CN114015626A - Lactobacillus plantarum-like bacteria with bacteriostatic function, composite preparation thereof and application - Google Patents

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus plantarum with an antibacterial function, a compound preparation of the lactobacillus plantarum and application of the lactobacillus plantarum. The preservation number of the lactobacillus plantarum is as follows: CCTCC NO: m20211339. The fermentation liquor of the strain has stronger bacteriostatic activity on pathogenic bacteria such as escherichia coli, staphylococcus aureus, listeria monocytogenes and the like, but has weaker bacteriostatic activity on lactic acid bacteria. Utilizes the bacteriostatic property of the strain and lactobacillus casei F209: (lactobacillus caseiF209) The CCTCC NO of the compound microbial inoculum is combined with the characteristics of quick fermentation and lactic acid production of M2018572 to carry out anaerobic fermentation on the aquatic feed, so that the feed quality can be improved, the disease incidence rate can be reduced, the culture benefit can be increased, and the compound microbial inoculum has a better application prospect.

Description

Lactobacillus plantarum-like bacteria with bacteriostatic function, composite preparation thereof and application

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus plantarum with an antibacterial function, a compound preparation of the lactobacillus plantarum and application of the lactobacillus plantarum.

Background

With the increasing intensification speed of the aquaculture industry and the popularization of high-density aquaculture modes, the aquaculture environment tends to worsen, so that aquatic animal diseases frequently occur in recent years, and huge losses are caused to the aquaculture industry. The problems of drug residue and drug resistance of germs caused by the fact that people use antibiotics in large quantities to reduce loss are increasingly prominent, serious threats are formed to human health and food safety, and antibiotic additives are limited or forbidden in many countries. Therefore, the micro-ecological preparation which is environment-friendly, safe and efficient becomes the focus of attention of people, and the product of the micro-ecological preparation is widely applied to the aquaculture industry after basic research at present, so that a better effect is achieved.

The probiotics refer to living microorganisms capable of promoting animal health in a certain concentration range, mainly comprise bacillus, lactic acid bacteria, saccharomycetes, photosynthetic bacteria and the like, and part of strains approved in China are applied to the breeding industry and the feed industry. Many studies have shown that probiotics primarily improve the intestinal flora structure of animals; enhancing the immunity of the animal body; promoting metabolism and growth of animals; purifying water quality, maintaining water environment stability and the like. Lactic acid bacteria are the most widely used probiotics, and can be tightly combined with intestinal mucosa cells through adhesins to fix and occupy the space on the surface of the intestinal mucosa; can also produce a plurality of bacteriostatic factors to inhibit the propagation of harmful bacteria; thereby improving the flora structure in intestinal tracts and improving the immunity of animal organisms; keep animal health and improve production capacity. The feed raw materials are fermented by using the lactic acid bacteria, so that the nutritive value of the feed can be effectively improved, and the production level is improved; the operation of inoculating lactobacillus to prepare the biological feed through anaerobic fermentation is relatively simple and convenient, the fermented feed has low pH, high acidity and good food calling property, and can promote digestion and absorption; the lactobacillus fermentation material does not need to be dried, has long storage time and good feeding effect.

Lactobacillus plantarum (Lactobacillus paracaseum) is a lactic acid bacterium that has passed the European Food Safety Agency (EFSA) safety qualification certificate (QPS), and has good bacteriostatic properties in addition to the probiotic function of Lactobacillus. The patent 'a strain of lactobacillus plantarum for reducing cholesterol and nitrite and a screening method thereof' (CN201610167712.1) introduces the excellent characteristics of lactobacillus plantarum GUFHSL-70 separated from mix the raw flesh of fish, meat and vegetables with salt or other flavouring fermented vegetables in the aspects of reducing nitrite and cholesterol, and the like lactobacillus plantarum stem fermentation liquor with the function of inhibiting pork decay in the 'property of lactobacillus plantarum-like antibacterial substances and the function thereof in pork fresh-keeping' introduced by Liu Changjian and the like, Liu Jie and the like discover that the lactobacillus plantarum L-ZS9 can form a biofilm to improve the cell adhesion capability, and the patent 'lactobacillus plantarum LuxS protein, the application thereof and lactobacillus plantarum recombinant bacteria' (CN202110139706.6) of Pinglan and the like successfully improve the expression of the LuxS protein to enhance the envelope adhesion function of the lactobacillus plantarum L-ZS 9. The research shows that the lactobacillus plantarum is expected to be applied to industries such as food health care, medicine and the like, but no report of the application of the lactobacillus plantarum in the breeding industry exists at present, and the research related to the compatibility and application of the lactobacillus plantarum and other probiotics is lacked. This may be related to the broad-spectrum bacteriostatic activity of lactobacillus plantarum, which not only has inhibitory effect on harmful bacteria such as putrefying bacteria, but also may inhibit the growth of other probiotics and reduce the probiotic effect.

