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Efficacy of potato resistant starch prepared by microwave–toughening treatment
Carbohydrate Polymers 192 (2018) 299–307
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Efficacy of potato resistant starch prepared by microwave–toughening treatment
T
You-Dong Lia,b,1, Tong-Cheng Xua,b,1, Jun-Xia Xiaoc, Ai-Zhen Zonga,b, Bin Qiua,b, Min Jiaa,b, ⁎ ⁎ Li-Na Liua,b, , Wei Liua,b, a
Institute of Agro-Food Science and Technology, Shandong Academy of Agricultural Sciences, Jinan 250100, PR China Key Laboratory of Agro-Products Processing Technology of Shandong Province, No. 202, Gongyebei Road, Jinan 250100, PR China c School of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Potato Resistant starch Microwave treatment Glycemic index Efficacy evaluation
Potato starch was treated by microwaving, toughening, and low-temperature aging to prepare resistant starch (RS). The functional properties of the resultant RS were evaluated and the effects of this microwave–toughening treatment (MTT) on the amylose content, RS content, digestive properties, pasting properties, morphological observation, crystal structure, and thermal performance of potato starch were determined. The optimal MTT parameters were microwaving at 300 W for 100 s, toughening at 55 °C for 16 h, and low-temperature aging at 4 °C for 18 h. After MTT, the amylose and RS contents of potato starch had increased from 26.08% and 11.54% to 35.06% and 27.09%, respectively. Furthermore, the pasting temperature increased from 66.8 °C to 76.36 °C, while the peak viscosity, trough viscosity, and final viscosity decreased significantly. After MTT, the potato starch surface had also changed significantly, and the crystallinity had increased from 32.43% to 51.36%. MTT starch had beneficial effects on fasting blood glucose, body weight, and organ index in mice. Furthermore, it had a protective effect on subcutaneous abdominal fat and liver tissue.
1. Introduction
amylopectin is a larger branched molecule with α-(1,4)- and α-(1,6)linkages (Yuan, Wang, Chen, Zhu, & Cao, 2015). Starch can be divided into three types according to digestion rate, namely, rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) (Englyst, Kingman, & Cummings, 1992). RDS can be completely digested and absorbed in the human small intestine, causing an increase in the blood glucose level (Pham & Nguyen, 2015). To control the sudden increase in blood sugar, pancreatic islets rapidly secrete insulin, causing the blood sugar levels to drop sharply. And long-term RDS intake can result in insulin resistance and type 2 diabetes (Kim & Lee, 2009). SDS is slowly digested in the human small intestine, which has potential health benefits for the blood sugar level and islets (Dong, 2012). RS is not digested, but fermented in the large intestine by human microflora to produce short-chain fatty acids. This causes a decrease in the intestinal pH value, which helps maintain intestinal peristalsis and protect intestinal mucosa (Fuentes-Zaragoza, Riquelme-Navarrete, Sánchez-Zapata, & Viuda-Martos, 2010). As RS has been shown to be effective at preventing diabetes and obesity, an increasing amount of research has focused on improving the RS content in functional foods (Patil, 2004).
The potato is a widely cultivated cash crop and the most prevalent food crop worldwide, providing energy to about 15% of the global population (Karim, Holmes, & Orfila, 2016). Potatoes are generally used to make popular consumer products, such as potato chips and mashed potato (Kondo, Higashi, Iwama, Ishihara, & Handa, 2012). Potato storage is problematic owing to shortcomings in technology, facilities, and industrial structure (Heltoft, Wold, & Molteberg, 2017). Potatoes are nutritious, containing starch (9–20%), protein (1.5–2.3%), fat (0.1–1.1%), crude fiber (0.6–0.8%), polyphenols (0.0071–0.031%), and other trace components (Kurilich, 2013). The saponin found in potato green skin dry powder can be used to control hyperglycemia (Jecht, 2013). The main ingredient in potatoes is starch, of which ∼80% is contained in the dry matter (Ciecko & Zolnowski, 2004). Potato starch is not only important in food, but also in pharmaceutical, chemicals, cosmetics, and textiles (Rodrigues & Emeje, 2012). Starch is composed of amylose and amylopectin. Amylose is a linear D-glucopyranose chain connected by α-l,4-glycosidic bonds that is slightly branched (Takeda, Shitaozono, & Hizukuri, 1990), while
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1
Corresponding authors at: Institute of Agro-Food Science and Technology, Shandong Academy of Agricultural Sciences, Jinan 250100, PR China. E-mail addresses: [email protected] (L.-N. Liu), [email protected] (W. Liu). These authors contributed equally to this work and should be considered co-first authors.
https://doi.org/10.1016/j.carbpol.2018.03.076 Received 5 December 2017; Received in revised form 20 March 2018; Accepted 22 March 2018 Available online 23 March 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
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Analysis Technology Co., Ltd. (Beijing, China). Heat-stable α-amylase (1400 U/g) and amyloglucosidase (3300 U/g) were purchased from Sigma–Aldrich (St. Louis, USA). Maltose standard product was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China).
RS can be divided into four types according to their origin, namely, physical embedding starch (RS1), resistant starch granules (RS2), aging of regenerated starch (RS3), and chemically modified starch (RS4) (Maziarz et al., 2017). Chemical modification is the most common method for enhancing RS content. Recently, many techniques have been studied for enhancing the RS content in starch, including hydrothermal, enzymatic hydrolysis, acid hydrolysis, and ultrasound methods. Arns et al. (2015) increased the RS content of wheat starch to 42.1% using a heat treatment method, which was then modified by Sun et al. (2015) to achieve further improvements. This improved RS content was due to starch gelatinization and regeneration. When starch is hydrothermally treated, the amylose double helix is destroyed (hydrogen bond dissociation) and amylopectin detaches from the main chain (Tester, 1989). These events, collectively referred to as “gelatinization”, are accompanied by a sharp increase in system viscosity, such as the gradual collapse of the grain structure (Yang & Rao, 1998). At low temperatures, recrystallization of the polymer chains causes degradation (Jang & Pyun, 1997). Mutungi, Rost, Onyango, Jaros, ànd Rohm (2009) improved the tapioca RS content from 21.4% to 67.3% by debranching with pullulanase, recrystallizing the linear dextran by incubation at 60 °C, and finally using heat-moisture treatment to broaden the endotherms and increased their enthalpies. Song, Park, and Shin (2015) increased the RS level by 114.5% through acid hydrolysis (pH 4.0) for 1 h under sonication. Sonication reduced the starch particle size and starch granules remained the same as in native starch, with a polygonal shape and A-type crystallinity after treatment. Microwave treatment could replace the traditional heat treatment process. Microwaves can induce the swelling stage of starch gelatinization and then the low temperature can age the starch. The molecular chains recombine through hydrogen bonds to form new crystals (Sjöqvist & Gatenholm, 2005). The toughening of starch is a physical modification under the action of heat and water. It refers to the starch modified in excess moisture (> 60% w/w) or moisture (40%–55% w/ w) and below starch gelatinization temperature of the initial temperature conditions for a period of time caused by changes in starch structure and properties. Toughening can reduce the degree of solubility and swelling, reduce the viscosity, and increase the paste temperature of starch, and does not produce harmful chemicals or cause genetic changes (Taguet, Bureau, Huneault, & Favis, 2014). During the toughening process, the starch crystals rearrange internally to increase the crystallinity, making them more resistant to enzymolysis (Yadav, Guleria, & Yadav, 2013). Microwave and toughening methods are safe and easy to perform, but few studies have applied this method to resistant starch production. The present study aims to use to improve the RS content in potato starch, as the raw material, through microwave and toughening technology, and observe the effect of microwave toughening on the starch structure characteristics and efficacy. Changes in eating habits and food composition have resulted in the incidence of chronic diseases, such as diabetes and cardiovascular diseases, increasing in recent years, with more people tending to consume low-calorie high-fiber foods for health purposes. The microwave and toughening techniques, and products of potato starch obtained from this study, have broad market prospects. These raw materials could be used to prepare diabetes-specific staple foods, and inspire new applications and development of potato starch in functional foods. Furthermore, this technology could promote industrial restructuring and ease problems associated with potato storage.
