Research On The Preparation Of Porous Starch By α-maltotriohydrolase In Collaboration With Amyloglucosidase And Its Application In Embedding Proanthocyanidins

Porous starch,also referred to as microporous starch,has been recognized for its high porosity,strong adsorption,large specific surface area,and high loading capacity,making it a suitable carrier for unstable and sensitive substances as well as an adsorbent for volatile substances.The process of enzymatic modification of granular starch has been widely adopted for the development of porous starch due to its gentle and efficient nature,as well as its environmental friendliness.Therefore,the present study aimed to compare and explore the hydrolytic mechanisms of various enzymes,including glucan 1,4-α-maltotriohydrolase(AMTS),porcine pancreatic α-amylase(PA),maltogenicα-amylase(MA),and amyloglucosidase(AMG),on granular starch in terms of their effects on morphology,internal cavity structure,short-range order,lamellar structure,relative crystallinity,and adsorption properties.Subsequently,we prepared porous starch via the cooperative action of AMTS and AMG and investigated the impact of the two enzymes’addition methods on the structure and function of porous starch.We identified the best preparation conditions for porous starch and used it as a carrier to encapsulate PC.The structural and functional properties of the resulting complexes were also systematically studied.In summary,our research aimed to identify optimal conditions for the preparation of a highly effective carrier for embedding PC.The primary focus of this research and its findings can be summarized as follows:(1)The structure and physicochemical properties of enzymatically hydrolyzed granular starch were examined to elucidate the action mechanism of AMTS.The granular starch hydrolyzed by AMTS,MA,and PA formed a large internal cavity structure with the extension of enzymatic hydrolysis time,and the short-range ordered structure,relative crystallinity,specific surface area,pore volume,and adsorption capacity first increased and then decreased.But the cavities created inside granular starch were smaller and the holes created by AMG hydrolysis were deep and narrow.In conclusion,AMTS primarily acts on the amorphous region of the granular starch,followed by the crystalline region using an"inside-out" centroidal hydrolysis mode.(2)By controlling variables,the further study was conducted to investigate the effects of the proportion of AMTS and AMG,as well as the sequence of enzyme action,on the structure and function of prepared porous starch.The yield of porous starch prepared by AMG treatment for 6h followed by AMTS treatment for 6 h(AM-AG),AMTS treatment for 6 h followed by AMG treatment for 6 h(AG-AM),and the use of composite enzymes with different addition ratios(AMTS:AMG 1:5-4:2)was lower than that of AMTS and AMG hydrolysis of granular starch under the optimal pH conditions.This indicates that the synergistic treatment of AMTS and AMG can improve the efficiency of preparing porous starch.Compared with raw starch and porous starch prepared by AMTS or AMG,the composite enzyme prepared porous starch has an increased specific surface area,pore volume,oil absorption rate,and water absorption rate.Moreover,according to laser confocal images,it can be observed that enzyme synergistic treatment is more conducive to the generation of cavity structures.In addition,compared with raw starch,the short-range order and relative crystallinity of porous starch increased,and the hydrolysis rate decreased in the simulated digestion in vitro,indicating that the structural stability of porous starch was improved,which was more conducive to the realization of targeted slow release in vivo.The three types of porous starch prepared by AM-AG,AMTS:AMG 2:4,and 3:3 have the best adsorption capacity and are the optimal treatment methods for preparing porous starch.Compared with the original starch,the oil absorption of porous starch prepared by AM-AG,AMTS:AMG 2:4,and 3:3 methods increased to 124.31%、117.81%、113.74%,respectively,and the water absorption increased to 156.45%、153.12%、143.09%,respectively.(3)The selection of porous starch with the highest adsorption capacity has allowed for efficient embedding of PC.Raw starch(CS-PC)and porous starch encapsulated anthocyanin complexes using AM-AG,AMTS:AMG 2:4 and 3:3(AMAG-PC,2:4-PC,3:3-PC)were found to have embedding quantities between 3.06-9.71 mg/g.SEM,FTIR,and XRD analysis have confirmed the amorphous state in which PC attaches to porous starch,connected through hydrogen bonding.As for storage stability,AMAG-PC>2:4-PC>3:3-PC>CS-PC with 81.6%,80.54%,79.14%,and 71.4%retention rates after 10 days at 37℃,respectively.Additionally,the scavenging ability of DPPH free radical was highest in AMAG-PC with 93.56%.Furthermore,the complex has demonstrated targeted and slow-release capabilities in simulated digestion in the gut and stomach.Ultimately,the use of porous starch prepared by AM-AG is the optimal carrier for embedding PC.