PLANT ESSENTIAL OIL AMINO ACID COMPOSITION AND PREPARATION METHOD THEREFOR

Abstract

The invention relates to a plant essential oil amino acid composition and a preparation method therefor. The composition of the present invention is a composition containing a plant essential oil and amino acids. The composition is obtained by mixing the plant essential oil with amino acids, the plant essential oil being selectively one or two of thymol and carvacrol. The composition of the present invention has a significantly higher melting point and lower volatility, thus significantly improving stability. When being used as an animal growth-promoting feed additive, the composition of the present invention has better palatability and lower death rate.

Claims

1. A composition comprising a plant essential oil and an amino acid, said composition is obtained by mixing a plant essential oil with an amino acid, said plant essential oil is one or two selected from the group consisting of thymol and carvacrol, said amino acid is one or two selected from the group consisting of L-proline and sarcosine.

2. The composition according to claim 1, wherein the plant essential oil and the amino acid is mixed at a molar ratio of 9-1:1-9.

3. The composition according to claim 1, wherein the plant essential oil is a mixture of thymol and carvacrol, and wherein the molar ratio of thymol to carvacrol is 9-1:1-9.

4. The composition according to claim 1, wherein the composition is obtained by mixing thymol with sarcosine at a molar ratio of 1:2, and wherein the composition has an X-ray powder diffraction pattern showing characteristic peaks at 2 angles of 6.40.2, 12.10.2, 12.40.2, 17.70.2, 20.20.2, 22.10.2, 23.00.2, 23.70.2 and 25.10.2.

5. The composition according to claim 1, wherein the composition is obtained by mixing thymol with L-proline at a molar ratio of 1:1, and wherein the composition has an X-ray powder diffraction pattern showing characteristic peaks at 2 angles of 6.20.2, 6.90.2, 12.50.2, 13.80.2, 18.00.2, 18.40.2, 18.70.2, 19.10.2, 19.30.2, 21.10.2 and 24.80.2.

6. The composition according to claim 1, wherein the composition is obtained by mixing carvacrol with L-proline at molar ratio of 1:1, and wherein the composition has an X-ray powder diffraction pattern showing characteristic peaks at 2 angles of 6.90.2, 11.50.2, 13.60.2, 13.90.2, 19.00.2, 19.20.2, 23.10.2 and 24.90.2.

7. The composition according to claim 1, wherein the composition is obtained by mixing carvacrol with L-proline at a molar ratio of 2:1, and wherein the composition has an X-ray powder diffraction pattern showing characteristic peaks at 2 angles of about 6.50.2, 11.00.2, 13.10.2, 13.20.2, 15.00.2, 15.50.2, 15.80.2, 17.50.2, 19.70.2, 20.50.2, 21.60.2, 21.90.2, 23.60.2, 25.10.2 and 26.70.2.

8. The composition according to claim 1, wherein the composition is obtained by mixing thymol, carvacrol and L-proline at a molar ratio of 1:1:2, and wherein the composition has an X-ray powder diffraction pattern showing characteristic peaks at 2 angles of about 6.20.2, 6.8+0.2, 12.50.2, 13.80.2, 18.40.2, 18.80.2, 19.40.2, 21.30.2, 24.80.2 and 25.00.2.

9. A method for preparing the composition according to claim 1, comprising a step of mixing the plant essential oil with the amino acid to obtain a composition comprising the plant essential oil and the amino acid.

10. The method according to claim 9, wherein the mixing comprises one of the following methods (1) and (2): Method (1): the plant essential oil and the amino acid are recrystallized in a solvent to obtain a precipitate, and the precipitate is dried to obtain a composition comprising the plant essential oil and the amino acid; and Method (2): the plant essential oil and the amino acid are mixed, added to a crushing equipment to make them fully contact by mechanical force, or a solution of the mixture is subjected to spray-drying, or the mixture is treated with an extruder, and after partial or complete reaction, a composition comprising the plant essential oil and the amino acid is formed, wherein the crushing equipment is selected from the group consisting of a ball mill, a pulverizer, a mixer and a stirring device.

11. A product comprising the composition according to claim 1, wherein the product is at least one of a health product, a food, a cosmetic, a medicine, a pharmaceutical excipient, or a feed.

