Liquid Enzyme Preparation and Preparation Method Thereof

Abstract

Provided are a liquid enzyme preparation of transglutaminase and a preparation method thereof, wherein the liquid enzyme preparation is a liquid preparation of transglutaminase EC2.3.2.13, and its components and amounts thereof are as follows: the liquid preparation of transglutaminase has an enzyme activity of 10-1000 u/ml, a water activity regulator is present in an amount of 30-80 w/v %, a redox potential regulator is present in an amount of 0.0075-1 w/v %, a food preservative is present in an amount of 0-0.1 w/v %, and a pH regulator is added to a final volume of 100%. The preparation method thereof comprises purification of an enzyme solution, mixing, sterilization, filling, and obtaining of a final product.

Claims

1. A liquid enzyme preparation, characterized in that the liquid enzyme preparation is a liquid enzyme preparation of transglutaminase EC2.3.2.13 having an enzyme activity of 10-1000 u/ml.

2. The liquid enzyme preparation according to claim 1, characterized in that its components and amounts thereof are as follows: the liquid preparation of transglutaminase has an enzyme activity of 10-1000 u/ml, a water activity regulator is present in an amount of 30-80 w/v %, a redox potential regulator is present in an amount of 0.0075-1 w/v %, a food preservative is present in an amount of 0-0.1 w/v %, and a pH regulator is added to a final volume of 100%.

3. The liquid enzyme preparation according to claim 1 or 2, characterized in that the liquid enzyme preparation has the following physicochemical properties: 1) having a pH of 5.0-9.0; 2) having a water activity A.sub.w of 0.89; and 3) having a redox potential of 400 mv to +50 mv.

4. The liquid enzyme preparation according to claim 3, characterized in that the liquid enzyme preparation preferably has the following physicochemical properties: 1) having a pH of 5.0-7.5; 2) having a water activity A.sub.w of 0.6-0.85; and 3) having a redox potential of 400 mv to 0 mv.

5. The liquid enzyme preparation according to claim 2, characterized in that the water activity regulator is selected from the group comprising of sorbitol, maltitol, propylene glycol, glycerol, xylitol, polyethylene glycol, trehalose, sucrose, maltose, isomaltose, maltodextrin, xylitol, and mannitol.

6. The liquid enzyme preparation according to claim 2, characterized in that the redox potential regulator comprises one or more of L-ascorbic acid and a salt thereof, L-serine and a salt thereof, L-cysteine and a salt thereof, reduced glutathione, tea polyphenol, soybean protein hydrolysate, wheat protein hydrolysate, casein hydrolysate, chitosan hydrolysate, bamboo leaf antioxidant, rosemary extract, liquorice antioxidant extract, superoxide dismutase, glucose oxidase, sodium bisulfite, sodium metabisulfite, potassium metabisulfite, sodium sulfite, sodium hyposulfite, phytic acid, etc.

7. The liquid enzyme preparation according to claim 2, characterized in that the food additive is selected from the group comprising of -polylysine, natamycin, lysozyme, Nisin, potassium sorbate, sodium dehydroacetate, sodium diacetate, methyl p-hydroxybenzoate, ethyl lauroyl arginate HCl, potassium metabisulfite, and sodium metabisulfite.

8. The liquid enzyme preparation according to claim 2, characterized in that the pH regulator is one of hydrochloric acid, sulfuric acid, acetic acid, lactic acid, citric acid, malic acid, phytic acid, phosphoric acid, nitric acid, oxalic acid, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, trisodium citrate, tripotassium citrate, sodium acetate, potassium acetate, sodium lactate, potassium lactate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, sodium phosphate, potassium phosphate, tap water, purified water, and mineral water, or a buffer system consisting of several pH regulators.

9. The liquid enzyme preparation according to claim 2, characterized in that the water activity regulator comprises one or more of sorbitol, maltitol, and glycerol; and the pH regulator is a buffer comprising phosphoric acid, acetic acid, lactic acid or citric acid, or a salt thereof.

10. A method for preparing the liquid enzyme preparation according to any one of claims 1-9, characterized by the following steps 1) purification of an enzyme solution: subjecting a crude enzyme solution to pressure filtration, microfiltration, and secondary ultrafiltration, to obtain a purified concentrated enzyme solution; 2) mixing: weighing the purified concentrated enzyme solution, a water activity regulator, and a redox potential regulator quantitatively, adding a food preservative if present, and mixing; adding a pH regulator to a final volume of 100%; after mixing homogeneously, testing and adjusting the liquid enzyme preparation for the following parameters: a pH of 5.0-9.0, a water activity A.sub.w of 0.89, and a redox potential of 400 mv to 50 mv; 3) bacteria removal: filtering the resulting mixture through a 0.1-0.22 m membrane for sterilization, followed by sterile filling; 4) package: packaging the liquid enzyme preparation by sterile filling or another relevant liquid package process.

