Cinnamon leaf zymolyte capable of improving immunocompetence as well as preparation method and application of cinnamon leaf zymolyte

Abstract

The invention discloses a cinnamon leaf zymolyte capable of improving immunocompetence as well as a preparation method and application of the cinnamon leaf zymolyte. According to the method, the damage rate of cinnamaldehyde in the cinnamon leaves in the fermentation process is reduced, and the immunological enhancement capability of the cinnamon leaves is improved. And the method is simple in process, low in cost, short in production period and practical, improves the feeding value and the immunomodulatory activity of the cinnamon leaves, and is convenient to popularize and apply in the green antibiotic-free livestock industry.

Description

Technical Field

[0001] This invention relates to the field of natural extract technology, and in particular to a cinnamon leaf hydrolysate that can enhance immunity, its preparation method, and its application. Background Technology

[0002] How to prevent and control livestock diseases in a green way has become a major challenge for the livestock industry. Research has found that many places have the habit of feeding animals with cinnamon leaves to improve their growth performance. Cinnamon leaves have a strong regenerative capacity and a large regeneration volume, but they are a major by-product of the cinnamon planting and processing industry and have been used as boiler fuel, resulting in a great waste of cinnamon resources.

[0003] Cinnamaldehyde is a naturally occurring aldehyde compound found in many plant essential oils, particularly in cinnamon bark volatile oil, where it constitutes 75%-90% of the total. Its molecular formula is C9H8O, and its molecular weight is 132.16. At room temperature, it is a yellow, viscous liquid with a strong spicy odor. The chemical structure of cinnamaldehyde is trans-phenylpropenal, containing conjugated double bonds and an aldehyde group, making it easily oxidized to cinnamic acid and unstable in strongly acidic and alkaline environments. It is sparingly soluble in water and glycerol, but readily soluble in organic solvents such as ethanol and ether, and can volatilize with steam. Extraction from natural sources is mainly achieved through steam distillation or supercritical CO2 extraction, but its application is limited by the difficulty and cost of separation and purification.

[0004] Cinnamon leaves contain a large number of anti-nutritional factors (cellulose, hemicellulose, pectin, and phytic acid, etc.), which is the main problem with feeding livestock with cinnamon leaves. Currently, the main methods for degrading / destroying anti-nutritional factors are microbial fermentation or enzymatic hydrolysis. Microbial fermentation has advantages such as a broad enzyme spectrum and low cost, but the selection cycle is long and difficult, and it easily degrades the effective components in the fermentation substrate, such as reducing cinnamaldehyde to cinnamyl alcohol. Enzymatic hydrolysis has the specificity to degrade / degrade anti-nutritional substances, but it is more expensive, has a narrower enzyme spectrum, and often requires multiple enzymes to work together for hydrolysis. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a cinnamon leaf hydrolysate that can enhance immunity.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned cinnamon leaf hydrolysate that can enhance immunity.

[0007] Another object of the present invention is to provide the application of the above-mentioned cinnamon leaf hydrolysate that can enhance immunity.

[0008] The objective of this invention is achieved through the following technical solution:

[0009] A method for preparing cinnamon leaf hydrolysate that can enhance immunity includes the following steps:

[0010] (1) Bacillus subtilis was activated and cultured. After the culture was completed, it was frozen and centrifuged. The supernatant was collected, filtered, and the exoenzyme solution was obtained.

[0011] (2) Dry the cinnamon leaves, crush them, sieve them, and sterilize them to obtain sterilized powdered cinnamon leaves.

[0012] (3) Mix the exoenzyme solution with sterilized powdered cinnamon leaves, let it stand for fermentation, filter after fermentation, take the filter residue, dry it, and sieve it to obtain cinnamon leaf fermentation product that can improve immunity.

[0013] The Bacillus subtilis mentioned in step (1) is Bacillus subtilis CICC No. 10089.

[0014] The cultivation conditions described in step (1) are 25℃~35℃ and 180~250r / min for 3~4 days.

[0015] The conditions for the refrigerated centrifugation in step (1) are 4°C and centrifugation at 3600 rpm for 30-40 min.

[0016] The sieving in step (2) is sieving through an 80-mesh sieve.

[0017] The ratio of the exocellular enzyme solution to the sterilized powdered cinnamon leaves in step (3) is 1g: 2-4mL.

[0018] The fermentation conditions described in step (3) are fermentation at room temperature for 2 to 4 days.

[0019] The sieving process described in step (3) is sieving through an 80-mesh sieve.

[0020] A cinnamon leaf hydrolysate that can enhance immunity was prepared according to the above preparation method.

[0021] The above-mentioned cinnamon leaf hydrolysate, which can enhance immunity, is used in the preparation of drugs that enhance immunity.

