2. Starch granule degrading enzymes

2. Starch granule degrading enzymes
Starch’s semi-crystalline nature, an ungelatinised starch granule is digested much more slowly by starch hydrolysing enzymes than a gelatinized granule or a starch solution. During hydrolysis a range of different morphologies may be observed, which range from pitting of small holes, shell formation, and surface erosion. The mode of enzymatic degradation is dependent both on the
Enzymatic treatment at sub-gelatinization temperature led to porous starch. Porous starch is attracting very much attention for its absorption and shielding ability in many food applications.
Highlight :
• α-amylase or amyloglucosidase action on corn starch was studied
• Pasting and thermal properties of porous starch depended on the enzyme used
• Porous starch was more susceptible to enzymatic digestion
Using enzyme: fungal α-amylase (AM) or amyloglucosidase (AMG)
α-Amylase can hydrolyze the (1→4) α-glucosidic bonds of starch in an endo-action. Hydrolysis occurs in a random fashion at any (1→4)-linkage within the starch chain to rapidly reduce the molecular size of starch and the viscosity of the starch solution during pasting.
Amyloglucosidase is an exo-acting enzyme that catalyzes the hydrolysis of both α-D-(1→4) and α-D-(1→6)-linkages from the non-reducing ends of the starch chain. Numerous researchers have investigated enzymatic hydrolysis of starches from cereals, roots, tubers, and legumes in terms of enzyme adsorption, action pattern, extent of hydrolysis, degree of crystallinity and hydrolysis products

Fig. Potato starch granules before (left) and after degradation by the amylases from Paenibacillus granivorans (middle) and Microbaterium aureum (right). (A5)
• Method
Method 1: Preliminary assays were carried out for optimizing enzymatic reactions (starch quantity and pH), and pH 4.0 was selected for AMG reaction and pH 6.0 in the case of AM modification. The quantity of enzymes was based on previous experiments, where the amount of enzyme required to hydrolyze 50% of the starch (15%, w/v) at 95ºC for 10 min was selected.
Starch (5.0 g) was suspended in 25 mL of 20mM NaH2PO4 buffer at pH 6.0 or in sodium acetate buffer at pH 4.0, those starch samples were referred as control-6 or control-4, respectively. For obtaining the enzymatic treated starches, enzymes (4 U of AMG /g starch and 5 U of AM /g starch) were added to the starch suspension. Samples were kept in a shaking water bath (50 rpm) at 50 ºC for 24 hours. Then, 50 mL of water were added and suspensions were homogenized. Samples were centrifuged for 15 min at 7,000×g and 4 ºC. Starches were washed again and centrifuged at the same conditions as before. Supernatants were pooled together and boiled in a water bath for 10 min to inactivate the enzymes before any further analyses (hydration properties and iodine binding values). Sediments containing starch were freeze-dried and kept at -25ºC for further thermal, biochemical and microstructural analyses. Four batches were prepared for each treatment (A2)
Method 2: Enzyme modification of starch was achieved using the enzyme α -amylase (Porcelain pancreatic amylase). 40% starch slurry was prepared by adding 40gm of starch to 100ml of distilled water, and enzyme (100µl) was added. The samples were allowed to stand at room temperature for 6 h. The flasks were then kept in the incubator at 50oC for 3h. After incubation, the supernatant was decanted and the starch was filtered and dried in the oven at 500 C. (A31)
3. Starch transferases
α 1,4-Glucanotransferases catalyse the cleavage of α-1,4 glucosidic bonds and the transfer of the newly formed reducing end group (donor) to a non-reducing saccharide unit (acceptor) with the formation of a new α -1,4 glucosidic bond. Donor and acceptor may originate from the same type of molecule or from different types of molecules. Several action patterns may be distinguished. Firstly, a single low molar mass maltooligosaccharide molecule may act both as donor and acceptor. In this case, cyclic saccharides are formed. Familiar examples are the cyclodextrins (CDs) with 6, 7, or 8 glucose units in the ring, which are formed by cyclodextrin glucosyltransferase. The net result of this type of activity is a decrease in the average molar mass. CDs are prepared by incubating a starch liquefact of DE < 10 with CGT-ase from Bacillus macerans (Riisgaard, 1990) or from thermostable microorganisms like Thermoanaerobacter species (Starnes, 1990). By addition of a complexing solvent, a crystalline inclusion complex is formed that can be separated from the mother liquor and refined by redissolution in water and recrystallisation. In a non-solvent process, the linear malto-oligosaccharides are degraded by amylolysis and cyclodextrin is recovered by partial evaporation and crystallisation. Like amylose, CDs have the ability to form crystalline complexes with a number of inclusion compounds. CDs have been widely explored as drug and flavour carriers (Szejtli, 1998). A novel development of randomly methylated α -CD is its use as an aid in emulsion polymerisation, e.g. in the surfactant-free production of polystyrene and polymethylmethacrylate latex particles in aqueous media (Storsberg et al., 2003). A different situation arises when the donor is transferred either to a different acceptor molecule or to a different side chain on the same molecule, e.g., in amylopectin. An interesting example is the so-called disproportioning enzyme or D-enzyme, also named amylomaltase because it catalyses the transfer of maltose from starch to glucose. A well known source of this enzyme is the potato, but a thermostable amylomaltase has also been isolated from Thermus thermophilus. Limited action of this enzyme on gelatinised starch leads to the progressive disappearance of amylose and the formation of amylopectin with a broader chain-length distribution. During this process the granular remnants dissolve completely and a low viscous molecular solution is obtained with a shift in iodine absorption maximum to lower wavelengths. Interestingly, these solutions form turbid rubber-like gels with a relatively high modulus on cooling at concentrations as low as 3%, which melt again on heating to c. 70oC. Amylomaltase-modified starch is a potential gelatin replacer in applications where gel transparency is not an issue. At higher conversions with this enzyme, cyclisation reactions on both linear and branched molecules occur, leading to relatively high molar mass starch products with a limited tendency to retrograde (Takaha et al., 1996, 1997).
