Enzyme composition for food manufacturing

Abstract

Provided is a collagenase composition having high storage stability, which can be used in a food production process while reducing the use of stabilizers and preservatives. The enzyme composition for food production according to the present invention contains collagenase and has substantially no contamination activity.

Description

Technical Field

[0001] The present invention relates to enzyme compositions for use in food production. Background Art

[0002] Collagen is a protein found in animal skin, tendons, bones, and cartilage, and accounts for the largest proportion of all proteins in the animal body. Collagen peptides, a degradation product of collagen, are obtained by breaking down collagen into structural units consisting of three amino acids, including glycine. Collagen peptides are highly absorbable in the body and are therefore widely used in foods, beverages, and supplements. Furthermore, due to their moisturizing properties, collagen peptides are also used as a cosmetic ingredient.

[0003] Patent Documents 1 and 2 disclose methods using collagenase that sequence-specifically degrades collagen as methods for producing collagen peptides, and Patent Documents 3 and 4 disclose the use of collagenase for the production of sausage casings for processed meat products and for tenderizing meat.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: International Publication No. 2021 / 200955

[0007] Patent Document 2: Japanese Patent Application Laid-Open No. 2017-537978

[0008] Patent Document 3: Japanese Patent Application Laid-Open No. 2020-074697

[0009] Patent Document 4: Japanese Patent Application Laid-Open No. 2016-052304 Summary of the Invention

[0010] Problems to be solved by the invention

[0011] The existing collagenase compositions used in the manufacturing process of food have insufficient storage stability. Usually, in enzyme preparations, in order to maintain enzyme activity and prevent corrosion, stabilizers and preservatives are sometimes added. However, if stabilizers and preservatives are mixed, the enzyme activity per unit enzyme composition decreases. In addition, these chemical substances are equivalent to designated additives with limited use in food applications. Therefore, due to the increased concern for food safety, they tend to be unpopular in the market. The object of the present invention is to provide a collagenase composition with high storage stability, which can reduce the use of stabilizers and preservatives and can be used in the manufacturing process of food.

[0012] Means for solving problems

[0013] The present invention relates to an enzyme composition for food production, comprising collagenase and having substantially no contaminating activity.

[0014] The contaminating activity is preferably protease activity.

[0015] When the protease activity is represented by P (U / g) and the collagenase activity is represented by C (U / g), the P / C value is preferably 2.0 or less.

[0016] The collagenase is preferably of bacterial origin.

[0017] The residual collagenase activity after storage at 40°C for one month is preferably 50% or more.

[0018] The food is preferably collagen peptide or processed meat food.

[0019] Furthermore, the present invention relates to a method for producing a food, comprising the step of processing a food material using the enzyme composition.

[0020] In addition, the present invention relates to a food comprising the above enzyme composition.

[0021] Effects of the Invention

[0022] The collagenase composition of the present invention has high storage stability and can reduce the production cost of food. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The collagenase activity after storage at 5°C is shown.

[0024] Figure 2 The collagenase activity after storage at 15°C is shown.

[0025] Figure 3 The collagenase activity after storage at 25°C is shown.

[0026] Figure 4 The results of a meat tenderization test are shown. DETAILED DESCRIPTION

[0027] <Enzyme Composition>

[0028] The present invention relates to an enzyme composition for food production, comprising collagenase and having substantially no contaminating activity.

[0029] <Collagenase>

[0030] Collagenase is an enzyme that breaks down collagen into structural units consisting of tripeptides containing glycine. Specific examples of tripeptides containing glycine include glycine-proline-hydroxyproline, glycine-proline-alanine, and glycine-alanine-hydroxyproline.

[0031] The origin of collagenase is not particularly limited, and examples thereof include microbial, animal, and plant-derived collagenases. Microbial sources are preferred from the perspective of ease of availability. Examples of microorganisms include bacteria and fungi, with bacteria being preferred.

