Propionic Acid, Butyric Acid, and Non-Pathogen Microbiome in Anemic Wistar Rat

Background. Anemia, as a global public health problem, needs to be solved. The previous study by the researcher found the potency of iron-fortified synbiotic fermented milk with tempeh extract as an alternative to overcome anemia. Objective. This study aims to determine the effects of iron-fortified synbiotic fermented milk with tempeh extract towards short-chain fecal fatty acids (SCFAs) and fecal microbiota. Method. The study was a randomized controlled trial with three groups, consisting of 8 anemic Wistar rats. The groups made into anemia within 17 days.The groups treated as follows: group NA: fermented milk with tempeh extract fortified by NaFeEDTA, group FE: fermented milk with tempeh extract fortified by FeSO4, and group KO: fermented milk with tempeh extract without fortification. SCFAs and microbiota of the rat’s feces determined using the Total Plate Count method. SCFAs were measured after the intervention, while fecal microbiota was measured before and after the intervention. One-way ANOVA was used to measure the difference between NA, FE, and KO groups with post hoc test Bonferroni. Results. There was a significant mean difference between propionic and butyric acid between NA and FE groups and the KO group. The highest Lactobacilli number was in the FE group, while Bifidobacteria and Enterobacteriaceae were highest in the KO group. Meanwhile, the NA group had the highest Escherichia coli number. Conclusion. Iron fortification has positive effects on increasing the production of SCFAs in the gut. Prebiotics and probiotics have positive effects on pathogenic bacteria. Further study is needed to determine the effects of iron-fortified synbiotic fermented milk with tempeh extract in human.


INTRODUCTION
Anemia is a global health problem either in a high income or middle-income country.
Anemia prevalence in Indonesia is still high, 21.7 percent. 1 Anemia considered having intergenerational impacts. It means that the effect of anemia can be passed on between generations.
In some periods, it can lower the brain function and immune system, lower the productivity, and indirectly correlate with the level of public welfare. 2,3 It is essential to cut the chain of anemia, mainly through some innovation about food and nutrition.
Fortification is a cost-effective way to improve micronutrient deficiencies. 4 Several studies suggested that iron supplementation and fortification on humans may increase hemoglobin concentration. 5 NaFeEDTA is one type of iron fortificants which recommended to be added on high phytate meal to increase iron absorption in the gut. 6 Compared with FeSO 4 , the absorption level of NaFeEDTA is 2-4 times higher.
Indonesian Ministry of Agriculture mentioned that the availability of soybeans were increasing from 7 million kg/capita/year in 2007 to 10 million kg/capita/year in 2011. 7 However, there are still many people who assume tempeh is a low social food. 8 Tempeh contains micronutrients which needed for hemoglobin syntheses such as vitamin B12, folic acid, protein, iron, Cu, and Zn. Every 100 g tempeh contains 20.8 g protein; 4 mg iron; 1.4 g fiber; and 155 mg calcium. 7,9 Tempeh is also known as a functional food to reduce the period of diarrhea. 10 Iron supplementation and fortification do have adverse effects. It enhances pathogenic gut microbiota and lowers lactobacilli number. 11 In the study of pigs, iron supplementation increases the coliform number in the gut. 12 Iron supplementation also modulates free radicals, thus interfere with the body's immune system. 13 The change of gut microbiota composition is due The experimental study aimed to determine the effect of the synbiotic milk towards gut microbiota and short-chain fatty acid (SCFA).

Study Design
The study is randomized controlled trial Information about the nutritional value of the ironfortified synbiotic fermented milk was in Table 1.

Hemoglobin and Body Weight Measurement
The photometric method (Diasys, Germany) used to measure the hemoglobin level of the rats. Hemoglobin was analyzed before and after 17 days of intervention using blood taken from sinus orbitalis. 25 The body weight of the rats examined using digital analytic scales and measured every three days.

