Patent Application
20210068432
The present invention relates to methods for producing a starch-containing food and the like. The present invention also relates to starch-containing foods produced by such a method.
There are various problems in the field of the food industry. For example, consumers' tastes are becoming more diverse and sophisticated, along with which the development of foods with new mouthfeel and flavor that have never existed before is desired. Since the quality of many foods is degraded over time, construction of means to reduce such time-related degradation of quality is also one of the major problems. Furthermore, since the amount of raw materials for food products is limited, establishment of a manufacturing method with minimal loss in the manufacturing process is also recognized as an important problem.
As one option for solving the above-mentioned problems in the field of the food industry, a means characterized by modifying the properties of food with an enzyme has been reported. For example, JP-B-5715346, which is incorporated herein by reference in its entirety, discloses a method for modifying a food by utilizing β-amylase. In addition, JP-B-4475276, which is incorporated herein by reference in its entirety, discloses a method for producing cooked rice food or processed wheat food by utilizing transglucosidase. Furthermore, JP-B-5944839, which is incorporated herein by reference in its entirety, discloses a method for producing an enzyme-treated starch grain that is resistant to aging, by utilizing 4-α-glucanotransferase.
Amylomaltase is known as one of the enzymes that can be used for producing foods. Amylomaltase (hereinafter sometimes to be referred to as “AM” in the present specification) is an enzyme that catalyzes the decomposition of amylose in starch and the elongation of amylopectin sugar chains, and is known to exist widely in nature. The organisms that produce amylomaltase include microorganisms such as Escherichia coli. For food processing, heat-resistant amylomaltase derived from the genus Thermus bacterium such as Thermus flavus, Thermus aquaticus and Thermus thermophilus (hereinafter sometimes to be referred to as “Tt” in the present specification) is generally used (see JP-B-5944839; JP-B-4187305; and Nguyen DH. et al., (2014) Modification of rice grain starch for lump-free cooked rice using thermostable disproportionating enzymes. Food Research International 63: 55-61, all of which are incorporated herein by reference in their entireties).
Corynebacterium glutamicum (hereinafter sometimes referred to as “Cg” or “Coryne” in the present specification), which is classified in the genus Corynebacterium, is also known as a microorganism that produce amylomaltase. Corynebacterium glutamicum is a microorganism isolated in Japan in 1957 as a microorganism that excretes L-glutamic acid into the medium. It is a non-motile, aerobic gram-positive bacterium belonging to the mycolic acid-containing actinomycete group and has no sporulation ability. At present, over 2 million tons of sodium L-glutamate (Umami component) is produced worldwide by a fermentation method using the bacteria. Corynebacterium glutamicum is used for producing many useful substances such as amino acids such as lysine, nucleic acids, and organic acids, in addition to glutamic acid. However, there is no finding as to the effect of amylomaltase derived from Corynebacterium glutamicum on edible starch or starch in food products. It is known that amylomaltase derived from the genus Streptomyces bacterium (e.g., Streptomyces avermitilis, Streptomyces cinnamoneus, Streptomyces griseus, Streptomyces thermoviolaceus, Streptomyces violaceoruber and the like) can be used as a food additive. Amylomaltase derived from Streptomyces is known to show a comparatively high amino acid identity (about 40%) with amylomaltase derived from Corynebacterium glutamicum, and is considered to have enzyme property similar to that of amylomaltase derived from Corynebacterium glutamicum. However, there is also no finding as to the effect of amylomaltase derived from the genus Streptomyces on edible starch or starch in food products.
When a heat-resistant enzyme is used in the manufacturing process for the purpose of modifying food, the temperature characteristic of the heat-resistant enzyme may pose a problem. For example, heat-resistant enzyme retains its activity even after the starch in the food is gelatinized, due to which it also modifies the gelatinized starch, possibly causing problems of scorching and the like (it is difficult to have the enzyme act only on ungelatinized starch). In addition, many heat-resistant enzymes have low activity in the temperature range (refrigerating to room temperature) often used during storage, mixing, and forming processes in food manufacturing processes. When they are allowed to act in these processes, the effect becomes low, and addition of a large amount of the enzyme becomes necessary. Furthermore, when the heat resistance of the enzyme is high, a high temperature, long-term step is required to deactivate the enzyme in the food manufacturing process, and an influence on the food ingredients is expected to be high. Depending on the kind of food, moreover, heating strength may be insufficient and the enzyme may not be deactivated.
Accordingly, it is one object of the present invention to provide novel methods for producing a starch-containing food and the like.
It is another object of the present invention to provide novel starch-containing foods produced by such a method.
These and other objects, which will become apparent during the following detailed description, have been achieved by the inventors' discovery that (1) CgAM acts only on ungelatinized starch because the enzyme is deactivated before the starch in the food is heat gelatinized, (2) CgAM has higher activity than TtAM in the temperature range (regrigelating to room temperature) that is often used in food manufacturing processes, and (3) CgAM is easily deactivated by heating, and the like. In addition to these, they have surprisingly found effects completely different from those of TtAM, for example, that (4) CgAM can suppress hydrogenation due to aging while maintaining the original physical properties of starch, (5) the rice added with CgAM shows improved stickiness and, while maintaining the stickiness, time-related degradation of mouthfeel can be suppressed, (6) the rice cooked with CgAM remarkably reduces the occurrence of scorching during cooking, and (7) the Δ blood glucose level AUC of rats that ate CgAM-modified sucrose or starch as measured 2 hr after eating is lower than that of rats that ate TtAM-modified sucrose or starch as measured 2 hr after eating. Based on such findings, they have conducted further studies and completed the present invention.
