A compound microbial agent, its preparation method, and its application in aquatic feed.

Abstract

This invention discloses a compound microbial agent, its preparation method, and its application in aquatic feed, belonging to the field of microbial technology. This invention provides a compound microbial powder based on *Bacillus belye* QCS-426, *Bacillus* H68, and *Lactobacillus plantarum* T80, which is then added to fish meal, soybean meal, and high-gluten flour to obtain the compound microbial agent, which is ultimately used to make aquatic feed. According to feeding experiments, compared with conventional aquatic feed, feeding the aquatic feed of this invention significantly improved the weight gain rate and specific growth rate of juvenile *Scorpionichthys schlegelii*, and reduced the feed conversion ratio. The above results indicate that mixing the compound microbial agent provided by this invention with basic aquatic feed enriches the nutrition of the aquatic feed, making it more conducive to the digestion and absorption of aquatic animals, and can significantly improve the growth performance of aquatic animals, showing broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of microbial applications, and in particular to a compound microbial agent, its preparation method, and its application in aquatic feed. Background Technology

[0002] Global consumption of edible fish is growing rapidly each year, and aquaculture's contribution to global fish production is increasing rapidly. To meet the rapidly growing demand for fish, aquaculture production practices need to be developed to a higher level in terms of both technology and practice.

[0003] The growing demand for aquaculture production is accompanied by many challenges, among which disease outbreaks are a major factor hindering the development of the aquaculture industry. For decades and even today, antibiotics have been frequently used to control and prevent diseases in aquaculture, promote aquatic product growth, and improve feed efficiency. However, most antibiotics only treat diseases and do not address the underlying problems, while also causing two major harms: antibiotic residues and antibiotic resistance.

[0004] Feed microbial preparations, as a green and safe alternative to antibiotics, have received significant attention in recent years due to their distinct mechanism of action. These preparations are made from natural beneficial microbial strains through fermentation and purification processes, offering advantages such as improved feed utilization, enhanced nutrition, increased animal growth, maintenance of intestinal microecological balance, and enhanced immunity in farmed products. Microbial preparations can also improve water quality in aquaculture and effectively reduce antibiotic residues and drug-resistant pollution, playing a crucial role in maintaining the health of aquatic organisms. However, single microbial preparations have limited functionality and poor environmental adaptability, making them difficult to meet actual aquaculture needs. Compound microbial preparations, through the rational combination of functionally complementary strains, such as Bacillus and Lactobacillus, can significantly enhance their environmental adaptability and overall efficacy. These preparations not only possess disease-resistant and growth-promoting functions but also improve the intestinal flora of aquatic products, thereby improving product quality and optimizing the aquaculture environment. They provide important technical support for the green development of aquaculture and represent an ideal future alternative to antibiotics. Summary of the Invention

[0005] The purpose of this invention is to provide a compound microbial agent, its preparation method, and its application in aquatic feed, to solve the problems existing in the prior art. This invention provides a compound microbial preparation suitable for aquaculture. This preparation, by optimizing the ratio of different microorganisms, aims to improve feed conversion efficiency, enhance the growth performance and immunity of farmed animals, and effectively reduce antibiotic use, thereby achieving the production of antibiotic-free aquatic products.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a compound microbial powder, comprising Bacillus velezensis QCS-426, Bacillus sp. H68, and Lactobacillus plantarum T80;

[0008] The Bacillus belyes QCS-426 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33327, deposited on January 8, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0009] The Bacillus H68 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33326, deposited on January 8, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0010] The Lactobacillus plantarum T80 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33328, deposited on January 7, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0011] Preferably, the total effective viable count of Bacillus vesiculosus QCS-426, Bacillus vesiculosus H68, and Lactobacillus plantarum T80 in the compound microbial powder is not less than 8.5-9.5 × 10⁻⁶. 10 cfu / g;

[0012] The ratio of the effective viable counts of the Bacillus belyssus QCS-426, the Bacillus H68, and the Lactobacillus plantarum T80 is (3-5):(3-5):(90-95).

