A high-yield digestive enzyme bacillus subtilis for promoting poultry nutrient absorption and improving meat quality and application thereof

Abstract

The present application relates to the technical field of microorganism, and discloses a high-yield digestive enzyme bacillus subtilis for promoting poultry nutrition absorption and improving meat quality and application thereof, the strain is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No:61711.The strain has the characteristics of high-yield amylase and protease, and can induce to improve lipase activity in poultry intestinal tract without producing lipase itself.The strain is prepared into a microbial preparation or a feed additive through a specific fermentation process, and is added into daily ration for feeding poultry at a concentration of 1.0*10 8 CFU / mL, which can not only significantly improve the body weight, dressing percentage and feed conversion rate of poultry, but also significantly improve meat quality, including improving meat color brightness and redness, reducing shear force and muscle fiber diameter to enhance tenderness, improving water binding capacity, and increasing the content of intermuscular fat and umami amino acids in muscle, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a high-yield digestive enzyme Bacillus subtilis that promotes nutrient absorption and improves meat quality in poultry, and its application. Background Technology

[0002] With the rapid development of the poultry farming industry, improving feed utilization and enhancing poultry meat quality has become a focus of industry attention. In the context of antibiotic-free farming, probiotics, as an alternative to antibiotics, are widely used in feed additives. Among them, Bacillus subtilis, due to its strong resistance and ability to produce various digestive enzymes, is one of the most widely used feed microorganisms.

[0003] However, existing feed-grade Bacillus subtilis mainly focuses on improving poultry growth performance (such as daily weight gain) or regulating gut microbiota balance to prevent disease, with relatively little research and application in specifically improving meat quality (such as tenderness, flavor, and intramuscular fat deposition). Furthermore, different strains exhibit significant differences in enzyme production characteristics; some strains, while producing a variety of enzymes, have low enzyme activity, or their metabolites cannot effectively regulate the host's own metabolic pathways. Currently, there is a lack of highly efficient strains on the market that can both promote efficient nutrient absorption through high-yield digestive enzyme production and significantly improve meat color, tenderness, and flavor deposition through specific metabolic regulatory mechanisms. Therefore, screening and developing Bacillus subtilis with specific superior traits (such as high production of amylase and protease, and the ability to induce host lipase activity) is of great significance for improving the economic benefits and product quality of poultry farming. Summary of the Invention

[0004] This invention provides a high-yield digestive enzyme Bacillus subtilis that promotes nutrient absorption and improves meat quality in poultry, and its application. While significantly promoting poultry growth and feed digestion, it can significantly reduce muscle shear force and increase intramuscular fat and umami amino acid content through specific metabolic regulation, thereby greatly improving the flavor and tenderness of the meat.

[0005] This invention provides a strain of Bacillus subtilis GX1578-1, which is classified as Bacillus subtilis GX1578-1 and has the accession number GDMCC No:61711.

[0006] The present invention also provides a microbial preparation comprising Bacillus subtilis GX1578-1 as described above.

[0007] The present invention also provides a method for preparing the microbial preparation as described above, comprising the following steps: inoculating Bacillus subtilis GX1578-1 into LB liquid medium and shaking culture at 37°C and 150 rpm for 18-24 hours to obtain bacterial solution.

[0008] The present invention also provides a feed additive comprising Bacillus subtilis GX1578-1 or a microbial preparation as described above, and a carrier or excipient.

[0009] The present invention also provides a poultry feed comprising the feed additives and basal diet described above.

[0010] The present invention also provides the application of Bacillus subtilis GX1578-1 as described in claim 1, the microbial preparation as described in claim 2, or the feed additive as described in claim 4 in poultry farming.

[0011] The present invention also provides a method for improving poultry meat quality, based on Bacillus subtilis GX1578-1 or a microbial preparation as described above, comprising the following steps: Bacillus subtilis GX1578-1 or a microbial preparation was added to the poultry diet and fed to poultry; wherein the concentration of Bacillus subtilis GX1578-1 added to the diet was 1.0×10⁸ CFU / mL.