2.2. Preparation of resistant starch 2.2.1. Preparation of microwave–toughening treatment (MTT) starch Potato resistant starch (P-RS) was prepared according to methods of Yang, Yang, and Ding (2008) and Sharma, Yadav, Singh, and Tomar (2015) with some modifications. Potato starch (100 g) was gelatinized with distilled water (220 mL) by microwaving at 300 W for 100 s (microwave process conducted in water bath at 45 °C; final temperature of water–starch solution, 80.4 °C). The supernatant was removed by centrifugation (3000 rpm, 15 min) and the sediment was washed thoroughly three times with distilled water. The sample moisture content was then adjusted to 60% (w/v) by spraying with the appropriate calculated amount of distilled water. The starch and water were then thoroughly mixed, placed in sealed containers, and heated on a constant-temperature shaking table at 55 °C for 16 h. The containers were then cooled to ambient temperature, opened, and then stored in a refrigerator at 4 °C for 18 h. The obtained starch was dried at 45 °C for 24 h, ground, and passed through a 100-mesh sieve for further analysis. 2.2.2. RS determination RS was determined according to the procedure of Goñi, García-Diz, Mañas, and Saura-Calixto (1996) with a slight modification. Treated potato starch (TPS) (0.5 g) was digested with excess amylase for 30 min. The reaction mixture was then centrifuged at 8000 rpm for 8 min. The resultant residue was collected and dispersed in 2 M KOH solution (6 mL) and stirred for 30 min at ambient temperature. The pH was adjusted to neutral using a citrate buffer solution and then adjusted to 4.4 using a 2 N HCl solution. Excess amyloglucosidase was added and the mixture was heated in a water bath at 60 °C for 45 min. The samples were centrifuged (8000 rpm, 8 min), and the supernatants were collected and made up to 100 mL using distilled water. The reducing sugar content was determined using the dinitrosalicylic acid (DNS) method. 2.3. Scanning electron microcopy (SEM) The surface structures and cross-sections of treated potato starch (TPS) and potato starch (PS) fractions were observed using field-emission scanning electron microscopy (FESEM). Starch samples were mounted on circular aluminum stubs with double sided carbon tape, coated with a thin platinum film under vacuum, and examined using FESEM (Supra 55VP microscope, Carl Zeiss, Oberkochen, Germany) at an accelerating potential of 3 kV. A starch granule cross section was prepared using a stainless steel razor blade approximately 2-μm thick (ST 300, Dorco Co., Seoul, Korea). 2.4. X-ray diffraction X-ray diffraction analysis was performed using an X-ray diffractometer (Model D5005, Bruker, Karlsruhe, Germany) operating at 40 kV and 40 mA producing CuK radiation with a wavelength of 1.54 Å scanning through the 2θ range of 3–30° and with a step time of 4 s. The relative crystallinity of the starches was calculated according to a previous report (Nara & Komiya, 1983) using peak-fitting software (Origin version 7.5, OriginLab, Northampton, MA, USA)
2. Materials and methods
2.5. Differential scanning calorimetry–thermogravimetric analysis (DSC–TGA)
2.1. Materials Potato starch (purity, 96.9%) was obtained from Gansu Mintian Food Co., Ltd. (Gansu, China). A blood glucose meter and blood glucose test strips was purchased from Roche Diagnostics GmbH (Mannheim, Germany). Amylose standard was purchased from Beijing Spectrum
The gelatinization characteristics and thermogravimetric properties of the products were measured using DSC–TGA (SDT Q600, TA Instruments, USA). Anhydrous starch samples (approximately 5 mg) 300
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experiment. The weight and food intake of the mice were recorded once a week.
were added to an aluminum DSC pan, which was then sealed, reweighed, and heated from 25 to 600 °C at 10 °C/min. An empty pan was used as a reference. The degree of gelatinization (DG) was calculated according to a literature method (Paola, Asis, & Aldao, 2003). The gelatinization enthalpy (DH) of the native and modified starch samples was used to calculate the DG using the following equation：
2.6.3.1. Changes in fasting blood glucose. The fasting blood glucose was measured after 12 h without water and feed. Specifically, scissors disinfected with medical alcohol were used to cut the mouse tail tip and the formed blood drops were quickly placed into the middle of the blood glucose meter test cassette top test area so that they were touching the detection zone. The test results were recorded after a few seconds. The test results and fasting blood glucose were measured again after six weeks.
DG (%) = (1 − ΔHg/ΔHt) × 100 where ΔHt and ΔHg are the gelatinization enthalpies of modified and native starch samples, respectively.
2.6.3.2. Effect of TPS on general signs and body weight of mice. The general signs and body weight of mice are intuitive indicators of TPS. During the experiment, the food intake and body weight of each group of mice were measured by weighing weekly, and the hair, feces, and mental state of each group were observed.
2.6. Animal experiments 2.6.1. Efficacy study of TPS Animal experiments were divided into two groups. The first group was a short-term experiment in which the effect of TPS on the glycemic index in mice was observed by gavage, and calculated using the area under the curve. The second group was a long-term experiment that explored the effect of TPS on mice on a high fat diet. Different starches were added to the diet to observe the effect of TPS on mouse symptoms through long-term feeding. The efficacy of TPS was evaluated by combining two experiments to provide a more robust scientific basis for the hypoglycemic effect of TPS. Fifty male Kunming mice (weight, 25 ± 2 g) were purchased from the Animal Experimental Center of Shandong University. During experiments, the mice were fed in an approved laboratory animal facility and maintained under controlled temperature (22–24 °C) and lighting (12-h light/dark cycle) for a 14-day adaptation period with a normal diet and weekly change of bedding and water. Fifty male C57BL/6J (B6) mice (SPF mouse; weight, 22 ± 2 g) were purchased from Nanjing Biomedical Research Institute of Nanjing University. Each squirrel cage of mice was independently ventilated. The mice were fed in an approved laboratory animal facility and maintained under controlled temperature (22–24 °C) and lighting (12-h light/dark cycle) for a 14-day adaptation period with a normal diet and weekly change of bedding and water. The experimental protocol was approved by the Animal Experiment Committee of China, in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals.
2.6.3.3. Determination of visceral organ index in mice. The mice were sacrificed after six weeks of feeding and the abdominal fat, liver, spleen, kidney, heart, brain, and brown fat were removed. The surface connective tissue was quickly separated in normal saline and weighed using the analytical balance after removing water. The organ index was calculated using the following formula: Organ index (%) = Organ weight (g)/Mouse weight (g) × 100
2.6.3.4. Observation of visceral tissue morphology. The abdominal subcutaneous fat and liver were weighed and placed in formalin, and a section of viscera was observed by hematoxylin–eosin (HE) staining. The test method involved a dozen steps, such as tissue fixation, drawing materials, dehydration, transparence, waxing, embedded, dressing wax, section, patch, baking sheet, wax removal, water addition, hematoxylin staining, differentiation, washing, back blue, eosin staining, dehydration, transparence, and seal sheet. The required sample size was 1.5 × 1.5 × 0.3 cm. When immersed in wax from low to high immersion, the temperature could not be higher than 65 °C. The baking sheet was treated at 65 °C for 20–30 min, the hematoxylin staining time needed to be controlled at 5–15 min, and hydrochloric acid differentiation within 30 s. Euphorbia staining took 1–3 min, and a drop of glacial acetic acid was added, which can play a role in promoting dye. With different concentrations of alcohol dehydration, xylene fully transparent with gum fragments.
2.6.2. Postprandial blood glucose of mice Ten animals (Kunming mice) per cage and five groups (groups I–V) were used. The mice in group I were given TPS, group II were given potato starch, group III were given corn starch, group IV were given RS4 (resistant starch type 4), and group V were given glucose by gavage. The water and diet were cut off 12 h before the experiment, and the fasting blood glucose of the mice was measured using a blood glucose meter by cutting their tails (as described in section 2.6.3.1). All mice received intragastric administration at a dose of 0.2 mL/10 g, and blood glucose was recorded within 2 h after intragastric administration to plot a blood glucose curve. The glycemic index was calculated as follows:
2.7. Statistical analyses Origin 8.5 (Origin Lab Corporation, USA) statistical software was used for statistical analysis. All experimental data were expressed as the means ± standard deviation (SD). Differences between the test subjects and model controls were evaluated using Student’s t-test. 3. Results and discussion 3.1. Preparation of microwave–toughening treatment (MTT) starch
GI (glycemic index) = (2 h curve area for starch)/(2 h curve area for glucose) × 100
3.1.1. Amylose and amylopectin contents The changes in amylose and amylopectin contents are shown in Fig. 1. Potato starch had an amylose content of 26.08% and amylopectin content of 73.92% before processing. After microwaving and toughening treatment, the amylose content had increased significantly to 35.06%. Amylose is a linear polymer composed of α-D-1,4-glycosidic bonds, while amylopectin has a highly branched bundle structure, with the main chain linked by α-1,4-glycosidic bonds, and the branched chains linked to the main chain via α-1,6-glycosidic bonds. Amylopectin is barely soluble in water, but easily gelatinized in hot water,
2.6.3. Effect of TPS on mouse signs in a long-term experiment Ten animals (C57BL/6J) per cage and five groups (groups A–E) were used. Each squirrel cage of mice was independently ventilated and all feed and water was sterilized. The mice in group A were fed a low-fat diet with corn starch, group B were fed a high-fat diet with corn starch, group C were fed a high-fat diet with potato starch, group D were fed a high-fat diet with MTT starch, and group E were fed a high-fat diet with RS4. The mice were fed a normal diet for two weeks before the 301
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Fig. 1. Effect of resistance enhancement on amylose and amylopectin contents in potato starch. Data are expressed as the mean ± standard deviation (n = 5); * indicate p < 0.05.
Fig. 3. Effect of resistance enhancement on the X-ray patterns of potato starch. (a) potato resistant starch and (b) standard potato starch.
3.1.2. RS contents The RS content in potato starch was 11.54% before treatment, but increased to 27.09% after microwave, toughening, and aging treatment. Microwave heating partly degrades amylopectin, and rapid rise of temperature and heating of the starch molecules between the hydrogen bond breakage molecular chain separation, starch particles swelling and gelatinization (Shi & Gao, 2011). Hydrothermal treatment further rearranged the toughened crystalline starch molecules, and the starch was recrystallized by aging after low-temperature treatment. Therefore, the anti-digestive capacity was increased and enhanced the RS content.
with loose branches separating from the main chain. The starch chains recombined into more stable amylose after low-temperature aging. The α-1,6-glycosidic bonds are destroyed by microwave heating, which removes branches from the main chain and, therefore, increases the amylose content (Chung, Liu, & Hoover, 2009). The fragments in Fig. 2(c) were formed by the recrystallization of starch branches. Diffraction peaks at about 2θ = 13°, 16°, 19°, and 34° were attributed to short-chain linear molecules in Fig. 3. As branching of the starch chain is reduced, the digestion site of the digestive enzyme in the human body is reduced. Therefore, increasing the amylose content can control starch digestibility.
