CN107164269B

CN107164269B - Lactobacillus paracasei, preparation and application of lactobacillus paracasei in pig feed

Info

Publication number: CN107164269B
Application number: CN201710418582.9A
Authority: CN
Inventors: 季海峰; 刘辉; 王四新; 张董燕; 王晶; 张伟; 王雅民
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2017-06-06
Filing date: 2017-06-06
Publication date: 2020-03-24
Anticipated expiration: 2037-06-06
Also published as: CN107164269A

Abstract

The invention discloses Lactobacillus paracasei (Lactobacillus paracasei) ZLP019 with the preservation number as follows: CGMCC No. 11532. The bacterium has good acid resistance, cholate resistance and bacteriostatic properties. The freeze-dried preparation prepared by the strain has high survival rate of viable bacteria, and is convenient to use in feed enterprises and farms. The preparation is directly added into the feed of the sow according to the proportion requirement, can improve the reproductive performance of the sow, improve the daily gain and the survival rate of suckling piglets, promote the intestinal health of the sow, reduce the emission of harmful bacteria in excrement and is beneficial to the environment-friendly breeding.

Description

Lactobacillus paracasei, preparation and application of lactobacillus paracasei in pig feed

Technical Field

The invention relates to the technical field of biological feed, and particularly relates to lactobacillus paracasei, a preparation and application thereof in pig feed.

Background

In the pig raising production, sows play an important role in breeding piglets and suckling piglets, and have long utilization years and great play effect. However, sows in large-scale pig farms often suffer from problems of constipation, decreased appetite, poor farrowing performance, low lactation yield, low piglet survival rate and the like, and the production performance and economic benefit of the farms are seriously affected.

The lactobacillus has the effects of regulating and controlling the micro-ecological balance of animal intestinal tracts, promoting the digestion and absorption of nutrient substances and promoting the health of organisms, is reasonably fed, and is expected to improve the comprehensive production capacity of sows.

Lactobacillus paracasei (Lactobacillus paracasei) is one of the groups of Lactobacillus, and is often applied to the production of dairy products, health foods, beverages, ice cream and the like. In recent years, the feed additive variety catalog (2013) of Ministry of agriculture allows lactobacillus paracasei to be applied to animal breeding, and promotes the application of lactobacillus paracasei in the aspect of feed breeding. However, the related lactobacillus paracasei preparation, the preparation method thereof and the advanced technology in the feed cultivation are few, and the patent application with the publication number of CN 103070287A discloses the steps of lactobacillus paracasei liquid fermentation and lactobacillus paracasei solid fermentation, and the fermented feed can reduce the diarrhea rate of weaned pigs, improve the growth speed of pigs and improve the feed conversion and utilization efficiency; patent application with publication number CN 103039699B discloses that lactobacillus paracasei CICC20296, bacillus subtilis ACCC10619 and saccharomyces cerevisiae CICC32236 are respectively subjected to liquid fermentation culture, fermentation liquor is mixed according to the volume ratio of 1:1:1, then solid fermentation culture is carried out, and the mixed fermentation liquor is added into weaned piglet feed according to a certain proportion, so that the production performance of weaned piglets is improved. The lactobacillus paracasei related in the patent is mainly used for preparing fermented feed, fermentation equipment is needed in the preparation process, the fermentation process consumes longer time, the production operation is more complicated, no preparation method related to the lactobacillus paracasei freeze-dried preparation exists at present, and no method related to the method that the freeze-dried preparation with stable quality is directly added into the feed in proportion and the reproductive performance of sows can be improved is provided.

Disclosure of Invention

The invention aims to solve the practical problems and requirements in production practice, and develops a novel lactobacillus paracasei which has strong acid resistance, bile salt resistance, harmful bacterium inhibition and the like.

Another object of the present invention is to provide a Lactobacillus paracasei preparation prepared from the Lactobacillus paracasei and a method for preparing the same.

Still another object of the present invention is to provide the use of said lactobacillus paracasei in a direct addition to pig feed in a proportional ratio.

