JP2019165727A - New application of lipase in dough - Google Patents

Abstract

To provide an agent capable of reducing kneading time in making a dough, an agent capable of reinforcing impact resistance of the fermented dough and an agent capable of increasing a ratio of added moisture in the dough and a production method of a dough using this and production method of food product as well.SOLUTION: The agent according to the present invention is a kneading time shortening agent for making a dough, an impact resistance reinforcement agent of a fermented dough and an addition rate increasing agent of moisture in the dough, each containing lipase as an active ingredient. The invention can contribute to increased productivity in production of dough or food product using this.SELECTED DRAWING: None

Description

  TECHNICAL FIELD The present invention relates to a novel use of lipase in dough, and in particular, a kneading time shortening agent for making dough, an impact resistance enhancing agent for fermented dough, and a moisture addition ratio increasing agent in dough, comprising lipase as an active ingredient And a method for producing dough and a method for producing food using the same.

  The dough (dough) is a food dough made by mixing water and other ingredients (sub-materials) as needed, with ingredients mainly composed of starch obtained from cereals and beans. It refers to a hard material with little fluidity and poor fluidity. The dough is cooked and cooked, such as baking, steaming, and frying. Specific examples include bread, donuts, pies, noodles, dumplings and shumai, spring rolls, buns, dumplings, cookies, Examples include baked goods such as rice crackers. The foods produced by heating these doughs have a wide product group as described above, the market size is large, and occupy an important position in the food industry.

  Therefore, techniques for improving the production process of dough and the food (final product) produced from the dough have been researched and developed. For example, Patent Document 1 discloses a dough containing phospholipase A as an active ingredient. The improved material is disclosed in Patent Document 2 as a bread quality improver having Rhizopus japonicus as its origin and lipase having an activity of 100,000 to 2,000,000 LUN / g as an active ingredient.

Japanese Patent Publication No. 60-30488 JP 2005-318833 A

  As described above, the dough is made by mixing water or water and sub-materials with a food mainly composed of starch and kneading. As is clear in the case of bread dough, which is a typical dough, this kneading process requires a great deal of time and the operating energy of a machine such as a mixer greatly affects the cost. Therefore, the present inventors have thought that if the chaos time can be shortened, it can greatly contribute to the reduction of the production cost or the improvement of the productivity.

  In addition, in the case of foods produced by fermenting doughs such as bread and yeast donuts, fermented dough will wither when shocked and lead to quality loss such as volume reduction in the final product. Need to be heated. However, it is difficult to completely remove the impact whether the line is manufactured by a machine in a large-scale factory or manually in a small store kitchen. Therefore, the present inventors can enhance the impact resistance of the fermented dough and reduce the deterioration of quality due to the impact, which can contribute to the improvement of productivity such as the reduction of mold loss and the quality of the final product. I thought it was possible.

  Moreover, it can be said that the thing with the lowest cost among the materials which comprise dough is water in many cases. Therefore, the present inventors considered that if the addition ratio of water can be increased in the blending of dough, it can greatly contribute to the reduction of production cost or the improvement of productivity.

  In this regard, the dough improving agent described in Patent Document 1 improves the dough extensibility, non-adhesiveness (adhesion to the machine) and moldability, and improves the film elongation and texture of the inner phase of the final bread. It improves and reduces the degree of aging. Moreover, the bread quality improving agent described in Patent Document 2 also improves the adhesion and extensibility of the dough to the equipment and softens the texture of the bread. That is, none of the prior arts solves the problems of shortening the kneading time at the time of dough production, enhancing the impact resistance of the fermented dough, or increasing the proportion of moisture added in the dough formulation.

  The present invention has been made to solve such problems, and is an agent that can shorten the chaos time during dough production, an agent that can enhance the impact resistance of fermented dough, and a dough An object of the present invention is to provide an agent capable of increasing the rate of addition of moisture, and a method for producing dough and a method for producing food using the same.

  As a result of diligent research, the inventors of the present invention have been able to shorten the chaos time for making dough, enhance the impact resistance of fermented dough, and add moisture in the dough by adding lipase to the dough material We found that the rate could be increased. Accordingly, the following inventions have been completed based on these findings.

(1) The kneading time shortening agent for dough preparation according to the present invention comprises lipase as an active ingredient.

