Lactobacillus paracasei pl4.4s and use thereof

Abstract

The application relates to the technical field of microorganisms, and particularly discloses Lactobacillus paracasei PL4.4s and application thereof. The Lactobacillus paracasei PL4.4s provided by the application is preserved in the China General Microbiological Culture Collection Center (CGMCC) and has a preservation number of CGMCC No. 32643 and a preservation time of November 15, 2024. The application also provides a fermentation product, a fermentation supernatant, a bacterial suspension, a bacterial agent prepared from the Lactobacillus paracasei PL4.4s and application of the fermentation product, the fermentation supernatant, the bacterial suspension and the bacterial agent in biological agents. The Lactobacillus paracasei PL4.4s provided by the application can resist acid, bile salts and artificial gastric juice, has a high lactic acid production rate and a high lactic acid production amount, and the fermentation product has antibacterial and antioxidant effects and has obvious inhibitory effects on enteropathogenic bacteria ETEC and SA.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms, in particular to a Lactobacillus paracasei PL4.4s and application thereof. BACKGROUND

[0002] Lactobacillus paracasei is a kind of probiotics widely used in food, medicine and health care fields, which has many functions such as regulating intestinal microecological balance and enhancing immunity. In recent years, with the increasing demand for health, more and more researches focus on the development of new functional probiotic strains and their applications. These researches not only promote the development of probiotic industry, but also bring significant value to improve the quality of human life.

[0003] In the existing technology, in order to meet the needs of different application scenarios, specific strains are usually screened to obtain probiotics with special functions. However, the functions of the known Lactobacillus paracasei are relatively single, and there are problems such as poor stability and weak tolerance to environmental changes. Especially in some specific application environments, such as high acidity or high temperature environment, the survival rate and activity of some Lactobacillus paracasei strains often decrease significantly, which limits the function of the strains.

[0004] Therefore, how to screen Lactobacillus paracasei strains with higher stability and adaptability has become a technical problem to be solved. SUMMARY

[0005] In order to overcome the problems of poor stability and weak tolerance to environmental changes of the existing Lactobacillus paracasei, the present application provides a Lactobacillus paracasei PL4.4s and application thereof.

[0006] In a first aspect, the present application provides a Lactobacillus paracasei PL4.4s, which adopts the following technical solution:

[0007] The Lactobacillus paracasei is Lactobacillus paracasei PL4.4s, which is preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC No.32643 and the preservation time of November 15, 2024.

[0008] The Lactobacillus paracasei PL4.4s provided in the application has excellent acid tolerance, bile salt tolerance and artificial gastric juice tolerance, and has a high lactic acid production rate and a high lactic acid production amount. When the Lactobacillus paracasei PL4.4s provided in the application is applied to biological feed, it has strong tolerance and can replace antibiotics to inhibit SA and ETEC. In addition, the Lactobacillus paracasei PL4.4s provided in the application is isolated and screened from the intestinal contents and feces of healthy piglets, and thus has a safe source and is suitable for the characteristics of the intestinal tract of animals, which is beneficial to the preparation and application of animal feed.

[0009] In the application, the Lactobacillus paracasei PL4.4s colony has a smooth surface, a neat edge, and a milky white color. An obvious calcium carbonate dissolution ring can be observed in the MRS solid culture medium containing calcium carbonate. After Gram staining, the strain is rod-shaped and exists in single or chain form, as shown in FIG. 1. Figure 2

[0010] It is found through experiments that the Lactobacillus paracasei PL4.4s has acid tolerance and can still survive under the condition of pH 2.5. The Lactobacillus paracasei PL4.4s has bile salt tolerance and can still survive under the condition of 0.3% high bile salt. The Lactobacillus paracasei PL4.4s has tolerance to artificial gastric and intestinal juice, and the survival rate can reach more than 80.53% after treatment with artificial gastric and intestinal juice. The extracellular fluid of the Lactobacillus paracasei PL4.4s after fermentation has an antioxidant effect, and the DPPH free radical clearance rate is 99.95%, the hydroxyl free radical clearance rate is 67.43%, and the superoxide anion free radical clearance rate is 64.86%. The extracellular fluid of the Lactobacillus paracasei PL4.4s after fermentation has an antibacterial effect, and the inhibition rate of pathogenic bacteria Enterotoxic Escherichia coli (ETEC) and / or Staphylococcus aureus (SA) is ≥99%.

[0011] In a second aspect, the application provides a fermentation product comprising the Lactobacillus paracasei PL4.4s.

[0012] ​The fermentation liquor obtained by fermenting Lactobacillus paracasei PL4.4s is rich in exopolysaccharide, protease, cellulase, pectinase and bacteriostatic substances, and has a bacteriostatic rate of greater than or equal to 99% on pathogenic bacteria such as enterotoxigenic Escherichia coli (ETEC) and / or Staphylococcus aureus (SA); and the bacteriostatic property is stable; in addition, the fermentation liquor also has good heat resistance and acid resistance, and can also resist various proteases (pepsin, trypsin, papain, protease K, etc.). The fermentation liquor can be used as an antibiotic substitute in biological feed and other biological agents, which can effectively prevent piglets from being infected with enterotoxigenic Escherichia coli and has great use significance.