[0012] The beneficial effects of this invention are as follows: The Bacillus subtilis GX1578-1 (GDMCC No: 61711) provided by this invention possesses excellent enzyme-producing characteristics, exhibiting strong amylase and protease production capabilities (D / d values ​​reaching 3.0 and 2.0, respectively). Although it does not produce lipase itself, it can significantly induce and enhance the activity of endogenous lipases in the poultry intestine, thereby comprehensively promoting the digestion and absorption of nutrients. Animal experiments have shown that this strain can significantly improve the growth performance of Guiliu Ma ducks, increase slaughter rate and eviscerated yield; more importantly, it can significantly improve meat quality, specifically by significantly increasing the brightness and redness of duck meat, significantly reducing muscle shear force and muscle fiber diameter (improving tenderness), increasing muscle water-holding capacity, and significantly increasing the content of protein, intramuscular fat, and umami amino acids (such as glutamic acid and aspartic acid) in the muscle, making the duck meat more tender and flavorful. Attached Figure Description

[0013] Figure 1 The figure shows the experimental results of GX15 producing digestive enzymes in Example 1 of the present invention.

[0014] Figure 2 This is a schematic diagram illustrating the effect of GX15 on the intestinal enzyme activity of Guiliu Ma Duck in Example 4 of the present invention.

[0015] Figure 3 This is a schematic diagram illustrating the effect of GX15 on the meat color of Guiliu Ma Duck in Embodiment 4 of the present invention.

[0016] Figure 4 This is a schematic diagram illustrating the effects of GX15 on pH, shear force, and water-holding capacity of Guiliu Ma Duck in Example 4 of the present invention.

[0017] Figure 5 This is a schematic diagram illustrating the effect of GX15 on the diameter of muscle fibers in Guiliu Ma ducks in Embodiment 4 of the present invention.

[0018] Figure 6 This is a schematic diagram illustrating the determination of protein content in GX15 Guiliu Ma Duck in Example 4 of the present invention.

[0019] Figure 7 This is a schematic diagram illustrating the determination of total amino acid content in Guiliu Ma Duck in Example 4 of the present invention.

[0020] Figure 8 This is a schematic diagram illustrating the determination of total essential amino acid content and the determination of the content of each major essential amino acid in Guiliu Ma Duck in Example 4 of the present invention.

[0021] Figure 9 This is a schematic diagram illustrating the determination of total umami amino acid content in Guiliu Ma Duck in Example 4 of the present invention.

[0022] Figure 10 This is a schematic diagram illustrating the determination of various umami amino acid contents in Guiliu Ma Duck in Example 4 of the present invention.

[0023] Figure 11 This is a schematic diagram illustrating the determination of intramuscular fat content in Guiliu Ma ducks in Example 4 of the present invention.

[0024] Figure 12 This is a schematic diagram illustrating the determination of fatty acid content in Guiliu Ma Duck in Example 4 of the present invention.

[0025] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0026] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0027] Strain Preservation Notes: The Bacillus subtilis involved in this invention is classified and named Bacillus subtilis. This strain was isolated and purified by the Guangxi Veterinary Research Institute and named GX1578-178-1. This strain has been deposited at the Guangdong Provincial Microbial Culture Collection Center, with accession number GDMCC No:61711, and the deposit date is June 7, 2021.

[0028] Example 1: Bacillus subtilis GX1578-1 and its enzyme production characteristics This embodiment illustrates the screening of the strains described in this invention and the identification of their digestive enzyme production characteristics.

[0029] 1. Amylase production test Starch plate preparation: Add 1% peptone, 0.5% beef extract, 0.5% NaCl, 1% starch, and 2% agar to sterile water, adjust the pH to 7.0, bring the volume to 1000mL, sterilize at 121℃ for 20min, and pour the plates for later use.

[0030] Inoculation and culture: GX1578-1 logarithmic phase bacterial suspension was inoculated on starch plates, with 3 replicates, and cultured at 37℃ inverted for 24-48 hours.

[0031] Results observation: After the culture was completed, 0.1% iodine solution was sprayed evenly onto the surface of the plate, and the plate was left to stand for 5 minutes. Observe whether a colorless transparent zone appears around the colony. If so, it indicates that the strain has the ability to produce amylase. Measure the "diameter of the transparent zone (D)" and the "colony diameter (d)", calculate the ratio (D / d), and judge the enzyme production ability. The larger the ratio, the stronger the amylase production ability.

[0032] 2. Protease production test Casein plate preparation: 1% peptone, 0.5% beef extract, 0.5% NaCl, 2% agar, adjust pH to 7.0, sterilize at 121℃ for 20 min. 1% casein, sterilized by filtration through a 0.22µm filter, is added at 40-50℃ before pouring the plates, and set aside.

