Lactobacillus casei and application thereof

Abstract

The invention provides lactobacillus casei and application thereof, the lactobacillus casei is separated and screened from the intestinal tract of apostichopus japonicus, has a promoting effect on the intestinal health of animals, and can increase the number of beneficial bacteria in the intestinal tract of the animals and inhibit the growth of harmful bacteria, so that the incidence rate of intestinal diseases of the animals is reduced; the disease resistance of animals can be improved, the immunity of the organism is enhanced, and the infection rate of viruses and bacteria on the animals is reduced. The lactobacillus casei is an endogenous intestinal probiotic, is not only suitable for growth and reproduction in a marine environment, but also has no danger to bred animals and human beings, and has high efficiency and safety in the aspect of application. The lactobacillus casei disclosed by the invention has the advantages of low cost, simplicity and convenience in operation and better commercial prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of aquatic microbial application, and particularly relates to a lactobacillus casei and application thereof. BACKGROUND

[0002] With the rise of the aquaculture industry in China and the changes in water quality environment and climate conditions during the process of intensive aquaculture, various diseases mainly caused by bacterial diseases occur during the process of aquaculture. These bacterial diseases are usually prevented and treated by antibiotics and chemicals, and the negative effects caused by them have attracted the attention of many scholars.

[0003] The serious pollution of water bodies and the surrounding environment caused by the abuse of broad-spectrum antibiotics and chemicals can also further lead to an increase in drug residues and drug-resistant bacteria in sea cucumbers. These drug-resistant pathogenic bacteria can be transmitted to humans through the food chain, leading to changes in the drug resistance of normal flora in the human body. Studies have reported that environmental antibiotics can not only cause chemical pollution, but also induce the production of resistant microorganisms and resistance genes in the environment and increase the risk of antibiotic resistance transmission. Some resistant microorganisms can directly or indirectly enter the human body through the food chain and other pathways, leading to an increase in human antibiotic resistance, thereby posing a certain degree of threat to human public health and seriously endangering human health.

[0004] In recent years, probiotics have been widely used in food animals and humans. As an effective substitute for antibiotics and chemicals, probiotics are expected to be used to prevent diseases and promote the growth of aquatic animals. Lactobacillus casei, as a natural and green feed additive, has various probiotic properties in the intestinal tract, such as reducing cholesterol, improving the intestinal environment, reducing blood sugar, reducing blood pressure, and the like. It can regulate the intestinal microbial flora structure by inhibiting the proliferation of intestinal pathogenic microorganisms, and can regulate immune function, stimulate immune cell activation and proliferation, and the like. Lactic acid bacteria have the potential to alleviate the problems in the sea cucumber industry due to their advantages of promoting the growth of aquatic animals, enhancing immunity, improving feed utilization, improving the environment, and ensuring the safety of aquatic products without drug residues, and are expected to promote the green and high-quality development of the sea cucumber industry. Therefore, lactic acid bacteria are highly valued in aquaculture. Further, lactic acid bacteria have become the focus of research on healthy aquaculture. SUMMARY

[0005] The technical problem solved by the present application is to provide a lactobacillus casei and application thereof, which can effectively improve the intestinal flora, prevent and control pathogenic microorganisms, and promote the growth ability of cultured organisms and enhance the immune function of the host as an additive.

[0006] Therefore, the present application provides a lactobacillus casei with a preservation number of CGMCC NO.34953.

[0007] Correspondingly, the application also provides application of the Lactobacillus casei with the preservation number of CGMCC NO.34953 in preparation of a microbial preparation.

[0008] Preferably, the microbial preparation is used for inhibiting pathogenic microorganisms, promoting growth ability of the cultivated organisms, and enhancing immune function of the host.

[0009] Preferably, the microbial preparation is a liquid or powder probiotic.

[0010] Preferably, the method comprises the following steps: Detecting absorbance of the Lactobacillus casei liquid after overnight culture at 620 nm; Mixing the appropriate liquid with the powdered sea cucumber basic feed, and oscillating and mixing to obtain the Lactobacillus casei probiotic feed.