[0013] Optionally, the preparation method of the fermentation liquor comprises the following steps: first, activating Lactobacillus paracasei PL4.4s to obtain a Lactobacillus paracasei PL4.4s seed solution; then inoculating the Lactobacillus paracasei PL4.4s seed solution into MRS liquid medium at a volume percentage of 0.5-10%, and oscillating and culturing for 24-48h to obtain the fermentation liquor.

[0014] Specifically, the preparation method of the fermentation liquor provided by the present application comprises the following steps:

[0015] (1) Lactobacillus paracasei PL4.4s seed solution preparation: inoculating Lactobacillus paracasei PL4.4s into MRS solid medium containing calcium carbonate, and statically culturing at 25-40℃ for 24-48h; screening single colonies with larger calcium-dissolving rings, inoculating into MRS liquid medium, and oscillating and culturing at 25-40℃ for 18-32h to obtain the Lactobacillus paracasei PL4.4s seed solution;

[0016] (2) Lactobacillus paracasei PL4.4s fermentation liquor preparation: inoculating the Lactobacillus paracasei PL4.4s seed solution into MRS liquid medium at a volume percentage of 0.5-10%, and oscillating and culturing for 24-48h to obtain the fermentation liquor.

[0017] In a third aspect, the present application provides a fermentation supernatant, which is the upper clear liquid obtained after centrifugation of the fermentation liquor.

[0018] It is found through experiments that the content of exopolysaccharide in the fermentation supernatant of Lactobacillus paracasei PL4.4s can reach 571.09±0.45μg / mL; the protease activity produced by Lactobacillus paracasei PL4.4s in one fermentation is 36.45±0.05U / mL, the cellulase activity is 26.55±0.11U / mL, and the pectinase activity is 61.38±0.09U / mL.

[0019] The application finds through experimental exploration that the fermentation supernatant of Lactobacillus paracasei PL4.4s has heat stability, and the antibacterial activity of the fermentation supernatant is not affected after being treated at high temperatures of 65 DEG C, 85 DEG C and 100 DEG C. The fermentation supernatant of Lactobacillus paracasei PL4.4s has acid resistance, and the antibacterial activity of the fermentation supernatant is not affected in a pH 3 environment. The fermentation supernatant of Lactobacillus paracasei PL4.4s does not contain hydrogen peroxide, and the antibacterial activity of the fermentation supernatant is not affected after being treated by catalase. The fermentation supernatant of Lactobacillus paracasei PL4.4s can resist multiple enzymes, and the antibacterial activity of the fermentation supernatant is not affected after being treated by protease (pepsin, trypsin, papain or proteinase K).

[0020] Optionally, the preparation method of the fermentation supernatant comprises the following steps: centrifuging the fermentation product at 8000-12000 rpm / min for 10-20 min, taking the supernatant, and obtaining the fermentation supernatant.

[0021] In a fourth aspect, the application provides a bacterial suspension, which comprises the Lactobacillus paracasei PL4.4s.

[0022] The application finds through experimental exploration that the bacterial suspension of Lactobacillus paracasei PL4.4s has a DPPH free radical scavenging rate of 99.95%, a hydroxyl free radical scavenging rate of 67.43%, and a superoxide anion free radical scavenging rate of 64.86%.

[0023] In a fifth aspect, the application provides a bacterial agent, which comprises the Lactobacillus paracasei PL4.4s.

[0024] In a sixth aspect, the application provides application of the Lactobacillus paracasei PL4.4s, the fermentation product, the fermentation supernatant, the bacterial suspension or the bacterial agent in biological preparations.

[0025] In summary, the application has the following beneficial effects:

[0026] 1. The application provides a Lactobacillus paracasei PL4.4s, which is preserved in the China General Microbiological Culture Collection Center (CGMCC) and has a preservation number of CGMCC No. 32643 and a preservation time of November 15, 2024.

[0027] 2. The Lactobacillus paracasei PL4.4s provided by the application has excellent acid resistance, bile salt resistance and artificial gastric juice resistance, and has a fast acid production rate, a high lactic acid production amount, and a 24h lactic acid production amount of 231.11 mmol / L.

[0028] 3. The fermentation product of Lactobacillus paracasei PL4.4s provided by the present application is rich in exopolysaccharide, protease, cellulase, pectinase and bacteriostatic substance, etc., and has a bacteriostatic rate of ≥99% on pathogenic bacteria such as enterotoxigenic Escherichia coli and / or Staphylococcus aureus, and the bacteriostatic property is stable; in addition, the fermentation product of Lactobacillus paracasei PL4.4s also has good heat resistance and acid resistance, and can also resist various proteases (pepsin, trypsin, papain, proteinase K, etc.). The fermentation product is used as an antibiotic substitute in biological feed and other biological agents, which can effectively prevent piglets from being infected with enterotoxigenic Escherichia coli, and has great use significance.

[0029] 4. The bacterial suspension of Lactobacillus paracasei PL4.4s provided by the present application has good antioxidant performance, and has a DPPH free radical clearance rate of 99.95%, a hydroxyl radical clearance rate of 67.43%, and a superoxide anion radical clearance rate of 64.86%.

