Sorting method of haliotis discus hannai probiotics and compound feed

Abstract

The invention belongs to the technical field of microorganism application and aquatic feed, and particularly discloses a sorting method of haliotis discus hannai probiotics, which is characterized in that the probiotics with strong adaptability and comprehensive functions are separated and screened from the intestinal tract of haliotis discus hannai, and the probiotics are naturally adapted to the intestinal tract environment of haliotis discus hannai, and have the effects of improving the intestinal tract environment of haliotis discus hannai and improving the intestinal tract environment of haliotis discus hannai. Meanwhile, digestive enzyme secretion capability and pathogenic bacterium inhibition capability are realized. On the other hand, the invention further discloses a compound feed containing the probiotics obtained through the sorting method, the feed can improve the growth performance of abalone and the feed utilization rate, and meanwhile the disease resistance is also enhanced.

Description

Technical Field

[0001] This invention relates to the fields of microbial application and aquatic feed technology, and more specifically to a method for sorting probiotics from abalone and a compound feed. Background Technology

[0002] Abalone farming faces two major constraints: First, growth is relatively slow. Although traditional formulated feed can meet basic nutritional needs, the feed conversion ratio of abalone is high, usually >2.0, resulting in a long farming cycle and high costs. Second, disease resistance is weak. Pathogenic Vibrio parahaemolyticus and other pathogenic Vibrio bacteria in the farming environment can easily cause large-scale mortality in abalone, especially during the high-temperature season, where disease mortality is high and seriously affects farming profits.

[0003] To address these issues, existing technologies often employ the addition of exogenous probiotics isolated from soil, seawater, or the intestines of other aquatic animals to feed. However, this approach has two major drawbacks: First, exogenous probiotics have poor adaptability to the intestinal microenvironment of abalone, weak colonization ability, and difficulty in long-term survival in the intestines, resulting in unstable effects. Second, most probiotics only possess a single function of inhibiting bacteria or promoting digestion, and cannot simultaneously achieve the dual goals of improving digestibility and resisting diseases. Summary of the Invention

[0004] To address the technical problems mentioned in the background section, this invention provides a method for sorting probiotics from wrinkled abalone. This method isolates and screens highly adaptable and functionally comprehensive probiotics from the abalone's own intestines. These probiotics are naturally adapted to the abalone's intestinal environment and simultaneously possess digestive enzyme secretion and pathogen inhibition capabilities, which is of great significance for promoting the high-quality development of the abalone farming industry. This invention also provides a formulated feed containing the probiotics obtained through the above sorting method, and the application of this formulated feed in wrinkled abalone farming.

[0005] As one aspect of the present invention, a method for sorting probiotics from *Abalone rotundifolia* is provided, comprising the following steps:

[0006] S1. Select several healthy wrinkled abalone and dissect them under aseptic conditions to remove their intestines.

[0007] S2, rinse the intestine with sterile phosphate buffer to remove residual intestinal contents, cut the intestine into pieces, add an equal volume of 0.9% sterile NaCl solution, and homogenize with a sterile homogenizer to obtain intestinal homogenate;

[0008] S3, mix the intestinal homogenate according to 10 -1 10 -2 10 -3 10 -4 10 -5 10-6 Serial dilutions were performed, with 0.1 mL of bacterial culture taken from each dilution and evenly spread on 2216E agar plates. Each dilution was replicated in triplicate and incubated at 28°C for 48 h.

[0009] S4. Select single colonies with different morphologies from 2216E agar plates, inoculate them into 2216E liquid medium, and culture at 28℃ and 150rpm for 24h with shaking. Repeat the streak purification three times to obtain the purified strain.

[0010] In the above-mentioned sorting method for abalone probiotics, the number of healthy abalone selected in S1 was 30, with an average weight of 4.62±0.19g.

[0011] In the above-mentioned sorting method for abalone probiotics, before dissecting and removing the intestines under aseptic conditions in step S1, the selected healthy abalone are anesthetized by soaking in 75% ethanol.

[0012] In the above sorting method for Abalone fasciatus, the intestines are flushed with sterile phosphate buffer at least 3 times in S2.