[0033] Inoculation and culture: In a clean bench, GX1578-1 logarithmic phase bacterial suspension was inoculated on casein plates, with 3 replicates, and cultured at 37℃ inverted for 24-48 hours.

[0034] Results observation: After the culture is completed, observe whether a clear zone appears around the colony. If so, it indicates that the strain has the ability to produce protease. Measure the "diameter of the clear zone (D)" and the "colony diameter (d)" and calculate the ratio (D / d). The larger the ratio, the stronger the protease production ability.

[0035] 3. Lipase production test Rhodamine B plate preparation: 1% peptone, 0.5% yeast extract, 0.5% NaCl, 1% olive oil, 0.001% Rhodamine B, 2% agar. Adjust the pH to 7.0, pour the plate, and set aside.

[0036] Inoculation and culture: GX1578-1 logarithmic phase bacterial suspension was inoculated on Rhodamine B plates, with 3 replicates, and cultured at 37℃ inverted for 48-72 h.

[0037] Results observation: After the culture is completed, observe under ultraviolet light to see if a colorless transparent ring appears on an orange-red background around the colony. If so, it indicates that the strain has the ability to produce lipase. Measure the "diameter of the transparent ring (D)" and the "colony diameter (d)" and calculate the ratio (D / d). The larger the ratio, the stronger the ability to produce lipase.

[0038] 4. Cellulase production test Preparation of sodium carboxymethyl cellulose (CMC-Na) medium: 10g sodium carboxymethyl cellulose, 5g peptone, 5g yeast extract, 5g NaCl, 1g KH2PO4, 0.5g MgSO4·7H2O, 20g agar, 1000mL distilled water. Adjust the pH to 7.0, sterilize at 121℃ for 20min, and pour the culture into plates for later use.

[0039] Inoculation and culture: Punch 3-4 wells (6 mm in diameter) on a CMC-Na plate using a sterile punch, with a spacing of ≥2 cm between wells. Add 100 µL of GX1578-1 logarithmic phase bacterial culture to each well using a pipette, with 3 replicates, and incubate at 37°C inverted for 48-72 h.

[0040] Results observation: After the culture was completed, Congo red solution was sprayed evenly on the surface of the plate, and the plate was left to stand for 15 minutes. The staining solution was then poured off, and 1 mol / L NaOH solution was added and soaked for 15 minutes. The NaOH solution was then discarded, and the appearance of a transparent zone around the well was observed. If a transparent zone appeared, it indicated that the strain had the ability to produce cellulase. The diameter of the transparent zone (D) and the diameter of the colony (d) were measured, and the ratio (D / d) was calculated. The larger the ratio, the stronger the cellulase production ability.

[0041] Analysis of experimental results as follows Figure 1 As shown, (A) represents the results of the GX15 amylase production test; (B) represents the results of the GX15 protease production test; (C) represents the results of the GX15 lipase production test; (D) represents the results of the GX15 cellulase production test; and (E) represents the comparison of the differences in the production of various digestive enzymes by GX15. Figure 1 The results represent the amylase, protease, lipase, and cellulase production of GX15 (i.e., GX1578-1), respectively. Figure 1 In sample A, after the culture was completed, a 0.1% iodine solution was sprayed. A clear, distinct transparent zone appeared around the GX15 colony, indicating that the amylase produced by this strain digested the starch on the plate. Furthermore, the D / d value reached 3.0, demonstrating its high amylase production capacity. Figure 1 In sample B, the GX15 colony exhibited a large clear zone, with a D / d value of 2.0, indicating a strong protease-producing ability. Figure 1 In D, small clear zones appeared around GX15 colonies, with a D / d value of 1.27, indicating moderate cellulase activity. However... Figure 1The absence of a clear zone in sample C indicates that GX15 cannot produce lipase.

[0042] The D / d value (the ratio of the diameter D of the clear zone / hydrolysis zone to the colony diameter d) is a core indicator for measuring the enzyme production capacity of a strain. The larger the ratio, the stronger the ability of the enzyme secreted by GX15 to decompose substrates (starch, casein, cellulose, etc.) and the higher the enzyme production activity. If no hydrolysis zone is formed (D=0), it is determined that there is no enzyme production capacity corresponding to the enzyme.

