Functional and Oxidative Stability of Traditional Kazakh Molded Smoked Ham Affect by Sea Buckthorn (Hippophae rhamnoides) Extract Addition

3. Point-by-point response to Comments and Suggestions for Authors

Comments 1: Lines 467-476, the optimization phase identifies 0.1% sea buckthorn extract as optimal, but the subsequent comparative experiment uses 3% extract without sufficient justification for this 30-fold increase. The manuscript mentions that "0.1% extract showed relatively high AV and POV, requiring a higher dose," but this reasoning lacks quantitative data (e.g., how AV/POV at 0.1% compares to industry standards, or why 3%—not 0.5% or 1%—was chosen).

Response 1: Thank you for pointing this out. We agree with this comment. Unfortunately, due to lack of sufficient funding and time in which the project had to be carried out, comparative scientifically sound experiments with sea buckthorn extract supplementation doses higher than 0.1% and lower than 3% were not conducted. We are deeply aware of this need and hope to have the opportunity in the future to conduct subsequent experiments through an appropriate design of the experimental setup in order to study parameters for lipid oxidation, color characteristics of the product, sensory properties determined by tasting or consumer preferences and to establish exactly what concentrations are most suitable. In order to make a more informed choice of concentration for the added sea buckthorn extract in the following comparative studies, a series of industrial tests were conducted with 0.5%, 1%, 2%, 3% and 4% added sea buckthorn extract to the injection brine. The changes in peroxide value (POV) and acid value (AV), as well as preferences in organoleptic characteristics and yield of the finished product were recorded by the team of the factory where the experiments were conducted. According to information from the manufacturer, the relative efficiency of the addition at a concentration of 0.5% is low, at 1% it is moderate; at 2% it is high, at 3% it is most effective, and at 4% it is comparable to 3%, but is overdosed. For these reasons, it was decided to conduct the next scientific experiments with the addition of 3% sea buckthorn extract.

In this regard, the following editorial changes were made to the text of the manuscript:

- on lines 998-998 on page 17 the term “optimal” was replaced by the term “appropriate dose”;

- General editorial changes were made to the conclusion and on lines 1417-1420 on page 24 the following phrase was inserted: “...do not lead to a significant improvement in oxidative stability and do not negatively affect the organoleptic properties of the product.”

- The conclusion on lines 1416-1428 of page 24 has been revised as follows: “It is therefore concluded that the addition of dry powder of sea buckthorn extract can be successfully applied in the production of traditional Kazakh molded smoked ham from horse and camel meat by stabilizing the oxidative stability of the product, but further studies are needed to determine whether concentrations of 0.5 to 3.0% (w/w) would not be more effective in terms of improving the sensory properties of the finished product.”

We hope that these explanations and editorial changes will satisfy the esteemed reviewer 1?

Comments 2: The stabilizer "Kf Stabinject 101" is described as containing "½ α-tocopherol and ½ L-ascorbic acid," but the units (e.g., % by weight, mg/kg) and its role in synergism with sea buckthorn extract are undefined. In Table 1, the stabilizer dosage (X2) is listed as 0, 0.05, and 0.1, but the unit (e.g., % of meat weight) is missing.

Response 2: Thank you for pointing out the shortcomings in question and the discrepancies. We agree with your remarks and comments and have taken them into account. We have made the following editorial changes to the text of the revised manuscript:

-          The preparation “KF Stabinject 101“ (manufactured by Koenigshof GmbH Lebensmitteladditive, Germany) is a multifunctional additive containing di- and triphosphates (E450 and E451), soy and milk proteins, thickeners (kappa-carrageenan E407 in combination with locust bean gum E410, guar gum E412 and xanthan gum E415), mono- and disaccharides, table salt and two antioxidants (α-tocopherol and L-ascorbic acid). “KF Stabinject 101” does not contain sodium nitrite and it is dosed additionally.

-          The exact ratios of the ingredients of “KF Stabinject 101” are a company secret. After laboratory analyses, for the purposes of the experiment, the exact concentrations of the two antioxidants that it contains were established, but since they are a company secret, we do not have permission from the manufacturer (copyright owner) to publish them.

-          The phosphate components in the additive promote the dissociation of the actomyosin complex and the activation of the water-binding capacity of muscle proteins. This ensures the formation of a dense, uniform texture of the product and a yield of approx. 180% of the finished product.

-          Proteins, carrageenan and thickeners stabilize the emulsion system of the ham matrix and prevent syneresis during heat treatment.

-          Mono- and disaccharides are reducing substances that stabilize the formed pink-red pigment of the ham.

-          The binary antioxidant mixture of α-tocopherol (vitamin E) E307 and L-ascorbic acid (vitamin C) E300 exhibits a synergistic effect. α-tocopherol stabilizes the lipid fraction of the ham, preventing the oxidation of unsaturated fatty acids, while L-ascorbic acid is a reducing agent, regenerating α-tocopherol and maintaining low levels of lipid hydroperoxides. The effectiveness of these two antioxidants is affected by heat differently. While α-tocopherol decomposes rapidly at 180°C, but is relatively thermally stable at 72°C (which is the temperature in the center of cooked ham), L-ascorbic acid is not particularly heat-resistant. It oxidizes quickly and loses its properties when exposed to heat and air, and is therefore used in combination with α-tocopherol. Therefore, in the experiments, a synergistic effect was sought by adding polyphenolic compounds from the sea buckthorn extract.

-          The use of a combined additive instead of separate additives simplifies dosing, ensures synergy of the components and technological reproducibility of yields, which is especially important when scaling up laboratory developments into industrial production.

-          the term "stabilizer" characterizing the preparation "Kf Stabinject 101" throughout the text of the article has been replaced with the term "additive", only stating that it contains α-tocopherol and L-ascorbic acid.

-          Editorial changes to the text are reflected on lines 225-231 of page 4, the composition of the additive "Kf Stabinject 101" is described in details.

