: þ1 706 542 1050. E-mail address: [email protected] (W.L. Kerr). 0023-6438/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2009.05.017
During freezing, there are also deleterious effects on dough structure. Electron microscopy studies (Esselink, Aalst, Maliepaard, Henderson, et al., 2003) showed a breakdown of the reticular gluten network when subjected to freezing and thawing, and related these to changes in the rheological properties of the dough. Ice recrystallization and water migration in the dough also affects the dough structure (Bache & Donald, 1998; Rojas, Rosell, Benedito de Barber, Pe´rez-Munuera, & Lluch, 2000). Extended frozen storage alters the gluten protein matrix (Berglund, Shelton, & Freeman, 1991; Varrianomarston, Hsu, & Mahdi, 1980), results in dough weakening, and causes less gas retention (Autio & Sinda, 1992; Hsu et al., 1979; Inoue & Bushuk, 1991). In addition, injury to yeast membranes caused by freezing and thawing release cellular chemicals that have deleterious effects on the dough structure. However, researchers have different opinions on the effects of specific leachates, mainly reducing compounds such as glutathione, on the gluten network (Casey & Foy, 1995; Inoue & Bushuk, 1991; Wolt & D’Appolonia, 1984). While several researchers have studied the effects of storage time or temperature on yeast viability and dough structure (El-Hady et al., 1996; Havet, Mankai, & Le Bail, 2000; Mazur, 1961), less has been done on the influence of freezing rate. Especially in frozen dough preparations, where freezing and sometimes prolonged frozen storage intervene between dough formation and bread baking, several factors still have not been fully investigated (Giannou & Tzia, 2007). In the present study, we examined the
J. Yi, W.L. Kerr / LWT - Food Science and Technology 42 (2009) 1474–1483
combined effects of freezing rate, storage temperature and storage time on dough and bread quality. The dough properties were evaluated based on dough extensibility and adhesiveness, and the viability of yeast assayed through measurements of gas production. Both volume and firmness were measured in the finished breads. Direct examination of the dough in the frozen state was accomplished using cryogenic scanning electron microscopy (cryo-SEM), allowing visualization of the ultra-structure of gluten-starch association and the state of gluten strands in the network. We also used time-domain NMR to investigate changes in water dynamics (Esselink, Aalst, Maliepaard, & Duynhoven, 2003; Roman-Gutierrez, Guilbert, & Cuq, 2002; Ruan et al., 1999). 2. Materials and methods 2.1. Dough preparation and freezing Bread dough was prepared from enriched, bleached hard winter wheat flour (Organic Select Artisan Flour, King Arthur Company, Norwich, Vermont) with 11.5 g/100 g protein content; Baker’s dry yeast (Fleischmann’s Yeast, Quebec, Canada); Dominion pure cane sugar (Dixie Crystal, Savannah, GA); non-iodized salt (Morton International, Inc., Chicago, IL); and soybean oil (Wesson Vegetable Oil, ConAgra Foods, Omaha, Nebraska). The basic formulation was (per 100 g flour weight): water (60 g/100 g), sugar (5 g/100 g), oil (5 g/100 g), yeast (3 g/100 g), and salt (2 g/100 g). The yeast was prehydrated with the water, and then all of the dough ingredients were placed in a 6-quart, 575 W mixer (Kitchen Aid Professional 600 Series, St. Joseph, MI) and mixed for 2 min at 120 rpm with a paddle, and for 8 min at 178 rmp with a dough hook. Once the dough was formed, it was separated into samples of 50 g, and shaped by hand into approximately 50 mm diameter circular slabs. Dough pieces were frozen in a built-in forced-air blast freezer located at the UGA Department of Food Science and Technology in Athens, GA. As part of the experimental design, the freezer was adjusted to give four different freezing rate schemes, as shown by the temperature-time plots in Fig. 1. Specific freezing times and rates depend on the conditions and final temperature. The average rate (and time) to reach 30 C were: Rate 1: 19 C/h (2.53 h); Rate 2: 41 C/h (1.18 h); Rate 3: 55 C/h (0.88 h); and Rate 4: 72 C/h (0.67 h). In terms of the time to go from the initial freezing point to 30 C, these times were: Rate 1: 2.27 h; Rate 2: 0.95 h; Rate 3: 0.62 h; and Rate 4: 0.45 h. After freezing to the desired temperature, dough samples were vacuum-packaged in plastic bags and placed at four different frozen storage temperatures (10, 20, 30 or 35 C), and stored for 30,
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60, 90, and 180 days. At each sampling period, 3 replications of the 16 treatments were withdrawn from the freezers and placed in an environmental chamber (Model HEC10R, HotPack, Warminster, PA) at 8 C and 35% RH in order to thaw. Thawed dough pieces were removed from the package, then placed in a chamber at 36 C and 85% RH for proofing. Dough samples were baked in a 5-rack gas convection oven (Blodgett BLD-DFG100, Burlington, Vermont) at 180 C for 15 min. After baking, bread samples were allowed to cool for approximately 1 h prior to subsequent measurements. 