Lactobacillus paracasei PL4.4 s and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses lactobacillus paracasei PL4.4 s and application thereof. The lactobacillus paracasei PL4.4 s provided by the invention is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.32643, and the preservation time is November 15, 2024; the invention also provides a fermentation product, a fermentation supernatant, a bacterial suspension and a microbial inoculum prepared from the lactobacillus paracasei PL4.4 s, and applications of the fermentation product, the fermentation supernatant, the bacterial suspension and the microbial inoculum in biological preparations. The lactobacillus paracasei PL4.4 s provided by the invention can resist acid, bile salt and artificial gastric juice, and is high in acid production rate and high in lactic acid yield; the fermented product also has antibacterial and antioxidant effects, and has an obvious inhibition effect on enteropathogenic bacteria ETEC and SA.

Description

Technical Field

[0001] This application relates to the field of microbial technology, and specifically relates to a Lactobacillus paracasei PL4.4s and its applications. Background Art

[0002] Lactobacillus paracasei is a probiotic widely used in the fields of food, medicine, and health care, with various functions such as regulating the balance of the intestinal microecology and enhancing immunity. In recent years, with the increasing demand for health, more and more research has focused on the development of new functional probiotic strains and their applications. These studies have not only promoted the development of the probiotic industry but also brought significant value to improving the quality of human life.

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

[0004] Therefore, how to screen Lactobacillus paracasei strains with higher stability and adaptability has become an urgent technical problem to be solved. Summary of the Invention

[0005] In order to overcome the problems of poor stability and weak ability to tolerate environmental changes existing in the existing Lactobacillus paracasei, this application provides a Lactobacillus paracasei PL4.4s and its applications.

[0006] In the first aspect, the Lactobacillus paracasei PL4.4s provided by this application adopts the following technical solution: A Lactobacillus paracasei, the Lactobacillus paracasei is Lactobacillus paracasei ( Lactobacillus paracasei ) PL4.4s, deposited in the China General Microbiological Culture Collection Center (CGMCC), with the deposit number CGMCC No. 32643, and the deposit date is November 15, 2024.

[0007] The Lactobacillus paracasei PL4.4s provided by this application has excellent acid resistance, bile salt resistance, and artificial gastric juice resistance, and has a fast acid production rate and a high lactic acid production; when the Lactobacillus paracasei PL4.4s provided by this 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 by this application is isolated and screened from the intestinal contents and feces of healthy piglets, so its source is safe and suitable for the characteristics of the animal intestine, which is beneficial to the preparation and application of animal feed.

[0008] In this application, the surface of the Lactobacillus paracasei PL4.4s colony is smooth, with neat edges, milky white in color. In the MRS solid medium containing calcium carbonate, an obvious calcium dissolution zone can be seen. After Gram staining, the strain is rod-shaped and exists in the form of single or chains, as Figure 2 shown.

[0009] Through experimental exploration, it is found in this application that Lactobacillus paracasei PL4.4s has acid resistance and can still survive under the condition of pH 2.5; Lactobacillus paracasei PL4.4s has bile salt tolerance and can still survive under the condition of 0.3% high bile salt; Lactobacillus paracasei PL4.4s can tolerate artificial gastrointestinal fluid, and after being treated with artificial gastrointestinal fluid, the survival rate can reach more than 80.53%; the extracellular fluid after fermentation by Lactobacillus paracasei PL4.4s has antioxidant effects, with the DPPH free radical scavenging rate of 99.95%, the hydroxyl free radical scavenging rate of 67.43%, and the superoxide anion free radical scavenging rate of 64.86%. The extracellular fluid after fermentation by Lactobacillus paracasei PL4.4s has antibacterial effects, and the antibacterial rate against the pathogenic bacteria Enterotoxic Escherichia coli (ETEC) and / or Staphylococcus aureus (SA) is ≥99%.

[0010] In the second aspect, this application provides a fermented product, including the Lactobacillus paracasei PL4.4s described above.

[0011] In this application, through the fermentation of Lactobacillus paracasei PL4.4s, the obtained fermented product is rich in extracellular polysaccharides, protease, cellulase, pectinase, antibacterial substances, etc. Its antibacterial rate against the pathogenic bacteria Enterotoxic Escherichia coli (ETEC) and / or Staphylococcus aureus (SA) is ≥99%; and the antibacterial property is stable; in addition, this fermented product also has good heat resistance and acid resistance, and can also tolerate a variety of proteases (pepsin, trypsin, papain, proteinase K, etc.). Applying the above fermented product as an antibiotic substitute to biological preparations such as biological feed can effectively prevent piglets from being infected with Enterotoxic Escherichia coli, and has great application significance.