[0013] The present invention also provides a method for preparing the aforementioned composite microbial powder, comprising the following steps:

[0014] The *Bacillus belyssioides* QCS-426 and *Bacillus* H68 were activated separately, and seed culture and fermentation expansion culture were carried out using LB liquid medium. The fermentation broth was centrifuged, the precipitate was collected, and 5-10% (w / w) of skim milk powder and 5-10% (w / w) of mannitol were added. The mixture was then freeze-dried under vacuum to obtain the bacterial powder of *Bacillus belyssioides* QCS-426 and the bacterial powder of *Bacillus* H68.

[0015] The *Lactobacillus plantarum* T80 was activated, and seed culture and fermentation expansion culture were carried out using MRS liquid medium. The fermentation broth was centrifuged, the precipitate was collected, and 5-10% (w / w) of skim milk powder and 5-10% (w / w) of mannitol were added. The mixture was then freeze-dried under vacuum to obtain the *Lactobacillus plantarum* T80 bacterial powder.

[0016] Weigh out 3-5 parts of the Bacillus berberis QCS-426 bacterial powder, 3-5 parts of the Bacillus berberis H68 bacterial powder, and 90-94 parts of the Lactobacillus plantarum T80 bacterial powder by weight, mix them evenly, and the compound microbial powder is obtained.

[0017] Preferably, the viable count of the Bacillus vesiculosus QCS-426 powder is 8.5-9.5 × 10⁻⁶. 10 cfu / g; the viable count in the Bacillus H68 bacterial powder is 8.5-9.5 × 10⁻⁶. 10 cfu / g; the viable count in the *Lactobacillus plantarum* T80 powder is 8.5-9.5 × 10⁻⁶. 10 cfu / g.

[0018] The present invention also provides a composite microbial agent, comprising the aforementioned composite microbial powder.

[0019] Preferably, by weight, it comprises the following components: 0.5-1.5 parts of the compound microbial powder, 19-21 parts of fish meal, 34-36 parts of soybean meal, and 41.5-46.5 parts of high-gluten flour.

[0020] The present invention also provides the application of the aforementioned compound microbial powder or the aforementioned compound microbial agent in the preparation of aquatic feed.

[0021] The present invention also provides an aquatic feed, comprising the aforementioned compound microbial agent.

[0022] Preferably, it also includes a basic feed; the mass ratio of the compound microbial agent to the basic feed is 1:2000.

[0023] This invention also provides a method for aquaculture, comprising the following steps:

[0024] The compound microbial powder and / or the compound microbial agent are applied to the aquaculture area.

[0025] And / or, feed the aquatic feed described above.

[0026] The present invention discloses the following technical effects:

[0027] This invention provides a compound microbial powder based on Bacillus belye QCS-426, Bacillus H68, and Lactobacillus plantarum T80, which is then added to fish meal, soybean meal, and high-gluten flour to obtain a compound microbial agent, which is ultimately used to produce aquatic feed. Feeding experiments showed that, compared with conventional aquatic feed, feeding the aquatic feed of this invention significantly improved the weight gain and specific growth rate of juvenile Scorpionichthys schlegelii, and reduced the feed conversion ratio. These results indicate that mixing the compound microbial agent provided by this invention with basic aquatic feed enriches the nutritional content of the feed, making it more digestible and absorbable for aquatic animals, and can significantly improve their growth performance, showing broad application prospects. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a colony morphology diagram of Bacillus belyss QCS-426.

[0030] Figure 2 This is a colony morphology diagram of Bacillus H68;

[0031] Figure 3 This is a colony morphology diagram of Lactobacillus plantarum T80.

[0032] Figure 4 The colony morphology of Bacillus belyss QCS-426 under a scanning electron microscope;

[0033] Figure 5 This is a scanning electron microscope image of the colony morphology of Bacillus H68.

[0034] Figure 6 The colony morphology of Lactobacillus plantarum T80 under a scanning electron microscope;

[0035] Figure 7 Phylogenetic tree diagram of Bacillus belyss QCS-426; where strain QCS-426 is Bacillus belyss QCS-426;

[0036] Figure 8 This is a phylogenetic tree diagram of Bacillus H68; where strain H68 is Bacillus H68.

