Effect of Ultrasound-Assisted Heat Treatment Combined with Trehalose and/or Glutamine Transaminase on Gel Characteristics of Unwashed Surimi

ZHANG Chang,LI Qiang,WANG Wei,YI Shumin,LI Xuepeng,LI Jianrong

(National &Local Joint Engineering Research Center of Storage,Processing and Safety Control Technology for Fresh Agricultural and Aquatic Products,National R &D Branch Center of Surimi and Surimi Products Processing,College of Food Science and Engineering,Bohai University,Jinzhou 121013,China)

Abstract: In this study,the effect of alginate and/or transglutaminase (TGase) in combination with ultrasonic treatment on the heat-induced gel quality,water-holding capacity (WHC) and myosin conformational changes of unwashed surimi was investigated.The results showed that the addition of trehalose (4.5%,m/m) and/or TGase (0.5%,m/m) together with ultrasonic treatment increased the gel strength by 35.11%-169.25%.Trehalose prevented protein unfolding while TGase had the opposite effect.However,ultrasonic treatment inhibited the catalytic activity of TGase,thereby reducing the gel strength to 4 737.23 g·mm,decreasing the relative content of free water to 19.52%,and alleviating excessive cross-linking of proteins.Furthermore,ultrasonic promoted protein unfolding even under the protection of trehalose.When added simultaneously,trehalose and TGase weakened their respective effects on protein structure.

Keywords: surimi gel;myosin;ultrasonic;trehalose;transglutaminase

Surimi is rinsed during the production process to remove fat,proteases and other components that can reduce the quality of surimi gel[1].However,such treatment causes the loss of protein,fat and other nutrients in fish.Surimi rinsing wastewater containing a large number of water-soluble proteins which have a high biological oxygen demand,and the growth and reproduction of nitrifying bacteria is inhibited,making it difficult to nitrify surimi rinsing wastewater[2-4].While the application of unrinsed surimi can improve this situation,it is important to find ways to enhance the gelation properties of no-rinse surimi.Current research on enhancing the gelation properties of surimi has been conducted in two general areas,gelation promoters and new processing techniques[5-6].

Transglutaminase (TGase) is commonly used as a gel promoter to enhance the gel properties of surimi.It catalyzes the formation ofε-(γ-Gln)-Lys bonds between theε-amino group of lysine and theγ-carboxy amide group on glutamine,thereby promoting cross-linking between proteins[7].Researchers have found that the addition of TGase to surimi gels of blue round trevally under different thermal gelation methods enhances the gel strength[8].However,excessive cross-linking between proteins caused by TGase can make the surimi gel brittle and reduce water retention[9].TGase is often used in conjunction with several new processing techniques.For example,when combined with high-pressure treatment,TGase can enhance surimi gel properties by promoting the formation of non-covalent bonds[10].In addition,the addition of TGase can improve the 3D printing performance of surimi[11].

Trehalose is a non-reducing disaccharide formed by 2 glucose molecules joined together by hemiacetal hydroxyl groups.It is a powerful stabilizer of proteins and cell membranes,which enhances cellular resistance to extreme environments[12].Wu Shengjun[13]discovered that at low temperatures,trehalose helped stabilize protein structures in surimi,slowing down protein freezing denaturation while increasing the amount of unfrozen water.Xu Shijie et al.[12]investigated the interaction between trehalose and myofibrillar proteins after freezing and found that trehalose could interact with myofibrillar proteins through non-covalent bonds,with the hydrophobic residues of myofibrillar proteins being more exposed.Jain et al.[14]summarized the interrelationships between trehalose and proteins during freezing,mainly including the vitrification theory,the preferential exclusion theory,and the water replacement theory.However,most research on the use of trehalose in surimi and surimi-related products has focused on cryoprotection,with little attention being paid to its interaction with proteins in surimi after heat treatment.

