阅读说明：本技术 一种鱼肉腐败菌氨基酸脱氨能力的检测方法 (Detection method for deamination capacity of fish putrefying bacteria amino acid ) 是由 罗永康 庄帅 沈慧星 于 2019-10-31 设计创作，主要内容包括：本发明公开了一种鱼肉腐败菌氨基酸脱氨能力的检测方法,包括：用磷酸盐缓冲液溶解待测氨基酸底物配制得到待测氨基酸的底物溶液,将含待测腐败菌的菌悬液加入到待测氨基酸的底物溶液中作为评价组,将含待测腐败菌的菌悬液加入到磷酸盐缓冲液中作为对照组,将无菌生理盐水加入到所述待测氨基酸的底物溶液中作为空白组,分别在相同条件下培养,随后检测得到评价组、对照组和空白组的游离氨浓度C<Sub>评价</Sub>、C<Sub>对照</Sub>和C<Sub>空白</Sub>,计算得到腐败菌对待测氨基酸的脱氨能力。本发明所提供的方法能广泛适用于多种鱼肉中各类腐败菌的氨基酸脱氨能力的检测；适用于定量检测和评价腐败菌对多种特定氨基酸的脱氨能力；适用于评价鱼肉腐败菌代谢利用游离氨基酸的能力。(The invention discloses a method for detecting the deamination capacity of fish putrefying bacteria amino acid, which comprises the following steps: dissolving an amino acid substrate to be detected by using a phosphate buffer solution to prepare a substrate solution of the amino acid to be detected, adding a bacterial suspension containing putrefying bacteria to be detected into the substrate solution of the amino acid to be detected as an evaluation group, adding the bacterial suspension containing the putrefying bacteria to be detected into the phosphate buffer solution as a control group, adding sterile physiological saline into the substrate solution of the amino acid to be detected as a blank group, respectively culturing under the same conditions, and then detecting to obtain the free ammonia concentration C of the evaluation group, the control group and the blank group Evaluation of 、C Control And C Blank space Calculating to obtain the ratio of putrefying bacteria to amino acid to be detectedAnd (4) deamination capability. The method provided by the invention can be widely applied to the detection of the amino acid deamination capability of various putrefying bacteria in various fish flesh; the method is suitable for quantitatively detecting and evaluating the deamination capability of the putrefying bacteria on various specific amino acids; is suitable for evaluating the ability of the fish putrefying bacteria to metabolize and utilize free amino acid.)

1. A detection method for deamination capacity of fish putrefying bacteria amino acid is characterized by comprising the following steps: dissolving an amino acid substrate to be detected by using a phosphate buffer solution to prepare a substrate solution of the amino acid to be detected, adding a bacterial suspension containing putrefying bacteria to be detected into the substrate solution of the amino acid to be detected as an evaluation group, adding the bacterial suspension containing the putrefying bacteria to be detected into the phosphate buffer solution as a control group, adding sterile normal saline into the substrate solution of the amino acid to be detected as a blank group, respectively culturing under the same conditions, and then detecting to obtain the free ammonia concentration C of the evaluation group, the control group and the blank group_{Evaluation of}、C_ControlAnd C_{Blank space}And calculating the deamination capacity of the putrefying bacteria to the amino acid to be detected.

2. The detection method according to claim 1, wherein the deamination ability of the putrefying bacteria to the amino acid to be detected is calculated by the following formula:

DMA＝(T×V)×(C_{evaluation of}-C_Control-C_{Blank space})/(M×TVC×t)；

In the formula:

DMA is the deamination capacity of putrefying bacteria amino acid, mu mol/h;

t is the dilution multiple when detecting the concentration of free ammonia;

v is the total volume of the reaction system when the concentration of the free ammonia is detected;

m is the molecular weight of free ammonia, g/mol;

TVC is the total number of bacterial colonies of the bacterial suspension, log CFU/mL;

t is the reaction time, h.

3. The detection method according to claim 1 or 2, wherein the free ammonia concentration is detected by indophenol blue method.