Aiming at the problems, the lactobacillus plantarum F153 with good bacteriostatic property is obtained by screening, and the lactobacillus plantarum F153 has strong bacteriostatic activity on pathogenic bacteria such as escherichia coli, staphylococcus aureus, bacillus subtilis vibrio parahaemolyticus, listeria monocytogenes and the like, but has weak bacteriostatic activity on lactic acid bacteria and saccharomycetes, and has the characteristics of inhibiting pathogenic bacteria and being compatible with other probiotics. The lactobacillus casei F209 with better fermentation acid production performance is co-fermented with another strain, the number of bacteria is high, the diameter of a bacteriostatic circle is large, and the lactobacillus casei can be directly mixed to ferment aquatic feeds. The fermentation method has simple process, low processing cost and easy popularization and application. The application test of aquaculture shows that the microbial inoculum can improve the activity of protease, lipase and amylase in the digestive tract of aquatic animals, promote the growth of the aquatic animals, reduce the feed coefficient, improve the survival rate and have better application potential.

Disclosure of Invention

The first purpose of the invention is to provide a Lactobacillus strain with bacteriostatic property and probiotic property, wherein the strain is Lactobacillus plantarum F153 with a preservation number of CCTCC NO: M20211339.

Another objective of the invention is to provide a compound Lactobacillus preparation, which comprises Lactobacillus plantarum F153(CCTCC NO: M20211339) and Lactobacillus casei F209(CCTCC NO: M2018572).

The last purpose of the invention is to provide the application of the compound lactobacillus in preparing feed.

In order to achieve the purpose, the invention adopts the following technical measures:

the applicant obtains a lactobacillus strain separated from pickled Chinese cabbage, the lactobacillus strain is identified as lactobacillus plantarum through a 16S rDNA gene sequence, the strain is delivered to the China center for type culture collection for preservation at 10 months and 28 days in 2021, and the classification and the naming are as follows: lactobacillus plantarum (Lactobacillus paracasei) F153 with deposit number: CCTCC NO: m20211339, address: wuhan university in Wuhan, China.

The Lactobacillus plantarum F153(Lactobacillus paraplantarum F153) is a gram-positive bacterium, rod-shaped and does not produce spores. The bacterial colony on MRS solid culture medium is small and circular, has smooth and moist surface, neat edge and light milky white color, and the diameter of the bacterial colony is about 3 mm.

The strain is simply dyed and observed under a microscope, the shape of the strain is rod-shaped, the strain is single, paired or short chain-shaped, the size of the strain is 0.7-1.0um multiplied by 4.0-5.5 mu m, and the strain has no flagellum. Producing L-lactic acid by using glucose homolactic fermentation; the optimum growth temperature is 37 ℃.

The Lactobacillus plantarum F153(Lactobacillus paracasei F153) fermentation liquor provided by the invention has strong bacteriostatic activity on pathogenic bacteria such as Escherichia coli, Staphylococcus aureus and Listeria monocytogenes, but has weak bacteriostatic activity on lactic acid bacteria.

A compound Lactobacillus preparation comprises Lactobacillus plantarum F153(CCTCC NO: M20211339) and Lactobacillus casei F209(CCTCC NO: M2018572).

The above-mentioned composite Lactobacillus preparation, preferably, has the effective bacteria concentration ratio of Lactobacillus plantarum (Lactobacillus paracasei) F153(CCTCC NO: M20211339) and Lactobacillus casei (Lactobacillus casei) F209(CCTCC NO: M2018572) as follows: 1-10: 1-10, and the total effective bacteria concentration is 5-15 hundred million CFU/ml.

