PEG-ACS/M-siRNA nanocomposite, application thereof, and method for reducing histamine content during fishmeal storage

Abstract

The present invention provides a PEG-ACS/M-siRNA nanocomposite application, and method for reducing the histamine content during fishmeal storage thereof. A small interfering ribonucleic acid (siRNA) is designed and prepared according to a histidine decarboxylase gene of Morganella morganii (Morganella morganii subsp. morganii KT), and the histidine decarboxylase gene has a sequence of SEQ ID No: 1. A PEG-ACS/M-siRNA nanocomposite is prepared by using a PEGylated arginine-modified chitosan as a carrier. A PEG-ACS/M-siRNA nanocomposite is added to fishmeal in a certain ratio. The method for reducing the histamine content during fishmeal storage has a significant inhibitory effect on the histamine content during fishmeal storage, and can reduce the histamine content in fishmeal by 49%-53%, which has great significance for the control of biogenic amines in fishmeal in the feed industry.

Claims

1. A PEGylated arginine-modified chitosan/small-interfering RNA (PEG-ACS/siRNA) nanocomposite, comprising a small-interfering RNA (siRNA), wherein the siRNA is designed according to a histidine decarboxylase gene of Morganella morganii (Morganella morganii subsp. morganii KT); and the histidine decarboxylase gene has the sequence of SEQ ID No: 1; and the PEG-ACS/siRNA nanocomposite is prepared by using a PEGylated arginine-modified chitosan (PEG-ACS) as a carrier.

2. The PEG-ACS/siRNA nanocomposite of claim 1, wherein the siRNA is double-stranded and comprises the sequence of: positive-sense strand: SEQ ID No: 2, antisense strand: SEQ ID No: 3.

3. The PEG-ACS/siRNA nanocomposite of claim 1, wherein the *PEG-ACS/siRNA nanocomposite has a particle diameter of 150-250 nm.

4. A method for storing fishmeal, comprising: administering a PEG-ACS/small-interfering RNA (siRNA) nanocomposite to store the fishmeal; wherein the PEG-ACS/siRNA nanocomposite comprises a siRNA; and wherein the siRNA is designed according to a histidine decarboxylase gene of Morganella morganii (Morganella morganii subsp. morganii KT); and the histidine decarboxylase gene has the sequence of SEQ ID No: 1; and the *PEG-ACS/siRNA nanocomposite is prepared by using a PEGylated arginine-modified chitosan as a carrier.

5. The method of claim 4, wherein 3-6 g of the PEG-ACS/siRNA nanocomposite is added per kilogram of the fishmeal.

6. A method for reducing a histamine content in fishmeal storage, wherein a PEG-ACS/small-interfering RNA (siRNA) nanocomposite is added to the fishmeal storage, wherein a PEG-ACS/siRNA nanocomposite is added to the fishmeal, at an addition ratio of 3-6 g of the PEG-ACS/siRNA nanocomposite per kilogram of the fishmeal; wherein the PEG-ACS/siRNA nanocomposite nanocomposite comprises a siRNA; and wherein the siRNA is designed according to a histidine decarboxylase gene of Morganella morganii (Morganella morganii subsp. morganii KT); and the histidine decarboxylase gene has the sequence of SEQ ID No: 1; and the PEG-ACS/siRNA nanocomposite is prepared by using a PEGylated arginine-modified chitosan as a carrier.

7. The method of claim 4, wherein the siRNA is double-stranded and comprises the sequence of: positive-sense strand: SEQ ID No: 2, antisense strand: SEQ ID No: 3.

8. The method of claim 4, wherein the PEGylated arginine-modified chitosan is prepared by the following steps: (1) preparation of an arginine-modified chitosan: dissolving a chitosan in a TEMED/HCl buffer solution to obtain a first solution, and then adding a 1-ethyl-3(3-dimethylaminopropyl) carbodiimide and a N-hydroxyl-succinimide coupling agent to the first solution to obtain a second solution; stirring the second solution evenly to obtain a third solution, adding an arginine containing chitosan amino with a molar amount of 50%-100% to the third solution to obtain a fourth solution, reacting the fourth solution under a magnetic stirring for 6-10 h at room temperature to obtain a fifth solution, and then dialyzing the fifth solution with demineralized water to obtain a sixth solution, and freeze-drying the sixth solution to obtain the arginine-modified chitosan; (2) preparation of the PEG-ACS: taking the arginine-modified chitosan, adding a PEG-SPA, reacting at room temperature and dialyzing with a dialysis bag with a molecular weight cutoff of 14,000, and removing an untreated PEG to obtain the PEGylated arginine-modified chitosan.

9. The method of claim 8, wherein the 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide and the chitosan are equimolar, and the N-hydroxy-succinimide coupling agent and the chitosan are equimolar.

