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Non-starch constituents influence the in vitro digestibility of naked oat (Avena nuda L.) starch
Food Chemistry 297 (2019) 124953
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Non-starch constituents influence the in vitro digestibility of naked oat (Avena nuda L.) starch Minyue Tanga, Luyu Wanga, Xuanxuan Chenga, Yanwen Wub, Jie Ouyanga,
T
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a Department of Food Science and Engineering, College of Biological Sciences and Technology, Beijing Key Laboratory of Forest Food Process and Safety, Beijing Forestry University, Beijing 100083, China b Beijing Center for Physical and Chemical Analysis, Beijing Food Safety Analysis and Testing Engineering Research Center, Beijing Academy of Science and Technology, Beijing 100089, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Naked oat In vitro digestibility Glycemic index β-Glucan Lipid Protein
The present study investigated the effects of proteins, lipids and β-glucan in naked oat flour (NOF) on the in vitro digestibility of starch. The content of rapidly digested starch (RDS) increased, and the content of resistant starch (RS) decreased in NOF after removing the non-starch constituents. The estimated glycemic index (eGI) of starch in NOF increased after the removal of the non-starch constituents, with a decreasing order of naked oat starch (NOS) > de-β-glucan flour > de-proteins flour > de-lipids flour > NOF. NOS was found to have an A-type crystalline pattern, but the removal of proteins or β-glucan rendered NOS a V-type crystalline pattern. The relative crystallinity decreased after removing non-starch constituents. The in vitro digestibility was positively correlated with the short-range molecular order and negatively correlated with the relative crystallinity. These results clearly illustrate the effects of non-starch constituents on the low digestibility of naked oat.
1. Introduction Naked oat (Avena nuda L.) originated from Mongolia and northern China, and it is different from oat (Avena sativa L.) in the growth environment, morphology and nutritional value. Oat is recognized as one of the most important grains in the world, because it contains a high content of proteins, lipids, dietary fiber (especially soluble fiber βglucan), minerals and vitamins. In addition, naked oat can lower serum lipids and blood pressure, prevent heart disease and regulate postprandial blood sugar (Liu, Li, et al., 2015). The consumption of naked oat by patients with type 2 diabetes mellitus can lower the risk of metabolic and cardiovascular diseases (Ma et al., 2013). As the main component in higher plants, starch is an important source of carbohydrates in human diet. There are three categories of starch in accordance with the rate and the degree of starch digestion, namely, rapidly digested starch (RDS), slowly digested starch (SDS) and resistant starch (RS) (Englyst, Kingman, & Cummings, 1992). In the small intestine, RDS can be rapidly digested and absorbed, causing the blood glucose level to rapidly rise, whereas SDS can be slowly digested and RS can not be digested. Instead, RS stabilizes the metabolism of glucose and promotes the growth of beneficial colonic flora to prevent colon cancer (Wang et al., 2014). The glycemic index (GI) is an indicator that reflects increases in the blood glucose level in response to
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carbohydrate foods (Brandmiller, 2007). Foods can be classified into three types according to the GI values, namely, low GI (≤55) foods, middle GI (56–69) foods and high GI (≥70) foods. The ingestion of low GI foods, such as oat, can help to reduce the risk of metabolic diseases (Brandmiller, 2007). The digestibility of starch is influenced by diverse internal and external conditions, including the size and shape of starch granules, the food processing method, physical and chemical modifications, the viscosity and food matrix components (Singh, Dartois, & Kaur, 2010). In foods, starches interact with proteins, fats and other constituents to achieve a strong effect on their digestion (Hu et al., 2018). For example, the presence of β-glucan in carbohydrate foods reduced the rate of starch digestion, thereby maintaining a low GI (Mäkeläinen et al., 2007). Considering the above, the present work aimed to explore the effects of non-starch constituents in naked oat flour (NOF), including endogenous proteins, lipids and β-glucan, on the in vitro digestibility of starch and further explore the mechanism for the effects of these constituents on starch digestibility. Furthermore, the physicochemical properties of naked oat starch (NOS) and NOF after removing nonstarch constituents were compared to study the relationship between starch digestibility and intrinsic factors, which can be helpful for understanding the mechanism behind the low digestibility of naked oat.
Corresponding author. E-mail address: [email protected] (J. Ouyang).
https://doi.org/10.1016/j.foodchem.2019.124953 Received 10 March 2019; Received in revised form 4 June 2019; Accepted 5 June 2019 Available online 06 June 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
Food Chemistry 297 (2019) 124953
M. Tang, et al.
2. Materials and methods
Hydrolysis rate (%) = Gt × 0.9/starch content (mg)
2.1. Materials and chemical regents
RDS, SDS and RS contents were obtained by the following equations:
Naked oat was harvested in Zhangjiakou, Hebei Province, China. The grains were milled to 80-mesh. Porcine pancreatic α-amylase (13 U/mg), β-glucanase (50 U/mg) and β-glucan were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Glucoamylase (≥100,000 U/g) was obtained from Beijing Aoboxing Biotechnology Co., Ltd. (Beijing, China). Alkaline protease (100,000–220,000 U/g) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Other chemicals were purchased from Beijing Chemical Reagent Co. (Beijing, China) and were analytical grade.
(1)
RDS (%) = (G20−FG) × 0.9/TS
(2)
SDS (%) = (G120 − G20) × 0.9/TS
(3)
RS (%) = [TS −(RDS + SDS)]/TS
(4)
where FG is the free glucose content (mg) of the sample (mg), G20 is the glucose released (mg) within 20 min, G120 is the glucose released (mg) within 120 min and TS is the total starch content of the sample (mg). According to the method of Goñi, Garcia-Alonso, and Saura-Calixto (1997), the in vitro digestibility of the sample was analyzed to simulate human intestinal digestion. The curve of starch hydrolysis obeyed a first-order reaction equation. The hydrolysis index (HI) of the sample was the percentage of the area under the hydrolysis curve (AUC) of each sample and that of white bread, and the estimated glycemic index (eGI) was determined by the following formula (Goñi et al., 1997):
2.2. Sample preparation Deproteinization of NOF. NOF (20 g) was added to 160 mL of carbonate buffer (0.02 M, pH 9.0), and then alkaline protease (0.2 g) was added and dissolved. The sample was continuously stirred at 45 °C for 1 h, then centrifuged at 4000×g for 15 min and the precipitate was hydrolyzed again. The precipitate was washed repeatedly with distilled water to adjust the pH to 7.0, and the deproteinized NOF (DP-NOF) was obtained by drying the precipitate overnight at 40 °C in DHG-9033B5III oven (Shanghai CIMO Medical Instrument Manufacturing Co., Ltd., China) (Ye et al., 2018). Degreasing of NOF. NOF (20 g) was mixed with petroleum ether (100 mL), and stirred continuously at room temperature for 4 h, then centrifuged at 4000×g for 15 min. The degreasing treatment was repeated two more times. The degreased NOF (DG-NOF) was obtained by drying the precipitate overnight at room temperature. De-β-glucan treatment of NOF. NOF (20 g) was added to 200 mL of sodium acetate buffer (0.1 M, pH 5.2), and then β-glucanase (0.016 g; stock, 50 U/mg) was added and dissolved. β-Glucan was hydrolyzed for 30 min at 55 °C. After centrifugation at 4000×g for 15 min, the precipitate was washed twice with distilled water. The precipitate was dried at 40 °C overnight in DHG-9033B5-III oven to obtain de-β-glucan NOF (Dβ-NOF) (Zhang, Luo, & Zhang, 2017). Extraction of naked oat starch (NOS). NOF was dissolved and homogenized with an NaOH solution (0.01 M) at a ratio of 1:8 (w/v), and then the pH was adjusted to 10.0. The solution was stirred for 2 h at 30 °C and then filtered. The filtrate was centrifuged at 4000×g for 15 min. The supernatant was discarded, and the precipitate was washed with distilled water three times, and then dried for 12 h at 40 °C in DHG-9033B5-III oven.
eGI = 39.71 + 0.549HI
(5)
2.5. Determination of solubility and swelling power The solubility and swelling power of the samples were determined based on the method of Kusumayanti, Handayani, and Santosa (2015) with minor modifications. The sample suspension (2%, w/v) was heated for 30 min at 95 °C and shaken every 1–2 min. The starch suspension was rapidly cooled and centrifuged at 4000×g for 15 min. The supernatant was weighed after drying to constant weight at 105 °C, and the sediment was weighed directly. The solubility and swelling power was calculated as follows:
Solubility (%) = (W2/W1) × 100
(6)
Swelling power (%) = [W3/[W1 × (100−solubility)]] × 100
(7)
where W1 is the weight of the dry sample (mg), W2 is the weight of soluble substance in the supernatant (mg) and W3 is the weight of sediment (mg). 2.6. X-ray diffraction pattern (XRD) analysis XRD analysis was carried out with an X-ray diffractometer (D8 ADVANCE; Bruker Corp., Karlsruhe, Germany) operating at 40 mA and 40 kV. The diffraction scan range (2θ) ranged from 5 to 40° with a scanning speed of 4°/min. The ratio of the sum of the area of each significant peak to the total AUC was calculated as the relative crystallinity (Rafiq, Jan, Singh, & Saxena, 2015).