Description

DESCRIPTION OF THE DRAWING

[0061] FIG. 1 is a scheme of the preparation procedure for a liquid enzyme preparation.

DETAILED EMBODIMENTS OF THE INVENTION

[0062] The following examples are provided in order to better understand the invention, the scope as claimed in the invention includes, but is not limited to, the contents described in the following examples, and any modifications and improvements made according to conventional knowledge in the art will be within the scope as claimed in the invention. The scheme is shown in FIG. 1.

Example 1

[0063] A crude transglutaminase solution was prepared by means of microbiological fermentation, which could be carried out by reference to the Patent (Patent Publication No: EP0379606B1): Streptomyces mobaraensis was used as the original strain, a few bacterial colonies in a state of good growth were picked with an inoculating loop, inoculated on a slant culture medium, and cultured at a constant temperature of 30 C. for 7 days. The activated strains were further inoculated in a seed culture medium, cultured at 30 C. for 48 h, added to a fermentation medium in an inoculation amount of 10%, and cultured at 30 C. for 48-72 h, thereby obtaining a crude transglutaminase solution. The crude enzyme solution was purified and concentrated by the following steps:

1) pressure filtration: at a pore size of 1 m, an operating pressure of 0.20 MPa, and a temperature of 20 C., obtaining a filtrate;
2) microfiltration: at a pore size of 0.25 m, an operating pressure of 0.25 MPa, and a temperature of 20 C., obtaining a filtrate;
3) ultrafiltration: at a molecular weight cutoff of 100 KDa, an operating pressure of 0.30 MPa, and a temperature of 20 C., obtaining a filtrate;
4) ultrafiltration: at a molecular weight cutoff of 10 KDa, an operating pressure of 0.30 MPa, and a temperature of 20 C., keeping the retenate (i.e. a purified concentrated enzyme solution).

[0064] The enzyme activity was determined by hydroxamic acid method (Peng Can, Stabilization Study of Microbial Transglutaminase Mt East China Normal University, 2007), (the same below), and a purified concentrated transglutaminase solution having an enzyme activity of 2500 u/ml was obtained. By measurement, it had a pH of 5.80, water activity of 0.95, and a redox potential of 60 mv. The solution is kept for later use.

Example 2

[0065] A crude transglutaminase solution was prepared by dissolving enzyme powder. A certain amount of pure water was weighed, and placed in a container equipped with a stirrer. A transglutaminase powder with an enzyme activity of 5000-8000 u/ml (an enzyme powder produced by Shanghai Kinry Food Ingredients Co., Ltd) was added slowly with stirring, so as to obtain a dissolved enzyme solution at a certain concentration. Bacteria were removed by microfiltration to obtain a clear transglutaminase solution with an enzyme activity of 2500 u/ml, i.e. a purified concentrated transglutaminase solution with an enzyme activity of 2500 u/ml. By measurement, it had a pH of 6.30, water activity of 0.95, and a redox potential of 58 mv. The solution is kept for later use.

Example 3

[0066] A method for preparing a liquid transglutaminase: the liquid enzyme in a total volume of 1000 ml was prepared as followed, and the raw materials were weighed in accordance with Table 11.

TABLE-US-00014 TABLE 11 Preparation of a liquid transglutaminase Formula Addition Added Composition Component level % amount Transglutaminase Purified 20% (v/v) 200 ml enzyme solution (Example 1) Water activity Glycerol 50% (w/v) 500 g regulator Potential L-cysteine 0.15% (w/v) 1.5 g regulator pH regulator 0.02M pH 6.0 / Adjusted to Sodium citrate the final buffer volume of 1000 ml