In order to realize the first object of the invention, a lactobacillus paracasei strain is provided, which is screened from healthy piglets and preserved in the general microbiological culture collection center of the China microbiological culture collection management committee (No. 3 of West Lu 1 of Beijing city Shangyu, institute of microbiology, China academy of sciences, postal code: 100101) within 10 months and 22 days of 2015, the strain is named as lactobacillus paracasei (Lactobacillus paracasei) ZLP019, and the preservation number is as follows: CGMCC No. 11532.

The Lactobacillus paracasei (Lactobacillus paracasei) ZLP019(CGMCC No.11532) is a gram-positive bacillus and is arranged singly or in pairs without spores. The colony on MRS plate culture medium is milky opaque, round, raised and regular.

The Lactobacillus paracasei (Lactobacillus paracasei) ZLP019(CGMCC No.11532) provided by the invention has the characteristics of acid resistance, cholate resistance and bacteriostasis besides the growth characteristics, and specifically comprises the following steps:

1) the survival rate of the lactobacillus paracasei after being acted for 2 hours in the environment with the pH value of 2 is 97.58%, the survival rate of the lactobacillus paracasei after being acted for 4 hours is 76.22%, the survival rate of the lactobacillus paracasei after being acted for 2 hours in the acidic environment with the pH values of 3 and 4 is more than 99%, and the survival rate of the lactobacillus paracasei after being acted for 4 hours is more than 85%.

2) The lactobacillus paracasei is incubated for 3 hours at 37 ℃ in 0.1% and 0.2% bile salt environments, and the survival rates are 82.25% and 56.08%, respectively.

3) The lactobacillus paracasei has a strong inhibiting effect on 3 common pig pathogenic bacteria, such as escherichia coli, salmonella, staphylococcus aureus and the like, and the diameter of an inhibition zone is 17-27 mm.

The second object of the invention provides a preparation of Lactobacillus paracasei of porcine origin, which contains Lactobacillus paracasei (Lactobacillus paracasei) ZLP019 with the accession number: CGMCC No. 11532.

Wherein, preferably, the viable count of the Lactobacillus paracasei (Lactobacillus paracasei) ZLP019 can be 1 × 10¹¹CFU/g and above.

The porcine lactobacillus paracasei preparation can be used as a feed additive.

The invention also provides a preparation method of the lactobacillus paracasei preparation, which comprises the steps of fermenting the lactobacillus paracasei ZLP019 in a liquid fermentation culture medium, centrifuging, emulsifying and freeze-drying the fermentation liquid, and preparing freeze-dried powder which is the lactobacillus paracasei preparation.

Preferably, the preparation method specifically comprises the following steps:

(1) fermentation culture: inoculating the lactobacillus paracasei ZLP019 into an MRS liquid culture medium in an inoculation amount of 1%, culturing for 16-24 hours at 37 ℃, and activating for 2 generations to obtain a seed culture;

inoculating the seed culture into a fermentation tank filled with a liquid culture medium at an inoculation amount of 1%, setting the temperature at 37 ℃ and the rotation speed at 100rpm, maintaining the constant pH at 5.5, starting feeding when the fermentation time reaches 14 hours, adding a feeding liquid, and then feeding once every 2 hours for 5 times;

when the pH value of the fermentation liquor is not reduced any more, stopping fermentation to obtain the fermentation liquor;

(2) centrifuging fermentation liquor: centrifuging the obtained fermentation liquor to obtain bacterial sludge;

(3) emulsification and freeze-drying: and (3) mixing the bacterial sludge obtained in the step (2) with a freeze-drying protective agent with the volume of 1/3 fermentation liquor, emulsifying by a high-shear dispersion emulsifying machine, subpackaging in a freezing tray with the thickness of 1.5-2.5 cm, pre-freezing for 3 hours, setting the condensation temperature of a vacuum freeze-drying machine to be-50 ℃ and the vacuum degree to be 10Pa, and freeze-drying for 24 hours to obtain the freeze-dried preparation containing lactobacillus paracasei.

Preferably, the liquid medium consists of the following concentrations of substances: 30g/L of glucose, 10g/L of peptone, 10g/L of beef extract, 5g/L of sodium acetate, 10g/L of yeast extract powder and K₂HPO₄·3H₂O2g/L, diammonium hydrogen citrate 2g/L, MgSO₄·7H₂O 0.5g/L，MnSO₄0.2g/L, Tween-801 ml/L, pH 6.5, sterilizing at 121 deg.C for 15 min.

