Evaluating the Therapeutic Potential of Catfish and Fermented Sweet Potato Chocolate Paste on Malnutrition: Effects on BMI, Blood Serum, and Faecal Based Gut Inflammation in Protein-Deficient Rats

Evaluating the Therapeutic Potential of Catfish and Fermented Sweet Potato Chocolate Paste on Malnutrition: Effects on BMI, Blood Serum, and Faecal Based Gut Inflammation in Protein-Deficient Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluating the Therapeutic Potential of Catfish and Fermented Sweet Potato Chocolate Paste on Malnutrition: Effects on BMI, Blood Serum, and Faecal Based Gut Inflammation in Protein-Deficient Rats Annis Catur Adi, Wizara Salisa, Emyr Reisha Isaura, Febi Dwirahmadi, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5694378/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The number of stunting cases in Indonesia that reached over 21% in 2023 has been an alarming issue. Protein and mineral insufficiency as well as intestinal inflammatory as determinants can lead to worsening condition. Providing additional food by combining animal and plant-based protein in the form of a ready-to-eat food is beneficial to alleviate stunting. This study aimed to ensure the standard quality, nutritional value, level of acceptance, and outcomes of the combination of catfish, fermented sweet potato, and isolated soy protein (ISP) flours in the form of chocolate paste for stunting biomarkers. The research process encompassed product development, evaluation of product and its compliance with the regulatory standards of food contaminants, nutritional content, hedonic tests (by the panelists), and pre-clinical testing in animal models of malnutrition induced by daily protein restriction (4% protein/day). Testing the effects of substituted products on experimental animals included monitoring growth rates (body weight and body mass index), blood serum profiles, and intestinal inflammation biomarkers through faecal samples. The findings implied that the use of catfish flour 3,3%, fermented sweet potato 9,8%, and ISP 3,3% on chocolate paste classified as a high-protein product with diverse arrays of minerals. This product fulfilled the requirements for additional food standards, and met the panelists' acceptance with a mean overall score of 4.08 (like). In malnourished rats, substituted chocolate paste proven to significantly affected (p < 0.05) blood serum (albumin, total protein, calcium, zinc, iron), also significantly preventing intestinal inflammation to increase (p < 0.01), by faecal calprotectin and myeloperoxidase (MPO) value. Biomarker profile catfish fermented sweet potato intestinal inflammation malnutrition Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION Stunting, health retardation due to multidimensional aspects such as prolonged nutrient deficiency, improper adapted consumption style in a household, limited access to health facilities, nutritious food, and clean water ( 1 ). In Indonesia, stunting is still a very serious nutritional problem. In 2023, the prevalence of stunting was 21.5% ( 2 ). Despite declining from the prior year which was 21.6% ( 3 ), it still neither meets the Indonesian Medium-Term National Development Plan 2020–2024 which is 14% ( 4 ) nor the WHO criterion which must not surpass 20% ( 5 ). The outcomes of stunting involve physical disability burden, increasing potential for chronic diseases, and socio-emotional and motor development delays ( 6 ). The adequacy of protein intake, animal and plant protein, plays a pivotal role in boosting growth and development. Protein acts as a stimulator which releases hormones for cell proliferation and differentiation ( 7 ). Previous research that has been conducted generally only focuses on the function of protein in relation to malnutrition, but has not yet implemented how product formulas containing a mixture of proteins from natural ingredients can affect growth. This is also projected to be a form of health independence through the implementation of SDGs 12 sustainable consumption and production. As a freshwater commodity, catfish, is a high source of protein and amino acid, affordable price, easy to cultivate, and minerals, especially a source of calcium that is easily absorbed by the body ( 8 – 10 ). Aside, plant protein is also needed because plant protein is a high-quality protein, it contains not only protein but also vitamins (A, C, B1, B2, B3) and minerals (potassium, calcium, magnesium, natrium, phosphorus, and iron) ( 11 – 12 ). Sweet potatoes that are fermented are known to increase protein content up to 5 times. Another vegetable protein chosen is isolated soy protein because it is composed of 97% protein ( 10 ). The success of stunting management through improving protein consumption can be assessed from the results of blood serum examination and analysis of inflammation in the digestive system. Inflammation can be caused by dysbiosis of the intestinal microbiota due to a lack of essential amino acids. In fact, intestinal microbiota affects child growth through the metabolic process and production of IGF-1, encouraging organ and systemic growth and skeletal growth mediators ( 13 ). Metabolites produced by intestinal microbiota such as Enterobacteriaceae and Firmicutes/Bacteroidetes are associated with intestinal inflammation found in stunted children. Diet determines the composition of the microbiota, such as a diet of vegetable and animal protein can increase Prevotella and Bacteroides in the intestine ( 14 ). The additional food that is widely available today is in the form of biscuits, so it often causes boredom and decreases acceptance. The evaluation output shows that the acceptance of biscuits in toddlers is only 32.2% ( 15 ). Providing additional food in new forms (other than biscuits) that contain complete protein and nutrients is an alternative solution for handling stunted children that is more practical, more desirable, and nutritious. This study aimed to develop an additional food based on local food containing animal and plant protein, and tested its effectiveness on low-protein induced experimental animals. MATERIALS AND METHODS Research design This study adopted a completely randomized experimental research design. The research encompassed product development, organolpetic testing, nutritional content analysis, and efficacy tests on experimental animals. Chocolate paste manufacturing process were carried out in Nutrition Laboratory of Nutrition Department at the Faculty of Public Health, Universitas Airlangga, located in Surabaya, East Java, Indonesia. The nutritional content analyses and food contamination tests were conducted at the Saraswanty Laboratory in Bogor, West Java, Indonesia. Further, preclinical testing on experimental animals was conducted in the Biochemical Laboratory at the Faculty of Medicine, University of Airlangga. Development of additional food Chocolate paste is a snack that has a delicious taste, simple ingredient composition, and is easy to make. The processing technique requires minimal heating, thus reducing the possibility of damaged nutrients. To make chocolate paste achieve the research objectives, ingredient substitutions were carried out. The substituted ingredients are catfish flour ( Clarias gariepinus ), fermented sweet potato ( Ipomoea batatas ) flour, and isolated soy protein. Fresh catfish purchased directly from catfish breeders in Bolorejo Village, Kauman District, Tulungagung, East Java, Indonesia, meanwhile the sweet potato from a farmer in Mojosari Village in the same district with catfish breeders. Initially, the process of producing catfish flour began with cleaning and washing the fresh catfish, separating the meat and the bone (including the head), softening by boiling it at high pressure, pressing and draining, drying (sun drying and roasting), flour milling, and filtering. Meanwhile, the process of producing fermented sweet potato flour began with pealing of the skin, washing, cutting, soaking with probiotics (12 hours, temperarture 30 o C), draining, drying using sunlight, and the last is milling using a machine (16). The chocolate paste production process adapted Indonesian patent number S00202416043, shown in Figure 1. Product characterization The Indonesian National Standard number 7934: 2014 (17) evaluated the product's characteristics, focusing on chocolate paste. This standard pertains with the evaluation of the physical properties of chocolate paste, metal composition, and other food contaminants such as insects, foreign objects, and bacterias. These tests were undertaken to ensure that the product complies with the established food safety standards. Nutrition value test The product's proximate composition was determined by standardized methods developed by the Association of Official Analytical Chemists (AOAC). In addition, the nutritional content, comprising 10 vitamins and 6 minerals, was also assessed through the Ultra Performance Liquid Chromatography (UPLC) method. Acceptability test The acceptability of the product was assessed using hedonic tests, examining four parameters including texture, aroma, color, and flavor. The untrained panelists involved in this test are 30 school-aged childrens (all participants are elementary school students) selected based on the following criteria: aged 6 to 12 years, no allergies to fish, dairies, chocolate, and gluten. The data were obtained by administering a questionnaire using a 5-point hedonic scale. The scale graded from 1 to 5, with 5 indicating a strongly like, to 1 indicating the least favorable preference (“strongly dislike”). Animal model and diet intervention Male Wistar Rattus norvegicus , weighing 100-150 g, were purchased from the Farma Veterinary Center (Pusvetma) Surabaya, and housed for 7 days without specific treatment for acclimatization. The animal models were then divided into five groups consisting of seven rats, the healthy group (K0), the low protein diet group (K1), and the three other groups were intervention groups (PP). During the malnutrition induction period, the K0 group received a standard diet, while the K1 and all PP groups received a low-protein diet. The standard diet contained 10.72% w/w protein, while the low protein diet contained 4% w/w protein (18-19). The conditioning of malnutrition was done for 21 days to mimic the protein deficiency. After 14 days, the animals in each group were tested for albumin and total protein levels to ensure their malnutrition induction. All rats continued the given diet until 14 days, while the PP groups received the following treatment; PP1: low protein diet with government supplementary biscuits, PP2: low protein diet with substituted chocolate paste, and PP3: standard protein diet with substituted chocolate paste. The dosage of biscuits is 120 g x 0.018 = 2.16 g/day, and chocolate paste formula is 40 g x 0.018 = 0.72 g/day. The number of 0.018 is obtained from the conversion of human doses to rat doses (20). Subsequently, the anthropometric measurement of each group was monitored until the intervention ended. Anthropometry measurement of experimental animals Body weight was measured by a digital weight scale. Body length (nose to anal) was measured by a ruler. Body mass index (BMI) was measured by dividing anthropometrical measurement results of body weight and body length. Blood serum analyses Blood serum was