[0012] Optionally, the preparation method of the fermented product includes the following steps: First, activate Lactobacillus paracasei PL4.4s to obtain the Lactobacillus paracasei PL4.4s seed liquid; then inoculate the Lactobacillus paracasei PL4.4s seed liquid into the MRS liquid medium according to the volume percentage of 0.5 - 10%, and shake culture for 24 - 48h to obtain the fermented product.

[0013] Specifically, the preparation method of the ferment provided by this application includes the following steps: (1) Preparation of Lactobacillus paracasei PL4.4s seed liquid: Inoculate the Lactobacillus paracasei PL4.4s strain into MRS solid medium containing calcium carbonate, and incubate it statically at 25 - 40 °C for 24 - 48 h. Screen single colonies with larger calcium dissolution circles, inoculate them into MRS liquid medium, and incubate them with shaking at 25 - 40 °C for 18 - 32 h to obtain Lactobacillus paracasei PL4.4s seed liquid; (2) Preparation of Lactobacillus paracasei PL4.4s ferment: Inoculate the Lactobacillus paracasei PL4.4s seed liquid into MRS liquid medium according to a volume percentage of 0.5 - 10%, and after incubating with shaking for 24 - 48 h, obtain the ferment.

[0014] In the third aspect, this application provides a fermentation supernatant, and the fermentation supernatant is the upper clarified liquid obtained after centrifuging the ferment described in claim 2.

[0015] Through experimental exploration, it is found in this application that in the fermentation supernatant of Lactobacillus paracasei PL4.4s, the content of exopolysaccharide can reach 571.09 ± 0.45 μg / mL; the protease activity produced by the first - stage fermentation of Lactobacillus paracasei PL4.4s is 36.45 ± 0.05 U / mL, the cellulase activity is 26.55 ± 0.11 U / mL, and the pectinase activity is 61.38 ± 0.09 U / mL.

[0016] Through experimental exploration, it is found in this application that the fermentation supernatant of Lactobacillus paracasei PL4.4s has thermal stability, and its antibacterial activity is not affected after being treated at high temperatures of 65 °C, 85 °C, and 100 °C. The fermentation supernatant of Lactobacillus paracasei PL4.4s has acid resistance, and its antibacterial activity is not affected in a pH3 environment. The fermentation supernatant of Lactobacillus paracasei PL4.4s does not contain hydrogen peroxide, and its antibacterial activity is not affected after being treated with catalase. The fermentation supernatant of Lactobacillus paracasei PL4.4s can tolerate a variety of enzymes, and its antibacterial activity is not affected after being treated with proteases (pepsin, trypsin, papain, or proteinase K).

[0017] Optionally, the preparation method of the fermentation supernatant includes the following steps: Centrifuge the ferment at 8000 - 12000 rpm / min for 10 - 20 min, take the supernatant to obtain the fermentation supernatant.

[0018] In the fourth aspect, this application provides a bacterial suspension, and the bacterial suspension includes the Lactobacillus paracasei PL4.4s described above.

[0019] Through experimental exploration, it is found in this application 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%.

[0020] In a fifth aspect, this application provides a bacterial agent, which includes the aforementioned Lactobacillus paracasei PL4.4s.

[0021] In a sixth aspect, this application provides the use of Lactobacillus paracasei PL4.4s, its fermentate, fermentation supernatant, bacterial suspension or bacterial agent in a biological preparation.

[0022] In summary, this application has the following beneficial effects: 1. This application provides a Lactobacillus paracasei ( Lactobacillus paracasei) PL4.4s, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), with the preservation number CGMCC No. 32643, and the preservation time is November 15, 2024.

[0023] 2. The Lactobacillus paracasei PL4.4s provided in this application has excellent acid resistance, bile salt resistance and artificial gastric juice resistance, and has a fast acid production rate and a high lactic acid production. The lactic acid production can reach 231.11 mmol / L in 24 h.

[0024] 3. The fermentate of the Lactobacillus paracasei PL4.4s provided in this application is rich in extracellular polysaccharides, protease, cellulase, pectinase and antibacterial substances, etc. Its antibacterial rate against the pathogenic bacteria enterotoxigenic Escherichia coli and / or Staphylococcus aureus is ≥99%, and the antibacterial property is stable; in addition, the fermentate of Lactobacillus paracasei PL4.4s also has good heat resistance and acid resistance, and can also tolerate a variety of proteases (pepsin, trypsin, papain, proteinase K, etc.). Applying the above fermentate as an antibiotic substitute in biological preparations such as biological feeds can effectively prevent piglets from being infected with enterotoxigenic Escherichia coli, which has great application significance.

[0025] 4. The bacterial suspension of the Lactobacillus paracasei PL4.4s provided in this application has good antioxidant properties, with 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%.

