CN104404107A

CN104404107A - Application of novel moderate temperature alpha-amylase

Info

Publication number: CN104404107A
Application number: CN201410713567.3A
Authority: CN
Inventors: 崔中利; 李周坤; 黄彦
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2014-11-28
Filing date: 2014-11-28
Publication date: 2015-03-11

Abstract

The invention discloses application of novel moderate temperature alpha-amylase, that is the invention discloses application of alpha-amylase AmyM of which the amino acid sequence is shown in SEQ ID No.1, for preparing syrup which is rich in maltohexaose through processingh. The syrup comprises the following components: more than 50% of oligosaccharide(higher than maltotetrose, 8-10% of maltotriose, lower than 6% of malt sugar, and lower than 35% of other kinds of oligosaccharide. When the syrup prepared from the amylase disclosed by the invention is compared with the conventional maltohexaose syrup, the glucose content is lower, the maltohexaose content is higher, the sugar components after enzyme treatment are relative single, and the application range is wider. Due to the good characteristic of the novel alpha-amylase AmyM disclosed by the invention, so that the preparation process of the oligosaccharide is simple, and products are easy to purify.

Description

A kind of application of Novel middle-temperature α-amylase

Technical field

The invention belongs to starch and β-amylose goods technology of preparing, food additive technology and starch-containing product treatment field, relate to a kind of application of Novel middle-temperature α-amylase, be specifically related to a kind of Novel middle-temperature α-amylase is rich in MALTOHAXAOASE syrup application in the various starch preparation of process.

Background technology

Oligomeric maltose refer generally to by 3 ?the polymer that forms of 9 monose, comprise trisaccharide maltose, maltotetrose, maltopentaose and MALTOHAXAOASE etc.Oligosaccharide malt syrup mainly adopts high-grade maize starch, the syrup obtained through amylorrhexis, and product sweetness is low, and mouthfeel is good.Through Oligomeric maltose prepared by enzymic hydrolysis, after edible, blood sugar rises slowly, and thermal value is low, has good health-care effect for diabetes, cardiovascular patient and obese person.The insulin response that Oligomeric maltose causes is steady, overcomes in motion the rebound hypoglycemic reaction taken single, double sugar and cause, has important application prospect in sports beverages.Produce high MALTOHAXAOASE content syrup due to diastatic character used, output is lower, and be rich in MALTOHAXAOASE syrup Off ジオリ go G67 in the market, the sugared total content of its MALTOHAXAOASE/seven only has about 40%.And other non-product MALTOHAXAOASE α ?the low six sugared content of its Fructus Hordei Germinatus of syrup prepared of amylase generally lower than 30%.

Malto-oligosaccharide (3-6 sugar) can stop the recrystallization of amylopectin in bread storage process, improves bread.In the flour products courses of processing such as bread at high temperature can there is gelatinization in starch granules, and in cooling/storage process, amylopectin wherein can crystallization again, thus causes bread hardening, reduced shelf-life.In the flour products such as bread, add warm amylase in high yield MALTOHAXAOASE can produce the Oligomeric maltoses such as a large amount of MALTOHAXAOASE, increase fermentation rate, improve volume and the quality of product, change the mouthfeel of flour products, the fresh keeping time of elongated surfaces goods.Nineteen fifty-five U.S. FDA and Britain 1963 respectively through after " general recognized as safe " (GeneralRecognized As Safe, GRAS) checking, fungal amylase allows to be used in bread industry as bread additives.But fungal amylase product is maltose, it suppresses the effect of amylopectin recrystallization poor.2009, come from the GRAS certification of maltotetrose enzyme by FDA of Pseudomonassaccharophila, the additive as baking bread was used.The Novel middle-temperature amylase of the high yield MALTOHAXAOASE that the present invention finds, due to its high yield MALTOHAXAOASE activity, has the potentiality that crop baking bread adds enzyme.

