Preparation of a probiotic metabolite combination preparation and its application in laying hens

Abstract

The present invention discloses the preparation of a probiotic metabolite combination preparation and its application to laying hens. The probiotics include Lactococcus lactis, Clostridium butyricum, and Bacillus velezensis. The Lactococcus lactis is Lactococcus lactis with the strain number FR01. The Clostridium butyricum is Clostridium butyricum with the strain number HZNDC-1. The Bacillus velezensis is Bacillus velezensis with the strain number BV-J11. All three strains are deposited in the China Center for Type Culture Collection, and the deposit numbers are: CCTCC NO: M 2022103, CCTCC: NO: M 2021874, and CCTCC NO: M 20231502 in sequence. The combined preparation does not contain live strains, solving the problem of cell loss during transportation and storage, and maximizing the effectiveness of the combined preparation in use. The combined preparation has excellent comprehensive effects in the application to laying hens, specifically manifested in improving the egg quality of laying hens, reducing the rates of dirty eggs and broken eggs, improving the fecal state of laying hens, and having the potential to extend the peak laying period of laying hens.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly, to the preparation of a probiotic metabolite combination preparation and its application in laying hens. Background Art

[0002] In recent years, the role of probiotics in animal feed has gradually emerged. Probiotics and their metabolites can balance the intestinal flora, maintain intestinal health, improve digestion and absorption, thereby improving the production performance of farmed livestock species, and are green and safe, reducing environmental protection risks. However, in the actual production practice of feed at present, most probiotic live preparations have weak stability during normal temperature transportation and storage, and are not tolerant to high temperature during the feed processing process, which limits their application scope.

[0003] The prior art CN118215404A discloses a feed additive composition, which comprises a direct-fed microbe (DFM) containing Bifidobacterium animalis subsp. lactis and / or Lactobacillus acidophilus or consisting essentially of the same; it also discloses that the above-mentioned Lactococcus lactis, Clostridium butyricum, and Bacillus velezensis can be used as probiotics for laying hen farming.

[0004] The present invention will develop a probiotic metabolite combination preparation, which all uses the metabolites of probiotics and has no live bacteria components. When used in the process of laying hen farming, it can greatly improve the production performance and effectively avoid the above problems existing in the actual application of live bacteria preparations. Summary of the Invention

[0005] The present invention first provides a Lactococcus lactis, which is Lactococcus lactis, and its strain number is FR01. It is preserved in the China Center for Type Culture Collection, and the preservation number is: CCTCC NO: M 2022103.

[0006] The present invention also provides a compound probiotic, which contains the above-mentioned Lactococcus lactis, Clostridium butyricum, and Bacillus velezensis;

[0007] The Clostridium butyricum is Clostridium butyricum, and its strain number is HZNDC-1. It is preserved in the China Center for Type Culture Collection, and the preservation number is: CCTCC: NO: M 2021874;

[0008] The Bacillus velezensis is Bacillus velezensis with the strain number BV-J11, which is deposited in the China Center for Type Culture Collection with the deposit number: CCTCC NO: M 20231502.

[0009] In some embodiments, the weight percentages of the Lactococcus lactis, Clostridium butyricum, and Bacillus velezensis are 6:2:2.

[0010] The present invention also provides a probiotic metabolite combination preparation, which is obtained by separately fermenting the above-mentioned compound probiotics.

[0011] In some embodiments, the weight percentages of the metabolites of Lactococcus lactis, Clostridium butyricum, and Bacillus velezensis in the probiotic metabolite combination preparation are 6:2:2.

[0012] The present invention also provides the use of the above-mentioned Lactococcus lactis or the above-mentioned compound probiotics in the preparation of poultry feed, poultry farming, or as a feed additive.

[0013] In some embodiments, the poultry is a laying hen.

[0014] The present invention also provides the use of the above-mentioned probiotic metabolite combination preparation in the preparation of poultry feed, poultry farming, or as a feed additive.

[0015] In some embodiments, the poultry is a laying hen.

[0016] Finally, the present invention provides a preparation method of a probiotic metabolite combination preparation, which includes the step of separately fermenting the above-mentioned compound probiotics.

[0017] Compared with the prior art, the present invention has at least the following beneficial effects:

[0018] 1. The combination preparation does not contain live strains, solves the problem of cell loss during transportation and storage, and maximally retains the effectiveness of the combination preparation.

