Cryoprotective Effects of Protein Hydrolysates Prepared from By-Products of Silver Carp (Hypophthalmichthys Molitrix) on Freeze-Thawed Surimi

Round  1

Reviewer 1 Report

Abstract section:

It is not understandable what PH-2 group is.

Introduction section:

I think it should be included a brief paragraph explaining the next issues:

-        Importance of including a cryoprotective compound in the production of surimi or other frozen products

-        What alternatives are there? Like SuSo or other. Why is it necessary to develop alternative products if there are already some efficient cryoprotective products?

-        Please define SuSo in the introduction

Not all these aspects are clear in the introduction.

Materials and methods:

-Consider changing the order of this section in the text. Change it and explain it, before the results.

-In lines 28-29, authors explained the methodology carry out to process the raw material. What do the authors want to say: “it was grinded with ice”? Was the raw material frozen?. Please, explain it better.

-Line 230, please indicate the E.C and the bacteria where the enzymes come from-Line 232: How many hydrolysates under each condition were elaborated?

-Line 233, what kind of equipment or procedure was used to degrease the samples?

-Line 236, Change concentrion by concentration

-Line 236, what percentage of humidity did the sample have before doing the hydrolysates?

-Line 236, Are you sure that the used concentration of Protamex and Alcalase are 2400 and 3000 U/g, respectively? I think it is very high considering it is common to use less than 1 AU/g. Please, detail what the substrate is in the relation between enzyme and substrate: g of protein or g of raw material

-Line 237-238, Why is different pHs and temperatures used to elaborate the hydrolysates with the enzymes?

-Line 239 and others: Please, unify along the text: 10000 or 10,000 not both (lines 236, 261 etc)

-Line 240: Please, indicate that “PH” are hydrolysates from Protamex and “AH” are from Alcalase

-Line 240: Please, indicate what solvent was used to dissolve the dried hydrolysates for futher analysis

-Line 241: Why is not the amount of protein in the raw material and the hydrolysates measured? Please, provide information about this issue.

Line 242: How many repetitions were carried out?

-Line 243: Please, indicate how the protein content was measured and how the total hydrolysis was carried out

-Line 243-244: “And 1 mol/L NaOH was used to maintain constant pH value during hydrolysis”. It is probably that this sentence belongs to the 3.2. Protein hydrolysates preparation section.

-Line 249-254. It is necessary to explain in more detail what chromatographic method was used to separate the hydrolysate, mobile phases, equipment, separation gradient etc.

-Line 255-257: It is necessary explain the methodology used

-Line 275: Change r/min by rpm/min

-Line 281-283: How was the total amount of actomyosin calculated? The folin method does not discriminate between actomyosin and other proteins, how do the authors know the amount of actomyosin in the solution?

-Line 288: Change pH7.0 by pH 7.0

Results:

Figure 1. In my opinion, it should be better and provide more information if authors change the figure 1 by the chromatographic pattern of the different standards. However, it could be eliminate if the authors include a figure showing the chromatographic separation of the two different hydrolysates.

Line 71-72: The research article would improve if the authors include a figure showing the chromatographic separation of the two different hydrolysates.

Line 76-77: Has the molecular weight distribution been of the sample without being hydrolyze?

Table 1: Please define what PH and AH is in the table

Lines 85-86: I think there is a mistake. The statistical analysis should be compared between rows, was not it? I mean for each parameter but comparing the two types of hydrolysates

Line 89: Please, define TAA in the text

Lines 87-104: Please, provide more information about other protein hydrolysates with a cryoprotective function.

Figure 2: Please, define what Suso, PH-2, PH-4 etc are.

Figure 2. It is difficult to identify the different samples in the graph, mainly the control group. Please, use different dotted lines too in order to improve the visualization of the different groups

Line 124-144: Please explain in more detail the relevance of measuring the Ca-ATPase activity

Table 3. Please, define what Suso, PH-2, PH-4 etc are.

In general, results should be a little more discussed. Please, compared with other published hydrolysates.

Author Response

Point 1: Abstract section: It is not understandable what PH-2 group is.

 Response 1: PH-2 group means “group with addition of 2 g hydrolysate prepared by Protamex hydrolysis”. We have added the explanation (shown in lines 19-20: ...group with addition of 2 g hydrolysate prepared by Protamex hydrolysis (PH-2)...)

