The Quality and Flavor Changes of Different Soymilk and Milk Mixtures Fermented Products during Storage

Round 1

Reviewer 1 Report

In this paper, the manuscript is dealing with the effect of different mixed fermentation methods on the quality and flavor of fermented soy milk during storage. The topic is interesting. However, there are several issues needed to be addressed. Please find a native English speaker to check the language.

Q1: Abstract need to be rewritten. The key point and the novelty of the study must be explained. In the current version, it only mentioned in a very broad view. Ln 11: Please include the specific details of research study presented in the manuscript.

Q2: Grammar problems. Ln 14: "Keep the number of viable count was greater than... " Ln 41 " A strain of Lactobacillus ..."is confusing. Ln 48" found that co-fermentation of soy milk and buttermilk mixtures..." Is confusing. Ln 231 "Including..." Is confusing.

Q3: Please check the writing throughout the manuscript and keep the writing in standard. Ln 98 and Ln 161, change"MSFM" to " MFSM"?

Q4: All the academic names of bacteria should be italics (Ln 41).

Q5: Ln 70: "According to previous studies.." Reference?

Q6: Ln 92: what is the function of "Recovered milk"? For the two groups, MFSM and SMFSM, do they both contain 8% sucrose? What is the major function of 8% sucrose?

Q7: Ln 119: Volatile aromatic compound section, was the method based on some literature method?

Q8: Ln 145: Frequency scanning part, what is the working volume? The details of this method were missing.

Q9: Ln 178: Are these pH are significantly different? Did you carry out the statistics here?

Q10: Ln 201, Ln 356, " different degrees and affect different species “，what do you mean?

Q11: Ln 219: "aggregate at the same time..." Is it your hypothesis? Please explain more and provide more information?

Q12: Ln 240:". And the flavor substance content..." Is confusing. Grammar problem?

Q13: Ln 250:" the 2-heptanone content of SMFSM samples was always higher..." Do you have some reasonable guessing? Please explain more with bacteria analysis.

Q14: Ln 329: what do you mean" the right amount..." ?

Q15: Ln 373:" G'>G". Please explain more. 

Q16: The conclusion section was not sufficient to effectively summarize the content of the entire manuscript. This section needs to be improved.

Author Response

Dear Professor:

First of all, thank you for carefully pointing out the inadequacies in our article so that we have the opportunity to improve. I have made changes to the article as per your request. Modifications are marked in red in the text.

In your comments and suggestions to the authors, you mentioned: In this paper, the manuscript deals with the effects of different mixed fermentation methods on the quality and flavor of fermented soybean milk during storage. This topic is interesting. However, there are several issues that need to be addressed. Please find a native English speaker to check the language.

This article found native English-speaking experts to revise the language.

Certificate is attached: Response 1

Now I will reply to the comments one by one.

Q1: The abstract needs to be rewritten. The key points and novelty of the study must be explained. In the current version, it's only mentioned in a very broad field. Ln 11: Please include specific details of the study in the manuscript.

A1: Rewrite the abstract from the original text and state the specific details of the study as required.

"This study aimed to explore the effects of two mixed soymilk fermentation methods on viable bacterial count, texture, rheological properties, stability, sensory quality and volatile flavor compounds. The results showed that compared with the prepared fermented soymilk, the Soymilk samples prepared by mixing soymilk and buttermilk before acidification, fermenting buttermilk separately and then mixing with soymilk after acidification can significantly reduce the post-acidification of fermented soymilk during storage. In addition, keep the viable count greater than 7 log cfu/mL. The water capacity does not change significantly until the 30th day. There is also no whey precipitation and good stability. In MFSM mode, it has higher aroma and legumes during storage than fermented soymilk. Volatile flavors throughout the storage period The variety is abundant, and the presence of acetylurea, 2-heptanone and (E,E)-3,5-octadien-2-one keeps the samples in good sensory flavor during storage. Another setting is MFSM method, The other is that lactic acid bacteria ferment milk alone at 42 °C for 12 h, acidify for 24 h, put it in a 4 °C refrigerator, then mix with soy milk in a ratio of 1.5:1, and store the mixture at 4 °C, set to SMFSM method. The quality and flavor of soymilk and milk blend products were investigated on days 0, 15 and 30 of the storage period. Acidity, pH, number of viable bacteria, viscosity, water holding capacity, texture, rheological properties were determined , sensory quality and changes in volatile flavor. The results showed that compared with the MFSM-fermented soybean milk and milk mixture, the acidification of the SMFSM-fermented soybean milk and milk mixture samples was significantly slowed down during storage, so the sensory quality of the product was on the 30th day of storage. Almost unaffected by acidity; in addition, the number of viable bacteria was greater than 7 log cfu/mL. The water holding capacity did not change significantly until the 30th day. There was also no whey precipitation, indicating good stability. The samples in SMFSM mode showed good stability during storage. Aromatic content and bean content were higher than fermented soymilk and milk blends in MFSM mode. A wide range of volatile flavors were found throughout storage, acetytoin, 2-heptanone and (E,E)-3,5-octanedi The presence of en-2-one keeps the sample good organoleptic flavor during storage.

