JP2787764B2 - Maltotetraose and foods and drinks containing it - Google Patents

Description

Description: BACKGROUND OF THE INVENTION The present invention relates to a novel maltotetrao
And foods and drinks containing it. [0002] Glucose and maltose are produced by converting starch into various types.
Manufactured industrially by saccharification with amylase
I have. In recent years, higher molecular maltooligos have been produced.
Research on various amylase for
Was. [0004] Above all, maltotetraose has a low sweetness.
It is attracting attention as a partially degraded starch with low taste and low sugar content.
It is desired to establish an industrial production method. [0005] Yeast for producing maltotetraose from starch
Element, ie, maltotetraose-forming amylase (EC
3.2.1.60) has been around for a long time
Pseudomonas stutz
eri) Produced from NRRL B-3389.
Are known. For example, US Pat. No. 3,654,082
And in the specification of John F.
And Rosary J. Ackerman (John
F. Robyt and Rosalie J. Acke
rman), Archives of Biochemist
Lee and Biophysics (Archives
of Biochemistry and Bioph
isics), Vol. 145, pp. 105-114 (1
971).
Michael John (Jurgen Schmidta)
nd Michael John)
Biophysica et al. (Biochimica et
Biophysica Acta), vol. 566, vol.
Reported on pages 88-99 (1979).
Yuki et al., Starch Science, Vol. 29, No. 2, 131-1-1
Page 37 (1982), Agricultural and
Biological chemistry (Agricultur)
al and Biological Chemist
ry), Vol. 46, No. 3, pp. 639-646 (1
982) and the same magazine, Vol. 47, No. 8, No. 1761
To 1768 (1983). [0006] However, the shrinkage found in these reports
Domonas Stutzeri NRRL B-3389 or
These maltotetraose-generating amylase have an optimal temperature,
Low stable temperature, insufficient heat resistance for industrial use
It is. In addition, this maltotetraose-forming amino acid
For example, Shoichi Kobayashi et al.
No. 7, in the first to fourth lines on page 2, upper right column,
The enzyme is Pseudomonas stutcheri (Pseudo
monas stutzeri)
Yes, but low productivity (2-5 IU / ml). "
Industrial use, as is evident from
Above is very disadvantageous. Means for Solving the Problems The inventors of the present invention have proposed a
Maltotetra can produce tetraose industrially advantageously
We continued our research on ausogenic amylase. As a result, surprisingly, Pseudomonas
New maltotetrao with improved heat resistance from Tuttsueri
Source amylase can be produced, and
To produce maltotetraose industrially
The present invention has been completed by establishing that it can be produced. Hereinafter, the maltotetraose forming catalyst of the present invention will be described.
The physicochemical properties of Millase will be described. (A) Action Acts on starch to mainly produce maltotetraose. (B) substrate-specific starch, amylose, amylopectin, glycogen and
And β-limit dextrin, and cyclodextrin
Phosphorus, Dextran, Bull Run and Ercinan
Has virtually no effect. (C) Optimum pH: pH 7.0 to about 7.5 at 40 ° C for 20 minutes. (D) Stable pH: pH 6.5 to 9.5 at 1 hour at 40 ° C. (E) Optimum temperature pH 7.0, around 50 ° C for 20 minutes. (F) Stability temperature: pH 7.0, 1 hour, to around 45 ° C. However, calci
Almost complete activity up to around 50 ° C in the presence of
And 70% activity around 55 ° C. (G) Inhibition and stabilization The activity is inhibited by mercury ions. Calcium ion
This improves the thermal stability. (H) Molecular weight SDS-polyacrylamide gel electrophoresis
6,000 ± 1,000. By ultracentrifugation analysis, 47,
2,000 ± 2,000. (I) Isoelectric point polyacrylamide gel The pI was determined by isoelectric focusing.
Near 4.8. (J) UV absorption near 275-280 nm. (K) Purification method It can be carried out according to a general enzyme purification method. That
A specific example will be described in a second embodiment. (1) Activity measurement method Enzyme was added to 5 ml of 1.0 w / v% soluble starch (pH 7.0)
Add 0.2 ml of the solution and react at 40 ° C for 20 minutes.
Stop reaction by adding Somogyi reagent
I let it. The generated reducing sugar is converted to Nelson-Somogyi (Nel).
son-Somogyi) method, one minute per minute.