[0067] The raw materials of the formula were weighed and mixed homogeneously, and after filtration through a 0.1 m sterile membrane, a light-yellow solution was obtained. By measurement, it had a redox potential of 100 m, water activity of 0.79, a pH of 6.0, and an enzyme activity of 498.1 u/ml. It was filled in ten of 100 mL PET opaque bottles, to finally obtain a transglutaminase preparation in a liquid form. After storage at room temperature for 180 days, the enzyme preparation of the formula was observed, the enzyme activity was measured, and the preservation rate of enzyme activity was calculated. By reference to GB4789.2-2010 National Food Safety Standard Food microbiological examination: Aerobic plate count, GB 4789.3 National Food Safety Standard Food microbiological examination: Enumeration of coliforms, GB/T 4789.38 National Food Safety Standard Food microbiological examination: Escherichia coli count, and GB 4789.4-2010 National Food Safety Standard Food microbiological examination: Salmonella, microbiological examination was carried out. The results showed that the liquid enzyme preparation had no significant change in appearance, flavor and the like. It had a preservation rate of enzyme activity of 86%, and its microorganism indexes meet the requirements in GB25594-2010 Standard. Therefore, it could be used for commercialization.

Example 4

[0068] The purified concentrated transglutaminase solution prepared in Example 2 was used to prepare a liquid preparation of transglutaminase in accordance with the formula in Table 12. After mixing homogenously, the pH, water activity, redox potential, and initial enzyme activity were measured in accordance with the relevant methods as described in Example 3. The liquid preparation of transglutaminase was then filtrated through 0.1 m sterile membrane, and was dispensed and packaged in PET bottles under sterile condition. After storage at room temperature for 180 days, the appearance of the liquid preparation of transglutaminase was observed, and the endpoint enzyme activity, microorganism indexes and the like were measured in accordance with the relevant methods as described in Example 3, and the preservation rate of enzyme activity was calculated.

TABLE-US-00015 TABLE 12 Preparation of a liquid transglutaminase Formula Addition Added Composition Component level % amount Transglutaminase Purified 4% (v/v) 4 L enzyme solution (Example 2) Water activity Glycerol 30% (w/v) 30 kg regulator Potential Reduced glutathione 0.1% (w/v) 100 g regulator pH regulator 0.02M pH 6.0 / Adjusted to Sodium citrate the final buffer volume of 100 L

[0069] The results showed that the transglutaminase preparation prepared by the method had good stability, had no significant and visible change in appearance, and had a preservation rate of enzyme activity of 82%. The microorganism indexes met the requirements in GB25594-2010 Standard. It could be used for commercialization. The use of filtration through 0.1 m sterile membrane and sterile filling could ensure the liquid enzyme preparation to have a preservation rate of enzyme activity of up to 80%, and could control the microorganism indexes in the liquid enzyme preparation of a formula with 30% water activity regulator during the period of storage.

Example 5

[0070]

TABLE-US-00016 TABLE 13 Preparation of a liquid transglutaminase Physicochemical parameters of the Results Formula enzyme preparation 180 d later Addition Added Enzyme activity: Appearance: Composition Component level % amount 99.5 u/ml No visible change Transglutaminase Purified 4% (v/v) 4 L pH: 6.0 concentrated enzyme solution (Example 2) Water activity Glycerol 30% (w/v) 30 kg Water Preservation rate regulator activity: 0.89 of enzyme activity: 84% Potential Reduced 0.1% (w/v) 100 g regulator glutathione Preservative Sodium 0.03% (w/v) 30 g Redox Microorganism dehydroacetate potential: 35 mv index: qualified pH regulator 0.02M pH 6.0 / Adjusted to Sodium citrate the final buffer volume of 100 L

[0071] After mixing said raw materials homogeneously, the mixture was directly filled into PET bottles under open environments. After storage at room temperature for 180 days, the transglutaminase preparation prepared by the method had good stability, had no significant and visible change in appearance, and had a preservation rate of enzyme activity of 84%. The microorganism indexes met the requirements in GB25594-2010 Standard, and it could be used for commercialization.

Example 6

[0072]

TABLE-US-00017 TABLE 14 Preparation of a liquid transglutaminase Physicochemical parameters of the Results Formula enzyme preparation 180 d later Addition Added Enzyme activity: Appearance: Composition Component level % amount 101 u/ml No visible change Transglutaminase Purified 4% (v/v) 4 L pH: 6.0 concentrated enzyme solution (Example 1) Water activity Glycerol 70%- 40% (w/v) 40 kg Water Preservation rate regulator maltitol 30% activity: 0.85 of enzyme Potential Reduced 0.1% (w/v) 100 g activity: 87% regulator glutathione Preservative Sodium 0.03% (w/v) 30 g Redox Microorganism dehydroacetate potential: 37 mv index: qualified pH regulator 0.02M pH 6.0 / Adjusted to Sodium citrate the final buffer volume of 100 L

[0073] The components as listed in Table 14 were mixed homogeneously to prepare a liquid preparation of transglutaminase, and the relevant parameters were measured. The liquid preparation of transglutaminase was filled into 1 L PET bottles under open conditions. After storage at room temperature for 180 days, the appearance of the liquid enzyme preparation was observed, and the enzyme activity and microorganism indexes were measured. The results showed that, after storage at room temperature for 180 days, the preservation rate of enzyme activity reached 87%, the microorganism indexes met the requirements, and thus a stable liquid preparation of transglutaminase was obtained.