[0013] The sorting method for *Abalone scabra* probiotics described above includes a step S5 after S4, which is a screening step for cellulase-producing strains. Specifically, the pure strains obtained in S4 are inoculated onto a screening medium containing 1% sodium carboxymethyl cellulose and cultured at 28°C for 48 hours. The strains are then stained with 0.1% Congo red solution for 1 hour and destained with 1 mol / L NaCl solution. Strains that can form clear zones are then screened to obtain cellulase-producing strains.

[0014] In the above-mentioned sorting method for *Abalone scabra* probiotics, S6, the step of screening for anti-Vibrio strains, is included after S5. Specifically, Vibrio harveyi is evenly spread on LB medium plates, and then 100 μL of the strain to be screened after purification in S4 is added. The plates are incubated at 28°C for 24 h, and strains with inhibition zone diameter ≥10 mm are screened to obtain anti-Vibrio strains.

[0015] In the above-mentioned sorting method for *Abalone contortus* probiotics, the concentration of *Vibrio harveyi* in S6 was 1.0 × 10⁻⁶. 7 CFU / mL.

[0016] As another aspect of the present invention, a compound feed comprising the above-mentioned wrinkled abalone probiotic is also provided, including a basic feed and wrinkled abalone probiotic.

[0017] The aforementioned feed containing abalone probiotics comprises, by weight percentage, 1% fishmeal, 7% wheat gluten, 25% soybean protein concentrate, 24% high-gluten flour, 2.4% fish oil + soybean oil, 1% compound vitamin A, 1% compound mineral B, 30% kelp powder, 2% calcium dihydrogen phosphate, 0.2% choline chloride, 0.01% ethoxyquinoline, 0.1% calcium propionate, and 6.29% shell powder.

[0018] In the aforementioned feed containing abalone probiotics, the preparation method of the basic feed is as follows: the raw materials of the basic feed are pulverized separately, passed through a 100-mesh sieve, mixed evenly according to the weight percentage, and water is added and stirred until homogeneous; the mixture is pressed into 3mm thick sheets using a double roller flaking machine, then cut into 30mm×20mm thin sheets, then steamed at 100℃ for 10 minutes, and finally dried at 60℃ until the moisture content is <8%, to obtain the basic sheet feed.

[0019] The above-mentioned feed containing abalone probiotics involves adding the abalone probiotics as follows: The abalone probiotics are inoculated into 2216E liquid medium, cultured at 28°C and 150 rpm for 24 hours, centrifuged at 4500 × g for 15 minutes to collect the bacterial cells, washed twice with sterile seawater, resuspended, and diluted to prepare bacterial suspensions of different concentrations. Based on feed weight, the bacterial suspensions are evenly sprayed onto the base flake feed to achieve a probiotic concentration of 10... 7 -10¹¹CFU / g;

[0020] The inoculated feed was dried in a 38℃ forced-air drying oven until the moisture content was <8%, then sealed and stored at -20℃. The inoculated feed was prepared once a week to ensure the activity of the bacteria.

[0021] This invention has at least the following technical effects:

[0022] (1) Strong strain adaptability and comprehensive functions: The wrinkled abalone probiotic of the present invention is derived from the intestine of wrinkled abalone, and is naturally adapted to the intestinal microenvironment of abalone. Its colonization ability is significantly better than that of exogenous probiotics. It can produce cellulase to improve feed digestibility. It can resist Vibrio and inhibit pathogenic microorganisms. Therefore, this strain has the dual function of both producing cellulase and resisting Vibrio, which solves the defect of traditional probiotics having only one function.

[0023] (2) Improve abalone growth performance and feed utilization: Adding abalone probiotics to feed can improve the weight gain rate and feed utilization of abalone, shorten the breeding cycle and reduce breeding costs.

[0024] (3) Enhance disease resistance: The challenge experiment showed that the cumulative mortality rate of abalone with wrinkled abalone probiotic added to the feed was reduced by Vibrio parahaemolyticus, thus reducing disease losses and improving the survival rate of aquaculture. Attached Figure Description

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. 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.

