Culture medium for detecting beer putrefying bacteria as well as preparation method and application of culture medium

Abstract

The invention provides a culture medium for detecting beer putrefying bacteria and a preparation method and application of the culture medium, and belongs to the field of beer detection, and every 1 L of the culture medium comprises the following components: 8-12 g of peptone, 8-12 g of beef extract, 2-6 g of yeast powder, 1-3 g of K2HPO4, 1-3 g of diammonium citrate, 7-10 g of sodium acetate trihydrate, 0.5-1.5 mL of Tween 80, 0.3-0.5 g of MgSO4. 7H2O, 0.3-0.5 g of MnSO4, and a proper amount of glucose, fructose, malic acid, pyruvic acid, catalase and methyl red-bromocresol green. The malic acid and the pyruvic acid are added into the culture medium, so that the effect of regulating and controlling putrefying bacteria horA and horC genes is achieved, the humulus lupulus compound resistance of the putrefying bacteria is improved, the growth capacity of the putrefying bacteria is further improved, the problem that harmful bacteria are difficult to grow due to missing detection in an existing culture medium method is solved, and the detection accuracy is improved.

Description

Technical Field

[0001] The invention relates to the technical field of beer detection, and in particular to a culture medium for detecting beer spoilage bacteria and a preparation method and application thereof. Background Art

[0002] The beer industry has a long production process and complex production links, and is prone to the entry of harmful microorganisms other than yeast. The presence of harmful bacteria in beer causes great damage to the beer industry, especially beer spoilage bacteria that are harmful to beer quality and do not require aerobic conditions to reproduce; although the hop compounds contained in beer can act as proton ion carriers and disperse transmembrane pH gradients to exert antibacterial effects and prevent Gram-positive bacteria (including most lactic acid bacteria) from growing in beer, the horA and horC carried by spoilage bacteria also give them hop resistance, which poses new challenges to the control of beer microorganisms. Only by quickly and accurately detecting spoilage bacteria in beer can the problem of and reduction of beer spoilage bacteria be solved, thereby improving the quality of beer.

[0003] In the prior art, lactic acid bacteria are considered to be the main destroyers among the anaerobic microorganisms that cause beer spoilage, and the commonly used detection methods for lactic acid bacteria are molecular biology methods and culture medium methods. Among them, the detection of molecular biology methods uses the 16SrRNA gene sequence analysis method to detect and identify it at the molecular biology level, but this method has high requirements for technicians and is difficult to operate. The culture medium method generally uses NBB-A solid culture medium to anaerobically culture the beer filter sample, and observes the color change of the NBB-A solid culture medium to determine whether it is lactic acid bacteria positive. However, the special environment of beer, especially the presence of hop compounds, may make it difficult for some lactic acid bacteria to grow to the detection limit in a short time. Therefore, the culture medium method may miss some lactic acid bacteria that are difficult to grow; these characteristics result in the lack of a method for qualitative detection of lactic acid bacteria with high accuracy, low technical requirements, and easy promotion. In addition, in addition to lactic acid bacteria, beer also contains a variety of other complex beer spoilage bacteria. Their growth will produce excessive acetic acid, butyric acid or lactic acid and other substances that seriously affect the flavor of beer, causing the beer to be contaminated and deteriorate. However, there are few detection methods for these beer spoilage bacteria in the existing technology, and the detection level is low. Summary of the invention

[0004] In view of the technical problems existing in the background technology, the present application provides a culture medium for detecting beer spoilage bacteria and its preparation method and application, aiming to solve the technical problems that the beer spoilage bacteria detection method in the prior art is difficult to detect a variety of complex spoilage bacteria and has poor detection accuracy.

[0005] In the first aspect, an embodiment of the present application provides a culture medium for detecting beer spoilage bacteria, wherein each 1L of the culture medium includes the following components: 8-12g of peptone, 8-12g of beef extract, 2-6g of yeast powder, 1-3g of K2HPO4, 1-3g of diammonium citrate, 7-10g of sodium acetate trihydrate, 0.5-1.5mL of Tween 80, 0.3-0.5g of MgSO4·7H2O, 0.3-0.5g of MnSO4, and appropriate amounts of glucose, fructose, malic acid, pyruvic acid, catalase, and methyl red-bromocresol green.