Example 7

[0074]

TABLE-US-00018 TABLE 15 Preparation of a liquid transglutaminase Physicochemical parameters of the Results Formula enzyme preparation 180 d later Addition Added Enzyme activity: Appearance: Composition Component level % amount 111 u/ml No visible change Transglutaminase Purified 4.4% (v/v) 4.4 L pH: 6.7 concentrated enzyme solution (Example 1) Water activity Glycerol 70%- 50% (w/v) 50 kg Water Preservation rate regulator Sorbitol 30% activity: 0.85 of enzyme Potential Wheat protein 1% (w/v) 1 kg activity: 89% regulator hydrolysate Preservative -polylysine 0.02% (w/v) 20 g Redox Microorganism pH regulator Tap water / Adjusted to potential: 32 mv index: qualified the final volume of 100 L

[0075] The components as listed in Table 15 were mixed homogeneously to prepare a liquid preparation of transglutaminase, and the relevant parameters were measured. The liquid preparation of transglutaminase was filled into 1 L PET bottles under open conditions. After storage at room temperature for 180 days, the appearance of the liquid enzyme preparation was observed, and the enzyme activity and microorganism indexes were measured. The results showed that, in the presence of a preservative, after storage at room temperature for 180 days, the preservation rate of enzyme activity reached 89%, and the microorganism indexes met the requirements, thus a stable liquid preparation of transglutaminase was obtained.

Example 8

[0076] The components as listed in Table 16 were mixed homogeneously to prepare a liquid preparation of transglutaminase, and the relevant parameters were measured. The liquid preparation of transglutaminase was filled into 1 L PET bottles under open conditions. After storage at room temperature for 180 days, the appearance of the liquid enzyme preparation was observed, and the enzyme activity and microorganism indexes were measured. The results showed that, after storage at room temperature for 180 days, the preservation rate of enzyme activity reached 88%, and the microorganism indexes met the requirements, thus a stable liquid preparation of transglutaminase was obtained, where the potential regulators and the water activity regulators were combined components, and a preservative was added.

TABLE-US-00019 TABLE 16 Preparation of a liquid transglutaminase Physicochemical parameters of the Results Formula enzyme preparation 180 d later Addition Added Enzyme activity: Appearance: Composition Component level % amount 101 u/ml No visible change Transglutaminase Purified 4% (v/v) 4 L pH: 6.6 concentrated enzyme solution (Example 1) Water activity Glycerol 70% 50% (w/v) 50 kg Water Preservation rate regulator Sorbitol 30% activity: 0.83 of enzyme Potential Sodium caseinate 1.0% (w/v) 1 kg activity: 89% regulator Chitosan 0.25% (w/v) 250 g hydrolysate Tea polyphenol 0.01% (w/v) 10 g Glucose oxidase / 5 u/ml Preservative Lysozyme 0.5% (w/v) 50 g Redox Microorganism pH regulator Tap water / Adjusted to potential: 38 mv index: qualified the final volume of 100 L

Example 9

[0077]

TABLE-US-00020 TABLE 17 Preparation of a liquid transglutaminase Physicochemical parameters of the Results Formula enzyme preparation 180 d later Addition Added Enzyme activity: Appearance: Composition Component level % amount 1000 u/ml No visible change Transglutaminase Purified 40% (v/v) 40 L pH: 6.2 concentrated enzyme solution (Example 1) Water activity Glycerol 50% (w/v) 50 kg Water Preservation rate regulator activity: 0.84 of enzyme Potential Sodium caseinate 1.0% (w/v) 1 kg activity: 88% regulator Chitosan 0.25% (w/v) 250 g hydrolysate Tea polyphenol 0.01% (w/v) 10 g Glucose oxidase / 5 u/ml Preservative Lysozyme 0.5% (w/v) 50 g Redox Microorganism pH regulator Purified water / Adjusted to potential: 42 mv index: qualified the final volume of 100 L

