阅读说明：本技术 一种促进乳源假单胞菌分泌胞外蛋白酶的方法 (Method for promoting milk-derived pseudomonas to secrete extracellular protease ) 是由 韩雪 王宇 张兰威 易华西 孙嘉蕾 陈曦 王淑梅 于 2021-09-26 设计创作，主要内容包括：本发明公开了一种促进乳源假单胞菌分泌胞外蛋白酶的方法,所述方法包括如下步骤：步骤一、将乳源添加剂加入到培养基中进行均质处理,然后灭菌；步骤二、将具有分泌蛋白酶AprX的乳源嗜冷假单胞菌按照0.5～1.5％(v/v)的接种量接种到培养基中活化2～4次,将活化好的乳源嗜冷假单胞菌接种到添加乳源添加剂的培养基中进行培养。该方法通过添加0.5～5％(w/v)的乳源添加剂能够使得假单胞菌分泌的蛋白酶活力提高3～5倍。(The invention discloses a method for promoting milk-derived pseudomonas to secrete extracellular protease, which comprises the following steps: step one, adding a milk source additive into a culture medium for homogenization treatment, and then sterilizing; and secondly, inoculating the milk-derived psychrophilic pseudomonas secreting the protease AprX into a culture medium according to the inoculation amount of 0.5-1.5% (v/v) for activation for 2-4 times, and inoculating the activated milk-derived psychrophilic pseudomonas into the culture medium added with the milk-derived additive for culture. According to the method, the activity of the protease secreted by the pseudomonas can be improved by 3-5 times by adding 0.5-5% (w/v) of milk source additive.)

1. A method for promoting secretion of extracellular protease by Pseudomonas lactis, which comprises the following steps:

step one, adding a milk source additive into a culture medium for homogenization treatment, and then sterilizing;

and secondly, inoculating the milk-derived psychrophilic pseudomonas secreting the protease AprX into a culture medium according to the inoculation amount of 0.5-1.5% (v/v) for activation for 2-4 times, and inoculating the activated milk-derived psychrophilic pseudomonas into the culture medium added with the milk-derived additive for culture.

2. The method for promoting secretion of extracellular protease by pseudomonas lactis according to claim 1, wherein the homogenization pressure is 10-20 MPa, and the homogenization time is 15-25 min.

3. The method for promoting secretion of extracellular protease by pseudomonas lactis according to claim 1, wherein the sterilization temperature is 121 ℃ and the sterilization time is 10-25 min.

4. The method for promoting secretion of extracellular protease by Pseudomonas lactis according to claim 1, wherein the culture medium is TSB culture medium.

5. The method for promoting secretion of extracellular protease by milk-derived pseudomonas according to claim 1, wherein the milk-derived additive is one or more of bovine casein, bovine whey protein, lactose and natural butter, and the addition amount is 0.5-5% (w/v).

6. The method for promoting secretion of extracellular protease by milk-derived pseudomonas as claimed in claim 5, wherein the content of α -casein in the bovine milk casein is 20-40%, the purity of bovine whey protein is 75-90%, and the salt content of natural butter is 0-20 mg/100 g.

7. The method for promoting secretion of extracellular protease by pseudomonas lactis according to claim 1, wherein the pseudomonas lactis is pseudomonas psychrophila having a secreted protease AprX isolated from raw milk.

8. The method for promoting secretion of extracellular protease by Pseudomonas lactis according to claim 7, wherein the Pseudomonas psychrophila is Pseudomonas fluorescens, Pseudomonas putida or Pseudomonas aeruginosa.

9. The method for promoting secretion of extracellular protease by pseudomonas lactis according to claim 1, wherein the culture temperature is 25 to 35 ℃, the culture time is 60 to 70 hours, and the stirring speed is 150 to 200 rpm.

Technical Field

The invention belongs to the field of dairy processing, and relates to a method for promoting milk-derived pseudomonas to secrete extracellular protease.

Background

The development of low-temperature storage and transportation greatly reduces the damage of microbial pollution to the quality of raw milk, and the low temperature can effectively inhibit the growth of conventional room-temperature bacteria such as lactic acid bacteria so as to prevent the influence of excessive growth on the quality of the milk. However, psychrophile, a group of bacteria that can grow and reproduce at low temperature, is a major factor of contaminating the quality of raw milk. The pseudomonas is the predominant psychrophile in the raw milk because of wide sources, strong growth capability under low temperature conditions and capability of secreting heat-resistant enzymes, wherein the pseudomonas fluorescens and the pseudomonas putida are the most common in the raw milk.