 Point 2: Introduction section: I think it should be included a brief paragraph explaining the next issues: 

- Importance of including a cryoprotective compound in the production of surimi or other frozen products

- What alternatives are there? Like SuSo or other. Why is it necessary to develop alternative products if there are already some efficient cryoprotective products?

- Please define SuSo in the introduction

Not all these aspects are clear in the introduction.

 Response 2: According to the suggestions of the reviewer, we have made some changes in the Introduction section (shown in lines 43-56: Recently, other studies have demonstrated that protein hydrolysates could reduce deterioration in quality of fish surimi or mince during frozen storage or repeated freeze-thaw treatments [5-13]. Although frozen storage is often used to preserve fish product, protein aggregation and denaturation could still occur and lead to potential decrease in product quality. Peptides in protein hydrolysate could slow the formation of ice crystals, thereby contributing to structural stabilization of proteins during frozen storage [6]. Carbohydrate-based cryoprotectants such as sucrose-sorbitol blend (SuSo) though are commonly used to maintain the quality of commercial fish product during frozen storage, impart a sensorially unfavorable sweetness to the product, along with the added caloric and glycemic values. These limitations of using carbohydrate-based cryoprotectants could render the end products unsuitable for many consumers and people with dietary restrictions (such as diabetics) [5, 14]. Therefore, protein hydrolysate is a preferred alternative cryoprotectant to SuSo, especially with its additional nutritional value (short peptides and free amino acids) in the absence of unwanted product sweetness and added caloric and glycemic value.)

 Point 3: Materials and methods: Consider changing the order of this section in the text. Change it and explain it, before the results.

 Response 3: We have changed the order of section (Materials and methods) and section  (Results and discussion) (shown in the manuscript). 

  Point 4: -In lines 28-29, authors explained the methodology carry out to process the raw material. What do the authors want to say: “it was grinded with ice”? Was the raw material frozen?. Please, explain it better.

 Response 4: The raw material was not frozen. We used by-products (fish meat leftovers on bones and heads) of silver carp provided by Hunan Yiyang Yihua Aquatic Products Co., Ltd (Yiyang, Hunan, China) to prepare hydrolysates. In order to prevent the fish protein denaturation in raw materials and inhibit the activities of a small amount of proteolytic enzymes existing in fish heads, thereby keeping the consistency of raw materials, we ground large amounts of supplied by-products into uniformity with addition of ice cubes to keep the ground temperature at < 10 °C. Then the ground by-products were sealed in polyethylene bags and stored at 40 ºC until use. Usually, we used the ground by-products within 24 h. So the consistencies of the raw materials were controlled. 

 Point 5: -Line 230, please indicate the E.C and the bacteria where the enzymes come from?

 Response 5: The enzyme information has been provided (shown in lines 76-77: The enzymes, Protamex (enzyme activity of 120,000 U/g, from Bacillus sp) and Alcalase (enzyme activity of 200,000 U/g, from Bacillus licheniformis)...)

 Point 6: -Line 232: How many hydrolysates under each condition were elaborated?

 Response 6: Under the conditions used in the experiment, we utilized by-products of silver carp to produce hydrolysates. The yield of the hydrolysates prepared by Protamex hydrolysis was about 42.10 % and the yield of the hydrolysates prepared by Alcalase hydrolysis was about 43.6%. we have provided the information (shown in lines 184-186).

 Point 7: -Line 233, what kind of equipment or procedure was used to degrease the samples?

 Response 7: The procedure was as follows: The ground by-products were defatted with isopropanol at 25 °C for 1 h at a ratio of 1:5 of raw material to solvent. The slurry was vacuum filtered and the filter cake was air-dried at room temperature. The dried material was ground to pass 80 meshes... We have provided the procedure (shown in lines 80-82).

 Point 8: -Line 236, Change concentrion by concentration

 Response 8: We have changed “concentrion” by “concentration” (shown in line 84).

 Point 9: -Line 236, what percentage of humidity did the sample have before doing the hydrolysates?

 Response 9: Defatted by-products was the sample to prepare the hydrolysates. The moisture of the sample was 3.72±0.61%. We have provided the proximate composition of defatted by-products and two hydrolysates (shown in Table 1).