2.2. Dough extensibility and adhesiveness Extensibility of the dough was measured using a texture analyzer (TA-XT2i, Texture Technologies Corp., Scarsdale, NY) with a modified Kieffer extensibility rig and 5 kg load cell. Approximately 50 g dough samples were molded into strips approximately 7 mm in diameter and 60 mm in length. All samples were left to rest on a grooved plate at 8 C for 20 min and 90% RH prior to testing (Anderssen, Bekes, Gras, Nikolov, & Wood, 2004). The dough was pulled at a crosshead speed of 3.3 mm/s. The resistance to extension (maximum force) and extensibility (distance to break) were calculated from the force–deformation curves. One advantage of the Kieffer dough extensibility rig is that it uses a micro-extension method involving a very small sample size. It correlates highly with methods such as the extensigraph as indicated by baking performance (Kieffer, Wieser, Henderson, & Graveland, 1998; Sharadanant & Khan, 2003; Suchy, Lukow, & Ingelin, 2000). Adhesiveness was measured using the texture analyzer with a modified Chen–Hoseney stickiness rig, with conditions as described by Chen and Hoseney (1995). The sample was placed in a cylindrical cell on the base of the texture analyzer, which was then enclosed by a lid with a perforated hole. A small amount of dough was extruded through the hole. The upper cylindrical probe was brought in contact with the exposed dough to adhere to it, and the probe was pulled away from the base at a speed of 1.7 mm/s. Both the maximum force and the area under the force–deformation curve required to separate the probe from the test sample were used as measures of adhesiveness. 2.3. Cryo scanning electron microscopy A Jeol JSM-5410 scanning electron microscope with a CT-500C cryo-unit (Oxford Instruments, Oxfordshire, UK) was used to investigate the microstructure of the frozen bread dough. Each frozen dough sample was placed on the cryo-specimen holder, placed in liquid nitrogen, and then transferred to the cryo-unit in the frozen state. The dough specimens were fractured, sublimated (10 min at 70 C) and sputter coated with gold (4 min at 0.2 kPa). The prepared dough specimen was transferred to the microscope where it was observed at 15 kV and 120 C. Micrographs were taken at 500, 1000 and 2000 magnification. 2.4. NMR measurements of dough
Fig. 1. Freezing protocols for frozen bread dough. Average rate (and time) to reach 30 C. Rate 1: 19 C/h (2.53 h); Rate 2: 41 C/h (1.18 h); Rate 3: 55 C/h (0.88 h); and Rate 4: 72 C/h (0.67 h).
Proton relaxation measurements were made using a 20 MHz Proton (1H) NMR spectrometer (Resonance Instruments, Whitney, UK). Approximately 5 g dough samples were taken from the center of the thawed dough. Three replicates were taken from each piece of dough. Each sample was placed in a 10 mm diameter glass tube then covered with parafilm. The glass tube was placed in a 10 C bath for 20 min. After 20 min, the glass tubes with dough specimens were placed in 18 mm diameter NMR tubes. Transverse (T2) relaxation curves were developed using the CPMG pulse sequence: 90x(s-180y s-echo)n. Acquisition parameters were set to a 90 pulse of 4.2 ms and a recycle delay of 2 s. A pulse spacing (s) of
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100 ms was chosen to exclude the fast decaying solid-like signal. All measurements were made at 25 C 1 C. Relaxation curves were analyzed using the WinFIT multi-exponential routine (Resonance Instruments, Whitney, UK). 2.5. Yeast activity Yeast activity was measured using AACC method 89-01 (2000) with slight modification. Dough pieces (10 g) and 100 g water were placed into a 250 ml glass reaction vessel, capped, and placed in a water bath at 36 1 C. A hose was connected to the vessel, leading to a volumetric manometer. Gassing power was determined from the volume of carbon dioxide gas trapped after 180 min. Each 10 g sample contained 0.163 g dry yeast.
Table 1 Maximum resistance to extension of frozen bread dough (force, g) after thawing. Values for unfrozen dough: 69.98 g. Storage day
Freezing rate
10 C
20 C
30 C
35 C
30
Rate Rate Rate Rate
1 2 3 4
49.68op 51.19pq 52.19r 52.75s
53.28st 54.01t 55.60vw 56.05wx
54.33tu 54.19t 56.99y 56.41xy
54.73uv 55.25v 55.74w 56.02wx
60
Rate Rate Rate Rate
1 2 3 4
47.01kl 47.62m 48.89no 48.70n
49.62o 50.38p 52.62rs 53.38st
51.77q 51.86qr 52.65rs 52.74s
51.11pq 52.25rs 51.45q 52.22rs
90
Rate Rate Rate Rate
1 2 3 4
43.78h 44.84hi 45.11i 45.71j
45.45ij 46.34jk 47.31lm 47.30lm
45.33i 47.50m 47.73 46.64k
45.38 47.73m 47.02kl 47.32lm
180
Rate Rate Rate Rate
1 2 3 4
29.17a 29.54ab 29.49a 29.71ab
30.15bc 33.68fg 32.64def 32.39de
31.99cd 33.19fg 32.82ef 32.67ef
32.01d 33.06f 33.12fg 32.13de
2.6. Bread volume Loaf volume and weight were determined after baking and cooling for 1 h at ambient temperature (AACC 10-05, 2000). A container was filled with a known volume of rapeseed. The amount of seed displaced when the loaf was introduced was measured in a graduated cylinder, and measured the loaf volume. 2.7. Bread crumb firmness The firmness of the bread samples was determined using a texture analyzer (TA-XT2i, Texture Technologies Corp., Scarsdale, NY) with a 36 mm radius cylinder probe according to AACC Method 74-10A (2000). Three center slices of each loaf were measured at 25 C. The compression rate was set at 1.7 mm/s. Firmness was the force required to compress the slices (20 mm thick) by 40% strain. 2.8. Statistical analysis ANOVA was used to analyze all data, based on a three-way mixed design. Fisher’s LSD was used to determine significant differences between samples. A p-value of less than 0.05 was considered significant. A response surface methodology was used to examine regions of minimal or maximal response, particularly to see if there were similar regions of optimal response for the various response variables. Models surfaces were fit using linear, squared, and crossed terms using the JMP 7 software (SAS, Cary, NC). Contour plots were developed to where the response reached 90% of its minimum or maximum value. 3. Results and discussion 3.1. Dough extensibility and adhesiveness
Storage temperature
Values followed by different superscript letters are significantly different at p < 0.05.
