Preparation, Structural Characterization, and Calcium Supplementation Activity of Lycium barbarum Peptide–Calcium Derived from Bovine Bones

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents an interesting and relevant study on the preparation and calcium supplementation activity of Lycium barbarum peptide–calcium derived from bovine bone calcium. The work addresses an important issue in nutrition science (calcium deficiency and bioavailability) and explores an innovative peptide–mineral chelate approach. The study is methodologically sound, and the pharmacological evaluation in a low-calcium mouse model provides valuable insights into the potential application of LBP–Ca as a functional food ingredient. Overall, the manuscript is promising and of potential interest to readers of Foods. That said, the current version of the manuscript requires several substantive and technical revisions to improve clarity, coherence, and scientific rigor:

1. The name Lycium barbarum must appear in italics, with barbarum in lowercase.
2. Lycium barbarum (including those in the Abstract, e.g., lines 20 and 26, and in the main text) should consistently be italicized.
3. Remove the acronym “ALP” of the abstract, as it is not used subsequently.
4. The current keywords are excessively long. Please shorten them and ensure that species names are properly formatted.
5. The figure inserted prior to the Introduction functions more as a graphical abstract than a scientific figure. It should be removed.
6. Unify the font style and size across the manuscript to ensure consistency.
7. Citations should be included at the first mention of relevant concepts. Moreover, references should be properly spaced, ensure that brackets are separated from the preceding word.
8. Add references regarding the use of citric acid as an extraction agent for calcium, as this is a key methodological point.
9. The abbreviation “LBPs” appears on line 83 without prior definition. Ensure acronyms are introduced before use. Additionally, “LBPs” is inconsistently written in full in line 85; please standardize usage.
10. In lines 88–94, the manuscript first outlines experimental steps and only then states the objective. For clarity, the objective should precede the description of the approach.
11. Specify the mode of administration of LBP–Ca (e.g., dissolved in water).
12. Report centrifugation speeds in relative centrifugal force (× g) rather than rpm.
13. Figures should appear after their first mention in the text, not before (e.g., Figure 3.1).
14. Table 1 is not referenced in the text and must be integrated appropriately.
15. Clarify that the positive control used is calcium gluconate.
16. In Section 3.5, it is stated that LBP–Ca exhibits the highest calcium concentration. However, the statistical analysis indicates no significant difference compared with the positive control. Please correct this interpretation.
17. The term “alkaline phosphatase (ALP)” is repeated in full on line 345 despite being defined earlier; use the acronym consistently.

Author Response

Comments 1 and 2：The name Lycium barbarum must appear in italics, with barbarum in lowercase.

Lycium barbarum (including those in the Abstract, e.g., lines 20 and 26, and in the main text) should consistently be italicized.

Response 1 and 2: Thank you for pointing this out. We have consistently italicized Lycium barbarum throughout the manuscript. Specific revisions include:

Page 1, Title, Line 3: “Lycium barbarum”

Page 1, Abstract, Line 20: “Lycium barbarum peptide-calcium chelate”

Page 1, Abstract, Line 26: “Lycium barbarum peptide”

Page 1, Keywords, Line 35: “Lycium barbarum peptide-calcium chelate”

Page 3, Introduction, Paragraph 1, Line 82: “Lycium barbarum”

Page 3, Introduction，Paragraph 1, Line 84: “Lycium barbarum peptide (LBP)”

Page 3, Introduction, Paragraph 2, Line 93: “Lycium barbarum peptide–calcium chelate”

Page 3, Section 2.1, Line 104: “Lycium barbarum polypeptide powder”

 

Comments 3：Remove the acronym “ALP” of the abstract, as it is not used subsequently.

Response 3: Thank you for this helpful comment. We agree that the acronym "ALP" is not used again in the Abstract and should be removed to improve clarity. We have replaced "ALP" with its full term, "alkaline phosphatase," in the Abstract (line 30).

