A proteolysate, its preparation and use

Abstract

The application provides a proteolysate and a preparation and application thereof. The preparation method of the proteolysate is as follows: after crushing animals and plants and adding water, high-pressure treatment, enzymolysis and low-temperature plasma treatment are sequentially performed to obtain the proteolysate; wherein the pressure of the high-pressure treatment is 100-400 MPa. The proteolysate obtained through high-pressure treatment in cooperation with low-temperature plasma treatment can significantly improve the salty taste of condiments (the proteolysate with a protein content of 20 mg / mL can increase the salty taste intensity of a 50 mmol / L NaCl solution by 45.6%-57.2%), and can be used as a high-quality salty taste enhancer, which is beneficial to guarantee the dietary health of consumers.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology. More specifically, it relates to a protease hydrolysate, its preparation, and its application. Background Technology

[0002] Sodium salt, as the most widely used condiment, plays an important role in people's daily lives, imparting saltiness to food and improving its overall flavor. However, excessive sodium intake increases the incidence of hypertension and cardiovascular disease. Therefore, sodium salt substitutes are gradually entering consumers' lives, such as commonly used metallic salts like potassium chloride and magnesium chloride, which can reduce sodium intake. However, these metallic salts have a strong bitter taste, making them difficult for consumers to accept, and potassium ions are unsuitable for consumers with renal insufficiency and hyperkalemia. Therefore, developing new low-sodium condiments with flavors similar to sodium salt has become a new goal for researchers, such as finding a condiment additive that enhances saltiness while reducing sodium intake. + Maintaining the saltiness of seasonings even with low concentrations, imparting saltiness to food, and improving the overall flavor of food is also a good way to protect consumers' dietary health.

[0003] After being hydrolyzed by proteases, animal and plant proteins can release small molecule peptides and free amino acids. Different processing techniques can help release more small molecule peptides and free amino acids and improve the characteristics of the hydrolysates, giving them a richer and more intense flavor. Some of these hydrolysates can be used as seasoning additives. However, the hydrolysates obtained by directly hydrolyzing proteins have limited effect on enhancing the saltiness of seasonings and cannot be used as high-quality saltiness enhancers.

[0004] Yang Xinwen et al. disclosed that low-temperature plasma treatment can cause chemical changes with proteins in food systems (Yang Xinwen, Niu Wenjun, Cheng Junhu, et al. Low-temperature plasma technology and its effects on food quality and microorganisms [J]. Food and Machinery, 2019, 35(9):199-203,215.DOI:10.13652 / j.issn.1003-5788.2019.09.038.), and Li Feng et al. disclosed that high pressure can improve the functional properties of proteins by changing their molecular structure. Furthermore, it can promote the hydrolysis of proteins by enzymes to generate new polypeptide products (Li Feng, Wei Ming, Wu Zhaomin, et al. Research progress on the effects of high pressure on protein structure, enzymatic hydrolysis and functional properties [J]. Anhui Agricultural Sciences, 2015, 43(10):3.). However, methods that can chemically change proteins, improve protein functional properties or promote the generation of new polypeptides may not necessarily enhance the saltiness of protein hydrolysates. Currently, there are no reports on using high pressure combined with low temperature plasma treatment to enhance the saltiness of protein hydrolysates. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing a method for preparing protease hydrolysates. By utilizing high-pressure synergistic low-temperature plasma treatment, the saltiness enhancement effect of the protease hydrolysates is significantly improved, thereby effectively enhancing the saltiness of seasonings. It is a high-quality saltiness enhancer that is beneficial to protecting consumers' dietary health.

[0006] Another object of the present invention is to provide a protease hydrolysate prepared by the above method.

[0007] Another object of the present invention is to provide the use of the above-mentioned proteolytic hydrolysate as and / or in the preparation of a salty flavor enhancer.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] This invention provides a method for preparing protease hydrolysate, which specifically involves: pulverizing animal or plant material, adding water, and then sequentially subjecting the mixture to high-pressure treatment, enzymatic hydrolysis, and low-temperature plasma treatment to obtain the protease hydrolysate;

[0010] The pressure of the high-pressure treatment is 100-400 MPa.

[0011] Preferably, the plant or animal has a protein content of more than 15%.

[0012] Preferably, the low-temperature plasma treatment is performed at a power of 30-40W and a temperature of -2-2℃ for 5-15 minutes. Most preferably, the low-temperature plasma treatment is performed at a power of 40W and a temperature of 0℃ for 5 minutes.

[0013] Preferably, the generating gas for the low-temperature plasma treatment is air.

[0014] More preferably, the gas flow rate is 3–5 L / min. Most preferably, it is 5 L / min.