[0022] The above-mentioned cinnamon leaf hydrolysate, which can enhance immunity, is used in the preparation of feed additives that enhance immunity.

[0023] The present invention has the following advantages and effects compared with the prior art:

[0024] This invention mainly includes methods for preparing exozyme solutions from microbial strains and methods for preparing fermented cinnamon leaves. Applying this method reduces the destruction rate of cinnamon aldehyde in cinnamon leaves during fermentation and enhances the immune-boosting ability of cinnamon leaves. Furthermore, this method is simple, low-cost, and has a short production cycle. It is a practical technical method that improves the feed value and immunomodulatory activity of cinnamon leaves, facilitating its promotion and application in the green, antibiotic-free livestock industry. Attached Figure Description

[0025] Figure 1 This is the result of the cinnamaldehyde retention rate determination in Example 3.

[0026] Figure 2 This is the result of the cell proliferation test of the laurel leaf fermentation product in Example 5.

[0027] Figure 3 This is the result of the cell phagocytosis rate test of the laurel leaf fermentation product in Example 5. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0029] Unless otherwise specified in the following implementation plan, the test conditions are generally as per standard test conditions or the test conditions recommended by the reagent company. Unless otherwise specified, all materials and reagents used are commercially available.

[0030] Example 1: Preparation of exocellular enzyme solution

[0031] (1) Activate Bacillus subtilis CICC No.10089 and culture it for 2-3 generations until it reaches an active state. Scrape off and inoculate it into a 1000 mL Erlenmeyer flask containing 300-400 mL of nutrient broth liquid culture medium.

[0032] (2) The bacteria were cultured at 20℃ and 200 rpm for 3 days. After the culture was completed, the bacteria were centrifuged at 4℃ and 3600 rpm for 30-40 minutes to remove most of the bacterial cells and obtain the supernatant. The supernatant was filtered through a 0.22 μm aqueous membrane to remove a small amount of cell spores and residual bacterial cells, thus obtaining the exoenzyme solution.

[0033] Example 2: Preparation of Cinnamon Leaf Powder

[0034] Fresh cinnamon leaves, free from insect infestation and mold, are evenly spread on a clean, well-ventilated bamboo mat or drying tray, about 2-3 cm thick. They are placed in a cool, well-ventilated area, turning them over every half hour to ensure even moisture loss and allowing them to air dry naturally. The leaves are then pulverized using a grinder and passed through a No. 5 pharmacopoeia sieve or an 80-mesh sieve to obtain uniformly sized powdered cinnamon leaves. Approximately 5g of the powdered cinnamon leaves is weighed and placed in a 100mL Erlenmeyer flask. The flask is then autoclaved at 121℃ for about 20 minutes. After cooling, the sterilized powdered cinnamon leaves are ready for use.

[0035] Example 3: Determination of Cinnamaldehyde Retention Rate

[0036] 3.1 Determination of Cinnamaldehyde Content

[0037] 3.1.1 Preparation of reference solution

[0038] Take an appropriate amount of cinnamaldehyde reference standard, accurately weigh it, and add methanol to prepare a solution containing 10 μg per 1 mL.

[0039] 3.1.2 Preparation of the test solution

[0040] Accurately weigh approximately 0.5g of cinnamon leaf powder and place it in a stoppered conical flask. Accurately add 25mL of methanol, weigh the flask, and sonicate for 10 minutes. Let it stand overnight, repeat the sonication process once more, and weigh the flask again. Make up the weight loss with methanol, shake well, and filter. Accurately measure 1mL of the filtrate, place it in a 25mL volumetric flask, add methanol to the mark, and shake well.

[0041] 3.1.3 Determination Method

[0042] Accurately pipette 10 μL each of the reference solution and the test solution into the liquid chromatograph and determine the result.

[0043] 3.2 Determination of Cinnamaldehyde Retention Rate

[0044] Under aseptic conditions, sterile powdered cinnamon leaves prepared in Example 2 were added to the exocytase solution prepared in Example 1, the uncentrifuged Bacillus subtilis culture solution, the nutrient broth liquid culture medium, and the cellulase solution (100 U / mL), respectively, at a mass-to-volume ratio of 1:4. The mixtures were then placed at room temperature and allowed to ferment for 2 days. The cinnamaldehyde content was then determined according to the method described in 3.1, and the retention rate of cinnamaldehyde was calculated using the following formula:

[0045]

[0046] Wherein, A1 is the peak area of ​​cinnamaldehyde measured in cinnamon leaves obtained by different treatment methods; A0 is the peak area of ​​cinnamaldehyde measured in cinnamon leaves without any treatment, and the retention rate of cinnamaldehyde is calculated based on the cinnamaldehyde content in the original sample.