10. The method of claim 4, wherein the preparation of the PEG-ACS/siRNA nanocomposite comprises the following steps: (a) preparation of a siRNA solution: dissolving the siRNA in DEPC water to prepare the siRNA solution at a concentration of 18-25 μM; (b) dissolving the PEG-ACS in a NaAc/HAc buffer solution to obtain a solution, incubating the solution and the siRNA solution at a constant temperature water bath at 50-55° C. for 10-15 min respectively, then quickly mixing the solution and the siRNA solution in an equal volume in a vortex mixer for 30-40 s to obtain the PEG ACS/siRNA nanocomposite.

11. The method of claim 4, wherein the PEG ACS/siRNA nanocomposite has a particle diameter of 150-250 nm.

12. The method of claim 6, wherein the siRNA is double-stranded and comprises the sequence of: positive-sense strand: SEQ ID No: 2, antisense strand: SEQ ID No: 3.

13. The method of claim 6, wherein the PEGylated arginine-modified chitosan is prepared by the following steps: preparation of an arginine-modified chitosan: dissolving a chitosan in a TENTED/HCl buffer solution to obtain a first solution, and then adding a 1-ethyl-3(3-dimethylaminopropyl) carbodiimide and a N-hydroxyl-succinimide coupling agent to the first solution; to obtain a second solution, adding an arginine containing chitosan amino with a molar amount of 50%-100% to the second solution to obtain a third solution, reacting the third solution under a magnetic stirring for 6-10 h at room temperature, and then dialyzing with demineralized water, and freeze drying to obtain the arginine-modified chitosan; (2) preparation of the PEG-ACS: taking the arginine-modified chitosan, adding a PEG SPA, reacting at room temperature and dialyzing with a dialysis bag with a molecular weight cutoff of 14,000, and removing an untreated PEG to obtain the PEGylated arginine-modified chitosan.

14. The method of claim 13, wherein the 1-ethyl-3(3-dimethylaminopropyl) carbodiimide and the chitosan are equimolar, and the N-hydroxy-succinimide coupling agent and the chitosan are equimolar.

15. The method of claim 6, wherein the preparation of the PEG-ACS/siRNA nanocomposite comprises the following steps: (a) preparation of a siRNA solution: dissolving the siRNA in DEPC water to prepare the siRNA solution at a concentration of 18-25 μM; (b) dissolving the PEG-ACS in a NaAc/HAc buffer solution to obtain a solution, incubating the solution and the siRNA solution at a constant temperature water bath at 50-55° C. for 10-15 min respectively, then quickly mixing the solution and the siRNA solution in an equal volume in a vortex mixer for 30-40 s to obtain the PEG ACS/siRNA nanocomposite.

16. The method of claim 6, wherein the PEG ACS/siRNA nanocomposite has a particle diameter of 150-250 nm.

17. The PEG-ACS/siRNA nanocomposite of claim 1, wherein the siRNA is double-stranded and comprises the sequence of: positive-sense strand: SEQ ID No: 4, antisense strand: SEQ ID No: 5.

18. The PEG-ACS/siRNA nanocomposite of claim 1, wherein the siRNA is double-stranded and comprises the sequence of: positive-sense strand: SEQ ID No: 6, antisense strand: SEQ ID No: 7.

19. The method of claim 4, wherein the siRNA is double-stranded and comprises the sequence of: positive-sense strand: SEQ ID No: 4, antisense strand: SEQ ID No: 5.

20. The method of claim 4, wherein the siRNA is double-stranded and comprises the sequence of: positive-sense strand: SEQ ID No: 6, antisense strand: SEQ ID No: 7.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the histamine content in fishmeal under different treatments in Embodiment 1;

(2) FIG. 2 shows the histamine content in fishmeal under different treatments in Embodiment 2;

(3) FIG. 3 shows the monthly increase of histamine in fishmeal under different treatments in Embodiment 1;

(4) FIG. 4 shows the monthly increase of histamine in fishmeal under different treatments in Embodiment 2; and

(5) FIG. 5 shows the changes in the expression level of the histidine decarboxylase gene with time using different siRNA carriers in Comparative Example 1 and Embodiment 1.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(6) The technical solutions of the present invention will be fully described below in conjunction with specific embodiments. In the embodiments of the present invention, different amounts of PEG-ACS/M-siRNA nanocomposite are added to the fishmeal, and the histamine content of the product is significantly reduced by 49%-53%.

Embodiment 1

(7) (1) Design of M-siRNA sequence.

(8) TABLE-US-00003 Positive-sense strand: (SEQ NO: 2) 5′-GGAUCGUAUUUCUGUUGAAAU-3′, antisense strand: (SEQ NO: 3) 5′-UUCAACAGAAAUACGAUCCAC-3′.

(9) (2) Preparation of M-siRNA.

