Lactobacillus brevis PMG and application thereof in yellow mealworm breeding

Abstract

The invention belongs to the crossing field of microbial technology and insect breeding, and relates to a lactobacillus brevis (PMG) strain and an application of the lactobacillus brevis (PMG) strain in yellow mealworm breeding. The lactobacillus brevis PMG is preserved in the China General Microbiological Culture Collection Center on January 14, 2025, the preservation address is No. 3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 33401. Compared with the conventional feed, the feed added with the lactobacillus brevis PMG has the advantages that the pupation rate of the tenebrio molitor can be remarkably increased, the increase amplitude reaches up to 49.20%, meanwhile, the feed promotes the feed conversion effect, the feed conversion rate is relatively reduced by 10.45%-18.30%, the breeding time of the tenebrio molitor is shortened, the breeding cost of the tenebrio molitor is reduced, and the feed has important application value.

Description

Technical Field

[0001] The present invention relates to the intersection of microbial technology and insect breeding, and specifically to a strain of Lactobacillus brevis PMG and application thereof in yellow mealworm breeding, and more specifically to a strain of Lactobacillus brevis PMG (Levilactobacillus brevis) and application thereof as a feed additive in improving the feed conversion rate and pupation rate of yellow mealworm. Background Art

[0002] Among insects, mealworm is one of the few candidate species with the potential for large-scale production. Mealworm is rich in nutrients such as protein, carbohydrates, amino acids, unsaturated fatty acids, vitamins and mineral elements, and has attracted widespread attention due to its economic value. Mealworm is a holometabolous species with a life cycle consisting of four main stages: egg, larva, pupa and adult. Mealworm larvae are widely used in animal feed due to their high protein characteristics, and studies have shown that the protein content of mealworm in the pupa and adult stages is higher than that in the larval stage. However, the breeding cost of raising larvae to the adult stage for harvesting is relatively high. Therefore, exploring effective methods to promote the growth and development of mealworm is of great significance to reduce breeding costs and improve breeding efficiency.

[0003] In this field, the application of lactic acid bacteria has shown great potential. Lactic acid bacteria are recognized as safe strains among probiotics and are widely used in animal feed. Lactic acid bacteria can improve animal intestinal absorption and metabolism, enhance immunity and prevent diseases, thereby promoting animal growth. Lactobacillus plantarum has been shown to promote the systemic growth of fruit flies by regulating hormone signals. Specifically, Lactobacillus plantarum can significantly reduce the development time from fruit fly eggs to pupae and increase their growth rate. This finding shows that the development of lactic acid bacteria suitable for insects and with the effect of promoting growth and development has important application value for the mealworm breeding industry.

[0004] In order to improve the breeding efficiency and reduce the breeding cost of mealworms, it is very important to improve the growth performance of mealworms, among which optimizing the feed formula is an important research direction. Existing patents such as CN117481275A and CN104814348A improve the growth of mealworms by providing optimized feed formulas and adding Chinese herbal medicine ingredients, respectively, which fully proves the importance of optimizing feed formulas for the growth of mealworms. However, the above patents all focus on the optimization of the feed formula itself, while ignoring the potential impact of microorganisms on the growth and reproduction of mealworms. Patent CN104342391A discloses a quadruple probiotic agent composed of Bifidobacterium bifidum, Clostridium butyricum, Bacillus licheniformis and Bacillus subtilis subsp. subtilis, and confirms that the agent can significantly promote the growth of yellow mealworms by improving the survival rate of yellow mealworms, and promoting its growth indicators such as body length, head width and 100 worm weight. However, the patent does not pay special attention to the use of probiotics to improve the feed conversion rate and pupation rate of yellow mealworms. After consulting relevant patent documents and research reports at home and abroad, it has not yet been found that there is a precedent for using short growth-promoting lactobacillus to improve the feed conversion rate and pupation rate of yellow mealworms. Summary of the invention

[0005] To this end, an embodiment of the present invention provides a strain of Lactobacillus breve PMG and its application in mealworm breeding. The present invention can significantly improve the pupation rate of mealworms by feeding feed containing Lactobacillus breve PMG, while promoting feed conversion, thereby effectively reducing the breeding time and breeding cost of mealworms, and has important application value.

