A kind of Lactobacillus paracasei LBR-002 and application thereof

Abstract

The invention provides a lactobacillus paracasei LBR-002 and an application thereof, belonging to the technical field of functional microbial fermentation and application, wherein the lactobacillus paracasei LBR-002 provided by the invention has a preservation number of CGMCC No.26775, has strong acid resistance, salt resistance, acid production ability, and has extremely strong fermentation conversion ability for traditional Chinese medicine and feed. The application effect is definite, safe and reliable, and the lactobacillus paracasei LBR-002 is suitable for the development of traditional Chinese medicine fermentation, feed additives, and fermented feed.

Description

Technical Field

[0001] The invention belongs to the technical field of functional microbial fermentation and application, and relates to a Lactobacillus paracasei LBR-002 and an application thereof. Background Art

[0002] As a probiotic, Lactobacillus paracasei plays an increasingly important role in livestock and aquaculture. In the field of aquaculture, Lactobacillus paracasei also shows its unique application value. It can help improve the intestinal microecology of aquatic animals, reduce the accumulation of harmful substances such as ammonia nitrogen and nitrite, thereby improving water quality and reducing the pathogen load in the breeding environment. For some high-density aquatic species, such as shrimp and crabs, the addition of Lactobacillus paracasei can help reduce the risk of disease outbreaks and increase survival rates and yields.

[0003] However, Lactobacillus paracasei also faces some challenges in practical applications: First, the survival rate and stability of Lactobacillus paracasei in aquatic environments are affected by multiple factors such as water quality, temperature, and pH value, which limits its effectiveness in practical applications; second, the aquaculture environment is complex and changeable, and Lactobacillus paracasei lacks sufficient stress resistance and is not able to adapt to adverse environments such as high salinity and low oxygen. Third, although Lactobacillus paracasei has a certain immunomodulatory effect, its immune enhancement effect in aquatic animals is not as significant as in mammals, and further research and optimization are needed.

[0004] As medicine or feed additive, Chinese medicine has the advantages of high safety, no drug resistance, and low residue. It can be used as a substitute for antibiotics for the prevention and treatment of diseases in aquatic animals, and has a positive effect on the healthy growth of aquatic animals. However, Chinese medicine takes a long time to take effect, and the effect of directly using Chinese medicine in aquatic animals needs to be strengthened. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a lactobacillus paracasei LBR-002 and an application thereof. The lactobacillus paracasei LBR-002 of the present invention has strong acid resistance, salt resistance and acid production ability, and has extremely strong fermentation conversion ability for Chinese medicine and feed. The application effect is definite, safe and reliable, and the lactobacillus paracasei LBR-002 is suitable for the development of Chinese medicine fermentation, feed additives and fermented feed.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides a Lactobacillus paracasei LBR-002, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration Committee, with a preservation date of March 9, 2023, a classification name of Lactobacillus paracasei, and a preservation number of CGMCC No.26775.

[0008] The Lactobacillus paracasei LBR-002 provided by the present invention was isolated from Sichuan bean paste originating from Pi County, Sichuan, purchased in Weifang City, Shandong Province, China on January 12, 2023.

[0009] The invention provides the Lactobacillus paracasei LBR-002, which has a suitable growth temperature of 8-45 DEG C, a pH value of 3.0-9.0, and a fermentation time of 16-72 hours.

[0010] Furthermore, the suitable growth temperature of the Lactobacillus paracasei LBR-002 provided by the present invention is 37° C., the pH value is 7.0, and the fermentation time is 24 hours.

[0011] The invention also provides application of the Lactobacillus paracasei LBR-002 in fermented traditional Chinese medicine.

[0012] Furthermore, the Lactobacillus paracasei LBR-002 fermented Chinese medicine is provided, wherein the suitable growth temperature of Lactobacillus paracasei LBR-002 is 37° C., the pH value is 7.0, and the fermentation time is 64 hours.

[0013] The invention also provides fermented feed containing the Lactobacillus paracasei LBR-002.

[0014] Furthermore, the Lactobacillus paracasei LBR-002 fermented feed is provided, wherein the Lactobacillus paracasei LBR-002 has a suitable growth temperature of 37° C., an initial pH value of 6.0, and a fermentation time of 72 hours.

[0015] The invention also provides a microecological preparation containing the Lactobacillus paracasei LBR-002.

