Kinetics of Formation of Butyric and Pyroglutamic Acid during the Shelf Life of Probiotic, Prebiotic and Synbiotic Yoghurt

Round 1

Reviewer 1 Report (Previous Reviewer 2)

Despite the authors having carried out a revision of the text in accordance with the issues raised during the review of the first version of the article, the relevance and utility of the study are questionable. The limitations of the practical utility of the findings, given the minimal increase in C4 and pGlu production in yogurt, are virtually ignored in the text and clearly overshadowed by claims of their relevance to human health.

Author Response

This work can offer opportunities for process optimization towards nutritional quality. The aim of the work was to study whether the content of pyroglutamic and butyric acid varied in yoghurt with different microbial formulations and during storage time. These two molecules have numerous beneficial properties, but bioaccessibility and bioavailability studies are needed to understand whether the quantities of C4 and pGlu present in yoghurt and taken daily may be able to produce beneficial effects on human health. On the other hand notable increases in C4 and pGlu could negatively affect the consumer acceptance of the yoghurt with rancid and bitter taste.
We have reiterated this point in the conclusion section.

Reviewer 2 Report (Previous Reviewer 1)

This study investigated the influence of probiotics (Lactobacillus acidophilus and Bifidobacteria) and/or prebiotics (1 and 3% inulin and fructo-oligosaccharides) on the content of butyric acid (C4) and pyroglutamic acid (pGlu) in yoghurt during the shelf-life period of 30 days of storage at 4 °C, which is creative and meaningful. While, the manuscript needed minor revision before publication.

1.      The generation of butyric acid (C4) and pyroglutamic acid (pGlu) may play important role on the taste and smell. Could you give an introduction or discussion of butyric acid and pyroglutamic acid on the taste and smell, especially how much amount of single or combined give negative effect on the yoghurt, which is need’s to be cared when producing in factory.

2.      Line 17：double “contained”

3.      Line 30 and 64: Please check whether the citation format of references are used correctly.

4.      Please explain the difference among intestinal microflora (Line 47), intestinal flora (Line 49) and gastrointestinal microbiota (Line 67). If the meanings expressed are same, it is better to use the same word for expression.

5.      As you mentioned on Line 54, the environmental tolerance of the strain is strain-specific, so it is best to polish your expression on Line 47 to 53. Not all the L. acidophilus and B. bifidum can colonize in the intestinal tract, and not all the L. bulgaricus and S. thermophilus cannot resist the gastrointestinal environment. Meanwhile, please include necessary references.

6.      Line 65-66: “Lactic acid bacteria, such as Lactobacillus plantarum, produce,instead, C4 in milk due to the activity of strain-specific lipase”, the sentence grammar should be modified.

7.      Line 70, 242, 269: The use of punctuation should be precise.

8.      Line 86: Add a comma between “Therefore” and “this”.

9.      Line 99: “1.0×106CFU/mL” should be modified.

10.  Line 155: “from 160 to 210℃” should be “from 160℃ to 210℃”.

11.  Line 156: “temperatures were 250 and 230 ° C” should be “temperatures were 250 ℃ and 230 ℃”.

12.  Line 120, 121 and126: “mL” instead of “ml”.

13.  Please read the author's guide and modify the consistent use of units in the article. Like “8,000 × g for 10 min” on Line 122 and “250 ° C for 10 minutes” on Line 151.

14.  Line 218, 237, 282, 286, 305, 315, etc.: Genus and species should be expressed in italics.

15.  Line 229, “tables 2” should be “Table 2”. And the expression of “tables” on Line 234 and Line 264 should be polished.

16.  Line 237-238: The use of italics should be precise.

English expression is ok, but some format problems, such as units, punctuation and other problems are need improved.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

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.

Round 1

Reviewer 1 Report

1. The data presented in this manuscript is not enough

2. What is the butyric and Pyroglutamic acid yield per strain of the starter?

3. Different probiotics and prebiotics can affect the content of butyric and Pyroglutamic acid, so whether it will affect the quality of yogurt.

4. Does the butyric and pyroglutamic acid tested in this paper have any influence on the quality and flavor of yogurt?

5. What are the butyric and pyroglutamic acid recommendations in yogurt and other fermented foods in different countries and regions?

 

Reviewer 2 Report

The manuscript "Kinetics of Formation of Butyric and Pyroglutamic Acid (pGlu) During the Shelf Life of Probiotic, Prebiotic, and Synbiotic Yoghurt" aims to demonstrate that the addition of some prebiotic/probiotic to regular yoghurts might influence the free butyric and pGlu content in the final product. The work is based on the assumption that butyric acid and pGlu have health benefits. However, the introduction is deficient regarding this topic, and additional significant references should be added. Although written in a straightforward and simple manner, a general revision of the text should be performed, particularly for the introduction (lines 28-38), which contains several short sentences that seem pasted in the text without considering the fluidity of the reading. Although some therapeutic potential is attributed to some types of yoghurts, the sentence in line 38 is too strong and generalist.

Regarding specific parts of the text:

·       Microorganism names are not italicized throughout the results and discussion.

·       Line 150 - The sentence seems incomplete.

·       Line 226 - "...a-aminoacyl..." - α-aminoacyl?

·       There are many butyrogenic bacterial species usually present in intestines, but this is omitted in the paper. This fact is relevant as it is not easy to understand the aim and relevance of the work performed. Is it important to produce yoghurts containing 15% more butyric acid when this represents only a few milligrams?

·       Are there any studies regarding the butyric acid dose that might have a biological effect in humans? In rats, at least, most of the studies use 200 mg/kg - 1g/kg (e.g., Zhang, L., Deng, M., Lu, A., Chen, Y., Chen, Y., Wu, C., et al. (2019). J. Cel Mol Med 23 (12), 8139–8150. doi:10.1111/jcmm.14684) to produce observable effects. This is roughly equivalent to the amount of butyric acid present in 20 kg of ProY yoghurt (needed for a rat of about 0.5 kg).

·       Considering the extremely low amount of butyric acid produced even in ProY yoghurts and that high hepatic clearance has been described for butyrate, do the authors still consider it relevant to produce yoghurts with the aim of supplementing the daily intake of butyric acid? Why?

·       Despite the authors referring to a statistically significant difference (p<0.05) between the amount of butyric acid present in ProY and SY in relation to TY yoghurts, the differences are very small (2.03-2.76 mg/100 g vs. 2.13-2.37 mg/100 g). The authors should indicate the p values.

Reviewer 3 Report

In the current version, the description of how to obtain the yoghurt contains serious shortcomings and therefore the article cannot be considered. Firstly, as a control yoghurt because the title refers to yoghurt, there should be a variant of UHT milk with the appropriate yoghurt culture. Secondly, it is not described whether these strains of L. acidophilus and Bifidobacterium were added as additional to the yogurt base or not? Because if there was only milk inoculated with L. acidophilus or Bifidobacterium, it cannot be called yogurt and fermented milk. Thirdly, the same applies to the addition of inulin. A serious methodological error because UHT milk cannot be considered as a control in this type of experiment. In its current form, the article cannot be considered for publication, only after filling the gaps in the methodology of the planned experiment can it be considered.

Comments for author File: Comments.pdf