4. The detection method according to claim 3, wherein the detection of the concentration of free ammonia comprises the following steps:

s1, diluting the cultured reaction solution by 10-50 times by adopting 0.005M sulfuric acid solution;

s2, uniformly mixing 2mL of the reaction solution diluted in the step S1, 100 mu L of mixed solution containing 5 wt% of salicylic acid and 5 wt% of sodium citrate, 20 mu L of 1 wt% of sodium nitroferricyanide solution and 20 mu L of 0.05M NaClO solution, and reacting for 1h in a dark place;

s3, putting 200 mu L of the reaction solution after the light-shielding reaction in the step S2 into a 96-well plate, and measuring the absorbance at the wavelength of 695 nm;

s4, establishing a standard curve by using the concentration of the free ammonia standard solution and the absorbance of each concentration standard solution at the wavelength of 695nm, which is measured by an indophenol blue method, and obtaining the concentration C of corresponding free ammonia in the solution to be measured according to the standard curve;

the standard curve form of absorbance-concentration is: y ═ aC + b, provided that R is²>0.995, wherein: c is free ammonia concentration in μ g/mL, Y is absorbance at 695nm wavelengthAnd (4) luminosity.

5. The detection method according to any one of claims 1 to 4,

the culture temperature is 30-35 ℃;

and/or the culture time is 36-54 h.

6. The detection method according to any one of claims 1 to 5, wherein the preparation of the substrate solution of the amino acid to be detected specifically comprises dissolving the substrate solution of the amino acid to be detected in a phosphate buffer solution of 0.045M, pH 7.50.50 to obtain a substrate solution of 4.0 to 6.5mM of the amino acid to be detected, sterilizing the substrate solution at 121 ℃ for 15 to 20min, adding oxidized nicotinamide adenine dinucleotide thereto after cooling, and controlling the final concentration of oxidized nicotinamide adenine dinucleotide to be 0.06 to 0.08 mM.

7. The detection method as claimed in any one of claims 1 to 6, wherein the preparation of the bacterial suspension containing the putrefying bacteria to be detected specifically comprises picking single colonies of the putrefying bacteria to be detected after separation and purification in tryptone soy broth, performing shake flask culture in water bath at 30 ℃ until the total number of the colonies of the broth reaches 9.0 to 10.0log CFU/mL, and adjusting the concentration of the bacterial suspension to 8.0 to 9.0log CFU/mL by using sterile physiological saline.

8. Use of the assay of any one of claims 1-7 for the detection of the deamination capacity of amino acids of fish spoilage bacteria.

Technical Field

The invention belongs to the fields of aquatic product storage and preservation technology and quality safety, and particularly relates to a method for detecting deamination capacity of fish putrefying bacteria amino acid.

Background

The fish meat is always an important component of daily diet of residents in China due to the characteristics of rich nutrition, easy digestion, delicious taste and the like; however, fish meat has high water activity and rich nutrition, so that the fish meat is extremely easy to decay and deteriorate due to the growth and propagation of spoilage bacteria in the process of storage and transportation, and the processing and consumption of fish meat products are influenced finally.

Research shows that putrefying bacteria in fish have strong amino acid deamination capability. The microorganisms can utilize the carbon skeleton of the deaminated amino acid to carry out energy metabolism and substance synthesis and promote the growth of the microorganisms by the deamination reaction of the amino acid; on the other hand, free ammonia generated by deamination is an important volatile off-flavor substance in fish meat and can seriously affect the sensory quality of the fish meat. In addition, amino acid deamination capacity and metabolic properties of different spoilage bacteria also vary greatly. Therefore, the amino acid deamination capability of various putrefying bacteria is widely researched and evaluated, and the method has important significance for researching the putrefaction mechanism of fish putrefying bacteria and designing and developing a fish quality control technology.

Disclosure of Invention

Aiming at the problems of the evaluation and the method of the putrefaction-causing capability of fish putrefactive bacteria in the prior art, the invention provides a method for detecting the deamination capability of amino acid of fish putrefactive bacteria.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for detecting the deamination capacity of fish putrefying bacteria amino acid comprises the following steps: dissolving an amino acid substrate to be detected by using a phosphate buffer solution to prepare a substrate solution of the amino acid to be detected, adding a bacterial suspension containing putrefying bacteria to be detected into the substrate solution of the amino acid to be detected as an evaluation group, adding the bacterial suspension containing the putrefying bacteria to be detected into the phosphate buffer solution as a control group, adding sterile normal saline into the substrate solution of the amino acid to be detected as a blank group, respectively culturing under the same conditions, and then detecting to obtain the free ammonia concentration C of the evaluation group, the control group and the blank group_{Evaluation of}、C_ControlAnd C_{Blank space}And calculating the deamination capacity of the putrefying bacteria to the amino acid to be detected.