[0006] In order to achieve the above purpose, the embodiment of the present invention provides the following technical solutions:

[0007] According to the first aspect of an embodiment of the present invention, the present invention provides a strain of Lactobacillus breve PMG, which is deposited in the Institute of Microbiology, Chinese Academy of Sciences on January 14, 2025, with a storage address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a storage number of CGMCC No. 33401.

[0008] According to a second aspect of an embodiment of the present invention, the present invention provides a bacterial agent containing the above-mentioned Lactobacillus breve PMG.

[0009] According to a third aspect of an embodiment of the present invention, the present invention provides a feed containing the growth-promoting Lactobacillus breve PMG as described above.

[0010] According to a fourth aspect of an embodiment of the present invention, the present invention provides a method for preparing a feed, wherein the Lactobacillus breve PMG as described above is expanded and cultured, and a bacterial suspension is prepared using a sodium chloride solution; the bacterial suspension is mixed with wheat bran to obtain the feed.

[0011] Furthermore, the conditions for the expanded culture are: inoculating Lactobacillus breve PMG into MRS broth culture medium, and fermenting and culturing for 16 to 32 hours at a temperature of 30 to 37° C. and a shaking speed of 200 to 250 rpm.

[0012] Further, the concentration of the sodium chloride solution is 0.6-0.9%;

[0013] The absorbance of the bacterial suspension at 600 nm is 0.55-0.60.

[0014] Furthermore, based on the mass of the wheat bran, the amount of the bacterial suspension added is 2.0-3.0×10 7 CFU / g.

[0015] According to a fifth aspect of an embodiment of the present invention, the present invention provides the use of the above-mentioned Lactobacillus breve PMG, the above-mentioned bacterial agent, and the above-mentioned feed in the breeding and production of mealworms.

[0016] Furthermore, the feed is fed to the mealworm larvae at an amount of 0.1 to 0.5 g per larva for 7 to 24 days.

[0017] Furthermore, the feed improves breeding efficiency and reduces costs by increasing the pupation rate of yellow mealworms and reducing feed conversion rate.

[0018] The embodiments of the present invention have the following advantages:

[0019] 1. The present invention innovatively isolates and obtains the PMG strain of breve growth-promoting Lactobacillus from the feces of yellow mealworms. The results of the feeding test show that compared with the control group, the survival rate of yellow mealworms only slightly decreases in the early stage of feeding, and has a higher survival rate in the middle and late stages of feeding, indicating that the PMG strain derived from the living environment of yellow mealworms themselves is easily accepted and utilized by yellow mealworm larvae, showing good ecological adaptability and growth-promoting performance.

[0020] 2. The breve growth-promoting Lactobacillus PMG provided by the present invention improves the conversion effect of feed, so that yellow mealworms can use less feed to achieve the same growth amount, thereby significantly reducing feed costs; the breve growth-promoting Lactobacillus PMG can also effectively promote the growth and development of yellow mealworm larvae, and increase the success rate of larvae to pupae transformation. The high pupation rate not only improves the output efficiency per unit breeding area, but also provides more sufficient raw material guarantee for subsequent breeding and deep processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0022] Figure 1 This is a graph showing the experimental results of the calcium dissolution circle of the strain PMG provided by the present invention;

[0023] Figure 2 The microscopic morphology of the strain PMG provided by the present invention after Gram staining;

[0024] Figure 3 A scanning electron microscope image of the strain PMG provided by the present invention;

[0025] Figure 4 To construct the phylogenetic tree of strain PMG based on the 16S rRNA gene sequence;

[0026] Figure 5 The pupation rate within 24 days of feeding yellow mealworms provided by the present invention;

[0027] Figure 6 The feed conversion rate within 24 days of feeding yellow mealworms provided by the present invention;

[0028] Figure 7 The survival rate of the mealworms fed with the present invention within 24 days;

[0029] Figure 8 The weight gain rate within 24 days of feeding yellow mealworms provided by the present invention. DETAILED DESCRIPTION

[0030] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] 1. Materials

[0032] Mealworm larvae (1.2±0.3 cm) were purchased from Lianyungang, Jiangsu. Wheat bran was from the State Administration of Grain and Material Reserves. MRS broth medium was purchased from Beijing Aoboxing Biotechnology Co., Ltd.