The feed liquid is glucose solution, and the concentration is 300 g/L.

Preferably, the freeze-drying protective agent comprises 14% of skim milk, 8% of trehalose, 5% of dextrin, 1.5% of L-cysteine, 1% of sodium glutamate, 0.5% of sodium carboxymethylcellulose and the balance of water in percentage by mass.

The invention also provides application of the prepared porcine lactobacillus paracasei preparation as an additive in feed.

Preferably, it is applied in pig feed, more preferably, in feed for sows in the late gestation and lactation period.

Further, the application is that a lactobacillus paracasei ZLP019 preparation is added into basic ration, and the number of viable bacteria of the lactobacillus paracasei ZLP019 in the complete feed per kilogram of sows is 2 multiplied by 10⁹CFU。

The invention also provides a freeze-drying protective agent which contains 14% of skim milk, 8% of trehalose, 5% of dextrin, 1.5% of L-cysteine, 1% of sodium glutamate, 0.5% of sodium carboxymethylcellulose and 70% of water in percentage by mass.

The invention has the beneficial effects that:

the Lactobacillus paracasei (Lactobacillus paracasei) ZLP019, CGMCC No.11532 has good acid resistance, cholate resistance and bacteriostasis characteristics. The lyophilized preparation prepared from the strain can be directly used as additive to be added into sow feed in late gestation and lactation period, and the viable count of Lactobacillus paracasei ZLP019 in the complete feed of lactating sow is 2 × 10⁹CFU/Kg, the average daily feed intake of the lactating sows is improved by 4.48 percent, and the constipation rate is reduced by 8.51 percent; the average daily gain of the suckling piglets is increased by 8.90 percent, the weaning survival rate is increased by 8.65 percent, and the diarrhea rate is reduced by 31.92 percent; the number of harmful bacteria such as escherichia coli, salmonella and staphylococcus aureus in the excrement of the sow is obviously reduced. Therefore, the lactobacillus paracasei ZLP019 can improve the reproductive performance of sows, improve the daily gain and weaning survival rate of suckling piglets and reduce the emission of harmful bacteria in excrement when the sows at the late gestation stage and the suckling stage are fed, and is beneficial to environment-friendly breeding.

Drawings

FIG. 1 shows the colony morphology of Lactobacillus paracasei ZLP 019.

FIG. 2 is a 16S rRNA gene evolutionary tree map of Lactobacillus paracasei ZLP 019.

FIG. 3 is a pheS gene phylogenetic tree map of Lactobacillus paracasei ZLP 019.

FIG. 4 is a growth curve of Lactobacillus paracasei ZLP 019.

Detailed Description

The present invention is described in detail below with reference to examples, it is to be understood that the scope of the claims of the present invention is not limited to the specific embodiments, which are provided as further examples of the present invention, and those skilled in the art can modify the specific embodiments of the present invention or make equivalent substitutions for some technical features with reference to the description of the present invention, and such modifications and substitutions without creative efforts should also fall within the scope of the claims appended to the present invention. Unless otherwise specified, the technical means used in the examples are conventional technical means well known to those skilled in the art.