2.3. Analysis of proximate constituents The content of total starch, crude protein, crude lipid and β-glucan were determined according to the AACC 76-13.01, 46–13.01, 30–25.01 and 32–22.01 methods (AACC, 2000), respectively. 2.4. In vitro starch digestibility
2.7. Fourier transform infrared spectroscopy (FTIR) analysis
According to the method of Englyst et al. (1992) with some modifications, the in vitro digestibility of the samples was determined. The sample (200 mg) was mixed with 15 mL of sodium acetate buffer (0.2 M, pH 5.2), and then 10 mL of the previously prepared enzyme solution [porcine pancreatic α-amylase (290 U/mL) and glucoamylase (15 U/mL)] was added. The mixture was shaken in a water-bathing constant temperature vibrator (SHA-A, Saidelisi Crop., Tianjin, China) at 37 °C at a speed of 150 revolutions/min. An aliquot (0.50 mL) was obtained at different times (10, 20, 40, 60, 90, 120 and 180 min), and the enzymes were inactivated by adding absolute ethanol (4.5 mL). Subsequently, the glucose content (Gt) was analyzed by the method of 3.5-dinitrosalicylic acid (DNS). The hydrolysis rate (%) was calculated using the following formula:
The finely ground samples were thoroughly mixed with dry potassium bromide powder (1:100, w/w) and tableted at 10,000 PSI. The spectrum was recorded using a Tensor27 FTIR spectrometer (Bruker Corp., Ettlingen, Germany) from 4000 to 400 cm−1 and at a resolution of 4 cm−1. 2.8. Statistical analysis All results were represented as mean ± standard deviation with triplicate experiments, and the data were subjected to a difference significance test using SPSS 23.0 software (IBM Corp., Armonk, NY, USA). 2
Food Chemistry 297 (2019) 124953
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NOF was 26.9% at 180 min, but that of Dβ-NOF increased to 32.4%. Due to the removal of lipids, proteins and β-glucan during the extraction process, the hydrolysis rate of NOS was greatly improved, and the final hydrolysis rate was 33.8%. The simultaneous removal of proteins and lipids in rice flour yielded the highest degree of hydrolysis, and the effect of proteins on hydrolysis was slightly higher than that of lipids (Ye et al., 2018). Furthermore, a significant increase was observed in the starch digestion rate when dextran-rich oat flour was treated with βglucanase, and β-glucan affected starch digestion in native form rather than in its extracted form (Zhang et al., 2017). Oats contain more proteins and lipids than other common grains. Proteins can effectively reduce starch digestibility by several mechanisms. Firstly, proteins can form a protection round the starch granules, thereby restricting the entry of enzymes into the substrates. Secondly, surface proteins can block the catalytic binding of enzymes on the starch granule exterior (Wang et al., 2014). Thirdly, α-amylase can partially bind to proteins, thereby reducing enzyme utilization (Bhattarai, Dhital, & Gidley, 2016). By contrast, the effect of lipids on starch digestibility is primarily due to the ability of lipids to form complexes with amylose, which is better able to resist the attack of amylase (Singh et al., 2010). Monoglycerides/free fatty acids are capable of forming complexes with amylose, but tri- or di-glycerides do not readily form complexes with amylose. Therefore, the types of lipids presented in naked oats are worthy of further investigation. Fatty acids in lipids rapidly form complexes with amylose under physiological conditions, promoting the formation of resistant complexes (Annor et al., 2013). Therefore, the proteins and lipids in naked oat interact with starch to form a barrier or complexes that makes naked oat hard to digest. The content of β-glucan in different grains greatly varies, among which barley and oat are the highest. A study has shown that β-glucan, particularly the extracted water-soluble fraction, can lower the digestion rate of starch by increasing the viscosity (Regand, Chowdhury, Tosh, Wolever, & Wood, 2011). At the same time, β-glucan can create a complex of adjacent proteins to form a robust structure that resists attack by amylase, thereby resulting in a decrease in starch digestibility. Therefore, the de-β-glucan treatment of NOF increased the degree of starch hydrolysis. Different treatments have caused different release of various nonstarch components. For example, the De-β-glucan treatment caused a decrease in protein content (from 11.69 to 8.46%) when removing the β-glucan, and deproteinization treatment caused the loss of a large amount of β-glucan (from 5.60 to 0.12%). Therefore, these components in naked oat may be closely linked to each other. When one component is hydrolyzed, other components may also be inevitably lost. The matrix formed by starch and non-starch components such as β-glucan, protein, and lipid in their ordered structure may be one of the key factor for the low starch digestibility (Zhang et al., 2017). In the extracted naked oat starch, the contents of lipid, protein and β-glucan declined to a great extent, and the digestibility of starch increased. This further demonstrated that the interaction among these substances and their effects on starch digestibility. The content of RDS, SDS and RS in NOF, DG-NOF, DP-NOF, Dβ-NOF and NOS are given in Table 2. The RDS content of the treated samples was higher than that of NOF, whereas the content of RS was decreased compared to NOF. The RDS increased from 15.1% in NOF to 23.0% in NOS, and the RS decreased from 76.0% in NOF to 68.3% in NOS. The decreasing order of the RDS content was NOS > Dβ-NOF > DPNOF > DG-NOF > NOF, whereas the RS content showed an opposite trend. These results were consistent with the in vitro digestion curve of the samples. Annor et al. (2013) studied the in vitro digestibility on deproteinized and degreased millet flour, millet flour and millet starch. They reported that proteins and lipids affected the digestibility of millet, and the effect of lipids was higher than that of proteins. The removal of lipids caused the RDS level of millet flour to increase from 11.48% to 20.07%, whereas the removal of proteins changed the RDS level to 14.21% (Annor et al., 2013).
Table 1 Proximate composition (g/100 g fresh weight) of NOF and removal of nonstarch constituents. Sample
Total starch
NOF DG-NOF DP-NOF Dβ-NOF NOS
71.51 75.81 78.70 70.36 91.19
± ± ± ± ±
0.34d 0.32c 0.21b 0.06e 0.41a
Crude protein
Crude lipid
11.69 ± 0.07a 9.38 ± 0.05b 1.52 ± 0.02d 8.46 ± 0.06c 0.85 ± 0.01e
5.19 0.30 4.12 1.80 1.53
± ± ± ± ±
0.04a 0.02e 0.08b 0.06c 0.03d
β-glucan 5.60 5.91 0.12 0.03 0.13
± ± ± ± ±
0.22b 0.18a 0.01c 0.02d 0.01c
Different superscripts mean significant differences in the same column (P < 0.05). NOF, naked oat flour; DG-NOF, degreased NOF; DP-NOF, deproteinized NOF; Dβ-NOF; de-β-glucan NOF; NOS, naked oat starch.