Q2: Grammar problem. Ln 14: "Maintaining viable counts greater than..." Ln 41 "Lactobacillus strains..." confusing. Ln 48 "Found a co-fermentation of soy milk and buttermilk blends..." confusing. Ln 231 "includes..." is confusing.

Answer 2: This part of the grammar has been revised in the original text. Thank you for your suggestion.

Line 14: As suggested by the reviewer, change the sentence "In addition, keep the number of viable cells greater than 7 log cfu/mL" to "The number of viable cells is greater than 7 log cfu/mL".

Line 41: According to the reviewer's suggestion, change "Yinzheng et al. screened Lactobacillus harbinger M1 strain with high yield of 2,3-butanedione and ethytoin and improved sensory quality of fermented soybean milk" to "Yinzheng" et al screened out a strain of Lactobacillus harbinis M1 [5], which improved the sensory quality of fermented soybean milk".

Line 48: As suggested by the reviewers, the sentence "Mitsuru Yoshida et al. [8] found that co-fermentation of soymilk and buttermilk mixtures with traditional lactic acid bacteria fermenters (Lactobacillus bulgaricus and Streptococcus thermophilus) and Bifidobacterium resulted in The samples fermented with soymilk and buttermilk mixture had better flavor than the samples fermented with soymilk alone, mainly due to hexanal and valeraldehyde, which caused unfavorable flavor" to "Yoshida Hikaru et al. [6] found that fermented with traditional lactic acid bacteria The samples fermented with soymilk and milk mixtures (Lactobacillus bulgaricus subsp. and Streptococcus thermophilus) and Bifidobacterium tasted better than the samples fermented with soymilk alone, mainly due to reduced hexanal and valeraldehyde production, resulting in an unpleasant flavor.

Line 231: According to the reviewer's suggestion, this sentence "includes 10 ketones, 9 acids, 8 aldehydes, 9 alcohols, 6 esters and 13 other compounds. The MFSM sample detected 23 volatile compounds on day 0, and on day 15. 26 kinds of volatile components were detected, and 16 kinds of volatile compounds were detected on the 30th. "Changed to" these compounds include 10 kinds of ketones, 9 kinds of acids, 8 kinds of aldehydes, 9 kinds of alcohols, 6 kinds of esters and 13 kinds of Other compounds. The MFSM samples contained 23 volatile compounds on day 0, 26 volatile compounds on day 15, and 16 volatile compounds on day 30.

Q3: Please check the writing of the entire manuscript and maintain the writing standard. Ln 98 and Ln 161, change "MSFM" to "MFSM"?

Response 3: Lines 98 and 161: "MSFM" has been changed to "MFSM" in the text.

Q4: The academic names of all bacteria should be in italics (Ln 41).

Response 4: Line 41: "Lactobacillus harbinger M1" has been changed to italics in the text. The names of other bacteria have been changed to italics.

Q5: Ln 70: "Based on previous research.." reference?

Response 5: Line 70: According to the previous study, it refers to the previous experimental part of this study. In the process optimization part, the optimal ratio of milk to soy milk was determined to be 1.5:1.