collected on the last day of the intervention. The rats were anesthetized, then their blood was taken from the heart. After the blood serum samples were collected, laboratory analysis was carried out. Total protein analysis was assessed using the Kjeldahl method. Albumin analysis was assessed using the Bromocresol green dye . Calcium, zinc, and iron were assessed using the Atomic Absorption Spectrometer (AAS). Fecal neopterin and calprotectin analyses Neopterin and calprotectin are measured to determine inflammatory conditions of the digestive system that affect nutrient absorption. Previously, we collected rat’s faecal samples along with blood serum sample collections. Faecals were extracted in phosphate buffer saline (PBS) to make the suspension and then centrifuged to separate the pellets and suspension. The concentration of neopterin and calprotectin were measured using enzyme-linked immunosorbent assay (ELISA) methods, following the manufacturers' instructions. The biomarkers were determined using Mouse ELISA Kits from BT LAB (Zhejiang, China). The stool suspensions that were previously prepared were thawed, vortexed, and centrifuged and the supernatant was used for analysis. Ethics approval and consent to participate The authors confirm that all methods were carried out by relevant guidelines and regulations of daily protein restriction which is adapted from other similar research (18-19). The animal experimental protocols were approved by the Health Research Ethical Clearance Commission of the Faculty of Dental Medicine at the University of Airlangga, under reference number 0522/HRECC.FODM/V/2024. The collection of the ingredients used in the study complies with local or national guidelines with no need for further affirmation. Specifically for the acceptance test using the hedonic test, we involved school-age children. Previously, an application for ethical protocols, including the informed consent document was made, which had been approved by the same Ethical Committee (Faculty of Dental Medicine at the University of Airlangga), number 0534/HRECC.FODM/V/2024. The informed consent document has explained the method of implementing the hedonic test (by tasting each product), and the possible side effects experienced such as diarrhea or vomiting. As a form of preventing side effects, participants’ criteria were applied (not having allergies), and as a form of overcoming side effects, researchers have collaborated with local health clinics. The document is used as a statement of participation, which is approved (signed) by all the parents/legal guardians of each student. Statistical analysis Before analyzing the differences, all data underwent an assessment to determine the normality and homogeneity of variance using the Shapiro-Wilk test, then continued by assessing the differences in data using the Anova test or the Kruskal-Wallis test. Specifically for groups that have significant differences, the Tukey and Mann-Whitney Anova tests will be continued to analyze further and find out which 2 groups have significant differences. All tests used a significance level of α = 0.05. Statistical analysis was performed using IBM Statistics SPSS 20 software. RESULTS AND DISCUSSION Characteristics of products Figure 2 depicts the visual of the chocolate paste from the combination of catfish flour and fermented sweet potato flours and the original chocolate paste. Visually there is no significant difference between the two products, both color are brown, only Table 1. Characteristics and food contamination test of chocolate paste from the combination of catfish and fermented sweet potato flours Table 1 outlines the food contamination test results, including chemical and biological contaminants. The results implied no chemical contaminants detected, such as lead, cadmium, tin, mercury, or arsenic, and likewise, the biological contaminants showed low levels of Escherichia coli, Staphylococcus aureus, Bacillus cereus , yeast mold and no harmful level of Salmonella sp. Nutritional value Table 2 presents the nutritional value of substitued chocolate paste formula compared to the standard of supplementary food for wasting toddlers and recommended dietary allowance (RDA) for toddlers (aged <6 years old) (22). The comparative results in both regulations showed protein content was already met even surpasses the requirements for supplementary food category and toddlers’ RDA. The ingredients used seem to corroborate its nutritional values. Table 2. Nutritional value of chocolate paste from the combination of catfish and fermented sweet potato flours per 100 g Catfish flour which has 46 g of protein (76.7%) considered a high-protein product according to the criteria outlined by the Indonesian Agency for Drug and Food Control (23) which declared that a product can be classified as a rich/high-protein if it contains minimum 21 g (35%) per 100 g of solid product. This emphasizes that catfish flour becomes one of the high-quality protein components that can elevate protein content in ready to eat products (24). The existence of catfish is very easy to find, because it is spread in many freshwater areas. It is not only in Southeast Asia, but also in greater Asia, South America, Africa, North America, North Europe, Australia, Madagascar, and New Guinea (39-40). Because the high yield of catfish is inversely proportional to its use, it is necessary to process fresh ingredients into semi-finished products, one of which is flour. so that the shelf life of the product is longer, and can be processed into various products. Not only does catfish flour contribute greatly to boosting the protein content, but fermented sweet potato flours and isolated soy protein are also validated in giving magnificent impact in terms of protein and arrays of minerals. The fermentation process in sweet potatoes formed a single cell protein by breaking down glucose into pyruvic acid through a glycolysis reaction which will then produce amino acids (25). Isolated soy protein has 21 g, making them also included as rich/high protein components (23). The combination of these ingredients has succeeded in creating a chocolate paste product that can meet the daily protein needs of children aged <6 years up to 25%. Acceptability ( organoleptic test results) A hedonic test was conducted to assess the organoleptic characteristics of the chocolate paste formula. Two formulas (control and substituted) were involved. Table 3 displays the score of each parameter. Regarding the findings presented in Table 3, the modification formula was found to have an overall average score of over 4.08 (like). Table 3. Acceptability of original/control and substituted chocolate paste The statistical analysis exhibited no resemblance in terms of color between control and substituted formula (p>0.05). Nevertheless, it was notable that the substituted formula had a lighter color appearance than the control formula (Figure 2). Basically, catfish flour and isolated soy protein have maillard reaction ability. The Maillard reaction is a chemical reaction between amino acids and reducing sugars to create melanoidins, the compounds that give browned food its distinctive flavor (26). However, because the main ingredient used is chocolate paste, the color appearance is not affected by the maillard reaction. Concerning its flavor, statistically, it revealed a significant distinction (p<0.05) among this pair (control formula and substituted formula). The high proportion of dry ingredients (catfish flour, fermented sweet potato flour, and isolated soy protein) added causes the chocolate paste substitution formula to have a lower sweetness level than the control formula. In the substitution formula, there is also the addition of fish oil which aims to increase the fatty acid content of the product, but has a side effect in the form of a fishy taste to the product. Apart from that, the panelists' preference scores are still in the liking category (>4.0). The aroma parameter has similarities with the assessment results for the flavor parameter that there was a significant difference (p4.0). The aroma of the original and substituted formulas was distinguished by the addition of fish oil. The addition of fish oil not only makes a significant difference in flavor, but also in the aroma. Meanwhile based on the texture, it visually conveys a difference among this pairs. From figure 2, it can be saw that the control formula has a smoother and oilier texture, while the substitution formula looks denser and a little drier. This is because the percentage of dry ingredients used in the substitution formula increases from the initial formula (control). Precedent research revealed that isolated soy protein can increase brittleness because its dryness (27). This resulted from the insufficiency of fat and oil bonded by protein (28). Catfish flour is also accountable for fracturability, depicting brittleness and easy-to-crack levels. Catfish flour has 2.17% ash content, while the regulation states that the maximum level of ash content is 1.5% (29). The higher the ash content, the higher the ability to absorb liquid, because the original minerals contained in flour can generally weaken the gluten structure formed in the dough/product (30). Overall, the organoleptic elements of chocolate paste from the combination of catfish flour, fermented sweet potato flour, and isolated soy protein obtained good scores, with average values is 4.08 of 5.00, indicating these formulas are likely well-accepted. The growth rate of the animal model during malnourished induction Based on the results of anthropometric measurements during the malnutrition induction period (Figure 3 & 4), it is known that there is a trend of decreasing body weight and BMI in the group given low-protein feed induction. While the K0 group (standard feed) still experienced an increase in body weight and BMI. The normal BMI limit for male rats is [0.45 ± 0.02–0.68 ± 0.05] g/cm2 and for female rats [0.4504–0.5044] g/cm2 (31). The average BMI of rats in the group given low-protein diet intervention was in the range of 0.44 – 0.46 g/cm2. This indicates that low-protein feed (4%/day) treatment for 21 days can cause rats to experience decreased growth to the point of experiencing a BMI below the normal limit. The results of substituted biscuits intervention on malnourished animals The body weight of all groups of rats during the intervention period showed an increase, both in the treated and untreated groups. The results of the BMI calculation also showed that all groups experienced a periodic increase, although in the K1 group, the increase tended to be flat, while in the intervention group with substituted chocolate paste (PP2 & PP3), the graph tended to increase sharply (Figure 4). The increase in BMI of rats in the intervention group (PP) still had a positive effect compared to the positive control group (K1), although statistically it did not show a significant difference (Table 4). This indicates that the treatment group (consumed supplementary food) was able to catch up on its growth delays and return to its normal BMI. Table 4. Analysis of BMI and blood serum on malnourished rats after intervention Statistical tests were also carried out on blood serum parameters, showed in Table 4. In contrast to the results of the difference test on BMI, all the