 Point 10: -Line 236, Are you sure that the used concentration of Protamex and Alcalase are 2400 and 3000 U/g, respectively? I think it is very high considering it is common to use less than 1 AU/g. Please, detail what the substrate is in the relation between enzyme and substrate: g of protein or g of raw material

 Response 10: The enzymes activity of Protamex and Alcalase are 120,000 U/g and 200,000 U/g, respectively (shown in lines 76-77). The amounts of enzyme are 0.020 g of Protamex/g of substrate and 0.015 g of substrate/g of raw material. We have changed the sentence by “...0.020 g of Protamex/g of substrate (enzyme/substrate was 2,400 U/g) and 0.015 g of Alcalase/g of raw material (enzyme/substrate was 3,000 U/g)...”(shown in lines 84-86)

 Point 11: -Line 237-238, Why is different pHs and temperatures used to elaborate the hydrolysates with the enzymes?

 Response 11: Based on our previous research, pH 7.0 and 50 ºC are the optimal pH values and temperatures for Protamex hydrolysis, and pH 8.5 and 60 ºC are the optimal pH values and temperatures for Alcalase hydrolysis. Therefore, we used different pHs and temperatures to elaborate the hydrolysates.  

 Point 12: -Line 239 and others: Please, unify along the text: 10000 or 10,000 not both (lines 236, 261 etc)

 Response 12: We have made changes according to the suggestions of the reviewer.

 Point 13: -Line 240: Please, indicate that “PH” are hydrolysates from Protamex and “AH” are from Alcalase

 Response 13: We have made the changes according to the suggestions of the reviewer (shown in lines 90-91).

 Point 14: -Line 240: Please, indicate what solvent was used to dissolve the dried hydrolysates for futher analysis

 Response 14: For zeta potential analysis, we used distilled water to dissolve the hydrolysates, and for analysis of molecular weight distribution, we used 0.1 mol/L Na₂SO₄ in 0.1 mol/L phosphate buffer (pH 6.7) to dissolve the hydrolysates, etc. We have added the solvent information to each testing method.

 Point 15: -Line 241: Why is not the amount of protein in the raw material and the hydrolysates measured? Please, provide information about this issue.

 Response 15: The amount of protein in the raw material (defatted by-products) have been provided in Table 1. Proximate composition of the hydrolysates were also measured, and we have provided the information in Table 1.

 Point 16: Line 242: How many repetitions were carried out?

 Response 16: Three independent tests were performed to verify the DH value.

 Point 17: -Line 243: Please, indicate how the protein content was measured and how the total hydrolysis was carried out

 Response 17: According to the suggestions of the reviewer, the testing methods of the protein content and degree of hydrolysis were indicated (shown in lines 94-101.)

 Point 18: -Line 243-244: “And 1 mol/L NaOH was used to maintain constant pH value during hydrolysis”. It is probably that this sentence belongs to the 3.2. Protein hydrolysates preparation section.

 Response 18: Yes, we have inserted the sentence “...and 1 mol/L NaOH was used to maintain constant pH value during hydrolysis” to lines 87-88.

 Point 19: -Line 249-254. It is necessary to explain in more detail what chromatographic method was used to separate the hydrolysate, mobile phases, equipment, separation gradient etc.

 Response 19: Yes, we have provided the detailed chromatographic method (shown in lines 110-115: The hydrolysates were dispersed in 0.1 mol/L Na₂SO₄ in 0.1 mol/L phosphate buffer (pH 6.7) and filtered through cellulose acetate membranes of 0.45 μm (Millipore, US) to remove insoluble particles before analysis. A Shimadzu HPLC system (Shimadzu Corporation, Japan) equipped with a TSKgel G3000 PWXL column and a Shimadzu ultraviolet detector were used. The hydrolysates were eluted by 0.1 mol/L Na₂SO₄ in 0.1 mol/L phosphate buffer (pH 6.7) at a flow rate of 1 mL/min and monitored at 220 nm at 25 °C.)

 Point 20: -Line 255-257: It is necessary explain the methodology used

 Response 20: Determination method of total and free amino acid composition has been provided (shown in lines 119-123: Total amino acid (TAA) and free amino acid (FAA) compositions were tested using an automatic amino acid analyzer (L-8900, Hitachi, Japan). The hydrolysates were hydrolyzed in 6 mol/L HCl at 110 °C for 24 h for the measurement of total amino acids. Tryptophan (Trp) was destroyed during HCl hydrolysis, therefore, the Trp content was not detected. Free amino acid composition was determined by analysis of the hydrolysates without prior HCl hydrolysis.) 

