Method for detecting thermostable amylase in raw materials of fermented food

Abstract

The present application relates to a kind of detection methods of high-temperature resistant amylase in fermented food raw materials, the process of detecting high-temperature resistant amylase in fermented food raw materials, the measured product is oxidized, after oxidation treatment, a portion is passivated and handled as the control group of detection, a portion is protected by enzyme and handled as the test group of detection, then respectively carry out enzymatic reaction, starch-iodine color reaction and detect absorbance, by the absorbance change rate of test group relative to control group, refer to standard interval, determine the content of high-temperature resistant amylase in measured product.The method of the present application can realize the detection of low limit high-temperature resistant amylase.

Description

Technical Field

[0001] This invention relates to the field of biochemical detection, and in particular to a method for detecting thermostable amylase in fermented food raw materials. Background Technology

[0002] Fermented semi-finished products such as brewed soy sauce, brewed vinegar, and yeast extracts are widely used as food ingredients in the preparation of starch-containing products like oyster sauce. Brewed soy sauce not only provides a rich source of amino acids for starch-containing products like oyster sauce but also significantly enhances their color and aroma. Brewed vinegar provides various organic acids, enriching the flavor of starch-containing products and ensuring their preservative properties. Fermented semi-finished products like yeast extracts also provide umami substances to starch-containing products like oyster sauce, increasing their richness. The production of these food ingredients involves fermentation cycles of varying lengths. During fermentation, the growth of miscellaneous microorganisms is unavoidable, including Bacillus licheniformis and Bacillus subtilis. Under specific conditions, Bacillus licheniformis and Bacillus subtilis may produce thermostable amylases. Thermostable amylases are a class of enzymes with good thermal stability, capable of rapidly hydrolyzing the α-1,4-glucosidic bonds of starch molecules at high temperatures, and hydrolyzing oligosaccharides or short-chain dextrins of different molecular weights. Yang Lei et al. isolated a thermoresistant *Bacillus licheniformis* strain from strong-aroma liquor mash, which can produce thermoresistant amylase with an activity of 40-50 U / mL. Li Zhongzheng et al. isolated strains with α-amylase activity ranging from 1.51 to 6.238 U / g from aged vinegar koji. Sun Changyan et al. analyzed the fermentation process of soybean paste koji and found that the enzyme activity of the koji could reach up to 13.2 U / g dry basis during fermentation, with the main enzyme-producing strains being *Aspergillus oryzae*, *Aspergillus niger*, and *Bacillus subtilis*. This suggests that fermented food ingredients, such as those used in brewing soy sauce, may contain a certain amount of thermoresistant amylase. While most of the amylase in these ingredients is inactivated after sterilization, a low limit of thermoresistant amylase may remain. Fermented ingredients with residual low levels of amylase, when applied to starch-based products, can cause irreversible damage to the product's shelf life, leading to dilution and water loss. Fermented food ingredients such as soy sauce are generally sterilized to commercial sterility before being supplied to downstream manufacturers. Since thermostable amylase can retain a certain level of activity after being treated at 105°C, conventional sterilization methods may not be sufficient to completely inactivate the thermostable amylase. Therefore, it is necessary to explore a method for detecting residual thermostable amylase in fermented food ingredients.

[0003] Current detection methods and target substances for thermostable amylases have certain limitations. For example, the detection activity limit for thermostable amylases in GB / T24401 α-amylase preparations is 60-65 U / mL, but in reality, residual enzyme activity below 0.01 U / mL can affect the shelf life of products with high starch content. The target substances for detection in the aforementioned national standard methods are also relatively limited, lacking complex interfering substances. Chinese invention patent application 201811589645.8 describes a method for detecting amylase in oyster sauce, but this method can only perform qualitative detection and cannot determine the amylase content in oyster sauce, nor can it specify a detection limit for the specific content. Similarly, Chinese invention patent applications CN115326638A and CN109459431A can only achieve qualitative detection, while Chinese invention patent application CN113834757A can only quantify to the order of 0.1 U / g. Therefore, how to detect low-limit heat-resistant amylases in fermented food raw materials is an urgent technical problem to be solved. Summary of the Invention

[0004] Based on this, the main objective of the embodiments of this application is to provide a method for detecting thermostable amylase in fermented food raw materials, which can achieve the detection of low-limit thermostable amylase.

[0005] This application provides a method for detecting thermostable amylase in fermented food raw materials, the method comprising the following steps:

[0006] The test sample and the oxidant are mixed to carry out an oxidation reaction, thus preparing an oxidized test sample;

[0007] One portion of the oxidized test sample is mixed with an enzyme inactivating agent to carry out an inactivation reaction, thus preparing a control group test sample; another portion of the oxidized test sample is mixed with an enzyme protectant to carry out a protective treatment, thus preparing an experimental group test sample.

[0008] The control group test sample and the experimental group test sample were respectively mixed with starch substrate solution and enzymatically hydrolyzed to prepare control group reaction solution and experimental group reaction solution;

[0009] The control group reaction solution and the experimental group reaction solution were respectively mixed with iodine solution to carry out a colorimetric reaction, and the control group colorimetric solution and the experimental group colorimetric solution were prepared.

[0010] The absorbance A1 of the control group colorimetric solution and the absorbance A2 of the experimental group colorimetric solution were measured respectively, and the absorbance change rate Y of the absorbance A2 relative to the absorbance A1 was calculated.

[0011] A standard range reflecting the relationship between the content of thermoresistant amylase and the rate of change of absorbance is prepared, and the range of thermoresistant amylase content in which the rate of change of absorbance Y is located is determined by referring to the standard range.

[0012] In some embodiments of this application, the detection method satisfies one or more of the following conditions ① to ③:

[0013] ① In the step of preparing the oxidized sample, the oxidation reaction conditions include: the concentration of the oxidant is 0.01 mol / L-0.05 mol / L;

[0014] ② The passivation reaction conditions include: the concentration of the passivating agent is 0.01 mol / L-0.05 mol / L; the conditions for the enzymatic hydrolysis reaction of the control group sample include: pH value of 3.5-4.5;

[0015] as well as,

[0016] ③The raw materials for fermented foods include crude soy sauce or vinegar.

[0017] In some embodiments of this application, the preparation of a standard range reflecting the relationship between the content of thermostable amylase and the rate of change in absorbance includes the following steps:

[0018] A standard sample with a preset thermo-resistant amylase content is prepared, wherein the preset thermo-resistant amylase content of multiple standard samples increases in a gradient from zero.

[0019] The multiple standard samples were respectively mixed with starch substrate solution and subjected to enzymatic hydrolysis to prepare multiple standard sample reaction solutions;

[0020] The multiple standard sample reaction solutions are respectively mixed with iodine solution to carry out colorimetric reactions and prepare multiple standard sample colorimetric solutions;

[0021] The absorbance of the multiple standard sample colorimetric solutions is detected. Taking the absorbance of the standard sample with a preset high-temperature amylase content of zero as a reference, the rate of change of the absorbance of other standard samples with preset high-temperature amylase content relative to the reference absorbance is calculated to create a standard range that reflects the relationship between the high-temperature amylase content and the rate of change of absorbance.

