Mostafa Gouda, Amr Moustafa, Laila Hussein, Mohammed Hamza
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1 Author s response to reviews Title: Three week dietary intervention using apricots, pomegranate juice or/and fermented sour sobya and impact on biomarkers of antioxidative activity, oxidative stress and erythrocytic glutathione transferase activity among adults Authors: Mostafa Gouda (goudarowing@yahoo.com) Amr Moustafa (amr.mostafa@fldc.cu.edu.eg) Laila Hussein (dr.lailahussein@yahoo.com) Mohamed Abd El-Hameed (mohamedhamza@agr.cu.edu.eg) Version: 1 Date: 22 Mar 2016 Author s response to reviews: Nutrition Journal NUTJ-D Three week dietary intervention using apricots, pomegranate juice or / and fermented sour sobya and impact on biomarkers of antioxidative activity, oxidative stress and erythrocytic glutathione transferase activity among adults Mostafa Gouda, Amr Moustafa, Laila Hussein, Mohammed Hamza Reviewer (1) --Manuscript Draft--
2 Manuscript Number: The authors are grateful to the reviewers for their comments and constructive critique provided. Modifications had been made according to the comments of the reviewer and hope will meet the satisfaction of all reviewers. Reviewer #1 comment: Quality of written English Needs some language corrections Authors response The language has been revised and improved so far. Reviewer #1 comment: Lines 116 and 117 : 24 years ± 0.96 Authors response corrected to ± 0.96 years Reviewer #1 comment: Lines 129 and 130 : In the text, the authors concentrated on the food items known to affect the polyphenol intake. And what about coffee and red wine Authors' response As a moslem country red wine drinking is unknown to almost all of the youths, because its drinking is prohibited by religion. Egypt is not a coffee drinking country. Reviewer #1 comment: Detailed description Lines 146 and 147 : The volunteers were divided into five groups each consisting of seven..this phrase is not enough to explain a randomized clinical trial Authors' response According to the reviewer comment, The methods regarding ingestion. A flow sheet was inserted to describe the methods regarding ingestion.
3 The study subjects were 35 healthy adults, who satisfied the inclusion criteria: young adults with age ranging between years, without metabolic diseases, not using any medication for the last 6 weeks, and with no signs of allergy or hypersensitivity to any food or material Design of the study: A randomized controlled trial was conducted and the protocol is outlined in the flow chart (1). At prefeeding, the subjects were assigned into five groups of equal number using computerbased randomization. Group (1) served as the control and didn't receive any supplement. Group (2) received daily 200 g (AF); group (3) a bottle containing 250 g of (PJ) equivalent to; group (4) a mixture of (PJ) (100 g) and (FS) (150 g) and the last group received (FS) 167 g. The volunteers in each group consumed the supplement daily between 5 6 pm. Blood and spot urine were collected at 8 a.m. at fasting state: at the beginning of the first intervention day; and at the end of 3 weeks dietary intervention day. Blood samples were used for biochemical analyses, and in combination with urine samples to ascertain treatment adherence. Reviewer #1 comment: Lines 276 to 279 Of the four test diets, the interaction between diet (different periods) and treatment (PGJ) was. The Bonferroni procedure Authors' response Statistical analysis This section was rewritten to satisfy the comments of the reviewers
4 Statistical analysis All results are expressed as the mean ±SEM. Differences in baseline characteristics were examined using an independent-samples paired Student's t test with BMI, age, and urinary total polyphenols as the dependent variables. Two way ANOVA were conducted using the general linear model procedure to assess the differences in the effect of the four dietary supplements (Apricots, pomegranate, mixture of pomegranate sour sobya or sour sobya ) and placebo on outcome measures. The models included the main effects of treatment, time and treatment time interaction. Fisher's least significant differences were used to account for multiple testing. was not carried out in the present study, but rather the least significant differences The Bonferroni procedure was not carried out in the present study, but rather the least significant differences Reviewer #1 comment: Results of two way ANOVA Authors response: Two way ANOVA table was included in the result section of the manuscript RESULTS AND DISCUSSION Reviewer #1 comment: groups? Results: Why Table 1 was not described according to randomization The ANOVA table shows no significant inter individual differences at baseline. Authors' response : According to the comments of the referee, Table 1 is presented