[0016] The beneficial effects of the present invention are mainly reflected in:

[0017] 1. Lactobacillus paracasei LBR-002 has the common characteristics and functions of Lactobacillus paracasei, and is more resistant to low temperature, acid and salt. It shows good tolerance in the digestive system and growth system of aquatic animals, can tolerate the low acid environment in the digestive tract, and quickly colonizes in the intestine, which helps to regulate the intestinal flora. In addition, it can grow normally in seawater, improve the breeding environment, and has the potential to be developed into a microecological preparation.

[0018] 2. Lactobacillus paracasei LBR-002 exhibits significant acid production ability, produces a large amount of organic acids, and has excellent potential for the transformation of fermented Chinese medicine. This strain can enhance the content of active ingredients in fermented Chinese medicine, convert macromolecules into small molecules, make it easier for farmed animals to absorb, improve the efficacy of Chinese medicine, and solve the problem of slow effect of Chinese medicine. At the same time, it helps prevent the corruption and deterioration of fermented Chinese medicine and feed. Therefore, this strain has a wide range of application potential in the fields of Chinese medicine fermentation and feed fermentation.

[0019] 3. Lactobacillus paracasei LBR-002 improves the quality of feed and the nutritional value of feed, promoting the growth of aquaculture animals in both directions.

[0020] 4. The fermentation product of Lactobacillus paracasei LBR-002 of the present invention is used as a probiotic and feed additive, and does not have the drug resistance and residue problems of antibiotic feed additives, and is safe to use. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Gram staining microscopic morphology of Lactobacillus paracasei LBR-002.

[0022] Figure 2 Effect of different initial pH values ​​on the growth of Lactobacillus paracasei LBR-002.

[0023] Figure 3 Effect of different fermentation temperatures on the growth of Lactobacillus paracasei LBR-002.

[0024] Figure 4 Effects of different fermentation times on the growth of Lactobacillus paracasei LBR-002. DETAILED DESCRIPTION

[0025] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0026] Example 1, screening, separation and identification of Lactobacillus paracasei LBR-002

[0027] 1. Screening, separation and purification of Lactobacillus paracasei LBR-002

[0028] 1g of sample was scraped from Sichuan bean paste, added to 9ml of sterile distilled water, vortexed for 2min to make a suspension, and then diluted and spread on MRS medium, a selective medium for lactic acid bacteria, and cultured anaerobically at 37°C for 36h. A single colony on the plate was picked, streaked again on the MRS medium plate, and cultured anaerobically at 37°C. The obtained colony was streaked and purified multiple times to obtain a pure colony. The single colonies were named LBL-001, LBL-002, LBL-003, ..., LBL-039 in sequence. The single colonies obtained by separation and purification were activated and cultured for 24 hours, and inoculated into MRS liquid culture media with pH values ​​of 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5 and 2.0 at an inoculum size of 1% by volume, and cultured anaerobically at 37°C for 24 hours. At the same time, the sterile culture medium with the corresponding pH value was used as the blank control group, and the absorbance at 600nm and pH value of each group of culture media were measured respectively.

[0029] The experimental results show that among the 39 bacterial strains screened by the applicant, only five strains, LBL-008, LBL-014, LBL-018, LBL-024 and LBL-033, can grow normally in MRS liquid culture medium with a pH value of 3.0-4.0 and have strong acid resistance.

[0030] The above 5 strains were inoculated with 1% of their volume into MRS liquid medium with a pH value of 7 and NaCl concentrations of 2%, 4%, 6%, 8%, 10%, and 12%, respectively, and cultured anaerobically at 37°C for 24 hours. At the same time, the sterile medium with the corresponding pH value was used as the blank control group, and the absorbance at 600nm and pH value of each group of medium were measured. The three strains of LBL-008, LBL-018, and LBL-033 can grow normally in 8%-12% MRS liquid medium and have strong salt tolerance. Among them, the LBL-033 strain has the strongest acid resistance. When the pH value is 3.0, the survival rate of live bacteria is still as high as 85%, and the acid production ability is also the strongest.

[0031] 2. Identification of Lactobacillus paracasei LBL-033

[0032] (1) Morphological characteristics and Gram staining

[0033] The LBL-033 strain was streaked and cultured several times, and the colony morphology was observed to be round, with neat edges, milky white or light color, and a smooth and raised surface.

[0034] Microscopic examination shows short or long rod-shaped pleomorphic bacilli, with varying lengths, but generally less than 1.5 μm in width. The two ends of the bacteria are flat and square, and the arrangement is mostly short or long chains. Sometimes spherical bacteria can also be seen. Gram staining is positive, without flagella and spores. Figure 1 .

