- Email: [email protected]
Synthesis and physico-chemical characterization of modified starches from banana (Musa AAB) and its biological activities in diabetic rats
International Journal of Biological Macromolecules 94 (2017) 500–507
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Synthesis and physico-chemical characterization of modified starches from banana (Musa AAB) and its biological activities in diabetic rats Chagam Koteswara Reddy a,b,∗∗ , M. Suriya a , P.V. Vidya a , Sundaramoorthy Haripriya a,∗ a b
Department of Food Science and Technology, Pondicherry University, Puducherry 605014, India School of Life Sciences and Biotechnology, Korea University, Seoul 136701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 30 June 2016 Received in revised form 25 July 2016 Accepted 17 October 2016 Available online 18 October 2016 Keywords: Amylose Banana Diabetes Modification Pullulanase Resistant starch
a b s t r a c t This study describes a simple method of preparation and physico-chemical properties of modified starches (type-3 resistant starches) from banana (Musa AAB), and the modified starches investigated as functional food with a beneficial effect on type-2 diabetes. RS3 was prepared using a method combined with debranching modification and physical modification; native and modifies starches were characterized by scanning electron microscope (SEM), powder X-ray diffraction (XRD), differential scanning calorimetry (DSC) and rapid visco analyzer (RVA). Use of the enzymatic and physical modification methodology, improved the yield of RS (26.62%) from Musa AAB. A reduced viscosity and swelling power; increased transition temperatures, water absorption capacity and solubility index with B-type crystalline pattern and loss of granular appearance were observed during the debranching modification and physical modification. The modified starches exhibited beneficial health effects in diabetic and HFD rats who consumed it. These results recommend that dietary feeding of RS3 was effective in the regulation of glucose and lipid profile in serum and suppressing the oxidative stress in rats under diabetic and HFD condition. This current study provides new bioactive starches, with potential applications in the food and non-food industries. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Now-a-days, the occurrence of obesity related problems are on the rise due to the modern life style, consumption of excess dietary fat and reduction in physical activities [1,2]. Obesity related issues also lead to complications like hyperlipidemia [3], non-alcoholic fatty liver disease [4], various cardiovascular diseases [2], and diabetes [5] in human beings. In general, diabetes is a form of metabolic disorder, which occurs due to the dietary intake of excess carbohydrates and lipids. In particular, type 2 diabetes mellitus (T2DM) is a common endocrine and metabolic disease [6]. T2DM is caused by an absolute or relative lack of insulin in the blood [7], resulting in metabolic abnormalities such as obesity, hypertension, low levels of high-density lipoprotein (HDL-C), elevated triglyceride (TG) levels, hyperglycemia and resistance to insulin [4]. In diabetes, major
∗ Corresponding author at: Department of Food Science and Technology, Pondicherry University, Puducherry 605014, India. ∗∗ Co-corresponding author at: School of Life Sciences and Biotechnology, Korea University, Seoul 136701, Republic of Korea. E-mail addresses: [email protected] (C.K. Reddy), [email protected] (S. Haripriya). http://dx.doi.org/10.1016/j.ijbiomac.2016.10.050 0141-8130/© 2016 Elsevier B.V. All rights reserved.
health problems are oxidative damage [8], dysfunction and eventual organ failure [9,10]. With improvement in social and economic environment in developed and developing countries, the occurrence of diabetes (T2DM) has rapidly increased over the years. Apart from genetic reasons, the dietary pattern of a person plays a key role in the occurrence of metabolic syndrome. A major reason could be the increased influence of western diet consumption which has an excess fat content in addition to poor minerals and fibre [11]. Due to the increased occurrence of diabetes in humans, current research is focused on the development of drugs for treatment and control of T2DM. Various drugs have been developed for the treatment and control of T2DM; however, the long-term usage of anti-diabetic drugs results into considerable side effects with the symptoms of hypoglycemia and malfunction of kidney and liver [7,10,12,13]. As medication cannot possibly have an alternative for treatment of T2DM, the focus should towards the prevention or delayed onset of diabetes by exploring the functional adjuncts responsible for it. Resistant starch (RS) is a new functional ingredient with a low glycemic index which is often employed in the development of functional food products. RS could be defined as the fraction of starch, which escapes digestion in the small intestine and undergoes fermentation in the large intestine in the presence of
C.K. Reddy et al. / International Journal of Biological Macromolecules 94 (2017) 500–507
microflora resulting into formation of short chain fatty acids, especially butyric acid [14,15]. RS can act as a dietary fiber with similar physiological effects [16]. Several reports suggested that the RS exhibits significant effect on physiological functions such as body weight management [17], prevention of gastrointestinal diseases [18], treatment of hyperglycemia and hypercholesterolemia, and enhancement of mineral absorption [19,20]. RS obtained various sources such as cereals (corn, rice and wheat), tubers (yam and potato) and pulses are widely used in the production of functional food products; and also various methods are involved in the preparation of RS from various sources including physical [7], chemical [2] and enzymatic [21] modification. Augmented the utility of RS with wider applications in food and non-food industries has increased the focus of research for the development of the RS from various food sources. Banana, is a common edible fruit of the genus Musa encompassed in the family Musaceae. Currently, worldwide production of banana is around 140 million tons, because of its extensive cultivation in tropical and sub-tropical regions of World. India ranks first in banana production and ‘Poovan’ (Musa AAB) cultivar is abundantly grown in Southern regions due to its higher adaptability to climatic conditions and cropping patterns [22,23]. The unripe bananas are highly consumed because of their high starch content. Due to the presence of high amount of starch, bananas are of great concern in the field of starch research. However, limited studies are available for the production of RS from Musa AAB using pullulanase enzyme and followed by its applications. From this basis, the objective of the present study was to the synthesis of resistant starch (RS3) from Musa AAB using a method of debranching modification followed by autoclaving and retrogradation; and evaluate their physico-chemical characteristics of native and modified starches, including morphological, physico-chemical, crystalline and thermal properties. Their biological activities (hypoglycemic and lipid lowering effects) of modified starches were also analysed using diabetes (T2DM) and obese rats fed with HFD. 2. Materials and methods 2.1. Materials Unripe poovan banana (Musa AAB) cultivar used in the study was obtained from the local harvest in the State of Tamil Nadu, India. Only unripe bananas were used in the study which did not have even small traces of yellow colour on its peel. Pullulanase from Bacillus acidopullulyticus (Promozyme 400 L) and Streptozotocin (STZ) were procured from Sigma-Aldrich Company, USA. Resistant starch assay kit was obtained from Megazyme International Ireland Limited, Ireland. Reagent kits of glucose, total cholesterol, TG, HDL-C, LDL-C, glutamate oxaloacetate transaminase (SGOT), glutamate pyruvate transaminase (SGPT) and alkaline phosphatase (ALP) were purchased from AGGAPE-diagnostics Pvt. Ltd, Kerala, India. All other chemicals and reagents were analytical grade. 2.2. Preparation of native starch Native starches from unripe poovan banana were extracted by Reddy et al. [22] with slight modifications as described. Raw poovan banana was peeled, washed and macerated for 2 to 3 min with sodium sulphite solution (1.22 g/L) using a waring hand blender (Prestige Hand Blender PHB 5.0, Bangalore, India). Homogenate was passed through 60 and 100 U.S mesh sieves using slurry and water (1:4 ratio). The filtrate was allowed to stand for 2 h at room temperature. After that supernatant was decanted off and then starch slurry was centrifuged (Eppendorf centrifuge, Model 5804, Mumbai, India) at 3000 rpm for 30 min. The white starch sediments were
501
dried in a hot air oven at 40 ◦ C for overnight, and ground to pass a sieve (100 U.S) for further analyses. 2.3. Preparation of resistant starch 2.3.1. Debranching of starch Debranching of native banana starch was performed based on the method described in Reddy et al. [21] with a slight modification. Starch sample (10 g) and 100 mL of sodium acetate buffer (0.1 M and pH 5.3) was mixed with pullulanase (40 U/g dry starch) and incubated in shaking water bath (Remi RSB-12, Mumbai, India) at 60 ◦ C for 10 h. The sample was heated in boiling water bath for 10 min to inactivate the enzyme. The starch gelatinization prior to enzymatic hydrolysis was performed with the sample in boiling water bath for 10 min, before adding the enzyme. 2.3.2. Preparation of resistant starch The starch samples, retrograded enzymatically hydrolysed native starch (REHNS) and retrograded enzymatically hydrolysed gelatinized starch (REHGS) in suspensions (10%, w/w dwb) were autoclaved at 121 ◦ C for 30 min, cooled to 4 ◦ C and stored at this temperature for 24 h. The samples were then freeze dried. In the samples, the RS content was determined using a Megazyme resistant starch assay kit with the description of Association of Official Analytical Chemists (AOAC) 2002.02. 2.4. Morphological characteristics The structure of the native and modified starch granules was observed by scanning electron microscope (SEM, HITACHI, S3400N, Tokyo, Japan). The powdered sample was sprinkled on double sided sticky tape placed on aluminium stubs and covered with carbon coating layer, observed, and photographed. 2.5. Colour and amylose content Colour of the native and modified starches was measured using Hunter Lab Colorimeter (D-25, Hunter Lab Associates Inc.) after standardisation using Hunter Lab colour standards. L* (lightness), a* (redness to greenness) and b* (yellowness to blueness) values of native and modified starches were analysed. Amylose content of native and modified starches was determined according to the method of Williams et al. [24]. 2.6. Pasting properties The pasting properties of native and modified starches were investigated with a Rapid Visco-Analyzer (RVA starch master 2, Newport Scientific, Warriewood, NSW, Australia) according to method of Reddy et al. [25]. 2.7. Water absorption capacity, solubility index and swelling power The water absorption capacity (WAC), water solubility index (WSI) and swelling power (SP) of native and modified starches were analysed respectively, according to the methods described by Reddy et al. [21]. 2.8. X-ray diffraction Powder X-ray diffraction (XRD) analysis of the native and the modified starches was carried out on powder X-ray diffractometer (Shimadzu XRD 7000) with Cu K␣ value of 1.54 radiation at step count of 2◦ /min, with a 2 range of 10◦ to 50◦ using a voltage of 40 kV and filament current 30 mA.
502
C.K. Reddy et al. / International Journal of Biological Macromolecules 94 (2017) 500–507
Fig. 1. Scanning electron micrographs of native and modified starches.