Fig. 2. Morphologies after resistance enhancement of (a,b) potato starch granules with ×200 and ×400 magnification, respectively, and (c,d) potato resistance starch granules with ×200 and ×400 magnification, respectively. 302
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3.2. Scanning electron microscopy (SEM)
Table 2 Effects of different starches on postprandial blood glucose in mice.
The SEM analysis of granules of native and treated starch is shown in Fig. 2. The native potato starch granules showed smooth surfaces and irregular polygonal shapes with no evidence of cracks by SEM. However, after treatment using the different methods, the granular shapes had changed. The surface of the general signs is intuitive indicators of treated potato starch (TPS) samples appeared to have an irregular, rough, and nonuniform morphology with relatively loose structures. The results in Fig. 2 indicate that microwave–toughening treatment (MTT) had a strong effect on the starch granules, breaking some of them down into smaller pieces, while the starch shell reformed into sheets (Van, Huong, Phi, & Tien, 2017). In the case of microwave heating, this was due to the starch rapidly expanding and swelling and then recrystallizing after aging. The irregular crystal structure was relatively dense and could effectively resist enzymatic hydrolysis to realize an increased resistance (Zavareze, Storck, Castro, Schirmer, & Dias, 2010).
Postprandial glucose (mmol/L) 0 min MTT starch (I) Potato starch (II) Corn starch (III) RS4 (IV) Glucose (V)
30 min
6.2 ± 1.20
a
60 min
8.11 ± 1.12
b
8.72 ± 1.94
120 min b
6.62 ± 2.43b
6.07 ± 0.81a
8.62 ± 1.53c
9.47 ± 1.35d
6.95 ± 1.53a
6.72 ± 3.10a
9.2 ± 4.22b
10.07 ± 4.05b
7.42 ± 3.91a
5.87 ± 0.91a 8.57 ± 1.72d
7.28 ± 1.32b 17.82 ± 1.36c
7.83 ± 1.91b 12.97 ± 1.22c
6.32 ± 1.94a 9.97 ± 0.81a
Each result represents the mean ± standard deviation (n = 10); different letters indicate significant differences (P < 0.05) between groups.
3.3. X-ray diffraction pattern and relative crystallinity X-ray diffraction patterns of the treated starches are shown in Fig. 3. There were three different types of crystal structure present: A-type, Btype, and C-type starch crystallites. Potato starch contains B-type starch crystallites. Compared with ordinary starch, the diffraction peak of RS still showed the B-type structure, with no significant change. The TPS Xray diffraction patterns were characteristic of B-type crystallites, with diffraction peaks at approximately 2θ = 13°, 16°, and 19°. Furthermore, as shown in Fig. 3, TPS samples had strong reflections at 5°, 24°, and 26°. This implied similar molecular orientation and packing of the double helices of native starch and MTT starch samples. Meanwhile, the degree of crystallinity increased from 32.43% to 51.36% after MTT, showing that the degree of crystallinity can improve starch anti-enzymolysis under certain conditions. Fig. 4. Effects of intake of various starches on mouse weight gain. Each result represents the mean ± standard deviation (n = 10); different letters indicate significant differences (P < 0.05) between groups. LFC, low-fat corn starch; HFC, high-fat corn starch; HFP, high-fat potato starch; HFR, high-fat potato resistant starch.
3.4. Thermal and thermogravimetric analyses Differential scanning calorimetry (DSC) was used to assess thermodynamic changes in the starch during gelatinization. The initial temperature (To), peak temperature (Tp), termination temperature (Tc), and enthalpy (ΔH) were obtained by measuring the heat absorbed in the sample gelatinization process. As destruction of the solid crystal structure requires more energy, the heat enthalpy represents the degree of difficulty for the sample to react. The thermal parameters of native and TPS were analyzed using DSC and are summarized in Table 1. Table 1 shows that, compared with the original starch, the ΔH of the potato starch was significantly increased, which indicated that the TPS had a more stable crystal structure, which was consistent with the XRD results. The To and Tc of native potato starch were 55.9 °C and 67.9 °C, respectively. After resistance enhancement, the To, Tp, and Tc had changed to 54.3 °C, 69.8 °C, and 77.6 °C, respectively. This showed the wider gelatinization temperature range. Amylose formed a double helix structure and the starch particles were recrystallized by heat treatment. As the crystallinity improved, a significant endothermic change was observed when RS was thermally analyzed.
Table 3 Effect of the intake of various starches on the fasting blood glucose level of mice. Fasting glucose (mmol/L) Pre-experiment LFC (A) HFC (B) HFP (C) HFR (D) RS4 (E)
8.32 8.23 8.54 8.11 8.03
± ± ± ± ±
Post-experiment
a
8.86 ± 0.82b 10.80 ± 1.02d 11.42 ± 0.96e 9.20 ± 0.32c 8.72 ± 0.56b
0.77 0.33a 0.88a 0.54a 0.45a
Each result represents the mean ± standard deviation (n = 10); different letters indicate significant differences (P < 0.05) between groups.
Table 1 Thermal parameters of native and resistance-enhanced potato starch. Samples Native starch MTT starch
To (°C)
Tp (°C) a
55.9 ± 0.24 54.3 ± 0.18b
Tc (°C) a
63.2 ± 0.55 69.8 ± 0.32b
△H (J/g)
Tc–To (°C) a
67.9 ± 0.42 77.6 ± 0.11b
a
12.0 ± 0.24 23.3 ± 0.55b
DG (%) a
8.1 ± 0.08 89.2 ± 0.84b
Each result represents the mean ± standard deviation (n = 5); different letters indicate significant differences (P < 0.05) between groups. 303
– 90.9 ± 0.48
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Table 4 Effect of the intake of various starches on the organ indexes of mice. Organ index (%) Abdominal fat LFC (A) HFC (B) HFP (C) HFR (D) RS4 (E)
2.91 4.91 6.52 2.89 3.12
± ± ± ± ±
a
0.45 0.84c 0.82d 0.34a 0.65b
Liver 3.72 3.79 3.87 3.58 3.88
Spleen ± ± ± ± ±
a
0.21 0.35a 0.37b 0.65a 0.48b
0.23 0.21 0.17 0.21 0.24
± ± ± ± ±
Renal a
0.08 0.10a 0.07a 0.07a 0.85a
1.11 1.32 0.94 1.12 1.53
± ± ± ± ±
Heart b
0.12 0.11b 0.09a 0.23b 0.09b
Brain a
0.46 ± 0.02 0.49 ± 0.02a 0.35 ± 0.09a 0.4 ± 0.03a 0.57 ± 0.01a
1.47 1.13 1.12 1.23 1.38
± ± ± ± ±
Brown fat b
0.08 0.07a 0.12a 0.08a 0.09b
0.35 0.38 0.24 0.31 0.51
± ± ± ± ±
0.01a 0.01a 0.03a 0.02a 0.01a
Each result represents the mean ± standard deviation (n = 10); different letters indicate significant differences (P < 0.05) between groups. LFC is low-fat corn starch; HFC is high-fat corn starch; HFP is high-fat potato starch; HFR is high-fat potato-resistant starch.
Fig. 5. Effect of intake of various starches on the HE staining of mouse liver (×200 magnification). (A) Low-fat corn starch, (B) high-fat corn starch, (C) high-fat potato starch, (D) high-fat potato resistant starch, and (E) phytic acid-modified starch.
blood glucose in group IV was the slowest, showing a more moderate trend, because the diet contained only a small amount of common starch, most of which was RS, resulting in a gentle and rapid decreased following a slight increase. Furthermore, the blood glucose of group I was between those of groups II and IV. This indicated that the RS content in treated potato starch was higher and the antienzymolysis ability was improved, along with digestion and absorption. Therefore, the maintained blood sugar level was low. The glycemic index of mice in groups IV, II, and III was 23.35 ± 6.02, 48.35 ± 8.66, and 45.21 ± 7.14, respectively, with a glucose curve of 100. Group I had a
3.5. Effect of TPS on glycemic index The effects of different starches on postprandial blood glucose in mice are shown in Table 2. Table 2 shows that each group of mice had a fasting blood glucose of 5.7–6.7 mmol/L, and that peak starch blood glucose was reached 30–60 min after gavage, while the blood glucose levels had generally dropped back to the fasting level after 120 min. Mice in group III showed the highest peak and an increasing amplitude of blood glucose due to corn starch containing starch that can be more rapidly digested and absorbed to increase the blood glucose. The rise in 304
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Fig. 6. Effect of intake of various starches on HE staining of mouse abdominal adipose (×200 magnification). (A) Low-fat corn starch, (B) high-fat corn starch, (C) high-fat potato starch, (D) high-fat potato resistant starch, and (E) phytic acid-modified starch.
controlled the weight of the mice, and a large amount of RS can also have anti-digestive and regulatory effects on weight control. However, the weight gain of mice in group D was lower than those in groups C and B (P < 0.05), and higher than those in groups A and E (P < 0.05), indicating that TPS could control the mouse body weight, as regulated by dietary fiber RS and controlling the intestinal microbes.