EXAMPLE 1 isolation, screening and characterization of Lactobacillus paracasei

Selecting healthy nursery pig, slaughtering, taking out intestinal tract under aseptic condition, scraping intestinal mucosa, mixing with aseptic normal saline, ten times diluting to 10^-5、10^-6、10^-70.1mL of each of the two solutions was applied to MRS plate medium and cultured in an anaerobic jar at 37 ℃ for 24-48 hours. Selecting milky white colony for primary screening, wherein the colony is gram-positive and H₂O₂The bacterial strain with negative enzyme test and negative catalase test can be preliminarily determined as Lactobacillus, the bacterial strain obtained by preliminary screening is subjected to characteristic analysis tests such as acid resistance, cholate resistance, bacteriostasis and the like, a Lactobacillus strain ZLP019 with excellent performance is finally screened out and is sent to the China general microbiological culture Collection center (CGMCC) for identification, and the bacterial strain is identified as Lactobacillus paracasei through comprehensive analysis of test data such as cell morphological characteristics, physiological and biochemical characteristics, a 16S rRNA sequence, a pheS gene sequence and the like. ZLP019 is a gram-positive bacilli, arranged singly or in pairs, without spores. The characteristics of the colonies on the MRS plate culture medium are shown in figure 1, and the colonies are milky opaque, round, raised and neat in edge. The identification results of the physiological and biochemical characteristics of the strain are shown in Table 1, 16S rRNA gene sequences such asSEQ ID No.1, and the pheS gene sequence is shown in SEQ ID No. 2. The 16S rRNA gene of the strain is subjected to evolutionary tree analysis, and the evolutionary tree map is shown in figure 2; the phylem tree analysis of the pheS gene of the strain is carried out, and the phylem of the phylem tree is shown in figure 3. The results show that the Lactobacillus paracasei (Lactobacillus paracasei) ZLP019 provided by the invention is different from the existing Lactobacillus paracasei and is a new strain.

TABLE 1 physiological and biochemical characteristics of Lactobacillus paracasei ZLP019

Experimental project	Results	Experimental project	Results	Experimental project	Results
						Glycerol	-	Inositol	-	Inulin	+
Erythritol and its preparation method	-	Mannitol	+	Melezitose	+
						D-arabinose	-	Sorbitol	+	Cotton seed candy	-
L-arabinose	-	α -methyl-D-mannoside	-	2-keto-gluconate	-
						D-ribose	+	α -methyl-D-glucoside	-	β methyl xyloside	-
D-xylose	-	N-formyl-glucamine	+	Xylitol, its preparation method and use	-
						L-xylose	-	Amygdalin	+	Adone alcohol	-
Glycogen	-	Arbutin	+	D-turanose	+
						D-galactose	+	Qiyeling (medicine for treating gynecopathy)	+	D-lyxose	-
D-glucose	+	Cellobiose	+	D-tagatose	+
						D-fructose	+	Maltose	+	D-fucose	-
D-mannose	+	Lactose	-	L-fucose	-
						L-sorbose	-	Melibiose	-	D-arabinitol	-
L-rhamnose	-	Sucrose	+	L-arabinitol	-
						Dulcitol	+	Trehalose	+	Starch	-

Note: positive when the sample is positive "+"; "-", negative.

The Lactobacillus paracasei strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (address: No. 3 of West Lu No.1 of North Chen of the south-oriented region of Beijing, China institute of microbiology, postal code: 100101) 10.22.10.2015, the strain name is Lactobacillus paracasei (Lactobacillus paracasei) ZLP019, and the preservation number is CGMCC No. 11532.

The MRS solid medium formula comprises: peptone 1g, beef extract 1g, yeast extract 0.5g, citric acid diamine 0.2g, glucose 2g, K₂HP0₄0.2g, 0.2g of sodium acetate, 800.1 ml of tween-1, 0.058g of magnesium sulfate, 0.02g of manganese sulfate, 2g of agar, 100m of water 1, pH 6-6.5, and autoclaving at 121 ℃ for 15 min.

EXAMPLE 2 characterization of Lactobacillus paracasei

1. Growth curves of Lactobacillus paracasei

Activated lactobacillus paracasei obtained in example 1 was inoculated into MRS liquid medium at an inoculation amount of 1%, and OD was detected using a full-automatic microbial growth curve analyzer₆₀₀Values, the results of which are shown in FIG. 2, wherein the abscissa represents the incubation time in hours and the ordinate represents the absorbance OD₆₀₀The value is obtained. Experimental results show that the lactobacillus paracasei grows rapidly in an MRS culture medium, enters a logarithmic phase within about 4 hours and enters a stationary phase within about 11 hours.

2. Determination of acid resistance

Activated lactobacillus paracasei is respectively inoculated into MRS liquid culture media with pH values of 2, 3 and 4 according to the inoculation amount of 1 percent, heat preservation is carried out for 2, 4 and 6 hours at 37 ℃, the number of live bacteria is measured by taking 0 hour as a control, the survival rate is calculated, and the result is shown in a table 2.

