Multifunctional culture medium for lactobacillus paracasei as well as preparation method and application of multifunctional culture medium

Abstract

The invention relates to the technical field of microbial culture media, in particular to a lactobacillus paracasei multifunctional culture medium and a preparation method and application thereof, and the lactobacillus paracasei multifunctional culture medium is prepared from the following components: spirulina platensis lysate, corn steep liquor, trehalose, glucose, Tween 80, dipotassium phosphate, sodium acetate, triammonium citrate, magnesium sulfate and manganese sulfate. The spirulina platensis lysate is prepared by lysing a spirulina platensis solution with the concentration of 110 g / L to 200 g / L; the pH value of the culture medium is 6.3 to 6.5; wherein 0.2 g to 0.8 g of corn steep liquor, 0.4 g to 1.6 g of trehalose, 5 g to 35 g of glucose, 0.4 ml to 1.6 ml of Tween 80, 0.5 g to 3.5 g of dipotassium phosphate, 2 g to 8 g of sodium acetate, 0.5 g to 3.5 g of triammonium citrate, 0.05 g to 0.35 g of magnesium sulfate and 0.02 g to 0.08 g of manganese sulfate are added into each liter of spirulina platensis lysate. According to the culture medium disclosed by the invention, rapid, high-density, high-activity, high-probiotic necessary characteristic and high-extracellular polysaccharide / extracellular protein yield culture of the lactobacillus paracasei in a batch culture mode is realized, and a foundation is laid for industrial application of the lactobacillus paracasei probiotics and the extracellular polysaccharide / extracellular protein.

Description

Technical Field

[0001] The invention relates to the technical field of microbial culture media, and in particular to a multifunctional Lactobacillus paracasei culture medium, a preparation method and application thereof. Background Art

[0002] Lactobacillus paracasei (formerly known as Lactobacillus paracasei) is a lactic acid bacterium with important industrial application value.

[0003] On the one hand, as a traditional probiotic, Lactobacillus paracasei has the function of regulating intestinal flora and promoting health. Further studies have found that Lactobacillus paracasei has the effect of treating diseases such as acute gout, hyperuricemia, colitis and constipation. However, probiotics need to exert their probiotic effects in the form of live bacteria, and they need to effectively pass through the gastrointestinal barrier and colonize in the intestines. They need to have five essential probiotic properties, such as good resistance to gastric acid, resistance to bile salts, cell surface hydrophobicity, and self-aggregation and co-aggregation capabilities. On the other hand, Lactobacillus paracasei produces a variety of extracellular polysaccharides and extracellular proteins, which have biological and pharmacological activities such as anti-tumor, antioxidant and immune regulation. Recent studies have found that the extracellular polysaccharides and extracellular proteins of Lactobacillus paracasei have prebiotic or postbiotic effects that promote health.

[0004] However, lactobacillus is a microorganism with extremely high nutritional requirement, and needs to add amino acid and B vitamins etc. in culture medium. Although multiple culture mediums have been developed in the prior art, the high density and high activity of lactobacillus paracasei have not yet been realized, not to mention the cultivation of high probiotic essential characteristics and high exopolysaccharide / exoprotein output, which are extremely low at present, have limited its industrial application. For example, the classical culture medium of lactobacillus---De Man, Rogosa, and Sharpe (MRS) culture medium, owing to producing growth inhibitors such as lactic acid during cultivation, has limited the cultivation of high density, high activity, high probiotic essential characteristics and high exopolysaccharide / exoprotein output of lactobacillus paracasei. For this reason, a kind of multifunctional culture medium is developed, the high density (specific yield), high activity and high probiotic essential characteristics of lactobacillus paracasei are realized, and the cultivation of high exopolysaccharide / exoprotein output is further realized in the same fermentation system, which is of great significance to the industrial application of its probiotics and exopolysaccharide / exoprotein. Summary of the Invention

[0005] The present invention aims to address the problem that existing culture media cannot achieve high-density, high-activity, high probiotic properties and high exopolysaccharide / exoprotein production of Lactobacillus paracasei, which is not conducive to the industrial application of Lactobacillus paracasei probiotics and exopolysaccharides / exoproteins. A multifunctional culture medium for Lactobacillus paracasei, a preparation method and application thereof are provided.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The invention provides a multifunctional culture medium for Lactobacillus paracasei. The culture medium consists of the following components: a lysate of Spirulina platensis, corn steep liquor, trehalose, glucose, Tween 80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate and manganese sulfate. The lysate of Spirulina platensis is prepared by lysing a 110g / L to 200g / L Spirulina platensis solution. The pH value of the culture medium is 6.3 to 6.5. Per liter of the lysate of Spirulina platensis contains the following: 0.2 to 0.8g of corn steep liquor, 0.4 to 1.6g of trehalose, 5 to 35g of glucose, 0.4 to 1.6ml of Tween 80, 0.5 to 3.5g of dipotassium hydrogen phosphate, 2 to 8g of sodium acetate, 0.5 to 3.5g of triammonium citrate, 0.05 to 0.35g of magnesium sulfate and 0.02 to 0.08g of manganese sulfate.

[0008] Preferably, in the culture medium, the lysate of Spirulina platensis is prepared by lysing 140g / L to 200g / L of Spirulina platensis solution; and each liter of Spirulina platensis lysate contains the following: 0.3 to 0.7g corn steep liquor, 0.6 to 1.4g trehalose, 10 to 30g glucose, 0.6 to 1.4ml Tween 80, 1.0 to 3.0g dipotassium hydrogen phosphate, 3 to 7g sodium acetate, 1.0 to 3.0g triammonium citrate, 0.10 to 0.30g magnesium sulfate, and 0.03 to 0.07g manganese sulfate.

[0009] More preferably, in the culture medium, the lysate of Spirulina platensis is prepared by lysing 170g / L to 200g / L of Spirulina platensis dry powder; and per liter of Spirulina platensis lysate, the following are added: 0.4-0.6g corn steep liquor, 0.8-1.2g trehalose, 15-25g glucose, 0.8-1.2ml Tween 80, 1.5-2.5g dipotassium hydrogen phosphate, 4-6g sodium acetate, 1.5-2.5g triammonium citrate, 0.15-0.25g magnesium sulfate, and 0.04-0.06g manganese sulfate.

[0010] In addition, the present invention also provides a method for preparing the multifunctional culture medium of Lactobacillus paracasei, comprising the following steps:

[0011] S1: preparing a lysate of Spirulina platensis by lysing the dry powder of Spirulina platensis in a water bath;

[0012] S2: adjusting the pH of the prepared Spirulina platensis lysate to 6.3-6.5;

[0013] S3: Weigh the volume of the pH-adjusted Spirulina platensis lysate, add corn steep liquor, trehalose, glucose, Tween 80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, and manganese sulfate in proportion, shake and mix, and sterilize under high-pressure steam conditions of at least 121° C. for at least 15 minutes to obtain the multifunctional culture medium for Lactobacillus paracasei.