2.9. Thermal properties The gelatinization temperatures of native and modified starches were measured with a differential scanning colorimeter (TA-Q20 DSC) according to the methods of Reddy et al. [25]. 2.10. Animals experiments 2.10.1. Experimental animals Male Sprague Dawley rats weighing 130–150 g and of 7–8 weeks age were obtained from Sri Ragavendra Enterprises, Bangalore, India. The animals were housed in polypropylene cages in a legalised laboratory animal facility with controlled temperature (∼24 ◦ C) and lighting (12 h day/night cycle) for a 7-day adaptation period. They were given the normal pellet diet (NPD) and drinking water ad libitum. The experimental procedures were approved by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. This present animal study was approved by Institutional Animal Ethical Committee (IAEC), Pondicherry University (PU/SLS/IAEC/2014/16), India. 2.10.2. Streptozotocin-induced diabetic rats The control group of rats were given a regular diet and the experimental group of rats were fed with a HFD (36.5 g of NPD, 31 g of lard oil, 25 g of casein, 1 g of cholesterol, 0.3 g of methionine, 6 g of vitamin and mineral mix, 0.1 g of yeast and 0.1 g of NaCl). After 4 weeks of HFD feeding, the experimental group rats were injected with STZ (35 mg/kg, dissolved in 0.01 M sodium citrate buffer, pH 4.4) intraperitoneally, while the control group rats were injected with the vehicle citrate buffer. The experimental animals which showed fasting blood glucose levels >140 mg/dl were categorised as diabetic. After adaptation period and STZ injection, rats showing elevated fasting blood glucose levels were randomly dispersed to different treatment groups. 2.10.3. Experimental design The animals were separated into 5 groups (n = 7) and treated for 8 weeks as follows. Rats in Group 1 (control group) were given NPD
for 8 weeks. The Group 2 rats were treated with HFD for 4 weeks followed by the STZ injection (35 mg/kg bw) and continued with the HFD for 4 more week (T2DM model). Group 3 rats were induced to T2DM as in group 2 and treated with the RS (30 g/100 g diet) for 4 weeks. Group 4 rats were given the HFD alone for 8 weeks without induction of diabetes in order to assess the biochemical changes associated with obesity. Group 5 rats were given HFD for the first 4 weeks followed by HFD diet with RS (30 g/100 g diet) for further 4 weeks. At the end of study, animals were kept fasted for overnight, anaesthetized and blood samples were collected with the abdominal aorta without heparin for serum. Organs like liver, pancreas, spleen, kidney and heart were excised, rinsed with cold isotonic saline and then stored at −80 ◦ C for biochemical analysis. 2.10.4. Estimation of serum glucose and lipid profile Serum glucose and lipid profiles (total cholesterol, TG, HDL-C and LDL-C) were examined by enzymatic colorimetric methods using commercially available kits (AGGAPE-diagnostics). From the experimental values of TG, the other cholesterol carrying lipoprotein (VLDL-C) were calculated using the following standard formula [26]. VLDL − C =
Triglycerides 5
2.10.5. Assessment of diagnostic enzymes of tissue damage in serum The levels of the SGOT, SGPT and ALP in serum were analysed using commercially available kits (AGGAPE-diagnostics). 2.10.6. Assessment of biochemical parameters in liver Glycogen content in liver was quantified according to the method of Sullivan et al. [27], TC and TG contents were examined by enzymatic colorimetric methods using commercially available kits (AGGAPE-diagnostics).
C.K. Reddy et al. / International Journal of Biological Macromolecules 94 (2017) 500–507
503
Table 1 Colour, amylose and resistant starch content of native and modified starches. Parameter
Colour parameters L* a* b* Amylose (%) Resistant starch (%)
Type NPBS
REHNS
REHGS
92.57 ± 0.16a 3.88 ± 0.43b 7.87 ± 0.33b 23.10 ± 1.12c 11.05 ± 0.45c
89.45 ± 0.19b 6.41 ± 0.44a 11.41 ± 0.41a 31.11 ± 1.35b 22.01 ± 0.24b
90.03 ± 0.15b 6.11 ± 0.21a 10.87 ± 0.59a 40.46 ± 0.95a 26.62 ± 0.37a
Values expressed are mean ± standard deviation. Means in the row with different superscript are significantly different at p ≤ 0.05.
2.10.7. Estimation of oxidative stress markers The enzymatic endogenous antioxidants such as superoxide dismutase (SOD) [28] and catalase [29]; non-enzymatic endogenous antioxidant like reduced glutathione (GSH) [30]; and oxidative stress marker such as lipid peroxidation (LPO) [31] were analysed in liver tissue homogenate (10%) using standard methods. 2.11. Statistical analysis Analytical determinations were carried out in triplicates and standard deviations were noted. The data was subjected to one way ANOVA to analyse the significant difference in all data followed by Tukey’s test using SPSS 18 software (SPSS Institute Inc., Cary, NC, USA). 3. Results and discussion 3.1. Morphological characteristics SEM images of the granules of native (NPBS) and modified starches (REHNS and REHGS) showed significant difference in their appearances (Fig. 1). NPBS granules had smooth surface with rod like and irregular oval shapes. REHNS and REHGS resembled an amorphous mass of cohesive structure, leading to the loss of granular appearance and irregular shape, as a result of the gelatinization process when the clumped starch granules form sponge like structure within the inner region of the retrograded starch [32]. Zhang and Jin [33] proposed that the variation in the structure of starch granules was due to the retrogradation of amylose chains which could have been initiated in a restructuring of the starch into a helical complex. These modification in the amorphous regions led to the rejection of the activity of alpha amylase on the retrograded starch and raised the density of crystalline structure [7,34].
Fig. 2. Pasting properties of native and modified starches.
(p ≤ 0.05) difference from REHNS (31.11%) and REHGS (40.46%). The percentage of RS in REHGS (26.62) was significantly (p ≤ 0.05) higher when compared with REHNS (22.01) and NPBS (11.05) owing to the increased amylose content in samples of REHNS and REHGS, which is the resulting retrograded starch of NPBS upon the treatment of pullulanase before and after gelatinization. Amylose content increased as RS content increased, it could be due to the breakdown of ␣ (1–6) linkage of amylopectin by pullulanase, which is changed into small chain linear polysaccharides like amylose molecules, which form strong gel network through retrogradation [35]. In general raw banana contain RS2 (granular starch) and during thermal process RS2 converts to RS3, which is due to recrystallinity of the short linear chains produced after debranching and retrogradation process [21,36]. 3.4. Pasting properties The pasting properties of native and modified starches were analysed using RVA and the results are presented in Table S1 and Fig. 2. The pasting temperature value of REHNS and REHGS are 69.4–63.3 ◦ C, respectively and is notably lesser than that of NPBS (83.06 ◦ C). Reduced pasting temperature was observed in the case of the REHNS and REHGS which indicates the destruction of starch granules during autoclaving [36]. REHNS and REHGS shown significantly (p ≤ 0.05) reduced pasting parameters including peak (819 cP & 743 cP), hold (737 cP & 264 cP), final (946 cP & 361 cP), and setback (203 cP & 69 cP) with increased breakdown (77 cP & 89 cP) viscosities when compared to what was
3.2. Colour Colour values (L*, a* and b*) are represented in Table 1 with significance (p ≤ 0.05) difference among native and modified starches. The L* value of starches ranged from 89.45 to 92.57 and the NPBS exposed maximum lightness with significant difference from REHNS and REHGS. a* and b* value of REHNS and REHGS differed significantly from NPBS and maximum a* (6.41) and b* (11.41) value observed for REHNS. These deviations in colour values may possible due to the caramelization that would have happened in small fragments; these small molecules are formed during debranching and autoclaving of starch granules. 3.3. Amylose and resistant starch content The increased levels of amylose and RS content in modified starches were observed and the obtained data are presented in Table 1. The amylose content of NPBS (23.10%) shown significant
Table 2 Water absorption capacity, solubility index and thermal properties of native and modified starches. Parameter
Type NPBS
REHNS
REHGS
WAC (%) Solubility index (%)
3.15 ± 0.13c 3.07 ± 0.05c
5.15 ± 0.16b 14.97 ± 0.65b
5.98 ± 0.12b 18.32 ± 0.98a
Thermal properties T0 (◦ C) TP (◦ C) TC (◦ C) H gel (J/g) R (◦ C)
62.08 ± 0.12c 69.63 ± 0.35c 82.35 ± 0.86c 15.06 ± 0.55c 21.04 ± 0.54c
64.39 ± 0.25b 73.61 ± 2.31b 89.51 ± 0.81b 19.39 ± 0.99b 25.11 ± 0.33b
69.32 ± 1.81a 82.27 ± 1.03a 97.91 ± 2.06a 25.83 ± 1.69a 28.59 ± 1.10a
WAC, water absorption capacity; T0 , onset temperature; TP , peak temperature; TC , conclusion temperature; Hgel, enthalpy of gelatinization; R, gelatinization range (TC -T0 ). Values expressed are mean ± standard deviation. Means in the row with different superscript are significantly different at p ≤ 0.05.
504
C.K. Reddy et al. / International Journal of Biological Macromolecules 94 (2017) 500–507
Fig. 3. (A) Swelling power and (B) X-ray diffraction patterns of native and modified starches.
observed in case of NPBS (1384 cP, 1333 cP, 1853 cP 489 cP & 4 cP). Reduced viscosity attribute could be as a result of debranching lead to increase the levels of small linear chain molecules and RS. The formation of starch gel ability reduced in REHNS and REHGS when compared with NPBS which is due to its autoclaving [37,38].
3.5. Water absorption capacity The WAC of native and modified starches shown significant (p ≤ 0.05) difference and the values ranged from 3.15 to 5.98 per cent (Table 2); and were in the following order: NPBS < REHNS ∼ REHGS. Increased WAC of REHNS and REHGS could be due to the gelatinisation caused by heating and autoclaving [35,39]. Reduced molecular weight of starch granules during debranching of starch granules lead to the formation of small molecules, which have more affinity with water molecules when compared to starch polymers [40]. The WAC depends upon the levels of intermolecular relationship among starches by forces like hydrogen and covalent bonding.