of frozen dough. Freezing rate 2 produced dough with the highest extensibility, while those from freezing rate 1 had the lowest extensibility. Dough from freezing rate 1 also had lower MRE than those from other freezing rates. Frozen storage temperature also had some effect on dough extensibility. With lower frozen storage temperature (closer to 35 C), the extensibility increased but there was no significant difference between rates 3 and 4 (the fastest rates). The interaction between the freezing rate and frozen storage temperature determined extensibility and MRE. For the first 30 days, dough prepared at freezing rate 4 (the fastest freezing) and stored at 35 C had the highest extensibility (81.0 mm); dough prepared at freezing rate 1 and stored at 10 C had the lowest extensibility (74.45 mm). After 180 days, dough prepared at freezing rate 2 and stored at 35 C had the highest extensibility (71.39 mm); those produced at freezing rate 1 and stored at 10 C had the lowest extensibility (49.94 mm). Decreases in maximum resistance, and in extensibility may indicate deterioration in the gluten network. Extensibility has been attributed to the gluten structure, and particularly to high molecular weight glutenins (Belton, 1999; Bushuk & Macritchie, 1989; Table 2 Extensibility of frozen bread dough (distance to peak, mm) after thawing. Values for unfrozen dough: 84.99 mm. Storage day
Freezing rate
Storage temperature 10 C
Tables 1 and 2 show the maximum resistance to extension (MRE, in g force) and extensibility (in mm) of dough subject to different freezing and storage conditions. For the control (a freshly made, unfrozen dough), MRE was 69.98 g with an extensibility of 84.99 mm. All dough samples subject to freezing had lower resistance and extensibility than the control. Both the MRE and the extensibility decreased with time from 0 to 180 days of frozen storage. For example, maximum resistance ranged from 49.68 to 56.99 g at 30 days, and from 29.17 to 33.68 g at 180 days. Similarly, extensibility ranged from 74.45 to 81.00 mm at 30 days, and from 49.94 to 71.39 mm at 180 days. Storage time had a greater influence on extensibility than temperature. For samples prepared at freezing rate 1 (the slowest rate), and held for 30 days, extensibility varied from 74.45 mm (at 10 C) to 76.84 mm (at 35 C). After 180 days of storage, extensibility ranged from 49.94 mm (at 10 C) to 70.04 mm (at 35 C). Freezing rate also affected the extensibility
qr
20 C vw
30 C wx
35 C
30
Rate Rate Rate Rate
1 2 3 4
74.45 76.12uv 75.86tuv 75.22rs
76.23 78.10AB 77.42xy 77.69yzA
77.19 79.58BC 80.18CD 79.95CD
76.84vw 80.92D 80.99D 81.00D
60
Rate Rate Rate Rate
1 2 3 4
69.81hi 72.90no 71.64lmn 70.96kl
73.37nop 76.09uv 75.23rs 75.30rs
75.28rs 77.45xy 76.93w 77.30wx
73.46op 78.81AB 77.49xy 77.93zA
90
Rate Rate Rate Rate
1 2 3 4
63.94c 67.10e 66.66de 65.75d
72.59mn 73.58op 73.74pq 74.63qr
73.94pq 75.76tu 74.93rs 75.63st
74.71r 75.81tuv 75.15rs 75.42st
180
Rate Rate Rate Rate
1 2 3 4
49.94a 52.43b 52.00b 52.42b
67.59ef 69.24g 68.38f 69.66gh
69.19g 71.07kl 70.02hij 70.78jkl
70.04ij 71.39lmn 70.30ij 70.48jk
Values followed by different superscript letters are significantly different at p < 0.05.
