Accepted Manuscript

Improving freeze tolerance of yeast and dough properties for enhancing frozen
dough quality - A review of effective methods

Wenhuang Luo, Da-Wen Sun, Zhiwei Zhu, Qi-Jun Wang

PII: S0924-2244(17)30504-6
DOI: 10.1016/j.tifs.2017.11.017
Reference: TIFS 2127

To appear in: Trends in Food Science & Technology

Received Date: 28 July 2017

Revised Date: 18 October 2017
Accepted Date: 28 November 2017

Please cite this article as: Luo, W., Sun, D.-W., Zhu, Z., Wang, Q.-J., Improving freeze tolerance of
yeast and dough properties for enhancing frozen dough quality - A review of effective methods, Trends
in Food Science & Technology (2018), doi: 10.1016/j.tifs.2017.11.017.

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 ACCEPTED MANUSCRIPT
Abstract

Background: Frozen dough technology could effectively extend the shelf life of bread to ensure
the freshness, which is widely used and gradually replace the traditional bread production.
However, during the production and storage of frozen dough, a series of problems could take
place, such as inhibition of yeast activity, damage of the structure of the dough, leading to the
deterioration of dough quality.
Scope and Approach: This review summarizes the factors that affect the final quality of frozen
dough, including yeast activity, dough structure and dough properties. Some effective methods
for improving freeze tolerance of yeast, dough structure and dough properties are discussed,
including addition of various additives, use of genetic engineering technique, optimization of
freezing and storage conditions, and employment of novel freezing technology.
Key Findings and Conclusions: The addition of additives can not only improve the freeze
tolerance of yeast but also maintain the rheological and thermophysical properties of dough.
Through the modification of gene, freeze tolerance and fermentation ability of yeast can be
improved. Optimizing freezing and storage conditions ensures the activity of yeast as well as
dough network structure so that freezing damage due to ice crystals can be minimized. In
addition, novel freezing technology such as ultrasound-assisted freezing can simultaneously
accelerate the freezing process as well as generate fine and uniform ice crystals, thus protecting
dough network structure.

Improving Freeze Tolerance of Yeast and Dough Properties for Enhancing

Frozen Dough Quality - A Review of Effective Methods

Wenhuang Luo, Da-Wen Suna b c d1, Zhiwei Zhua b c, Qi-Jun Wanga b c

1 Corresponding author. Tel: +353-1-7167342, Fax: +353-1-7167493, E-mail: dawen.sun@ucd.ie,

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a
School of F ood Science and Engineering, South China University of Technology, Guangzhou 510641, China b Academy of

Contemporary Food Engineering, South China University of Technology, Guangzhou Higher Education

Mega Center, Guangzhou 510006, China c Engineering and Technological Research Centre of Guangdong Province on Intelligent

Sensing and Process Control of Cold Chain Foods, Guangzhou Higher Education Mega Centre, Guangzhou 510006, China d Food

Refrigeration and Computerized Food Technology, University College Dublin, National University of Ireland, Agriculture and

Food Science Centre, Belfield, Dublin 4, Ireland

Abstract

Background: Frozen dough technology could effectively extend the shelf life of bread to

ensure the freshness, which is widely used and gradually replace the traditional bread

production. However, during the production and storage of frozen dough, a series of

problems could take place, such as inhibition of yeast activity, damage of the structure of the

Website: www.ucd.ie/refrig;www.ucd.ie/sun.
 ACCEPTED MANUSCRIPT

dough, leading to the deterioration of dough quality.

Scope and Approach: This review summarizes the factors that affect the final quality of frozen

dough,

including yeast activity, dough structure and dough properties. Some effective methods for

improving freeze tolerance of yeast, dough structure and dough properties are discussed,

including addition of various additives, use of genetic engineering technique, optimization of

freezing and storage conditions, and employment of novel freezing technology.

Key Findings and Conclusions: The addition of additives can not only improve the freeze

tolerance of yeast but also maintain the rheological and thermophysical properties of dough.

Through the modification of gene, freeze tolerance and fermentation ability of yeast can be

improved. Optimizing freezing and storage conditions ensures the activity of yeast as well as

dough network structure so that freezing damage due to ice crystals can be minimized. In

addition, novel freezing technology such as ultrasound-assisted freezing can simultaneously

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accelerate the freezing process as well as generate fine and uniform ice crystals, thus

protecting dough network structure.

Keywords: frozen dough, yeast, quality, freezing, ice crystals

1. Introduction

Bread is one of the most widely consumed foods all over the world. However, starch

retrogradation causes a series of physicochemical reactions, shortening the shelf life (Steffolani et

al,

2012), while the loss of moisture increases the hardness of bread (Selomulyo & Zhou, 2007).