The high protein content of surimi offers great processing potential[15].Proteins are denatured by heat treatment,causing the surimi to form a gel with viscoelastic properties[8].Due to simplicity of operation and low cost,water bath heating is usually used to prepare surimi gels.With the emergence of new processing technologies,combined processing based on water bath heating usually enhances the quality of surimi products[8,16].Ultrasound technology is a new and promising non-thermal processing technique that has gained interest in studies related to protein gels,such as surimi.This technology induces thermal and cavitation effects,generating high-pressure impacts,shear,and turbulence,which in turn break chemical bonds and generate free radicals[17].According to the method of He Xueli et al.[18],ultrasonic treatment enhances the unfolding of theα-helix structure of proteins,resulting in stronger hydrophobic interactions and denser texture of surimi gels.Furthermore,Ye Yuehua et al.[19]suggested that combined microwave-ultrasound treatment could be an effective way to produce new low-sodium surimi gel products.Although the use of ultrasound technology in surimi and surimi products is gradually becoming more widespread,there are still few reports on the use of ultrasound technology in combination with other additives to investigate the effects of ultrasound treatment interventions on different additive-surimi protein interactions.

The aim of this research was to examine the impact of ultrasound-assisted,low-temperature (40 ℃) heat treatment on the gel properties of unrinsed sea bass surimi sols containing trehalose and/or TGase.Additionally,myosin in unrinsed surimi was studied to explore how ultrasoundassisted treatment affects the gel properties from the perspective of protein conformational changes.The aim is to further enrich the theoretical basis for the application of ultrasound technology in surimi products,as well as to contribute to a certain extent to the development of green processing of surimi products.

1 Materials and Methods

1.1 Materials,chemicals,and reagents

Chilled sea bass weighing approximately 1 000 g were purchased from Linxi Road Aquatic Products Market (Jinzhou,China).Trehalose with a purity of 98% was produced by Dezhou Huiyang Biotechnology Co.,Ltd.(Dezhou,China).TGase with an activity of 1×106U was produced by Taixing Dongsheng Food Technology Co.,Ltd.(Taixing,China).

1.2 Instruments and equipment

Vacuum-freezing chopper was purchased from Stephan Machinery Co.,Hameln,Germany;NMI20 low-field nuclear magnetic resonance (LF-NMR) analyzer was purchased from Suzhou Niumag Analytical Instrument Co.,Suzhou,China;TA-XT texture analyzer was purchased from Stable Micro Systems Ltd.,Godalming,UK;Cold field emission scanning electron microscopy (SEM) was purchased from Hitachi Ltd.,Tokyo,Japan;UV-2550 UV spectrophotometer was purchased from Shimadzu Co.,Tokyo,Japan.

1.3 Method

1.3.1 Preparation of unrinsed surimi

The chilled sea bass was decapitated,scaled and gutted,then rinsed with water for meat collection and fine filtration.The obtained unrinsed surimi was divided into two parts,namely the group without trehalose (NT) and the group with 4.5% trehalose (WT).

1.3.2 Preparation of surimi gel

The control group (C),the ultrasonic treatment group (U),the TGase-containing group (G),and the TGase-containing ultrasonic treatment group (UG) surimi gel were prepared by NT respectively.The surimi gels of the trehalose-containing group (T),trehalose-containing ultrasonic treatment group (UT),TGase and trehalose-containing group (GT) and ultrasonic treatment group containing TGase and trehalose (UGT) were prepared by WT.The gel samples of each group were prepared as follows:

1) C&T.The surimi was chopped for 3 min with a vacuum-freezing chopper.Then,2.5% table salt was added,followed by ice water to adjust the moisture content to 78%,and then chopped again.Packed the resulting surimi sol into the nylon casing and sealed both ends.Then,water bath at 40 ℃ for 30 min,and then water bath at 90 ℃ for 20 min.Finally,the prepared samples were taken out and placed in tap water at room temperature to cool to room temperature,and stored at 4 ℃ overnight for the determination of related indicators.