J. Yi, W.L. Kerr / LWT - Food Science and Technology 42 (2009) 1474–1483
Lindsay & Skerritt, 1999). While disulfide bonds help to maintain the gluten network and provide resistance to stretching, several theories have been developed to explain dough elasticity (Belton, 1999; MacRitchie & Lafiandra, 1997). Our results indicate that relatively rapid freezing and low storage temperatures result in the least changes to the gluten structure. Most deleterious were slow freezing rates, high storage temperature, and prolonged storage times. Rapid freezing and low storage temperatures promote the creation of a greater number of small ice crystals, due to enhanced nucleation and crystal growth rates. Low freezing rate promotes the growth of relatively large crystals. It has been suggested that ice crystals physically disrupt and damage the gluten network, and dehydrate the gluten so as to cause irreversible structural changes. Extensibility determines the ability of the dough to extend during gas production by yeast during proofing. Excessively high extensibility results in weak and slack dough, which collapses during the proofing stage or while baking in the oven (Sharadanant & Khan, 2003). MRE measures the ability of the dough to retain gas and subsequently to form springy bread. A very low resistance to extension results in poor gas retention and lower loaf volume. A very high resistance to extension also results in a lower loaf volume because the tough dough is not capable of proofing to an optimum height with the gas produced by the yeast. Contrary to our findings, Inoue and Bushuk (1991) reported that extensibility increased with storage time for one week but no clear trend was observed. However, Sharadanant and Khan (2003) reported that extensibility increased with extended storage time and maximum resistance to extension decreased with storage time. Table 3 shows the adhesiveness of dough samples prepared at different freezing rates, storage temperatures and storage times. Adhesiveness of unfrozen dough was 69.08 g. Adhesiveness of all samples from frozen dough were higher than that of control, indicating more sticky dough. In general, dough adhesiveness increased with storage time. For example, at 30 days adhesiveness ranged from 74.77 to 80.77 g, while at 180 days values ranged from 93.25 to 115.63 g. Storage temperature was not a significant factor for samples stored at 30 and 60 days, but was for samples stored at 90 and 180 days. At 90 and 180 days, samples stored at lower temperatures were less adhesive. Freezing rate was also a significant factor for adhesiveness, except for samples stored at 30 days. Samples prepared at freezing rate 2 were less adhesive; those at freezing rate 1 had the highest adhesiveness. In general, adhesiveness values followed a similar Table 3 Adhesiveness of frozen bread dough (tension force, g) after thawing. Values for unfrozen dough: 69.08 g. Storage day
Freezing rate
Storage temperature 10 C fg
20 C cd
30 C
35 C
ef
30
Rate Rate Rate Rate
1 2 3 4
79.47 75.04b 77.49c 78.73def
78.03 77.20c 81.00hi 80.77ghi
79.30 78.22def 80.10gh 79.28ef
78.07cd 75.12b 74.77a 77.82cd
60
Rate Rate Rate Rate
1 2 3 4
82.78klm 77.88cd 82.21jk 82.01jk
88.39mn 78.81ef 83.37lm 82.37jkl
87.70mn 78.16de 80.30gh 82.00jk
83.15lm 81.63ij 82.41kl 80.93hi
90
Rate Rate Rate Rate
1 2 3 4
108.45BC 105.86zA 107.84B 106.24A
97.48x 91.99p 92.36qr 92.37qr
94.37tu 90.83o 91.97p 92.00pq
93.15rs 91.63p 92.41qr 93.43rs
180
Rate Rate Rate Rate
1 2 3 4
115.63E 114.47DE 115.52E 113.74D
109.60C 98.83y 108.69BC 105.32z
97.23x 93.25rs 94.44uv 94.25tu
95.19vw 94.34tu 94.02st 93.46rst
Values followed by different superscript letters are significantly different at p < 0.05.
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dependence as extensibility. Samples stored at 35 C for short times (30 days) were more extensible (with higher force) and less adhesive. Samples stored for long time at low temperature (10 C) were less extensible (with lower maximum force) and were more adhesive. Adhesiveness is related to the force required to separate a material from a dissimilar surface. As discussed previously, freezing of dough and frozen storage may cause separation of water from the gluten network, particularly as water is redistributed due to ice recrystallization. This water may be more feely assessable at surfaces, leading to increased adhesiveness. Samples prepared at freezing rate 2 were less adhesive than those at other freezing rates. Several researchers have reported that dead yeast cells produce reducing substances that cause disruption of molecular bonds in the gluten network. It will be shown in a subsequent section that highest yeast activity incurred for samples prepared at freezing rate 2. One possibility is that fewer reducing substances are produced at freezing rate 2, as there are more viable yeast cells, and consequently less deterioration of the gluten network. This in turn results in less migration of water after freezing and thawing, and thus less adhesiveness. 