[0035] (2) Molecular Biological Identification

[0036] The DNA of the strain LBL-033 was used as a template, 16SrRNA universal primers were used for amplification, and the amplified fragments were sequenced. The 16SrDNA sequencing results of the strain LBL-033 were compared with the sequences in GenBank. The results showed that the strain LBL-033 and Lactobacillus paracasei The homology was the highest, so the strain LBL-033 was determined to be Lactobacillus paracasei.

[0037] The applicant named the screened LBL-033 strain as Lactobacillus paracasei LBR-002 for strain preservation. The depository unit is the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC); address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; deposit date: March 9, 2023; the deposit number of Lactobacillus paracasei LBR-002 is: CGMCC No.26775.

[0038] Example 2, Lactobacillus paracasei LBR-002 growth performance determination

[0039] Lactobacillus paracasei LBR-002 was inoculated with 1% inoculum in MRS liquid medium at different initial pH values ​​and the OD 600 To draw a growth curve for the determination of indicators, see Figure 2 ; Lactobacillus paracasei LBR-002 was inoculated with 3% inoculum in MRS liquid medium, and the OD 600 Draw growth curves for the determination indicators. Figure 3 , Figure 4 .Depend on Figure 2 It can be seen that Lactobacillus paracasei LBR-002 can grow at pH values ​​of 3.0-9.0, and the most suitable initial pH value for growth is 7.0; Figure 3 It can be seen that Lactobacillus paracasei LBR-002 can grow at 6℃-53℃, and the most suitable growth temperature is 37℃; Figure 4 It can be seen that the growth hysteresis period of Lactobacillus paracasei LBR-002 is about 6 hours from the beginning of culture, and then it enters the logarithmic growth period, during which the bacteria increase exponentially. When cultured for 24 hours, the OD 600The maximum value was reached, and 24 to 72 hours was the stable growth period of Lactobacillus paracasei LBR-002. The bacteria grew slowly and gradually entered the decline period after 72 hours.

[0040] Example 3: Evaluation of the Acid Production Capacity of Lactobacillus paracasei LBR-002

[0041] In order to further verify the acid production ability of Lactobacillus paracasei LBR-002, the applicant inoculated equal amounts of Lactobacillus paracasei LBR-002 and the control strain Lactobacillus paracasei ATCC27216 into MRS liquid culture medium with a pH value of 7.0, cultured at 37°C, and measured the pH value of the culture medium every 4 hours. The specific results are shown in Table 1.

[0042] Table 1 Changes in pH value of culture medium of Lactobacillus paracasei LBR-002 strain at different culture times

[0043]

[0044] As can be seen from the data in Table 1, after culturing for 12 h, the pH value of the culture medium inoculated with the control strain Lactobacillus paracasei ATCC27216 was reduced from 7.0 to 5.54, while the pH value of the culture medium inoculated with Lactobacillus paracasei LBR-002 was reduced from 7.0 to 5.12; after culturing for 24 h, the pH value of the culture medium inoculated with the control strain Lactobacillus paracasei ATCC27216 was reduced to 4.42, while the pH value of the culture medium inoculated with Lactobacillus paracasei LBR-002 was reduced to 3.14; thus, it is illustrated that the Lactobacillus paracasei LBR-002 provided by the present invention has a stronger acid production ability and a faster acid production speed than the control strain.

[0045] Example 4: Evaluation of the fermentation conversion ability of Lactobacillus paracasei LBR-002 on traditional Chinese medicine

[0046] (1) Preparation of fermentation culture medium for traditional Chinese medicine

[0047] The combined Chinese medicines: licorice, dandelion, houttuynia cordata, honeysuckle, are used in a ratio of 2:2:2:1, crushed and passed through a 40-mesh sieve, added with water to cover the Chinese medicines, soaked for 1 hour, boiled until concentrated twice, and the final concentration of the Chinese medicine extract liquid is 0.5g / ml, and the supplementary culture medium components are added in proportion according to volume, and sterilized by high-pressure steam at 121°C for 20min for use.

[0048] Supplementary medium ingredients (g / L):

[0049] Peptone 10.0, beef extract powder 5.0, yeast extract powder 4.0, glucose 20.0, dipotassium hydrogen phosphate 2.0, triammonium citrate 2.0, sodium acetate 5.0, magnesium sulfate 0.2, manganese sulfate 0.05.