A third situation arises when a different linkage type is formed during the transfer reaction. The action of starch branching enzyme (SBE) involves breaking of an -1,4-linkage and the concomitant formation of an -1,6-linkage. Thermostable branching enzymes have been isolated from a limited number of sources, e.g., Bacillus stearothermophilus.
The main effect is an overall reduction of amylopectin chain length, thereby reducing the propensity of starch to retrograde and increasing the solubility of starch in water. The fact that this action also results in the elimination of amylose and is not accompanied by the formation of maltose as a by-product makes SBE more useful than α-amylase. Involvement of a newly formed reducing end group into an α-1,6 linkage on the same molecule will also lead to the formation of cyclic molecules (Takata et al., 1996, 1997). (A5)

Kết quả (Việt) 1:

2. bột hạt làm giảm đi enzyme Tính chất tinh thể bán của tinh bột, một ungelatinised tinh bột hạt tiêu hóa chậm nhiều hơn của tinh bột hydrolysing enzyme hơn một hạt gelatinized hoặc một giải pháp tinh bột. Trong quá trình thủy phân một loạt các khác nhau morphologies có thể được quan sát thấy, mà phạm vi từ rỗ nhỏ lỗ, vỏ hình thành, và xói mòn bề mặt. Các chế độ của enzym suy thoái là phụ thuộc cả hai trên cácEnzym để điều trị ở nhiệt độ tiểu gelatinization dẫn tới xốp tinh bột. Tinh bột xốp thu hút rất nhiều sự chú ý cho sự hấp thụ của nó và bảo vệ các khả năng trong nhiều ứng dụng thực phẩm. Đặc trưng:• Α-amylase hoặc amyloglucosidase hành động trên tinh bột ngô được nghiên cứu• Dán và nhiệt thuộc tính của tinh bột xốp phụ thuộc vào men tiêu hóa được sử dụng• Xốp tinh bột là dễ bị enzyme tiêu hóa Bằng cách sử dụng enzym: nấm α-amylase (AM) hoặc amyloglucosidase (AMG)Α-Amylase có thể hydrolyze các trái phiếu α-glucosidic (1→4) của tinh bột trong một hành động endo. Thủy phân xảy ra một cách ngẫu nhiên tại bất kỳ (1→4)-các liên kết trong chuỗi tinh bột để nhanh chóng giảm kích thước phân tử của tinh bột và độ nhớt của các giải pháp tinh bột trong dán. Amyloglucosidase là một loại enzyme exo tác catalyzes thủy phân α-D-(1→4) và α - D-(1→6)-các liên kết từ phòng không giảm đầu của chuỗi tinh bột. Nhiều nhà nghiên cứu đã điều tra các enzym thủy phân tinh bột ngũ cốc, rễ, củ và đậu trong điều khoản của enzym hấp phụ, mô hình hành động, mức độ thủy phân, mức độ crystallinity và thủy phân các sản phẩm Fig. Potato starch granules before (left) and after degradation by the amylases from Paenibacillus granivorans (middle) and Microbaterium aureum (right). (A5)• MethodMethod 1: Preliminary assays were carried out for optimizing enzymatic reactions (starch quantity and pH), and pH 4.0 was selected for AMG reaction and pH 6.0 in the case of AM modification. The quantity of enzymes was based on previous experiments, where the amount of enzyme required to hydrolyze 50% of the starch (15%, w/v) at 95ºC for 10 min was selected.Starch (5.0 g) was suspended in 25 mL of 20mM NaH2PO4 buffer at pH 6.0 or in sodium acetate buffer at pH 4.0, those starch samples were referred as control-6 or control-4, respectively. For obtaining the enzymatic treated starches, enzymes (4 U of AMG /g starch and 5 U of AM /g starch) were added to the starch suspension. Samples were kept in a shaking water bath (50 rpm) at 50 ºC for 24 hours. Then, 50 mL of water were added and suspensions were homogenized. Samples were centrifuged for 15 min at 7,000×g and 4 ºC. Starches were washed again and centrifuged at the same conditions as before. Supernatants were pooled together and boiled in a water bath for 10 min to inactivate the enzymes before any further analyses (hydration properties and iodine binding values). Sediments containing starch were freeze-dried and kept at -25ºC for further thermal, biochemical and microstructural analyses. Four batches were prepared for each treatment (A2)Phương pháp 2: Enzyme phân loại tinh bột đã đạt được bằng cách sử dụng enzym α-amylase (sứ tuyến tụy amylase). 