[0043] As shown in Table 1 below, GX15 has a very strong ability to produce amylase, a relatively strong ability to produce protease, a moderate ability to produce cellulase, but no ability to produce lipase.

[0044] Table 1. Digestive enzyme production capacity of Bacillus subtilis GX15

[0045] Example 2: Microbial preparations and their preparation methods This embodiment describes the preparation process of the microbial preparation of the present invention, namely the bacterial culture preparation process.

[0046] Strain resuscitation: The purified and preserved Bacillus subtilis GX1578-1 was taken out of the -40℃ freezer and slightly thawed. A loopful was then used to dab a sterile inoculation loop and streak it in three zones of an LB agar plate. The plate was then incubated at 37℃ for 18-24 hours.

[0047] Preparation of bacterial culture: Pick a single colony from the streak line of an LB agar plate and transfer it to 10 mL of LB liquid medium. Incubate at 37°C and 150 rpm for 18-24 hours for enrichment. This bacterial culture is the microbial preparation of this invention and can be used directly for subsequent applications or further processing.

[0048] Example 3: Feed additives and poultry feed This embodiment describes a method for preparing Bacillus subtilis as described in Example 1 or 2 into feed additives and poultry feed. In practical applications, the GX15 bacterial solution prepared in Example 2 is used as an additive. When preparing poultry feed, the GX15 bacterial solution is adjusted to a concentration of 10⁸ CFU / mL and uniformly mixed into the diet (basal diet). The basal diet is a combination of carriers or supplements and nutrients that is acceptable in food science or animal husbandry.

[0049] Example 4: Application and feeding methods in improving poultry meat quality and promoting nutrient absorption. This embodiment verifies the application effect of GX1578-1 in improving growth performance, digestion and absorption, and meat quality in Guiliu Ma Duck farming.

[0050] 1. Animal Experiment Design Purchase 60 one-day-old Guiliu Ma Ducks, divide them into 2 groups of 30 each: (1) Experimental group (BS+Sty / GX15): The GX15 bacterial solution was adjusted to a concentration of 108 CFU / mL and evenly distributed in the diet according to the method in Example 3.

[0051] (2) Control group (CON): Normal feeding without any additives. After reaching 60 days of age, duodenal contents were collected, mixed with extract, centrifuged, and the supernatant was used as the test sample. Enzyme activity was determined according to the purchased kit. Pectoral and leg muscles were sent to the testing center for relevant indicator testing.

[0052] 2. Test Results (1) Impact on the production performance of Guiliu Ma Duck The results are shown in the table below. After adding GX15, the ducks' 60-day body weight increased, the feed conversion ratio decreased, and the slaughter performance indicators (eviscerated yield, breast muscle yield, and leg muscle yield) all improved.

[0053]

[0054] (2) Effects on intestinal enzyme activity of Guiliu Ma ducks like Figure 2 As shown, Figure 2 The activities of amylase, protease, and lipase were demonstrated. The intestinal enzyme activities in the experimental group were significantly higher than those in the control group, indicating that the addition of GX15 Bacillus subtilis significantly enhanced the intestinal digestive function of Guiliu Ma Ducks, and this difference was statistically significant (p < 0.05). Notably, although in vitro experiments showed that GX15 does not produce lipase, the lipase activity in the experimental group was significantly higher than that in the control group in animal experiments.

[0055] (3) Effects on meat quality of 60-day-old Guiliu Ma ducks like Figure 3 As shown, the experimental group's duck meat had significantly higher brightness and redness than the control group, and significantly lower yellowness, indicating that adding GX15 Bacillus subtilis can significantly improve the color of duck meat. Figure 3 middle, Figure 3 A shows a significant increase in flesh-colored brightness. Figure 3 B shows a significant increase in the redness of the flesh. Figure 3C indicates a significant decrease in the yellowness of the meat. Within a suitable range, a significant increase in brightness (L) indicates that the duck meat in the experimental group has a brighter and more uniform color, without dullness or paleness, reflecting good water retention of muscle fibers, less water loss, and a more attractive appearance. A significant increase in redness (a), if a* is within the suitable range of 5-8, indicates that the myoglobin content in the duck meat is reasonable, the bleeding after slaughter is sufficient, the degree of oxidation is low, and the meat color presents a natural reddish hue, reflecting both the freshness of the duck meat and suggesting better oxygen supply and metabolic status in the muscles. A significant decrease in yellowness indicates more uniform fat distribution or a lower degree of fat oxidation, resulting in fresher meat.