2) U&UT.Based on the C&T preparation method,sonication at 40 Hz and 360 W was used to assist the water bath at 40 ℃ for 30 min.

3) G>.Based on the C&T preparation method,0.5%(m/m) TGase was mixed into ice water for chopping.

4) UG&UGT.Contains all the steps of the above surimi gel preparation method.

1.3.3 Analysis of surimi gel properties

1.3.3.1 LF-NMR analysis

Measurements were performed using a LF-NMR analyzer according to the method described by He Xueli et al.[18].Parameter setting: spectrometer frequency (SF)=22 MHz,pulse 90° (P1)=14 μs,sampling bandwidth (SW)=100 kHz,echocnt=8 000,number sampling (NS)=4,echo time (TE)=200 μs,pulse 180° (P2)=80 μs,repetition time (TR)=800 μs,regulate analog gain 1 (RG1)=20 db,regulate digital gain 1 (DRG1)=3.Finally,using the instrument’s software reproduces the obtained CPMG exponential decay curve to obtain aT2map.Three replicates were set for each group of samples.

1.3.3.2 Determination of gel strength

The puncture test was performed using a TA-XT texture analyzer according to the method described by Zhu Yajun et al.[20].The analyses were performed in quintuplicates using a P/5S model probe (a spherical plunger with a diameter of 5 mm) at pre-test,test,and post-test speeds of 1 mm/s,compression shape of 50%,and trigger force of 15 g.Gel strength (g·mm) was expressed by breaking force (g)multiplied by deformation (mm).For each group of samples,10 replicates were conducted and the highest and lowest values were excluded from the obtained results.

1.3.3.3 Observation of microstructure

Referring to the method of He Xueli et al.[21],the microstructure of surimi gel samples was observed at 1 000 ×using cold field emission SEM.

1.3.4 Extraction of myosin and pretreatment of samples

The back muscles of sea bass were cut into minced meat,and then the myosin in minced meat was extracted by referring to the method of Xu Yongxia et al.[22].Protein concentration was determined by the biuret method[23].Using the extracted myosin as raw material,the group setting and processing method were the same as the surimi gel.

1.3.5 Analysis of myosin aggregation behavior

1.3.5.1 Determination of fluorescence spectra

Myosin samples were processed and protein intrinsic fluorescence was measured by the method of Xu Yongxia et al.[24].Test conditions: sensitivity 3;scanning speed high;excitation wavelength (λex) slit width 5 nm;emission wavelength (λem)slit width 10 nm;currentλex295 nm;λemrange from 320 to 400 nm;scan time 1 min.

1.3.5.2 Determination of turbidity

The OD600nmof the samples was measured by a UV spectrophotometer according to the method described by Yi Shumin et al.[25]and 3 parallels were set for each group of samples.

1.3.5.3 Determination of reactive sulfhydryl groups

The content of reactive sulfhydryl groups in the samples was characterized according to the method described by Yi Shumin et al.[26],and 3 parallels were set for each group of samples.

1.3.5.4 Determination of protein secondary structure

The myosin solution was diluted to 1 mg/mL with 20 mmol/L Tris-HCl buffer (containing 0.5 mol/L NaCl,pH 7.0),and the secondary structure of the protein was characterized by Raman spectroscopy as described by Yi Shumin et al.[27].Three parallels were set for each group of samples.

1.4 Data analysis

Except for the microstructure results were analyzed by Image J software,all other experimental data were analyzed by IBM SPSS 23.0 software (SPSS Inc.,Chicago,USA),and Origin 8.0 was used for graphing.Data are presented as mean ±standard deviation.Significant differences between means were found atP<0.05 by Duncan’s multivariate analysis.