We thank reviewer 1 for their comments on Table 1. We acknowledge the comments and have added the units of measurement in Table 1. The units of measurement for acid value (AV) are mg KOH/g fat extracted from the product, formally mg KOH/g, for peroxide value (POV) meq O₂/kg fat, formally meq O₂/kg and for TBARS - mg MDA/kg sample of finished product (ham), formally mg MDA/kg. Editorial changes to the text are reflected in lines 405-412 on page 7 of the updated text in the manuscript.

Comments 3: The sensory analysis mentions a "10-member trained panel" and a "5-ball hedonic scale" but provides no details on panel training (e.g., training duration, reference standards used), sensory attributes evaluated (beyond cross-sectional surface), or statistical methods for analyzing sensory data.

Response 3: Thank you for your remarks and recommendations. Completely new text was insert in this section: “A trained group of 10 participants completed three one-hour training sessions, using reference samples characterizing different levels of color, uniformity and moisture of the cross-sectional surface, following the guidelines of Meilgaard [48]. Sensory evaluated scores of the cross-sectional surface were assessed by 5-point hedonic scale, where 1 = strongly dislikes and 5 = strongly likes. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test at a significance level of p < 0.05. The analysis was performed using SPSS version 26 software.”

Editorial changes to the text are reflected in lines 537-624 on pages 9-10 of the updated text in the manuscript.

Comments 4: The study states that protein carbonyl content was "below the limit of detection (LoD)" but does not report the LoD of the method (Mercier et al., 1994) or explain why protein oxidation was not initiated (e.g., whether the smoking process or low storage temperature inhibited it). This is critical because horse/camel meat’s high iron content is typically pro-oxidative for both lipids and proteins.

Response 4: Thank you, the reviewer 1 for the question and remark.

-          The Limit of detection of the used procedure is 0.010 nmol DNPH/g protein. As presented by Mercier et al., (1994) also Estévez, (2011) and Cheng et al., (2021), protein carbonyls content can vary from 3 - 5 nmol DNPH/mg protein and also from 0.2 - 1 nmol DNPH/mg protein (Vlahova-Vangelova et., 2014; Balev et al., 2019). For these reasons, we kindly decline the comment and recommendation]

-          The explanation why protein oxidation was not initiated have been added to the text of the manuscript. Editorial changes to the text are reflected in lines 1252-1258 on page 22 of the updated text in the manuscript: „This is a logical explanation for the reduction of free amine nitrogen levels established by us and for the relatively low levels of TBARS, as well as for the practical absence of protein carbonyls. The low levels of the TBARS (low molecule wight oxidized derivatives of the lipids) and Protein carbonyls (oxidized derivatives of the muscle proteins) from the ham matrix could be explained by penetration and interaction with phenolic compounds penetrating as a result of the prolonged smoking with goose smoke. “

Comments 5: The discussion compares results with Papuc et al. (2010) and Anchidin et al. (2024) but fails to address discrepancies (e.g., Papuc et al. reported sea buckthorn polyphenols inhibit lipid peroxidation in ground pork, but this study finds minimal effects on TBARS). Additionally, no comparison is made with studies on sea buckthorn in horse/camel meat products (e.g., Makangali et al., 2019, who used sea buckthorn seed powder in camel meat smoked products).

Response 5: We thank reviewer 1 for the suggested opportunities for improvement. We consider the three sources shown by reviewer 1 and after careful analysis of all data our answer is following:

Initially, we are left with the impression that the esteemed reviewer 1 is right to speak of inconsistencies. A careful in-depth reading of the full texts of the original articles reveals that there are significant differences, both in the design of the experiments and in the object of study, as well as in the indicators that were studied, and in the analytical procedures applied by the different authors.

In our study, the effect of adding a sea buckthorn powder extract obtained from dried cake of sea buckthorn pomace, was determined. The extraction was with 70% ethanol and was ultrasonically assisted. The extract was concentrated and ground in a ball mill into powder. The object of our study is cooked and smoked molded ham from horse and camel meat, the technology of which includes injecting the pieces of meat with 70% brine, tumbling under vacuum, cooking and smoking with thick smoke at a relatively high temperature, until reaching 73C in the center. Our study lasts 30 days during refrigerated storage of a vacuum-packed finished product.

In the study by Papuc et al. (2010) a 60% ethanol extract of dried sea buckthorn berries was used. The extraction was carried out by a solid-liquid procedure in a Soxhlet apparatuses. The obtained alcoholic extract was evaporated on a rotary vacuum evaporator and the resulting residue was dissolved in ethanol at dilutions of 1:9, 2:8, 3:7, 4:6 and 5:5 (v:v). The object of the study was fresh beef and pork minced meat.

In the study by Anchidin et al. (2024) sea buckthorn oil was used. The study involved three batches of chilled pork tenderloin injected with 1%, 3% and 5% sea buckthorn oil. The meat quality was analyzed in terms of antioxidant capacity, physicochemical and microbiological quality, as well as sensory perception. Consumers preferred the batch injected with 3% sea buckthorn oil due to its more balanced taste.

In the study by Makangali et al. (2019), an additive of 5, 10 and 15% sea buckthorn seed powder was used, which was compared with additives of wheat and rice flour. The object of the study was the filling mass (minced meat) for a cooked-smoked molded meat product from camel and beef. Sea buckthorn seed powder was characterized as a source of tocopherol 62.15 ± 2.13 mg/100 g, carotenoids 4.21 ± 0.22 mg/100 g and flavonoids 1.54 ± 0.06%. The quality of the minced meat (filling mass) for the cooked-smoked delicacy was analyzed (it is not described how it was packaged and whether it was stored?). The structural, mechanical and color characteristics of the finished product, its pH and yield of the finished product, as well as the so-called color stability measured in % (The analysis method is not described in the article, nor is any information presented on how the stability in question was established or calculated!?!). An increase in: the yield of the finished product by 6.8%; the water content by 9.35%; the moisture-binding capacity by 2%; the ability to retain fat by 3.5%; the shear strength is 5.8 N/m. The pH of the filling mass for the meat product has increased from 6.0 to 6.4 - 6.8 in the three samples of the addition of sea buckthorn seed powder. There is no information that the indicators of acid and peroxide value, TBARS, protein carbonyls, PHAN, etc., which are tracked in dynamics in our study, have been studied.