12. The composition according to claim 1, wherein the plant essential oil and the amino acid is mixed at a molar ratio of 2-1:1-2.

13. The composition according to claim 1, wherein the plant essential oil is a mixture of thymol and carvacrol, and wherein the molar ratio of thymol to carvacrol is 2-1:1-2.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIG. 1 is the X-ray powder diffraction (XRPD) pattern of Composition 1;

[0084] FIG. 2 is the differential scanning calorimetry (DSC) pattern of Composition 1;

[0085] FIG. 3 is the X-ray powder diffraction (XRPD) pattern of Composition 2;

[0086] FIG. 4 is the differential scanning calorimetry (DSC) pattern of Composition 2;

[0087] FIG. 5 is the X-ray powder diffraction (XRPD) FIG. of Composition 3;

[0088] FIG. 6 is the differential scanning calorimetry (DSC) pattern of Composition 3;

[0089] FIG. 7 is the X-ray powder diffraction (XRPD) pattern of Composition 4;

[0090] FIG. 8 is the differential scanning calorimetry (DSC) pattern of Composition 4;

[0091] FIG. 9 is the X-ray powder diffraction (XRPD) pattern of Composition 5;

[0092] FIG. 10 is the differential scanning calorimetry (DSC) pattern of Composition 5.

MODE OF THE INVENTION

[0093] The present invention will be further described below in conjunction with specific examples, but the present invention is not limited by the specific examples.

[0094] The experimental methods used in the following examples were conventional methods unless otherwise specified.

[0095] The materials and reagents used in the following examples could be obtained from commercial sources unless otherwise specified.

[0096] Thymol and carvacrol were purchased from Nanjing Jiulonghui Spices Co., Ltd.; sarcosine and L-proline were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0097] The ball mill was purchased from Shanghai Jingxin Industrial Development Co., Ltd.

[0098] The pulverizer was purchased from Xinlongwei Machinery Parts Co., Ltd.

Detection Instrument and Analysis Method:

[0099] For X-ray powder diffraction (XRPD) method, instrument model: Bruker D8 advance, target: Cu Ku (40 kV, 40 mA), the standard sample that comes with the instrument was used to correct the peak position of the instrument before use. The acquisition software is Diffrac Plus XRD Commander, and the analysis software is MDI Jade 6.0. The samples were tested at room temperature, and the samples to be tested were placed on an organic glass slide. Distance from sample to detector: 30 cm, scanning range: 3-40 (20 value), scanning step: 0.02, step length: 0.1 seconds/step.

[0100] For differential scanning calorimetry (DSC) method, instrument model: TA DSC Q2000, temperature range: 50-200 C., scan rate: 10 C./min, nitrogen flow rate: 50 mL/min.

[0101] For thermogravimetric analysis (TGA) method, instrument model: Netzsch TG 209F3 thermogravimetric analyzer detection, temperature: 80 C. for 100 minutes, purge gas: 25 mL/min.

[0102] The industry standard NY/T 3137-2017 of the Ministry of Agriculture was adopted as the content analysis method for thymol and carvacrol.

PREPARATION EXAMPLE

Preparation Example 1: Preparation of Composition 1

[0103] The preparation process of Composition 1 was as follows. 5 mmol of thymol and 10 mmol of sarcosine were added at a molar ratio of 1:2 to 20 mL of methanol. The mixture was stirred at 60 C. for 1 h, and then recrystallized to obtain a white precipitate. The precipitate was filtered and put in a vacuum drying oven to be dried at room temperature to obtain Composition 1 formed by thymol and sarcosine, with a yield of 86%.

[0104] Alternatively, 5 mmol of thymol and 10 mmol of sarcosine were added to a ball mill at a molar ratio of 1:2 and vibrated at a vibrating frequency of 30 Hz for a vibrating time of 20 minutes to obtain Composition 1 formed by thymol and sarcosine, which was a crystalline powder with good fluidity, with a yield of 98%.

[0105] Alternatively, 1 mol of thymol and 2 mol of sarcosine were added to a pulverizer at a molar ratio of 1:2 and pulverized for 2 minutes to obtain Composition 1 formed by thymol and sarcosine, which was a crystalline powder with good fluidity, with a yield of 95%.