TABLE 2 acid resistance of Lactobacillus paracasei (%)

The results show that the survival rate of the lactobacillus paracasei is 97.58% after 2 hours of action in an acidic environment with the pH value of 2 and 76.22% after 4 hours; the survival rate of the plant after being acted for 2 hours in the acidic environment with pH values of 3 and 4 reaches more than 99 percent, and the survival rate of the plant after being acted for 4 hours reaches more than 85 percent. The lactobacillus paracasei has strong acid resistance and can well resist the gastric acid environment of animals.

2. Determination of the bile salt resistance

Activated lactobacillus paracasei is respectively inoculated into MRS liquid culture medium containing 0.1%, 0.2% and 0.3% of pig bile salt according to the inoculation amount of 1%, the temperature is kept at 37 ℃ for 3 and 6 hours, the viable count of 0 hour is used as a control, the survival rate is calculated, and the result is shown in table 3.

TABLE 3 determination of the bile salt resistance of Lactobacillus paracasei (%)

The results show that the bacterium acts for 3 hours in 0.1% bile salt environment, and the survival rate is 82.25%; the product acts for 3 hours in the environment of 0.2% bile salt, and the survival rate is 56.08%; the survival rate of the medicine is only 10.47 percent when the medicine is acted for 3 hours at the concentration of 0.3 percent of bile salt. The lactobacillus paracasei has higher tolerance to 0.1 percent of bile salt.

3. Bacteriostasis test

The results of the inhibition effect of the lactobacillus paracasei fermentation broth on 3 common pig pathogenic bacteria were detected by the oxford cup method and are shown in table 4.

TABLE 4 Lactobacillus paracasei bacteriostasis test

As can be seen from the results, the lactic acid bacteria are resistant to these 3 harmful bacteria: escherichia coli (C838901, available from China institute of veterinary drugs), Salmonella choleraesuis (C78-4, available from China institute of veterinary drugs), and Staphylococcus aureus (C56023, available from China institute of veterinary drugs) have strong inhibitory effects, and the diameter of the antibacterial zone reaches 17-27 mm.

4. Susceptibility test

The L.paracasei was subjected to drug sensitivity tests with 26 antibiotics (OXOID drug sensitive paper sheets) and the results are shown in Table 5.

TABLE 5 Lactobacillus paracasei susceptibility test

Note: s is sensitive; i is moderately sensitive; r is drug resistance.

As can be seen from Table 5, the bacterium is sensitive to ampicillin, tetracycline, amoxicillin, azithromycin, polymyxin E, cefoperazone, cefotaxime, ceftriaxone, enrofloxacin, amikacin, gentamicin, clindamycin, rifampin, florfenicol, chloramphenicol and the like, moderately sensitive to ofloxacin, and resistant to antibiotics such as neonomine, erythromycin, bacitracin, cephalexin, ceftizoxime, ciprofloxacin, norfloxacin, kanamycin, streptomycin, nystatin and the like.

EXAMPLE 3 preparation of Lactobacillus paracasei preparation

1. Fermentation culture of lactobacillus paracasei

Inoculating the Lactobacillus paracasei (Lactobacillus paracasei) ZLP019(CGMCC No.11532) obtained in the embodiment 1 into an MRS liquid culture medium by the inoculation amount of 1%, culturing for 16-24 hours at 37 ℃, and activating for 2 generations in this way to obtain a seed culture; inoculating the seed culture into a 50L fermentation tank filled with 30L of liquid culture medium at an inoculation amount of 1%, setting the temperature to be 37 ℃, rotating speed to be 100rpm, maintaining constant pH to be 5.5, starting feeding when the fermentation time reaches 14 hours, adding 200mL of feeding liquid into the fermentation tank once, and then feeding once every 2 hours for 5 times; when the pH of the fermentation broth no longer decreased, the fermentation was terminated.

Wherein, the liquid culture medium comprises the following components: 30g/L of glucose, 10g/L of peptone, 10g/L of beef extract, 5g/L of sodium acetate, 10g/L of yeast extract powder and K₂HPO₄·3H₂O2g/L, diammonium hydrogen citrate 2g/L, MgSO₄·7H₂O 0.5g/L，MnSO₄0.2g/L, Tween-801 ml/L, pH 6.5, sterilizing at 121 deg.C for 15 min.