5 Reviewer #1 comment: The methods regarding ingestion intakes are not described in the method section Authors' response : appropriate manner. The methods regarding ingestion intakes had been written in more Reviewers # 1 comments: Discussion: The authors did not discuss the limitation of their study. Authors' response: Limitations were added to the Discussion section : Evidence obtained from the present trial is based on a healthy adult population group, may not be generalizable to individuals with a disease or specific risk factors. Inability to apply molecular markers or food-specific signatures such as metabolomic analysis is a further limitation due to protocol limitation. Reviewer #2 comments Authors wrote that natural polyphenols are considered beneficial for human health. But what about their prooxidant activities Authors response To the best of our knowledge, there is no mention in the literature about any prooxidant activities of apricots, pomegranate juice or fermented sobya. The in vitro antioxidative activities of the three food products had been reported recently (2015) by our research group. According to the antioxidative activities obtained, the present randomized control trial was carried out. Fouad M T, Moustafa A, Hussein L, Romeilah R, Gouda M (2015). In-Vitro antioxidant and antimicrobial activities of selected fruit and vegetable juices and fermented dairy products commonly consumed in Egypt. Res J Pharmac, Biol Cheml Sc 6:
6 Reviewer #2 comments: Subjects and methods: Thirty five human subjects were enrolled into the study. How many women and men were included into each group? Author response: They were five men and two women in each group Reviewer #2 comments: What about the female sex hormones (oestrogens) and their antioxidant properties. The women were non pregnant and non lactating women. Previous clinical trials to assess the beneficial effects of dietary intervention with pomegranate juice included both men and women in their studies and the results were based on pooled data of both sexes (Reference). (Tsang C, Smail N F, Almoosawi S, Davidson I, Al-Dujaili E A S (2012). Intake of polyphenol-rich pomegranate pure juice influences urinary glucocorticoids, blood pressure and homeostasis model assessment of insulin resistance in human volunteers J Nutr Sci. 1: e9. Reviewer #2 comments: Results of two-way ANOVA should be presented more particularly Authors' comment : Two way ANOVA was included to the results Reviewer # 2 comment Discussion : Authors should provide study limitation Authors response Limitation had been added to the discussion section Evidence obtained from the present trial is based on a healthy adult population group, may not be generalizable to individuals with a disease or specific risk factors. Inability to apply molecular markers or food-specific signatures such as metabolomic analysis is a further limitation due to protocol limitation.
7 Reviewer #3 comments: Quality of written English Not suitable for publication unless extensively edited Authors response : The manuscript had been extensively edited. Reviewer #3 comments: Introduction section. Abbreviations used should be clearly indicated.. Authors response : All abbreviations had been clearly indicated Reviewer #3 comments: The used units need to be revised and adapted to the International System units Authors response : The units used were adapted to the International System units Reviewer #3 comments: Material and method section: A more detailed and organized description of the dietary intervention is needed Authors response : The Material and method section was written according to the comment and a flow shart was included. Reviewer #3 comments: The description of the experimental group is unclear Authors response : The experimental group are described appropriately. Reviewer #3 comments: (as for example creatinine analysis) is excessively long and should be abbreviated. Authors response : The description of creatinine analysis had been abbreviated.