[0006] In the technical solution of the embodiment of the present application, by adding malic acid and pyruvic acid to the culture medium, the horA and horC genes of the spoilage bacteria are regulated, thereby increasing their resistance to hop compounds, thereby improving the growth ability of the spoilage bacteria in the same environment, avoiding the problem of missing some harmful bacteria that are difficult to grow in the existing culture medium method, and increasing the accuracy of detection.

[0007] In some embodiments, the content of malic acid and pyruvic acid in the culture medium is 0.1 to 0.3 g, preferably 0.2 g. The base liquid of the culture medium is a mixture of deionized water and beer, and the volume ratio of deionized water to beer is 1: (0.8 to 1.2). The concentration of catalase in the culture medium is 50 to 80 U / mL. The content of glucose and fructose is 8 to 12 g, respectively. The methyl red-bromocresol green is prepared by mixing 0.1 to 0.3 g / L of methyl red and 0.2 to 0.4 g / L of bromocresol green.

[0008] In this embodiment, by adding glucose and fructose to the culture medium, the types of carbon sources consumed by the growth and metabolism of spoilage bacteria are increased, providing growth energy for a variety of spoilage bacteria. At the same time, the addition of catalase also activates the growth characteristics of beer spoilage bacteria, so that some spoilage bacteria that are in a metabolizable but non-growing state in the harsh environment of beer can be metabolized and grown; the addition of sodium acetate trihydrate can inhibit the growth of Escherichia coli and reduce the possibility of false positive test results.

[0009] In a second aspect, an embodiment of the present application provides a method for preparing a culture medium for detecting beer spoilage bacteria, wherein peptone, beef extract, yeast powder, K2HPO4, diammonium citrate, sodium acetate trihydrate, Tween 80, MgSO4·7H2O, MnSO4, glucose, fructose, malic acid, pyruvic acid, methyl red-bromocresol green, deionized water and beer are mixed in different contents, sterilized, and catalase is added to obtain a culture medium for detecting beer spoilage bacteria.

[0010] In the technical solution of the embodiment of the present application, by using sterilized beer to replace part of the deionized water in the culture medium, an environment close to the original growth is provided for the beer spoilage bacteria; the culture medium increases the growth rate of a variety of beer spoilage bacteria from the source, expands the detection range, and enables the detection method to observe changes in colonies on the surface of the non-woven fabric within 48 hours, thereby shortening the detection time of beer spoilage bacteria.

[0011] In the third aspect, an embodiment of the present application provides an application of a culture medium for detecting beer spoilage bacteria. After filtering the beer to be tested with a bacteria filter membrane, the bacteria filter membrane is tightly attached to the upper surface of a hydrophilic non-woven fabric containing the culture medium, and anaerobic culture is carried out for not less than 48 hours. During the culture process, the color change of the non-woven fabric is observed to determine whether the beer to be tested contains spoilage bacteria; the hydrophilic contact angle of the hydrophilic non-woven fabric is 0 to 40°, and the water absorption rate is 1500% to 1700%.

[0012] In the technical solution of the embodiment of the present application, the above-mentioned culture medium for detecting beer spoilage bacteria is used, thereby increasing the growth rate of beer spoilage bacteria, thereby increasing the detection efficiency, and providing a new method for detecting spoilage bacteria in beer. In addition, when the culture medium is used to detect beer spoilage bacteria, a hydrophilic non-woven fabric with excellent adsorption is used. After saturated infiltration of the culture medium, the culture medium is evenly filled in the non-woven fabric, providing a favorable growth environment for beer spoilage bacteria, and the structure of the hydrophilic non-woven fabric increases the speed of supplying nutrients to the colony; the hydrophilic fiber structure of the non-woven fabric plays a role in locking the acid produced by the spoilage bacteria during the growth process, thereby increasing the sensitivity of the color change indicator, reducing the amount of culture medium used, and saving detection costs. The bacterial filter membrane is tightly attached to the surface of the hydrophilic non-woven fabric, which enriches the beer spoilage bacteria and is also conducive to the rapid growth of the colony.

