Exploration of Co-Inoculation of Lactiplantibacillus plantarum and Kazachstania bulderi for Potential Use in Mushroom Pleurotus eryngii Pickle Fermentation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

General

Overall, the paper is well written and experimental parts competently performed.

Some critical advices/suggestions:

Line 79-80: Are these results or are they previous results? If so, please clarify and cite (if applicable). 

Line 87: what was the liquid used? Water?please clarify

Fig 1A: what is Ck? Please add it to the figure caption

It will be interesting if you add in the introduction or discussion section a little bit more information on whether this fermented product is commonly consumed in China, or if it is similar to other fermented foods . Also, it will be interesting to know  depending on the volatile compounds, what aromas are expected or not in this kind of food, or if there are other studies regarding sensory quality of this product.

Author Response

Response to Reviewer 1 Comments

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted changes in the re-submitted files.

Comments 1: Line 79-80: Are these results or are they previous results? If so, please clarify and cite (if applicable).

Response 1: Yes, these are the results that we have not published. We isolated 46 bacteria strains and 6 yeast strains from homemade Sichuan Paocai. Most of bacteria belong to Lactiplantibacillus plantarum and Levilactobacillus brevis. The 6 yeast strains belong to Kazachstania bulderi and Rhodotorula mucilaginosa. We selected these two strains to performed the experiments that exhibited in the manuscript after tested the sensory characteristics of P. eryngii fermented by single lactic acid bacteria, single yeast, combination of lactic acid bacteria and yeast by using electronic nose. We already submitted the sensory data to a Chinese journal and still in the under review station, so we had not paste these data into our manuscript and cite any reference. We changed sentence “P. eryngii fermented by the above two strains had better sensory tasting,” to “Our preliminary experimental results indicated that P. eryngii fermented by the above two strains had better sensory tasting,” and highlighted that with red color (Page 4, 74-75).

Comments 2: Line 87: What was the liquid used? Water? Please clarify

Response 2: Thank you for pointing this out. “liquid” represents the mixture with a final concentration of 2% salt (w/w) and 1% sucrose. In order to express that clearly, we changed the sentence “Next, the mushrooms were cut into slices, and 150 g of the treated mushrooms were placed into screw-capped glass jars…” to “Next, the mushrooms were cut into slices, and 150 g of the treated mushrooms were placed into screw-capped glass jars, then added 150 mL water with a final concentration of 2% salt (w/v) and 1% sucrose (w/v).” and highlighted that with red color in revised manuscript (Page 4, Lines 78-80).

Comments 3: Fig 1A: What is CK? Please add it to the figure caption

Response 3: Thank you for pointing this out. CK in Fig 1A represents the mushrooms that treated as the other groups except without any inoculation. We agree with this comment. Therefore, we added it to the figure caption and highlighted that with red color.

Comments 4: It will be interesting if you add in the introduction or discussion section a little bit more information on whether this fermented product is commonly consumed in China, or if it is similar to other fermented foods. Also, it will be interesting to know depending on the volatile compounds, what aromas are expected or not in this kind of food, or if there are other studies regarding sensory quality of this product.

Response 4: Thank you for pointing this out. In fact, fermented mushrooms are mainly seen in Europe, and they are not common in the Asian region. In Europe, the way of fermenting mushrooms is mostly natural fermentation in family workshops, and more products are made through methods such as salting and pickling but not fermentation. However, considering that there are countless fermented food products produced worldwide through fermentation techniques, such as Sichuan pickled vegetables, Korean pickled vegetables, ham, sourdough, and vinegar, among others. Therefore, we hope to develop new and healthy mushroom products through fermentation. Although we did not introduce these fermented foods in detail in the manuscript, we provided a brief introduction through literature citations (references 7-28) when discussing fermentation technology in Introduction part. Moreover, due to the differences in taste preferences among people from different regions, it is difficult to judge the superiority or inferiority of certain flavor substances. Generally speaking, organic acids, alcohols, esters, certain aldehydes, and ketones are all important components of flavor substances in fermented foods. We also discussed these volatile compounds in manuscript (Page 13, Lines 270-278). Of course, we also believe that exploring the composition of flavor substances in different fermented products and identifying their key flavor compounds is a very meaningful endeavor in fermented foods. We will also conduct some exploratory experiments in this aspect based on your suggestions.

