Influence of Kluyveromyces lactis and Enterococcus faecalis on Obtaining Lactic Acid by Cheese Whey Fermentation

Response to Reviewer 1 Comments

Comments 1: [Small letter]

Response 1: Thank you for pointing this out.  We agree with this comment. Therefore, we have written as follow: Lactobacillus delbrueckii subsp. bulgaricus (page number: 2, paragraph: 3, and line: 65)

Comments 2: [This part is for the conclusion. At the end of the introduction, the aim of the study as well as the hypothesis must be pointed out.]

Response 2: We are not agreeing because in the template of this journal it is indicated that at the end of the introduction, “briefly mention the main aim of the work and highlight the principal conclusions”, so that is the reason why we put the main results.

Comments 3: [This part of the manuscript is written in a very complex style , making it difficult to follow. Therefore, I suggest to the authors to somehow simplify this section.

Response 3:  It was changed:

2.1. Isolation, biochemical and molecular identification

“The taxonomy marker genes were amplified using PCR technology (RNAr16S for bacteria and the ITS gene for yeast), and the amplicons were sequenced by the Sanger method. These sequences were compared with the NCBI database using the “ BLAST ®” blastn suite software.” (page number: 4, paragraph: 1, and line: 155)

2.3.1. Batch fermentation with strains in suspension using Enterococcus faecalis and Kluyveromyces lactis.

“In order to deproteinize the cheese whey (to eliminate not only the proteins but also the fats), it was put in an autoclave at 121 °C and 1.03 bar pressure during 15 minutes. After it was taken to a 60 sieve (0.050 nm) to eliminate the greatest amount of proteins, then it was centrifuged at 4500 RPM for 10 minutes to lastly go through vacuum filtration.” (page number: 4, paragraph: 4, and line: 169)

2.3.2. Batch fermentation with strains immobilized using Enterococcus faecalis and Kluyveromyces lactis.

Batch fermentation using strains immobilized in alginate beads was done in deproteinized and proteinized cheese whey. The culture medium was composed only by the deproteinized and proteinized cheese whey without adding other additives. This due that in preliminary tests it was observed that the alginate beads lost their stability, so it was decided to use the cheese whey without any additives. (page number: 4, paragraph: 7, and line: 186)

After the biomass solution was mixed in 2.5% sodium alginate solution (1 ml of biomass in 25 ml of sodium alginate) and then drip added to a 4% CaCl₂ solution during 1 h for its stabilization, these conditions for the Kluyveromyces lactis. At 5% CaCl₂ and 3% sodium alginate solution for 1 h of stabilization for the Enterococcus faecalis. (page number: 4, paragraph: 8, and line: 196)

Comments 4: [This sentence must be rephrased].

Response 4: It was changed:

“2.2. Determination of optimal microorganism growth conditions

It is important to know the optimal growth conditions of both strains so the growth temperature were tested at 4, 8, 20, 25, 29, 32, 34, 35, 36, 37 and 45 ºC, the pH at 4, 7 and 9 and growth inhibition by increasing the saline concentration (NaCl) at 1, 2, 4, 6, 8, 10, 12 and 13%.” (page number: 4, paragraph: 2, and line: 158-161)

Comments 5: [Use a unit bar].

Response 5: It was changed:

“it was put in an autoclave at 121 °C and 1.03 bar pressure during 15 minutes.” (page number: 4, paragraph: 4, and line: 169)

Comments 6: [Too long sentence].

Response 6: It was changed:

“2.3.2. Batch fermentation with strains immobilized using Enterococcus faecalis and Kluyveromyces lactis.

The culture medium was composed only by the deproteinized and proteinized cheese whey without adding other additives. This due that in preliminary tests it was observed that the alginate beads lost their stability, so it was decided to use the cheese whey without any additives.” (page number: 4, paragraph: 7, and line: 185-188)

Response to Reviewer 2 Comments

Comments 1: [The are a lot of grammar and writing issues that must be fixed. Sentence structure is a big issue. Most sentences were very long (in many cases 4-line sentences) which sometimes makes it hard to understand. So, the manuscript should be Carefully edited and proofread]

2.1. Isolation, biochemical and molecular identification

“The taxonomy marker genes were amplified using PCR technology (RNAr16S for bacteria and the ITS gene for yeast), and the amplicons were sequenced by the Sanger method. These sequences were compared with the NCBI database using the “ BLAST ®” blastn suite software.” (page number: 4, paragraph: 1, and line: 155)