Due to the incomparability of the results discussed in the Discussion section by Papuc et al. (2010), Anchidin et al. (2024) and Makangali et al. (2019) with our data, it is not correct to make a comparison and draw any general conclusions. So we would not like to talk about inconsistencies with the mentioned articles? Maybe the style of the discussion we wrote is not correct? After careful discussion, we made some style edits in the text of the Discussion section: see lines 1387 - 1412 on page 24.

Comments 6: The study notes that 3% extract causes ham discoloration, which may affect consumer acceptance, but does not propose solutions. Additionally, the cost-effectiveness of using 3% extract (a relatively high dosage) in industrial production is not discussed.

Response 6: We thank reviewer 1 for the suggested opportunities for improvement. We accept them and have made the corresponding editorial changes. A new paragraph with similar reasoning and conclusions has been added in the text of the revised manuscript. See lines 1416-1428 on pages 24.

The cost-effectiveness of using additives in this case 3% sea buckthorn extract (relatively high dose) in industrial production is an economic indicator and is not the subject of a technological study. However, it should be noted that the results of this experiment were implemented in practice and the product is now produced with a 3% input dose. This is practical proof that the doses in question are not an obstacle to the cost of the finished product and allow for pricing that makes this type of ham competitive on the market.

Comments 7: Figures 2, 3, and 4 lack detailed legends (e.g., Figure 2’s axes are labeled "x2, x3" without defining what x2/x3 represent; Figure 4’s "Desirability profile" has unlabeled y-axes).

Response 7: We thank reviewer 1 for his constructive criticism.

We acknowledge the comments and have taken action to address the deficiencies.

Editorial changes to the text are reflected in lines 788-812 on pages 12-14 of the updated text in the manuscript.

Comments 1: Lines 44–132 (Introduction): Please consider adding a concise gap analysis, specifically what has not been demonstrated for molded smoked ham from horse/camel, and why polyphenols would be beneficial in a nitrite-cured, cooked-smoked matrix (heme chemistry and pro-oxidant iron).

Response 1: We thank reviewer 2 for pointing this out. We agree with this comment. On lines 49-69 of page 2 of the updated text in the manuscript, a paragraph was inserted. It stating that there is an increased risk of pigment and lipid oxidation in traditional Kazakhstan molded smoked horse/camel ham, drawing attention to the pro-oxidant effect of heme iron and why polyphenols would be beneficial in the matrix of this type of meat product.

Comments 2: Line 130–132, please, explicitly state the primary hypothesis and pre-registered primary endpoints (e.g., TBARS at day 30).

Response 2: We thank reviewer 2 for the suggestion for improvement. We accept it and in this regard. We have made the following additions and changes to the text indicated on lines 202-210 on page 4 of the updated text in the manuscript. A new sentence has been added in which the primary hypothesis is justified and the expected results after 30 days of refrigerated storage of the finished product are specifically stated. As a result, some revision to the formulation of the purpose of the study has also been made.

Comments 3: Line 141–144, please, correct “fat (piece size: 1–2 cm) was added in a ratio of 1:0.1–0.2 (fat: meat)” to a conventional expression (e.g., 10–20% fat relative to meat), and resolve whether pieces were cut or minced; “minced to size 5–6 cm thick” is likely 5–6 mm or “chunks of 5–6 cm”.

Response 3: We thank the esteemed reviewer for his comments and recommendations.

We accept them and have made the editorial changes to the text from lines 202 to 203 on page 4 of the updated text in the manuscript.

Comments 4: Lines 145–147 would be beneficial to specify the exact composition of “Kf Stabinject 101” (including the percentage of α-tocopherol, L-ascorbic acid, and carriers), the manufacturer, lot number, and whether it contains other additives (e.g., phosphates).

Response 4: We thank the esteemed reviewer for the suggestions for clarifications. We accept the suggestion without specifying the batch number, because a number of experiments were conducted with different batches of meat and additives. On the other hand, we did not encounter a practice in research articles to publish data on batch numbers of salting materials, especially since this is specific production information maintained in records by the HACCP system.

The preparation “KF Stabinject 101“ (manufactured by Koenigshof GmbH Lebensmitteladditive, Germany) is a multifunctional additive containing di- and triphosphates (E450 and E451), soy and milk proteins, thickeners (kappa-carrageenan E407 in combination with locust bean gum E410, guar gum E412 and xanthan gum E415), mono- and disaccharides, table salt and two antioxidants (α-tocopherol and L-ascorbic acid). KF Stabinject 101 does not contain sodium nitrite and it is dosed additionally.

The exact ratios of the ingredients of KF Stabinject 101 are a company secret.

After laboratory analyses, for the purposes of the experiment, the exact concentrations of the two antioxidants that it contains were established, but since they are a company secret, we do not have permission from the manufacturer (copyright owner) to publish them.

The phosphate components in the additive promote the dissociation of the actomyosin complex and the activation of the water-binding capacity of muscle proteins. This ensures the formation of a dense, uniform texture of the product and a yield of approx. 180% of the finished product.

Proteins, carrageenan and thickeners stabilize the emulsion system of the ham matrix and prevent syneresis during heat treatment.

Mono- and disaccharides are reducing substances that stabilize the formed pink-red pigment of the ham.