The amount of the enzyme that cuts the glucosidic bond in chromol is 1 unit.
did. Among the above physicochemical properties, the main properties
About Agricultural and Biological Deer
Le Chemistry Vol. 47, No. 8, 1761-
Pseudomo reported on page 1768 (1983)
Machine from eggplant Stutzeri NRRL B-3389
Compared to the properties of rutotetraose producing amylase,
It is as follows. (1) At the optimum pH, the enzyme of the present invention
While the pH range is as wide as 7.0 to 7.5,
It from NRRL B-3389 has a pH around 8.0
It is. (2) At a stable pH, the enzyme of the present invention has a pH of 6.5 to
While the pH range is as wide as 9.5, NRRL
From B-3389 at around pH 6.5 to 8.5
is there. (3) At the optimum temperature, the enzyme of the present invention has a high temperature of around 50 ° C.
Whereas from NRRL B-3389 is
It is around 45 ° C. (4) At a stable temperature, the enzyme of the present invention has a temperature around 45 ° C.
High temperature up to around 50 ° C with coexisting ions
Whereas that from NRRL B-3389 is 40 ° C.
It is up to the neighborhood. (5) In terms of molecular weight, SDS-polyacrylamide gel
In the electrophoresis method, the enzyme of the present invention is 46,000 ± 1,
NRRL B-3389
It is 55,000. (6) The isoelectric point of the enzyme of the present invention is around 4.8.
In contrast, that from NRRL B-3389 was up to 5.6.
Or 5.3. As described above, the maltotetrao of the present invention
Production amylase is derived from NRRL B-3389.
Compared to this, it has better pH stability and heat resistance, and is industrially
Novel maltotetraose-producing amylor that can be used conveniently
You can say it. The present invention provides a maltotetraose-producing amylor.
The microorganism that produces zeo is not limited. The example shown below
Domonas Stutzeri MO-19 or its variants
A heterologous strain or the like can be advantageously used in the present invention. Pseudomonas stutzeri MO-1
9 was found and isolated from soil in Kayo-cho, Kamifusa-gun, Okayama Prefecture
It is a thing, February 16, 1987,
Deposited with the National Institute of Advanced Industrial Science and Technology and received the microorganism accession number FER
Deposited at MBP-1682. Hereinafter, Pseudomonas stutzeri M
The mycological properties of O-19 are described. A. Morphological properties (a) Cell shape and size Bacillus, 0.5 to 0.6 × 1.0 to 1.2 μm FIG. 1 shows an electron micrograph (× 10,000). (B) Presence or absence of cell polymorphism (c) Presence or absence of motility (d) Epithelial state of flagella One (e) spore, spore spore, spore sac does not form (f) B.) Gram stain negative (g) Acid-fast negative Cultural properties (a) Gravy agar plate culture (28 ° C., 2 days) Bacteria grow rapidly and form 1-3 mm colonies.
Colonies are opaque, moist, brownish-yellow
It is circular and the surface is smooth. (B) Gravy agar slant culture (28 ° C., 2 days) The growth of bacteria is moderate. Colonies are opaque and moist
Moist, brownish-yellow, feathery, with flat ridges
One. No soluble dye is formed. (C) Liquid broth culture (28 ° C, 2 days) One day of culture is slightly turbid, but 2 days of culture is strongly turbid
And grow well. The surface forms a film, gas,
No pigment is formed. (D) Broth puncture culture (28 ° C, 2 days) Growing in a chain at the puncture site. (E) Broth gelatin puncture Culture gelatin does not liquefy. (F) Litmus milk (28 ° C., 5 days) It becomes alkaline by 5 days of culture, and milk coagulates. C. Physiological properties (a) Reduction of nitrate Positive (b) Denitrification reaction Positive (c) MR test Negative (d) VR test Negative (e) Indole formation Negative (f) Hydrogen sulfide formation Positive (g) Starch degradation (H) Use of citric acid Use (i) Use of inorganic nitrogen Ammonium salt, nitrate
(J) Formation of dye used together with acid salt Soluble dye is formed
No (k) Oxidase-positive (l) Catalase-positive (m) Growth range Good growth at pH 4-7, pH 2 or less or pH 8 or less
Does not grow on. It grows well at temperatures between 16 and 35 ° C,
Does not grow below 42 ° C or above 42 ° C. (N) Attitude to oxygen Aerobic (o) OF test Oxidized (o) (p) Presence of acid and gas generation from saccharides D-glucose, D-mannose, lactose, maltose,
Acid is generated from starch, but L-arabinose, D
-Xylose, D-fructose, sucrose, glycerol
No acid is produced from the D-Mannose to gas
Gas, but gas is produced from the other sugars.
Not. (Q) Growth pH pH 7.7 (proteose peptone glucose culture
Ground) (r) Cellulose degradation negative Negative for the above mycological properties
Manual of Deterministic Bacteriology
ー (Bergeys Manual of Dete
rmative Bacteriology) 7th
Edition (1957) and the eighth edition (1974)
Classified as Pseudomonas stutzeri
And this bacterium was used in Pseudomonas stutzeri MO-19.