3.2. Microstructure of frozen bread dough Cryo-SEM was used to visualize the microstructure of frozen dough (in the frozen state). The frozen dough structure at 90 days showed the typical structure of starch granules embedded in a gluten network (Figs. 2–5). All frozen dough samples had voids among the gluten network and starch granules. The gluten strands composing the network appeared to be quite damaged after 90 days. Dough produced at freezing rate 1 and 2 (the slowest rates) were less uniform and had thinner strands. Dough produced at freezing rates 3 and 4 (more rapid freezing) showed less disrupted structure. Storage temperature had a more notable effect on the gluten network in the dough. With lower storage temperature (closer to 35 C), the dough was more intact and had a more uniform gluten network. Regardless of freezing rate, dough stored at 35 C had less disrupted gluten strands than that stored at 10 C. Microstructural observations help explain the observed rheological properties of dough. As noted, dough stored at higher temperatures and for longer times was less extensible, and more adhesive, than dough stored at low temperatures for shorter times. Lower extensibility can be attributed to the thinner, more disrupted bonding in the gluten. As previously discussed, this disrupted structure is also less able to hold water, and thus more adhesive. Dough microstructure has been investigated by several researchers. Ribotta, Perez, Leon, and Anon (2004) studied the effect of emulsifiers on frozen bread dough and described the effects of extended frozen storage on the protein matrices. After long storage time, the gluten matrix was quite damaged. Rojas et al. (2000) described dough as a continuous gluten matrix, with starch granules scattered among the protein network. Discontinuities were observed in the lax matrix surrounding the starch granules under greater magnification. Berglund et al. (1991) found that after 24 weeks frozen storage, the gluten matrix had thinner strands, seemed more disrupted, and was separated from the starch granules. Nicolas et al. (2003) observed that during freezing, ice crystals appear to compress the gluten, leading to a significant phase separation between the gluten and ice. 3.3. NMR relaxometry Pulsed 1H NMR was used to investigate the relaxation characteristics of dough systems subject to different freezing rate and storage conditions. Table 4 shows the transverse relaxation times
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Fig. 2. Cryo scanning electron microscopy dough pieces stored for 90 days at 10 C and frozen at (a) Rate 1, (b) Rate 2, (c) Rate (3) and (d) Rate 4.
for the thawed dough samples. The T2 relaxation time increased somewhat with increased frozen storage time at all storage temperatures. At 30 days, T2 values ranged from 15.39 to 16.45 ms, while at 180 days T2 values ranged from 17.56 to 18.71 ms. In most cases, the T2 times were smallest for samples at freezing rate 2. Frozen storage temperature also had a significant, but small, effect on transverse relaxation times. Samples stored at lower
temperature had somewhat shorter T2 values. For example, at 30 days samples held at 10 C had values ranging from 16.28 to 16.42, while at 35 C values ranged from 15.39 to 15.93 ms. The amount and state of water play an important role in the preparation and properties of wheat flour dough and their products. Ruan et al. (1999) used several methods for presentation and analysis of relaxation time measurement of protons in dough, and
Fig. 3. Cryo scanning electron microscopy dough pieces stored for 90 days at 20 C and frozen at (a) Rate 1, (b) Rate 2, (c) Rate (3) and (d) Rate 4.
J. Yi, W.L. Kerr / LWT - Food Science and Technology 42 (2009) 1474–1483
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Fig. 4. Cryo scanning electron microscopy dough pieces stored for 90 days at 30 C and frozen at (a) Rate 1, (b) Rate 2, (c) Rate (3) and (d) Rate 4.
suggested there is a continuous distribution of protons having different relaxation times in heterogeneous systems such as dough (Esselink, Aalst, Maliepaard, & Duynhoven, 2003; Esselink, Aalst, Maliepaard, Henderson, et al., 2003; Ruan et al., 1999). Timedomain NMR techniques have demonstrated that at high water contents, water is no longer bound in the physical sense. However,
the T2 relaxation times of this water population are still small due to hydrogen exchange with surface hydroxyl groups. In gluten, generally much longer T2 relaxation times are observed, and this has been attributed to a higher degree of water mobility (Cherian & Chinachoti, 1996; Esselink, Aalst, Maliepaard, & Duynhoven, 2003; Esselink, Aalst, Maliepaard, Henderson, et al., 2003).
Fig. 5. Cryo scanning electron microscopy dough pieces stored for 90 days at 35 C and frozen at (a) Rate 1, (b) Rate 2, (c) Rate (3) and (d) Rate 4.
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Table 4 Transverse relaxation time (T2, ms) of frozen bread dough after thawing. Storage day
Freezing rate
Storage temperature 10 C
20 C
30 C
35 C
30
Rate Rate Rate Rate
1 2 3 4
16.42 16.35ef 16.28d 16.35ef
16.32 16.01bc 16.25cd 16.45f
16.01 16.35ef 16.37ef 16.21cd
15.93 15.52a 15.66ab 15.39a
60
Rate Rate Rate Rate
1 2 3 4
16.91j 16.76h 16.82hi 16.89ij
16.98jk 16.59g 16.87ij 16.93j
16.84i 16.43f 16.79h 17.06kl
16.73h 16.42f 16.80h 16.62g
90
Rate Rate Rate Rate
1 2 3 4
17.84o 18.01pq 18.20rs 17.61m
17.96pq 17.65m 17.82o 17.67mn
17.50m 17.85o 17.15l 17.51m
17.80o 17.73n 17.60m 17.84o
180
Rate Rate Rate Rate
1 2 3 4
18.59tu 18.46t 18.71w 18.54t
17.56m 18.15r 18.30s 18.03q
18.65uv 18.31st 18.00pq 17.89op
18.00pq 17.78no 17.62m 17.91pq
Values followed by different superscript letters are significantly different at p < 0.05.
