Zhou 2013

Starch/Sta¨rke 2013, 65, 509–516

DOI 10.1002/star.201200166

509

RESEARCH ARTICLE

Effect of resistant starch structure on short-chain fatty acids production by human gut microbiota fermentation in vitro Zhongkai Zhou1, Xiaohong Cao1 and Julia Y. H. Zhou2 1 2

School of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin, P. R. China School of Pharmacy and Medical Science, University of South Australia, Adelaide, Australia

Resistant starch (RS) from uncooked and cooked high amylose starch (HAS) was used to investigate the effect of RS structure on the production of short-chain fatty acids (SCFA) using a static anaerobic in vitro system. The pH value of culture correlated well to the production of total SCFA after the fermentation (R2 ¼ 0.969). Most importantly, fermentation of RS from thermally treated starch under no moisture condition produced the highest concentration of SCFA and the greatest ratios of butyrate/acetate and butyrate/total SCFA, followed by the fermentation of RS from uncooked HAS. FTIR analysis suggests that all the RS exhibited a relatively higher organized structure compared to its corresponding original starch. The analysis of molecular structure by HPLC showed that the most pronounced difference in molecular composition of RS between the group favoring greater butyrate production and the group supporting a lower butyrate production was that larger molecules (amylopectin fraction) still existed in the former group but not in the latter group. Thus, it can be concluded that RS structure, in particular molecular structure is one of the key factors manipulating SCFA production in amount and proportion.

Received: August 4, 2012 Revised: September 24, 2012 Accepted: September 27, 2012

Keywords: Butyrate / Gut microbiota fermentation / Molecular structure / RS / Short-chain fatty acids

1

Introduction

Abbreviations: HAS, high amylose starch; HTT, hydrothermal treatment; NTT, non-moisture thermal treatment; SCFA, shortchain fatty acid; SE-HPLC, size-exclusion high performance liquid chromatography

RS as a substrate for stimulating colonic fermentation and promoting production of short-chain fatty acids (SCFA) [2–5]. In terms of production, acetic, propionic, and butyric are the major SCFA produced in the human colon. Physiological studies found that SCFA can modulate colonic muscular activity and stimulate blood flow to the colon, and they also appear to lower the risk of pathogen overgrowth [6, 7]. Of the principal SCFA, butyrate is thought to be pivotal for human colonic health, as it has been shown to promote growth of normal colonocytes and enhance apoptosis in colorectal cancer cells in vitro [8–11]. There is some evidence that RS, relative to other potential substrates such as non-starch polysaccharides, favors increasing production of butyrate [12–15]. However, this may not be a characteristic of all RSs [7]. For instance, starches from various botanical sources were fermented differently by the microbiota of the large intestine, producing gas and SCFA in different proportions and at different

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RS is defined as the ‘‘total amount of starch, and the products of starch degradation that resists digestion in the small intestine of healthy people’’ [1]. In the large intestine RS acts as a substrate for microbial fermentation to produce a number of metabolites that are considered beneficial for the host. In recent years, an increasing number of studies have focused on the importance of

Correspondence: Dr. Zhongkai Zhou, School of Food Engineering and Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, P. R. China E-mail: [email protected] Fax: þ86-22-60601371

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rates depending on their origin [16]. These differences may be due in part to the experimental method used, sample preparation, type, amount, and structure of RS, and on the duration of feeding. Nevertheless, under tightly controlled experimental conditions it seems that the nature of the starch plays a very important role in modulating bacterial fermentation, especially concerning the amount and type of SCFA that are produced [17]. Quantitative data on the relationship between dietary starch and the metabolic activity of the colonic microbiota are still very limited due largely to the inaccessability of intestinal contents of humans. Much information on the role of RS as a fermentative substrate derives from studies that have involved the use of human fecal inocula to ferment indigestible polysaccharides in vitro as a method of quantifying the production of SCFA and lactate [5, 15, 18, 19]. A major deficiency is lack of knowledge of the relationship between starch/RS structure (both in physical and chemical properties), SCFA production and its composition, and the large bowel microbiota profile. This is crucial because the effects of RS appear to be mediated largely through fermentation products rather than physical bulking. In this study, starch chemical and physical properties were investigated using size-exclusion high performance liquid chromatography (SE-HPLC) and FTIR. Previous studies [20, 21] indicated that some of the bonds in FTIR spectrum were sensitive to changes in the degree of arrangements at molecular level. Ordered and amorphous structures of starch are represented at 1047 and 1022 cm1 bands of the infrared spectrum. In order to express the relative organization of starch structure, the ratio of absorbance at 1047 and 1022 cm1 was used. Hence, these could be used as a tool to study starch structural organization. Thus, the objective of this study was to investigate the relationship between RS structure (physical and chemical

properties) and its physiological functionality (e.g. SCFA production). In this paper, high amylose starch (HAS) in uncooked (native) and cooked forms are used to study the influence of starch structure on the production of SCFA using a static anaerobic in vitro system inoculated with a fresh fecal slurry to simulate large bowel fermentation in humans.