[0026] 5. The Lactobacillus paracasei PL4.4s provided in this application is isolated and screened from the intestinal contents and feces of healthy piglets. Therefore, its source is safe and it is suitable for the characteristics of the animal intestine, which is beneficial to the preparation and application of animal feeds. Description of the Drawings

[0027] Figure 1 It is a phylogenetic tree constructed based on the 16S rDNA sequence of Lactobacillus paracasei strain PL4.4s; Figure 2 It is an observation diagram of Lactobacillus paracasei strain PL4.4s after Gram staining; Figure 3 It is the growth curve and the change of acid production rate of Lactobacillus paracasei strain PL4.4s; Figure 4 It is an observation diagram of the antibacterial circle test result of Lactobacillus paracasei strain PL4.4s against enterotoxigenic Escherichia coli; Figure 5 It is an observation diagram of the antibacterial circle test result of Lactobacillus paracasei strain PL4.4s against Staphylococcus aureus. Specific embodiments

[0028] The following further elaborates on this application in conjunction with the accompanying drawings and examples. It should be specifically noted that: for those not specifying specific conditions in the following examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can all be obtained from ordinary commercial sources.

[0029] This application provides a Lactobacillus paracasei strain PL4.4s, which is isolated and screened from the intestinal contents and feces of healthy piglets. Through 16S rDNA identification, this strain is Lactobacillus paracasei, named Lactobacillus paracasei strain PL4.4s, and is deposited in the China General Microbiological Culture Collection Center (CGMCC), with the deposit number CGMCC No. 32643, and the deposit date: November 15, 2024.

[0030] This application provides a fermentation supernatant, and its preparation method is as follows: (1) Preparation of Lactobacillus paracasei strain PL4.4s seed liquid: Inoculate the Lactobacillus paracasei strain PL4.4s into an MRS solid medium containing calcium carbonate, and statically culture it at 25 - 40 °C for 24 - 48 h. Screen single colonies with larger calcium dissolution circles, inoculate them into an MRS liquid medium, and shake-culture them at 25 - 40 °C for 18 - 32 h to obtain the Lactobacillus paracasei strain PL4.4s seed liquid; (2) Preparation of Lactobacillus paracasei strain PL4.4s fermented product: Inoculate the Lactobacillus paracasei strain PL4.4s seed liquid into an MRS liquid medium according to a volume percentage of 0.5 - 10%, and after shaking-culturing for 24 - 48 h, obtain the fermented product.

[0031] (3) Preparation of Lactobacillus paracasei strain PL4.4s fermentation supernatant: Centrifuge the fermented product at 8000 - 12000 rpm / min for 10 - 20 min, take the supernatant to obtain the fermentation supernatant.

[0032] The culture media and raw material sources involved in the embodiments of the present application are as follows: MRS solid medium: Peptone 10.0 g / L, beef powder 5.0 g / L, yeast powder 4.0 g / L, glucose 20.0 g / L, magnesium sulfate 0.2 g / L, sodium acetate 5.0 g / L, ammonium citrate tribasic 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate monohydrate 0.05 g / L, Tween 80 1.0 g / L, and agar 15.0 g / L, pH 6.2 ± 0.2; MRS broth medium: Peptone 10.0 g / L, beef powder 10.0 g / L, yeast powder 5.0 g / L, glucose 20.0 g / L, magnesium sulfate 0.1 g / L, sodium acetate 5.0 g / L, ammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate monohydrate 0.05 g / L, and Tween 80 1.0 g / L, pH 6.2 ± 0.2; Acid-resistant MRS broth medium: Peptone 10.0 g / L, beef powder 10.0 g / L, yeast powder 5.0 g / L, glucose 20.0 g / L, magnesium sulfate 0.1 g / L, sodium acetate 5.0 g / L, ammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate monohydrate 0.05 g / L, and Tween 80 1.0 g / L, pH 2.0 / 3.0; Bile salt-resistant MRS broth medium: Peptone 10.0 g / L, beef powder 10.0 g / L, yeast powder 5.0 g / L, glucose 20.0 g / L, magnesium sulfate 0.1 g / L, sodium acetate 5.0 g / L, ammonium citrate 2.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, manganese sulfate monohydrate 0.05 g / L, and Tween 80 1.0 g / L, 3 / 5 g of porcine bile salt;

[0033] The enterotoxigenic Escherichia coli (model number ATCC35401) and Staphylococcus aureus (model number ATCC29213) which are the indicator bacteria involved in the embodiments of the present application are commercially available; The artificial gastrointestinal fluid involved in the embodiments of the present application is purchased from Beijing Regene Biotechnology Co., Ltd.; The lactic acid (LD) test kit involved in the embodiments of the present application is purchased from Nanjing Jiancheng Bioengineering Institute; The catalase, pepsin, trypsin, and proteinase K involved in the embodiments of the present application are purchased from Shanghai Yuanye Bio-Technology Co., Ltd.; The calcium carbonate involved in the embodiments of the present application is purchased from Sinopharm Chemical Reagent Co., Ltd.; The intestinal contents and fresh feces of piglets involved in the embodiments of the present application are collected from Faku Kangda Ecological Agriculture Co., Ltd.; In the embodiments of the present application, the activation of the strain refers to the process of streaking the strain from the preservation tube onto a solid medium plate and growing single colonies.