glycemic index of 32.64 ± 11.25, which was slightly higher than that of positive control group IV, but lower than those of groups II and III (P < 0.05). 3.6. Effect of TPS on body weight Before the experiment, the mice were shiny, energetic, and had good appetites. As the feeding time was extended, the mice of groups B and C appeared obese, with dull hair, a lack of movement, gathering together, frequent micturition, and so on. Group D also showed dull hair, a loss of appetite, and other phenomena, but remained lively. Groups A and E had smooth hair smooth, remained lively, and showed a normal performance state. The average intake of each mouse was 2.5 g/ d, and the energy intake of each group was relatively uniform. Changes in the body weight of mice over six weeks are shown in Fig. 4. The body weight of each group increased over time. Among them, groups C and B showed the fastest weight gain. This was because the high fat diet, containing 40% fat, can provide a lot of heat, while 30% of starch consumption is used to provide energy, resulting in fat accumulation in the mice and the body weight of the two groups increasing significantly. The body weight of mice in groups A and E was not significantly different, and the increase was significantly smaller than that in groups C and B (P < 0.01). The lower calorie diet
3.7. Effects of TPS on fasting blood glucose Changes in the blood glucose for each experimental group before and after feeding are shown in Table 3. After six weeks of feeding, the fasting blood glucose levels of each group were higher than those before feeding. The difference between groups B and C was significant. There was no significant difference in the fasting blood glucose before and after intervention for groups A and E. Compared with groups B and C, group D effectively controlled postprandial blood glucose. This showed that MTT potato starch effectively controlled the blood sugar level through long-term intake. 3.8. Effects of TPS on organ indices The effects of different diets on the mouse visceral index are shown in Table 4. The different starch feeds had a significant impact on the 305
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Acknowledgements
visceral index. The abdominal fat indexes of groups B and C were significantly higher than those of groups A, D, and E (P < 0.05). The liver index of group D was lower than that of other groups (P < 0.05). There were no significant differences among the liver indexes of the groups. The spleen, kidney, and heart indexes of group C were significantly lower than those of the other groups (P < 0.05), indicating visceral atrophy or growth obstruction during feeding for group C. The liver index of group D was significantly lower than that of group A, with no significant differences in the other organ indexes. There was no significant difference among the cardiac indexes of each group (P > 0.05). This indicated that MTT potato starch feed effectively controlled the health of mouse organs.
This work was supported by the Science and Technology Major Project of Shandong Province [grant number 2015ZDZX05005], and the Major Agricultural Application Technology Innovation in Shandong, P.R.China. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.carbpol.2018.03.076. References
3.9. Effects of TPS on splanchnic tissues Arner, E., Westermark, P. O., Spalding, K. L., Britton, T., Ryden, M., Frisen, J., et al. (2010). Adipocyte turnover: Relevance to human adipose tissue morphology. Diabetes, 59(1), 105–112. Arns, B., Bartz, J., Radunz, M., Evangelho, J. A. D., Pinto, V. Z., & Zavareze, E. D. R. (2015). Impact of heat-moisture treatment on rice starch, applied directly in grain paddy rice or in isolated starch. LWT – Food Science and Technology, 60(2), 708–713. Chung, H. J., Liu, Q., & Hoover, R. (2009). Impact of annealing and heat-moisture treatment on rapidly digestible, slowly digestible and resistant starch levels in native and gelatinized corn, pea and lentil starches. Cabohydrate Polymers, 75(3), 436–447. Ciecko, Z., & Zolnowski, A. (2004). The effect of NPK fertilization on tuber yield and starch content in potato tubers. Annales UMCS, 59(4), 399–406. Deng, Y. J., Zhang, Y. P., Yang, Q. H., Han, L., Liang, Y. J., He, Y. F., et al. (2016). Effects of berberine on hepatic sirtuin 1–uncoupling protein 2 pathway in non–alcoholic fatty liver disease rats induced by high–fat diet. Chinese Herbal Medicines, 8(4), 359–365. Dong, S. (2012). Characteristics and antioxidant activity of hydrolyzed β–lactoglobulin–glucose Maillard reaction products. Food Research International, 46(01), 55–61. Englyst, H. N., Kingman, S. M., & Cummings, J. H. (1992). Classification and measurement of nutritionally important starch fractions. European Journal of Clinical Nutrition, 46, 333–350. Fuentes-Zaragoza, E., Riquelme-Navarrete, M. J., Sánchez-Zapata, E., & Viuda-Martos, M. (2010). Resistant starch as functional ingredient: A review. Food Research International, 43(04), 931–942. Goñi, I., García-Diz, L., Mañas, E., & Saura-Calixto, F. (1996). Analysis of resistant starch: A method for foods and food products Source. Food Chemistry, 56(4), 445–449. Heltoft, P., Wold, A. B., & Molteberg, E. L. (2017). Maturity indicators for prediction of potato (solanum tuberosum, l.) quality during storage. Postharvest Biology & Technology, 129, 97–106. Jang, J. K., & Pyun, Y. R. (1997). Effect of moisture level on the crystallinity of wheat starch aged at different temperatures. Starch – Stärke, 49(07), 272–277. Jecht, M. (2013). Latent autoimmune diabetes of adults in China. Der Diabetologe, 9(2), 149–150. Karim, A., Holmes, M., & Orfila, C. (2016). Inhibitory effect of chlorogenic acid on digestion of potato starch. Food Chemistry, 58(8), 498–504. Kim, J. S., & Lee, Y. S. (2009). Antioxidant activity of Maillard reaction products derived from aqueous glucose/glycine, diglycine, and triglycine model systems as a function of heating time. Food Chemistry, 116(01), 227–232. Kondo, Y., Higashi, C., Iwama, M., Ishihara, K., & Handa, S. (2012). Bioavailability of vitamin C from mashed potatoes and potato chips after oral administration in healthy Japanese men. British Journal of Nutrition, 107(6), 885. Kurilich, A. C. (2013). The role of potatoes and potato components in cardiometabolic health: A review. Annals of Medicine, 45(7), 467. Maziarz, M. P., Preisendanz, S., Juma, S., Imrhan, V., Prasad, C., & Vijayagopal, P. (2017). Resistant starch lowers postprandial glucose and leptin in overweight adults consuming a moderate–to–high–fat diet: A randomized–controlled trial. Nutrition Journal, 16(01), 14. Mutungi, C., Rost, F., Onyango, C., Jaros, D., & Rohm, H. (2009). Crystallinity, thermal and morphological characteristics of resistant starch type iii produced by hydrothermal treatment of debranched cassava starch. Starch – Stärke, 61(11), 634–645. Nara, S., & Komiya, T. (1983). Studies on the relationship between water-satured state and crystallinity by the diffraction method for moistened potato starch. Starch – Stärke, 35(12), 407–410. Paola, R. D. D., Asis, R., & Aldao, M. A. J. (2003). Evaluation of the degree of starch gelatinization by a new enzymatic method. Starch – Starke, 55(9), 403–409. Patil, S. K. (2004). Resistant starches as low–carb ingredients – Current applications and issues. Cereal Foods World, 49, 292–294. Pham, V. H., & Nguyen, T. H. (2015). My and Nguyen Thi Lan Phi. Impact of acid and heat?moisture treatment combination on physicochemical characteristics and resistant starch contents of sweet potato and yam starches. Starch – Stärke, 66(11), 1013–1021. Rodrigues, A., & Emeje, M. (2012). Recent applications of starch derivatives in nanodrug delivery. Carbohydrate Polymers, 87(2), 987–994. Sharma, M., Yadav, D. N., Singh, A. K., & Tomar, S. K. (2015). Effect of heat–moisture treatment on resistant starch content as well as heat and shear stability of pearl millet starch. Agricultural Research, 4(04), 411–419. Shi, M. M., & Gao, Q. Y. (2011). Physicochemical properties, structure and in vitro
To observe the effect of TPS on the mouse liver, the liver tissue was observed by sectioning and HE staining, and the results are shown in Fig. 5. The liver tissue of high-fat groups B and C showed more serious cytoplasm swelling, nuclear condensation, local cell necrosis, and tissue inflammation than other groups. Similar variability has been reported previously (Deng et al., 2016) for rat livers. Groups D and E also showed mild cytoplasm swelling, nuclear condensation, and inflammation in some areas. The liver tissue of group A was relatively complete and had a normal cell structure without damage and lesions. Furthermore, a large number of fat vacuoles in the liver tissue were dissolved by alcohol during staining of the group C liver, and to a lesser extent in the liver tissue of group B. This suggested that mice fed with potato and corn starch high-fat diets showed a more severe fatty liver. Compared with groups B and C, the liver tissue of group D had less fat vacuoles and showed good health. Excessive food intake can alter the hepatic metabolic balance drastically, which can precipitate fatty liver disease, causing fat accumulation in the liver (Suzuki, Shinjo, Arai, Kanai, & Goda, 2014). Therefore, MTT potato starch had a significant effect on liver protection and controlling fatty livers in mice. The abdominal subcutaneous fat of the mice was also observed in the same experiment, as shown in Fig. 6. The results showed that subcutaneous abdominal adipocytes in group C were severely damaged and that cytoplasm atrophy was accompanied by massive cell necrosis. This indicated that the adipose tissue of the mice was damaged by a high-fat potato starch feed. The adipose tissue of mice in groups A, B, and E maintained a completely regular cell morphology. Adipose tissue may contain few large adipocytes (hypertrophy) or many small adipocytes (hyperplasia) (Arner et al., 2010). Too high an energy intake leads to hypertrophy and hyperplasia of fat cells, and the size of fat cells can also be associated with insulin secretion, with insulin and fat cell hypertrophy being negatively correlated. Group D was partially deformed and the cytoplasm was swollen, but maintained the integrity of fat cells better than group C. 4. Conclusions This study aimed to provide useful insight for research in the potato industry. Microwave–toughening treatment (MTT) starch samples were prepared and examined for their RS formation, structural properties, and efficacy of treated potato starch (TPS). After treatment, the amylose and RS contents were improved to 35.06% and 27.09%, respectively. This study found that the TPS had a low glycemic index, and was beneficial for controlling fasting blood glucose, body weight, organ index, and fatty liver, owing to the improved crystalline performance and thermal stability. Long-term dietary intervention in mice showed the sensitivity of mice to increased insulin. Therefore, the TPS prepared can be used to produce staple foods and special medical foods a low glycemic index for patients with diabetic nephropathy. Our research not only reduces the cost of industrial production and uses easily controlled technology, but can also solve the problem of potato storage through deep starch processing. 306
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University of Strathclyde PhD Thesis. Van, H. P., Huong, N. T., Phi, N. T., & Tien, N. N. (2017). Physicochemical characteristics and in vitro digestibility of potato and cassava starches under organic acid and heatmoisture treatments. International Journal of Biological Macromolecules, 95, 299. Yadav, B. S., Guleria, P., & Yadav, R. B. (2013). Hydrothermal modification of Indian water chestnut starch: Influence of heat–moisture treatment and annealing on the physicochemical, gelatinization and pasting characteristics. LWT – Food Science and Technology, 53(1), 211–217. Yang, W. H., & Rao, M. A. (1998). Complex viscosity–temperature mastercurve of cornstarch dispersion during gelatinisation. Journal of Food Process Engineering, 21(3), 191–207. Yang, G., Yang, B., & Ding, X. L. (2008). Effects of microwave irradiation on formation of resistant starch. Food Science, 10, 38–46. Yuan, H. B., Wang, W. J., Chen, D. Y., Zhu, X. P., & Cao, S. Q. (2015). Characterization of resistant starch from Se–rich rice flour and its anti–diabetic effect in diabetic ICR mice. Starch – Stärke, 68, 106–111. Zavareze, E. R., Storck, C. R., Castro, L. A. S., Schirmer, M. A., & Dias, A. R. G. (2010). Effect of heat-moisture treatment on rice starch of varying amylose content. Food Chemistry, 121(2), 358–365.