2.3.1. Batch fermentation with strains in suspension using Enterococcus faecalis and Kluyveromyces lactis.

“In order to deproteinize the cheese whey (to eliminate not only the proteins but also the fats), it was put in an autoclave at 121 °C and 1.03 bar pressure during 15 minutes. After it was taken to a 60 sieve (0.050 nm) to eliminate the greatest amount of proteins, then it was centrifuged at 4500 RPM for 10 minutes to lastly go through vacuum filtration.” (page number: 4, paragraph: 4, and line: 169)

2.3.2. Batch fermentation with strains immobilized using Enterococcus faecalis and Kluyveromyces lactis.

Batch fermentation using strains immobilized in alginate beads was done in deproteinized and proteinized cheese whey. The culture medium was composed only by the deproteinized and proteinized cheese whey without adding other additives. This due that in preliminary tests it was observed that the alginate beads lost their stability, so it was decided to use the cheese whey without any additives. (page number: 4, paragraph: 7, and line: 186)

After the biomass solution was mixed in 2.5% sodium alginate solution (1 ml of biomass in 25 ml of sodium alginate) and then drip added to a 4% CaCl₂ solution during 1 h for its stabilization, these conditions for the Kluyveromyces lactis. At 5% CaCl₂ and 3% sodium alginate solution for 1 h of stabilization for the Enterococcus faecalis. (page number: 4, paragraph: 8, and line: 196)

Comments 2: [It has been mentioned that “cheese whey is a waste”, however, there is a huge industry that uses cheese whey to make whey protein concentrate, lactose, etc.  So, it is better to change it to a byproduct instead of waste.]

Response 2: Agree. We have changed the word “waste”.

“[updated text in the manuscript if necessary]”

Featured Application: In this work, the cheese whey, which is a byproduct from the cheese industry, was used to obtain lactic acid by biotechnological processes (using strains isolated from the same cheese whey). The lactic acid has several uses in the cosmetic, food, medical, textile and manufacture industries. Therefore, the lactic acid could have multiple uses but we want to use as raw material for biopolymers for the manufacture of bioplastics in a future research. (page number: 1, paragraph: 1, and line: 14).

Abstract: Cheese whey is a byproduct from cheese industry that causes high pollution in the environment but its high lactose content can be used as a source to obtain lactic acid. (page number:1, paragraph: 1, and line:19).

Comments 3: [Lines 38-40, Cheese whey is usually used as food for animals or discarded without any treatment, and due to its composition, it generates huge contamination in the environment such as water eutrophication, and soil contamination, among others. Furthermore, considering that 90% of the milk used in cheese production is discarded as cheese whey. There are wrong statements. You must search and find out how valuable cheese whey is and how much profit is obtained from the cheese whey industry.]

Response 3: Agree we have changes that data but it is important to mention that there is another situation here in Latinamerican, we have this issue due the cheese whey discarded. So, that is the main reason why this research was made.

“1. Introduction

In the production of cheese, a by-product is generated, which is a yellowish liquid called cheese whey that is composed of proteins, lactose, lipids and mineral salts [1]. In Larinamerican the cheese whey is usually used as food for animals or discarded without any treatment and due to its composition it generates a huge contamination in the environment such as water eutrophication, soil contamination, among others [2]. Furthermore, considering that 9 to 10 L of cheese whey is generated from 1 kg of cheese production that if is discarded without any treatment [1], so it is important to see what other uses it can be given.” (page number:1, paragraph: 4, and line:36-43).

Comments 4: [All the first paragraphs must be changed (not accurate information)].

Response 4: Agree. We have changed and use another reference.

“In the production of cheese, a by-product is generated, which is a yellowish liquid called cheese whey that is composed of proteins, lactose, lipids and mineral salts [1]. In Larinamerican the cheese whey is usually used as food for animals or discarded without any treatment and due to its composition it generates a huge contamination in the environment such as water eutrophication, soil contamination, among others [2]. Furthermore, considering that 9 to 10 L of cheese whey is generated from 1 kg of cheese production that if is discarded without any treatment [1], so it is important to see what other uses it can be given.” (page number:1, paragraph: 4, and line:36-43).