That is, the present invention provides the following:
(1) A method for producing a starch-containing food, comprising reacting an actinomycete-derived amylomaltase with starch in a raw material.
(2) The production method of (1), wherein the actinomycete is the genus Corynebacterium or the genus Streptomyces.
(3) The production method of (1) or (2), wherein the actinomycete is selected from the group consisting of Corynebacterium glutamicum, Streptomyces avermitilis, Streptomyces cinnamoneus, Streptomyces griseus, Streptomyces thermoviolaceus, and Streptomyces violaceoruber.
(4) The production method of any of (1) to (3), wherein the starch-containing food is selected from the group consisting of a rice processing food, a wheat processing food, a potato processing food, a corn processing food, a tapioca processing food, and a processing food containing one or more kinds of starch extracted from rice, wheat, potato, corn, or tapioca.
(5) The production method of any of (1) to (4), wherein the starch-containing food comprises sucrose.
(6) A method for producing a starch-containing food product, comprising reacting an actinomycete-derived amylomaltase with starch in a raw material, wherein the amylomaltase has the amino acid sequence shown in SEQ ID NO: 1, 3, 4, 5, or 6, or an amino acid sequence not less than 90% identical to the amino acid sequence.
(7) A method for modifying property of a starch-containing food, comprising reacting an actinomycete-derived amylomaltase with starch in a raw material.
(8) The method of (7), wherein the actinomycete is the genus Corynebacterium or the genus Streptomyces.
(9) The method of (7) or (8), wherein the actinomycete is selected from the group consisting of Corynebacterium glutamicum, Streptomyces avermitilis, Streptomyces cinnamoneus, Streptomyces griseus, Streptomyces thermoviolaceus, and Streptomyces violaceoruber.
(10) The production method of any of (7) to (9), wherein the starch-containing food is selected from the group consisting of a rice processing food, a wheat processing food, a potato processing food, a corn processing food, a tapioca processing food, and a processing food containing one or more kinds of starch extracted from rice, wheat, potato, corn, or tapioca.
(11) The production method of any of (7) to (10), wherein the starch-containing food comprises sucrose.
(12) A method for modifying property of a starch-containing food product, comprising reacting an actinomycete-derived amylomaltase with starch in a raw material, wherein the amylomaltase has the amino acid sequence shown in SEQ ID NO: 1, 3, 4, 5, or 6, or an amino acid sequence not less than 90% identical to the amino acid sequence.
(13) An agent for modifying property of a starch-containing food, comprising an actinomycete-derived amylomaltase.
(14) The agent of (13), wherein the actinomycete is the genus Corynebacterium or the genus Streptomyces.
(15) The agent of (13) or (14), wherein the actinomycete is selected from the group consisting of Corynebacterium glutamicum, Streptomyces avermitilis, Streptomyces cinnamoneus, Streptomyces griseus, Streptomyces thermoviolaceus, and Streptomyces violaceoruber.
(16) The agent of any of (13) to (15), wherein the starch-containing food is selected from the group consisting of a rice processing food, a wheat processing food, a potato processing food, a corn processing food, a tapioca processing food, and a processing food containing one or more kinds of starch extracted from rice, wheat, potato, corn, or tapioca.
(17) The agent of any of (13) to (16), wherein the starch-containing food comprises sucrose.
(18) An agent for modifying property of a starch-containing food, comprising an amylomaltase having the amino acid sequence shown in SEQ ID NO: 1, 3, 4, 5, or 6, or an amino acid sequence not less than 90% identical to the amino acid sequence.
In one embodiment of the present invention, the present invention provides the following:
(1A) A method for producing a starch-containing food, comprising reacting an amylomaltase derived from the genus Corynebacterium or the genus Streptomyces with starch in a raw material.
(2A) The production method of (1A), wherein the starch-containing food is selected from the group consisting of a rice processing food, a wheat processing food, a potato processing food, a corn processing food, a tapioca processing food, and a processing food containing one or more kinds of purified starch extracted from rice, wheat, potato, corn, or tapioca.
(3A) The production method of (1A) or (2A), wherein the amylomaltase has the amino acid sequence shown in SEQ ID NO: 1, 3, 4, 5, or 6, or an amino acid sequence not less than 90% identical to the amino acid sequence.
(4A) A method for modifying property of a starch-containing food, comprising reacting an amylomaltase derived from the genus Corynebacterium or the genus Streptomyces with starch in a raw material.
(5A) The production method of (4A), wherein the starch-containing food is selected from the group consisting of a rice processing food, a wheat processing food, a potato processing food, a corn processing food, a tapioca processing food, and a processing food containing one or more kinds of purified starch extracted from rice, wheat, potato, corn, or tapioca.
(6A) The production method of (4A) or (5A), wherein the amylomaltase has the amino acid sequence shown in SEQ ID NO: 1, 3, 4, 5, or 6, or an amino acid sequence not less than 90% identical to the amino acid sequence.
(7A) An agent for modifying property of a starch-containing food, comprising an amylomaltase derived from the genus Corynebacterium or the genus Streptomyces.
(8A) The agent of (7A), wherein the starch-containing food is selected from the group consisting of a rice processing food, a wheat processing food, a potato processing food, a corn processing food, a tapioca processing food, and a processing food containing one or more kinds of purified starch extracted from rice, wheat, potato, corn, or tapioca.
(9A) The agent of (7A) or (BA), wherein the amylomaltase has the amino acid sequence shown in SEQ ID NO: 1, 3, 4, 5, or 6, or an amino acid sequence not less than 90% identical to the amino acid sequence.
