Preparation Of White Kidney Bean α-amylase Inhibitor And Its Application In Instant Porridge With Low GI

White kidney bean α-amylase inhibitor(α-AI) was a natural substance preventing and controlling diabetes and obesity, while trypsin inhibitor(TI) and phytohemagglutinin(PHA) in beans affected its physiological functions and biological safety. Extract of white kidney bean α-AI with low TI activity and PHA activity had a good prospect to be applied in food. This paper was to explore an industrial process for the preparation of α-AI reducing TI and PHA activities. Following the investigation on the physical and chemical discrepancies of α-AI, TI and PHA, the crude extract of α-AI was prepared by ultra-filtration, heat treatment and spray drying and then was applied into instant porridge.Firstly, white kidney bean A10 was determined to be the best breed for the preparation of α-AI by comparing the extraction rates of α-AI, TI and PHA from 12 kinds of beans in China. The impacts of various processing methods, for example, blanching beans, heating water extract, enzymatic hydrolysis and isoelectrical precipitation, and simulated gastrointestinal treatment, on the activities of α-AI, TI and PHA were investigated. The acidic and alkaline stability of α-AI, TI and PHA was relatively good at temperature lower than 50℃. At high temperature(>70℃), both thermostability and acidic and alkaline stability of α-AI were lower than TI and PHA. Only when the condition was 70℃/pH 3.0 did α-AI and PHA own the similar stability and their activity losses were 60.00% and 53.78%, respectively. α-AI, TI and PHA were resistant to Flavourzyme and Neutrase, while Alcalase inactivated all of them. During isoelectrical precipitation, α-AI rarely precipitated and TI(>71.62%) and PHA(>80.49%) tended to stay in the supernatant. Simulated intestinal treatment had no significant effect on the activities of α-AI, TI and PHA(P>0.05). During simulated gastric treatment, the acidic condition was the main factor that affected α-AI, TI and PHA. Among them, the loss(38.37%) of α-AI activity was the greatest. Pepsin did not affect α-AI significantly(P>0.05), while it caused losses in TI(13.33%) and PHA(10.51%).Secondly, parameters for preparing water extract of white kidney bean were optimized: the ratio of material to liquid was 1:5, flour mesh was 80, extraction temperature was room temperature, extraction time was 2 h, pH was 6.4. Preliminary purified protein was obtained from water extract by hydrolysis using Flavourzyme, isoelectrical precipitation at pH 3.6 and 70% ethanol precipitation. During the process, the purification folds of α-AI, TI and PHA were 3.70, 2.82 and 3.50, respectively. Analyzing the protein by SDS-PAGE, the relative molecular weight(about 1.5×10~4) of TI was obviously different from α-AI(5.67×10~4) and PHA(>6.0×10~4). Ultra-filtration was applied to separate TI from water extract and the optimized parameters were as follows: pH of water extract was 10.0; inlet pressure was 0.04 MPa; inlet temperature was 40℃; protein concentration was 24.31 mg/mL. Compared with water extract, the specific activity of TI in the trapped fluid reduced by 90.02% and those of α-AI and PHA increased by 171% and 184%, respectively.Finally, crude extract of α-AI was prepared by ultra-filtration and heat treatment. The activities of α-AI, TI and PHA in the extract were 162.85 U/g, 13.33×10~3 U/g and 14.04 U/g, respectively. During the pilot test for preparing crude extract, those activities in the extract were 96.91 U/g, 7.97×10~3 U/g and 11.88 U/g, respectively. Compared with Phase 2, the physiological function and biological safety of the crude extract obtained by the process were reliable. The inhibitory properties of the crude extract were also studied. The inhibition of α-AI on α-amylase was non-competitive and the variation of starch did not affect the inhibition. With the extension of preincuation time, the inhibition significantly increased(P<0.05). When α-amylase activity exceeded 1.125 U, the inhibition decreased significantly(P<0.05). As the addition of crude extract in rice porridge increased, the GI of rice porridge continuously decreased. When the addition of the crude extract was 3%(w/w), the GIs of instant porridge(rice porridge, oat porridge, barley porridge and lotus seed porridge) were all lower than 50, which indicated these porridge belonged to the category of low GI food.