2 Results and Analysis

2.1 LF-NMR spectra

The ability to research changes in water mobility during food processing by measuring the relaxation of hydrogen protons using NMR techniques has been widely used in studies related to surimi gels[6,28].The relaxation profiles of the different treatment groups are shown in Fig.1A.Three distinct characteristic peaks are visible with relaxation times at 1–10 (T21),30–200 (T22),and 200–1 000 ms (T23).These represent bound water,immobilized water,and free water,respectively[29].The content of different types of moisture is represented by the peak area of each relaxation wave[30]shown in Fig.1B.The significantly lower bound water content in the U group compared to the C group is consistent with the findings of Zhang Ziye et al.[31].This may be because the binding between proteins becomes tighter.The free water in T group is further increased by trehalose to bind to myofibrillar proteins through hydrogen bonds or electrostatic interactions,replacing the water molecules originally bound to myofibrillar proteins on the hand,and reducing the electrostatic repulsion between proteins to bind them more tightly on the other[12].The amount of free water in the UT group was increased by both ultrasonic treatment and trehalose.When TGase was added to the gel system,it caused excessive cross-linking between proteins,resulting in a tighter structure that reduces the space for water to separate from the gel system[9].In the UG group,however,the ultrasound-assisted treatment was able to significantly(P<0.05) reduce the free water content compare with G group.The catalytic action of TGase occurs mainly during the heat treatment below 40 ℃[32],and the high-intensity ultrasonic treatment in this temperature range may have promoted the dispersion of myosin,facilitated the unfolding of myosin and exposed the reactive groups[33].Thus,ultrasonic treatment may have somewhat inhibited the excessive cross-linking caused by TGase catalysis.When both TGase and trehalose were present in the gel system,the ultrasound-assisted treatment (UGT group) did not induce a positive effect in terms of water distribution.Trehalose was known to chelate Ca2+necessary for TGase catalysis and inhibit TGase catalysis[34].The high-temperature cavitation bubbles formed by ultrasonic treatment were able to cause chemical bond breakage through thermal decomposition and bubble rupture impacts and cleave water molecules to produce free radicals[17].The increased free water content in the UGT group may be due to the synergistic effect between trehalose,TGase and ultrasound-assisted treatment.Further studies are needed to fully understand this process.

Fig.1 Effects of trehalose and TGase on water distribution in heat-induced gels of unwashed surimi under ultrasonic treatment

2.2 Gel strength

Gel strength is an important indicator for evaluating the quality of surimi products.It incorporates the breaking force and deformation of the gel and is often used to measure the textural properties of the gel[35-36].The breaking force,deformation and gel strength of the different treatment groups were shown in Fig.2.It was found that the gel strength of the U group was significantly higher than that of the C group(P<0.05).Several studies have confirmed that the strength of surimi gels is positively affected by ultrasound-assisted treatment.Gao Xia et al.[33]discovered that high-intensity sonication may have promoted myosin dispersion,which facilitated myosin unfolding and exposure of reactive groups.Su Jing et al.[37]proposed that enhanced mass transfer through acoustic waves could lead to the exposure of hydrophobic residues within the protein,resulting in an irreversible denaturation of the protein.The ultrasound-assisted treatment further enhanced the gel strength when trehalose was present.The UT group was 67.63% stronger than the T group and 101.96% stronger than the C group.The reason may be that the bound of trehalose to myofibrillar proteins reduced the absolute value of myofibrillar potential and electrostatic repulsion between proteins,thus promoting the formation of hydrophobic interactions[12].In addition,ultrasoundassisted treatment promoted the exposure of hydrophobic residues,which further facilitated binding between proteins through hydrophobic interactions.The effect of TGase on gel strength of surimi was well recognized,but after ultrasoundassisted treatment,the gel strength decreased from 5 225.36(G group) to 4 737.23 g·mm (UG group).This was consistent with the results of the analysis of moisture distribution.This may be due to the fact that ultrasound-assisted treatment at the low-temperature treatment stage (below 40 ℃) promoted protein dispersion and weakened the excessive cross-linking catalyzed by TGase on the one hand[32-33];on the other hand,it may be due to the fact that ultrasound-assisted treatment induced a conformational change of TGase,which affected the enzyme activity[16].Trehalose chelated Ca2+was required for TGase catalysis and inhibited it[34],resulting in 18.76%lower gel strength and 16.00% lower breaking force in the GT group compared to the G group.However,no significant reduction (P>0.05) in the deformation was observed,which may be due to a reduction in the TGase-catalyzed formation of the covalent bondε-(γ-Glu)-Lys and an increased formation of hydrophobic interactions by the action of trehalose,which were forces on which protein aggregation depends.Chen Bo et al.[38]suggested that hydrophobic interactions focus on improved gel matrix,while disulfide bonds focus on increased stiffness of interfacial protein membranes.As mentioned previously,ultrasound-assisted treatment promoted the exposure of hydrophobic residues,resulting in a 10.43% increased gel strength and a significantly increased deformation alone in the UGT group (P<0.05).