[0015] Preferably, the mass ratio of water to the pulverized plant and animal matter is 1.8–2.2:1. Most preferably, it is 2:1.

[0016] Preferably, the high-pressure treatment time is 15 to 25 minutes. Most preferably, it is 20 minutes.

[0017] Preferably, the high-pressure treatment temperature is 20–30°C. Most preferably, it is 25°C.

[0018] Preferably, the enzyme used in the enzymatic hydrolysis is a protease.

[0019] More preferably, the enzyme includes one or more of papain, trypsin, or flavor protease.

[0020] More preferably, the enzyme mass is 1.8‰ to 2.2‰ of the mass of the crushed animal or plant material. Most preferably, it is 2‰.

[0021] Preferably, the enzymatic hydrolysis is performed at 45–55°C for 3.5–4.5 hours. Most preferably, it is performed at 50°C for 4 hours.

[0022] Preferably, after enzymatic hydrolysis, the enzyme is inactivated by boiling for 15–25 minutes. Most preferably, it is 20 minutes.

[0023] More preferably, the enzyme is further centrifuged after inactivation.

[0024] More preferably, the centrifugation is performed at 4500–5000g and 3–5°C for 18–22 min. Most preferably, it is performed at 4800g and 4°C for 20 min.

[0025] Most preferably, the centrifugation is followed by vacuum filtration.

[0026] Preferably, the plant or animal is one or more of the following: culled laying hens, bream, or peas.

[0027] Preferably, when the animal or plant is a culled laying hen, the grinding process is as follows: after removing the skin, bones, and fat from the culled laying hen, cut it into small pieces and then grind it into minced meat.

[0028] The proteolytic enzyme obtained by high-pressure synergistic low-temperature plasma treatment has excellent saltiness enhancement effect and can effectively enhance the saltiness of seasonings. It is a high-quality saltiness enhancer and is beneficial to protecting consumers' dietary health. Therefore, the proteolytic enzyme prepared by the above method, and its application as and / or in the preparation of saltiness enhancers, should also be within the protection scope of this invention.

[0029] The present invention has the following beneficial effects:

[0030] 1. The present invention obtains proteolytic enzymes through high-pressure synergistic low-temperature plasma treatment, which can significantly enhance the saltiness (the proteolytic enzymes with a protein content of 20 mg / mL can increase the saltiness intensity of 50 mmol / L NaCl solution by 45.6% to 57.2%) and umami flavor of seasonings. It can be used as a high-quality saltiness enhancer and umami enhancer, which is beneficial to protecting consumers' dietary health.

[0031] 2. The protease hydrolysate of the present invention is simple to prepare, the raw materials are readily available, the cost is low, it contains a large number of small molecule peptides, has high nutritional value, is easily absorbed and utilized by the human body, and has the antioxidant and antibacterial effects of the small molecule peptides themselves. Attached Figure Description

[0032] Figure 1 This is the result of the electronic tongue detection.

[0033] Figure 2 This is a statistical graph showing the molecular weight distribution of peptides. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0035] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0036] Example 1: Preparation of protease hydrolysate

[0037] S1. Frozen culled laying hens (purchased from Guangdong Wens Food Group Co., Ltd.) were placed in a 4℃ freezer for 12 hours to thaw. After the thawed culled laying hens were skinned, deboned, and degreased, they were cut into small pieces and then minced through a meat grinder with a perforated plate diameter of 6mm to obtain chicken meat paste.

[0038] S2. Take 20g of minced chicken, add 40g of deionized water, mix well, vacuum pack in a bag, and place in a high-pressure device to be treated under high pressure at 25℃ and 100MPa for 20min.

[0039] S3. After high pressure treatment, add papain (enzyme mass is 2‰ of the meat mass), and enzymatically hydrolyze in a water bath constant temperature shaker at 50℃ for 4 hours. Then boil for 20 minutes to inactivate the enzyme, cool to 25℃, and then centrifuge at 4800g and 4℃ for 20 minutes. Filter to obtain the enzymatic hydrolysis product.

[0040] S4. Place the enzymatic hydrolysis product obtained in S3 into a low-temperature plasma device and perform low-temperature plasma treatment for 15 min at a power of 30 W and a temperature of 0 °C. The generated gas is air, and the gas flow rate is 3 L / min.

[0041] Example 2 Preparation of protease hydrolysate

[0042] Same as Example 1, except that the pressure of the high-pressure treatment is 200MPa; the low-temperature plasma treatment is performed at a power of 40W and a temperature of 0℃ for 5 minutes, and the generated gas is air with a gas flow rate of 5L / min.