[0034] The present application will be further described in detail below in conjunction with examples, performance detection tests and attached drawings. Example 1 (I) Isolation, screening and detection of strains

[0035] (1) Enrichment: Take 10 g of the sample and enrich it in 100 mL of MRS broth medium, and culture it at 37 °C for 24 - 48 h.

[0036] (2) Isolation: Take 1 mL of the enriched bacterial solution and place it in 10 mL of MRS broth medium (pH 2.5), culture it at 37 °C for 12 h, then take 1 mL and place it in 10 mL of 0.3% bile salt MRS broth medium, culture it at 37 °C for 12 h, dilute it and coat it on a CaCO3 MRS plate, select the colonies with large calcium dissolution circles and fast growth, streak and purify them on an MRS solid medium, and preserve the purified strains in glycerol at -80 °C.

[0037] Biological identification of the target strain: Inoculate the isolated strain into MRS broth medium, culture it at 37 °C for 18 - 32 h, conduct morphological identification and 16S rDNA molecular biological identification, and the strain identification is carried out by BGI Tech Solutions Co., Ltd. Analyze the sequencing results by BLAST in the National Center for Biotechnology Information (NCBI) database in the United States, and construct a phylogenetic tree using MEGA 5.0 software, as Figure 1 shown.

[0038] A total of 105 colonies with calcium dissolution circles were screened from the healthy piglet intestines and fresh feces. Morphological identification showed that 26 strains were Gram-positive bacilli. The 16S rDNA sequences of the 26 strains were amplified and sequenced, and the sequences were compared by BLAST of GenBank. 4 strains belonged to the genus Lactobacillus paracasei and had 99% similarity with Lactobacillus paracasei. They were named Lactobacillus paracasei PL4.3s, Lactobacillus paracasei PL4.4s, Lactobacillus paracasei PLp2s, and Lactobacillus paracasei PLp4s respectively. (II) Determination and analysis of the acid production rate and acid production ability of 4 strains

[0039] The acid production rate and acid production ability of the above 4 strains were determined and analyzed, and the specific process is as follows: Inoculate the strain into 100 mL of sterile MRS broth medium at an inoculation amount of 2%, culture at 37 °C, take samples at 0, 2, 4, 8, 12, 16, 20, and 24 hours respectively, measure the OD600 value, and record the pH value at each time period; measure the lactic acid concentration after 24 h, and perform 3 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 below. The growth curves and changes in acid production rates of the 4 strains are as Figure 3 shown.

[0040] Table 1 Lactic acid content produced by 4 Lactobacillus paracasei strains

[0041] According to Figure 3 and the test results in Table 1, it can be seen that Lactobacillus paracasei PL4.4s has the best growth rate and acid production ability, and its lactic acid content is the highest after 24 h, reaching 231.11 mmol / L. (III) Tolerance analysis of Lactobacillus paracasei PL4.4s strain

[0042] Examine the tolerance ability of Lactobacillus paracasei PL4.4s strain to the gastrointestinal tract. The specific process is as follows: Centrifuge and wash to obtain the cells of Lactobacillus paracasei PL4.4s, resuspend the cells with an equal volume of artificial animal gastric juice to simulate the digestive effect of gastric juice, culture at 37 °C and 80 rpm for 4 h, then centrifuge the artificial gastric juice to obtain the digested cells, and then resuspend with the same volume of artificial animal intestinal juice. The culture conditions are the same as those of gastric juice, culture for 5 h, take samples to count the viable bacteria, and calculate the survival rate. The results are shown in Table 2 below.

[0043] Table 2 Results of the test of Lactobacillus paracasei PL4.4s tolerance to artificial gastrointestinal juice

[0044] According to the test results in Table 2, it can be seen that PL4.4s of this application can still maintain more than 80% viable bacteria after 4 h of gastric juice culture and 5 h of intestinal juice treatment under the influence of test factors such as centrifugation and resuspension. Therefore, it shows that the Lactobacillus paracasei PL4.4s strain provided in this 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. (IV) Determination of the antibacterial effect of Lactobacillus paracasei PL4.4s strain

[0045] Detect the antibacterial effect of Lactobacillus paracasei PL4.4s strain. The specific process is as follows: The inhibitory zone diameter of Lactobacillus paracasei strain PL4.4s provided in this application against enterotoxigenic Escherichia coli and Staphylococcus aureus was determined by the Oxford cup plate method. Four parallel tests were conducted respectively, and the average value of the inhibitory zone diameter was calculated. The average value was taken, and the results are as Figures 4 - 5 shown in Table 3 below.