Fig.2 Effects of trehalose and TGase on the strength of heat-induced gels of unwashed surimi gel under ultrasonic treatment

2.3 Microstructure

The microstructure of the unrinsed surimi gel was observed using cold field emission SEM for each treatment group,as shown in Fig.3.Compared to the C group,the microstructure of the U group was more ordered and homogeneous.He Xueli et al.[18]found that low-temperature(40 ℃) ultrasound-assisted treatment combined with a hightemperature (90 ℃) water bath resulted in a more uniform and smooth surface structure of surimi gels,regardless of the salt content (1.0% or 2.5%).This may be related to the fact that the ultrasound-assisted treatment in the low-temperature phase promotes protein dispersion and facilitates the unfolding of myosin and the exposure of reactive groups,resulting in a more ordered structure of the gel[33].In all other treatment groups,ultrasound-assisted treatment was able to make the microstructure of the surimi gel flatter and denser (UT,UG,and UGT groups).This suggests that the cavitation effect brought by the ultrasonic treatment was not diminished by the presence of trehalose and TGase.However,in the UGT group,there was a significant bulge and reduced surface flatness.As analyzed previously,the TGase-catalyzed formation ofε-(γ-Glu)-Lys competes with the hydrophobic interactions formed by trehalose-ultrasound promotion.The gel system appears to be cross-linked to varying degrees and eventually becomes inhomogeneous in texture while leading to high free water content.

Fig.3 Changes in the microstructure of heat-induced gels of unwashed surimi with different treatments (× 1 000)

2.4 Fluorescence quenching

Endogenous amino acids such as tryptophan (Trp),tyrosine (Tyr) and phenylalanine (Phe) in proteins can produce fluorescence emission spectra at certain excitation wavelengths[39].Fluorescence emission spectra of proteins are commonly used to reflect changes in the tertiary structure of proteins.The effects of trehalose,TGase and ultrasoundassisted treatment on the endogenous fluorescence intensity of heat-treated myosin were shown in Fig.4.Each treatment group’s myosin showed a fluorescence emission peak at around 330 nm.The samples that were not subjected to ultrasound-assisted treatment had peak intensities ranked from highest to lowest as T group >GT group >C group >G group.This suggested that trehalose promoted the unfolding of myosin tertiary structure,while TGase inhibited the unfolding of myosin tertiary structure,and the promotional effect of trehalose was stronger than the inhibitory effect of TGase.Previous studies have generally concluded that trehalose plays a key role in maintaining the tertiary structure of proteins[13,34,40].However,this conclusion was mainly drawn from the cryoprotection of proteins.During heat treatment,thermodynamic effects were amplified,which may trigger the unfolding of protein tertiary structure due to trehalose.However,further validation is needed to confirm this claim.The catalytic effect of TGase occurs mainly during heat treatment below 40 ℃,which was also the temperature range in which the unfolding of myosin mainly occurred.In this temperature range,TGase-catalyzed cross-linking of proteins inhibits the unfolding of myosin structures.