2

Materials and methods

2.1 Starch sample A high amylose maize starch (HAS, Hi-maizeTM, National Starch and Chemical Company, NSW, Australia) was used in this study. High amylose starch varieties (e.g. HAS) usually associated have a higher RS content than conventional starch varieties which are significantly degraded during digestion in the human gut, and a very limited fraction can reach the colon. Thus, it was only possible to obtain sufficient amount of RS from HAS and its residual was processed for physical and chemical analyses.

2.2 Starch treatment/sample preparation The preparations of non-moisture thermal treatment (NTT) starch and hydrothermal treatment (HTT) starch were developed in this study. The methodologies for sample preparations were briefly described in Table 1. Under different thermal treatment conditions, four groups of starches were obtained (Table 1). Prior to all analyses, all samples were freeze-dried after preparation and were ground using a Cyclone Sample Mill (UDY Corporation, Fort Collins, CO) and passed through a 0.5 mm sieve screen.

Table 1. Sample preparation: starch thermal treatment under different conditions Sample group

Sample preparation

Group 1 Group 2

Native (uncooked). Starch was thermally treated in an oven (at 1358C for 3.5 h). Starch was hydrothermally treated in an autoclave (at 1218C for 20 min) once. Starch was hydrothermally treated in an autoclave (at 1218C for 20 min) once. Starch was hydrothermally treated in an autoclave (at 1218C for 20 min) for 2 times. Starch was hydrothermally treated in an autoclave (at 1218C for 20 min) for 3 times.

Group 3 Group 4

Native (uncooked). Thermal treatment under no moisture condition (NTT)a). Hydrothermal treatment at a lower moisture content (25%) (HTT)b). Hydrothermal treatment at a higher moisture content (60%) (HTT).

Sample no. 1 2 3 4 5 6

a) NTT: thermal treatment under no moisture conditions. b) HTT: thermal treatment under moisture conditions. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Starch/Sta¨rke 2013, 65, 509–516

2.3 Starch digestion and RS preparation Two hundred milligrams of starch material from uncooked and cooked forms (see Table 1) was pre-digested using an in vitro system developed by CSIRO (Human Nutrition, Adelaide, Australia) to simulate human small intestinal starch digestion. Briefly, samples were mixed with artificial saliva and the resultant bolus incubated with pancreatic and gastric enzymes at physiological pH and temperature. After incubation, the supernatant was removed by centrifuge and the resultant RS was recovered and used as substrate for fermentation. The amount of residual starch in the digesta (RS) was determined using Megazyme assay kit (GOPOD method, manufacture instruction). After the digestion, around 90 mg of RS substrate from each treated HAS was collected and added to the fermentors.

2.4 FTIR for analysis of starch structural organization FTIR spectrum of each starch sample was recorded on a Varian spectrometer (Model: Excalibur 3100) equipped with a cooled deuterated triglycine sulfate (DTGS) detector. The measurement was performed on a MIRacleTM attenuated total reflectance (ATR) crystal plate with Digital Readout High Pressure Clamp (Pike Technologies, USA). Sample was directly loaded on the plate and scanned in the range of 3600–600 cm1 at a resolution of 4 cm1. Prior to recording, the spectra were transformed against an empty cell as background. Spectra were ATR deconvoluted and baseline corrected using Varian Resolutions Pro software.

