Cheese and Yogurt By-Products as Valuable Ingredients for the Production of Prebiotic Oligosaccharides

Round 1

Reviewer 1 Report

The manuscript is clearly presented and very well written. I have a few points for improvement:

L. 92-95: Add related references.

L. 143-145: How were the optimal conditions determined? Is it information from the manufacturer?

Table 1: Data retrieved from previous study? The methodology implies that it was carried out in this study. This point needs to be clarified.

L. 210-211: I don't see this behavior at all the concentrations tested in Figure 1... I suggest describing the results and discussing them individually for each concentration.

Table 2: I don't think that data from previous studies should be included in the table. I suggest removing it and just discussing it in the text.

Conclusion: The conclusion needs to be rewritten. Remove objectives and numerical repetition of results. There should only be a very general summary of the results obtained (preferably without numerical description) and the answer found to the proposed objective.

Author Response

Reviewer #1

The manuscript is clearly presented and very well written. I have a few points for improvement:

Reply: We thank Reviewer #1 for their comments on our manuscript. Please see below are point to point response.

1. 92-95: Add related references.

Reply: Related referenced have been added in Lines 101-102.

1. 143-145: How were the optimal conditions determined? Is it information from the manufacturer?

Reply: The optimal reaction conditions for the two lactases employed in this study have been determined in our previous study by Limnaios et al. (2023). This has been clarified in Line 158.

Table 1: Data retrieved from previous study? The methodology implies that it was carried out in this study. This point needs to be clarified.

Reply: The results for acid whey presented in Table 1 were indeed retrieved from our previous study by Limnaios et al. (2023) for the purpose of a comprehensive comparative study. Appropriate modifications have been made in the methodology as for this to be clarified (Lines 127-149).

1. 210-211: I don’t see this behavior at all the concentrations tested in Figure 1... I suggest describing the results and discussing them individually for each concentration.

Reply: In most cases, especially when high enzyme loads were applied in whey with low initial lactose content, the y_GOS exhibited an initial increase in the onset of the transgalactosylation, followed by reaching a peak, and subsequently, a decline. This pattern indicates the disintegration of the generated GOS, which can be elucidated by the catalytic mechanism of the enzymatic reaction. When low enzyme loads were applied in highly concentrated whey, for example when 0.13 U/mL of the K. lactis lactase were applied in concentrated SW with c_lac(0) equal to 10 or 15 % w/v (Figure B₁ and C₁), the aforementioned peak was not observed during the eight-hour enzymatic reaction, owing to the low reaction rate that leads to maximization of the y_GOS at longer reaction times. This has now been clarified in Lines 226-227 and 238-242.

Table 2: I don't think that data from previous studies should be included in the table. I suggest removing it and just discussing it in the text.

Reply: The results from Limnaios et al. (2023) were not just presented in Table 2 as they were included in an overall statistical analysis with the current data in order to discuss any significant differences between the two lactases applied in both acid and sweet whey. The justification of the inclusion of the respective data for AW was also included in the text, in Lines 258-261. If these data are not reproduced, this analysis cannot be clearly demonstrated. Although we understand the reviewer’s position on repeating clearly marked previous data, we consider this to be of added value for the reader.

Conclusion: The conclusion needs to be rewritten. Remove objectives and numerical repetition of results. There should only be a very general summary of the results obtained (preferably without numerical description) and the answer found to the proposed objective.

Reply: The conclusions have been extensively changed according to the reviewer’s comments. The aim of the study and most numerical descriptions have been removed and only the necessary information from the results has been retained to support the answer to the proposed objective, i.e. the efficient utilization of the two dairy by-products for the production of high added and nutritional value prebiotics, offering a practical and environmentally friendly solution for addressing the issue of sweet and acid whey management.

Author Response File: Author Response.pdf

Reviewer 2 Report

This study investigated the production of prebiotic galactooligosaccharide from

 sweet and acid whey by using two commercially accessible biocatalysts from

 Kluyveromyces lactis and Aspergillus oryzae.

·         It is meaningful work, but there are some questions and/or suggestions the authors may consider.

·         There are huge industrial applications for getting whey protein concentrate, whey protein isolate, and lactose from Cheese and yogurt by-products (whey), however, these big and important applications were not mentioned in the introduction, why?

·         The statistical analysis was not used to support the discussion of the results in most of the discussion.

·         In table one no statistical analysis is shown, it should be included in the table.

·         Were the quality characteristics of the final product obtained by these processing methods? Which one has better physicochemical properties?