[0106] The samples prepared in Preparation Example 1 were characterized by X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) methods. The results of the samples obtained in the above three preparation processes were consistent, as shown in FIGS. 1 and 2. FIG. 1 is the X-ray powder diffraction (XRPD) pattern of Composition 1; and FIG. 2 is the differential scanning calorimetry (DSC) pattern of Composition 1.

Preparation Example 2: Preparation of Composition 2

[0107] The preparation process of Composition 2 was as follows.

[0108] 10 mmol of thymol and 10 mmol of L-proline were added at a molar ratio of 1:1 to 10 mL of ethanol. The mixture was stirred at 40 C. for 1 h, and then recrystallized to obtain a white precipitate. The precipitate was filtered and put in a vacuum drying oven to be dried at room temperature to obtain Composition 2 formed by thymol and L-proline, with a yield of 92%.

[0109] Alternatively, 10 mmol of thymol and 10 mmol of L-proline were added to a ball mill at a molar ratio of 1:1 and vibrated at a vibrating frequency of 30 Hz for a vibrating time of 20 minutes to obtain Composition 2 formed by thymol and L-proline, which was a crystalline powder with good fluidity, with a yield of 98%.

[0110] Alternatively, 1 mol of thymol and 1 mol of L-proline were added to a pulverizer at a molar ratio of 1:1 and pulverized for 2 minutes to obtain Composition 2 formed by thymol and L-proline, which was a crystalline powder with good fluidity, with a yield of 94%.

[0111] The samples prepared in Preparation Example 2 were characterized by X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) methods. The results of the samples obtained in the above three preparation processes were consistent, as shown in FIGS. 3 and 4. FIG. 3 is the X-ray powder diffraction (XRPD) pattern of Composition 2; and FIG. 4 is the differential scanning calorimetry (DSC) pattern of Composition 2.

Preparation Example 3: Preparation of Composition 3

[0112] The preparation process of Composition 3 was as follows.

[0113] 10 mmol of carvacrol and 10 mmol of L-proline were added at a molar ratio of 1:1 to 10 mL of ethanol. The mixture was stirred at 40 C. for 1 h, and then recrystallized to obtain a white precipitate. The precipitate was filtered and put in a vacuum drying oven to be dried at room temperature to obtain Composition 3 formed by carvacrol and L-proline, with a yield of 94%.

[0114] Alternatively, 10 mmol of carvacrol and 10 mmol of L-proline were added to a ball mill at a molar ratio of 1:1 and vibrated at a vibrating frequency of 30 Hz for a vibrating time of 20 minutes to obtain Composition 3 formed by carvacrol and L-proline, which was a crystalline powder with good fluidity, with a yield of 98%.

[0115] Alternatively, 10 mol of carvacrol and 10 mol of L-proline were added to a pulverizer at a molar ratio of 1:1 and pulverized for 2 minutes to obtain Composition 3 formed by carvacrol and L-proline, which was a crystalline powder with good fluidity, with a yield of 94%.

[0116] The samples prepared in Preparation Example 3 were characterized by X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) methods. The results of the samples obtained in the above three preparation processes were consistent, as shown in FIGS. 5 and 6. FIG. 5 is the X-ray powder diffraction (XRPD) pattern of Composition 3; and FIG. 6 is the differential scanning calorimetry (DSC) pattern of Composition 3.

Preparation Example 4: Preparation of Composition 4

[0117] The preparation process of Composition 4 was as follows.

[0118] 10 mmol of carvacrol and 5 mmol of L-proline were added at a molar ratio of 2:1 to 10 mL of ethanol. The mixture was stirred at 40 C. for 1 h, and then recrystallized to obtain a white precipitate. The precipitate was filtered and put in a vacuum drying oven to be dried at room temperature to obtain Composition 4 formed by carvacrol and L-proline, with a yield of 82%.

[0119] Alternatively, 10 mmol of carvacrol and 5 mmol of L-proline were added to a ball mill at a molar ratio of 2:1 and vibrated at a vibrating frequency of 30 Hz for a vibrating time of 20 minutes to obtain Composition 4 formed by carvacrol and L-proline, which was a crystalline powder with good fluidity, with a yield of 98%.