3.6. Water solubility index The WSI of modified starches (Table 2) showed an increasing trend, where the NPBS showed the least solubility. Native starch (NPBS) hardly dissolved in the water, whereas the debranched starches had short-chain molecules with high solubility. The values ranged from 3.07 to 18.32 per cent and the maximum solubility was observed in REHGS. Starch granule solubility is greatly affected by the quantity of amylose content and structure of starch granule. The increased starch solubility could be due to the variations in the molecular structure of starch [41], reduced molecular weight of starch granules and increased formation of short chain molecules from starch granules [42]. 3.7. Swelling power Swelling power is a degree of starch hydration capacity and the extent of the interaction between starch granules within the amorphous and crystalline areas. SP of native and modified starches was shown in Fig. 3A. From the results, we noted that with an increase in temperature the SP of native and modified starches was gradu-
C.K. Reddy et al. / International Journal of Biological Macromolecules 94 (2017) 500–507
505
Table 3 Effect of resistant starch on glucose and lipid profile in serum. Groups
Glucose (mg/dl)
Cholesterol (mg/dl)
Triglycerides (mg/dl)
HDL-C (mg/dl)
LDL-C (mg/dl)
VLDL-C (mg/dl)
Control HFD HFD + RS T2DM T2DM + RS
105.64 ± 8.72 152.42 ± 9.31* 111.05 ± 8.41** 506.74 ± 53.01# 332.59 ± 42.97##
47.61 ± 10.71 66.93 ± 10.42* 50.97 ± 10.19** 86.89 ± 9.32# 52.24 ± 12.71##
115.25 ± 3.52 300.56 ± 15.62* 220.00 ± 12.60** 464.89 ± 25.20# 271.45 ± 15.29##
31.82 ± 1.96 28.02 ± 0.73* 35.63 ± 0.83** 22.96 ± 1.13# 30.56 ± 0.96##
7.73 ± 1.12 50.89 ± 5.65* 33.58 ± 2.24** 76.96 ± 9.90# 22.15 ± 1.17##
22.13 ± 0.91 91.82 ± 2.36* 53.92 ± 2.33** 61.89 ± 4.28# 45.07 ± 2.60##
Values expressed are mean ± standard deviation from seven rats in each group. # Significantly different from Control, P≤0.05; ## Significantly different from T2DM, P≤0.05; * Significantly different from Control, P≤0.05; ** Significantly different from HFD, P≤0.05.
ally increased. At 50 ◦ C, the SP values ranged from 3.42 (NPBS) to 3.25 per cent (REHGS). There was no significant difference in the SP between native and modified starches until 70 ◦ C. Upon increasing the temperature to 80 ◦ C and 90 ◦ C there was a noticeable reduction of SP was observed in native and modified starches; and NPBS shown maximum SP at 90 ◦ C (12.23%), and at the same temperature the lowest SP was shown by REHGS (7.17%) and REHNS (7.98%). The swelling capacity of starch granules is predominantly influenced by the quantity of amylopectin in starch molecules [43]. Reduced the SP of modified starches may be due to the destruction of starch granules during debranching, autoclaving and retrogradation [21].
ference. After STZ injection, the levels of fasting blood glucose in the two diabetic groups (T2DM and T2DM + RS) were significantly higher than those control group, and no significant difference between STZ injected groups, this reveals that a diabetic rat model had been successfully established. However, without STZ injection, increased glucose levels were noted in HFD groups and it may be due to the consumption of excess HFD. From the results (Table 3), after 4 weeks treatment with RS3, significantly (p ≤ 0.05) declined the serum glucose levels of the diabetic (T2DM + RS) and HFD + RS groups was observed when compared with T2DM and HFD groups. These results advised that RS can regulate the carbohydrate metabolism and decrease the serum glucose levels of diabetic rats.
3.8. X-ray diffraction 3.10. Effect of resistant starch on hyperlipidemia The X-ray diffraction patterns of native and modified starches are shown in Fig. 3B. The diffractogram of NPBS showed highest peak at 17.1◦ 2 and two minor peaks at 15◦ and 23◦ 2 of diffraction angles and showed B-type crystalline pattern. The REHNS and REHGS had peaks at 16.85◦ and 16.95◦ 2 reflects the B-type crystalline pattern. These changes in the crystalline pattern of starches may be due to the process of debranching, autoclaving and retrogradation [35]. In general, retrogradation of starch at low temperature leads to the formation of B-type crystallinity [44]; and starches with B-type crystalline pattern are highly resistant to enzymatic hydrolysis. The crystalline pattern of starches could be influenced by the chain length of amylopectin, growth and cultivation conditions [45]. 3.9. Thermal properties The gelatinisation temperatures includes T0 , Tp , Tc , and enthalpy (Hgel) of NPBS were 62.08 ◦ C, 69.63 ◦ C, 82.35 ◦ C and 15.06 J/g, respectively (Table 2). REHNS and REHGS showed significantly (p ≤ 0.05) improved gelatinisation temperatures and enthalpy including T0 (64.39 ◦ C & 69.32 ◦ C), Tp (73.61 ◦ C & 82.27 ◦ C), Tc (89.51 ◦ C & 97.91 ◦ C) and enthalpy (19.39 J/g & 25.83 J/g), respectively. Amplified enthalpy was observed for REHNS and REHGS reflects the amount of double helical and crystalline order of starch; and these are influenced by ratio of amylose and amylopectin and amylopectin chain length distribution [45]. Enhanced amount of RS in REHNS and REHGS showed more thermal stability than NPBS. The discrepancy in the gelatinization temperature range (R-value) between starches may be due to the existence of crystallites of variable stability within crystalline domains of its starch granules [46]. REHGS having highest R-value followed by REHNS while the lowest value noted for NPBS. Variations in gelatinization temperatures and R-value among native and modified starches may be possible due to the difference in amylose and amylopectin content, amount of crystallinity and stability of crystal region in starch granules. 3.9.1. Effect of resistant starch on serum glucose Generally, HFD has been shown to induce insulin resistance, hyperinsulinaemia and dyslipidaemia in rats and increase the risk of developing T2DM in obese cases [47]. Initially, all rats showed similar glucose levels in serum without significant (p ≤ 0.05) dif-
Diabetes is generally accompanied by the existence of symptoms of high cholesterol [7]. Table 3 data shows the levels of serum lipid profile of rats. The total cholesterol, TG, and LDLC levels were significantly elevated, whereas the HDL-C levels significantly decreased in the STZ induced diabetic and HFD (without STZ) groups as compared to the control group. However, after 4 weeks treatment with RS3, we observed that significantly (P ≤ 0.05) decreased serum total cholesterol, TG, LDL-C, VLDL-C levels, whereas increased HDL-C in treated groups when compared with diabetic and HFD groups. These results confirmed that the modified starches (RS3) from Musa AAB had anti-hyperpidemia activity. These results are reliable with previously reported studies that confirmed the biological activity of RS4 from corn [2] and RS3 from rice [7] and yam [5]. 3.11. Effect of resistant starch on organ indices The organ indices of the rats from each group index were assessed and are shown in Table S2. The liver indices of HFD fed rats did not show any significant difference from control group; liver indices of T2DM group significantly (P ≤ 0.05) differed from control group. Liver index is a key factor in obese and hypertensive patients [48]. These findings specifies that RS can effectively control liver in obese and diabetic rats. From the results the indices of spleen, kidney and heart of HFD fed, diabetic and treated groups did not show any significant difference (P ≤ 0.05) when compared with control group. Pancreas indices of diabetic group shown significant difference (P ≤ 0.05) with control group; no significant difference from treated groups. These findings indicates that RS can effectively control diabetic liver in rats. 3.12. Assessment of diagnostic enzymes of tissue damage in serum The levels of liver marker enzymes such as SGOT, SGPT and ALP in serum of control and diabetic rats were analysed and the data are presented in Table 4. After STZ injection, the levels of liver marker enzymes in serum were significantly (P ≤ 0.05) raised in diabetic (T2DM) and hypercholesterolemic (HFD) groups when compared with control group. After 4 weeks treatment with RS3, there was
506
C.K. Reddy et al. / International Journal of Biological Macromolecules 94 (2017) 500–507
Table 4 Effect of resistant starch on liver marker enzymes in serum.
3.15. Effect of resistant starch on markers of oxidative stress in liver
Groups
SGOT (U/L)
SGPT (U/L)
ALP (U/L)
Control HFD HFD + RS T2DM T2DM + RS
108.71 ± 4.65 134.71 ± 9.52* 109.48 ± 8.49** 150.24 ± 7.63# 113.35 ± 6.32##
56.14 ± 3.99 69.63 ± 1.23* 57.63 ± 2.23** 93.92 ± 4.01# 77.52 ± 2.32##
112.41 ± 8.23 417.56 ± 8.96* 307.95 ± 14.32** 747.72 ± 11.23# 335.13 ± 9.56##
Values expressed are mean ± standard deviation from seven rats in each group. # Significantly different from Control, P ≤ 0.05; ## Significantly different from T2DM, P ≤ 0.05; * Significantly different from Control, P ≤ 0.05; ** Significantly different from HFD, P ≤ 0.05.
Table 5 Effect of resistant starch on glycogen, cholesterol and triglycerides in liver. Groups
Glycogen (g/100 g)
Cholesterol (mg/dl)
Triglycerides (mg/dl)
Control HFD HFD + RS T2DM T2DM + RS
2.72 ± 0.05 2.95 ± 0.15* 2.77 ± 0.15 0.53 ± 0.08# 1.48 ± 0.06##
75.80 ± 10.71 185.49 ± 10.42* 169.08 ± 10.19** 270.06 ± 9.32# 172.25 ± 12.71##
275.50 ± 23.45 428.05 ± 33.55* 315.21 ± 36.12** 691.23 ± 35.12# 330.29 ± 24.15##
Values expressed are mean ± standard deviation from seven rats in each group. # Significantly different from Control, P ≤ 0.05; ## Significantly different from T2DM, P ≤ 0.05; * Significantly different from Control, P ≤ 0.05; ** Significantly different from HFD, P ≤ 0.05.
a significant effect on liver marker enzymes in serum; the levels of SGOT, SGPT and ALP were significantly (P ≤ 0.05) decreased in serum of treated groups, and these values are comparable with control group.
3.13. Effect of resistant starch on glycogen content in liver In general, liver is an insulin sensitive organ which shows a key role in the regulation of glucose homeostasis by storing glycogen. During diabetes, under a state of hepatic insulin resistance the capacity of insulin to regulate the glucose production from the liver is reduced which in turn lead to the expression of hyperglycemia [49,50]. The content of glycogen in liver of diabetic and HFD fed rats were analysed and the results is shown in Table 5. During diabetes, the decreased level of glycogen content was observed in diabetic (T2DM) group, shows significant (P ≤ 0.05) difference against control group. After 4 weeks of treatment with RS3 from poovan banana starch exhibited a significant effect on diabetic group, whereas increased the levels of glycogen content in liver of treated group (T2DM + RS).