Therefore traditional breads on the market generally have a short shelf life. The development of

frozen dough can minimize the effects of these problems. The production of frozen dough

involves
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mixing the flour, water and ingredients, kneading and molding, and finally freezing the dough

(Wang
2

et al., 2015), and normally the process requires rapid fermentation. Since yeast can be fully

activated only after prolonged fermentation, the yeast would be susceptible to freezing and

thawing damage. Compared to the rapid-fermented dough before freezing, the freeze tolerance

of yeast in unleavened dough is better, but the firmness, flavor and color of the baked bread

are not as good as former (Ayati et al, 2016). Meanwhile, flour with high gluten content

should be selected as it was shown that using high levels (9.5%-11%) of gluten, the frozen

dough was more resistant to freezing damage and the loaf volume, texture, color and hardness

of the final products similar to the fresh one (Kondakci et al., 2015). Furthermore, the

kneading time is usually longer, especially for dough with high sugar content, so as to make

the gluten network more homogeneous (Ohgren et al., 2016).

Frozen dough has great benefit to both manufacturer and consumers. For the
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manufacturer, frozen dough can not only facilitate their handing, trading, and retail ability, but

can also greatly improve the shelf life of the dough. For the consumers, frozen dough allows

consumers to taste fresh bread anytime and anywhere (Rashidi et al., 2016). However, while

freezing extends the shelf life of the dough, the freezing and storage process can also cause

decline in the quality of frozen dough, such as damaging the structure of the gluten network

and lowering the yeast fermentation activity, consequently affecting the quality of the final

products.

This review intends to introduce the change in the activity of yeast and the structure and

properties of dough as affected by freezing and frozen storage. Effective methods to improve

freeze tolerance of yeast and dough structure and properties are summarized, which includes

using additives, employing genetic engineering technique, and optimizing the freezing rate and

storage condition. Although several reviews on frozen dough have been published (Akbarian

et al., 2015; Selomulyo &

3
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Zhou, 2007), they mainly focus on the use of additives, and information on using genetic

engineering and optimizing freezing conditions is limited. Therefore, it is hoped that the current

review will enhance further understanding of these methods and promote their applications in the

bakery industry.

2. Effects of Freezing on Quality of Frozen Dough

The quality of frozen dough depends on CO2 production ability of yeast and CO2 retention

capacity of bread after fermentation. Decreasing yeast viability and destruction of dough network

structure are regarded as two major factors that lead to the deterioration of dough quality while

both are caused by ice crystals. The effects of ice crystals could be multiple: mechanical action of

ice crystals may affect the structure and properties of the dough (Baierschenk et al., 2005), and

the larger the ice crystals, the greater the possible damage to the dough structure; the formation of

intracellular ice crystals would pierce the cell membrane leading to yeast death (Acker &
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Mcgann, 2003); and finally, the formation of extracellular ice crystals would increase the

intracellular osmotic pressure, leading to dehydration of yeast cells and the decrease in their

viability (Devireddy et al., 2000). Therefore for obtaining better bread, it is necessary to control

the formation of ice crystals (Yi & Kerr, 2009). There are two ways to control ice crystals, one is

to add protective agents to inhibit the formation and growth of ice crystals, and another is to

control the freezing rate to produce fine ice crystals with a more uniform distribution.

2.1 Yeast activity

Yeast generally refers to a variety of single cell fungi that can ferment sugar and is widely

distributed in nature, which mainly grows in acidic, moist, sugar-containing environments and

can survive both in the aerobic and anaerobic conditions. In anaerobic environment, yeast

converts carbohydrates into carbon dioxide and ethanol. Yeast must have water to survive, and

the optimum growth temperature for it is between 20-30 oC while its viability will be greatly
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inhibited if the temperature is below 0oC or higher than 40°C. Yeast used in frozen dough system

normally refers to Saccharomyces cerevisiae (Ji et al., 2016) and there are three types available

for using in frozen dough production, including fresh yeast (FY), dry yeast (DY) and bulk liquid

yeast, of which DY is the most common type. Some studies (Oda et al., 1986; Wolt, 1984) found

that DY is as efficient as FY and the fermentation performance could be better than fresh one,

leading to the reduction in pre-fermentation time and resulting in a more stable frozen dough.

However when storage for more than 20 weeks, the time for DY proofing is always longer than

the fresh one (Wolt, 1984).

Many studies reported that yeast viability is one of a key factor influencing the quality of the

frozen dough (Akbarian et al, 2015; Meziani et al, 2012). Yeast viability in frozen dough has a

significant relationship with proofing time, loaf volume, bread firmness, and bread porosity

because its reduction may result in a decrease in yeast gas production and prolong the

fermentation time. The freeze tolerance of yeast is very low and yeast activity is affected by sub-
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zero temperatures. In addition, freezing yeast in the dough system is likely more damaging than

freezing yeast directly, because yeast would suffer from kinds of stresses such as freeze-thaw,

high osmotic pressure and oxidative stress (Tsolmonbaatar et al., 2016). During freezing and

subsequent frozen storage, yeast activity is inhibited, and many of the yeast cells die (Ribotta et

al, 2003). Therefore, the temperature

5

during frozen storage should be properly controlled.