[0046] Figure 4 Figure for observing the results of the inhibitory zone test of Lactobacillus paracasei PL4.4s against enterotoxigenic Escherichia coli; Figure 5 Figure for observing the results of the inhibitory zone test of Lactobacillus paracasei PL4.4s against Staphylococcus aureus; Table 3 Detection results of the antibacterial activity of Lactobacillus paracasei PL4.4s

[0047] According to the detection results as Figures 4 - 5 shown in Table 3, it can be seen that Lactobacillus paracasei PL4.4s has good antibacterial activity against both enterotoxigenic Escherichia coli and Staphylococcus aureus, and the inhibitory zone diameters against Escherichia coli and Staphylococcus aureus are 24.77 mm and 28.38 mm respectively. Example 2

[0048] Example 2 provides a fermentation supernatant, and its preparation method is as follows: (1) Preparation of Lactobacillus paracasei PL4.4s seed liquid: Inoculate the Lactobacillus paracasei PL4.4s strain into MRS solid medium containing calcium carbonate, and incubate statically at 37 °C for 36 h. Screen single colonies with larger calcium dissolution circles, inoculate them into MRS liquid medium, and incubate with shaking at 37 °C for 24 h to obtain Lactobacillus paracasei PL4.4s seed liquid; (2) Preparation of Lactobacillus paracasei PL4.4s fermentation product: Inoculate the Lactobacillus paracasei PL4.4s seed liquid into MRS liquid medium according to a volume percentage of 2%, and after incubating with shaking for 36 h, obtain the fermentation product.

[0049] (3) Preparation of Lactobacillus paracasei PL4.4s fermentation supernatant: Centrifuge the fermentation product at 10000 rpm / min for 10 min, take the supernatant, and filter it through a 0.22 μm filter to obtain the fermentation supernatant. (I) Detection of antibacterial rate

[0050] The antibacterial property of the fermentation supernatant obtained in Example 2 was detected, and the specific process is as follows: (1) Take the indicator bacteria liquid (Escherichia coli and Staphylococcus aureus) and add it to LB liquid medium (generally, 1 mL of bacteria liquid is added to 100 mL of LB liquid medium), measure the absorbance at 600 nm, and make its absorbance approximately OD600 = 0.05; (2) Add the fermentation supernatant to 10 mL of LB liquid medium (containing the indicator bacteria) at a volume ratio of 1%, mix well, and make 3 parallel groups; at the same time, set up a blank control A 空 (without adding the fermentation supernatant), culture at 200 r / min in a shaking incubator at 37 °C for 12 h, use a spectrophotometer to measure the OD value of each sample at a wavelength of 600 nm, record the readings (zero with sterile LB medium), and calculate the antibacterial rate of each fermentation supernatant. The detection results are shown in Table 4 below; the calculation formula for the antibacterial rate is as follows: Antibacterial rate (%) = [(A 空 - A 空0 ) - (A 样 - A 样0 ) / (A 空 - A 空0 )] × 100%.

[0051] Among them, A 样0 represents the initial OD600 of the mixture of the fermentation supernatant and LB medium (containing indicator bacteria) at 0 h, A 样 represents the OD600 of the mixture after 12 h of culture, A 空0 represents the initial OD600 of the blank control group at 0 h, and A 空 represents the OD600 of the mixture in the blank control group after 12 h of culture.

[0052] Table 4 Detection results of the antibacterial rate of the fermentation supernatant of Lactobacillus paracasei PL4.4s

[0053] According to the detection results in Table 4, the fermentation supernatant of Lactobacillus paracasei PL4.4s showed a high inhibitory effect on both the inhibition rate of enterotoxigenic Escherichia coli and the inhibition rate of Staphylococcus aureus. (II) Detection of antibacterial stability

[0054] Examine the effects of hydrogen peroxide, temperature, protease, and acid-base pH on the antibacterial performance of the fermentation supernatant, and the results are shown in Table 5 below.

[0055] (1) Hydrogen peroxide: Add 1.5 mg / mL of catalase to the fermentation supernatant of Lactobacillus paracasei PL4.4s. The control group is not treated with catalase. Incubate in a water bath at 37 °C for 2 h to eliminate the effect of hydrogen peroxide in the fermentation broth. Use ETEC and SA as indicator bacteria. Add the fermentation supernatant of PL4.4s to 10 mL of LB liquid medium (containing indicator bacteria) at a volume ratio of 1%, mix well, make three parallel groups, and measure the antibacterial ability. The specific method refers to the detection of the antibacterial rate in the above antibacterial experiment of Lactobacillus paracasei PL4.4s strain.