Although the conventional heat sterilization method of raw milk can effectively inactivate psychrophile, the secreted enzyme has heat resistance and partial activity is remained. The residual enzyme activity can slowly decompose milk components in the preservation period of the milk products, so that the milk products have adverse effects of gel, precipitation, fat floating and the like. The metalloprotease AprX secreted by pseudomonas is the most widely studied psychrophile protease at present, and the AprX has strong heat resistance and strong hydrolysis capacity on casein.

The research on the secretion of protease by psychrophile has important significance on the hydrolysis of milk protein, so that the improvement of the secretion of protease by psychrophile has important effect on the subsequent obtaining of a pure protease product. The separation and purification of the conventional psychrophile protease are performed by using synthetic media such as TSB, LB and the like. Numerous studies show that the milk culture medium can promote the activity of the protease secreted by the psychrophile, meanwhile, the protease secretion of thalli in different culture media can be influenced due to the difference of the synthetic culture medium and milk components, and the protease secreted in milk cannot be represented by the protease in the synthetic culture medium. Therefore, considering that the milk components have the promotion effect on the secretion of the psychrophile protease, the influence of different milk components on the activity of the psychrophile protease is researched, the components having the promotion effect on the activity of the psychrophile protease are found, and the determination of the optimal proportion has important significance on the research of the psychrophile protease.

Disclosure of Invention

The invention aims to provide a method for promoting milk-derived pseudomonas to secrete extracellular protease, which can improve the activity of the protease secreted by pseudomonas by 3-5 times by adding 0.5-5% (w/v) of milk-derived additive.

The purpose of the invention is realized by the following technical scheme:

a method for promoting secretion of extracellular protease by milk-derived pseudomonas comprises the following steps:

step one, adding a milk source additive into a culture medium for homogenization treatment, then sterilizing, controlling the homogenization pressure to be 10-20 MPa, the homogenization time to be 15-25 min, the sterilization temperature to be 121 ℃, the time to be 10-25 min, and the culture medium to be a TSB culture medium (tryptone 17g/L, soybean peptone 3g/L, sodium chloride 5g/L, dipotassium hydrogen phosphate 2.5g/L, and glucose 2.5 g/L);

inoculating the milk-derived psychrophilic pseudomonas secreting the protease AprX into a culture medium according to the inoculation amount of 0.5-1.5% (v/v) for activating for 2-4 times, inoculating the activated milk-derived psychrophilic pseudomonas into the culture medium added with the milk-derived additive for culturing, controlling the culture temperature to be 25-35 ℃, the culture time to be 60-70 h, and the stirring speed to be 150-200 rpm.

In the present invention, the strain used is a milk-derived psychrophilic pseudomonas having an ability to secrete the protease AprX isolated from a raw milk, for example: pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida and the like.

In the invention, the milk-derived additive is one or more of cow milk casein (the content of alpha-casein is 20-40%), cow whey protein (the purity is 75-90%), lactose or natural butter (the salt content is 0-20 mg/100g), the optimal addition amount is 0.5-5% (w/v), and when the milk-derived additive is the optimal addition amount, the extracellular protease secreted by the psychrophilic pseudomonas strain has the strongest activity, can reach 4.5-6.0U/mL, and is 3-5 times of that without the milk-derived additive.

Compared with the prior art, the invention has the following advantages:

1. the addition of the milk source additive can improve the activity of extracellular protease secreted by the psychrophilic pseudomonas by 3-5 times, and the activity reaches the level when 12% (w/v) skim milk is used as a culture medium.

2. The zymogram experiment shows that the protease for promoting the secretion of the pseudomonas psychrophila has the molecular weight of about 47kDa, is a representative heat-resistant metallo-protease AprX secreted by the pseudomonas, has strong hydrolysis effect on casein, and preferentially hydrolyzes beta-casein or kappa-casein in the casein.

3. Compared with skim milk, the milk-derived additive can obviously reduce the protein content in the pseudomonas psychrophila fermentation liquor, and is beneficial to the separation and purification of extracellular protease secreted by the strain.