·         Figure 3 shows two characteristic chromatograms, what is the logic of including these chromatograms in the manuscript?

Author Response

Reviewer #2:

This study investigated the production of prebiotic galactooligosaccharide from sweet and acid whey by using two commercially accessible biocatalysts from Kluyveromyces lactis and Aspergillus oryzae. It is meaningful work, but there are some questions and/or suggestions the authors may consider.

Reply: We thank Reviewer #2 for their comments on our manuscript. Please see below are point to point response.

There are huge industrial applications for getting whey protein concentrate, whey protein isolate, and lactose from Cheese and yogurt by-products (whey), however, these big and important applications were not mentioned in the introduction, why?

Reply: This was an omission on our behalf. Now the important applications regarding the acquisition of protein concentrate, whey protein isolate, and lactose from dairy industry by-products are now mentioned in Lines 67-73, according to the reviewer’s suggestions.

The statistical analysis was not used to support the discussion of the results in most of the discussion.

Reply: Several additions have been made in the discussion for the results of the statistical analysis to be exploited better in the text. The statistical analysis of the results regarding the maximum GOS yields achieved in each reaction has been used to discuss the statistically similar values of the maximum GOS yields achieved with different enzyme loads for the same enzyme and whey type, as well as for the statistically different maximum GOS yields achieved with the same enzyme in different whey type (Lines 243-255 and 314-339).

In table one no statistical analysis is shown, it should be included in the table.

Reply: One way Analysis of Variation (ANOVA) has been performed for the data of Table 1 and statistical analysis is now shown, according to the reviewer’s recommendations. The composition of SW and AW was significantly different (p < 0.05) for all measured compounds besides Na⁺, which is now mentioned in Lines 198-200.

Were the quality characteristics of the final product obtained by these processing methods? Which one has better physicochemical properties?

Reply: The quality characteristics of the final GOS solution obtained by the processing methods described in the manuscript were not evaluated, since this was not within the scope of the present study. The physicochemical properties of the obtained GOS products as well as the incorporation of the produced GOS from sweet and acid whey into food products and the analysis of their quality and shelf life could be an interesting objective of a future study.

Figure 3 shows two characteristic chromatograms, what is the logic of including these chromatograms in the manuscript?

Reply: The two characteristic chromatograms of Figure 3 were included for the visualization of the different GOS profile achieved by applying a different enzyme in a different substrate and to support the discussion of section 3.3, as now mentioned in Lines 367-368.

Author Response File: Author Response.pdf

Reviewer 3 Report

Cheese and yoghurt by-products as valuable ingredients for the production of prebiotic oligosaccharides

Abstract:

“The growing global market of dairy products has led to the need for alternative approaches regarding sweet and acid whey valorization, the primary by-product of cheese and strained yogurt production. In this context, prebiotic galactooligosaccharides can be produced enzymatically from whey, using commercially available β-galactosidases. A comparative study was conducted to assess the production of galactooligosaccharides from sweet and acid whey, employing two commercial β-galactosidases from Aspergillus oryzae and Kluyveromyces lactis. The study considered the initial lactose content and enzyme load as variables. Maximum yields of galactooligosaccharides in concentrated sweet whey (15 % w/v initial lactose) and non-concentrated acid whey (3.1 % w/v initial lactose) reached 34.4 and 14.7 % with β-galactosidase from Kluyveromyces lactis (0.13 U/mL), respectively. The corresponding galactooligosaccharide yields for β-galactosidase from Aspergillus oryzae were equal to and 27.4 and 24.8 % in concentrated sweet and acid whey (15 % w/v initial lactose) with enzyme loads of 2 and 1 U/mL, respectively. Concerning the profile of the produced galactooligosaccharides, the Kluyveromyces lactis lactase hydrolyses lactose more rapidly, resulting in higher levels of allolactose and lower levels of 6-galactosyl-lactose, compared to the lactase from Aspergillus oryzae.”

The abstract is generally well-written, but there are a few areas where clarity and conciseness could be improved:

The phrase "concentrated sweet whey (15 % w/v initial lactose)" is repeated in the description of the yields for both β-galactosidases. Consider condensing this information to avoid redundancy.

The sentences in the last paragraph are lengthy and could be broken down for better readability. Consider separating information about maximum yields, the β-galactosidase from Kluyveromyces lactis, and the corresponding yields for the β-galactosidase from Aspergillus oryzae into distinct sentences.