[0030] 5. Lactobacillus paracasei PL4.4s provided by the present application is isolated and screened from the intestinal contents and feces of healthy piglets, so it is safe in origin and suitable for the characteristics of animal intestinal tract, which is conducive to the preparation and application of animal feed. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is a phylogenetic tree of Lactobacillus paracasei PL4.4s strain based on 16S rDNA sequence;

[0032] Figure 2 is an observation diagram of Lactobacillus paracasei PL4.4s after Gram staining;

[0033] Figure 3 is the growth curve and change of acid production rate of Lactobacillus paracasei PL4.4s strain;

[0034] Figure 4 is an observation diagram of the inhibition zone test result of Lactobacillus paracasei PL4.4s on enterotoxigenic Escherichia coli;

[0035] Figure 5 is an observation diagram of the inhibition zone test result of Lactobacillus paracasei PL4.4s on Staphylococcus aureus. DETAILED DESCRIPTION

[0036] The present application is further described in detail below in combination with the drawings and examples, and it is particularly pointed out that: in the following examples, the specific conditions are not specified, and the conventional conditions or the conditions recommended by the manufacturer are used, and the raw materials used in the following examples can be obtained from ordinary market sales unless otherwise specified.

[0037] The application provides a Lactobacillus paracasei PL4.4s, which is isolated and screened from intestinal contents and feces of healthy piglets, and is identified as Lactobacillus paracasei through 16S rDNA, and is named as Lactobacillus paracasei PL4.4s, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No. 32643, and the preservation time is November 15, 2024.

[0038] The application provides a fermentation supernatant, and a preparation method thereof is as follows:

[0039] (1) Seed liquid preparation of Lactobacillus paracasei PL4.4s: inoculate Lactobacillus paracasei PL4.4s strains into MRS solid culture medium containing calcium carbonate, and perform static culture at 25-40 DEG C for 24-48 h; select single colonies with larger calcium-dissolving rings, inoculate into MRS liquid culture medium, and perform oscillation culture at 25-40 DEG C for 18-32 h to obtain seed liquid of Lactobacillus paracasei PL4.4s.

[0040] (2) Fermentation product preparation of Lactobacillus paracasei PL4.4s: inoculate the seed liquid of Lactobacillus paracasei PL4.4s into MRS liquid culture medium at a volume percentage of 0.5-10%, and perform oscillation culture for 24-48 h to obtain a fermentation product.

[0041] (3) Preparation of fermentation supernatant of Lactobacillus paracasei PL4.4s: centrifuge the fermentation product at 8000-12000 rpm / min for 10-20 min, take the supernatant to obtain the fermentation supernatant.

[0042] The culture medium and raw material sources involved in the examples of the application are as follows:

[0043] MRS solid culture medium: 10.0 g / L of proteose peptone, 5.0 g / L of beef powder, 4.0 g / L of yeast powder, 20.0 g / L of glucose, 0.2 g / L of magnesium sulfate, 5.0 g / L of sodium acetate, 2.0 g / L of triammonium citrate, 2.0 g / L of potassium phosphate dibasic, 0.05 g / L of manganese sulfate, 1.0 g / L of Tween 80 and 15.0 g / L of agar, pH 6.2±0.2;

[0044] MRS broth culture medium: 10.0 g / L of proteose peptone, 10.0 g / L of beef powder, 5.0 g / L of yeast powder, 20.0 g / L of glucose, 0.1 g / L of magnesium sulfate, 5.0 g / L of sodium acetate, 2.0 g / L of ammonium citrate, 2.0 g / L of potassium phosphate dibasic, 0.05 g / L of manganese sulfate and 1.0 g / L of Tween 80, pH 6.2±0.2;

[0045] Acid-resistant MRS broth medium: 10.0 g / L of proteose peptone, 10.0 g / L of beef extract, 5.0 g / L of yeast extract, 20.0 g / L of glucose, 0.1 g / L of magnesium sulfate, 5.0 g / L of sodium acetate, 2.0 g / L of ammonium citrate, 2.0 g / L of potassium phosphate dibasic, 0.05 g / L of manganese sulfate, and 1.0 g / L of Tween 80, pH 2.0 / 3.0;

[0046] Cholate-resistant MRS broth medium: 10.0 g / L of proteose peptone, 10.0 g / L of beef extract, 5.0 g / L of yeast extract, 20.0 g / L of glucose, 0.1 g / L of magnesium sulfate, 5.0 g / L of sodium acetate, 2.0 g / L of ammonium citrate, 2.0 g / L of potassium phosphate dibasic, 0.05 g / L of manganese sulfate, and 1.0 g / L of Tween 80, 3 / 5 g of porcine cholate.

[0047] The indicator bacteria involved in the embodiments of the present application, enterotoxigenic Escherichia coli (model number ATCC35401) and Staphylococcus aureus (model number ATCC29213), are obtained from common markets;

[0048] The artificial gastrointestinal fluid involved in the embodiments of the present application is purchased from Beijing Regen Biotechnology Co., Ltd.;

[0049] The lactic acid (LD) test kit involved in the embodiments of the present application is purchased from Nanjing Jiancheng Biological Engineering Institute;

[0050] The catalase, pepsin, trypsin, and protease K involved in the embodiments of the present application are purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0051] The calcium carbonate involved in the embodiments of the present application is purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.;

[0052] The intestinal contents and fresh feces of piglets involved in the embodiments of the present application are collected from Faguo Kangda Ecological Agriculture Co., Ltd.;

[0053] The strain activation referred to in the embodiments of the present application refers to the process of streaking the strain from a preservation tube into a solid culture medium plate and growing single colonies.