[0050] (2) Lactobacillus paracasei LBR-002 was inoculated into a fermentation Chinese medicine medium at a volume ratio of 5%, the pH value was adjusted to 7.0 with 30% sodium hydroxide solution, the fermentation temperature was 37°C, and the fermentation was performed at 100 r / min for 64 h.

[0051] The dandelion, houttuynia cordata, and honeysuckle in the fermented Chinese medicine culture medium contain flavonoid active ingredients, and licorice contains glycyrrhizic acid and glycyrrhetinic acid. The fermented Chinese medicine culture medium without inoculation of any strain was used as the control group, and the fermented Chinese medicine culture medium with Lactobacillus paracasei LBR-002 added was used as the treatment group. The final pH value of the control group was 5.42, and the pH value of the treatment group was 3.86. The content of the effective ingredients in the fermented Chinese medicine culture medium was determined, and the specific results are shown in Table 2.

[0052] Table 2 Effects of Lactobacillus paracasei LBR-002 on some active ingredients of fermented Chinese medicine

[0053]

[0054] From Table 2, we can see that compared with the control group, the effective ingredients of Chinese medicine fermented by Lactobacillus paracasei LBR-002 are 1-8 times higher than those of unfermented Chinese medicine, which shows that the Lactobacillus paracasei LBR-002 provided by the present invention has a very strong fermentation and conversion ability for Chinese medicine, and releases the effective ingredients that are difficult to dissolve in Chinese medicine. Taking licorice as an example, the intestinal metabolism and pharmacokinetic studies of licorice show that glycyrrhizic acid is slowly converted into glycyrrhetinic acid in the intestine. After oral administration of glycyrrhizic acid, glycyrrhetinic acid is mainly detected in plasma, while glycyrrhizic acid is not detected. The effective ingredient glycyrrhizic acid of licorice fermentation for 24h increases by 1.6 times, and the more easily absorbed glycyrrhetinic acid fermentation for 48h increases by 8 times, converting glycyrrhizic acid into more easily absorbed glycyrrhetinic acid.

[0055] At the same time, the two groups were subjected to accelerated tests: they were placed under the same conditions of temperature 40℃±2℃ and relative humidity 75%±5% for 6 months. The control group was initially a dark brown uniform liquid with a bitter taste of Chinese medicine cooking. In the first month, floccules and a large amount of sediment appeared. In the second month, the color changed significantly, stratification occurred, and a corrupt smell appeared. In the third month, it was corrupt and slightly appeared cyan mold-like substances. In the fourth month, it was completely corrupted and a large amount of cyan bacteria appeared. The treatment group was initially a light brown uniform liquid with a lactic acid taste and the fragrance of Chinese medicine. In the first month, a slight precipitate appeared that dispersed with a shake, and the color and smell did not change. In the second month, the precipitate, color, and smell did not change significantly. There were no significant changes in the third and sixth months. From the results of the accelerated test, we can see that Lactobacillus paracasei LBR-002 has a preservative and preservation effect on fermented Chinese medicine, and it can be used as a feed additive.

[0056] According to the results of the control test and the accelerated test, Lactobacillus paracasei LBR-002 has a strong acid-producing ability, produces a large amount of organic acid, and has a strong fermentation conversion ability in fermented Chinese medicine, which can effectively increase the content of active ingredients in fermented Chinese medicine and convert large molecules into small molecules. At the same time, it has the function of a natural preservative, effectively preventing the fermented Chinese medicine from spoiling and deteriorating, and can be widely used in the field of Chinese medicine fermentation.

[0057] Example 5: Effect of Lactobacillus paracasei LBR-002 on the quality of fermented feed

[0058] The suspension of Lactobacillus paracasei LBR-002 was streaked on MRS solid medium and cultured at 37℃ for 36h to isolate single colonies. A single colony was picked and inoculated in MRS liquid medium and cultured at 37℃ and 100r·min-1 for 2d to obtain the seed solution for fermentation; Lactobacillus paracasei LBR-002 was inoculated into MRS liquid medium at an inoculum of 1% and cultured at 37℃ for 24h; the above bacterial solution was inoculated into a roughage consisting of 15% wheat bran and 35% soybean meal at an inoculum of 5% according to the mass fraction, and sterile water was added to adjust the final moisture to 50%. The mixture was placed in a closed fermentation bag with an initial pH of 6.0 and cultured at 37℃. The above roughage without any strain inoculation was used as the control group. Samples were taken after 72h of fermentation to determine the pH value, viable count, total acid, crude protein and acid-soluble protein content of the fermented feed. The specific results are shown in Table 3.

