Chitosan composition and method of manufacturing the same

Abstract

A method of manufacturing a chitosan composition comprising steps performed in the following specific order: (i) preparing materials; (ii) creating a chitin ingredient from shrimp shells; (iii) creating a chitin ingredient from oyster mushroom; (iv) creating a chitin mixture by homogenously mixing the chitin ingredient from shrimp shells with the chitin ingredient from oyster mushroom; (v) loading the chitin mixture into the centrifugal spray drying device having predetermined specifications to create the chitosan composition; and (vi) packaging and preservation.

Claims

1. A method of manufacturing a chitosan composition comprising steps performed in the following specific order: (i) preparing materials including: a microorganism solution, a fruit vinegar, an enzyme solution, and a rice alcohol has alcohol range 30%-50%; in which, prepare the microorganism solution by performing steps (A) to (E): (A) creating a first suspension solution comprising performing in a specific order from (a1) to (c1): (a1) activating Bacillus sp. TV11 on the Nutrient agar medium, incubating at 37 C. for a period of 36-48 hours to obtain an activated Bacillus sp. TV11; (b1) inoculating a single colony of the activated Bacillus sp. TV11 into a test tube containing 10 ml of Nutrient broth medium, shaking at 200 rpm for 24 hours at 37 C. to obtain an increased first biomass solution; and (c1) inoculating the increased first biomass solution into Nutrient broth medium at a ratio of (1-3): 100, shaking at 200 rpm for 24 hours at 37 C. to obtain the first suspension solution; (B) creating a second suspension solution comprising performing in a specific order from (a2) to (c2): (a2) activating Lactobacillus sp. T432 on the Man Rogosa Sharpe (MRS) agar medium, incubating at 37 C. for a period of 36-48 hours to obtain an activated Lactobacillus sp. T432; (b2) inoculating a single colony of the activated Lactobacillus sp. T432 into a test tube containing 10 ml of MRS broth medium, shaking at 200 rpm for 24 hours at 37 C. to obtain an increased second biomass solution; and (c2) inoculating the increased second biomass solution into MRS broth medium at a ratio of (1-3): 100, shaking at 200 rpm for 24 hours at 37 C. to obtain the second suspension solution; (C) creating a third suspension solution comprising performing in a specific order from (a3) to (c3): (a3) activating Lactobacillus plantarum VTCC 431 on the MRS agar medium, incubating at 37 C. for a period of 36-48 hours to obtain an activated Lactobacillus plantarum VTCC 431; (b3) inoculating a single colony of the activated Lactobacillus plantarum VTCC 431 into a test tube containing 10 ml of MRS broth medium, shaking at 200 rpm for 24 hours at 37 C. to obtain an increased third biomass solution; and (c3) inoculating the increased third biomass solution into MRS broth medium at a ratio of (1-3): 100, shaking at 200 rpm for 24 hours at 37 C. to obtain the third suspension solution; (D) creating a fourth suspension solution comprising performing in a specific order from (a4) to (c4): (a4) activating Lactobacillus bulgaricus VTCC 703 on the MRS agar medium, incubating at 37 C. for a period of 36-48 hours to obtain an activated Lactobacillus bulgaricus VTCC 703; (b4) inoculating a single colony of the activated Lactobacillus bulgaricus VTCC 703 into a test tube containing 10 ml of MRS broth medium, shaking at 200 rpm for 24 hours at 37 C. to obtain an increased fourth biomass solution; and (c4) inoculating the increased fourth biomass solution into MRS broth medium at a ratio of (1-3): 100, shaking at 200 rpm for 24 hours at 37 C. to obtain the fourth suspension solution; (E) mixing (1-2) parts of the first suspension solution with (1-2) parts of the second suspension solution, (1-2) parts of the third suspension solution, and (1-2) parts of the fourth suspension solution to obtain the microorganism solution; in which, prepare the fruit vinegar by performing steps from (A) to (G) including: (A) selecting and preparing fruits by a predetermined quality guideline, includes selecting said fruits that have a Brix level of at highest 2; in which fruits are selected from the group consisting of ambarella (Spondias dulcis), plum (Prunus salicina), apricot (Prunus armeniaca (L.)), crocodile fruit (Dracontomelon duperreanum), star gooseberry (Phyllanthus acidus (L.)), and a combination thereof; (B) performing visual inspection to select undamaged fruits; (C) washing fruits, then cold-squeezing fruits to obtain a fruit juice; (D) creating a foundation solution by mixing the fruit juice and the coconut water in ratio of 1:3; (E) pasteurizing the foundation solution at 85 C. for 4 minutes, then let it cool, and adjusting the Brix to 6 to obtain a pasteurized solution; (F) adding ethanol 95% at a ratio of 3% (v/v) to the pasteurized solution, adjusting the pH to 5 by