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Fermentation characteristics of resistant starch from maize prepared by the enzymatic method in vitro
International Journal of Biological Macromolecules 51 (2012) 1185–1188
Contents lists available at SciVerse ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Fermentation characteristics of resistant starch from maize prepared by the enzymatic method in vitro Huanxin Zhang a,b , Xueming Xu a , Zhengyu Jin a,∗ a b
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China School of Food Science and Technology, Jiangsu Animal Husbandry and Veterinary College, Taizhou 225300, China
a r t i c l e
i n f o
Article history: Received 8 August 2012 Received in revised form 22 August 2012 Accepted 25 August 2012 Available online 1 September 2012 Keywords: Resistant starch Fermentation in vitro Short-chain fatty acids
a b s t r a c t To investigate the fermentation characteristics of resistant starch prepared by hydrolysing maize starch with ␣-amylase and pullulanase, fresh faecal extracts from healthy humans and infants were used as a fermentation model of human intestines in vitro. The RS was fermented for a certain period of time under the simulated condition of the large intestines (anaerobic and 37 ◦ C). The concentration of shortchain fatty acids in the fermented product as determined by gas chromatography was used as an index to characterise the fermentation effect. The results showed that the concentration of short-chain fatty acids, especially butyric acid, in the fermented product gradually increased with increased fermentation time and RS content. However, the concentration of short-chain fatty acids in the fermented product from healthy infant faecal extracts, especially butyric acid, was much higher than that from healthy adult faecal extracts. It suggested that the model of RS-produced acids was affected by the fermentation extract source, i.e., by the existence of microbial flora. The production model of acids demonstrated that maize RS prepared by the enzymatic method can be a promising ingredient of functional foods. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Dietary fibre has been recognised as a functional food ingredient for more than 20 years. It acts either directly as a consequence of its chemical composition and physicochemical properties [1], or indirectly via fermentation by colonic microflora [2]. Some health benefits related to the consumption of dietary fibre include decreased glycemic index value of food products and decreased colonic pH. The fibre portion fermented by the intestinal microflora also produces a range of short-chain fatty acids, especially butyrate, which is thought to provide a degree of protection against bowel cancer, reduce gall stone formation, exert hypocholesterolemic effects, inhibit fat accumulation, and increase mineral absorption [3,4]. In recent years, resistant starch (RS) has been classified as a functional fibre. RS is the sum of starch and its degradation products not absorbed in the small intestine of healthy individuals. Englyst et al. [5] have classified RS into three major groups, namely, RS1, RS2, and RS3. RS1 is physically inaccessible starch, such as that found in plant tissue structures. RS2 is condensed and partially crystalline native (uncooked) starch granules. RS3 is mainly retrograded or recrystallised amylose [6,7], which forms in cooked products after
∗ Corresponding author. Tel.: +86 510 85913922; fax: +86 510 85919189. E-mail addresses: [email protected] (H. Zhang), [email protected] (Z. Jin). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.08.031
cooling (e.g., in breads, corn flakes, or potatoes). More recently, a fourth type (RS4) has been produced by chemical modifications [8], such as conversion, substitution, or cross-linking. Such modifications prevent the digestion of RS4 by blocking access to enzymes and forming atypical linkages, e.g., 1 → 2, 1 → 3, 1 → 4, and 1 → 6. From the four RS types, many food processes decrease or eliminate RS1 and RS2 but have the potential to generate RS3. RS3 is particularly interesting because it retains its indigestibility when added as an ingredient to processed foods, and has many advantages over traditional insoluble fibres. It is a natural-white source of dietary fibre, has a bland flavour, has a better appearance and texture, imparts a less gritty mouthfeel, and masks flavour less than other typical insoluble fibres [9]. It is mainly used as a functional ingredient in low-moisture food products [10], particularly in bakery products such as bread, muffins, and breakfast cereals [11,12]. In the last decade, many studies on the fermentation characteristics of dietary fibre in vitro and in vivo have been conducted [13–15]. The in vitro research model uses extracted intestinal microflora as the intestinal environment. The short-chain fatty acid content of the fermented products is determined to evaluate the fermentability of dietary fibre by intestinal microflora [16–18]. However, information on the fermentation characteristics of RS is limited [19,20]. The RS used in this study was prepared from maize starch by combining ␣-amylase and pullulanase in our previous work [21]. We used extracts from fresh faeces of healthy adults and healthy