[0032] As bacteria, actinomycetes and subtilis can be mentioned. As actinomycetes, Streptomyces, Actinomyces, Nocardia and Rhodococcus can be mentioned. As subtilis, Bacillus and Lysinibacillus can be mentioned. As fungi, filamentous fungi, basidiomycetes and yeast can be mentioned. Among these, preferably bacteria are derived, more preferably actinomycetes are derived, further preferably Streptomyces are derived, and particularly preferably Streptomyces violaceoruber is derived. In the collagenase preparation as the modifier of the present invention, the above-mentioned collagenase can contain only one kind or two or more kinds.

[0033] The collagenase may be purified from plants, animals, or microorganisms as its source, or may be purified after mass production using genetic recombination techniques. In addition, wild-type collagenase or mutant collagenase may be used.

[0034] As a method for obtaining collagenase, when collagenase accumulates in the cells of the source organism, the tissue and cells are broken and a cell-free extract is obtained by centrifugation. The collagenase obtained by purifying the cell-free extract as the starting material and appropriately combining common protein purification methods such as salting-out, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, and affinity chromatography can be used. When collagenase is secreted and produced outside the cell by microorganisms, it can be purified from the culture medium.

[0035] The collagenase activity in the enzyme composition of the present invention is preferably more than 10 units (U), more preferably more than 50 units (U), and further preferably more than 100 units (U) per 1g of enzyme composition. The higher the upper limit, the more preferred it is, and there is no particular limitation, but it is usually less than 5000 units (U) per 1g of enzyme composition. In addition, the weight of the collagenase in the enzyme composition of the present invention is preferably 0.01 to 20% by weight, more preferably 0.1 to 10% by weight per 1g of enzyme composition. Here, the activity of the collagenase is determined as follows: according to the assay method described in ANALYTICAL BIOCHEMISTRY, 136, 446-450 pages (1984), an enzyme is added to an AZO collagen aqueous solution (0.9% AZOCOLL substrate (manufactured by SIGMA-ALDRICH)), and the reaction is carried out at 37°C and pH 7.5 for 10 minutes. The increase in OD595nm in 1 minute is taken as 1 unit (U), thereby determining the activity of the collagenase.

[0036] <Pollution Activity>

[0037] The enzyme composition has substantially no contaminating activity. Contaminating activity refers to enzyme activity other than collagenase activity that may impair collagenase activity, and examples include protease activity, amylase activity, and lipase activity. Of these, it is preferred that the enzyme composition has substantially no protease activity. Here, "substantially no contaminating activity" means that the residual collagenase activity after storage at 40°C for one month is 50% or greater, preferably 60% or greater, and more preferably 65% or greater.

[0038] The term "protease activity" is sometimes interpreted as including all proteolytic activities. However, the contaminating protease activity in the present invention refers to activities that hydrolyze proteins other than collagenase activity.

[0039] In the enzyme composition, the method for preparing an enzyme composition having substantially no contaminating activity is not particularly limited, and examples thereof include the following methods: a method using collagenase having inherently low contaminating activity; a method using collagenase having reduced contaminating activity by introducing mutations; a method reducing contaminating activity by purification; and a method reducing contaminating activity by mixing optional components.

[0040] Regarding the contaminating protease activity in the enzyme composition, when the protease activity is set to P (U / g) and the collagenase activity is set to C (U / g), the value of P / C is preferably 3.5 or less, more preferably 3 or less, further preferably 2.5 or less, further more preferably 2 or less, and particularly preferably 1.5 or less. Here, regarding protease activity, according to the determination method described in the 2018 9th edition of the Food Additives Official Book, pages 887-888, the enzyme is added to a bovine casein aqueous solution (0.6% casein, bovine milk, free of carbohydrates and fatty acids (manufactured by Merck)), and under the conditions of 30°C, pH 7.5, and 10 minutes, the amount of enzyme that increases in the amount of enzyme that produces a Folin reagent colorimetric substance equivalent to 1 μg of L-tyrosine in 1 minute is taken as 1 U.

[0041] The enzyme composition of the present invention has substantially no contaminating activity and therefore has high storage stability. The residual collagenase activity of the enzyme composition after storage at 40°C is preferably 50% or more, more preferably 60% or more, and even more preferably 65% or more.