[0014] Preferably, in the above preparation method, the preparation of the Spirulina platensis lysate in step S1 comprises the following steps:

[0015] S1-1: Soaking Spirulina platensis in double-distilled water to obtain a Spirulina platensis solution;

[0016] S1-2: preheating the Spirulina platensis solution at 60° C. for 30 minutes, and then lysing the Spirulina platensis in a water bath at 100° C. for 1 to 1.5 hours;

[0017] S1-3: Centrifuging the lysed Spirulina platensis solution at 8000-10,000 g for 15-25 minutes to obtain the Spirulina platensis lysate. That is, centrifuging the lysed Spirulina platensis solution at a centrifugal force of 8000-10,000 times the acceleration of gravity for 15-25 minutes to obtain the Spirulina platensis lysate.

[0018] Preferably, in the above preparation method, the specific operation of adjusting the pH in step S2 is to adjust the pH of the Spirulina platensis lysate to 6.3-6.5 using 0.5-1.5 mol / L HCl.

[0019] The present invention also provides a culturing method for culturing Lactobacillus paracasei using the culture medium. The culturing method comprises the following steps: inoculating a Lactobacillus paracasei GY-1 seed liquid with an OD600 of 0.5-1.5 onto the culture medium according to any one of claims 1 to 3, wherein the volume ratio of the Lactobacillus paracasei GY-1 seed liquid to the culture medium is (1-3):100; placing the inoculated culture medium in an anaerobic tank, and culturing the culture medium in a constant temperature shaking incubator at a temperature of 37±5°C and a rotation speed of 100-200 rpm for 48 hours.

[0020] Preferably, in the above culture method, the volume ratio of the Lactobacillus paracasei GY-1 seed solution to the culture medium is 2:100; and the culture conditions are 37° C. and 150 rpm.

[0021] In addition, the Lactobacillus paracasei obtained by culturing the culture medium can be used as a probiotic.

[0022] Furthermore, the culture method can be applied to prepare Lactobacillus paracasei exopolysaccharide or exoprotein.

[0023] In summary, due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0024] (1) The culture medium provided by the present invention can realize rapid and high-density culture of Lactobacillus paracasei in a batch culture mode;

[0025] (2) The culture medium provided by the present invention can achieve high-activity cultivation of Lactobacillus paracasei in a batch culture mode;

[0026] (3) The culture medium provided by the present invention can achieve the high probiotic characteristics necessary for the cultivation of Lactobacillus paracasei in a batch culture mode;

[0027] (4) The culture medium provided by the present invention can achieve high exopolysaccharide / exoprotein production cultivation of Lactobacillus paracasei in batch culture mode;

[0028] (5) The culture medium provided by the present invention is low in cost and does not rely on the required instruments and equipment for other culture modes (e.g., continuous culture, fed-batch culture, and dialysis culture). The multifunctional culture medium provided by the present invention has a high output, good activity, and strong probiotic essential properties for the cultured Lactobacillus paracasei. Its exopolysaccharide / exoprotein output is high, which can significantly reduce the cost of Lactobacillus paracasei in laboratory or industrial production applications. Therefore, the present invention provides a high-quality and low-cost multifunctional culture medium for the industrial application of Lactobacillus paracasei probiotics and exopolysaccharides / exoproteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The figures are comparative diagrams showing the effects of different concentrations of Spirulina platensis lysate on the growth of Lactobacillus paracasei in Examples 1 to 3 of the present invention; wherein A is a growth curve, ***, P<0.001, **, P<0.01, and a t-test was performed with the lysate of a 200 mg / L Spirulina platensis solution; B is the OD600 after 20 hours of culture, and the differences among the multiple groups were analyzed using the Tukey post hoc test in a one-way analysis of variance (ANOVA). Different lowercase letters indicate statistically significant differences when P<0.05.

[0030] Figure 2The figures are comparative diagrams showing the effects of different concentrations of corn steep liquor on the growth of Lactobacillus paracasei in Examples 3 to 5 of the present invention; wherein A is a growth curve, ***, P<0.001, **, P<0.01, ns, P>0.05, and a t-test was performed with 0.05% (w / v) corn steep liquor; B is the OD600 after 20 hours of culture; differences among the multi-level groups were analyzed using the Tukey post hoc test in a one-way analysis of variance (ANOVA), and different lowercase letters indicate statistical differences when P<0.05.

[0031] Figure 3 The figures are comparative diagrams showing the effects of different concentrations of trehalose on the growth of Lactobacillus paracasei in Examples 4 and 6 to 8 of the present invention; wherein A is a growth curve, ***, P<0.001, **, P<0.01, ns, P>0.05, and a t-test was performed with 0.10% (w / v) trehalose; B is the OD600 after 20 hours of culture, and the differences among the multi-level groups were analyzed using the Tukey post hoc test in a one-way analysis of variance (ANOVA), and different lowercase letters indicate that there was a statistical difference when P<0.05;

[0032] Figure 4 The figure is a comparison of the culture effect of Lactobacillus paracasei cultured in the culture medium of Example 9 of the present invention and the MRS culture medium of Comparative Example 1; wherein A is a growth curve; B is a photograph of a test tube cultured for 20 hours; C is an OD600 value for 20 hours of culture; D is the maximum growth rate; E is the incubation period; F is the average growth rate within the 20-hour culture time; G is the number of viable cells; B is the number of viable cells per unit OD600; ***, P < 0.001, *, P < 0.05, t test was performed with the culture medium of the present invention;

[0033] Figure 5 : This is a comparison chart of the essential properties of the Lactobacillus paracasei probiotic cultured in the culture medium of Example 9 of the present invention and the MRS culture medium of Comparative Example 1; A is gastric acid resistance; B is bile salt resistance; C is cell surface hydrophobicity; D is self-aggregation ability; E is co-aggregation ability; *, P < 0.05, t test was performed with the culture medium of the present invention;

[0034] Figure 6 This is a comparison chart of the exopolysaccharide / exoprotein yields of Lactobacillus paracasei cultured in the culture medium of Example 9 of the present invention and in the MRS culture medium of Comparative Example 1; A is the total yield of free exopolysaccharides; B is the total yield of cell-bound exopolysaccharides; C is the total yield of free extracellular proteins; D is the total yield of cell-bound extracellular proteins; *, P<0.05, t-test was performed with the culture medium of the present invention. DETAILED DESCRIPTION

[0035] The principles and features of the present invention are described below. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. Where the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through regular channels.

[0036] Description of the main sources of drugs or materials:

[0037] Spirulina platensis powder was purchased from Hunan Zhengdi Biological Resources Development Co., Ltd.

[0038] Lactobacillus paracasei GY-1 was isolated, purified and identified by the inventor team and deposited on June 12, 2023 at the General Microbiology Center of China Culture Collection Administration, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 27600.