(2) The fermented dough impact resistance enhancer according to the present invention comprises lipase as an active ingredient.

(3) The water addition ratio increasing agent in the dough according to the present invention contains lipase as an active ingredient.

(4) In the agent according to the present invention, the lipase preferably has the following amino acid sequence (a) or (b):
(A) In the amino acid sequence of SEQ ID NO: 1 (full length 269 amino acids), an amino acid sequence into which any one amino acid mutation selected from those listed in Tables 1 to 5 below is introduced,
(B) An amino acid sequence having 90% or more sequence identity with (a), having an amino acid mutation corresponding to the amino acid mutation introduced in (a), and having lipase activity.

(5) The manufacturing method of the dough which concerns on this invention has the process of mixing the agent in any one of Claims 1-4 with the material which comprises dough.

(6) The manufacturing method of the foodstuff which concerns on this invention has the process of heating the dough containing the agent in any one of Claims 1-4.

  According to the kneading time shortening agent for dough preparation according to the present invention, a dough production method or a food production method using the same, the kneading time at the time of dough preparation can be shortened. Therefore, in manufacturing dough or food using the dough, it can greatly contribute to reduction of production cost or improvement of productivity.

  According to the fermented dough impact resistance enhancer according to the present invention, a dough production method or a food production method using the same, the impact resistance of the fermented dough is enhanced, the volume of the final product is reduced by impact, and the texture of the inner layer And the deterioration of food texture can be reduced. Therefore, in the manufacture of dough or food using the dough, it can greatly contribute to the improvement of productivity such as the reduction of mold-off products and the quality of the final product.

According to the water addition ratio increasing agent in the dough, the dough manufacturing method or the food manufacturing method using the same, the water addition ratio can be increased in the dough blending.
That is, a product having the same quality can be manufactured even if a large amount of moisture is added to the same amount of material such as grain flour, so that more products can be manufactured from the same amount of material other than water. I can say that. Therefore, in manufacturing dough or food using the dough, it can greatly contribute to reduction of production cost or improvement of productivity.

It is a graph (dough graph) which shows the relationship between the chaos time and the power consumption of a mixer at the time of producing the dough (sample 1) which does not add lipase, and the dough (sample 2, sample 3) which added lipase. Bread dough without addition of lipase, bread without any impact after final fermentation (sample 1), bread dough without addition of lipase with impact after final fermentation (sample 2), and dough with addition of lipase, final fermentation It is a photograph which shows each shape and internal phase of the bread (sample 3) which applied the impact later. (A) is a bread (sample 1) manufactured with bread dough that does not add lipase and does not increase moisture, and bread (sample 2) that is manufactured with bread dough with added lipase and increased moisture. It is a bar graph which shows volume. (B) is a table showing numerical values of volume, weight and specific volume of Sample 1 and Sample 2.

  Hereinafter, the kneading time shortening agent for dough preparation according to the present invention, the impact resistance enhancer of fermented dough, the additive ratio of moisture addition in the dough, and the method for producing dough and the method for producing food using the same explain.

  The kneading time shortening agent for dough preparation according to the present invention, the impact resistance enhancing agent for fermented dough, and the moisture content increasing agent in the dough (hereinafter sometimes collectively referred to as “the agent of the present invention”). In any case, lipase is an active ingredient. Moreover, the manufacturing method of the dough concerning this invention has the process of mixing the agent of this invention with the material which comprises dough. Moreover, the manufacturing method of the foodstuff which concerns on this invention has the process of heating the dough containing the agent of this invention.

  Lipase is an enzyme that catalyzes the reaction of hydrolyzing glycerol esters (neutral fats, triglycerides) into fatty acids and glycerol (Iwanami Biology Encyclopedia, 4th edition, 9th edition, 2005, Iwanami Shoten). In the present invention, as the lipase, a commercially available lipase preparation for bread can be used. Examples of such commercially available products include “Lipase A Amano 6”, “Lipase DF Amano 15” (above, Amano Pharmaceutical), “Denabak RICH (bread)” (Nagase Seikagaku Corporation), “Lipopan 50BG” (Novozymes) and the like can be mentioned.

