Resistant starch type 3 (RS3) produced from high amylose food sources through retrogradation or enzymatic process is known to have physiological function as dietary fiber. Fermentation of RS3 by colonic microorganisms produced SCFA (acetate, propionate, and butyrate), maintain ed the health of colon, balance of gut microbiota, preventing inflammatory bowel diseases (IBD) and colon cancer. RS3 in this study was produced from IR-42 and Inpari-16 broken rice by enzymatic treatment (combination of amylase-pullulanase). The Resistant Starch was fermented for 12 and 24 h by colonic microbiota (extracted from healthy human subject), <i> Clostiridium butyricum </i> BCC-B2571, or <i> Eubacterium rectale </i> DSM 17629. SCFA produced was analyzed by gas chromatography. Treatment by amylase-pullulanase combination was advantageous to increase their RS3 content. The result showed that after enzymatic process, the RS3 content of IR-42 (41.13%) was not significantly different (p < 0.05) from that of Inpari-16 (37.70%). High concentration of acetate (82.5 mM) and propionate (7.5 mM) were produced by colonic microbiota after 12 h fermentation and best concentration of butyrate (6.8 mM) was produced by colonic microbiota after 24 h fermentation. It is clear that utilization of colonic microbiota rather than single strain was better in the production of SCFA.
Healthy digestive system is increasingly important, in line with changes in diet and lifestyles. Imbalanced diet, such as not enough consuming dietary fiber, can harm the colon health that can lead to colon cancer. In healthy individuals, composition of the gut microbiota is very diverse, which is beneficial for colonic health. However, a loss of diversity combined with emerging imbalances between the proportions of bacterial strains can have severe consequences. Disruption of the equilibrium is called dysbiosis, associated with diarrhea, inflammatory bowel diseases (IBD), colorectal cancer as well as certain liver diseases and allergies, and nutrition-related conditions such as obesity, type 2 diabetes and celiac disease. Altered compositions of intestinal microbiota also affect the central nervous system as gut and brain are connected by a multitude of communication pathways used by bacterial metabolites and transmitters [
Dietary fiber intake can reduce risk of inflammatory bowel disease, cardiovascular disease, colon cancer, obesity and diabetes [
Resistant starch (RS) refers to starch and starch degradation products that escape from digestion in the small intestine of healthy individuals. Resistant starch, not digested in the small intestine, has physiological function as dietary fibers.Some types of resistant starch (RS1, RS2, and RS3) are fermented by the colonic microbiota and produce metabolite such as short chain fatty acids (SCFA): acetate, propionate, butyrate, and lactate. SCFAs is involved in many factors related to the health of colon,including the composition of gut microbiota, regulation of the immune system, inhibition of pathogens, intestinal motility, energy recovery, metabolic syndrome, bowel disorders, and colon cancer [
Applications of resistant starch in food products as prebiotics and food ingredients and their consumption are expected to maintain the health of colon, balance of gut microbiota and prevent colon cancer. Various studies had been conducted to produce RS flour. Basically, RS3 can be produced from high starch materials such as rice, sweet potato, banana, cassava, etc. Rice is food source, largely composed of starch. Rice milling will produce broken rice at considerable amount and currently, broken rice utilization is still limited, even regarded as waste or consumed as animal feed. Potential of the broken rice’s to be developed as RS, can be the solution to increase its economical value. Brokenrice production in Indonesia reached about 16% of MPD (milled rice), or about 11.4 million tons annually [
Zhao and Lin [
In our study, RS3 was made through combination of retrogradation (interaction between amylose fractions) and enzymatic hydrolysis (amylase-pullulanase). Then, RS3 was fermented by either colonic microbiota, or individual bacteria: Clostridium butyricum BCC-B2571, and Eubacterium rectale DSM 17629. The aim of this research were to find out the effect of these bacterial fermentation on the SFCA compositions.