[0026] Figure 1 The morphology of the pure strain obtained after dilution, plating, and single-colony purification in Example 1;

[0027] Figure 2 The results are from the cellulase-producing strains obtained after functional screening in Example 1;

[0028] Figure 3 This is the result of the anti-Vibrio strain obtained after functional screening in Example 1;

[0029] Figure 4 This is the molecular identification result of the strain in Example 1;

[0030] Figure 5 The results are from the virus challenge experiment in Example 3. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0032] Example 1:

[0033] As one aspect of the present invention, a wrinkled abalone intestinal probiotic BW-23 is provided.

[0034] (a) The characteristics of the BW-23 are as follows:

[0035] Strain source: It was isolated from the intestines of 30 healthy abalone with an average weight of 4.62 ± 0.19 g and named BW-23.

[0036] Molecular identification: Genomic DNA was extracted from the strain and amplified and sequenced using 16S rRNA gene primers 27F / 1492R. The sequence was submitted to GenBank, accession number PV366890. By BLAST comparison, the homology with the standard strain of Bacillus pumilus was ≥99%. Combined with morphological characteristics, it was identified as Bacillus pumilus.

[0037] Morphological characteristics: When cultured on 2216E agar medium at 28℃ for 48h, the colonies are round with neat edges, smooth and moist surface, and 1-2mm in diameter;

[0038] Functional characteristics: Cellulase activity: When cultured on a selection medium containing sodium carboxymethyl cellulose, it can form a clear zone; Antibacterial activity: It has a significant inhibitory effect on pathogenic Vibrio harveyi, with an inhibition zone diameter ≥12mm.

[0039] (II) The isolation and screening method for probiotic BW-23 is as follows:

[0040] Experimental materials include samples, culture media, reagents, and instruments.

[0041] Sample: Healthy wrinkled abalone (average weight 4.62 ± 0.19g);

[0042] Culture media: 2216E modified medium (5.0g peptone, 1.0g yeast extract, 0.1g ferric citrate, 19.45g sodium chloride, 5.98g magnesium chloride, 3.24g sodium sulfate, 1.8g calcium chloride, 0.6g potassium chloride, 0.16g sodium carbonate, 0.08g potassium bromide, 0.02g boric acid, 0.01g sodium silicate, 0.01g disodium hydrogen phosphate, 15.0g agar, pH 7.6±0.2), sodium carboxymethyl cellulose screening medium (10g sodium carboxymethyl cellulose, 5g peptone, 1g yeast extract, 20g agar, 1000mL seawater, pH 7.6-7.8), LB medium;

[0043] Reagents: sterile PBS, 0.9% sterile NaCl, 75% ethanol, 20% sterile glycerol, Congo red (0.1%), NaCl solution (1 mol / L);

[0044] Instruments: sterile homogenizer, constant temperature incubator, shaker, centrifuge, sequencer.

[0045] 1. Sample pretreatment: Thirty healthy wrinkled abalone with an average weight of 4.62 ± 0.19 g were selected. After anesthetizing by soaking in 75% ethanol, the intestines were dissected and removed under aseptic conditions. The intestines were rinsed three times with sterile phosphate-buffered saline (PBS) to remove residual intestinal contents. The intestines were then cut into small pieces and an equal volume of 0.9% sterile NaCl solution was added. The mixture was homogenized using a sterile homogenizer to obtain an intestinal homogenate.

[0046] 2. Dilution plating and single colony purification:

[0047] The dilution coating process involves subjecting the intestinal homogenate to 10... -1 Up to 10 -6Six serial dilutions were performed. 0.1 mL of bacterial culture from each dilution was evenly spread on 2216E agar plates. Each dilution was replicated in triplicate and incubated at 28°C for 48 h.

[0048] Single colony purification was performed by picking single colonies with different morphologies from the plate and inoculating them into 2216E liquid medium. The culture was carried out at 28°C with shaking at 150 rpm for 24 hours. The streak purification was repeated three times. The morphology of the purified strains was as follows: Figure 1 As shown.