[0078] The components as listed in Table 17 were mixed homogeneously to prepare a liquid preparation of transglutaminase, and the relevant parameters were measured. The liquid preparation of transglutaminase was filled into 1 L PET bottles under open conditions. After storage at room temperature for 180 days, the appearance of the liquid enzyme preparation was observed, and the enzyme activity and microorganism indexes were measured. The results showed that, after storage at room temperature for 180 days, the preservation rate of enzyme activity reached 88%, and the microorganism indexes met the requirements, thus a stable liquid preparation of transglutaminase with an enzyme activity of 1000 u/ml was obtained.

Example 10

[0079] The components as listed in Table 18 were mixed homogeneously to prepare a liquid preparation of transglutaminase, and the relevant parameters were measured. The liquid preparation of transglutaminase was filled into 1 L PET bottles under open conditions. After storage at room temperature for 180 days, the appearance of the liquid enzyme preparation was observed, and the enzyme activity and microorganism indexes were measured. The results showed that, after storage at room temperature for 180 days, the preservation rate of enzyme activity reached 81.5%, and the microorganism indexes met the requirements, thus a stable liquid preparation of transglutaminase with an enzyme activity of 10 u/ml and a pH of 5.5 was obtained.

TABLE-US-00021 TABLE 18 Preparation of a liquid transglutaminase Physicochemical parameters of the Results Formula enzyme preparation 180 d later Addition Added Enzyme activity: Appearance: Composition Component level % amount 10 u/ml No visible change Transglutaminase Purified 0.4% (v/v) 0.4 L pH: 5.5 concentrated enzyme solution (Example 1) Water activity Glycerol 50% (w/v) 50 kg Water Preservation rate regulator activity: 0.83 of enzyme Potential Sodium bisulfite 0.02% (w/v) 20 g activity: 81.5% regulator Soybean protein 1% (w/v) 1 kg hydrolysate Bamboo leaf 0.05% (w/v) 50 g antioxidant Sodium 0.5% (w/v) 500 g D-isoascorbate Preservative Methyl 0.25% (w/v) 250 g Redox Microorganism parahydroxybenzoate potential: 60 mv index: qualified pH regulator 0.1M HCl 0.1% (v/v) 0.1 L Purified water / Adjusted to the final volume of 100 L

Example 11

[0080] The components as listed in Table 19 were mixed homogeneously to prepare a liquid preparation of transglutaminase, and the relevant parameters were measured. The liquid preparation of transglutaminase was filled into 1 L PET bottles under open conditions. After storage at room temperature for 180 days, the appearance of the liquid enzyme preparation was observed, and the enzyme activity and microorganism indexes were measured. The results showed that, after storage at room temperature for 180 days, the preservation rate of enzyme activity reached 81%, and the microorganism indexes met the requirements, thus a stable liquid preparation of transglutaminase with an enzyme activity of 99.8 u/ml and a pH of 8.0 was obtained.

TABLE-US-00022 TABLE 19 Preparation of a liquid transglutaminase Physicochemical parameters of the Results Formula enzyme preparation 180 d later Addition Added Enzyme activity: Appearance: Composition Component level % amount 99.8 u/ml No visible change Transglutaminase Purified 4% (v/v) 4 L pH: 8.0 concentrated enzyme solution (Example 1) Water activity Glycerol 50% (w/v) 50 kg Water Preservation rate regulator activity: 0.83 of enzyme Potential Wheat protein 1.0% (w/v) 1 kg activity: 80.5% regulator hydrolysate Preservative Sodium diacetate 0.5% (w/v) 500 g pH regulator NaOH 0.05% (w/v) 50 g Redox Microorganism Purified water / Adjusted to potential: 32 mv index: qualified the final volume of 100 L

Example 12

[0081] The components as listed in Table 20 were mixed homogeneously to prepare a liquid preparation of transglutaminase, and the relevant parameters were measured. The liquid preparation of transglutaminase was filled into 1 L PET bottles under open conditions. After storage at room temperature for 180 days, the appearance of the liquid enzyme preparation was observed, and the enzyme activity and microorganism indexes were measured. The results showed that, after storage at room temperature for 180 days, the preservation rate of enzyme activity reached 81%, and the microorganism indexes met the requirements, thus a stable liquid preparation of transglutaminase with an enzyme activity of 100 u/ml, a pH of 6.0 and a redox potential of 400 mv was obtained. Since sodium borohydride in the preparation does not meet the requirement in Food Hygiene Regulations, the liquid enzyme preparation of this formula can only be applied for the purpose of scientific research or non-food addition.