[0011] Preferably, the method further comprises: Detecting absorbance of the Lactobacillus casei liquid after overnight culture at 620 nm.

[0012] Preferably, the oscillating and mixing is performed at 30 DEG C.

[0013] Preferably, the oscillating and mixing is performed at a speed of 180 r / min.

[0014] Preferably, the oscillating and mixing is performed for 2 h.

[0015] Preferably, the effective bacterial concentration of the Lactobacillus casei strain is 1x108 CFU / g.

[0016] The application provides a Lactobacillus casei with the preservation number of CGMCC NO.34953, which is preserved in the China General Microbiological Culture Collection Center on June 19, 2025. The application also provides application of the Lactobacillus casei with the preservation number of CGMCC NO.34953 in preparation of a microbial preparation. The strain is isolated and screened from the intestine of Apostichopus japonicus, can promote healthy growth of the Apostichopus japonicus, improve intestinal flora, can effectively resist infection of the pathogenic bacteria Photobacterium damsel, and enhance immune function of the organism. The Lactobacillus casei is an endogenous intestinal probiotic, is suitable for growth and reproduction in a marine environment, and is safe for cultured animals and humans, and has high efficiency and safety in application. The Lactobacillus casei has low production cost, simple process, and good commercial prospect.

[0017] Biological preservation instruction Classification and naming: Lactobacillus casei Lactobacillus casei). It was preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 19, 2025, located at No. 1, Beichen West Road, Yard 3, Beijing City, Chaoyang District, Institute of Microbiology of Chinese Academy of Sciences. The preservation number is CGMCC NO. 34953. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiment or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can also be obtained according to the provided drawings without creative labor for those skilled in the art.

[0019] Figure 1 The growth curve of Lactobacillus casei provided for Example 1 is shown in the figure, the culture medium is MRS broth, and the culture temperature is 37℃; Figure 2 The growth curve of Lactobacillus casei provided for Example 1 under different temperature conditions is shown in the figure, and the culture medium is MRS broth; Figure 3 The growth curve of Lactobacillus casei provided for Example 1 under different pH conditions is shown in the figure, the culture medium is MRS broth, and the culture temperature is 37℃; Figure 4 The growth curve of Lactobacillus casei provided for Example 1 under different sea salt addition conditions is shown in the figure, the culture medium is MRS broth, and the culture temperature is 37℃; Figure 5 The cumulative survival rate graph of Lactobacillus casei provided for this embodiment 2 applied to Apostichopus japonicus culture is shown in the figure. DETAILED DESCRIPTION

[0020] In order to further understand the present application, the preferred embodiments of the present application will be described below in combination with examples, but it should be understood that these descriptions are only for further illustrating the features and advantages of the present application, and are not limitations of the claims of the present application.

[0021] The embodiment of the present application discloses a kind of Lactobacillus casei, and its preservation number is CGMCC NO.34953.The Lactobacillus casei CGMCC NO.34953 of the present application is preserved in China General Microbiological Culture Collection Center, and the preservation date is June 19, 2025, and the address of preservation unit is Beijing City, Chaoyang District, Beichen West Road, Yard 3.

[0022] Correspondingly, the application also provides application of Lactobacillus casei with a preservation number of CGMCC NO.34953 in preparation of a microbial preparation. The microbial preparation is used for inhibiting pathogenic microorganisms, promoting growth ability of a cultivated organism and enhancing immune function of a host. As a preferred solution, the microbial preparation is a liquid or powder probiotic, i.e., the Lactobacillus casei is prepared into a bacterial liquid or a bacterial powder.

[0023] Preferably, the present application adopts the following method to detect the concentration of the bacterial body: an enzyme label instrument method is adopted, and the absorbance (OD620) is used to represent the concentration of the bacterial body. The OD620 value of the bacterial body after the strain grows for a certain time is measured, and the OD620 value of the background is subtracted to obtain the value of the measured bacterial body. The number of the bacterial body is measured by a coating plate counting method, and the required concentration of the bacterial liquid is determined according to the OD620 value and the number of the bacterial body in the bacterial liquid, so as to prepare a liquid probiotic.