The feed liquid is glucose solution with concentration of 300g/L, and is sterilized at 115 ℃ for 15min for later use.

2. Centrifuging the fermentation broth

Separating thalli from the fermentation liquor obtained in the step 1 by using a tubular centrifuge under the condition of 19000rpm to obtain bacterial sludge;

3. preparation of lyophilized preparation

Mixing the bacterial sludge with a freeze-drying protective agent with the volume of 1/3 fermentation liquor, emulsifying for 30min by adopting a high-shear dispersion emulsifying machine at 3000r/min, and freeze-drying to obtain the lactobacillus paracasei freeze-dried preparation.

The freeze-drying condition is that the emulsified bacterial suspension is sub-packaged in a freezing tray, the thickness is 1.5-2.5 cm, pre-freezing is carried out for 3 hours, the condensation temperature is-50 ℃, the vacuum degree is 10Pa, and the freeze-drying time is 24 hours. The freeze-drying protective agent comprises the following substances in percentage by mass: 14% of skim milk, 8% of trehalose, 5% of dextrin, 1.5% of L-cysteine, 1% of sodium glutamate, 0.5% of sodium carboxymethylcellulose and the balance of water. By using the formula, the freeze-drying protection rate of the lactobacillus paracasei can reach 88.20 percent. Wherein, the calculation formula of the freeze-drying protection rate is as follows: freeze-drying protection rate (%) - (viable count of lyophilized powder × weight of lyophilized powder)/(viable count of pre-lyophilized suspension × volume of pre-lyophilized suspension) × 100.

Taking the prepared lactobacillus paracasei freeze-dried preparation, carrying out gradient dilution by using sterile normal saline, adopting an MRS plate culture medium to culture and measure the viable count, and measuring the viable count of the lactobacillus paracasei in the freeze-dried powder to be 1.20 multiplied by 10¹¹CFU/g。

4. Determination of storage stability of Lactobacillus paracasei lyophilized preparation

Vacuum packaging the prepared lactobacillus paracasei lyophilized preparation with aluminum foil bags, storing at-18 deg.C for 1 month, 6 months, and 12 months, taking out bacteria powder, performing gradient dilution with sterile normal saline, and measuring viable count with MRS plate culture medium. As can be seen from Table 6, after the lactobacillus paracasei freeze-dried preparation is stored at low temperature for 12 months, the survival rate of the live bacteria can still reach 83.66%, and the quality of the preparation is stable.

TABLE 6 measurement of storage Properties of Lactobacillus paracasei lyophilized preparation

Example 4 application of Lactobacillus paracasei preparation in sow production

1. Test animals and groups

100 healthy, nearly fetal-time and 85-day-pregnant big binary sows are selected and randomly divided into a control group and a test group of 2 groups, each group has 50 repetitions, and each repetition has 1 sow. The control group is fed normally, and the test group is added with the porcine lactobacillus paracasei jelly on the basis of the daily ration of the feeding baseDry preparation (complete feed with final viable bacteria concentration of 2 × 10)⁹CFU/Kg). Wherein, the basal diet of the sow is prepared according to the pig feeding standard (2004), and the measured values of the specific nutrient components are shown in the table 7. The sows are raised in the same colony house, and feeding management, immunization programs and the like are executed according to a unified system of the pig farm. The test time is from the 85 th day of gestation of the sow to the weaning of the suckling piglet at the age of 28 days.

Table 7 actual value of nutrient content (%) -based ration

3. Test management

During the test period, the growth condition of the swinery is observed every day, and the feed consumption, the farrowing, litter weight, diarrhea and the like of the sow are recorded; after the test is finished, 10 parts of fresh sow manure samples are taken in each group, and the viable count of lactic acid bacteria, escherichia coli, staphylococcus aureus and the like in the manure is measured. Data statistics single-factor analysis of variance was performed using SPSS 18.0 statistical software, and results are expressed as mean ± standard deviation.

4. Results and analysis

4.1 test results of porcine lactobacillus paracasei preparation on reproductive performance of sows are shown in Table 8.