[0039] The main instruments and equipment used in the following embodiments and experimental examples are as follows:

[0040] Varioskan LUX multifunctional microplate reader, Thermo Fisher Scientific Inc., USA;

[0041] Electronic analytical balance (model QUINTIX124-1CN), Beijing Sartorius Instrument Systems Co., Ltd.;

[0042] OxoidAnaeroJar anaerobic jar, Thermo Fisher Scientific Inc., USA;

[0043] Constant temperature oscillating incubator, Shanghai Boxun Medical Biological Instrument Co., Ltd.

[0044] The invention provides a multifunctional culture medium for Lactobacillus paracasei. The culture medium consists of the following components: a lysate of Spirulina platensis, corn steep liquor, trehalose, glucose, Tween 80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate and manganese sulfate. The lysate of Spirulina platensis is prepared by lysing a 110g / L to 200g / L Spirulina platensis solution. The pH value of the culture medium is 6.3 to 6.5. Per liter of the lysate of Spirulina platensis contains the following: 0.2 to 0.8g of corn steep liquor, 0.4 to 1.6g of trehalose, 5 to 35g of glucose, 0.4 to 1.6ml of Tween 80, 0.5 to 3.5g of dipotassium hydrogen phosphate, 2 to 8g of sodium acetate, 0.5 to 3.5g of triammonium citrate, 0.05 to 0.35g of magnesium sulfate and 0.02 to 0.08g of manganese sulfate.

[0045] As a preferred embodiment, in the culture medium, the lysate of Spirulina platensis is prepared by lysing 140g / L to 200g / L of Spirulina platensis solution; and per liter of Spirulina platensis lysate, the following are added: 0.3-0.7g corn steep liquor, 0.6-1.4g trehalose, 10-30g glucose, 0.6-1.4ml Tween 80, 1.0-3.0g dipotassium hydrogen phosphate, 3-7g sodium acetate, 1.0-3.0g triammonium citrate, 0.10-0.30g magnesium sulfate, and 0.03-0.07g manganese sulfate.

[0046] More preferably, in the culture medium, the lysate of Spirulina platensis is prepared by lysing 170g / L to 200g / L of Spirulina platensis dry powder; and per liter of Spirulina platensis lysate, the following are added: 0.4-0.6g corn steep liquor, 0.8-1.2g trehalose, 15-25g glucose, 0.8-1.2ml Tween 80, 1.5-2.5g dipotassium hydrogen phosphate, 4-6g sodium acetate, 1.5-2.5g triammonium citrate, 0.15-0.25g magnesium sulfate, and 0.04-0.06g manganese sulfate.

[0047] In addition, the present invention also provides a method for preparing the multifunctional culture medium of Lactobacillus paracasei, comprising the following steps:

[0048] S1: Prepare Spirulina platensis lysate by lysing Spirulina platensis powder in a water bath, specifically:

[0049] S1-1: Soaking the dried Spirulina platensis powder in double-distilled water to obtain a Spirulina platensis solution;

[0050] S1-2: preheating the Spirulina platensis solution at 60° C. for 30 minutes, and then lysing the Spirulina platensis in a water bath at 100° C. for 1 to 1.5 hours;

[0051] S1-3: Centrifuging the lysed Spirulina platensis solution at 8000-10,000 g (under a centrifugal force of 8000-10,000 times the acceleration of gravity) for 15-25 minutes to obtain the Spirulina platensis lysate.

[0052] S2: adjusting the pH of the prepared Spirulina platensis lysate to 6.3-6.5 using 0.5-1.5 mol / L HCl;

[0053] S3: Weigh the volume of the pH-adjusted Spirulina platensis lysate, add corn steep liquor, trehalose, glucose, Tween 80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, and manganese sulfate in proportion, shake and mix, and sterilize under high-pressure steam conditions of at least 121° C. for at least 15 minutes to obtain the multifunctional culture medium for Lactobacillus paracasei.

[0054] In addition, the method for culturing Lactobacillus paracasei using the multifunctional Lactobacillus paracasei culture medium comprises the following steps: inoculating a Lactobacillus paracasei GY-1 seed liquid having an OD600 of 0.5 to 1.5 onto the culture medium according to any one of claims 1 to 3, wherein the volume ratio of the Lactobacillus paracasei GY-1 seed liquid to the culture medium is (1 to 3):100; placing the inoculated culture medium in an anaerobic jar, and culturing the culture medium in a constant temperature shaking incubator at a temperature of 37±5°C and a rotation speed of 100 to 200 rpm for 48 hours.

[0055] Preferably, in the above culture method, the volume ratio of the Lactobacillus paracasei GY-1 seed solution to the culture medium is 2:100; and the culture conditions are 37° C. and 150 rpm.

[0056] In order to express the present invention more clearly, the present invention is further described below through specific examples.

[0057] Example 1

[0058] This embodiment relates to a Lactobacillus paracasei culture medium, which is composed of the following components: a Spirulina platensis lysate, glucose, Tween 80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, and manganese sulfate; wherein the Spirulina platensis lysate is prepared by lysing a 100 g / L Spirulina platensis solution, and each liter of the Spirulina platensis lysate contains the following: 20 g glucose, 1 ml Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate, and the pH is 6.4.

[0059] In addition, the preparation method of the described Lactobacillus paracasei culture medium that the present embodiment also relates to comprises the steps:

[0060] S1: Prepare a lysate of Spirulina platensis by lysing the dry powder of Spirulina platensis in a water bath, specifically:

[0061] S1-1: Soaking Spirulina platensis in double-distilled water to obtain a Spirulina platensis solution;

[0062] S1-2: preheating the Spirulina platensis solution at 60° C. for 30 minutes, and then lysing the Spirulina platensis in a water bath at 100° C. for 1 hour;

[0063] S1-3: centrifuging the lysed Spirulina platensis solution at 10,000 g for 20 minutes to obtain the Spirulina platensis lysate;

[0064] S2: adjusting the pH of the prepared Spirulina platensis lysate to 6.4 using 0.5-1.5 mol / L HCl;

[0065] S3: Weigh the volume of the pH-adjusted Spirulina platensis lysate, add other components of the culture medium in proportion, shake and mix, and sterilize under high-pressure steam conditions of at least 121° C. for at least 15 minutes to obtain the Lactobacillus paracasei culture medium.

[0066] This embodiment also relates to a method for culturing Lactobacillus paracasei using the Lactobacillus paracasei culture medium, comprising the following steps: inoculating a Lactobacillus paracasei GY-1 seed liquid with an OD600 of 1 onto the culture medium, wherein the volume ratio of the Lactobacillus paracasei GY-1 seed liquid to the culture medium is 2:100; placing the inoculated culture medium in an anaerobic jar, and culturing it in a constant temperature shaking incubator at a temperature of 37° C. and a rotation speed of 150 rpm for 48 hours; and taking samples at 0, 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 hours to measure the OD600 values.