[0022] Optionally, in the step of preparing the standard sample reaction solution, the conditions for the enzymatic hydrolysis reaction include: a temperature of 60℃-80℃ and a time of 0.5h-12h.

[0023] Optionally, a standard sample with a preset thermostable amylase content is prepared, comprising the following steps:

[0024] Provide basic standard raw materials, which are fermented food raw materials that do not contain heat-resistant amylase or whose amount of heat-resistant amylase does not affect shelf life;

[0025] The basic standard raw materials and oxidant are mixed and subjected to an oxidation reaction to prepare a blank sample;

[0026] The blank sample and a predetermined amount of thermoresistant amylase were mixed to prepare a standard sample with a predetermined thermoresistant amylase content.

[0027] Optionally, in the step of preparing the blank sample, the oxidation reaction conditions include: the concentration of the oxidant is 0.01 mol / L-0.05 mol / L.

[0028] In some embodiments of this application, the screening step of the basic standard raw materials includes:

[0029] Mixed fermented food ingredients and oyster sauce, the initial viscosity of the mixture and the viscosity after the storage period were tested, and the fermented food ingredients corresponding to the mixture whose viscosity after the storage period did not change by more than 5% relative to the initial viscosity were selected as the basic standard ingredients.

[0030] Optionally, the storage temperature is 50℃-70℃, and the shelf life is 25-35 days;

[0031] Optionally, the volume ratio of the fermented food ingredients to oyster sauce is 1:(45-55).

[0032] In some embodiments of this application, the preset thermostable amylase content includes 0 U / mL to 10 U / mL; optionally, the preset thermostable amylase content includes 0 U / mL, 0.01 U / mL, 0.1 U / mL, 1 U / mL, 5 U / mL and 10 U / mL.

[0033] In some embodiments of this application, the passivation reaction conditions further include one or more of the conditions shown in (A) and (B):

[0034] (A) The passivating agent is selected from zinc sulfate, aluminum chloride, or a combination thereof; and,

[0035] (B) Rotation speed is 45r / min-55r / min, and time is 5min-15min.

[0036] In some embodiments of this application, the conditions for protection processing include one or more of the conditions shown in (I) to (III):

[0037] (I) The protective agent is calcium chloride;

[0038] (II) The concentration of the protective agent is 0.01 mol / L to 0.05 mol / L; and,

[0039] (III) Rotation speed is 45r / min-55r / min, and time is 5min-15min.

[0040] In some embodiments of this application, the oxidation reaction conditions further include one or more of the conditions shown in (1) and (2):

[0041] (1) The oxidant is selected from ferric chloride, nitric acid, or a combination thereof; and,

[0042] (2) The rotation speed is 45r / min-55r / min and the time is 5min-15min.

[0043] In some embodiments of this application, the detection method satisfies one or more of the conditions shown in A) to C):

[0044] A) The conditions for the enzymatic hydrolysis reaction of the test samples in the test group include: pH value of 6.5-7.5, temperature of 60℃-80℃, and time of 0.5h-12h;

[0045] B) The conditions for the enzymatic hydrolysis reaction of the control group test sample also include: a temperature of 60℃-80℃ and a time of 0.5h-12h; and,

[0046] C) The conditions for the enzymatic hydrolysis reaction of the multiple standard samples include: pH value of 6.5-7.5, temperature of 60℃-80℃, and time of 0.5h-12h.

[0047] In some embodiments of this application, the detection method satisfies one or more of the conditions shown in i) to ii):

[0048] i) The concentration of soluble starch in the starch substrate solution is 2 g / mL-6 g / mL; the volume ratio of the control group sample to the starch substrate solution is 1:(3-5); the volume ratio of the test group sample to the starch substrate solution is 1:(3-5); the volume ratio of the standard sample to the starch substrate solution is 1:(3-5); and,

[0049] ii) The concentration of potassium iodide in the iodine solution is 0.01 mol / L-0.02 mol / L, the volume ratio of the control group reaction solution to the iodine solution is 1:(3-7), the volume ratio of the test group reaction solution to the iodine solution is 1:(3-7), and the volume ratio of the standard sample reaction solution to the iodine solution is 1:(3-7).

[0050] Compared to traditional technologies, the beneficial effects of this application include:

[0051] This application describes a method for detecting thermostable amylase in fermented food ingredients. The sample is oxidized, and after oxidation, one sample is passivated (control group) and the other is enzyme-protected (experimental group). Enzymatic hydrolysis and starch-iodine colorimetric reactions are then performed, and absorbance is measured. The content of thermostable amylase in the sample is determined by comparing the absorbance change rate of the experimental group with that of the control group, and referring to a standard range. This method enables the detection of low-limit thermostable amylase. Attached Figure Description

[0052] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0053] Figure 1 This is a flowchart illustrating the testing method of this application. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the invention. The purpose of providing these embodiments and examples is to enable a more thorough and complete understanding of the disclosure of the present invention. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present invention, and the equivalent forms obtained also fall within the protection scope of this application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for descriptive purposes only and is not intended to be limiting of the invention.

[0056] the term

[0057] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0058] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0059] In this invention, terms such as "multiple", "various", "multiple times", and "multi-source" are used, and unless otherwise specified, they refer to a quantity greater than or equal to 2. For example, "one or more" means one or more types.

[0060] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0061] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this invention, solve the technical problem of this invention, and achieve the expected technical effect of this invention.

[0062] In this document, terms such as “preferred,” “better,” “more suitable,” and “ideal” are merely used to describe implementation methods or examples that yield better results, and should be understood not to limit the scope of protection of this invention.

[0063] In this invention, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0064] In this invention, "optionally," "optionally," and "optional" mean that they are optional, that is, they are selected from either "with" or "without." If multiple "options" appear in a technical solution, unless otherwise specified and there are no contradictions or mutual constraints, each "option" is independent.

[0065] In this invention, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on the quantity.

[0066] In this invention, the technical features described in an open-ended manner include both closed-ended technical solutions composed of the listed features and open-ended technical solutions that include the listed features.

[0067] In this invention, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0068] Unless otherwise specified, the temperature parameters in this invention can be either constant temperature treatment or variations within a certain temperature range. It should be understood that the constant temperature treatment allows temperature fluctuations within the precision range controlled by the instrument. Fluctuations are permitted within ranges such as ±5℃, ±4℃, ±3℃, ±2℃, and ±1℃.

[0069] In this invention, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0070] All references to this invention are incorporated herein by reference as if each document were individually incorporated by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, the referenced documents involved in this invention are incorporated in their entirety and for all purposes. When references are made in this invention, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When references are made in this invention, examples and preferred embodiments of the relevant technical features cited may also be incorporated herein by reference, but only to the extent that they enable the implementation of this invention. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptively based on the description in this application.

[0071] The composition of brewed soy sauce and vinegar is relatively complex. These fermentation products contain various organic acids and amino acids. Among these organic acids, there may be reducing organic acids such as formic acid, oxalic acid, and ascorbic acid. When using the iodine-starch principle for detection, these reducing substances will cause the complex between iodine and starch to decolorize, making it impossible to determine the reaction endpoint and resulting in undetectable positive results. Thermase detection of thermostable amylase generally requires ultra-high temperature enzyme inactivation treatment of blank samples in the blank group (control group). This treatment process often requires high-end equipment, and for dark-colored fermented semi-finished products like brewed soy sauce, the color of the sample changes significantly before and after treatment. This color change will also affect the absorbance of the test, causing the blank control group and the experimental group to not maintain a single variable, leading to deviations in the test results.