[0043] Example 3 Preparation of protease hydrolysate

[0044] Same as Example 1, except that the low-temperature plasma treatment is performed at a power of 40W and a temperature of 0℃ for 10 minutes, and the generated gas is air with a gas flow rate of 4L / min.

[0045] Comparative Example 1

[0046] Same as Example 1, except that low-temperature plasma treatment is not performed.

[0047] Comparative Example 2

[0048] Same as Example 2, except that low-temperature plasma treatment is not performed.

[0049] Comparative Example 3

[0050] Similar to Example 2, the difference is that high-pressure treatment is not performed; that is, after adding water to the minced meat, enzymes are added directly for subsequent enzymatic hydrolysis.

[0051] Comparative Example 4

[0052] Similar to Example 2, except that no low-temperature plasma treatment or high-pressure treatment is performed.

[0053] Experimental Example 1: Determination of the saltiness of protease hydrolysate

[0054] I. Sensory Evaluation

[0055] (1) Evaluation Method

[0056] Twenty evaluators were randomly selected from the sensory evaluation personnel database. The appearance, odor, taste, and fishy smell of the protein hydrolysates of Examples 1-3 and Comparative Examples 1-4 were first evaluated using the simple descriptive test method. Then, the enhancement effect of the protein hydrolysates of Examples 1-3 and Comparative Examples 1-4 on the saltiness of 50 mmol / L NaCl solution was evaluated using the quantitative estimation method.

[0057] The evaluation methods for the appearance, odor, taste, and fishy smell of protein hydrolysates are as follows:

[0058] S1. First, through certain training, all sensory evaluators can use the same concepts to describe the appearance, smell, taste, and fishy smell of the samples;

[0059] S2. During the sensory evaluation, the protein hydrolysate of Comparative Example 4 was used as a control. Each sensory evaluator wrote down the characteristics of each sample during the evaluation. After all evaluations were completed, a discussion was held under the chairmanship of the group leader to draw a comprehensive conclusion, and the results are shown in Table 1.

[0060] The method for evaluating the effect of protease hydrolysate on enhancing the saltiness of 50 mmol / L NaCl solution is as follows:

[0061] S1. First, by assessing the area of ​​geometric figures, sensory evaluators are guided to grasp the basic concept of the quantitative estimation method. Then, a series of sodium chloride solutions with gradients of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mmol / L are prepared, and the evaluators are allowed to taste the sodium chloride solutions of the series gradient concentrations to understand the saltiness intensity of sodium chloride solutions of different concentrations.

[0062] S2. Water was added to the protein hydrolysates of Examples 1-3 and Comparative Examples 1-4 respectively, so that the protein content of all samples was 20 mg / mL. Then, NaCl was added to remove the Na+ from the solution. + The concentration was adjusted to 50 mmol / L, and the evaluators tasted each sample and gave the specific saltiness intensity value (mmol / L NaCl). The results recorded by all the evaluators were then averaged to obtain the results shown in Table 1.

[0063] It should be noted that sensory evaluators should not consume other foods or beverages with pungent tastes or be overly fatigued 1 hour before conducting sensory evaluation experiments; the samples to be tested should be numbered using three random combinations of numbers; and in the evaluation experiment of saltiness enhancement, evaluators should only assess the saltiness intensity of the sample solution and not consider other sensory characteristics.

[0064] (2) Evaluation Results

[0065] Table 1. Sensory evaluation results of protein hydrolysates

[0066]

[0067] As shown in Table 1, the protease hydrolysates of Examples 1-3 are significantly superior to those of Comparative Examples 1-4 in terms of appearance, odor, taste, and fishy smell. Furthermore, the protease hydrolysates of Examples 1-3 with a protein content of 20 mg / mL can increase the saltiness intensity of a 50 mmol / L NaCl solution to 72.8-78.6 mmol / L, an increase of 45.6%-57.2% compared to the 50 mmol / L NaCl solution. Therefore, the protease hydrolysates obtained by high-pressure synergistic low-temperature plasma treatment in this invention can serve as a high-quality saltiness enhancer, significantly improving the saltiness of seasonings.

[0068] II. Electronic Tongue Analysis

[0069] (1) Analytical methods

[0070] Using a 50 mmol / L sodium chloride solution as a reference, the taste signals of 50 mmol / L NaCl solutions (with a protein content of 20 mg / mL in each solution) containing protein hydrolysates from Examples 1-3 and Comparative Examples 1-4 were detected using the TS-5000Z taste analysis system from Insent Corporation, Japan. The four taste sensors of the TS-5000Z system interacted with the taste-producing substances, causing changes in the potential of the artificial lipid membrane. This potential was transmitted as the sensor output signal to a computer for analysis, thereby evaluating the umami, saltiness, bitterness, astringency, and strong flavor of the samples. These four taste sensors were: umami sensor AAE (reflecting umami and strong flavor), saltiness sensor CTO (reflecting saltiness), bitterness sensor CO0 (reflecting bitterness), and astringency sensor AE1 (reflecting astringency). Both the taste sensors and the reference electrode were activated before use to ensure data stability.