[0042] <Optional Ingredients>

[0043] In the enzyme composition, in addition to collagenase, other components that can be usually contained in the enzyme composition can also be contained to a degree that does not hinder the effect of the present invention. As such components, excipients, pH adjusting agents, preservatives, thickening polysaccharides, emulsifiers, inorganic salts, amino acids, peptides or proteins, solvents can be enumerated. The content of these components is not particularly limited and can be selected by those skilled in the art in any amount. It should be noted that, as shown in the examples, in order to solve the problem of the application, it is not necessary to include pH adjusting agents, preservatives, thickening polysaccharides, emulsifiers, inorganic salts, amino acids, peptides or proteins.

[0044] Examples of the excipient include dextrin, trehalose, and cereal flours such as rice flour and wheat flour.

[0045] Examples of pH adjusters include ascorbic acid, acetic acid, dehydroacetic acid, lactic acid, citric acid, gluconic acid, succinic acid, tartaric acid, fumaric acid, malic acid, and adipic acid; sodium (Na) salts, calcium (Ca) salts, and potassium (K) salts of these organic acids; and sodium (Na) salts and potassium (K) salts of carbonic acid, phosphoric acid, and pyrophosphoric acid; and inorganic acids.

[0046] Examples of preservatives include propionic acid, propionates, sulfites, benzoates, sorbic acid, sorbates, etc. Examples of salts include sodium (Na) salts, calcium (Ca) salts, potassium (K) salts, and polyamines.

[0047] Examples of thickening polysaccharides include modified starch, gums, alginic acid, alginic acid derivatives, pectin, carrageenan, curdlan, pullulan, gelatin, cellulose derivatives, agar, tamarind gum, psyllium gum, and glucomannan.

[0048] Examples of the emulsifier include glycerol fatty acid esters, polyglycerol fatty acid esters, sucrose fatty acid esters, propylene glycol fatty acid esters, sorbitan fatty acid esters, lecithin, enzymatically degraded lecithin, and saponin.

[0049] Examples of the inorganic salts include common salt, ammonium sulfate, sodium sulfate, calcium chloride, and polyphosphates.

[0050] Examples of the amino acid include aspartic acid, threonine, serine, asparagine, glutamic acid, glutamine, proline, glycine, alanine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, tryptophan, and arginine.

[0051] Examples of peptides or proteins include collagen peptides, milk protein, soy protein, casein, egg protein, wheat protein, corn protein, buckwheat protein, seaweed or microalgae-derived proteins, pea protein, animal protein, fish and shellfish protein, yeast or yeast-derived proteins, enzymatic hydrolysates of these proteins, fish peptides, soy peptides, whey peptides, sardine peptides, etc. These peptides can contribute to the storage stability of collagenase by serving as substrates for contaminating activity or functioning as inhibitors of contaminating activity.

[0052] As the solvent, water, polyols, etc. can be mentioned. Specific examples of polyols include glycerol, sorbitol, propylene glycol, polyvinyl alcohol, pentaerythritol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, etc. These solvents can be used alone or in combination of two or more. As a mixed solvent, a mixture of water and a solvent other than water is preferred, and a mixture of water and glycerol is more preferred. The weight ratio of (water): (solvent other than water) is preferably 2:1 to 1:8, more preferably 1:1 to 1:4. In addition, when using a solvent, the weight ratio of enzyme: solvent is preferably set to 2:1 to 1:2.

[0053] The form of the enzyme composition is not particularly limited, and examples thereof include liquid, paste, powder, and granular forms. Liquid or paste-like enzyme compositions are significantly affected by contaminating activity, and therefore, substantially lacking contaminating activity tends to significantly improve storage stability. Powdered or granular enzyme compositions tend to be less affected by contaminating activity.

[0054] When the enzyme composition is in a liquid or paste form, the pH is preferably 5.5 to 9.0, more preferably 5.5 to 6.5.