[0013] In some embodiments, if the color of the hydrophilic non-woven fabric changes from green to yellow during the culture process, the beer to be tested contains beer spoilage bacteria. If the color remains green, the beer to be tested does not contain beer spoilage bacteria. The fiber diameter of the hydrophilic non-woven fabric is 3 to 7 cm; the filter membrane is a nanofiber membrane with a pore size of 200 to 250 nm, and the fiber diameter of the filter membrane is 3 to 7 cm; the hydrophilic non-woven fabric includes one of cotton non-woven fabric, polyurethane non-woven fabric, and polyester-cotton blended non-woven fabric.

[0014] In this embodiment, the indicator is directly added to the culture medium. As the spoilage bacteria grow and metabolize, the filter membrane will slowly change color. The entire color change process can be observed. Whether the spoilage bacteria grow faster or slower, they can all be observed without missing any detection.

[0015] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solution of the present application, the following is a brief introduction to the drawings used in the present application. Obviously, the drawings described below are only some embodiments of the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 This is a schematic diagram of the preparation and application process of the culture medium for detecting beer spoilage bacteria in the embodiments of the present application;

[0018] Figure 2 These are color development pictures of the hydrophilic nonwoven fabrics after anaerobic culture in Examples 1 to 2 of the present application and Comparative Example 1. DETAILED DESCRIPTION

[0019] The following embodiments of the technical solution of the present application are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" in the specification and claims of this application and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0021] In the description of the embodiments of the present application, the technical terms "first", "second", etc. are only used to distinguish different objects, and cannot be understood as indicating or implying relative importance or implicitly indicating the number, specific order or primary and secondary relationship of the indicated technical features. In the description of the embodiments of the present application, the meaning of "multiple" is more than two, unless otherwise clearly and specifically defined.

[0022] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0023] In the description of the embodiments of the present application, the term "and / or" is only a description of the association relationship of the associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.

[0024] In the description of the embodiments of the present application, the term "multiple" refers to more than two (including two). Similarly, "multiple groups" refers to more than two groups (including two groups), and "multiple pieces" refers to more than two pieces (including two pieces).

[0025] In the prior art, the special environment of beer, especially the presence of hop compounds, may make it difficult for some lactic acid bacteria to grow to the detection limit in a short period of time. Therefore, the culture medium method may miss some lactic acid bacteria that are difficult to grow; these characteristics lead to the lack of a method for qualitative detection of lactic acid bacteria with high accuracy, low technical requirements, and easy promotion. In addition, in addition to lactic acid bacteria, beer also contains a variety of other complex beer spoilage bacteria, whose growth will produce excessive acetic acid, butyric acid or lactic acid and other substances that seriously affect the flavor of beer, causing beer to be contaminated and deteriorated. However, there are few detection methods for these beer spoilage bacteria in the prior art, and the detection level is low.

[0026] In order to solve the technical problem that the beer spoilage bacteria detection method in the prior art is difficult to detect a variety of complex spoilage bacteria and the detection accuracy is poor, the present application provides a culture medium for detecting beer spoilage bacteria and its preparation method and application, wherein the culture medium for detecting beer spoilage bacteria, by adding malic acid and pyruvic acid to the culture medium, plays a role in regulating the horA and horC genes of the spoilage bacteria, increases its hop compound resistance, and thus improves the growth ability of the spoilage bacteria in the same environment, avoids the problem of missing some harmful bacteria with difficulty in growth in the existing culture medium method, and increases the accuracy of the detection. By adding glucose and fructose to the culture medium, the types of carbon sources consumed by the growth metabolism of the spoilage bacteria are increased, providing growth energy for a variety of spoilage bacteria. At the same time, the addition of catalase also activates the growth characteristics of beer spoilage bacteria, so that some spoilage bacteria that are in a metabolizable and non-growing state in the harsh environment of beer can be metabolized and grown; the addition of sodium acetate trihydrate can inhibit the growth of Escherichia coli and reduce the possibility of false positive test results. Furthermore, when the culture medium for detecting beer spoilage bacteria is used to detect beer spoilage bacteria, the growth rate of beer spoilage bacteria is increased, and the growth of some spoilage bacteria that are difficult to grow due to the special environment of beer is promoted, thereby improving the accuracy and efficiency of beer spoilage bacteria detection; and the application method has low technical requirements, simple operation and low cost, and has broad promotion and application prospects in the detection of beer spoilage bacteria in breweries.