 

Comments 4：The current keywords are excessively long. Please shorten them and ensure that species names are properly formatted.

Response 4: Thank you for pointing this out. We have shortened the keywords and ensured that all species names are correctly italicized.

Page 1, Keywords section (Lines 35–36): Original: Bovine bone; extraction method; Lycium barbarum peptide; Lycium barbarum peptide-calcium chelate (LBP-Ca); preparation technology; bone microstructure; valuability of calcium supplementation activity.

Revised: Bovine bone; Lycium barbarum peptide-calcium chelate; characterization; calsium supplementation activity.

 

Comments 5：The figure inserted prior to the Introduction functions more as a graphical abstract than a scientific figure. It should be removed.

Response 5: We have removed the figure that appeared before the Introduction (previously located on Page 2). The manuscript now follows standard scientific structure.

 

Comments 6：Unify the font style and size across the manuscript to ensure consistency.

Response 6: We have standardized the font style (Palatino Linotype) and size (10 pt) throughout the manuscript to ensure uniformity.

 

Comments 7：Citations should be included at the first mention of relevant concepts. Moreover, references should be properly spaced, ensure that brackets are separated from the preceding word.

Response 7: Thank you for this suggestion. We have carefully reviewed the manuscript and ensured that all citations are placed at the first mention of the relevant concepts. Additionally, we have standardized the spacing between words and brackets throughout the text.

First, in the Introduction section, we have added key citations to support the rationale for using citric acid and to highlight the limitations of conventional calcium supplements. Specifically, the sentences now read:

"Among various extraction methods, citric acid offers unique advantages due to its tricarboxylic structure. It can chelate calcium ions to form soluble calcium citrate, which not only prevents precipitation but also improves safety by reducing corrosion and maintaining protein integrity [8-10]. Thus, citric acid extraction of bovine bone calcium provides a feasible pathway for preparing bioavailable calcium sources. However, conventional calcium supplements such as calcium citrate and calcium gluconate are still limited by poor intestinal absorption and susceptibility to inhibition from dietary antagonists, leading to unsatisfactory bioavailability [11, 12]."(Page 2, Lines 55-64)

Second, we have supplemented the Methods and Results sections with appropriate citations to validate the experimental procedures and data interpretation. This includes:

In Section 2.6.3 (Particle Size and Zeta Potential Analysis): "…Samples were equilibrated for 30 seconds before analysis [25]." (Page 4, Line 156)

In Section 2.9 (Bone Densitometry Analysis): "…a rotation step of 0.7° [28]." (Page 5, Line 194)

In Section 3.4.2 (FTIR Analysis), multiple citations have been added to support the spectral assignments: "As shown in Fig. 7, the broad absorption band in LBP spanning 3500–3100 cm⁻¹, centered at 3441.7 cm⁻¹ with a shoulder at 3283.5 cm⁻¹, is characteristic of N-H and O-H stretching vibrations, indicative of extensive hydrogen bonding.  Upon chelation, this region transformed into a sharper band at 3512 cm⁻¹ and a broadened band at 3435.1 cm⁻¹.  This shift suggests that Ca²⁺ coordination competitively disrupts the native hydrogen-bonding network, forming new N–Ca or O–Ca linkages [29].The most critical evidence comes from the amide I region.  The peak at 1654.9 cm⁻¹ in LBP, assigned to C=O stretching vibrations, underwent a significant shift to 1604.3 cm⁻¹ in LBP-Ca.  This substantial 50 cm⁻¹ redshift is a classic indicator that the carbonyl oxygen atoms are directly involved in chelating Ca²⁺ ions, as the electron density of the C=O bond is altered upon coordination [30].Further evidence was found in the fingerprint region.  The bands in LBP at 1156.0 cm⁻¹ and 1015.3 cm⁻¹, associated with C-O-C stretching or P-O vibrations (if phosphorylation is present), shifted to 1136.2 cm⁻¹ and 1076.9 cm⁻¹ in LBP-Ca, respectively.  This implies the participation of hydroxyl, ether, or potentially phosphate groups in the chelation process [31].  Crucially, the appearance of new absorption peaks at 843.9 cm⁻¹ and around 600 cm⁻¹ in LBP-Ca can be assigned to Ca-O coordination stretching and metal-oxygen bending vibrations, providing definitive evidence for the formation of covalent bonds between calcium and the peptide [32]." (Page 11, Lines 305-322)