[0067] The OD600 values ​​are obtained by subtracting the OD600 of the corresponding culture medium from the OD600 of the sample, reflecting the growth of Lactobacillus paracasei, and are measured using a Shimadzu UV-2700 with a wavelength of 600 nm.

[0068] Note: The above experiments were repeated 3 times.

[0069] Example 2

[0070] Compared with Example 1, this example is the same as Example 1 except that the lysate of Spirulina platensis is prepared by lysing 150 g / L Spirulina platensis solution.

[0071] Example 3

[0072] Compared with Example 1, this example is the same as Example 1 except that the lysate of Spirulina platensis is prepared by lysing 200 g / L Spirulina platensis solution.

[0073] Figure 1 The comparison diagram of the effects of different concentrations of Spirulina platensis lysate on the growth effect of Lactobacillus paracasei in Examples 1 to 3 is shown in FIG. Figure 1 It can be seen that when the lysate of Spirulina platensis was prepared by lysing 200 g / L of Spirulina platensis solution (ie, Example 3), the culture effect of Lactobacillus paracasei was the best ( Figure 1 A), the time to reach the maximum growth is the shortest, only 20h ( Figure 1 A); Maximum OD600 ( Figure 1 B), is 1.92.

[0074] Example 4

[0075] The difference between this embodiment and Example 3 is that the components of the culture medium are different. 0.05% (w / v) corn steep liquor is added to the culture medium of this embodiment. Other than that, the other components are the same as those of Comparative Example 3.

[0076] Specifically, the components of the Lactobacillus paracasei culture medium described in the present embodiment and the proportional relationship of each component are as follows:

[0077] The Lactobacillus paracasei culture medium prepared in this example consists of the following components: Spirulina platensis lysate, corn steep liquor, glucose, Tween 80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, and manganese sulfate; wherein, per liter of Spirulina platensis lysate, the following components are added: 0.5 g corn steep liquor, 20 g glucose, 1 ml Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.20 g magnesium sulfate, and 0.05 g manganese sulfate; the Spirulina platensis lysate is prepared by lysing a 200 g / L Spirulina platensis solution.

[0078] Example 5

[0079] Compared with Example 4, this embodiment differs only in the amount of corn steep liquor. In this embodiment, the amount of corn steep liquor added is 1% (w / v). That is, in this embodiment, the proportion of the components in the Lactobacillus paracasei culture medium is as follows: per liter of Spirulina lysate, there are added: 10g corn steep liquor, 1.5g trehalose, 20g glucose, 1ml Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.20g magnesium sulfate, and 0.05g manganese sulfate; except for this, the rest are the same as in Example 4.

[0080] Figure 2 3 to 5 of the present invention, different contents of corn steep liquor on the growth effect of Lactobacillus paracasei; Figure 2 It can be seen that in Examples 3 to 5, when the lysate of Spirulina platensis used in the culture medium is prepared by lysing 200 g / L of Spirulina platensis solution and the amount of corn steep liquor added to the culture medium components is 0.05% (w / v) (i.e., Example 4), the culture effect of Lactobacillus paracasei is the best ( Figure 2 A), the maximum OD600 is the highest ( Figure 2 B), is 2.1.

[0081] Example 6

[0082] The difference between this embodiment and embodiment 4 lies in the different components of the culture medium. In this embodiment, 0.05% (w / v) trehalose is added to the culture medium. Other than that, the other components are the same as those in embodiment 4.

[0083] Specifically, the components of the Lactobacillus paracasei culture medium described in the present embodiment and the proportional relationship of each component are as follows:

[0084] The Lactobacillus paracasei culture medium prepared in this example consists of the following components: Spirulina platensis lysate, corn steep liquor, trehalose, glucose, Tween 80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, and manganese sulfate; wherein, per liter of Spirulina platensis lysate, the following components are added: 0.5 g corn steep liquor, 0.5 g trehalose, 20 g glucose, 1 ml Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.20 g magnesium sulfate, and 0.05 g manganese sulfate; the Spirulina platensis lysate is prepared by lysing a 200 g / L Spirulina platensis solution.

[0085] Example 7

[0086] The difference between this embodiment and Example 6 is only the amount of trehalose used. In this embodiment, the amount of trehalose added is 0.10% (w / v). That is, in this embodiment, the proportion of the components in the Lactobacillus paracasei culture medium is as follows: per liter of Spirulina lysate, there are added: 0.5g corn steep liquor, 1g trehalose, 20g glucose, 1ml Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.20g magnesium sulfate, and 0.05g manganese sulfate. Apart from this, the rest is the same as in Example 6.

[0087] Example 8

[0088] The difference between this embodiment and Example 6 is only the amount of trehalose used. In this embodiment, the amount of trehalose added is 0.15% (w / v). That is, in this embodiment, the proportion of the components in the Lactobacillus paracasei culture medium is as follows: per liter of Spirulina apical lysate, there are added: 0.5g corn steep liquor, 1.5g trehalose, 20g glucose, 1ml Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.20g magnesium sulfate, and 0.05g manganese sulfate. Apart from this, the rest is the same as in Example 6.

[0089] Figure 3 This is a comparison diagram of the effects of different concentrations of trehalose on the growth effect of Lactobacillus paracasei in Examples 4 and 6 to 8 of the present invention. Figure 3 It can be seen that in Examples 4, 6 and 8, when the lysate of Spirulina platensis was prepared by lysing 200 g / L of Spirulina platensis solution, 0.05% (w / v) corn steep liquor was added to the culture medium, and then 0.10% (w / v) trehalose was added (Example 7), the culture effect of Lactobacillus paracasei was the best ( Figure 3 A), the maximum OD600 is the highest ( Figure 3 B), is 2.15.

[0090] Example 9

[0091] This embodiment relates to a multifunctional culture medium for Lactobacillus paracasei, comprising the following components: a lysate of Spirulina platensis, corn steep liquor, trehalose, glucose, Tween 80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, and manganese sulfate; the lysate of Spirulina platensis is prepared by lysing a 200 g / L solution of Spirulina platensis; the pH of the culture medium is 6.4; and per liter of the lysate of Spirulina platensis, the following components are added: 0.5 g corn steep liquor, 1 g trehalose, 20 g glucose, 1 ml Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.20 g magnesium sulfate, and 0.05 g manganese sulfate.

[0092] The preparation method of a multifunctional culture medium of Lactobacillus paracasei and the method for culturing Lactobacillus paracasei involved in this embodiment are the same as those in Example 1.

[0093] Among the above Examples 1-9, Examples 6 to 9 are specific implementations of the scheme provided in this application. Due to the different components of the culture medium, the culture effects of Lactobacillus paracasei are also different. Among them, the culture medium prepared with the components of Example 7 and Example 9 has the best culture effect of Lactobacillus paracasei.