The binary antioxidant mixture of α-tocopherol (vitamin E) E307 and L-ascorbic acid (vitamin C) E300 exhibits a synergistic effect. α-tocopherol stabilizes the lipid fraction of the ham, preventing the oxidation of unsaturated fatty acids, while L-ascorbic acid is a reducing agent, regenerating α-tocopherol and maintaining low levels of lipid hydroperoxides. The effectiveness of these two antioxidants is affected by heat differently. While α-tocopherol decomposes rapidly at 180°C, but is relatively thermally stable at 72°C (which is the temperature in the center of cooked ham), L-ascorbic acid is not particularly heat-resistant. It oxidizes quickly and loses its properties when exposed to heat and air, and is therefore used in combination with α-tocopherol. Therefore, in the experiments, a synergistic effect was sought by adding polyphenolic compounds from the sea buckthorn extract.

The use of a combined additive instead of separate additives simplifies dosing, ensures synergy of the components and technological reproducibility of yields, which is especially important when scaling up laboratory developments into industrial production.

Editorial changes to the text are reflected on lines 205 - 210 on page 4 of the updated text in the manuscript.

Comments 5: Line 149–151, please, define the basis of the 3% extract dose (w/w of meat? of brine? of final product).

Response 5: Thank you for the question.

The 3% extract dosage means 3 kg/100 L brine. The sea buckthorn extract dosage of 3% was selected based on a series of preliminary experiments examining the effect of various extract concentrations (3, 5, 7, 9, 11, 13, and 15%) on the sensory properties of the finish product. All values are presented as a mass fraction relative to the meat weight. Based on an assessment of taste, flavour, texture, and color, 3% was found to be the optimal concentration, providing the best combination of improved sensory properties without excessive flavor intensity or texture changes. Higher extract concentrations resulted in lower sensory scores due to the excessive dominance of sea buckthorn's flavor and color characteristics. Thus, the choice of 3% was based on empirical selection aimed at achieving maximum sensory quality while maintaining technological and economic feasibility. On line 214 of page 4 of the updated text in the manuscript “kg/ 100 L brine” was added.

Comments 6: Line 153, please consider stating the actual brine to meat ratio used (70% injection target vs. achieved uptake) and report yield pick-up (± SD).

Response 6: Thank you for your comment.

A 70% target injection to meat means that 70 L of brine was injected in 100 kg of meat. This type of forced pressure salting and subsequent vacuum tumbling contributes to high yields of brine to meat input. Yields of the order of 75 ± 0.1 % were observed in the finished product, meaning that from 170 kg meat injected with brine, equal of 127.5 ± 1.7 kg of finished product is obtained.

The following additions and editorial changes have been made to the text from lines 217 - 219 on page 4 of the updated text in the manuscript

Comments 7: Line 155–159, I recommend giving tumbler model settings (rpm), drum load %, and whether tumbling was intermittent; also report mold dimensions (diameter/length) and pressing pressure.

Response 7: Thank you for the recommendations to present the tumbling parameters in great detail.

The dimensions of the matrix (diameter/length) are presented above in the text - lines 202-203 of page 4 of the corrected version of the manuscript. The meat is chopped into pieces measuring 5 - 6 cm.

On lines - 217-241 of pages 4 and 5 of the corrected text of the manuscript, the amendments and additions indicated by the reviewer have been made.

Comments 8: Line 160–168, please, clarify thermal process: give internal temperature curve (time to 73 °C), oven humidity, and whether nitrosylation (color fix) step preceded smoking.

Response 8: We thank reviewer 2 for the suggestions, but we believe that we have described the steps of the thermal process in sufficient depth, namely: steam cooking, drying and hot smoking. The internal temperature curves were recorded on the microprocessor devices of the steam cooking cabins used, but we did not take these records. In the available literature on a similar topic, we did not find a presentation of time-temperature curves of the process. The duration of cooking depends on many factors and is a variable value. On the other hand, until the specified temperature of 73°C is reached, cooking is not stopped, in order to eliminate the risk of microbiological danger. The relative humidity in the cabin during steam cooking is 100% and this is a textbook truth. We do not consider it appropriate to report it.

During hot air drying, the aim is to dry the surface of the cooked hams in order to properly carry out the subsequent hot smoking process and deposit the smoke particles on a non-moist surface. This is exactly the nitrosylation step (color fixation) before smoking the hams. Here, the relative humidity of the air is kept as low as possible. However, it is a function of the relative humidity of the outside air sucked in from the outside environment. During smoking, a dense smoke-air mixture with a relative humidity of 65 - 68% is used.

Comments 9: Line 165–167, please consider specifying wood species for smoke and smoke source (friction, smoldering, liquid smoke), smoke density, and total phenols exposure.

Response 9: Thank you. We accept the reviewer’s 2 recommendations and modified the text - see lines 251-265, page 5 to emphasize this point.

The alder wood for smoking was traditionally used in Kazakhstan for its balanced lignin composition and low resin content. Alder produces smoke with a high level of phenolic compounds (not determined in this study), which are responsible for the pleasant aroma and have a pronounced antioxidant effect. Thus, ensures an optimal balance of aroma, flavor, and protective properties, an essential element of the smoking process.

The smoke was produced by smoldering alder wood chips, which produces a dense smoke with a rich composition of phenols and other aromatic components. Smoldering ensures uniform exposure of the smoke to the product's surface, improves sensory characteristics, and extends shelf life due to the antimicrobial and antioxidant effects of phenolic compounds. A smoke generator was used by smoldering a layer of moistened alder wood sawdust. The smoke was with a density of 1.3 - 1.5 g/cm³ and a speed of movement of 2 - 3 m/s.

Comments 10: Line 169–172, I recommend reporting package film OTR/WVTR and shrink conditions; “vacuum shrink film” should include brand/specs to interpret microbiology and oxidation.

Response 10: Thank you. We accept the reviewer’s 2 recommendations and added new text on lines 269 - 284, page 5.