In addition to triglyceride degrading activity (lipase activity), lipases are known to have phospholipid degrading activity (phospholipase activity) and digalactosyl diglyceride degrading activity (DGDG degrading activity) (Patent No. 1). No. 4723087; Example 5, Example 13). Furthermore, it is known that a lipase having a high degradation activity for a long-chain triglyceride, which is a C _{16 to} C ₂₀ long-chain fatty acid ester, is also present (Japanese Patent No. 4723087; Example 5, Example 7). In the present invention, such lipase having phospholipase activity, lipase having DGDG degradation activity, or lipase having high degradation activity of long chain triglycerides can be suitably used.

Specific examples of lipases having phospholipase activity include those having the following amino acid sequences (a) or (b);
(A) In the amino acid sequence of SEQ ID NO: 1 (full length 269 amino acids), an amino acid sequence into which any one amino acid mutation selected from those listed in Tables 1 to 5 above is introduced,
(B) An amino acid sequence having 90% or more sequence identity with (a), having an amino acid mutation corresponding to the amino acid mutation introduced in (a), and having lipase activity.

  Here, SEQ ID NO: 1 is the amino acid sequence of a lipase from the strain DSM 4109 of the filamentous fungus Humicola lanuginosa (hereinafter sometimes referred to as “DSM4109 lipase”). Therefore, the lipase having the amino acid sequence (a) means a mutant lipase obtained by introducing any one of 129 amino acid mutations described in Tables 1 to 5 into DSM4109 lipase. . The lipase having the amino acid sequence (b) means a mutant lipase having high similarity to (a) in terms of amino acid sequence and enzyme activity.

  SEQ ID NO: 1 is equivalent to the amino acid sequence at positions 1 to 269 of SEQ ID NO: 2 described in US Pat. No. 5,869,438. Further, 129 kinds of amino acid mutations described in Tables 1 to 5 are listed in “Tables 1 to 5” described in “Claims” of Japanese Patent No. 4723087, or “Detailed Description of the Invention”. It is equal to the amino acid mutation described in “Table 6 to Table 11”.

In the present invention, the lipase activity, phospholipase activity, DGDG degradation activity and long chain triglyceride degradation activity can be confirmed according to a conventional method, for example, by the following method.
<Lipase activity>
A substrate is prepared by emulsifying tributyrin (glycerin tributyrate) with gum arabic (emulsifier). The substrate is hydrolyzed at 30 ° C. and pH 7, and the release of butyric acid is followed by pH stat titration. One unit of lipase activity (1 LU) is the amount of enzyme that liberates 1 μmol of butyric acid per minute under these conditions.

《Phospholipase activity》
Phospholipase activity (PHLU) is measured using lecithin as a substrate. That is, 50 μL of diluted lipase in 50 μL of a buffer (4% Triton X-100 and 50 mM HEPES buffer containing 5 mM calcium chloride) containing 4% L-alpha-phosphatidylcholine (plant lecithin, Avanti) as a substrate. The sample is added and the substrate is hydrolyzed at 30 ° C. and pH 7 for 10 minutes, incubated at 95 ° C. for 5 minutes to stop the reaction, and then centrifuged at 7000 rpm for 5 minutes. Subsequently, after the reaction of the reagent A and the reagent B of the NEFA C kit (Wako Chemicals), the free fatty acid is measured by measuring the absorption at 550 nm using an HP 8452A diode array spectrophotometer. . Use oleic acid as the standard sample. 1 PHLU is the amount of enzyme that liberates 1 μmol of fatty acid per minute under these conditions.

<< DGDG degradation activity >>
A monolayer of digalactosyl diglyceride (DGDG) (Sigma catalog number D4651) is chloroformated on the surface of a buffer solution (containing 10 mM NaOAc, 1 mM CaCl ₂ and 10 mM β-cyclodextrin, 25 ° C., pH 5.5). Spread from solution. After leaving the monolayer to evaporate chloroform, the surface pressure is adjusted to 15 mN / m. A solution containing about 60 μg of enzyme is injected through a monolayer into the subphase of the reaction compartment in the “zero-order trough” (syringe with surface area 2230 mm ² and reaction volume 56570 mm ³ ). . As insoluble substrate molecules are hydrolyzed into more water-soluble reaction products (in the presence of β-cyclodextrin), an increased number of movable barriers compressing the monolayer to maintain a constant surface pressure Enzyme activity is represented by the rate. If the barrier moves at less than 1 mm / min, DGDG degradation activity is considered positive.