[0027] For the convenience of explanation, the following embodiments are described by taking a culture medium for detecting beer spoilage bacteria in an embodiment of the present application as an example.

[0028] In a first aspect, an embodiment of the present application provides a culture medium for detecting beer spoilage bacteria, wherein each 1L of the culture medium includes the following components: 8-12g of peptone, 8-12g of beef extract, 2-6g of yeast powder, 1-3g of K2HPO4, 1-3g of diammonium citrate, 7-10g of sodium acetate trihydrate, 0.5-1.5mL of Tween 80, 0.3-0.5g of MgSO4·7H2O, 0.3-0.5g of MnSO4, and appropriate amounts of glucose, fructose, malic acid, pyruvic acid, catalase, and methyl red-bromocresol green.

[0029] In the technical solution of the embodiment of the present application, by adding malic acid and pyruvic acid to the culture medium, the horA and horC genes of the spoilage bacteria are regulated, thereby increasing their resistance to hop compounds, thereby improving the growth ability of the spoilage bacteria in the same environment, avoiding the problem of missing some harmful bacteria that are difficult to grow in the existing culture medium method, and increasing the accuracy of detection.

[0030] Hop compounds in beer give beer bitterness and play an antibacterial role by acting as proton ion carriers and dispersing transmembrane pH gradients, which can prevent Gram-positive bacteria (including most lactic acid bacteria) from growing in beer. The genes horA and horC that lactic acid bacteria carry can give them the ability to resist hop compounds. The HorA gene is activated by ATP to excrete toxic hop compounds, and HorC transports drugs by relying on proton motive force (PMF), giving bacteria resistance to various drugs (including hop compounds). Bacteria produce PMF in the process of decomposing malic acid, and then synthesize ATP. Bacteria can directly produce ATP when metabolizing pyruvate; therefore, the addition of malic acid and pyruvate can help regulate HorA and HorC genes, activate these two groups, increase the resistance of spoilage bacteria to hop compounds, improve the growth ability of spoilage bacteria, and increase detection accuracy.

[0031] Further, in some embodiments, the content of malic acid and pyruvic acid in the culture medium is 0.1 to 0.3 g, preferably 0.2 g. The base liquid of the culture medium is a mixture of deionized water and beer, and the volume ratio of deionized water to beer is 1: (0.8 to 1.2). The concentration of catalase in the culture medium is 50 to 80 U / mL. The content of glucose and fructose is 8 to 12 g, respectively. Methyl red-bromocresol green is prepared by mixing 0.1 to 0.3 g / L of methyl red and 0.2 to 0.4 g / L of bromocresol green.

[0032] In the technical solution of the embodiment of the present application, by adding glucose and fructose to the culture medium, the types of carbon sources consumed by the growth and metabolism of spoilage bacteria are increased, providing growth energy for a variety of spoilage bacteria. At the same time, the addition of catalase also activates the growth characteristics of beer spoilage bacteria, so that some spoilage bacteria that are in a metabolizable but non-growing state in the harsh environment of beer can be metabolized and grown; the addition of sodium acetate trihydrate can inhibit the growth of Escherichia coli and reduce the possibility of false positive test results.

[0033] In a second aspect, an embodiment of the present application provides a method for preparing a culture medium for detecting beer spoilage bacteria, wherein peptone, beef extract, yeast powder, K2HPO4, diammonium citrate, sodium acetate trihydrate, Tween 80, MgSO4·7H2O, MnSO4, glucose, fructose, malic acid, pyruvic acid, methyl red-bromocresol green, deionized water and beer are mixed in different contents, sterilized, and catalase is added to obtain a culture medium for detecting beer spoilage bacteria.

