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 1; peer review: awaiting peer review]

Incorporating eels and tempe can replace and complement the content of proteins, macronutrients, and micronutrients, which may be Nutritional Status, Protein Diet, Preclinical Trial, Eel, Tempe, IGF-1 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.


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.

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 AE 1.39 ug/100 g) and unsaturated fatty acids (20.83 AE 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 AE 2.2 o C) 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  Table 1.

Sample collection and preparation
At the end of the intervention, the rats were fasted overnight and given ketamine 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.
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.