<Decomposition activity of long-chain triglycerides>
Lipase activity (SLU) against triolein is measured using olive oil as a substrate. That is, 2.5 mL of substrate (olive oil emulsion (Sigma catalog number 800-1) was mixed with 12.5 mL of buffer (5 mM Tris buffer containing 40 mM NaCl and 5 mM calcium chloride) and 0.5 mL of Diluted lipase sample is added and the substrate is hydrolyzed at 30 ° C., pH 9, and the amount of oleic acid released by pH stat titration is followed.1 unit of lipase activity (1 SLU) is Is the amount of enzyme that liberates 1 mole of oleic acid per minute.

  The definition of the amino acid variation | mutation described in Table 1-Table 5 is demonstrated. Amino acids are represented by a single letter code. For example, G91A indicates that G (glycine) is substituted with A (alanine) at position 91. For example, T267A and Q indicate that T (threonine) is substituted with A or Q (glutamine) at position 267. E1E, D, and A indicate that E (glutamic acid) is left as it is or substituted with D (aspartic acid) or A at position 1. T267stop indicates a stop codon. That is, it shows deletion of all amino acids (ie, T267, C268 and L269) downstream (carboxyl terminal side) from position 267. 270P, 271V indicates the extension of PV toward the carboxyl terminus (ie, at new positions 270 and 271). -G266 indicates a deletion of G at position 266. Parentheses indicate that the change is optional. SPIRR indicates N-terminal extension. D266 indicates substitution with any amino acid except D at position 266. E1SPPCGRRP indicates substitution of E at position 1 with SPPCGRRP, ie, peptide addition at the amino terminus. T267GS indicates that T is replaced with GS at position 267, or in other words, indicates substitution T267G and insertion of S between G267 and G268.

  The identity of the amino acid sequence can be confirmed according to a conventional method. For example, FASTA (http://www.genome.JP/tools/fasta/), Basic local alignment search tool (BLAST; http: // www. ncbi.nlm.nih.gov.), Position-Specific Iterated BLAST (PSI-BLAST; http://www.ncbi.nlm.nih.gov.) and the like. Note that “identity” indicates coincidence and is used interchangeably with “identity”.

  Mutant lipase can be obtained according to a conventional method, and examples of such a method include a chemical synthesis method and a method using a gene recombination technique. In the chemical synthesis method, for example, the Fmoc method (fluorenylmethyloxycarbonyl method), the tBoc method (t-butyloxycarbonyl method), etc., based on the amino acid sequence information of (a) or (b) described above. In addition to the synthesis according to the synthesis method, it can also be synthesized using various commercially available peptide synthesizers.

  Further, in the method using the gene recombination technique, the mutant lipase may be expressed in a suitable expression system. That is, DNA encoding the amino acid sequence of the mutant lipase is inserted into an appropriate vector to obtain a recombinant vector, and then the recombinant vector is introduced into an appropriate host to obtain a transformant. And it can obtain by culturing the obtained transformant and expressing mutant lipase. Here, the DNA encoding the amino acid sequence of the mutant lipase can be synthesized using various commercially available DNA synthesizers based on the nucleotide sequence information, and the DNA encoding the DSM4109 lipase can be used as a template. Can be obtained by performing polymerase chain reaction (PCR).

  For example, first, a DNA primer encoding an amino acid mutation to be introduced is designed, and the DNA primer is used to perform PCR using a DNA encoding DSM4109 lipase as a template. DNA encoding a mutant lipase can be obtained.

  In the present invention, the lipase is used by mixing with the material constituting the dough. Examples of the timing for adding the lipase include a step of mixing various materials composing the dough. The amount of lipase added is not particularly limited, and examples thereof include 1000 to 12000 U, 2000 to 10,000 U, or 3000 to 6000 U per kg of flour. In this case, the unit of enzyme activity is 10 U when the enzyme is liberated 1 μmol of fatty acid per minute when the olive oil emulsion is allowed to act for 30 minutes at 37 ° C. and pH 6.0.