[0047] See results Figure 1 As shown, there are certain differences in the retention rates of cinnamaldehyde in cinnamon leaves obtained by treatment with exozyme solution, Bacillus subtilis bacterial solution, nutrient broth liquid culture medium, and cellulase solution. Cellulase solution, exozyme solution, and culture medium have relatively high retention rates of cinnamaldehyde, around 100%, but Bacillus subtilis bacterial solution has a relatively low retention rate of cinnamaldehyde, only around 30%. This indicates that cellulase, culture medium, and Bacillus subtilis exozyme have a relatively small destructive effect on cinnamaldehyde, while Bacillus subtilis contains certain substances that can destroy cinnamaldehyde.

[0048] Example 4: Preparation of Cinnamon Leaf Fermented Hydrolysate

[0049] 4.1 Preparation of Cinnamon Leaf Fermentation Product 1

[0050] Under aseptic conditions, the sterile powdered cinnamon leaves prepared in Example 2 were mixed with the exoenzyme solution 1 prepared in Example 1 at a ratio of 1g:3mL. The mixture was placed at room temperature and allowed to ferment for 3 days. Excess exoenzyme solution on the surface of the fermented cinnamon leaves was removed by vacuum filtration. The filter residue was spread evenly on a drying dish or drying tray with a thickness of about 1-2 cm and vacuum dried at 30-40°C. The residue was turned over every 3-4 hours until a constant weight was achieved. The dried product was then removed and ground through a No. 5 pharmacopoeia sieve or an 80-mesh sieve to obtain cinnamon leaf fermentation product 1.

[0051] 4.2 Preparation of Cinnamon Leaf Fermentation Product 2

[0052] Following the preparation method in 4.1, the fermentation time was adjusted to 0 days, and the remaining steps were the same, to prepare cinnamon leaf fermentation product 2.

[0053] 4.3 Preparation of Cinnamon Leaf Fermented Hydrolysate 3

[0054] Following the preparation method in 4.1, the fermentation time was adjusted to 6 days, while the remaining steps remained the same, and cinnamon leaf fermentation product 3 was prepared.

[0055] Example 5 Macrophage proliferation and phagocytosis test

[0056] 5.1 Preparation of aqueous extract of cinnamon leaf fermentation product

[0057] Weigh 5g of cinnamon leaf hydrolysate, soak it in 50mL of 90% ethanol for 10min to remove the pigment components, filter and dry the cinnamon leaves, add 100mL of double-distilled water, and extract in a 70℃ water bath for 60min, shaking constantly to ensure complete extraction. After extraction, filter to obtain the cinnamon leaf hydrolysate extract mother liquor. Take 5mL of the mother liquor, place it in a 100mL volumetric flask, add double-distilled water to the mark, shake well, and filter through a 0.22μm filter membrane to obtain a 2.5mg / mL crude cinnamon leaf hydrolysate extract for later use.

[0058] 5.2 Macrophage proliferation test

[0059] The proliferative activity of RAW 264.7 macrophages was detected using the CCK-8 assay. Macrophages with a density of 2 × 10⁻⁶ cells were used. 5 A suspension of RAW 264.7 single cells in logarithmic growth phase was seeded into 96-well plates (100 μL / well) and incubated overnight in a 5% CO2 incubator at 37°C. The supernatant was discarded. Experimental groups, background groups (cell-free), positive control groups (with LPS), and blank control groups (with PBS) were set up, with three replicates for each group. 100 μL of complete culture medium was added to each well. 100 μL of LPS (0.1 μg / mL) was added to each well in the positive control group, and 1-3 μL of the cinnamon hydrolysate aqueous extract prepared in Example 4 was added to each well in the experimental groups. After incubation for 24 h, 10 μL of LCK-8 solution was added to each well, and incubation was continued for 40 min. The absorbance (A) at 450 nm was measured using a microplate reader. The incubation was repeated three times, and cell proliferation activity was calculated using the following formula:

[0060]

[0061] Where A1 is the absorbance measured after incubation with samples or LPS of different mass concentrations for 24 hours; A2 is the background PBS; and A3 is the absorbance measured after incubation with PBS for 24 hours.

[0062] Experimental results are as follows Figure 2 As shown, the cell proliferation rate test of cinnamon leaf hydrolysates 1-3 revealed that cinnamon leaves treated with Bacillus subtilis exoenzyme had different degrees of proliferative effects on macrophages. In terms of macrophage proliferation, cinnamon leaf hydrolysate 1, which was fermented for 3 days, showed a certain degree of increased activity compared to cinnamon leaf hydrolysates 2 and 3. This indicates that cinnamon leaf hydrolysates prepared by exoenzyme solution can enhance the in vitro immune activity of cinnamon leaves, and that the fermentation conditions need to be controlled to achieve better results.