A method for preparing compound lactobacillus preparation comprises inoculating mixed seed solution of Lactobacillus plantarum F153(CCTCC NO: M20211339) and Lactobacillus casei F209(CCTCC NO: M2018572) (effective bacteria concentration ratio of two bacteria in the mixed seed solution is 1: 0.5-1.5) into fermentation culture medium (inoculation amount is 0.5-5%) for mixed culture or mixing seed solution after independent culture;

the fermentation medium comprises the following components in parts by weight:

1-3 parts of brown sugar, 3-9 parts of glucose, 0.5-5 parts of yeast extract, 0.01-0.2 part of sodium hydroxide and 100 parts of water, and fermenting for 24-48 hours at 35-40 ℃ after inoculation.

The compound lactobacillus (Lactobacillus plantarum F153 and Lactobacillus casei F209) is applied to the preparation of feed, and the feed is livestock feed or aquatic feed.

The method is that 0.5-5 parts of composite lactic acid bacteria liquid is added with water to be diluted to 80-120 parts and then mixed with 200 parts of complete feed, and the mixture is anaerobically fermented for more than 7 days in the environment with the temperature of more than 20 ℃, and then is extruded and granulated at low temperature, and is put into a breathing bag for low-temperature sealing storage, and is added into the aquatic feed according to a certain proportion for direct feeding, and the feeding is preferably carried out after 20-40% of fermented feed is added into the complete feed of the crayfish and is mixed uniformly.

When used in livestock feed, chickens are preferred.

Compared with the prior art, the invention has the following advantages:

1. the lactobacillus plantarum F153 has strong bacteriostatic activity on pathogenic bacteria such as escherichia coli, staphylococcus aureus, bacillus subtilis vibrio parahaemolyticus, listeria monocytogenes and the like, but has weak bacteriostatic activity on lactic acid bacteria and saccharomycetes, and has the characteristics of inhibiting pathogenic bacteria and being compatible with other probiotics.

2. Generally, the bacteriostasis of the lactobacilli such as lactobacillin of the lactobacillus is enhanced along with the reduction of pH, and the bacteriostasis capability can be improved by reducing the pH through the co-fermentation of the lactobacillus plantarum and other lactobacillus with strong acid production capability, so that the application value of the lactobacillus plantarum is improved. The two lactic acid bacteria are compounded, the lactobacillus casei F209 is rapidly fermented to produce lactic acid, and the lactobacillus plantarum F153 has strong antibacterial and disease-resistant capabilities and synergistic effects, so that the health-care effect is enhanced;

3. the application effect of the compound lactobacillus preparation in aquatic products is superior to that of poultry cultivation, and the yield and the survival rate can be obviously improved by feeding crayfish after aquatic feed is fermented.

Drawings

FIG. 1 shows the analysis of the single fermentation and co-fermentation processes of Lactobacillus plantarum F153 and Lactobacillus casei F209.

Detailed Description

The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available. The lactobacillus casei F209 of the present invention has been disclosed in CN 110804571B.

Example 1:

screening and identification of the Lactobacillus plantarum F153-like strain:

1. preparation of a culture medium:

BCP culture medium peptone 5 g; 3g of yeast extract; 5g of lactose; 20g of agar; 10ml of 0.5 percent bromocresol purple; 1000ml of distilled water; the pH value is 6.8-7.0.

MRS liquid medium: 20g/L of glucose; peptone 10 g/L; 5g/L of yeast extract; 10g/L of beef extract powder; 2g/L of diammonium citrate; 5g/L of sodium acetate; tween 801 g/L; dipotassium phosphate 2 g/L.

Solid medium 1.5% agar was added based on liquid medium and all media were sterilized at 115 ℃ for 20 min.

2. Screening: the method comprises the steps of putting 5-10g of various fermented foods such as spicy cabbage, pickled pepper, chopped hot pepper, fermented grains and pickled Chinese cabbage in 100ml of physiological saline, shaking at 150rpm for 15min, taking 1ml of culture solution for gradient dilution, coating a BCP (brain-cell plasma) plate, culturing at 37 ℃ for 48h, selecting single colonies with yellow colony peripheries, carrying out streaking separation culture, and selecting 256 single colonies in total. The obtained single colony is transferred into an MRS liquid culture medium, cultured for 48h at 37 ℃, and subjected to MRS slant preservation, and simultaneously, 16 strains with good bacteriostatic action on Escherichia coli K88 are obtained by primary screening through an agar dot diffusion cross-antagonism experiment. And then culturing the 16 strains of bacteria, respectively carrying out an Oxford cup quantitative diffusion inhibition Escherichia coli K88 test, and comparing the inhibition effect according to the size of the inhibition zone to obtain a lactobacillus F153 with a better effect of fermentation liquor on inhibiting Escherichia coli K88.

