Brevibacterium lactofermentum PMG and application thereof in tenebrio molitor breeding

Abstract

The present application belongs to the cross field of microbial technology and insect breeding, and relates to a strain of Levilactobacillus brevis PMG and application of the strain in breeding of Tenebrio molitor. The Levilactobacillus brevis PMG is preserved in the China General Microbiological Culture Collection Center on January 14, 2025, and the address of the center is No. 1, Xibaixili, Chaoyang District, Beijing, and the preservation number is CGMCC No. 33401. Compared with conventional feed, the feed added with the Levilactobacillus brevis PMG can significantly improve the pupation rate of Tenebrio molitor, and the increase is up to 49.20%. Meanwhile, the feed promotes the feed conversion effect, and the feed conversion rate is relatively reduced by 10.45% to 18.30%, thereby reducing the breeding time and breeding cost of Tenebrio molitor, and having important application value.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of microbial technology and insect farming, specifically to a strain of Lactobacillus short-lived growth (PMG) and its application in mealworm farming, and more specifically, to a strain of Lactobacillus short-lived growth (PMG) and its application as a feed additive in improving the feed conversion rate and pupation rate of mealworms. Background Technology

[0002] Among insects, the yellow mealworm is one of the few candidate species with large-scale production potential. Rich in nutrients such as protein, carbohydrates, amino acids, unsaturated fatty acids, vitamins, and minerals, the yellow mealworm has attracted widespread attention due to its economic value. It undergoes holometabolous metamorphosis, with its life cycle consisting of four main stages: egg, larva, pupa, and adult. Yellow mealworm larvae are widely used in animal feed due to their high protein content, and studies have shown that the protein content is even higher in the pupa and adult stages than in the larval stage. However, raising larvae to adulthood for harvest is relatively costly. Therefore, exploring effective methods to promote the growth and development of yellow mealworms is of great significance for reducing farming costs and improving farming efficiency.

[0003] In this field, the application of lactic acid bacteria has shown great potential. Lactic acid bacteria are recognized as safe strains of probiotics and are widely used in animal feed. They can improve intestinal absorption and metabolism, enhance immunity, and prevent disease, thereby promoting animal growth. *Lactobacillus plantarum* has been shown to promote the overall growth of fruit flies by regulating hormone signaling. Specifically, *Lactobacillus plantarum* can significantly reduce the development time from egg to pupa and increase the growth rate of fruit flies. This finding indicates that developing lactic acid bacteria suitable for insects and with growth-promoting effects has important application value for the mealworm farming industry.

[0004] To improve the breeding efficiency and reduce the breeding cost of mealworms, improving their growth performance is crucial, and optimizing feed formulation is an important research direction. Existing patents such as CN117481275A and CN104814348A improve mealworm growth by providing optimized feed formulations and adding traditional Chinese medicine ingredients, respectively, fully demonstrating the importance of optimized feed formulation for mealworm growth. However, the above patents focus on optimizing the feed formulation itself, while neglecting 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 this agent can significantly promote the growth of mealworms by improving their survival rate and increasing their body length, head width, and weight per 100 insects. However, this patent does not specifically focus on using probiotics to improve the feed conversion rate and pupation rate of mealworms. A review of relevant domestic and international patent literature and research reports has revealed no precedent for using *Lactobacillus short-lived* to improve the feed conversion rate and pupation rate of mealworms. Summary of the Invention

[0005] Therefore, this invention provides a strain of Lactobacillus brevis p.M. (PMG) and its application in mealworm farming. This invention can significantly improve the pupation rate of mealworms by feeding them feed containing Lactobacillus brevis p.M. (PMG), while also promoting feed conversion, thereby effectively reducing the farming time and cost of mealworms, and has important application value.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] According to a first aspect of the present invention, the present invention provides a strain of Lactobacillus short-lived PMG, which was deposited on January 14, 2025 at the Institute of Microbiology, Chinese Academy of Sciences, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33401.

[0008] According to a second aspect of the present invention, the present invention provides a bacterial agent containing *Lactobacillus shortiflora* PMG as described above.