Fig.4 Effects of trehalose and TGase on the fluorescence spectrum of myosin under ultrasonic-assisted heating

Interestingly,when ultrasound-assisted treatment intervened,the fluorescence peaks of each sample were ranked from highest to lowest intensity as UG group >U group >UGT group >UT group.This was the opposite of the samples that did not undergo ultrasound-assisted treatment.However,the effect of trehalose was still stronger than that of TGase.Research conducted by Cao Hongwei et al.[16]suggested that microwave treatment could cause the TGase molecule to change its conformation and unfold,exposing more tryptophan residues to a more polar environment on the surface of the molecule.This may account for the increased peak fluorescence of TGase in combination with ultrasound-assisted treatment.The mode of action between trehalose and proteins remained uncertain,and there were 3 well-known theories,namely vitrification theory(physical defense),preferential exclusion theory (indirect action with water as a medium) and water replacement theory(direct action with proteins)[14].Ultrasound-assisted treatment substantially reduced the peak intensity of the fluorescence of the trehalose-containing myosin solution.This could be attributed to the cavitation effect of ultrasound disrupting the interrelationships between trehalose and myosin.The simultaneous presence of trehalose and TGase neutralized their respective effects,probably due to competition for Ca2+[34].

2.5 Turbidity

Turbidity provides an initial response to the aggregation of lysed myosin[41].The turbidity of the myosin solution for each treatment group was shown in Fig.5.The addition of trehalose significantly increased the turbidity of the protein solution.This was due to the fact that trehalose bound to myosin,decreasing the absolute value of myosin charge,thus reducing solubility and making binding between proteins easier through hydrophobic interactions[12].Additionally,interactions between trehalose and myosin (e.g.,vitrification theory assumes and water replacement theory)[14]may have increased the solute volume in the solution.The results of reactive sulfhydryl groups showed that after heat treatment of myosin,trehalose also promoted the formation of disulfide bonds.This may also contribute to the increased turbidity.Numerous studies have shown that TGase promoted protein aggregation and thus increased solution turbidity[42-43],but the G group did not show higher turbidity.It was hypothesized that the smaller size of the polymers could be due to excessive cross-linking of the proteins by the TGase.As the polymer formed,the amount of solute in solution decreased and the distance between the proteins increased,thus slowing down further cross-linking between the proteins.Ultrasoundassisted treatment reduced the turbidity of protein solutions in the presence or absence of trehalose and TGase (U,UT,UG,and UGT groups).Chen Jiahui et al.[44]concluded that ultrasound disrupted the protein structure,increased the scattering surface area,and consequently reduced the turbidity of protein samples.In this regard,Zhang Ziye et al.[31]hold a similar view.In agreement with the previous section,the neutralization between trehalose and TGase remained appropriate for the illustration of myosin turbidity,regardless of the intervention of ultrasound-assisted treatment.

Fig.5 Effects of trehalose and TGase on the turbidity of myosin under ultrasonic-assisted heating

