Effect of eel and tempe composite flour supplementation on the nutritional status biomarkers of rats with a restricted protein diet: Data from a preclinical trial

Feny Mentang; Nurmeilita Taher; Fahrul Nurkolis; William Ben Gunawan; Vincentius Mario Yusuf; Nindy Sabrina; Faqrizal Ria Qhabibi; Nelly Mayulu; Nurpudji Astuti Taslim; Happy Kurnia Permatasari

doi:10.12688/f1000research.122646.2

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Revised

Effect of eel and tempe composite flour supplementation on the nutritional status biomarkers of rats with a restricted protein diet: Data from a preclinical trial

[version 2; peer review: 1 approved, 1 approved with reservations]

Feny Mentang ¹, Nurmeilita Taher¹, Fahrul Nurkolis², [...] William Ben Gunawan³, Vincentius Mario Yusuf⁴, Nindy Sabrina⁵, Faqrizal Ria Qhabibi⁴, Nelly Mayulu⁶, Nurpudji Astuti Taslim⁷, Happy Kurnia Permatasari⁸

Feny Mentang ¹, Nurmeilita Taher¹, [...] Fahrul Nurkolis², William Ben Gunawan³, Vincentius Mario Yusuf⁴, Nindy Sabrina⁵, Faqrizal Ria Qhabibi⁴, Nelly Mayulu⁶, Nurpudji Astuti Taslim⁷, Happy Kurnia Permatasari⁸

PUBLISHED 27 Apr 2023

Author details Author details

¹ Fishery Products Technology Study Program, Faculty of Fisheries and Marine Sciences, Sam Ratulangi University, Manado, North Sulawesi, 95115, Indonesia
² Biological Sciences Department, State Islamic University of Sunan Kalijaga Yogyakarta (UIN Sunan Kalijaga Yogyakarta), Yogyakarta, Central Java, 55281, Indonesia
³ Nutrition Sciences Department, Faculty of Medicine, Diponegoro University, Semarang, Central Java, 50275, Indonesia
⁴ Medical Science Study Program, Faculty of Medicine, Brawijaya University, Malang, East Java, 65145, Indonesia
⁵ Department of Nutrition, Dietetics, and Food, Faculty of Medicine, Nursing, and Health Sciences, Monash University, Wellington Rd, Clayton VIC 3800, Australia
⁶ Nutrition and Food, Faculty of Medicine, Sam Ratulangi University, Manado, North Sulawesi, 95115, Indonesia
⁷ Clinical Nutrition, Faculty of Medicine, Hasanuddin University, Makassar, South Sulawesi, 90245, Indonesia
⁸ Department of Biochemistry and Biomolecular, Faculty of Medicine, Brawijaya University, Malang, East Java, 65145, Indonesia

Feny Mentang
Roles: Formal Analysis, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Nurmeilita Taher
Roles: Formal Analysis, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Fahrul Nurkolis
Roles: Conceptualization, Funding Acquisition, Project Administration, Writing – Original Draft Preparation, Writing – Review & Editing

William Ben Gunawan
Roles: Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Vincentius Mario Yusuf
Roles: Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Nindy Sabrina
Roles: Supervision, Validation, Writing – Review & Editing

Faqrizal Ria Qhabibi
Roles: Formal Analysis, Visualization, Writing – Review & Editing

Nelly Mayulu
Roles: Supervision, Validation, Writing – Review & Editing

Nurpudji Astuti Taslim
Roles: Supervision, Validation, Writing – Review & Editing

Happy Kurnia Permatasari
Roles: Supervision, Validation, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Agriculture, Food and Nutrition gateway.

This article is included in the Data: Use and Reuse collection.

Abstract

Incorporating eels and tempe can replace and complement the content of proteins, macronutrients, and micronutrients, which may be related to curative effects for malnutrition. In addition, converting the ingredients into a form of flour can increase their shelf life and nutrient concentration. Therefore, an in vivo approach was undertaken to explore further the nutritional status value of biomarkers in malnourished male rats (Rattus norvegicus) after Eel and Tempe Composite (ETC) flour supplementation. Data was collected from blood samples (both plasma and serum) of rats in all groups, and the appropriate biomarkers were analyzed. The final data presented in this study is openly available and can be further analyzed using statistical means to determine the dose of ETC flour as the basis of clinical trials, which other researchers can reproduce. This data may also be valuable for those interested in using different analytical methods to research the same questions or even new preclinical studies focusing solely on nutritional status biomarker analysis methods, including clinical trial prospects.