[0009] According to a third aspect of the present invention, the present invention provides a feed containing *Lactobacillus short-lived* PMG as described above.

[0010] According to a fourth aspect of the present invention, the present invention provides a method for preparing feed, wherein *Lactobacillus short-lived* PMG as described above is cultured on a large scale, and a bacterial suspension is prepared using sodium chloride solution; the bacterial suspension is mixed with wheat bran to obtain the feed.

[0011] Furthermore, the conditions for the expanded culture are as follows: Lactobacillus short-lived PMG is inoculated into MRS broth medium and fermented for 16 to 32 hours at a temperature of 30 to 37°C and a shaking speed of 200 to 250 rpm.

[0012] Furthermore, the concentration of the sodium chloride solution is 0.6% to 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 microbial suspension added is 2.0–3.0 × 10⁻⁶. 7 CFU / g.

[0015] According to a fifth aspect of the present invention, the present invention provides the application of the Lactobacillus short-life PMG, the bacterial agent, and the feed as described above in the production of yellow mealworms.

[0016] Furthermore, the feed is given to mealworm larvae at a rate of 0.1–0.5 g / larva for 7–24 days.

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

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

[0019] 1. This invention innovatively isolates and obtains the short-lived Lactobacillus PMG strain from mealworm feces. The results of the feeding experiment show that, compared with the control group, the survival rate of mealworms only decreased slightly in the early stage of feeding, and had a high survival rate in the middle and late stages of feeding. This indicates that the PMG strain derived from the mealworm's own living environment is easily accepted and utilized by mealworm larvae, showing good ecological adaptability and growth-promoting performance.

[0020] 2. The *Lactobacillus short-lived* PMG provided by this invention improves feed conversion efficiency, enabling mealworms to achieve the same growth with less feed, thereby significantly reducing feed costs. *Lactobacillus short-lived* PMG can also effectively promote the growth and development of mealworm larvae and increase the success rate of larval transformation into pupae. The high pupation rate not only improves the output efficiency per unit breeding area but also provides a more sufficient raw material guarantee for subsequent breeding and deep processing. Attached Figure Description

[0021] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0022] Figure 1 The experimental results of the calcium dissolution zone of strain PMG provided by this invention are shown in the figure.

[0023] Figure 2 The Gram-stained microscopic morphology of the strain PMG provided by this invention;

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

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

[0026] Figure 5 The pupation rate of mealworms fed to this invention within 24 days;

[0027] Figure 6 The feed conversion rate of mealworms provided by this invention within 24 days;

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

[0029] Figure 8 The weight gain rate of mealworms fed to this invention within 24 days. Detailed Implementation

[0030] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] 1. Materials

[0032] Yellow mealworm larvae (1.2±0.3cm) were purchased from Lianyungang, Jiangsu Province. Wheat bran was obtained from the National Food and Strategic Reserves Administration. MRS broth culture 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.; Autoclave (HIRAYAMA HVA-110), Hirayama Seisakusho Co., Ltd., Japan; Electronic balance (MS3002TS / 02), Mettler Toledo Instruments (Shanghai) Co., Ltd.; Benchtop high-speed refrigerated centrifuge (5430R), Eppendorf China Co., Ltd.; Thermostatic shaker (New Brunswick) TM 42R), Eppendorf China Co., Ltd.; Intelligent Artificial Climate Chamber (PRX-450B), Ningbo Saif Experimental Instrument Co., Ltd.; Spectrophotometer (Nanodrop) TM 2000c), Thermo Fisher Scientific; Scanning Electron Microscope (JSM-IT700HR InTouchScope) TM ), Nippon Electronics Co., Ltd.

[0035] Example 1

[0036] Isolation, screening and identification of Lactobacillus short-lived PMG

[0037] (1) Pretreatment of mealworm excrement suspension

[0038] Weigh 1.5g of mealworm excrement from Beijing, Sichuan, Yunnan, Guangdong and Shandong respectively, and place them in 50mL sterile centrifuge tubes. Then, add 30mL of 0.85% NaCl solution to each centrifuge tube, let it swell, shake and mix for 20 minutes, and let it stand for 2 minutes to obtain the mealworm excrement suspension.