Broken rice IR-42 and Inpari-16 were obtained from the Indonesia Center for RiceResearch, Sukamandi, Indonesia. Two types of starch degradation enzymes were from Novozymes. Enzyme used were: alpha-amilase (Liquozymes® Supra) 135 KNU/g and Pullulanase (Dextrozymes® DX 1.5X) 510 NPUN/g.
Colonic microbiota was extracted from feces of healthy adult subject, 30 - 50 years, who did not take antibiotics for at least 3 months and had no history of gastrointestinal disease. Feces (10 g) as dissolved in 90 mL of BPW was vortexed 30 seconds, then it was filtered. The filtrate was distributed into serum bottle (contained 100 mL of medium) and flushed with CO2. Pureculture of C. butyricum BCC-B2571 was obtained from Culture Collection of Indonesia Research Center for Veterinary Sciences (IVETRI), Indonesia. Eubacterium rectale DSM 17629 was obtained from DSMZ, Germany. The basal medium for colonic microbiota and C. butyricum BCC-B2571 consist of (g/L): yeast extract 3, beef powder 10, peptone 10, glucose 5, soluble starch 1, NaCl 5, Na-acetate 3 and cysteine hydrochloride 0.5. The pH was adjusted to 6.8. The basal medium for Eubacterium rectale DSM 17629 contained (g/L): tryptone 5, bacteriological peptone 5, yeast extract 10, beef extract 5, glucose 5, Tween 80 1 mL, resazurin 0.001, CaCl2 0.01, MgSO4 0.02, K2HPO4 0.04, KH2PO4 0.04, NaHCO3 0.4, NaCl 0.08, Vitamin K1 0.0002. The pH was adjusted to 7.0.
Rice was extracted byalkaline solution [
Rice starch was processed into RS3 following Kim et al. [
Moisture, ash, and crude fat were analyzed following AOAC [
RS3 content was analyzed according to Goni et al. [
Growth medium 20 mL, with RS3 (2%), was distributed in the serum bottles flushed with CO2, sealed with a rubber and sterilized at 121˚C 15 min. The medium was inoculated with 1 mL of 24 h pre-cultured bacterial strain (at about 109 CFU/mL), and incubated under anaerobic condition at 37˚C in water bath. Fermentation was carried out for 12 and 24 h, (three replications). In another in vitro fermentation, glucose (control) was used as the only carbon source (concentration 2%).
Gas production (mL) was measured by channeling the gas in the serum bottle to expand into glass syringe. The pH of the cultures was determined by pH meter.
The fermentation media was centrifuged (3000 g, 10 min), the supernatant was filtered with a membrane (0.45 µm) and stored at 4˚C until use. Samples (1 mL) was injected into gas chromatography (Agilent Technologist, 7890A GC System) equipped with a flame ionization detector (FID) and HP Innowax 19091-136 column (60 m × 0.250 mm). The carrier gas (H2) was run at speed 1.8 mL/min. The oven temperature was maintained at 90˚C for 0.5 min, and then increased to 110˚C at a rate of 10˚C/min, increased to 170˚C at a rate of 5˚C/min and finally increased to 210˚C at a rate of 20˚C/min. Injector and detector temperatures were 275˚C. SCFA mixture containing acetate, propionate and butyrate at specific concentration were used as standard.
All data were expressed as means ± SE from three independent trials. Differences between the mean values of multiple groups were analyzed by one-way analysis of variance (ANOVA). Duncan test was carried out to compare the data between treatments, independent t-test. Pearson correlation coefficients, p < 0.05 was considered a significant different, and SPSS 22 software was applied to analyze the data.
Chemical composition of the extracted rice starch is shown in
Chemical composition | IR-42 | Inpari-16 |
---|---|---|
Amylose (%) | 34.09 ± 0.17 | 28.28 ± 0.13 |
Moisture (%) | 8.28 ± 0.15 | 6.99 ± 0.25 |
Ash (%) | 0.15 ± 0.05 | 0.09 ± 0.001 |
Crude Fat (%) | 0.29 ± 0.04 | 0.26 ± 0.02 |
methods for maintaining high amylose content. The high starch (amylose) content of rice is considered to have better oppurtunity to be processed into RS3.