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babies as models of the large intestine microbial environment to evaluate the fermentation properties of maize RS. We analysed the short-chain fatty acid content of the fermented products to examine the fermentation behaviour of maize RS under different environments. The objective of this work was to investigate the fermentation characteristics of maize RS in human intestine in vitro. 2. Materials and methods 2.1. Materials Native maize starch was obtained from the Carbohydrate Research Center, Jiangnan University, China. Pullulanase was purchased from Genencor Bio-Products Co. Ltd. (Wuxi, China). The enzymatic activity was 400 ASPU/g as determined by reaction with pullulan (1.0%) buffered with sodium acetate (pH 5.0) at 50 ◦ C. ␣-Amylase was obtained from Sigma–Aldrich Trading Co. Ltd. (Shanghai, China). The activity of ␣-amylase was 1400 U/g. Enzymatic activity was determined by reaction with soluble starch (1.0%) buffered with sodium acetate (pH 4.4) at 37 ◦ C [22]. Highperformance liquid chromatography-grade methanol was acquired from Thermo Fisher Scientific (USA). The fatty acid standards of formic acid, acetic acid, propionic acid, and butyric acid were purchased from Sigma Chemical Co. All other chemicals were reagent grade. 2.2. Preparation of maize RS Maize starch (20%, w/v) was pre-gelatinised with distilled water by stirring for 20 min at 80 ◦ C/and pH 6.0. The starch paste was treated with thermostable ␣-amylase under the following conditions: temperature, 90 ◦ C; pH, 5.5; time, 15 min; and amount of ␣-amylase, 4 U/g. After adding pullulanase, the mixture was incubated in a shaking water bath at 46 ◦ C for 12 h, cooled at room temperature, and then stored at 4 ◦ C overnight. The mixture was treated with thermostable ␣-amylase (1%, w/w), incubated in a 95 ◦ C boiling water bath for 45 min, and centrifuged at 4000 × g for 15 min. After discarding the supernatant, the residue was suspended in 100% ethanol (1:4 residue:ethanol) and shaken for 15 min. Ethanol was removed by centrifugation (4000 × g, 15 min), and the above procedure was repeated two more times. The residue was dried overnight at 40 ◦ C, ground in a centrifugal mill to pass through a 0.5 mm sieve, and stored in airtight containers at room temperature until use. 2.3. Fermentation of RS in vitro 2.3.1. Collection and preparation of human faeces Human faeces were anaerobically collected from 10 healthy adults and 10 healthy babies who had received no antibiotic treatment for 1 month and no abdominal diarrhea and enteritis. About 40 g of faecal sample was dissolved in 250 mL of phosphate buffer (0.1 mol/L, pH 6.5) and homogenate for 2 min to produce a 160 g/L suspension. The suspension was filtered through a nylon sieve to remove particulate matter. The collection and preparation was finished within 1 h. 2.3.2. Fermentation in vitro Approximately 5 mL of faecal suspension from healthy adults and healthy babies were transferred to 5 mL of phosphate buffer containing 2.0, 4.0, and 8.0 g/L RS. Phosphate buffer without RS was used as a blank control. The mixtures were anaerobically cultured with continuous vibration (50 r/min) at 37 ◦ C for 6, 12, and 24 h, respectively. The test tube was removed and vigorous mixing was performed for further analysis.
2.4. Gas chromatographic analysis of short-chain fatty acids The short-chain fatty acids were analysed by gas chromatography (GC) according to the method of Drzikova et al. [17] with slight modifications. About 3 mL of the fermentation broth was centrifuged (4 ◦ C, 10,000 × g, 10 min). The supernatant (100 L) was mixed with 280 L of HClO4 solution (0.36 mol/L) and 270 L of NaOH solution (1 mol/L) with vigorous shaking. About 50 L of phosphoric acid (5 mol/L) was added, and the mixture was again vigorously shaken. About 300 L of methanol solution (containing 1% H2 SO4 ) was added and refluxed at 70 ◦ C for 30 min. Thereafter, 300 L of cold hexane was added to extract the short-chain fatty acid methyl ester. About 2 L of the organic layer was analysed by gas chromatography, and the determination was performed in triplicate. GC analysis was carried out as follows: 2 L of the hexane solution of methylated fatty acids was injected into a GC unit (Agilent 6890N, USA) equipped with an auto sampler (HP 7673) using a flame ionisation detector (FID) and capillary column (HP5; 30 m × 0.32 mm × 0.25 m) with nitrogen as the carrier gas at the flow rate of 0.6 mL/min. The split ratio was 50:1, the injection temperature was 250 ◦ C, and the FID temperature was 250 ◦ C. The temperature of the column was initially 100 ◦ C, increased at a rate of 2 ◦ C/min to 200 ◦ C, and held at 200 ◦ C for 20 min. The short-chain fatty acid standards were analysed by the same method. The concentrations of formic acid and acetic acid were 20 mmol/L, whereas those of propionic acid and butyric acid were 10 mmol/L. The fatty acid methyl esters were identified by comparison with standards and quantified by the area percentage of each fatty acid methyl ester. 2.5. Statistical analysis All experiments were performed in triplicate, and data were expressed as means ± SE. Statistical analysis was carried out using the data analysis tool pack of the software Origin 7.5. 