(10) (3) Preparation of ACS: 0.5 g of chitosan is weighed and dissolved in a TEMED/HCl buffer solution (pH 5.0), and then 567 mg of EDC and 340 mg of NHS coupling agents are added to the solution; after being stirred evenly, the arginine containing chitosan amino with a molar amount of 80% is added, reacting under a magnetic stirring for 8 h at room temperature, and then dialyzed with demineralized water and freeze-dried to obtain the arginine-modified chitosan.

(11) (4) Preparation of PEG-ACS: 2.5 mL of 10 mg/mL arginine-modified chitosan is taken and added with 25 mg of PEG-SPA to react at room temperature for 4 h. The PEGylated arginine-modified chitosan is obtained after the unreacted PEG is removed through dialysis using a molecular weight cutoff of 14,000, and then is freeze-dried for use.

(12) (5) Preparation of siRNA solution: siRNA is dissolved in the DEPC water to prepare the siRNA solution at a concentration of 20 μM and stored at −20° C. for use.

(13) (6) Preparation of PEG-ACS/M-siRNA: PEG-ACS is dissolved in the NaAc/HAc buffer solution (pH 5.5) to a mass fraction of 0.02%. The PEG-ACS solution and the siRNA solution are respectively incubated in a constant temperature water bath at 50° C. for 10 min, then mixed in an equal volume, and quickly mixed in a vortex mixer for 30 s to obtain the PEG-ACS/M-siRNA nanocomposite.

(14) (7) The average particle size of the nanocomposite is measured by a dynamic light scattering method to be 217 nm. The prepared composite is frozen at −20° C. for 4 h and then freeze-dried for 24 h to obtain the lyophilized powder for use.

(15) (8) 5.5 g of PEG-ACS/M-siRNA nanocomposite per kilogram of fishmeal is added.

(16) (9) High-performance liquid chromatography (HPLC) is used to detect the histamine content in the fishmeal, and a blank control is performed. The results are shown in Table 1 and FIG. 1.

(17) TABLE-US-00004 TABLE 1 Histamine content in the fishmeal under different treatments (mg/100 g) Storage time/month January February March April May June Control 43.64 47.52 52.08 58.01 65.24 76.83 group Treatment 20.43 22.56 24.98 27.78 31.58 36.81 group Storage time/month July August September October November December Control 93.97 109.52 121.29 129.73 134.67 138.75 group Treatment 45.72 53.25 58.49 62.37 65.55 67.71 group

(18) As shown in Table 1, during the process of storage, the histamine content in the fishmeal increased continuously, and the standard of feed deterioration was achieved in August (the amine content of the secondary fishmeal was ≤1000 mg/kg). The fishmeal added with PEG-ACS/M-siRNA nanocomposite did not reach the deterioration standard even in December, and the experimental results showed that the fishmeal could be effectively extended. Combined with the results, a further comparative analysis showed that the inhibition of histamine in fishmeal by the PEG-ACS/M-siRNA nanocomposite reached 51%-53%.

(19) As shown in FIG. 1, during the process of storage, the histamine content in the fishmeal increased, and the PEG-ACS/M-siRNA nanocomposite significantly reduced the histamine content in the fishmeal in February, March, and May and significantly reduced the histamine content in the fishmeal in other months. The PEG-ACS/M-siRNA nanocomposite has obvious inhibitory effects on the histamine synthesis during the process of fishmeal storage.

(20) As shown in FIG. 3, during the process of storage, the histamine content in the fishmeal increased, and the amount of histamine was relatively increased in July and August. The PEG-ACS/M-siRNA nanocomposite has obvious inhibitory effects on the histamine synthesis during the process of fishmeal storage.

Embodiment 2

(21) (1) Design of M-siRNA sequence.

(22) TABLE-US-00005 Positive-sense strand: (SEQ NO: 2) 5′-GGAUCGUAUUUCUGUUGAAAU-3′, antisense strand: (SEQ NO: 3) 5′-UUCAACAGAAAUACGAUCCAC-3′.

(23) (2) Preparation of M-siRNA.

(24) (3) Preparation of ACS: 0.8 g of chitosan is weighed and dissolved in a TEMED/HCl buffer solution (pH 4.5), and then 900 mg of EDC and 550 mg of NHS coupling agent are added to the solution; after being stirred evenly, the arginine containing chitosan amino with a molar amount of 100% is added, reacting under a magnetic stirring for 7 h at room temperature, and then dialyzed with demineralized water and freeze-dried to obtain the arginine-modified chitosan.

(25) (4) Preparation of PEG-ACS: 3 mL of 8 mg/mL arginine-modified chitosan is taken and added with 24 mg of PEG-SPA to react at room temperature for 4 h. The PEGylated arginine-modified chitosan is obtained after the unreacted PEG is removed through dialysis using a molecular weight cutoff of 14,000, and then is freeze-dried for use.