Biochemical identification of strain F153: strain F153, which is a gram-positive bacterium without spores, was briefly stained with crystal violet and observed by light microscopy. Grow into round, smooth and moist surface, neat edge, light milky small colony on MRS solid culture medium, the diameter is about 3 mm. The strain produces L-lactic acid by fermenting glucose homolactic acid; the optimum growth temperature is 37 ℃.

16s rDNA identification of the strains: f153 single colonies were picked and amplified with the universal primers and the amplified products were subjected to 16SrDNA sequencing analysis. The result shows that the sequence has the highest similarity with the Lactobacillus paraplantarum16S rDNA sequence, and reaches 99 percent, and the combined colony morphology can determine that the strain is the Lactobacillus plantarum like strain, which is named as the Lactobacillus plantarum F153. The strain is delivered to China center for type culture Collection for preservation at 28 months 10 and 2021, and is classified and named: lactobacillus plantarum (Lactobacillus paracasei) F153 with deposit number: CCTCC NO: m20211339, address: wuhan university in Wuhan, China.

Example 2:

bacteriostatic experiment of lactobacillus plantarum F153:

taking 50ml of lactobacillus plantarum F153-like fermentation liquor cultured for 24 hours in an MRS liquid culture medium, centrifuging at 10000rpm for 10min, separating thalli, adding 10ml of water for ultrasonic crushing after the thalli is resuspended for 2 times, and respectively carrying out an Oxford cup quantitative diffusion bacteriostasis test (the external diameter of an Oxford cup is 7.5mm) by using the fermentation liquor supernatant and the crushed bacteria liquid. The pH of MRS medium was adjusted with lactic acid to be the same as that of lactic acid bacteria supernatant as Control (CK). The antibacterial subjects are Escherichia Coli (Escherichia Coli K88), Staphylococcus aureus (Staphylococcus aureus ATCC 27217), Listeria monocytogenes (Listeria monocytogenes), Vibrio parahaemolyticus (Vibrio parahaemolyticus), Lactobacillus casei Zhang (Lactobacillus casei), Pediococcus acidilactici (Pediococcus acidilactici), Enterococcus faecalis (Enterococcus faecis), Lactobacillus acidophilus (Lactobacillus acidophilus), Bacillus subtilis (Bacillus subtilis), Candida tropicalis (Candida utilis) and Saccharomyces cerevisiae (Saccharomyces cerevisiae).

Table 1: bacteriostatic test result of lactobacillus plantarum F153-like

By comparing the diameters of inhibition zones of the supernatant, the broken thallus liquid and the CK in the table 1 to the indication bacteria, the supernatant of the fermentation liquor of the lactobacillus plantarum F153 and the broken thallus liquid have an inhibition effect on escherichia coli, staphylococcus aureus, vibrio parahaemolyticus, listeria monocytogenes and bacillus subtilis, and the inhibition effect of the supernatant of the fermentation liquor is obviously higher than that of the broken cell liquid; only the supernatant of the fermentation liquor has a relatively obvious inhibiting effect on pediococcus acidilactici; the fermentation broth supernatant and the broken bacterium liquid have weak bacteriostatic ability on lactobacillus casei, enterococcus faecalis, lactobacillus acidophilus, saccharomyces cerevisiae and candida tropicalis.

The results show that substances with strong bacteriostatic activity exist in the metabolite secreted to the extracellular by the lactobacillus plantarum F153, so the diameter of the bacteriostatic circle of the thallus crushing liquid is larger than that of the supernatant. The lactobacillus plantarum F153 has poor bacteriostatic effect on pediococcus acidilactici, lactobacillus casei Zhang, enterococcus faecalis, lactobacillus acidophilus, saccharomyces cerevisiae and candida tropicalis, so the lactobacillus plantarum F153 has the potential of compatibility and use with the lactobacillus and the saccharomycetes.