3. Results and discussion 3.1. Analysis of short-chain fatty acids in the fermentation broth GC analysis is well established as a useful tool for determining the quality and quantity of short-chain fatty acids in various industrial applications. Fig. 1 shows that the short-chain fatty acid standards had four major chromatographic peaks at 4.4, 4.7, 5.1, and 8.0 min, representing formic, acetic, propionic, and butyric acids, respectively. The fermentation profiles of RS are shown in Fig. 2. There were also four peaks in the profiles from 4 min to 10 min. Compared with the GC results of the short-chain fatty acid standards, four typical fatty acids (formic, acetic, propionic, and butyric acids) were identified in the fermentation products of RS. Hence, the model of standards effectively distinguished the short-chain fatty acids from the fermentation products of RS. 3.2. RS fermentation in the simulated adult intestinal environment The substrate composition is known to affect the production of fermentation metabolites and the composition of colonic microflora, which together exert beneficial health effects [23]. RS fermentation in the simulated adult intestinal environment is shown in Table 1. The total amount of short-chain fatty acids in the fermentation products gradually increased with increased fermentation time. The total amount of short-chain fatty acids in the fermentation products also gradually increased with increased RS content. The total amount of short-chain fatty acids markedly
H. Zhang et al. / International Journal of Biological Macromolecules 51 (2012) 1185–1188
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related to the prevention of rectal cancer. These facts prompted us to focus on butyric acid in this study. As shown in Table 1, a variety of short-chain fatty acids in the fermentation products increased with increased fermentation time and RS content. However, the increase rates differed among the various short-chain fatty acids. The increase rates of formic, acetic, and propionic acids were smaller than that of butyric acid. The concentration of butyric acid increased from 2.71 mmol/L to 6.78 mmol/L with increased RS content from 2.0 g/L to 8.0 g/L after 6 h of fermentation. The concentration of butyric acid increased from 5.26 mmol/L to 9.86 mmol/L with increased RS from 2.0 g/L to 8.0 g/L after 12 h of fermentation. The concentration of butyric acid increased from 5.87 mmol/L to 10.23 mmol/L with increased RS content from 2.0 g/L to 8.0 g/L after 24 h of fermentation. Butyric acid was the main product after 12 h of fermentation. 3.3. RS fermentation in the simulated baby intestinal environment Fig. 1. Chromatogram of a standard mixture of short-chain fatty acids (1, formic acid; 2, acetic acid; 3, propionic acid; 4, butyric acid).
Fig. 2. Chromatogram of the short-chain fatty acids in the RS fermentation products (1, formic acid; 2, acetic acid; 3, propionic acid; 4, butyric acid).
increased from 6 h to 12 h, but the increase was smaller after 12 h, implying that the most of the short-chain fatty acids were produced within 12 h. Given that butyric acid can induce intestinal epithelial cell growth and cell differentiation, it can also enhance the immune effect. Butyric acid can also induce colon cancer cell cracking, thus preventing the spread of cancer by post-modification-induced gene expression and normal cell decline. Thus, butyric acid is closely
RS fermentation in the simulated baby intestinal environment is shown in Table 2. The total amount of short-chain fatty acids in fermentation products gradually increased with increased fermentation time. The total amount of short-chain fatty acids in the fermentation products also gradually increased with increased RS. The total amount of short-chain fatty acids markedly increased from 6 h to 12 h, but the increase was smaller after 12 h, implying that most of the short-chain fatty acids were produced within 12 h. The variety of short-chain fatty acids in the fermentation products increased with increased fermentation time and RS content, but the increases varied. The development trends of the short-chain fatty acids in the fermentation product were very similar to those shown in Table 1. Interestingly, the total shortchain fatty acid contents of the fermentation products from the baby faecal extracts were higher than those from the adult faecal extracts. Most importantly, the butyric acid content of RS fermented from baby faecal extracts was greatly improved compared with that from healthy adult faecal extracts. The butyric acid content of the fermentation broth increased from 7.42 mmol/L to 11.45 mmol/L with increased RS concentration after 6 h of fermentation. After 12 h of fermentation, the butyric acid content of the fermentation broth increased from 9.58 mmol/L to 19.58 mmol/L. The butyric acid content of the fermentation broth increased from 10.67 mmol/L to 21.48 mmol/L after 24 h of fermentation time. Most of the butyric acid was produced after 12 h of fermentation. Surprisingly, the highest level of butyric acid in the healthy adult faecal extracts fermented for 6 h was 6.78 mmol/L, whereas that in the healthy infant faecal extracts was almost double at 11.45 mmol/L. The highest level of butyric acid in the healthy adult faecal extracts after 12 h was 9.86 mmol/L, whereas that in the healthy infant faecal extracts was also double at 19.58 mmol/L.
Table 1 Fermentation effect of RS from healthy adult faecal extracts. Concentration of resistant starch (g/L)
Time (h)