[0072] Furthermore, the detection effectiveness of high-temperature amylase is currently low, with the most precise detection method only able to detect up to 0.1 U / mL. These highly accurate methods require a relatively long detection time, while methods with shorter detection times cannot meet current detection requirements in terms of accuracy.

[0073] This application pretreats semi-finished soy sauce crude oil or brewed vinegar with oxidizing substances such as ferric chloride and dilute nitric acid, ensuring that the semi-finished product to be tested has been oxidized before testing and will not interfere with the subsequent reaction between the substrate and the indicator.

[0074] The control group of pre-treated semi-finished products was further inactivated by using zinc sulfate and aluminum chloride solutions to inhibit the activity of thermostable amylase. In contrast, the experimental group was treated with calcium chloride solution to maintain enzyme activity in reaction with the substrate and improve the enzymatic hydrolysis efficiency of residual amylase.

[0075] By adjusting the substrates of the test samples in the control and experimental groups, the control group underwent further inactivation treatment of the thermostable amylase to inhibit its enzyme activity; in addition, the experimental group adjusted the optimal pH to ensure that the amylase maintained its optimal activity and improved the enzymatic hydrolysis efficiency of residual amylase.

[0076] By adjusting the enzymatic hydrolysis temperature, time, and substrate concentration, the enzymatic hydrolysis reaction is promoted and the reaction efficiency is improved. A reference range is prepared by adding a series of known concentrations of thermostable amylase aqueous buffer solutions as blank substrates, and the blank group and the reference range are used to achieve semi-quantitative judgment of the detection results.

[0077] The method of this application can perform semi-quantitative detection of the content of heat-resistant amylase in vinegar, oyster sauce, etc., with a detection precision of 0.01 U / mL and rapid results within 12 hours, which greatly improves the detection efficiency of heat-resistant amylase. Therefore, it can be used for rapid detection and acceptance of heat-resistant amylase in fermented semi-finished materials, so as to avoid affecting the raw material receiving efficiency of fermented semi-finished products.

[0078] The technical solution of this application includes:

[0079] This application provides a method for detecting thermostable amylase in fermented food raw materials, the method comprising the following steps:

[0080] The test sample and the oxidant are mixed to carry out an oxidation reaction, thus preparing an oxidized test sample;

[0081] One portion of the oxidized test sample is mixed with an enzyme inactivating agent to carry out an inactivation reaction, thus preparing a control group test sample; another portion of the oxidized test sample is mixed with an enzyme protectant to carry out a protective treatment, thus preparing an experimental group test sample.

[0082] The control group test sample and the experimental group test sample were respectively mixed with starch substrate solution and enzymatically hydrolyzed to prepare control group reaction solution and experimental group reaction solution;

[0083] The control group reaction solution and the experimental group reaction solution were respectively mixed with iodine solution to carry out a colorimetric reaction, and the control group colorimetric solution and the experimental group colorimetric solution were prepared.

[0084] The absorbance A1 of the control group colorimetric solution and the absorbance A2 of the experimental group colorimetric solution were measured respectively, and the absorbance change rate Y of the absorbance A2 relative to the absorbance A1 was calculated.

[0085] A standard range reflecting the relationship between the content of thermoresistant amylase and the rate of change of absorbance is prepared, and the range of thermoresistant amylase content in which the rate of change of absorbance Y is located is determined by referring to the standard range.

[0086] In one example, the detection method satisfies one or more of the conditions shown in ① to ④ below:

[0087] ① In the step of preparing the oxidized sample, the conditions for the oxidation reaction include: the concentration of the oxidant is 0.01 mol / L-0.05 mol / L (for example, 0.01, 0.02, 0.03, 0.04, 0.05 mol / L);

[0088] ② The passivation reaction conditions include: the concentration of the passivating agent is 0.01 mol / L-0.05 mol / L (e.g., 0.01, 0.02, 0.03, 0.04, 0.05 mol / L); the conditions for the enzymatic hydrolysis reaction of the control group sample include: a pH value of 3.5-4.5 (e.g., 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5);

[0089] ③ To prepare a standard range reflecting the relationship between the content of heat-resistant amylase and the rate of change in absorbance, the following steps are included:

[0090] A standard sample with a preset thermo-resistant amylase content is prepared, wherein the preset thermo-resistant amylase content of multiple standard samples increases in a gradient from zero.

[0091] The multiple standard samples were respectively mixed with starch substrate solution and subjected to enzymatic hydrolysis to prepare multiple standard sample reaction solutions;

[0092] The multiple standard sample reaction solutions are respectively mixed with iodine solution to carry out colorimetric reactions and prepare multiple standard sample colorimetric solutions;

[0093] The absorbance of the multiple standard sample colorimetric solutions is detected. Taking the absorbance of the standard sample with a preset high-temperature amylase content of zero as a reference, the rate of change of the absorbance of other standard samples with preset high-temperature amylase content relative to the reference absorbance is calculated to create a standard range that reflects the relationship between the high-temperature amylase content and the rate of change of absorbance.

[0094] Optionally, in the step of preparing the standard sample reaction solution, the conditions for the enzymatic hydrolysis reaction include: a temperature of 60℃-80℃ (e.g., 60, 62, 64, 68, 70, 72, 74, 76, 78, 80℃) and a time of 0.5h-12h (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12h);

[0095] as well as,

[0096] ④ The raw materials for fermented foods include crude soy sauce or vinegar.

[0097] In one example, the preparation of a standard sample with a predetermined content of thermostable amylase includes the following steps:

[0098] Provide basic standard raw materials, which are fermented food raw materials that do not contain heat-resistant amylase or whose amount of heat-resistant amylase does not affect shelf life;

[0099] The basic standard raw materials and oxidant are mixed and subjected to an oxidation reaction to prepare a blank sample;

[0100] The blank sample and a predetermined amount of thermoresistant amylase were mixed to prepare a standard sample with a predetermined thermoresistant amylase content.

[0101] Optionally, in the step of preparing the blank sample, the oxidation reaction conditions include: the concentration of the oxidant is 0.01 mol / L to 0.05 mol / L (e.g., 0.01, 0.02, 0.03, 0.04, 0.05 mol / L).

[0102] In one example, the screening steps for the basic standard raw materials include:

[0103] Mixed fermented food ingredients and oyster sauce, the initial viscosity of the mixture and the viscosity after the storage period were tested, and the fermented food ingredients corresponding to the mixture whose viscosity after the storage period did not change by more than 5% relative to the initial viscosity were selected as the basic standard ingredients.

[0104] Optionally, the storage temperature is 50℃-70℃ (e.g., 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70℃), and the shelf life is 25-35 days (e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 days).

[0105] Optionally, the volume ratio of the fermented food ingredients to oyster sauce is 1:(45-55) (e.g., 1:45, 1:46, 1:47, 1:48, 1:49, 1:50, 1:51, 1:52, 1:53, 1:54, 1:55).