[0063] 5.3 Macrophage phagocytosis test

[0064] The phagocytic capacity of RAW 264.7 cells was assessed using the neutral red assay. Cells were arranged at a density of 2 × 10⁻⁶. 5A suspension of RAW 264.7 single cells in logarithmic growth phase was seeded into 96-well plates (100 μL / well) and incubated overnight in a 5% CO2, 37°C incubator. The supernatant was discarded, and experimental groups, a background group (cell-free), a positive control group (with LPS), and a blank control group (with PBS) were set up, with three replicates for each group. 100 μL of complete culture medium was added to each well, 100 μL of LPS (0.1 μg / mL) was added to each well in the positive control group, and 1-3 μL of the cinnamon leaf hydrolysate prepared in Example 4 were added to each well in the experimental groups. Incubate in an incubator for 24 hours, discard the supernatant, wash each well twice with PBS, then add 100 μL of 0.05% neutral red solution to each well, incubate for 1 hour, discard the solution in the wells, wash twice with PBS, spin dry, add 100 μL of cell lysis buffer (glacial acetic acid-anhydrous ethanol 1:1) to each well, lyse at room temperature for 20 minutes, and detect at 540 nm using a microplate reader. Calculate the cell phagocytosis rate using the following formula:

[0065]

[0066] Where A1 is the absorbance measured after adding samples of different mass concentrations or incubating with LPS for 24 hours; A2 is the absorbance measured after adding PBS and incubating for 24 hours.

[0067] Experimental results are as follows Figure 3 As shown, the phagocytic rate test of cinnamon leaf hydrolysate for 1-3 days revealed that cinnamon leaves not treated with Bacillus subtilis exoenzyme significantly inhibited macrophage phagocytosis, while cinnamon leaves treated with Bacillus subtilis exoenzyme for 3 days showed significant macrophage phagocytosis. However, after 6 days of treatment, the effect on macrophage phagocytosis was not significant, indicating that cinnamon leaves hydrolyzed by this method can enhance the in vitro immune activity of cinnamon leaves.

[0068] Examples 3, 4, and 5 demonstrate that the fermentation of cinnamon leaves using this method not only improves the retention rate of cinnamaldehyde during the fermentation process but also enhances the immune activity of cinnamon leaves.

[0069] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing cinnamon leaf hydrolysate that can enhance immunity, characterized in that... Includes the following steps: (1) Bacillus subtilis was activated and cultured. After the culture was completed, it was frozen and centrifuged. The supernatant was collected, filtered, and the exoenzyme solution was obtained. (2) Dry the cinnamon leaves, crush them, sieve them, and sterilize them to obtain sterilized powdered cinnamon leaves. (3) Mix the exoenzyme solution with sterilized powdered cinnamon leaves, let it stand for fermentation, filter after fermentation, take the filter residue, dry it, and sieve it to obtain cinnamon leaf fermentation product that can improve immunity.

2. The method for preparing cinnamon leaf fermented hydrolysate capable of enhancing immunity according to claim 1, characterized in that: The Bacillus subtilis mentioned in step (1) is Bacillus subtilis CICC No. 10089.

3. The method for preparing cinnamon leaf fermented hydrolysate capable of enhancing immunity according to claim 1, characterized in that: The culture conditions described in step (1) are 25℃~35℃ and 180~250r / min for 3~4 days; The conditions for the refrigerated centrifugation in step (1) are 4°C and centrifugation at 3600 rpm for 30-40 min.

4. The method for preparing cinnamon leaf fermented hydrolysate capable of enhancing immunity according to claim 1, characterized in that: The sieving in step (2) is sieving through an 80-mesh sieve.

5. The method for preparing cinnamon leaf fermented hydrolysate capable of enhancing immunity according to claim 1, characterized in that: The ratio of the exocellular enzyme solution to the sterilized powdered cinnamon leaves in step (3) is 1g: 2-4mL.

6. The method for preparing cinnamon leaf fermented hydrolysate capable of enhancing immunity according to claim 1, characterized in that: The fermentation conditions described in step (3) are fermentation at room temperature for 2 to 4 days.

7. The method for preparing cinnamon leaf fermented hydrolysate capable of enhancing immunity according to claim 1, characterized in that: The sieving process described in step (3) is sieving through an 80-mesh sieve.

8. A cinnamon leaf hydrolysate that can enhance immunity, characterized in that... It is prepared according to any one of the preparation methods described in claims 1 to 8.

9. The use of the cinnamon leaf hydrolysate of claim 8, which can enhance immunity, in the preparation of a drug that enhances immunity.

10. The use of the cinnamon leaf hydrolysate of claim 8, which can enhance immunity, in the preparation of an immune-enhancing feed additive.

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