Organic acids (mmol/L) Formic acid
2.0
4.0
8.0 Data were expressed as x ± SD (n = 3).
6 12 24 6 12 24 6 12 24
12.54 15.63 15.47 13.15 16.65 17.14 12.57 15.83 15.67
± ± ± ± ± ± ± ± ±
0.68 1.23 0.78 0.27 0.34 0.18 1.83 0.84 0.52
Acetic acid 5.72 7.66 7.59 6.57 9.26 10.13 6.18 9.13 10.56
± ± ± ± ± ± ± ± ±
0.24 0.84 0.54 0.92 0.53 0.28 0.38 0.43 0.22
Propionic acid 7.27 9.62 9.75 11.15 12.46 13.71 12.43 13.37 13.12
± ± ± ± ± ± ± ± ±
0.76 1.72 0.93 0.45 0.38 0.62 0.75 1.18 0.63
Butyric acid 2.71 5.26 5.87 5.74 9.26 10.73 6.78 9.86 10.23
± ± ± ± ± ± ± ± ±
0.25 0.28 0.62 0.46 0.33 0.44 0.36 0.24 0.38
Total acid 28.24 38.17 38.67 36.62 47.58 51.71 37.97 48.18 49.58
± ± ± ± ± ± ± ± ±
1.75 3.36 2.37 2.72 1.48 2.78 2.74 3.56 3.28
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Table 2 The fermentation effect of maize resistant starch by health babies faecal extractions. Concentration of resistant starch (g/L)
2.0
4.0
8.0
Time (h)
Organic acids (mmol/L) Formic acid
6 12 24 6 12 24 6 12 24
16.73 18.46 19.52 17.65 19.72 21.28 19.68 22.57 22.74
± ± ± ± ± ± ± ± ±
0.48 0.43 0.37 0.47 0.94 0.85 0.75 0.62 0.34
Acetic acid 13.27 14.58 15.65 12.48 15.67 14.83 14.37 16.64 17.14
± ± ± ± ± ± ± ± ±
0.72 0.78 0.57 0.56 0.73 0.34 0.68 0.36 1.78
Propionic acid 15.56 17.27 19.16 17.15 19.52 20.18 18.23 19.05 19.83
± ± ± ± ± ± ± ± ±
0.45 0.64 0.87 0.46 0.40 0.37 0.47 0.78 1.32
Butyric acid 7.42 9.58 10.67 8.74 13.25 15.73 11.45 19.58 21.48
± ± ± ± ± ± ± ± ±
0.38 0.52 0.95 0.61 0.83 0.74 0.78 1.28 0.67
Total acid 52.98 59.89 65.00 56.02 68.16 72.02 63.73 77.84 81.19
± ± ± ± ± ± ± ± ±
1.78 2.13 2.47 2.42 2.63 2.19 2.45 3.14 3.78
Data were expressed as x ± SD (n = 3).
Hence, the microbial flora in the infant faeces was more effective in fermenting maize RS. Lebet et al. [1] using healthy adult faeces as fermenters and dietary fibre as a fermentation substrate, have found that with increased fermentation time, the short-chain fatty acids in the fermentation products gradually increased, but the total shortchain fatty acid content slowly increased after 12 h. Mette et al. [24] have studied the impact of RS-produced short-chain fatty acid concentrations in pig intestine. The results show that the total amount of short-chain fatty acids, especially butyric acid, in pig intestine significantly increased with increased RS content in the diet. These results are similar to those of the present work. 4. Conclusions The fermentation behaviour of RS in vivo was evaluated under a simulated environment using microbial flora extracted from fresh faeces of healthy adults and babies in vitro. The short-chain fatty acids of the fermentation products gradually increased with increased fermentation time and RS content. The butyric acid content increased significantly. The amount of short-chain fatty acids, especially butyric acid, of the fermentation products from fresh healthy baby faecal extracts was significantly higher than that from fresh adult faecal extracts. These results suggested that the model of RS-produced acids was affected by the fermentation extract source, i.e., by the existence of microbial flora. The production model of acids demonstrated that maize RS prepared by the enzymatic method can be a promising ingredient of functional foods. Acknowledgements This work was financially supported by the National Natural Science Foundation (nos. 20976070 and 31071490), the Jiangsu
Animal Husbandry and Veterinary College (project no. YB201004), the Jiangsu Provincial Qing Lan Project. References [1] V. Lebet, E. Arrigoni, R. Amadò, LWT – Food Science and Technology 31 (1998) 473–479. [2] J.H. Cummings, G.T. Andmacfarlane, Journal of Applied Bacteriology 70 (1991) 443–459. [3] C.W.C. Kendall, A. Esfahani, D.J.A. Jenkins, Food Hydrocolloids 24 (2010) 42–48. [4] C.K. Miller, M.D. Gutshcall, D.C. Mitchell, Journal of the American Dietetic Association 109 (2009) 319–324. [5] H.N. Englyst, S.M. Kingman, J.H. Cummings, European Journal of Clinical Nutrition 46 (1992) 33–50. [6] C.S. Berry, Journal of Cereal Science 4 (1986) 301–314. [7] S.G. Ring, J.M. Gee, M. Whittam, P. Oxford, I.T. Johnson, Food Chemistry 28 (1988) 97–109. [8] A. Laurentín, M. Cárdenas, J. Ruales, E. Pérez, J. Tovar, Journal of Agricultural and Food Chemistry 51 (2003) 5510–5515. [9] M.G. Sajilata, R.S. Singhal, P.R. Kulkarni, Comprehensive Reviews in Food Science and Food Safety 5 (2006) 1–17. [10] R. Vue, S. Waring, Food Australia 50 (1998) 615–621. [11] R. Baixauli, T. Sanz, A. Salvador, S.M. Fiszman, Journal of Cereal Science 47 (2008) 502–509. [12] M.G. Sajilata, R.S. Singhal, Carbohydrate Polymers 59 (2005) 131–151. [13] L. Brown, B. Rosner, W.W. Willett, F.M. Sacks, American Journal of Clinical Nutrition 