(26) (5) Preparation of siRNA solution: siRNA is dissolved in the DEPC water to prepare the siRNA solution at a concentration of 18 μM and stored at −20° C. for use.

(27) (6) Preparation of PEG-ACS/M-siRNA: PEG-ACS is dissolved in the NaAc/HAc buffer solution (pH 5.5) to a mass fraction of 0.02%. The PEG-ACS solution and the siRNA solution are respectively incubated in a constant temperature water bath at 55° C. for 15 min, then mixed in an equal volume, and quickly mixed in a vortex mixer for 40 s to obtain the PEG-ACS/M-siRNA nanocomposite.

(28) (7) The average particle size of the nanocomposite is measured by a dynamic light scattering method to be 215 nm. The prepared composite is frozen at −20° C. for 5 h and then freeze-dried for 20 h to obtain the lyophilized powder for use.

(29) (8) 4 g of PEG-ACS/M-siRNA nanocomposite per kilogram of fishmeal is added.

(30) (9) High-performance liquid chromatography (HPLC) is used to detect the histamine content in the fishmeal, and a blank control is performed. As shown in Table 2 and FIG. 2, the inhibition of the histamine in the fishmeal storage by PEG-ACS/M-siRNA nanocomposite is about 49%-51%.

(31) TABLE-US-00006 TABLE 2 Histamine content in fishmeal under different treatments (mg/100 g) Storage time/month January February March April May June Control 44.68 48.81 53.5 59.51 67.03 79.42 group Treatment 22.17 24.28 26.64 29.75 33.89 39.53 group Storage time/month July August September October November December Control 97.53 113.82 126.05 134.81 139.64 143.89 group Treatment 48.39 55.89 61.75 65.66 68.93 71.02 group

(32) As shown in Table 2, during the process of storage, the histamine content in the fishmeal increased continuously, and the standard of feed deterioration was achieved in August (the amine content of the secondary fishmeal was ≤1000 mg/kg). The fishmeal added with PEG-ACS/M-siRNA nanocomposite did not reach the deterioration standard even in December, and the experimental results showed that the fishmeal could be effectively extended. Combined with the results, a further comparative analysis showed that the inhibition of histamine in fishmeal by the PEG-ACS/M-siRNA nanocomposite reached 49%-51%.

(33) As shown in FIG. 2, during the process of storage, the histamine content in the fishmeal increased, and the PEG-ACS/M-siRNA nanocomposite significantly reduced the histamine content in the fishmeal in January, February and March and significantly reduced the histamine content in the fishmeal in other months. The PEG-ACS/M-siRNA nanocomposite has obvious inhibitory effects on the histamine synthesis during the process of fishmeal storage.

(34) As shown in FIG. 4, during the process of storage, the histamine content in the fishmeal increased, and the amount of histamine was relatively increased in July and August. The PEG-ACS/M-siRNA nanocomposite has obvious inhibitory effects on the histamine synthesis during the process of fishmeal storage.

Comparative Example 1

(35) Experimental steps using chitosan as a carrier:

(36) (1) Design of M-siRNA sequence.

(37) (2) Preparation of M-siRNA.

(38) (3) Preparation of CS solution: CS is dissolved in NaAc/HAc buffer solution (pH=4.5) to obtain 1 mg/mL of CS solution, and the pH is adjusted to 5.5 with a NaOH solution.

(39) (5) Preparation of siRNA solution: siRNA is dissolved in the DEPC water to prepare the siRNA solution at a concentration of 18-25 LM and stored at −20° C. for use.

(40) (4) Preparation of CS/M-siRNA nanocomposite: the CS solution and the si RNA solution are respectively incubated in a constant temperature water bath at 50-55° C. for 10-15 min, then mixed in an equal volume, and quickly mixed in a vortex mixer for 30-40 s to obtain the CS/M-siRNA nanoparticle.

(41) (5) The particle size of the nanocomposite is measured by a dynamic light scattering method between 170-240 nm. The prepared composite is frozen at −20° C. for 3-6 h and then freeze-dried for 20-26 h to obtain the lyophilized powder for use.

(42) (6) The CS/M-siRNA freeze-dried powder is added to fishmeal at a ratio of 3.5-5 g of nanocomposite per kilogram of fishmeal.

(43) As shown in FIG. 5, when the chitosan is used as a carrier for transmitting siRNA, the expression of histidine decarboxylase can be reduced to some extent, but it has disadvantages such as inefficiency and poor targeting. When the PEGylated arginine-modified chitosan is used as a carrier for transmitting siRNA, the expression level of histidine decarboxylase is significantly decreased. The PEG-ACS/M-siRNA nanocomposite shows a high efficiency and strong targeting.