[0024] The Lactobacillus casei of the present application is derived from endogenous Lactobacillus casei CGMCC NO.34953 in the intestinal tract of Apostichopus japonicus, and the strain not only has the effect of inhibiting pathogenic bacteria, but also can enhance the non-specific immune level of the cultivated organism and the ability to resist infection of pathogenic bacteria.

[0025] The strain information of the Lactobacillus casei CGMCC NO.34953 of the present application is as follows: 1. Source. The strain is isolated from the intestinal tract of Apostichopus japonicus, can promote healthy growth of Apostichopus japonicus, improve intestinal flora, can effectively resist infection of Photobacterium damsel, and enhance immune function of the organism.

[0026] 2. Morphological characteristics. Gram-positive bacteria, facultative anaerobic, do not produce capsules and spores, the colony morphology on the MRS solid culture medium is 1-2mm in diameter, the colony is smooth, round, convex and has a neat edge after being cultured at 36℃±1℃ for 48h.

[0027] 3. Physiological and biochemical characteristics. The strain is negative in arabinose reaction, positive in cellobiose reaction, positive in esculin reaction, positive in glucose reaction, positive in lactose reaction, negative in maltose reaction, positive in mannitol reaction, positive in mannose reaction, positive in melezitose reaction, negative in raffinose reaction, negative in rhamnose reaction, negative in ribose reaction, negative in sorbitol reaction, negative in sucrose reaction, positive in trehalose reaction, negative in xylose reaction, negative in peroxidase reaction, negative in nitrate reduction reaction, positive in gelatin liquefaction experiment reaction, negative in indole experiment reaction and negative in hydrogen sulfide experiment reaction.

[0028] The 16S rDNA gene sequence of the Lactobacillus casei is shown in SEQ ID No.1.

[0029] The Lactococcus lactis CGMCC NO.34953 has the following characteristics: 1、The probiotic feed prepared from the Lactococcus lactis can promote the healthy growth of Apostichopus japonicus, improve intestinal flora, effectively resist the infection of pathogenic bacteria Vibrio, and enhance the immune function of the body.

[0030] 2、The Lactococcus lactis has low production cost, simple process, and good commercial prospect.

[0031] 3、The Lactococcus lactis is derived from the intestinal tract of Apostichopus japonicus in the sea, is an endogenous intestinal probiotic, is suitable for growth and reproduction in a marine environment, and does not exist dangerous to farmed animals and humans, and has high efficiency and safety in application.

[0032] In order to further understand the present application, the technical solutions provided by the present application are described in detail below in combination with examples, and the protection scope of the present application is not limited by the following examples.

[0033] The raw materials used in the embodiments of the present application are all commercially available.

[0034] The Lactococcus lactis used in the embodiments of the present application is preserved in the China General Microbiological Culture Collection Center, and the preservation date is June 19, 2025, and the preservation number is CGMCC NO.34953. The strain is isolated and screened from the intestinal tract of Apostichopus japonicus.

[0035] Example 1

[0036] Bacterial concentration detection: The enzyme label instrument method is used to represent the concentration of the bacteria by absorbance (OD620). The OD620 value of the strain after a certain time of growth is measured, and the OD620 value of the background is subtracted to obtain the number of bacteria.

[0037] The number of bacteria is determined by the coating plate counting method, and the OD620 is related to the number of bacteria in the bacterial solution.

[0038] The MRS solid culture medium and the MRS liquid culture medium are purchased from Haibo Biotechnology Co., Ltd., and the MRS liquid culture medium is MRS broth, which is prepared according to the use method in the instruction manual.

[0039] Figure 1 The growth curve of the Lactococcus lactis provided in the present embodiment is shown in the figure. The culture medium is MRS broth, and the culture temperature is 37 DEG C. Figure 2 The growth curve of the Lactococcus lactis provided in the present embodiment under different temperature conditions is shown in the figure, and the culture medium is MRS broth. Figure 3The growth chart of Lactobacillus casei provided for this example under different pH conditions, the culture medium is MRS broth, and the culture temperature is 37℃. Figure 4 The growth chart of Lactobacillus casei provided for this example under different sea salt addition conditions, the culture medium is MRS broth, and the culture temperature is 37℃.