It was named. The maltotetraose-producing amylor of the present invention
The cultivation for the production of zeolites is in accordance with the usual cultivation method for bacteria.
Then, liquid culture, solid culture and the like are performed. Liquid culture
In this case, it may be allowed to stand still, but shake culture or aeration and stirring
Culture is preferred. As the medium used for the culture,
Contains nutrients required by Pseudomonas bacteria
Carbon source, nitrogen source, inorganic salt, etc.
As appropriate, synthetic medium, semi-synthetic medium, natural medium, etc. containing
To be elected. In particular, the maltotetraose-forming amino acids of the present invention
To increase the production of lase, starch, amylopecti
Starch, such as starch, amylose, and partially hydrolyzed starch
A medium containing a lucium salt or the like is preferable. Generally, the pH is adjusted to neutral or slightly alkaline.
Inoculated with bacteria belonging to the genus Pseudomonas in
Culture is performed at 0 to 35 ° C. for about 0.5 to 5 days. The culture obtained as described above is
Usually used as it is or to remove cells from the culture
The removed supernatant or filtrate is used. If necessary, ammonium sulfate and sodium sulfate
Salting out method using thorium, acetone, ethanol, etc.
Precipitation method, gel filtration method, ion exchange chromatography
For chromatography, affinity chromatography, etc.
It is possible to purify and use these enzymes in a conventional manner.
Therefore, fixed by carrier binding method, cross-linking method, inclusive method, etc.
It can also be advantageously implemented to use it. [0025] Maltotetraose-producing amiller of the present invention
To produce high maltotetraose content
Industrially, starch, amylopectin, amylo
Starch and partially hydrolyzed starch, etc.
Preferably. Also, if necessary, contain starch
In the production of food and drink
Activated amylase, maltotetraose in food and drink
To prevent the aging of starchy food and drink
You can extend your holding. Generally, about 5 to 45% w / w
In the powdery solution, the maltotetraose-forming amylase of the present invention
Zeze is added at a rate of about 1 to 20 units per gram of starchy material,
pH 5 ~ 9, reaction temperature about 40 ~ 70 ° C, 0.5 ~
Let react for 3 days. At this time, the reaction solution is treated with calcium chloride or the like.
Calcium salt with a concentration of about 0.0005 to 0.05 molar
To produce maltotetraose-producing amilla
Improve the heat resistance of the enzyme and make the reaction easier
Can also be advantageously implemented. The maltotetraose content in the reaction product
In order to increase the acid content as much as possible, the starch acid or α-
The degree of liquefaction by the millase is as low as
Less than, preferably less than DE6, malt
Preferred to act on tetraose-forming amylase
No. In the reaction, maltotetraose
Amylases, along with other starch-related enzymes, such as
For example, cyclomaltodextrin glucano transfer
(EC 2.4.1.19), α-amylase (EC
3.2.1.1), β-amylase (EC 3.2.1.1.
2), glucoamylase (EC 3.2.1.3), α-
Glucosidase (EC 3.2.1.20), Pullulana
(EC 3.2.1.41), isoamylase (EC
3.2.1.68) and the like,
Change the composition of high traose content,
It is also free to increase the source content. Above all,
In combination with starch debranching enzymes such as pullulanase and isoamylase
The maltotetraose content is from 60 to
An increase to 80% w / w can also be advantageously implemented. The thus obtained maltotetraau
Malt with a solid content of about 40 to 80 w / w% per solid
Using a high content of tetraose as a raw material, by fractionation, etc.
Remove contaminating sugars, dextrin, etc.
It is also free to further increase the Laose content. The fractionation method is described, for example, in JP-A-48-4
U.S. Patent No. 647, U.S. Pat.
Utilization of organic precipitants described in JP-A-102854,
Strongly acidic clicks described in JP-A-59-148794
The use of on-exchange resin is appropriately selected, and if necessary, 9
High purity maltotetraose high content of 8w / w% or more
It is easy to collect things. Next, these maltotetraose-rich contents
The material is usually filtered, decolorized with activated carbon and H-type,
Through purification process such as desalting with OH type ion exchange resin
Then, it is concentrated to a syrup-like product. If necessary, further
It is free to be dried and made into a powdery product. The thus obtained maltotetraau
High-content materials usually include maltotetraose as a solid
40% w / w or more. Further, reduction of maltotetraose high content
Higher content of maltotetriol, which is more chemically stable
The production of a product can also be carried out advantageously. For example, a high maltotetraose content is
Make an aqueous solution with a concentration of about 40 to 60% and put it in an autoclave.
About 8-10% of Raney nickel as catalyst
Then, while stirring, raise the temperature to 90 to 140 ° C.