Example 3:

co-fermentation experiment of lactobacillus plantarum F153 and lactic acid bacteria:

the mixed culture of the Lactobacillus plantarum F153 and the Pediococcus acidilactici, the Lactobacillus casei Zhang, the enterococcus faecalis, the Lactobacillus acidophilus, the Lactobacillus plantarum, the Bacillus coagulans and the Lactobacillus casei F209 was compared with the culture of the Lactobacillus plantarum F153 alone.

YPD medium (1% yeast extract powder, 2% peptone, 2% glucose), pH-adjusted to 7.0, 500ml triangular flask 300ml, heating to 115 deg.C and sterilizing for 20 min. After cooling, inoculating 5ml of each of lactobacillus plantarum F153 and other lactic acid bacteria (the effective bacteria concentration ratio is 1: 1, the total bacteria concentration of the seed liquid is 6-15 hundred million CFU/ml, performing plate counting after culturing for 24h at 37 ℃, and measuring the pH and the bacteriostatic ability of the fermentation liquid, wherein the bacteriostatic experimental method is the same as that of example 2, the bacteriostatic material is the supernatant of the fermentation liquid, and the bacteriostatic experimental object is Escherichia Coli K88.

Table 2: experimental result of co-fermentation of lactobacillus plantarum F153 and lactic acid bacteria

The results are shown in table 2: after the lactobacillus plantarum F153 and other lactic acid bacteria except the lactobacillus casei F209 are co-fermented, the diameter of the inhibition zone of the supernatant is reduced to different degrees, and the pH of the fermentation liquor is reduced to different degrees. However, the influence of the co-fermentation on the total viable count is different, the total count of the lactobacillus plantarum F153 after co-fermentation with lactobacillus acidophilus, bacillus coagulans and enterococcus faecalis is reduced, and the total count of the lactobacillus plantarum after co-fermentation with pediococcus acidilactici, two strains of lactobacillus casei and lactobacillus plantarum is increased. From the results, the co-fermentation of the lactobacillus plantarum F153-like and the lactobacillus casei F209 can improve the viable count, greatly increase the pH value of the fermentation liquor, improve the bacteriostatic ability of the fermentation liquor and possibly improve the bacteriostatic effect due to low pH. In addition, the co-fermentation effect of the lactobacillus plantarum F153 also has strain specificity, and the lactobacillus plantarum F153 and the lactobacillus casei Zhang are co-fermented, so that the diameter of a bacteriostatic zone is reduced. The results show that the co-fermentation of the lactobacillus plantarum F153 and the lactobacillus casei F209 is most suitable and has the value of co-fermentation application.

Example 4:

single fermentation and co-fermentation experiments of lactobacillus plantarum F153 and lactobacillus casei F209:

preparing a fermentation medium: dissolving 10g brown sugar, 30g glucose and 20g yeast extract powder in 1000ml water, adjusting pH to 7.0, heating to 115 deg.C, and sterilizing for 20 min.

Seed liquid: the YPD medium (1% yeast extract powder, 2% peptone and 2% glucose) is sterilized at 115 deg.C for 20min, cooled to room temperature, inoculated with 1% lactobacillus, and cultured at 37 deg.C for 24 hr to obtain lactobacillus seed solution.

Fermentation was carried out using a 5L fermentor. Preparing 3L of fermentation medium according to the formula, controlling the temperature at 37 ℃ in the fermentation process, stirring at 50rpm, inoculating the seed liquid into the single fermentation group according to the inoculation amount of 3%, inoculating the two lactobacillus seed liquids into the co-fermentation group according to the inoculation amounts of 1.5%, and finishing the fermentation after 36 h. Sampling every 6h during the fermentation process to determine the concentration of reducing sugar, pH, the number of lactic acid bacteria and the size of the bacteriostatic circle of the fermentation liquid. The bacteriostasis experiment method is the same as that in example 2, the bacteriostasis material is supernatant of fermentation liquor, and the bacteriostasis experiment object is Escherichia Coli (Escherichia Coli K88).