3. Results and discussion 3.1. Proximate constituents of NOF and removal of non-starch constituents The content of total starch, crude protein, crude lipid and β-glucan in NOF was 71.51%, 11.69%, 5.19% and 5.60% (fresh weight) (Table 1), respectively, and the moisture content was approximately 10%. The results were consistent with previous studies, which showed the protein content in naked oat to be 11–19% (Mohamed, Biresaw, Xu, Hojilla-Evangelista, & Rayas-Duarte, 2009). The content of β-glucan and lipid in naked oat was 3–8% and 1.42–11.52%, respectively, which was far higher than those of other cereal crops (Hu, Zheng, Li, Xu, & Zhao, 2014). NOF was individually underwent degreasing, deproteinization and de-β-glucan treatment, and NOS was also extracted. The lipid removal rate of DG-NOF reached 94.2%, and the protein removal rate of DP-NOF was 87.0%. In Dβ-NOF, the β-glucan content decreased from 5.60 to 0.03%, whereas the lipid content decreased from 5.19 to 1.80%. Virtually all non-starch constituents in NOS were removed. Similarly, the degreasing rate of millet flour was 94.38%, and the protein content of millet treated with proteases decreased from 8.35 to 2.29% (Annor, Marcone, Bertoft, & Seetharaman, 2013). 3.2. In vitro digestibility of NOF and removal of non-starch constituents The in vitro hydrolysis curves of NOF, DG-NOF, DP-NOF, Dβ-NOF and NOS were compared in Fig. 1. The hydrolysis rate of all samples increased rapidly within 20 min, and then it slowed down from 20 to 120 min, with a decreasing order of NOS > Dβ-NOF > DP-NOF > DG-NOF > NOF. It can be concluded that the intrinsic constituents in NOF had significant effects on starch digestibility. β-Glucan had the greatest impact, followed by proteins and lipids. The hydrolysis rate of
Fig. 1. Enzymatic hydrolysis curves of NOF and removal of non-starch constituents. NOF, naked oat flour; DG-NOF, degreased NOF; DP-NOF, deproteinized NOF; Dβ-NOF, de-β-glucan NOF; NOS, naked oat starch. 3
Food Chemistry 297 (2019) 124953
M. Tang, et al.
Table 2 Digestibility parameters of NOF and removal of non-starch constituents. Sample
RDS (g/g starch)
NOF DG-NOF DP-NOF Dβ-NOF NOS
15.1 15.8 16.7 21.5 23.0
± ± ± ± ±
SDS (g/g starch)
b
RS (g/g starch)
b
8.8 ± 0.4 9.6 ± 0.8b 11.2 ± 0.2a 9.1 ± 0.2b 8.7 ± 0.1b
0.1 0.9b 0.8b 0.9a 0.3a
76.0 74.6 72.1 69.5 68.3
± ± ± ± ±
k × 10−2
C∞
a
0.4 0.2b 0.4c 0.4d 0.2d
26.9 28.0 30.4 32.4 33.8
± ± ± ± ±
c
0.2 0.3c 0.5b 1.4ab 0.9a
1.4 1.5 1.7 1.8 1.8
± ± ± ± ±
HI c
0.1 0.1c 0.0b 0.0a 0.1ab
50.8 54.4 61.4 67.3 69.5
eGI ± ± ± ± ±
e
0.2 0.6d 0.8c 0.2b 0.3a
67.6 69.6 73.4 76.7 77.9
± ± ± ± ±
0.1e 0.3d 0.4c 0.3d 0.2a
Different superscripts mean significant differences in the same column (P < 0.05). RDS is rapidly digestible starch; SDS is slowly digestible starch; RS is resistant starch; C∞ is the equilibrium percentage of starch hydrolyzed after 180 min; k is the reaction kinetic constant; HI is the hydrolysis index; eGI is the estimated glycemic index. NOF, naked oat flour; DG-NOF, degreased NOF; DP-NOF, deproteinized NOF; Dβ-NOF, de-β-glucan NOF; NOS, naked oat starch.
small pores in the starch granules, adversely affecting the penetration of digestive enzymes, thus reducing the rate of starch hydrolysis. A previous study has shown that the surface components (glucan + protein + lipid) of starch in wheat and corn can combine to form a complex, which performs a vital role in limiting the speed and extent of swelling of the particles (Debet & Gidley, 2006). The in vitro digestibility and swelling power of rice starch from different varieties increased after surface lipids and proteins were removed (Hu et al., 2018).
Naked oat is a traditional Chinese grain, which is generally considered to be difficult to digest. The eGI of NOF was 67.6, indicating that it belongs to the middle GI food category (Table 2). A previous study has shown that some common grains, such as rice (91.8), corn (89.2) and wheat (87.3), are high-GI foods (Kaur, Gill, & Karwasra, 2018). The decreasing order of the eGI of naked oat samples was NOS > Dβ-NOF > DP-NOF > DG-NOF > NOF. The proteins, lipids and β-glucan in NOF were the internal factors responsible for the slow digestion of naked oat. Starch in food always forms complex matrices with lipids and proteins, causing intrinsic differences in human postprandial glycemic responses and leading to different GI (Parada & Santos, 2016). The GI of oat starch supplemented with β-glucan of high molecular weight decreased from 88.3 to 68.4, indicating that β-glucan could inhibit the digestibility of starch, and the molecular weight was negatively related to the GI (Kim & White, 2013).
3.4. Crystal properties of NOF and removal of non-starch constituents Starch can be classified into four crystal types: A, B, C and V, and cereal starches are usually A-type (Liu, Xu, et al., 2015). The diffraction angles (2θ) of all naked oat samples had strong diffraction peaks at 15°, 17°, 18° and 23°, which were typical A-type diffraction characteristics (Fig. 2). The diffraction peak intensity of DP-NOF and Dβ-glucan increased at a 2θ of 20°, which corresponds to the V-type crystal pattern and relates to the presence of an amylose-lipid complex (Shah, Masoodi, Gani, & Ashwar, 2018). Thus, deproteinization and de-βglucan treatment may promote the formation of the complex of amylose-lipid. NOF had the highest crystallinity (32.38%) and NOS had the lowest crystallinity (22.73%), with a decreasing order of NOF > DGNOF > DP-NOF > Dβ-NOF > NOS. The peak intensity of samples after different treatments also decreased. In general, the high crystallinity of starch indicates more crystalline regions within the particles (Li et al., 2011). In addition, the crystallinity was also affected by many factors, such as the crystal size, the orientation of double helices, amount of crystalline regions (amylopectin content and chain length
3.3. Solubility and swelling power of NOF and removal of non-starch constituents Swelling power and solubility are good indicators to measure the degree of interaction between amorphous and crystalline regions of starch chains (Kusumayanti et al., 2015). Solubility represents a percentage of molecules that released from swelling starch granules. The solubility of NOF was the lowest (12.0%) and increased when the nonstarch constituents were removed (Table 3). The different removal treatments resulted in the disintegration and structural weakening of the starch granules, and the solubility of the starch increased (Shi, Gu, Wu, Yu, & Gao, 2013). It is generally considered that the presence of constituents will hinder the entry of water molecules into the starch granules, thereby inhibiting the starch solubility. The level of swelling power reflects the hydration ability of the starch. The expansion of starch granules can promote the attack of the internal structure by digestive enzymes, thereby increasing the rate of starch hydrolysis (Shi et al., 2013). The decreasing order of swelling power was NOS > Dβ-NOF > DP-NOF > DG-NOF > NOF. The result indicated that proteins, lipids and β-glucan significantly (P < 0.05) inhibited the swelling power of starch granules. Therefore, the existence of non-starch constituents in NOF may slow the digestion of starch. At the same time, the low swelling power was related to the Table 3 Solubility, swelling power, short-range ordered index of NOF and removal of non-starch constituents. Sample
Solubility (%)
NOF DG-NOF DP-NOF Dβ-NOF NOS
12.0 18.5 18.5 18.8 20.1
± ± ± ± ±
0.7c 0.2b 0.3b 0.1ab 0.2a
Swelling power (%) 13.9 18.0 20.1 21.5 26.4
± ± ± ± ±
0.1d 0.6c 1.0b 0.2b 0.2a
IR ratio of 1047/1022 cm−1 1.083 1.120 1.193 1.197 1.335
± ± ± ± ±
0.009d 0.002c 0.008b 0.012b 0.013a
Fig. 2. X-ray diffraction spectra of NOF and removal of non-starch constituents. NOF, naked oat flour; DG-NOF, degreased NOF; DP-NOF, deproteinized NOF; Dβ-NOF, de-β-glucan NOF; NOS, naked oat starch.