2.5 SE-HPLC for analysis of RS molecular composition A 10-mL aliquot of aqueous dimethylsulfoxide (90:10, DMSO/water, v/v) was added to 50 mg of freeze-dried RS in 25 mL test tube. Each tube was capped and placed in a boiling water bath for 60 min and then cooled to room temperature before centrifuging at 2095  g for 15 min. The supernatant was collected into a separate 75 mL polypropylene centrifuge tube and the dissolved starch precipitated by adding 30 mL of 95% ethanol. The tube was then centrifuged (2095  g for 15 min), the supernatant discarded and the starch precipitate stored for analysis. The RS precipitate (around 30 mg), prepared as described above, was redissolved in 0.6 mL of 0.2 M sodium hydroxide solution and mixed vigorously for approximately 30 s. The solution was neutralized by addition of sodium acetate buffer (0.6 mL; 0.05 M, pH 4.0) before adding ion-exchange resin (0.25 g, BioRad ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

511 AG1 501-X8, USA) and incubating at 408C for 2 h with occasional shaking. After centrifuged at 10 000 rpm for 10 min, the clear supernatant was collected for HPLC analysis. The HPLC system comprised a GBC pump (LC 1150, GBC Instruments, Vic, Australia) equipped with Auto Sampler (GBC, LC1610) and Evaporative Light Scattering (ELS) Detector (ALLTech, USA). The UltrahydrogelTM 250 column and guard column (Waters, 7.8 mm  300 mm, made in Japan) were maintained at 358C. Ammonium acetate buffer (0.05 M; pH 5.2) was used as mobile phase at a flow rate of 0.6 mL/min. A 40 mL-aliquot of the supernatant was used for injection. Conditions for ELS detector operation were: tube temperature: 1158C; N2 gas flow rate: 2.0 L/min; gain: 16; impactor: on.

2.6 In vitro fermentation by human gut bacteria A 24 h in vitro anaerobic static batch fermentation system was used to investigate SCFA production of each RS. Fresh human fecal material was used as inoculum. Samples were collected from two separate individuals, both of whom were apparently healthy with no history of antibiotic treatment over the preceding 3-month period, consuming their usual diet. Feces were processed soon after defecation and a 10% w/v inoculum prepared. Briefly, equivalent amounts of homogenized stool from each donor were combined, mixed thoroughly, and then 150 mL of prereduced phosphate buffer added. The slurry was mixed and the resultant suspension constantly stirred as aliquots of the homogenate were removed and dispensed into individual pre-sterilized McCartney vials. Quadruplicate incubations were set up for the blank and each test. In the case of the blank, no additional substrate was added to the vials. Substrates were hydrated for 1 h at 48C in 9 mL of fermentation media. Fermentation medium contained the following (per liter of distilled water): 2.5 g trypticase, 125 mL micromineral solution (132 g CaCl2  2H2O, 100 g MnCl2  4H2O, 10 g CoCl2  6H2O and 80 g FeCl3  6H2O per liter distilled water.), 250 mL buffer solution (4 g (NH4)HCO3 and 35 g NaHCO3 per liter distilled water), 250 mL macromineral solution (5.7 g Na2HPO4 and 0.6 g MgSO4  7H2O per liter of distilled water) and 1.25 mL resazurine solution 0.1% w/v. To 1L of fermentation medium 33.5 mL of reducing solution (6.25 g cysteine hydrochloride, 6.25 g Na2S  9H2O and 40 mL NaOH 1 M per liter of distilled water), was added and the resulting solution sterilized at 1218C for 15 min. The pH of fermentation media was adjusted to 7.2. Each fermentation vial was inoculated with 1 mL of fecal slurry containing 1  1010 viable bacteria/mL to achieve a final concentration of 1  109 viable bacteria per ferment. Each vial was then sparged with nitrogen gas and capped then sealed with paraffin as an additional www.starch-journal.com

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precaution to ensure no leakage. Vials were incubated in a hybridization oven (XTRON HI 2002, Bartelt Instruments) at 378C and rocked gently (19 rpm) over a 24 h period, after which they were frozen immediately and stored. Samples were defrosted and processed; subsamples from each vial were taken for total and individual SCFAs, DL-lactate and pH.