variables in the blood serum sample test were found to have significant differences (p <0.05) between groups, which means that the provision of supplementary food affected its blood serum levels. Normal levels of albumin in rats are 3.3 - 4.2 g / dL, total protein 6.0 - 8.2 g / dL, calcium 8.5-10.9 mg / dL (20, 32-35). Substitution of catfish and fermented sweet potato flour in chocolate paste (PP2 & PP3) found to have significant differences compared to the control positive (malnutrition) group (K1), these indicates that the substituted ingredients were effective in improving blood serum values (albumin, total protein, and calcium) and maintained their levels within normal limits. Meanwhile the group with supplementary food in the form of government biscuit showed almost the same results with the chocolate paste intervention, except for the albumin serum levels, which is still low (below normal limits). Faecal calprotectin, myeloperoxidase, and neopterin as malnutrition biomarkers after intervention period In figure 5, the increase in growth after the addition of additional food was confirmed by the measurement of intestinal inflammation biomarkers, calprotectin, myeloperoxidase (MPO), and neopterin. From the results of the difference test between groups showed a significant difference (p<0.05) in calprotectin and MPO, especially the chocolate paste treatment group (PP2 & PP3) compared to the positive control (malnutrition) group (K1), whereas when compared to the normal group (K0) there was no significant difference (p>0.05), so it was assumed that the conditions of the groups tended to be similar. Calprotectin and neopterin are biomarkers associated with intestinal inflammation, which reflect the severity of the cell-mediated immune response in the intestine. There is no definite limit to determining intestinal inflammation for each biomarker, but a significant increase in levels indicates inflammation (36-38). The limitation of this study found in the rat malnutrition model used. It may reflect human malnutrition which caused by nutrient deficiencies, but may not fully reflect the complexity of human malnutrition, which involves environmental factors, and chronic infections. However, considering that this substitute food innovation emphasizes nutritional content and the use of local ingredients, the expected output results also focus on parameters directly related to nutrients. In addition, preclinical testing on experimental animals is at least an initial stage of proving the theory which can then be continued with clinical trials on humans by adjusting the factors experienced by stunted toddlers. CONCLUSIONS The research findings indicate that chocolate paste from the combination of catfish, and fermented sweet potato flours offers a sufficient supply of nutrients, particularly protein, calcium, and fat acid (omega), also in line with supplementary food regulations for wasting toddlers. The findings of the organoleptic test reveal that the panelists expressed positive preferences (like) for both formulas. The substituted chocolate paste demonstrated consistent and standardized quality, as outlined in SNI 7934: 2014, and are free from any food contaminants. Based on the efficacy test on malnourished rats, substituted chocolate paste proven to be able to increase body weight and BMI from a malnourished condition to be equivalent to a healthy condition, and significantly affect albumin, total protein, calcium, zinc, and iron serum levels so that they have the same condition as the normal group. Furthermore, substitution of chocolate paste ingredients can influence intestinal inflammatory biomarkers, by prevented an increase in calprotectin and MPO levels. Declarations DATA AVAILABILITY The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. RESEARCH FUNDING Airlangga University financially supported this project under the Airlangga Superior Research scheme with contract number 373/UN3.LPPM/PT.01.03/2024. 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(2015) ‘The Effect Of Mangifera Foetida Leaves Extract Towards The Levels Of Albumin And Total Protein Serum In Protein Energy Malnutrition-Induced Rats’, Penelitian Gizi dan Makanan 38(2):133–8. Drake, T. M., & Gupta, V. (2022). Calcium . Treasure Island (FL): StatPearls Publishing. UCLA. (2013). Serum Chemistry Reference Range – Mouse . Los Angeles: Division of Laboratory Animal Medicine. R. M. Ayling and K. Kok, ‘Fecal Calprotectin’, Adv Clin Chem, vol. 87, pp. 161–190, Jan. 2018, doi: 10.1016/bs.acc.2018.07.005 . T. Pang, S. T. Leach, T. Katz, A. S. Day, and C. Y. Ooi, ‘Fecal biomarkers of intestinal health and disease in children’, Jan. 01, 2014, Frontiers Media S.A. doi: 10.3389/fped.2014.00006 . B. Seethaler et al. , ‘Biomarkers for assessment of intestinal permeability in clinical practice’, Am J Physiol Gastrointest Liver Physiol, vol. 321, no. 1, pp. G11–G17, Jul. 2021, doi: 10.1152/AJPGI.00113.2021 . Lundberg, John G. and John P. Friel. 2003. Siluriformes. Catfishes. Version 20 January 2003 (under construction). http://tolweb.org/Siluriformes/15065/2003.01 . 20 in The Tree of Life Web Project, http://tolweb.org/ Bruton, M. (1996). Alternative life-history strategies of catfishes. Aquatic Living Resources, 9 : 35–41, doi: https://doi.org/10.1051/ALR%3A1996040 Tables Table 1. Characteristics and food contamination test of chocolate paste from the combination of catfish and fermented sweet potato flours Parameter Unit Standard* Result Keterangan Taste - Khas, normal Normal, sweet Fulfilled Smell - Khas, normal Normal Fulfilled Color - Khas, normal Chocolate Fulfilled Ash content % - 2.32 Fulfilled Water content % - 1.07 Fulfilled Metal contamination: Lead (Pb) mg / kg 0.0018 Not detected Fulfilled Cadmium (Cd) mg / kg Max. 0.5 0.06 Fulfilled Tin (Sn) mg / kg 0.0016 Not detected Fulfilled Mercury (Hg) mg / kg Max. 0.03 0.02 Fulfilled Arsenic (As) mg / kg 0.0003 Not detected Fulfilled Microbes contamination: Total Plate Number colony / g Max. 1x10 4 1.0x10 4 Fulfilled Coliform APM/g - 4.3 Fulfilled Escherichia coli APM / g <3 0 Fulfilled Salmonella sp. /25 g Negative/25 g Negative Fulfilled Staphylococcus aureus colony / g Max. 1x10 2 <10 Fulfilled Bacillus cereus colony / g 2.1x10 3 2.1x10 3 Fulfilled Yeast mold colony / g <10 <10 Fulfilled *Source: Indonesia National Standard for Chocolate Paste 7934: 2014 (16) Table 2. Nutritional value of chocolate paste from the combination of catfish and fermented sweet potato flours per 100 g Nutrients Nutritional Value (100 g) Standards a (100 g) Status Nutritional Value per serving (40 g) RDA for <6 years b %Fulfillment c Energy (kcal) 562.7 Min. 400 Fulfilled 225.1 1400.0 16.1 Protein (g) 10.9 8 – 12 Fulfilled 4.4 25.0 17.4 Fat (g) 35.3 10 – 18 Fulfilled 14.1 50.0 28.2 Carbohydrate (g) 50.6 Not set Fulfilled 20.2 220.0 9.2 Vitamin A (mcg) 0.0 200 – 400 Not Fulfilled 0.0 450.0 0.0 Vitamin D (mcg) 47.4 5 – 10 Fulfilled 19.0 15.0 126.4 Vitamin E (mg) 7.4 3 – 6 Fulfilled 3.0 7.0 42.3 Vitamin K (mcg) 7.1 4 – 6 Fulfilled 2.8 20.0 14.2 Vitamin B1 (mg) 0.0 0.25 – 0.5 Not Fulfilled 0.0 0.6 0.0 Vitamin B2 (mg) 0.2 0.3 – 0.6 Not Fulfilled 0.1 0.6 13.3 Vitamin B3 (mg) 0.4 2.5 – 5 Not Fulfilled 0.2 8.0 2.0 Vitamin B6 (mg) 0.0 0.2 – 0.4 Not Fulfilled 0.0 0.6 0.0 Vitamin B12 (mcg) 0.0 0.35 – 0.7 Fulfilled 0.0 1.5 0.0 Folic acid (mcg) 390.7 60 – 120 Fulfilled 156.3 200.0 78.1 Iron (mg) 7.8 4 – 7.5 Fulfilled 3.1 10.0 31.2 Iodine (mcg) 72.3 60 – 120 Fulfilled 28.9 120.0 24.1 Zinc (mg) 1.9 2 – 3.75 Not Fulfilled 0.8 5.0 15.2 Calcium (mg) 351.5 225 – 450 Fulfilled 140.6 1000.0 14.1 Selenium (mcg) 0.0 7 – 14 Not Fulfilled 0.0 21.0 0.0 Phosphor (mg) 260.7 180 – 275 Fulfilled 104.3 500.0 20.9 Fluoride (mg) 4.3 Max. 0.25 Not Fulfilled 1.7 1.0 172.0 Sodium (mg) 86.4 Max. 300 Fulfilled 34.6 900.0 3.8 Omega 3 (g) 0.3 0.4 – 0.6 Fulfilled 0.1 0.9 13.3 Omega 6 (g) 2.7 1.7 – 2.9 Fulfilled 1.1 10.0 10.8 Omega 9 (g) 9.3 Not set Fulfilled 3.7 Not set - Note: a ) Regulation of the Indonesian Minister of Health Number 51 Year 2016 (22). b ) Regulation of the Indonesian Minister of Health Number 28 Year 2019 (21). c ) The percentage of fulfillment is obtained from the nutritional value per serving (40 g) divided by the RDA for <6 years. Table 3. Acceptability of original/control and substituted chocolate paste Parameter Control Substituted Color 3.90 3.93 Flavor 4.59 4.07* Smell 4.62 4.03* Texture 4.21 4.31 Overall mean 4.33 4.08 *)Significantly different (p-value <0.05) using Kruskal-Wallis test Table 4. Analysis of BMI and blood serum on malnourished rats after intervention Group BMI Albumin Total Protein Calcium Zinc Iron Mean ±SD Mean ±SD Mean ±SD Mean ±SD Mean ±SD Mean ±SD K0 0.50 0.05 3.58 b 0.42 6.43 b 0.18 8.55 b 0.63 125.04 b 5.22 170.26 5.86 K1 0.45 0.07 1.77 a 0.32 5.57 a 0.31 5.65 a 0.73 116.40 a 4.93 161.84 6.64 PP1 0.47 0.03 3.03 a b 0.28 6.47 b 0.25 9.12 a b 0.28 132.85 a b 3.97 172.52 b 3.90 PP2 0.49 0.06 3.23 b 0.28 6.85 b 0.38 9.63 ab 0.56 131.68 ab 1.87 175.04 b 0.87 PP3 0.49 0.10 3.47 b 0.40 7.02 ab 0.43 9.87 ab 0.34 128.21 b 2.35 175.94 b 4.34 Sig. 0.761 0.001* 0.000* 0.000* 0.000* 0.016* *)significantly different (p<0.05) between groups a )significantly different (p<0.05) compared to K0 b )significantly different (p<0.05) compared to K1 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 06 May, 2025 Editor assigned by journal 06 May, 2025 Reviews received at journal 28 Apr, 2025 Reviewers agreed at journal 24 Apr, 2025 Reviews received at journal 22 Apr, 2025 Reviewers agreed at journal 22 Apr, 2025 Reviewers invited by journal 22 Apr, 2025 Submission checks completed at journal 17 Apr, 2025 First submitted to journal 24 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5694378","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":446350872,"identity":"bab69022-53a7-4c03-8c72-374f2170408b","order_by":0,"name":"Annis Catur Adi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIie2RMUvEMBTHXwnUJdg1LtevkCK46X2VysF1qdCx4GDgwFvqJ/BLCILzC4FzKc6B3KDLzb3NSXwtPXRI1dEhvyXh8X7h/14AAoF/Co5n9Frj9woD+aPCqUW2f1VgVGJxUA54leT+Tut3uEjn4mpXY/sE0q3eTE0VOGIPPkVsX3LDYZE1ojizaB3I7UbqliqKxZU3kC2lofBRI5ax6zpSbC61ApYD495gKSkUDOe9UuGgFB0pN5OKJAU54GWvwBCsf0SBmVSyPhjNsmjaHRPYOn5iy0or+ZzdTswys+XpnjZ2vl4vow43bnZsi8e9qq/TJDHejQ1EH193Pgamb5rsDwQCgcBvfAISxGSGUgKYugAAAABJRU5ErkJggg==","orcid":"","institution":"Universitas Airlangga","correspondingAuthor":true,"prefix":"","firstName":"Annis","middleName":"Catur","lastName":"Adi","suffix":""},{"id":446350875,"identity":"4e10038f-b9d4-4135-acf2-bb47eea9331f","order_by":1,"name":"Wizara Salisa","email":"","orcid":"","institution":"Rumah Inovasi Natura","correspondingAuthor":false,"prefix":"","firstName":"Wizara","middleName":"","lastName":"Salisa","suffix":""},{"id":446350879,"identity":"72da5a0f-5520-49b0-a80c-fe5b8794d5d6","order_by":2,"name":"Emyr Reisha Isaura","email":"","orcid":"","institution":"Universitas Airlangga","correspondingAuthor":false,"prefix":"","firstName":"Emyr","middleName":"Reisha","lastName":"Isaura","suffix":""},{"id":446350882,"identity":"1c743494-d403-46ec-80ce-96fea8a622bf","order_by":3,"name":"Febi Dwirahmadi","email":"","orcid":"","institution":"Griffith University","correspondingAuthor":false,"prefix":"","firstName":"Febi","middleName":"","lastName":"Dwirahmadi","suffix":""},{"id":446350891,"identity":"ac26dc62-20dc-47d6-a7ca-18b78163c587","order_by":4,"name":"Heni Rachmawati","email":"","orcid":"","institution":"Bandung Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Heni","middleName":"","lastName":"Rachmawati","suffix":""}],"badges":[],"createdAt":"2024-12-22 14:53:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5694378/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5694378/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82049041,"identity":"b6f41300-d5f1-4643-8e0c-cdc779f8f960","added_by":"auto","created_at":"2025-05-06 09:49:38","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":80907,"visible":true,"origin":"","legend":"\u003cp\u003eThe making process of substituted chocolate paste formula\u003c/p\u003e","description":"","filename":"Figure1.Themakingprocessofsubstitutedchocolatepasteformula.