According to the present invention, a starch-containing food that has a preferable mouthfeel different in quality from existing sugar-modifying enzymes, is less susceptible to time-related degradation, and is less likely to raise blood glucose level after eating can be produced, and a starch-containing food provided with the above-mentioned properties and free from a problem specific to existing heat-resistant amylomaltase (e.g., scorching on cookware) in some cooking styles can be produced.
A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same become better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:
The present invention is explained in detail in the following.
The present invention provides a method for producing a starch-containing food, including reacting an actinomycete-derived amylomaltase with starch in a raw material (hereinafter sometimes to be referred to as “the production method of the present invention”).
Amylomaltase (EC number: 2.4.1.25) is an enzyme that catalyzes a chemical reaction that transfers a portion of 1,4-α-glucan chain to 4-OH group of glucose or another α-glucan. When the substrate is sufficiently large, amylomaltase causes an intramolecular transfer, and the resultant product has a cyclic structure.
In the present specification, the “α-glucan” is α-1,4-glucan (polysaccharide with a chain-like structure and maltose as a constitutional disaccharide unit), or α-1,4-glucan having an α-1,6-branched structure. The “α-glucan” nonlimitatively includes amylose, amylopectin, starch and glycogen, waxy starch, high amylose starch, soluble starch, dextrin, starch hydrolyzate, enzyme-synthesized amylopectin by phosphorylase and the like.
In the present specification, the “cyclic glucan” includes cyclic α-1,4-glucan having α-1,4-glucoside bond alone, and branched type cyclic glucan having both α-1,4-glucoside bond and α-1,6-glucoside bond. The “branched type” means having at least one glucoside bond other than α-1,4-bond. Examples of the branched type cyclic glucan include an inner branched type cyclic glucan containing a branched structure having an α-1,6-bond inside the cyclic structure, and an outer branched type cyclic glucan having α non-cyclic structure part in addition to the cyclic structure.
The amylomaltase used in the present invention may be amylomaltase derived from the genus Corynebacterium or the genus Streptomyces, or a mutant thereof. In the present specification, the “amylomaltase derived from the genus Corynebacterium or the genus Streptomyces” means an amylomaltase produced by a bacterium (wild-type or variant strain) classified into the genus Corynebacterium or the genus Streptomyces, or an amylomaltase obtained by a genetic engineering method using the amylomaltase gene of a bacterium (wild-type or variant strain) classified into the genus Corynebacterium or the genus Streptomyces. Therefore, a recombinant amylomaltase protein expressed by a host transformed or transduced with the amylomaltase gene (wild-type or variant) obtained from a bacterium classified into the genus Corynebacterium or the genus Streptomyces also falls under the “amylomaltase derived from the genus Corynebacterium or the genus Streptomyces”.
Examples of the origin of amylomaltase preferably used in the production method of the present invention include, but are not limited to, Corynebacterium glutamicum, Streptomyces avermitilis, Streptomyces cinnamoneus, Streptomyces griseus, Streptomyces thermoviolaceus, and Streptomyces violaceoruber. Examples of the amylomaltase derived from Corynebacterium glutamicum include amylomaltase having the amino acid sequence shown in SEQ ID NO: 1. Examples of the amylomaltase derived from Streptomyces avermitilis include amylomaltase having the amino acid sequence shown in SEQ ID NO: 3. Examples of the amylomaltase derived from Streptomyces cinnamoneus include amylomaltase having the amino acid sequence shown in SEQ ID NO: 4. Examples of the amylomaltase derived from Streptomyces griseus include amylomaltase having the amino acid sequence shown in SEQ ID NO: 5. Examples of the amylomaltase derived from Streptomyces violaceoruber include amylomaltase having the amino acid sequence shown in SEQ ID NO: 6. Examples of the variants thereof include amylomaltase having an amino acid sequence which is the amino acid sequence shown in SEQ ID NO: 1, 3, 4, 5 or 6 wherein one or more amino acids are added, deleted, inserted or substituted. Amylomaltase having one or more mutations may include any mutation or modification as long as it has the equivalent (or more) enzymological properties as a wild-type amylomaltase derived from Corynebacterium glutamicum, Streptomyces avermitilis, Streptomyces cinnamoneus, Streptomyces griseus, Streptomyces thermoviolaceus, or Streptomyces violaceoruber. Amylomaltase having one or more mutations in the amino acid sequence shown in SEQ ID NO: 1, 3, 4, 5, or 6 has an amino acid sequence has identity of generally not less than 70%, preferably not less than 80%, more preferably not less than 90%, further preferably not less than 95%, further more preferably not less than 98%, particularly preferably not less than 99%, with respect to the amino acid sequence shown in SEQ ID NO: 1, 3, 4, 5, or 6, and each also has the equivalent (or more) amylomaltase activity as the corresponding wild-type amylomaltase. In the present specification, the “identity” of an amino acid sequence or a base sequence refers to the degree of appearance of the same amino acid (base when base sequences are compared) between two sequences. The “identity” of a sequence can be easily determined by those skilled in the art by a method known per se.
The above-mentioned amylomaltase having the amino acid sequence shown in SEQ ID NO: 1, 3, 4, 5, or 6, or mutant amylomaltase having one or more mutations in the amino acid sequence shown in SEQ ID NO: 1, 3, 4, 5, or 6 can be produced by a method known per se. In one embodiment, amylomaltase can be prepared by inserting a base sequence (SEQ ID NO: 2) encoding an amylomaltase having the amino acid sequence shown in SEQ ID NO: 1 into an appropriate gene expression vector, expressing the vector in a protein overexpression system known per se such as Escherichia coli, and purifying the same using an appropriate means. A mutant amylomaltase can be produced by first modifying a part of the base sequence shown in SEQ ID NO: 2 by using a genetic engineering method such as site-directed mutagenesis, inserting same into a gene expression vector, expressing the vector in a protein overexpression system known per se such as Escherichia coli, and purifying same.