[0055] <Method for producing enzyme composition>

[0056] As long as the enzyme composition contains collagenase and does not substantially have contaminating activity, its preparation method is not particularly limited. For example, in the case of obtaining a solid enzyme composition, the collagenase is dissolved in a solvent such as water, and then freeze-dried to obtain a powdered enzyme. Optional ingredients can be added to the powdered enzyme as needed and mixed using a mixer. In addition, when the collagenase is dissolved in a solvent such as water, the optional ingredients can be dissolved simultaneously. As mixers, container rotary types, container fixed types, composite types, etc. can be cited, which can be appropriately selected according to the target activity value and amount, the type of excipient. In the case of obtaining a liquid enzyme composition, the enzyme and the required optional ingredients are dissolved in a solvent.

[0057] <Food Manufacturing>

[0058] The enzyme composition of the present invention is used in food production. The food is not particularly limited as long as it is made from a food material containing collagen, and examples include collagen peptides, processed meat foods, and processed fish foods. Examples of processed meat foods include sausages, hamburgers, aged meat, and livestock meat extracts. Examples of processed fish foods include chikuwa fish rolls, kamaboko (fish cakes), stewed fish, and fish extracts.

[0059] Specific examples of collagen peptides produced using the enzyme composition of the present invention include glycine-proline-hydroxyproline, glycine-proline-alanine, glycine-alanine-hydroxyproline, etc. The collagen peptides produced using the enzyme composition of the present invention can be suitably used as active ingredients in functional foods, functional beverages, cosmetics, and pharmaceuticals.

[0060] In processed meat foods produced using the enzyme composition of the present invention, the texture can be improved by decomposing collagen and gelatin. For example, foods containing large amounts of collagen and gelatin may gel when cooled, reducing the texture. However, the enzyme composition of the present invention can prevent the formation of gel.

[0061] The food product is produced by a method including a step of processing the food material with an enzyme composition. In the step of processing the food material with the enzyme composition, the collagenase in the enzyme composition is allowed to act on the collagen contained in the food material.

[0062] The food material is not particularly limited as long as it contains collagen, and examples include bones, skin, tendons, cartilage of animal origin, and bones, skin, meat, cartilage, and fish scales of fish origin. Examples of animals include cattle, pigs, and chickens. Examples of fish include tuna, sea bream, mackerel, trevally, sardines, salmon, bonito, yellowtail, saury, and tilapia. When using the enzyme composition of the present invention to produce collagen peptides, collagen purified from these materials or gelatin obtained by partial decomposition can be used.

[0063] The conditions for allowing collagenase to act are not particularly limited, but the temperature is preferably 30 to 60° C., more preferably 40 to 50° C. The pH is preferably 6.0 to 9.0, more preferably 7.0 to 8.0. The treatment time is preferably 1 to 24 hours.

[0064] Specific examples of methods for processing food materials using the enzyme composition of the present invention include methods in which food materials, preferably meat, are treated with a pickling solution containing the enzyme composition of the present invention. The collagenase activity in the pickling solution is preferably 0.001 to 100 U / mL, more preferably 0.005 to 50 U / mL, and even more preferably 0.01 to 10 U / mL. The pickling solution may further contain salts such as sodium chloride, sodium bicarbonate, sodium nitrate, sodium nitrite, magnesium chloride, calcium chloride, calcium lactate, potassium nitrate, and potassium nitrite, as well as thickening polysaccharides such as modified starches, gums, alginic acid, alginic acid derivatives, pectin, carrageenan, curdlan, pullulan, gelatin, cellulose derivatives, agar, tamarind gum, psyllium gum, and glucomannan, at a concentration preferably of 0.01 to 100% by weight, more preferably 0.05 to 50% by weight. Regarding the amount of the enzyme composition used with respect to the meat, the collagenase activity is preferably 0.0005 to 50 U, more preferably 0.002 to 5 U per 1 g of the meat.

[0065] The method for treating the meat with the pickling liquid is not particularly limited, as long as the pickling liquid comes into contact with the meat. Examples include spraying the meat with the pickling liquid, immersing the meat in the pickling liquid, and injecting the pickling liquid into the meat with a syringe. The pickling liquid treatment time is preferably within 100 hours, more preferably 1 to 80 hours. The temperature during the pickling liquid treatment is preferably 2 to 20°C, more preferably 5 to 15°C. Before or after treatment with the pickling liquid, the meat can be physically tenderized using methods such as a tenderizer or tumbler, or by incision with a knife or similar method.