Finally, we have significantly enhanced the Discussion section by incorporating relevant references to substantiate our claims regarding the safety profile of citric acid and the structural characterization of the LBP-Ca chelate. Key additions include:The safety and practicality of citric acid: "As a widely used acidity regulator in the food industry, citric acid is one of the few food additives designated as 'Generally Recognized as Safe' (GRAS) [39]." (Page 17, Lines 442-443) and "…proving to be an ideal solvent for LBP-Ca preparation [40]." (Page 17, Lines 443-446)

The interpretation of UV-Vis and FTIR data: "The hyperchromic effect in the UV spectrum signifies Ca²⁺-induced electronic polarization within the amide chromophore [41, 42].", "…confirms the primary role of carbonyl oxygen atoms [43]…", "…suggest additional coordination through amino, hydroxyl, and possibly phosphate groups [44].", and "…definitive fingerprint of successful chelate formation… [45]." (Page 17, Lines 458-468)

The discussion on particle size, zeta potential, and SEM morphology: "…consistent with findings in other peptide-calcium systems [46, 47].", "…enhance colloidal stability and reduce aggregation propensity [48].", and "…leading to the formation of larger, more porous aggregates [49, 50]." (Page 18, Lines 469-485)

 

Comments 8：Add references regarding the use of citric acid as an extraction agent for calcium, as this is a key methodological point.

Response 8: Thank you for this valuable suggestion. We agree that citric acid extraction is a pivotal aspect of our methodology. Accordingly, we have added key references in the Introduction (Page 2, Paragraph 2, Lines 55-64) to better support and contextualize our choice of citric acid as a calcium extraction agent. The revised text now reads:

 "Among various extraction methods, citric acid offers unique advantages due to its tricarboxylic structure. It can chelate calcium ions to form soluble calcium citrate, which not only prevents precipitation but also improves safety by reducing corrosion and maintaining protein integrity [8-10].  Thus, citric acid extraction of bovine bone calcium provides a feasible pathway for preparing bioavailable calcium sources. However, conventional calcium supplements such as calcium citrate and calcium gluconate are still limited by poor intestinal absorption and susceptibility to inhibition from dietary antagonists, leading to unsatisfactory bioavailability[11, 12]."

The added and key existing citations are as follows:

[8] Lambros et al. (2022): Highlights the multifunctional role and safety of citric acid as a GRAS (Generally Recognized as Safe) substance.

[9] Vavrusova & Skibsted (2014): Provides context on calcium bioavailability and the advantages of citrate complexes.

[10] Zhang et al. (2023): Demonstrates a direct application of citric acid in extracting calcium from cattle bone for peptide-chelate preparation.

[11] An et al. (2022): This review on calcium-peptide chelates clearly outlines the absorption challenges faced by conventional calcium supplements, which justifies our research aim to develop a superior alternative.

[12] Hua et al. (2019): This study provides experimental evidence on the enhanced calcium absorption mediated by peptide-chelate complexes, directly supporting the rationale behind our subsequent development of the LBP-Ca chelate.

 

Comments 9：The abbreviation “LBPs” appears on line 83 without prior definition. Ensure acronyms are introduced before use. Additionally, “LBPs” is inconsistently written in full in line 85; please standardize usage.