TABLE 8 Effect of porcine Lactobacillus paracasei preparation on reproductive Performance of sows

Note: the difference between the same shoulder marks represents significant difference, and the difference between the lower case letters represents significant difference (P < 0.05).

Compared with a control group, the individual weight of piglets in the test group at birth is improved by 7.01 percent (P <0.05), the individual weight of weaning is improved by 8.33 percent (P <0.05), the survival rate of weaning is improved by 8.65 percent (P <0.05), the daily weight gain of suckling piglets is improved by 8.90 percent (P <0.05), the daily feed intake of suckling sows is improved by 4.48 percent (P <0.05), the diarrhea rate of suckling piglets is reduced by 31.92 percent, and the constipation rate of suckling sows is reduced by 8.51 percent. Therefore, the porcine lactobacillus paracasei preparation can improve the reproductive performance of sows, promote the health of piglets and improve the survival rate when the sows at the later gestation stage and the lactation stage are fed with the porcine lactobacillus paracasei preparation.

4.2 Effect of porcine-derived Lactobacillus paracasei preparation on fecal microbiology of lactating sows result 9.

TABLE 9 Effect of porcine Lactobacillus paracasei preparation on fecal microbiota of lactating sows

As can be seen from Table 9, the number of lactic acid bacteria in the feces of the sows in the test group was significantly increased (P <0.05), and the number of Escherichia coli and Salmonella was significantly decreased (P <0.05) as compared with the control group. Therefore, when the porcine lactobacillus paracasei preparation is fed to sows, harmful bacteria in excrement can be reduced, and the porcine lactobacillus paracasei preparation has positive effects on intestinal health and improvement of breeding environment.

In conclusion, the lactobacillus paracasei preparation is used for feeding sows in the later gestation period and the lactation period, can improve the reproductive performance of the sows, improve the daily gain and weaning survival rate of suckling piglets, promote the intestinal health of the sows, reduce the emission of harmful bacteria in excrement and is beneficial to environment-friendly breeding.

SEQUENCE LISTING

<110> agriculture and forestry academy of sciences of Beijing City

<120> lactobacillus paracasei, preparation and application thereof in pig feed

<130>

<160>2

<170>PatentIn version 3.5

<210>1

<211>1401

<212>DNA

<213> 16S rRNA gene fragment of Lactobacillus paracasei (Lactobacillus paracasei) ZLP019

<400>1

ttgatgatcg gtgcttgcac cgagattcaa catggaacga gtggcggacg ggtgagtaac 60

acgtgggtaa cctgccctta agtgggggat aacatttgga aacagatgct aataccgcat 120

agatccaaga accgcatggt tcttggctga aagatggcgt aagctatcgc ttttggatgg 180

acccgcggcg tattagctag ttggtgaggt aatggctcac caaggcgatg atacgtagcc 240

gaactgagag gttgatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg 300

cagcagtagg gaatcttcca caatggacgc aagtctgatg gagcaacgcc gcgtgagtga 360

agaaggcttt cgggtcgtaa aactctgttg ttggagaaga atggtcggca gagtaactgt 420

tgtcggcgtg acggtatcca accagaaagc cacggctaac tacgtgccag cagccgcggt 480

aatacgtagg tggcaagcgt tatccggatt tattgggcgt aaagcgagcg caggcggttt 540

tttaagtctg atgtgaaagc cctcggctta accgaggaag cgcatcggaa actgggaaac 600

ttgagtgcag aagaggacag tggaactcca tgtgtagcgg tgaaatgcgt agatatatgg 660

aagaacacca gtggcgaagg cggctgtctg gtctgtaact gacgctgagg ctcgaaagca 720

tgggtagcga acaggattag ataccctggt agtccatgcc gtaaacgatg aatgctaggt 780

gttggagggt ttccgccctt cagtgccgca gctaacgcat taagcattcc gcctggggag 840

tacgaccgca aggttgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat 900

gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatctt tttgatcacc 960

tgagagatca ggtttcccct tcgggggcaa aatgacaggt ggtgcatggt tgtcgtcagc 1020

tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttatg actagttgcc 1080

cagcatttag ttgggcactc tagtaagact gccggtgaca aaccggagga aggtggggat 1140

gacgtcaaat catcatgccc cttatgacct gggctacaca cgtgctacaa tggatggtac 1200

aacgagttgc gagaccgcga ggtcaagcta atctcttaaa gccattctca gttcggactg 1260

taggctgcaa ctcgcctaca cgaagtcgga atcgctagta atcgcggatc agcacgccgc 1320

ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac accatgagag tttgtaacac 1380