[0054] The present application is further described in detail below in combination with the embodiments, performance detection tests, and the description of the accompanying drawings.

[0055] Embodiment 1

[0056] (I) Isolation, screening, and detection of strains

[0057] (1) Enrichment: 10 g of sample is enriched in 100 mL of MRS broth medium, and cultured at 37°C for 24-48 h.

[0058] (2) Isolation: Take 1 mL of enriched bacteria solution and place it in 10 mL of MRS broth medium (pH 2.5), incubate at 37°C for 12 h, then take 1 mL and place it in 10 mL of 0.3% bile salt MRS broth medium, incubate at 37°C for 12 h, dilute and spread on CaCO3 MRS plates, select colonies with large calcium-dissolving rings and fast growth, and purify the colonies on MRS solid medium, and store the purified strains at -80°C in glycerol.

[0059] Biological identification of target strains: inoculate the isolated strains in MRS broth medium, incubate at 37°C for 18-32 h, perform morphological identification and 16S rDNA molecular biological identification, and send the strains to Huada Gene Technology Co., Ltd. for identification. Perform BLAST analysis of the sequencing results in the National Center for Biotechnology Information (NCBI) database, and construct a phylogenetic tree using MEGA5.0 software, as shown in Figure 1

[0060] A total of 105 colonies with calcium-dissolving rings were screened from the intestinal tracts of healthy piglets and fresh feces, and morphological identification showed that 26 of them were gram-positive bacilli. The 26 strains were subjected to 16S rDNA sequence amplification and sequencing, and the sequences were compared using BLAST of GenBank. Four of the strains belonged to the Lactobacillus paracasei genus, with 99% similarity to Lactobacillus paracasei. They were named Lactobacillus paracasei PL4.3s, Lactobacillus paracasei PL4.4s, Lactobacillus paracasei PLp2s, and Lactobacillus paracasei PLp4s, respectively.

[0061] (II) Determination and analysis of the acid production rate and acid production capacity of the four strains

[0062] The acid production rate and acid production capacity of the four strains were determined and analyzed as follows:

[0063] Inoculate the strains into 100 mL of sterile MRS broth medium at a 2% inoculation amount, incubate at 37°C, take samples at 0, 2, 4, 8, 12, 16, 20, and 24, measure the OD600 value, and record the pH value at each time point; measure the lactic acid concentration after 24 h, with three replicates for each strain. The lactic acid concentration was measured using a lactic acid (LD) test kit, and the results are shown in Table 1. The growth curves of the four strains and the changes in the acid production rate are shown in Figure 3

[0064] Table 1 Lactic acid content produced by the four Lactobacillus paracasei strains

[0065] Strain No. PL4.3s PL4.4s PLp2s PLp4s Lactic acid content (mmol / L) 188.88±0.38 231.11±0.25 177.67±0.39 187.18±0.41

[0066] According to Figure 3 ​​The detection results of Table 1 and Table 2 show that the growth rate and acid production ability of Lactobacillus paracasei PL4.4s are optimal, and the lactic acid content after 24 hours is the highest, reaching 231.11 mmol / L.

[0067] (III) Tolerance analysis of Lactobacillus paracasei PL4.4s strain

[0068] The tolerance of Lactobacillus paracasei PL4.4s strain to gastrointestinal tract was investigated, and the specific process was as follows:

[0069] The Lactobacillus paracasei PL4.4s strain was centrifuged and washed, and then resuspended in an equal volume of artificial animal gastric juice to simulate the digestion of gastric juice. After 4 hours of culture at 37°C and 80 rpm, the artificial gastric juice was centrifuged to obtain the digested bacterial cells, which were then resuspended in an equal volume of artificial animal intestinal juice and cultured under the same conditions as the gastric juice for 5 hours. The viable cell count was determined, and the survival rate was calculated. The results are shown in Table 2.

[0070] Table 2: Results of artificial gastric and intestinal juice tolerance test of Lactobacillus paracasei PL4.4s

[0071]

[0072] According to the detection results of Table 2, the PL4.4s of the present application can still maintain more than 80% of the viable cell count after 4 hours of gastric juice culture and 5 hours of intestinal juice treatment under the influence of test factors such as centrifugation and resuspension. Therefore, it is shown that the Lactobacillus paracasei PL4.4s strain provided by the present application has strong tolerance to the gastrointestinal tract and can successfully pass through the stomach and intestines to reach the small intestine and play a role.

[0073] (IV) Determination of the antibacterial effect of Lactobacillus paracasei PL4.4s strain

[0074] The antibacterial effect of Lactobacillus paracasei PL4.4s strain was detected, and the specific process was as follows:

[0075] The Lactobacillus paracasei PL4.4s strain provided by the present application was used to determine the diameter of the inhibition zone against enterotoxigenic Escherichia coli and Staphylococcus aureus using the Oxford cup plate method. Four parallel tests were performed, the average value of the inhibition zone diameter was calculated, and the average value was taken. The results are shown in Figure 4-5 and Table 3 below.