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5694378/v1/9344163e418c54988ad033c7.jpeg"},{"id":82050532,"identity":"ac43eb68-5867-47cd-be0f-4181e6a45bce","added_by":"auto","created_at":"2025-05-06 09:57:38","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":88517,"visible":true,"origin":"","legend":"\u003cp\u003eThe final product of chocolate paste formulas\u003c/p\u003e","description":"","filename":"Figure2.Thefinalproductofchocolatepasteformulas.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5694378/v1/05520695a8a06132573bb895.jpeg"},{"id":82049045,"identity":"cf228850-b64b-4594-8439-fc83f64bd996","added_by":"auto","created_at":"2025-05-06 09:49:38","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":41596,"visible":true,"origin":"","legend":"\u003cp\u003eRat’s growth rate based on body weight during malnutrition induction (M) to intervention (I)\u003c/p\u003e","description":"","filename":"Figure3.RatsgrowthratebasedonbodyweightduringmalnutritioninductionMtointerventionI.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5694378/v1/3fbac05c43d9128c63c9191c.jpeg"},{"id":82047321,"identity":"e64809a4-646f-4fb3-91ac-f6aeb4db6a7b","added_by":"auto","created_at":"2025-05-06 09:41:38","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":45249,"visible":true,"origin":"","legend":"\u003cp\u003eRat’s growth rate based on body mass index during malnutrition induction (M) to intervention (I)\u003c/p\u003e","description":"","filename":"Figure4.RatsgrowthratebasedonbodymassindexduringmalnutritioninductionMtointerventionI.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5694378/v1/55bb1ec6dea94c000d172bdf.jpeg"},{"id":82047337,"identity":"35c8bbaa-6c77-46f0-90cb-4167f3efad24","added_by":"auto","created_at":"2025-05-06 09:41:38","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":173341,"visible":true,"origin":"","legend":"\u003cp\u003eFaecal calprotectin, MPO, and neopterin of rats after intervention period\u003c/p\u003e\n\u003cp\u003eNote: *)significantly different between groups; \u003csup\u003ea\u003c/sup\u003e)significantly different compared to K0; \u003csup\u003eb\u003c/sup\u003e)significantly different compared to K1\u003c/p\u003e","description":"","filename":"Figure5.FaecalcalprotectinMPOandneopterinofratsafterinterventionperiod.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5694378/v1/a781d2b1389c82a1cbd2971f.jpeg"},{"id":82053526,"identity":"751beda8-52c5-4603-bcc7-0ce3f336c0c0","added_by":"auto","created_at":"2025-05-06 10:13:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1367586,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5694378/v1/ebafc846-e45a-42d7-9d89-a687b05ae150.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluating the Therapeutic Potential of Catfish and Fermented Sweet Potato Chocolate Paste on Malnutrition: Effects on BMI, Blood Serum, and Faecal Based Gut Inflammation in Protein-Deficient Rats","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eStunting, health retardation due to multidimensional aspects such as prolonged nutrient deficiency, improper adapted consumption style in a household, limited access to health facilities, nutritious food, and clean water (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In Indonesia, stunting is still a very serious nutritional problem. In 2023, the prevalence of stunting was 21.5% (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Despite declining from the prior year which was 21.6% (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), it still neither meets the Indonesian Medium-Term National Development Plan 2020\u0026ndash;2024 which is 14% (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) nor the WHO criterion which must not surpass 20% (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The outcomes of stunting involve physical disability burden, increasing potential for chronic diseases, and socio-emotional and motor development delays (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe adequacy of protein intake, animal and plant protein, plays a pivotal role in boosting growth and development. Protein acts as a stimulator which releases hormones for cell proliferation and differentiation (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Previous research that has been conducted generally only focuses on the function of protein in relation to malnutrition, but has not yet implemented how product formulas containing a mixture of proteins from natural ingredients can affect growth. This is also projected to be a form of health independence through the implementation of SDGs 12 sustainable consumption and production.\u003c/p\u003e \u003cp\u003eAs a freshwater commodity, catfish, is a high source of protein and amino acid, affordable price, easy to cultivate, and minerals, especially a source of calcium that is easily absorbed by the body (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Aside, plant protein is also needed because plant protein is a high-quality protein, it contains not only protein but also vitamins (A, C, B1, B2, B3) and minerals (potassium, calcium, magnesium, natrium, phosphorus, and iron) (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Sweet potatoes that are fermented are known to increase protein content up to 5 times. Another vegetable protein chosen is isolated soy protein because it is composed of 97% protein (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe success of stunting management through improving protein consumption can be assessed from the results of blood serum examination and analysis of inflammation in the digestive system. Inflammation can be caused by dysbiosis of the intestinal microbiota due to a lack of essential amino acids. In fact, intestinal microbiota affects child growth through the metabolic process and production of IGF-1, encouraging organ and systemic growth and skeletal growth mediators (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Metabolites produced by intestinal microbiota such as Enterobacteriaceae and Firmicutes/Bacteroidetes are associated with intestinal inflammation found in stunted children. Diet determines the composition of the microbiota, such as a diet of vegetable and animal protein can increase Prevotella and Bacteroides in the intestine (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe additional food that is widely available today is in the form of biscuits, so it often causes boredom and decreases acceptance. The evaluation output shows that the acceptance of biscuits in toddlers is only 32.2% (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Providing additional food in new forms (other than biscuits) that contain complete protein and nutrients is an alternative solution for handling stunted children that is more practical, more desirable, and nutritious. This study aimed to develop an additional food based on local food containing animal and plant protein, and tested its effectiveness on low-protein induced experimental animals.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eResearch design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study adopted a completely randomized experimental research design. The research encompassed product development, organolpetic testing, nutritional content analysis, and efficacy tests on experimental animals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eChocolate paste manufacturing process were carried out in Nutrition Laboratory of Nutrition Department at the Faculty of Public Health, Universitas Airlangga, located in Surabaya, East Java, Indonesia. The nutritional content analyses and food contamination tests were conducted at the Saraswanty Laboratory in Bogor, West Java, Indonesia. Further, preclinical testing on experimental animals was conducted in the Biochemical Laboratory at the Faculty of Medicine, University of Airlangga.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDevelopment of additional food\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChocolate paste is a snack that has a delicious taste, simple ingredient composition, and is easy to make. The processing technique requires minimal heating, thus reducing the possibility of damaged nutrients. \u003ca id=\"_anchor_1\" href=\"#_msocom_1\" language=\"JavaScript\" name=\"_msoanchor_1\"\u003e\u003c/a\u003eTo make chocolate paste achieve the research objectives, ingredient substitutions were carried out. The substituted ingredients are catfish flour (\u003cem\u003eClarias gariepinus\u003c/em\u003e), fermented sweet potato (\u003cem\u003eIpomoea batatas\u003c/em\u003e) flour, and isolated soy protein.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFresh catfish purchased directly from catfish breeders in Bolorejo Village, Kauman District, Tulungagung, East Java, Indonesia, meanwhile the sweet potato from a farmer in Mojosari Village in the same district with catfish breeders. Initially, the process of producing catfish flour began with cleaning and washing the fresh catfish, separating the meat and the bone (including the head), softening by boiling it at high pressure, pressing and draining, drying (sun drying and roasting), flour milling, and filtering. Meanwhile, the process of producing fermented sweet potato flour began with pealing of the skin, washing, cutting, soaking with probiotics (12 hours, temperarture 30\u003csup\u003eo\u003c/sup\u003eC), draining, drying using sunlight, and the last is milling using a machine (16). The chocolate paste production process adapted Indonesian patent number S00202416043, shown in Figure 1.\u003ca id=\"_anchor_2\" href=\"#_msocom_2\" language=\"JavaScript\" name=\"_msoanchor_2\"\u003e\u003c/a\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProduct characterization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Indonesian National Standard number 7934: 2014 (17) evaluated the product\u0026apos;s characteristics, focusing on chocolate paste. This standard pertains with the evaluation of the physical properties of chocolate paste, metal composition, and other food contaminants such as insects, foreign objects, and bacterias. These tests were undertaken to ensure that the product complies with the established food safety standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNutrition\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003evalue\u0026nbsp;test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe product\u0026apos;s proximate composition was determined by standardized methods developed by the Association of Official Analytical Chemists (AOAC). In addition, the nutritional content, comprising 10 vitamins and 6 minerals, was also assessed through the Ultra Performance Liquid Chromatography (UPLC) method. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcceptability test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe acceptability of the product was assessed using hedonic tests, examining four parameters including texture, aroma, color, and flavor. The untrained panelists involved in this test are 30 school-aged childrens (all participants are elementary school students) selected based on the following criteria: aged 6 to 12 years, no allergies to fish, dairies, chocolate, and gluten. The data were obtained by administering a questionnaire using a 5-point hedonic scale. The scale graded from 1 to 5, with 5 indicating a strongly like, to 1 indicating the least favorable preference (\u0026ldquo;strongly dislike\u0026rdquo;).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimal model and diet intervention\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale Wistar \u003cem\u003eRattus norvegicus\u003c/em\u003e, weighing 100-150 g, were purchased from the Farma Veterinary Center (Pusvetma) Surabaya, and housed for 7 days without specific treatment for acclimatization. The animal models were then divided into five groups consisting of seven rats, the healthy group (K0), the low protein diet group (K1), and the three other groups were intervention groups (PP). During the malnutrition induction period, the K0 group received a standard diet, while the K1 and all PP groups received a low-protein diet. The standard diet contained 10.72% w/w protein, while the low protein diet contained 4% w/w protein (18-19). The conditioning of malnutrition was done for 21 days to mimic the protein deficiency. After 14 days, the animals in each group were tested for albumin and total protein levels to ensure their malnutrition induction. All rats continued the given diet until 14 days, while the PP groups received the following treatment; PP1: low protein diet with government supplementary biscuits, PP2: low protein diet with substituted chocolate paste, and PP3: standard protein diet with substituted chocolate paste. The dosage of biscuits is 120 g x 0.018 = 2.16 g/day, and chocolate paste formula is 40 g x 0.018 = 0.72 g/day. The number of 0.018 is obtained from the conversion of human doses to rat doses (20). Subsequently, the anthropometric measurement of each group was monitored until the intervention ended.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnthropometry measurement of experimental animals\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBody weight was measured by a digital weight scale. Body length (nose to anal) was measured by a ruler. Body mass index (BMI) was measured by dividing anthropometrical measurement results of body weight and body length.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood serum analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood serum was collected on the last day of the intervention. The rats were anesthetized, then their blood was taken from the heart. After the blood serum samples were collected, laboratory analysis was carried out. Total protein analysis was assessed using the \u003cem\u003eKjeldahl\u003c/em\u003e method. Albumin analysis was assessed using the\u003cem\u003e\u0026nbsp;Bromocresol green dye\u003c/em\u003e. Calcium, zinc, and iron were assessed using the\u003cem\u003e\u0026nbsp;\u003c/em\u003eAtomic Absorption Spectrometer (AAS).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFecal neopterin and calprotectin analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNeopterin and calprotectin are measured to determine inflammatory conditions of the digestive system that affect nutrient absorption. Previously, we collected rat\u0026rsquo;s faecal samples along with blood serum sample collections. Faecals were extracted in phosphate buffer saline (PBS) to make the suspension and then centrifuged to separate the pellets and suspension. The concentration of neopterin and calprotectin were measured using \u003cem\u003eenzyme-linked immunosorbent assay\u003c/em\u003e (ELISA) methods, following the manufacturers\u0026apos; instructions. The biomarkers were determined using Mouse ELISA Kits from BT LAB (Zhejiang, China). The stool suspensions that were previously prepared were thawed, vortexed, and centrifuged and the supernatant was used for analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that all methods were carried out by relevant guidelines and regulations of daily protein restriction which is adapted from other similar research (18-19). The animal experimental protocols were approved by the Health Research Ethical Clearance Commission of the Faculty of Dental Medicine at the University of Airlangga, under reference number 0522/HRECC.FODM/V/2024. The collection of the ingredients used in the study complies with local or national guidelines with no need for further affirmation.\u003c/p\u003e\n\u003cp\u003eSpecifically for the acceptance test using the hedonic test, we involved school-age children. Previously, an application for ethical protocols, including the informed consent document was made, which had been approved by the same Ethical Committee (Faculty of Dental Medicine at the University of Airlangga), number 0534/HRECC.FODM/V/2024. The informed consent document has explained the method of implementing the hedonic test (by tasting each product), and the possible side effects experienced such as diarrhea or vomiting. As a form of preventing side effects, participants\u0026rsquo; criteria were applied (not having allergies), and as a form of overcoming side effects, researchers have collaborated with local health clinics. The document is used as a statement of participation, which is approved (signed) by all the parents/legal guardians of each student.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBefore analyzing the differences, all data underwent an assessment to determine the normality and homogeneity of variance using the Shapiro-Wilk test, then continued by assessing the differences in data using the Anova test or the Kruskal-Wallis test. Specifically for groups that have significant differences, the Tukey and Mann-Whitney Anova tests will be continued to analyze further and find out which 2 groups have significant differences. \u003ca id=\"_anchor_5\" href=\"#_msocom_5\" language=\"JavaScript\" name=\"_msoanchor_5\"\u003e\u003c/a\u003e All tests used a significance level of \u0026alpha; = 0.05. Statistical analysis was performed using IBM Statistics SPSS 20 software.\u003c/p\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cp\u003e\u003cstrong\u003eCharacteristics of products\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure 2 depicts the visual of the chocolate paste from the combination of catfish flour and fermented sweet potato flours and the original chocolate paste. Visually there is no significant difference between the two products, both color are brown, only\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 1. Characteristics and food contamination test of chocolate paste from the combination of catfish and fermented sweet potato flours\u003c/p\u003e\n\u003cp\u003eTable 1 outlines the food contamination test results, including chemical and biological contaminants. The results implied no chemical contaminants detected, such as lead, cadmium, tin, mercury, or arsenic, and likewise, the biological contaminants showed low levels of \u003cem\u003eEscherichia coli, Staphylococcus aureus, Bacillus cereus\u003c/em\u003e, yeast mold and no harmful level of \u003cem\u003eSalmonella sp.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNutritional value\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTable 2 presents the nutritional value of substitued chocolate paste formula compared to the standard of supplementary food for wasting toddlers and recommended dietary allowance (RDA) for toddlers (aged \u0026lt;6 years old) (22). The comparative results in both regulations showed protein content was already met even surpasses the requirements for supplementary food category and toddlers\u0026rsquo; RDA. The ingredients used seem to corroborate its nutritional values.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2. Nutritional value of chocolate paste from the combination of catfish and fermented sweet potato flours per 100 g\u003c/p\u003e\n\u003cp\u003eCatfish flour which has 46 g of protein (76.7%) considered a high-protein product according to the criteria outlined by the Indonesian Agency for Drug and Food Control (23) which declared that a product can be classified as a rich/high-protein if it contains minimum 21 g (35%) per 100 g of solid product. This emphasizes that catfish flour becomes one of the high-quality protein components that can elevate protein content in ready to eat products (24). The existence of catfish is very easy to find, because it is spread in many freshwater areas. It is not only in Southeast Asia, but also in greater Asia, South America, Africa, North America, North Europe, Australia, Madagascar, and New Guinea (39-40). Because the high yield of catfish is inversely proportional to its use, it is necessary to process fresh ingredients into semi-finished products, one of which is flour. so that the shelf life of the product is longer, and can be processed into various products.\u003c/p\u003e\n\u003cp\u003eNot only does catfish flour contribute greatly to boosting the protein content, but fermented sweet potato flours and isolated soy protein are also validated in giving magnificent impact in terms of protein and arrays of minerals. The fermentation process in sweet potatoes formed a single cell protein by breaking down glucose into pyruvic acid through a glycolysis reaction which will then produce amino acids (25). Isolated soy protein has 21 g, making them also included as rich/high protein components (23). The combination of these ingredients has succeeded in creating a chocolate paste product that can meet the daily protein needs of children aged \u0026lt;6 years up to 25%.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcceptability (\u003c/strong\u003e\u003cstrong\u003eorganoleptic test results)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA hedonic test was conducted to assess the organoleptic characteristics of the chocolate paste formula. Two formulas (control and substituted) were involved. Table 3 displays the score of each parameter. Regarding the findings presented in Table 3, the modification formula was found to have an overall average score of over 4.08 (like). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 3. Acceptability of original/control and substituted chocolate paste\u003c/p\u003e\n\u003cp\u003eThe statistical analysis exhibited no resemblance in terms of color between control and substituted formula (p\u0026gt;0.05). Nevertheless, it was notable that the substituted formula had a lighter color appearance than the control formula (Figure 2). Basically, catfish flour and isolated soy protein have maillard reaction ability. The Maillard reaction is a chemical reaction between amino acids and reducing sugars to create melanoidins, the compounds that give browned food its distinctive flavor (26). However, because the main ingredient used is chocolate paste, the color appearance is not affected by the maillard reaction.