[0033] 2. Instruments

[0034] Clean bench (AIRTECH VS-840K-Ⅱ), Suzhou Antai Air Technology Co., Ltd.; high pressure sterilizer (HIRAYAMA HVA-110), Hirayama Manufacturing Co., Ltd.; electronic balance (MS3002TS / 02), Mettler-Toledo Instruments (Shanghai) Co., Ltd.; desktop high-speed refrigerated centrifuge (5430R), Eppendorf China Co., Ltd.; constant temperature shaker (NewBrunswick TM 42R), Eppendorf China Co., Ltd.; Intelligent artificial climate chamber (PRX-450B), Ningbo Saifu Experimental Instrument Co., Ltd.; Spectrophotometer (Nanodrop TM 2000c), Thermo Fisher Scientific Inc.; Scanning Electron Microscope (JSM-IT700HR InTouchScope TM ), Japan Electronics Corporation.

[0035] Example 1

[0036] Isolation, Screening and Identification of Lactobacillus breve PMG

[0037] (1) Pretreatment of insect sand (mealworm feces) bacterial suspension

[0038] 1.5 g of mealworm feces from Beijing, Sichuan, Yunnan, Guangdong and Shandong were weighed and placed in 50 mL sterile centrifuge tubes. Then, 30 mL of 0.85% NaCl solution was added to each centrifuge tube, and the mixture was swollen and shaken for 20 minutes, and then allowed to stand for 2 minutes to obtain a worm sand suspension.

[0039] (2) Preparation of culture medium

[0040] MRS broth medium (g / L): peptone 10.0 g, beef extract powder 8.0 g, yeast extract powder 4.0 g, glucose 20 g, dipotassium hydrogen phosphate 2.0 g, diammonium hydrogen citrate 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.04 g, Tween 80 1.0 g, add distilled water to make up to 1 L, pH = 5.7.

[0041] Improved MRS solid medium (g / L): Add 15 g agar and 20 g calcium carbonate to the above medium, pH = 6.2.

[0042] (3) Isolation, purification and microscopic morphology of lactic acid bacteria from insect sand

[0043] 1 mL of the worm sand suspension was taken and inoculated into 10 mL of MRS broth medium, and cultured at 37°C and 220 rpm for 48 hours. The bacterial suspension was diluted by the ten-fold incremental dilution method, and the dilution degree was selected as 10-5 , 10 -6 , 10 -7 The bacterial suspension was spread on the modified MRS solid medium and the plates were placed in a 37°C biochemical incubator for 48 hours.

[0044] like Figure 1 As shown, a colony with a calcium-dissolving ring was selected and further isolated and purified on MRS solid medium, transferred to 2 mL of MRS broth medium, and cultured at 37°C and 220 rpm for 48 hours. Then, a single colony was purified using the plate streak method on the modified MRS solid medium, and the operation was repeated three times to obtain a pure culture. The pure culture was subjected to Gram staining, and the results were as follows: Figure 2 Strains that stained positive were cultured and used in subsequent experiments as indicated.

[0045] Inoculate the above-screened strains at a 1% inoculation rate into 2 ml of MRS broth medium, and culture at 37°C and 220rpm for 48 hours. Use a centrifuge to centrifuge at 4000rpm and 4°C for 10 minutes to collect the bacterial precipitate. Discard the supernatant and retain the bacterial precipitate for subsequent processing. Use sterile 30%, 50%, 70%, 90% and 100% ethanol concentration solutions to perform gradient dehydration on the bacteria. In each step, resuspend the bacterial precipitate in the corresponding concentration of alcohol, mix gently, and then collect the bacteria by centrifugation again to ensure that the surface of the bacteria is completely dry. Spread a small amount of dried bacteria on tin foil to ensure that the bacteria are evenly distributed and spray gold. The gold-sprayed bacterial sample was placed under a scanning electron microscope (SEM) for observation, and the results are as follows Figure 3 As shown, the bacteria are rod-shaped and have a relatively regular shape.