[0076] Figure 4 The observation chart of the inhibition zone test results of Lactobacillus paracasei PL4.4s against enterotoxigenic Escherichia coli is shown in Figure 1. Figure 5 The observation chart of the inhibition zone test results of Lactobacillus paracasei PL4.4s against Staphylococcus aureus is shown in Figure 2.

[0077] Table 3: Results of antibacterial detection experiment of Lactobacillus paracasei PL4.4s

[0078] Strain No. E. coli (mm) S. aureus (mm) PL4.4s 24.77±0.32 28.88±0.24

[0079] According to Figure 4-5 As shown in the test results in Table 3, Lactobacillus paracasei PL4.4s has good antibacterial activity against enterotoxigenic Escherichia coli and Staphylococcus aureus, and the diameters of the inhibition zones against Escherichia coli and Staphylococcus aureus are 24.77 mm and 28.38 mm, respectively.

[0080] Example 2

[0081] Example 2 provides a fermentation supernatant, the preparation method of which is as follows:

[0082] (1) Preparation of Lactobacillus paracasei PL4.4s seed liquid: Lactobacillus paracasei PL4.4s was inoculated into MRS solid medium containing calcium carbonate, incubated at 37°C for 36 h, single colonies with larger calcium dissolution zones were selected, inoculated into MRS liquid medium, and cultured at 37°C with shaking for 24 h to obtain Lactobacillus paracasei PL4.4s seed liquid;

[0083] (2) Preparation of Lactobacillus paracasei PL4.4s fermentation product: Lactobacillus paracasei PL4.4s seed liquid was inoculated into MRS liquid culture medium at a volume percentage of 2%, and the culture was shaken for 36 hours to obtain the fermentation product.

[0084] (3) Preparation of Lactobacillus paracasei PL4.4s fermentation supernatant: The fermented product was centrifuged at 10,000 rpm / min for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter to obtain a fermentation supernatant.

[0085] (1) Antibacterial rate detection

[0086] The antibacterial activity of the fermentation supernatant obtained in Example 2 was tested, and the specific process was as follows:

[0087] (1) Take the indicator bacteria solution (Escherichia coli and Staphylococcus aureus) and add it to LB liquid medium (generally, add 1 mL of the bacterial solution to 100 mL of LB liquid medium) and measure the absorbance at 600 nm until the absorbance is approximately OD600 = 0.05;

[0088] (2) The fermentation supernatant was added to 10 mL of LB liquid medium (with indicator bacteria added) at a volume ratio of 1% and mixed to make three parallel groups; at the same time, a blank control A was set up. 空 (without adding fermentation supernatant), culture at 37°C in a shaker at 200 r / min for 12 h, measure the OD value of each sample at a wavelength of 600 nm using a spectrophotometer, record the reading (zeroed with sterile LB medium), and calculate the inhibition rate of each fermentation supernatant. The test results are shown in Table 4 below; the calculation formula for the inhibition rate is as follows:

[0089] Bacteriostatic rate (%) = [(A 空 -A 空0 )-(A 样 -A 样0 ) / (A 空 -A 空0 )] x 100%.

[0090] Wherein, A 样0 represents the initial OD600 of the fermentation supernatant and LB medium (containing indicator bacteria) mixture 0h, A 样 represents the OD600 of the mixture after 12h culture, A 空0 represents the initial OD600 of the blank control group 0h, A 空 represents the OD600 of the mixture of the blank control group after 12h culture.

[0091] Table 4 Detection results of bacteriostatic rate of L. paracasei PL4.4s fermentation supernatant

[0092] Strain No. Enterotoxigenic E. coli inhibition rate (%) S. aureus inhibition rate (%) PL4.4s 99.99±0.01 99.99±0.01

[0093] According to the detection results in Table 4, the fermentation supernatant of L. paracasei PL4.4s showed very high inhibition effect on ETEC and S. aureus.

[0094] (II) Bacteriostatic stability detection

[0095] The effects of hydrogen peroxide, temperature, protease, and acidic and alkaline pH on the bacteriostatic performance of the fermentation supernatant were investigated, and the results are shown in Table 5 below.

[0096] (1) Hydrogen peroxide: 1.5 mg / mL catalase was added to the fermentation supernatant of L. paracasei PL4.4s, and the control group was not treated with catalase. After 2h water bath at 37℃, the effect of hydrogen peroxide in the fermentation broth was excluded. ETEC and SA were used as indicator bacteria, and the fermentation supernatant of PL4.4s was added to 10 mL of LB liquid medium (containing indicator bacteria) at a volume ratio of 1%. Three parallel groups were prepared for determination of bacteriostatic ability. The specific method refers to the bacteriostatic rate detection in the above-mentioned bacteriostatic experiment of L. paracasei PL4.4s strain.

[0097] (2) Temperature: The supernatant of L. paracasei PL4.4s was placed in a water bath at 60°C, 80°C and 100°C for 20 min, respectively. The supernatant of L. paracasei PL4.4s without high temperature treatment was used as a blank control. ETEC and SA were used as indicator bacteria. The supernatant of L. paracasei PL4.4s was added to 10 mL of LB liquid medium (containing indicator bacteria) at a volume ratio of 1%, and three parallel groups were prepared for determination of antibacterial ability. The specific method refers to the detection of antibacterial rate in the above-mentioned antibacterial experiment of L. paracasei PL4.4s strain.