In the present specification, the activity of amylomaltase is measured and defined as follows. That is, an amylomaltase solution is added to a 30 mM Tris-HCl buffer (pH 7.5) containing 0.05% potato starch and 0.05% maltose, and the mixture is reacted for a certain period of time in a water bath at 30° C. to 70° C. (which may vary depending on the kind of amylomaltase), and heated at 96° C. for 5 min to discontinue the reaction. 0.1 ml of this reaction mixture and 1 ml of iodine solution (0.02% iodine, 0.2% potassium iodide) are mixed and the absorbance at 600 nm is measured. A value obtained by subtracting the absorbance at the time of enzyme addition from the absorbance of the blank in which milli-Q water is mixed instead of the enzyme solution was used as the activity value, and the amount of enzyme necessary for reducing the absorbance at 600 nm by 1 per minute was used as 1 unit (U).
The optimum temperature of Corynebacterium glutamicum-derived amylomaltase used in the production method of the present invention is 30° C. to 38° C. The optimum temperature of amylomaltase derived from the genus Streptomyces used in the production method of the present invention is 45° C. to 55° C. (e.g., 50° C.). In the present specification, the “optimum temperature” means the temperature at which the activity is highest when amylomaltase is allowed to act for 10 min at each temperature in 30 mM Tris-HCl buffer (pH 7.5) in the presence of 0.05% potato starch and 0.05% maltose. The optimum temperature of the amylomaltase derived the genus Thermus thermophilus is about 70° C.
The optimum pH of the Corynebacterium glutamicum-derived amylomaltase used in the production method of the present invention is 6 to 7. The optimum pH of the amylomaltase derived from the genus Streptomyces used in the production method of the present invention is 4 to 9 (e.g., 7). In the present specification, the “optimum pH” means the pH at which the activity is highest when amylomaltase is allowed to act for 10 min at 37° C. in 30 mM acetate buffer (pH3 to 5.5) or 30 mM phosphate buffer (pH 6 to 7) or 30 mM Tris-HCl buffer (pH 7.5 to 10) in the presence of 0.1% potato starch and 0.05% maltose.
Regarding the heat resistance of the Corynebacterium glutamicum-derived amylomaltase used in the production method of the present invention, the enzyme is stable at not more than 40° C. As for the heat resistance of the amylomaltase derived from the genus Streptomyces used in the production method of the present invention, it is stable at not more than 50° C. In the present specification, the “heat resistance” means that amylomaltase does not lose activity in 30 mM Tris-HCl buffer (pH 7.5) for 10 min.
The pH stability of the Corynebacterium glutamicum-derived amylomaltase used in the production method of the present invention is 6 to 8. The pH stability of the amylomaltase derived from the genus Streptomyces used in the production method of the present invention is 4 to 9. In the present specification, the “pH stability” means that amylomaltase does not lose activity in a buffer (25° C.) for about 18 hr.
The amount of the amylomaltase derived from the genus Corynebacterium glutamicum or amylomaltase derived from the genus Streptomyces used in the production method of the present invention is not particularly limited as long as a desired effect can be obtained. It may be generally 0.00001 to 10000 U, preferably 0.0001 to 1000 U, more preferably 0.001 to 100 U, further preferably 0.01 to 10 U, particularly preferably 0.1 to 1 U, per 1 g of starch in the raw material. In one embodiment, when the starch-containing food produced by the production method of the present invention is cooked rice, generally 0.00001 to 10000 U, preferably 0.0001 to 1000 U, more preferably 0.001 to 100 U, further preferably 0.01 to 10 U, particularly preferably 0.1 to 1 U, of amylomaltase can be added per 1 g of dried rice before boiling.
The timing of adding the amylomaltase derived from the genus Corynebacterium or Streptomyces is not particularly limited as long as a desired effect is obtained. The timing of adding the enzyme may be appropriately set in consideration of the kind of food to be produced and the cooking procedure thereof, the kind of starch raw material to be used, the preference of the consumer and the like, and is not particularly limited. Therefore, the enzyme may be added at any time before cooking, during cooking, after cooking, or before eating the starch-containing food. In one embodiment, it is preferable to add and mix amylomaltase to the raw material to produce or process a food, or allow the enzyme to act on the starch in the raw material by adding and mixing amylomaltase to the food under manufacturing or processing. In a specific example when producing cooked rice, dry rice is washed with water, the washed rice is mixed with water and the amylomaltase derived from the genus Corynebacterium or Streptomyces and stood at room temperature (10° C. to 30° C.) for a certain period of time (e.g., 0.5 to 2 hr), and thereafter cooked by a general method, whereby the cooked rice having the desired effect of the present invention can be prepared.
In the production method of the present invention, the temperature and time at which the amylomaltase derived from the genus Corynebacterium or Streptomyces is allowed to act on starch in the raw material are not particularly limited as long as a desired effect is obtained. The action temperature of the enzyme may be appropriately set in consideration of the amount of the enzyme to be used, the kind of food to be produced and the cooking procedure thereof, the kind of starch raw material to be used, the preference of the consumer and the like, and is not particularly limited. However, it should be taken into consideration that the optimal temperature of the amylomaltase derived from the genus Corynebacterium or Streptomyces is lower than that of amylomaltase derived from a heat resistant bacterium. Therefore, in one embodiment, the temperature of food at the time of addition of the enzyme may be generally 1 to 100° C., preferably 5 to 50° C., more preferably 10 to 45° C., further preferably 20 to 40° C., particularly preferably 30 to 37° C. While the action time of the enzyme is not particularly limited, it may be generally 0.1 to 48 hr, preferably 0.2 to 36 hr, more preferably 0.5 to 24 hr, further preferably 0.8 to 20 hr, particularly preferably 1 to 18 hr. For example, when cooked rice is produced with addition of 0.1 to 1 U amylomaltase per 1 g of dried rice, the washed rice is mixed with water and the amylomaltase derived from the genus Corynebacterium or Streptomyces, and modified with the enzyme for 0.1 to 18 hr at 4 to 40° C., preferably 0.25 to 6 hr at 10 to 38° C., more preferably 0.5 to 1 hr at 20 to 37° C., and thereafter cooked by a general method, whereby the cooked rice having the desired effect of the present invention can be prepared.