[0106] In one example, the preset thermostable amylase content includes 0 U / mL to 10 U / mL (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 U / mL); optionally, the preset thermostable amylase content includes 0 U / mL, 0.01 U / mL, 0.1 U / mL, 1 U / mL, 5 U / mL, and 10 U / mL.

[0107] In one example, the passivation reaction conditions also include one or more of the conditions shown in (A) and (B):

[0108] (A) The passivating agent is selected from zinc sulfate, aluminum chloride, or a combination thereof; and,

[0109] (B) Rotation speed of 45 r / min-55 r / min (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 r / min), time of 5 min-15 min (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 min).

[0110] In one example, the protection processing conditions include one or more of the conditions shown in (I) to (III):

[0111] (I) The protective agent is calcium chloride;

[0112] (II) The concentration of the protective agent is 0.01 mol / L to 0.05 mol / L (e.g., 0.01, 0.02, 0.03, 0.04, 0.05 mol / L); and,

[0113] (III) Rotation speed is 45r / min-55r / min (e.g. 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55r / min), and time is 5min-15min (e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15min).

[0114] In one example, the oxidation reaction conditions also include one or more of the conditions shown in (1) and (2):

[0115] (1) The oxidant is selected from ferric chloride, nitric acid, or a combination thereof; and,

[0116] (2) The rotation speed is 45r / min-55r / min (e.g. 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55r / min), and the time is 5min-15min (e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15min).

[0117] In one example, the detection method satisfies one or more of the conditions shown in A) to C):

[0118] A) The conditions for the enzymatic hydrolysis reaction of the test samples in the test group include: pH value of 6.5-7.5 (e.g., 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5), temperature of 60℃-80℃ (e.g., 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80℃), and time of 0.5h-12h (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12h);

[0119] B) The conditions for the enzymatic hydrolysis reaction of the control group test sample further include: a temperature of 60℃-80℃ (e.g., 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80℃), and a time of 0.5h-12h (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12h); and,

[0120] C) The conditions for the enzymatic hydrolysis reaction of the multiple standard samples include: pH value of 6.5-7.5 (e.g., 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5), temperature of 60℃-80℃ (e.g., 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80℃), and time of 0.5h-12h (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12h).

[0121] In one example, the detection method satisfies one or more of the conditions shown in i) to ii):

[0122] i) The concentration of soluble starch in the starch substrate solution is 2 g / mL-6 g / mL (e.g., 2, 3, 4, 5, 6 g / mL), the volume ratio of the control group sample to the starch substrate solution is 1:(3-5) (e.g., 1:3, 1:3.5, 1:4, 1:4.5, 1:5), the volume ratio of the test group sample to the starch substrate solution is 1:(3-5) (e.g., 1:3, 1:3.5, 1:4, 1:4.5, 1:5), and the volume ratio of the standard sample to the starch substrate solution is 1:(3-5) (e.g., 1:3, 1:3.5, 1:4, 1:4.5, 1:5); and,

[0123] ii) The concentration of potassium iodide in the iodine solution is 0.01 mol / L-0.02 mol / L, the volume ratio of the control group reaction solution to the iodine solution is 1:(3-7) (e.g., 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7), the volume ratio of the test group reaction solution to the iodine solution is 1:(3-7) (e.g., 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7), and the volume ratio of the standard sample reaction solution to the iodine solution is 1:(3-7) (e.g., 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7).

[0124] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0125] Fermented food ingredients are generally sterilized before being supplied to downstream manufacturers. Sterilized ingredients typically do not contain or retain only low levels of thermostable amylase. This application requires screening fermented food ingredients to identify a group of fermented foods whose residual thermostable amylase levels have no significant impact on product shelf life as basic standard ingredients. The following examples and comparative examples use crude soy sauce or vinegar as basic standard ingredients.

[0126] Screening of basic standard raw materials:

[0127] Select different batches of soy sauce crude oil or vinegar, take 10mL of each and add it back to oyster sauce products of the same specification. Observe the shelf life of the product at 50℃ for 28 days and observe the change in viscosity (cp) of the oyster sauce during the shelf life. If the viscosity change rate does not exceed 5%, it is determined that the product does not contain heat-resistant amylase or that the residual amount of heat-resistant amylase has no effect on the shelf life. The basic standard raw materials are screened by this method.

[0128] Table 1. Results of screening basic standard raw materials from different batches of soy sauce crude oil and vinegar.

[0129]

[0130]

[0131] Based on the table above, the viscosity change rate of soy sauce batch 1, vinegar batch 1, and vinegar batch 3 did not exceed 5%, indicating that these three batches of fermented food raw materials do not contain heat-resistant amylase or the residual amount of heat-resistant amylase has no impact on shelf life, and can be used as basic standard raw materials. The raw materials screened by this method are suitable for subsequent comparative examples and verification.

[0132] Example 1 (in combination) Figure 1 ):

[0133] (1) Experiment 1: Verification of the effectiveness of oxidant

[0134] ① Select batch 1 of the above-mentioned soy sauce crude oil, add one portion to ferric chloride solution to a concentration of 0.01 mol / L, and react on a shaker at a frequency of 50 r / min for 10 min to oxidize the reducing substances such as formic acid, oxalic acid and ascorbic acid in the soy sauce crude oil. One portion is not oxidized.

[0135] ② Select a specific soluble starch and prepare a substrate solution of 4 g / mL using a pH 6.5 phosphate buffer solution. Take the oxidized and unoxidized soy sauce crude oil from ① and mix it with the substrate solution in a 70℃ water bath shaker for 5 min.

[0136] ③ Take 1 mL of the reaction solution from ② and react it with 5 mL of 0.012 mol / L dilute iodine solution to develop a colorimetric reaction. Immediately detect the absorbance at 660 nm.

[0137] ④ The effectiveness of the oxidation treatment can be judged by observing whether the absorbance of starch changes after reacting with iodine.

[0138] Table 2. Verification of Oxidation Efficacy

[0139] 0.01 mol / L ferric chloride treatment <![CDATA[0.654±0.011 a ]]> <![CDATA[0.650±0.007 a ]]> Unfaded Unprocessed <![CDATA[0.648±0.009 a ]]> <![CDATA[0.475±0.021 b ]]> fade

[0140] Note: Absorbance is indicated as mean ± standard deviation; different letters in the same column of absorbance indicate significant differences, while the same letter indicates no significant differences.

[0141] Based on the table above: Soy sauce crude oil batch 1 was used as the basic standard raw material. After treatment with ferric chloride, the influence of reducing substances was effectively avoided. The starch substrate reacted well with iodine, the absorbance did not change significantly, and the color did not fade after development. However, without ferric chloride treatment, reducing substances interfered with the reaction between starch and iodine, causing the complex to fade and the absorbance to change significantly. The above results prove the effectiveness of the oxidant treatment.

[0142] (2) Verification of passivation effectiveness

[0143] ① Select batch 1 of the above-mentioned oxidized soy sauce crude oil and divide it into three equal parts. Take a thermoresistant amylase with an effective concentration of 80000 U / mL as tested by GB / T 24401 α-amylase preparation. Add thermoresistant amylase to two samples to 10 U / mL as the test group and the control group. The other sample is untreated as the blank group.

[0144] ② Add zinc sulfate to the experimental group to a concentration of 0.01 mol / L, while the other two groups were left untreated. Both groups were reacted on a shaker at a frequency of 50 r / min for 10 min.