 Point 21: -Line 275: Change r/min by rpm/min

 Response 21: We have changed r/min by rpm/min (shown in line 141).

 Point 22: -Line 281-283: How was the total amount of actomyosin calculated? The folin method does not discriminate between actomyosin and other proteins, how do the authors know the amount of actomyosin in the solution?

 Response 22: The protein extraction method in section 2.5 (lines 136-149) is the method to extract actomyosin. We extracted the actomyosin from unfrozen and freeze-thawed surimi, therefore the protein concentration of each sample solution measured by Folin-phenol method was the actomyosin concentration.

 Point 23: -Line 288: Change pH7.0 by pH 7.0

 Response 23: We have changed pH7.0 by pH 7.0 (shown in line 154).

 Point 24: Results: Figure 1. In my opinion, it should be better and provide more information if authors change the figure 1 by the chromatographic pattern of the different standards. However, it could be eliminate if the authors include a figure showing the chromatographic separation of the two different hydrolysates.

 Response 24: According the suggestions of the reviewer, we have included the chromatogram of molecular weight distribution of the two different hydrolysates (shown in Figure 1b).

 Point 25: Line 71-72: The research article would improve if the authors include a figure showing the chromatographic separation of the two different hydrolysates.

 Response 25: Yes, we have included the chromatogram of molecular weight distribution of the two different hydrolysates (shown in Figure 1b).

 Point 26: Line 76-77: Has the molecular weight distribution been of the sample without being hydrolyze?

 Response 26: In order to confirm the properties of the protein in the raw materials, we used 2% SDS to extract the total proteins in the raw materials in our previous studies. The molecular weight distribution of the proteins was obtained. Possible macromolecules were existed in the proteins, thus a Shodex protein KW-804 Column (Shodex Separation and HPLC Group, Tokyo, Japan, exclusion limit) was used to separate the total proteins. Relative proportions (%) of the first three elution peaks with retention time around 6.0, 6.8 and 7.9 min were about 6.72%, 26.26% and 7.78%, respectively. These fractions were estimated to have molecular weight larger than 1,000 kDa, which were very large macromolecular myofibrillar protein complexes polymerized by disulfide bond and hydrophobic interactions. The other five peaks were also detected around the retention time of 9.5, 10.3, 11.8, 12.1 and 12.5 min, whose Mw were estimated as 151, 53, 6.9, 5.2 and 2.8 kDa, respectively. Compared to the molecular weight distribution of the two different hydrolysates, the differences were rather obvious. And the results also demonstrated that both Protamex and Alcalase were efficient enzyme choices for preparing hydrolysates from by-products of silver carp. This study was focused on the characterization of the hydrolysates prepared from the raw materials and the cryoprotective ability of the two types of hydrolysates. So we didn’t include the chromatogram of molecular weight distribution of the protein without being hydrolyzed.

Point 27: Table 1: Please define what PH and AH is in the table

 Response 27: We have defined what PH and AH are (shown in lines 230-231).

 Point 28: Lines 85-86: I think there is a mistake. The statistical analysis should be compared between rows, was not it? I mean for each parameter but comparing the two types of hydrolysates

 Response 28: Yes, in the SPSS software, the statistical analysis should be compared between rows. We compared the two types of hydrolysates in the software and listed  the results of statistical analysis in the table. And we have made some changes about the definition (shown in lines 232-233: Different letters in the same column indicate significant differences at P<0.05.).< span="">

 Point 29: Line 89: Please, define TAA in the text

 Response 29: TAA is abbreviation of total amino acid. We have changed it by total amino acid.

 Point 30: Lines 87-104: Please, provide more information about other protein hydrolysates with a cryoprotective function.