[0034] In the technical solution of the embodiment of the present application, by using sterilized beer to replace part of the deionized water in the culture medium, an environment close to the original growth is provided for the beer spoilage bacteria; the culture medium increases the growth rate of a variety of beer spoilage bacteria from the source, expands the detection range, and enables the detection method to observe changes in colonies on the surface of the non-woven fabric within 48 hours, thereby shortening the detection time of beer spoilage bacteria.

[0035] In the third aspect, the embodiment of the present application provides an application of a culture medium for detecting beer spoilage bacteria. After filtering the beer to be detected with a bacterial filter membrane, the bacterial filter membrane is closely attached to the upper surface of a hydrophilic non-woven fabric containing the culture medium, and anaerobic culture is performed for not less than 48 hours. During the culture process, the color change of the non-woven fabric is observed to determine whether the beer to be detected contains spoilage bacteria; the hydrophilic contact angle of the hydrophilic non-woven fabric is 0-40°, and the water absorption rate is 1500%-1700%. In practical applications, when beer is detected using a culture medium for detecting beer spoilage bacteria, a small range of yellow can be observed in the non-woven fabric after 36 hours of anaerobic culture, but because its original green and yellow are close and difficult to distinguish, in order to improve the detection accuracy, the present application sets the culture time to not less than 48 hours, so that the human eye can more easily observe the color change.

[0036] In the technical solution of the embodiment of the present application, the above-mentioned culture medium for detecting beer spoilage bacteria is used, thereby increasing the growth rate of beer spoilage bacteria, thereby increasing the detection efficiency, and providing a new method for detecting spoilage bacteria in beer. In addition, when the culture medium is used to detect beer spoilage bacteria, a hydrophilic non-woven fabric with excellent adsorption is used. After saturated infiltration of the culture medium, the culture medium is evenly filled in the non-woven fabric, providing a favorable growth environment for beer spoilage bacteria, and the structure of the hydrophilic non-woven fabric increases the speed of supplying nutrients to the colony; the hydrophilic fiber structure of the non-woven fabric plays a role in locking the acid produced by the spoilage bacteria during the growth process, thereby increasing the sensitivity of the color change indicator, reducing the amount of culture medium used, and saving detection costs. The bacterial filter membrane is tightly attached to the surface of the hydrophilic non-woven fabric, which enriches the beer spoilage bacteria and is also conducive to the rapid growth of the colony.

[0037] In some embodiments, if the color of the hydrophilic non-woven fabric changes from green to yellow during the culture process, the beer to be tested contains beer spoilage bacteria. If the color remains green, the beer to be tested does not contain beer spoilage bacteria. The fiber diameter of the hydrophilic non-woven fabric is 3 to 7 cm; the filter membrane is a nanofiber membrane with a pore size of 200 to 250 nm, and the fiber diameter of the filter membrane is 3 to 7 cm; the hydrophilic non-woven fabric includes one of cotton non-woven fabric, polyurethane non-woven fabric, and polyester-cotton blended non-woven fabric.

[0038] In this embodiment, the indicator is directly added to the culture medium. As the spoilage bacteria grow and metabolize, the filter membrane will slowly change color. The entire color change process can be observed. Whether the spoilage bacteria grow faster or slower, they can all be observed without missing any detection.

[0039] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. If specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used without specifying the manufacturer are all conventional products that can be obtained commercially.

[0040] Example 1

[0041] See also Figure 1 As shown, this embodiment provides a culture medium for detecting beer spoilage bacteria and a preparation method and application thereof, comprising the following steps:

[0042] S1. Preparation of culture medium: 10 g of peptone, 10 g of beef extract, 4 g of yeast powder, 2 g of K2HPO4, 2 g of diammonium citrate, 8.29 g of sodium acetate trihydrate, 10 g of glucose, 10 g of fructose, 0.2 g of malic acid, 0.2 g of pyruvic acid, 1 mL of Tween 80, 0.41 g of MgSO4·7H2O, 0.4 g of MnSO4, deionized water, methyl red-bromocresol green (0.2 g of methyl red, 0.3 g of bromocresol green) and 1000 mL of a mixture of beer (the volume ratio of deionized water to beer is 1:1) are mixed, sterilized, and then an appropriate amount of catalase is added to prepare a culture medium; wherein the concentration of catalase in the culture medium is 70 U / mL, and the pH value is 5.76;