[0094] Therefore, the present invention is tested based on other performances of the multifunctional culture medium of Lactobacillus paracasei provided by the present invention, specifically as follows:

[0095] Comparative Example 1

[0096] This comparative example relates to a method for culturing Lactobacillus paracasei using De Man, Rogosa, and Sharpe (MRS) culture medium, comprising the following steps:

[0097] Step 1: Prepare MRS medium: 10 g tryptone, 5 g beef extract, 4 g yeast extract, 20 g glucose, 1 ml Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, add double-distilled water to 1000 ml, mix, adjust the pH to 6.4, and sterilize at 121°C for 15 min.

[0098] Step 2: inoculating Lactobacillus paracasei seed solution with an OD600 of 1 into MRS culture medium, with a volume ratio of Lactobacillus paracasei seed solution to MRS culture medium of 2:100;

[0099] Step 3: Place the inoculated culture medium in an anaerobic jar and culture it in a constant temperature shaking incubator at a temperature of 37°C and a rotation speed of 150 rpm for 48 hours. Sampling is performed at 0, 2, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, and 48 hours to determine the OD600 value.

[0100] Note: The above experiments were repeated 3 times.

[0101] The OD600 value, which reflects the growth of Lactobacillus paracasei, was calculated by subtracting the OD600 of the culture medium from the OD600 obtained from the sample. Measurements were made using a Shimadzu UV-2700 at a wavelength of 600 nm. Growth curves were analyzed using the grofit package, using a model-free smoothing spline curve (with the parameter "smooth.gc" set to 0). Two key growth parameters were extracted from each fitted growth curve: the maximum growth rate (OD600 / hour), expressed as the steepest slope, and the incubation period (hours).

[0102] The Lactobacillus paracasei culture effect of embodiment 9 is compared with that of comparative example 1, and the result is as follows Figure 4 As shown. Figure 4 It can be seen that the culture effect of the multifunctional culture medium of Lactobacillus paracasei provided by the present invention is significantly higher than that of the MRS culture medium at any time, and it only takes 20 hours to reach the maximum culture amount, while the MRS culture medium takes 24 hours to reach the maximum culture amount. Figure 4 A); Test tube photography ( Figure 4 B) The results show that the multifunctional culture medium for Lactobacillus paracasei provided by the present invention realizes high-density culture of Lactobacillus paracasei; the OD600 value of the maximum culture amount of Lactobacillus paracasei cultivated in the multifunctional culture medium for Lactobacillus paracasei provided by the present invention is about 2.15, which is much higher than the maximum culture amount of Lactobacillus paracasei cultivated in the MRS culture medium (OD600 value is about 1.33) ( Figure 4 C); The maximum growth rate of Lactobacillus paracasei cultured in the multifunctional culture medium of Lactobacillus paracasei provided by the present invention is significantly higher than that of MRS culture medium ( Figure 4 D), the incubation period was significantly shorter than that of MRS culture medium ( Figure 4 E), the average growth rate within 20 h was significantly higher than that in MRS medium ( Figure 4 F).

[0103] Comparative Example 2

[0104] This comparative example evaluated the viable counts of Lactobacillus paracasei in Example 9 and Comparative Example 1 by the dilution spread plate method.

[0105] The dilution spread plate method comprises the following steps: taking 1 mL of the culture solution obtained for 20 hours in Example 9 and Comparative Example 1, performing a 10-fold dilution, and evenly applying 100 μL of the diluted culture solution on a plate using a glass spreader; incubating in a constant temperature incubator at 37°C for 48 hours, counting the number of colonies; and calculating the number of viable bacteria at dilutions between 30 and 300 colonies.

[0106] Note: The above experiments were repeated 3 times.

[0107] The viable counts of Lactobacillus paracasei cultivated in Example 9 and Comparative Example 1 were compared, and the results were as follows: Figure 4 As shown. Figure 4 It can be seen that the number of viable Lactobacillus paracasei cultured in the multifunctional Lactobacillus paracasei culture medium provided by the present invention is 1.72×10 11 CFU / ml, which is 70.07 times the number of viable Lactobacillus paracasei cultured by MRS ( Figure 4 G), achieving high activity culture of Lactobacillus paracasei; the viable count of Lactobacillus paracasei per OD600 cultured in the multifunctional Lactobacillus paracasei culture medium provided by the present invention is 2.45×10 9 CFU / mL, which is 43.75 times the viable bacterial count per OD600 of Lactobacillus paracasei cultured by MRS ( Figure 4 H), illustrating that the multifunctional culture medium of Lactobacillus paracasei provided by the present invention can cultivate and maintain more viable bacteria.

[0108] Comparative Example 3

[0109] This comparative example evaluates the high gastric acid resistance culture property of the multifunctional culture medium of Lactobacillus paracasei provided by the present invention by comparing the gastric acid resistance of Lactobacillus paracasei in Example 9 and Comparative Example 1.

[0110] Here are the steps:

[0111] Step 1: 100 μL of the 20-hour culture medium obtained in Example 9 and Comparative Example 1 was mixed with 900 μL of simulated gastric fluid (125 mM NaCl, 7 mM KCl, 45 mM NaHCO3 and 3 g / L pepsin), and the final pH was adjusted to 2.0±0.1 with 1 M HCl.

[0112] Step 2: Incubate anaerobically in a constant temperature shaking incubator at 37°C and 110 rpm for 2 h;

[0113] Step 3: Centrifuge at 4000 g for 15 min at 4°C, collect the precipitate, and resuspend in 100 μL of fresh MRS culture medium;

[0114] Step 4: Dilute the resuspension 10-fold with 0.85% NaCl solution. Use a glass applicator to evenly spread 100 μL of the diluted bacterial solution on the plate; incubate in a constant temperature incubator at 37°C for 48 hours and count the number of colonies; calculate the number of viable bacteria at a dilution between 30 and 300, which is set as CFU. t The viable bacterial count of the bacterial solution that was not treated with simulated gastric acid was quantified using the same method and was designated as CFU0.

[0115] Step 5: Calculate the gastric acid resistance (Survival rate) according to the following formula:

[0116]

[0117] Note: The above experiments were repeated 3 times.

[0118] The gastric acid resistance of Lactobacillus paracasei of embodiment 9 is compared with that of comparative example 1, and the result is as follows Figure 5 As shown in A. Figure 5 A shows that the survival rate of Lactobacillus paracasei cultured in the multifunctional culture medium of Lactobacillus paracasei provided by the present invention in simulated gastric acid is significantly higher than that of Lactobacillus paracasei cultured in MRS culture medium, indicating that the multifunctional culture medium of Lactobacillus paracasei provided by the present invention enhances the gastric acid resistance of Lactobacillus paracasei.