2.7 SCFA and lactate analyses Two milliliters of ferment was centrifuged at 2851  g for 15 min. One milliliter of suspension was mixed with 50 mL of 0.1 M 2-ethylbutyric acids as internal standard and extracted by the addition of 500 mL of concentrated HCl and 2 mL of diethyl ether solution. Samples were vortexed for 1 min and the layers separated by centrifuging for 10 min at 2095  g. The ether layer was then transferred to a clean tube and capped. The extraction was repeated by adding another 1 mL of diethyl ether to the initial tube, vortexing for 1 min followed by centrifuging at 2095  g for 10 min. The second ether layer was then removed and combined with the first. Hundred microliters N-methyl-N-t-butyldimethylsilyltrifluoroacetamide (MTBSTFA) was added to 800 mL of the combined ether extracts. The reaction mixture was heated to 808C for 20 min to form t-butyldimethylsilyl (TBDMS) derivative, and left at room temperature for a further 24 h to ensure complete derivatization of lactic acid. Derivatives were analyzed using a gas chromatograph (6890 Agilent) fitted with a flame ionization detector, split injector and a Alltech AT-1 30 m  0.25 mm capillary column with 0.1 mm film thickness. Injector and detector temperatures were 2758C with the column temperature programmed from 638C for 3 min to 1908C at 108C/min. Helium was the carrier gas (head pressure 100 kPa). 0.2 mL injections were made in the split mode (50:1 split). The standard solution contained (mmol/L): acetate, 30; propionate, 20; isobutyrate, 5; n-butyrate, 20; isovalerate, 5; n-valerate, 5; lactate, 10. The internal standard was 2-ethylbutyric acid (100 mmol/L).

2.8 Statistical analysis RS is quantified as the difference between total starch and readily digestible starch after digestion. All of the sample analyses were performed and expressed as means  SEM of 3 replicates. Experimental data were subjected to analysis of variance using Genstat 5 (release 4.1). Differences in fermentation properties of RS from uncooked and cooked starches were assessed using the least significant differences (LSD) of means (5% level). The difference was considered as significant at p < 0.05. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

3

Results and discussion

3.1 IR spectrum of RS made from cooked and uncooked starches FTIR ratio of 1047/1022 cm1 for each starch sample was list in Table 2. Previous studies suggested that FTIR spectrum was sensitive to the changes of starch conformation during various thermal treatments, specifically in the range of 1300–900 cm1 [20, 21]. The band 1047 cm1 is composed of two overlapping bands, positioned at 1040 and 1053 cm1. During starch molecule’s re-association, the 1040 cm1 band appears very quickly; however the 1053 cm1 band requires a longer time to form. The intensity of the 1047 cm1 band increases as crystallinity of starch increases. As an example, the higher the crystallinity of starch the greater the intensity of the 1047 cm1 band, whereas the intensity of the 1022 cm1 band is characterized by reduction of crysallinity of starch. Thus, Absorbance at 1047 and 1022 cm1 was associated with the ordered and amorphous structures of the starch, respectively [20–22]. The ratio of absorbance at these particular wavelengths (1047 and 1022 cm1) might be used to express the relative organization of starch structure. In this study, it was found that starch molecules treated under a higher moisture content more likely tend to reorganize and then form a more organized structure than starch treated under a lower moisture content. Compared to uncooked status, there was a slight increase in starch structural organization after three times HTTs (Fig. 1). Meanwhile, increasing HTT cycle can also enhance starch structural organization at a higher moisture condition (i.e. sample no. 6>sample no. 5>sample no. 4; Table 2). Table 2. Change in FTIR Ratio of absorbance at 1047/ 1022 cm1 before and after digestions FTIR ratio: 1047/1022 cm1)b) After digestion Substrate Sample Before digestion no. (i.e. original starch) (i.e. RS) groupa) Group 1 Group 2 Group 3 Group 4

1 2 3 4 5 6

0.844 0.815 0.755 0.769 0.833 0.855

     

0.03 0.04 0.0 0.03 0.04 0.0

1.25 1.41 1.91 1.97 2.03 2.01

     

0.03 0.03 0.07 0.06 0.06 0.07

a) Group 1: uncooked/native HAS; group 2: HAS was thermally treated in an oven; group 3: HAS was hydrothermally treated in an autoclave at 25% moisture condition; group 4: HAS was hydrothermally treated in an autoclave at 60% moisture condition. b) Data expressed as mean  SEM of triplicate determinations for each sample. www.starch-journal.com

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0.5 0.4

3.2 Molecular composition of RS Cooked HAS Native HAS

Absorbance

0.3 0.2 0.1 0.0 -0.1 1100

1080

1060

1040

1020

1000

980

Wavenumber (cm-1)

960

940

Figure 1. Difference in FTIR spectra between native/ uncooked HAS (sample no. 1) and cooked HAS (sample no. 6).