acetic acid 1.5%, then adding Acetobacter aceti bacteria in ratio of 1.2% (v/v), and fermenting for 7 days to obtain a solution after fermentation; and (G) filtering the solution after fermentation to obtain the fruit vinegar; in which, prepare the enzyme solution by performing steps from (A) to (B) including: (A) creating an enzyme preparation by homogeneously mixing (1-3) parts of a protease ingredient with 1 part of a lipase ingredient; and (B) creating the enzyme solution by homogeneously mixing (3-5) parts of the enzyme preparation with (5000-10000) parts of the water; in which, prepare the rice alcohol has alcohol range 30%-50% by fermenting a homogeneous mixture twice, then distilling and aging the alcohol using an alcohol aged machine or alcohol aging equipment; wherein the homogeneous mixture includes 600 parts of a cooked rice mixture, and (1-2) parts of a wine yeast ingredient; the cooked rice mixture is cooked from 3 parts glutinous rice with 1 part plain rice, 0.2 part shelled green beans, and 4.5 parts water; wherein the wine yeast ingredient includes 11 parts of a rice flour, (0.6-0.8) parts of an extracted herbal, and (0.001-0.1) parts of a yeast ingredient; the extracted herbal is extracted from a herbal mixture crushed/chopped, and soaked in solvent, or saturated brine solution; the herbal mixture comprising: Myristica fragrans Houtt., Curcuma aromatica Salisb., Atractylodes macrocephala Koidz., Mentha arvensis L., Amomum aromaticum Roxb., Glycyrrhiza uralensis Fisch., Foeniculum vulgare Mill., Illicium verum Hook. f., and Cortex Cinnamomi cassiae; the yeast ingredient is listed in Table 3; (ii) creating a chitin ingredient from shrimp shells comprising performing in a specific order from (a) to (c): (a) collecting a molting shell of shrimp, then washing to remove impurities, and soaking with HCl solution in a ratio of 1: (3-5) (w/v) for 15-20 days, then filtering to remove liquid, washing twice with the rice alcohol has alcohol range 30%-50 to obtain a first temporary mixture; (b) treating the first temporary mixture to obtain a basic solution including: dissolving a quicklime (CaO) ingredient in solution concentrated HCl contains 40% (concentrated grade) with combined stirring at 50 rpm for 5 minutes to obtain a solution 1; admixing the first temporary mixture to the solution 1 with combined stirring at 50 rpm for 5 minutes, then stop stirring and let stand for 7-10 days at 28 C.-40 C. to obtain the basic solution; wherein a ratio of the first temporary mixture and the quicklime (CaO) ingredient is 1: (2-5) (w/w); wherein a ratio of the quicklime (CaO) ingredient and the solution concentrated HCl contains 40% (concentrated grade) is 1: (2-5) (w/v); (c) admixing 1 part of the enzyme solution at step (i) into 10 parts of the basic solution, then stop stirring and let stand for 12-18 hours to obtain the chitin ingredient from shrimp shells; (iii) creating a chitin ingredient from oyster mushroom comprising performing in a specific order from (a) to (d): (a) collecting oyster mushrooms, then washing to remove impurities, and soaking with the fruit vinegar at step (i) in a ratio of 1: (3-5) (w/v) for 15-20 days, then filtering to remove liquid, and washing twice with the rice alcohol has alcohol range 30%-50% to obtain a second temporary mixture; wherein the fruit vinegar has a concentration of 35%-55%; (b) treating the second temporary mixture to obtain a basic temporary solution including: dissolving the quicklime (CaO) ingredient in solution concentrated HCl contains 40% (concentrated grade) with combined stirring at 50 rpm for 5 minutes to obtain the solution 1; admixing the second temporary mixture to the solution 1 with combined stirring at 50 rpm for 5 minutes, then stop stirring and let stand for 7-10 days at 28 C.-40 C. to obtain the basic temporary solution; wherein a ratio of the second temporary mixture and the quicklime (CaO) ingredient is 1: (3-5) (w/w); wherein the ratio of the quicklime (CaO) ingredient and the solution concentrated HCl contains 40% (concentrated grade) is 1: (2-5) (w/v); (c) admixing the microorganism solution at step (i) into the basic temporary solution at a ratio of (1-2): 10 (w/v) to obtain a foundation temporary solution; and (d) adjusting pH of the foundation temporary solution reached 6.8-7.2, then fermenting at 30 C.-40 C. for 125-135 hours to obtain the chitin temporary mixture from oyster mushroom; (iv) creating a chitin mixture by homogenously mixing (1-10) parts of the chitin ingredient from shrimp shells at step (ii) with 1 part of the chitin ingredient from oyster mushroom at step (iii); (v) loading the chitin mixture into the centrifugal spray drying device having predetermined specifications to create the chitosan composition; in which predetermined specifications including: a rotate speed of 1200 rpm for 3-5 hours at temperature 20 C.-28 C.; and (vi) packaging and preservation.