 Response 30: We have provided more information about other protein hydrolysates with cryoprotective ability (shown in lines 235-255: Results from amino acid analysis of PH and AH are shown in Table 3. Both hydrolysates contained similar amounts of total amino acid. The total contents of charged and hydrophilic amino acid residues (including Glu, Asp, Lys, Pro, Gly, Ser, Thr, Arg and His) in both hydrolysates were up to about 68% of the total amino acid contents, which were relative to ice affinity and cryoprotective activity of antifreeze proteins [23]. About 27% of acidic amino acid residues (Glu and Asp) were found in both hydrolysates. The relative content of Glu, which contains strongly polar hydroxyl groups that are favorable for cryoprotective properties, in both hydrolysates reached about 15% [21]. PH and AH were also rich in Pro residues (about 13%) which also contributed to ice affinity [23]. Sericin hydrolysates (molecular weight of less than 3 kD) with cryoprotective activity were also rich in the amino acids Ser, Asp, Gly, Thr and Glu [24]. In ice-binding proteins from arctic yeast, aligned Thr/Ser/Ala residues were found to be critical for binding of ice [25]. The content of basic amino acid (Lys) in both hydrolysates were about 9%, which enhanced the stability if hydrogen bonds between the hydrolysate and the ice crystal. The total content of hydrophobic amino acid residues (including Phe, Met, Leu, Ile and Val) in PH was a little higher than that in AH. Previous researchers have verified that hydrophobic amino acid residues in fish protein hydrolysate helped to retain textures, water-binding properties and proportion of unfrozen water  of frozen fish mince [5]. Free amino acid composition results of PH and AH indicated that PH had higher free amino acid content than AH due to the exo- and endo-protease property of Protamex. PH also contained significantly higher content of free Lys (P<0.05), which has been reported to be cryoprotective [26]. Free Lys, combined with Arg, Asp and Glu, preferentially hydrated vulnerable proteins and bound free water, thereby enhancing cryoprotective abilities [26]. )

 Point 31: Figure 2: Please, define what Suso, PH-2, PH-4 etc are.

 Response 31: We have defined what SuSo, PH-2, PH-4 etc. are (shown in lines 279-282: SuSo (surimi added with sucrose-sorbitol blend); PH-2, PH-4 and PH-6 (surimi with addition of 2, 4 and 6 g of the hydrolysate prepared by Protamex hydrolysis, respectively); AH-2, AH-4 and AH-6 (surimi with addition of 2, 4 and 6 g of the hydrolysate prepared by Alcalase hydrolysis, respectively); and control (surimi without cryoprotectant).)

 Point 32: Figure 2. It is difficult to identify the different samples in the graph, mainly the control group. Please, use different dotted lines too in order to improve the visualization of the different groups

 Response 32: According to the suggestions of the reviewer, in Figure 2, 3, 4 and 5, we have used different dotted lines to improve the visualization of the different groups.

 Point 33: Line 124-144: Please explain in more detail the relevance of measuring the Ca-ATPase activity

 Response 33: We have provided the detailed reasons for measuring the Ca²⁺-ATPase activity (shown in lines 285-288: Myosin (combines with actin to form actomyosin) accounts for 50% of fish myofibrillar protein. The active site of Ca²⁺-ATPase is located in the globular head of myosin [29]. Thus the Ca²⁺- ATPase activity is a good indicator of the integrity of actomyosin molecule and protein freeze denaturation.)

 Point 34: Table 3. Please, define what Suso, PH-2, PH-4 etc are.

 Response 34: We have defined what SuSo, PH-2, PH-4 etc. are (shown in lines 393-396: SuSo (surimi added with sucrose-sorbitol blend); PH-2, PH-4 and PH-6 (surimi with addition of 2, 4 and 6 g of the hydrolysate prepared by Protamex hydrolysis, respectively); AH-2, AH-4 and AH-6 (surimi with addition of 2, 4 and 6 g of the hydrolysate prepared by Alcalase hydrolysis, respectively); and control (surimi without cryoprotectant).)

 Point 35: In general, results should be a little more discussed. Please, compared with other published hydrolysates.

 Response 35: According to the suggestions of the reviewer, the results have been compared with other published hydrolysates (shown in lines 273-275, 299-305, 323-329, 354-360, 387-390).

Author Response File: Author Response.pdf

Reviewer 2 Report

The experimental characterization is satisfactory, even if the authors do not try to discuss the results obtained more in depth.

English is enough but a rereading of a native speaker can eliminate the various imperfections scattered throughout the text, which in some places make understanding difficult.