3.14. Effect of resistant starch on cholesterol and triglyceride levels in liver The levels of cholesterol and TG in liver of diabetic and HFD fed rats was analysed and the data are shown in Table 5. During diabetes, significantly (P ≤ 0.05) elevated the levels of cholesterol and TG were observed in diabetic (T2DM) and HFD fed groups when compared with control group. After four weeks of treatment with RS3 there was a significant change in the diabetic group, and lead to reduce the levels of cholesterol and triglycerides in liver of treated groups (T2DM + RS and HFD + RS). Reduced cholesterol and TG in liver designates that the hypocholesterolemic effect of RS may be due to the interference of intestinal cholesterol and bile acid absorption [51].
Enhanced the formation of free radicals and impaired antioxidant defence system result in oxidative stress, which in turn plays a significant role in the pathogenesis of diabetes and its complications [52]. To maintain oxidative stress and normalise the destructive potential of free radicals, the cells have established a highly complex antioxidant protection system, which includes antioxidant enzymes such as SOD, GSH, Catalase, and that catalyse free radicals-quenching reactions [53]. The levels of oxidative stress markers such as SOD, catalase, GSH and TBARS in liver of control and diabetic group rats were analysed and the resulted data shown in Table S3. From the results, it could be noted that significantly (P ≤ 0.05) increased the levels of lipid peroxidation (LPO) and decreased levels of antioxidants such as SOD, catalase and GSH were noted in liver of diabetic and HFD fed rats. In the diabetic group and HFD fed rats, treated with RS3 from poovan banana starch showed positive significant observations along with raised levels of SOD, catalase, GSH and reduced TBARS (lipid peroxidation) in liver of diabetic group. These finding demonstrated that there was severe oxidation reaction in hyperlipidemia rats and the modified starches (RS3) improved the in-vivo antioxidative defense status of the rats; it could lead to decreased oxidative stress under HFD condition. 4. Conclusion The present study demonstrates that by debranching with pullulanase and employing the autoclaving and retrogradation techniques the extraction of RS3 from Musa AAB starch can be brought about in an efficient manner. Use of the pullulanase, increases the yield of RS3 and amylose content in the native banana (Musa AAB) starch. The results suggest RS3 shows highest thermal stability and reduced viscosity when compared with native starch. Which is one of the desirable functional property of starch in food formulations. The RS3 had beneficial health effects in diabetic rats. The results exhibited that dietary feeding of RS3 was effective in the regulation of glucose and lipid profile in serum and suppressing oxidative stress in rats under diabetic and HFD condition. Finally, RS3 from poovan banana starch showed hypoglycemic effect and it can be used as functional ingredient in food product development. Acknowledgements The authors is grateful to the help rendered by technologists and technicians of SEM, DSC, and powder XRD and other necessary instruments at the Central Instrumentation Facility, Pondicherry University, Puducherry. This project is financially supported by the Department of Food Science and Technology, Pondicherry University, Pondicherry, India. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2016. 10.050. References [1] M.-N. Woo, S.-H. Bok, M.-K. Lee, H.-J. Kim, S.-M. Jeon, G.-M. Do, S.K. Shin, T.Y. Ha, M.-S. Choi, J. Med. Food 11 (2008) 169–178. [2] K.Y. Lee, S.H. Yoo, H.G. Lee, Starch-Stärke 64 (2012) 78–85. [3] M.-N. Woo, S.-H. Bok, M.-S. Choi, Food Chem. Toxicol. 47 (2009) 2076–2082. [4] K.R. Coenen, A.H. Hasty, Am. J. Physiol.—Endocrinol. Metab. 293 (2007) E492–E499. [5] H. Huang, Q. Jiang, Y. Chen, X. Li, X. Mao, X. Chen, L. Huang, W. Gao, Food Hydrocolloids 55 (2016) 244–253.
C.K. Reddy et al. / International Journal of Biological Macromolecules 94 (2017) 500–507 [6] J.E. Shaw, R.A. Sicree, P.Z. Zimmet, Diabetes Res. Clin. Pract. 87 (2010) 4–14. [7] Y. Zhou, S. Meng, D. Chen, X. Zhu, H. Yuan, Carbohydr. Polym. 103 (2014) 81–86. [8] T. Kullisaar, M. Zilmer, M. Mikelsaar, T. Vihalemm, H. Annuk, C. Kairane, A. Kilk, Int. J. Food Microbiol. 72 (2002) 215–224. [9] T.H. Huang, G. Peng, B.P. Kota, G.Q. Li, J. Yamahara, B.D. Roufogalis, Y. Li, Toxicol. Appl. Pharmacol. 207 (2005) 160–169. [10] B. Sharma, C. Balomajumder, P. Roy, Food Chem. Toxicol. 46 (2008) 2376–2383. [11] M.-A. Cornier, D. Dabelea, T.L. Hernandez, R.C. Lindstrom, A.J. Steig, N.R. Stob, R.E. Van Pelt, H. Wang, R.H. Eckel, Endocr. Rev. 29 (2008) 777–822. [12] C.-S. Niu, W. Chen, H.-T. Wu, K.-C. Cheng, Y.-J. Wen, K.-C. Lin, J.-T. Cheng, J. Agric. Food Chem. 58 (2010) 12031–12035. [13] J. Posuwan, P. Prangthip, V. Leardkamolkarn, U. Yamborisut, R. Surasiang, R. Charoensiri, R. Kongkachuichai, Food Chem. 138 (2013) 501–508. [14] S. Haralampu, Carbohydr. Polym. 41 (2000) 285–292. [15] H.N. Englyst, S. Kingman, J. Cummings, Eur. J. Clin. Nutr. 46 (1992) S33–50. [16] I. Björck, N.G. Asp, Trends Food Sci. Technol. 5 (1994) 213–218. [17] D. Lösel, R. Claus, J. Vet. Med. Ser. A 52 (2005) 209–212. [18] J.H. Cummings, E.R. Beatty, S.M. Kingman, S.A. Bingham, H.N. Englyst, Br. J. Nutr. 75 (1996) 733–747. [19] F. Brouns, B. Kettlitz, E. Arrigoni, Trends Food Sci. Technol. 13 (2002) 251–261. [20] L. Yonekura, H. Suzuki, Eur. J. Nutr. 44 (2005) 384–391. [21] C.K. Reddy, S. Pramila, S. Haripriya, J. Food Sci. Technol. 52 (2015) 1594–1601. [22] C.K. Reddy, S. Haripriya, P. Vidya, J. Food Sci. Technol. 52 (2015) 7289–7296. [23] P. Selvamani, K. Manivannan, R.J. Mohan, Bot. Res. Int. 2 (2009) 103–106. [24] P. Williams, F. Kuzina, I. Hlynka, Cereal Chem. 47 (1970) 411–421. [25] C.K. Reddy, P. Vidya, S. Haripriya, Int. J. Biol. Macromol. 81 (2015) 1039–1045. [26] W.T. Friedewald, R.I. Levy, D.S. Fredrickson, Clin. Chem. 18 (1972) 499–502. [27] M.A. Sullivan, S. Li, S.T. Aroney, B. Deng, C. Li, E. Roura, B.L. Schulz, B.E. Harcourt, J.M. Forbes, R.G. Gilbert, Carbohydr. Polym. 118 (2015) 9–15. [28] S. Marklund, G. Marklund, Eur. J. Biochem. 47 (1974) 469–474. [29] L. Goth, Clin. Chim. Acta 196 (1991) 143–151. [30] G.L. Ellman, Arch. Biochem. Biophys. 82 (1959) 70–77.