"According to a previous study, the ratio of milk to soy milk mixture was 1.5:1, and the product quality was the best. Therefore, soy milk and milk were mixed well at a ratio of 1.5:1" to "In this study, the ratio of milk and soy milk mixture was The ratio is 1.5: 1. One way is to mix milk and soy milk in a 1.5: 1 ratio.

Q6: Ln 92: What is the function of "recovered milk"? For both MFSM and SMFSM groups, do they both contain 8% sucrose? What is the main function of 8% sucrose?

Response 6: Line 92: This is an expression error, "reclaimed milk" has been changed to "milk". Both MFSM and SMFSM samples contained the same amount of sucrose. 8% refers to the proportion of sucrose in milk. It mainly provides sweetness for mixed fermented dairy products, so that the products have good sensory quality, and also provides carbon source for the growth of lactic acid bacteria.

Q7: Ln 119: Volatile Aromatics section, is this method based on some literature method?

Response 7: Line 119: Reference method for the determination of volatile compounds [15] Dan, T.; Ren Wen; Liu Yan; et al. Characteristics of volatile flavor compounds and fermentation characteristics of Lactobacillus bulgaricus-fermented milk. Front Microbiol 2019, 10 , 2183, doi:10.3389/fmicb.2019.02183. The text adds to this.

Q8: Ln 145: Frequency sweep part, what is the working volume? Details for this method are missing.

Response 8: Line 145: The working volume of the frequency sweep section is 2 mL of sample, which has been supplemented in the article.

Q9: Line 178: Are these pH values ​​significantly different? Did you do the stats here?

Response 9: Line 178: This section provides statistical analysis between and within groups. For the same sample, there were significant differences in pH between samples prepared at the same storage time (p < 0.5) and those prepared by different mixed fermentation methods (p < 0.5).

Q10: Ln 201, Ln 356, "in varying degrees and affecting different species", what do you mean?

Response 10: Line 201: Thanks for the suggestion. Differences between strains are not studied in this section, so this section has been deleted. "These results show that different mixed fermentation methods have different degrees of influence on the growth of lactic acid bacteria during storage, and also have different effects on different types of lactic acid bacteria" to "These results show that different mixed fermentation methods affect the lactic acid bacteria during storage. growth".

Line 356: Here is an expression error. “have different effects” was changed into “have great effects”

Q11: Ln 219: "aggregate at the same time..." Is it your hypothesis? Please explain more and provide more information?

Response 11：On the basis of the literature, I did make a hypothesis here. Both soy protein and casein contribute to the gel network formation, the literature describes that milk protein can aggregate before soy protein or both, and if casein and soy protein aggregate at the same time, they can be more uniformly dispersed throughout the gel network, of course, there are some other factors that affect the gel properties of proteins, such as: the presence of fat globules, which in turn are affected by homogenization pressure and number of homogenizations. Therefore, the formation of protein gel network is influenced by several factors, and in this study, the results showed that the viscosity and water holding capacity of MFSM samples were always higher than that of SMFSM samples, so I guess that the casein and soy protein aggregated simultaneously to make the samples form a stable gel structure. In the next study, I will explore the mechanism of casein and soy protein gel formation in more depth.

[30] Barakat, H.; Mohamed, A.; Gemiel, D.G.; et al. Microstructural, Volatile Compounds, Microbiological and Organoleptical Characteristics of Low-Fat Buffalo Milk Yogurt Enriched with Whey Protein Concentrate and Ca-Caseinate During Cold Storage. Fermentation 2021, 7, 250-271.

Q12: Ln 240:". And the flavor substance content..." Is confusing. Grammar problem?

Response 12：Line 240: This is a grammar problem and has been changed to "and the content of flavor substances". The language of the entire manuscript was polished.

Q13: Ln 250:" the 2-heptanone content of SMFSM samples was always higher..." Do you have some reasonable guessing? Please explain more with bacteria analysis.