69 (1999) 30–42. [14] T.S. Kahlon, C.L. Woodruff, Cereal Chemistry 80 (2003) 260–263. [15] S. Kalra, S. Jood, Journal of Cereal Science 31 (2001) 141–145. [16] B. Drzikova, G. Dongowski, E. Gebhardt, A. Habel, Food Chemistry 90 (2005) 181–192. [17] A.C. Nilsson, E.M. Ostman, J.J. Holst, I.M.E. Bjorck, Journal of Nutrition 138 (2008) 732–739. [18] M.L. Sudha, R. Vetrimani, K. Leelavathi, Food Chemistry 100 (2007) 1365–1370. [19] A.O. Elkhalifa, B. Schiffler, R. Bernhard, Molecular Nutrition & Food Research 48 (2004) 91–94. [20] S. Mahadevamma, T.R. Shamala, R.N. Tharanathan, International Journal of Food Sciences and Nutrition 55 (2004) 399–405. [21] H.X. Zhang, Z.Y. Jin, Carbohydrate Polymers 86 (2011) 1610–1614. [22] Y. Chen, S. Huang, Z. Tang, X. Chen, Z. Zhang, Carbohydrate Polymers 85 (2011) 272–275. [23] A. Eva, J. Francisca, K. Beat, R. Isabelle, H.S. Monique, M. Leo, A. Rennato, Food Research International 35 (2002) 475–481. [24] S.H. Mette, E.K.B. Knud, Livestock Science 108 (2007) 175–177.
Contents lists available at SciVerse ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Fermentation characteristics of resistant starch from maize prepared by the enzymatic method in vitro Huanxin Zhang a,b , Xueming Xu a , Zhengyu Jin a,∗ a b
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China School of Food Science and Technology, Jiangsu Animal Husbandry and Veterinary College, Taizhou 225300, China
a r t i c l e
i n f o
Article history: Received 8 August 2012 Received in revised form 22 August 2012 Accepted 25 August 2012 Available online 1 September 2012 Keywords: Resistant starch Fermentation in vitro Short-chain fatty acids
a b s t r a c t To investigate the fermentation characteristics of resistant starch prepared by hydrolysing maize starch with ␣-amylase and pullulanase, fresh faecal extracts from healthy humans and infants were used as a fermentation model of human intestines in vitro. The RS was fermented for a certain period of time under the simulated condition of the large intestines (anaerobic and 37 ◦ C). The concentration of shortchain fatty acids in the fermented product as determined by gas chromatography was used as an index to characterise the fermentation effect. The results showed that the concentration of short-chain fatty acids, especially butyric acid, in the fermented product gradually increased with increased fermentation time and RS content. However, the concentration of short-chain fatty acids in the fermented product from healthy infant faecal extracts, especially butyric acid, was much higher than that from healthy adult faecal extracts. It suggested that the model of RS-produced acids was affected by the fermentation extract source, i.e., by the existence of microbial flora. The production model of acids demonstrated that maize RS prepared by the enzymatic method can be a promising ingredient of functional foods. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Dietary fibre has been recognised as a functional food ingredient for more than 20 years. It acts either directly as a consequence of its chemical composition and physicochemical properties [1], or indirectly via fermentation by colonic microflora [2]. Some health benefits related to the consumption of dietary fibre include decreased glycemic index value of food products and decreased colonic pH. The fibre portion fermented by the intestinal microflora also produces a range of short-chain fatty acids, especially butyrate, which is thought to provide a degree of protection against bowel cancer, reduce gall stone formation, exert hypocholesterolemic effects, inhibit fat accumulation, and increase mineral absorption [3,4]. In recent years, resistant starch (RS) has been classified as a functional fibre. RS is the sum of starch and its degradation products not absorbed in the small intestine of healthy individuals. Englyst et al. [5] have classified RS into three major groups, namely, RS1, RS2, and RS3. RS1 is physically inaccessible starch, such as that found in plant tissue structures. RS2 is condensed and partially crystalline native (uncooked) starch granules. RS3 is mainly retrograded or recrystallised amylose [6,7], which forms in cooked products after
∗ Corresponding author. Tel.: +86 510 85913922; fax: +86 510 85919189. E-mail addresses: [email protected] (H. Zhang), [email protected] (Z. Jin). 0141-8130/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2012.08.031