[0039] (2) Preparation of culture medium

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

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

[0042] (3) Isolation, purification and microscopic morphological observation of lactic acid bacteria in insect sand

[0043] Take 1 mL of the insect sand suspension and inoculate it into 10 mL of MRS broth medium. Incubate at 37°C and 220 rpm for 48 hours. Dilute the bacterial suspension using a tenfold serial dilution method, selecting a dilution of 10...-5 10 -6 10 -7 The bacterial suspensions were spread onto modified MRS solid medium. The plates were then incubated at 37°C for 48 hours.

[0044] like Figure 1 As shown, a colony with a calcification zone was selected and further purified on MRS solid medium. The colony was then transferred to 2 mL of MRS broth and incubated at 37°C and 220 rpm for 48 hours. Next, a single colony was purified using the streak plate method on modified MRS solid medium, repeated three times to obtain a pure culture. Gram staining was performed on the pure culture, and the results are shown below. Figure 2 As shown, the strains that stained positive were further cultured and used in subsequent experiments.

[0045] The screened bacterial strains were inoculated at a 1% inoculum into 2 ml of MRS broth and cultured at 37°C and 220 rpm for 48 hours. The bacterial pellet was collected by centrifugation at 4000 rpm and 4°C for 10 minutes. The supernatant was discarded, and the bacterial pellet was retained for subsequent processing. The bacterial cells were sequentially dehydrated using sterile 30%, 50%, 70%, 90%, and 100% ethanol solutions. In each step, the bacterial pellet was resuspended in the corresponding concentration of ethanol, gently mixed, and then centrifuged again to ensure complete drying of the bacterial surface. A small amount of dried bacterial cells were evenly distributed on aluminum foil and then sputtered with gold. The gold-sputtered bacterial samples were observed under a scanning electron microscope (SEM). The results are shown below. 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 sequence

[0047] Using the bacterial genomic DNA as a template, the 16S rDNA sequence of the target strain was amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GTACCTTGTACGACT-3'). The PCR amplification conditions were: 94℃ pre-denaturation for 2 min, followed by 98℃ denaturation for 10 s, 55℃ annealing for 30 s, and 68℃ extension for 45 s, repeated 35 times, followed by a final extension at 68℃ for 4 min. The corresponding amplified products were sequenced by Beijing Bomeide Gene Technology Co., Ltd., and the 16S rRNA 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 16S rRNA gene sequence of this strain had a high similarity (99.86%) to the standard strain sequence of the known species *Levilactobacillus brevis* ATCC 14869. To further verify the phylogenetic relationship of the strains, we used MEGA 6.0 software and constructed a phylogenetic tree using the neighbor-joining method (e.g., ...). Figure 4 (As shown). In the phylogenetic tree, strain PMG belongs to the same branch as the representative strain of *Levilactobacillus brevis*, KI271266. This indicates that strain PMG is closely related to known *Levilactobacillus brevis* strains in terms of evolution, further supporting the classification conclusion that the strain obtained through screening belongs to *Levilactobacillus brevis*.

[0050] The strain was deposited under the name: *Lactobacillus brevis* PMG, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on January 14, 2025, with accession number CGMCC No. 33401.

[0051] Example 2

[0052] Preparation of Lactobacillus short-lived PMG strain suspension

[0053] *Lactobacillus short-lived* PMG strain was inoculated into MRS broth and cultured continuously at 37°C and 220 rpm for 24 hours to promote growth and reproduction. After culturing, 1 ml of the bacterial culture was collected and centrifuged at 4°C and 6000 rpm to collect the bacterial cells. The collected cells were then washed twice with 0.85% sterile sodium chloride solution to remove impurities and metabolic products from the culture medium. Finally, the absorbance (OD) of the washed bacterial cells was calculated using 0.85% sterile sodium chloride solution. 600nm The bacterial suspension with a concentration of 0.55-0.60 was analyzed using the plate count method, and the total bacterial count was found to be 2.6 × 10⁻⁶. 8 CFU / ml.