Data in Table 2 demonstrated that all RS had a higher ratio of 1047/1022 cm1 compared to the starch before digestion (i.e. ranging from 1.25 to 2.03 after digestion versus ranging from 0.755 to 0.855 before digestion). Because the IR ratio for RS exceeds 1 suggesting that every RS exhibited a relatively higher organized structure due to the removal of the digestible starch fraction by digestion [23]. RS from thermal treated starch, irrespective of moisture content was of greater structural organization than RS from uncooked (Table 2).

The difference in molecular composition among the RS substrates was examined and their chromatographs were presented in Fig. 2a–c. The pronounced feature of the structural profile among the substrates was the difference in the ratio of amylopectin/amylose. For instance, RS from uncooked HAS had the highest amylopectin fraction in its molecular profile, followed by RS from NTT sample. However, for molecular profile of RS from HTT samples (both group 3 and group 4), their amylopectin fraction was completely absent, irrespective of moisture content during HTT. Figure 2 indicated that thermal treatment altered starch structure from exterior to interior, subsequently influencing RS molecular structure. The results also implied thermal treatment on starch, either with or without moisture, greatly enhanced enzymatic susceptibility for both amylose and amylopectin fractions, in particular for amylopectin fraction. The obtained RS with different molecular structure is useful to study the effect of molecular structure on its physiological property.

3.3 pH of ferment culture and total SCFA production In vitro systems have been used as an alternative to invasive techniques to investigate RS fermentation. Static 24 h batch fermentations were set up as described previously using substrates consisting of

Figure 2. Difference in molecular composition of RS from uncooked HAS (the curve on the bottom) and RS from thermally treated starch (the curve on the top) (a: uncooked HAS versus NTT sample; b: uncooked HAS versus HTT sample at 25% moisture condition (sample no. 3); c: uncooked HAS versus HTT sample at 60% moisture condition (sample no. 5). NTT: thermally treated under no moisture condition; HTT: thermally treated under moisture condition. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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RS from different treatment of HAS. Quadruplicate fermentations were inoculated with a human fecal microbiota suspension and incubated for 24 h under anaerobic conditions at 378C (as described in materials and method). The culture medium had a pH of 7.2 at time zero. After fermentation, all the cultures showed a decrease in pH value (Table 3), but the magnitude of the reduction in pH value was markedly different depending on the RS resources. Generally, the fall in pH value observed for fermentation of RS from NTT sample was greater than that of RS from HTT sample. This difference might be due to the higher concentration of net total SCFA, in particular acetate produced by fermentation, because acetate is the major SCFA in each ferment and it has a lower pKa (4.74) than other SCFA in cultures. The pH value of batch culture correlated very well to the production of net total SCFA after the fermentation (R2 ¼ 0.969; Fig. 3). Acidification of the large bowel lumen is believed to be advantageous for the host in that it is less favorable for the growth of intestinal pathogens and may also reduce the production and activity of potential toxins [24]. The net total SCFA production, estimated as mmol/L of each fermented culture, is summarized in Table 3. The effect of RS resources on SCFA production showed a similar pattern to that of pH. Briefly, RS from NTT sample produced the highest SCFA concentration, followed by RS from uncooked HAS, and then RS from HTT samples. Increase of thermal treatment cycles under higher moisture content (group 4 samples) could significantly improve SCFA production (Table 3; p < 0.001). Results obtained in this study suggested that RS from either NTT sample or uncooked HAS was the best ferment substrate for SCFA production.

Figure 3. Relationship between culture pH value and net total SCFA concentration after gut bacterial fermentation of each RS sample.

3.4 Principal SCFA concentration and proportion The principal SCFAs in all instances were acetic, propionic and butyric (Table 3). Other SCFAs were also detected in cultures, including iso-butyric, iso-valeric, and valeric acids, but in much lower concentrations and were not reported in this study. Of the principal SCFA, acetic acid was present at the highest concentration in all ferments. Although no significant difference in acetate production was found between the fermentation of RS from uncooked HAS and RS from NTT sample, fermentation of RS from uncooked HAS produced the highest acetate concentration in all instances. Fermentation of RS from NTT sample produced the highest concentration of butyrate, followed by the fermentation of RS from uncooked HAS, whereas HTT groups

Table 3. Culture pH value and net total SCFA and lactate concentrations after 24 h fermentation SCFA (mmol/L)b) Group Group 1 Group 2 Group 3 Group 4

Sample no.a)

pH

1 2 3 4 5 6

5.55 5.26 5.99 6.27 6.00 5.90

Acetic      

0.3 0.1 0.1 0.4 0.3 0

23.44 20.66 18.93 12.43 15.79 21.47

Propionic      

0.63 2.38 1.63 5.13 2.52 1.11

4.34 9.40 2.83 3.74 3.65 4.03

     