Pressure 20-150kg / cm ² To complete hydrogenation
After that, the Raney nickel is removed and then the malt
Activated carbon, iron, etc.
Purify with on-exchange resin and concentrate to a syrup-like product
Or further dried to a powdered product. The maltote try thus produced
Maltetriteol is usually used for high tall content.
Contains 40 w / w% or more per form. Maltotetra produced by the method described above
The high content of aose can be used as a low-sweetness sweetener,
Ee-imparting agent, viscosity modifier, humectant, illuminating agent, adhesive,
Perfume preservative, crystal inhibitor, candy dripping inhibitor, starch
As an anti-aging agent, and as a supplement for nutrition
Widely used for food and drink. For example, the content of maltotetraose-rich material
How to use such as heating the wrap product and applying it to the food surface
In this case, its curing and drying speed is faster than other sugar sweeteners.
It has the characteristic of less dripping. Moreover, on the surface
The lighting and gloss are good. Also, use as a sweetener can be used as a single product.
As well as, if necessary, other sweeteners, like
For example, glucose, maltose, isomerized sugar, sugar, honey,
Maple sugar, sorbitol, maltitol, para
Tinose, dihydrochalcone, stevioside, α-gly
Cosyl Stevioside, Lakanka Sweets, Glycyrrh
, Α-glycosylglycyrrhizin, L-asparatyl
L-phenylalanine methyl ester, saccharin,
Free to use in combination with glycine, alanine, etc.
It is. [0040] The maltotetraose-rich content includes acidity and salt.
Various items with other tastes such as kara, astringency, umami, and bitterness
Because it is in harmony with quality and has high heat resistance,
It can be used freely for seasoning, taste improvement, and seasoning. For example, soy sauce, powdered soy sauce, miso, powdered taste
Miso, Moromi, Hishio, Furikake, Mayonnaise, Dressi
Vinegar, vinegar, three tablespoons vinegar, powdered sushi vinegar, Chinese ingredients, Tentsuyu,
Noodle soup, sauce, ketchup, grilled meat sauce, curry
C, stew mushroom, soup mushroom, dashi mushroom, complex seasoning,
Mirin, new mirin, table syrup, coffee sugar
-It can be used freely as various seasonings. Also, for example, rice crackers, hail,
Shi, mochi, manju, uiro, bean jam, yokan, boiled sheep
Various Japanese sweets such as sweets, nishikidama, jelly, castella, candy,
Bread, biscuits, crackers, cookies, pies, puri
Noodles, butter cream, custard cream, and cream
, Waffle, sponge cake, donut, chocolate
Cheese, chewing gum, caramel, candy, etc.
Ice of various Western confectionery, ice cream, sherbet
Confectionery, fruit syrup pickles, syrups such as ice honey, noodles,
Processed cereals such as cooked rice and artificial meat, flower pace
, Peanut paste, fruit paste, spread
Paste, jam, marmalade, syrup
Fruits such as pickles and sugar-fruits, processed foods of vegetables,
Pickles such as tararazuke, senmaizuke, and aratsukizuke, takuan
Pickles such as pickles and Chinese cabbage pickles, ham and sausage
Meat products such as fish, fish ham, fish sausage, kama
Fish meat products such as Boko, Chikuwa, Tempura, sea urchin, squid salt
Spicy, vinegared kombu, Sakisume, Fugu mirin dried, etc.
Manufactured from delicacies, seaweed, wild vegetables, soy sauce, small fish, shellfish, etc.
Tsukuda stew, boiled beans, potato salad, kelp, etc.
Dairy products such as soy vegetables, processed cheese, powdered milk, fish meat,
Meat, fruit, vegetable bottles, canned foods, synthetic wine, fruit wine,
Alcoholic beverages such as Western liquor, coffee, cocoa, juice, carbonated drinks
Beverages such as beverages, lactic acid drinks, lactic acid bacteria drinks, etc.
Box, hot cake mix, instant juice, instant co
Coffee, instant shiroko, instant soup, etc.
Sweeteners, taste improvers, and property improvers for various foods and beverages
And can be used freely. In addition, normal consumption of these foods and drinks
For sick people who are difficult to reach, frail people after illness, infants and the elderly
As a result, the maltotetriol-rich material of the present invention
This means that maltotetraose-rich substances with good digestion and absorption
Nutrition supplements such as tube feeding liquid, dialysis nutrition, baby food, etc.
It can also be implemented advantageously. At this time, a liquid nutritional supplement is manufactured and
It can be used as is, or it can be used in powders, granules, etc.
Manufacture solid nutritional supplements of water, saline, fruit juice,
It can be used by dissolving it in milk. Further, for example, a substance having a high maltotetraose content may be used.