Keywords

Nutraceuticals, Nutritional Status, Protein Diet, Preclinical Trial, Eel, Tempe, IGF-1

Corresponding author: Feny Mentang

Competing interests: No competing interests were disclosed.

Grant information: This work has been supported by an award grant from The International Institute of Knowledge Management (TIIKM) Sri Lanka with the funding number ICOPH 2020 A 410.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2023 Mentang F et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Mentang F, Taher N, Nurkolis F et al. Effect of eel and tempe composite flour supplementation on the nutritional status biomarkers of rats with a restricted protein diet: Data from a preclinical trial [version 2; peer review: 1 approved, 1 approved with reservations]. F1000Research 2023, 11:1031 (https://doi.org/10.12688/f1000research.122646.2) First published: 12 Sep 2022, 11:1031 (https://doi.org/10.12688/f1000research.122646.1) Latest published: 27 Apr 2023, 11:1031 (https://doi.org/10.12688/f1000research.122646.2)

Revised Amendments from Version 1

Added details on the dosage of anesthetized rats

To read any peer review reports and author responses for this article, follow the "read" links in the Open Peer Review table.

Abbreviations

BW: Body Weight

CIOMS: Council for International Organizations of Medical Sciences

ETC: Eel and Tempe Composite

IGF-1: Insulin-Like Growth Factor 1

NP: Normal-Protein

RBP: Retinol-Binding Protein

RP: Restricted-Protein

Introduction

Prolonged protein undernutrition can result in malnutrition, anemia, and stunting (Wu, 2016). Malnutrition can manifest through many clinical parameters and biomarkers. The most-reported blood biomarkers of malnutrition are total protein and hemoglobin (Zhang et al., 2017), while serum insulin-like growth factor 1 (IGF-1) have been underreported (Keller, 2019). There is strong evidence that functional meals can help prevent and mitigate the effects of malnutrition (Ahmed et al., 2022), presumably by improving malnutrition biomarkers.

Agricultural products such as soybean (Glycine max L.) tempe is the potential source of protein and other nutrients which may improve malnutrition, anemia, and gut health due to their folate, iron, vitamin B12, probiotics, and isoflavone content (Ahnan-Winarno et al., 2021). In addition to tempe, one of the fishery products that can be used as an alternative protein-rich functional food is eel fish (Monopterus albus) which has immense nutritional contents that is high in vitamin A, vitamin E, and unsaturated fatty acids (eicosapentaenoic acid and docosahexaenoic acid) (Nafsiyah et al., 2018). A recent review also highlighted the role of unsaturated fatty acids on protein metabolism, synthesis, and muscle growth (López-Seoane et al., 2022), which are crucial in a state of malnutrition.

Combining eel and tempe may substitute and complement the protein, macronutrients, and micronutrient content, which may be linked with their malnutrition ameliorating effects. Moreover, turning an ingredient into the form of flour may increase its shelf life and nutrient concentration (Tomasi et al., 2015). Therefore, an in vivo approach was performed to further explore the value of nutritional status biomarkers on malnourished rats after a supplementation of Eel and Tempe Composite (ETC) flour. A review study by Ahmed et al. (2022) stated that only a few of the studies reported a strong link between functional foods and malnutrition.

In this study, the data was collected from the blood samples (both plasma and serum) of rats in all treatment groups, and the appropriate biomarkers were analyzed, accordingly. The final data presented in this study can be further analyzed using statistical means to define an ETC flour dose as a basis for the clinical trial, which other researchers could reproduce. This data may also be valuable for those interested in using other analytical methods to research the same research questions or even new preclinical studies focusing solely on biomarker nutrition status analysis methods, including clinical trial prospects.

Methods

Ethics statement

The protocol for this preclinical study refers to the Animal Research: Reporting in vivo Experiments (ARRIVE) Guidelines by the Institutional Animal Care and Use Committee. See the Reporting guidelines section (Nurkolis and Gunawan, 2022a) for the completed checklist. The study also adheres to the Declaration of Helsinki by The Council for International Organizations of Medical Sciences (CIOMS). This study protocol has been registered at Preclinical Trials Europe preclinicaltrials.eu (PCTE0000271). Ethical approval was obtained from the Ethics Committee at Educational General Hospital, Sam Ratulangi University (approval number 039/EC/KEPK-KANDOU/III/2022). Every effort was taken to keep the experimental animals as pain-free as possible throughout the trial and by maintaining laboratory conditions at room temperature and noise-free.