[0056] (2) Temperature: The fermentation supernatant of Lactobacillus paracasei PL4.4s was placed in a water bath at 60 °C, 80 °C, and 100 °C for 20 min respectively. The fermentation supernatant without high-temperature treatment was used as a blank control. Using ETEC and SA as indicator bacteria, the fermentation supernatant of PL4.4s was added to 10 mL of LB liquid medium (indicator bacteria had been added) at a volume ratio of 1%, and three parallel groups were made to determine the antibacterial ability. The specific method refers to the detection of the antibacterial rate in the antibacterial experiment of Lactobacillus paracasei PL4.4s strain described above.

[0057] (3) Protease: The fermentation supernatant of Lactobacillus 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) for 2 h respectively. The enzyme concentration was 3 mg / mL for all. After the treatment, the fermentation supernatant was adjusted back to the original pH value. Using the same pathogenic bacteria as indicator bacteria above, the fermentation supernatant of PL4.4s was added to 10 mL of LB liquid medium (indicator bacteria had been added) at a volume ratio of 1%, and three parallel groups were made to determine the antibacterial ability.

[0058] (4) Acid-base pH: The fermentation supernatant was adjusted to pH 3 and pH 6.5 respectively with 0.1 mol / L NaOH to test its antibacterial activity.

[0059] Table 5 Detection results of the antibacterial rate of the fermentation supernatant of Lactobacillus paracasei PL4.4s under different treatment conditions

[0060] According to the detection results in Table 5, when the fermentation supernatant of Lactobacillus paracasei PL4.4s was treated with catalase, it was found that the antibacterial rate of the fermentation supernatant against Escherichia coli was still ≥99%, and the antibacterial rate against Staphylococcus aureus was still ≥99%. This indicates that hydrogen peroxide is not produced during the fermentation process of Lactobacillus paracasei PL4.4s. After the fermentation supernatant of Lactobacillus paracasei PL4.4s was heat-treated at 65°C, 85°C, and 100°C for 20 min respectively, the antibacterial rate of each fermentation supernatant against Escherichia coli was still ≥99%, and the antibacterial rate against Staphylococcus aureus was still ≥99%. This shows that the fermentation supernatant of Lactobacillus paracasei PL4.4s has very good heat resistance. When the fermentation supernatant of Lactobacillus paracasei PL4.4s was treated with pepsin, trypsin, proteinase K, and papain respectively, the results showed that the antibacterial rate of each fermentation supernatant against Escherichia coli was still ≥99%, and the antibacterial rate against Staphylococcus aureus was still ≥99%. This indicates that the fermentation supernatant of Lactobacillus paracasei PL4.4s can tolerate a variety of enzymes. After the pH of the fermentation supernatant of Lactobacillus paracasei PL4.4s was adjusted to 3, it was found that the antibacterial rate of the fermentation supernatant of Lactobacillus paracasei PL4.4s against Escherichia coli was still ≥99%, and the antibacterial rate against Staphylococcus aureus was still ≥99%. This indicates 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 was adjusted to 6.5, it was found that the antibacterial activity of the fermentation supernatant of Lactobacillus paracasei PL4.4s decreased significantly, and the antibacterial rate was only about 20%. This indicates that the fermentation supernatant of Lactobacillus paracasei PL4.4s has poor alkali resistance. Example 3

[0061] Example 3 provides a fermentation supernatant, and its preparation method is as follows: (1)Preparation of Lactobacillus paracasei PL4.4s seed solution: Inoculate the strain of Lactobacillus paracasei PL4.4s into MRS solid medium containing calcium carbonate, and statically culture at 37°C for 4 h to obtain the Lactobacillus paracasei PL4.4s seed solution; (2)Preparation of Lactobacillus paracasei PL4.4s fermented product: Inoculate the Lactobacillus paracasei PL4.4s seed solution into MRS liquid medium according to 2% (v / v), and oscillate and culture at 37°C for 48 h to obtain the fermented product.

[0062] (3)Preparation of Lactobacillus paracasei PL4.4s fermentation supernatant: Centrifuge the fermented product at 5000 rpm / min for 20 min, collect the supernatant to obtain the fermentation supernatant. Detection of exopolysaccharide content

[0063] Extract the exopolysaccharide in the above fermentation supernatant and detect the exopolysaccharide content. The specific process is as follows: (1) Trichloroacetic acid treatment: Slowly add trichloroacetic acid with a concentration of 800 mg / mL to the fermentation supernatant to make its final concentration 40 mg / mL. Let it stand at 4°C for 8 h, then centrifuge at 10000 rpm and 4°C for 10 min. Discard the precipitate and collect the supernatant.