[0037] Figure 9 This is a phylogenetic tree diagram of Lactobacillus plantarum T80; where strain T80 is Lactobacillus plantarum T80.

[0038] Figure 10 The growth curve of Bacillus belyss QCS-426;

[0039] Figure 11 This is a growth curve of Bacillus H68;

[0040] Figure 12 This is a growth curve of Lactobacillus plantarum T80. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] The culture medium formulations involved in the embodiments of the present invention are as follows:

[0047] LB medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, bring to a final volume of 1L with deionized water, pH 7.0, sterilize at 121℃ for 20min.

[0048] LB solid medium: 10g tryptone, 5g yeast extract, 10g sodium chloride, 15g agar, bring to a final volume of 1L with deionized water, pH 7.0, sterilize at 121℃ for 20min.

[0049] MRS medium: 10g peptone, 8g beef extract, 4g yeast extract, 20g glucose, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 5g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate, 1.0g Tween 80, bring to a final volume of 1L with deionized water, pH 5.7, sterilize at 118℃ for 15min.

[0050] MRS solid medium: 10g peptone, 8g beef extract, 4g yeast extract, 20g glucose, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 5g sodium acetate, 0.2g magnesium sulfate, 0.04g manganese sulfate, 1.0g Tween 80, 15g agar. Adjust the volume to 1L with deionized water, pH 6.2, and sterilize at 121℃ for 20min.

[0051] Example 1

[0052] I. Identification and Properties of Strains

[0053] 1. Observation of strain morphology

[0054] (1) Activation of strains

[0055] Bacillus belye QCS-426 and Bacillus H68 were streaked on LB solid medium and cultured at 37°C for 18 h. Lactobacillus plantarum T80 was inoculated on MRS solid medium and cultured at 30°C for 24 h.

[0056] (2) Morphological observation

[0057] Bacillus belye QCS-426 and Bacillus H68 were streaked on LB solid medium and cultured at 37°C for 18 h. Lactobacillus plantarum T80 was inoculated on MRS solid medium and cultured at 30°C for 24 h. Colony morphology was then observed.

[0058] (3) Liquid culture

[0059] Single colonies of Bacillus belye QCS-426 and Bacillus H68 were picked and cultured in LB medium at 37°C and 180 rpm for 12 h. Single colonies of Lactobacillus plantarum T80 were picked and cultured in MRS medium at 30°C for 12 h.

[0060] (4) Scanning electron microscopy observation

[0061] Take 1 mL each of Bacillus belyss QCS-426, Bacillus H68 and Lactobacillus plantarum T80 bacterial suspensions, centrifuge, fix overnight with 2.5% glutaraldehyde, elute with ethanol in a gradient, and then transfer a small amount of bacterial suspension onto a silicon wafer, sputter gold and observe under a scanning electron microscope.

[0062] 2. Strain identification

[0063] The 16S rRNA gene of the strains was amplified by PCR. The sequencing results of Bacillus belyss QCS-426, Bacillus H68 and Lactobacillus plantarum T80 were submitted to NCBI GenBank. The sequence analysis was performed using the BLAST program, and a phylogenetic tree was drawn based on the results.

[0064] 3. Growth curve of the strain

[0065] Bacillus belye QCS-426 and Bacillus H68 were streaked on LB solid medium and cultured at 37°C for 18 h. Lactobacillus plantarum T80 was inoculated on MRS solid medium and cultured at 30°C for 24 h.

[0066] Determination of seed liquid OD 600 Value, add seed culture to the culture medium respectively, so that the initial OD 600 The value was 0.02. The cells were incubated at the optimal temperature, and the OD was measured every 2 hours. 600 value.