The results are shown in FIG. 1: the results show that the fermentation process of the lactobacillus plantarum F153 is compared with the fermentation process of the lactobacillus casei F209, the pH of the fermentation liquor in the same fermentation time and at the end is high (4.21VS 3.54), the lactic acid content is low (13.98mg/ml VS 22.68mg/ml), the diameter of the inhibition zone is large (25.40mm VS 16.40mm), and the viable count is low (10.56 hundred million CFU/ml VS 16.56 hundred million CFU/ml), which indicates that the lactobacillus plantarum F153 has weak acid production capacity by fermentation and strong metabolic capacity of synthesizing the antibacterial substances compared with the lactobacillus casei F209.

After co-fermentation, the diameter of a bacteriostatic circle of the lactobacillus plantarum F153 and the lactobacillus casei F209 is slightly larger than that of the lactobacillus plantarum F153 in single fermentation (27.68mm VS 25.40mm), the number of viable bacteria is slightly smaller than that of the lactobacillus casei F209 in single fermentation (15.52 hundred million CFU/ml VS 16.56 hundred million CFU/ml), the content of lactic acid is 17.68mg/ml, and the lactic acid content is between that of the two lactobacillus in single fermentation. The results show that the single fermentation of the two lactic acid bacteria can simultaneously improve the yield and the bacteriostatic ability, and the two lactic acid bacteria can be fermented in a synergistic manner in production to quickly produce acid and improve the bacteriostatic ability.

Example 5:

the application of the composite lactic acid bacteria liquid preparation in the turbot feed comprises the following steps:

selecting a healthy turbot with the body length of 5-6cm, temporarily culturing the turbot in a laboratory of a sea water fine breeding center for one week, randomly dividing the turbot into 5 groups, repeating the 3 groups, and culturing the turbot in a circulating water aquarium, wherein the daily feeding amount is 5-10% of the weight, and the feeding is carried out for 2 times.

The 5 groups and feeds were: blank control group: basal ration; antibiotic group: basal diet + 50g amoxicillin antibiotics per ton; mixed bacteria fermentation group: mixing lactobacillus casei F209 and lactobacillus plantarum F153 to ferment the feed; lactobacillus casei F209 fermentation group: lactobacillus casei F209 fermented feed; lactobacillus plantarum F153-like fermentation group: lactobacillus plantarum F153-like fermented feed.

Preparing fermented feed: diluting 1kg of lactobacillus liquid with water to 100kg, mixing with 200kg of complete shrimp feed, performing anaerobic fermentation at 28-33 ℃ for 72h, performing low-temperature extrusion granulation, and packaging in a breathing bag for low-temperature sealed storage. The lactobacillus liquid is single lactobacillus casei F209 or lactobacillus plantarum F153, and the viable count is 5-10 hundred million CFU/ml; the lactobacillus liquid can also be prepared by mixing lactobacillus casei F209 and lactobacillus plantarum F153-like fermentation liquid according to the ratio of viable count of 1:2, wherein the viable count is 5-10 hundred million CFU/ml.

The grouped experimental fishes are cultured in a circulating water aquarium, and water is changed for 2 times every day, wherein 1/2 is changed every time. The bait is thrown twice a day, the feeding amount is 7-8 and 16-17 points respectively, and the feeding amount is based on the standard of satiation. The feed fed by each box of fish is weighed before and after feeding. And (3) absorbing excrement before feeding each time, absorbing residual baits after feeding, filtering, drying and storing the residual baits for calculating the food intake. The feeding conditions are constant (the water temperature is 18 +/-1 ℃, the salinity is 31 +/-0.2), and the experimental period is 60 days.

The results shown in Table 3 show that the lactobacillus fermented feed can obviously improve the survival rate and the specific growth rate of the juvenile turbot and reduce the feed coefficient, the survival rate of the lactobacillus plantarum F153-like fermentation group is higher than that of the lactobacillus casei F209 fermentation group, but the specific growth rate is lower, and the performance of the mixed fermentation group is better than that of single fermentation.

Table 3: experiment for 60d survival rate and specific growth rate of turbot (n is 6; X + -SE)

Randomly selecting 6 turbots from each group for dissection after the experiment is finished, taking stomach, cecum and intestine for enzyme activity detection,

the used kits are all produced from Nanjing to build up bioengineering research.

Results shown in Table 4 show that the three groups of lactobacillus fermented feed fermentation groups can improve the activity of protease in the digestive tract of the juvenile turbot, the digestive tract protease activity of the mixed fermentation group is relatively highest, the digestive tract protease activity of the lactobacillus casei F209 fermentation group is higher than that of the lactobacillus plantarum F153-like fermentation group, and the digestive tract protease activity of the antibiotic group is lowest.