Response 13：Line 250: Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus make a great contribution to the volatile flavor of fermented milk. Lactobacillus can produce pyruvic acid by metabolizing lactose, and pyruvic acid can produce ketones. SMFSM samples are prepared by lactic acid bacteria alone fermenting milk, acidified and then mixed with soy milk. Lactobacillus can metabolize more lactose when fermenting in a separate milk system, so more ketones may be produced. There are many ketones in the sample after being mixed with soy milk, but no literature shows which ketone is specific. Therefore, it is speculated that 2-heptanone may be one of them. Reference [30] Barakat, H.; Mohamed, A.; Gemiel, D.G.; et al. Microstructural, Volatile Compounds, Microbiological and Organoleptical Characteristics of Low-Fat Buffalo Milk Yogurt Enriched with Whey Protein Concentrate and Ca-Caseinate During Cold Storage. Fermentation 2021, 7, doi:10.3390/fermentation7040250.

Q14: Ln 329: what do you mean" the right amount..." ?

Response 14：Line 329:" the right amount..."was changed into " but the SMFSM samples showed better flavor".

Q15: Ln 373:" G'>G". Please explain more.

Response 15：Line 373" G'>G"，it has been explained in the text with reference to references. G ′ and G ″ respectively represent the degree of elasticity and viscosity of gel [34]. The samples of both mixed fermentation methods exhibited a significant increase in G′>G″, which is typical of weakly elastic gels and is consistent with the results of Haykuhi [35] et al. for both G′ and G″ with increasing frequency during storage.

[34] Mishra, S.; Mishra, H.N. Effect of Synbiotic Interaction of Fructooligosaccharide and Probiotics on the Acidification Profile, Textural and Rheological Characteristics of Fermented Soy Milk. Food and Bioprocess Technology 2012, 6, 3166-3176.

[35] Charchoghlyan, H.; Bae, J.E.; Kwon, H.; et al. Rheological Properties and Volatile Composition of Fermented Milk Prepared by Exopolysaccharide-Producing Lactobacillus Acidophilus N.V. Er2 317/402 Strain Narine. Biotechnology and Bioprocess Engineering 2017, 22, 327-338, doi:10.1007/s12257-017-0065-8.

Q16: The conclusion section was not sufficient to effectively summarize the content of the entire manuscript. This section needs to be improved.

Response 16：The conclusion has been modified in the original text.

“The mixing method of fermented milk and soymilk has a large influence on the quality and flavor of fermented soymilk. And it was found that SMFSM could significantly slow down the acidification of fermented soymilk during storage, so that the sensory of the product was almost not affected by acidity after 30 days of storage. And keep the number of live bacteria greater than 7 log cfu/mL, and have good adhesion, water holding capacity at the 30th day compared with 0th did not change significantly, no whey precipitation, with good stability. It had higher aromatic and soy flavors during storage than the fermented soymilk in MFSM mode. The rich variety of volatile flavors and the presence of Acetoin, 2-Heptanone, and (E, E)-3,5-Octadien-2-one throughout the storage period allowed the samples to maintain a good sensory flavor during storage.” was changed into “This study shows that the acidity of soymilk and milk mixtures prepared by the two mixed fermentation methods during storage increases, the pH decreases, and the number of viable bacteria can be maintained above 7 log cfu/mL, providing good probiotic proper-ties. The number of Lactobacillus bulgaricus subspecies in the MFSM samples on the 0th day was lower than that of the SMFSM samples; however, the number of viable bacteria in the MFSM samples was higher than that of the SMFSM samples, and the lactic acid bacteria were still in the logarithmic growth period and vigorous at 70 °T in the MFSM samples. The MFSM samples were prepared in such a way that the plant and animal proteins were more tightly bound and the system was more stable, resulting in products with a higher water holding capacity. However, the SMFSM method requires fermentation of milk to a high acidity and then mixing with soymilk. The SMFSM method could significantly slow down the acidification of fermented soymilk and milk mixtures during storage so that the product exhibited good adhesiveness, and the water holding capacity on the 30th day exhibited no significant change from that on the 0th day. The SMFSM samples had higher aromatic and soy flavors during storage than the fermented soymilk and milk mixtures in MFSM mode. Throughout the storage period, the content of 2-heptanone in the SMFSM samples was always higher than that of the MFSM samples, and on the 0th day, the con-tent of 2-heptanone in the SMFSM samples was 2.1 times higher than that of the MFSM samples. On the 15th day, it was 1.47 times higher, and on the 30th day, no 2-heptanone was detected in the MFSM samples. The higher content of 2-heptanone can bring good flavor to the fermented products, which is well liked by people.”