In the technical scheme, the deamination capacity of the putrefying bacteria on the amino acid to be detected is calculated by adopting the following formula:

DMA＝(T×V)×(C_{evaluation of}-C_Control-C_{Blank space})/(M×TVC×t)；

In the formula:

DMA is the deamination capacity of putrefying bacteria amino acid, mu mol/h;

t is the dilution multiple when detecting the concentration of free ammonia;

v is the total volume of the reaction system when the concentration of the free ammonia is detected;

m is the molecular weight of free ammonia, g/mol;

TVC is the total number of bacterial colonies of the bacterial suspension, log CFU/mL;

t is the reaction time, h.

Further, in the above technical solution, the detection of the concentration of the free ammonia is performed by indophenol blue method.

Specifically, in the above technical solution, the detecting of the free ammonia concentration specifically includes the following steps:

s1, diluting the cultured reaction solution by 10-50 times by adopting 0.005M sulfuric acid solution;

s2, uniformly mixing 2mL of the reaction solution diluted in the step S1, 100 mu L of mixed solution containing 5 wt% of salicylic acid and 5 wt% of sodium citrate, 20 mu L of 1 wt% of sodium nitroferricyanide solution and 20 mu L of 0.05M NaClO solution, and reacting for 1h in a dark place;

s3, putting 200 mu L of the reaction solution after the light-shielding reaction in the step S2 into a 96-well plate, and measuring the absorbance at the wavelength of 695 nm;

s4, establishing a standard curve by using the concentration of the free ammonia standard solution and the absorbance of each concentration standard solution at the wavelength of 695nm, which is measured by an indophenol blue method, and obtaining the concentration C of corresponding free ammonia in the solution to be measured according to the standard curve;

the standard curve form of absorbance-concentration is: y ═ aC + b, provided that R is²>0.995. In the formula: c is the free ammonia concentration in μ g/mL and Y is the absorbance at 695 nm.

Still further, in the above technical solution, the temperature of the cultivation is 30-35 ℃.

Still further, in the above technical scheme, the culture time is 36-54 h.

Still further, in the above technical solution, the preparation of the substrate solution of the amino acid to be detected specifically comprises dissolving the substrate solution of the amino acid to be detected with 0.045M, pH 7.50.50 phosphate buffer solution to obtain 4.0-6.5mM, sterilizing at 121 ℃ for 15-20min, cooling, adding oxidized nicotinamide adenine dinucleotide, and controlling the final concentration of oxidized nicotinamide adenine dinucleotide to be 0.06-0.08 mM.

Still further, in the above technical solution, the preparation of the bacterial suspension containing the spoilage bacteria to be detected specifically comprises picking a single colony of the spoilage bacteria to be detected after separation and purification in tryptone soy broth, performing shake flask culture in water bath at 30 ℃ until the total number of colonies of the broth reaches 9.0-10.0log CFU/mL, and adjusting the concentration of the bacterial suspension to 8.0-9.0log CFU/mL with sterile physiological saline.

The invention also provides application of the detection method in detection of the deamination capacity of the amino acid of the fish spoilage bacteria.

The invention has the advantages that:

(1) the method provided by the invention is not limited by the source and the type of the putrefying bacteria, and can be widely applied to the detection of the amino acid deamination capability of various putrefying bacteria in various fish flesh;

(2) the method provided by the invention is suitable for quantitatively evaluating the deamination capability of the putrefying bacteria on various specific amino acids;

(3) the method provided by the invention is suitable for evaluating the ability of the fish spoilage bacteria to metabolize and utilize free amino acids.

Drawings

FIG. 1 shows the comparison results of amino acid deamination ability of Pseudomonas elman menticensis and Shewanella pultrefaciens in the examples of the present invention.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the present invention, but not to limit the scope of the invention, which is defined by the claims.

Unless otherwise specified, the test reagents and materials used in the examples of the present invention are commercially available.

Unless otherwise specified, the technical means used in the examples of the present invention are conventional means well known to those skilled in the art.