[0046] (4) Molecular biological identification of strains based on 16S rRNA sequences

[0047] Using the genomic DNA of the strain as a template, the 16S rDNA sequence of the target strain was amplified on a PCR instrument using the universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GTACCTTGTACGACT-3') for amplifying bacterial 16S rDNA. The reaction conditions for PCR amplification were 94°C pre-denaturation for 2 minutes, then 98°C denaturation for 10 seconds, 55°C annealing for 30 seconds, 68°C extension for 45 seconds, and 68°C extension for 4 minutes after 35 cycles. The corresponding amplified product was sequenced by Beijing Bomed Gene Technology Co., Ltd., and the 16SrRNA gene sequence is shown in SEQ ID NO: 1.

[0048]

[0049] Sequence alignment analysis was performed using the EzBioCloud database platform. The alignment results showed that the 16SrRNA gene sequence of the strain was highly similar to the standard strain sequence of the known species Levilactobacillus brevis ATCC 14869, with a similarity of up to 99.86%. To further verify the phylogenetic relationship of the strain, we used MEGA 6.0 software and selected the Neighbor-Joining method to construct a phylogenetic tree (e.g. Figure 4 In the phylogenetic tree, strain PMG and the representative strain KI271266 of Levilactobacillus brevis were in the same branch, which indicated that strain PMG was closely related to the known strains of Levilactobacillus brevis in evolution, further supporting the classification conclusion that the strains obtained by screening belonged to Levilactobacillus brevis.

[0050] The strain was preserved under the name of Levilactobacillus brevis PMG, the preservation unit was General Microbiology Center of China Microbiological Culture Collection Administration, the preservation address was No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the preservation date was January 14, 2025, and the preservation number was CGMCC No.33401.

[0051] Example 2

[0052] Preparation of suspension of Lactobacillus breve PMG

[0053] The PMG strain of Lactobacillus breve was inoculated into MRS broth medium for cultivation. Under the temperature condition of 37°C, the shaking speed was set to 220rpm, and continuous cultivation was carried out for 24 hours to promote the growth and reproduction of the strain. After the cultivation is completed, 1ml of the bacterial liquid sample was taken and centrifuged at 4°C and 6000rpm to collect the bacterial cells. Then, the collected bacterial cells were washed twice with 0.85% sterile sodium chloride solution to remove impurities and metabolites in the culture medium. Finally, the washed bacterial cells were prepared into absorbance values ​​(OD 600nm ) of the bacterial suspension was 0.55-0.60, and the total number of colonies in the bacterial suspension obtained by the plate counting method was 2.6×10 8 CFU / ml.

[0054] Example 3

[0055] Preparation of mealworm feed

[0056] The bacterial suspension prepared in Example 2 was added to the wheat bran, and the bacterial suspension and the wheat bran content were mixed at a volume mass ratio of 1:10, so that the bacterial concentration in the wheat bran was 2.6×10 7 CFU / g, and mix by vortexing.

[0057] Application Example 1

[0058] Experiment on feeding of mealworm larvae

[0059] The mealworm feed prepared in Example 3 was used as the experimental group feed, and the mealworm larvae of 1.2 ± 0.3 cm in length were fed according to the amount of 0.25 g / only, and the control group was fed with wheat bran without adding PMG bacteria. The experiment set up three parallel groups, each group including 400 mealworm larvae, and the feed was changed daily to maintain the activity of the strain, and the feeding was continued for 24 days. During the 24-day feeding period, the various growth indicators of the mealworm were continuously observed and the number of pupations per day was recorded to evaluate the long-term effect of the strain on the growth performance of the mealworm, and the pupation rate, feed conversion rate, survival rate and weight gain rate of the mealworm were calculated and obtained.

[0060] See also Figure 5 Compared with the control group, the pupation rate of the experimental group of mealworms increased significantly after the 24-day feeding period. Specifically, the pupation rate of the experimental group of mealworms increased by 49.20% compared with the control group. This result shows that adding Lactobacillus breve PMG to the feed can effectively promote the pupation process of mealworms. This discovery provides a new strategy for the feeding of mealworms, which helps to improve the efficiency and quality of mealworms as a protein source.