[0040] Example 2 Application of Lactobacillus casei probiotic feed Step 1) Preparation of probiotic feed: The overnight culture of Lactobacillus casei was detected for absorbance at 620 nm, and the appropriate bacterial solution was mixed with powdered sea cucumber basic feed. The effective bacterial concentration of the strain was 1×108 CFU / g after 2h of shaking mixing at 30℃ and 180 r / min on a shaking table.

[0041] Step 2) Apostichopus japonicus culture: After 2 days of starvation treatment, 180 healthy Apostichopus japonicus were domesticated with basic feed for 5 days and randomly divided into two groups: the experimental group and the control group, with 90 in each group. The Apostichopus japonicus was cultured in a 0.3×0.25×0.3m water tank for 24h aeration, and the quantitative feeding was adopted with a daily feeding amount of about 3% of the body weight. The Apostichopus japonicus was cultured in flowing water, and the remaining feed and excrement were removed with a siphon before feeding. The experimental group was fed with Lactobacillus casei feed, and the control group was fed with basic feed. The Apostichopus japonicus was observed for feeding and state every day, and the test period was 8 weeks.

[0042] Step 3) Intestinal immune indicators of Apostichopus japonicus: The intestinal mixed samples of 3 Apostichopus japonicus from each tank in each group were taken for aseptic grinding at the 8th week of the test, and the intestinal acid phosphatase, alkaline phosphatase, superoxide dismutase, and catalase were detected. As shown in Table 1, the contents of acid phosphatase, alkaline phosphatase, superoxide dismutase, and catalase were significantly higher than those of the control group, indicating that the Lactobacillus casei prepared in Example 1 can improve the intestinal non-specific immune factors of Apostichopus japonicus.

[0043] Table 1 Effect of Lactobacillus casei strain on intestinal immune enzyme activity of Apostichopus japonicus Acid phosphatase Alkaline phosphatase SOD Catalase Experimental group 101.72 ± 5.51 a ]] 148.02 ± 22.79 a ]] 42.04 ± 1.37 a ]] 26.95 ± 0.5 5a ]] Control group 49.74 ± 9.62 b ]] 83.95 ± 13.30 b ]] 30.87 ± 1.36 b ]] 20.78 ± 1.50 b ]] Step 4) Relative weight gain rate detection: The body weight was measured every two weeks, and the weight was weighed to the 8th week. The relative weight gain rate (Weight gain ratio, WGR, %) = 100×(final body weight-initial body weight) / initial body weight. As shown in Table 2, the body weight gain of the experimental group of Apostichopus japonicus was significantly better than that of the control group, and the relative weight gain rate of each group was calculated as follows: the experimental group was 42.77%, and the control group was 17.14%. This indicates that Lactobacillus casei can promote the growth of Apostichopus japonicus.

[0044] Table 2 is the body weight chart of Lactobacillus casei provided in the present embodiment for the cultivation of Apostichopus japonicus. The experiment is to feed Lactobacillus casei, and the control group is to feed the control group with basic feed.

[0045] Table 2 Effect of Lactobacillus casei on the growth of Apostichopus japonicus Indexs XP-7 group control group Initial weight (g) 11.75±0.23 11.73±0.21 Final weight (g) 21.73±0.08b 24.33±0.71a Weight gain (g) 9.98 12.6 Weight gain rate (%) 84.96±1.34b 107.33±1.10a Specific growth rate (% / d) 1.03±0.01b 1.22±0.01a Step 5) High-throughput sequencing analysis of intestinal flora composition: At the end of the cultivation experiment, 9 Apostichopus japonicus were randomly selected, 3 biological replicates for each treatment, and the intestinal microbial diversity was analyzed by high-throughput sequencing. In the door level: the experimental CGMCC NO.: 34953 group and the control group were mainly Pseudomonadota, Verrucomicrobia and Bacteroidetes; in the genus level, the beneficial flora in the intestines of Apostichopus japonicus in the CGMCC NO.: 34953 group increased, and the proportion of Vibrio was within the healthy range (0.44%); the harmful flora in the intestines of Apostichopus japonicus in the control group was dominant, and the proportion of beneficial flora decreased. It is shown that after feeding Lactobacillus casei, the amount of harmful bacteria in the intestines of Apostichopus japonicus decreases significantly, the amount of beneficial bacteria increases obviously, and the composition of intestinal flora is improved. The flora structure analysis of the present embodiment is shown in Tables 3-4.