[31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53]
507
H. Ohkawa, N. Ohishi, K. Yagi, Anal. Biochem. 95 (1979) 351–358. W.S. Ratnayake, D.S. Jackson, Carbohydr. Polym. 67 (2007) 511–529. H. Zhang, Z. Jin, Carbohydr. Polym. 86 (2011) 1610–1614. M.-M. Shi, Q.-Y. Gao, Carbohydr. Polym. 84 (2011) 1151–1157. L.F. Polesi, S.B.S. Sarmento, Starch-Stärke 63 (2011) 226–235. L.F. Polesi, S. Sarmento, C.M. Franco, Int. J. Food Sci. Technol. 46 (2011) 2257–2264. S.I. Shin, C.J. Lee, D.-I. Kim, H.A. Lee, J.-J. Cheong, K.M. Chung, M.-Y. Baik, C.S. Park, C.H. Kim, T.W. Moon, J. Cereal Sci. 45 (2007) 24–33. T. Gelencsér, V. Gál, M. Hódsági, A. Salgó, Food Bioprocess Technol. 1 (2008) 171–179. H. Köksel, A. Basman, K. Kahraman, S. Ozturk, Int. J. Food Prop. 10 (2007) 691–702. P. Rayas-Duarte, J. Rupnow, J. Food Sci. 58 (1993) 389–394. S. Govindasamy, O. Campanella, C. Oates, Carbohydr. Polym. 30 (1996) 275–286. H.-J. Chung, R. Hoover, Q. Liu, Int. J. Biol. Macromol. 44 (2009) 203–210. R.F. Tester, W.R. Morrison, Cereal Chem. 67 (1990) 551–557. R. Eerlingen, M. Deceuninck, J. Delcour, Cereal Chem. 70 (1993) 345–350. A. Gunaratne, R. Hoover, Carbohydr. Polym. 49 (2002) 425–437. T. Vasanthan, R. Bhatty, Cereal Chem. 73 (1996) 199–207. T. Handa, K. Yamaguchi, Y. Sono, K. Yazawa, Biosci. Biotechnol. Biochem. 69 (2005) 1186–1188. Y. Zhang, R. Du, L. Wang, H. Zhang, Eur. Food Res. Technol. 231 (2010) 151–158. M.D. Michael, R.N. Kulkarni, C. Postic, S.F. Previs, G.I. Shulman, M.A. Magnuson, C.R. Kahn, Mol. Cell 6 (2000) 87–97. S.C. Hung, W.H. Anderson, D.R. Albers, M.L. Langhorst, S.A. Young, J. Diabetes 3 (2011) 158–167. C. Morand, M.A. Levrat, C. Besson, C. Demigne, C. Remesy, J. Nutr. Biochem. 5 (1994) 138–144. A. Maritim, A. Sanders, R.J. Watkins, J. Biochem. Mol. Toxicol. 17 (2003) 24–38. H.M. Hassan, Free Radic. Biol. Med. 5 (1988) 377–385.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Synthesis and physico-chemical characterization of modified starches from banana (Musa AAB) and its biological activities in diabetic rats Chagam Koteswara Reddy a,b,∗∗ , M. Suriya a , P.V. Vidya a , Sundaramoorthy Haripriya a,∗ a b
Department of Food Science and Technology, Pondicherry University, Puducherry 605014, India School of Life Sciences and Biotechnology, Korea University, Seoul 136701, Republic of Korea
a r t i c l e
i n f o
Article history: Received 30 June 2016 Received in revised form 25 July 2016 Accepted 17 October 2016 Available online 18 October 2016 Keywords: Amylose Banana Diabetes Modification Pullulanase Resistant starch
a b s t r a c t This study describes a simple method of preparation and physico-chemical properties of modified starches (type-3 resistant starches) from banana (Musa AAB), and the modified starches investigated as functional food with a beneficial effect on type-2 diabetes. RS3 was prepared using a method combined with debranching modification and physical modification; native and modifies starches were characterized by scanning electron microscope (SEM), powder X-ray diffraction (XRD), differential scanning calorimetry (DSC) and rapid visco analyzer (RVA). Use of the enzymatic and physical modification methodology, improved the yield of RS (26.62%) from Musa AAB. A reduced viscosity and swelling power; increased transition temperatures, water absorption capacity and solubility index with B-type crystalline pattern and loss of granular appearance were observed during the debranching modification and physical modification. The modified starches exhibited beneficial health effects in diabetic and HFD rats who consumed it. These results recommend that dietary feeding of RS3 was effective in the regulation of glucose and lipid profile in serum and suppressing the oxidative stress in rats under diabetic and HFD condition. This current study provides new bioactive starches, with potential applications in the food and non-food industries. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Now-a-days, the occurrence of obesity related problems are on the rise due to the modern life style, consumption of excess dietary fat and reduction in physical activities [1,2]. Obesity related issues also lead to complications like hyperlipidemia [3], non-alcoholic fatty liver disease [4], various cardiovascular diseases [2], and diabetes [5] in human beings. In general, diabetes is a form of metabolic disorder, which occurs due to the dietary intake of excess carbohydrates and lipids. In particular, type 2 diabetes mellitus (T2DM) is a common endocrine and metabolic disease [6]. T2DM is caused by an absolute or relative lack of insulin in the blood [7], resulting in metabolic abnormalities such as obesity, hypertension, low levels of high-density lipoprotein (HDL-C), elevated triglyceride (TG) levels, hyperglycemia and resistance to insulin [4]. In diabetes, major
∗ Corresponding author at: Department of Food Science and Technology, Pondicherry University, Puducherry 605014, India. ∗∗ Co-corresponding author at: School of Life Sciences and Biotechnology, Korea University, Seoul 136701, Republic of Korea. E-mail addresses: [email protected] (C.K. Reddy), [email protected] (S. Haripriya). http://dx.doi.org/10.1016/j.ijbiomac.2016.10.050 0141-8130/© 2016 Elsevier B.V. All rights reserved.
health problems are oxidative damage [8], dysfunction and eventual organ failure [9,10]. With improvement in social and economic environment in developed and developing countries, the occurrence of diabetes (T2DM) has rapidly increased over the years. Apart from genetic reasons, the dietary pattern of a person plays a key role in the occurrence of metabolic syndrome. A major reason could be the increased influence of western diet consumption which has an excess fat content in addition to poor minerals and fibre [11]. Due to the increased occurrence of diabetes in humans, current research is focused on the development of drugs for treatment and control of T2DM. Various drugs have been developed for the treatment and control of T2DM; however, the long-term usage of anti-diabetic drugs results into considerable side effects with the symptoms of hypoglycemia and malfunction of kidney and liver [7,10,12,13]. As medication cannot possibly have an alternative for treatment of T2DM, the focus should towards the prevention or delayed onset of diabetes by exploring the functional adjuncts responsible for it. Resistant starch (RS) is a new functional ingredient with a low glycemic index which is often employed in the development of functional food products. RS could be defined as the fraction of starch, which escapes digestion in the small intestine and undergoes fermentation in the large intestine in the presence of
C.K. Reddy et al. / International Journal of Biological Macromolecules 94 (2017) 500–507
microflora resulting into formation of short chain fatty acids, especially butyric acid [14,15]. RS can act as a dietary fiber with similar physiological effects [16]. Several reports suggested that the RS exhibits significant effect on physiological functions such as body weight management [17], prevention of gastrointestinal diseases [18], treatment of hyperglycemia and hypercholesterolemia, and enhancement of mineral absorption [19,20]. RS obtained various sources such as cereals (corn, rice and wheat), tubers (yam and potato) and pulses are widely used in the production of functional food products; and also various methods are involved in the preparation of RS from various sources including physical [7], chemical [2] and enzymatic [21] modification. Augmented the utility of RS with wider applications in food and non-food industries has increased the focus of research for the development of the RS from various food sources. Banana, is a common edible fruit of the genus Musa encompassed in the family Musaceae. Currently, worldwide production of banana is around 140 million tons, because of its extensive cultivation in tropical and sub-tropical regions of World. India ranks first in banana production and ‘Poovan’ (Musa AAB) cultivar is abundantly grown in Southern regions due to its higher adaptability to climatic conditions and cropping patterns [22,23]. The unripe bananas are highly consumed because of their high starch content. Due to the presence of high amount of starch, bananas are of great concern in the field of starch research. However, limited studies are available for the production of RS from Musa AAB using pullulanase enzyme and followed by its applications. From this basis, the objective of the present study was to the synthesis of resistant starch (RS3) from Musa AAB using a method of debranching modification followed by autoclaving and retrogradation; and evaluate their physico-chemical characteristics of native and modified starches, including morphological, physico-chemical, crystalline and thermal properties. Their biological activities (hypoglycemic and lipid lowering effects) of modified starches were also analysed using diabetes (T2DM) and obese rats fed with HFD. 2. Materials and methods 2.1. Materials Unripe poovan banana (Musa AAB) cultivar used in the study was obtained from the local harvest in the State of Tamil Nadu, India. Only unripe bananas were used in the study which did not have even small traces of yellow colour on its peel. Pullulanase from Bacillus acidopullulyticus (Promozyme 400 L) and Streptozotocin (STZ) were procured from Sigma-Aldrich Company, USA. Resistant starch assay kit was obtained from Megazyme International Ireland Limited, Ireland. Reagent kits of glucose, total cholesterol, TG, HDL-C, LDL-C, glutamate oxaloacetate transaminase (SGOT), glutamate pyruvate transaminase (SGPT) and alkaline phosphatase (ALP) were purchased from AGGAPE-diagnostics Pvt. Ltd, Kerala, India. All other chemicals and reagents were analytical grade. 2.2. Preparation of native starch Native starches from unripe poovan banana were extracted by Reddy et al. [22] with slight modifications as described. Raw poovan banana was peeled, washed and macerated for 2 to 3 min with sodium sulphite solution (1.22 g/L) using a waring hand blender (Prestige Hand Blender PHB 5.0, Bangalore, India). Homogenate was passed through 60 and 100 U.S mesh sieves using slurry and water (1:4 ratio). The filtrate was allowed to stand for 2 h at room temperature. After that supernatant was decanted off and then starch slurry was centrifuged (Eppendorf centrifuge, Model 5804, Mumbai, India) at 3000 rpm for 30 min. The white starch sediments were
501
dried in a hot air oven at 40 ◦ C for overnight, and ground to pass a sieve (100 U.S) for further analyses. 2.3. Preparation of resistant starch 2.3.1. Debranching of starch Debranching of native banana starch was performed based on the method described in Reddy et al. [21] with a slight modification. Starch sample (10 g) and 100 mL of sodium acetate buffer (0.1 M and pH 5.3) was mixed with pullulanase (40 U/g dry starch) and incubated in shaking water bath (Remi RSB-12, Mumbai, India) at 60 ◦ C for 10 h. The sample was heated in boiling water bath for 10 min to inactivate the enzyme. The starch gelatinization prior to enzymatic hydrolysis was performed with the sample in boiling water bath for 10 min, before adding the enzyme. 2.3.2. Preparation of resistant starch The starch samples, retrograded enzymatically hydrolysed native starch (REHNS) and retrograded enzymatically hydrolysed gelatinized starch (REHGS) in suspensions (10%, w/w dwb) were autoclaved at 121 ◦ C for 30 min, cooled to 4 ◦ C and stored at this temperature for 24 h. The samples were then freeze dried. In the samples, the RS content was determined using a Megazyme resistant starch assay kit with the description of Association of Official Analytical Chemists (AOAC) 2002.02. 2.4. Morphological characteristics The structure of the native and modified starch granules was observed by scanning electron microscope (SEM, HITACHI, S3400N, Tokyo, Japan). The powdered sample was sprinkled on double sided sticky tape placed on aluminium stubs and covered with carbon coating layer, observed, and photographed. 2.5. Colour and amylose content Colour of the native and modified starches was measured using Hunter Lab Colorimeter (D-25, Hunter Lab Associates Inc.) after standardisation using Hunter Lab colour standards. L* (lightness), a* (redness to greenness) and b* (yellowness to blueness) values of native and modified starches were analysed. Amylose content of native and modified starches was determined according to the method of Williams et al. [24]. 2.6. Pasting properties The pasting properties of native and modified starches were investigated with a Rapid Visco-Analyzer (RVA starch master 2, Newport Scientific, Warriewood, NSW, Australia) according to method of Reddy et al. [25]. 2.7. Water absorption capacity, solubility index and swelling power The water absorption capacity (WAC), water solubility index (WSI) and swelling power (SP) of native and modified starches were analysed respectively, according to the methods described by Reddy et al. [21]. 2.8. X-ray diffraction Powder X-ray diffraction (XRD) analysis of the native and the modified starches was carried out on powder X-ray diffractometer (Shimadzu XRD 7000) with Cu K␣ value of 1.54 radiation at step count of 2◦ /min, with a 2 range of 10◦ to 50◦ using a voltage of 40 kV and filament current 30 mA.
502
C.K. Reddy et al. / International Journal of Biological Macromolecules 94 (2017) 500–507
Fig. 1. Scanning electron micrographs of native and modified starches.