Livestock, poultry, and other domestic animals such as honey, silkworms, and fish.
To improve the taste of feed, feed, etc.
You can also. In addition, tobacco, toothpaste, lipstick,
Cream, oral medicine, troche, liver oil drop, mouthwash
As you can taste the taste of cooling agents, oral tablets, gargle, etc.
Sweeteners for tastes, cosmetics, pharmaceuticals, etc.
It can also be used as an agent and a quality improving agent. As described above, the food and drink of the present invention
Is not only a taste-able oral ingestion,
It also refers to nutritional supplements such as tube fed liquids. Further, the maltothe of the present invention may be added to these foods and drinks.
The method of incorporating the high content of traose is based on
It may be included in the process until completion.
Sum, kneading, dissolving, melting, dipping, penetrating, spraying, coating, spraying
Known methods such as fog and injection are appropriately selected. Hereinafter, maltote used in the present invention
The production method of laose-forming amylase is described as a reference example.
In both cases, examples of the present invention and excellent effects will be described. Reference Example 1 Maltotetraose-forming amylase Soluble starch 2% w / v, corn steep
Liquor 0.5% w / v, Polypeptone 0.2% w / v
%, Dipotassium phosphate 0.2w / v%, calcium chloride
Liquid medium comprising 0.05% w / v% of water and water
Was adjusted to pH 7.2, and 100 ml each of this was
l Place in a shake flask and automatize at 120 ° C for 20 minutes
After craving, Pseudomonas Stutzeri MO
Inoculate -19 loops of -19 at a time and shake at 27 ° C for 3 days.
Nourished. Amylate producing maltotetraose in culture solution
The activity was about 250 units per ml. The culture solution is centrifuged and the resulting supernatant enzyme
The liquid is useful for the production of maltotetraose high content from starch
It can be used conveniently. Reference Example 2 Maltotetraose-forming amylase Liquefied starch 4% w / v, corn steep
Liquor 1.0% w / v, Holipeptone 0.5% w / v
%, Ammonium nitrate 0.2w / v%, dipotassium phosphate
0.2w / v%, calcium chloride dihydrate 0.05w
/ V% and water in a 30 l jar
Add 15 liters to the mentor, adjust to pH 7.0, and add
After sterilizing at 0 ° C for 20 minutes, Pseudomonas sututsu
Inoculated at 1 v / v% of a seed culture of
For 4 days. Maltotetrao in culture
The amylase activity is about 220 units per ml.
Was. The culture solution is subjected to membrane filtration, and the obtained filtrate is salted out with ammonium sulfate.
And collecting 0.2 to 0.4 saturated sediment fractions and dialysis,
Next, anion exchange chromatography (Toyo Soda Co., Ltd.)
Shikisha Manufacturing, trade name, DEAE-Toyopearl-1
650s, and furthermore, Pharma, Sweden
Company A, using the product name Mono Q)
High purity gel showing a single band on acrylamide gel electrophoresis
The orthotetraose-forming amylase solution was collected. According to this purification step, the specific activity is about 4.3.
Recovery of maltotetraose-forming amylase
The rate was about 48%. The obtained maltotetraose-forming
Milase has a specific activity of 680 ± 60 (unit / mg protein)
And has the following physicochemical properties. (A) Action Acts on starch to mainly produce maltotetraose. (B) substrate-specific starch, amylose, amylopectin, glycogen and
And β-limit dextrin, and cyclodextrin
Phosphorus, dextran, pullulan and ercinnan
Has virtually no effect. (C) Optimum pH: pH 7.0 to about 7.5 at 40 ° C for 20 minutes. (D) Stable pH: pH 6.5 to 9.5 at 1 hour at 40 ° C. (E) Optimum temperature pH 7.0, around 50 ° C for 20 minutes. (F) Stability temperature: pH 7.0, 1 hour, to around 45 ° C. However, calci
Almost complete activity up to around 50 ° C in the presence of
And about 70% of the activity remains at around 55 ° C. (G) Inhibition and stabilization The activity is inhibited by mercury ions. Calcium ion
This improves the thermal stability. (H) Molecular weight SDS-polyacrylamide gel electrophoresis
6,000 ± 1,000. Ultra-centrifugal analysis, specific volume
× When density = 0.75, 47,000 ± 2,000
0. (I) Isoelectric point polyacrylamide gel The pI was determined by isoelectric focusing.
Near 4.8. (J) UV absorption near 275-280 nm. The production of high purity maltotetraose
10 mM Tris containing about 1.0 w / v% amylase
The hydrochloric acid buffer (pH 8.0) was left at 4 ° C. for one month.