[0043] S2. After filtering 250 mL of the beer to be tested with a filter membrane with an average pore size of 220 nm, 1.5 mL of the culture medium prepared in step S1 is added dropwise to a hydrophilic non-woven fabric (fiber diameter is 5.5 cm), and the filter membrane is tightly attached to the upper surface of the hydrophilic non-woven fabric. Anaerobic culture is performed for 72 hours, and the color change is observed to determine whether the beer to be tested contains beer spoilage bacteria; if the color changes from green to yellow, the beer to be tested contains beer spoilage bacteria, and if the color is green, the beer to be tested does not contain beer spoilage bacteria; the hydrophilic non-woven fabric is made of pure cotton non-woven fabric with a contact angle of 0° and a water absorption rate of 1677%.

[0044] Example 2

[0045] This embodiment provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that in step S2, the anaerobic culture time is 48 hours, and the rest is roughly the same as Example 1, which will not be repeated here.

[0046] Comparative Example 1

[0047] Comparative Example 1 provides a culture medium for detecting beer spoilage bacteria and a preparation method and application thereof. Compared with Example 1, the difference is that in step S2, the anaerobic culture time is 24 hours, and the rest is roughly the same as Example 1, which will not be repeated here.

[0048] See also Figure 2As shown, the hydrophilic non-woven fabrics after anaerobic culture of Examples 1 to 2 and Comparative Example 1 are color pictures, wherein (a) is Comparative Example 1, (b) is Example 2, and (c) is Example 1. It can be seen from the figure that after 24 hours of culture, the color of the non-woven fabric of Comparative Example 1 is still close to green, and the detection of anaerobic acid-producing spoilage bacteria cannot be achieved; at 48 hours, the color of the non-woven fabric of Example 2 turns light yellow, indicating that there are beer spoilage bacteria on the surface of the non-woven fabric; and at 72 hours of culture, Example 1 changes from a small range of light yellow to a more conspicuous large range of yellow, indicating that the beer spoilage bacteria grow and reproduce, and the acid produced causes the pH of the non-woven fabric to decrease, achieving a more obvious detection of anaerobic acid-producing beer spoilage bacteria.

[0049] Comparative Example 2

[0050] Comparative Example 2 provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that in step S2, a traditional MRS culture medium (without adding catalase and beer) is used instead of the culture medium of Example 1, and the rest is roughly the same as Example 1 and will not be repeated here.

[0051] Comparative Example 3

[0052] Comparative Example 3 provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that in step S1, glucose is not added to the culture medium, and the rest is roughly the same as Example 1, which will not be repeated here.

[0053] Comparative Example 4

[0054] Comparative Example 4 provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that in step S1, fructose is not added to the culture medium, and the rest is roughly the same as Example 1, which will not be repeated here.

[0055] Comparative Example 5

[0056] Comparative Example 5 provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that in step S1, malic acid is not added to the culture medium, and the rest is roughly the same as Example 1, which will not be repeated here.

[0057] Comparative Example 6

[0058] Comparative Example 6 provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that in step S1, pyruvic acid is not added to the culture medium, and the rest is roughly the same as Example 1, which will not be repeated here.

[0059] Comparative Example 7

[0060] Comparative Example 7 provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that in step S1, no catalase is added to the culture medium, and the rest is roughly the same as Example 1, which will not be repeated here.

[0061] Comparative Example 8

[0062] Comparative Example 8 provides a culture medium for detecting beer spoilage bacteria, and its preparation method and application. Compared with Example 1, the difference is that in step S1, beer is not used in the culture medium to replace part of the deionized water. The rest is roughly the same as Example 1 and will not be repeated here.

[0063] Comparative Example 9

[0064] Comparative Example 9 provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that the content of glucose in the culture medium is 50 g, and the rest is roughly the same as Example 1, which will not be repeated here.

[0065] Comparative Example 10

[0066] Comparative Example 10 provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that the fructose content in the culture medium is 50 g, and the rest is roughly the same as Example 1, which will not be repeated here.