In general, longer relaxation times indicate the presence of more mobile water, or at least water that has less restricted rotational freedom. In our studies, T2 values were greatest at 180 days storage at 10 C, and least at 30 days storage at 35 C. At 180 days at 10 C, we also found the least dough extensibility and maximum dough stickiness, while at 30 days storage at 35 C we found maximum extensibility and the least stickiness. This suggests that changes in T2 values are most related to changes in the gluten and starch network. As discussed previously, greater changes in the gluten structure and separation of water occur at high storage temperatures and longer storage times. This greater mobility of water once removed from the vicinity of gluten could well explain the increased T2 values. 3.4. Yeast activity and gas production Table 5 presents the total gas production for thawed dough slurries after 180 min at 36 C. Yeast activity in frozen dough produced under all freezing and storage conditions was lower than that for an unfrozen control. For example, total gas was 112 ml/g
Table 5 Yeast activity (ml CO2 gas/g dry yeast) in frozen dough after thawing. Values for unfrozen dough: 147.0 ml/g dry yeast. Storage day
Freezing rate
Storage temperature 10 C qr
20 C s
30 C q
35 C
30
Rate Rate Rate Rate
1 2 3 4
75.6 84.0s 61.2mn 58.8lm
86.4 95.4t 64.8op 60.0no
74.4 74.4q 57.0lm 55.2k
73.2q 73.2q 55.2k 56.4klm
60
Rate Rate Rate Rate
1 2 3 4
64.8op 72.0q 54.0k 48.6gh
75.6qr 84.6s 56.4klm 51.6ij
63.0no 64.2nop 49.2hi 49.2hi
61.2mn 62.4n 46.8fg 48.0gh
90
Rate Rate Rate Rate
1 2 3 4
51.0ij 56.4klm 43.2ef 40.8d
55.8kl 63.0no 50.4hij 50.4hij
48.6gh 55.8kl 42.6ef 41.4de
47.4fgh 54.6k 41.4de 41.4de
180
Rate Rate Rate Rate
1 2 3 4
43.2ef 46.8fg 36.6bc 34.2a
47.4fgh 51.6ij 41.4de 39.6bcd
41.4de 47.4fgh 36.0ab 35.4a
40.8d 46.2f 34.8a 36.0ab
Values followed by different superscript letters are significantly different at p < 0.05.
dry yeast for unfrozen dough as compared to 95.4 ml/g for frozen dough produced under freezing rate 2 and stored at 20 C for 30 days. In general, the total gas production decreased with longer storage periods. The highest gas production at 30 days was obtained from frozen dough produced at freezing rate 2 and stored at 20 C storage temperature (95.4 ml/g), and the lowest from dough produced at freezing rates 3 and 4 (most rapid freezing) and stored at 30 or 35 C (55.2–57.0 ml/g). Dough frozen at rates 1 and 2 (relatively slow freezing) and stored at 10 and 20 C had higher gas production for all frozen dough samples. This same relative gas production was maintained for all storage periods. However, the total gas volume obtained was lower at longer storage periods at all freezing rates and storage temperatures. The highest gas volume for frozen dough was 84.6 ml/g at 60 days storage, 63.0 ml/g at 90 days, and 51.6 ml/g ml at 180 days; the lowest volume was 46.8 ml/g at 60 days, 40.8 ml/g at 90 days, and 34.2 ml/ g at 180 days. Greatest volumes were obtained from dough produced at freezing rate 2. In almost all cases, maximum gassing power was observed for dough samples held at 20 C, particularly at freezing rates 2 or 1 (41 C or 19 C/h). This temperature is often recommended for storage in industrial settings, although the reasons for this are somewhat empirical (Trevedi, Hauser, Nagodawithana, & Reed, 1989). It has been observed by several researchers that yeast activity is diminished by freezing and storage (Autio & Sinda, 1992; El-Hady et al., 1996; Neyreneuf & Delpuech, 1993; Wolt & D’Appolonia, 1984). Fluctuations in temperature have been found to be especially damaging (Berglund et al., 1991; El-Hady et al., 1996; Inoue & Bushuk, 1991). In addition, yeast may be less viable after freezing (Baguena et al., 1991). Several factors influence yeast activity in frozen dough, including the influence of temperature on metabolism, osmotic stress incurred as ice forms, the ability of yeast to form osmoregulating or other protective compounds, and the effects that freeze-damage of the gluten network may play in limiting diffusion of nutrients and byproducts to and from metabolizing yeast. Lower temperature per se should improve the survival of yeast. However, in the frozen state, temperature determines the amount of ice present in the system, and the rate of freezing determines how the freezing process ensues. Early work (Mazur, 1970; Mazur & Schmidt, 1968) showed that optimal survival of Saccharomyces cerevisiae occurs at cooling rates of 7 C/min. It was suggested that water in yeast cells remains supercooled down to 10 C, as the cell membrane prevents extracellular ice from propagating into the cell. At relatively slow freezing rates this allows time for water to diffuse from the cytoplasm to external spaces, due to the osmotic gradient. Concentration of intercellular solutes and electrolytes leads to dehydration and changes in ionic strength and pH that affect the metabolic functions of the cell and integrity of the cell membrane. At rates faster than 10 C/min, less time is allowed for cell dehydration, and intercellular freezing is reached more quickly. This also contributes to freeze concentration of solutes in the cell. Interestingly, much of the cell water is frozen at 20 C. One might expect that faster freezing rates and lower storage temperatures would be more beneficial to yeast survival. However, Mazur (1970) postulated that in these conditions, intercellular ice is subject to recrystallization during warming, and that this leads to direct damage of cell membranes. Alternately, Muldrew and McGann (1990) speculated that rapid cooling leads to greater osmotic pressure gradients across the membrane, rupturing the membrane and allowing ice to propagate into the cell. In any event, thawing is also a critical determinant of yeast survival. For example, survival in slowly cooled-slowly warmed systems is greater than in slowly cooled-rapidly warmed systems (Mazur & Schmidt, 1968).