[0066] When using the enzyme composition of the present invention to produce collagen peptides, after the collagenase is allowed to act, the collagen peptides can be purified as needed by filtration, ion exchange, activated carbon treatment, etc. In addition, there is no problem in ingesting the mixture of collagen peptides and the enzyme composition into the body, and there is no need to purify the collagen peptides. When the enzyme composition is ingested, it is digested and absorbed in the body in the same way as the collagen peptides. When it is necessary to inactivate the collagenase, it can be allowed to act on the food material and then heated to 70°C or above.

[0067] Example

[0068] The present invention will be described below with reference to Examples, but the present invention is not limited to the following Examples. Hereinafter, "parts" or "%" means "parts by weight" or "% by weight" respectively, unless otherwise specified.

[0069] The following enzymes were used in the following tests. The blending amounts (%) described below are values relative to the weight of each substance.

[0070] Collagenase (NAGAZYME-01, manufactured by Nagase ChemteX, 210 units (U) / g)

[0071] Protease (Nagase ChemteX, product name: Bioprase SP-20FG, 100,000 units (U) / g)

[0072] (1) Example 1, Comparative Examples 1 to 3

[0073] (1-1) Production of enzyme composition

[0074] Liquid enzyme compositions were prepared by mixing collagenase in a 50% glycerol solution so as to achieve the activities shown in Table 1. In Comparative Examples 1 to 3, protease was mixed in addition to collagenase.

[0075] (1-2) Accelerated stability test

[0076] Each enzyme composition was stored at 40° C. for one month. The activity of the enzyme composition before and after storage was measured by the following method, and the relative activity was calculated with the activity immediately after production being set as 100%. The results are shown in Table 1.

[0077] [Table 1]

[0078] Example 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Collagenase activity C (U / g) 219 213 224 276 Protease activity P (U / g) 291 844 5058 41059 P / C Ratio 1.3 4.0 22.6 148.7 Residual activity after accelerated stability test (%) 66.9％ 16.4％ 2.3％ 28.2％

[0079] Collagenase activity was determined by reacting AZO collagen (collagen impregnated with azo dye) as a substrate at 37°C and pH 7.5 for 10 minutes. The increase in OD595nm per minute was defined as 1U.

[0080] As shown in Table 1, in Comparative Examples 1 to 3, the residual activity after the accelerated test was reduced to approximately 30% or less.

[0081] In Example 1, the residual activity was maintained at more than 65%.

[0082] (2) Example 2, Comparative Examples 4 to 6

[0083] (2-1) Production of enzyme composition

[0084] Liquid enzyme compositions were prepared by mixing collagenase in a 50% glycerol solution so as to achieve the activities shown in Table 2. In Comparative Examples 4 to 6, protease was mixed in addition to collagenase.

[0085] [Table 2]

[0086] Example 2 Comparative Example 4 Comparative Example 5 Comparative Example 6 Collagenase activity C (U / g) 186 186 185 177 Protease activity P (U / g) 253 712 4883 46604 P / C Ratio 1.4 3.8 26.4 263.1

[0087] (2-2) Storage stability test

[0088] Each enzyme composition was stored at 5°C, 15°C, and 20°C for 15 months. The collagenase activity of the enzyme composition before and after storage was measured by the method described in (1-2), and the relative activity was calculated with the activity immediately after production being set as 100%. The results are shown in Figures 1 to 3 .

[0089] like Figures 1 to 3 As shown, in Comparative Examples 4 to 6, the residual activity was significantly reduced at 15°C and 25°C. In Comparative Examples 5 and 6, the residual activity was significantly reduced even at 5°C, where the reactivity of the protease is low. In Example 2, the residual activity was maintained at 65% or more even after storage at 25°C for 15 months.

[0090] (3) Example 3, Comparative Examples 7-8

[0091] (3-1) Production of enzyme composition

[0092] Liquid enzyme compositions were prepared by mixing collagenase in a 50% glycerol solution so as to achieve the activities shown in Table 3. In Comparative Examples 7 and 8, protease was mixed in addition to collagenase.