In the production of Resistant starch, therice starch was gelatinized and retrograded before hydrolyzed by enzymes. Gelatinization change the granular structure so that the starch became more accessible to the enzyme action. Storage at 4˚C induced retrogradation, crystallization and formation of the starch matrix which had undergone gelatinization. Alpha-amylase hydrolyzes (1,4)-α-D-glycosidic bond of the rice starch and produces linear oligosaccharide, maltose and glucose. The short linear oligosaccharide, maltose and glucose were removed during RS process, while the rest of its α-dextrin will be used for RS formation. Pullulanase hydrolyzes (1,6)-α-D-glycosidic of the amylopectin and produces linear oligosaccharides, maltose and glucose.Most oligosaccharides are able to form RS3 structure [
RS content of the starch extracted from rice IR-42 was higher than that of Inpari-16 (
The effect of resistant starch on pHs during in vitro fermentation by 12 and 24 his shown in
showed that fermentation of RS rice (1%) treated by amylase and pullulanase, resulted in pH 4.5 after 48 h fermentation when C. butyricum BCC-B2571 or E. rectale DSM 17629 were used. Acetate, propionate, and butyrate suppressed both growth and toxin production by C. difficile at concentrations as low as 10 mM, and these effects are pH dependent [
The effect of resistant starch on gas produced after 12 and 24 h fermentation is shown in
SCFA profile resulted from 12 and 24 h fermentation by different microbes is shown in
The main product after 24 h fermentation of RS3 for each microbe was acetate (18.09 to 63.28 mM), followed by butyrate (4.80 to 6.84 mM) and propionate (3.45 to 6.27 mM). Molar (mM) of acetate:propionate:butyric after 24 h fermentation by colonic microbiota were 63.28:6.27:6.84 in medium supplemented with RS3 IR-42 and 48.64:3.45:4.86 in medium supplemented with RS3 Inpari-16. Molar (mM) of acetate:propionate:butyrate after 24 h fermentation by C. butyricum BCC-B2571 were 59.45:4.53:6.39 in medium supplemented with RS3 IR-42 and 35.06:3.98:4.80 in medium supplemented with RS3 Inpari-16. Molar (mM) of acetate:propionate:butyrate after 24 h fermentation by E. rectale DSM 17629 were 28.27:5.74:6.48 in medium supplemented with RS3 IR-42 and 18.09:4.97:3.59 in medium supplemented with RS3 Inpari-16.
Our study showed that after 12 h fermentation, C. butyricum BCC B2571 produced higher butyrate (p < 0.05) in medium supplemented with RS3 IR42
(6.78 mM) than in medium supplemented with RS3 Inpari-16 (2.51 mM). Meanwhile after 24 hfermentation, the colonic microbiota produced butyrate higher (p < 0.05) in medium supplemented with RS3 IR42 (6.84 mM) than in medium supplemented with RS3 Inpari-16 (4.86 mM).
In medium supplemented with RS Inapri-16, production of acetate, propionate, and butyrate by colonic microbiota and C. butyricum BCC-B2571 showed different molarity after 12 and 24 h fermentation. RS3 Inpari-16 fermented by colonic microbiota and C. butyricum BCC B2571, produced higher acetate, propionate, and butyrate significantly after 24 h fermentation compared with at 12 h fermentation.