  In addition, in this invention, as a main material which comprises a dough, foods mainly consisting of starch, such as water and flour (wheat flour, rice flour, barley flour, rye flour, corn flour, potato flour, tef flour, fly flour, kinako flour) , Soybean powder, chickpea powder, pea powder, mung bean powder, buckwheat flour, amaranth powder, potato starch, litter powder, tapioca powder, chestnut powder, acorn powder, etc.). In addition to the above, the dough can be blended with arbitrary auxiliary materials. Examples of such auxiliary materials include yeast, fats and oils, seasonings such as sweeteners and salt, dairy products, eggs, gluten, Various food additives, dough improving agents and the like can be mentioned.

  In this invention, the heating method at the time of manufacturing food by heating dough is not specifically limited, For example, dough is heated by arbitrary methods, such as baking (baking), steaming (steaming), and frying (oil tone). be able to.

  Hereinafter, although this invention is demonstrated based on each Example, the technical scope of this invention is not limited to the characteristic shown by these Examples. In this example, the lipase used was a mutant lipase in which any one of 129 amino acid mutations described in Tables 1 to 5 was introduced into DSM4109 lipase.

<Example 1> Verification of effect of lipase: Effect of shortening kneading time for making dough By putting the material in a spiral mixer (Kemper) with the composition shown in Table 6, mixing at low speed for 3 minutes and then at high speed for 10 minutes The dough (bread dough) of samples 1 to 3 was prepared. During this chaos, the power consumption (watts, W) of the mixer was measured over time, and this was used as an index for evaluating the elasticity of the dough. That is, it can be evaluated that the greater the power, the greater the elasticity of the dough. The measurement results (dough graph) of the power at each chaos time are shown in FIG. 1, and the power at the chaos time of 640 seconds, 660 seconds, 680 seconds and 700 seconds are shown in Table 7, respectively.

  As shown in FIG. 1 and Table 7, when comparing the chaos time to reach 277.4 watts, which is the maximum power of Sample 1, it was 680 seconds for Sample 1, while it was a little less than 660 seconds for Sample 2. In sample 3, it was about 650 seconds. That is, in the case where lipase was added, the time required for the dough to acquire the elasticity required for breadmaking was shorter than in the case where lipase was not added. Moreover, the said time was the tendency to become short, so that the addition amount of lipase is large. From this result, it became clear that lipase can shorten the chaotic time for making dough. Note that the time difference between sample 1 and samples 2, 3 is a significant time reduction when a large amount of dough is continuously mixed in industrial production, for example, a large-scale bread factory. This greatly contributes to reducing production costs and improving productivity.

<Example 2> Verification of effect of lipase: Impact resistance enhancing effect of fermented dough With the formulation shown in Table 8, samples 1 to 3 (five for each sample) bread were produced by the medium seed method. Specific manufacturing procedures are shown in the following [1] to [9]. However, Sample 2 and Sample 3 were impacted by dropping them from a height of 30 cm in the state of being put in a mold after the final fermentation step of [8] and before the firing step of [9]. . For sample 1, the impact was not applied.

《Procedure for making bread by the middle seed method》
[1] Medium seed mixing: Medium seed materials shown in Table 8 were put into a mixer (Aikosha Seisakusho) and mixed for 3 minutes at low speed and then for 3 minutes at medium speed to prepare a medium seed dough.
[2] Medium seed fermentation: The medium seed dough was fermented by placing it at 28 ° C. and 75% humidity for 4 hours.
[3] Main rice cake: Medium fermented dough after fermentation and the main rice ingredients shown in Table 8 (excluding margarine) are put in a mixer (Aikosha Seisakusho), 3 minutes at low speed, 3 minutes at medium speed, then high speed The mixture was kneaded for 1 minute, and after adding the margarine, the koji was kneaded for 2 minutes at low speed, 3 minutes at medium speed, and then 1 minute at high speed.
[4] Primary fermentation: Fermentation was carried out by placing the main body dough at 28 ° C. and 75% humidity for 30 minutes.
[5] Division: The dough after the primary fermentation was divided into 450 g per ball and rounded.
[6] Bench time: The rolled dough was left at room temperature for 20 minutes to rest.
[7] Molding: The rested dough was passed through a molder (Kotobuki baking machine), molded into a roll mold, and placed in a mold.
[8] Final fermentation: The dough after molding was fermented by placing it at 38 ° C. and a humidity of 85% for 60 minutes.
[9] Baking: The fermented dough was baked for 25 minutes at 220.degree.