Different superscripts means significant differences in the same column (P < 0.05). NOF, naked oat flour; DG-NOF, degreased NOF; DP-NOF, deproteinized NOF; Dβ-NOF, de-β-glucan NOF; NOS, naked oat starch. 4
Food Chemistry 297 (2019) 124953
M. Tang, et al.
removal of non-starch constituents (Table 3). This may have been due to the disruption of the parallel alignment of the crystals by the different treatments (Shah, Masoodi, Gani, & Ashwar, 2016). The amorphous components in starch were preferentially degraded or degraded faster, which resulted in an increase in the degree of order within starch (Sevenou, Hill, Farhat, & Mitchell, 2002). Furthermore, the correlation coefficient (r) between 1047/1022 cm−1 and eGI and RS was 0.908 and −0.918, respectively. Similarly, the positive correlation between 1047/ 1022 cm−1 and the digestibility of chestnut starch after cooking was also observed (Bao, Li, Wu, & Ouyang, 2018). In addition, a previous study showed that the correlation coefficient between the 1047/ 1022 cm−1 and relative crystallinity was −0.162, which indicated that the short-range molecular order observed by FTIR was not correlated with the crystallinity observed by XRD (Li et al., 2018). 4. Conclusion Naked oat is a medium eGI food, and the eGI of NOF increased after the removal of proteins, lipids and β-glucan. The solubility and swelling power of NOF without non-starch constituents increased, indicating that the presence of constituents hindered the entry of water and enzymes into the starch granules. The crystalline structure of NOF was Atype, whereas the crystalline structure of DP-NOF and Dβ-NOF was Vtype. The relative crystallinity decreased after removing non-starch constituents and showed a negative correlation with in vitro digestibility. The removal of non-starch constituents resulted in an increase of starch short-range molecular order, which was significantly correlated with eGI. These results are helpful for understanding the role of nonstarch constituents in the low digestibility of naked oat, where endogenous complexes between starch and other constituents may be formed. Further research will be carried out in the binding models between starch and other constituents, and inhibitory effect of endogenous bioactive components (saponins, polyphenols, etc.) in naked oat on the activity of amylase.
Fig. 3. FTIR spectra of NOF and removal of non-starch constituents. NOF, naked oat flour; DG-NOF, degreased NOF; DP-NOF, deproteinized NOF; DβNOF, de-β-glucan NOF; NOS, naked oat starch.
distribution of amylopectin), and the extent of interaction between double helices (Rafiq et al., 2015). Physical and chemical treatments during sample processing can cause damage to the particles, destroy the internal structure of the starch and undermine the combination of starch and other substances. The samples may undergo reorientation of crystallite, reduction of large particle crystals, and/or destroy the crystalline regions and decrease the double helices within starch, thereby resulting in a decrease in its crystallinity. Starch with higher crystallinity usually contains higher RS content because enzymes can’t easily enter into the ordered crystal structure (Shaikh, Ali, Mustafa, & Hasnain, 2019). The change in crystallinity was significantly negatively correlated (r = −0.995) with the eGI of the naked oat samples. Similarly, the eGI and crystallinity of starch in naturally air-dried chestnut was negatively correlated (Zhao, Zhang, Wu, Liu, & Ouyang, 2018).
Declaration of Competing Interest There are no conflicts to declare.
3.5. FTIR of NOF and removal of non-starch constituents Acknowledgements The FTIR spectra of different samples were similar (Fig. 3). However, the protein absorption peaks of NOS and DP-NOF at 1535 cm−1 disappeared, which represented the stretching vibration of CeN and NeH in the protein (Hernández-Martínez et al., 2013) and indicated a decrease in the protein content, which was consistent with the previous results of chemical measurements. Furthermore, a broad and strong absorption appeared at 3400 cm−1, which assigned to the OeH stretching vibration in or between molecules. The absorption peak at 2927 cm−1 was due to the asymmetric stretching vibration of CeCH2eC, and the absorption peak at 1649 cm−1 was attributed to the C]O stretching vibration in the aldehyde group (Flores-Morales, Jiménez-Estrada, & Mora-Escobedo, 2012; Amir & Kumar, 2007). The characteristic peaks in the range of 1200–1020 cm−1 were assigned to the stretching vibration of CeO and CeC in the anhydroglucose ring. In the range of 930–900 cm−1, the absorption was due to the skeleton vibration of the α-1,4 glycosidic bond (CeOeC). The absorption peak at 767 cm−1 was corresponded to the ring-symmetric stretching vibration of the pyran ring, and the absorption peaks from 620 to 527 cm−1 corresponded to the pyran ring skeleton vibration (Pigorsch, 2009; Wang et al., 2015). The short-range molecular order of starch was obtained by the ratio of 1047/1022 cm−1, which indicated the proportional relationship between the ordered structure and the amorphous structure in starches, and the larger ratio represented the higher degree of inside order within the particles (Varatharajan, Hoover, Liu, & Seetharaman, 2010). The 1047/1022 cm−1 of NOF was 1.083, and the value increased after the
The authors are grateful for the support of the Fundamental Research Funds to the Central Universities of China (2015ZCQ-SW-04), Innovation and Entrepreneurship Training Program for College Students of Beijing Forestry University, China (S201810022049), and Beijing Natural Science Foundation (2182020). References AACC (2000). Approved methods of the American association of cereal chemists. St. Paul, MN, USA: AACC International. Amir, M., & Kumar, S. (2007). Synthesis and evaluation of anti-inflammatory, analgesic, ulcerogenic and lipid peroxidation properties of ibuprofen derivatives. Acta Pharmaceutica, 57, 31–45. Annor, G. A., Marcone, M., Bertoft, E., & Seetharaman, K. (2013). In vitro starch digestibility and expected glycemic index of kodo millet (Paspalum scrobiculatum) as affected by starch-protein-lipid interactions. Cereal Chemistry, 90, 211–217. Bao, W., Li, Q., Wu, Y., & Ouyang, J. (2018). Insights into the crystallinity and in vitro, digestibility of chestnut starch during thermal processing. Food Chemistry, 269, 244–251. Bhattarai, R. R., Dhital, S., & Gidley, M. J. (2016). Interactions among macronutrients in wheat flour determine their enzymic susceptibility. Food Hydrocolloids, 61, 415–425. Brandmiller, J. (2007). The glycemic index as a measure of health and nutritional quality: An Australian perspective. Cereal Foods World, 52, 41–44. Debet, M. R., & Gidley, M. J. (2006). Three classes of starch granule swelling: Influence of surface proteins and lipids. Carbohydrate Polymers, 64, 452–465. Englyst, H. N., Kingman, S. M., & Cummings, J. H. (1992). Classification and measurement of nutritionally important starch fractions. European Journal of Clinical Nutrition, 46, S33–S50. Flores-Morales, A., Jiménez-Estrada, M., & Mora-Escobedo, R. (2012). Determination of the structural changes by FT-IR, Raman, and CP/MAS 13C NMR spectroscopy on
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Microstructure control for glycemic response modulation. Critical Reviews in Food Science & Nutrition, 56, 2362–2369. Pigorsch, E. (2009). Spectroscopic characterisation of cationic quaternary ammonium starches. Starch-Stärke, 61, 129–138. Rafiq, S. I., Jan, K., Singh, S., & Saxena, D. C. (2015). Physicochemical, pasting, rheological, thermal and morphological properties of horse chestnut starch. Journal of Food Science & Technology, 52, 5651–5660. Regand, A., Chowdhury, Z., Tosh, S. M., Wolever, T. M. S., & Wood, P. (2011). The molecular weight, solubility and viscosity of oat beta-glucan affect human glycemic response by modifying starch digestibility. Food Chemistry, 129, 297–304. Sevenou, O., Hill, S. E., Farhat, I. A., & Mitchell, J. R. (2002). Organisation of the external region of the starch granule as determined by infrared spectroscopy. International Journal of Biological Macromolecules, 31, 79–85. Shah, A., Masoodi, F. A., Gani, A., & Ashwar, B. A. (2016). In-vitro digestibility, rheology, structure, and functionality of RS3 from oat starch. Food Chemistry, 212, 749–758. Shah, A., Masoodi, F. A., Gani, A., & Ashwar, B. (2018). Dual enzyme modified oat starch: Structural characterisation, rheological properties, and digestibility in simulated GI tract. International Journal of Biological Macromolecules, 106, 140–147. Shaikh, F., Ali, T. M., Mustafa, G., & Hasnain, A. (2019). Comparative study on effects of citric and lactic acid treatment on morphological, functional, resistant starch fraction and glycemic index of corn and sorghum starches. International Journal of Biological Macromolecules, 135, 314–327. Shi, M., Gu, F., Wu, J., Yu, S., & Gao, Q. (2013). Preparation, physicochemical properties, and in vitro digestibility of cross-linked resistant starch from pea starch. Starch-Stärke, 65, 947–953. Singh, J., Dartois, A., & Kaur, L. (2010). Starch digestibility in food matrix: A review. Trends in Food Science & Technology, 21, 168–180. Varatharajan, V., Hoover, R., Liu, Q., & Seetharaman, K. (2010). The impact of heatmoisture treatment on the molecular structure and physicochemical properties of normal and waxy potato starches. Carbohydrate Polymers, 81, 466–475. Wang, S., Wang, J., Zhang, W., Li, C., Yu, J., & Wang, S. (2015). Molecular order and functional properties of starches from three waxy wheat varieties grown in China. Food Chemistry, 181, 43–50. Wang, S., Luo, H., Zhang, J., Zhang, Y., He, Z., & Wang, S. (2014). Alkali-induced changes in functional properties and in vitro digestibility of wheat starch: The role of surface proteins and lipids. Journal of Agricultural and Food Chemistry, 62, 3636–3643. Ye, J., Hu, X., Luo, S., Mcclements, D. J., Liang, L., & Liu, C. (2018). Effect of endogenous proteins and lipids on starch digestibility in rice flour. Food Research International, 106, 404–409. Zhang, J., Luo, K., & Zhang, G. (2017). Impact of native form oat β-glucan on starch digestion and postprandial glycemia. Journal of Cereal Science, 73, 84–90. Zhao, J., Zhang, Y., Wu, Y., Liu, L., & Ouyang, J. (2018). Physicochemical properties and in vitro digestibility of starch from naturally air-dried chestnut. International Journal of Biological Macromolecules, 117, 1074–1080.