In addition, studies also proved that the freeze tolerance of yeast is related to the trehalose

content, which is an effective protective agent to maintain the integrity of the cell membrane and

prevent the internal structure from being damaged in a wide range of freezing conditions (Shima

& Takagi, 2009; Yokoigawa et al., 2006). Since the production of carbon dioxide is only related

to yeast, the volume of carbon dioxide produced can be used to determine the viability of yeast.

2.2 Structure of frozen dough

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Dough network structure, which can lead to the deterioration of final quality of the products

if damage, is another essential factor for the quality of bread. The function of the dough network

structure is similar to yeast, which affects the retention ability of CO 2, proofing time and loaf

volume. The structure may also affect bread sensory properties such as firmness and chewiness

and is tight after rapid thawing, as gluten matrix inlays with many small spherical starch

granules. However, after several weeks of storage, starch granules have the tendency to separate

from the gluten, indicating the damage of the network structure (Akbarian et al., 2015).

Generally, the structure and the content of the damaged starch of the frozen dough can be

observed by low temperature scanning electron microscopy (SEM). Fig. 1 shows the images of

starch granules under SEM. As shown in Fig. 1, the normal starch surface has some ridges, but it

looks very smooth and the starch granules are closely connected. However, the surface of the

damaged starch granules is relatively rough. The pimple and many clumps formed by distorted

granules can be clearly

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observed in Fig. 1. The content of the damaged starch increases after freezing and during frozen
6

storage. An increase in the content leads to an increase in water absorption capacity, eventually

causing water flowing out of the gluten matrix (Zounis et al., 2002). A high level of damaged

starch would make the bread more sticky and resistant to deformations, but at the same time, due

to the increase in the competing of water between gluten and damaged starch, it would result in a

weaker development of gluten, cause a decline in elastic and extensible level (Barrera et al.,

2016). However a proper content of the damaged starch would be more beneficial to frozen

dough. Ma et al. (2016) compared the influence of different contents (range from 9.3% to 30%)

of damaged starch on dough quality and showed that a proper damaged starch content (about

15.7%) improved the farinograph properties (including water absorption, development time,

stability time, falling number and gluten index) of flour, and the quality of steam bread.

The distribution of ice crystals can be observed by various microscopy techniques. Early

studies showed that ice crystals were mainly distributed at the external of gas cells (Gan et al.,
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1990). Unlike freezing of other porous foods, Efj et al. (2003) revealed some ice crystals in gas

pores after several weeks of storage, which was considered as the damage to dough network

structure.

2.3 Properties of frozen dough

Rheological properties of the dough mainly include hardness, extensibility and stickiness,

which reflect the deformation of dough (Jia et al., 2014). The rheological properties of dough are

closely related to the quality of the baked bread. It is difficult to proof for dough that has large

tensile resistance and small extensibility, resulting in an undesirable volume of baked bread.

Various rheological parameters, including the storage modulus, loss modulus and loss tangent

can be
7

measured by a rheometer. In these parameters, storage modulus is used to indicate the elastic

composition of the dough, loss modulus shows the viscous element, and loss tangent represents
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the

increase in the ratio of the latter to the former (Adams et al.,2017). In the production of bread

made

from frozen dough, manufactures generally prefer bread with similar hardness, extensibility and

stickiness to fresh bread.

Thermophysical properties of dough include effective thermal conductivity, apparent

specific heat, ice melting enthalpy, freezable water fraction and ice fraction. These parameters

can be used not only to simulate the heat transfer process but also to calculate the size of the ice

crystals in frozen dough.

The most common and simplest way to measure the thermal conductivity is the unsteady

method, in which a thermal probe is inserted into the dough, and the sample is cooled to different

temperatures and then held for at least 30 min. After the temperature is stable, direct-current

power supply is switched on to allow passing a 500 mA current and the change of temperature
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with time is recorded. The thermal conductivity can then be expressed as (Kumcuoglu et al.,

2007):
k _ QIn(t2-t0)/(t1-t0) (1)
()
4n(T2-T1)

where k is the thermal conductivity of frozen dough (W m-1 K-1), Q is the energy provided by the

power (W m-1), t1 and t2 are the initial and termination times, respectively, and T1 and T2 are the

temperatures corresponding to t1 and t2.

The apparent specific heat of the frozen dough is the sum of the proportions of the specific

heat of each phase plus the energy released by melting of the ice (Miles et al., 1983):
Cp Cp X Cp X (2)
app _ w • w + i • i + Cps • Xs + L ^

where Cp app (J kg-1 °C-1) is the apparent specific, Cpw, Cpi, and Cps are the specific heat of liquid

water, ice and solid, respectively, xw, xi and xs are the mass fraction of liquid water, ice and solid,

respectively, L is ice fusion latent heat (J/kg).

The thermal conductivity and the ice melting enthalpy (AH) can also be determined directly by

differential scanning calorimetry (DSC). Based on the content of freezable water, the ice fraction
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in

frozen dough can be calculated. In general, increasing freezable water content may result in

changing

the distribution of water in dough, therefore water has less contact with protein and starch,

leading to

possible destruction of the frozen dough system (Adams et al., 2017).