[0064] (2) Absolute ethanol treatment: Add absolute ethanol to the supernatant according to a volume ratio of 1:4. Let it stand overnight at 4°C, then centrifuge at 10000 rpm and 4°C for 10 min. Collect the precipitate at the bottom; dissolve the precipitate in distilled water at 60°C, centrifuge at 10000 rpm for 10 min, discard the insoluble precipitate at the bottom, and collect the supernatant.

[0065] (3) Dialysis: Load the supernatant into a dialysis bag with a molecular weight cut-off of 8 KDa, dialyze for 2 h, change the dialysis fluid, and then change the dialysis fluid every 6 h. Dialyze continuously for 2 d; finally, collect the polysaccharide solution in the dialysis bag and vacuum freeze-dry to obtain the extracellular polysaccharide.

[0066] (4) Detection: First, dry glucose by blowing air for 2 h. After cooling, take 100 mg of glucose and put it into a 1 L volumetric flask, shake well, and make up the volume; add components to the reaction system as shown in the following table. After it cools, measure the absorbance at a wavelength of 490 nm. Do 3 parallels for each group; draw a standard curve with the glucose content (μg / mL) as the abscissa and the absorbance value (A490) as the ordinate. Measure the absorbance of the extracellular polysaccharide at 490 nm in the same way as above, and calculate the extracellular polysaccharide yield of the strain through the regression equation.

[0067] After detection, the content of extracellular polysaccharide secreted by Lactobacillus paracasei PL4.4s is 571.09 ± 0.45 μg / mL. Therefore, it shows that the Lactobacillus paracasei PL4.4s provided in this application can secrete extracellular polysaccharide and can obtain a fermentation supernatant with a relatively high content of extracellular polysaccharide. Example 4

[0068] Example 4 provides a fermentation supernatant, and its preparation method is as follows: (1) Preparation of Lactobacillus paracasei PL4.4s seed solution: Inoculate the Lactobacillus paracasei PL4.4s strain into MRS solid medium containing calcium carbonate, culture it statically at 37°C for 36 h, screen single colonies with larger calcium dissolution circles, inoculate them into MRS liquid medium, and culture them with shaking at 37°C for 24 h to obtain the Lactobacillus paracasei PL4.4s seed solution; (2) Preparation of Lactobacillus paracasei PL4.4s fermentation product: Inoculate the Lactobacillus paracasei PL4.4s seed solution into MRS liquid medium according to a volume percentage of 2%, and culture it with shaking for 48 h to obtain the fermentation product.

[0069] (3) Preparation of the fermentation supernatant of Lactobacillus paracasei PL4.4s: Centrifuge the fermented product at 5000 rpm / min for 20 min, take the supernatant to obtain the fermentation supernatant. Detection of enzyme production characteristics

[0070] Dilute the fermentation supernatant obtained in Example 4, and then measure the enzyme activity, specifically as follows: Determination of cellulase activity: Determine the cellulase activity in the crude enzyme solution by the spectrophotometer method for the determination of cellulase activity in feed additives according to NY / T 912-2004.

[0071] Determination of protease activity: Determine the protease activity in the crude enzyme solution by the method for the determination of protease activity according to GB / T 28715-2012.

[0072] Determination of pectinase activity: Determine the pectinase activity in the crude enzyme solution by the method for the determination of pectinase preparation according to QB 1502-92.

[0073] After detection, in the fermentation supernatant obtained in Example 4, the protease activity is 36.45±0.05 U / mL, the cellulase activity is 26.55±0.11 U / mL, and the pectinase activity is 61.38±0.09 U / mL. Therefore, it shows that the primary fermentation of Lactobacillus paracasei PL4.4s provided by this application can produce protease, cellulase and pectinase. Applying it to fermented feed is more conducive to the digestion and absorption of livestock and poultry, and improves the growth and health of the animal body. Example 5

[0074] Example 5 provides a bacterial suspension of Lactobacillus paracasei PL4.4s, and its preparation method is as follows: (1) Preparation of the seed liquid of Lactobacillus paracasei PL4.4s: Inoculate the strain of Lactobacillus paracasei PL4.4s into the MRS solid medium containing calcium carbonate, statically culture at 37°C for 36 h, screen the single colony with a larger calcium dissolution circle, inoculate it into the MRS liquid medium, and shake culture at 37°C for 24 h to obtain the seed liquid of Lactobacillus paracasei PL4.4s; (2) Preparation of the bacterial suspension of Lactobacillus paracasei PL4.4s: Inoculate the seed liquid of Lactobacillus paracasei PL4.4s into the MRS liquid medium according to the volume percentage of 2%, and after shaking culture for 48 h, obtain the bacterial suspension. Determination of antioxidant capacity