[0120] Alternatively, 10 mol of carvacrol and 5 mol of L-proline were added to a pulverizer at a molar ratio of 2:1 and pulverized for 2 minutes to obtain Composition 4 formed by carvacrol and L-proline, which was a crystalline powder with good fluidity, with a yield of 96%.

[0121] The samples prepared in Preparation Example 4 were characterized by X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) methods. The results of the samples obtained in the above three preparation processes were consistent, as shown in FIGS. 7 and 8. FIG. 7 is the X-ray powder diffraction (XRPD) pattern of Composition 4; and FIG. 8 is the differential scanning calorimetry (DSC) pattern of Composition 4.

Preparation Example 5: Preparation of Composition 5

[0122] The preparation process of composition 5 was as follows.

[0123] 5 mmol of thymol, 5 mmol of carvacrol and 10 mmol of L-proline were added to a ball mill at a molar ratio of 1:1:2 and vibrated at a vibrating frequency of 30 Hz for a vibrating time of 20 minutes to obtain Composition 5, which was a crystalline powder with good fluidity, with a yield of 98%.

[0124] Alternatively, 5 mmol of thymol, 5 mmol of carvacrol and 10 mmol of L-proline were added to a pulverizer at a molar ratio of 1:1:2 and pulverized for 2 minutes to obtain Composition 5, which was a crystalline powder with good fluidity, with a yield of 95%.

[0125] The samples prepared in Preparation Example 5 were characterized by X-ray powder diffraction (XRPD) and differential scanning calorimetry (DSC) methods. The results of the samples obtained in the above two preparation processes were consistent, as shown in FIGS. 9 and 10. FIG. 9 is the X-ray powder diffraction (XRPD) pattern of Composition 5; and FIG. 10 is the differential scanning calorimetry (DSC) pattern of Composition 5.

TEST EXAMPLES

Test Example 1

[0126] The melting points of Composition 1, Composition 2, Composition 3 and Composition 4 were obtained from the DSC patterns, and the results were shown in the table below.

TABLE-US-00001 TABLE 1 Sample Thymol Carvacrol Composition 1 Composition 2 Composition 3 Composition 4 Melting point ( C.) 50 2 129 123 133 80

Test Example 2

[0127] Thymol and/or carvacrol and L-proline and/or sarcosine were added to a ball mill according to the compositions shown in Table 2, and vibrated at a vibrating frequency of 30 Hz for a vibrating time of 20 minutes to obtain the compositions. The compositions were subjected to thermogravimetric analysis (TGA), kept at 80 C. for 100 minutes, and the vapor pressure of thymol and/or carvacrol and the compositions thereof were calculated according to the formula:

[00001]$p={}^{-1}\left(-dm/\mathrm{dt}\right){\left(2\mathrm{RT}/M\right)}^{1/2}$ [0128] wherein m is the mass of the sample at time t, dm/dt is the weight loss rate per unit area, p is the saturated vapor pressure, a is the vaporization constant, T is the absolute temperature, R is the gas constant, and M is the molecule mass of the evaporated substance. Under vacuum condition, =1. can be considered as a constant when a protective gas is present.

[0129] The greater the vapor pressure, the stronger the volatility. It can be seen from Table 2 that the vapor pressure was significantly reduced after formation of the compositions. Therefore, the volatility could be effectively reduced, thereby significantly improving the stability during processing and storage. The compositions of the invention significantly reduced the vapor pressures of thymol and carvacrol, so that the volatilization was reduced and the stability was improved. It can be seen that the compositions of thymol and/or carvacrol and sarcosine and/or proline can significantly reduce the vapor pressures of thymol and/or carvacrol, so that the volatilization was reduced and the stability was improved.