[0067] Example 3

[0068] This embodiment provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that the content of malic acid in the culture medium is 0.1 g, and the rest is substantially the same as Example 1, which will not be repeated here.

[0069] Example 4

[0070] This embodiment provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that the content of malic acid in the culture medium is 0.3 g, and the rest is substantially the same as Example 1, which will not be repeated here.

[0071] Example 5

[0072] This embodiment provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that the content of pyruvic acid in the culture medium is 0.1 g, and the rest is roughly the same as Example 1, which will not be repeated here.

[0073] Example 6

[0074] This embodiment provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that the content of pyruvic acid in the culture medium is 0.3 g, and the rest is roughly the same as Example 1, which will not be repeated here.

[0075] Comparative Example 11

[0076] Comparative Example 11 provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that the content of malic acid in the culture medium is 5 g, and the rest is roughly the same as Example 1, which will not be repeated here.

[0077] Comparative Example 12

[0078] Comparative Example 12 provides a culture medium for detecting beer spoilage bacteria, and its preparation method and application. Compared with Example 1, the difference is that the content of pyruvic acid in the culture medium is 5 g, and the rest is roughly the same as Example 1, which will not be repeated here.

[0079] Comparative Example 13

[0080] Comparative Example 13 provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that the concentration of catalase in the culture medium is 30 U / mL, and the rest is roughly the same as Example 1, which will not be repeated here.

[0081] Comparative Example 14

[0082] Comparative Example 14 provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that the concentration of catalase in the culture medium is 90 U / mL, and the rest is roughly the same as Example 1, which will not be repeated here.

[0083] Comparative Example 15

[0084] Comparative Example 15 provides a culture medium for detecting beer spoilage bacteria, and its preparation method and application. Compared with Example 1, the difference is that the culture medium contains only 1000 mL of beer and no deionized water. The rest is roughly the same as Example 1 and will not be repeated here.

[0085] Comparative Example 16

[0086] Comparative Example 16 provides a culture medium for detecting beer spoilage bacteria, and its preparation method and application. Compared with Example 1, the difference is that the volume ratio of deionized water to beer in the mixed liquid is 1:3, and the rest is roughly the same as Example 1, which will not be repeated here.

[0087] Comparative Example 17

[0088] Comparative Example 17 provides a culture medium for detecting beer spoilage bacteria and its preparation method and application. Compared with Example 1, the difference is that the volume ratio of deionized water to beer in the mixed liquid is 3:1, and the rest is roughly the same as Example 1, which will not be repeated here.

[0089] The number of colonies on the surface of the non-woven fabrics after anaerobic culture of Examples 1 to 6 and Comparative Examples 1 to 17 was subjected to colorimetric detection, and the results were shown in Table 1 below; wherein, in Table 1, "large range of yellow" means that the yellow area occupies more than 60% of the total area of ​​the sample, "small range of yellow" means that the yellow area occupies 10% to 30% of the total area of ​​the sample, and "extremely small range of yellow" means that the yellow area occupies less than 10% of the total area of ​​the sample.

[0090] Table 1 Color characterization of samples of Examples 1 to 6 and Comparative Examples 1 to 17

[0091]

[0092]

[0093] As shown in Table 1, adding appropriate amounts of glucose, fructose, malic acid, pyruvic acid, catalase and beer to the culture medium can promote the growth and metabolism of beer spoilage bacteria. The improved culture medium in Example 1 only needs anaerobic culture for 48 hours to obtain accurate detection results; high concentrations of glucose, fructose, malic acid, pyruvic acid and catalase will inhibit the growth of spoilage bacteria, and a high proportion of alcohol will also inhibit the growth of spoilage bacteria. It takes a longer culture time to develop color than the solution in Example 1, and the detection efficiency is low.

[0094] It should be noted that the beer to be tested in the above embodiments and comparative examples are all from the same batch of beer, and in order to further verify the feasibility of this solution, under laboratory conditions, spoilage bacteria samples extracted from beer were directly used for repeatability verification, and experimental results with the same rules as those in the above embodiments and comparative examples were obtained.