cooling (e.g., in breads, corn flakes, or potatoes). More recently, a fourth type (RS4) has been produced by chemical modifications [8], such as conversion, substitution, or cross-linking. Such modifications prevent the digestion of RS4 by blocking access to enzymes and forming atypical linkages, e.g., 1 → 2, 1 → 3, 1 → 4, and 1 → 6. From the four RS types, many food processes decrease or eliminate RS1 and RS2 but have the potential to generate RS3. RS3 is particularly interesting because it retains its indigestibility when added as an ingredient to processed foods, and has many advantages over traditional insoluble fibres. It is a natural-white source of dietary fibre, has a bland flavour, has a better appearance and texture, imparts a less gritty mouthfeel, and masks flavour less than other typical insoluble fibres [9]. It is mainly used as a functional ingredient in low-moisture food products [10], particularly in bakery products such as bread, muffins, and breakfast cereals [11,12]. In the last decade, many studies on the fermentation characteristics of dietary fibre in vitro and in vivo have been conducted [13–15]. The in vitro research model uses extracted intestinal microflora as the intestinal environment. The short-chain fatty acid content of the fermented products is determined to evaluate the fermentability of dietary fibre by intestinal microflora [16–18]. However, information on the fermentation characteristics of RS is limited [19,20]. The RS used in this study was prepared from maize starch by combining ␣-amylase and pullulanase in our previous work [21]. We used extracts from fresh faeces of healthy adults and healthy
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babies as models of the large intestine microbial environment to evaluate the fermentation properties of maize RS. We analysed the short-chain fatty acid content of the fermented products to examine the fermentation behaviour of maize RS under different environments. The objective of this work was to investigate the fermentation characteristics of maize RS in human intestine in vitro. 2. Materials and methods 2.1. Materials Native maize starch was obtained from the Carbohydrate Research Center, Jiangnan University, China. Pullulanase was purchased from Genencor Bio-Products Co. Ltd. (Wuxi, China). The enzymatic activity was 400 ASPU/g as determined by reaction with pullulan (1.0%) buffered with sodium acetate (pH 5.0) at 50 ◦ C. ␣-Amylase was obtained from Sigma–Aldrich Trading Co. Ltd. (Shanghai, China). The activity of ␣-amylase was 1400 U/g. Enzymatic activity was determined by reaction with soluble starch (1.0%) buffered with sodium acetate (pH 4.4) at 37 ◦ C [22]. Highperformance liquid chromatography-grade methanol was acquired from Thermo Fisher Scientific (USA). The fatty acid standards of formic acid, acetic acid, propionic acid, and butyric acid were purchased from Sigma Chemical Co. All other chemicals were reagent grade. 2.2. Preparation of maize RS Maize starch (20%, w/v) was pre-gelatinised with distilled water by stirring for 20 min at 80 ◦ C/and pH 6.0. The starch paste was treated with thermostable ␣-amylase under the following conditions: temperature, 90 ◦ C; pH, 5.5; time, 15 min; and amount of ␣-amylase, 4 U/g. After adding pullulanase, the mixture was incubated in a shaking water bath at 46 ◦ C for 12 h, cooled at room temperature, and then stored at 4 ◦ C overnight. The mixture was treated with thermostable ␣-amylase (1%, w/w), incubated in a 95 ◦ C boiling water bath for 45 min, and centrifuged at 4000 × g for 15 min. After discarding the supernatant, the residue was suspended in 100% ethanol (1:4 residue:ethanol) and shaken for 15 min. Ethanol was removed by centrifugation (4000 × g, 15 min), and the above procedure was repeated two more times. The residue was dried overnight at 40 ◦ C, ground in a centrifugal mill to pass through a 0.5 mm sieve, and stored in airtight containers at room temperature until use. 2.3. Fermentation of RS in vitro 2.3.1. Collection and preparation of human faeces Human faeces were anaerobically collected from 10 healthy adults and 10 healthy babies who had received no antibiotic treatment for 1 month and no abdominal diarrhea and enteritis. About 40 g of faecal sample was dissolved in 250 mL of phosphate buffer (0.1 mol/L, pH 6.5) and homogenate for 2 min to produce a 160 g/L suspension. The suspension was filtered through a nylon sieve to remove particulate matter. The collection and preparation was finished within 1 h. 2.3.2. Fermentation in vitro Approximately 5 mL of faecal suspension from healthy adults and healthy babies were transferred to 5 mL of phosphate buffer containing 2.0, 4.0, and 8.0 g/L RS. Phosphate buffer without RS was used as a blank control. The mixtures were anaerobically cultured with continuous vibration (50 r/min) at 37 ◦ C for 6, 12, and 24 h, respectively. The test tube was removed and vigorous mixing was performed for further analysis.