[0098] (3) Protease: The supernatant of L. paracasei PL4.4s was treated with pepsin (reaction conditions: 37°C, pH 3), trypsin (reaction conditions: 37°C, pH 8), proteinase K (reaction conditions: 37°C, pH 8), and papain (reaction conditions: 50-60°C, pH 6.7) at a concentration of 3 mg / mL for 2 h. After treatment, the supernatant was adjusted back to the original pH value. The same pathogenic bacteria as described above were used as indicator bacteria. The supernatant of L. paracasei PL4.4s was added to 10 mL of LB liquid medium (containing indicator bacteria) at a volume ratio of 1%, and three parallel groups were prepared for determination of antibacterial ability.

[0099] (4) Acid-base pH: The supernatant was adjusted to pH 3 and pH 6.5 with 0.1 mol / L NaOH, and its antibacterial activity was tested.

[0100] Table 5 Detection results of antibacterial rate of L. paracasei PL4.4s supernatant under different treatment conditions

[0101] / E. coli (%) S. aureus (%) Untreated 99.99±0.03 99.98±0.01 Peroxidase 99.86±0.19 99.69±0.09 65°C, 20 min 99.97±0.28 99.93±0.22 85°C, 20 min 99.85±0.32 99.89±0.21 100°C, 20 min 99.65±0.14 99.67±0.26 Pepsin 99.86±0.23 99.88±0.45 Trypsin 99.94±0.34 99.89±0.38 Proteinase K 99.87±0.17 99.89±0.24 Papain 99.86±0.31 99.91±0.25 PH 3 99.89±0.46 99.58±0.37 PH 6.5 19.88±0.64 24.25±0.56

[0102] According to the detection results of Table 5, it is found that the fermentation supernatant of Lactobacillus paracasei PL4.4s treated by catalase still has an antibacterial rate of ≥99% against Escherichia coli and an antibacterial rate of ≥99% against Staphylococcus aureus, indicating that the fermentation process of Lactobacillus paracasei PL4.4s does not produce hydrogen peroxide. After the fermentation supernatant of Lactobacillus paracasei PL4.4s is treated at high temperatures of 65°C, 85°C and 100°C for 20 min, the antibacterial rate of each fermentation supernatant against Escherichia coli is still ≥99%, and the antibacterial rate against Staphylococcus aureus is still ≥99%, indicating that the fermentation supernatant of Lactobacillus paracasei PL4.4s has very good heat resistance. After the fermentation supernatant of Lactobacillus paracasei PL4.4s is treated by pepsin, trypsin, proteinase K and papain, respectively, the antibacterial rate of each fermentation supernatant against Escherichia coli is still ≥99%, and the antibacterial rate against Staphylococcus aureus is still ≥99%, indicating that the fermentation supernatant of Lactobacillus paracasei PL4.4s can resist multiple enzymes. After the pH of the fermentation supernatant of Lactobacillus paracasei PL4.4s is adjusted to 3, it is found that the antibacterial rate of the fermentation supernatant of Lactobacillus paracasei PL4.4s against Escherichia coli is still ≥99%, and the antibacterial rate against Staphylococcus aureus is still ≥99%, indicating that the fermentation supernatant of Lactobacillus paracasei PL4.4s has good acid resistance; after the pH of the fermentation supernatant of Lactobacillus paracasei PL4.4s is adjusted to 6.5, the antibacterial activity of the fermentation supernatant of Lactobacillus paracasei PL4.4s is significantly reduced, and the antibacterial rate is only about 20%, indicating that the fermentation supernatant of Lactobacillus paracasei PL4.4s has poor alkali resistance.

[0103] Example 3

[0104] Example 3 provides a fermentation supernatant, and a preparation method thereof is as follows:

[0105] (1) Preparation of Lactobacillus paracasei PL4.4s seed liquid: inoculate Lactobacillus paracasei PL4.4s strain into MRS solid medium containing calcium carbonate, and incubate at 37°C for 4 h to obtain Lactobacillus paracasei PL4.4s seed liquid.

[0106] (2) Preparation of Lactobacillus paracasei PL4.4s fermentation product: inoculate Lactobacillus paracasei PL4.4s seed liquid into MRS liquid medium at a volume percentage of 2%, and incubate at 37°C for 48 h to obtain fermentation product.

[0107] (3) Preparation of Lactobacillus paracasei PL4.4s fermentation supernatant: centrifuge the fermentation product at 5000 rpm / min for 20 min, collect the supernatant, and obtain fermentation supernatant.

[0108] Extracellular polysaccharide content detection

[0109] The exopolysaccharide in the fermentation supernatant is extracted, and the exopolysaccharide content is detected, and the specific process is as follows:

[0110] (1) Trichloroacetic acid treatment: slowly add trichloroacetic acid with a concentration of 800 mg / mL to the fermentation supernatant to make the final concentration 40 mg / mL, stand at 4°C for 8h, then centrifuge at 10000 rpm, 4°C for 10 min, discard the precipitate, and collect the supernatant.