Some parts should be corrected or better described:

Hydrolysates characterization

- "ζ potentials": should be explained what it is and what its measurement is for

- the elution profiles from the TSK-gel G3000 PWXL column should be shown, to better follow the description of the various fractions

- the calibration curve in Figure 1 should be interpolated with a straight line

Amino acid analysis of hydrolysates

- in Table 2 should be indicated the respective % values, to better follow the description of the results

- the experimental description is completely missing in the Materials and methods (section 3.3.4. Amino acid composition)

Surface hydrophobicity of actomyosin

- the description of the curves in Figure 5 (lines 180-182) should be more detailed, referring to the Freeze-thaw cycles

Textural properties of heat-set unfrozen and freeze-thawed surimi gels

- as regards the statistical treatment of the data in Table 3, the meaning of the symbols a, b, c, A, etc ... is not clear at all; moreover, some of these (d, e, f, g) are not used in the table

- the TPA test is not explained in the Materials and methods section

Author Response

Point 1: The experimental characterization is satisfactory, even if the authors do not try to discuss the results obtained more in depth. English is enough but a rereading of a native speaker can eliminate the various imperfections scattered throughout the text, which in some places make understanding difficult. 

 Response 1: According to the suggestions of the reviewer, we have added some information to discuss the results throughout Section 3 (Results and discussion), and this manuscript has been thoroughly edited by a native English speaker from an editing company. Editing Certificate has been provided (please see the attachment).

Point 2: Some parts should be corrected or better described:

Hydrolysates characterization

- "ζ potentials": should be explained what it is and what its measurement is for

- the elution profiles from the TSK-gel G3000 PWXL column should be shown, to better follow the description of the various fractions

- the calibration curve in Figure 1 should be interpolated with a straight line

 Response 2: We have changed "ζ potentials" by “zeta potential values”, and the role of its measurement has been explained (shown in lines 192-195: Zeta potential value is an indicator of the surface charge of the hydrolysate in solution, which can reflect the stability and possible binding ability of the hydrolysate to ice crystal and/or protein via hydrogen bond and electrostatic interaction [20].)

    We have provided the elution profiles of the hydrolysates (shown in Figure 1b) and description of the various fractions (shown in lines 205-217: There were a small amount of larger molecules (Mw ≥ 7500 Da) in PH and AH , about 7.7±1.6 and 5.1±0.9 %, respectively. Larger molecules and their proportion in the hydrolysates are crucial factors that affect gelling properties [4]. PH contained peptides at Mw of 2027 Da, which was absent in AH, at a relative proportion that reached 32.6±2.8%. Compared to PH, AH exhibited higher proportions of peptides at Mw of about 1420 and 286-780 Da (P<0.05), which comprised of 50.6±5.2% and 39.1±2.6% of the hydrolysates, respectively. The difference in molecular weight distribution of PH and AH could be attribute to the varied specificities of Protamex and Alcalase for peptide bonds adjacent to certain amino acid residues. The results also indicated that short peptides constituted a dominant proportion in both PH and AH. The molecular weight of the hydrolysates or peptides, after being added to fish mince, showed significant impacts on the formation and growth of ice crystals in frozen mince, and on their interactions with fish myofibrillar proteins [5, 6, 21]. For example, short peptides can easily attach to the surface of ice and inhibit ice crystallization, therefore, acting as effective cryoprotective agents [22].). The calibration curve in Figure 1 has been interpolated with a straight line (shown in Figure 1a).

 Point 3: Amino acid analysis of hydrolysates

- in Table 2 should be indicated the respective % values, to better follow the description of the results

- the experimental description is completely missing in the Materials and methods (section 3.3.4. Amino acid composition) 