Additional clarifications

We changed the corresponding email address [email protected] to [email protected] in the manuscript in order to communicate easily, and highlighted that with red color in revised manuscript (Page 1).

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is interesting and deals with the current topic of producing affordable and valuable food products through fermentation technologies. The manuscript tackles the influence of the co-culture of Lactiplantibacillus plantarum and Kazachstania bulderi on some characteristics such as some relevant physicochemical properties and the volatile flavor profile of the fermented mushroom Pleurotus eryngii.

The subject of the study is of considerable technological and practical interest, with real potential to be upscaled, therefore it deserves a more in-depth dissertation.

However, some minor aspects are recommended to be analyzed and addressed by authors:

-the authors should explain / mark if PL and PY belong to the name of the microorganisms or these are used as codes.

-line 33 – it is about factory or industrial cultivation of mushrooms?

-line 38 – could authors be more specific / clear as regards the remark “to promote the healthy and sustainable growth of the industry”?

-line 90 – the meaning / wording of the sentence “All the treatments were inoculated into the P. eryngii and fermented at 25 °C for 7 days” is not fully clear. It is about the inoculation of a treatment ?

-lines 313-316 – it is recommended to clarify the sentence “showed the types of volatile flavor compounds increased, but the types of the unique substances decreased”, as well as the following one.

-lines 329-330 – there are some minor typos into the following text segments: 3-methyl-1-butanol.and hexanal had VIP scores > 2.0; According to GB 2760-96, the two compounds mainly used …

-line 330 – it is necessary to explain what GB 2760-96 represents and to verify if it is a reference. In this case, it should be cited.

 

Author Response

Response to Reviewer 2 Comments

Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted changes in the re-submitted files.

Comments 1: the authors should explain / mark if PL and PY belong to the name of the microorganisms or these are used as codes.

Response 1: PL and PY in manuscript are used as codes to represent Lactiplantibacillus plantarum PC-004 and Kazachstania bulderi PY-1, respectively. These two strains were isolated from homemade Sichuan Paocai and collected in the China Center for Type Culture Collection (CCTCC), with the accession numbers of CCTCC NO: 20231963 and CCTCC NO: 20231962, respectively. Thank you for pointing this out. We agree with this comment. In order to eliminate the ambiguity arising from this in the manuscript, we changed the expression in the revised manuscript and highlighted with red color. Page 1, Lines 9 – 10: “This study investigated the impact of Lactiplantibacillus plantarum PL and Kazachstania bulderi PY” was changed to “This study investigated the impact of Lactiplantibacillus plantarum PC-004 (PL) and Kazachstania bulderi PY-1 (PY)”. Page 4, Line 69: “L. plantarum PL and K. bulderi PY were used in this study” was changed to “L. plantarum PC-004 (PL) and K. bulderi PY-1 (PY) were used in this study”.

Comments 2: line 33 – it is about factory or industrial cultivation of mushrooms?

Response 2: It is not about factory or industrial cultivation of mushrooms. Here we mainly wanted to point out that the imbalance between the huge production of P. eryngii and the single form of consumption. Thank you for pointing this out. We agree with this comment. So we deleted the sentence “P. eryngii is cultivated on a large scale and has become the second-largest mushroom variety in factory cultivation. However,” and changed that to “In China, the production of P. eryngii is high, but the consumption forms of P. eryngii are primarily limited to fresh and dried products because of high perishability of fresh edible mushrooms, which have a high water content, a fast respiration rate, and a tendency to spoil quickly due to the presence of microflora”. We highlighted that with red color in revised manuscript (Page 2, Lines 31-33).

Comments 3: line 38 – could authors be more specific / clear as regards the remark “to promote the healthy and sustainable growth of the industry”?

Response 3: Well, we think that the imbalance between the high production volume and limited consumption forms of king oyster mushrooms is harmful for its healthy and sustainable growth. What we want to express is that that will be fixed by developing new processing technologies and products. Thank you for pointing this out. We agree with this comment. In order to express that clearly, we changed the sentence “Therefore, it is necessary to develop processing technologies and value-added products to promote the healthy and sustainable growth of the industry.” to “Therefore, it is necessary to increase the consumption patterns of king oyster mushrooms by developing new processing technologies and value-added products, so to address the imbalance between its high production volume and limited consumption forms.”. We also highlighted that with red color in revised manuscript (Page 2, Lines 34-37).