Comments 11: Line 177–181, please, check grind size: ≤ 3 µm is implausible for pomace with simple milling; confirm intended ≤ 3 mm or provide milling method achieving micron scale.

Response 11: We thank reviewer 2 for his constructive criticism. We have considered it and made a substantial addition to the text of the revised manuscript at lines 303–306 on page 6.

Comments 12: Line 182–191, please, clarify whether extraction solvent was 70% ethanol or water ethanol at other steps; define solvent: solid basis (1:5 w/v), extract mass balance, and whether extract was standardized (e.g., mg GAE/g).

Response 12: We thank reviewer 2 for his constructive criticism. We have considered it and made a substantial addition to the text of the revised manuscript on lines 308-320 of page 6.

Comments 13: Line 186–188, please, correct rotary evaporation conditions: 0.101 MPa (825 mmHg) is not vacuum; provide absolute pressure (kPa) or mbar, bath temperature, and end point solvent % (GC).

Response 13: We are grateful to the esteemed reviewer 2 for the inaccuracies indicated.

We accept the remark and have made the corresponding changes in the text, which after revising the text of the manuscript are reflected in lines 313-314 of page 6.

Comments 14: Line 189–193, it would be beneficial to provide extract yield basis (g extract / g dry pomace), moisture/ash in final powder, and a minimal chemical characterization (TPC, water activity).

Response 14: We are grateful to the esteemed reviewer 2 for the indicated inaccuracies.

We accept the remark and made the changes in the text, which after revising the text of the manuscript are reflected on lines 316-320 on page 6.

Comments 15: Line 194–200, please, re title the section: the design in Table 1 optimizes formulation doses, not extraction parameters.

Response 15: We are grateful to reviewer 2 for pointing out the discrepancy. We accept the remark. We have changed the title as follows: Optimization of the formulation doses of sea buckthorn and the additive "Kf Stabinject 101" to study their synergism at very low application concentrations. We have numbered the subheadings in this section with 2.2.1 etc. for greater clarity for the reader and readability. The editorial changes in the text are reflected on lines 327 - 330 on page 6 of the updated text in the manuscript.

Comments 16: Line 201–214, please, justify the factor ranges (0.025–0.10% extract; 0–0.10% stabilizer) with pretests and say whether these are % of meat mass.

Response 16: We are grateful to reviewer 2 for the question. Here is our response:

The concentration ranges of the sea buckthorn extract (0.025–0.10%) and the additive “Kf Stabinject 101” (0–0.10%) were selected based on preliminary tests, including an assessment of their synergistic effect on the quality of the meat product, shelf life and sensory characteristics of the ham. Our hypothesis is that such low concentrations would provide a good balance between functional efficiency and preservation of the taste and textural properties of the finished product, while minimizing the cost of additives and seeking a strong synergistic effect between the two preparations. The values ​​are given as a percentage of the meat raw material mass. The editorial changes in the text are reflected on lines 360 – 369 on page 7 of the updated text in the manuscript.

Comments 17: Line 215–226, I recommend fixing the generic models to two factors (X1, X2) and removing unused variables (x3, x4); also state the coded to natural variable mapping explicitly.

Response 17: We thank reviewer 2 for the recommendation. We accept the remark. We have removed the unused variables x3, x4. Editorial changes to the text are reflected in lines 379 - 382 on page 7 of the updated text in the manuscript.

Comments 18: Line 229–231 (Table 1), please, define units for AV (mg KOH/g fat or product?) and TBARS (mg MDA/kg product), and verify all numbers (e.g., TBARS 0.23–1.20) align with the narrative.

Response 18: We thank reviewer 2 for their comments. We acknowledge the comments and have added the units of measurement in Table 1. The units of measurement for acid value (AV) are mg KOH/g fat extracted from the product, formally mg KOH/g, for peroxide value (POV) meq O₂/kg fat, formally meq O₂/kg and for TBARS - mg MDA/kg sample of finished product (ham), formally mg MDA/kg. Editorial changes to the text are reflected in lines 385-390 on page 7 of the updated text in the manuscript.

Comments 19: Line 239–256, please consider reporting colorimeter aperture, illuminant/observer, calibration tile values, and sample presentation (bloom time, surface prep) per AMSA 2023.

Response 19: Thank you for your remark. The following information was included in the text: “A Konica Minolta colorimeter CR-410 (Konica Minolta Holding, New Jersey, USA) equipped with a 50 mm aperture, illuminant D65, and a 2◦ standard observer was used to assess the lightness (L*), redness (a*) and yellowness (b*) on the cross-sectional surface of smoked ham [5]. Hams were incubated for 1 h at room temperature, and 1 cm thick ham slices were cut. Color was measured immediately without blooming. Calibration was performed against white reference standard No. 18833116 (Y = 94.3, x = 0.3134 and y = 0.3197). [5]. For each time interval, Instrumental colour (L*, a*, b*) was measured in seven independent samples (n = 7), each of which was analyzed in duplicate (2 measurements). Editorial changes to the text are reflected in lines 415-423 on page 8 of the updated text in the manuscript.

Comments 20: Line 242–245, I recommend adding the number of independent samples (n) and technical replicates per time point for pH and all physicochemical tests.

Response 20: Thank you for your remark.

For each time interval, pH and all physicochemical parameters were measured on seventh independent samples (n = 7), each of which was analyzed in duplicate (2 measurements). Results are presented as mean ± standard deviation.

Editorial changes to the text are reflected in lines 426-432 on page 8 of the updated text in the manuscript.

Comments 21: Line 246–255 (TPA), please, report sample orientation (fiber direction), test temperature, and number of cubes per batch (n) and per ham.

Response 21: Thank you for your remark. We find some of your remarks inappropriate. Most of the clarifications you requested are described in the text. The test temperature is the usual one at room conditions (+22 – +25℃) and the number of cubes per batch (n) and per ham are specified in the cited method. Similar information has been added to the text, which has undergone a new edition. Editorial changes to the text are reflected in lines 433-476 on pages 8-9 of the updated text in the manuscript.