[0067] 4. Strain preservation

[0068] Bacillus velezensis QCS-426 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33327, deposited on January 8, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0069] Bacillus sp. H68 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33326, deposited on January 8, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0070] Lactobacillus plantarum T80 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33328, deposited on January 7, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0071] II. Preparation of Compound Microbial Agents

[0072] 1. Preparation of microbial inoculum powder

[0073] (1) Preparation method of Bacillus belyss QCS-426 and Bacillus H68 bacterial powder

[0074] Both strains of Bacillus belyss were prepared using the same powder preparation method:

[0075] Bacillus belye was inoculated onto LB solid medium and cultured at 37°C for 18 hours.

[0076] A single colony of Bacillus belye was taken from LB solid medium and inoculated into LB liquid medium. The culture was carried out at 37°C and 180 r / min for 12 h to obtain Bacillus belye seed culture.

[0077] The seed culture was inoculated into LB liquid medium at a volume ratio of 1%, and cultured at 37°C and 180 r / min for 12 h to obtain Bacillus belyssus fermentation broth.

[0078] The Bacillus berberis fermentation broth was centrifuged to obtain bacterial sludge. Skim milk powder and mannitol were added at a mass ratio of 5-10%, and the mixture was then freeze-dried under vacuum to obtain bacterial powder. The viable cell count in the bacterial powder was 8.5-9.5 × 10⁻⁶. 10 cfu / g.

[0079] (2) Preparation method of Lactobacillus plantarum T80 powder

[0080] Lactobacillus plantarum was inoculated into MRS solid medium and cultured at 30°C for 24 hours.

[0081] A single colony of *Lactobacillus plantarum* was taken from MRS solid medium and inoculated into MRS liquid medium. The culture was then incubated at 37°C for 18 hours to obtain *Lactobacillus plantarum* seed culture.

[0082] The seed culture was inoculated into MRS liquid medium at a volume ratio of 1%, and cultured at 30°C for 36 hours to obtain the fermentation broth of Lactobacillus plantarum.

[0083] The fermentation broth of *Lactobacillus plantarum* was centrifuged to obtain bacterial sludge. Skim milk powder and mannitol were added at a mass ratio of 5-10%, and the mixture was then freeze-dried under vacuum to obtain bacterial powder. The viable count in the bacterial powder was 8.5-9.5 × 10⁻⁶. 10 cfu / g.

[0084] 2. Preparation of compound microbial inoculants

[0085] Weigh out 0.3-0.5g of Bacillus vesiculosus QCS-426 powder, 0.3-0.5g of Bacillus vesiculosus H68 powder and 9.0-9.5g of Lactobacillus plantarum T80 powder, mix them evenly to obtain compound microbial powder.

[0086] The compound microbial agent is prepared by mixing 0.5-1.5g of compound microbial powder, 19-21g of fish meal, 34-36g of soybean meal and 41.5-46.5g of high-gluten flour.

[0087] 3. Preparation of feed additives for the control group

[0088] Weigh out 19-21g of fish meal, 34-36g of soybean meal, and 43-47g of high-gluten flour and mix them to prepare the feed additive for control group 1.

[0089] Weigh out 0.3-0.5g of Bacillus vesiculosus QCS-426 powder, 0.3-0.5g of Bacillus vesiculosus H68 powder, 19-21g of fish meal, 34-36g of soybean meal, and 42-46.4g of high-gluten flour, mix them, and prepare the feed additive for control group 2.

[0090] III. Application of Compound Microbial Agents in Aquatic Feed

[0091] The basic feed components for aquatic animals of this invention are: 200g fish meal, 200g peanut meal, 100g chicken meal, 100g soybean meal, 100g corn flour, 100g wheat flour, 60g rice bran, 25g squid meal, 30g sodium alginate, 20g protein powder, 20g shrimp shell powder, and 20g soybean lecithin oil.

[0092] The prepared compound microbial agent was mixed with the basic feed for aquaculture animals. 0.5g of the agent was added to every 1kg of feed to form the experimental group. The blank group was fed with feed without any added substances. The control group was fed with feed containing the above-mentioned control group feed additive (addition amount was 0.5g / kg). Each group consisted of 80 juvenile Scorpionfish, fed 3 times a day for a total of 60 days.