At this time, crystals precipitated. Micrograph of this crystal (× 22
0) is shown in FIG. Further, the production of this high-purity maltotetraose
Using Amylase, Journal of Biological
Chemistry (Journal of Biolo)
medical Chemistry), Vol. 256, No. 7
990 to 7997 (1981),
Edman degradation by gas-phase protein sequencer
The degradation products were identified by high performance liquid chromatography.
Was. As a result, two amino acids from the N-terminal amino acid of this enzyme
The amino acid sequence of up to 0 is aspartic acid-glutamic acid.
-Alanine-glycine-lysine-serine-proline-
Asparagine-alanine-valine-arginine-tyrosi
N-histidine-glycine-glycine-aspartic acid
-Glutamic acid-isoleucine-isoleucine-leucine
-(However, any amino acid residue is an L-amino acid residue
Is shown. ). The high-purity mask obtained in this reference example
The orthotetraose-forming amylase is also the enzyme solution of Reference Example 1.
As in the above case, maltotetraose high content from starch
It can be used advantageously for the production of property. Example 1 High content of maltotetraose 1 part by weight of corn starch was added in an amount of 0.1 per starch.
α-amylase (Novo, Denmark)
3.0 parts by weight of water containing Tamamir 60L)
After stirring and adjusting the pH to 6.5, the suspension was mixed with 95
~ 100 ° C, gelatinization and liquefaction occur simultaneously,
When the reaction progressed to DE 4.5, 5 minutes at 120 ° C
For a while and cool it rapidly to 60 ° C.
Maltotetraose-forming amylase obtained by the method of Example 1.
Solution (supernatant) was added at a rate of 4 units per gram of starch,
Saccharification was performed at 6.5 at 55 ° C. for 46 hours. The saccharified product is heated to inactivate the enzyme, and filtered.
Then, decolorize with activated carbon and convert to H-type and OH-type ion exchange resins.
Desalted and concentrated to give a syrup-like malt with a water content of 25% w / w.
High content of tetratetraose at 95% based on raw starch solids
Obtained in yield. This product contains about 2% glucose per solid.
Having DE of about 24 and having maltotetraose
Contains about 55% per solid and its sweetness
Was about 25% of that of sugar. This product has a low sweetness, a low sugar content and good digestion and absorption.
As a sweetener, body-imparting agent, viscosity modifier, preservative
Wetting agents, illuminants, adhesives, fragrances, crystallization inhibitors, cans
Widely used in food and drink as an anti-sagging agent for Dee
You. Example 2 Maltotetraose High Content One part by weight of potato starch was added at 0.01 w / starch.
Α-amylase (Nagase Seikagaku Corporation)
Company, trade name, neospitase)
After stirring and mixing to adjust the pH to 6.0, the suspension was diluted with 85 to
Keep at 90 ° C, gelatinization and liquefaction occur simultaneously.
Heat to 0 ° C for 5 minutes to keep DE below 1.0,
Rapidly cooled to 55 ° C and adjusted to pH 7.0.
Manufactured by Hayashibara Biochemical Research Institute, trade name Pullulanase (EC
3.2.1.41) and high purity obtained by the method of Example 2.
Each maltotetraose-forming amylase solution was added to starch
Add 150 units and 8 units per gram,
Saccharification was carried out at 7.0 and 50 ° C. for 36 hours. The saccharified product was purified and concentrated in the same manner as in Example 1.
And then spray-dried to obtain a powdery mar having a water content of less than 1 w / w%.
About 93% of high content of tetratetraose per raw starch solid
In a yield of This product contains about 2% glucose per solid.
Having DE of about 24 and having maltotetraose
Contains about 76% per solid and its sweetness
Was 25% of that of sugar. This product has low sweetness, low sugar content and good digestion and absorption.
As a sweetener, it is widely used in food and drink as in the case of Example 1.
Can be used. Example 3 Maltotetraose High Content Maltotetraose Obtained by the Method of Example 2
Using high acid cation exchange resin,
Increased totetraose content. The maltotetrao produced by the method of Example 4
The raw sugar content was adjusted to a concentration of 60 w / w% to prepare a raw sugar solution. The resin is an alkali metal type strongly acidic cation exchange resin.
Exchange resin (manufactured by Tokyo Organic Chemical Industry Co., Ltd., trade name XT-
1007, Na ⁺ Mold, degree of cross-linking 6%).
Connect 4 stainless steel columns with 4cm jacket
The total length of the resin layer was set to 20 m. Column temperature to 55 ° C
While maintaining, add 5 v / v% of the raw sugar solution to the resin.
Hot water of 55 ° C. was flowed through the solution at SV 0.13, and fractionated.
Maltotetraose with a maltotetraose content of 90% or more
The high-content fraction was collected, purified and concentrated as in Example 3.