Comments 22: Line 256–261, please, state centrifuge g and temperature correctly and total protein yield (mg/mL) to interpret protein assays.

Response 22:  Thank you for your remark. The following information was included in the text: “Protein extraction was performed according to the procedure described by Khan [37], with minor modifications. Meat samples (2.5 g) were homogenized with 48.5 cm³ of phosphate-buffered saline (49 mM Na₂HPO₄·7H₂O, 4.5 mM NaH₂PO₄·H₂O, 23 mM KCl), adjusted to an ionic strength of 0.55. The homogenate was conditioned for 12 hours at 0–4 °C and subsequently centrifuged at 1000 × g for 15 min.” The total protein yield was not measured with a direct assay.

Editorial changes to the text are reflected in lines 478-484 on page 9 of the updated text in the manuscript.

Comments 23: Line 262–273, it would be beneficial to state whether AV/POV/TBARS were measured on extracted fat or whole product, and to harmonize units with later tables.

Response 23: Thank you for your question.

The acid value (AV) of extracted fat was determined according to ISO 660:2020 [38] and was measured on extracted fat.

The peroxide value (POV) of extracted fat was determined spectrophotometrically by method describe by Shantha and Decker [39] and was measured on extracted fat.

The 2-thiobarbituric acid reactive substances (TBARS) of hams were determined by the method of Botsoglou et al. [40] modified by Moraru Manea et al. [41] and was measured on whole product.

Editorial changes to the text are reflected in lines 486-492 on page 9 of the updated text in the manuscript.

Comments 24: Line 274–281, please, define the ham extraction protocol for TPC/DPPH/FRAP (solvent, mass of sample, extraction time) and express TPC as mg GAE/kg (not “mg GA/kg”).

Response 24: Thank you for your remark.

Extraction of phenolic compound. Five grams of sample (ground ham) were homogenised with 20 mL of 50% methanol at 20,000 rpm for 30 sec and incubated 60 min at room temperature (~22–25°C). Mixture was centrifuge (10,000 x g) for 10 min, followed by addition of 20 mL of 70% acetone, mixed and left to stand for 60 min at room temperature. Diluted to 50 mL with distilled water and centrifuge again (10,000 x g) for 10 min, collect the supernatant.

The total phenolic content (TPC) was determination is based on the reaction of Folin-Ciocalteu reagent and phenolic compounds described by Vardakas et al. [44].

The radical scavenging activity against 1,1-diphenyl-2-picrylhydrazil (DPPH) radical is performed according to Brand-Williams et al. [45] modified by Dinkova et al. [46].

The transition metals-chelating activity against ferric (Fe3+) ions - FRAP assay was conducted according to the method of Benzie and Strain [47] modified by Dinkova et al. [46].

Editorial changes to the text are reflected in lines 499-516 on page 9 of the updated text in the manuscript.

TPC were express as mg GAE/kg (not “mg GA/kg”) in the table of the Results and Discussion section.

Comments 25: Line 283–287 (Sensory), I recommend moving the sensory method details earlier and clarifying the exact attribute “cross sectional surface color”, panel training/calibration, serving order, and statistics (linearity of 5 point hedonic data).

Response 25: Thank you for your remarks and recommendations. Completely new text was insert in this section: “A trained group of 10 participants completed three one-hour training sessions, using reference samples characterizing different levels of color, uniformity and moisture of the cross-sectional surface, following the guidelines of Meilgaard [48]. Sensory evaluated scores of the cross-sectional surface were assessed by 5-point hedonic scale, where 1 = strongly dislikes and 5 = strongly likes. Data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test at a significance level of p < 0.05. The analysis was performed using SPSS version 26 software.”

Editorial changes to the text are reflected in lines 517-591 on pages 9-10 of the updated text in the manuscript.

Comments 26: Line 288–297 (Microbiology), please, align the days sampled with Table 12 (1/15/30 vs. 5/10/15/30), specify limits of detection (CFU/g), and reconcile Salmonella test mass (0.1 g in text vs. 25 g in Table 12).

Response 26: Thank you for your remark. We agree with them and we made several changes in the text and in Table 12. Salmonella analysis was conducted according to ISO 6579-1:2017 using a 25g sample weight. The detection limit was 1 CFU per 25g of product, which meets regulatory requirements. The indication of the sample weight in the text as 0.1g is an error and does not reflect the actual test method.

Editorial changes to the text are reflected in lines 592-603 on page 10 of the updated text in the manuscript.

Comments 27: Line 299–307, please, correct “Reflection surface method” to “Response surface methodology (RSM)” and provide model diagnostics (lack of fit test, residual plots).

Response 27: Thank you for your remark. Somehow a ridiculous terminological error was made. On line 606 of page 10, the term “Reflecting Surface Method” was corrected to “Reflecting Surface Methodology (RSM)”. Specifics regarding model diagnostics (non-conformity test and residual plots) are described in the cited source. For these reasons and due to the very large volume of the article, please allow us to refrain from describing them in detail.

Comments 28: Line 313–315, I recommend harmonizing acceptability constraints with your measurement units (e.g., POV < 6 meq O₂/kg earlier vs. measured ~1 meq later).

Response 28: Thank you for your remark. We accept the comment and have harmonized the acceptability limits.

Editorial changes to the text are reflected on line 619 of page 10 of the updated text in the manuscript, the limit has been corrected to "Y2 (POV) < 1 meq O₂/kg;"

Comments 29: Line 316–319, please, specify exact two-way ANOVA model, interaction terms, post hoc corrections, and n at each cell; where p values are reported, ensure they’re two sided and verify distribution assumptions or use non parametric tests.