[0019] 2. The combination preparation has excellent comprehensive effects in the application of laying hens, specifically manifested in improving the egg quality of laying hens, reducing the rates of dirty eggs and broken eggs, improving the fecal state of laying hens, and having the possibility of extending the peak laying period of laying hens.

[0020] Depository description

[0021] The Lactococcus lactis FR01 was isolated from fermented sufu naturally fermented in Shennongjia Forest Region. It was sent to the China Center for Type Culture Collection for preservation on January 21, 2022. The taxonomic name is Lactococcus lactis FR01, and it is preserved in the China Center for Type Culture Collection with the preservation number CCTCC NO: M2022103. The address is Wuhan University, No. 16 Luojiashan Road, Wuchang District, Wuhan City, Hubei Province, China.

[0022] The Clostridium butyricum HZNDC-1 was isolated from the bovine intestine. It was sent to the China Center for Type Culture Collection for preservation on July 13, 2021. The taxonomic name is Clostridium butyricum HZNDC-1, and the preservation number is CCTCC: NO: M 2021874. The address is Wuhan University, Wuhan, Hubei, China. See CN115094010 B.

[0023] The Bacillus velezensis BV-J11 was isolated from the chicken intestine. It was sent to the China Center for Type Culture Collection for preservation on August 17, 2023. The taxonomic name is Bacillus velezensis BV-J11, and the preservation number is CCTCC: NO: M 20231502. The address is Wuhan University, Wuhan, Hubei, China. See CN117343876B. Description of the Drawings

[0024] Figure 1 The effect of the probiotic metabolite combination preparation on the fecal morphology of laying hens; Note: (A) is the fecal state before the experiment; (B) is the fecal state at the end of the experiment; (C) is the state after the feces before the experiment are diluted with water; (D) is the state after the feces at the end of the experiment are diluted with water. Detailed Embodiments

[0025] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the drawings and specific embodiments.

[0026] Example 1: Preparation Method of Metabolites of Lactococcus lactis FR01

[0027] Liquid agar medium (MRS): Peptone 10 g / L, Beef extract powder 10 g / L, Yeast extract 5 g / L, Dipotassium hydrogen phosphate 2 g / L, Diammonium citrate 2 g / L, Sodium acetate 5 g / L, Glucose 20 g / L, Tween 80 1 mL / L, Magnesium sulfate 0.5 g / L, Manganese sulfate 0.2 g / L, Agar powder 15 g / L. pH 6.2 - 6.4, Sterilize at 101 Kpa, 121 °C for 15 min and reserve for use.

[0028] The Lactococcus lactis FR01 was isolated from the naturally fermented sufu in Shennongjia Forest Region and was deposited at the China Center for Type Culture Collection on January 21, 2022. Its taxonomic name is: Lactococcus lactis FR01, and it is deposited at the China Center for Type Culture Collection with the deposit number: CCTCC NO: M2022103, Address: Wuhan University, Wuhan, Hubei, China.

[0029] Pick up a loop of purified single colony of Lactococcus lactis and inoculate it into MRS medium at 1%, then place it in a 37 °C incubator and statically culture for 18 h to prepare a seed solution. Inoculate the seed solution of Lactococcus lactis into MRS medium at 2% and culture it in a 5 L fermenter for 24 h. Control the temperature to be constantly 37 °C, the pH to be constantly 6.0 (add 10% NaOH to regulate the pH), the rotation speed to be 120 rpm. When fermentation starts for 12 h, add 5% glucose at one time.

[0030] After fermentation ends, adjust the temperature to rise to 50 °C for 2 h to make the cells autolyze, centrifuge at 4000 rpm for 10 min to collect the supernatant; add 10% (w / v) maltodextrin as a carrier and mix evenly, pour it into a freeze-drying tray to make the liquid level height 1 - 2 cm, and then place it at -80 °C for pre-freezing for 6 h; transfer the pre-frozen fermentation broth to a vacuum freeze dryer (Beijing Songyuan, LGJ-30FD) and dry for 36 - 60 h to obtain the freeze-dried powder of Lactococcus lactis metabolites.