The freezable water fraction can be calculated by (Laaksonen & Roos, 2000):

-
fw=T$$ x100% (3)

Any where fw is the freezable water of frozen dough (%), AH is the ice melting

enthalpy in frozen dough

(J), AHi is the latent heat of fusion for pure water (334 J/g), m is the mass of water in frozen

dough

(g).
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-
The ice fraction can be expressed as (Hamdami et al., 2004): f = 1 - - A
$

(4)
v
AH ’

where f is the ice fraction in frozen dough, AHr is the exothermic enthalpy for temperature from

T0 to T1.

3. Techniques to Improve the Quality of Frozen Dough

In order to improve the quality of frozen dough, several techniques and methods have been

developed, which include the addition of additives, such as antifreeze proteins (AFPs) and
9

hydrocolloids, genetic engineering technique, freezing rate and storage condition optimization,

and novel freezing technologies such as ultrasound-assisted freezing (UAF).

3. 1 Adding additives
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Common additives used in frozen dough include hydrocolloids, AFPs, ice nucleation agents

and other dough improvers. Effects of different additives on frozen dough system are shown in

Table 1. In early studies (Lee et al., 2004; Ribotta et al., 2004), most were about using lipid-

related emulsifiers including diglycerides and sucrose esters. The addition of emulsifiers was

able to prevent the retrogradation of the starch effectively due to the interaction with starch and

minimized migration of moisture between gluten and starch (Ribotta et al., 2004). Nowadays

studies focus more on novel additives such as ice structuring proteins (ISPs) or AFPs and ice

nucleation proteins agents.

AFPs, also called ISPs, is a series of proteins that can improve the freeze resistance of

organism. Zhang et al. (2007) added Daucus carota antifreeze protein (DcAFP) (a leucine- rich

repeat protein) to frozen dough and indicated that DcAFP could increase the freeze tolerance of

yeast by protecting against freezing damage to cell membranes, controlling the distribution of ice

crystals and inhibiting the formation of large ice crystals. Xu et al. (2009) showed that the
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addition of ISPs from winter wheat significantly affected the thermal properties of frozen dough,

such as decreased peak of freezing point, reduced content of freezable water, lowered effective

thermal conductivity and decreased peak of apparent specific heat in the frozen range. In another

study, Ding et al. (2015) demonstrated that barley antifreeze protein could decrease the melting

enthalpy and reduce the

amount of freezable water during freezing and frozen storage. Wang et al. (2016) found that

water
10

extractable arabinoxylan had a function similar to AFPs, in addition, it also had the ability to

prevent disulfide bonds from being depolymerized, because breakage of disulfide bonds could

lead to depolymerization of glutenin macromolecule polymers (GMP). Therefore soluble high

molecular and low molecular weight proteins were generated in dough samples, whereas low

content of low molecular weight proteins and high content of GMP were generated in the frozen

dough with extractable arabinoxylan added. Thus water extractable arabinoxylan has the ability
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to lower the loss of yeast activity and to protect the dough from ice crystal stress.

As for ice nucleation agents, their main function is to minimize the degree of supercooling

and inhibit the formation of large ice crystals. Extracellular ice nucleators (ECINs), extracted

from Erwinia herbicola, are mainly composed of protein, polysaccharide, polyamine and lipid

(Lorv et al, 2014). Shi et al. (2013b) added extracellular ice nucleators into frozen dough and

obtained the bread comparable to fresh one due to less damage of yeast cells during freezing. Fig.

2 shows the appearance of fresh bread as compared with that made with frozen dough with and

without adding ECINs. Compared with the fresh bread, bread made with frozen dough showed

lower loaf volume, and the addition of ECINs produced better bread than without ECINs. In

addition, the color of the bread with ECINs was similar to the fresh. Shi et al. (2013a) also

reported that zein-based ice nucleation films (INFs) would increase temperature of ice nucleation

from -15 °C to -6.7 °C, decrease the proportion of water loss and lower the damage to dough in

freeze-thaw cycles.
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The addition of hydrocolloids can minimize negative effects of freeze-thaw cycles on frozen

dough and maintain its rheology properties. During frozen storage, recrystallization of free water

can

lead to the formation of large ice crystals. As hydrophilic colloid can combine with the gluten

protein
11

and bound water to form a hydrophilic complex, the addition of hydrocolloids can therefore

increase the water holding capacity of the dough, thus reducing the migration of moisture in the

dough. In addition, hydrocolloids can decrease water activity because they can bind water in their

structure (Akbarian et al., 2015). Sim et al. (2012) revealed that adding locust bean gum from

Ceratonia siliqua seeds could avoid the recrystallization of free water and modify the melting

properties of a frozen dough, as locust bean gum has a strong water holding capacity and can

control the migration of moisture in the dough. In another work, the frozen dough added with

tragacanth exhibited a strong ability to absorb water and had better sensory quality (Gharaie et
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al, 2015).