Fecal Short-Chain Fatty Acids and Microflora Analysis
Short-chain fatty acids (SCFA) counted from the rats' feces. The measurement of SCFA post-intervention using High-Performance Liquid Chromatography (HPLC) method. 26,27 The number of gut microbiota were analyzed twice, pre-and post-intervention. There were four types of gut microbiota assessed, Lactobacilli, Bifidobacteria, Enterobacteria, and Escherichia coli. Gut microbiota was analyzed using Total Plate Count (TPC) following the method by Mailoa et al. 28 Lactobacilli were determined by MRS medium agar, Bifidobacteria by Bifidobacterium agar, Enterobacteria by MacConkey agar, and Escherichia coli by Tryptone Bile X-Glucuronide (TBX) agar.

Statistical Analysis
Data analysis used software SPSS to determine the difference between the three groups. One-way ANOVA was used to measure the difference between NA, FE, and KO groups with post hoc test Bonferroni. 29

RESULTS
Body weight of the rats increased in all groups. It was measured before the intervention began and after the hemoglobin depletion and found not significantly different, p>0.05 (Table 2).  Table 3 shows hemoglobin concentration of the rats before and after given the intervention.
From the results, the level of hemoglobin after the intervention was the same between the three groups.
We also measured fecal short chain fatty acid (SCFA) concentration by the intervention.
Three types of SCFA which were acetic acid, butyric acid, and propionic acid were analyzed. In the present study, it was found a significant mean difference of propionic and butyric acid concentration between the three groups. However, there was no significant mean difference of acetic acid concentration between the three groups. From the study, SCFA concentration was higher in the group given ironfortified synbiotic fermented milk with tempeh extract compared to synbiotic fermented milk with tempeh extract without fortification (Table 4).  stated that iron influenced butyrate production pathway and the gene of butyrate production in the gut. Yilmaz and Li 14 concluded that in irondeficient conditions, the Bacteroides decreased while Lactobacilli and Enterobacteriaceae increased, which caused lower propionate and butyrate production. However, different results showed in the human study by Jaeggi et al, 36 who mentioned that iron did not have any effect on fecal SCFA concentration since 95-99 percent of SCFA produced by bacterial fermentation were absorbed in the colon.
Fructooligosaccharides, the prebiotics added to fermented milk with tempeh extract, function to transform ferric ion into a ferrous form in the colon and the probiotics. 21 The ferrous form is more readily to absorb than ferric. Beside of it, prebiotic helps the probiotic against pathogen bacteria. The result proved that the hemoglobin level increased in all groups. Although before the intervention, the hemoglobin level is different between NA and FE and KO group, after the intervention, the hemoglobin level was the same. It suggested that the consumption of synbiotic fermented milk with tempeh extracts with or without iron fortification was able to increase the rat's hemoglobin concentration. Perez-Conesa et al, 37 explained that iron absorption mainly

DISCUSSION
NaFeEDTA is a yellow, pale iron powder that has good solubility in the pH 7 solution. The stability of NaFeEDTA made it suitable to use in the long-stored foods or were made at high temperatures. The EDTA protects iron compounds from the formation of insoluble iron like iron-phytate. EDTA is influenced by pH to work. 30,31 Unlike the NaFeEDTA, FeSO 4 is more unstable, mainly when use in the phytate rich foods or stored in a long time. It is because FeSO 4 does not have chelating capacity, which could protect the fortificant from forming other compounds. 32 Short-chain fatty acid (SCFA) is a fermented product of undigested fiber by gut microbiota in the colon. The main types of SCFAs are acetic, butyric, and propionic acid. 33 The present study suggests a significant difference of propionic and butyric acid between NaFeEDTA and FeSO 4 fortified synbiotic fermented milk with tempeh extract. SCFA concentration is higher in the group of iron-fortified compared with unfortified group.
A study by Dostal et al, 34 in rats suggested that availability of colonic iron modulated gut microbiota metabolites and the production of SCFA. It was supported by Louis and Flint 35

RECOMMENDATION
Further study is needed to determine the effects of iron-fortified synbiotic fermented milk with tempeh extract in human.