\u003c/p\u003e\n\u003cp\u003eConcerning its flavor, statistically, it revealed a significant distinction (p\u0026lt;0.05) among this pair (control formula and substituted formula). The high proportion of dry ingredients (catfish flour, fermented sweet potato flour, and isolated soy protein) added causes the chocolate paste substitution formula to have a lower sweetness level than the control formula. In the substitution formula, there is also the addition of fish oil which aims to increase the fatty acid content of the product, but has a side effect in the form of a fishy taste to the product. Apart from that, the panelists\u0026apos; preference scores are still in the liking category (\u0026gt;4.0).\u003c/p\u003e\n\u003cp\u003eThe aroma parameter has similarities with the assessment results for the flavor parameter that there was a significant difference (p\u0026lt;0.05) between the two products, but both received an average preference rating of like (\u0026gt;4.0). The aroma of the original and substituted formulas was distinguished by the addition of fish oil. The addition of fish oil not only makes a significant difference in flavor, but also in the aroma.\u003c/p\u003e\n\u003cp\u003eMeanwhile based on the texture, it visually conveys a difference among this pairs. From figure 2, it can be saw that the control formula has a smoother and oilier texture, while the substitution formula looks denser and a little drier. This is because the percentage of dry ingredients used in the substitution formula increases from the initial formula (control). Precedent research revealed that isolated soy protein can increase brittleness because its dryness (27). This resulted from the insufficiency of fat and oil bonded by protein (28). Catfish flour is also accountable for fracturability, depicting brittleness and easy-to-crack levels. Catfish flour has 2.17% ash content, while the regulation states that the maximum level of ash content is 1.5% (29). The higher the ash content, the higher the ability to absorb liquid, because the original minerals contained in flour can generally weaken the gluten structure formed in the dough/product (30).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOverall, the organoleptic elements of chocolate paste from the combination of catfish flour, fermented sweet potato flour, and isolated soy protein obtained good scores, with average values is 4.08 of 5.00, indicating these formulas are likely well-accepted.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe growth rate of the animal model during malnourished induction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the results of anthropometric measurements during the malnutrition induction period (Figure 3 \u0026amp; 4), it is known that there is a trend of decreasing body weight and BMI in the group given low-protein feed induction.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhile the K0 group (standard feed) still experienced an increase in body weight and BMI. The normal BMI limit for male rats is [0.45 \u0026plusmn; 0.02\u0026ndash;0.68 \u0026plusmn; 0.05] g/cm2 and for female rats [0.4504\u0026ndash;0.5044] g/cm2 (31). The average BMI of rats in the group given low-protein diet intervention was in the range of 0.44 \u0026ndash; 0.46 g/cm2. This indicates that low-protein feed (4%/day) treatment for 21 days can cause rats to experience decreased growth to the point of experiencing a BMI below the normal limit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe results of substituted biscuits intervention on malnourished animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe body weight of all groups of rats during the intervention period showed an increase, both in the treated and untreated groups. The results of the BMI calculation also showed that all groups experienced a periodic increase, although in the K1 group, the increase tended to be flat, while in the intervention group with substituted chocolate paste (PP2 \u0026amp; PP3), the graph tended to increase sharply (Figure 4). The increase in BMI of rats in the intervention group (PP) still had a positive effect compared to the positive control group (K1), although statistically it did not show a significant difference (Table 4). This indicates that the treatment group (consumed supplementary food) was able to catch up on its growth delays and return to its normal BMI.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 4. Analysis of BMI and blood serum on malnourished rats after intervention\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStatistical tests were also carried out on blood serum parameters, showed in Table 4. In contrast to the results of the difference test on BMI, all the variables in the blood serum sample test were found to have significant differences (p \u0026lt;0.05) between groups, which means that the provision of supplementary food affected its blood serum levels. Normal levels of albumin in rats are 3.3 - 4.2 g / dL, total protein 6.0 - 8.2 g / dL, calcium 8.5-10.9 mg / dL (20, 32-35). Substitution of catfish and fermented sweet potato flour in chocolate paste (PP2 \u0026amp; PP3) found to have significant differences compared to the control positive (malnutrition) group (K1), these indicates that the substituted ingredients were effective in improving blood serum values (albumin, total protein, and calcium) and maintained their levels within normal limits. Meanwhile the group with supplementary food in the form of government biscuit showed almost the same results with the chocolate paste intervention, except for the albumin serum levels, which is still low (below normal limits).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFaecal calprotectin, myeloperoxidase, and neopterin as malnutrition biomarkers after intervention period\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn figure 5, the increase in growth after the addition of additional food was confirmed by the measurement of intestinal inflammation biomarkers, calprotectin, myeloperoxidase (MPO), and neopterin. From the results of the difference test between groups showed a significant difference (p\u0026lt;0.05) in calprotectin and MPO, especially the chocolate paste treatment group (PP2 \u0026amp; PP3) compared to the positive control (malnutrition) group (K1), whereas when compared to the normal group (K0) there was no significant difference (p\u0026gt;0.05), so it was assumed that the conditions of the groups tended to be similar.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCalprotectin and neopterin are biomarkers associated with intestinal inflammation, which reflect the severity of the cell-mediated immune response in the intestine. There is no definite limit to determining intestinal inflammation for each biomarker, but a significant increase in levels indicates inflammation (36-38).\u003c/p\u003e\n\u003cp\u003eThe limitation of this study found in the rat malnutrition model used. It may reflect human malnutrition which caused by nutrient deficiencies, but may not fully reflect the complexity of human malnutrition, which involves environmental factors, and chronic infections. However, considering that this substitute food innovation emphasizes nutritional content and the use of local ingredients, the expected output results also focus on parameters directly related to nutrients. In addition, preclinical testing on experimental animals is at least an initial stage of proving the theory which can then be continued with clinical trials on humans by adjusting the factors experienced by stunted toddlers.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThe research findings indicate that chocolate paste from the combination of catfish, and fermented sweet potato flours offers a sufficient supply of nutrients, particularly protein, calcium, and fat acid (omega), also in line with supplementary food regulations for wasting toddlers. The findings of the organoleptic test reveal that the panelists expressed positive preferences (like) for both formulas. The substituted chocolate paste demonstrated consistent and standardized quality, as outlined in SNI 7934: 2014, and are free from any food contaminants. Based on the efficacy test on malnourished rats, substituted chocolate paste proven to be able to increase body weight and BMI from a malnourished condition to be equivalent to a healthy condition, and significantly affect albumin, total protein, calcium, zinc, and iron serum levels so that they have the same condition as the normal group. Furthermore, substitution of chocolate paste ingredients can influence intestinal inflammatory biomarkers, by prevented an increase in calprotectin and MPO levels.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRESEARCH FUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAirlangga University financially supported this project under the Airlangga Superior Research scheme with contract number 373/UN3.LPPM/PT.01.03/2024.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAurora, W.I.D., 2021. 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Ayling and K. Kok, \u0026lsquo;Fecal Calprotectin\u0026rsquo;, Adv Clin Chem, vol. 87, pp. 161\u0026ndash;190, Jan. 2018, doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/bs.acc.2018.07.005\u003c/span\u003e\u003cspan address=\"10.1016/bs.acc.2018.07.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eT. Pang, S. T. Leach, T. Katz, A. S. Day, and C. Y. Ooi, \u0026lsquo;Fecal biomarkers of intestinal health and disease in children\u0026rsquo;, Jan. 01, 2014, Frontiers Media S.A. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fped.2014.00006\u003c/span\u003e\u003cspan address=\"10.3389/fped.2014.00006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB. Seethaler \u003cem\u003eet al.\u003c/em\u003e, \u0026lsquo;Biomarkers for assessment of intestinal permeability in clinical practice\u0026rsquo;, Am J Physiol Gastrointest Liver Physiol, vol. 321, no. 1, pp. G11\u0026ndash;G17, Jul. 2021, doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1152/AJPGI.00113.2021\u003c/span\u003e\u003cspan address=\"10.1152/AJPGI.00113.2021\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLundberg, John G. and John P. Friel. 2003. Siluriformes. Catfishes. 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Aquatic Living Resources, \u003cem\u003e9\u003c/em\u003e: 35\u0026ndash;41, doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1051/ALR%3A1996040\u003c/span\u003e\u003cspan address=\"10.1051/ALR%3A1996040\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1. Characteristics and food contamination test of chocolate paste from the combination of catfish and fermented sweet potato flours\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"600\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameter\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eUnit\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eStandard*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eResult\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eKeterangan\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eTaste\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eKhas, normal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eNormal, sweet\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eSmell\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eKhas, normal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eNormal\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eColor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eKhas, normal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eChocolate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eAsh content\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e2.