There are some flour materials that can be also used as additives to improve the rheological

properties of dough. Bae et al. (2014) reported that adding whole-grain wheat flour rich in

dietary fibers (11.84%) into frozen dough could generate strong hydration properties with better

pasting properties, thus improving baking properties. Jia et al. (2017) indicated that adding 10%

waxy wheat flour to replace the regular wheat flour could significantly reduce the content of the

damaged starch in frozen dough, increase the gelatinization temperature and crystallinity of

starch because of the prohibition of the migration and redistribution of water molecules.

On the other hand, the effects of additives on frozen dough also depend on the amount of

additives, formula, and processing conditions, and each additive should have an optimum

amount. Giannou and Tzia (2016) compared the effects of different levels of vital wheat gluten in

white flour or whole-wheat flour on frozen dough, and showed that white flour with 4% or 6%

and whole-wheat flour with 4% or 5% levels of vital wheat gluten were most effective to the
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stability of frozen dough.

Ortolan et al. (2015) used 5% extruded wheat flour or pre-gelatinized cassava starch to replace

the
12

wheat flour, and indicated that the proofing and baking times were reduced significantly. Park

et al. (2016) also demonstrated that adding 6% fructo-oligosaccharide or isomalto-

oligosaccharide achieved the best baking quality such as proof volume and bread volume,

however, adding more than 6% would gradually decrease the baking quality.

3.2 Genetic engineering technique

As mentioned previously, yeast would suffer from all kinds of stresses in the process of

producing bread with frozen dough (Lin et al., 2015; Sasano et al., 2013; Tsolmonbaatar et al.,

2016). Under these stresses, the viability of yeast is reduced. The viability of yeast is strongly

related to the total intracellular compound content such as trehalose, glutamic acid, arginine,
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proline and glycerol (Shi et al., 2014). It is generally believed that the freezing tolerance of

yeast is mainly related to the content of trehalose and proline, and trehalose plays the most

important role (Dong et al., 2016). Trehalose is a non-reducing disaccharide appearing in

many organisms such as bacteria, fungi, animals and plants. It acts as a pressure-protected

proteins and biofilms through the combination with phospholipids to resist unfavorable

conditions. Because of this function, trehalose can be used as a cryoprotectant for protecting

the yeast from freezing damage (Shima & Takagi, 2009). On the other hand, trehalose can also

be a carbon source stored in organism, and thus can act as an energy supplement for cells at the

initial stage of fermentation. When yeast cells are exposed to freezing stresses, they

accumulate quickly a large amount of trehalose (Blomberg, 2000). As for proline, it has been

shown that it can combine with intracellular free water to form strong hydrogen bonds to

reduce ice nucleation and cells dehydration. High proline content of yeast contains high level

of superoxide
13
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dismutase and low level of reactive oxygen species, proline can therefore prevent intracellular

substances from oxidation, leading to a high level fermentation ability in frozen dough (Sasano et

al., 2012b). Furthermore, when the level of trehalose is stable in yeast cells, an appropriate

proline concentration is important to protect the yeast from damage by freeze-thaw stress (Kaino

et al., 2008).

Intracellular trehalose and proline concentrations can both be controlled by synthesizing and

hydrolyzing enzymes in yeast. If the content of synthetase can be increased and the content of

hydrolase be reduced, more trehalose and proline will accumulate in yeast. Therefore, genetic

engineering has been studies for this purpose. Table 2 summarizes the applications of genetic

engineering on yeast cell employed in frozen dough. For trehalose hydrolase, there are two types:

neutral trehalase and vacuolar acid trehalase (Nwaka & Holzer, 1997). Among them, the neutral

trehalase mainly regulates the hydrolysis of trehalose. The hydrolysis of trehalose is controlled

by the NTH1 gene (Zhang et al., 2010) while the synthesis of trehalose in yeast cells is controlled
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by MAL62 and TSP1 genes. Sun et al. (2016) and Tan et al. (2014) reported that the over-

expression of single gene (MAL62 or TSP1) in yeast would increase the accumulation of

trehalose, resulting in the promotion of the yeast viability under freezing stress, moreover, the

over-expression of the MAL62 gene and the deleting of NTH1 gene could further enhance the

freeze tolerance of yeast and improve the performance of fermentation. Similar experimental

result was reported by Dong et al. (2016), who deleted the NTH1 gene and obtained better yeast

for resisting freezing damage. At the same time, they also proved that PUT1 gene could control

the synthesis of proline oxidase and deleting both NTH1 and PUT1 genes showed higher freeze

tolerance and better fermentation performance, as

14

compared with deleting only one of them (Dong et al., 2016). In addition, the content of proline

in yeast could be increased by expressing the PR01-l150T and Mpr1-F65L genes (Sasano et al.,

2012a).

The P0G1 gene can promote compressive capacity of cells. Demae et al. (2007) illustrated
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that the over-expression of P0G1 gene made yeast cells better resistance to LiCl, NaCl, high

temperature and high glucose stresses. Sasano et al. (2013) over-expressed the P0G1 gene in

yeast cells, leading to drastically improved leavening performance in high sucrose conditions,

while removing the P0G1 gene significantly enhanced freeze-thaw stress tolerance of yeast.