[0049] 3. Function Filtering:

[0050] Screening of cellulase-producing strains: Pure strains were inoculated onto selection medium containing 1% sodium carboxymethyl cellulose and cultured at 28°C for 48 hours. After staining with 0.1% Congo red solution for 1 hour, the staining was followed by destaining with 1 mol / L NaCl solution. Strains capable of forming a clear zone were screened. For example, strain BW-23 can form a clear zone in strains such as... Figure 2 As shown;

[0051] Screening for anti-Vibrio strains: Vibrio harveyi (concentration 1.0 × 10⁻⁶) was selected. 7 The CFU / mL concentration was evenly spread onto LB agar plates, and then 100 μL of the strain to be screened was added. The plates were incubated at 28°C for 24 hours. Strains with inhibition zones ≥10 mm in diameter were screened. The results are as follows: Figure 3 As shown, BW-23 can inhibit Vibrio harveyi;

[0052] 5. Molecular identification: Genomic DNA of the purified single strain was extracted and amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') for the 16S rRNA gene. The amplified products were sequenced and compared with the NCBI database to identify the strain species.

[0053] Among them, such as Figure 4 As shown, the 16S rRNA sequence of BW-23 has 99.3% homology with the standard strain of Bacillus pumilus, and it was identified as Bacillus pumilus.

[0054] 6. Preservation: The strain identified as Bacillus pumilus BW-23 was resuspended in 2216E liquid medium containing 20% ​​sterile glycerol and stored at -80°C.

[0055] Conclusion: A strain of intestinal probiotic from the wrinkled abalone, possessing both high cellulase activity and strong anti-Vibrio function, was obtained and named Bacillus pumilus BW-23.

[0056] Example 2:

[0057] The instruments used in this embodiment include: a pulverizer, a 100-mesh sieve, a double-roller tablet press, a slicer, a cooking device, a drying oven, and a forced-air drying oven.

[0058] As another aspect of the present invention, the application of probiotic BW-23 in abalone farming is also provided. Specifically, BW-23 is added to the compound feed of wrinkled abalone to form a functional compound feed.

[0059] The basic feed formula for this functional compound feed, by weight, is shown in the table below:

[0060]

[0061] In the table above, multivitamins a and complex minerals b Specifically as follows:

[0062] Multivitamins a ( / kg): Thiamine, 120.0 mg; Riboflavin, 100.0 mg; Folic acid, 30.0 mg; Niacin, 800.0 mg; Vitamin B6, 40 mg; Calcium pantothenate, 200.0 mg; Inositol, 4000.0 mg; Biotin, 12.0 mg; Vitamin C, 4000.0 mg; Vitamin B12, 0.18 mg; Vitamin A, 100,000 IU; Vitamin D, 2000 IU; Vitamin E, 450 mg; Vitamin K3, 80 mg; Vitamin D3, 1000 IU;

[0063] Complex minerals b ( / kg): NaCl, 0.4 g; MgSO4·7H2O, 6.0 g; NaH2PO4·2H2O, 10.0 g; KH2PO4, 12.8 g; Ca(H2PO4)2·H2O, 8.0 g; Fe-citrate, 1.0 g; 0.4 mg; KIO3, 1.2 mg; Mn SO4·H2O, 64.8 mg; CuSO4·5H2O, 12.4 mg; Na2SeO3·5H2O, 0.61 mg.

[0064] The specific preparation steps for the above-mentioned functional compound feed are as follows:

[0065] Basic feed preparation: Crush the above basic feed ingredients separately, pass them through a 100-mesh sieve, mix them evenly in proportion, add water and stir until homogeneous; press the mixture into 3mm thick flakes using a double roller flaking machine, then cut them into 30mm×20mm thin slices, then steam them at 100℃ for 10 minutes, and finally dry them at 60℃ until the moisture content is <8% to obtain basic flake feed.

[0066] Preparation of probiotic-enriched feed: Probiotic BW-23 was inoculated into 2216E liquid medium and cultured at 28℃ and 150 rpm for 24 h. The bacterial cells were collected by centrifugation at 4500×g for 15 min, washed twice with sterile seawater, and resuspended to prepare bacterial suspensions of different concentrations. Based on feed weight, the bacterial suspensions were evenly sprayed onto basic feed tablets to achieve a probiotic concentration of 10 in the feed. 7 -10¹¹CFU / g;

[0067] Specifically, take 100g of basic feed and spray at a concentration of 10. 12 10 mL of bacterial suspension with CFU / mL was added to bring the probiotic concentration in the feed to 10. 11 CFU / g; Similarly, by adjusting the spraying amount, a probiotic concentration of 10 was prepared. 7 10 8 10 9 10 10 The feed was CFU / g, the control group was the basal feed, and the feed was sprayed with an equal amount of sterile seawater.