2.9. Thermal properties The gelatinization temperatures of native and modified starches were measured with a differential scanning colorimeter (TA-Q20 DSC) according to the methods of Reddy et al. [25]. 2.10. Animals experiments 2.10.1. Experimental animals Male Sprague Dawley rats weighing 130–150 g and of 7–8 weeks age were obtained from Sri Ragavendra Enterprises, Bangalore, India. The animals were housed in polypropylene cages in a legalised laboratory animal facility with controlled temperature (∼24 ◦ C) and lighting (12 h day/night cycle) for a 7-day adaptation period. They were given the normal pellet diet (NPD) and drinking water ad libitum. The experimental procedures were approved by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. This present animal study was approved by Institutional Animal Ethical Committee (IAEC), Pondicherry University (PU/SLS/IAEC/2014/16), India. 2.10.2. Streptozotocin-induced diabetic rats The control group of rats were given a regular diet and the experimental group of rats were fed with a HFD (36.5 g of NPD, 31 g of lard oil, 25 g of casein, 1 g of cholesterol, 0.3 g of methionine, 6 g of vitamin and mineral mix, 0.1 g of yeast and 0.1 g of NaCl). After 4 weeks of HFD feeding, the experimental group rats were injected with STZ (35 mg/kg, dissolved in 0.01 M sodium citrate buffer, pH 4.4) intraperitoneally, while the control group rats were injected with the vehicle citrate buffer. The experimental animals which showed fasting blood glucose levels >140 mg/dl were categorised as diabetic. After adaptation period and STZ injection, rats showing elevated fasting blood glucose levels were randomly dispersed to different treatment groups. 2.10.3. Experimental design The animals were separated into 5 groups (n = 7) and treated for 8 weeks as follows. Rats in Group 1 (control group) were given NPD
for 8 weeks. The Group 2 rats were treated with HFD for 4 weeks followed by the STZ injection (35 mg/kg bw) and continued with the HFD for 4 more week (T2DM model). Group 3 rats were induced to T2DM as in group 2 and treated with the RS (30 g/100 g diet) for 4 weeks. Group 4 rats were given the HFD alone for 8 weeks without induction of diabetes in order to assess the biochemical changes associated with obesity. Group 5 rats were given HFD for the first 4 weeks followed by HFD diet with RS (30 g/100 g diet) for further 4 weeks. At the end of study, animals were kept fasted for overnight, anaesthetized and blood samples were collected with the abdominal aorta without heparin for serum. Organs like liver, pancreas, spleen, kidney and heart were excised, rinsed with cold isotonic saline and then stored at −80 ◦ C for biochemical analysis. 2.10.4. Estimation of serum glucose and lipid profile Serum glucose and lipid profiles (total cholesterol, TG, HDL-C and LDL-C) were examined by enzymatic colorimetric methods using commercially available kits (AGGAPE-diagnostics). From the experimental values of TG, the other cholesterol carrying lipoprotein (VLDL-C) were calculated using the following standard formula [26]. VLDL − C =
Triglycerides 5
2.10.5. Assessment of diagnostic enzymes of tissue damage in serum The levels of the SGOT, SGPT and ALP in serum were analysed using commercially available kits (AGGAPE-diagnostics). 2.10.6. Assessment of biochemical parameters in liver Glycogen content in liver was quantified according to the method of Sullivan et al. [27], TC and TG contents were examined by enzymatic colorimetric methods using commercially available kits (AGGAPE-diagnostics).
C.K. Reddy et al. / International Journal of Biological Macromolecules 94 (2017) 500–507
503
Table 1 Colour, amylose and resistant starch content of native and modified starches. Parameter
Colour parameters L* a* b* Amylose (%) Resistant starch (%)
Type NPBS
REHNS
REHGS
92.57 ± 0.16a 3.88 ± 0.43b 7.87 ± 0.33b 23.10 ± 1.12c 11.05 ± 0.45c
89.45 ± 0.19b 6.41 ± 0.44a 11.41 ± 0.41a 31.11 ± 1.35b 22.01 ± 0.24b
90.03 ± 0.15b 6.11 ± 0.21a 10.87 ± 0.59a 40.46 ± 0.95a 26.62 ± 0.37a
Values expressed are mean ± standard deviation. Means in the row with different superscript are significantly different at p ≤ 0.05.
2.10.7. Estimation of oxidative stress markers The enzymatic endogenous antioxidants such as superoxide dismutase (SOD) [28] and catalase [29]; non-enzymatic endogenous antioxidant like reduced glutathione (GSH) [30]; and oxidative stress marker such as lipid peroxidation (LPO) [31] were analysed in liver tissue homogenate (10%) using standard methods. 2.11. Statistical analysis Analytical determinations were carried out in triplicates and standard deviations were noted. The data was subjected to one way ANOVA to analyse the significant difference in all data followed by Tukey’s test using SPSS 18 software (SPSS Institute Inc., Cary, NC, USA). 3. Results and discussion 3.1. Morphological characteristics SEM images of the granules of native (NPBS) and modified starches (REHNS and REHGS) showed significant difference in their appearances (Fig. 1). NPBS granules had smooth surface with rod like and irregular oval shapes. REHNS and REHGS resembled an amorphous mass of cohesive structure, leading to the loss of granular appearance and irregular shape, as a result of the gelatinization process when the clumped starch granules form sponge like structure within the inner region of the retrograded starch [32]. Zhang and Jin [33] proposed that the variation in the structure of starch granules was due to the retrogradation of amylose chains which could have been initiated in a restructuring of the starch into a helical complex. These modification in the amorphous regions led to the rejection of the activity of alpha amylase on the retrograded starch and raised the density of crystalline structure [7,34].
Fig. 2. Pasting properties of native and modified starches.
(p ≤ 0.05) difference from REHNS (31.11%) and REHGS (40.46%). The percentage of RS in REHGS (26.62) was significantly (p ≤ 0.05) higher when compared with REHNS (22.01) and NPBS (11.05) owing to the increased amylose content in samples of REHNS and REHGS, which is the resulting retrograded starch of NPBS upon the treatment of pullulanase before and after gelatinization. Amylose content increased as RS content increased, it could be due to the breakdown of ␣ (1–6) linkage of amylopectin by pullulanase, which is changed into small chain linear polysaccharides like amylose molecules, which form strong gel network through retrogradation [35]. In general raw banana contain RS2 (granular starch) and during thermal process RS2 converts to RS3, which is due to recrystallinity of the short linear chains produced after debranching and retrogradation process [21,36]. 3.4. Pasting properties The pasting properties of native and modified starches were analysed using RVA and the results are presented in Table S1 and Fig. 2. The pasting temperature value of REHNS and REHGS are 69.4–63.3 ◦ C, respectively and is notably lesser than that of NPBS (83.06 ◦ C). Reduced pasting temperature was observed in the case of the REHNS and REHGS which indicates the destruction of starch granules during autoclaving [36]. REHNS and REHGS shown significantly (p ≤ 0.05) reduced pasting parameters including peak (819 cP & 743 cP), hold (737 cP & 264 cP), final (946 cP & 361 cP), and setback (203 cP & 69 cP) with increased breakdown (77 cP & 89 cP) viscosities when compared to what was
3.2. Colour Colour values (L*, a* and b*) are represented in Table 1 with significance (p ≤ 0.05) difference among native and modified starches. The L* value of starches ranged from 89.45 to 92.57 and the NPBS exposed maximum lightness with significant difference from REHNS and REHGS. a* and b* value of REHNS and REHGS differed significantly from NPBS and maximum a* (6.41) and b* (11.41) value observed for REHNS. These deviations in colour values may possible due to the caramelization that would have happened in small fragments; these small molecules are formed during debranching and autoclaving of starch granules. 3.3. Amylose and resistant starch content The increased levels of amylose and RS content in modified starches were observed and the obtained data are presented in Table 1. The amylose content of NPBS (23.10%) shown significant
Table 2 Water absorption capacity, solubility index and thermal properties of native and modified starches. Parameter
Type NPBS
REHNS
REHGS
WAC (%) Solubility index (%)
3.15 ± 0.13c 3.07 ± 0.05c
5.15 ± 0.16b 14.97 ± 0.65b
5.98 ± 0.12b 18.32 ± 0.98a
Thermal properties T0 (◦ C) TP (◦ C) TC (◦ C) H gel (J/g) R (◦ C)
62.08 ± 0.12c 69.63 ± 0.35c 82.35 ± 0.86c 15.06 ± 0.55c 21.04 ± 0.54c
64.39 ± 0.25b 73.61 ± 2.31b 89.51 ± 0.81b 19.39 ± 0.99b 25.11 ± 0.33b
69.32 ± 1.81a 82.27 ± 1.03a 97.91 ± 2.06a 25.83 ± 1.69a 28.59 ± 1.10a
WAC, water absorption capacity; T0 , onset temperature; TP , peak temperature; TC , conclusion temperature; Hgel, enthalpy of gelatinization; R, gelatinization range (TC -T0 ). Values expressed are mean ± standard deviation. Means in the row with different superscript are significantly different at p ≤ 0.05.
504
C.K. Reddy et al. / International Journal of Biological Macromolecules 94 (2017) 500–507
Fig. 3. (A) Swelling power and (B) X-ray diffraction patterns of native and modified starches.
observed in case of NPBS (1384 cP, 1333 cP, 1853 cP 489 cP & 4 cP). Reduced viscosity attribute could be as a result of debranching lead to increase the levels of small linear chain molecules and RS. The formation of starch gel ability reduced in REHNS and REHGS when compared with NPBS which is due to its autoclaving [37,38].
3.5. Water absorption capacity The WAC of native and modified starches shown significant (p ≤ 0.05) difference and the values ranged from 3.15 to 5.98 per cent (Table 2); and were in the following order: NPBS < REHNS ∼ REHGS. Increased WAC of REHNS and REHGS could be due to the gelatinisation caused by heating and autoclaving [35,39]. Reduced molecular weight of starch granules during debranching of starch granules lead to the formation of small molecules, which have more affinity with water molecules when compared to starch polymers [40]. The WAC depends upon the levels of intermolecular relationship among starches by forces like hydrogen and covalent bonding.