digestion of resistant starch from waxy rice starch. Cabohydrate Polymers, 84(3), 1151–1157. Sjöqvist, M., & Gatenholm, P. (2005). The effect of starch composition on structure of foams prepared by microwave treatment. Journal of Polymers & the Environment, 13(1), 29–37. Song, J., Park, J., & Shin, M. (2015). The effects of annealing and acid hydrolysis on resistant starch level and the properties of cross-linked rs4 rice starch. Starch – Stärke, 63(3), 147–153. Sun, Q., Nan, C., Dai, L., & Xiong, L. (2015). Effect of heat-moisture treatment with maltitol on physicochemical properties of wheat starch. LWT-Food Science and Technology, 62, 319–324. Suzuki, T., Shinjo, S., Arai, T., Kanai, M., & Goda, N. (2014). Hypoxia and fatty liver. World Journal of Gastroenterology, 20(41), 15087–15097. Taguet, A., Bureau, M. N., Huneault, M. A., & Favis, B. D. (2014). Toughening mechanisms in interfacially modified hdpe/thermoplastic starch blends. Carbohydrate Polymers, 114(114), 222–229. Takeda, Y., Shitaozono, T., & Hizukuri, S. (1990). Structures of sub-fractions of corn amylose. Carbohydrate Research, 199(2), 207–214. Tester, R. F. (1989). The swelling and gelatinisation properties of cereal starchesGlasgow:
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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Efficacy of potato resistant starch prepared by microwave–toughening treatment
T
You-Dong Lia,b,1, Tong-Cheng Xua,b,1, Jun-Xia Xiaoc, Ai-Zhen Zonga,b, Bin Qiua,b, Min Jiaa,b, ⁎ ⁎ Li-Na Liua,b, , Wei Liua,b, a
Institute of Agro-Food Science and Technology, Shandong Academy of Agricultural Sciences, Jinan 250100, PR China Key Laboratory of Agro-Products Processing Technology of Shandong Province, No. 202, Gongyebei Road, Jinan 250100, PR China c School of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Potato Resistant starch Microwave treatment Glycemic index Efficacy evaluation
Potato starch was treated by microwaving, toughening, and low-temperature aging to prepare resistant starch (RS). The functional properties of the resultant RS were evaluated and the effects of this microwave–toughening treatment (MTT) on the amylose content, RS content, digestive properties, pasting properties, morphological observation, crystal structure, and thermal performance of potato starch were determined. The optimal MTT parameters were microwaving at 300 W for 100 s, toughening at 55 °C for 16 h, and low-temperature aging at 4 °C for 18 h. After MTT, the amylose and RS contents of potato starch had increased from 26.08% and 11.54% to 35.06% and 27.09%, respectively. Furthermore, the pasting temperature increased from 66.8 °C to 76.36 °C, while the peak viscosity, trough viscosity, and final viscosity decreased significantly. After MTT, the potato starch surface had also changed significantly, and the crystallinity had increased from 32.43% to 51.36%. MTT starch had beneficial effects on fasting blood glucose, body weight, and organ index in mice. Furthermore, it had a protective effect on subcutaneous abdominal fat and liver tissue.
1. Introduction
amylopectin is a larger branched molecule with α-(1,4)- and α-(1,6)linkages (Yuan, Wang, Chen, Zhu, & Cao, 2015). Starch can be divided into three types according to digestion rate, namely, rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) (Englyst, Kingman, & Cummings, 1992). RDS can be completely digested and absorbed in the human small intestine, causing an increase in the blood glucose level (Pham & Nguyen, 2015). To control the sudden increase in blood sugar, pancreatic islets rapidly secrete insulin, causing the blood sugar levels to drop sharply. And long-term RDS intake can result in insulin resistance and type 2 diabetes (Kim & Lee, 2009). SDS is slowly digested in the human small intestine, which has potential health benefits for the blood sugar level and islets (Dong, 2012). RS is not digested, but fermented in the large intestine by human microflora to produce short-chain fatty acids. This causes a decrease in the intestinal pH value, which helps maintain intestinal peristalsis and protect intestinal mucosa (Fuentes-Zaragoza, Riquelme-Navarrete, Sánchez-Zapata, & Viuda-Martos, 2010). As RS has been shown to be effective at preventing diabetes and obesity, an increasing amount of research has focused on improving the RS content in functional foods (Patil, 2004).
The potato is a widely cultivated cash crop and the most prevalent food crop worldwide, providing energy to about 15% of the global population (Karim, Holmes, & Orfila, 2016). Potatoes are generally used to make popular consumer products, such as potato chips and mashed potato (Kondo, Higashi, Iwama, Ishihara, & Handa, 2012). Potato storage is problematic owing to shortcomings in technology, facilities, and industrial structure (Heltoft, Wold, & Molteberg, 2017). Potatoes are nutritious, containing starch (9–20%), protein (1.5–2.3%), fat (0.1–1.1%), crude fiber (0.6–0.8%), polyphenols (0.0071–0.031%), and other trace components (Kurilich, 2013). The saponin found in potato green skin dry powder can be used to control hyperglycemia (Jecht, 2013). The main ingredient in potatoes is starch, of which ∼80% is contained in the dry matter (Ciecko & Zolnowski, 2004). Potato starch is not only important in food, but also in pharmaceutical, chemicals, cosmetics, and textiles (Rodrigues & Emeje, 2012). Starch is composed of amylose and amylopectin. Amylose is a linear D-glucopyranose chain connected by α-l,4-glycosidic bonds that is slightly branched (Takeda, Shitaozono, & Hizukuri, 1990), while
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1
Corresponding authors at: Institute of Agro-Food Science and Technology, Shandong Academy of Agricultural Sciences, Jinan 250100, PR China. E-mail addresses: [email protected] (L.-N. Liu), [email protected] (W. Liu). These authors contributed equally to this work and should be considered co-first authors.
https://doi.org/10.1016/j.carbpol.2018.03.076 Received 5 December 2017; Received in revised form 20 March 2018; Accepted 22 March 2018 Available online 23 March 2018 0144-8617/ © 2018 Elsevier Ltd. All rights reserved.
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Analysis Technology Co., Ltd. (Beijing, China). Heat-stable α-amylase (1400 U/g) and amyloglucosidase (3300 U/g) were purchased from Sigma–Aldrich (St. Louis, USA). Maltose standard product was purchased from Aladdin Reagent Co., Ltd. (Shanghai, China).