[0054] Example 3

[0055] Preparation of mealworm feed

[0056] The microbial suspension prepared in Example 2 was added to wheat bran, and the microbial suspension and wheat bran were mixed at a volume-to-mass ratio of 1:10 to achieve a microbial concentration of 2.6 × 10⁻⁶ in the wheat bran. 7 CFU / g, mixed using a stirring vortex method.

[0057] Application Example 1

[0058] Rearing experiment of mealworm larvae

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

[0060] See Figure 5 Compared to the control group, the experimental group of mealworms showed a significantly higher pupation rate after a 24-day rearing period. Specifically, the pupation rate of the experimental group increased by 49.20% compared to the control group. This result indicates that adding *Lactobacillus short-lived* PMG to the feed can effectively promote the pupation process of mealworms. This finding provides a new strategy for feeding mealworms, helping to improve the efficiency and quality of mealworms as a protein source.

[0061] Feed conversion ratio (FCR) refers to the amount of feed an animal needs to consume to achieve a unit increase in body weight. It is a key indicator of feed utilization efficiency in animal production; a lower FCR value means higher feed utilization efficiency, lower breeding costs, and higher economic benefits. See also... Figure 6 The experimental group supplemented with *Lactobacillus brevis* PMG had a lower feed conversion ratio than the control group. On days 8, 16, and 24, the relative decreases in feed conversion ratio were 12.27%, 10.45%, and 18.30%, respectively. Therefore, supplementing with *Lactobacillus brevis* PMG can significantly improve feed conversion efficiency, thereby helping to reduce breeding costs, which is of great significance for improving the economic benefits of the aquaculture industry.

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

[0063] See Figure 8 Overall, there was no significant difference in weight gain rate between the experimental and control groups, indicating that the two groups were essentially consistent in terms of growth and development. However, at approximately 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 control group grew faster than the experimental group at a specific growth stage, but this difference did not persist throughout the entire experimental period and was therefore insufficient to affect the overall comparison of weight gain rates. This may mean that the addition of *Lactobacillus short-lived* PMG has a positive effect on improving survival rate, but its effect on weight gain is limited, or that this effect varies at different growth stages.

[0064] Pupation is a crucial developmental stage in holometabolous insects, transitioning from larva to adult, and is a highly metabolic process requiring repeated molting. The above findings indicate that *Lactobacillus brevis* PMG, as a lactic acid bacterium, has a significant impact on the growth and development of mealworms. Specifically, the addition of *Lactobacillus brevis* PMG not only improves feed conversion ratio and promotes sustained weight gain in mealworms, but also effectively increases their survival rate, thereby promoting their overall growth and development. These characteristics suggest that *Lactobacillus brevis* 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 with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0066]

[0067]

Claims

1. A strain of short-lived lactobacillus (Levilactobacillus brevis) PMG, characterized in that, The *Lactobacillus short-lived* PMG was deposited on January 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33401.

2. An inoculum containing the short-lived Lactobacillus PMG as described in claim 1.

3. A feed containing the short-lived Lactobacillus PMG as described in claim 1.

4. A method for preparing feed, characterized in that, The *Lactobacillus short-lived* PMG strain as described in claim 1 is cultured on a large scale, and a bacterial suspension is prepared using 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 as follows: *Lactobacillus short-lived* PMG is inoculated into MRS broth medium and fermented for 16-32 hours at a temperature of 30-37°C and a shaking speed of 200-250 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 microbial suspension added is 2.0~3.0×10⁻⁶. 7 CFU / g.

8. The application of the short-lived Lactobacillus PMG of claim 1, the bacterial agent of claim 2, and the feed of claim 3 in the production of yellow mealworms.

9. The application according to claim 8, characterized in that, The feed is given to mealworm larvae at a rate of 0.1-0.5g / larva for 7-24 days.

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

Images