[0093] In all the above groups, the breeding conditions should be kept as the same as possible. At the beginning of the experiment, the weight and length of each fish should be weighed. At the end of the experiment, the weight and length of each fish should be measured, and the weight gain rate, specific growth rate, feed conversion ratio, visceral-to-body ratio, liver-to-body ratio, intestinal-to-body ratio, and condition factor should be calculated.

[0094] IV. Experimental Results

[0095] 1. Observation of bacterial colony morphology

[0096] The colony morphology of Bacillus belyss QCS-426, Bacillus H68, and Lactobacillus plantarum T80 is as follows: Figures 1-3 As shown.

[0097] 2. Morphological observation of the strain using scanning electron microscopy

[0098] The morphology of Bacillus belyss QCS-426, Bacillus H68 and Lactobacillus plantarum T80 under a scanning electron microscope is as follows: Figures 4-6 As shown.

[0099] 3. Strain identification

[0100] Phylogenetic tree of Bacillus belyss QCS-426, Bacillus H68 and Lactobacillus plantarum T80 as follows Figures 7-9 As shown.

[0101] 4. Growth curve of the strain

[0102] The growth curves of Bacillus belyss QCS-426, Bacillus H68, and Lactobacillus plantarum T80 are as follows: Figures 10-12 As shown.

[0103] 5. Viable bacteria count of the inoculant

[0104] The viable cell count in Bacillus vesiculosus QCS-426 and Bacillus vesiculosus H68 bacterial powders was 8.5-9.5 × 10⁻⁶. 10 CFU / g; The viable count in Lactobacillus plantarum T80 bacterial powder is 8.5-9.5 × 10⁻⁶. 10 cfu / g

[0105] 6. Effects of compound microbial inoculants on the growth of juvenile Scorpionfish (Scorpionfish schlegelii)

[0106] Compared with the control group, the experimental group of juvenile Scorpionfish fed with the compound microbial agent described in this invention showed a 44.94% increase in weight gain, a 17.46% increase in specific growth rate, and a 31.09% decrease in feed conversion ratio. The effects were highly significant and significantly superior to the control group. This demonstrates that mixing the compound microbial agent provided by this invention with aquaculture feed results in a richer nutritional profile, better digestibility and absorption by aquatic animals, and a significant improvement in their growth performance, indicating broad application prospects.

[0107] Table 1. Effects of different diet groups on juvenile Scorpionfish (Scorpionfish schlegelii)

[0108]

[0109]

[0110] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a compound microbial powder or compound microbial agent in the preparation of aquatic feed that promotes the growth of juvenile Scorpionfish, characterized in that, The compound microbial powder includes Bacillus velezensis QCS-426, Bacillus sp. H68, and Lactobacillus plantarum T80; The Bacillus belyes QCS-426 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33327, deposited on January 8, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The Bacillus H68 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33326, deposited on January 8, 2025, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The Lactobacillus plantarum T80 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33328, deposit date of January 7, 2025, and address of No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The total viable count of *Bacillus vesiculosus* QCS-426, *Bacillus vesiculosus* H68, and *Lactobacillus plantarum* T80 in the compound microbial powder is not less than 8.5-9.5 × 10⁻⁶. 10 cfu / g; The effective viable count ratio of the Bacillus vesiculus QCS-426, the Bacillus vesiculus H68, and the Lactobacillus plantarum T80 is (3-5):(3-5):(90-95); The compound microbial agent comprises the following components in parts by weight: The compound microbial powder contains 0.5-1.5 parts, fish meal 19-21 parts, soybean meal 34-36 parts, and high-gluten flour 41.5-46.5 parts; The compound microbial agent is mixed with the basic feed to obtain an aquatic feed that promotes the growth of juvenile scorpionfish.

2. An aquatic feed, characterized in that, Includes the compound microbial agent described in claim 1.

3. The aquatic feed as described in claim 2, characterized in that, It also includes basic feed; The mass ratio of the compound microbial agent to the basic feed is 1:2000.

4. A method for aquaculture, characterized in that, Includes the following steps: The compound microbial powder and / or the compound microbial agent as described in claim 1 are applied to the aquaculture area; And / or, feed the aquatic feed as described in claim 2 or 3.

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