[0145] ③ Select a specific soluble starch and prepare a 4 g / mL substrate solution using phosphate buffer solutions at pH 3.5 and pH 6.5 respectively. Mix the substrate with the solution in a 70℃ water bath shaker for 0.5 h according to Table 3.

[0146] ④ Take 1 mL of the reaction solution from ③ and react it with 5 mL of 0.012 mol / L dilute iodine solution to develop a colorimetric reaction. Immediately detect the absorbance at 660 nm.

[0147] ⑤ The effectiveness of passivation treatment can be determined by observing whether the absorbance changes after starch reacts with iodine.

[0148] Table 3. Verification of passivation effectiveness

[0149] experimental group 1U / mL 0.01 mol / L zinc sulfate treatment 3.5 <![CDATA[0.630±0.009 a ]]> control group 1U / mL Unprocessed 6.5 <![CDATA[0.251±0.005 b ]]> Blank group 0U / mL Unprocessed 6.5 <![CDATA[0.634±0.011 a ]]>

[0150] Note: Absorbance is indicated as mean ± standard deviation; different letters in the same column of absorbance indicate significant differences, while the same letter indicates no significant differences.

[0151] Based on the table above: both the experimental group and the control group had thermostable amylase added compared to the blank group. However, the absorbance of the experimental group, which was passivated with zinc sulfate, did not change significantly compared to the blank group, while the absorbance of the control group, which was not passivated with zinc sulfate, changed significantly compared to the blank group, thus proving the effectiveness of zinc sulfate passivation treatment.

[0152] ⑥ The rate of change in absorbance of the passivated test group compared with the blank group was within 5%, which was determined to be of no effect on absorbance.

[0153] (3) Standard interval formulation

[0154] ① Taking soy sauce crude oil as an example, select soy sauce crude oil batch 1; add ferric chloride solution to soy sauce crude oil batch 1 to a concentration of 0.01 mol / L, and react on a shaker at a frequency of 50 r / min for 10 min to oxidize the reducing substances such as formic acid, oxalic acid and ascorbic acid in the soy sauce crude oil.

[0155] ② Take the blank sample treated in ① and add heat-resistant amylase of known concentration according to national standards to a concentration of 0-10 U / mL to prepare a standard sample.

[0156] ③ Select a specific soluble starch and prepare a 4 g / mL substrate solution using pH 6.5 phosphate buffer. Add 0.01 mol / L calcium chloride for protection. Mix 10 mL of the oxidized standard sample from step ② with 40 mL of the substrate solution and react in a 60℃ water bath with a shaker for 12 h. Take 1 mL of the incubated reaction solution and react it with 5 mL of 0.012 mol / L dilute iodine solution for color development. Measure the absorbance of the standard samples with different thermostable amylase contents. Use the sample with an enzyme content of 0 U / mL as the blank sample. The absorbance change rate = (blank sample absorbance - absorbance of standard samples with different enzyme contents) / blank sample absorbance * 100%. Construct a standard range based on the relationship between the corresponding enzyme content and the corresponding absorbance change rate.

[0157] The results are shown in the table below:

[0158] Table 4. Standard Range of Change Rate of Temperature-Resistant Amylase Content and Absorbance in Crude Soy Sauce

[0159] 0U / mL <![CDATA[0.636±0.006 a ]]> 0.00％ 0.01 U / mL <![CDATA[0.587±0.004 b ]]> 7.60％ 0.1 U / mL <![CDATA[0.476±0.004 c ]]> 25.12％ 1U / mL <![CDATA[0.248±0.008 d ]]> 60.94％ 5U / mL <![CDATA[0.127±0.007 e ]]> 80.08％ 10U / mL <![CDATA[0.004±0.001 f ]]> 99.37％

[0160] Note: Absorbance is indicated as mean ± standard deviation; different letters in the same column of absorbance indicate significant differences, while the same letter indicates no significant differences.

[0161] (4) Test for amylase content in crude soy sauce containing heat-resistant amylase

[0162] ① Take 100 mL of the fermented crude oil to be tested, add ferric chloride solution to a concentration of 0.01 mol / L, and react on a shaker at a frequency of 50 r / min for 10 min to oxidize the reducing substances in the soy sauce crude oil.

[0163] ② Take the oxidized crude oil from ① and divide it into two portions, each 50 mL. Add 0.01 mol / L zinc sulfate to one portion for passivation treatment as the control group powder, and add 0.01 mol / L calcium chloride to the other portion for protection treatment as the experimental group. Place the two groups of samples on a shaker and react at a frequency of 50 r / min for 10 min.

[0164] ③ Prepare phosphate buffer solutions with pH 3.5 and pH 6.5. Select specific soluble starches and prepare substrate solutions with a soluble starch concentration of 4 g / mL using the two sets of phosphate buffer solutions. Take 10 mL of samples from the control group and the experimental group and mix them with 40 mL of pH 3.5 starch substrate solution and pH 6.5 starch substrate solution, respectively. Place them in a 60℃ water bath shaker for 12 h of mixed enzymatic hydrolysis.

[0165] ④ Take 1 mL of the control group and experimental group solutions from ③ after enzymatic hydrolysis, and react them with 5 mL of 0.012 mol / L dilute iodine solution to develop a colorimetric reaction. Immediately detect the absorbance at 660 nm.

[0166] Take batch 3 of the above-mentioned crude soy sauce and test its heat-resistant amylase content according to the standard method including the above steps. The absorbance change rate and judgment results are shown in the table below:

[0167] Table 5. Results of Amylase Content Detection in Crude Oil Containing High-Temperature Resistant Amylase

[0168] Soy sauce crude oil batch 3 0.639 0.556 12.99％ 0.01-0.1 U / mL

[0169] Based on the table above, the absorbance change rate corresponding to crude soy sauce batch 3 is 12.99%. Referring to the standard range in Table 4, 12.99% falls between 7.60% and 25.12%. The concentrations of thermostable amylase corresponding to 7.60% and 25.12% are 0.01 U / mL and 0.1 U / mL, respectively. Therefore, the concentration of thermostable amylase contained in crude soy sauce batch 3 is between 0.01 U / mL and 0.1 U / mL.

[0170] Example 2 (in combination) Figure 1 ):

[0171] (1) Experiment 1: Verification of the effectiveness of oxidant

[0172] ① Select batch 1 of the above-mentioned vinegar, add one portion to dilute nitric acid solution to a concentration of 0.05 mol / L, and react on a shaker at a frequency of 50 r / min for 10 min to oxidize the reducing substances in the vinegar. The other portion is not oxidized.

[0173] ② Select a specific soluble starch and prepare a substrate solution of 4 g / mL using a pH 7.5 phosphate buffer solution. Take the crude oil from ① that has undergone oxidation treatment and the untreated crude oil and mix them with the substrate solution in an 80℃ water bath shaker for 5 min.

[0174] ③ Take 1 mL of the reaction solution from ② and react it with 5 mL of 0.012 mol / L dilute iodine solution to develop a colorimetric reaction. Immediately detect the absorbance at 660 nm.

[0175] ④ The effectiveness of the oxidation treatment can be judged by observing whether the absorbance of starch changes after reacting with iodine.