Comments 5: [Lines 49-51, “as well as, currently there is a high demand to produce polylactic acid (PLA) that is a biopolymer that is classified as environmentally friendly. So, lactic acid has increased its demand as a raw material to produce polylactic acid (PLA)” Why?].

Response 5: We have explained that lactic acid has increased its demand because it is used to produce polylactic acid (PLA) that is the raw material to manufacture biopolymers, as you can see in the following paragraph:

“Lactic acid is a product of high importance due to its wide applications, mainly in the pharmaceutical, cosmetic, chemical and food industry [3], as well as, currently there is a high demand to produce polylactic acid (PLA) that is a biopolymer that is classified as environmentally friendly. So, lactic acid has increased its demand as a raw material to produce polylactic acid (PLA) [4].”

Comments 6: [Line 166,  “untreated (proteinized) and treated (deproteinized) cheese” how the cheese was made without protein “(deproteinized) cheese”?].

Response 6: It is explained the procedure to deproteinized the cheese whey:

“2.3.1. Batch fermentation with strains in suspension using Enterococcus faecalis and Kluyveromyces lactis.

In order to deproteinize the cheese whey (to eliminate not only the proteins but also the fats), it was put in an autoclave at 121 °C and 1.03 bar pressure during 15 minutes. After it was taken to a 60 sieve (0.050 nm) to eliminate the greatest amount of proteins, then it was centrifuged at 4500 RPM for 10 minutes to lastly go through vacuum filtration.” (page number:4, paragraph: 3, and line:165-171).

Comments 7: [Line 189, what is “ina pellet”?].

Response 7: We have modified “ina pellet”, it was a miswriting, the corrected words are: “in a pellet”(page number: 4, paragraph: 7, and line: 190.]. The pellet is the sediment obtained after the centrifugation process.

Comments 8: [What does “an acidity of 13.09 ± 1.54 g/L of lactic acid”  in line 298 mean?].

Response 8: The acidity is a form to quantify the lactic acid through the title of acidity. So to clarify this procedure we have modified in methods:

“2.4.1.  Acidity analysis

A known volume from the fermentation broth was titrated with 0.1 N NaOH solution and 2 drops of phenolphthalein (indicator solution). This is a way to determine the lactic acid production.“ (page number: 5, paragraph:3, and line:212-214.].

Comments 9: [Line 302, “In addition, it contains other components that may affect the assimilation of lactose.” What are these other components?].

Response 9: The other components that are discarded in a deproteinization process are the proteins and fats. So it was modifies that text:

“In addition, it contains other components that may affect the assimilation of lactose as the proteins and fats.” (page number:8, paragraph: 2, and line: 304-305.].

Comments 10: [The format of the charts should be the same].

Response 10: Agree. We have modified the size of the titles (Palatino Linotype, 11).

Response to Reviewer 3 Comments

Comments 1: [Line 37 - The authors should cite more relevant reference for this statement, such as peer reviewed paper published in the scientific journal]

Response 1: Thank you for pointing this out.  We agree with this comment so we have changed the reference for another one that belongs to an article.

Comments 2: [Line 52 - The authors should cite more relevant reference for this statement, such as peer reviewed paper published in the scientific journal.]

Response 2: We have changed that reference.

Comments 3: [Line 152 - The authors should provide primer sequences used for amplification of genomic DNA.]

Response 3:   We added as an extra material the sequences obtained using the BLAST ® » blastn suite.

Comments 4: [The sample labeling is a bit confusing and hard to follow. The authors should provide explanations (in the figure caption) for the labels of the samples such as ES1_LA, ES2_LA, ES1_pH, ES2_pH in the Figure 1. Same to do with the other figures in the manuscript].

Response 4: We have put their menanings in Table 1 and 2. Also ES comes from E.faecalis Suspended, EI from E.faecalis Immobilized and KS from K. lactis suspended and KI from K. lactis Immobilized.

Comments 5: [Line 448 and 516 – The reference authors provided is not accessible by the provided link. Anyway, the reference is a thesis and the authors should cite more relevant reference for this statement, such as peer reviewed paper published in the scientific journal].

Response 5: The reference that belongs to a thesis has been changed.

Comments 6: [After the first appearance, the authors should use short names of investigated strains (E. faecalis and K. lactis)]

Response 6: We have used those terms but at the beginning we wrote the full name.