The result confirmed that proportion and content of SCFA was dependent on the bacterial strain used and type of the resistant starch (RS content). The
Resistant starch | SCFA after fermentation | Colonic microbiota | C. butyricum BCC B2571 | E. rectale DSM 17629 |
---|---|---|---|---|
RS3 IR-42 | Acetate (mM) 12 h | 82.47 | 74.93 | 21.62 |
Acetate (mM) 24 h | 63.28 | 59.45 | 28.27 | |
p-value | 0.037 | 0.042 | 0.010 | |
Propionate (mM) 12 h | 6.27 | 6.10 | 5.33 | |
Propionate (mM) 24 h | 7.45 | 4.53 | 5.74 | |
p-value | 0.043 | |||
Butyrate (mM) 12 h | 6.44 | 6.68 | 5.37 | |
Butyrate (mM) 24 h | 6.84 | 6.39 | 6.48 | |
p-value | ||||
RS3 Inpari-16 | Acetate (mM) 12 h | 32.04 | 19.18 | 18.68 |
Acetate (mM) 24 h | 48.64 | 35.06 | 18.09 | |
p-value | 0.000 | 0.025 | ||
Propionate (mM) 12 h | 2.45 | 1.95 | 5.67 | |
Propionate (mM) 24 h | 3.45 | 3.98 | 4.97 | |
p-value | 0.000 | 0.000 | ||
Butyrate (mM) 12 h | 0.89 | 2.51 | 5.65 | |
Butyrate (mM) 24 h | 4.86 | 4.80 | 5.38 | |
p-value | 0.001 | 0.000 |
aOnly significant independent t-test reported (p < 0.05).
butyrate produced in our study was higher than previously reported (produced by colonic microbiota) [
In our study, accumulation of acetate implied that the butyrate was produced via butyryl-CoA transferase. Miller and Wolin [
The capability of RS3 rice as prebiotic was supported by the fact that RS3 was metabolized by the tested microbes and in the production of SCFA. Resistant starch improve metabolic activity of the gut microbiota by increasing the production of SCFA and, thus supported the growth of beneficial species in the healthy individual [
Propionate reduces food intake and increases satiety viaaugmentation of the satiety hormone leptin, and through activation of GPCR [
Butyrate is an energy source of the intestinal epithelial cells (growth and differentiation) and can increase mucin production which may result in changes on bacterial adhesion [
Significant positive correlations were observed between concentrations of SCFA (acetate, propionate, and butyrate) and colonic microbiota or C. butyricum BCC-B2571 after 12 h fermentation (
Treatment with amilase-pullulanase increased RS content of IR-42 from 19% to 41% and Inpari-16 from 10% to 37%. Fermentation of RS3 IR-42 and Inpari-16 by colonic microbiota, C. butyricum BCC-B2571, and E. rectale DSM 17629, produced SCFA with different molar ratio. Time fermentation affected molar
Microbe | 12 h | 24 h | ||||
---|---|---|---|---|---|---|
Acetate | Propionate | Butyrate | Acetate | Propionate | Butyrate | |
Colonic microbiota | 0.991b | 0.966b | 0.972 | 0.986 | ||
C. butyricum BCC-B2571 | 0.975 | 0.991 | 0.991 | 0.976 | 0.912 | |
E. rectale DSM 17629 | 0.975 |
aOnly significant correlations reported (p < 0.05), bp < 0.01.
ratio of SCFA production. Fermentation of C. butyricum BCC-B2571 in medium supplemented with RS3 IR42 produced molar of acetate:propionate:butyrate, 74.93 mM:6.10 mM:6.78 mM after 12 h fermentation. Fermentation of colonic microbiota in medium supplemented with RS3 IR42 produced molar of acetate:propionate:butyrate, 63.28 mM:6.27 mM:6.84 mM after 24 h fermentation. Both SCFA profile produced high butyrate. Our study showed that RS3 IR-42 had potential in the production of butyrate.
The research was supported by Indonesian Center for Agricultural Postharvest Research Development, Indonesia Center for Rice Research, Sukamandi, and Ministry of Research and Technology, Republic of Indonesia.
Hutabarat, D.J.C., Zakaria, F.R., Purwani, E.Y. and Suhartono, M.T. (2018) SCFA Profile of Rice RS Fermentation by Colonic Microbiota, Clostridium butyricum BCC B2571, and Eubacterium rectale DSM 17629. Advances in Bioscience and Biotechnology, 9, 90-106. https://doi.org/10.4236/abb.2018.92008