Production of ETC flour

A total of 1,000 g of fresh Asian swamp eel (Monopterus albus) were purchased live from a local market in Jakarta. The live eels were placed in a bucket measuring 21 cm in diameter with a volume capacity of 5 L (pH 5.5) and acclimatized for 24 hours in a normal light-dark cycle before being euthanized. The Asian swamp eel were euthanised using a slaughter process such as fish processing and cleaned (including removal of the bone) and steamed for 10 minutes. After drying for 12 hours at 60 °C, the meat was grounded using a meat mincer. Then, 1,000 g of soy-based tempe was chopped and steamed for 20 minutes. Before being ground, the tempe sample was steamed and then baked for 12 hours at 60 °C. Both eel and tempe were filtered using a 60-mesh filter. According to the specifications of a previously published preliminary study (Ngadiarti et al., 2022), a formulation with the highest level of folic acid (vitamin B9; 1258.53 ± 1.39 ug/100 g) and unsaturated fatty acids (20.83 ± 0.12%) was prepared by combining eel and tempe flour samples in 1: 3.5 ratio. A complete formulation of ETC flour and the results of proximate amino acids, unsaturated fatty acids, and folate contents were presented by Ngadiarti et al. (2022). The ETC formulations were conducted by Ngadiarti et al. (2022) at the Laboratory of the Ministry of Health Polytechnic Jakarta II (Poltekkes Kemenkes Jakarta II), Jakarta 12540, Indonesia (Ngadiarti et al., 2022).

Source of animals

Thirty male Albino Wistar rats (Rattus norvegicus) weighing 40.5 – 5.4 g, aged 3 – 4 weeks, were put in cages under typical laboratory settings (27 ± 2.2 ^oC) with normal light and dark cycles (12/12 hours). Before the experiment, all rats were acclimatized for a 10-day period. The minimum sample size was determined using the Frederer formula, with the equation = (t-1) (r-1)≥15. t = treatment, r = replication. t in this study is 3, then = (3-1) (r-1) ≥ 15; 2(r-1) ≥ 15; 2r-2 ≥ 15; r ≥ 8.5. So the minimum sample for each group is 8.5. The research team chose 10 (just in case there were rats that needed to be excluded, such as those experiencing moody eating and drinking) (Federer, 1966).

Experimental design

According to de Oliveira et al. (2011), all rats were supplemented with restricted-protein diet treatment (RP; 4% w/w protein) for four weeks and separated into three groups, with 10 rats per group. Shuffled blinded envelopes simple randomization (Taken at random from the population). We consider no confounders in this study since they were controlled by choosing the gender of the rats (male, not mixed) and the trials in the laboratory were carried out by a professional veterinary in animal studies. The allocation process and experiment were conducted by NM with the assistance of a professional veterinary. The assessment of the outcomes and data analysis were done by FN and FM. Rats that experienced moody eating and drinking and exhibited stress were excluded. Group C (control) was given an ad libitum diet of normal protein (NP; 23% w/w protein), but did not supplement with ETC. Rats in groups A and B got the same RP diet for 4 weeks but were orally given ETC dosages of 100 and 200 mg/kgBW. These dosages were based on the lower and higher limits for stomach safety in rats (Eichenbaum et al., 2011). Blood samples were taken and analyzed for their protein, hemoglobin, and IGF-1 serum levels. The flowchart of this study is presented in detail in Figure 1.

Figure 1. Study design (malnourished/undernourished rats).

IGF-1, insulin-like growth factor 1.

Primary outcomes for this preclinical trial are defined as:

1. Protein (mg/dL)
2. Hemoglobin (mg/dL)
3. IGF-1 (pg/mL)

Secondary outcomes for this preclinical trial define as:

1. Body Weight or BW (g) = Initial Body Weight (g); Final Body Weight (g) and Weight Gain (g/day)
2. Erythrocytes (×10⁶/uL)
3. Haematocrit (%)
4. Retinol-Binding Protein (RBP, ng/mL)

The details of feed composition are shown in Table 1.

Table 1. Composition of normal and restricted-protein diets.