[0068] Drying and storage: Dry the inoculated feed in a 38℃ forced-air drying oven until the moisture content is <8%, seal the package and store at -20℃. Prepare the inoculated feed once a week to ensure bacterial activity.

[0069] Example 3:

[0070] This embodiment will study the effects of different concentrations of probiotic feed on the growth performance and disease resistance of abalone.

[0071] 1. Experimental Design

[0072] Experimental subject: Abalone fasciatus (initial weight 8.19 ± 0.13 g, purchased from Fujian Zhongxin Yongfeng Industry Co., Ltd.);

[0073] 2. Experimental grouping: 6 groups were set up, with 3 replicates in each group, and 60 abalone in each replicate.

[0074] P0 (control group): basic feed (without bacterial solution);

[0075] P1: Basic feed + 10 7 CFU / g BW-23;

[0076] P2: Basic feed + 10 8CFU / g BW-23;

[0077] P3: Basic feed +10 9 CFU / g BW-23;

[0078] P4: Basic feed +10 10 CFU / g BW-23;

[0079] P5: Basic feed +10 11 CFU / g BW-23;

[0080] 3. Aquaculture conditions: Indoor recirculating water system, water temperature 18℃ (temperature controlled), salinity 27-28‰, pH 8.05-8.18, dissolved oxygen > 6.5mg / L; feed at 18:00 every day at 3% of the total abalone biomass, clean up uneaten feed and feces at 8:00 the next day, and record the number of deaths;

[0081] 4. Breeding cycle: 120 days;

[0082] 5. Challenge experiment: After the aquaculture was completed, 15 abalone were taken from each group (3 replicates), and 0.1 mL of Vibrio parahaemolyticus suspension (concentration 1.0×107 CFU / mL) was injected intramuscularly. They were then fasted for 7 days, and the cumulative mortality rate was recorded.

[0083] 6. Detection indicators:

[0084] Growth performance indicators: Initial weight (IW), final weight (FW); initial shell length (ISL), final shell length (FSL); weight gain rate (WGR) = (FW - IW) / IW × 100%;

[0085] Daily growth of shells (DISL) = (FSL - ISL) / number of days of cultivation × 1000 (μm / day);

[0086] Feed conversion ratio (FCR) = Dry weight of feed / (FW - IW);

[0087] Survival rate (SR) = (Number of surviving abalone / Initial number of abalone) × 100%;

[0088] Disease resistance index: Cumulative mortality rate (CM) = Number of dead abalone / Number of abalone initially challenged × 100%.

[0089] 7. Experimental Results

[0090] Growth performance and feed utilization outcomes:

[0091]

[0092] Note: Different lowercase letters after the data in the same column indicate significant differences (P < 0.05).

[0093] As shown in the table, compared with the control group (P0), the probiotic-added groups showed significantly increased FW, WGR, and DISL (P < 0.05), and significantly decreased FCR; among them, the P3 group (10 9 The CFU / g ratio showed the best performance, with WGR reaching 88.91%, FCR reduced to 1.60, and DISL reaching 75.43 μm / day.

[0094] See the results of the challenge experiment. Figure 5 :

[0095] As shown in the figure, the cumulative mortality rate of abalone in the probiotic-added groups (except P1) was significantly lower than that in the control group (P < 0.05). Among them, the cumulative mortality rate of group P5 was the lowest, indicating that the probiotics had the strongest anti-Vibrio protective effect on abalone at this concentration. However, considering the parameters of weight gain rate, daily shell growth, and feed conversion ratio, the probiotic concentration of P3 had a better overall effect.

[0096] In summary, the probiotics and their application scheme of this invention provide an effective way to solve the problems of "long cycle, frequent disease outbreaks, and high costs" in the cultivation of wrinkled abalone, and have important economic and ecological value for promoting the sustainable development of my country's abalone farming industry.