 Response 3: The respective % values of each amino acid and free amino acid have been calculated and shown in Table 3. The results have been discussed more (shown in lines 236-255: The total contents of charged and hydrophilic amino acid residues (including Glu, Asp, Lys, Pro, Gly, Ser, Thr, Arg and His) in both hydrolysates were up to about 68% of the total amino acid contents, which were relative to ice affinity and cryoprotective activity of antifreeze proteins [23]. About 27% of acidic amino acid residues (Glu and Asp) were found in both hydrolysates. The relative content of Glu, which contains strongly polar hydroxyl groups that are favorable for cryoprotective properties, in both hydrolysates reached about 15% [21]. PH and AH were also rich in Pro residues (about 13%) which also contributed to ice affinity [23]. Sericin hydrolysates (molecular weight of less than 3 kD) with cryoprotective activity were also rich in the amino acids Ser, Asp, Gly, Thr and Glu [24]. In ice-binding proteins from arctic yeast, aligned Thr/Ser/Ala residues were found to be critical for binding of ice [25]. The content of basic amino acid (Lys) in both hydrolysates were about 9%, which enhanced the stability if hydrogen bonds between the hydrolysate and the ice crystal. The total content of hydrophobic amino acid residues (including Phe, Met, Leu, Ile and Val) in PH was a little higher than that in AH. Previous researchers have verified that hydrophobic amino acid residues in fish protein hydrolysate helped to retain textures, water-binding properties and proportion of unfrozen water  of frozen fish mince [5]. Free amino acid composition results of PH and AH indicated that PH had higher free amino acid content than AH due to the exo- and endo-protease property of Protamex. PH also contained significantly higher content of free Lys (P<0.05), which has been reported to be cryoprotective [26]. Free Lys, combined with Arg, Asp and Glu, preferentially hydrated vulnerable proteins and bound free water, thereby enhancing cryoprotective abilities [26]. )

     The experimental description has been provided (shown in lines 120-123: The hydrolysates were hydrolyzed in 6 mol/L HCl at 110 °C for 24 h for the measurement of total amino acids. Tryptophan (Trp) was destroyed during HCl hydrolysis, therefore, the Trp content was not detected. Free amino acid composition was determined by analysis of the hydrolysates without prior HCl hydrolysis.)

 Point 4: Surface hydrophobicity of actomyosin

- the description of the curves in Figure 5 (lines 180-182) should be more detailed, referring to the Freeze-thaw cycles

 Response 4: We have provided detailed description of Figure 5 (shown in lines 341-348: As shown in Figure 5, increased surface hydrophobicity of actomyosin was observed in all samples. However, in the PH, AH and SuSo groupss, increase in surface hydrophobicity was retarded after six freeze-thaw cycles in comparison with a sharp increase in the control group (P<0.05). Overall, the control group showed 245.7% increase in surface hydrophobicity of actomyosin by the second freeze-thaw cycle, and 320.2% increase after six freeze-thaw cycles. While the total increases in surface hydrophobicity of SuSo, PH-2, PH-4, PH-6, AH-2, AH-4 and AH-6 groups were 124.8, 110.4, 120.8, 284.5, 191.0, 274.8 and 323.5 %, respectively. 

And in lines 354-361: SuSo group exhibited continual increase in surface hydrophobicity throughout six freeze-thaw treatments. In the hydrolysate groups, after the initial increase, surface hydrophobicity sharply decreased after four freeze-thaw cycles and then increased further. Benjakul and Sutthipan had revealed that exposure of aliphatic and aromatic amino acid residues to protein molecular surface could lead to increases in surface hydrophobicity [31]. It’s possible that during the freeze-thaw treatments, peptides and/or free amino acids in the hydrolysates interact with actomyosin via hydrophobic interactions, inducing a decrease in surface hydrophobicity. Our findings also supported that the hydrolysates and SuSo might adopt different mechanisms of cryoprotection.)

Point 5: Textural properties of heat-set unfrozen and freeze-thawed surimi gels

- as regards the statistical treatment of the data in Table 3, the meaning of the symbols a, b, c, A, etc ... is not clear at all; moreover, some of these (d, e, f, g) are not used in the table

- the TPA test is not explained in the Materials and methods section

 Response 5: We have made some changes about the definition of statistical analysis in Table 3 (shown in lines 397-400: ^{a, b, c} represent significant differences of samples in the same column (P<0.05); ^{A, B} represent significant difference of the same sample before and after freeze-thaw treatment (UF and FT) (P<0.05)); ^* indicates significant differences between freeze-thaw treated samples and unfrozen control sample (P<0.05).) 

    The TPA test has been explained in section 2.7 (shown in lines 172-176: Texture profile analysis (TPA) of the gel was performed on a texture analyzer (TA.XTPlus, Stable Micro Systems, UK) using a P/36R probe with a test speed of 1 mm/s. The gel was subjected to two-cycle compression at strain of 50%. A total of 10 replicates were tested for each sample. From the resulting curves, hardness, springiness, cohesiveness and chewiness of the gel were determined.)

Author Response File: Author Response.pdf

Round  2

Reviewer 1 Report

The drafting of the manuscript, the explanation of the different methodologies and the visualization of the graphs have improved greatly.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.