[0095] Example 7

[0096] The present embodiment provides a culture medium for detecting beer spoilage bacteria, and a preparation method and application thereof. Compared with Example 1, the difference is that in step S1, 20 g / L of starch and 100 U / L of amylase are added to the culture medium, and the starch is added before sterilization and the amylase is added after sterilization. The rest is substantially the same as Example 1 and will not be repeated here.

[0097] By investigating the color development and culture time of the sample in Example 7, it was found that by adding amylase and starch, while the anaerobic harmful bacteria consumed glucose, glucose nutrition was continuously and durably provided to the harmful bacteria without changing the osmotic pressure inside and outside the culture solution, thereby further promoting the reproduction and growth rate of beer spoilage bacteria.

[0098] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and the same effect as the technical solution of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the main purpose of the present application, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A culture medium for detecting beer spoilage bacteria, characterized in that: Each 1L of culture medium includes the following components: 8-12g of peptone, 8-12g of beef extract, 2-6g of yeast powder, 1-3g of K2HPO4, 1-3g of diammonium citrate, 7-10g of sodium acetate trihydrate, 0.5-1.5mL of Tween 80, 0.3-0.5g of MgSO4·7H2O, 0.3-0.5g of MnSO4, and appropriate amounts of glucose, fructose, malic acid, pyruvic acid, catalase, and methyl red-bromocresol green.

2. The culture medium for detecting beer spoilage bacteria according to claim 1, characterized in that: The contents of malic acid and pyruvic acid are 0.1-0.3 g, preferably 0.2 g.

3. The culture medium for detecting beer spoilage bacteria according to claim 1, characterized in that: The base liquid of the culture medium is a mixture of deionized water and beer, and the volume ratio of the deionized water to the beer is 1:(0.8-1.2).

4. The culture medium for detecting beer spoilage bacteria according to claim 1, characterized in that: The concentration of catalase in the culture medium is 50-80 U / mL.

5. The culture medium for detecting beer spoilage bacteria according to claim 1, characterized in that: The contents of glucose and fructose in the culture medium are 8-12 g respectively.

6. The culture medium for detecting beer spoilage bacteria according to claim 1, characterized in that: The methyl red-bromocresol green is prepared by mixing 0.1-0.3 g / L of methyl red and 0.2-0.4 g / L of bromocresol green.

7. A method for preparing a culture medium for detecting beer spoilage bacteria according to any one of claims 1 to 6, characterized in that: Peptone, beef extract, yeast powder, K2HPO4, diammonium citrate, sodium acetate trihydrate, Tween 80, MgSO4·7H2O, MnSO4, glucose, fructose, malic acid, pyruvic acid, methyl red-bromocresol green, deionized water and beer were mixed in different amounts, sterilized, and then catalase was added to prepare a culture medium for detecting beer spoilage bacteria.

8. Use of a culture medium for detecting beer spoilage bacteria according to any one of claims 1 to 6, characterized in that: After filtering the beer to be tested with a bacteria filter membrane, the bacteria filter membrane is closely attached to the upper surface of a hydrophilic non-woven fabric containing a culture medium, and anaerobic culture is carried out for no less than 48 hours. During the culture process, the color change of the non-woven fabric is observed to determine whether the beer to be tested contains spoilage bacteria; the hydrophilic contact angle of the hydrophilic non-woven fabric is 0-40°, and the water absorption rate is 1500%-1700%.

9. The use of the culture medium for detecting beer spoilage bacteria according to claim 8, characterized in that: If the color of the hydrophilic nonwoven fabric changes from green to yellow during the culture process, the beer to be tested contains beer spoilage bacteria. If the color remains green, the beer to be tested does not contain beer spoilage bacteria.

10. The use of the culture medium for detecting beer spoilage bacteria according to claim 8, characterized in that: The fiber diameter of the hydrophilic non-woven fabric is 3 to 7 cm; the bacterial filter membrane is a nanofiber membrane with a pore size of 200 to 250 nm, and the fiber diameter of the bacterial filter membrane is 3 to 7 cm; the hydrophilic non-woven fabric includes one of cotton non-woven fabric, polyurethane non-woven fabric, and polyester-cotton blended non-woven fabric.

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