[0111] (2) Anhydrous ethanol treatment: add anhydrous ethanol to the supernatant at a volume ratio of 1:4, 4°C overnight, centrifuge at 10000 rpm, 4°C for 10 min, collect the bottom precipitate; dissolve the precipitate in 60°C distilled water, centrifuge at 10000 rpm for 10 min, discard the insoluble precipitate at the bottom, and collect the supernatant.

[0112] (3) Dialysis: load the supernatant into a dialysis bag with a molecular weight cut-off of 8KDa, dialyze for 2h, replace the dialysate, then replace it every 6h, and dialyze continuously for 2d; finally collect the polysaccharide solution in the dialysis bag, vacuum freeze-dry to obtain exopolysaccharide.

[0113] (4) Detection: first dry the glucose for 2h, then take 100mg of glucose in a 1L volumetric flask, shake well, and dilute to volume; add the reaction system according to the table below, and measure the absorbance at 490nm after cooling; each group is done in triplicate; draw a standard curve with glucose content (μg / mL) as the abscissa and absorbance value (A490) as the ordinate. The exopolysaccharide yield of the strain is calculated by the regression equation.

[0114] After detection, the content of exopolysaccharide secreted by Lactobacillus paracasei PL4.4s is 571.09±0.45 μg / mL. Therefore, it is proved that the Lactobacillus paracasei PL4.4s provided by the present application can secrete exopolysaccharide, and can obtain fermentation supernatant with high exopolysaccharide content.

[0115] Example 4

[0116] Example 4 provides a fermentation supernatant, and the preparation method is as follows:

[0117] (1) Seed liquid preparation of Lactobacillus paracasei PL4.4s: inoculate Lactobacillus paracasei PL4.4s strain into MRS solid medium containing calcium carbonate, stand at 37°C for 36h, select single colonies with larger calcium-dissolving rings, inoculate into MRS liquid medium, and shake culture at 37°C for 24h to obtain Lactobacillus paracasei PL4.4s seed liquid.

[0118] (2) Preparation of L. paracasei PL4.4s fermentation product: The seed liquid of L. paracasei PL4.4s was inoculated into MRS liquid medium at a volume percentage of 2%, and after 48h of shaking culture, a fermentation product was obtained.

[0119] (3) Preparation of L. paracasei PL4.4s fermentation supernatant: The fermentation product was centrifuged at 5000rpm / min for 20min, and the supernatant was taken to obtain the fermentation supernatant.

[0120] Enzyme production property detection

[0121] The fermentation supernatant obtained in Example 4 was diluted, and then enzyme activity determination was performed, as follows:

[0122] (1) Cellulase activity determination: The cellulase activity in the crude enzyme liquid was determined according to the spectrophotometric method for determination of cellulase activity of feed additives in NY / T 912-2004.

[0123] (2) Protease activity determination: The protease activity in the crude enzyme liquid was determined according to the method for determination of protease activity in GB / T 28715-2012.

[0124] (3) Pectinase activity determination: The pectinase activity in the crude enzyme liquid was determined according to the method for determination of pectinase preparation in QB 1502-92.

[0125] It was detected that the protease activity in the fermentation supernatant obtained in Example 4 was 36.45±0.05U / mL, the cellulase activity was 26.55±0.11U / mL, and the pectinase activity was 61.38±0.09U / mL. Therefore, it is proved that the L. paracasei PL4.4s primary fermentation provided in the present application can produce protease, cellulase and pectinase, and the application thereof in fermented feed is more conducive to the digestion and absorption of livestock and poultry, and improves the growth and health of animal body

[0126] Example 5

[0127] Example 5 provides a L. paracasei PL4.4s bacterial suspension, and the preparation method thereof is as follows:

[0128] (1) Preparation of L. paracasei PL4.4s seed liquid: The L. paracasei PL4.4s strain was inoculated into MRS solid medium containing calcium carbonate, and was placed and cultured at 37℃ for 36h. Single colonies with larger calcium-dissolving rings were selected, inoculated into MRS liquid medium, and shaken and cultured at 37℃ for 24h to obtain the L. paracasei PL4.4s seed liquid.

[0129] (2) Preparation of L. paracasei PL4.4s bacterial suspension: The seed liquid of L. paracasei PL4.4s was inoculated into MRS liquid medium at a volume percentage of 2%, and after 48h of shaking culture, a bacterial suspension was obtained.