Response 9: Thank you for pointing out this oversight. We have carefully reviewed the text and corrected the issue. The acronym "LBP" for Lycium barbarum peptides is now properly defined upon its first occurrence in the manuscript. Specifically, on line 83 (Page 3, Paragraph 3), the text has been revised from "Lycium barbarum peptides(LBPs)" to "Lycium barbarum peptides (LBP)" to clearly introduce the acronym. Furthermore, we have standardized the usage throughout the document, ensuring that the acronym "LBP" is used consistently after its definition.

 

Comments 10：In lines 88–94, the manuscript first outlines experimental steps and only then states the objective. For clarity, the objective should precede the description of the approach.

Response 10: We have reorganized the text to state the objective before describing the experimental approach for better clarity. The specific revisions are located in the second paragraph of the Introduction on Page 3, Lines 89-95: The original order has been adjusted. The objective is now stated first: "In this study, calcium ion was extracted from bovine bones under optimized conditions using citric acid and subsequently utilized to prepare Lycium barbarum peptide–calcium chelate (LBP–Ca)." This is followed by the description of the methods: "The structural features of LBP–Ca were characterized by multiple spectroscopic and microscopic techniques. The calcium supplementation activity was evaluated in a low-calcium mouse model."

 

Comments 11：Specify the mode of administration of LBP–Ca (e.g., dissolved in water).

Response 11: We have clarified the mode of administration in Section 2.7.1 (Page 5, Line 173-176): "All calcium supplements (calcium gluconate, calcium citrate, and LBP–Ca) were administered by oral gavage in aqueous solution at a dose equivalent to the Recommended Daily Allowance of 800 mg Ca per 60 kg body weight (39.99 mg Ca/kg). "

 

Comments 12：Report centrifugation speeds in relative centrifugal force (× g) rather than rpm.

Response 12: Thank you for this important clarification. We agree that reporting centrifugation speed in relative centrifugal force (× g) is the standard practice as it ensures reproducibility independent of the centrifuge rotor type. We have revised the manuscript accordingly.

In Section 2.7.2 (Sample Collection and Analysis), the sentence has been updated from:"Serum was obtained by centrifugation at 2,236 × g for 15 minutes..."(Page 5, Line 180-181)

 

Comments 13：Figures should appear after their first mention in the text, not before (e.g., Figure 3.1).

Response 13: Thank you for this valuable comment.  We sincerely apologize for the oversight in the figure placement.  We have thoroughly reviewed the entire manuscript and have reordered all figures to ensure that each one now appears in the text after its first citation.

This correction has been applied to all figures throughout the manuscript.  For instance:

Figure 3 is now positioned after its first mention in Section 3.1.

Figure 4 now appears after its introduction in Section 3.2.

Figure 5 is placed after its initial citation in Section 3.3.

The same logical order has been applied to all subsequent figures (Figures 6-11).

 

Comments 14：Table 1 is not referenced in the text and must be integrated appropriately.

Response 14: Thank you for bringing this to our attention. We apologize for the oversight. The table in question, which details the Box-Behnken experimental design and results, was indeed not properly cited in the original submission.In the revised manuscript, this table has been renumbered as Table 3 and is now appropriately referenced in the text. A citation has been added in Section 3.3 to introduce the table to the reader.Specifically, the following sentence has been added:"The calcium content under different extraction conditions and in the final products was summarized in Table 3, highlighting the efficiency of the optimized acid extraction process."(Page 10, Line 275-277)

 

Comments 15：Clarify that the positive control used is calcium gluconate.

Response 15: Thank you for this suggestion.  We agree that explicitly stating the identity of the positive control enhances the clarity of the experimental design.We have revised the manuscript to clearly indicate that calcium gluconate was used as the positive control. In Section 2.7.1 (Animals and Experimental Design), the description of the groups has been updated from:

"…three calcium supplementation groups (calcium gluconate as the positive control, calcium citrate and LBP–Ca)."(Page 5, Line 172-173)

 

Comments 16：In Section 3.5, it is stated that LBP–Ca exhibits the highest calcium concentration. However, the statistical analysis indicates no significant difference compared with the positive control. Please correct this interpretation.