Thank you again for your scientific and rigorous advice, so that we can continue to grow on the road of scientific research. Thank you very much.

Kind regards,

Ms. Lina Zong

Author Response File: Author Response.docx

Reviewer 2 Report

Представленный материал представляет собой интересный и перспективный обзор брожения.

Однако автору необходимы существенные коррективы для указанной статьи.

Комментарии представлены ниже.

1. Неудачное название статьи. Среди объектов исследования нет соевого молока, а есть только смеси соевого и коровьего молока. Называть смесь 1,5:1 просто соевым молоком неправильно. Это необходимо учитывать в пунктах 3 и 4 статьи.

2. строки 23-28. Неудачное начало вступления с информацией именно о йогуртах. Данные по кисломолочным напиткам лучше представить в целом.

3. пункт 2.2.2. Чем обусловлен выбор соотношения коровьего и соевого молока для смеси. Это нужно объяснить.

4. пункт 2.2.2. Для чего смесь в методе МССМ заквашивали до 70°Т, а коровье молоко в методе СМЖМ сквашивали в течение суток без контроля кислотности. Это нужно объяснить.

5. Необходимо навести порядок в описании и их анализе. Вязкость в опасности 2.3.2 является характеристикой текстуры продукта. Его следует ожидать в версиях 2.2.7 и 2.2.8.

Author Response

Dear Professor:

First of all, thank you for carefully pointing out the inadequacies in our article so that we have the opportunity to improve.

Now I will reply to the comments one by one.

1.Неудачное название статьи.Среди объектов исследования нет соевого молока, а есть только смеси соевого и коровьего молока.Называть смесь 1,5:1 просто соевым молоком неправильно.Это необходимо учитывать в пунктах 3 и 4 статьи.

Response 1: The title of the article "Effects of different mixed fermentation methods on the quality and flavor of fermented soybean milk during storage" was changed to "Changes in quality and flavor of fermented products of different soybean milk and milk mixtures during storage"

2.строки 23-28.Неудачное начало вступления с информацией именно о йогуртах.Данные по кисломолочным напиткам лучше представить в целом.

Response 2: Thank you for your question. Taking into account the continuity of the article, the deletion of this paragraph does not affect the preamble of the article. Therefore, this paragraph has been deleted.

3. пункт 2.2.2.Чем обусловлен выбор соотношения коровьего и соевого молока для смеси.Это нужно объяснить.

Response 3: The mixing ratio of milk and soy milk is determined by the results of the optimization of the process conditions in the early stage of this experiment.

The certificate is in the attachment.

An article on process optimization has been submitted to the Journal of Food Industry Science and Technology.

The certificate is in the attachment.

4. пункт 2.2.2.Для чего смесь в методе МССМ заквашивали до 70°Т, а коровье молоко в методе СМЖМ сквашивали в течение суток без контроля кислотности.Это нужно объяснить.

Response 4: SMFSM samples were acidified for 24 h after fermented milk, and then mixed with soy milk to prepare. The reason for post-acidification milk is that it can be fermented to 100°T, and then post-acidified for 24 hours to reach 110°T, and then mixed with soy milk to achieve the desired 70°T acidity of the sample. Soymilk blending can improve product stability.

5. Необходимо навести порядок в описании и их анализе.Вязкость в опасности 2.3.2 является характеристикой текстуры продукта.Его следует ожидать в версиях 2.2.7 и 2.2.8.

Response 5:

2.2.3. "Determination of texture characteristics of fermented soybean milk by a texture analyzer (TMS Pro, TMS, USA)" has been deleted. This part is covered in Section 2.2.7.

2.2.7 and 2.2.8, make a brief description and analysis.

2.2.7 Put the glass bottle containing 100 mL of sample on the rack and measure its hardness, adhesion, cohesion, stickiness and chewiness.

2.2.8 Determine the energy storage modulus (G′) and loss modulus (G) of the sample during storage.

Thank you again for your scientifically rigorous advice, allowing us to continue to grow on the road of scientific research. thanks.

warm regards

Ms. Zong Lina

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

accept in present form