Components	Control/Normal protein diet (NP; 23% Protein)	Restricted-protein diet (RP; 4% protein)
Sucrose	12.72	20.00
Cornstarch	52.75	64.25
Casein (88% protein)	23.33	4.55
Mixture of mineral salts^*	3.20	3.20
Vitamin mixture^*	1.60	1.60
Soybean oil	4.80	4.80
Fish oil	1.60	1.60
Total energy (Cal/100 g)	382	382

* The combination of salts and vitamins used in diet formulation is referred to AIN 93 recommendation (de Oliveira et al., 2011).

Sample collection and preparation

At the end of the intervention, the rats were fasted overnight and given ketamine anesthesia. The dose used was ketamine at a dose of 75 mg/kg Body Weight and referred to https://www.research.uky.edu/division-laboratory-animal-resources/recommended-anesthesia. To allow clotting at room temperature, a 2.5 mL blood sample is obtained from liver tissue and kept in a dry, clean tube without anticoagulants. Hemoglobin, hematocrit, and erythrocyte levels were determined from the blood plasma samples based on the method introduced by Arnaud et al. (2017) (see also the Biomedical analysis section for a brief description). The sample was then centrifuged for 20 minutes at 3,000 rpm to extract the protein, IGF-1, and Retinol-Binding Protein (RBP) from the blood serum for further analysis.

Biomedical analysis

A fresh blood plasma sample was used to evaluate the hemoglobin levels using the Rat Hemoglobin ELISA Kit (#AB157733). The sample was rinsed in a 1% phosphate buffer salt (PBS, pH 7.4) solution until the liquid cleared. The sample was then concentrated for 20 minutes at 3,000 rpm to produce the supernatant component. Protein levels and IGF-1 were measured with The Rat Protein Assay ELISA Kit (#MBS3808613) and The Rat IGF-1 ELISA Kit (#MBS268050) at a wavelength of 450 nm. Erythrocyte and hematocrit levels are measured using the Rat Erythrocyte Protoporphyrin ELISA Kit (#RTES01121).

Dataset

This in vivo research was successfully carried out, and the raw data from the research can be accessed via the Underlying data section (Nurkolis and Gunawan, 2022b).

Table 2 proved that undernourished rats supplemented with a higher dose of ETC flour (group B) would have a higher protein, hemoglobin, and IGF-1 serum than groups A and C. This data suggests that ETC flour may improve nutritional status biomarkers via a dose-dependent manner.

Table 2. Primary outcomes data.

Groups	A	B	C
Protein (mg/dL)	5.04 ± 0.66	5.85 ± 0.55	3.68 ± 0.54
Hemoglobin (mg/dL)	19.22 ± 0.39	21.31 ± 0.80	15.99 ± 0.79
IGF-1 (pg/mL)	281.87 ± 3.81	321.04 ± 2.07	36.53 ± 4.35

The data presented in Table 3 revealed a higher increase in weight gain in group B than in group A and C. All secondary outcomes and characteristics, except for food intake, were best in group B. These findings reinforce the previous statement that ETC flour works in a dose-dependent manner.

Table 3. Secondary outcomes and characteristics of rats.

Groups	A	B	C (Normal/NP)
Initial Body Weight (g)	44.72 ± 4.51	43.70 ± 3.44	42.37 ± 1.14
Final Body Weight (g)	161.26 ± 7.14	162.51 ±5.81	155.77 ± 4.49
Weight Gain (g/day)	4.16 ± 0.21	4.24 ± 0.21	4.05 ± 0.18
Food Intake (g)	6.30 ± 0.54	5.81 ± 0.64	5.65 ± 0.58
Water Intake (mL)	5.53 ± 0.49	5.92 ± 0.37	5.54 ± 0.56
¹FER (%)	66.44 ± 5.61	73.90 ± 9.28	72.52 ± 9.20
Erythrocytes (×10⁶/uL)	6.38 ± 0.63	7.55 ± 0.95	5.47 ± 0.66
Haematocrit (%)	56.04 ± 4.57	61.19 ± 4.51	48.09 ± 2.46
²RBP (ng/mL)	6420.02 ± 168.96	7597.92 ± 211.52	5315.94 ± 122.00

1 Food Efficiency Ratio (FER, %) = (Body Weight Gain (g/day) /Food Intake (g/day)) × 100.

2 Retinol-Binding Protein (RBP, ng/mL).

Data availability

Underlying data

Figshare: RAW Data for An Eel and Tempe Composite Flour Supplementation on the Nutritional Status Biomarkers of Rats with A Restricted Protein Diet.xlsx, https://doi.org/10.6084/m9.figshare.19782784.v1 (Nurkolis and Gunawan, 2022b).