TABLE-US-00023 TABLE 20 Preparation of a liquid transglutaminase Physicochemical parameters of the Results Formula enzyme preparation 180 d later Addition Added Enzyme activity: Appearance: Composition Component level % amount 100 u/ml No visible change Transglutaminase Purified 4% (v/v) 4 L pH: 6.0 concentrated enzyme solution (Example 1) Water activity Glycerol 50% (w/v) 50 kg Water Preservation rate regulator activity: 0.84 of enzyme Potential Sodium 0.055% (w/v) 55 g activity: 80.1% regulator borohydride Preservative Sodium diacetate 0.05% (w/v) 50 g Redox Microorganism pH regulator NaOH 0.005% (w/v) 5 g potential: 400 mv index: qualified Purified water / Adjusted to the final volume of 100 L

Example 13

[0082] The components as listed in Table 21 were mixed homogeneously to prepare a liquid preparation of transglutaminase, and the relevant parameters were measured. The liquid preparation of transglutaminase was filled into 1 L PET bottles under open conditions. After storage at room temperature for 180 days, the appearance of the liquid enzyme preparation was observed, and the enzyme activity and microorganism indexes were measured. The results showed that, after storage at room temperature for 180 days, the preservation rate of enzyme activity reached 82%, and the microorganism indexes met the requirements, thus a stable liquid preparation of transglutaminase with an enzyme activity of 50 u/ml, a pH of 6.0 and a redox potential of 50 mv was obtained.

TABLE-US-00024 TABLE 21 Preparation of a liquid transglutaminase Physicochemical parameters of the Results Formula enzyme preparation 180 d later Addition Added Enzyme activity: Appearance: Composition Component level % amount 50 u/ml No visible change Transglutaminase Purified 2% (v/v) 2 L pH: 6.0 concentrated enzyme solution (Example 1) Water activity Glycerol 50% (w/v) 50 kg Water Preservation rate regulator activity: 0.85 of enzyme Potential Wheat protein 0.05% (w/v) 50 g activity: 82% hydrolysate regulator L-cysteine HCl 0.01% (w/v) 10 g Preservative Lysozyme 0.05% (w/v) 50 g Redox Microorganism pH regulator 0.1M pH 6.0 / Adjusted to potential: 50 mv index: qualified Phosphate buffer the final volume of 100 L

Example 14

[0083]

TABLE-US-00025 TABLE 22 Preparation of a liquid transglutaminase Physicochemical Results Formula parameters 180 d later Addition Added Enzyme activity: Appearance: Composition Component level % amount 100 u/ml No visible change Transglutaminase Purified 4% (v/v) 4 L pH: 6.0 concentrated enzyme solution (Example 1) Water activity Glycerol 50% (w/v) 50 kg Water Preservation rate regulator activity: 0.85 of enzyme Potential Wheat protein 0.1% (w/v) 100 g activity: 84% hydrolysate regulator L-cysteine HCl 0.03% (w/v) 30 g Preservative Lysozyme 0.05% (w/v) 50 g Redox Microorganism pH regulator 0.02M pH 6.0 / Adjusted to potential: 0 mv index: qualified Phosphate buffer the final volume of 100 L

[0084] The components as listed in Table 22 were mixed homogeneously to prepare a liquid preparation of transglutaminase, and the relevant parameters were measured. The liquid preparation of transglutaminase was filled into 1 L PET bottles under open conditions. After storage at room temperature for 180 days, the appearance of the liquid enzyme preparation was observed, and the enzyme activity and microorganism indexes were measured. The results showed that, after storage at room temperature for 180 days, the preservation rate of enzyme activity reached 84%, and the microorganism indexes met the requirements, thus a stable liquid preparation of transglutaminase with an enzyme activity of 100 u/ml, a pH of 6.0 and a redox potential of 0 mv was obtained.

Example 15

[0085] The components as listed in Table 23 were mixed homogeneously to prepare a liquid preparation of transglutaminase, and the relevant parameters were measured. The liquid preparation of transglutaminase was filled into 1 L PET bottles under open conditions. After storage at room temperature for 180 days, the appearance of the liquid enzyme preparation was observed, and the enzyme activity and microorganism indexes were measured. The results showed that, after storage at room temperature for 180 days, the preservation rate of enzyme activity reached 89%, and the microorganism indexes met the requirements, thus a stable liquid preparation of transglutaminase with an enzyme activity of 50 u/ml, water activity of 0.61, and a redox potential of 140 mv was obtained. No preservative was comprised in the formula.