And a syrup-like maltotetraau having a water content of 25 w / w%
High content was obtained in a yield of about 60% based on the raw sugar solids. This product uses maltotetraose per solid
It contains about 93%, and its sweetness is that of sugar
Was about 25%. This product has low sweetness, low sugar content and good digestion and absorption.
As a sweetener, it is widely used in food and drink as in the case of Example 3.
Can be used. Example 4 Sweetener A syrup-like maltote obtained by the method of Example 3
Α-Glycosyl stevio in 1 part by weight of high content of traose
Sid (manufactured by Toyo Seika Co., Ltd., trade name α-G suite)
Syrup-shaped sweetener obtained by uniformly mixing 0.02 parts by weight
Has excellent sweetness, about twice the sweetness of sugar,
Lolly is about 1/2 of sugar per sweetness. Therefore, the book
Sweeteners consume less calories as a low-calorie sweetener
People, such as obese, diabetic, etc.
Suitable for sweetening low-calorie foods and drinks
You. The present sweetener is also acidified by cariogenic bacteria.
Less insoluble glucans
Also suitable for sweetening foods that suppress tooth decay
It is. Example 5 Custard cream 500 parts by weight of corn starch,
Malt tetraose high content 400 weight obtained by the method
Parts, maltose 500 parts by weight and salt 5 parts by weight,
Mix thoroughly through a sieve and add 1,400 parts by weight of chicken eggs
And gradually add 5,000 parts by weight of boiling milk
To a low heat, continue stirring, and
When the starch is completely gelatinized and becomes entirely translucent
Turn off the heat, cool it and add a small amount of vanilla flavor.
This produced a custard cream. This product has a smooth, glossy and strong sweetness
It is just delicious. Example 6 Hard candy Syrup-like maltote obtained by the method of Example 3
90 parts by weight of sugar mixed with 70 parts by weight of high traose content
Dissolve and heat under reduced pressure until water content is less than about 2% w / w
Concentrate and add 0.15 parts by weight of citric acid and a small amount
Mix the lemon flavor and the flavor, then follow the usual method
Molded to produce hard candy. This product has low hygroscopicity and does not easily sag
It is a crisp hard candy. Example 7 Tsukudani [0086] Sand removal, acid treatment and slicing according to a conventional method.
250 parts by weight of kelp, 212 parts by weight of soy sauce, amino acid solution
300 parts by weight, 40 parts by weight of sugar and the method of Example 1
20 parts by weight of the obtained syrup-like maltotetraose high content
Add 12 grams of sodium glutamate while simmering
And 8 parts by weight of caramel and 21 parts by weight of taste
We made kelp Tsukudani. This product not only tastes and smells, but also
It's an appetizing kelp tsukudani in minutes. Example 8 Betara Pickles Syrup-like sweetener 4 obtained by the method of Example 4
Parts by weight, licorice preparation 0.05 parts by weight, malic acid 0.008
Parts by weight, sodium glutamate 0.07 parts by weight, sol
0.03 parts by weight of potassium borate and 0.2 parts by weight of pullulan
The mixed portions were uniformly mixed to produce pickled mushrooms. 30 kg of radish is reduced with salt according to a conventional method.
Pickle, then pickle with sugar,
Pickled in a seasoning liquid prepared with 4kg of base
did. This product has good color luster and aroma, and has moderate sweetness.
The crispness was also good. Example 9 Fluid-feeding by tube feeding Maltotetrapowder obtained by the method of Example 2
550 parts by weight of high aose content, anhydrous crystalline maltose (Hayashi
Hara Co., Ltd., registered trademark Fine Tose) 50 weight
Parts, dried egg yolk 190 parts by weight, skim milk powder 209 parts by weight, salt
4.4 parts by weight of sodium chloride, 1.85 parts by weight of potassium chloride
Parts, magnesium sulfate 4 parts by weight, thiamine 0.01 parts by weight
Parts, 0.1 parts by weight of sodium ascorbate, vitamins
E acetate 0.6 parts by weight and nicotinamide 0.0
A formulation consisting of 4 parts by weight is prepared. 25 g each of this composition was laminated with aluminum
Filled in heat-sealed parcels and heat-sealed to provide a solid nutritional supplement
Was manufactured. This product does not need to be stored at low temperature and can be stored at room temperature.
It is stable for a long time, and has good fluidity and dissolution and dispersibility. This product is about 150 to 300 ml per bag
Dissolve in water to make a tube-fed liquid food.