[0059] Table 3 Effect of Lactobacillus paracasei LBR-002 on the quality of fermented feed

[0060]

[0061] As can be seen from the data of Table 3, compared with the control group, crude protein and acid-soluble protein contents in the fermentation group feed after fermentation by Lactobacillus paracasei LBR-002 have increased by 81.48% and 86.74% respectively, the organic acid content has significantly increased, and the pH has significantly decreased. Thus, it can be explained that the Lactobacillus paracasei LBR-002 provided by the invention can degrade macromolecular substances such as partial protein and fat, generate small molecules such as organic acid and soluble polypeptide, form a biological feed with rich nutrition, good palatability and high viable bacteria content, and significantly improve the palatability and nutritional value of fermented feed.

[0062] At the same time, the two groups were subjected to accelerated tests: placed under the same conditions of temperature 40℃±2℃ and relative humidity 75%±5%, the control group showed signs of deterioration on the 55th day and completely deteriorated in the 3rd month; the fermentation group had no obvious changes in color and odor until the 6th month, the color began to change in the 9th month, and the odor did not change significantly, and the color changed from yellow to brown in the 12th month, and the odor did not change significantly. Considering that the shelf life of raw soybean meal is 6 months, the fermentation of Lactobacillus paracasei LBR-002 greatly extends the shelf life of feed made from soybean meal.

[0063] According to the results of control tests and accelerated tests, Lactobacillus paracasei LBR-002 significantly improved the quality of fermented feed and can be widely used in the field of feed fermentation.

[0064] Example 6: Application of Lactobacillus paracasei LBR-002 fermented feed in sea cucumber farming.

[0065] At a sea cucumber breeding farm in Weihai of a certain group, on January 20, 2023, there were two mature sea cucumber ponds, each with 20,000 sea cucumbers. The two sea cucumber ponds were divided into a control group and an experimental group. The two groups had the same management conditions and basically the same growth conditions of sea cucumbers. The control group used daily sea cucumber feed, and the experimental group used daily sea cucumber feed supplemented with 5% of the fermented feed of Lactobacillus paracasei LBR-002 in Example 5, with a mass ratio of 95:5. After harvesting in spring, the experimental group produced 7% more sea cucumbers than the control group, which greatly improved the breeding efficiency.

[0066] The invention discloses a Lactobacillus paracasei LBR-002 and an application thereof. The above embodiments are only used to illustrate the technical scheme of the invention rather than to limit the same. Although the invention is described in detail with reference to the above embodiments, it is still possible for a person skilled in the art to modify the technical scheme recorded in the above embodiments, or to perform equivalent replacement on some of the technical features thereof. However, these modifications or replacements do not make the essence of the corresponding technical scheme deviate from the spirit and scope of the technical scheme claimed to be protected by the invention.

Claims

1. A Lactobacillus paracasei LBR-002, characterized in that: The Lactobacillus paracasei LBR-002 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, with a preservation date of March 9, 2023, a classification name of Lactobacillus paracasei, and a preservation number of CGMCC No.26775.

2. Lactobacillus paracasei LBR-002 according to claim 1, is characterized in that: The Lactobacillus paracasei LBR-002 is isolated from Sichuan bean paste originating from Pi County, Sichuan in Weifang City, Shandong Province, China.

3. Lactobacillus paracasei LBR-002 according to claim 1, is characterized in that: The suitable growth temperature of the Lactobacillus paracasei LBR-002 is 8-45°C, the pH value is 3.0-9.0, and the fermentation time is 16-72h.

4. Lactobacillus paracasei LBR-002 according to claim 3, is characterized in that: The suitable growth temperature of the Lactobacillus paracasei LBR-002 is 37°C; the pH value is 7.0; and the fermentation time is 24 hours.

5. application of Lactobacillus paracasei LBR-002 according to claim 1 in fermented Chinese medicine.

6. Lactobacillus paracasei LBR-002 fermented Chinese medicine according to claim 5, is characterized in that: The suitable growth temperature of the Lactobacillus paracasei LBR-002 is 37° C., the pH value is 7, and the fermentation time is 64 hours.

7. A fermented feed containing the Lactobacillus paracasei LBR-002 according to claim 1.

8. Lactobacillus paracasei LBR-002 fermented feed according to claim 7, is characterized in that: The suitable growth temperature of the Lactobacillus paracasei LBR-002 is 37° C., the initial pH value is 6, and the fermentation time is 72 hours.

9. A microecological preparation containing the Lactobacillus paracasei LBR-002 according to claim 1.

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