2.4. Gas chromatographic analysis of short-chain fatty acids The short-chain fatty acids were analysed by gas chromatography (GC) according to the method of Drzikova et al. [17] with slight modifications. About 3 mL of the fermentation broth was centrifuged (4 ◦ C, 10,000 × g, 10 min). The supernatant (100 L) was mixed with 280 L of HClO4 solution (0.36 mol/L) and 270 L of NaOH solution (1 mol/L) with vigorous shaking. About 50 L of phosphoric acid (5 mol/L) was added, and the mixture was again vigorously shaken. About 300 L of methanol solution (containing 1% H2 SO4 ) was added and refluxed at 70 ◦ C for 30 min. Thereafter, 300 L of cold hexane was added to extract the short-chain fatty acid methyl ester. About 2 L of the organic layer was analysed by gas chromatography, and the determination was performed in triplicate. GC analysis was carried out as follows: 2 L of the hexane solution of methylated fatty acids was injected into a GC unit (Agilent 6890N, USA) equipped with an auto sampler (HP 7673) using a flame ionisation detector (FID) and capillary column (HP5; 30 m × 0.32 mm × 0.25 m) with nitrogen as the carrier gas at the flow rate of 0.6 mL/min. The split ratio was 50:1, the injection temperature was 250 ◦ C, and the FID temperature was 250 ◦ C. The temperature of the column was initially 100 ◦ C, increased at a rate of 2 ◦ C/min to 200 ◦ C, and held at 200 ◦ C for 20 min. The short-chain fatty acid standards were analysed by the same method. The concentrations of formic acid and acetic acid were 20 mmol/L, whereas those of propionic acid and butyric acid were 10 mmol/L. The fatty acid methyl esters were identified by comparison with standards and quantified by the area percentage of each fatty acid methyl ester. 2.5. Statistical analysis All experiments were performed in triplicate, and data were expressed as means ± SE. Statistical analysis was carried out using the data analysis tool pack of the software Origin 7.5. 3. Results and discussion 3.1. Analysis of short-chain fatty acids in the fermentation broth GC analysis is well established as a useful tool for determining the quality and quantity of short-chain fatty acids in various industrial applications. Fig. 1 shows that the short-chain fatty acid standards had four major chromatographic peaks at 4.4, 4.7, 5.1, and 8.0 min, representing formic, acetic, propionic, and butyric acids, respectively. The fermentation profiles of RS are shown in Fig. 2. There were also four peaks in the profiles from 4 min to 10 min. Compared with the GC results of the short-chain fatty acid standards, four typical fatty acids (formic, acetic, propionic, and butyric acids) were identified in the fermentation products of RS. Hence, the model of standards effectively distinguished the short-chain fatty acids from the fermentation products of RS. 3.2. RS fermentation in the simulated adult intestinal environment The substrate composition is known to affect the production of fermentation metabolites and the composition of colonic microflora, which together exert beneficial health effects [23]. RS fermentation in the simulated adult intestinal environment is shown in Table 1. The total amount of short-chain fatty acids in the fermentation products gradually increased with increased fermentation time. The total amount of short-chain fatty acids in the fermentation products also gradually increased with increased RS content. The total amount of short-chain fatty acids markedly
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related to the prevention of rectal cancer. These facts prompted us to focus on butyric acid in this study. As shown in Table 1, a variety of short-chain fatty acids in the fermentation products increased with increased fermentation time and RS content. However, the increase rates differed among the various short-chain fatty acids. The increase rates of formic, acetic, and propionic acids were smaller than that of butyric acid. The concentration of butyric acid increased from 2.71 mmol/L to 6.78 mmol/L with increased RS content from 2.0 g/L to 8.0 g/L after 6 h of fermentation. The concentration of butyric acid increased from 5.26 mmol/L to 9.86 mmol/L with increased RS from 2.0 g/L to 8.0 g/L after 12 h of fermentation. The concentration of butyric acid increased from 5.87 mmol/L to 10.23 mmol/L with increased RS content from 2.0 g/L to 8.0 g/L after 24 h of fermentation. Butyric acid was the main product after 12 h of fermentation. 3.3. RS fermentation in the simulated baby intestinal environment Fig. 1. Chromatogram of a standard mixture of short-chain fatty acids (1, formic acid; 2, acetic acid; 3, propionic acid; 4, butyric acid).
Fig. 2. Chromatogram of the short-chain fatty acids in the RS fermentation products (1, formic acid; 2, acetic acid; 3, propionic acid; 4, butyric acid).
increased from 6 h to 12 h, but the increase was smaller after 12 h, implying that the most of the short-chain fatty acids were produced within 12 h. Given that butyric acid can induce intestinal epithelial cell growth and cell differentiation, it can also enhance the immune effect. Butyric acid can also induce colon cancer cell cracking, thus preventing the spread of cancer by post-modification-induced gene expression and normal cell decline. Thus, butyric acid is closely
RS fermentation in the simulated baby intestinal environment is shown in Table 2. The total amount of short-chain fatty acids in fermentation products gradually increased with increased fermentation time. The total amount of short-chain fatty acids in the fermentation products also gradually increased with increased RS. The total amount of short-chain fatty acids markedly increased from 6 h to 12 h, but the increase was smaller after 12 h, implying that most of the short-chain fatty acids were produced within 12 h. The variety of short-chain fatty acids in the fermentation products increased with increased fermentation time and RS content, but the increases varied. The development trends of the short-chain fatty acids in the fermentation product were very similar to those shown in Table 1. Interestingly, the total shortchain fatty acid contents of the fermentation products from the baby faecal extracts were higher than those from the adult faecal extracts. Most importantly, the butyric acid content of RS fermented from baby faecal extracts was greatly improved compared with that from healthy adult faecal extracts. The butyric acid content of the fermentation broth increased from 7.42 mmol/L to 11.45 mmol/L with increased RS concentration after 6 h of fermentation. After 12 h of fermentation, the butyric acid content of the fermentation broth increased from 9.58 mmol/L to 19.58 mmol/L. The butyric acid content of the fermentation broth increased from 10.67 mmol/L to 21.48 mmol/L after 24 h of fermentation time. Most of the butyric acid was produced after 12 h of fermentation. Surprisingly, the highest level of butyric acid in the healthy adult faecal extracts fermented for 6 h was 6.78 mmol/L, whereas that in the healthy infant faecal extracts was almost double at 11.45 mmol/L. The highest level of butyric acid in the healthy adult faecal extracts after 12 h was 9.86 mmol/L, whereas that in the healthy infant faecal extracts was also double at 19.58 mmol/L.