Table 4: effect of different treatments of the feed on the digestive tract protease Activity of turbot (n ═ 6; X + -SE)

Results shown in Table 5 show that the three groups of lactobacillus fermented feed fermentation groups can improve the activity of lipase in the digestive tract of the juvenile turbot, the digestive tract lipase activity of the mixed bacteria fermentation group is relatively highest, the digestive tract lipase activity of the lactobacillus casei F209 fermentation group is higher than that of the lactobacillus plantarum F153-like fermentation group, and the digestive tract lipase activity of the blank group is lowest.

Table 5: effect of different treatments of the feed on the Activity of turbot digestive Lipase (n ═ 6; X. + -. SE)

The results shown in Table 6 show that the three groups of lactobacillus fermented feed fermentation groups can improve the activity of amylase in the digestive tract of the juvenile turbot, the digestive tract amylase activity of the mixed fermentation group is similar to and highest than that of the lactobacillus casei F209 fermentation group, the digestive tract amylase activity of the lactobacillus plantarum F153-like fermentation group is slightly lower than that of the blank group, and the digestive tract amylase activity of the antibiotic group is similar.

Table 6: effect of different treatments of the feed on the Activity of the digestive tract Amylase of turbot (n ═ 6; X. + -. SE)

After the experiment is finished, starvation treatment is carried out on juvenile turbots for 24h, 6 turbots are randomly selected from each group, blood (3-4 mL) is taken from tail veins of each group, a low-temperature centrifuge is used for centrifuging (10000 r/min, 10min), and then serum is taken for measuring immunological indexes including Lysozyme (LZM), Catalase (CAT), superoxide dismutase (SOD) activity and total antioxidant capacity (T-AOC), and the kit is built by Nanjing for bioengineering research and production.

Table 7: effect of different treatments of the feed on the Activity of the digestive tract Amylase of turbot (n ═ 6; X. + -. SE)

Results shown in Table 7 show that the three groups of lactobacillus fermented feed fermentation groups can improve the activities of Lysozyme (LZM), Catalase (CAT), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in the serum of the juvenile turbot, compared with the blank control group and the antibiotic group, the four groups of lactobacillus plantarum F153-like fermentation group and lactobacillus casei F209-like fermentation group have the highest enzyme activities.

In general, the fermented feed of lactobacillus casei F209 and lactobacillus plantarum F153 can improve the activities of digestive tract protease, amylase and lipase, promote digestion and absorption, further improve the growth rate, improve the activities of Lysozyme (LZM), Catalase (CAT), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in the serum of juvenile turbot and enhance the immunity related capacity of the turbot by feeding the fermented feed, and the mixed-strain fermented feed has better growth promoting effect and immunity improving effect on the turbot than the single-strain fermented feed.

Example 6:

the application of the compound lactobacillus liquid preparation in the broilers comprises the following steps:

80 white feather broilers of 20 days old are divided into 4 groups, each group comprises 20 white feather broilers, and the 4 groups and feeds respectively comprise: blank control group: basal commodity ration; mixed bacteria fermentation group: mixing lactobacillus casei F209 and lactobacillus plantarum F153 to ferment the feed; lactobacillus casei F209 fermentation group: lactobacillus casei F209 fermented feed; lactobacillus plantarum F153-like fermentation group: lactobacillus plantarum F153-like fermented feed. The feed was prepared as in example 5. Growth performance was measured after 30 days of culture and 5 animals were slaughtered and slaughter performance was measured for each group.

Table 8: effect of different treatments of feed on growth of broiler chickens

The results in Table 8 show that the lactobacillus fermented feed can increase the daily average feed intake of broilers, improve the average daily gain and reduce the feed-weight ratio, but the difference between the groups is not obvious.

Table 9: influence of different treatments of feed on slaughter performance of broiler chickens

The results in Table 9 show that the dressing percentage, the pectoral muscle rate, the leg muscle rate and the total thorax rate of each group have no obvious difference, the dressing performance of the broiler chicken cannot be obviously improved by the lactobacillus-fermented feed, and the growth promoting effect of the lactobacillus plantarum F153 on the broiler chicken is not obvious.