retrograded starch of maize tortillas. Carbohydrate Polymers, 87, 61–68. Goñi, I., Garcia-Alonso, A., & Saura-Calixto, F. (1997). A starch hydrolysis procedure to estimate glycemic index. Nutrition Research, 17, 427–437. Hernández-Martínez, M., Gallardo-Velázquez, T., Osorio-Revilla, G., Almaraz-Abarca, N., Ponce-Mendoza, A., & Vásquez-Murrieta, M. S. (2013). Prediction of total fat, fatty acid composition and nutritional parameters in fish fillets using MID-FTIR spectroscopy and chemometrics. LWT-Food Science and Technology, 52, 12–20. Hu, X. Z., Zheng, J. M., Li, X. P., Xu, C., & Zhao, Q. (2014). Chemical composition and sensory characteristics of oat flakes: A comparative study of naked oat flakes from China and hulled oat flakes from western countries. Journal of Cereal Science, 60, 297–301. Hu, P., Fan, X., Lin, L., Wang, J., Zhang, L., & Wei, C. (2018). Effects of surface proteins and lipids on molecular structure, thermal properties, and enzymatic hydrolysis of rice starch. Food Science Technology, 38, 84–90. Kaur, H., Gill, B. S., & Karwasra, B. L. (2018). In vitro digestibility, pasting, and structural properties of starches from different cereals. International Journal of Food Properties, 21, 70–85. Kim, H. J., & White, P. J. (2013). Impact of the molecular weight, viscosity, and solubility of β-glucan on in vitro oat starch digestibility. Journal of Agricultural and Food Chemistry, 61, 3270–3277. Kusumayanti, H., Handayani, N. A., & Santosa, H. (2015). Swelling power and water solubility of cassava and sweet potatoes flour. Procedia Environmental Sciences, 23, 164–167. Li, P., Dhital, S., Zhang, B., He, X., Fu, X., & Huang, Q. (2018). Surface structural features control in vitro digestion kinetics of bean starches. Food Hydrocolloids, 85, 343–351. Li, W., Zhang, F., Liu, P., Bai, Y., Gao, L., & Shen, Q. (2011). Effect of high hydrostatic pressure on physicochemical, thermal and morphological properties of mung bean (Vigna radiata L.) starch. Journal of Food Engineering, 103, 388–393. Liu, R., Li, J., Wu, T., Li, Q., Meng, Y., & Zhang, M. (2015). Effects of ultrafine grinding and cellulase hydrolysis treatment on physicochemical and rheological properties of oat (Avena nuda L.) β-glucans. Journal of Cereal Science, 65, 125–131. Liu, Y., Xu, Y., Yan, Y., Hu, D., Yang, L., & Shen, R. (2015). Application of Raman spectroscopy in structure analysis and crystallinity calculation of corn starch. Starch -Stärke, 67, 612–619. Ma, X., Gu, J., Zhang, Z., Jing, L., Xu, M., Dai, X., et al. (2013). Effects of Avena nuda L. on metabolic control and cardiovascular disease risk among Chinese patients with diabetes and meeting metabolic syndrome criteria: Secondary analysis of a randomized clinical trial. European Journal of Clinical Nutrition, 67, 1291–1297. Mäkeläinen, H., Anttila, H., Sihvonen, J., Hietanen, R. M., Tahvonen, R., Salminen, E., et al. (2007). The effect of β-glucan on the glycemic and insulin index. European Journal of Clinical Nutrition, 61, 779–785. Mohamed, A., Biresaw, G., Xu, J. Y., Hojilla-Evangelista, M. P., & Rayas-Duarte, P. (2009). Oats protein isolate: Thermal, rheological, surface and functional properties. Food Research International, 42, 107–114. Parada, J., & Santos, J. L. (2016). Interactions among starch, lipids, and proteins in foods:
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Non-starch constituents influence the in vitro digestibility of naked oat (Avena nuda L.) starch Minyue Tanga, Luyu Wanga, Xuanxuan Chenga, Yanwen Wub, Jie Ouyanga,
T
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a Department of Food Science and Engineering, College of Biological Sciences and Technology, Beijing Key Laboratory of Forest Food Process and Safety, Beijing Forestry University, Beijing 100083, China b Beijing Center for Physical and Chemical Analysis, Beijing Food Safety Analysis and Testing Engineering Research Center, Beijing Academy of Science and Technology, Beijing 100089, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Naked oat In vitro digestibility Glycemic index β-Glucan Lipid Protein
The present study investigated the effects of proteins, lipids and β-glucan in naked oat flour (NOF) on the in vitro digestibility of starch. The content of rapidly digested starch (RDS) increased, and the content of resistant starch (RS) decreased in NOF after removing the non-starch constituents. The estimated glycemic index (eGI) of starch in NOF increased after the removal of the non-starch constituents, with a decreasing order of naked oat starch (NOS) > de-β-glucan flour > de-proteins flour > de-lipids flour > NOF. NOS was found to have an A-type crystalline pattern, but the removal of proteins or β-glucan rendered NOS a V-type crystalline pattern. The relative crystallinity decreased after removing non-starch constituents. The in vitro digestibility was positively correlated with the short-range molecular order and negatively correlated with the relative crystallinity. These results clearly illustrate the effects of non-starch constituents on the low digestibility of naked oat.
1. Introduction Naked oat (Avena nuda L.) originated from Mongolia and northern China, and it is different from oat (Avena sativa L.) in the growth environment, morphology and nutritional value. Oat is recognized as one of the most important grains in the world, because it contains a high content of proteins, lipids, dietary fiber (especially soluble fiber βglucan), minerals and vitamins. In addition, naked oat can lower serum lipids and blood pressure, prevent heart disease and regulate postprandial blood sugar (Liu, Li, et al., 2015). The consumption of naked oat by patients with type 2 diabetes mellitus can lower the risk of metabolic and cardiovascular diseases (Ma et al., 2013). As the main component in higher plants, starch is an important source of carbohydrates in human diet. There are three categories of starch in accordance with the rate and the degree of starch digestion, namely, rapidly digested starch (RDS), slowly digested starch (SDS) and resistant starch (RS) (Englyst, Kingman, & Cummings, 1992). In the small intestine, RDS can be rapidly digested and absorbed, causing the blood glucose level to rapidly rise, whereas SDS can be slowly digested and RS can not be digested. Instead, RS stabilizes the metabolism of glucose and promotes the growth of beneficial colonic flora to prevent colon cancer (Wang et al., 2014). The glycemic index (GI) is an indicator that reflects increases in the blood glucose level in response to
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carbohydrate foods (Brandmiller, 2007). Foods can be classified into three types according to the GI values, namely, low GI (≤55) foods, middle GI (56–69) foods and high GI (≥70) foods. The ingestion of low GI foods, such as oat, can help to reduce the risk of metabolic diseases (Brandmiller, 2007). The digestibility of starch is influenced by diverse internal and external conditions, including the size and shape of starch granules, the food processing method, physical and chemical modifications, the viscosity and food matrix components (Singh, Dartois, & Kaur, 2010). In foods, starches interact with proteins, fats and other constituents to achieve a strong effect on their digestion (Hu et al., 2018). For example, the presence of β-glucan in carbohydrate foods reduced the rate of starch digestion, thereby maintaining a low GI (Mäkeläinen et al., 2007). Considering the above, the present work aimed to explore the effects of non-starch constituents in naked oat flour (NOF), including endogenous proteins, lipids and β-glucan, on the in vitro digestibility of starch and further explore the mechanism for the effects of these constituents on starch digestibility. Furthermore, the physicochemical properties of naked oat starch (NOS) and NOF after removing nonstarch constituents were compared to study the relationship between starch digestibility and intrinsic factors, which can be helpful for understanding the mechanism behind the low digestibility of naked oat.