TABLE-US-00002 TABLE 2 Compositions and vapor pressures (interaction at molecular level is present between thymol or carvacrol and the amino acid in the compositions) vapor pressure Composition (molar ratio) (Pa) at 80 C. thymol 333 carvacrol 242 Composition 1 thymol and sarcosine 1:2 35 Composition 2 thymol and L-proline 1:1 43 Composition 3 carvacrol and L-proline 1:1 21 Composition 4 carvacrol and L-proline 2:1 163 Composition 5 thymol-carvacrol-proline1:1:2 30 Composition 6 thymol-carvacrol-sarcosine 1:1:2 36 Composition 7 thymol-proline 2:1 169 Composition 8 thymol-proline 1:2 84 Composition 9 carvacrol-proline 1:2 40 Composition 10 thymol-sarcosine 2:1 263 Composition 11 thymol-sarcosine 1:3 66 Composition 12 carvacrol-sarcosine 1:2 188 Composition 13 thymol-sarcosine 1:9 39 Composition 14 thymol-sarcosine 9:1 81 Composition 15 thymol-proline 1:9 46 Composition 16 thymol-proline 9:1 97 Composition 17 carvacrol-proline 1:9 27 Composition 18 thymol-carvacrol-proline 1:1:18 42

Test Example 3

[0130] This experiment adopted a single factor design, and 4 treatments were set up: Treatment 1: no essential oil was added to the basal diet; Treatment 2: 30 mg/kg of Composition 5 was added to the basal diet; Treatment 3: 60 mg/kg of Composition 5 was added to the basal diet; Treatment 4:120 mg/kg of Composition 5 was added to the basal diet.

[0131] 384 1-day-old meat ducks were divided into 4 treatment groups, each treatment had 6 replicates, and each replicate had 16 ducks; the basal diet was corn soybean meal; the test period was 35 days, which was divided into two stages of 1-14 day-old and 15-35 day-old for feeding.

[0132] As shown in Table 3, compared with the treatment 1 wherein the plant essential oil composition was not added, the death rate was significantly reduced in the treatments 2-3, and the death rate was 0 when the addition amount reached 120 mg/kg. It can be seen that the advantages of using the composition provided by the present invention as animal feed additives are that the Composition provided by the present invention is less volatile, has better palatability and improved stability, and fully exerts antibacterial properties in animals and reduces the death rate while maintaining quality and quantity. It can be shown from the test results in meat ducks that the composition provided by the present invention has the effect of significantly reducing the death rate.

TABLE-US-00003 TABLE 3 Test results in meat ducks Item Treatment 1 Treatment 2 Treatment 3 Treatment 4 Death rate for 8.3% 7.3% 4.2% 0% 1-35 days

Test Example 4

[0133] The accelerated stability advantage of Composition 5 was examined.

[0134] The sources of the tested samples: the sample of Composition 5 prepared in Preparation Example 5 above and the commercially available mixed essential oil additive coating product A (containing 25% thymol and 25% carvacrol).

[0135] Under the condition of 40 C./75% RH, the contents of thymol and carvacrol in Composition 5 were 25.7%/25.5% respectively at day 0, and the contents of thymol and carvacrol in the additive product A were 26.1%/26.2% respectively. After 2 months, the contents of thymol and carvacrol in Composition 5 were 25.6%/25.2%, respectively, and the contents of thymol and carvacrol in the additive product A were 20.6%/20.7%, respectively.

[0136] After 2 months of accelerated testing, the retention rate was 99.2% for Composition 5, while both thymol and carvacrol in the product A were unstable, with a retention rate of 79.0%, indicating that the composition of the present invention had better storage stability.

TABLE-US-00004 TABLE 4 Comparison of storage stability with the commercially available product A Day 0 One month Two months 40 C./75% RH thymol carvacrol thymol carvacrol thymol carvacrol Composition 5 25.7% 25.5% 25.9% 25.1% 25.6% 25.2% Product A 26.1% 26.2% 21.3% 21.3% 20.6% 20.7%

Test Example 5

[0137] The stability advantage of Composition 5 during granulation was examined.

[0138] Sources of the tested samples: the sample of Composition 5 prepared in Preparation Example 5 above and the commercially available mixed essential oil additive product B (containing 8.5% thymol and 8.5% carvacrol).

[0139] Granulation processing was performed referring to the meat duck feed of the Chinese duck feeding standard, and the retention rate of the essential oil was 53% for the product B, while the retention rate was 72.3% for Composition 5. It can be seen that the essential oil in the composition of the present invention had better processing stability.