3.6. Water solubility index The WSI of modified starches (Table 2) showed an increasing trend, where the NPBS showed the least solubility. Native starch (NPBS) hardly dissolved in the water, whereas the debranched starches had short-chain molecules with high solubility. The values ranged from 3.07 to 18.32 per cent and the maximum solubility was observed in REHGS. Starch granule solubility is greatly affected by the quantity of amylose content and structure of starch granule. The increased starch solubility could be due to the variations in the molecular structure of starch [41], reduced molecular weight of starch granules and increased formation of short chain molecules from starch granules [42]. 3.7. Swelling power Swelling power is a degree of starch hydration capacity and the extent of the interaction between starch granules within the amorphous and crystalline areas. SP of native and modified starches was shown in Fig. 3A. From the results, we noted that with an increase in temperature the SP of native and modified starches was gradu-
C.K. Reddy et al. / International Journal of Biological Macromolecules 94 (2017) 500–507
505
Table 3 Effect of resistant starch on glucose and lipid profile in serum. Groups
Glucose (mg/dl)
Cholesterol (mg/dl)
Triglycerides (mg/dl)
HDL-C (mg/dl)
LDL-C (mg/dl)
VLDL-C (mg/dl)
Control HFD HFD + RS T2DM T2DM + RS
105.64 ± 8.72 152.42 ± 9.31* 111.05 ± 8.41** 506.74 ± 53.01# 332.59 ± 42.97##
47.61 ± 10.71 66.93 ± 10.42* 50.97 ± 10.19** 86.89 ± 9.32# 52.24 ± 12.71##
115.25 ± 3.52 300.56 ± 15.62* 220.00 ± 12.60** 464.89 ± 25.20# 271.45 ± 15.29##
31.82 ± 1.96 28.02 ± 0.73* 35.63 ± 0.83** 22.96 ± 1.13# 30.56 ± 0.96##
7.73 ± 1.12 50.89 ± 5.65* 33.58 ± 2.24** 76.96 ± 9.90# 22.15 ± 1.17##
22.13 ± 0.91 91.82 ± 2.36* 53.92 ± 2.33** 61.89 ± 4.28# 45.07 ± 2.60##
Values expressed are mean ± standard deviation from seven rats in each group. # Significantly different from Control, P≤0.05; ## Significantly different from T2DM, P≤0.05; * Significantly different from Control, P≤0.05; ** Significantly different from HFD, P≤0.05.
ally increased. At 50 ◦ C, the SP values ranged from 3.42 (NPBS) to 3.25 per cent (REHGS). There was no significant difference in the SP between native and modified starches until 70 ◦ C. Upon increasing the temperature to 80 ◦ C and 90 ◦ C there was a noticeable reduction of SP was observed in native and modified starches; and NPBS shown maximum SP at 90 ◦ C (12.23%), and at the same temperature the lowest SP was shown by REHGS (7.17%) and REHNS (7.98%). The swelling capacity of starch granules is predominantly influenced by the quantity of amylopectin in starch molecules [43]. Reduced the SP of modified starches may be due to the destruction of starch granules during debranching, autoclaving and retrogradation [21].
ference. After STZ injection, the levels of fasting blood glucose in the two diabetic groups (T2DM and T2DM + RS) were significantly higher than those control group, and no significant difference between STZ injected groups, this reveals that a diabetic rat model had been successfully established. However, without STZ injection, increased glucose levels were noted in HFD groups and it may be due to the consumption of excess HFD. From the results (Table 3), after 4 weeks treatment with RS3, significantly (p ≤ 0.05) declined the serum glucose levels of the diabetic (T2DM + RS) and HFD + RS groups was observed when compared with T2DM and HFD groups. These results advised that RS can regulate the carbohydrate metabolism and decrease the serum glucose levels of diabetic rats.
3.8. X-ray diffraction 3.10. Effect of resistant starch on hyperlipidemia The X-ray diffraction patterns of native and modified starches are shown in Fig. 3B. The diffractogram of NPBS showed highest peak at 17.1◦ 2 and two minor peaks at 15◦ and 23◦ 2 of diffraction angles and showed B-type crystalline pattern. The REHNS and REHGS had peaks at 16.85◦ and 16.95◦ 2 reflects the B-type crystalline pattern. These changes in the crystalline pattern of starches may be due to the process of debranching, autoclaving and retrogradation [35]. In general, retrogradation of starch at low temperature leads to the formation of B-type crystallinity [44]; and starches with B-type crystalline pattern are highly resistant to enzymatic hydrolysis. The crystalline pattern of starches could be influenced by the chain length of amylopectin, growth and cultivation conditions [45]. 3.9. Thermal properties The gelatinisation temperatures includes T0 , Tp , Tc , and enthalpy (Hgel) of NPBS were 62.08 ◦ C, 69.63 ◦ C, 82.35 ◦ C and 15.06 J/g, respectively (Table 2). REHNS and REHGS showed significantly (p ≤ 0.05) improved gelatinisation temperatures and enthalpy including T0 (64.39 ◦ C & 69.32 ◦ C), Tp (73.61 ◦ C & 82.27 ◦ C), Tc (89.51 ◦ C & 97.91 ◦ C) and enthalpy (19.39 J/g & 25.83 J/g), respectively. Amplified enthalpy was observed for REHNS and REHGS reflects the amount of double helical and crystalline order of starch; and these are influenced by ratio of amylose and amylopectin and amylopectin chain length distribution [45]. Enhanced amount of RS in REHNS and REHGS showed more thermal stability than NPBS. The discrepancy in the gelatinization temperature range (R-value) between starches may be due to the existence of crystallites of variable stability within crystalline domains of its starch granules [46]. REHGS having highest R-value followed by REHNS while the lowest value noted for NPBS. Variations in gelatinization temperatures and R-value among native and modified starches may be possible due to the difference in amylose and amylopectin content, amount of crystallinity and stability of crystal region in starch granules. 3.9.1. Effect of resistant starch on serum glucose Generally, HFD has been shown to induce insulin resistance, hyperinsulinaemia and dyslipidaemia in rats and increase the risk of developing T2DM in obese cases [47]. Initially, all rats showed similar glucose levels in serum without significant (p ≤ 0.05) dif-
Diabetes is generally accompanied by the existence of symptoms of high cholesterol [7]. Table 3 data shows the levels of serum lipid profile of rats. The total cholesterol, TG, and LDLC levels were significantly elevated, whereas the HDL-C levels significantly decreased in the STZ induced diabetic and HFD (without STZ) groups as compared to the control group. However, after 4 weeks treatment with RS3, we observed that significantly (P ≤ 0.05) decreased serum total cholesterol, TG, LDL-C, VLDL-C levels, whereas increased HDL-C in treated groups when compared with diabetic and HFD groups. These results confirmed that the modified starches (RS3) from Musa AAB had anti-hyperpidemia activity. These results are reliable with previously reported studies that confirmed the biological activity of RS4 from corn [2] and RS3 from rice [7] and yam [5]. 3.11. Effect of resistant starch on organ indices The organ indices of the rats from each group index were assessed and are shown in Table S2. The liver indices of HFD fed rats did not show any significant difference from control group; liver indices of T2DM group significantly (P ≤ 0.05) differed from control group. Liver index is a key factor in obese and hypertensive patients [48]. These findings specifies that RS can effectively control liver in obese and diabetic rats. From the results the indices of spleen, kidney and heart of HFD fed, diabetic and treated groups did not show any significant difference (P ≤ 0.05) when compared with control group. Pancreas indices of diabetic group shown significant difference (P ≤ 0.05) with control group; no significant difference from treated groups. These findings indicates that RS can effectively control diabetic liver in rats. 3.12. Assessment of diagnostic enzymes of tissue damage in serum The levels of liver marker enzymes such as SGOT, SGPT and ALP in serum of control and diabetic rats were analysed and the data are presented in Table 4. After STZ injection, the levels of liver marker enzymes in serum were significantly (P ≤ 0.05) raised in diabetic (T2DM) and hypercholesterolemic (HFD) groups when compared with control group. After 4 weeks treatment with RS3, there was
506
C.K. Reddy et al. / International Journal of Biological Macromolecules 94 (2017) 500–507
Table 4 Effect of resistant starch on liver marker enzymes in serum.
3.15. Effect of resistant starch on markers of oxidative stress in liver
Groups
SGOT (U/L)
SGPT (U/L)
ALP (U/L)
Control HFD HFD + RS T2DM T2DM + RS
108.71 ± 4.65 134.71 ± 9.52* 109.48 ± 8.49** 150.24 ± 7.63# 113.35 ± 6.32##
56.14 ± 3.99 69.63 ± 1.23* 57.63 ± 2.23** 93.92 ± 4.01# 77.52 ± 2.32##
112.41 ± 8.23 417.56 ± 8.96* 307.95 ± 14.32** 747.72 ± 11.23# 335.13 ± 9.56##
Values expressed are mean ± standard deviation from seven rats in each group. # Significantly different from Control, P ≤ 0.05; ## Significantly different from T2DM, P ≤ 0.05; * Significantly different from Control, P ≤ 0.05; ** Significantly different from HFD, P ≤ 0.05.
Table 5 Effect of resistant starch on glycogen, cholesterol and triglycerides in liver. Groups
Glycogen (g/100 g)
Cholesterol (mg/dl)
Triglycerides (mg/dl)
Control HFD HFD + RS T2DM T2DM + RS
2.72 ± 0.05 2.95 ± 0.15* 2.77 ± 0.15 0.53 ± 0.08# 1.48 ± 0.06##
75.80 ± 10.71 185.49 ± 10.42* 169.08 ± 10.19** 270.06 ± 9.32# 172.25 ± 12.71##
275.50 ± 23.45 428.05 ± 33.55* 315.21 ± 36.12** 691.23 ± 35.12# 330.29 ± 24.15##
Values expressed are mean ± standard deviation from seven rats in each group. # Significantly different from Control, P ≤ 0.05; ## Significantly different from T2DM, P ≤ 0.05; * Significantly different from Control, P ≤ 0.05; ** Significantly different from HFD, P ≤ 0.05.
a significant effect on liver marker enzymes in serum; the levels of SGOT, SGPT and ALP were significantly (P ≤ 0.05) decreased in serum of treated groups, and these values are comparable with control group.
3.13. Effect of resistant starch on glycogen content in liver In general, liver is an insulin sensitive organ which shows a key role in the regulation of glucose homeostasis by storing glycogen. During diabetes, under a state of hepatic insulin resistance the capacity of insulin to regulate the glucose production from the liver is reduced which in turn lead to the expression of hyperglycemia [49,50]. The content of glycogen in liver of diabetic and HFD fed rats were analysed and the results is shown in Table 5. During diabetes, the decreased level of glycogen content was observed in diabetic (T2DM) group, shows significant (P ≤ 0.05) difference against control group. After 4 weeks of treatment with RS3 from poovan banana starch exhibited a significant effect on diabetic group, whereas increased the levels of glycogen content in liver of treated group (T2DM + RS).