Summary of the invention

The object of the invention is for the deficiencies in the prior art, a kind of application of Novel middle-temperature α-amylase is provided.

Object of the present invention realizes by following technical scheme:

α-amylase AmyM of the present invention is the applying date is on February 4th, 2013; the α ?Isosorbide-5-Nitrae amylase excision enzyme protected is asked in the Chinese invention patent that the patent No. is called for " ZL 201310043628.5 " name " a kind of α-amylase and gene thereof, engineering bacteria containing this gene and application thereof ".This diastatic preparation method refers to " ZL 201310043628.5 " patent specification embodiment 1 ~ 2.

Aminoacid sequence for the α-amylase AmyM shown in SEQ ID NO.1 process starch prepare the application of being rich in MALTOHAXAOASE syrup.

Wherein, described syrup components consists of: the above oligosaccharide contg more than 50% of maltotetrose; Trisaccharide maltose content 8-10%, maltose content is lower than 6% (comprising 6%), and other oligose are lower than 35% (comprising 35%), and the above oligose of described maltotetrose is the oligose that 4 ~ 7 monose polymerizations are formed; Other described oligose are the oligose of eight and above monose polymerization formation.

Seven sugar and the consisting of of following oligose in described syrup: G1-glucose 3-5%; G2-maltose 5-7%; G3-trisaccharide maltose 6-8%; G4-maltotetrose 7-9%; G5-maltopentaose 8-10%; G6-MALTOHAXAOASE 55-60%; G7-Fructus Hordei Germinatus seven sugar about 4-6%.

The preferred corn in source of described starch, wheat, potato.

Aminoacid sequence is preparing the application in sports beverages, fermented drink and/or beer fermentation for the α-amylase AmyM enzymatic starch product shown in SEQ ID NO.1.

Malto-oligosaccharide can reach the low sugariness effect of food when not changing the original flavor of food.Its moisturizing, hygroscopic property stabilization energy maintain suitable moisture, prevent starch retrogradation in baked product, maintain the flexibility of food, extend the shelf life.The α-amylase of high yield MALTOHAXAOASE of the present invention has good using value preparing in low dextrose, high MALTOHAXAOASE content syrup, and can extend the shelf-lives of roasted flour products as additive.

Beneficial effect

The patent No. is that " ZL 201310043628.5 " has the work of high enzyme although disclose α-amylase, and middle temperature characteristics, salt tolerance and satisfactory stability, does not disclose the composition of its product.This amylase of discovery pleasantly surprised in follow-up study of contriver has aforesaid plurality of advantages incessantly, and in its enzymolysis product, MALTOHAXAOASE occupies very large ratio, more than 50%.And the syrup that amylase of the present invention obtains is compared with MALTOHAXAOASE syrup with existing maltose syrups, production of maltotetraose syrup, glucose content is lower, after ferment treatment, sugar component is relatively single, applicability is wider, and the good characteristic of Novel α amylase AmyM owing to the present invention relates to, make the preparation process of oligose simple, the easy purifying of product.

Accompanying drawing explanation

Fig. 1 utilizes application number to be that the α-amylase process uncooked amylum product HPLC that " 201310043628.5 " relate to analyzes

Fig. 2 utilizes application number to be that oligosaccharide compositions HPLC in " 201310043628.5 " α-amylase process uncooked amylum after product of relating to analyzes

A figure is Fructus Hordei Germinatus oligose standard substance HPLC analysis chart; B figure is oligosaccharide compositions HPLC analysis chart in hydrolysate after AmyM process uncooked amylum, and wherein M1 is glucose, and M2 is maltose, and M3 is trisaccharide maltose, and M4 is maltotetrose, and M5 is maltopentaose, and M6 is MALTOHAXAOASE, and M7 is Fructus Hordei Germinatus seven sugar.