TABLE-US-00026 TABLE 23 Preparation of a liquid transglutaminase Physicochemical Results Formula parameters 180 d later Addition Added Enzyme activity: Appearance: Composition Component level % amount 50 u/ml No visible change Transglutaminase Purified 2% (v/v) 2 L pH: 6.0 concentrated enzyme solution (Example 1) Water activity Glycerol 70% (w/v) 70 kg Water Preservation rate regulator activity: 0.61 of enzyme Potential L-cysteine HCl 0.12% (w/v) 120 g activity: 89% regulator Preservative No Redox Microorganism pH regulator pH 6.0 Acetate / Adjusted to potential: 140 mv index: qualified buffer the final volume of 100 L

Example 16

[0086] The components in the formula of Example 15 were mixed homogeneously to prepare a liquid preparation of transglutaminase, and the relevant parameters were measured. The liquid preparation of transglutaminase was filled into 1 L opaque PET bottles under open conditions. After storage at room temperature for 180 days, the appearance of the liquid enzyme preparation was observed, and the enzyme activity and microorganism indexes were measured. The results showed that, after storage at room temperature for 180 days, the preservation rate of enzyme activity reached 88%, and the microorganism indexes met the requirements, thus a stable liquid preparation of transglutaminase with an enzyme activity of 101 u/ml, water activity of 0.85, and a redox potential of 38 mv was obtained. The package for the enzyme preparation was an opaque material, and had no significant effect on the enzyme stability.

Example 17

[0087] After storage at room temperature for 180 days, the liquid preparation of transglutaminase prepared in Example 7 was measured to have an enzyme activity of 98.79 u/ml. It was used in the production of sausage. The particular formula and process were shown in Table 24-1. A commercially available transglutaminase preparation in a powder form (from Kinry Biotech (Jinan) Co., Ltd.) was used as control. It was measured to have an enzyme activity of 100 u/g before use.

TABLE-US-00027 TABLE 24-1 Use of liquid, powder transglutaminase in production of sausage Control without Liquid enzyme Powder enzyme addition of Item preparation (A) control(B) enzyme(C) Enzyme dose 150 ml 150 g Lean pork 40 kg 40 kg 40 kg Chicken 10 kg 10 kg 10 kg breast Pork fat 17 kg 17 kg 17 kg lining Edible salt 1.6 kg 1.6 kg 1.6 kg Composite 250 g 250 g 250 g phosphate Sodium 200 g 200 g 200 g caseinate Gourmet 500 g 500 g 500 g powder Spice 5 kg 5 kg 5 kg I + G 20 g 20 g 20 g Carrageenan 300 g 300 g 300 g Soy protein 1.8 kg 1.8 kg 1.8 kg isolates Corn starch 5 kg 5 kg 5 kg Ice water 14 kg 14 kg 14 kg Sodium 1 kg 1 kg 1 kg lactate Sodium 30 g 30 g 30 g isoascorbate White sugar 3 kg 3 kg 3 kg Pork soluble 300 g 300 g 300 g meal Processing In accordance with the formula above, the raw procedure materials, meat and fat lining, were defrosted, and then minced to a size of 0.5 cm. All the raw materials were subjected to the rolling and rubbing in a vacuum tumbler. The rolling and rubbing were performed in the following manner: rolling and rubbing for 10 min, resting for 2 min, in a total period of 60 min. The resultant meat thus obtained was poured into a sausage stuffer. After sausage stuffing was finished, the sausage was dried at 60 C. for 25 min, and cooked at 80 C. for 30 min. The quality of the product was evaluated after cooling.

[0088] After the sausage was made, the final product was tested for the indexes such as gel strength, elasticity and sensory score. The experimental results were shown in Table 24-2.

TABLE-US-00028 TABLE 24-2 Effect of a transglutaminase preparation in a liquid form and a powder form on indexes of sausage Item Sample A Sample B Sample C Gel strength (g/cm.sup.2) 463 396 244 Elasticity (mm) 2.502 2.071 1.105 Cooking loss (%) 2.8% 2.9% 4% Sensory score 92.1 87.7 75.6 (maximum score: 100)

[0089] The results of experimental test showed that the liquid preparation of transglutaminase had a good effect in the sausage application, and had a significantly improved performance indexes of sausage products such as gel strength and elasticity, as compared to the powder transglutaminase preparation at the same dose. Cooling loss was somewhat but not significantly improved, and the sensory evaluation was significantly improved.