Response 29: Thank you for your remark. We accept it. To assess the influence of the factors "storage time" and "sample type" on the studied parameters, a two-way analysis of variance (ANOVA with fixed effects) was used, taking into account the main effects and their interaction. The sample size was n = 3 independent samples in each cell. The assumptions of normal distribution (Shapiro–Wilk test) and homogeneity of variances (Levene's test) were preliminarily checked. All p values were two-sided. For multiple comparisons, the Tukey test was used with a significance level of p < 0.05. In cases where assumptions were violated, nonparametric tests were used. Editorial changes to the text are reflected in lines 623-631 on page 10 of the updated text in the manuscript.

Comments 30: Line 322–331 (Table 2), please, check the “coefficient of variation” values (reported 1–11% appear inconsistent with SD/mean) and confirm whether variables are approximately normal.

Response 30: Thank you for your question. Our answer is following: The coefficients of variation (CV) were recalculated and confirmed the acceptable level of variability (1–11%). CV was defined as the ratio of the standard deviation to the mean, expressed as a percentage. The normality of the variable distribution was tested using the Shapiro–Wilk test (p > 0.05 for all parameters), confirming the feasibility of using parametric analysis methods. In cases of deviation from normality, appropriate nonparametric tests were used.

Comments 31: Line 343 (Table 3), please, replace “special stationary points” with “stationary points” and ensure coordinates/response values match Table 1.

Response 31: Thank you for your remark.

The term “special stationary points” has been replaced with “stationary points”.

Editorial changes to the text are reflected in line 690 on page 11 of the updated text in the manuscript. We have verified and confirm that the response coordinates/values ​​are consistent with Table 1.

Comments 32: Line 356–362 (Eq. 3), please, reconcile the regression model with Table 4: include main effects for X2 if significant, and avoid odd mixed higher order terms unless justified and supported by model selection criteria.

Response 32: We thank reviewer 2 for his comments. We have carefully reconciled the regression model with the data presented in Table 4 and found that the included main effects for X2 are significant. The two odd second-order mixed terms are justified and supported by the model selection criteria and cannot be removed. Editorial changes to the text are reflected in line 740-742 on page 14 of the updated text in the manuscript.

Comments 33: Line 372–379 (Fig. 3 caption), it would be beneficial to define the coded factors (“A”, “B”, etc.), the reference line (Bonferroni corrected alpha), and units of standardized effects for Pareto charts.

Response 33: We thank the esteemed reviewer 2 for his comments and recommendations. We accept them and below the text of the figures we have defined the coded factors (“A”, “B”, etc.), the reference line (corrected by Bonferroni alpha), and the units for standardized effects for the Pareto diagrams. Editorial changes to the text are reflected in line 730-735 on page 14 of the updated text in the manuscript.

Comments 34: Line 381–399 (Y2/POV), please, ensure Eq. 4 includes all significant terms from Table 5 and report adjusted R²; avoid reporting “multiple R” as negative.

Response 34: We thank the reviewer for the suggestions. We ensured that equation 4 includes all significant terms from Table 5 and reported the adjusted R²; “multiple R” as negative (- 466.0317*X12 - 315.8095*X1*X2) was removed. See line 752, page 14 of the text of the revised manuscript.

Comments 35: Line 400–406, please, correct the TBARS prose: Y3min is 0.23, control is 1.20 (Table 1), and 4.8 belongs to sensory score, not TBARS; fix all downstream references.

Response 35: We thank the reviewer for the inaccuracies noted and for the constructive suggestions. We have corrected the TBARS wording: Y3min to 0.23, and the control group to 1.20. See line 788 – 791 on page 15 of the text of the revised manuscript.

Comments 36: Line 411–418 (Eq. 5), I recommend providing 95% CIs for coefficients and verifying sign/direction with the plotted surfaces in Figure 2c.

Response 36: We thank the reviewer for constructive suggestions. The confidence intervals (CI) for the coefficients and signs have been checked and confirmed (line 797 on page 15). The direction of the plotted surfaces in Figure 2c has been changed. See line 711-719, pages 12-13 of the text of the revised manuscript.

Comments 37: Line 431–438 (Eq. 6), please, correct the notation “Y2 (peroxide value)” to Y4 (SACSS) in the caption and equation header.

Response 37: We thank the reviewer for the inconsistency noted. It has been corrected and the notation “Y2 (peroxide number)” to Y4 (SACSS) in the caption and header of equation (6) has been corrected. See line 842, page 16 of the text of the revised manuscript.

Comments 38: Line 458–476, please consider tempering the transition sentence: you concluded optimal 0.1%, then chose 3% because “relatively high AV/POV” persisted—present data supporting the saturation argument and predefine the 3% test as a separate validation

Response 38: We thank the reviewer for the recommendation. The text of this paragraph has been softened and corrected. See lines 874-875, page 17 of the revised manuscript. We do not consider it necessary to redefine the 3% test as a separate validation.

Comments 39: Line 482–499 (Table 8 + text), please, verify numbers and language: a* decreased, L* and b* increased; remove the subjective “not ‘good’ news” and quantify practical significance (ΔE*).

Response 39: We thank the reviewer for the inaccuracy noted and the suggestion for improvement made. Both have been taken into account and the appropriate corrections have been made. See lines 933 and 940 on page 18 of the revised manuscript.

Comments 40: Line 500–505 (pH), please, report absolute pH values at each timepoint (mean ± SD, n) and avoid percentage changes for pH

Response 40: We thank the reviewer for his suggestions and opportunities for improvement. These are accepted and corrections have been made. See lines 950-955 on page 18 of the revised manuscript.

Comments 41: Line 506–517 (TPA), I recommend correcting statements that use “significantly (p > 0.05)”; if not significant, say “no significant difference (p > 0.05)”.

Response 41: We thank the reviewer for the comment and the suggested improvement. These have been accepted and corrections have been made. See line 960 on page 18 of the revised manuscript.

