Side dishes were heated in a 1100 W microwave for two minutes to mimic participant food preparation, except for the couscous and potato salad side dishes, which were to be served cold. Reheated side dishes were analyzed when dishes were warm; couscous and potato salad were analyzed chilled. Each sample was then ground evenly using an electric meat grinder (Aobosi, Guangzhou, China), on the “Fine” setting. RS was then immediately quantified by the Megazyme Resistant Starch Assay Kit (Megazyme, Bray, Ireland), according to the kit instructions. The Megazyme RS Assay Kit is able to analyze RS types 1, 2, and 3 and has a standard deviation of 3% and 3–5% between days according to the manufacturer. RS determination was done in triplicate for each study dish. Grams of RS were calculated by taking the percentage of RS wet weight and multiplying by the serving weight (in grams) of each dish.
Rs assay kit
Manufactured by Megazyme
Sourced in Ireland
The RS assay kit is a laboratory equipment product designed for the determination of rapidly digestible starch (RS) content. It provides a standardized method for quantifying the RS component in food and feed samples.
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15 protocols using rs assay kit
1
Quantifying Resistant Starch in Heated Sides
2
Starch Biochemical Trait Analysis
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Biochemical traits measured were total starch content, amylose content (AC), resistant starch (RS) and hydrolysis index (HI). Total starch contents were determined on the basis of the AACC International (AACC Method 76–13.01) method. AC was determined through high performance size exclusion liquid chromatography as described by Demeke et al. [74 (link)]. The RS content was estimated on dry weight basis following Goni et al. [75 (link)] using the Megazyme RS assay kit (Cat#K-RSTAR; Megazyme International Ireland Ltd., Ireland). In vitro starch hydrolysis rate and HI were determined according to Goni et al. [61 (link)]. In vitro enzymatic hydrolysis with different time points (0, 30, 60, 120 and 240 min) were carried out to predict the rate of starch digestibility which is measured as HI by comparing the rate of digestibility of white bread. This is considered to be in vitro equivalent of GI estimate. All determinations were done in three biological replicates and two independent observations for each replicate.
3
Gamma-induced Rice Mutant for Resistant Starch
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The cultivar ADT 43 was chosen for inducing mutations which is a short duration variety derived from a cross between IR 50 and Improved White Ponni (IWP). To create an ADT43 rice mutant population, gamma radiation (Tamil Nadu Agricultural University-Radiation facility) was used and LD50 value (250 Gy) was determined using kill curve analysis. Fixed LD50 was used to treat and to develop the 5,000 M1 plants (Agasimani et al., 2013 (link)). Then the 3,000 M2 families were raised and three plants from each family have been tagged. The seeds were harvested in a single plant basis and a portion of seeds was processed to white rice and used for biochemical analysis (data not shown). The RS content of M2 to M6 generation was estimated on dry weight basis following Goni et al. (1996) (link) using the Megazyme RS assay kit (Cat#K-RSTAR; Megazyme International Ireland Ltd., Ireland). Identified mutant (γ278) line with increased RS was used for further molecular studies to identify the functional mutations.
4
Enzymatic Modification of Pea Starch
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Pea starch (PS) was provided by Liangrun Food Co., Ltd. (Xinxiang, Henan, China). Amylose percentage of PS is 14.3%, and its amylose/amylopectin ratio is 0.17. Relatively crystallinity of PS is 26.2%, and its X-ray diffraction pattern is C type, showing diffraction peaks at 5.7°, 15.2°, 17.2°, and 23.1°. The enzyme (PulY103A) was prepared, and its activity was verified according to the method of Liu et al. [12 (link)]. The Megazyme RS assay kit was purchased from Megazyme International (Wicklow, Ireland). Artificial gastric juice (containing pepsin) and artificial small intestinal fluid (containing pancreatin and amyloglucosidase) were provided by Yuanye Co., Ltd. (Shanghai, China).