It is used by administering it to the cavity, esophagus, stomach and intestine. Example 10 Tablet Aspirin 50 parts by weight according to the method of Example 2
6 parts by weight of the obtained powdery maltotetraose high content and
After thoroughly mixing 8 parts by weight of corn starch,
Then, it is tableted with a tableting machine and has a thickness of 5.25 mm, 1 tablet
680 mg tablets were produced. This product has low hygroscopicity and sufficient physical strength
And the disintegration in water was very good. As is clear from the above, the present invention
Akira elucidates novel maltotetraose-generating amylase
And a method of manufacturing the same. The new monate tetraose-producing amylor
Ze has a wide pH range at the optimum pH and stable pH.
High temperature at optimum temperature and stable temperature
High enzyme production from starch, maltotetra
It has been found that it can be used advantageously for the production of high aus content
However, its industrial significance is extremely large. Further, the maltote produced in this manner is
The high content of traose is used as a sweetener with low sweetness,
With body-imparting agents, viscosity modifiers, humectants, illuminating agents, etc.
In addition, it is widely used in food and drink as a nutritional supplement.
What can be done is a big feature.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 Pseudomonas stutzeri MO-19 (FERM-BP) magnified 10,000 times with an electron microscope
1168 is a photograph showing an example of the shape of the cell. FIG. 2 is a photograph showing an example of a crystal of maltotetraose-generating amylase which is magnified 220 times with a microscope.

──────────────────────────────────────────────────の Continued on front page (51) Int.Cl. ⁶ Identification code FI (C12N 9/26 C12R 1:38)

Claims (1)

(57) [Claims] A maltotetraose-rich substance obtained by reacting starch with maltotetraose-forming amylase having the following physicochemical properties. (A) Action Acts on starch to mainly produce maltotetraose. (B) acting on starch, amylose, amylopectin, glycogen and β-limit dextrin, and cyclodextrin, dextran, pullulan and ercinnan
Has virtually no effect. (C) Optimum pH: pH 7.0 to about 7.5 at 40 ° C for 20 minutes. (D) Stable pH: pH 6.5 to 9.5 at 1 hour at 40 ° C. (E) Optimum temperature pH 7.0, around 50 ° C for 20 minutes. (F) Stability temperature: pH 7.0, 1 hour, to around 45 ° C. However, in the presence of calcium ions, the activity remains almost completely up to around 50 ° C., and about 70% remains around 55 ° C. (G) Inhibition and stabilization The activity is inhibited by mercury ions. Calcium ions improve thermal stability. (H) Molecular weight SDS-polyacrylamide gel electrophoresis
6,000 ± 1,000. By ultracentrifugation analysis, 47,
2,000 ± 2,000. (I) Isoelectric point polyacrylamide gel The pI was determined by isoelectric focusing.
Near 4.8. (J) UV absorption near 275-280 nm. 2. The maltotetraose-forming amylase has aspartic acid-glutamine-alanine-glycine-lysine-serine-proline-asparagine-alanine-valine- from the N-terminus as the amino acid sequence of the N-terminal region. The high content maltotetraose according to item (1). 3. The maltotetraose-rich material according to claim (1) or (2), wherein maltotetraose-forming amylase is allowed to act together with other starch-related enzymes. 4. Claim (1) wherein the high maltotetraose content is in the form of syrup or powder.
Item, high content of maltotetraose according to item (2) or (3). 5. The starch, food was allowed containing maltotetraose rich product obtained allowed act maltotetraose-forming amylase having the following physicochemical properties. (A) Action It acts on starch to produce mainly maltotetraose. (B) acting on starch, amylose, amylopectin, glycogen and β-limit dextrin, and cyclodextrin, dextran, pullulan and ercinnan
Has virtually no effect. (C) Optimum pH: pH 7.0 to about 7.5 at 40 ° C for 20 minutes. (D) Stable pH: pH 6.5 to 9.5 at 1 hour at 40 ° C. (E) Optimum temperature pH 7.0, around 50 ° C for 20 minutes. (F) Stability temperature: pH 7.0, 1 hour, to around 45 ° C. However, in the presence of calcium ions, the activity remains almost completely up to around 50 ° C., and about 70% remains around 55 ° C. (G) Inhibition and stabilization The activity is inhibited by mercury ions. Calcium ions improve thermal stability. (H) Molecular weight SDS-polyacrylamide gel electrophoresis
6,000 ± 1,000. 47,000 ± 2,000 by ultracentrifugation analysis. (I) Isoelectric point polyacrylamide gel The pI was determined by isoelectric focusing.
Near 4.8. (J) UV absorption near 275-280 nm. 6. A patent characterized in that maltotetraose-forming amylase has aspartic acid-glutamine-alanine-glycine-lysine-serine-proline-asparagine-alanine-valine- from the N-terminal as the amino acid sequence of the N-terminal region. The food or drink according to claim (5).