[0119] Comparative Example 4

[0120] This comparative example evaluates the high bile salt resistance culture property of the multifunctional culture medium for Lactobacillus paracasei provided by the present invention by comparing the bile salt resistance of Lactobacillus paracasei in Example 9 and Comparative Example 1.

[0121] Here are the steps:

[0122] Step 1: 100 μL of the culture medium obtained for 20 hours in Example 9 and Comparative Example 1 was mixed with 900 μL of bile salt solution (3 g / L ox bile powder, 0.2 M sodium bicarbonate, pH 6.3).

[0123] Step 2: Incubate anaerobically in a constant temperature shaking incubator at 37°C and 110 rpm for 2 h;

[0124] Step 3: Centrifuge at 4000 g for 15 min at 4°C, collect the precipitate, and resuspend in 100 μL of fresh MRS culture medium;

[0125] Step 4: Dilute the resuspension 10-fold with 0.85% NaCl solution. Use a glass spreader to evenly spread 100 μL of the diluted bacterial solution onto a plate. Incubate in a constant temperature incubator at 37°C for 48 hours and count the colonies. Calculate the viable bacterial count at the dilution that produces a colony count between 30 and 300, which is designated as CFUt. Quantify the viable bacterial count of the bacterial solution not treated with bile salts using the same method, which is designated as CFU0.

[0126] Step 5: Calculate the bile salt resistance (Survival rate) according to the following formula:

[0127]

[0128] Note: The above experiments were repeated 3 times.

[0129] The bile salt resistance of Lactobacillus paracasei of Example 9 is compared with that of Comparative Example 1. Figure 5 As shown in B. Figure 5 B shows that the survival rate of Lactobacillus paracasei cultured in the culture medium provided by the present invention in bile salt solution is significantly higher than that of Lactobacillus paracasei cultured in MRS culture medium, indicating that the culture medium provided by the present invention enhances the bile salt resistance of Lactobacillus paracasei.

[0130] Comparative Example 5

[0131] This comparative example evaluates the high cell surface hydrophobicity culture characteristic of the multifunctional culture medium for Lactobacillus paracasei provided by the present invention by comparing the cell surface hydrophobicity of Lactobacillus paracasei in Example 9 and Comparative Example 1.

[0132] Here are the steps:

[0133] Step 1: 5 mL of the culture medium obtained after 20 hours in Example 9 and Comparative Example 1 was centrifuged at 4000 g for 5 minutes to obtain a cell pellet;

[0134] Step 2: Resuspend the cell pellet in PBS (pH 7.20) and centrifuge at 4000 g for 5 min. Repeat twice to ensure thorough washing of the pellet.

[0135] Step 3: The cell pellet was resuspended in PBS solution (pH 7.20) and diluted to an OD600 of 0.4, which was designated as A0.

[0136] Step 4: Add 1 mL of chloroform to 3 mL of diluted bacterial solution and vortex vigorously for 1 min to completely mix the chloroform and bacterial solution;

[0137] Step 5: After standing at room temperature for 30 minutes, the aqueous phase was aspirated and the OD600 value of the aqueous phase was measured, which was set as A. t ;

[0138] Step 6: Calculate the cell surface hydrophobicity (CSH) according to the following formula:

[0139]

[0140] Note: The above experiments were repeated 3 times.

[0141] The hydrophobicity of the cell surface of Lactobacillus paracasei of Example 9 is compared with that of Comparative Example 1. Figure 5 As shown in C. Figure 5 C shows that the hydrophobicity of the cell surface of Lactobacillus paracasei cultured in the multifunctional culture medium of Lactobacillus paracasei provided by the present invention is significantly higher than that of Lactobacillus paracasei cultured in MRS culture medium, indicating that the multifunctional culture medium of Lactobacillus paracasei provided by the present invention enhances the cell surface hydrophobicity of Lactobacillus paracasei.

[0142] Comparative Example 6

[0143] This comparative example evaluates the high self-aggregation culture characteristics of the multifunctional culture medium of Lactobacillus paracasei provided by the present invention by comparing the self-aggregation ability of Lactobacillus paracasei in Example 9 and Comparative Example 1.

[0144] Here are the steps:

[0145] Step 1: 5 mL of the culture medium obtained after 20 hours in Example 9 and Comparative Example 1 was centrifuged at 4000 g for 5 minutes to obtain a cell pellet;

[0146] Step 2: Resuspend the cell pellet in PBS (pH 7.20) and centrifuge at 4000 g for 5 min. Repeat twice to ensure thorough washing of the pellet.

[0147] Step 3: Resuspend the obtained cell pellet with 5 mL of PBS solution (pH 7.40), measure OD600, and set it as A0;

[0148] Step 4: Gently vortex and stir for 10 seconds, let it stand at room temperature for 1-3 hours; carefully aspirate the supernatant and measure OD600, which is recorded as A. t ;

[0149] Step 5: Calculate the auto-aggregation capability using the following formula:

[0150]

[0151] Note: The above experiments were repeated 3 times.

[0152] The self-aggregation ability of Lactobacillus paracasei in 1-3 hour of Example 9 is compared with that of Comparative Example 1. Figure 5 As shown in D. Figure 5D shows that the self-aggregation ability of Lactobacillus paracasei cultured in the multifunctional culture medium of Lactobacillus paracasei provided by the present invention is significantly higher than that of Lactobacillus paracasei cultured in MRS culture medium, indicating that the multifunctional culture medium of Lactobacillus paracasei provided by the present invention enhances the self-aggregation ability of Lactobacillus paracasei.

[0153] Comparative Example 7

[0154] This comparative example evaluates the high coaggregation culture characteristics of the multifunctional culture medium of Lactobacillus paracasei provided by the present invention by comparing the coaggregation abilities of Lactobacillus paracasei in Example 9 and Comparative Example 1.

[0155] Here are the steps:

[0156] Step 1: 2.5 mL of the culture medium obtained for 20 hours in Example 9 and Comparative Example 1 was mixed with 2.5 mL of Staphylococcus aureus solution (OD600 of about 0.86); the mixture was centrifuged at 4000 g for 5 minutes to obtain a cell pellet;

[0157] Step 2: Resuspend the cell pellet in PBS (pH 7.20) and centrifuge at 4000 g for 5 min. Repeat twice to ensure thorough washing of the pellet.

[0158] Step 3: Resuspend the obtained cell pellet with 5 mL of PBS solution (pH 7.40), measure OD600, and set it as A0;

[0159] Step 4: Gently vortex and stir for 10 seconds, let it stand at room temperature for 1-3 hours; carefully aspirate the supernatant and measure OD600, which is recorded as A. t ;

[0160] Step 5: Calculate the co-aggregation capability using the following formula:

[0161]

[0162] Note: The above experiments were repeated 3 times.