8 Reviewer #3 comments: Results should be organized with the same scheme Authors response : The comments of the referee were considered in organizing the Results text and tables Reviewer #3 comments: In the Table 1 some parameters that are not described in Materials and Methods section "Estimated energy intake".. Authors response : The comments of the referee were considered Reviewer #3 comments: Discussion section : it is a bit chaotic and should be rewritten following the presented results Authors response : The comments of the referee were considered Reviewer #3 comments: Citations should be uniformed following the journal style Authors response : The comments of the referee were considered Reviewer #3 comments: English language should be deeply revised Authors response : The comments of the referee were considered Reviewer #4 comments: Table 2: Intake of Lactobacillus counts in group 4. Authors response : Thanks to the referee. His comments were rechecked and the bacteria counts corrected Reviewer #4 comments: The study design suffers from poor rationale and insufficient number of subjects Authors response : The sample size was adequate and comparable to those reported earlier by other investigators. The following article is an example.
9 van Baarlen P, Troost F, van der Meer C, Hooiveld G, Boekschoten M, Brummer R J M, Kleerebezem M (2011). Human mucosal in vivo transcriptome responses to three lactobacilli indicate how probiotics may modulate human cellular pathways. Proceed Natl Acad Sc Reviewer #4 comments: the duration of dietary intervention Authors response : The majority of the dietary interventions reported in the literature were terminated after three weeks. Kindly note that such studies are expensive in terms of time and money. The following articles are typical examples Ahlroos T, Tynkkynen S. (2008)..Quantitative strain-specific detection of Lactobacillus rhamnosus GG in human faecal samples by real-time PCR.J Applied Microbiol 106 : Reviewer #4 comments: The parameters studied to achieve the objective are incoherent and inconclusive Authors response : Realistic and meaningful effects were obtained with the measured outcomes. In the present study, the guide to designing and conducting studies involving probiotic applications in human participants. Important strengths of the study are the use of standardized, validated instrument to measure and linear regression models were conducted to predict the best fit. Shane A L, Cabana M D, Vidry S, Merenstein D, Hummelen R, Ellis C L, Heimbach J T, Hempel S, Lynch S V, Sanders M E, Tancredi D (2010). Guide to designing, conducting, publishing and communicating results of clinical studies involving probiotic applications in human participants Gut Microbes 1:4,
10 Introduction and Scope The seventh Annual Conference of the International Scientific Association for Probiotics and Prebiotics (ISAPP) was held from November 4 5, 2009 in Irvine, California. ISAPP is an inter national collaboration of scientists and representatives from industry who exchange views and discuss topics related to sci entific application of probiotics and prebiotics. The discussions of the 16 member work group titled, Parameters for Designing, Publishing and Communicating Human Studies on Human Probiotics formed the basis of this review. The growing interest in the application of probiotics to the maintenance and improvement of human health is evidenced by a recent National Library of Medicine database search using the keywords probiotic, human, clinical trial spanning the dates 1992 to Over 1,186 peerreviewed publications identified; of which 338 were review articles. Due to the interest in clinical research studies in humans involving probiotics, the ISAPP work ing group set out to identify the challenges of designing, conduct ing and interpreting results from human probiotic studies and to develop strategies to serve as standards for future endeavors. This review will discuss study design, target populations, selec tion of placebo and probiotic microorganism(s), duration of fol low up, outcome and endpoint measurements, safety assessments and regulatory considerations. The issues associated with each of The heterogeneity of human clinical trials to assess the effectiveness of probiotics presents challenges regarding interpretation and comparison. Evidence obtained from clinical trials among a population with a disease or specific risk factors may not be generalizable to healthy individuals. The evaluation of interventions in healthy persons requires careful selection of outcomes due to the absence of health indicators and the low incidence of preventable conditions. Given the tremendous resources invested in such trials, development of consistent approaches to assessing the effectiveness of probiotics would be beneficial. Furthermore, the reporting, presentation and communication of results may also affect the validity of the scientific evidence obtained from a trial. This review outlines the challenges associated with the design, implementation, data analysis and interpretation of clinical trials in humans involving probiotics. Best practices related to their design are offered along with recommendations for enhanced collaboration to advance research in this emerging field. *Correspondence to: Andi L. Shane; ashane@emory.edu
11 Submitted: 03/31/10; Revised: 06/15/10; Accepted: 06/16/10 Previously published online:
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