Example 18

[0090] The liquid transglutaminase product prepared in Example 5 (99.5 u/mL) was used in the production of Chiba tofu. Commercially available powder enzyme preparations, Product A (transglutaminase, manufacturer: Taixing Dongsheng Bio-Tech Co., Ltd., Type TG-TI, Batch No: B20150926, nominal enzyme activity: 116 u/g, actual enzyme activity measured in our laboratory: 108 u/g), and Product B (transglutaminase, manufacturer: Taixing Yiming Biological Co. Ltd., Type TG-B, Batch No: 20150824, nominal enzyme activity: 110 u/g, actual enzyme activity measured in our laboratory: 100 u/g) were used as controls. The manufacturer of soy protein isolates was Shandong Yuwang Group; Cassava denatured starch was of Rose Brand; soybean oil was provided by COFCO Corporation; I+G and gravy salt were purchased from METRO supermarket; and Control C was a powder enzyme preparation Biobond TG-I produced by Kinry Biotech (Jinan) Co., Ltd., actual enzyme activity measured: 110 u/ml. Chiba tofu was prepared according to the formula and the process as shown in Table 25-1.

TABLE-US-00029 TABLE 25-1 Use of liquid and powder transglutaminase in the production of Chiba tofu Sample from Control Control Control Item Example 14 A B C Enzyme dose 200 ml 200 g 200 g 200 g Soy protein 15 kg 15 kg 15 kg 15 kg Cassava 3 kg 3 kg 3 kg 3 kg denatured starch Edible 15 kg 15 kg 15 kg 15 kg soybean oil Edible salt 200 g 200 g 200 g 200 g Gourmet powder 200 g 200 g 200 g 200 g Ice water 70 kg 70 kg 70 kg 70 kg (1:4) Processing In accordance with the formula, soy protein procedure isolate was added to a 250 L chopping pot, ice water was added, and a transglutaminase preparation was added in a specified amount. After high-speed chopping at 2800 rpm/min for 2 min, soy protein was completely swollen. Soybean oil was added, and high-speed chopping was further performed for 2-3 min, and the slurry was completely emulsified and turned white. Modified starch, edible salt and gourmet powder were added, and high-speed chopping was further performed for 1 min. The slurry turned into a milky white, homogeneous emulsified slurry. The emulsified slurry was placed in a 80 cm 60 cm 8 cm (length width depth) stainless steel tray. The tray was covered with a plastic cloth, and kept in a 50 C. steam box for 1 h, and then was further kept for 1 h in the steam box with the temperature increased to 90 C. The steel tray was taken out, and the product was cut into slices after air cooling.

[0091] Chiba tofu produced by using different transglutaminase preparations were tested for gel strength, elasticity, color and luster, mouthfeel, etc. The results were shown in Table 25-2.

TABLE-US-00030 TABLE 25-2 Evaluation on quality of Chiba tofu produced by liquid and powder transglutaminase Sample from Test items Example 14 Control A Control B Control C Gel strength 1036 958 946 963 (g/cm2) Elasticity (mm) 7.188 6.771 6.282 6.845 Color milky white milky white milky white milky white Mouthfeel Excellent Excellent slightly Excellent soft Texture exquisite exquisite exquisite exquisite Flavor Pure taste Pure taste Pure taste Pure taste and palatable and palatable Resistant Excellent Excellent Good Excellent to boiling water Change after No visible No visible No visible No visible freezing ice crystal ice crystal ice crystal ice crystal at 18 C. for 48 h

[0092] The results of experimental test showed that the liquid preparation of transglutaminase was comparable to the powder transglutaminase preparations in the application in Chiba tofu, and the powder preparation could be entirely replaced by the liquid preparation during the production of Chiba tofu. The Chiba tofu thus produced had its quality improved to some extent. The liquid preparation of transglutaminase had commercial value.

[0093] The above examples are only some preferred embodiments and application examples of the invention, and the invention are not limited to them. For a person skilled in the art in the technical field to which the invention pertains, various changes and modifications may be readily made to the invention. Any amendment, equivalent substitutions, improvement to the methods and the like within the concept and principle of the invention are included in the protection scope of the invention.