[0031] Example 2: Effects of several Lactococcus lactis on the ovarian development of laying hens

[0032] Select 900 18-week-old XinHaiLan Grey laying hens in the rearing period with similar physical conditions and good health, and randomly divide them into 6 groups, namely the control group, strain 1-5 groups (where strain 1 is FR01), with 6 replicates in each group and 25 hens in each replicate. The experimental period is 8 weeks. The control group is fed a basal diet, and the experimental groups are respectively added with 240 g / t of 5 different Lactococcus lactis metabolites in the basal diet. The experimental chicken flock is kept in a closed chicken house, with free access to food and water. The feeding management and immunization program are the same as the daily management of the chicken farm. After the experiment, 2 hens are randomly selected from each replicate for slaughter, that is, 12 laying hens in each group. The ovaries are taken for weighing, and the large white follicles, small yellow follicles, and large yellow follicles are counted and statistically analyzed.

[0033] Table 1 Effects of 5 strains of Lactococcus lactis on ovarian development of laying hens

[0034] Item Control Group Group of Strain 1 (FR01) Group of Strain 2 Group of Strain 3 Group of Strain 4 Group of Strain 5 P Value Ovary (g / kg) 37.03±3.19 42.19±2.17 38.81±3.41 37.43±3.35 37.66±3.11 38.00±2.79 0.065 LWF (2 - 5 mm) <![CDATA[31.37±2.28 b > <![CDATA[35.79±1.15 a > <![CDATA[32.77±2.45 b > <![CDATA[34.71±2.00 b > <![CDATA[32.33±2.15 b > <![CDATA[32.14±2.25 b > 0.007 SYF (6 - 8 mm) 10.38±0.71 10.09±0.61 9.96±0.87 9.97±0.84 10.61±0.80 10.47±0.58 0.536 LYF (8 - 12 mm) 66.86±2.41 65.84±2.85 65.33±4.04 66.01±4.17 67.72±2.81 67.03±2.88 0.812

[0035] Note: Different superscript letters in the same row indicate significant differences (P<0.05); the same superscript letters or no letter annotation indicate no significant differences (P>0.05). LWF: large white follicles; SYF: small yellow follicles; LYF: large yellow follicles.

[0036] As can be seen from Table 1, after 8 weeks of the experiment, compared with the control group, the group of strain 1 (FR01) had a tendency to increase the ovarian weight of laying hens, and at the same time, it could significantly increase the number of large white follicles, and had no effect on the number of yellow follicles; the other groups had no effect on ovarian development indexes such as ovarian weight, the number of large white follicles, small yellow follicles, and large yellow follicles. It shows that the metabolite of Lactococcus lactis FR01 has the potential to extend the peak laying period of laying hens.

[0037] Example 3: Preparation of a probiotic metabolite combination preparation

[0038] The preparation method of the Lactococcus lactis metabolite is shown in Example 1.

[0039] The Clostridium butyricum HZNDC-1 was isolated from bovine intestine and sent to the China Center for Type Culture Collection for preservation on July 13, 2021. Its taxonomic name is: Clostridium butyricum HZNDC-1, and the preservation number is: CCTCC: NO: M 2021874, address: Wuhan University, Wuhan, Hubei, China. See CN115094010 B.

[0040] The preparation method of the Clostridium butyricum metabolite:

[0041] (1) Preparation of solid medium: Ready-made TSC medium (Beijing Aoboxing, 02-351); Preparation of liquid medium: 10 g of beef extract, 10 g of peptone, 5 g of yeast extract powder, 5 g of glucose, 5 g of sodium chloride, 1000 ml of distilled water, pH 7 ± 0.2, sterilized at 115 °C for 20 min and reserved for use.

[0042] (2) Pick and culture single colonies: Pick the Clostridium butyricum HZNDC-1 on the plate with a pipette tip and inoculate it into 20 ml of liquid medium, and culture it anaerobically at 37 °C for 24 h to obtain a single colony culture solution.

[0043] (3) Subculture: Take 2 ml of the activated bacterial solution and suspend it in a conical flask containing 300 ml of liquid medium, pH 7 ± 0.2, and culture it anaerobically at 37 °C for 24 h to obtain a bacterial solution with a bacterial content of 108 cfu / mL.

[0044] (4) Inoculate the above-obtained bacterial solution into the fermentation medium for fermentation at an inoculation amount of 2%. After the fermentation is completed, the Clostridium butyricum fermentation broth is obtained.

[0045] After the fermentation is completed, centrifuge at 4000 rpm for 10 min to collect the supernatant; add 10% (w / v) of maltodextrin as a carrier and mix evenly, pour it into a freeze-drying tray to make the liquid level height 1-2 cm, and then place it at -80 °C for pre-freezing for 6 h; transfer the pre-frozen fermentation broth to a vacuum freeze-dryer (Beijing Songyuan, LGJ-30FD) and dry it for 36-60 h to obtain the freeze-dried powder of the Clostridium butyricum metabolite.