In the production method of the present invention, the pH environment when the amylomaltase derived from the genus Corynebacterium or the genus Streptomyces is acted on starch in the raw material is adjusted in advance to, for example, pH 6 to pH 7 as described above in consideration of the optimum pH and pH stability of the enzyme by using, where necessary, a pH adjustor that can be added to food.
The starch-containing food produced by the production method of the present invention includes any food containing starch. The origin of the starch contained in the starch-containing food is not particularly limited, and may be one or more selected from the group consisting of rice starch, wheat starch (including wheat, barley, rye and the like), potato starch, ocarina starch, corn starch, soybean starch, and tapioca starch. Specific examples of the starch-containing food include, but are not limited to, rice processing food, wheat processing food, potato processing food, corn processing food, tapioca processing food, and a processing food containing one or more kinds of starch extracted from rice, wheat, potato, corn, or tapioca. In the present specification, examples of the rice processing food include, but are not limited to, cooked rice and processed product thereof (festive red rice, pilaf, cooked rice with fish and vegetables mixed in advance (takikomigohan), rice porridge, risotto, rice ball, sushi, rice cake, rice-cake sweets etc.), rice noodle and processed product thereof and the like. Examples of the wheat processing food include, but are not limited to, noodles such as pasta, ramen, Japanese wheat noodle and the like, bread, bread dough such as pizza, naan and the like, confectionery such as cookie, cake and the like, and the like. Examples of the potato processing food include, but are not limited to, potato salad, fried potato, boiled potato, mashed potato, potato snacks such as potato chips and the like, and the like. Examples of the corn processing food include, but are not limited to, taco, tortilla, arepa, doughs thereof and the like. Examples of the tapioca processing food include, but are not limited to, steamed dumpling containing tapioca, purine containing tapioca, and the like. Examples of the processing food containing one or more kinds of purified starch extracted from rice, wheat, potato, corn, or tapioca include, but are not limited to, boiled fish paste, crab stick, sausage, hamburg and the like.
The present invention also provides a method for modifying property of a starch-containing food, including reacting an actinomycete-derived amylomaltase with starch in a raw material (hereinafter sometimes to be referred to as “the method of the present invention”).
The amylomaltase of actinomycete to be used in the method of the present invention, the amount thereof to be added, the timing of addition, the reaction temperature, the reaction pH, the preparation method, etc., and the kind of the starch-containing food are all the same as those explained in “1. Production method of starch-containing food”.
The present invention also provides an agent for modifying property of a starch-containing food product, including an actinomycete-derived amylomaltase (hereinafter sometimes to be referred to as “the agent of the present invention”).
The amount of the actinomycete-derived amylomaltase (e.g., amylomaltase derived from the genus Corynebacterium or Streptomyces) contained in the agent of the present invention is not particularly limited. It may be any as long as the amylomaltase can be added at a proportion of generally 0.00001 to 10000 U, preferably 0.0001 to 1000 U, more preferably 0.001 to 100 U, further preferably 0.01 to 10 U, particularly preferably 0.1 to 1 U, per 1 g of starch in the raw material.
The agent of the present invention may contain components other than the amylomaltase derived from the genus Corynebacterium or Streptomyces. Examples of such component include, but are not limited to, excipient, buffering agent, suspension, stabilizer, preservative, antiseptic and seasoning and the like.
The dosage form of the agent of the present invention is not particularly limited, and may be a solid form such as powder form, granular form or the like, a liquid form, or a paste form.
Other aspects of the agent of the present invention such as use conditions and the like can be appropriately set by those of ordinary skill in the art by reference to the conditions explained in “1. Production method of starch-containing food product”.
Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.
To a 10% suspension of rice starch was added Corynebacterium glutamicum-derived amylomaltase or Thermus thermophilus-derived amylomaltase (hereinafter sometimes referred to as “CgAM” or “TtAM” and the like) at 50 U/g starch, and the mixture was reacted at 37° C. for 1 hr while stirring. The mixture was heated at 98° C. for 10 min, and cooled to 50° C. The starch gel after cooling was molded into a cylindrical shape having a diameter of 5 mm and a height of 20 mm by using a plastic cylindrical tube and gelled by storing at 5° C. for 1 to 7 days. The obtained starch gel was taken out of the tube and formed into a cylindrical shape having a diameter of 5 mm and a height of 5 mm by using a razor blade, and then subjected to a compression test using a texture analyzer (TA-XT Plus). The gel strength was measured on day 1, day 3 and day 7. The results are shown in
As shown in
Dried rice (150 g) was washed, water and amylomaltase derived from each microorganism (1.0 U/g dried rice) were added to adjust to 325 g, and the mixture was stood at room temperature (20° C.) for 1 hr. This was cooked with a rice cooker SR-13GP (Panasonic). The obtained cooked rice was taken out of the rice cooker, transferred to a container with a plastic cover, and stood at room temperature for 1 hr. Then, sensory evaluation was performed by four professional panels.