[0075] Determine the antioxidant capacity of the bacterial suspension obtained in Example 5, and the specific process is as follows: (1) DPPH radical scavenging ability Take 2 mL of the bacterial suspension and add 2 mL of a 0.2 mmol / L DPPH absolute ethanol solution, designated as Ai. Mix well thoroughly and react in the dark at room temperature for 30 min. After centrifuging at 3000×g for 10 min, take the supernatant and measure the absorbance Ai at 517 nm, calculate the DPPH free radical scavenging rate, and perform parallel measurements 3 times for each group and take the average value. Aj is 2 mL of the bacterial suspension + 2 mL of absolute ethanol, and Ac is 2 mL of absolute ethanol + 2 mL of DPPH. The calculation formula for the DPPH free radical scavenging rate is as follows: DPPH free radical scavenging rate (%) = (1 - (Ai - Aj) / Ac) × 100% (2)Hydroxyl radical scavenging ability Take 1 mL of 2.5 mmol / L o-phenanthroline, sequentially add 1 mL of PBS buffer solution and 1 mL of distilled water, mix well thoroughly, and then add 1 mL of 2.5 mmol / L FeSO 4 and 1 mL of 20 mmol / L H 2 O 2 , react at 37 °C for 60 min, take the supernatant, and then measure the absorbance A of the control group (A 对 ) at 536 nm. Use 1 mL of distilled water to replace 1 mL of H 2 O 2 as the blank group and measure the absorbance A of the blank group (A 空 ). Use 1 mL of the bacterial suspension to replace 1 mL of distilled water as the sample group and measure the absorbance A of the sample group (A 样 ). Calculate the hydroxyl radical scavenging rate, perform parallel measurements 3 times for each group and take the average value. The calculation formula for the hydroxyl radical scavenging rate is as follows: Hydroxyl radical scavenging rate (%) = (A 样 - A 对 ) / (A 空 - A 对 ) × 100% (3)Superoxide anion scavenging ability Mix 0.5 mL of the bacterial suspension with 1.5 mL of Tris-HCl solution (pH 8.0), react at 25 °C for 20 min, then add 200 μL of 25 mmol / L pyrogallol and react for 5 min, and then add 250 μL of HCl to terminate the reaction. Centrifuge at 3000×g for 10 min, take the supernatant and measure the absorbance A of the sample group (A 样 ) at 325 nm. Use 0.5 mL of distilled water to replace 0.5 mL of the bacterial suspension as the control group and measure the absorbance A of the control group (A 对 ). Calculate the superoxide anion free radical scavenging rate, perform parallel measurements 3 times for each group and take the average value. The calculation formula for the superoxide anion free radical scavenging rate is as follows: Superoxide anion free radical scavenging rate (%) = (1 - A样 / A 对 ) × 100% The test results are shown in Table 6 below.

[0076] Table 6 Determination results of the antioxidant capacity of Lactobacillus paracasei PL4.4s

[0077] According to the test results in Table 6, the scavenging rate of the cell suspension of Lactobacillus paracasei PL4.4s against DPPH free radicals is 99.95%, the scavenging rate against hydroxyl free radicals is 67.43%, and the scavenging rate against superoxide anion free radicals is 64.86%. Therefore, it shows that the Lactobacillus paracasei PL4.4s provided in this application has good antioxidant effects and can be used to prepare preparations such as biological feeds, which has good practical significance.

[0078] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A Lactobacillus paracasei, characterized in that The Lactobacillus paracasei is Lactobacillus paracasei ( Lactobacillus paracasei PL4.4s is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC), with the deposit number CGMCC No. 32643 and the deposit date is November 15, 2024.

2. A fermented product, characterized in that: The fermented product comprises the Lactobacillus paracasei PL4.4s according to claim 1.

3. The fermentation product according to claim 2, characterized in that The method for preparing the fermented product comprises the following steps: First, Lactobacillus paracasei PL4.4s is activated to obtain Lactobacillus paracasei PL4.4s seed solution; then, the Lactobacillus paracasei PL4.4s seed solution is inoculated into MRS liquid culture medium at a volume percentage of 0.5-10%, and shaken culture is performed for 24-48 hours to obtain a fermentation product.

4. A fermentation supernatant, characterized in that: The fermentation supernatant is the upper clear liquid obtained after centrifugation of the fermentation product according to claim 2.

5. The fermentation supernatant according to claim 4, characterized in that The method for preparing 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.

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

7. A bacterial agent, characterized in that: The bacterial agent includes the Lactobacillus paracasei PL4.4s described in claim 1.

8. Use of the Lactobacillus paracasei PL4.4s as claimed in claim 1, the fermented product as claimed in claim 2 or 3, the fermentation supernatant as claimed in claim 4 or 5, the bacterial suspension as claimed in claim 6 or the bacterial agent as claimed in claim 7 in a biological preparation.

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