This project contains the following underlying data:

- RAW Data for An Eel and Tempe Composite Flour Supplementation on the Nutritional Status Biomarkers of Rats with A Restricted Protein Diet.xlsx (The measured serum protein, hemoglobin, and IGF-1 levels from each group of rats observed in this study).

Reporting guidelines

Figshare: ARRIVE Checklist for ‘Effect of eel and tempe composite flour supplementation on the nutritional status biomarkers of rats with a restricted protein diet: Data from a preclinical trial’, https://doi.org/10.6084/m9.figshare.19799731.v2 (Nurkolis and Gunawan, 2022a).

Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).

Acknowledgements

Special thanks to Professor Dr. Nurpudji Astuti Taslim, MD., MPH, Sp.GK (K) (Chairman of the Indonesian Association of Clinical Nutrition Physicians), for the motivation and supervision in conducting this research during the pandemic.

References

Ahmed M, Vasas D, Hassan A, et al.: The impact of functional food in prevention of malnutrition. PharmaNutrition. 2022; 19: 100288. Publisher Full Text
Ahnan-Winarno AD, Cordeiro L, Winarno FG, et al.: Tempeh: A semi centennial review on its health benefits, fermentation, safety, processing, sustainability, and affordability. Compr. Rev. Food Sci. Food Saf. 2021; 20: 1717–1767. PubMed Abstract | Publisher Full Text
Arnaud F, Higgins A, McCarron R, et al.: Determination of methemoglobin and hemoglobin levels in small volume samples. Artif Cells Nanomed. Biotechnol. 2017 Feb; 45(1): 58–62. PubMed Abstract | Publisher Full Text
de Oliveira JC , Scomparin DX, Andreazzi AE, et al.: Metabolic Imprinting by Maternal Protein Malnourishment Impairs Vagal Activity in Adult Rats. J. Neuroendocrinol. 2011; 23(2): 148–157. PubMed Abstract | Publisher Full Text
Eichenbaum G, Damsch S, Looszova A, et al.: Impact of gavage dosing procedure and gastric content on adverse respiratory effects and mortality in rat toxicity studies. J. Appl. Toxicol. 2011 May; 31(4): 342–354. PubMed Abstract | Publisher Full Text
Federer WT: Randomization and sample size in experimentation.1966.Reference Source
Keller U: Nutritional Laboratory Markers in Malnutrition. J. Clin. Med. 2019; 8(6): 775. PubMed Abstract | Publisher Full Text
López-Seoane J, Jiménez SL, Del Coso J, et al.: Muscle hypertrophy induced by N-3 PUFA supplementation in absence of exercise: a systematic review of randomized controlled trials. Crit. Rev. Food Sci. Nutr. 2022 Jan 28; 1–1.
Nafsiyah I, Nurilmala M, Abdullah A: Nutrient Composition of Eel Anguilla bicolor bicolor and Anguilla marmorata. J. Pengolah. Hasil Pernikan. Indones. 2018; 21: 504. Publisher Full Text
Ngadiarti I, Nurkolis F, Handoko MN, et al.: Physicochemical Properties and Nutrient Content of Tempe Flour Enriched Eel Flour. Open Access Maced. J. Med. Sci. 2022 Jan. 18; 10(A): 552–556. Publisher Full Text
Nurkolis F, Gunawan WB: Arrive Checklist for An Eel and Tempe Composite Flour Supplementation on the Nutritional Status Biomarkers of Rats with a Restricted Protein Diet. figshare. Online resource.2022a. Publisher Full Text
Nurkolis F, Gunawan WB: RAW Data for An Eel and Tempe Composite Flour Supplementation on the Nutritional Status Biomarkers of Rats with A Restricted Protein Diet.xlsx. figshare [Dataset].2022b. Publisher Full Text
Tomasi N, Pinton R, Dalla Costa L, et al.: New ‘solutions’ for floating cultivation system of ready-to-eat salad: A review. Trends Food Sci. Technol. 2015; 46(2): 267–276. Publisher Full Text
Wu G: Dietary protein intake and human health. Food Funct. 2016 Mar; 7(3): 1251–1265. Publisher Full Text
Zhang Z, Pereira SL, Luo M, et al.: Evaluation of Blood Biomarkers Associated with Risk of Malnutrition in Older Adults: A Systematic Review and Meta-Analysis. Nutrients. 2017; 9(8): 829. PubMed Abstract | Publisher Full Text

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