[0163] The coaggregation ability of Lactobacillus paracasei in 1-3 hour of Example 9 is compared with that of Comparative Example 1. Figure 5 As shown in E. Figure 5 E shows that the coaggregation ability of Lactobacillus paracasei cultured in the multifunctional culture medium of Lactobacillus paracasei provided by the present invention is significantly higher than that of Lactobacillus paracasei cultured in MRS culture medium, indicating that the multifunctional culture medium of Lactobacillus paracasei provided by the present invention enhances the coaggregation ability of Lactobacillus paracasei.

[0164] Comparative Example 8

[0165] In this comparative example, the free exopolysaccharide yields of Lactobacillus paracasei in Example 9 and Comparative Example 1 were compared by weighing the crude extracts to evaluate the high free exopolysaccharide yield culture characteristics of the multifunctional culture medium for Lactobacillus paracasei provided by the present invention.

[0166] Here are the steps:

[0167] Step 1: Centrifuge 20 mL of the culture medium obtained after 20 hours in Example 9 and Comparative Example 1 at 10,000 g and 4° C. for 10 minutes, and collect the supernatant for extraction of free exopolysaccharides;

[0168] Step 2: Treat the culture supernatant with 20 mL of 4% (w / v) trichloroacetic acid (TCA); allow to precipitate at 4°C for 6 hours, then centrifuge at 10,000 g for 10 minutes at 4°C; mix the supernatant with three volumes of pre-chilled anhydrous ethanol, vigorously stir, and incubate at 4°C overnight; then centrifuge at 10,000 g for 10 minutes at 4°C to collect the free exopolysaccharide precipitate; resuspend the free exopolysaccharide precipitate in double-distilled water, dialyze, freeze-dry, and weigh. The same procedure is performed on a blank culture medium, and the dry weight of the free exopolysaccharide extracted from the blank culture medium is subtracted to obtain the yield of free exopolysaccharide in the culture medium of the present invention and in the MRS culture medium.

[0169] Example 9 compared the exopolysaccharide dry weight of Lactobacillus paracasei with that of Comparative Example 1. Figure 6 As shown. Figure 6 It can be seen that the total yield of free extracellular polysaccharide in the culture medium provided by the present invention is 4.16 times that of the total yield in MRS culture medium ( Figure 6 A), indicating that the multifunctional culture medium for Lactobacillus paracasei provided by the present invention realizes the high-yield cultivation of free exopolysaccharides of Lactobacillus paracasei.

[0170] Comparative Example 9

[0171] In this comparative example, the cell-bound exopolysaccharide yields of Lactobacillus paracasei in Example 9 and Comparative Example 1 were compared by weighing the crude extracts to evaluate the high cell-bound exopolysaccharide yield culture characteristics of the multifunctional culture medium for Lactobacillus paracasei provided by the present invention.

[0172] Here are the steps:

[0173] Step 1: 20 mL of the culture medium obtained after 20 hours in Example 9 and Comparative Example 1 was centrifuged at 10,000 g and 4° C. for 10 minutes to collect the cell pellet for extraction of cell-bound exopolysaccharides;

[0174] Step 2: Wash the cell pellet twice with normal saline and resuspend it in 20 mL of 1 M NaCl solution, sonicate at 40 watts and 4°C for 3 minutes; centrifuge at 10,000 rpm and 4°C for 10 minutes, and take the supernatant; treat the culture supernatant with 20 mL of 4% (w / v) trichloroacetic acid (TCA); after precipitation at 4°C for 6 hours, centrifuge at 10,000 g and 4°C for 10 minutes; mix the supernatant with three volumes of pre-cooled anhydrous ethanol, stir vigorously and place at 4°C overnight; then centrifuge at 10,000 g and 4°C for 10 minutes to collect the cell-bound exopolysaccharide precipitate; resuspend the cell-bound exopolysaccharide precipitate with double-distilled water, dialyze, freeze-dry and weigh.

[0175] Example 9 compared the dry weight of Lactobacillus paracasei cell-bound exopolysaccharide with that of Comparative Example 1. Figure 6 As shown. Figure 6 It can be seen that the total yield of cell-bound extracellular polysaccharide in the multifunctional culture medium of Lactobacillus paracasei provided by the present invention is 1.94 times that in the MRS culture medium ( Figure 6 B), indicating that the multifunctional culture medium for Lactobacillus paracasei provided by the present invention realizes the high-yield cultivation of Lactobacillus paracasei cell-bound exopolysaccharide.

[0176] Comparative Example 10

[0177] In this comparative example, the free extracellular protein yields of Lactobacillus paracasei in Example 9 and Comparative Example 1 were compared by weighing the crude extracts to evaluate the high free extracellular protein yield culture characteristics of the multifunctional culture medium for Lactobacillus paracasei provided by the present invention.

[0178] Here are the steps:

[0179] Step 1: Centrifuge 20 mL of the culture medium obtained after 20 hours in Example 9 and Comparative Example 1 at 10,000 g and 4° C. for 10 minutes, and collect the supernatant for extraction of free extracellular proteins;

[0180] Step 2: Treat the culture supernatant with 20 mL of 4% (w / v) trichloroacetic acid (TCA); allow to precipitate at 4°C for 6 hours, then centrifuge at 10,000g for 10 minutes at 4°C; resuspend the free extracellular protein precipitate in double-distilled water, dialyze, freeze-dry, and weigh. A blank culture medium is subjected to the same procedures, and the dry weight of the free extracellular protein extracted from the blank culture medium is subtracted to obtain the free extracellular protein yield obtained by culturing the Lactobacillus paracasei multifunctional culture medium and the MRS culture medium provided by the present invention.

[0181] Note: The above experiments were repeated 3 times.

[0182] The extracellular protein dry weight of Example 9 and Comparative Example 1 is compared, and the result is as follows: Figure 6 As shown. Figure 6 It can be seen that the total output of free extracellular protein in the multifunctional culture medium of Lactobacillus paracasei provided by the present invention is 10.09 times that in the MRS culture medium ( Figure 6 C), indicating that the multifunctional culture medium for Lactobacillus paracasei provided by the present invention realizes the high-yield cultivation of free extracellular protein of Lactobacillus paracasei.

[0183] Comparative Example 11

[0184] In this comparative example, the cell-bound extracellular protein yields of Lactobacillus paracasei in Example 9 and Comparative Example 1 were compared by weighing the crude extracts to evaluate the high cell-bound extracellular protein yield culture characteristics of the multifunctional culture medium for Lactobacillus paracasei provided by the present invention.

[0185] Here are the steps:

[0186] Step 1: 20 mL of the culture medium obtained after 20 hours in Example 9 and Comparative Example 1 was centrifuged at 10,000 g and 4° C. for 10 min to collect the cell pellet for extraction of cell-bound extracellular proteins;

[0187] Step 2: The cell pellet was washed twice with physiological saline and resuspended in 20 mL of 5 M LiCl solution and treated at 37°C for 1 hour; centrifuged at 10,000 rpm and 4°C for 10 minutes, and the supernatant was obtained; the culture supernatant was treated with 20 mL of 4% (w / v) trichloroacetic acid (TCA); after precipitation at 4°C for 6 hours, the supernatant was centrifuged at 10,000 g and 4°C for 10 minutes, and the supernatant was obtained; the collected supernatant was dialyzed, freeze-dried, and weighed to obtain the dry weight of the crude cell-bound extracellular protein extract.