[0061] Feed conversion ratio (FCR) refers to the amount of feed consumed by animals to obtain a unit of weight gain. It is a key indicator to measure the efficiency of feed utilization in animal production. A lower FCR value means higher feed utilization efficiency, lower breeding costs and higher economic benefits. Figure 6 The feed conversion rate of the experimental group supplemented with Lactobacillus breve PMG was lower than that of the control group. On the 8th, 16th and 24th days, the relative decreases in feed conversion rate were 12.27%, 10.45% and 18.30%, respectively. Therefore, the addition of Lactobacillus breve PMG can significantly improve the feed conversion effect, thereby helping to reduce breeding costs, which is of great significance for improving the economic benefits of the breeding industry.

[0062] See also Figure 7During the 24-day feeding period, the survival rate of mealworms showed significant differences compared with the control group in different time periods. Specifically, in the initial stage of the experiment (0-4 days), the survival rate of mealworms decreased slightly compared with the control group, decreasing by 0.27%. However, in the subsequent time periods, the survival rate of mealworms was higher than that of the control group, and the percentage increase ranged from 4.26% to 6.22%. This phenomenon indicates that after the adaptation period, mealworms have enhanced their adaptability to experimental conditions and show stronger survival ability than the control group. These data not only reveal the response of mealworms to experimental conditions, but are also of great significance for understanding their biological characteristics and optimizing breeding conditions.

[0063] See also Figure 8 , there was no significant difference in weight gain rate between the experimental and control groups overall, indicating that the two groups were basically consistent in growth and development. However, at about 20 days, the weight gain rate of the control group was significantly higher than that of the experimental group. This phenomenon may indicate that the growth rate of the control group exceeded that of the experimental group at a specific growth stage, but this difference did not persist throughout the experimental period and was therefore not sufficient to affect the overall weight gain comparison. This may mean that the addition of Lactobacillus breve PMG has a positive effect on improving survival rate, but has a limited effect on weight gain, or that this effect varies at different growth stages.

[0064] Pupation is a key developmental stage for holometabolous insects to transition from larvae to adults. It is a highly metabolic process that requires larvae to molt repeatedly. The above content shows that the addition of Lactobacillus breve PMG, as a lactic acid bacteria, has a significant effect on the growth and development of mealworms. Specifically, the addition of Lactobacillus breve PMG can not only improve the feed conversion rate and promote the continuous growth of mealworm weight, but also effectively improve the survival rate of mealworms, thereby promoting their overall growth and development. These characteristics indicate that Lactobacillus breve PMG has potential commercial application value in mealworm farming, especially in improving farming efficiency and reducing costs.

[0065] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

[0066]

[0067]

Claims

1. A strain of Lactobacillus breve PMG, characterized in that: The breve lactobacillus PMG was deposited in the General Microbiology Center of the China Culture Collection Administration on January 14, 2025, with the deposit address being No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC No. 33401.

2. A bacterial agent containing the breve growth-promoting Lactobacillus PMG according to claim 1.

3. A feed containing the breve growth-promoting Lactobacillus PMG according to claim 1.

4. A method for preparing a feed, characterized in that: The Lactobacillus breve PMG as claimed in claim 1 is expanded and cultured, and a bacterial suspension is prepared using a sodium chloride solution; the bacterial suspension is mixed with wheat bran to obtain the feed.

5. The method for preparing feed according to claim 4, characterized in that: The conditions for the expanded culture are: inoculating Lactobacillus breve PMG into MRS broth culture medium, and fermenting and culturing for 16 to 32 hours at a temperature of 30 to 37° C. and a shaking speed of 200 to 250 rpm.

6. The method for preparing feed according to claim 4, characterized in that: The concentration of the sodium chloride solution is 0.6-0.9%; The absorbance of the bacterial suspension at 600 nm is 0.55-0.

60.

7. The method for preparing feed according to claim 4, characterized in that: Based on the mass of the wheat bran, the amount of the bacterial suspension added is 2.0-3.0×10 7 CFU / g.

8. Use of the breve lactobacillus PMG according to claim 1, the bacterial agent according to claim 2, and the feed according to claim 3 in the breeding and production of mealworms.

9. The use according to claim 8, characterized in that: The feed is fed to the yellow mealworm larvae in an amount of 0.1 to 0.5 g per larva for 7 to 24 days.

10. The use according to claim 9, characterized in that: The feed improves breeding efficiency and reduces costs by increasing the pupation rate of yellow mealworms and reducing feed conversion rate.

Images