Corresponding author. E-mail address: [email protected] (J. Ouyang).
https://doi.org/10.1016/j.foodchem.2019.124953 Received 10 March 2019; Received in revised form 4 June 2019; Accepted 5 June 2019 Available online 06 June 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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2. Materials and methods
Hydrolysis rate (%) = Gt × 0.9/starch content (mg)
2.1. Materials and chemical regents
RDS, SDS and RS contents were obtained by the following equations:
Naked oat was harvested in Zhangjiakou, Hebei Province, China. The grains were milled to 80-mesh. Porcine pancreatic α-amylase (13 U/mg), β-glucanase (50 U/mg) and β-glucan were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). Glucoamylase (≥100,000 U/g) was obtained from Beijing Aoboxing Biotechnology Co., Ltd. (Beijing, China). Alkaline protease (100,000–220,000 U/g) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Other chemicals were purchased from Beijing Chemical Reagent Co. (Beijing, China) and were analytical grade.
(1)
RDS (%) = (G20−FG) × 0.9/TS
(2)
SDS (%) = (G120 − G20) × 0.9/TS
(3)
RS (%) = [TS −(RDS + SDS)]/TS
(4)
where FG is the free glucose content (mg) of the sample (mg), G20 is the glucose released (mg) within 20 min, G120 is the glucose released (mg) within 120 min and TS is the total starch content of the sample (mg). According to the method of Goñi, Garcia-Alonso, and Saura-Calixto (1997), the in vitro digestibility of the sample was analyzed to simulate human intestinal digestion. The curve of starch hydrolysis obeyed a first-order reaction equation. The hydrolysis index (HI) of the sample was the percentage of the area under the hydrolysis curve (AUC) of each sample and that of white bread, and the estimated glycemic index (eGI) was determined by the following formula (Goñi et al., 1997):
2.2. Sample preparation Deproteinization of NOF. NOF (20 g) was added to 160 mL of carbonate buffer (0.02 M, pH 9.0), and then alkaline protease (0.2 g) was added and dissolved. The sample was continuously stirred at 45 °C for 1 h, then centrifuged at 4000×g for 15 min and the precipitate was hydrolyzed again. The precipitate was washed repeatedly with distilled water to adjust the pH to 7.0, and the deproteinized NOF (DP-NOF) was obtained by drying the precipitate overnight at 40 °C in DHG-9033B5III oven (Shanghai CIMO Medical Instrument Manufacturing Co., Ltd., China) (Ye et al., 2018). Degreasing of NOF. NOF (20 g) was mixed with petroleum ether (100 mL), and stirred continuously at room temperature for 4 h, then centrifuged at 4000×g for 15 min. The degreasing treatment was repeated two more times. The degreased NOF (DG-NOF) was obtained by drying the precipitate overnight at room temperature. De-β-glucan treatment of NOF. NOF (20 g) was added to 200 mL of sodium acetate buffer (0.1 M, pH 5.2), and then β-glucanase (0.016 g; stock, 50 U/mg) was added and dissolved. β-Glucan was hydrolyzed for 30 min at 55 °C. After centrifugation at 4000×g for 15 min, the precipitate was washed twice with distilled water. The precipitate was dried at 40 °C overnight in DHG-9033B5-III oven to obtain de-β-glucan NOF (Dβ-NOF) (Zhang, Luo, & Zhang, 2017). Extraction of naked oat starch (NOS). NOF was dissolved and homogenized with an NaOH solution (0.01 M) at a ratio of 1:8 (w/v), and then the pH was adjusted to 10.0. The solution was stirred for 2 h at 30 °C and then filtered. The filtrate was centrifuged at 4000×g for 15 min. The supernatant was discarded, and the precipitate was washed with distilled water three times, and then dried for 12 h at 40 °C in DHG-9033B5-III oven.
eGI = 39.71 + 0.549HI
(5)
2.5. Determination of solubility and swelling power The solubility and swelling power of the samples were determined based on the method of Kusumayanti, Handayani, and Santosa (2015) with minor modifications. The sample suspension (2%, w/v) was heated for 30 min at 95 °C and shaken every 1–2 min. The starch suspension was rapidly cooled and centrifuged at 4000×g for 15 min. The supernatant was weighed after drying to constant weight at 105 °C, and the sediment was weighed directly. The solubility and swelling power was calculated as follows:
Solubility (%) = (W2/W1) × 100
(6)
Swelling power (%) = [W3/[W1 × (100−solubility)]] × 100
(7)
where W1 is the weight of the dry sample (mg), W2 is the weight of soluble substance in the supernatant (mg) and W3 is the weight of sediment (mg). 2.6. X-ray diffraction pattern (XRD) analysis XRD analysis was carried out with an X-ray diffractometer (D8 ADVANCE; Bruker Corp., Karlsruhe, Germany) operating at 40 mA and 40 kV. The diffraction scan range (2θ) ranged from 5 to 40° with a scanning speed of 4°/min. The ratio of the sum of the area of each significant peak to the total AUC was calculated as the relative crystallinity (Rafiq, Jan, Singh, & Saxena, 2015).
2.3. Analysis of proximate constituents The content of total starch, crude protein, crude lipid and β-glucan were determined according to the AACC 76-13.01, 46–13.01, 30–25.01 and 32–22.01 methods (AACC, 2000), respectively. 2.4. In vitro starch digestibility
2.7. Fourier transform infrared spectroscopy (FTIR) analysis
According to the method of Englyst et al. (1992) with some modifications, the in vitro digestibility of the samples was determined. The sample (200 mg) was mixed with 15 mL of sodium acetate buffer (0.2 M, pH 5.2), and then 10 mL of the previously prepared enzyme solution [porcine pancreatic α-amylase (290 U/mL) and glucoamylase (15 U/mL)] was added. The mixture was shaken in a water-bathing constant temperature vibrator (SHA-A, Saidelisi Crop., Tianjin, China) at 37 °C at a speed of 150 revolutions/min. An aliquot (0.50 mL) was obtained at different times (10, 20, 40, 60, 90, 120 and 180 min), and the enzymes were inactivated by adding absolute ethanol (4.5 mL). Subsequently, the glucose content (Gt) was analyzed by the method of 3.5-dinitrosalicylic acid (DNS). The hydrolysis rate (%) was calculated using the following formula:
The finely ground samples were thoroughly mixed with dry potassium bromide powder (1:100, w/w) and tableted at 10,000 PSI. The spectrum was recorded using a Tensor27 FTIR spectrometer (Bruker Corp., Ettlingen, Germany) from 4000 to 400 cm−1 and at a resolution of 4 cm−1. 2.8. Statistical analysis All results were represented as mean ± standard deviation with triplicate experiments, and the data were subjected to a difference significance test using SPSS 23.0 software (IBM Corp., Armonk, NY, USA). 2
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NOF was 26.9% at 180 min, but that of Dβ-NOF increased to 32.4%. Due to the removal of lipids, proteins and β-glucan during the extraction process, the hydrolysis rate of NOS was greatly improved, and the final hydrolysis rate was 33.8%. The simultaneous removal of proteins and lipids in rice flour yielded the highest degree of hydrolysis, and the effect of proteins on hydrolysis was slightly higher than that of lipids (Ye et al., 2018). Furthermore, a significant increase was observed in the starch digestion rate when dextran-rich oat flour was treated with βglucanase, and β-glucan affected starch digestion in native form rather than in its extracted form (Zhang et al., 2017). Oats contain more proteins and lipids than other common grains. Proteins can effectively reduce starch digestibility by several mechanisms. Firstly, proteins can form a protection round the starch granules, thereby restricting the entry of enzymes into the substrates. Secondly, surface proteins can block the catalytic binding of enzymes on the starch granule exterior (Wang et al., 2014). Thirdly, α-amylase can partially bind to proteins, thereby reducing enzyme utilization (Bhattarai, Dhital, & Gidley, 2016). By contrast, the effect of lipids on starch digestibility is primarily due to the ability of lipids to form complexes with amylose, which is better able to resist the attack of amylase (Singh et al., 2010). Monoglycerides/free fatty acids are capable of forming complexes with amylose, but tri- or di-glycerides do not readily form complexes with amylose. Therefore, the types of lipids presented in naked oats are worthy of further investigation. Fatty acids in lipids rapidly form complexes with amylose under physiological conditions, promoting the formation of resistant complexes (Annor et al., 2013). Therefore, the proteins and lipids in naked oat interact with starch to form a barrier or complexes that makes naked oat hard to digest. The content of β-glucan in different grains greatly varies, among which barley and oat are the highest. A study has shown that β-glucan, particularly the extracted water-soluble fraction, can lower the digestion rate of starch by increasing the viscosity (Regand, Chowdhury, Tosh, Wolever, & Wood, 2011). At the same time, β-glucan can create a complex of adjacent proteins to form a robust structure that resists attack by amylase, thereby resulting in a decrease in starch digestibility. Therefore, the de-β-glucan treatment of NOF increased the degree of starch hydrolysis. Different treatments have caused different release of various nonstarch components. For example, the De-β-glucan treatment caused a decrease in protein content (from 11.69 to 8.46%) when removing the β-glucan, and deproteinization treatment caused the loss of a large amount of β-glucan (from 5.60 to 0.12%). Therefore, these components in naked oat may be closely linked to each other. When one component is hydrolyzed, other components may also be inevitably lost. The matrix formed by starch and non-starch components such as β-glucan, protein, and lipid in their ordered structure may be one of the key factor for the low starch digestibility (Zhang et al., 2017). In the extracted naked oat starch, the contents of lipid, protein and β-glucan declined to a great extent, and the digestibility of starch increased. This further demonstrated that the interaction among these substances and their effects on starch digestibility. The content of RDS, SDS and RS in NOF, DG-NOF, DP-NOF, Dβ-NOF and NOS are given in Table 2. The RDS content of the treated samples was higher than that of NOF, whereas the content of RS was decreased compared to NOF. The RDS increased from 15.1% in NOF to 23.0% in NOS, and the RS decreased from 76.0% in NOF to 68.3% in NOS. The decreasing order of the RDS content was NOS > Dβ-NOF > DPNOF > DG-NOF > NOF, whereas the RS content showed an opposite trend. These results were consistent with the in vitro digestion curve of the samples. Annor et al. (2013) studied the in vitro digestibility on deproteinized and degreased millet flour, millet flour and millet starch. They reported that proteins and lipids affected the digestibility of millet, and the effect of lipids was higher than that of proteins. The removal of lipids caused the RDS level of millet flour to increase from 11.48% to 20.07%, whereas the removal of proteins changed the RDS level to 14.21% (Annor et al., 2013).