[0056] like Figure 4 As shown, the pH in the experimental group was significantly higher than that in the control group (p < 0.0001). Figure 4 A), shear force decreased significantly (p < 0.0001) Figure 4 B), the hydraulic system was significantly improved (p < 0.01 at 24h). Figure 4 C) indicates that the addition of GX15 significantly improved the pH, tenderness, and water retention of duck meat.

[0057] like Figure 5 As shown, the experimental group's duck breast muscle ( Figure 5 A) Leg muscle fiber diameter ( Figure 5 C) were significantly lower than the control group ( Figure 5 (B,5D) indicates that adding GX15 can significantly improve the tenderness of duck meat and make the meat quality better.

[0058] (4) Determination of nutritional components Depend on Figure 6-12 As shown, the addition of GX15 significantly improved the protein, amino acid, intramuscular fat content, and fatty acid content of Guiliu Ma Duck meat.

[0059] like Figure 6 As shown, the protein content of the GX15 group was significantly higher than that of the CON group. Figure 7 As shown, the total amino acid content of the GX15 group was significantly higher than that of the CON group. Figure 8 As shown on the left, the total essential amino acid content of the GX15 group was significantly higher than that of the CON group. Figure 8 The right side further illustrates the determination of the content of each major essential amino acid (Lys, Met, Thr, Leu, Ile, Val), with Lys, Met, Thr, and Ile showing significantly increased contents. Figure 9 As shown, the total umami amino acid content of group GX15 was significantly higher than that of group CON. Figure 10 As shown, the content of each umami amino acid (Glu, Gly, Ala, Arg, Asp) was determined, and all were significantly increased. Figure 11As shown, the intramuscular fat content in the GX15 group was significantly higher than that in the CON group. Figure 12 As shown, the effect of GX15 on the proportion of fatty acids, including lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid, is demonstrated.

[0060] 3. Conclusion Bacillus subtilis GX15 possesses excellent digestive enzyme production characteristics, exhibiting high amylase production (D / d=3.0), strong protease activity (D / d=2.0), and moderate cellulase activity (D / d=1.27), but lacking lipase production. Adding it to the diet of Guiliu Ma ducks at a concentration of 10^8 CFU / mL significantly enhances intestinal digestive enzyme activity and optimizes meat quality: resulting in brighter meat color (increased brightness and redness, decreased yellowness), significantly improved pH and water-holding capacity, decreased shear force and muscle fiber diameter, and improved tenderness and water retention; simultaneously, it significantly increases the content of nutrients such as protein, amino acids, and intramuscular fat in the muscle. This strain effectively promotes nutrient absorption and optimizes meat quality, providing a practical microbial additive option for high-quality and efficient Guiliu Ma duck farming, with broad application prospects.

[0061] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, apparatus, article, or method that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, apparatus, article, or method. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, apparatus, article, or method that includes that element.

[0062] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A strain of Bacillus subtilis GX1578-1, characterized in that, The classification name of Bacillus subtilis is Bacillus subtilis GX1578-1, and the accession number is GDMCC No:61711.

2. A microbial preparation, characterized in that, It contains Bacillus subtilis GX1578-1 as described in claim 1.

3. A method for preparing the microbial preparation of claim 2, characterized in that, Includes the following steps: Bacillus subtilis GX1578-1 was inoculated into LB liquid medium and cultured with shaking at 37°C and 150 rpm for 18-24 hours to obtain bacterial culture.

4. A feed additive, characterized in that, It comprises Bacillus subtilis GX1578-1 as described in claim 1 or the microbial preparation as described in claim 2, as well as a carrier or excipient.

5. A poultry feed, characterized in that, It includes the feed additive and basal diet as described in claim 4.

6. The application of Bacillus subtilis GX1578-1 as described in claim 1, the microbial preparation as described in claim 2, or the feed additive as described in claim 4 in poultry farming.

7. A method for improving poultry meat quality, characterized in that, Based on Bacillus subtilis GX1578-1 according to claim 1 or the microbial preparation according to claim 2, the process includes the following steps: Bacillus subtilis GX1578-1 or a microbial preparation was added to poultry diets and fed to poultry; wherein the concentration of Bacillus subtilis GX1578-1 in the diet was 1.0 × 10⁻⁶. 8 CFU / mL.

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