Fig. 3 utilizes application number to be that oligosaccharide compositions TLC in " 201310043628.5 " α-amylase process uncooked amylum after product of relating to analyzes

Wherein 1 ?7 for being respectively glucose, maltose, trisaccharide maltose, maltotetrose, maltopentaose, MALTOHAXAOASE, Fructus Hordei Germinatus seven saccharide, 8 ?10 for Zulkovsky starch was through α-amylase AmyM process 10 minutes, the product after 30 minutes and 1 hour forms

Fig. 4 commercialization amylase BAN800 and Novel α amylase contrast treatment uncooked amylum after product HPLC analyzes

Wherein a figure: Novel α amylase different time (1.5h, 3h, 5h, 12h) processes uncooked amylum HPLC schemes; B schemes: commercialization amylase BAN800 and Novel α amylase 3h process uncooked amylum HPLC schemes; C schemes: commercialization amylase BAN800 and Novel α amylase 12h process uncooked amylum HPLC schemes.

Embodiment

Embodiment 1 Xin Xing α ?diastatic preparation

Refer to Chinese invention patent 201310043628.5 specification sheets embodiment 1 ~ 2.

Embodiment 2. Xin Xing α ?amylase activity measure and product thin layer chromatographic analysis

1.0ml 0.5% Zulkovsky starch (50mM pH7.0Tris ?HCl damping fluid) (being first heated to 50 DEG C) adds 1 μ l enzyme liquid (stoste dilute 10 times after), after reacting 10min under 50 DEG C of conditions, after adding 2ml DNS and constant volume to 3ml, boiling water bath 5min, cooling, in OD ₅₄₀lower its light absorption value of mensuration, control group is heat inactivation 1 μ l enzyme liquid (stoste dilute 10 times after), processes the same.According to glucose standard curve calculate survey diastatic activity, the enzyme amount needed for reducing sugar that under 50 DEG C of conditions, per minute produces 1 μm of ol is defined as a unit of activity.

Amylase AmyM process starch 10 minutes, utilize thin plate chromatography (TLC) to analyze its product component after 30 minutes and 1 hour, TLC condition is: developing agent is propyl carbinol: methyl alcohol: water=4:2:1, and developer is H ₂sO ₄/ methyl alcohol (1:1, v/v), color condition be 90 DEG C dry 10min, the results are shown in Figure 3, find this enzyme act on starch after primary product be MALTOHAXAOASE.

Embodiment 3. Xin Xing α ?amylase hydrolyzes starch produce Oligomeric maltose based on MALTOHAXAOASE

By waxy corn starch (being not limited to waxy corn), be made into the suspension of 8%, with phosphate buffer solution, pH value is adjusted to 6, be placed in 99 DEG C of thermostatic water-circulator bath pots to complete gelatinization, add after afterwards the starch paste of complete gelatinization being cooled to 50 DEG C α-amylase AmyM amylase α ?the addition of amylase be 5% (being equivalent to 100U every g dry starch matrix) of starch on dry basis, and collect 3 respectively, the product of 12h, be warmed up to 100 DEG C of insulation 20min after enzymolysis 12h to go out enzyme, the product of collection uses the analysis of KS803 post.Chromatographic column be Shodex KS ?803 (8mm × 300mm), detector is Composition distribution.Sample preparation becomes the solution of 2g/L, and cross the film of 0.45 μm, take ultrapure water as moving phase, sample size 20 μ L, flow velocity is 0.8mL/min, column temperature 70 DEG C, and detector temperature is 50 DEG C.

Product analysis after W-Gum enzymolysis finds (Fig. 1), more than tetrose, and 52.24%; Trisaccharide, 8.14%, disaccharides, 5.94%; Other oligosaccharides, 33.68%.By analyzing the relative content (Fig. 2) of its concrete malto-oligosaccharide component to enzymolysis product HPLC, find G1 ?glucose, 3.9%; G2 ?maltose, 6.2%; G3 ?trisaccharide maltose, 8.1%; G4 ?maltotetrose, 8.3%; G5 ?maltopentaose, 8.8%; G6 ?MALTOHAXAOASE, 59.4%; G7 ?Fructus Hordei Germinatus seven sugar, 5.3%.