Table 1 Fermentation effect of RS from healthy adult faecal extracts. Concentration of resistant starch (g/L)
Time (h)
Organic acids (mmol/L) Formic acid
2.0
4.0
8.0 Data were expressed as x ± SD (n = 3).
6 12 24 6 12 24 6 12 24
12.54 15.63 15.47 13.15 16.65 17.14 12.57 15.83 15.67
± ± ± ± ± ± ± ± ±
0.68 1.23 0.78 0.27 0.34 0.18 1.83 0.84 0.52
Acetic acid 5.72 7.66 7.59 6.57 9.26 10.13 6.18 9.13 10.56
± ± ± ± ± ± ± ± ±
0.24 0.84 0.54 0.92 0.53 0.28 0.38 0.43 0.22
Propionic acid 7.27 9.62 9.75 11.15 12.46 13.71 12.43 13.37 13.12
± ± ± ± ± ± ± ± ±
0.76 1.72 0.93 0.45 0.38 0.62 0.75 1.18 0.63
Butyric acid 2.71 5.26 5.87 5.74 9.26 10.73 6.78 9.86 10.23
± ± ± ± ± ± ± ± ±
0.25 0.28 0.62 0.46 0.33 0.44 0.36 0.24 0.38
Total acid 28.24 38.17 38.67 36.62 47.58 51.71 37.97 48.18 49.58
± ± ± ± ± ± ± ± ±
1.75 3.36 2.37 2.72 1.48 2.78 2.74 3.56 3.28
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Table 2 The fermentation effect of maize resistant starch by health babies faecal extractions. Concentration of resistant starch (g/L)
2.0
4.0
8.0
Time (h)
Organic acids (mmol/L) Formic acid
6 12 24 6 12 24 6 12 24
16.73 18.46 19.52 17.65 19.72 21.28 19.68 22.57 22.74
± ± ± ± ± ± ± ± ±
0.48 0.43 0.37 0.47 0.94 0.85 0.75 0.62 0.34
Acetic acid 13.27 14.58 15.65 12.48 15.67 14.83 14.37 16.64 17.14
± ± ± ± ± ± ± ± ±
0.72 0.78 0.57 0.56 0.73 0.34 0.68 0.36 1.78
Propionic acid 15.56 17.27 19.16 17.15 19.52 20.18 18.23 19.05 19.83
± ± ± ± ± ± ± ± ±
0.45 0.64 0.87 0.46 0.40 0.37 0.47 0.78 1.32
Butyric acid 7.42 9.58 10.67 8.74 13.25 15.73 11.45 19.58 21.48
± ± ± ± ± ± ± ± ±
0.38 0.52 0.95 0.61 0.83 0.74 0.78 1.28 0.67
Total acid 52.98 59.89 65.00 56.02 68.16 72.02 63.73 77.84 81.19
± ± ± ± ± ± ± ± ±
1.78 2.13 2.47 2.42 2.63 2.19 2.45 3.14 3.78
Data were expressed as x ± SD (n = 3).
Hence, the microbial flora in the infant faeces was more effective in fermenting maize RS. Lebet et al. [1] using healthy adult faeces as fermenters and dietary fibre as a fermentation substrate, have found that with increased fermentation time, the short-chain fatty acids in the fermentation products gradually increased, but the total shortchain fatty acid content slowly increased after 12 h. Mette et al. [24] have studied the impact of RS-produced short-chain fatty acid concentrations in pig intestine. The results show that the total amount of short-chain fatty acids, especially butyric acid, in pig intestine significantly increased with increased RS content in the diet. These results are similar to those of the present work. 4. Conclusions The fermentation behaviour of RS in vivo was evaluated under a simulated environment using microbial flora extracted from fresh faeces of healthy adults and babies in vitro. The short-chain fatty acids of the fermentation products gradually increased with increased fermentation time and RS content. The butyric acid content increased significantly. The amount of short-chain fatty acids, especially butyric acid, of the fermentation products from fresh healthy baby faecal extracts was significantly higher than that from fresh adult faecal extracts. These results suggested that the model of RS-produced acids was affected by the fermentation extract source, i.e., by the existence of microbial flora. The production model of acids demonstrated that maize RS prepared by the enzymatic method can be a promising ingredient of functional foods. Acknowledgements This work was financially supported by the National Natural Science Foundation (nos. 20976070 and 31071490), the Jiangsu
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