2.6 Reactive sulfhydryl groups

Disulfide bonds can be formed between reactive sulfhydryl groups by oxidation to maintain the protein gel structure[45].The unfolding of the protein molecule’s heavy chain,the breaking of the original disulfide bond,or the release of the sulfhydryl groups embedded in the molecule,can lead to an increase in the amount of reactive sulfhydryl groups[46].The effect of ultrasonic treatment and the 3 additives on the content of reactive sulfhydryl groups in sea bass myosin gels was shown in Fig.6.There was a negative correlation between the number of active sulfhydryl groups and turbidity in each treatment group.The addition of trehalose and/or TGase did not significantly affect the content of active sulfhydryl groups in samples without ultrasound-assisted treatment (P>0.05),and there was only a significant difference between T and G groups(P<0.05).As mentioned earlier,the reduced reactive sulfhydryl groups in the T group may formed more disulfide bonds,leading to an increased turbidity.In contrast,the presence of TGase may have inhibited the formation of disulfide bonds.In this regard,Li Qiang et al.[8]examined the role of TGase using different thermogenic gelations and found a reduction in the disulfide bond content in the presence of TGase.When ultrasound-assisted treatment intervened,the content of reactive sulfhydryl groups was increased in samples from all treatment groups (UG group >UGT group >UT group >U group).The high shear effect of ultrasound was capable of destroying the original structure of proteins[44].This process broke the existing disulfide bonds,exposed the active sulfhydryl groups within the molecule,and reduced the turbidity of the protein samples,thus facilitating the unfolding of the proteins.The elevation of active sulfhydryl content became significant(P<0.05) in the presence of trehalose and/or TGase,especially in UT and UGT groups.This is probably due to the effect of ultrasound-assisted treatment and TGase on trehalose-myosin,which not only released the active sulfhydryl group but also reduced the turbidity.And the elevation in the UG group was undoubtedly due to the combined effect of TGase and ultrasound-assisted treatment.

Fig.6 Effects of trehalose and TGase on the reactive sulfhydryl group content of myosin under ultrasonic-assisted heating

2.7 Protein secondary structure

Transitions in protein secondary structure are thought to be a marker of protein gelation denaturation,withα-helix toβ-sheet transitions predominating[47].As shown in Fig.7,compared to the C group,samples from all treatment groups except the UG group showed varying degrees of reduction in the relative content ofα-helix structures,as well as an increase in the relative content ofβ-sheet structures,which echoed the gel strength findings.

Fig.7 Effects of trehalose and TGase on the secondary structure of myosin under ultrasonic-assisted heating

When ultrasound-assisted treatment was not involved,trehalose had the least effect on the secondary structure of myosin.During frozen storage,trehalose enhanced the stability of proteins against environmental stresses[14,48].In the present studies,the effect of trehalose on the protein’s mode of action may have been affected by thermodynamic effects.These effects prevented trehalose from stabilizing

the tertiary structure of the protein but did not seem to affect the stabilizing effect of trehalose on the protein’s secondary structure.In contrast,while the relative content ofα-helix in the G group showed a greater decrease,both the relative content ofβ-sheet andβ-turn exhibited a significant increase.In this regard,Huang Jianlian et al.[49]and Liang Feng et al.[50]have similarly found that the presence of TGase can facilitate the transition from theαto theβstructure of the protein.

When ultrasound-assisted treatment intervened,the most transformation of protein secondary structure occurs in the U group compared to the C group.This is due to the high shear forces generated by sonication during the low-temperature treatment phase,which promoted the dispersion of myosin while also disrupting the original structure of the protein[33,44].Trehalose may weaken the effect of ultrasound-assisted treatment on protein structure due to its protective effect on protein secondary structure (UT and UGT groups).However,in the UG group,the ultrasonication did not further promote the secondary structure transition but rather inhibited it.This is consistent with the results for gel strength.It is possible that the protein dispersion promoted by sonication weakens the catalytic effect of TGase and also causes excessive crosslinking under changes in TGase activity[16,32-33].

3 Conclusion

Ultrasound-assisted low-temperature heat treatment increased gel strength and free water content of unbleached surimi.This effect was enhanced by the addition of trehalose and TGase,but the pathways of action of the two were different.Despite trehalose preventing the unfolding of the protein structure during sonication,it has the added benefit of increasing the gel strength of the surimi.This is achieved by reducing protein solubility and promoting the formation of hydrophobic and electrostatic interactions,which leads to protein aggregation.In addition,ultrasound-assisted treatment inhibited the catalytic effect of TGase to some extent and slowed down the excessive cross-linking induced by TGase.When both trehalose and TGase were present,the two inhibited each other and the binding to ultrasound was subsequently neutralized.The effect of ultrasonic treatment on additive-surimi interactions was not equal and depended on the pathway of action of the selected additive on the surimi.Therefore,when using ultrasonication with additives,careful selection of the additives is essential.