[0176] Table 6. Verification of Oxidation Effectiveness

[0177] Treatment with 0.05 mol / L dilute nitric acid <![CDATA[0.580±0.008 a ]]> <![CDATA[0.572±0.009 a ]]> Unfaded Unprocessed <![CDATA[0.575±0.012 a ]]> <![CDATA[0.475±0.021 b ]]> fade

[0178] Note: Absorbance is indicated as mean ± standard deviation; different letters in the same column of absorbance indicate significant differences, while the same letter indicates no significant differences.

[0179] Based on the table above: Batch 1 of vinegar was the basic standard raw material. After treatment with dilute nitric acid, the influence of reducing substances was effectively avoided. The starch substrate reacted well with iodine, the absorbance did not change significantly, and the color did not fade after development. However, without dilute nitric acid treatment, reducing substances interfered with the reaction between starch and iodine, causing the complex to fade and the absorbance to change significantly. The above results prove the effectiveness of the oxidant treatment.

[0180] (2) Verification of passivation effectiveness

[0181] ① Select batch 1 of the above-mentioned oxidized vinegar and divide it into three equal parts. Take the thermoresistant amylase with an effective concentration of 80000 U / mL as detected by GB / T 24401 α-amylase preparation. Add the thermoresistant amylase to two samples to 10 U / mL as the test group and the control group. The other sample is untreated as the blank group.

[0182] ② Add aluminum chloride to the experimental group to a concentration of 0.05 mol / L, while the other two groups were left untreated. Both groups were reacted on a shaker at a frequency of 50 r / min for 10 min.

[0183] ③ Select a specific soluble starch and prepare substrate solutions of 4 g / mL using phosphate buffer solutions at pH 4.5 and pH 7.5 respectively. Mix the substrate with the solution in a water bath shaker at 80℃ for 0.5 h according to Table 7.

[0184] ④ Take 1 mL of the reaction solution from ③ and react it with 5 mL of 0.012 mol / L dilute iodine solution to develop a colorimetric reaction. Immediately detect the absorbance at 660 nm.

[0185] ⑤ The effectiveness of passivation treatment can be determined by observing whether the absorbance changes after starch reacts with iodine.

[0186] Table 7. Verification of passivation effectiveness

[0187] experimental group 1U / mL 0.05 mol / L aluminum chloride treatment 4.5 0.567±0.007a control group 1U / mL Unprocessed 7.5 0.222±0.013b Blank group 0U / mL Unprocessed 7.5 0.581±0.010a

[0188] Note: Absorbance is indicated as mean ± standard deviation; different letters in the same column of absorbance indicate significant differences, while the same letter indicates no significant differences.

[0189] Based on the table above: both the experimental group and the control group had thermostable amylase added compared to the blank group. However, the absorbance of the experimental group, which was passivated with aluminum chloride, did not change significantly compared to the blank group, while the absorbance of the control group, which was not passivated with aluminum chloride, changed significantly compared to the blank group, thus proving the effectiveness of aluminum chloride passivation treatment.

[0190] ⑥ The rate of change in absorbance of the passivated test group compared with the blank group was within 5%, which was determined to be of no effect on absorbance.

[0191] (3) Standard interval formulation

[0192] ① Taking vinegar as an example, select batch 1 of vinegar; add dilute nitric acid solution to the vinegar to a concentration of 0.05 mol / L, and react on a shaker at a frequency of 50 r / min for 10 min to oxidize the reducing substances in the vinegar.

[0193] ② Take the blank sample treated in ① and add heat-resistant amylase of known concentration according to national standards to six standard samples with a concentration of 0-10 U / mL.

[0194] ③ Select a specific soluble starch and prepare a 4 g / mL substrate solution using pH 7.5 phosphate buffer. Add 0.05 mol / L calcium chloride for protection. Mix 10 mL of the oxidized standard sample from step ② with 40 mL of the substrate solution and react in an 80℃ water bath with a shaker for 12 h. Take 1 mL of the incubated reaction solution and react it with 5 mL of 0.012 mol / L dilute iodine solution for color development. Measure the absorbance of the standard samples with different concentrations of thermostable amylase. Use the sample with an enzyme content of 0 u / mL as a blank sample. The absorbance change rate = (blank sample absorbance - absorbance of standard samples with different enzyme contents) / blank sample absorbance * 100%. Construct a standard range based on the relationship between the corresponding enzyme content and the corresponding absorbance change rate.

[0195] The results are shown in the table below:

[0196] Table 8. Standard Range of Change in Absorbance Rate of Thermoresistant Amylase Content in Vinegar

[0197] a 0U / mL <![CDATA[0.558±0.004 a ]]> 0.00％ b 0.01 U / mL <![CDATA[0.518±0.002 b ]]> 7.22％ c 0.1 U / mL <![CDATA[0.478±0.001 c ]]> 14.44％ d 1U / mL <![CDATA[0.220±0.001 d ]]> 60.65％ e 5U / mL <![CDATA[0.117±0.003 e ]]> 79.05％ f 10U / mL <![CDATA[0.010±0.002 f ]]> 98.15％

[0198] Note: Absorbance is indicated as mean ± standard deviation; different letters in the same column of absorbance indicate significant differences, while the same letter indicates no significant differences.

[0199] (4) Test for the content of thermoresistant amylase in vinegar containing thermoresistant amylase

[0200] ① Take 100 mL of the vinegar to be tested, add dilute nitric acid solution to a concentration of 0.05 mol / L, and react on a shaker at a frequency of 50 r / min for 10 min to oxidize the reducing substances in the vinegar.

[0201] ② Take the crude oil from ① and divide it into two portions, each 50 mL. One portion was added with 0.01 mol / L aluminum chloride for passivation treatment as the control group, and the other portion was added with 0.05 mol / L calcium chloride for protection treatment as the experimental group. The two groups were placed on a shaker and reacted at a frequency of 50 r / min for 10 min.

[0202] ③ Prepare pH 4.5 and pH 7.5 phosphate buffer solutions. Select a specific soluble starch and prepare a substrate solution of 4 g / mL with the two phosphate buffer solutions. Take 10 mL of the control group and the experimental group samples and mix them with 40 mL of pH 4.5 starch substrate solution and pH 7.5 starch substrate solution respectively. Place them in an 80℃ water bath shaker for 8 h of mixed enzymatic hydrolysis.

[0203] ④ Take 1 mL of the control group and experimental group solutions from ③ after enzymatic hydrolysis, and react them with 5 mL of 0.012 mol / L dilute iodine solution to develop a colorimetric reaction. Immediately detect the absorbance at 660 nm.

[0204] Take batch 2 of the above-mentioned vinegar and test its heat-resistant amylase content according to the standard method including the above steps. The absorbance change rate and judgment results are shown in the table below:

[0205] Table 9. Detection results of thermoresistant amylase content in vinegar containing thermoresistant amylase

[0206] Vinegar Batch 2 0.559 0.453 18.96％ 0.1-1 U / mL

[0207] Based on the table above, the absorbance change rate corresponding to vinegar batch 2 is 18.96%. Referring to the standard range in Table 8, 18.96% falls between 14.44% and 60.65%. The concentrations of thermostable amylase corresponding to 14.44% and 60.65% are 0.1 U / mL and 1 U / mL, respectively. Therefore, the concentration of thermostable amylase contained in vinegar batch 2 is between 0.1 U / mL and 1 U / mL.