[0130] Antioxidant capacity assay

[0131] Antioxidant capacity assay was performed on the bacterial suspension obtained from Example 5, in accordance with the following procedure:

[0132] (1) DPPH radical scavenging capacity

[0133] Take 2 mL of bacterial suspension, add 2 mL of 0.2 mmol / L DPPH anhydrous ethanol solution, which is Ai, mix well, and react for 30 min at room temperature in the dark. After centrifugation at 3000 x g for 10 min, measure the absorbance Ai of the supernatant at 517 nm, and calculate the DPPH radical scavenging rate. Each group is measured in triplicate, and the average value is taken. Aj is 2 mL of bacterial suspension + 2 mL of anhydrous ethanol, and Ac is 2 mL of anhydrous ethanol + 2 mL of DPPH. The calculation formula of DPPH radical scavenging rate is as follows:

[0134] DPPH radical scavenging rate (%) = (1 - (Ai - Aj) / Ac) x 100%

[0135] (2) Hydroxyl radical scavenging capacity

[0136] Take 1 mL of 2.5 mmol / L phenanthroline, add 1 mL of PBS buffer, 1 mL of distilled water in turn, mix well, and then add 1 mL of 2.5 mmol / L FeSO4 and 1 mL of 20 mmol / L H2O2, and react for 60 min at 37°C. Take the supernatant, and then measure the absorbance A(A 对 ) of the control group at 536 nm. Use 1 mL of distilled water instead of 1 mL of H2O2 as the blank group, and measure the absorbance A(A 空 ) of the blank group. Use 1 mL of bacterial suspension instead of 1 mL of distilled water as the sample group, and measure the absorbance A(A 样 ) of the sample group. Calculate the hydroxyl radical scavenging rate, and each group is measured in triplicate, and the average value is taken. The calculation formula of hydroxyl radical scavenging rate is as follows:

[0137] Hydroxyl radical scavenging rate (%) = (A(A 样 -A(A 对 )) / (A(A 空 -A(A 对 )) x 100%

[0138] (3) Superoxide anion radical scavenging capacity

[0139] Mix 0.5 mL bacterial suspension with 1.5 mL Tris-HCl solution (pH 8.0), after reaction at 25℃ for 20 min, add 200 μL, 25 mmol / L o-phenanthroline, react for 5 min, then add 250 μL HCl to terminate the reaction, centrifuge at 3000 x g for 10 min, take the supernatant to measure the absorbance A (A 样 ) of the sample group at 325 nm. Use 0.5 mL distilled water instead of 0.5 mL bacterial suspension as a control group, measure the absorbance A (A 对 ) of the control group. Calculate the superoxide anion radical scavenging rate, measure 3 times for each group in parallel, and take the average value. The calculation formula of the superoxide anion radical scavenging rate is as follows:

[0140] Superoxide anion radical scavenging rate (%) = (1-A 样 / A 对 ) x 100%

[0141] The detection results are shown in Table 6.

[0142] Table 6 Determination results of the antioxidant capacity of L. paracasei PL4.4s

[0143] / Lactobacillus paracasei PL4.4s DPPH free radical scavenging rate (%) 99.95 Hydroxyl radical scavenging rate (%) 67.43 Superoxide anion radical scavenging rate (%) 64.86

[0144] According to the detection results in Table 6, the DPPH radical scavenging rate of the bacterial suspension of L. paracasei PL4.4s is 99.95%, the hydroxyl radical scavenging rate is 67.43%, and the superoxide anion radical scavenging rate is 64.86%. Therefore, it is shown that the L. paracasei PL4.4s provided in the application has good antioxidant effect, and can be used for preparing biological feed and other preparations, and has good practical significance.

[0145] Although the present application has been described in detail in the foregoing with general description and specific embodiments, some modifications or improvements can be made on the basis of the present application, which is obvious to those skilled in the art. Therefore, these modifications or improvements made on the basis of not deviating from the spirit of the present application, all belong to the scope of the present application claimed.

Claims

1. A Lactobacillus paracasei ( Lactobacillus paracasei ) PL4.4s, characterized in that, The Lactobacillus paracasei is preserved in China General Microbiological Culture Collection Center (CGMCC) with a preservation number of CGMCC No. 32643 and a preservation time of November 15, 2024.

2. A ferment, characterized by, The fermentate comprises the Lactobacillus paracasei PL4.4s of claim 1.

3. The ferment according to claim 2, characterized in that, The preparation method of the fermentate comprises the following steps: First, the Lactobacillus paracasei PL4.4s is activated to obtain a Lactobacillus paracasei PL4.4s seed solution; then the Lactobacillus paracasei PL4.4s seed solution is inoculated into MRS liquid medium at a volume percentage of 0.5-10 %, and is subjected to oscillation culture for 24-48 h to obtain the fermentate.

4. A fermentation supernatant, characterized in that, The fermentation supernatant is a supernatant obtained by centrifugation of the fermentate of claim 2.

5. The fermentation supernatant according to claim 4, characterized in that, The preparation method of the fermentation supernatant comprises the following steps: centrifuging the fermentate at 8000-12000 rpm / min for 10-20 min, and taking the supernatant to obtain the fermentation supernatant.

6. A bacterial suspension, characterized in that, The bacterial suspension comprises the Lactobacillus paracasei PL4.4s of claim 1.

7. An inoculant characterized in that, The bacterial agent comprises the Lactobacillus paracasei PL4.4s of claim 1.

8. Use of Lactobacillus paracasei PL4.4s according to claim 1, of the ferment according to claim 2 or 3, of the fermentation supernatant according to claim 4 or 5, of the bacterial suspension according to claim 6 or of the bacterial agent according to claim 7 as a biological agent, characterized in that, The biological agent has antioxidant properties and tolerance to artificial gastric and intestinal juice, can produce acid and lactic acid, extracellular polysaccharide, protease, cellulase and pectinase, and can effectively inhibit enterotoxigenic Escherichia coli and / or Staphylococcus aureus.

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