40% tinh bột bùn đã được chuẩn bị bằng cách thêm 40gm của tinh bột đến 100ml nước cất, và men tiêu hóa (100µl) đã được bổ sung. Các mẫu đã được cho phép để đứng ở nhiệt độ phòng trong 6giờ. Các bình được sau đó giữ trong các vườn ươm tại 50oC cho 3h. Sau khi ủ bệnh, supernatant decanted và tinh bột được lọc và khô trong lò ở 500 C. (A31)3. tinh bột transferasesα 1,4-Glucanotransferases catalyse the cleavage of α-1,4 glucosidic bonds and the transfer of the newly formed reducing end group (donor) to a non-reducing saccharide unit (acceptor) with the formation of a new α -1,4 glucosidic bond. Donor and acceptor may originate from the same type of molecule or from different types of molecules. Several action patterns may be distinguished. Firstly, a single low molar mass maltooligosaccharide molecule may act both as donor and acceptor. In this case, cyclic saccharides are formed. Familiar examples are the cyclodextrins (CDs) with 6, 7, or 8 glucose units in the ring, which are formed by cyclodextrin glucosyltransferase. The net result of this type of activity is a decrease in the average molar mass. CDs are prepared by incubating a starch liquefact of DE < 10 with CGT-ase from Bacillus macerans (Riisgaard, 1990) or from thermostable microorganisms like Thermoanaerobacter species (Starnes, 1990). By addition of a complexing solvent, a crystalline inclusion complex is formed that can be separated from the mother liquor and refined by redissolution in water and recrystallisation. In a non-solvent process, the linear malto-oligosaccharides are degraded by amylolysis and cyclodextrin is recovered by partial evaporation and crystallisation. Like amylose, CDs have the ability to form crystalline complexes with a number of inclusion compounds. CDs have been widely explored as drug and flavour carriers (Szejtli, 1998). A novel development of randomly methylated α -CD is its use as an aid in emulsion polymerisation, e.g. in the surfactant-free production of polystyrene and polymethylmethacrylate latex particles in aqueous media (Storsberg et al., 2003). A different situation arises when the donor is transferred either to a different acceptor molecule or to a different side chain on the same molecule, e.g., in amylopectin. An interesting example is the so-called disproportioning enzyme or D-enzyme, also named amylomaltase because it catalyses the transfer of maltose from starch to glucose. A well known source of this enzyme is the potato, but a thermostable amylomaltase has also been isolated from Thermus thermophilus. Limited action of this enzyme on gelatinised starch leads to the progressive disappearance of amylose and the formation of amylopectin with a broader chain-length distribution. During this process the granular remnants dissolve completely and a low viscous molecular solution is obtained with a shift in iodine absorption maximum to lower wavelengths. Interestingly, these solutions form turbid rubber-like gels with a relatively high modulus on cooling at concentrations as low as 3%, which melt again on heating to c. 70oC. Amylomaltase-modified starch is a potential gelatin replacer in applications where gel transparency is not an issue. At higher conversions with this enzyme, cyclisation reactions on both linear and branched molecules occur, leading to relatively high molar mass starch products with a limited tendency to retrograde (Takaha et al., 1996, 1997). A third situation arises when a different linkage type is formed during the transfer reaction. The action of starch branching enzyme (SBE) involves breaking of an -1,4-linkage and the concomitant formation of an -1,6-linkage. Thermostable branching enzymes have been isolated from a limited number of sources, e.g., Bacillus stearothermophilus.The main effect is an overall reduction of amylopectin chain length, thereby reducing the propensity of starch to retrograde and increasing the solubility of starch in water. The fact that this action also results in the elimination of amylose and is not accompanied by the formation of maltose as a by-product makes SBE more useful than α-amylase. Involvement of a newly formed reducing end group into an α-1,6 linkage on the same molecule will also lead to the formation of cyclic molecules (Takata et al., 1996, 1997). (A5)