RS can be divided into four types according to their origin, namely, physical embedding starch (RS1), resistant starch granules (RS2), aging of regenerated starch (RS3), and chemically modified starch (RS4) (Maziarz et al., 2017). Chemical modification is the most common method for enhancing RS content. Recently, many techniques have been studied for enhancing the RS content in starch, including hydrothermal, enzymatic hydrolysis, acid hydrolysis, and ultrasound methods. Arns et al. (2015) increased the RS content of wheat starch to 42.1% using a heat treatment method, which was then modified by Sun et al. (2015) to achieve further improvements. This improved RS content was due to starch gelatinization and regeneration. When starch is hydrothermally treated, the amylose double helix is destroyed (hydrogen bond dissociation) and amylopectin detaches from the main chain (Tester, 1989). These events, collectively referred to as “gelatinization”, are accompanied by a sharp increase in system viscosity, such as the gradual collapse of the grain structure (Yang & Rao, 1998). At low temperatures, recrystallization of the polymer chains causes degradation (Jang & Pyun, 1997). Mutungi, Rost, Onyango, Jaros, ànd Rohm (2009) improved the tapioca RS content from 21.4% to 67.3% by debranching with pullulanase, recrystallizing the linear dextran by incubation at 60 °C, and finally using heat-moisture treatment to broaden the endotherms and increased their enthalpies. Song, Park, and Shin (2015) increased the RS level by 114.5% through acid hydrolysis (pH 4.0) for 1 h under sonication. Sonication reduced the starch particle size and starch granules remained the same as in native starch, with a polygonal shape and A-type crystallinity after treatment. Microwave treatment could replace the traditional heat treatment process. Microwaves can induce the swelling stage of starch gelatinization and then the low temperature can age the starch. The molecular chains recombine through hydrogen bonds to form new crystals (Sjöqvist & Gatenholm, 2005). The toughening of starch is a physical modification under the action of heat and water. It refers to the starch modified in excess moisture (> 60% w/w) or moisture (40%–55% w/ w) and below starch gelatinization temperature of the initial temperature conditions for a period of time caused by changes in starch structure and properties. Toughening can reduce the degree of solubility and swelling, reduce the viscosity, and increase the paste temperature of starch, and does not produce harmful chemicals or cause genetic changes (Taguet, Bureau, Huneault, & Favis, 2014). During the toughening process, the starch crystals rearrange internally to increase the crystallinity, making them more resistant to enzymolysis (Yadav, Guleria, & Yadav, 2013). Microwave and toughening methods are safe and easy to perform, but few studies have applied this method to resistant starch production. The present study aims to use to improve the RS content in potato starch, as the raw material, through microwave and toughening technology, and observe the effect of microwave toughening on the starch structure characteristics and efficacy. Changes in eating habits and food composition have resulted in the incidence of chronic diseases, such as diabetes and cardiovascular diseases, increasing in recent years, with more people tending to consume low-calorie high-fiber foods for health purposes. The microwave and toughening techniques, and products of potato starch obtained from this study, have broad market prospects. These raw materials could be used to prepare diabetes-specific staple foods, and inspire new applications and development of potato starch in functional foods. Furthermore, this technology could promote industrial restructuring and ease problems associated with potato storage.
2.2. Preparation of resistant starch 2.2.1. Preparation of microwave–toughening treatment (MTT) starch Potato resistant starch (P-RS) was prepared according to methods of Yang, Yang, and Ding (2008) and Sharma, Yadav, Singh, and Tomar (2015) with some modifications. Potato starch (100 g) was gelatinized with distilled water (220 mL) by microwaving at 300 W for 100 s (microwave process conducted in water bath at 45 °C; final temperature of water–starch solution, 80.4 °C). The supernatant was removed by centrifugation (3000 rpm, 15 min) and the sediment was washed thoroughly three times with distilled water. The sample moisture content was then adjusted to 60% (w/v) by spraying with the appropriate calculated amount of distilled water. The starch and water were then thoroughly mixed, placed in sealed containers, and heated on a constant-temperature shaking table at 55 °C for 16 h. The containers were then cooled to ambient temperature, opened, and then stored in a refrigerator at 4 °C for 18 h. The obtained starch was dried at 45 °C for 24 h, ground, and passed through a 100-mesh sieve for further analysis. 2.2.2. RS determination RS was determined according to the procedure of Goñi, García-Diz, Mañas, and Saura-Calixto (1996) with a slight modification. Treated potato starch (TPS) (0.5 g) was digested with excess amylase for 30 min. The reaction mixture was then centrifuged at 8000 rpm for 8 min. The resultant residue was collected and dispersed in 2 M KOH solution (6 mL) and stirred for 30 min at ambient temperature. The pH was adjusted to neutral using a citrate buffer solution and then adjusted to 4.4 using a 2 N HCl solution. Excess amyloglucosidase was added and the mixture was heated in a water bath at 60 °C for 45 min. The samples were centrifuged (8000 rpm, 8 min), and the supernatants were collected and made up to 100 mL using distilled water. The reducing sugar content was determined using the dinitrosalicylic acid (DNS) method. 2.3. Scanning electron microcopy (SEM) The surface structures and cross-sections of treated potato starch (TPS) and potato starch (PS) fractions were observed using field-emission scanning electron microscopy (FESEM). Starch samples were mounted on circular aluminum stubs with double sided carbon tape, coated with a thin platinum film under vacuum, and examined using FESEM (Supra 55VP microscope, Carl Zeiss, Oberkochen, Germany) at an accelerating potential of 3 kV. A starch granule cross section was prepared using a stainless steel razor blade approximately 2-μm thick (ST 300, Dorco Co., Seoul, Korea). 2.4. X-ray diffraction X-ray diffraction analysis was performed using an X-ray diffractometer (Model D5005, Bruker, Karlsruhe, Germany) operating at 40 kV and 40 mA producing CuK radiation with a wavelength of 1.54 Å scanning through the 2θ range of 3–30° and with a step time of 4 s. The relative crystallinity of the starches was calculated according to a previous report (Nara & Komiya, 1983) using peak-fitting software (Origin version 7.5, OriginLab, Northampton, MA, USA)
2. Materials and methods
2.5. Differential scanning calorimetry–thermogravimetric analysis (DSC–TGA)
2.1. Materials Potato starch (purity, 96.9%) was obtained from Gansu Mintian Food Co., Ltd. (Gansu, China). A blood glucose meter and blood glucose test strips was purchased from Roche Diagnostics GmbH (Mannheim, Germany). Amylose standard was purchased from Beijing Spectrum
The gelatinization characteristics and thermogravimetric properties of the products were measured using DSC–TGA (SDT Q600, TA Instruments, USA). Anhydrous starch samples (approximately 5 mg) 300
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experiment. The weight and food intake of the mice were recorded once a week.
were added to an aluminum DSC pan, which was then sealed, reweighed, and heated from 25 to 600 °C at 10 °C/min. An empty pan was used as a reference. The degree of gelatinization (DG) was calculated according to a literature method (Paola, Asis, & Aldao, 2003). The gelatinization enthalpy (DH) of the native and modified starch samples was used to calculate the DG using the following equation：
2.6.3.1. Changes in fasting blood glucose. The fasting blood glucose was measured after 12 h without water and feed. Specifically, scissors disinfected with medical alcohol were used to cut the mouse tail tip and the formed blood drops were quickly placed into the middle of the blood glucose meter test cassette top test area so that they were touching the detection zone. The test results were recorded after a few seconds. The test results and fasting blood glucose were measured again after six weeks.
DG (%) = (1 − ΔHg/ΔHt) × 100 where ΔHt and ΔHg are the gelatinization enthalpies of modified and native starch samples, respectively.
2.6.3.2. Effect of TPS on general signs and body weight of mice. The general signs and body weight of mice are intuitive indicators of TPS. During the experiment, the food intake and body weight of each group of mice were measured by weighing weekly, and the hair, feces, and mental state of each group were observed.
2.6. Animal experiments 2.6.1. Efficacy study of TPS Animal experiments were divided into two groups. The first group was a short-term experiment in which the effect of TPS on the glycemic index in mice was observed by gavage, and calculated using the area under the curve. The second group was a long-term experiment that explored the effect of TPS on mice on a high fat diet. Different starches were added to the diet to observe the effect of TPS on mouse symptoms through long-term feeding. The efficacy of TPS was evaluated by combining two experiments to provide a more robust scientific basis for the hypoglycemic effect of TPS. Fifty male Kunming mice (weight, 25 ± 2 g) were purchased from the Animal Experimental Center of Shandong University. During experiments, the mice were fed in an approved laboratory animal facility and maintained under controlled temperature (22–24 °C) and lighting (12-h light/dark cycle) for a 14-day adaptation period with a normal diet and weekly change of bedding and water. Fifty male C57BL/6J (B6) mice (SPF mouse; weight, 22 ± 2 g) were purchased from Nanjing Biomedical Research Institute of Nanjing University. Each squirrel cage of mice was independently ventilated. The mice were fed in an approved laboratory animal facility and maintained under controlled temperature (22–24 °C) and lighting (12-h light/dark cycle) for a 14-day adaptation period with a normal diet and weekly change of bedding and water. The experimental protocol was approved by the Animal Experiment Committee of China, in accordance with the National Institute of Health Guidelines for the Care and Use of Laboratory Animals.