Response 16: Thank you for this critical observation.  You are correct.  While the mean serum calcium level in the LBP-Ca group was numerically the highest, the statistical analysis (indicated by the same superscript letter 'a' in Table 4) clearly shows that there is no significant difference between the LBP-Ca group and the positive control (calcium gluconate) group.We sincerely apologize for this misinterpretation. The text in Section 3.5 has been corrected to accurately reflect the statistical findings. The sentence has been revised from:

"The LBP-Ca group exhibited the highest serum calcium concentration..." to:"The LBP-Ca group exhibited a serum calcium concentration (123.0 ± 24.5 μmol/dL) that was comparable to the positive control (calcium gluconate) group (114.5 ± 19.8 μmol/dL), with both groups showing significantly higher levels than the model and calcium citrate groups (p < 0.05)."(Page 14, Line 356-359)

 

Comments 17：The term “alkaline phosphatase (ALP)” is repeated in full on line 345 despite being defined earlier; use the acronym consistently.

Response 17: Thank you for pointing out this inconsistency. We agree that after its initial definition, the acronym should be used consistently throughout the manuscript to maintain clarity and conciseness.

We have revised the text on line 365 (located in Section 3.5) accordingly. The phrase has been changed from:"Serum alkaline phosphatase (ALP), a marker of bone turnover..." to: "Serum ALP, a marker of bone turnover..." Additionally, we have performed a thorough check to ensure that the acronym "ALP" is used consistently everywhere in the text after its first full definition in the Methods section.(Page 14, Line 365)

 

4. Response to Comments on the Quality of English Language

We have thoroughly reviewed the manuscript and corrected any grammatical errors, improved sentence structure, and ensured overall clarity and readability. All changes are highlighted in the revised document.

 

5. Additional clarifications

We confirm that all authors have approved the revisions and that no conflicts of interest exist. We thank the reviewers for their valuable input, which has greatly enhanced the quality of our manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

I reviewed the manuscript titled “The Study On Preparation Technology And Calcium Supplementation Activity Of Lycium Barbarum Peptide-Calcium Originated From Bovine Bone Calcium”. The tile should be revised focusing on the major content and provide eye-catching title

Graphical abstract is very poor.

Lycium barbarum. Sciemtific name should be in Italics

 

2.9. Bone Densitometry Analysis: provide reference

2.6.3. Particle Size and Zeta Potential Analysis: provide citation

2.5. Preparation of Peptide-Calcium Chelate (LBP-Ca): provide citation

Fig 3: details on the statistical significance and error bar info should be provided along with the caption.

Figure 4: x axis titles should be provided for better understanding

The content is acid extracted bone is high from Table 1. However, since the extraction process is using acid, how it can be applied for food application or as supplements??

Fig5: notations, such as A, B, C are missing

Discussion should be improved with more scientific literature support . This section is very weak.

Figg10 should be presented a better way

Table5: Different lowercase letters (a, b ).. it is a to c; not a,b

More details/discussion should be provided for the increase in particle size for LBP-CA in fig 8(particle size)

Figure 7. FTIR spectral difference should be details clearly and explain with literature support

Conclusions should be revised to reflect the content of the manuscript covered

Overall, results presentation and discussion is very weak. I suggest authors to improve this section

Author Response

Thank you very much for taking the time to review this manuscript. We sincerely appreciate your valuable comments and suggestions, which have significantly improved the quality of our work. We have carefully addressed all points raised, and the corresponding revisions are highlighted in the re-submitted manuscript. Below, we provide a point-by-point response to each comment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The revised manuscript has been thoroughly reviewed, and all comments and suggestions have been addressed appropriately. The manuscript is now in accordance with the reviewer’s recommendations and is deemed ready for publication.

Reviewer 2 Report

Comments and Suggestions for Authors

The quality of the manuscript is now improved. In my opinion, this version can be accepted for publication.