5
Measuring Resistant Starch in Crops
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RS was measured using the Megazyme RS Assay kit (Megazyme, Co., Wicklow, Ireland), which is widely used for RS determination in crops, according to the manufacturer’s instructions. The grain samples were treated with 10 mg/mL pancreatic α-amylase and 3 U/mL amyloglucosidase (AMG) enzyme to hydrolyze and solubilize the non-RS. After terminating the enzymatic reaction by adding 99% ethanol solution, RS was recovered as a pellet by centrifugation at 1,500 g for 10 min and the supernatant was decanted. RS in the pellet was dissolved in 2 mol/L KOH before the pellet was washed twice with 50% ethanol solution. Subsequently, starch in the solution was hydrolyzed quantitatively into glucose with AMG. D-Glucose was measured with glucose oxidase/peroxidase reagent at 510 nm against the reagent blank. The percentage of RS was calculated using the formula provided by the manufacturer. All analyses were performed in triplicate.
6
Starch Resistant Starch Analysis
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RS content was measured using the Megazyme RS assay kit (Megazyme, Co. Wicklow, Ireland) according to manufacturer’s instructions; this kit has been widely used for RS determination in crops (McCleary et al. 2002 (link)). The apparent amylose content (AAC) of starch was measured using a simplified I2/KI assay as described by Yang et al. (2006) (link). Four standard samples, each with a different AAC (1.2%, 11.2%, 16.8% and 26.8%), were procured from the China National Rice Research Institute (CNRRI, China). The experiments were performed in triplicate.
7
Increasing Resistant Starch in Corn Starch
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The heating-cooling cycle to increase the RS (type 3) content of corn starch was as follows; steam heating at over 100°C for 30 min, and cooled at 4°C for 24 hours. The RS content in rice, corn starch, and experimental diets was measured using Megazyme RS assay kit (K-RSTAR, Megazyme, CT, USA), according to the manufacturer’s manual. The RS content of rice and corn starch was 2.33% and 6.69%, respectively, and the degree of starch degradation by amylase enzyme in vitro was 97.0% and 92.5%, respectively. The rice and corn starch with increased RS content were used as raw ingredients for each experimental diet.
Diets were prepared according to a previously described method with minor modifications [21 (link)]. The raw materials of the diets were commercially available as food products that were mixed, heated, molded, cut, dried, and cooled. The experimental diets used to evaluate nutrient digestibility were prepared separately using the same method; each experimental diet was supplemented with 0.5% chromium oxide.Table 1 lists the formulation ratios and chemical compositions of the diets used in this study.
Diets were prepared according to a previously described method with minor modifications [21 (link)]. The raw materials of the diets were commercially available as food products that were mixed, heated, molded, cut, dried, and cooled. The experimental diets used to evaluate nutrient digestibility were prepared separately using the same method; each experimental diet was supplemented with 0.5% chromium oxide.
8
Resistant Starch Analysis in Rice
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RS contents were analyzed in cooked rice (mashed and un-mashed) and rice flour (raw and gelatinized). To prepare cooked rice, 1 g polished rice was washed twice with distilled water and cooked with 1.5 volumes of water (w/w of dry polished rice) in 15 mL plastic test tubes using a rice cooker (NS-WF10, Zojirushi, Osaka, Japan). Then, 250 mg mashed and un-mashed cooked rice were transferred to new tubes. To prepare rice flour, polished rice was ground to a fine powder using a mortar and pestle, and sieved through a 100 μm colander. For the raw rice flour test, 100 mg rice flour was transferred to a tube. For the gelatinized rice flour test, 100 mg rice flour was boiled with two volumes of water (w/w) for 10 mins and the resulting rice pastes were ground again with a pestle and mortar. Samples were digested in 4 mL digestive enzymes using the RS assay kit (Megazyme, Bray, Ireland) containing porcine pancreatic α-amylase (120 U) and amyloglucosidase (12 U) at 37 °C for 16 h by continuous shaking. RS contents were measured as described previously (Tsuiki et al. 2016 (link)).
9
Resistant Starch Measurement in Rice Flour
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The RS of cooked rice flour was measured according to the Association of Official Analytical Collaboration International (AOAC) method (2002.02), with a slight modification, using an RS assay kit (Megazyme, Ltd., Wicklow, Ireland). Each sample (100 mg; dry matter) was digested with pancreatin and amyloglucosidase at 37 °C for 6 h, and the glucose content was measured using a spectrophotometer at 510 nm.
10
Measuring Cecal Resistant Starch
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