TABLE-US-00005 TABLE 5 Comparison of retention rate with commercially available product B after granulation Group Composition 5 Product B Theoretical added value 60 34 (thymol + carvacrol, ppm) Measured value in feed 43.4 18.1 (thymol + carvacrol, ppm) Retention rate of 72.3% 53% essential oil in feed

Test Example 6

[0140] This experiment adopted a single factor design, and 4 treatments were set up: Treatment 1: no essential oil was added to the basal diet; Treatment 2: 30 mg/kg of Composition 5 was added to the basal diet; Treatment 3: 60 mg/kg of Composition 5 was added to the basal diet; Treatment 4:120 mg/kg of Composition 5 was added to the basal diet.

[0141] 640 1-day-old broilers were divided into 4 treatment groups, each treatment had 8 replicates, and each replicate had 20 broilers; the basal diet was corn soybean meal; the test period was 42 days, which was divided into two stages of 1-21 day-old and 22-42 day-old for feeding.

[0142] As shown in Table 6, compared with the treatment 1 wherein the plant essential oil composition was not added, the body weights were significantly increased in the treatments 2-4, and the body weight was increased by 6% when the addition amount was 30 mg/kg. It can be seen that the advantages of using the composition provided by the present invention as an animal feed additive are that the composition provided by the present invention is less volatile, has better palatability and improved stability, and fully exerts antibacterial properties in animals and reduces the death rate while maintaining quality and quantity. It can be shown from the test results in broilers that it has the effect of significantly increasing the body weight.

TABLE-US-00006 TABLE 6 test results in broilers Item Treatment 1 Treatment 2 Treatment 3 Treatment 4 SEM P Average weight on day 1 (g) 43.05 43.15 43.08 42.98 0.09 0.321 Average weight on day 42 (g) 2704.66.sup.b 2866.82.sup.a 2809.22.sup.a 2856.01.sup.a 49.56 0.026 Average daily weight gain for 1-42 days (g) 63.37.sup.b 67.23.sup.a 65.86.sup.a 66.98.sup.a 1.18 0.026 Average feed intake (g) 116.52 120.67 119.37 119.16 2.20 0.354 Feed conversion ratio for 1-42 days 1.84 1.80 1.81 1.78 0.02 0.029 For the data in the same row, different lowercase letters on the shoulders indicate significant differences (p < 0.05), and the same or no letters indicate no significant differences (p > 0.05).

Test Example 7

[0143] This experiment adopted a single factor design, and 4 treatments were set up: Treatment 1: no essential oil was added to the basal diet; Treatment 2: 30 mg/kg composition 5 was added to the basal diet; Treatment 3: 60 mg/kg Composition 5 was added to the basal diet; Treatment 4:120 mg/kg composition 5 was added to the basal diet.

[0144] For a 28-day feeding experiment, 96 weaned piglets were divided into 4 treatment groups, each treatment had 4 replicates, and each replicate had 6 piglets; the body weight, feed intake, and daily diarrhea situation were recorded for each replicate at day 1 and 28 respectively, and feed conversion ratio and diarrhea rate were calculated.

[0145] As shown in Table 7, compared with Treatment 1 wherein the plant essential oil composition was not added, when the test was ended on day 28, in the treatment group 3 wherein the addition amount was 60 mg/kg, the body weight was significantly increased, the feed conversion ratio was significantly decreased, and the body weight was increased by 15%, and the feed conversion ratio was reduced by 22%.

TABLE-US-00007 TABLE 7 Experimental results in weaned piglets Item Treatment 1 Treatment 2 Treatment 3 Treatment 4 SEM P Body weight/kg Day 1 7.90 7.88 7.88 7.93 0.2 0.998 Day 28 13.27.sup.b 14.40.sup.ab 15.63.sup.a 13.85.sup.ab 0.67 0.118 Average daily feed intake ADFI/(g/d) Days 1-28 391 413 428 361 0.04 0.647 Average daily weight gain ADG/(g/d) Days 1-28 179 213 263 198 35 0.399 Feed conversion ratio F/G Days 1-28 2.19.sup.a 2.01.sup.ab 1.71.sup.b 1.85.sup.ab 0.14 0.14 For the data in the same row, different lowercase letters on the shoulders indicate significant differences (p < 0.05), and the same or no letters indicate no significant differences (p > 0.05).

[0146] The above description are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto, any changes or substitutions that can be conceived by those skilled in the art within the technical scope disclosed in the present invention without creative efforts shall be covered by the protection scope of the present invention.