The sentence "The corresponding galactooligosaccharide yields for β-galactosidase from Aspergillus oryzae were equal to and 27.4 and 24.8 %..." is a bit unclear. It could be rewritten for better clarity, possibly by specifying which conditions correspond to the mentioned yields.

In the last sentence, "hydrolyses" should be "hydrolyzes."

Introduction:

Will need some more info regarding the high protein fermented products

Results and discussion:

It requires more literature to discuss more options on the high protein fermented products

Conclusion:

The conclusion appears to be generally well-written, but there are a few areas where clarity and precision can be improved.

Minor revision!

Author Response

Reviewer #3:

Cheese and yoghurt by-products as valuable ingredients for the production of prebiotic oligosaccharides

Abstract:

“The growing global market of dairy products has led to the need for alternative approaches regarding sweet and acid whey valorization, the primary by-product of cheese and strained yogurt production. In this context, prebiotic galactooligosaccharides can be produced enzymatically from whey, using commercially available β-galactosidases. A comparative study was conducted to assess the production of galactooligosaccharides from sweet and acid whey, employing two commercial β-galactosidases from Aspergillus oryzae and Kluyveromyces lactis. The study considered the initial lactose content and enzyme load as variables. Maximum yields of galactooligosaccharides in concentrated sweet whey (15 % w/v initial lactose) and non-concentrated acid whey (3.1 % w/v initial lactose) reached 34.4 and 14.7 % with β-galactosidase from Kluyveromyces lactis (0.13 U/mL), respectively. The corresponding galactooligosaccharide yields for β-galactosidase from Aspergillus oryzae were equal to and 27.4 and 24.8 % in concentrated sweet and acid whey (15 % w/v initial lactose) with enzyme loads of 2 and 1 U/mL, respectively. Concerning the profile of the produced galactooligosaccharides, the Kluyveromyces lactis lactase hydrolyses lactose more rapidly, resulting in higher levels of allolactose and lower levels of 6-galactosyl-lactose, compared to the lactase from Aspergillus oryzae.”

The abstract is generally well-written, but there are a few areas where clarity and conciseness could be improved.

Reply: We thank Reviewer #3 for their comments on our manuscript. Please see below are point to point response.

The phrase "concentrated sweet whey (15 % w/v initial lactose)" is repeated in the description of the yields for both β-galactosidases. Consider condensing this information to avoid redundancy.

Reply: The mentioned phrase has been changed as proposed to avoid redundancy.

The sentences in the last paragraph are lengthy and could be broken down for better readability. Consider separating information about maximum yields, the β-galactosidase from Kluyveromyces lactis, and the corresponding yields for the β-galactosidase from Aspergillus oryzae into distinct sentences.

Reply: The sentences mentioned cannot be broken down any further since they are already separated as proposed. One sentence is about Kluyveromyces lactis (Lines 16-19) and one about Aspergillus oryzae (Lines 19-21).

The sentence "The corresponding galactooligosaccharide yields for β-galactosidase from Aspergillus oryzae were equal to and 27.4 and 24.8 %..." is a bit unclear. It could be rewritten for better clarity, possibly by specifying which conditions correspond to the mentioned yields.

 Reply: This sentence has been changed for better clarity. The enzyme loads that correspond to each whey type have been mentioned in detail.

In the last sentence, "hydrolyses" should be "hydrolyzes."

 Reply: The typo mentioned has been corrected.

Introduction: Will need some more info regarding the high protein fermented products.

Reply: As recommended also by Reviewer #2, more info has been added in the introduction regarding the exploitation options of the high protein contend of the whey in order to produce protein concentrate and isolate, a practice that has been used for several years from the dairy industries in order to partially manage with the huge amounts of whey discarded annually (Lines 67-73).

Results and discussion: It requires more literature to discuss more options on the high protein fermented products.

Reply: This study aims to comparatively investigate the GOS production from the bioconversion of lactose of sweet and acid whey, the two major by-products discarded during the production of high protein fermented products, like cheese and strained yoghurt. Two commercial lactases from the fungus A. oryzae and the yeast K. lactis were used to define the optimum process conditions resulting in the GOS yield maximization. The fermented products from which the by-products derive were not the scope of the present study and therefore, the discussion on the literature focuses on the option of the valorization of the by-product, i.e. sweet whey.

Conclusion: The conclusion appears to be generally well-written, but there are a few areas where clarity and precision can be improved.

Reply: The conclusions have been extensively changed according to the reviewer’s comments to reduce numerical repetitions from the results and discussion section and to improve clarity and precision.

Author Response File: Author Response.pdf