Sasano et al. (2010) also revealed that over-expression of the MPR2 gene in yeast cells reduced

the intracellular oxidation levels, leading to enhancing the air-drying stress tolerance. Lin et al.

(2015) over-expressed the SNR84 gene in yeast, which exhibited a higher freeze resistance and

improved fermentation capacity in high sucrose containing dough. Nakagawa et al. (2017)

reported that the over-expression of self-cloning PDE2 gene made yeast better freezing stress

tolerance, which had a better genetic trait.

The above studies are helpful for ameliorating the quality of frozen dough, however

consumers are more receptive to product without foreign gene. Therefore, investigations have

been conducted for gene mutation (Ando & Nakamura, 2016; Tsolmonbaatar et al., 2016). For
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example, Tsolmonbaatar et al. (2016) reported that gene PR01 mutations caused intracellular

proline accumulation, leading to enhanced resistance to freeze-thaw and osmotic pressure. As

gene mutation technology is safer, it is thus expected to be more widely applied in future.

3.3 Optimization of freezing rate and storage conditions

As discussed above, the freezing rate and the temperature of frozen storage can seriously

affect the quality of frozen dough, mainly due to their effects on the activity of yeast, and the

nucleation and growth of ice crystals. During freezing, a slow freezing rate tends to form large

ice crystals, which can cause serious damage to tissue cells. For frozen dough system, rapid

freezing can increase the extensibility of the dough and reduce the damage to the microstructure

of the dough network, but in the meantime, rapid freezing could also have negative effect on

yeast activity (Ban et al., 2016; Selomulyo & Zhou, 2007), therefore the freezing rate should be

properly controlled. Slow freezing allows plenty of time for water to flow out of the cells to form
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extracellular ice, causing dehydration of yeast cells (Nakamura et al., 2009). Ayati et al. (2016)

showed that the activity of yeast increased and then decreased with the increase of the freezing

rate. Ban et al. (2016) indicated that an appropriate freezing rate was necessary to guarantee the

yeast viability while also ensure forming small ice crystals to protect the structure of the dough.

Recent studies (Silvas-Garcia et al., 2016) have proved that lower freezing rate is more

beneficial to the microstructure of frozen dough than fast freezing after storage for several weeks.

Fig. 3 compares the microstructures of wheat flour and dough under low freezing rate (-

0.14oC/min) and high freezing rate (-1.75oC/min) with 2, 4 and 8 weeks of storage, respectively.

As shown in Fig.

3, starch granules (S) and gluten matrix protein (P) separation occurred after storage for 8 weeks

with slow freezing, and the gaps between the starch granules were not obvious, however with fast

freezing, the starch granules and gluten matrix protein were isolated after storage for 2 weeks,

and the gaps between the starch granules were obvious after 8 weeks of storage (Silvas-Garcia et
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al.,

2016). Gerardo-RodrlGuez et al. (2017) also chose an appropriate freezing rate (-0.14oC/min) and
16 concentration of trehalose to make the bread from frozen dough with comparable fresh bread quality.

The storage temperature and time can affect the growth of ice crystals and recrystallization

of water in frozen dough. Normally, the ice content does not change during storage. The

generation of the large ice crystals during storage resulted from temperature fluctuations that

make small ice crystals dissolved and the surface of the dough pores would adsorb a large

amount of water molecules (Baier-Schenk et al., 2005). This change may not be obvious for

short storage time, however, with the prolonging of storage, large ice crystals could gradually

form. Normally, the suitable storage temperature for frozen dough is between -18 and -22 oC

(Kenny et al., 2001; Leray et al., 2010) and any temperature fluctuations during storage should

be avoided as fluctuations in temperature may cause the frozen dough to undergo freezing and

thawing cycles. With repeated freeze-thaw cycles, due to water migration, ice recrystallization,

and mechanical stress during water transform to ice, the flexibility and CO2 retention capacity of
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the dough are deteriorated, causing the change in high molecular weight glutenin in dough and

resulting in poor extensibility, which also affects the activity of yeast. Phimolsiripol et al. (2008)

reported that the fluctuation of frozen storage temperature decreased dough quality, which was

reflected as reduced capacity of CO2 production and retention.

With the increase in storage time, the quality of dough would gradually decline. Fig. 4

shows the images of the size and distribution of ice crystals in the dough at different storage

times. The ice crystals produced under fast freezing conditions using liquid nitrogen were small

and did not distribute in the pore of the dough, while most of the ice crystals were distributed in

the pore wall of the dough if air blast freezing was used (C and D). Fig. 4 also shows that some

ice crystals gathered

17

together after storage for one day (E and F); after five months of storage (G-J), the sizes of ice

crystals were further increased and the ice crystals were no longer distributed on the pore walls,

but inside the pore, meanwhile, large cube (I) and spherical (J) shapes of ice crystals could be
 ACCEPTED MANUSCRIPT

observed in the pores (Baier-Schenk et al., 2005).