[0046] The Bacillus velezensis BV-J11 was isolated from chicken intestine and sent to the China Center for Type Culture Collection for preservation on August 17, 2023. The taxonomic name is: Bacillus velezensis BV-J11, and the preservation number is: CCTCC: NO: M 20231502, address: Wuhan University, Wuhan, Hubei, China. See CN117343876B.

[0047] The preparation method of the metabolite of Bacillus velezensis:

[0048] (1) Pick a loop of purified single colonies and inoculate them into YPD liquid medium, shake culture at 220 rpm at 37 °C for 24-36 h, observe the cell morphology under a microscope, and collect the fermentation broth after the spores are completely mature; add 10%-20% of maltodextrin to the fermentation broth and mix evenly; spray-dry the fermentation broth (Shanghai Yacheng YC-015) to obtain the spore dry powder of Bacillus velezensis BV-J11.

[0049] (2) Preparation of the dry powder of the metabolite of Bacillus velezensis BV-J11: Pick a loop of purified single colonies and inoculate them into YPD liquid medium. Incubate with shaking at 220 rpm at 37 °C for 24 - 36 h, and collect the fermentation broth; Pour the fermentation broth into a freeze-drying tray so that the liquid level height is 1 - 2 cm, and then place it at -80 °C for pre-freezing for 6 h; Transfer the pre-frozen fermentation broth to a vacuum freeze-dryer (LGJ-30FD, Songyuan, Beijing), and dry for 36 - 60 h to obtain the freeze-dried powder of the fermentation broth of Bacillus velezensis BV-H11C3. Take the above freeze-dried powder of the fermentation broth, add ethyl acetate at a ratio of 1:60 (w / v), extract three times, each extraction for 24 h, and collect the extraction solution of the metabolites in total; Concentrate the extraction solution through a rotary evaporator to obtain a concentrated metabolite. Dilute the concentrated metabolite 100 times with maltodextrin, which is the dry powder of the metabolite of Bacillus velezensis BV-J11.

[0050] Liquid medium (YPD): Yeast extract powder 10 g, peptone 20 g, glucose 20 g, distilled water 1000 mL, pH 7.2 ± 0.2, sterilize at 115 °C for 20 min for standby.

[0051] The probiotic metabolite preparation contains the following components by weight percentage: metabolite of Lactococcus lactis 60%, metabolite of Clostridium butyricum 20%, metabolite of Bacillus velezensis 20%.

[0052] Example 4: Application of a probiotic metabolite combined preparation in laying hens

[0053] Select 600 34-week-old Xin Hailan Grey laying hens with similar body conditions and good health in the same house, and randomly divide them into 2 groups, namely the control group and the experimental group, with 6 replicates in each group and 50 hens in each replicate. The experimental period is 60 days. The control group is fed with the basal diet, and the experimental group is added with the probiotic metabolite prepared in Example 2 at 400 g per ton of feed. The experimental chicken flock is kept in a closed chicken house, with free access to food and water. The feeding management and its immunization program are the same as the daily management of the chicken farm, and eggs are regularly picked at 15:00 every afternoon. During the experiment, observe the mental state, feeding situation, fecal state, death and culling situation, and eggshell appearance quality of the chicken flock every day, and record the number of eggs laid, broken eggs, dirty eggs, weight of each box of eggs, and death and culling number in each group; Collect eggs in the last 3 days of the experiment to detect egg quality; Collect blood after fasting for 6 h on the last day of the experiment to detect plasma biochemical indexes. Calculate the average daily feed intake, egg production rate, egg weight, feed-to-egg ratio, broken egg rate, dirty egg rate, death and culling rate during the whole period, and egg quality and plasma biochemical indexes in the last stage of the experiment, as shown in Table 2 - Table 4.