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eWater content\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e1.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 600px;\"\u003e\n \u003cp\u003eMetal contamination:\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eLead (Pb)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003emg / kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.0018\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eNot detected\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eCadmium (Cd)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003emg / kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eMax. 0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eTin (Sn)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003emg / kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.0016\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eNot detected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eMercury (Hg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003emg / kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eMax. 0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eArsenic (As)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003emg / kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0.0003\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eNot detected\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"5\" valign=\"top\" style=\"width: 600px;\"\u003e\n \u003cp\u003eMicrobes contamination:\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eTotal Plate Number\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003ecolony / g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eMax. 1x10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e1.0x10\u003csup\u003e4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eColiform\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eAPM/g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e4.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eEscherichia coli\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eAPM / g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u0026lt;3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eSalmonella sp.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u0026nbsp;/25 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eNegative/25 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eStaphylococcus aureus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003ecolony / g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eMax. 1x10\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u0026lt;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eBacillus cereus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003ecolony / g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e2.1x10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e2.1x10\u003csup\u003e3\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eYeast mold\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003ecolony / g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u0026lt;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003e\u0026lt;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 120px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*Source: Indonesia National Standard for Chocolate Paste 7934: 2014 (16)\u003c/p\u003e\n\u003cp\u003eTable 2. Nutritional value of chocolate paste from the combination of catfish and fermented sweet potato flours per 100 g\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"600\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eNutrients\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003eNutritional Value\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(100 g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003eStandards\u003csup\u003ea\u003c/sup\u003e (100 g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eStatus\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003eNutritional Value per serving\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(40 g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eRDA for \u0026lt;6 years\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e%Fulfillment\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eEnergy (kcal)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e562.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003eMin. 400\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e225.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e1400.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e16.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eProtein (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e10.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e8 \u0026ndash; 12\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e4.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e25.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e17.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eFat (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e35.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e10 \u0026ndash; 18\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e14.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e50.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e28.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eCarbohydrate (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e50.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003eNot set\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e20.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e220.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e9.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eVitamin A (mcg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e200 \u0026ndash; 400\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eNot Fulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e450.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eVitamin D (mcg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e47.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e5 \u0026ndash; 10\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e19.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e15.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e126.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eVitamin E (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e7.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e3 \u0026ndash; 6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e3.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e7.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e42.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eVitamin K (mcg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e7.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e4 \u0026ndash; 6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e2.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e20.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e14.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eVitamin B1 (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0.25 \u0026ndash; 0.5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eNot Fulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eVitamin B2 (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0.3 \u0026ndash; 0.6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eNot Fulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e13.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eVitamin B3 (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e2.5 \u0026ndash; 5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eNot Fulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e8.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e2.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eVitamin B6 (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0.2 \u0026ndash; 0.4\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eNot Fulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eVitamin B12 (mcg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0.35 \u0026ndash; 0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e1.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eFolic acid (mcg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e390.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e60 \u0026ndash; 120\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e156.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e200.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e78.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eIron (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e4 \u0026ndash; 7.5\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e3.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e31.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eIodine (mcg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e72.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e60 \u0026ndash; 120\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e28.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e120.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e24.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eZinc (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e1.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e2 \u0026ndash; 3.75\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eNot Fulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e5.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e15.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eCalcium (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e351.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e225 \u0026ndash; 450\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e140.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e1000.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e14.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eSelenium (mcg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e7 \u0026ndash; 14\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eNot Fulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e21.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003ePhosphor (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e260.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e180 \u0026ndash; 275\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e104.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e500.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e20.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eFluoride (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e4.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003eMax. 0.25\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eNot Fulfilled\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e172.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eSodium (mg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e86.