J. Yi, W.L. Kerr / LWT - Food Science and Technology 42 (2009) 1474–1483
The direct effect of temperature on yeast cell membranes also cannot be discounted. Lower temperature can lead to phase transitions and loss of fluidity of the lipid bilayer, and alter proteinbilayer interactions (Morris & Clark, 1987). Thermotropic transitions have been measured in a variety of unicellular organisms and various ‘‘lesions’’ observed during freezing. The membrane can become leaky at low temperature. For example, liposomal models progressively released more glucose at temperatures between 0 and 25 C (Morris & McGrath, 1981). The amount released and response to temperature depended on the lipid composition of the membrane. A few other factors should also be considered in understanding survival and activity of yeast in frozen dough. First, most of the above studies are done in isolated systems. Yeast in dough will be subject to a different physical and osmotic environment than in compressed yeast (Ribotta et al., 2003). Optimal freezing rates in 2dough are typically below 2 C/min as compared to 7 C/min in isolated yeast cultures (Gelinas, Deaudelin, & Grenier, 1995). In our work, rates around 0.86–1.2 C/min provided optimal yeast activity. In addition, S. cerevisiae have developed mechanisms to combat stress conditions. The accumulation of trehalose in the cell seems to be particularly important in combating the osmotic stresses of freezing and drying, as well as stabilizing the cell membrane (Hino, Mihara, Nakashima, & Takano, 1990; Majara, Oconnor-Cox, & Axcell, 1996; Van Dijck, Colavizza, Smet, & Thevelein, 1995). Indeed, much work is ongoing in producing S. cerevisiae with elevated trehalose, as by disrupting the ATH1 gene that produces trehalose-hydrolyzing enzyme (Kim, Alizadeh, Harding, Hafner-Gravink, & Klionsky, 1996). In addition, membrane proteins such as aquaporin also help transport water across the yeast cell membrane, and it is thought that this controlled efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage (Tanghe et al., 2002). Rapid freezing may limit the ability of yeast to adapt to freezing stress. On the whole, relatively slow freezing rates are optimal for maintaining yeast activity after thawing (El-Hady et al., 1996; Havet et al., 2000; Lorenz, 1974). 3.5. Bread volume Table 6 shows the volume of bread made from the various frozen dough treatments. The volume of bread made from unfrozen control dough was 137.8 ml. All breads made from frozen dough
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had lower volumes. Typically, greatest loaf volumes were attained from dough frozen at rate 2. However, freezing rate was not a strong determinant for loaf volume. For example, after 10 C and 30 days, loaf volume varied only from 111.7 to 113.3 ml, while at 35 C it varied from 91.7 to 94.7 ml. Similarly, for dough stored for 180 days at 10 C loaf volume varied from 86.3 to 90.0 ml. Greatest overall loaf volume (119.0 ml) was seen with bread made from dough frozen at rate 2, and stored at 20 C for 30 days, the same conditions found for greatest yeast activity. Lowest volumes were found with bread made from dough frozen at rates 3 or 4 and stored at 30 or 35 C (83.7–84.0 ml at 180 days). There was no significant difference between freezing rate 3 and 4 for all storage times. Loaf volumes for all breads decreased with increased dough storage time. For example, at 10 C volumes decreased from 111.7 to 113.3 ml at 10 C and 30 days to 86.3–90.0 ml after 180 days. At storage temperatures of 30 and 35 C, the effect of storage time was less pronounced. After 180 days, bread volumes from samples frozen at rate 2, and held at 10 or 20 C, were slightly higher than the other treatments, but there were no significant differences amongst volumes for all other samples held for 180 days. Storage temperature also had a major influence on subsequent loaf volume. In general, samples held at 20 C produced bread with greatest loaf volume, although storage temperature effects interacted with storage time as described above. Again, this seems to reflect, at least in part, the effects of treatment on yeast activity.
3.6. Bread crumb firmness Table 7 shows crumb firmness for bread made from frozen dough. Measured firmness was 4.03 N for bread made from unfrozen control dough. For that made from frozen/thawed dough, crumb firmness increased significantly with frozen storage time of the dough. For example, at 20 C firmness values ranged from 5.99 to 6.88 N at 30 days, and from 7.76 to 8.04 N at 180 days. In general, bread made from dough frozen at rates 1 and 2 was less firm than bread made from dough frozen at rates 3 and 4 for all storage times. The storage temperature also had a significant effect on bread firmness. All dough samples stored at 10 and 20 C produced bread that was less firm than other samples for all storage times. At 30 and 60 days, bread made from dough stored at 10 C was less firm than that from dough stored at 20 C.