[0054] Table 2 Effects of the probiotic metabolite combined preparation on the laying performance of laying hens

[0055] Item Control Group Experimental Group P Value Average Daily Feed Intake (g / bird / d) 122.07±2.12 120.67±1.99 0.264 Egg Production Rate (%) 95.83±2.83 97.64±2.04 0.230 Egg Weight (g / egg) 55.88±1.46 58.16±1.68 0.031 Feed - Egg Ratio 2.17±0.02 2.09±0.03 0.072 Broken Egg Rate (%) 0.94±0.02 0.54±0.03 ＜0.001 Dirty Egg Rate (%) 5.03±0.09 2.89±0.12 ＜0.001 Mortality and Culling Rate (%) 4.00±1.26 1.67±0.75 0.003

[0056] Effect of the probiotic metabolite combination preparation described in Table 3 on the egg quality of laying hens

[0057]

[0058]

[0059] Effect of the probiotic metabolite combination preparation described in Table 4 on the plasma biochemical indexes of laying hens

[0060] Item Control Group Experimental Group P Value TB (umol / L) 5.28±1.01 6.95±1.03 0.011 TP (g / L) 59.32±4.54 60.12±3.27 0.743 ALB (g / L) 16.99±1.43 17.38±0.72 0.606 AST (U / L) 174.51±15.36 171.59±6.22 0.673 ALT (U / L) 2.88±1.05 2.34±0.58 0.319 ALP (U / L) 236.75±106.91 170.08±81.18 0.214 TC (mmol / L) 2.70±0.56 3.64±0.62 0.018 TG (mmol / L) 13.13±4.29 21.05±6.40 0.066 GLU (mmol / L) 13.50±1.31 13.92±0.70 0.593 CA (nmol / L) 6.78±0.42 6.76±0.49 0.935 P (nmol / L) 1.77±0.40 2.21±0.41 0.071 CREA (umol / L) 8.41±2.25 8.99±1.02 0.601 HDL (mmol / L) 0.92±0.14 1.25±0.19 0.084 LDL (mmol / L) 0.66±0.18 0.90±0.19 0.036 UA (mmol / L) 223.05±63.47 279.22±61.56 0.127 CK (U / L) 640.03±111.16 641.92±97.78 0.996 LDH (U / L) 191.25±35.28 188.26±34.69 0.902

[0061] Note: TB: total bilirubin; TP: total protein; ALB: albumin; AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphatase; TC: total cholesterol; TG: triglyceride; GLU: blood glucose; CA: blood calcium; P: blood phosphorus; CREA: creatinine; HDL: high-density lipoprotein; LDL: low-density lipoprotein; UA: uric acid; CK: creatine kinase; LDH: lactate dehydrogenase.

[0062] As can be seen from Tables 2-4, compared with the control group, adding the probiotic metabolite combination preparation can increase the egg production rate by 1.90%, increase the egg weight by 4.08%, reduce the feed-to-egg ratio by 4.21%, reduce the cracked egg rate and dirty egg rate by 42.55% and 42.54% respectively, and reduce the mortality and culling rate by 58.25%; significantly increase the eggshell weight, eggshell thickness and eggshell strength (P < 0.01), and there is a tendency to increase the egg weight, egg yolk color and Haugh unit (P < 0.1); significantly increase the contents of TB, TC and LDL (P < 0.05), and there is a tendency to increase the contents of TG, P and HDL (P < 0.1). In summary, adding the probiotic metabolite combination preparation can increase the egg production rate and egg weight, reduce the feed-to-egg ratio, cracked egg rate, dirty egg rate and mortality and culling rate; improve the egg quality; and may improve the above indexes by regulating blood lipid metabolism.

[0063] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A Lactococcus lactis, characterized in that The lactococcus lactis is Lactococcus lactis ( Lactococcuslactis ), the strain number is FR01, and it is deposited in China Center for Type Culture Collection with the deposit number: CCTCCNO:M2022103.

2. A composite probiotic, characterized in that: The composite probiotics contain Lactococcus lactis, Clostridium butyricum and Bacillus Velezii as claimed in claim 1; The butyric Clostridium is butyric Clostridium ( Clostridiumbutyricum ), the strain number is HZNDC-1, which is deposited in China Center for Type Culture Collection with the deposit number: CCTCC: NO: M 2021874; The Velez Bacillus is Velez Bacillus ( Bacillus velezensis ), the strain number is BV-J11, which is deposited in China Center for Type Culture Collection with the deposit number: CCTCC NO: M 20231502.

3. The composite probiotic according to claim 2, characterized in that The weight percentage of Lactococcus lactis, Clostridium butyricum and Bacillus Velez is 6:2:

2.

4. Use of the Lactococcus lactis according to claim 1 or the composite probiotic according to any one of claims 2 to 3 in preparing poultry feed or poultry breeding or as a feed additive.

5. The use according to claim 4, characterized in that: The poultry is laying hens.

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