The sensory evaluation was performed for three items of “softness”, “pebbly feel”, and “stickiness”. In the present specification, the “softness” means that the resistance felt when masticating cooked rice is small, “pebbly feel” means that the grains remain strong during masticating, and “stickiness” means that the rice grains are attached to each other. The evaluation criteria were as follows. With the “softness”, “pebbly feel”, and “stickiness” of cooked rice prepared without adding enzymes as a standard (0 point), the degree of strength in each item was evaluated from −2 to +2 points (in increments of 0.5 point). Samples other than the standard product (enzyme-free) were presented in blind. The score was an average of the scores of the four professional panels. The results are shown in Table 1.
Cooked rice was prepared under the same conditions as in Example 2. This rice was left standing for 18 hr in a thermostatic tank set to 20° C. and then subjected to sensory evaluation. The conditions and method for sensory evaluation were the same as those used in Example 2. The results are shown in Table 2.
As shown in Table 2, the softness of cooked rice without addition of enzyme decreased by preservation at 20° C. for 18 hr and the stickiness thereof became low. When TtAM was added, the softness and stickiness were low immediately after cooking rice, and changes in the properties were small after preservation at 20° C. for 18 hr. On the other hand, when CgAM was added, softness and stickiness increased immediately after cooking rice, and the softness and stickiness decreased somewhat after preservation at 20° C. for 18 hr. Compared with TtAM, however, the properties were generally maintained close to those of cooked rice prepared without addition of enzyme on the day of cooking.
Cooked rice was prepared under the same conditions as in Example 2, and the amount of scorching was examined. Note that the amount of scorching refers to the weight of the portion of rice that remained sticking to the inner pot when the inner pot containing the cooked rice was taken out of the rice cooker immediately after completion of cooking rice and inverted to drop the cooked rice. The results are shown in Table 3 and
As shown in Table 3 and
To confirm a time-related degradation-suppressive effect of CgAM on a wheat starch-containing food, plain bread blended with CgAM was prepared, and the degree of degradation of the mouthfeel of the plain bread after preservation for a given period after the preparation was verified. To be specific, water was weighed in a metal container attached to a breadmaker (MK SEIKO CO., LTD., HBK-100), and a predetermined amount of CgAM (test section 1: no addition, test section 2: 0.01 U/g wheat flour, test section 3: 0.1 U/g wheat flour, test section 4: 1.0 U/g wheat flour) was added. Then a pre-mixed mixture of hard flour, sugar, skim milk, and salt was added thereto. Water and then dry yeast and shortening were added, and bread was prepared by kneading and baking according to the predetermined program (plain bread, baking color: normal). The actual amount of each component is shown in the following Table 4. After completion of baking, the bread was cooled by allowing to stand at room temperature for 1.5 hr, then sliced to a thickness of 2 cm, enclosed in a vinyl bag with a chuck, and stored at a temperature of 10° C. and a humidity of 50% for 2 days.
66%
The mouthfeel (“drying”) of the plain bread stored under the aforementioned conditions was evaluated by sensory evaluation by three professional panels. The evaluation criteria used in the sensory evaluation were as follows.
X: strongly dried
Δ: dried
◯: a little dried
⊙: moist
The results are shown in the following Table 5.
As shown in Table 5, in the bread containing CgAM, the dryness of the plain bread was suppressed even after 2 days from baking.
Potato flakes (“potato flakes”, Taimou Kabushiki Kaisha) were weighed (40 g in one test section) and mixed with 120 g of water. CgAM was added thereto and the mixture was stirred with a spatula until uniformity. The obtained mixture was stood at room temperature for 30 min. After standing for 30 min, the mixture was divided into 3 packs by 50 g each and tightly sealed with a vacuum sealer. The tightly sealed packs were heated in boiling water for 30 min, and then cooled with running water to about 25° C. The pack was opened and the content was dispensed into a 24 well microplate. This was stored in a refrigerator (5° C.) and subjected to a compression test using a texture analyzer on the day of dispensing, one day later, one week later, and two weeks later and the hardness of the paste was measured.
The amount of CgAM used in this Example was 0.1 U or 1.0 U per 1 g of the dried potato flakes.
The measurement conditions of the texture analyzer used for the property measurement are as follows.
apparatus: texture analyzer (“TA-XT Plus” (EKO INSTRUMENTS CO., LTD.)) diameter 5 mm stainless spherical plunger measurement conditions: compression rate 1 mm/sec, the center of the model potato salad filled in a plate was compressed (penetrated) 50% and the maximum stress was recorded (N=3).
The results are shown in the following Table 6 (compression stress).
The numerical value is maximum stress (mean) by 50% compression, and the unit is g.
As shown in Table 6, CgAM was shown to suppress time-course hardening of the model potato salad.
Amylomaltase derived from the genus Streptomyces and Corynebacterium glutamicum-derived amylomaltase are known to have relatively high amino acid identity and their functions are expected to be similar. The following experiment was conducted to demonstrate same.
To 10 mM phosphate buffer (pH 7) was added rice starch (SIGMA) to give a 10% suspension, amylomaltase derived from CgAM, TtAM or Streptomyces avermitilis (hereinafter sometimes to be referred to as “SaAM”) was further added at 50 U/g starch, and the mixture was reacted at 37° C. for 1 hr while stirring. The mixture was heated at 98° C. for 10 min to deactivate the enzyme, and cooled to 50° C. The starch gel after cooling was dispensed to a cylindrical tube having a diameter of 5 mm and gelled by storing at 5° C. for 1 to 7 days. The obtained starch gel was taken out of a refrigerator at the time points of one day, 3 days and 7 days later with the preparation day of the starch paste as day 0, and formed into a cylindrical shape having a diameter of 5 mm and a height of 5 mm by using a razor blade. It was placed on the stage of a texture analyzer (TA-XT Plus) such that the cut surface is up and down, and subjected to a compression test in which the gel strength was measured.