2.6.3.3. Determination of visceral organ index in mice. The mice were sacrificed after six weeks of feeding and the abdominal fat, liver, spleen, kidney, heart, brain, and brown fat were removed. The surface connective tissue was quickly separated in normal saline and weighed using the analytical balance after removing water. The organ index was calculated using the following formula: Organ index (%) = Organ weight (g)/Mouse weight (g) × 100
2.6.3.4. Observation of visceral tissue morphology. The abdominal subcutaneous fat and liver were weighed and placed in formalin, and a section of viscera was observed by hematoxylin–eosin (HE) staining. The test method involved a dozen steps, such as tissue fixation, drawing materials, dehydration, transparence, waxing, embedded, dressing wax, section, patch, baking sheet, wax removal, water addition, hematoxylin staining, differentiation, washing, back blue, eosin staining, dehydration, transparence, and seal sheet. The required sample size was 1.5 × 1.5 × 0.3 cm. When immersed in wax from low to high immersion, the temperature could not be higher than 65 °C. The baking sheet was treated at 65 °C for 20–30 min, the hematoxylin staining time needed to be controlled at 5–15 min, and hydrochloric acid differentiation within 30 s. Euphorbia staining took 1–3 min, and a drop of glacial acetic acid was added, which can play a role in promoting dye. With different concentrations of alcohol dehydration, xylene fully transparent with gum fragments.
2.6.2. Postprandial blood glucose of mice Ten animals (Kunming mice) per cage and five groups (groups I–V) were used. The mice in group I were given TPS, group II were given potato starch, group III were given corn starch, group IV were given RS4 (resistant starch type 4), and group V were given glucose by gavage. The water and diet were cut off 12 h before the experiment, and the fasting blood glucose of the mice was measured using a blood glucose meter by cutting their tails (as described in section 2.6.3.1). All mice received intragastric administration at a dose of 0.2 mL/10 g, and blood glucose was recorded within 2 h after intragastric administration to plot a blood glucose curve. The glycemic index was calculated as follows:
2.7. Statistical analyses Origin 8.5 (Origin Lab Corporation, USA) statistical software was used for statistical analysis. All experimental data were expressed as the means ± standard deviation (SD). Differences between the test subjects and model controls were evaluated using Student’s t-test. 3. Results and discussion 3.1. Preparation of microwave–toughening treatment (MTT) starch
GI (glycemic index) = (2 h curve area for starch)/(2 h curve area for glucose) × 100
3.1.1. Amylose and amylopectin contents The changes in amylose and amylopectin contents are shown in Fig. 1. Potato starch had an amylose content of 26.08% and amylopectin content of 73.92% before processing. After microwaving and toughening treatment, the amylose content had increased significantly to 35.06%. Amylose is a linear polymer composed of α-D-1,4-glycosidic bonds, while amylopectin has a highly branched bundle structure, with the main chain linked by α-1,4-glycosidic bonds, and the branched chains linked to the main chain via α-1,6-glycosidic bonds. Amylopectin is barely soluble in water, but easily gelatinized in hot water,
2.6.3. Effect of TPS on mouse signs in a long-term experiment Ten animals (C57BL/6J) per cage and five groups (groups A–E) were used. Each squirrel cage of mice was independently ventilated and all feed and water was sterilized. The mice in group A were fed a low-fat diet with corn starch, group B were fed a high-fat diet with corn starch, group C were fed a high-fat diet with potato starch, group D were fed a high-fat diet with MTT starch, and group E were fed a high-fat diet with RS4. The mice were fed a normal diet for two weeks before the 301
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Fig. 1. Effect of resistance enhancement on amylose and amylopectin contents in potato starch. Data are expressed as the mean ± standard deviation (n = 5); * indicate p < 0.05.
Fig. 3. Effect of resistance enhancement on the X-ray patterns of potato starch. (a) potato resistant starch and (b) standard potato starch.
3.1.2. RS contents The RS content in potato starch was 11.54% before treatment, but increased to 27.09% after microwave, toughening, and aging treatment. Microwave heating partly degrades amylopectin, and rapid rise of temperature and heating of the starch molecules between the hydrogen bond breakage molecular chain separation, starch particles swelling and gelatinization (Shi & Gao, 2011). Hydrothermal treatment further rearranged the toughened crystalline starch molecules, and the starch was recrystallized by aging after low-temperature treatment. Therefore, the anti-digestive capacity was increased and enhanced the RS content.
with loose branches separating from the main chain. The starch chains recombined into more stable amylose after low-temperature aging. The α-1,6-glycosidic bonds are destroyed by microwave heating, which removes branches from the main chain and, therefore, increases the amylose content (Chung, Liu, & Hoover, 2009). The fragments in Fig. 2(c) were formed by the recrystallization of starch branches. Diffraction peaks at about 2θ = 13°, 16°, 19°, and 34° were attributed to short-chain linear molecules in Fig. 3. As branching of the starch chain is reduced, the digestion site of the digestive enzyme in the human body is reduced. Therefore, increasing the amylose content can control starch digestibility.
Fig. 2. Morphologies after resistance enhancement of (a,b) potato starch granules with ×200 and ×400 magnification, respectively, and (c,d) potato resistance starch granules with ×200 and ×400 magnification, respectively. 302
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3.2. Scanning electron microscopy (SEM)
Table 2 Effects of different starches on postprandial blood glucose in mice.
The SEM analysis of granules of native and treated starch is shown in Fig. 2. The native potato starch granules showed smooth surfaces and irregular polygonal shapes with no evidence of cracks by SEM. However, after treatment using the different methods, the granular shapes had changed. The surface of the general signs is intuitive indicators of treated potato starch (TPS) samples appeared to have an irregular, rough, and nonuniform morphology with relatively loose structures. The results in Fig. 2 indicate that microwave–toughening treatment (MTT) had a strong effect on the starch granules, breaking some of them down into smaller pieces, while the starch shell reformed into sheets (Van, Huong, Phi, & Tien, 2017). In the case of microwave heating, this was due to the starch rapidly expanding and swelling and then recrystallizing after aging. The irregular crystal structure was relatively dense and could effectively resist enzymatic hydrolysis to realize an increased resistance (Zavareze, Storck, Castro, Schirmer, & Dias, 2010).
Postprandial glucose (mmol/L) 0 min MTT starch (I) Potato starch (II) Corn starch (III) RS4 (IV) Glucose (V)
30 min
6.2 ± 1.20
a
60 min
8.11 ± 1.12
b
8.72 ± 1.94
120 min b
6.62 ± 2.43b
6.07 ± 0.81a
8.62 ± 1.53c
9.47 ± 1.35d
6.95 ± 1.53a
6.72 ± 3.10a
9.2 ± 4.22b
10.07 ± 4.05b
7.42 ± 3.91a
5.87 ± 0.91a 8.57 ± 1.72d
7.28 ± 1.32b 17.82 ± 1.36c
7.83 ± 1.91b 12.97 ± 1.22c
6.32 ± 1.94a 9.97 ± 0.81a
Each result represents the mean ± standard deviation (n = 10); different letters indicate significant differences (P < 0.05) between groups.
3.3. X-ray diffraction pattern and relative crystallinity X-ray diffraction patterns of the treated starches are shown in Fig. 3. There were three different types of crystal structure present: A-type, Btype, and C-type starch crystallites. Potato starch contains B-type starch crystallites. Compared with ordinary starch, the diffraction peak of RS still showed the B-type structure, with no significant change. The TPS Xray diffraction patterns were characteristic of B-type crystallites, with diffraction peaks at approximately 2θ = 13°, 16°, and 19°. Furthermore, as shown in Fig. 3, TPS samples had strong reflections at 5°, 24°, and 26°. This implied similar molecular orientation and packing of the double helices of native starch and MTT starch samples. Meanwhile, the degree of crystallinity increased from 32.43% to 51.36% after MTT, showing that the degree of crystallinity can improve starch anti-enzymolysis under certain conditions. Fig. 4. Effects of intake of various starches on mouse weight gain. Each result represents the mean ± standard deviation (n = 10); different letters indicate significant differences (P < 0.05) between groups. LFC, low-fat corn starch; HFC, high-fat corn starch; HFP, high-fat potato starch; HFR, high-fat potato resistant starch.
3.4. Thermal and thermogravimetric analyses Differential scanning calorimetry (DSC) was used to assess thermodynamic changes in the starch during gelatinization. The initial temperature (To), peak temperature (Tp), termination temperature (Tc), and enthalpy (ΔH) were obtained by measuring the heat absorbed in the sample gelatinization process. As destruction of the solid crystal structure requires more energy, the heat enthalpy represents the degree of difficulty for the sample to react. The thermal parameters of native and TPS were analyzed using DSC and are summarized in Table 1. Table 1 shows that, compared with the original starch, the ΔH of the potato starch was significantly increased, which indicated that the TPS had a more stable crystal structure, which was consistent with the XRD results. The To and Tc of native potato starch were 55.9 °C and 67.9 °C, respectively. After resistance enhancement, the To, Tp, and Tc had changed to 54.3 °C, 69.8 °C, and 77.6 °C, respectively. This showed the wider gelatinization temperature range. Amylose formed a double helix structure and the starch particles were recrystallized by heat treatment. As the crystallinity improved, a significant endothermic change was observed when RS was thermally analyzed.
Table 3 Effect of the intake of various starches on the fasting blood glucose level of mice. Fasting glucose (mmol/L) Pre-experiment LFC (A) HFC (B) HFP (C) HFR (D) RS4 (E)
8.32 8.23 8.54 8.11 8.03
± ± ± ± ±
Post-experiment
a
8.86 ± 0.82b 10.80 ± 1.02d 11.42 ± 0.96e 9.20 ± 0.32c 8.72 ± 0.56b
0.77 0.33a 0.88a 0.54a 0.45a
Each result represents the mean ± standard deviation (n = 10); different letters indicate significant differences (P < 0.05) between groups.