4. The milk source additive can enable the secretion of the protease by the psychrophile to be closer to the secretion condition of the protease in milk, and can be applied to the research on the psychrophile protease and the harm of the psychrophile protease to dairy products and a control method.

5. The principle that the milk-derived additive can promote the psychrophilic pseudomonas to secrete the extracellular protease activity is that the milk-derived additive can promote the expression of a protease gene aprX, and the difference multiple reaches 15-20 times.

Drawings

FIG. 1 is a diagram showing the ability of Pseudomonas fluorescens to produce extracellular protease in TSB and skim milk.

FIG. 2 is a graph showing the effect of different milk-derived additive ratios on the activity of Pseudomonas aeruginosa secreting extracellular protease.

FIG. 3 is a diagram showing the hydrolysis of lactoprotein by Pseudomonas fluorescens extracellular protease secretion.

FIG. 4 is an zymogram of Pseudomonas fluorescens in TSB according to the present invention.

FIG. 5 is an enzyme spectrum of Pseudomonas fluorescens after the addition of the milk-derived additive of the present invention.

FIG. 6 is a graph showing the effect of the milk-derived additive of the present invention on the expression of the Pseudomonas putida protease gene aprX.

Detailed Description

The technical solution of the present invention is further described below with reference to the accompanying drawings, but not limited thereto, and any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be covered by the protection scope of the present invention.

Example 1

Growth capacity and protease activity of pseudomonas fluorescens in milk medium and TSB medium:

the pseudomonas fluorescens (with the preservation number of CGMCC No.14540) secreting protease AprX is inoculated into TSB according to the inoculation amount of 1% (v/v) for activation for 2 times, the activated pseudomonas fluorescens are respectively inoculated into TSB culture medium (tryptone 17g/L, soybean peptone 3g/L, sodium chloride 5g/L, dipotassium hydrogen phosphate 2.5g/L, glucose 2.5g/L) and skim milk according to the inoculation amount of 1% (v/v) and stored for 5 days at the temperature of 4 ℃, and the bacterial number and the protease activity are measured every 1 day. The data in FIG. 1a show that P.fluorescens has similar growth curves in TSB medium and skim milk medium, indicating that skim milk does not promote P.fluorescens growth. As shown in FIG. 1b, the activity of the protease secreted by P.fluorescens in skim milk medium reached a maximum of 2.0U/mL at day 6, which was significantly greater than 0.6U/mL in TSB, indicating that the milk component can promote P.fluorescens secreted protease activity.

Example 2

Effect of milk-derived additives on pseudomonas aeruginosa exoprotease secretion:

milk-derived additive (bovine whey protein (purity 80%): natural butter (salt content 5mg/100g) ═ 1: 2(w/w)) was added to TSB medium at a ratio of 2% (w/v), homogenized at 10MPa for 20min, and sterilized at 121 ℃ for 15 min. Inoculating 1% (v/v) of activated pseudomonas aeruginosa (obtained by separating from raw milk in the black river region, and the separation method and the strain identification result are disclosed in doctor paper of Haerbin Industrial university, 2017, construction of diversity research on psychrophile in raw milk and LAMP rapid detection method), and culturing at 30 ℃ and 160rpm for 60h, and then determining the protease activity. As shown in figure 2, the extracellular protease activity of the strain added with the milk-derived additive reaches 6U/mL, which is 5 times that of the strain without the milk-derived additive.

Example 3

Effect of milk-derived additives on pseudomonas putida protease gene aprX expression:

1% (w/v) milk-derived additive (bovine whey protein (purity 85%): lactose: native butter (salt content 1mg/100g) ═ 1: 2: 1(w/w)) was added to the TSB medium, homogenized at 15MPa for 20min, and sterilized at 121 ℃ for 20 min. Activated pseudomonas putida (obtained by separating raw milk in the Heihe area, and the separation method and strain identification result are disclosed in the doctor paper of Haerbin Industrial university, 2017, the construction of the diversity research on psychrophile in raw milk and the LAMP rapid detection method) is inoculated according to the inoculation amount of 1% (v/v), and then cultured for 70h under the conditions of 35 ℃ and 100rpm, and the expression condition of aprX gene is determined. As shown in fig. 6, 1% of milk-derived additives were able to promote the expression of aprX gene with a 15-fold expression.