The texture analyzer used for the property measurement and the measurement conditions are as follows.
apparatus: texture analyzer (“TA-XT Plus” (EKO INSTRUMENTS CO., LTD.)) diameter 15 mm acrylic cylindrical plunger, stainless stage
measurement conditions: compression rate 0.5 mm/sec, the maximum stress (g) obtained when starch gel piece was compressed 90% was recorded (N=6-9).
The results are shown in
Amylose (BAR-5K-1, GLICO NUTRITION CO., LTD.) was added to dimethyl sulfoxide to give a 10% solution, and the solution was diluted with milli-Q water to prepare a 1% amylose solution. To the prepared 1% amylose solution (500 μl) was added 50 μL of each amylomaltase solution (SaAM, CgAM, or TtAM) adjusted to 1 U/mL. The mixture was reacted at SaAM: 50° C., CgAM: 37° C., TtAM: 70° C. for 60 min. Then, each reaction mixture was heated at 100° C. for 10 min to deactivate the enzyme and cooled to room temperature. After cooling, each reaction mixture was diluted with milli-Q water such that the amylose concentration was 0.1%, and the distribution of sugar chain length of the amylose degradant contained in the diluted reaction mixture was analyzed using ion chromatography (Dionex). The relative value of the peak area of each sugar chain to the total peak area was calculated. The results are shown in
As shown in
Each rice starch (1.5 g) was added to 28.5 ml of 50 mM phosphate buffer (pH 6.0) to prepare a 5% starch suspension. The obtained starch suspension was placed in a standing pouch, and the starch was gelatinized by heating at 100° C. for 15 min (gelatinized starch solution 1). In addition, 1.5 g of glucose (sometimes referred to as “G1”) or sucrose (sometimes referred to as “suc”) was added to 28.5 ml of 50 mM phosphate buffer (pH 6.0) to prepare a 5% G1 solution or suc solution and the solution was dispensed into a 1.5 mL tube. Then, gelatinized starch solution 1 (500 μL) and G1 solution or Suc solution or so milli-Q water (500 μL) were mixed to give a mixed solution (1 mL).
Next, 2.5 U/mL CgAM solution and 2.5 U/mL TtAM solution were prepared. The obtained enzyme solutions (100 μL) were each added (enzyme 100 U per 1 g of starch) to the mixed solution (1 mL) adjusted as mentioned above. After addition of the enzyme solution, the reaction mixture added with CgAM was heated to 30° C., and the reaction mixture added with TtAM was heated to 50° C. The control added with milli-Q water instead of the enzyme solution was heated to 30° C. After 24 hr, each enzyme in the reaction mixture was deactivated by heating at 100° C. for 10 min. Each reaction mixture was subjected to TLC analysis later.
TLC analysis was performed as follows. As a standard, 2 μL of three kinds of 0.5% sugar solutions were spotted. The three kinds of sugar solutions used as the standards were as follows.
sugar solution 1: glucose (G1), maltose (G2), maltotriose (G3), maltotetraose (G4), maltopentaose (G5), maltohexaose (G6) and maltoheptaose (G7) mixture
sugar solution 2: sucrose solution (suc)
sugar solution 3: α cyclodextrin (αCD) and β cyclodextrin (βCD) mixture
The reaction mixture after the enzyme deactivation was 5-fold diluted with milli-Q water to adjust rice starch, G1, or Suc to 0.5%, and then 2 μL was spotted.
The reaction mixture was developed once, and the composition of the solvent for development was n-butanol:pyridine:milli-Q water (MQ)-6:4:1. For detection, color was developed by spraying 20% sulfuric acid/EtOH on the carrier and then heating same at 110° C. for about 10 min. The results are shown in
As shown in
Dextrin (Matsutani Chemical Industry Co., Ltd. Paindex #100) was dissolved with milli-Q water to give a 5% solution. Sucrose (JUNSEI CHEMICAL CO., LTD. reagent special grade) was similarly dissolved with milli-Q water to give a 5% solution. These were mixed at a 1:1 quantitative ratio, and the CgAM solution was added at 100 U per 1 g of dextrin. The same amount of milli-Q water was added to the control. The mixture was stood in a water bath at 30° C. for 24 hr to perform an enzyme reaction. Thereafter, the enzyme was deactivated by heating in a hot-water bath at 100° C. for 10 min. The dextrin-sucrose solution after the enzyme reaction was stored in a freezer at −80° C.
By the method described below, the blood glucose level after administration of starch was measured using rats and a suppressive effect on an increase in the blood glucose level was evaluated. The test substance was thawed with running water on the day of the blood glucose measurement test and used for the test. According to the following measurement method of the blood glucose level and the test schedule of
Various glucose loading tests on rats have been performed. In this test, the glucose loading test disclosed in JP-A-2005-328776, which is incorporated herein by reference in its entirety, was modified and performed.
animal species and lineage: rat, Slc:Wistar (SPF)
manufacturer: Japan SLC, Inc.
sex: male
age at arrival: 6 weeks of age
quarantine, acclimation: Animals are acclimatized from arrival to grouping. Quarantine is conducted for up to day 7 with the arrival date as day 0. General condition is observed every day.
temperature: 22±3° C.
humidity: 50±20%
lighting time: 12 hr/day
kind: Labo MR stock solid feed (Nosan Corporation) or CRF-1 (Oriental Yeast Co., Ltd.)
feeding method: freely given except during the fasting period.
kind: tap water
water feed method: freely given throughout the test period.