[0208] Comparative Example 1:

[0209] ① Select batch 1 of the above-mentioned soy sauce crude oil, add one portion to ferric chloride solution to a concentration of 0.001 mol / L, and react on a shaker at a frequency of 50 r / min for 10 min to oxidize the reducing substances such as formic acid, oxalic acid and ascorbic acid in the soy sauce crude oil. One portion is not oxidized.

[0210] ② Select a specific soluble starch and prepare a substrate solution of 4 g / mL using a pH 6.5 phosphate buffer solution. Take the crude oil from ① that has undergone oxidation treatment and the untreated crude oil and mix them with the substrate solution in a 70℃ water bath shaker for 5 min.

[0211] ③ Take 1 mL of the reaction solution from ② and react it with 5 mL of 0.012 mol / L dilute iodine solution to develop a colorimetric reaction. Immediately detect the absorbance at 660 nm.

[0212] ④ The effectiveness of the oxidation treatment can be judged by observing whether the absorbance of starch changes after reacting with iodine.

[0213] Table 10. Verification of Oxidation Efficacy

[0214] 0.001 mol / L ferric chloride treatment <![CDATA[0.653±0.006 a ]]> <![CDATA[0.465±0.003 b ]]> fade Unprocessed <![CDATA[0.652±0.003 a ]]> <![CDATA[0.450±0.019 b ]]> fade

[0215] Note: Absorbance is indicated as mean ± standard deviation; different letters in the same column of absorbance indicate significant differences, while the same letter indicates no significant differences.

[0216] This comparative example is the comparative example of Example 1. The conditions of the oxidation treatment were modified in this comparative example, and the concentration of ferric chloride in the oxidation treatment was reduced. All other parameters were exactly the same. Only the concentration of ferric chloride oxidant was changed, and the absorbance changed significantly, indicating that the oxidant needs to be controlled within a certain concentration range.

[0217] Comparative Example 2:

[0218] ① Select batch 1 of the above-mentioned vinegar, add ferric chloride solution to a concentration of 0.05 mol / L, and react on a shaker at a frequency of 50 r / min for 10 min to oxidize the reducing substances in the vinegar.

[0219] ② Select batch 1 of the above-mentioned oxidized vinegar and divide it into three equal parts. Take a thermoresistant amylase with an effective concentration of 80000 U / mL as detected by GB / T 24401 α-amylase preparation. Add thermoresistant amylase to two samples to 1 U / mL as the test group and the control group. The other sample is untreated as the blank group.

[0220] ② Add aluminum chloride to the experimental group to a concentration of 0.001 mol / L, while the other two groups were left untreated. Both groups were then reacted on a shaker at a frequency of 50 r / min for 10 min.

[0221] ③ Select a specific soluble starch and prepare substrate solutions of 4 g / mL using phosphate buffer solutions of pH 5.5 and pH 7.0 respectively. Mix the substrate with the solution in a 70℃ water bath shaker for 0.5 h according to Table 3.

[0222] ④ Take 1 mL of the reaction solution from ③ and react it with 5 mL of 0.012 mol / L dilute iodine solution to develop a colorimetric reaction. Immediately detect the absorbance at 660 nm.

[0223] ⑤ The effectiveness of passivation treatment can be determined by observing whether the absorbance changes after starch reacts with iodine.

[0224] Table 11. Verification of passivation effectiveness

[0225] experimental group 1U / mL 0.001 mol / L aluminum chloride treatment 5.5 <![CDATA[0.482±0.005 a ]]> control group 1U / mL Unprocessed 7.0 <![CDATA[0.218±0.006 b ]]> Blank group 0U / mL Unprocessed 7.0 <![CDATA[0.577±0.009 c ]]>

[0226] Note: Absorbance is indicated as mean ± standard deviation; different letters in the same column of absorbance indicate significant differences, while the same letter indicates no significant differences.

[0227] This comparative example is the comparative example of Example 2. The passivation treatment conditions were modified in this comparative example, the reagent concentration was reduced and the pH of the substrate was increased. The other parameters were basically the same. Only the passivation conditions were changed. The absorbance of the experimental group changed significantly, indicating that the passivation strength was insufficient and the activity of the thermostable amylase was not completely inhibited.

[0228] Comparative Example 3:

[0229] ① Select batch 1 of the above-mentioned soy sauce crude oil, add ferric chloride solution to a concentration of 0.05 mol / L, and react on a shaker at a frequency of 50 r / min for 10 min to oxidize the reducing substances such as formic acid, oxalic acid and ascorbic acid in the soy sauce crude oil.

[0230] ② Take the blank sample treated in ① and add heat-resistant amylase of known concentration according to national standards to six standard samples with a concentration of 0-10 U / mL.

[0231] ③ Select a specific soluble starch, prepare a substrate solution of 4 g / mL using pH 6.5 phosphate buffer, add 0.05 mol / L calcium chloride for protection, and mix 10 mL of the standard sample from ② with 40 mL of the substrate solution. React in a 50℃ water bath shaker for 10 min.

[0232] ④ Take 1 mL of the reaction solution from ③ and react it with 5 mL of 0.012 mol / L dilute iodine solution to develop a colorimetric reaction. Immediately detect the absorbance at 660 nm.

[0233] ⑤ Measure the absorbance of different standard samples, using the enzyme content of 0 U / mL as the blank sample. The absorbance change rate = (absorbance of blank sample - absorbance of crude oil with different enzyme contents) / absorbance of blank sample * 100%. Establish a standard range based on the relationship between the corresponding enzyme content and the corresponding absorbance change rate.

[0234] The results are shown in the table below:

[0235] Table 12. Standard Range of Enzyme Content-Absorbance Change Rate in Crude Soy Sauce

[0236] a 0U / mL <![CDATA[0.635±0.007 a ]]> 0.00％ b 0.01 U / mL <![CDATA[0.627±0.002 a ]]> 1.19％ c 0.1 U / mL <![CDATA[0.595±0.003 b ]]> 6.29％ d 1U / mL <![CDATA[0.468±0.020 c ]]> 26.38％ e 5U / mL <![CDATA[0.254±0.004 d ]]> 60.05％ f 10U / mL <![CDATA[0.159±0.004 e ]]> 74.96％

[0237] This comparative example is the comparative example of Example 1. The reaction temperature and reaction time after oxidation and protection treatment were revised, the reaction temperature was reduced to 50°C and the reaction time was shortened to 10 min. After these two changes, the absorbance of the 0.01 U / mL standard sample was not significantly different from that of the blank control group, indicating that the reaction conditions have a significant effect on the change in absorbance and the detection limit is closely related to the reaction conditions.