[0188] Note: The above experiments were repeated 3 times.

[0189] The extracellular protein dry weight of Example 9 and Comparative Example 1 is compared, and the result is as follows: Figure 6 As shown. Figure 6 It can be seen that the total yield of cell-bound extracellular protein in the multifunctional culture medium of Lactobacillus paracasei provided by the present invention is 3.09 times the total yield in the MRS culture medium ( Figure 6 D), indicating that the multifunctional culture medium for Lactobacillus paracasei provided by the present invention realizes the high-yield cultivation of Lactobacillus paracasei cell-bound extracellular proteins.

[0190] To sum up, the lactobacillus paracasei multifunctional culture medium provided by the invention can quickly obtain high density, high activity, high probiotics essential characteristic and high exopolysaccharide / exoprotein cultivation of lactobacillus paracasei under batch culture mode.The lactobacillus paracasei multifunctional culture medium provided by the invention is low in cost, does not rely on the required instrument and equipment of other culture modes (such as continuous culture, fed-batch culture and dialysis culture), and the lactobacillus paracasei multifunctional culture medium provided by the invention cultivates the lactobacillus paracasei output height, good activity and probiotics essential characteristic strong, its exopolysaccharide / exoprotein output height can significantly reduce the cost of lactobacillus paracasei in laboratory or industrialized production application.Therefore, the present invention is lactobacillus paracasei probiotic application and exopolysaccharide / exoprotein industry application, provides high-quality and low-cost multifunctional culture medium.

[0191] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0192] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

[0193] The above description is a detailed description of the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. Any equivalent changes or modifications completed under the technical spirit suggested by the present invention should fall within the patent scope covered by the present invention.

Claims

1. A multifunctional culture medium for Lactobacillus paracasei, characterized in that The culture medium comprises the following components: a lysate of Spirulina platensis, corn steep liquor, trehalose, glucose, Tween 80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, and manganese sulfate; the lysate of Spirulina platensis is prepared by lysing a 110 g / L to 200 g / L Spirulina platensis solution; the pH value of the culture medium is 6.3 to 6.5; and per liter of the lysate of Spirulina platensis, the following components are added: 0.2 to 0.8 g corn steep liquor, 0.4 to 1.6 g trehalose, 5 to 35 g glucose, 0.4 to 1.6 ml Tween 80, 0.5 to 3.5 g dipotassium hydrogen phosphate, 2 to 8 g sodium acetate, 0.5 to 3.5 g triammonium citrate, 0.05 to 0.35 g magnesium sulfate, and 0.02 to 0.08 g manganese sulfate.

2. a multifunctional culture medium for Lactobacillus paracasei according to claim 1, is characterized in that, In the culture medium, the lysate of Spirulina platensis is prepared by lysing a 140g / L to 200g / L Spirulina platensis solution; and per liter of the lysate of Spirulina platensis, the following are added: 0.3 to 0.7g corn steep liquor, 0.6 to 1.4g trehalose, 10 to 30g glucose, 0.6 to 1.4ml Tween 80, 1.0 to 3.0g dipotassium hydrogen phosphate, 3 to 7g sodium acetate, 1.0 to 3.0g triammonium citrate, 0.10 to 0.30g magnesium sulfate, and 0.03 to 0.07g manganese sulfate.

3. according to claim 2 a kind of multifunctional culture medium of Lactobacillus paracasei, it is characterized in that, In the culture medium, the lysate of Spirulina platensis is prepared by lysing 170g / L to 200g / L of dry powder of Spirulina platensis; and per liter of the lysate of Spirulina platensis, the following are added: 0.4-0.6g corn steep liquor, 0.8-1.2g trehalose, 15-25g glucose, 0.8-1.2ml Tween 80, 1.5-2.5g dipotassium hydrogen phosphate, 4-6g sodium acetate, 1.5-2.5g triammonium citrate, 0.15-0.25g magnesium sulfate, and 0.04-0.06g manganese sulfate.

4. according to the preparation method of each described Lactobacillus paracasei multifunctional culture medium among the claim 1-3, it is characterized in that, The following steps are involved: S1: preparing a lysate of Spirulina platensis by lysing the dry powder of Spirulina platensis in a water bath; S2: adjusting the pH of the prepared Spirulina platensis lysate to 6.3-6.5; S3: Weigh the volume of the pH-adjusted Spirulina platensis lysate, add corn steep liquor, trehalose, glucose, Tween 80, dipotassium hydrogen phosphate, sodium acetate, triammonium citrate, magnesium sulfate, and manganese sulfate in proportion, shake and mix, and sterilize under high-pressure steam conditions of at least 121° C. for at least 15 minutes to obtain the multifunctional culture medium for Lactobacillus paracasei.

5. The preparation method according to claim 4, characterized in that The preparation of the Spirulina platensis lysate in step S1 comprises the following steps: S1-1: Soaking the dried Spirulina platensis powder in double-distilled water to obtain a Spirulina platensis solution; S1-2: preheating the Spirulina platensis solution at 60° C. for 30 minutes, and then lysing the Spirulina platensis in a water bath at 100° C. for 1 to 1.5 hours; S1-3: Centrifuging the lysed Spirulina platensis solution at 8000-10,000 g for 15-25 min to obtain the Spirulina platensis lysate.

6. The preparation method according to claim 4, characterized in that The specific operation of adjusting the pH in step S2 is to adjust the pH of the Spirulina platensis lysate to 6.3-6.5 using 0.5-1.5 mol / L HCl.

7. A method for cultivating Lactobacillus paracasei, characterized in that, The method comprises the following steps: inoculating Lactobacillus paracasei GY-1 seed liquid with an OD600 of 0.5-1.5 onto the culture medium according to any one of claims 1 to 3, wherein the volume ratio of the Lactobacillus paracasei GY-1 seed liquid to the culture medium is (1-3):100; placing the inoculated culture medium in an anaerobic tank, and culturing in a constant temperature shaking incubator for 48 hours under the conditions of a temperature of 37±5°C and a rotation speed of 100-200 rpm.

8. The culture method according to claim 7, characterized in that The volume ratio of the Lactobacillus paracasei GY-1 seed liquid to the culture medium is 2:100; and the culture conditions are 37° C. and 150 rpm.

9. The application of the Lactobacillus paracasei obtained by cultivating the culture method described in any one in claim 7-8 in probiotics.

10. Use of the culture method according to any one of claims 7-8 in preparing Lactobacillus paracasei exopolysaccharide or extracellular protein.

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