Comments 4: line 90 – the meaning / wording of the sentence “All the treatments were inoculated into the P. eryngii and fermented at 25 °C for 7 days” is not fully clear. It is about the inoculation of a treatment?

Response 4: Here we wanted to express that all fermentation including the PY/PL/PYL fermented P. eryngii and the control group were treated at 25 °C for 7 days. Thank you for pointing this out. We agree with this comment. In order to express that clearly, we changed the sentence “After cooling to room temperature, the starter cultures were added with on initial viable cell counts of lactic acid bacteria (7.00 log CFU/mL) and yeast (6 log CFU/mL). All the treatments were inoculated into the P. eryngii and fermented at 25 °C for 7 days.” to “After cooling to room temperature, the starter cultures, including single LAB strain (PL), single yeast strain (PY) and the mixed starter culture (PYL) that be composed of LAB and yeast, were added with on initial viable cell counts of LAB (7.00 log CFU/mL) and yeast (6 log CFU/mL). Without any inoculation as control. All treatments fermented at 25 °C for 7 days.” and highlighted that with red color in revised manuscript (Page 4, Lines 80-84).

Comments 5: lines 313-316 – it is recommended to clarify the sentence “showed the types of volatile flavor compounds increased, but the types of the unique substances decreased”, as well as the following one.

Response 5: Here, we wanted to emphasize that the co-fermented oyster mushroom had more flavor compounds. However, the specific flavor compounds that are only associated with co-fermentation actually decrease. In other words, the flavor substances in co-fermented P. eryngii might predominantly derived from the superposition of flavor substances fermented by the lactic acid bacteria and yeast, rather than new formations by these two strains during fermentation. This also implied that co-fermentation can enhance the complexity of flavor compounds in the fermented products. Thank you for pointing this out. We agree with this comment. In order to express that clearly and simply, we changed the sentence “Co-fermentation, compared with the mono-fermentation, showed the types of volatile flavor compounds increased, but the types of the unique substances decreased. Moreover, there were much more common compounds between the co-fermentation and the two mono-fermentations, but not between the latter two.” to “Compared to the mono-fermentations, co-fermentation exhibited more types of flavor compounds. The common substances between co-fermentation and mono-fermentations more than that between the two mono-fermentation groups implied that the flavor substances in co-fermented P. eryngii might predominantly derived from the superposition of flavor substances fermented by the lactic acid bacteria and yeast, rather than new formations by these two strains during fermentation. This also showed that co-fermentation can enhance the complexity of flavor compounds in the fermented products.” (Page 13, Lines 277-284).

Comments 6: lines 329-330 – there are some minor typos into the following text segments: 3-methyl-1-butanol.and hexanal had VIP scores > 2.0; According to GB 2760-96, the two compounds mainly used …

Response 6: Thank you for pointing this out. We agree with this comment. We already corrected the errors. Specifically, we deleted the period after butanol, and highlighted the sentence with red color (Page 14, Lines 289-290).

Comments 7: line 330 – it is necessary to explain what GB 2760-96 represents and to verify if it is a reference. In this case, it should be cited.

Response 7: A Thank you for pointing this out. We agree with this comment. By reviewing the literature, we found that these two ingredients (3-methyl-1-butanol and hexanal) are mainly used for the preparation of apple, banana and tomato flavors. However, due to our negligence, there were issues with the unclear expression and inaccurate citation of the standard. We have corrected this error and highlighted that with red color in revised manuscript (Page 14, Lines 290-291; Pages 21-22, Lines 460-461). Specifically, sentence “According to GB 2760-96, the two compounds mainly used for the preparation of apple, banana and tomato flavors.” was changed to “According to "National food safety standard - Standard for uses of food additives" (GB 2760-2014) [48], the two compounds mainly used for the preparation of apple, banana and tomato flavors.”. We also cited the standard at the end of the revised manuscript.

Additional clarifications

We changed the corresponding email address [email protected] to [email protected] in the manuscript in order to communicate easily, and highlighted that with red color in revised manuscript (Page 1).