Table 1 Proximate composition (g/100 g fresh weight) of NOF and removal of nonstarch constituents. Sample
Total starch
NOF DG-NOF DP-NOF Dβ-NOF NOS
71.51 75.81 78.70 70.36 91.19
± ± ± ± ±
0.34d 0.32c 0.21b 0.06e 0.41a
Crude protein
Crude lipid
11.69 ± 0.07a 9.38 ± 0.05b 1.52 ± 0.02d 8.46 ± 0.06c 0.85 ± 0.01e
5.19 0.30 4.12 1.80 1.53
± ± ± ± ±
0.04a 0.02e 0.08b 0.06c 0.03d
β-glucan 5.60 5.91 0.12 0.03 0.13
± ± ± ± ±
0.22b 0.18a 0.01c 0.02d 0.01c
Different superscripts mean significant differences in the same column (P < 0.05). NOF, naked oat flour; DG-NOF, degreased NOF; DP-NOF, deproteinized NOF; Dβ-NOF; de-β-glucan NOF; NOS, naked oat starch.
3. Results and discussion 3.1. Proximate constituents of NOF and removal of non-starch constituents The content of total starch, crude protein, crude lipid and β-glucan in NOF was 71.51%, 11.69%, 5.19% and 5.60% (fresh weight) (Table 1), respectively, and the moisture content was approximately 10%. The results were consistent with previous studies, which showed the protein content in naked oat to be 11–19% (Mohamed, Biresaw, Xu, Hojilla-Evangelista, & Rayas-Duarte, 2009). The content of β-glucan and lipid in naked oat was 3–8% and 1.42–11.52%, respectively, which was far higher than those of other cereal crops (Hu, Zheng, Li, Xu, & Zhao, 2014). NOF was individually underwent degreasing, deproteinization and de-β-glucan treatment, and NOS was also extracted. The lipid removal rate of DG-NOF reached 94.2%, and the protein removal rate of DP-NOF was 87.0%. In Dβ-NOF, the β-glucan content decreased from 5.60 to 0.03%, whereas the lipid content decreased from 5.19 to 1.80%. Virtually all non-starch constituents in NOS were removed. Similarly, the degreasing rate of millet flour was 94.38%, and the protein content of millet treated with proteases decreased from 8.35 to 2.29% (Annor, Marcone, Bertoft, & Seetharaman, 2013). 3.2. In vitro digestibility of NOF and removal of non-starch constituents The in vitro hydrolysis curves of NOF, DG-NOF, DP-NOF, Dβ-NOF and NOS were compared in Fig. 1. The hydrolysis rate of all samples increased rapidly within 20 min, and then it slowed down from 20 to 120 min, with a decreasing order of NOS > Dβ-NOF > DP-NOF > DG-NOF > NOF. It can be concluded that the intrinsic constituents in NOF had significant effects on starch digestibility. β-Glucan had the greatest impact, followed by proteins and lipids. The hydrolysis rate of
Fig. 1. Enzymatic hydrolysis curves of NOF and removal of non-starch constituents. NOF, naked oat flour; DG-NOF, degreased NOF; DP-NOF, deproteinized NOF; Dβ-NOF, de-β-glucan NOF; NOS, naked oat starch. 3
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Table 2 Digestibility parameters of NOF and removal of non-starch constituents. Sample
RDS (g/g starch)
NOF DG-NOF DP-NOF Dβ-NOF NOS
15.1 15.8 16.7 21.5 23.0
± ± ± ± ±
SDS (g/g starch)
b
RS (g/g starch)
b
8.8 ± 0.4 9.6 ± 0.8b 11.2 ± 0.2a 9.1 ± 0.2b 8.7 ± 0.1b
0.1 0.9b 0.8b 0.9a 0.3a
76.0 74.6 72.1 69.5 68.3
± ± ± ± ±
k × 10−2
C∞
a
0.4 0.2b 0.4c 0.4d 0.2d
26.9 28.0 30.4 32.4 33.8
± ± ± ± ±
c
0.2 0.3c 0.5b 1.4ab 0.9a
1.4 1.5 1.7 1.8 1.8
± ± ± ± ±
HI c
0.1 0.1c 0.0b 0.0a 0.1ab
50.8 54.4 61.4 67.3 69.5
eGI ± ± ± ± ±
e
0.2 0.6d 0.8c 0.2b 0.3a
67.6 69.6 73.4 76.7 77.9
± ± ± ± ±
0.1e 0.3d 0.4c 0.3d 0.2a
Different superscripts mean significant differences in the same column (P < 0.05). RDS is rapidly digestible starch; SDS is slowly digestible starch; RS is resistant starch; C∞ is the equilibrium percentage of starch hydrolyzed after 180 min; k is the reaction kinetic constant; HI is the hydrolysis index; eGI is the estimated glycemic index. NOF, naked oat flour; DG-NOF, degreased NOF; DP-NOF, deproteinized NOF; Dβ-NOF, de-β-glucan NOF; NOS, naked oat starch.
small pores in the starch granules, adversely affecting the penetration of digestive enzymes, thus reducing the rate of starch hydrolysis. A previous study has shown that the surface components (glucan + protein + lipid) of starch in wheat and corn can combine to form a complex, which performs a vital role in limiting the speed and extent of swelling of the particles (Debet & Gidley, 2006). The in vitro digestibility and swelling power of rice starch from different varieties increased after surface lipids and proteins were removed (Hu et al., 2018).
Naked oat is a traditional Chinese grain, which is generally considered to be difficult to digest. The eGI of NOF was 67.6, indicating that it belongs to the middle GI food category (Table 2). A previous study has shown that some common grains, such as rice (91.8), corn (89.2) and wheat (87.3), are high-GI foods (Kaur, Gill, & Karwasra, 2018). The decreasing order of the eGI of naked oat samples was NOS > Dβ-NOF > DP-NOF > DG-NOF > NOF. The proteins, lipids and β-glucan in NOF were the internal factors responsible for the slow digestion of naked oat. Starch in food always forms complex matrices with lipids and proteins, causing intrinsic differences in human postprandial glycemic responses and leading to different GI (Parada & Santos, 2016). The GI of oat starch supplemented with β-glucan of high molecular weight decreased from 88.3 to 68.4, indicating that β-glucan could inhibit the digestibility of starch, and the molecular weight was negatively related to the GI (Kim & White, 2013).