[0238] The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0239] The above-described embodiments are merely illustrative of several implementation methods of the present invention, facilitating a detailed and specific understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Furthermore, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various alterations or modifications to the present invention, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims of the present invention. Therefore, the scope of protection of this invention patent should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for detecting thermostable amylase in fermented food raw materials, characterized in that, The detection method consists of the following steps: The test sample and oxidant are mixed and subjected to an oxidation reaction to prepare an oxidized test sample; the concentration of the oxidant is 0.01 mol / L-0.05 mol / L. One sample of the oxidized test sample is mixed with an enzyme passivating agent to carry out a passivation reaction, and a control group test sample is prepared; the concentration of the passivating agent is 0.01 mol / L-0.05 mol / L. Another sample of the oxidized test sample was mixed with an enzyme protectant and subjected to protective treatment to prepare the test sample group; The control group test sample and the experimental group test sample were respectively mixed with starch substrate solution and subjected to enzymatic hydrolysis to prepare control group reaction solution and experimental group reaction solution; the conditions for enzymatic hydrolysis of the control group test sample included: pH value of 3.5-4.5 and temperature of 60℃-80℃; the conditions for enzymatic hydrolysis of the experimental group test sample included: pH value of 6.5-7.5 and temperature of 60℃-80℃. The control group reaction solution and the experimental group reaction solution were respectively mixed with iodine solution to carry out a colorimetric reaction, and the control group colorimetric solution and the experimental group colorimetric solution were prepared. The absorbance A1 of the control group colorimetric solution and the absorbance A2 of the experimental group colorimetric solution were measured respectively, and the absorbance change rate Y of the absorbance A2 relative to the absorbance A1 was calculated. Establish a standard range that reflects the relationship between the content of thermostable amylase and the rate of change of absorbance, and determine the range of thermostable amylase content in which the rate of change of absorbance Y falls by referring to the standard range; The raw materials for the fermented food are crude soy sauce or vinegar.

2. The method for detecting thermostable amylase in fermented food raw materials according to claim 1, characterized in that, To establish a standard range reflecting the relationship between the content of thermostable amylase and the rate of change in absorbance, the following steps are included: A standard sample with a preset thermo-resistant amylase content is prepared, wherein the preset thermo-resistant amylase content of multiple standard samples increases in a gradient from zero. The multiple standard samples were respectively mixed with starch substrate solution and subjected to enzymatic hydrolysis to prepare multiple standard sample reaction solutions; The multiple standard sample reaction solutions are respectively mixed with iodine solution to carry out colorimetric reactions and prepare multiple standard sample colorimetric solutions; The absorbance of the multiple standard sample colorimetric solutions is detected. Taking the absorbance of the standard sample with a preset high-temperature amylase content of zero as a reference, the rate of change of absorbance of other standard samples with preset high-temperature amylase content relative to the reference absorbance is calculated to create a standard range that reflects the relationship between the high-temperature amylase content and the rate of change of absorbance.

3. The method for detecting thermostable amylase in fermented food raw materials according to claim 2, characterized in that, In the steps of preparing the standard sample reaction solution, the conditions for the enzymatic hydrolysis reaction include: a temperature of 60℃-80℃ and a time of 0.5h-12h.

4. The method for detecting thermostable amylase in fermented food raw materials according to claim 2, characterized in that, The preparation of a standard sample with a predetermined content of heat-resistant amylase includes the following steps: Provide basic standard raw materials, which are fermented food raw materials that do not contain heat-resistant amylase or whose amount of heat-resistant amylase does not affect shelf life; The basic standard raw materials and oxidant are mixed and subjected to an oxidation reaction to prepare a blank sample; The blank sample and a preset amount of thermoresistant amylase are mixed to prepare a standard sample with a preset thermoresistant amylase content.

5. The method for detecting thermostable amylase in fermented food raw materials according to claim 4, characterized in that, In the step of preparing the blank sample, the oxidation reaction conditions include: the concentration of the oxidant is 0.01 mol / L-0.05 mol / L.

6. The method for detecting thermostable amylase in fermented food raw materials according to claim 4, characterized in that, The screening steps for the basic standard raw materials include: Fermented food ingredients and oyster sauce were mixed, and the initial viscosity and viscosity of the mixture after the storage period were tested. Fermented food ingredients corresponding to the mixture whose viscosity after the storage period did not change by more than 5% relative to the initial viscosity were selected as the basic standard ingredients.

7. The method for detecting thermostable amylase in fermented food raw materials according to claim 6, characterized in that, The storage temperature is 50℃-70℃, and the shelf life is 25-35 days.

8. The method for detecting thermostable amylase in fermented food raw materials according to claim 6, characterized in that, The volume ratio of the fermented food ingredients to oyster sauce is 1:(45-55).

9. The method for detecting thermostable amylase in fermented food raw materials according to claim 2, characterized in that, The preset high-temperature resistant amylase content includes 0 U / mL-10 U / mL.

10. The method for detecting thermostable amylase in fermented food raw materials according to claim 9, characterized in that, The preset thermostable amylase content includes 0 U / mL, 0.01 U / mL, 0.1 U / mL, 1 U / mL, 5 U / mL and 10 U / mL.

11. The method for detecting thermostable amylase in fermented food raw materials according to any one of claims 1 to 10, characterized in that, The passivation reaction also includes one or more of the conditions shown in (A) and (B): (A) The passivating agent is selected from zinc sulfate, aluminum chloride, or a combination thereof; as well as, (B) Rotation speed is 45r / min-55r / min, and time is 5min-15min.

12. The method for detecting thermostable amylase in fermented food raw materials according to any one of claims 1 to 10, characterized in that, The conditions for protection processing include one or more of the conditions shown in (I) to (III): (I) The protective agent is calcium chloride; (II) The concentration of the protective agent is 0.01 mol / L to 0.05 mol / L; and, (III) Rotation speed is 45 r / min - 55 r / min, and time is 5 min - 15 min.

13. The method for detecting thermostable amylase in fermented food raw materials according to any one of claims 1 to 10, characterized in that, The conditions for the oxidation reaction also include one or more of the conditions shown in (1) and (2): (1) The oxidant is selected from ferric chloride, nitric acid, or a combination thereof; and, (2) The rotation speed is 45r / min - 55r / min, and the time is 5min - 15min.

14. The method for detecting thermostable amylase in fermented food raw materials according to any one of claims 1 to 10, characterized in that, The detection method satisfies one or more of the conditions shown in A) to C): A) The conditions for the enzymatic hydrolysis reaction of the test samples in the experimental group include: time of 0.5h-12h; B) The conditions for the enzymatic hydrolysis reaction of the control group test sample also include: a time of 0.5 h–12 h; and, C) The conditions for the enzymatic hydrolysis reaction of the multiple standard samples include: pH value of 6.5-7.5, temperature of 60℃-80℃, and time of 0.5h-12h.

15. The method for detecting thermostable amylase in fermented food raw materials according to any one of claims 1 to 10, characterized in that, The detection method satisfies one or more of the conditions shown in i) to ii): i) The concentration of soluble starch in the starch substrate solution is 2 g / mL-6 g / mL; the volume ratio of the control group sample to the starch substrate solution is 1:(3-5); the volume ratio of the test group sample to the starch substrate solution is 1:(3-5); and the volume ratio of the standard sample to the starch substrate solution is 1:(3-5); and, ii) The concentration of potassium iodide in the iodine solution is 0.01 mol / L - 0.02 mol / L, the volume ratio of the control group reaction solution to the iodine solution is 1:(3-7), the volume ratio of the test group reaction solution to the iodine solution is 1:(3-7), and the volume ratio of the standard sample reaction solution to the iodine solution is 1:(3-7).

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