The breads of Sample 1 to Sample 3 were cooled at room temperature for 60 minutes, then cut in the short direction, the shape and the internal phase were observed, and photographs were taken. The photograph is shown in FIG. Further, the weight and volume (cm ³ ) were measured, the specific volume (cm ³ / g) was calculated, and the average value (5 average values for each sample) was obtained. Furthermore, each of the five experienced panelists ate each sample and evaluated the “crumb (inner phase) texture” and “clam (inner phase) hardness” in the following three stages based on sample 1. did. “Clam texture” +: finer, ±: comparable, −: coarser. “Clam hardness” +: softer, ±: comparable, −: harder. The results are shown in Table 9.

  As shown in FIG. 2 and Table 9, the volume and specific volume were remarkably small in sample 2 as compared to sample 1, but almost the same in sample 3. That is, when an impact is applied to the fermented dough before baking, the volume (bulge) is remarkably reduced, and it has been clarified that addition of lipase can prevent the volume (bulge) from decreasing. In addition, the texture of crumb was coarser (−) in sample 2 than in sample 1, whereas it was as fine (±) as in sample 3. Furthermore, the hardness of the crumb was harder in Sample 2 (−) compared to Sample 1 (−), whereas it was softer in Sample 3 (+). That is, when an impact is applied to the fermented dough before baking, the texture of the crumb becomes rough and the texture becomes hard, and it has been clarified that the degradation of the crumb can be prevented by adding lipase. These results revealed that lipase can enhance the impact resistance of fermented dough.

<Example 3> Verification of effect of lipase: Effect of increasing water addition ratio Sample 1 and Sample 2 breads (eight per sample) were produced by the medium seed method with the formulation shown in Table 10. The specific production procedure was the same as [1] to [9] in Example 2. However, [3] Chaos after the introduction of margarine in the main process was 1 minute at low speed, 2 minutes at medium speed, and then 2 minutes at high speed. [5] The divided weight of the dividing step was 380 g per ball, [8] the time of the final fermentation step was about 45 minutes, [9] the lower flame of the firing step was 200 ° C., and the upper flame was 190 ° C.

After the breads of Sample 1 and Sample 2 were cooled at room temperature for 60 minutes, the weight and volume (cm ³ ) were measured, the specific volume (cm ³ / g) was calculated, and the average value (8 average values for each sample) ) The result is shown in FIG. Moreover, each sample was eaten in five skilled panels, and the tactile sensation (moistness) and the texture (softness and fluffyness) were evaluated in the following four stages based on the sample 1. The results are shown in Table 11.
“Moistness” ++: Remarkably moist, +: More moist, ±: Same level, −: Dryer.
“Soft” ++: remarkably soft, +: softer, ±: comparable, −: harder.
“Fretting” ++: Remarkably flirting, +: Reducing more, ±: Equivalent, −: More crisp.

  As shown in FIG. 3, the volume, weight, and specific volume of bread were the same for sample 1 and sample 2. Moreover, as shown in Table 11, the tactile sensation is more moist than the sample 1 in the sample 2, and the softness of the texture is remarkably softer than the sample 1 in the sample 2, and the fluffy feeling of the texture is Sample 1 and sample 2 were equivalent. In other words, the sample 2 bread with the increased amount of water had the same volume as the sample 1 bread, and had a touch and texture equivalent to or higher than those of the sample 1. From this result, it became clear that lipase can increase the rate of water addition in the dough.

Claims (6)

  A kneading time shortening agent for making dough, containing lipase as an active ingredient.   A fermented dough impact resistance enhancer comprising lipase as an active ingredient.   An additive for increasing the amount of water added in dough, comprising lipase as an active ingredient. The agent according to any one of claims 1 to 3, wherein the lipase has the following amino acid sequence (a) or (b):
(A) In the amino acid sequence of SEQ ID NO: 1 (full length 269 amino acids), an amino acid sequence into which any one amino acid mutation selected from those listed in Tables 1 to 5 below is introduced,
(B) An amino acid sequence having 90% or more sequence identity with (a), having an amino acid mutation corresponding to the amino acid mutation introduced in (a), and having lipase activity.
  The manufacturing method of a dough which has the process of mixing the agent in any one of Claims 1-4 with the material which comprises a dough.   The manufacturing method of foodstuff which has a process of heating the dough containing the agent in any one of Claims 1-4.