The animals to be used for the test are selected from the animals that did not show any abnormalities in the observation of general conditions during the quarantine/acclimation period. The animals are used at 7 weeks of age. The body weight is measured on the final day of quarantine/acclimation, and animals are assigned to 6 to 10 animals/group by the stratified randomization method and using the obtained body weight as an index.
Fasting is started from the evening of one day before the glucose loading test and performed overnight.
The vein at the tip of the tail is incised using a scalpel blade under unanesthesia. The test (blood glucose level) is performed using blood leaking from the incision surface. The measurement is performed using a self-checking glucometer “ACCU-CHEK” (Roche DC) or “glutest Neo” (Sanwa Kagaku Kenkyusho), and the blood glucose level displayed on the measuring instrument is recorded. This is used as the fasting blood glucose level. The same measuring instrument was used for the tests on the same day.
The calculation of the evaluation items was performed as follows.
Fasting blood glucose level was used as the blood glucose level at 0 minute.
Value obtained by subtracting the blood glucose level at 0 minute from the blood glucose level at each measurement time was used as “Δblood glucose level (mg/dL)”.
The highest value of the A blood glucose level at each measurement time was used as “ΔCmax (mg/dL)” of each individual.
The value obtained by calculating the area under the A blood glucose level increase curve was used as “A blood glucose level AUC (mg/dL·min)”. The calculation method was based on the method of Japanese Association for the Study of Glycemic Index.
The suppressive effect on the elevation of blood glucose level was evaluated from the value of Δ blood glucose level AUC of the test substance administration group based on the Δ blood glucose level AUC of the control group as 100.
The test results are shown in
α non-glutinous rice starch (My Alpha K: Joetsu Starch Co., Ltd.) was weighed by 10 g, and 80 g of milli-Q water was added. The mixture was transferred into a standing pouch (Lami Zip Stand Type: produced by SEISANNIPPONSHA LTD.), sealed with a heat sealer, and starch was completely gelatinized by heating at 100° C. for 15 min in a thermostatic tank. During gelatinization, the mixture was stirred so that the starch would not become lumps. The starch paste for the control and CgAM was cooled to 37° C., and the starch paste for TtAM was cooled to 50° C., and 10 mL of CgAM solution or TtAM solution prepared such that the amount of each AM added was 1 U per 1 g of starch was added. Milli-Q water (10 mL) was added to the control. Thereafter, they were immediately sealed with a sealer and placed in a thermostatic tank. The control and CgAM addition section were stood at 37° C., and the TtAM addition section was stood at 50° C. for 60 min to cause enzyme reaction. After the enzyme reaction, they were heated at 100° C. for 15 min in a thermostatic tank to deactivate the enzyme. The enzyme-treated starch was cooled to room temperature and frozen at −80° C.
The test was performed according to the method of Example 10.
The measurement was performed according to the method of Example 10.
The test results are shown in
Hitomebore from Miyagi prefecture was used as the raw material. For brown rice, the rice harvested on the same day by one manufacturer was ensured, and polished on the same day. The rice was placed in a shading vacuum bag containing an oxygen absorber and kept in a refrigerator at 5° C. until the test.
The polished rice was allowed to return to room temperature by 30 min before weighing on the day of cooking rice. The polished rice was weighed using an electronic balance (US6002S, METTLER TOLEDO Co., Ltd.). The polished rice in a sieve was gently stirred clockwise 10 times in tap water in a bowl. The tap water was replaced and the same operation was repeated 5 times. After washing the rice, the rice was picked up in a sieve, transferred into a rice cooking pot, tap water was added so that the water content was 150% on an electronic balance, and CgAM was further added in an amount of 1 U per 1 g of raw rice. After immersing at room temperature for 1 hr, the pot was set in a rice cooker (SR-03GP: Panasonic Corporation) and the rice was cooked. Immediately after cooking rice, the rice cooking pot was turned over on a tray and the cooked rice was taken out. The rice near the pot wall was removed and moved to the end of the bat with a spatula. The cooked rice was flattened with the spatula, wrapped lightly with a small gap on the rice, and then roughly cooled at room temperature for 15 min. The rice was put in UNI-PACK (SEISANNIPPONSHA LTD), smoothed to a thickness of about 2 cm, and frozen in a freezer at −80° C. The next day, the rice was freeze-dried using a freeze-dryer (FDU-2100: TOKYO RIKAKIKAI CO, LTD). After freeze-drying was confirmed, the rice was pulverized using a mixer mill (MM301: Verder Scientific Co. Ltd.). The pulverization sample was dispensed into a standing pouch, tightly sealed, and stored at room temperature.
The test was performed according to the method of Example 10. The test substance was suspended in milli-Q water and subjected to the test.
The measurement was performed according to the method of Example 10. The test substance was administered orally at a total amount of sugar in the test substance of 2 g/20 mL/kg.
The test results are shown in
According to the present invention, a starch-containing food that has a preferable mouthfeel, is less susceptible to time-related degradation, and is less likely to raise blood glucose level can be produced without the problem possessed by the existing heat-resistant amylomaltase. Therefore, it is highly beneficial in the food producing industry.
Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.
As used herein the words “a” and “an” and the like carry the meaning of “one or more.”
Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that, within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.
All patents and other references mentioned above are incorporated in full herein by this reference, the same as if set forth at length.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018-058931 | Mar 2018 | JP | national |
This application is a continuation of International Patent Application No. PCT/JP2019/012670, filed on Mar. 26, 2019, and claims priority to Japanese Patent Application No. 2018-058931, filed on Mar. 26, 2018, both of which are incorporated herein by reference in their entireties.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/JP2019/012670 | Mar 2019 | US |
| Child | 17032555 | US |