ccgaagccgg tggcgtaacc c 1401

<210>2

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<213> pheS Gene sequence

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caagtccaat gcaggcgcgg acaatggaaa agcacgactt taccaaagga ccgctgaaaa 60

tgattagccc tggggtggtt tatcgacgtg atgacgacga tgctactcat agccatcagt 120

ttcaccagat ggaaggactc gtcattgaca agcatataac catggctgat ctaaagggaa 180

ccttgttggc catgtgccaa cacgtgtttg gtaaagatcg gacaattcgc ttgcggccaa 240

gttattttcc atttacggag ccatccgttg aagttgatgt ttcctgtttt cgttgcggcg 300

gtaaaggttg cccggtttgc aaatataccg gttggattga agtgttaggt gccggcatgg 360

tgcatcccaa tgtgctacgg gcagcgaaca ttga 394

Claims

1. A Lactobacillus paracasei (Lactobacillus paracasei) strain ZLP019 has a preservation number of CGMCCNo.11532.

2. A lactobacillus paracasei preparation characterized by comprising the lactobacillus paracasei ZLP019 according to claim 1.

3. A feed additive characterized by comprising the lactobacillus paracasei preparation as claimed in claim 2.

4. A preparation method of a lactobacillus paracasei preparation is characterized in that lactobacillus paracasei ZLP019 as claimed in claim 1 is subjected to fermentation culture in a liquid fermentation medium to obtain a fermentation liquid, and the fermentation liquid is subjected to centrifugation, emulsification and freeze-drying to prepare freeze-dried powder which is the lactobacillus paracasei preparation.

5. The preparation method according to claim 4, which specifically comprises the steps of:

inoculating the seed culture into a fermentation tank filled with a liquid culture medium at an inoculation amount of 1%, setting the temperature to be 37 ℃ and the rotating speed to be 100rpm, maintaining the constant pH to be 5.5, beginning to supplement the feed liquid when the fermentation time reaches 14 hours, and then supplementing the feed liquid once every 2 hours for 5 times;

(2) centrifuging the fermentation liquor: centrifuging the fermentation liquor obtained in the step (1) to obtain bacterial sludge;

(3) emulsification and freeze-drying: and (3) mixing the bacterial sludge obtained in the step (2) with 1/3 freeze-drying protective agent with the volume of the fermentation liquid, emulsifying by a high-shear dispersion emulsifying machine, subpackaging in a freezing tray with the thickness of 1.5-2.5 cm, pre-freezing for 3 hours, setting the condensation temperature of a vacuum freeze-drying machine to be-50 ℃ and the vacuum degree to be 10Pa, and freeze-drying for 24 hours to obtain the lactobacillus paracasei preparation.

6. The method according to claim 5, wherein the liquid medium is composed of the following substances in concentrations: 30g/L of glucose, 10g/L of peptone, 10g/L of beef extract, 5g/L of sodium acetate, 10g/L of yeast extract powder and K₂HPO₄·3H₂O2g/L, diammonium hydrogen citrate 2g/L, MgSO₄·7H₂O 0.5g/L，MnSO₄0.2g/L, Tween-801 ml/L, pH 6.5;

the feed liquid is a glucose solution, and the concentration is 300 g/L;

the freeze-drying protective agent comprises 14% of skim milk, 8% of trehalose, 5% of dextrin, 1.5% of L-cysteine, 1% of sodium glutamate, 0.5% of sodium carboxymethylcellulose and the balance of water.

7. Use of a lactobacillus paracasei preparation according to claim 2 in feed.

8. Use according to claim 7, wherein the feed is a pig feed.

9. Use according to claim 8, wherein the preparation of lactobacillus paracasei is added to a basal ration such that the number of viable bacteria of the lactobacillus paracasei per kg of sow complete feed is 2 x 10⁹CFU。