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003eMax. 300\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e34.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e900.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e3.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eOmega 3 (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e0.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e0.4 \u0026ndash; 0.6\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e0.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e0.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e13.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eOmega 6 (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e2.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003e1.7 \u0026ndash; 2.9\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e1.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003e10.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e10.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 120px;\"\u003e\n \u003cp\u003eOmega 9 (g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e9.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 87px;\"\u003e\n \u003cp\u003eNot set\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 60px;\"\u003e\n \u003cp\u003eFulfilled\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 84px;\"\u003e\n \u003cp\u003e3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 77px;\"\u003e\n \u003cp\u003eNot set\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eNote:\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e) Regulation of the Indonesian Minister of Health Number 51 Year 2016 (22).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e) Regulation of the Indonesian Minister of Health Number 28 Year 2019 (21).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u003c/sup\u003e) The percentage of fulfillment is obtained from the nutritional value per serving (40 g) divided by the RDA for \u0026lt;6 years.\u003c/p\u003e\n\u003cp\u003eTable 3. Acceptability of original/control and substituted chocolate paste\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"515\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003eParameter\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003eSubstituted\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003eColor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e3.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e3.93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003eFlavor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e4.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e4.07*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003eSmell\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e4.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e4.03*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003eTexture\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e4.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e4.31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 179px;\"\u003e\n \u003cp\u003eOverall mean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e4.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003e4.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*)Significantly different (p-value \u0026lt;0.05) using Kruskal-Wallis test\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 4. Analysis of BMI and blood serum on malnourished rats after intervention\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"659\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eBMI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eAlbumin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003eTotal Protein\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003eCalcium\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003eZinc\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003eIron\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 49px;\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026plusmn;SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 52px;\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 44px;\"\u003e\n \u003cp\u003e\u0026plusmn;SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 49px;\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 47px;\"\u003e\n \u003cp\u003e\u0026plusmn;SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 45px;\"\u003e\n \u003cp\u003e\u0026plusmn;SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 69px;\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 43px;\"\u003e\n \u003cp\u003e\u0026plusmn;SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 61px;\"\u003e\n \u003cp\u003eMean\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 46px;\"\u003e\n \u003cp\u003e\u0026plusmn;SD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 1px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003eK0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e0.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e3.58\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44px;\"\u003e\n \u003cp\u003e0.42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e6.43\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e8.55\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e0.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 69px;\"\u003e\n \u003cp\u003e125.04\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 43px;\"\u003e\n \u003cp\u003e5.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e170.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46px;\"\u003e\n \u003cp\u003e5.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 1px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003eK1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e0.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 52px;\"\u003e\n \u003cp\u003e1.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 44px;\"\u003e\n \u003cp\u003e0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 49px;\"\u003e\n \u003cp\u003e5.57\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 47px;\"\u003e\n \u003cp\u003e0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 53px;\"\u003e\n \u003cp\u003e5.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n 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\u003c/td\u003e\n \u003ctd style=\"width: 61px;\"\u003e\n \u003cp\u003e175.94\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 46px;\"\u003e\n \u003cp\u003e4.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 1px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 53px;\"\u003e\n \u003cp\u003eSig.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.761\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.001*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 96px;\"\u003e\n \u003cp\u003e0.000*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 98px;\"\u003e\n \u003cp\u003e0.000*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 112px;\"\u003e\n \u003cp\u003e0.000*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" style=\"width: 108px;\"\u003e\n \u003cp\u003e0.016*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e*)significantly different (p\u0026lt;0.05) between groups\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003e)significantly different (p\u0026lt;0.05) compared to K0\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003e)significantly different (p\u0026lt;0.05) compared to K1\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"discoverfood","sideBox":"Learn more about [Discover Food](https://www.springer.com/44187)","snPcode":"","submissionUrl":"","title":"Discover Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Biomarker profile, catfish, fermented sweet potato, intestinal inflammation, malnutrition","lastPublishedDoi":"10.21203/rs.3.rs-5694378/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5694378/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe number of stunting cases in Indonesia that reached over 21% in 2023 has been an alarming issue. Protein and mineral insufficiency as well as intestinal inflammatory as determinants can lead to worsening condition. Providing additional food by combining animal and plant-based protein in the form of a ready-to-eat food is beneficial to alleviate stunting. This study aimed to ensure the standard quality, nutritional value, level of acceptance, and outcomes of the combination of catfish, fermented sweet potato, and isolated soy protein (ISP) flours in the form of chocolate paste for stunting biomarkers. The research process encompassed product development, evaluation of product and its compliance with the regulatory standards of food contaminants, nutritional content, hedonic tests (by the panelists), and pre-clinical testing in animal models of malnutrition induced by daily protein restriction (4% protein/day). Testing the effects of substituted products on experimental animals included monitoring growth rates (body weight and body mass index), blood serum profiles, and intestinal inflammation biomarkers through faecal samples. The findings implied that the use of catfish flour 3,3%, fermented sweet potato 9,8%, and ISP 3,3% on chocolate paste classified as a high-protein product with diverse arrays of minerals. This product fulfilled the requirements for additional food standards, and met the panelists' acceptance with a mean overall score of 4.08 (like). In malnourished rats, substituted chocolate paste proven to significantly affected (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) blood serum (albumin, total protein, calcium, zinc, iron), also significantly preventing intestinal inflammation to increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), by faecal calprotectin and myeloperoxidase (MPO) value.\u003c/p\u003e","manuscriptTitle":"Evaluating the Therapeutic Potential of Catfish and Fermented Sweet Potato Chocolate Paste on Malnutrition: Effects on BMI, Blood Serum, and Faecal Based Gut Inflammation in Protein-Deficient Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 09:41:33","doi":"10.21203/rs.3.rs-5694378/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-06T10:51:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-06T10:50:29+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-28T04:54:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"163406510671548668727535783274339216167","date":"2025-04-24T04:41:05+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-22T12:29:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"212711392588927568570387358592607303864","date":"2025-04-22T12:24:50+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-22T04:07:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-17T12:09:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Food","date":"2025-03-24T05:41:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"discoverfood","sideBox":"Learn more about [Discover Food](https://www.springer.com/44187)","snPcode":"","submissionUrl":"","title":"Discover Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dc13c8be-d5f3-4654-9bc7-401b07ee0b3c","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-05-19T11:08:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-06 09:41:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5694378","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5694378","identity":"rs-5694378","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}