Comments 42: Line 541–548, please, address the decrease in AV over storage in the experimental sample (1.16 → 0.40 mg KOH/g): discuss potential analytical or matrix interferences (e.g., antioxidant acids consuming KOH) and consider re testing with fat extraction before AV measurement.

Response 42: We thank the reviewer for the remark and suggestion. Following this, we contacted our colleagues in Kazakhstan. It turned out that the manufacturer had a batch of ham stored for 30 days and the experiment was repeated. It turned out that a laboratory error had been made in the initially performed analyses. The established value for AV is significantly higher - 2.40 ± 0.13 mg KOH/g. This gave us reason to accept your remark and make the corresponding corrections in Table 10 and in the text. See line 1016 on page 20 and lines from page of the revised manuscript.

Comments 43: Line 558–562, it would be beneficial to correct the reported percentage difference: 0.99 vs. 1.32 meq is ~25% lower, not “almost 16%”.

Response 43: Thank you for your remark. The suggestion has been accepted and the change has been made. See line 1039 on page 21 of the revised manuscript.

Comments 44: Line 565–577, please, ensure consistency in TBARS conclusions (factor Extract was not significant) and align with Table 10 p values

Response 44: We thank the esteemed reviewer 2 for his critical remarks. We have carefully checked the statistical data; the numbers reflected in Table 10 regarding TBARS from the paragraph indicated in the reviewer's question. We did not find inconsistency in the conclusions, as the reviewer claims. There is probably some confusion. Factor Addition of sea buckthorn extract and Interfactor interaction from Table 10 have P - value 0.781 and resp. 0.493 > 0.05. This means that neither Factor Addition of sea buckthorn extract nor Interfactor interaction have a statistically significant effect on the changes in TBARS values. It follows that the differences in mean fall within the SEM deviation. Therefore, the wording of the sentence in line 1092 on page 22 is correct and we do not need to change it.

Comments 45: Line 578–587, please, interpret FAN decreases carefully; decreasing free amines during storage is counterintuitive if proteolysis dominates—consider Maillard binding, smoke phenols interactions, or measurement variance.

Response 45: We thank the esteemed reviewer 2 for the critical comments and remarks. Indeed, the decrease in free amines during storage is illogical at first glance.

However, the following few features should be taken into account in the production of this type of traditional Kazakhstan ham.

1)                  The center of the ham reaches and exceeds 60℃ relatively soon – within 1 - 1.5 h after the start of heat treatment. All proteolytic enzymes from the sarcoplasmic fraction of muscle cells are thermally denatured at a similar temperature and are practically deactivated during the further 3-4 hours hot smoking and cooking to 73℃. Therefore, conditions for the occurrence of proteolytic processes and accumulation of low molecular weight amino derivatives of proteins do not exist.

2)                  It is very likely that the resulting free amines interact with secondary products of lipid oxidation and form products of the Maillard reaction during the smoking and cooking of ham. This is a logical explanation for the reduction of free amine nitrogen levels established by us and for the relatively low levels of TBARS, as well as for the practical absence of protein carbonyls.

3)                  The low levels of the last two categories of oxidized derivatives of the lipid and protein fractions from the ham matrix could be explained by penetration and interaction with phenolic compounds penetrating as a result of the prolonged smoking with goose smoke.

4)                  As can be seen from the data for FAN in Table 10, the factor of addition of sea buckthorn extract does not have a statistically significant effect on the FAN indicator, because the P - value is 0.316, which is greater than 0.05. Therefore, the dispersion of the measurement is large. A paragraph with similar reasoning and discussion has been added in the text of the revised manuscript. See lines 1099-1114 on page 22.

Comments 46: Line 588–594, I recommend acknowledging the limitation of sensitivity of the carbonyl assay and LOD; “below detection limit” across all days could reflect limited assay sensitivity for this matrix.

Comments 47: Line 597–604 (Table 11), please, change “mg GA/kg” to mg GAE/kg (gallic acid equivalents) and specify extraction solvent/conditions for the ham matrix.

Response 47: We thank reviewer 2 for his comments and suggestions. The extraction of phenolic compounds is described in detail on lines 499–508 of page 9. We have corrected the dimensionality of the total phenolic content (TPC) and it is expressed as mg GAE/kg. See line 1140 on page 23.

Comments 48: Line 606–616 (Table 12), please, add detection limits (e.g., <10 CFU/g), specify method validation/controls, and align days with the Methods.

Response 48: We thank reviewer 2 for his critical comments and suggestions. In Table 12, detection limits (<10 CFU/g) have been added and method controls have been indicated. Days have also been aligned with methods. See line 1154-1155 on page 23.

Comments 49: Line 617–635 (Discussion), please consider removing extensive phytochemical enumeration that is not measured here, and instead explain mechanisms relevant to cured meat (nitrite, ascorbate, polyphenols) and the observed a* decrease.

Response 49: We thank reviewer 2 for his critical comments and suggestions. In the Discussion section, we have removed the extensive phytochemical listing and instead added an explanation of the mechanisms associated with cured meat (nitrites, ascorbate, polyphenols) and the observed decrease in a*. See lines 1157-1160 on page 23 and 1224-1232 on page 24 of the revised manuscript text.

Comments 50: Line 637–647, please, correct “horse and pork ham” to camel; ensure all species references match the study.

Response 50: We thank reviewer 2 for pointing out the inaccuracy. There was a ridiculous spelling error, writing pork instead of camel meat. This error has been corrected. See line 1222 on page 24 of the revised manuscript text.

Comments 51: Line 655–668 (Conclusions), I recommend resolving the contradiction between “minor effect” and “stabilize oxidative stability”; present a cautious conclusion and align future work doses (you suggest 0.2–1.0% despite first concluding 0.1% optimal).

Response 51: We thank reviewer 2 for the suggested opportunities for improvement. We accept them and have made the corresponding editorial changes. A new paragraph with similar reasoning and conclusions has been added in the text of the revised manuscript. See lines 1248-1260 on page 24.