[0097] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for sorting probiotics from *Abalone fasciatus*, characterized in that, It includes the following steps: S1. Select several healthy wrinkled abalone and dissect them under aseptic conditions to remove their intestines. S2, rinse the intestine with sterile phosphate buffer to remove residual intestinal contents, cut the intestine into pieces, add an equal volume of 0.9% sterile NaCl solution, and homogenize with a sterile homogenizer to obtain intestinal homogenate; S3, mix the intestinal homogenate at 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 Serial dilutions were performed, with 0.1 mL of bacterial culture taken from each dilution and evenly spread on 2216E agar plates. Each dilution was replicated in triplicate and incubated at 28°C for 48 h. S4. Select single colonies with different morphologies from 2216E agar plates, inoculate them into 2216E liquid medium, and culture at 28℃ and 150rpm for 24h with shaking. Repeat the streak purification three times to obtain the purified strain.

2. The sorting method for *Abalone fasciatus* probiotics according to claim 1, characterized in that, The number of healthy wrinkled abalone selected in S1 was 30, with an average weight of 4.62±0.19g.

3. The sorting method for *Abalone fasciatus* probiotics according to claim 1, characterized in that, Before dissecting and removing the intestines under aseptic conditions, S1 also includes the step of anesthetizing the selected healthy wrinkled abalone by soaking it in 75% ethanol.

4. The sorting method for *Abalone fasciatus* probiotics according to claim 1, characterized in that, In S2, the intestines are flushed with sterile phosphate buffer at least three times.

5. The sorting method for *Abalone fasciatus* probiotics according to claim 1, characterized in that, S4 is followed by S5, a step for screening cellulase-producing strains. Specifically, the strains obtained in S4 are inoculated on a screening medium containing 1% sodium carboxymethyl cellulose and cultured at 28°C for 48 hours. They are then stained with 0.1% Congo red solution for 1 hour, destained with 1 mol / L NaCl solution, and strains that can form a clear zone are screened to obtain cellulase-producing strains.

6. The sorting method for *Abalone fasciatus* probiotics according to claim 1, characterized in that, S6, following S5, is a step for screening anti-Vibrio strains. Specifically, Vibrio harveyi is evenly spread on LB agar plates, then 100 μL of the strain to be screened is added, and the plates are incubated at 28°C for 24 h. Strains with inhibition zone diameter ≥10 mm are screened to obtain anti-Vibrio strains.

7. The sorting method for *Abalone fasciatus* probiotics according to claim 1, characterized in that, The concentration of Vibrio harveyi in S6 was 1.0 × 10⁻⁶. 7 CFU / mL.

8. A compound feed, characterized in that, It comprises abalone probiotics obtained by the sorting method of abalone probiotics according to any one of claims 1-7 and a basic feed.

9. The compound feed according to claim 8, characterized in that, The preparation method of the basic feed is as follows: the raw materials of the basic feed are pulverized separately, passed through a 100-mesh sieve, mixed evenly according to the weight percentage, and water is added and stirred until homogeneous; the mixture is pressed into 3mm thick flakes using a double roller flaking machine, then cut into 30mm×20mm thin slices, then steamed at 100℃ for 10 minutes, and finally dried at 60℃ until the moisture content is <8%, to obtain the basic flake feed.

10. The compound feed according to claim 9, characterized in that, The method for adding probiotics to *Abalone rubra* is as follows: Inoculate *Abalone rubra* probiotics into 2216E liquid medium, incubate at 28℃ and 150 rpm for 24 h, collect the bacterial cells by centrifugation at 4500×g for 15 min, wash twice with sterile seawater, resuspend, and dilute to prepare bacterial suspensions of different concentrations. Based on feed weight, evenly spray the bacterial suspensions onto the basic flake feed to achieve a probiotic concentration of 10 in the feed. 7 -10¹¹CFU / g; The inoculated feed was dried in a 38℃ forced-air drying oven until the moisture content was <8%, then sealed and stored at -20℃. The inoculated feed was prepared once a week to ensure the activity of the bacteria.

Images