Table 6 Volume of bread (ml) made from frozen dough. Values of bread from unfrozen dough: 137.8 ml.
Table 7 Firmness (force, N) of bread made from frozen bread dough. Values of bread from unfrozen dough: 4.03 N.
Storage day
storage day
Freezing rate
Storage temperature 10 C w
20 C
o
Freezing rate
35 C no
Storage temperature 10 C
tu
35 C
1 2 3 4
111.7 113.3xy 113.3xy 112.7wx
115.3 119.0A 113.3xy 113.0wxy
94.3 95.3pq 93.7mno 94.7op
94.0 94.7op 91.7jk 92.0jkl
30
Rate Rate Rate Rate
1 2 3 4
5.00 4.56a 5.78c 5.91c
6.12 5.99cd 6.47e 6.88fg
10.60 10.76tu 10.46stu 10.38rs
9.05no 8.92no 9.33p 9.52pqr
60
Rate Rate Rate Rate
1 2 3 4
108.7tu 109.3uv 109.0u 108.0t
113.3x 116.3z 111.7w 112.7wx
92.3kl 93.7mno 91.3ij 91.7jk
92.3kl 93.3mn 91.3ij 91.3ij
60
Rate Rate Rate Rate
1 2 3 4
5.93c 5.87c 6.47e 6.16de
6.80f 6.64f 6.90g 6.95gh
11.81wx 12.18yz 12.29yzA 11.74w
11.40v 11.68vw 11.42v 11.75w
90
Rate Rate Rate Rate
1 2 3 4
104.7s 107.3t 101.0r 102.0r
109.0u 112.0w 107.7t 107.7t
92.0jkl 92.7lm 88.7e 88.7e
90.3ghi 90.7hi 88.3de 88.7e
90
Rate Rate Rate Rate
1 2 3 4
8.00kl 7.71k 9.07op 8.86mno
7.28hij 7.12hi 6.96gh 7.35ij
12.26yz 12.36zA 12.04xy 12.40zAB
12.39zA 11.91wxy 11.92wxy 12.38zA
180
Rate Rate Rate Rate
1 2 3 4
88.7e 91.3ij 87.0c 87.7cd
88.0cde 89.3fg 85.3ab 84.0a
86.0b 89.0ef 84.0a 83.7a
180
Rate Rate Rate Rate
1 2 3 4
8.03kl 8.04kl 7.76k 8.04kl
12.48AB 12.73CD 12.34zA 12.94CD
12.91CD 12.77BCD 12.60BC 12.82CD
8.78mn 8.46lm 10.18rs 9.73qr
cd
30 C
Rate Rate Rate Rate
Values followed by different superscript letters are significantly different at p < 0.05.
ab
20 C
30
88.3de 90.0gh 86.3bc 87.7cd
z
30 C
Values followed by different superscript letters are significantly different at p < 0.05.
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J. Yi, W.L. Kerr / LWT - Food Science and Technology 42 (2009) 1474–1483
optimize bread quality. Prolonged storage times lead to detrimental changes in both gluten structure and yeast viability.
References
Fig. 6. Contour plots for elasticity (E), yeast activity (Y), and bread volume (V) at 90% of their maximum value; and adhesiveness (A) and firmness (F) at 90% of their minimum value. Arrows show direction toward more optimal values.
One usually assumes that a less firm, tenderer bread is most desirable for consumers. As noted, firmness of bread from the control was lower than that from frozen dough. Crumb firmness arises from the integrity of the gluten network, the degree of crosslinking, the amount of gas incorporated, and from other structural components of the bread. Ribbotta, Perez, Leon & Anon (2004) reported similar results to the ones reported in this work with frozen storage temperature. With longer frozen storage time, the firmness increased, which may come from depolymerization of glutenin and higher retrogradation of amylopectin in bread made from frozen dough.
3.7. Response surface Fig. 6 shows a response surface for the various quality measurements as a function of freezing rate and frozen storage temperature. The overlapping contour lines show each variable at 90% of their optimal levels, with arrows pointing to more optimal regions. Both volume and firmness depended primarily on storage temperature, and storage temperatures below 20 C were most beneficial. Elasticity and adhesiveness depended on both storage temperature and freezing rate, and generally lower temperatures and higher freezing rates were beneficial. Yeast activity also depended on temperature and freezing rate, but in this case lower temperatures and slower freezing rates were more beneficial. An optimal region for all attributes (shaded in grey) occurs at temperatures approximately between 15 and 20 C and centered around freezing rate 2.
4. Conclusions The quality of bread made from frozen dough depends on the rate of freezing, storage temperature, and the length of time stored. Faster freezing and lower storage temperatures promote less damage to the gluten network, thus help retain elastic properties of the dough. However, relatively lower freezing rates and storage temperatures promote yeast viability and gassing power. As such, a compromise in freezing rate and storage temperature is needed to
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