3.4 Novel freezing technologies

In controlling ice crystal formation and distribution during dough freezing, novel freezing

technologies could be used (Sun, 2016). Among them ultrasound-assisted freezing (UAF) has

been investigated for promoting ice nucleation and crystal growth. UAF can decrease the degree

of supercooling, increase heat transfer efficiency, and produce a large number of fine ice crystals

with better distribution (Cheng et al., 2014). The mechanism of the UAF is mainly related to the

function of the ultrasound cavitation. Cavitation is an important phenomenon during sonication,

which produces a large number of cavitation bubbles and such bubbles could act as the seeding

sites for ice nucleation. In addition, the collapse of the bubbles would produce momentarily high

pressure, breaking large dendritic ice crystals into small fragments to promote secondary ice

nucleation, thus producing small ice crystals with uniform distribution (Zhang et al., 2015).
 ACCEPTED MANUSCRIPT

Hu et al. (2013) revealed that using UAF, the total freezing time of dough was reduced by

more than 11% at 288 W and 360 W levels, in the meantime, the elasticity and sensory properties

of the dough were also increased significantly owing to the fine ice crystals. Parameters of UAF

also have effects on microbial viability. Kiani et al. (2013) reported that UAF could increase the

viability of

Lactic acid bacteria cells in the temperature range from -4 to -2 oC, furthermore, ultrasound

irradiation
18

resulted in a further increase in cell viability during phase change period. As the novel freezing

technologies are emerging methods, their applications in dough freezing is very limited and

further studies should be encouraged in this area.

4. Conclusions and Future Prospects

Frozen dough can overcome problems of traditional bread, such as starch retrogradation and
 ACCEPTED MANUSCRIPT

short shelf life. However, in the production of frozen dough, reduction of yeast activity and the

destruction of dough network structure are inevitable, resulting in decline of the baking quality of

dough. This review discusses main factors affecting the quality of frozen dough, and suggests

effective methods for minimizing the quality loss of the frozen dough.

Using additives is a method to increase the freeze tolerance of yeast and improve the

rheological and thermophysical properties of dough, so that final products close to fresh bread

could be made. Breeding yeast has higher trehalose and proline content, thus the freeze tolerance

and fermentation capacity of the yeast could be enhanced. 0ptimizing freezing rate and avoiding

temperature fluctuation could maintain yeast activity and prevent the recrystallization of

moisture in frozen dough. In addition, novel freezing technologies can accelerate the freezing

process and improve heat transfer and mass transfer, and ultrasound assisted freezing has been

shown to have positive effects on dough properties and cell viability, however few studies are

available and future research should focus on this area.

 ACCEPTED MANUSCRIPT

Although frozen dough has been widely studied, few studies focus on the micro perspective

of

frozen dough, on the other hand, there is little research on physical and chemical reactions of

certain
19

components in frozen dough, and little information is available on the interaction between

additives and components in dough, all of which require further research attention. In addition,

the safety of genetic engineering technique and the mechanism of action of certain genes are still

unclear, which also require further studies.

Up to now, the above methods have been mainly used alone. These methods could be

combined to further improve yeast viability, protect dough network structure, and provide final

products with fresh-like taste. However no study has been attempted yet to verify the

effectiveness and viability of the concept, and thus future investigation is needed.
 ACCEPTED MANUSCRIPT
Acknowledgement

The authors are grateful to the International S&T Cooperation Program of China

(2015DFA71150) for its support. This research was also supported by the Collaborative

Innovation Major Special Projects of Guangzhou City (201508020097, 201604020007,

201604020057), the Guangdong Provincial Science and Technology Plan Projects

(2015A020209016, 2016A040403040), the Key Projects of Administration of Ocean and

Fisheries of Guangdong Province (A201401C04), the National Key Technologies R&D Program

(2015BAD19B03), the China Postdoctoral Science Foundation (2017M612672), the

Fundamental Research Funds for the Central Universities (2017MS067), the International and

Hong Kong-Macau-Taiwan Collaborative Innovation Platform of Guangdong Province on

Intelligent Food Quality Control and Process Technology & Equipment (2015KGJHZ001), the

Guangdong Provincial R & D Centre for the Modern Agricultural Industry on Non-destructive

Detection and Intensive Processing of Agricultural Products, the Common Technical Innovation

Team of Guangdong Province on Preservation and Logistics of Agricultural

Products (2016LM2154), and the Innovation Centre of Guangdong Province for Modern

Agricultural
 ACCEPTED MANUSCRIPT
20

Science and Technology on Intelligent Sensing and Precision Control of Agricultural Product

Qualities.