Choose commercialization amylase BAN800 to test as a comparison simultaneously, by above-mentioned waxy corn starch, be made into the suspension of 8%, with phosphate buffer solution, pH value is adjusted to 6, be placed in 99 DEG C of thermostatic water-circulator bath pots to complete gelatinization, add after afterwards the starch paste of complete gelatinization being cooled to 70 DEG C BAN800 α ?amylase, amylase addition and subsequent detection analytical procedure the same, the results are shown in Figure 4.

Product analysis finds that commercialization amylase BAN800 (purchased from Novozymes Company) hydrolysate is more complicated, grows oligose less at 3h, just tends to stablize at 12h, its G6 ?MALTOHAXAOASE output be starkly lower than amylase of the present invention.

Embodiment 4. utilize Xin Xing α ?amylase preparation be rich in Fructus Hordei Germinatus MALTOHAXAOASE syrup

Utilize Xin Xing α ?amylase preparation be rich in Fructus Hordei Germinatus MALTOHAXAOASE syrup and can realize in existing starch machining process.In material-compound tank, powder slurry breast is transferred to the outstanding slurry of 25% ~ 35% starch, pH value Na ₂cO ₃be transferred to 5.0 ?7.0, with pump, powder stock pump is entered Jet liquefier after feed liquid stirs, directly meet and carry out gelatinization spraying wherein powder slurry and steam, drop temperature control 110 ?115 DEG C, from injector, gelatinization feed liquid is out cooled to 50 DEG C, amylase AmyM is added, isothermal holding 3 hours by the amount of 100U/ gram of dry starch.After terminal, feed liquid is sprayed heating 80 DEG C of enzymes that go out.First use filter deslagging filtering, then add activated carbon decolorizing half an hour, carry out decolorization filtering, then liquid glucose is carried out secondary decolourization, after decolouring, carry out ion-exchange.Product requirement is concentrated into by from the five effect falling film evaporators of the liquid glucose after friendship.

Sample and product analysis is carried out to the MALTOHAXAOASE syrup of preparation, find that this syrup MALTOHAXAOASE component accounts for about 53% of total component, other oligosaccharide compositions account for about 4% of total component, other components about 43%, illustrate that this syrup main ingredient is MALTOHAXAOASE, for being rich in MALTOHAXAOASE syrup.

Claims

1. aminoacid sequence for the α-amylase AmyM shown in SEQ ID NO.1 process starch prepare the application of being rich in MALTOHAXAOASE syrup.

2. application according to claim 1, is characterized in that described syrup components consists of: the above oligosaccharide contg more than 50% of maltotetrose, trisaccharide maltose content 8-10%, and maltose content is lower than 6%, and other oligosaccharide contgs are lower than 35%; The above oligose of described maltotetrose is the oligose that 4 ~ 7 monose polymerizations are formed; Other described oligose are the oligose of eight and above monose polymerization formation.

3. application according to claim 1, to is characterized in that in described syrup consisting of of seven sugar and following oligose: G1-glucose 3-5%; G2-maltose 5-7%; G3-trisaccharide maltose 6-8%; G4-maltotetrose 7-9%; G5-maltopentaose 8-10%; G6-MALTOHAXAOASE 55-60%; G7-Fructus Hordei Germinatus seven sugar about 4-6%.

4. application according to claim 1, is characterized in that described starch source is in corn, wheat, potato.

5. aminoacid sequence is preparing the application in sports beverages, fermented drink and/or beer fermentation for the α-amylase AmyM enzymatic starch product shown in SEQ ID NO.1.

6. aminoacid sequence is the application of α-amylase AmyM in bread is baked and banked up with earth shown in SEQ ID NO.1.