[0046] Table 3 Intestinal flora structure composition of XP-7 group and control group at door level Phylum XP-7 group control group Pseudomonadota 50.04% 83.36% Niemann-Matthewia 26.27% 6.65% Bacteroidetes 17.72% 5.89% Planctomycetes 2.29% 2.52% Actinobacteria 2.03% 0.45% Firmicutes 0.83% 0.53% Dependentiae 0.25% 0.27% Bdellovibrio 0.13% 0.08% Patescibacteria 0.11% 0.05% Others 0.12% 0.01% Unclassified 0.16% 0.10% Genus 0.01% 0.04% Table 4 Intestinal flora structure composition of XP-7 group and control group at genus level XP-7 group control group Vibrio Erythrobacter 0.43% 35.92% Pseudomonas 22.58% 6.01% Sporohylium 0.07% 27.65% Hymenobacter 12.33% 4.29% Persicirhabdus 4.41% 1.62% Acinetobacter 2.96% 0.33% Erythropolis 0.01% 1.91% Leucothrix 0.31% 0.87% Nocardia 0.81% 0.06% Others 0.46% 0.31% Unclassified 10.46% 6.64% Figure 5 45.11% 14.33% Step 6) Disease resistance of Apostichopus japonicus: At the sixth week, the experimental group and the control group were attacked by Photobacterium damsel. The pathogenic bacteria Photobacterium damsel was enriched and cultured in 2216E liquid medium, and after centrifugation and PBS washing, the bacterial solution was adjusted to the required concentration (1x107CFU / mL). 30 Apostichopus japonicus were randomly divided into three biological replicates in each group and placed in the cultivation water containing the pathogenic bacteria. The survival rate of each treatment group within 7 days after soaking infection was recorded. The experimental group was 53.33%, and the control group was 16.67%. Therefore, Lactobacillus casei CGMCC NO. 34953 can improve the resistance of Apostichopus japonicus to Photobacterium damsel infection. ​The lactobacillus casei provided in the embodiment is applied to the cumulative survival rate graph of the Apostichopus japonicus culture. The experimental group is fed with the lactobacillus casei, and the control group is fed with the basic feed.

[0047] The above description of the embodiments is only used to help understand the method of the present application and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

[0048] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present application. Various modifications to the embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A Lactobacillus casei, characterized in that, The preservation number is CGMCC NO. 34953.

2. Use of Lactobacillus casei with a preservation number of CGMCC NO. 34953 in the preparation of a microbial preparation.

3. Use according to claim 2, characterized in that, The microbial preparation is used for inhibiting pathogenic microorganisms, promoting the growth ability of cultivated organisms, and enhancing the immune function of a host.

4. Use according to claim 2, characterized in that, The microbial preparation is a liquid or powder probiotic.

5. Use according to claim 2, characterized in that, The method comprises the following steps: The overnight culture of Lactobacillus casei is detected for absorbance at 620 nm. The appropriate bacterial solution is mixed with powdered sea cucumber basic feed to obtain Lactobacillus casei probiotic feed.

6. Use according to claim 5, characterized in that, The method further comprises the following steps: The overnight culture of Lactobacillus casei is detected for absorbance at 620 nm.

7. Use according to claim 5, characterized in that, The temperature of the shock mixing is 30 DEG C.

8. Use according to claim 5, characterized in that, The speed of the shock mixing is 180 r / min.

9. Use according to claim 5, characterized in that, The time of the shock mixing is 2 h.

10. Use according to claim 5, characterized in that, The effective bacterial concentration of the Lactobacillus casei strain is 1x108 CFU / g.

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