[0093] [Table 3]

[0094] Example 3 Comparative Example 7 Comparative Example 8 Collagenase activity C (U / g) 186 177 126 Protease activity P (U / g) 253 4651 32226 P / C Ratio 1.4 26.2 255.5

[0095] (3-2) Meat tenderization test

[0096] The components shown in Table 4 were mixed in water to prepare pickling liquids containing the enzyme compositions of Example 3 and Comparative Examples 7 and 8.

[0097] [Table 4]

[0098] weight% Enzyme composition 0.5 salt 1.5 Sodium bicarbonate 1 calcium lactate 0.5

[0099] Commercially available pork tenderloin was pre-treated using a Jaccard meat tenderizer. After cutting only the lean portion, the meat was cut into multiple 30g slices. 30g of the marinade containing the enzyme composition of Example 3 and Comparative Examples 7-8, or a marinade without an enzyme composition (Reference Example 2), was dispensed into bags. Two slices of meat (total 60g) were placed in each bag and tumbled at 10°C for 2 hours. After tumbling, each sample was stored at 10°C.

[0100] After being stored at 10°C for 3 days, the meat slices were oven-baked at 200°C for 10 minutes (5 minutes per side). After cooling to room temperature, the breaking stress (N) was measured using a physical property analyzer (SUN RHEO METER CR-500DX, manufactured by Sun Scientific Co., Ltd.). The same treatment as in Reference Example 1 was performed on the meat before treatment with the pickling solution, and the breaking stress (N) was measured.

[0101] Generally speaking, edible meat has a moderate taste when the breaking stress is 10 to 20 N. Figure 4 As shown, the meat treated with the pickling solution containing the enzyme composition of Example 3 had an appropriate breaking stress. The meat treated with the pickling solution containing the enzyme composition of Comparative Examples 7 and 8 was excessively tenderized, and the breaking stress was reduced.

[0102] The present disclosure (1) relates to an enzyme composition for food production, which contains collagenase and has substantially no contaminating activity.

[0103] The present disclosure (2) relates to the enzyme composition described in the present disclosure (1), wherein the contaminating activity is protease activity.

[0104] The present disclosure (3) relates to the enzyme composition described in the present disclosure (1) or (2), wherein, when protease activity is represented by P (U / g) and collagenase activity is represented by C (U / g), the value of P / C is 2.0 or less.

[0105] The present disclosure (4) relates to the enzyme composition according to any one of the present disclosures (1) to (3), wherein the collagenase is a bacterial-derived collagenase.

[0106] The present disclosure (5) relates to the enzyme composition according to any one of the present disclosures (1) to (4), wherein the residual collagenase activity after storage at 40° C. for one month is 50% or more.

[0107] The present disclosure (6) relates to the enzyme composition according to any one of the present disclosures (1) to (5), wherein the food is a collagen peptide or a processed meat food.

[0108] The present disclosure (7) relates to a method for producing a food, comprising the step of processing a food material using the enzyme composition described in any one of the present disclosures (1) to (6).

[0109] The present disclosure (8) relates to a food comprising the enzyme composition according to any one of the present disclosures (1) to (6).

Claims

An enzyme composition for food production, comprising collagenase and having substantially no contaminating activity.

2. The enzyme composition according to claim 1, wherein The contaminating activity is protease activity.

3. The enzyme composition according to claim 1 or 2, wherein When protease activity is defined as P and collagenase activity is defined as C, the value of P / C is 2.0 or less, and the units of both P and Q are U / g.

4. The enzyme composition according to claim 1 or 2, wherein Collagenase is a collagenase of bacterial origin.

5. The enzyme composition according to claim 1 or 2, wherein The residual collagenase activity after storage at 40°C for one month was 50% or more.

6. The enzyme composition according to claim 1 or 2, wherein The food is collagen peptide or processed meat food.

7. A method for producing a food, comprising the step of processing a food material using the enzyme composition according to claim 1 or 2. A food comprising the enzyme composition according to claim 1 or 2.

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