0.08 1.07 0.30 0.41 0.16 0.29

Butyric 8.15 14.55 3.38 2.20 3.27 4.95

     

0.42 1.88 0.45 1.62 0.72 0.38

Lactic

Total

Nil Nil 0.02  0.03 Nil Nil Nil

35.93 44.58 25.17 18.32 22.66 30.41

     

0.35 3.56 1.39 6.80 3.28 1.11

Butyric/ Acetic (100)

Butyric/ total SCFA (%)

34.77 70.43 17.86 17.70 20.71 23.06

22.68 32.64 13.43 12.01 14.43 16.28

a) Sample 1: native HAS; sample 2: HAS was thermally treated in an oven; sample 3: HAS was hydrothermally treated at 25% moisture condition; sample 4: HAS was hydrothermally treated at 60% moisture condition once; sample 5: HAS was hydrothermally treated at 60% moisture condition twice; sample 6: HAS was hydrothermally treated at 60% moisture condition for three times. b) Data were quoted as mean  SEM of quadruplicate determinations. ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Starch/Sta¨rke 2013, 65, 509–516 produced the lowest concentration of butyrate during fermentation (Table 3). This result is consistent with the report from Saito et al. [25] studied in an animal trial. For samples in group 4, starches were hydrothermally treated for different times (once, twice and three times, respectively), and their fermentation data (Table 3) showed that butyrate production was greatly enhanced with increasing treatment times. Fermentation of RS from NTT sample produced the greatest propionate concentration as well among all the samples (Table 3). Compared to acetate, propionate concentration was much lower, which might be due to only a limited number of gut bacterial species producing propionate. Many of the known propionate producing bacteria included Selenomonas, Mitsuokella, Megamonas, Megasphaera, Veillonella etc., which are capable of utilizing lactate and converting it largely to acetate and propionate. Nevertheless, in some studies it was also found that the fermentation of RS could promote the formation of propionic acid [26, 27]. Most importantly, ratios of butyrate/acetate and butyrate/total SCFA were greatly enhanced during the fermentation of RS from NTTsample, followed by the fermentation of RS from uncooked HAS. The much lower in those two ratios (Table 3) obtained from the fermentation of RS from HTT groups suggested that starch which underwent different gelatinization/retrogradation process could modulate bacterial fermentation pattern, subsequently influencing metabolites of ferments (e.g. SCFA). Furthermore, the analysis of molecular profile by HPLC showed that the most pronounced difference in molecular structure of RS between the groups favoring greater butyrate production (i.e. NTT sample and uncooked HAS sample) and the groups supporting a lower butyrate production (i.e. HTT samples, both group 3 and group 4) was that the amylopectin fraction still existed in the molecular profile of former groups (although it is not a dominant molecular fraction in the profile) but absent in the molecular profile of the latter groups. It seems that molecular structure of RS plays an important role manipulating gut bacterial fermentation behavior, subsequently influencing SCFA production in amount and proportion.

4

Conclusions

Fermentation of RS by human gut bacteria resulted in the production of SCFA and a concomitant decline in pH. A significant correlation was observed between the pH value of batch culture and the net production of total SCFA after the fermentation. This study indicated gut microbiota fermentation patterns can be modulated by RS structure. RS from NTT sample seems to be the most favorable fermentation substrate for the production of ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

515 SCFA, particularly butyric acid, followed by the fermentation of RS from uncooked HAS. Their fermentation was characterized by the highest production of total SCFA, and the greatest ratios of butyrate/acetate and butyrate/total SCFA. Structural analyses by FTIR suggested that these two sources of RS have a higher absorption ratio at 1047/1022 cm1 indicating a higher organized structure because of removing less-organized region in starch by digestion. The analysis of molecular structure by HPLC showed that these two sources of RS contain larger molecular composition (amylopectin fraction) in their molecular profiles, which was assumed to be one of key factors promoting butyric/SCFA production. The authors wish to thank Nutrition Analysis Lab staff at CSIRO Human Nutrition, Adelaide, Australia, particularly Michelle Vuaran for her excellent work conducting fermentation experiment and SCFA analysis. Special thanks to Drs Anthony Bird & David Topping for the discussion of the project plan, data interpretation and reviewing the manuscript. The authors have declared no conflict of interest.

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