Table 1 Thermal parameters of native and resistance-enhanced potato starch. Samples Native starch MTT starch
To (°C)
Tp (°C) a
55.9 ± 0.24 54.3 ± 0.18b
Tc (°C) a
63.2 ± 0.55 69.8 ± 0.32b
△H (J/g)
Tc–To (°C) a
67.9 ± 0.42 77.6 ± 0.11b
a
12.0 ± 0.24 23.3 ± 0.55b
DG (%) a
8.1 ± 0.08 89.2 ± 0.84b
Each result represents the mean ± standard deviation (n = 5); different letters indicate significant differences (P < 0.05) between groups. 303
– 90.9 ± 0.48
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Table 4 Effect of the intake of various starches on the organ indexes of mice. Organ index (%) Abdominal fat LFC (A) HFC (B) HFP (C) HFR (D) RS4 (E)
2.91 4.91 6.52 2.89 3.12
± ± ± ± ±
a
0.45 0.84c 0.82d 0.34a 0.65b
Liver 3.72 3.79 3.87 3.58 3.88
Spleen ± ± ± ± ±
a
0.21 0.35a 0.37b 0.65a 0.48b
0.23 0.21 0.17 0.21 0.24
± ± ± ± ±
Renal a
0.08 0.10a 0.07a 0.07a 0.85a
1.11 1.32 0.94 1.12 1.53
± ± ± ± ±
Heart b
0.12 0.11b 0.09a 0.23b 0.09b
Brain a
0.46 ± 0.02 0.49 ± 0.02a 0.35 ± 0.09a 0.4 ± 0.03a 0.57 ± 0.01a
1.47 1.13 1.12 1.23 1.38
± ± ± ± ±
Brown fat b
0.08 0.07a 0.12a 0.08a 0.09b
0.35 0.38 0.24 0.31 0.51
± ± ± ± ±
0.01a 0.01a 0.03a 0.02a 0.01a
Each result represents the mean ± standard deviation (n = 10); different letters indicate significant differences (P < 0.05) between groups. LFC is low-fat corn starch; HFC is high-fat corn starch; HFP is high-fat potato starch; HFR is high-fat potato-resistant starch.
Fig. 5. Effect of intake of various starches on the HE staining of mouse liver (×200 magnification). (A) Low-fat corn starch, (B) high-fat corn starch, (C) high-fat potato starch, (D) high-fat potato resistant starch, and (E) phytic acid-modified starch.
blood glucose in group IV was the slowest, showing a more moderate trend, because the diet contained only a small amount of common starch, most of which was RS, resulting in a gentle and rapid decreased following a slight increase. Furthermore, the blood glucose of group I was between those of groups II and IV. This indicated that the RS content in treated potato starch was higher and the antienzymolysis ability was improved, along with digestion and absorption. Therefore, the maintained blood sugar level was low. The glycemic index of mice in groups IV, II, and III was 23.35 ± 6.02, 48.35 ± 8.66, and 45.21 ± 7.14, respectively, with a glucose curve of 100. Group I had a
3.5. Effect of TPS on glycemic index The effects of different starches on postprandial blood glucose in mice are shown in Table 2. Table 2 shows that each group of mice had a fasting blood glucose of 5.7–6.7 mmol/L, and that peak starch blood glucose was reached 30–60 min after gavage, while the blood glucose levels had generally dropped back to the fasting level after 120 min. Mice in group III showed the highest peak and an increasing amplitude of blood glucose due to corn starch containing starch that can be more rapidly digested and absorbed to increase the blood glucose. The rise in 304
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Fig. 6. Effect of intake of various starches on HE staining of mouse abdominal adipose (×200 magnification). (A) Low-fat corn starch, (B) high-fat corn starch, (C) high-fat potato starch, (D) high-fat potato resistant starch, and (E) phytic acid-modified starch.
controlled the weight of the mice, and a large amount of RS can also have anti-digestive and regulatory effects on weight control. However, the weight gain of mice in group D was lower than those in groups C and B (P < 0.05), and higher than those in groups A and E (P < 0.05), indicating that TPS could control the mouse body weight, as regulated by dietary fiber RS and controlling the intestinal microbes.
glycemic index of 32.64 ± 11.25, which was slightly higher than that of positive control group IV, but lower than those of groups II and III (P < 0.05). 3.6. Effect of TPS on body weight Before the experiment, the mice were shiny, energetic, and had good appetites. As the feeding time was extended, the mice of groups B and C appeared obese, with dull hair, a lack of movement, gathering together, frequent micturition, and so on. Group D also showed dull hair, a loss of appetite, and other phenomena, but remained lively. Groups A and E had smooth hair smooth, remained lively, and showed a normal performance state. The average intake of each mouse was 2.5 g/ d, and the energy intake of each group was relatively uniform. Changes in the body weight of mice over six weeks are shown in Fig. 4. The body weight of each group increased over time. Among them, groups C and B showed the fastest weight gain. This was because the high fat diet, containing 40% fat, can provide a lot of heat, while 30% of starch consumption is used to provide energy, resulting in fat accumulation in the mice and the body weight of the two groups increasing significantly. The body weight of mice in groups A and E was not significantly different, and the increase was significantly smaller than that in groups C and B (P < 0.01). The lower calorie diet
3.7. Effects of TPS on fasting blood glucose Changes in the blood glucose for each experimental group before and after feeding are shown in Table 3. After six weeks of feeding, the fasting blood glucose levels of each group were higher than those before feeding. The difference between groups B and C was significant. There was no significant difference in the fasting blood glucose before and after intervention for groups A and E. Compared with groups B and C, group D effectively controlled postprandial blood glucose. This showed that MTT potato starch effectively controlled the blood sugar level through long-term intake. 3.8. Effects of TPS on organ indices The effects of different diets on the mouse visceral index are shown in Table 4. The different starch feeds had a significant impact on the 305
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Acknowledgements
visceral index. The abdominal fat indexes of groups B and C were significantly higher than those of groups A, D, and E (P < 0.05). The liver index of group D was lower than that of other groups (P < 0.05). There were no significant differences among the liver indexes of the groups. The spleen, kidney, and heart indexes of group C were significantly lower than those of the other groups (P < 0.05), indicating visceral atrophy or growth obstruction during feeding for group C. The liver index of group D was significantly lower than that of group A, with no significant differences in the other organ indexes. There was no significant difference among the cardiac indexes of each group (P > 0.05). This indicated that MTT potato starch feed effectively controlled the health of mouse organs.
This work was supported by the Science and Technology Major Project of Shandong Province [grant number 2015ZDZX05005], and the Major Agricultural Application Technology Innovation in Shandong, P.R.China. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.carbpol.2018.03.076. References
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To observe the effect of TPS on the mouse liver, the liver tissue was observed by sectioning and HE staining, and the results are shown in Fig. 5. The liver tissue of high-fat groups B and C showed more serious cytoplasm swelling, nuclear condensation, local cell necrosis, and tissue inflammation than other groups. Similar variability has been reported previously (Deng et al., 2016) for rat livers. Groups D and E also showed mild cytoplasm swelling, nuclear condensation, and inflammation in some areas. The liver tissue of group A was relatively complete and had a normal cell structure without damage and lesions. Furthermore, a large number of fat vacuoles in the liver tissue were dissolved by alcohol during staining of the group C liver, and to a lesser extent in the liver tissue of group B. This suggested that mice fed with potato and corn starch high-fat diets showed a more severe fatty liver. Compared with groups B and C, the liver tissue of group D had less fat vacuoles and showed good health. Excessive food intake can alter the hepatic metabolic balance drastically, which can precipitate fatty liver disease, causing fat accumulation in the liver (Suzuki, Shinjo, Arai, Kanai, & Goda, 2014). Therefore, MTT potato starch had a significant effect on liver protection and controlling fatty livers in mice. The abdominal subcutaneous fat of the mice was also observed in the same experiment, as shown in Fig. 6. The results showed that subcutaneous abdominal adipocytes in group C were severely damaged and that cytoplasm atrophy was accompanied by massive cell necrosis. This indicated that the adipose tissue of the mice was damaged by a high-fat potato starch feed. The adipose tissue of mice in groups A, B, and E maintained a completely regular cell morphology. Adipose tissue may contain few large adipocytes (hypertrophy) or many small adipocytes (hyperplasia) (Arner et al., 2010). Too high an energy intake leads to hypertrophy and hyperplasia of fat cells, and the size of fat cells can also be associated with insulin secretion, with insulin and fat cell hypertrophy being negatively correlated. Group D was partially deformed and the cytoplasm was swollen, but maintained the integrity of fat cells better than group C. 4. Conclusions This study aimed to provide useful insight for research in the potato industry. Microwave–toughening treatment (MTT) starch samples were prepared and examined for their RS formation, structural properties, and efficacy of treated potato starch (TPS). After treatment, the amylose and RS contents were improved to 35.06% and 27.09%, respectively. This study found that the TPS had a low glycemic index, and was beneficial for controlling fasting blood glucose, body weight, organ index, and fatty liver, owing to the improved crystalline performance and thermal stability. Long-term dietary intervention in mice showed the sensitivity of mice to increased insulin. Therefore, the TPS prepared can be used to produce staple foods and special medical foods a low glycemic index for patients with diabetic nephropathy. Our research not only reduces the cost of industrial production and uses easily controlled technology, but can also solve the problem of potato storage through deep starch processing. 306
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