3.14. Effect of resistant starch on cholesterol and triglyceride levels in liver The levels of cholesterol and TG in liver of diabetic and HFD fed rats was analysed and the data are shown in Table 5. During diabetes, significantly (P ≤ 0.05) elevated the levels of cholesterol and TG were observed in diabetic (T2DM) and HFD fed groups when compared with control group. After four weeks of treatment with RS3 there was a significant change in the diabetic group, and lead to reduce the levels of cholesterol and triglycerides in liver of treated groups (T2DM + RS and HFD + RS). Reduced cholesterol and TG in liver designates that the hypocholesterolemic effect of RS may be due to the interference of intestinal cholesterol and bile acid absorption [51].
Enhanced the formation of free radicals and impaired antioxidant defence system result in oxidative stress, which in turn plays a significant role in the pathogenesis of diabetes and its complications [52]. To maintain oxidative stress and normalise the destructive potential of free radicals, the cells have established a highly complex antioxidant protection system, which includes antioxidant enzymes such as SOD, GSH, Catalase, and that catalyse free radicals-quenching reactions [53]. The levels of oxidative stress markers such as SOD, catalase, GSH and TBARS in liver of control and diabetic group rats were analysed and the resulted data shown in Table S3. From the results, it could be noted that significantly (P ≤ 0.05) increased the levels of lipid peroxidation (LPO) and decreased levels of antioxidants such as SOD, catalase and GSH were noted in liver of diabetic and HFD fed rats. In the diabetic group and HFD fed rats, treated with RS3 from poovan banana starch showed positive significant observations along with raised levels of SOD, catalase, GSH and reduced TBARS (lipid peroxidation) in liver of diabetic group. These finding demonstrated that there was severe oxidation reaction in hyperlipidemia rats and the modified starches (RS3) improved the in-vivo antioxidative defense status of the rats; it could lead to decreased oxidative stress under HFD condition. 4. Conclusion The present study demonstrates that by debranching with pullulanase and employing the autoclaving and retrogradation techniques the extraction of RS3 from Musa AAB starch can be brought about in an efficient manner. Use of the pullulanase, increases the yield of RS3 and amylose content in the native banana (Musa AAB) starch. The results suggest RS3 shows highest thermal stability and reduced viscosity when compared with native starch. Which is one of the desirable functional property of starch in food formulations. The RS3 had beneficial health effects in diabetic rats. The results exhibited that dietary feeding of RS3 was effective in the regulation of glucose and lipid profile in serum and suppressing oxidative stress in rats under diabetic and HFD condition. Finally, RS3 from poovan banana starch showed hypoglycemic effect and it can be used as functional ingredient in food product development. Acknowledgements The authors is grateful to the help rendered by technologists and technicians of SEM, DSC, and powder XRD and other necessary instruments at the Central Instrumentation Facility, Pondicherry University, Puducherry. This project is financially supported by the Department of Food Science and Technology, Pondicherry University, Pondicherry, India. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ijbiomac.2016. 10.050. References [1] M.-N. Woo, S.-H. Bok, M.-K. Lee, H.-J. Kim, S.-M. Jeon, G.-M. Do, S.K. Shin, T.Y. Ha, M.-S. Choi, J. Med. Food 11 (2008) 169–178. [2] K.Y. Lee, S.H. Yoo, H.G. Lee, Starch-Stärke 64 (2012) 78–85. [3] M.-N. Woo, S.-H. Bok, M.-S. Choi, Food Chem. Toxicol. 47 (2009) 2076–2082. [4] K.R. Coenen, A.H. Hasty, Am. J. Physiol.—Endocrinol. Metab. 293 (2007) E492–E499. [5] H. Huang, Q. Jiang, Y. Chen, X. Li, X. Mao, X. Chen, L. Huang, W. Gao, Food Hydrocolloids 55 (2016) 244–253.
C.K. Reddy et al. / International Journal of Biological Macromolecules 94 (2017) 500–507 [6] J.E. Shaw, R.A. Sicree, P.Z. Zimmet, Diabetes Res. Clin. Pract. 87 (2010) 4–14. [7] Y. Zhou, S. Meng, D. Chen, X. Zhu, H. Yuan, Carbohydr. Polym. 103 (2014) 81–86. [8] T. Kullisaar, M. Zilmer, M. Mikelsaar, T. Vihalemm, H. Annuk, C. Kairane, A. Kilk, Int. J. Food Microbiol. 72 (2002) 215–224. [9] T.H. Huang, G. Peng, B.P. Kota, G.Q. Li, J. Yamahara, B.D. Roufogalis, Y. Li, Toxicol. Appl. Pharmacol. 207 (2005) 160–169. [10] B. Sharma, C. Balomajumder, P. Roy, Food Chem. Toxicol. 46 (2008) 2376–2383. [11] M.-A. Cornier, D. Dabelea, T.L. Hernandez, R.C. Lindstrom, A.J. Steig, N.R. Stob, R.E. Van Pelt, H. Wang, R.H. Eckel, Endocr. Rev. 29 (2008) 777–822. [12] C.-S. Niu, W. Chen, H.-T. Wu, K.-C. Cheng, Y.-J. Wen, K.-C. Lin, J.-T. Cheng, J. Agric. Food Chem. 58 (2010) 12031–12035. [13] J. Posuwan, P. Prangthip, V. Leardkamolkarn, U. Yamborisut, R. Surasiang, R. Charoensiri, R. Kongkachuichai, Food Chem. 138 (2013) 501–508. [14] S. Haralampu, Carbohydr. Polym. 41 (2000) 285–292. [15] H.N. Englyst, S. Kingman, J. Cummings, Eur. J. Clin. Nutr. 46 (1992) S33–50. [16] I. Björck, N.G. Asp, Trends Food Sci. Technol. 5 (1994) 213–218. [17] D. Lösel, R. Claus, J. Vet. Med. Ser. A 52 (2005) 209–212. [18] J.H. Cummings, E.R. Beatty, S.M. Kingman, S.A. Bingham, H.N. Englyst, Br. J. Nutr. 75 (1996) 733–747. [19] F. Brouns, B. Kettlitz, E. Arrigoni, Trends Food Sci. Technol. 13 (2002) 251–261. [20] L. Yonekura, H. Suzuki, Eur. J. Nutr. 44 (2005) 384–391. [21] C.K. Reddy, S. Pramila, S. Haripriya, J. Food Sci. Technol. 52 (2015) 1594–1601. [22] C.K. Reddy, S. Haripriya, P. Vidya, J. Food Sci. Technol. 52 (2015) 7289–7296. [23] P. Selvamani, K. Manivannan, R.J. Mohan, Bot. Res. Int. 2 (2009) 103–106. [24] P. Williams, F. Kuzina, I. Hlynka, Cereal Chem. 47 (1970) 411–421. [25] C.K. Reddy, P. Vidya, S. Haripriya, Int. J. Biol. Macromol. 81 (2015) 1039–1045. [26] W.T. Friedewald, R.I. Levy, D.S. Fredrickson, Clin. Chem. 18 (1972) 499–502. [27] M.A. Sullivan, S. Li, S.T. Aroney, B. Deng, C. Li, E. Roura, B.L. Schulz, B.E. Harcourt, J.M. Forbes, R.G. Gilbert, Carbohydr. Polym. 118 (2015) 9–15. [28] S. Marklund, G. Marklund, Eur. J. Biochem. 47 (1974) 469–474. [29] L. Goth, Clin. Chim. Acta 196 (1991) 143–151. [30] G.L. Ellman, Arch. Biochem. Biophys. 82 (1959) 70–77.
[31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53]
507
H. Ohkawa, N. Ohishi, K. Yagi, Anal. Biochem. 95 (1979) 351–358. W.S. Ratnayake, D.S. Jackson, Carbohydr. Polym. 67 (2007) 511–529. H. Zhang, Z. Jin, Carbohydr. Polym. 86 (2011) 1610–1614. M.-M. Shi, Q.-Y. Gao, Carbohydr. Polym. 84 (2011) 1151–1157. L.F. Polesi, S.B.S. Sarmento, Starch-Stärke 63 (2011) 226–235. L.F. Polesi, S. Sarmento, C.M. Franco, Int. J. Food Sci. Technol. 46 (2011) 2257–2264. S.I. Shin, C.J. Lee, D.-I. Kim, H.A. Lee, J.-J. Cheong, K.M. Chung, M.-Y. Baik, C.S. Park, C.H. Kim, T.W. Moon, J. Cereal Sci. 45 (2007) 24–33. T. Gelencsér, V. Gál, M. Hódsági, A. Salgó, Food Bioprocess Technol. 1 (2008) 171–179. H. Köksel, A. Basman, K. Kahraman, S. Ozturk, Int. J. Food Prop. 10 (2007) 691–702. P. Rayas-Duarte, J. Rupnow, J. Food Sci. 58 (1993) 389–394. S. Govindasamy, O. Campanella, C. Oates, Carbohydr. Polym. 30 (1996) 275–286. H.-J. Chung, R. Hoover, Q. Liu, Int. J. Biol. Macromol. 44 (2009) 203–210. R.F. Tester, W.R. Morrison, Cereal Chem. 67 (1990) 551–557. R. Eerlingen, M. Deceuninck, J. Delcour, Cereal Chem. 70 (1993) 345–350. A. Gunaratne, R. Hoover, Carbohydr. Polym. 49 (2002) 425–437. T. Vasanthan, R. Bhatty, Cereal Chem. 73 (1996) 199–207. T. Handa, K. Yamaguchi, Y. Sono, K. Yazawa, Biosci. Biotechnol. Biochem. 69 (2005) 1186–1188. Y. Zhang, R. Du, L. Wang, H. Zhang, Eur. Food Res. Technol. 231 (2010) 151–158. M.D. Michael, R.N. Kulkarni, C. Postic, S.F. Previs, G.I. Shulman, M.A. Magnuson, C.R. Kahn, Mol. Cell 6 (2000) 87–97. S.C. Hung, W.H. Anderson, D.R. Albers, M.L. Langhorst, S.A. Young, J. Diabetes 3 (2011) 158–167. C. Morand, M.A. Levrat, C. Besson, C. Demigne, C. Remesy, J. Nutr. Biochem. 5 (1994) 138–144. A. Maritim, A. Sanders, R.J. Watkins, J. Biochem. Mol. Toxicol. 17 (2003) 24–38. H.M. Hassan, Free Radic. Biol. Med. 5 (1988) 377–385.