3.4. Crystal properties of NOF and removal of non-starch constituents Starch can be classified into four crystal types: A, B, C and V, and cereal starches are usually A-type (Liu, Xu, et al., 2015). The diffraction angles (2θ) of all naked oat samples had strong diffraction peaks at 15°, 17°, 18° and 23°, which were typical A-type diffraction characteristics (Fig. 2). The diffraction peak intensity of DP-NOF and Dβ-glucan increased at a 2θ of 20°, which corresponds to the V-type crystal pattern and relates to the presence of an amylose-lipid complex (Shah, Masoodi, Gani, & Ashwar, 2018). Thus, deproteinization and de-βglucan treatment may promote the formation of the complex of amylose-lipid. NOF had the highest crystallinity (32.38%) and NOS had the lowest crystallinity (22.73%), with a decreasing order of NOF > DGNOF > DP-NOF > Dβ-NOF > NOS. The peak intensity of samples after different treatments also decreased. In general, the high crystallinity of starch indicates more crystalline regions within the particles (Li et al., 2011). In addition, the crystallinity was also affected by many factors, such as the crystal size, the orientation of double helices, amount of crystalline regions (amylopectin content and chain length
3.3. Solubility and swelling power of NOF and removal of non-starch constituents Swelling power and solubility are good indicators to measure the degree of interaction between amorphous and crystalline regions of starch chains (Kusumayanti et al., 2015). Solubility represents a percentage of molecules that released from swelling starch granules. The solubility of NOF was the lowest (12.0%) and increased when the nonstarch constituents were removed (Table 3). The different removal treatments resulted in the disintegration and structural weakening of the starch granules, and the solubility of the starch increased (Shi, Gu, Wu, Yu, & Gao, 2013). It is generally considered that the presence of constituents will hinder the entry of water molecules into the starch granules, thereby inhibiting the starch solubility. The level of swelling power reflects the hydration ability of the starch. The expansion of starch granules can promote the attack of the internal structure by digestive enzymes, thereby increasing the rate of starch hydrolysis (Shi et al., 2013). The decreasing order of swelling power was NOS > Dβ-NOF > DP-NOF > DG-NOF > NOF. The result indicated that proteins, lipids and β-glucan significantly (P < 0.05) inhibited the swelling power of starch granules. Therefore, the existence of non-starch constituents in NOF may slow the digestion of starch. At the same time, the low swelling power was related to the Table 3 Solubility, swelling power, short-range ordered index of NOF and removal of non-starch constituents. Sample
Solubility (%)
NOF DG-NOF DP-NOF Dβ-NOF NOS
12.0 18.5 18.5 18.8 20.1
± ± ± ± ±
0.7c 0.2b 0.3b 0.1ab 0.2a
Swelling power (%) 13.9 18.0 20.1 21.5 26.4
± ± ± ± ±
0.1d 0.6c 1.0b 0.2b 0.2a
IR ratio of 1047/1022 cm−1 1.083 1.120 1.193 1.197 1.335
± ± ± ± ±
0.009d 0.002c 0.008b 0.012b 0.013a
Fig. 2. X-ray diffraction spectra of NOF and removal of non-starch constituents. NOF, naked oat flour; DG-NOF, degreased NOF; DP-NOF, deproteinized NOF; Dβ-NOF, de-β-glucan NOF; NOS, naked oat starch.
Different superscripts means significant differences in the same column (P < 0.05). NOF, naked oat flour; DG-NOF, degreased NOF; DP-NOF, deproteinized NOF; Dβ-NOF, de-β-glucan NOF; NOS, naked oat starch. 4
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removal of non-starch constituents (Table 3). This may have been due to the disruption of the parallel alignment of the crystals by the different treatments (Shah, Masoodi, Gani, & Ashwar, 2016). The amorphous components in starch were preferentially degraded or degraded faster, which resulted in an increase in the degree of order within starch (Sevenou, Hill, Farhat, & Mitchell, 2002). Furthermore, the correlation coefficient (r) between 1047/1022 cm−1 and eGI and RS was 0.908 and −0.918, respectively. Similarly, the positive correlation between 1047/ 1022 cm−1 and the digestibility of chestnut starch after cooking was also observed (Bao, Li, Wu, & Ouyang, 2018). In addition, a previous study showed that the correlation coefficient between the 1047/ 1022 cm−1 and relative crystallinity was −0.162, which indicated that the short-range molecular order observed by FTIR was not correlated with the crystallinity observed by XRD (Li et al., 2018). 4. Conclusion Naked oat is a medium eGI food, and the eGI of NOF increased after the removal of proteins, lipids and β-glucan. The solubility and swelling power of NOF without non-starch constituents increased, indicating that the presence of constituents hindered the entry of water and enzymes into the starch granules. The crystalline structure of NOF was Atype, whereas the crystalline structure of DP-NOF and Dβ-NOF was Vtype. The relative crystallinity decreased after removing non-starch constituents and showed a negative correlation with in vitro digestibility. The removal of non-starch constituents resulted in an increase of starch short-range molecular order, which was significantly correlated with eGI. These results are helpful for understanding the role of nonstarch constituents in the low digestibility of naked oat, where endogenous complexes between starch and other constituents may be formed. Further research will be carried out in the binding models between starch and other constituents, and inhibitory effect of endogenous bioactive components (saponins, polyphenols, etc.) in naked oat on the activity of amylase.
Fig. 3. FTIR spectra of NOF and removal of non-starch constituents. NOF, naked oat flour; DG-NOF, degreased NOF; DP-NOF, deproteinized NOF; DβNOF, de-β-glucan NOF; NOS, naked oat starch.
distribution of amylopectin), and the extent of interaction between double helices (Rafiq et al., 2015). Physical and chemical treatments during sample processing can cause damage to the particles, destroy the internal structure of the starch and undermine the combination of starch and other substances. The samples may undergo reorientation of crystallite, reduction of large particle crystals, and/or destroy the crystalline regions and decrease the double helices within starch, thereby resulting in a decrease in its crystallinity. Starch with higher crystallinity usually contains higher RS content because enzymes can’t easily enter into the ordered crystal structure (Shaikh, Ali, Mustafa, & Hasnain, 2019). The change in crystallinity was significantly negatively correlated (r = −0.995) with the eGI of the naked oat samples. Similarly, the eGI and crystallinity of starch in naturally air-dried chestnut was negatively correlated (Zhao, Zhang, Wu, Liu, & Ouyang, 2018).
Declaration of Competing Interest There are no conflicts to declare.
3.5. FTIR of NOF and removal of non-starch constituents Acknowledgements The FTIR spectra of different samples were similar (Fig. 3). However, the protein absorption peaks of NOS and DP-NOF at 1535 cm−1 disappeared, which represented the stretching vibration of CeN and NeH in the protein (Hernández-Martínez et al., 2013) and indicated a decrease in the protein content, which was consistent with the previous results of chemical measurements. Furthermore, a broad and strong absorption appeared at 3400 cm−1, which assigned to the OeH stretching vibration in or between molecules. The absorption peak at 2927 cm−1 was due to the asymmetric stretching vibration of CeCH2eC, and the absorption peak at 1649 cm−1 was attributed to the C]O stretching vibration in the aldehyde group (Flores-Morales, Jiménez-Estrada, & Mora-Escobedo, 2012; Amir & Kumar, 2007). The characteristic peaks in the range of 1200–1020 cm−1 were assigned to the stretching vibration of CeO and CeC in the anhydroglucose ring. In the range of 930–900 cm−1, the absorption was due to the skeleton vibration of the α-1,4 glycosidic bond (CeOeC). The absorption peak at 767 cm−1 was corresponded to the ring-symmetric stretching vibration of the pyran ring, and the absorption peaks from 620 to 527 cm−1 corresponded to the pyran ring skeleton vibration (Pigorsch, 2009; Wang et al., 2015). The short-range molecular order of starch was obtained by the ratio of 1047/1022 cm−1, which indicated the proportional relationship between the ordered structure and the amorphous structure in starches, and the larger ratio represented the higher degree of inside order within the particles (Varatharajan, Hoover, Liu, & Seetharaman, 2010). The 1047/1022 cm−1 of NOF was 1.083, and the value increased after the
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