[0071] (2) Analysis Results

[0072] Test results as follows Figure 1 As shown, compared with the 50 mmol / L sodium chloride solution (reference solution) without added protease hydrolysate, the saltiness and umami of the 50 mmol / L sodium chloride solution with added protease hydrolysates from Examples 1-3 and Comparative Examples 1-4 were improved. The saltiness enhancement effect of the protease hydrolysates from Examples 1-3 was significantly better than that of Comparative Examples 1-4. Furthermore, compared with Comparative Examples 2 and 3, the protease hydrolysates from Examples 1-3 also significantly reduced the astringency.

[0073] III. Determination of Peptide Molecular Weight Distribution

[0074] (1) Measurement method

[0075] The peptide molecular weight distribution of the proteolytic hydrolysates of Examples 1-3 and Comparative Examples 1-4 was determined by high-performance liquid chromatography (HPLC). Specific conditions were as follows: a TSKG2000SWXL (7.8 × 300 mm, 5 μm) gel column; a mobile phase of 0.125% (v / v) trifluoroacetic acid solution and acetonitrile at a volume ratio of 80:20; a flow rate of 1.0 mL / min; and a detection wavelength of 220 nm. Peptide standards were cytochrome C (12384 Da), aprotinin (6511 Da), somatostatin (1637 Da), Asp-Phe-Pro-Ala-Leu (561 Da), and Leu-Val-Phe (377 Da). The linear equation fitted to the logarithm of the peptide standard molecular weight versus retention time was: y = -2.587x + 17.002 (R²). 2 =0.99), where y is the logarithm of the molecular weight of the peptide standard; x is the retention time.

[0076] (2) Measurement results

[0077] Statistical results of peptide molecular weight distribution are as follows: Figure 2 As shown, the release of 500-1000 Da and <500 Da peptides in the protein hydrolysates of Examples 1-3 was significantly greater than that in Comparative Examples 1-4. This indicates that the processing method of the present invention can significantly promote the release of 1000 Da and <500 Da peptides in the protein hydrolysates. Since the <1000 Da peptides are mainly associated with saltiness or saltiness enhancement, the significant improvement in saltiness enhancement effect of the method on the protein hydrolysates may be related to the promotion of the release of <1000 Da peptides.

[0078] In summary, the proteolytic enzyme obtained by high-pressure synergistic low-temperature plasma treatment can significantly enhance the saltiness (the proteolytic enzyme with a protein content of 20 mg / mL can increase the saltiness intensity of 50 mmol / L NaCl solution by 45.6% to 57.2%) and umami flavor of seasonings. It can be used as a high-quality saltiness enhancer and umami enhancer, which is beneficial to protecting consumers' dietary health.

[0079] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a protease hydrolysate, characterized in that, The plant and animal products are crushed, water is added, and then subjected to high-pressure treatment, enzymatic hydrolysis, and low-temperature plasma treatment in sequence to obtain the protein hydrolysate. The plants and animals mentioned are culled laying hens; the high-pressure treatment is performed at a pressure of 100–400 MPa for 15–25 min; the enzyme used in the enzymatic hydrolysis is papain; the enzymatic hydrolysis is performed at 45–55 °C for 3.5–4.5 h; the low-temperature plasma treatment is performed at a power of 30–40 W and a temperature of -2–2 °C for 5–15 min, with air as the generated gas and a gas flow rate of 3–5 L / min.

2. The preparation method according to claim 1, characterized in that, The mass ratio of water to the crushed plant and animal matter is 1.8–2.2:

1.

3. The preparation method according to claim 1, characterized in that, The high-pressure treatment temperature is 20–30 °C.

4. The preparation method according to claim 1, characterized in that, The enzyme mass is 1.8‰ to 2.2‰ of the mass of the crushed animal and plant matter.

5. The method of claim 1, wherein After enzymatic hydrolysis, the enzyme is inactivated by boiling for 15–25 minutes.

6. The preparation method according to claim 5, characterized in that, After enzyme inactivation, centrifugation is performed.

7. The preparation method according to claim 6, characterized in that, The centrifugation was performed at 4500–5000 g and 3–5 °C for 18–22 min.

8. The preparation method according to claim 6, characterized in that, After centrifugation, the material is further filtered.

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