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640 Table 1. Effects of different additives on frozen dough system
Additives Mechanism Results Quality of final product
Diglycerides Interaction with starch, prevention of the
Sucrose esters
migration of moisture between gluten and starch Preventing the rétrogradation of the starch and Increase in volume and reduction in firmnes
production of high protein breads of bread

Increased freezing tolerance of yeast and

Ice structuring proteins Absorbed to the non-bottom plane of ice Color of crumb close to fresh bread,
improved thermophysical properties of frozen
from winter wheat and preventing growth of the ice crystals and relatively uniform texture structure
dough

Minimized supercooling of water and

Extracellular ice nucleators Inhabiting the formation of large ice crystals so Increase in volume and elasticity of bread
(ECINs) that the freezing tolerance of reduction in crumb hardening, and brea
modulating the rate of phase transition
extracted from Erwinia yeast and the structure of the frozen dough could color similar to the fresh
herbicola be enhanced

Locust bean gum from Increased water holding capacity of the

Ceratonia siliqua seeds
Tragacanth
Whole-grain wheat flour
Waxy wheat flour
dough, and decreased activity of water due to Preventing the recrystallization of moisture and Increase in moisture content of bread and
water in hydrocolloids competing with the maintaining the rheological properties of frozen better sensory quality
protein and starch in frozen dough dough
Combining with the moisture and prohibiting the Reduced freezable water content and improved Increase in volume, firmness and antioxidan
migration and redistribution of water frozen dough with strong hydration properties activity of bread
and pasting properties

641 Table2. Genetic engineering applied to yeast cell employed in frozen dough
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nes Conditions Results Re
or TSP1 Overexpression of the MAL62 or Increased accumulation of trehalose resulting in promoting the Sun et al. (
TSP1 gene yeast viability under freezing stress
d PUT1 Deleting both NTH1 and PUT1 Higher freeze tolerance and fermentation performance Dong
gene
1150T Overexpression of PRO 1-1150T Improved content of the proline and fermentation ability Sasano
-F65L and mpr1-F65L gene
G1 Overexpression of the POG1 gene Improved leavening performance in high sucrose condition Sasano
Deletion of the POG1 gene Increased freeze tolerance of yeast
R2 Overexpression of MPR2 gene Reduced intracellular oxidation levels leading to enhance the air- Sasano
drying
R84 Overexpression of the SNR84 Higher freeze resistancestress tolerancefermentation capacity in
and improved Lin e
gene
high sucrose containing dough and freeze-thaw conditions
E2 Overexpression of PDE2 gene Improved freezing stress tolerance and better genetic trait Nakagaw
O1 PRO1 gene mutation Increased tolerance to freeze-thaw and high sucrose stresses Tsolmonba

642

Figure caption

Fig. 1. Images of starch granules under scanning electron microscopy (Barrera et al., 2013). A is

normal starch, and B, C and D are damaged starch with damaging percentage of 13.3%,

34.1%, and 72.9%, respectively. The top row of images show the magnification of 8 kx of

the whole starch granules and the bottom row of images show the magnification of 40 kx of
 ACCEPTED MANUSCRIPT

the granules surface.

Fig. 2. The bread appearance of fresh, with and without addition of ECINs (Shi et al., 2013b). A

is fresh bread, B and C are the bread made from frozen dough without or with ECINs

addition after three freeze-thaw cycles.

Fig. 3. Microstructure of wheat flour and frozen dough at different freezing rate (Silvas-Garcia et

al., 2016). (a) and (b) represent microstructure of wheat flour at slow freezing rate and fast

freezing rate respectively, (c, e, g) and (d, f, h) show the microstructure of frozen dough at

slow freezing rate and fast freezing rate respectively for 2, 4 and 8 weeks of storage.

Fig. 4. Images of the size and distribution of ice crystals at different storage times (Baier-Schenk

et al., 2005). (A) and (B) show ice crystals in dough under the condition of liquid nitrogen

freezing, (C-J) show the ice crystals in dough under air-blast freezing for one hour, one

day, and five months of storage.

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662

663

663 Fig. 1. Images of starch granules under scanning electron microscopy (Barrera et al.,

2013). A is

664 normal starch, and B, C and D are damaged starch with damaging percentage of

13.3%,

665 34.1%, and 72.9%, respectively. The top row of images show the magnification of 8

kx of
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666 the whole starch granules and the bottom row of images show the magnification of 40

kx of

667 the granules surface.

669
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668 Fig. 2. The bread appearance of fresh, with and without addition of ECINs (Shi et al.,
2013b). A

669 is fresh bread, B and C are the bread made from frozen dough without or with ECINs

670 addition after three freeze-thaw cycles.

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679

678 Fig. 3. Microstructure of wheat flour and frozen dough at different freezing rate (Silvas-

Garcia et

679 al., 2016). (a) and (b) represent microstructure of wheat flour at slow freezing rate

and fast

680 freezing rate respectively, (c, e, g) and (d, f, h) show the microstructure of frozen

dough at

681 slow freezing rate and fast freezing rate respectively for 2, 4 and 8 weeks of storage.
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691 Fig. 4. Images of the

size and distribution

of ice crystals at different storage times (Baier-Schenk

692 et al., 2005). (A) and (B) show ice crystals in dough under the condition of liquid

nitrogen

693 freezing, (C-J) show the ice crystals in dough under air-blast freezing for one hour, one

day,
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694 and five months of storage.

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Highlights

• Effective methods for enhancing frozen dough quality are reviewed

• Additives improve freeze tolerance of yeast and maintain dough property
• Gene modification enhances freeze tolerance and fermentation ability of yeast
• Optimizing freezing and storage conditions ensures the activity of yeast
• Novel freezing technology protects dough network structure