Example 7:

the application of the compound lactobacillus in the crayfish feed comprises the following steps:

adding 20% and 50% of fermented feed into the shrimp complete feed to serve as experimental groups, and carrying out crayfish breeding experiments by taking the fermented feed as a control group without adding the fermented feed; 100 plus 150 healthy shrimp seedlings per jin are put in 3 months in 2021, and 30 jin are put in each shrimp pond. Feeding a complete material added with 20% of fermentation material by using two ponds as an experimental group 1; the two ponds are used as an experimental group 2, full-value materials added with 50% of fermentation materials are fed, the two ponds are used as a control group, and full-value materials not added with the fermentation materials are fed. The feeding period is three months, the management mode is the same, and the feed feeding amount per mu of shrimp pond is the same every day (the fermented feed is calculated on a dry basis).

Preparing fermented feed: diluting 1kg of composite lactobacillus liquid (the viable count of lactobacillus casei F209 and lactobacillus plantarum F153 is 20 hundred million and 12 hundred million CFU/ml respectively) with water to 100kg, mixing with 200kg of complete shrimp feed, performing anaerobic fermentation at 25-30 ℃ for 7 days, performing low-temperature extrusion granulation, and packaging in a breathing bag for low-temperature sealed storage.

Table 10: experimental result of complete feed cultivation of shrimp by fermenting composite lactobacillus liquid preparation

The culture results are shown in table 10: the yield per mu of the crayfish added with 20% of the fermented feed in the experimental group can reach 199kg averagely, and is improved by 23.22% compared with the average yield per mu of 161.5kg in the control group; the weight of the experimental group of crayfish can reach 42.05g, and the weight of the experimental group of crayfish is increased by 18.62 percent compared with the weight of the control group of crayfish which is 35.45 g; the average survival rate is also improved from 78.65% to 86.76%. The yield per mu of the crayfish added with 40 percent of fermented feed can reach 221.5kg averagely, and is improved by 37.15 percent compared with the average yield per mu of 161.5kg of a control group; the weight of the experimental group of crayfish can reach 44.10g, and the weight of the experimental group of crayfish is increased by 24.40% compared with the weight of the control group of crayfish which is 35.45 g; the average survival rate is improved to 88.22%. The mixed fermented feed containing lactobacillus casei F209 and lactobacillus plantarum F153 added into the crayfish feed can obviously improve the weight gain and the survival rate of crayfish, and the effect of 50% of the added amount of the fermented feed is better than that of 20% of the added amount of the fermented feed, so that the breeding benefit is better.

Claims (9)

1. Separated lactobacillus plantarum (A)Lactobacillus paraplantarum) The preservation number of the lactobacillus plantarum is as follows: CCTCC NO: m20211339.

2. The use of a lactobacillus plantarum as defined in claim 1 for the preparation of a bacterial bacteriostatic agent, said bacterium being: escherichia coli (Escherichia Coli K88) Staphylococcus aureus (1)Staphylococcus aureusATCC 27217), Listeria monocytogenes (L.) (Listeria monocytogenes) Vibrio parahaemolyticus: (Vibrio parahaemolyticus) Bacillus subtilis preparation (B)Bacillus subtilis）。

3. A complex lactic acid bacteria preparation comprising the Lactobacillus plantarum and Lactobacillus casei of claim 1 (L.), (L.casei)lactobacillus casei）F209。

4. The compound lactic acid bacteria preparation of claim 3, wherein the effective bacteria concentration ratio of the lactobacillus plantarum and lactobacillus casei F209 is as follows: 1-10: 1-10.

5. The method for producing the composite lactic acid bacterium preparation according to claim 4, comprising inoculating the mixed seed solution of Lactobacillus plantarum and Lactobacillus casei F209 according to claim 1 to a fermentation medium and performing mixed culture;

the fermentation medium comprises the following components in parts by weight: 1-3 parts of brown sugar, 3-9 parts of glucose, 0.5-5 parts of yeast extract, 0.01-0.2 part of sodium hydroxide and 100 parts of water.

6. Use of the complex lactic acid bacteria of claim 3 in the preparation of aquaculture feed.

7. The use of the compound lactic acid bacteria of claim 3 in the preparation of livestock and poultry feed.

8. The use of claim 6, wherein the water is: crayfish, weever, turbot.

9. The use according to claim 7, wherein the livestock and poultry are chickens.

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