2. The method of claim 1, wherein characterized in that the washing temperature of the distilled water is 55 C.-60 C.

3. The method of claim 1, wherein the molting shell of shrimp is a molting shell of shrimp from 60-180 days old shrimp.

4. The method of claim 3, wherein the molting shell of shrimp is the molting shell of shrimp from 61-90 days old shrimp.

5. The method of claim 4, wherein the molting shell of shrimp is the molting shell of shrimp from 71-80 days old shrimp.

6. The method of claim 1, wherein the molting shell of shrimp is selected from the group consisting of litopenaeus vannamei (Penaeus vannamei), Penaeus monodon, Penaeus Merguiensis, Macrobrachium rosenbergii, Metapenaeus ensis, Macrobrachium lanchesteri, Fenneropenaeus Merguiensis, Penaeus Semisulcatus, and a combination thereof.

7. The method of claim 1, wherein the rice alcohol has alcohol range 45%-50%.

8. The method of claim 1, wherein oyster mushrooms are selected from the group consisting of Pleurotus pulmonarius, Pleurotus cf. floridanus, Pleurotus ostreatus, Pleurotus citrinopileutus, and a combination thereof.

9. The method of claim 1, wherein the wine yeast ingredient includes 11 parts of the rice flour, (0.6-0.8) parts of the extracted herbal, and (0.01-0.1) parts of the yeast ingredient.

10. The method of claim 9, wherein the wine yeast ingredient includes 11 parts of the rice flour, 0.7 parts of the extracted herbal, and (0.01-0.1) parts of the yeast ingredient.

11. The method of claim 1, wherein at step (ii) the ratio of the first temporary mixture and the quicklime (CaO) ingredient is 1:3 (w/w).

12. The method of claim 1, wherein at step (iii) the ratio of the second temporary mixture and the quicklime (CaO) ingredient is 1:4 (w/w).

13. The method of claim 1, wherein at step (iv) mixing (1-7) parts of the chitin ingredient from shrimp shells with 1 part of the chitin ingredient from oyster mushroom obtain to the chitin mixture.

14. The method of claim 13, wherein at step (iv) mixing (1-5) parts of the chitin ingredient from shrimp shells with 1 part of the chitin ingredient from oyster mushroom obtain to the chitin mixture.

15. The method of claim 14, wherein at step (iv) mixing (1-3) parts of the chitin ingredient from shrimp shells with 1 part of the chitin ingredient from oyster mushroom obtain to the chitin mixture.

16. The method of claim 1, wherein at step (v) time is 5 hours.

17. The method of claim 1, wherein the method further comprising a step drying the chitosan composition at 70 C. for 5-10 minutes before the step (vi) packaging and preservation.

18. The method of claim 1, wherein at step (v) temperature is 25 C.

19. The method of claim 1, wherein at step (v) the chitosan composition has a pH of 7-8.5.

20. The method of claim 19, wherein at step (v) the chitosan composition has a pH of 8-8.5.

Description

BRIEF DESCRIPTION OF THE DRAWING

(1) The accompanying drawings, which are incorporated in and form a part of this specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

(2) FIG. 1 is a flowchart illustrating a method of manufacturing the chitosan composition according to an embodiment of the present invention;

(3) FIG. 2 is a flowchart illustrating a process for preparing the microorganism solution according to an embodiment of the present invention; and

(4) FIG. 3 is a flowchart illustrating a process for preparing the fruit vinegar according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

(5) Reference will now be made in detail to the preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the preferred embodiments, it will be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to cover alternatives, modifications, and equivalents, which may be included within the spirit and scope of the invention as defined by the appended claims.

(6) Furthermore, in the following detailed description of the present invention, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be obvious to one of ordinary skill in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, components, and circuits have not been described in detail so as not to unnecessarily obscure aspects of the present invention.

(7) It should also be noted that the term dissolve/dissolving/dissolved is used in the invention understood to mean the uniform distribution, or complete dissolution of, substances present in a solution/mixture.

(8) One embodiment of the invention is now described with reference to FIG. 1. FIG. 1 illustrating a specific process of manufacturing the chitosan composition 100 (method 100) based on the above principle in accordance with an exemplary embodiment of the present invention. In particular, method 100 includes the following steps:

(9) At step 110, prepare materials including: a microorganism solution, a fruit vinegar, an enzyme solution, and a rice alcohol that has alcohol range 30%-50%.

(10) According to the embodiment of the invention, the microorganism solution obtained from a process 200 will be described in detail later below. The fruit vinegar obtained from a process 300 will be described in detail later below.

(11) According to the embodiment of the invention, prepare the enzyme solution by performing steps from (A) to (B) including: (A) creating an enzyme preparation by homogeneously mixing (1-3) parts of a protease ingredient with 1 part of a lipase ingredient; and (B) creating the enzyme solution by homogeneously mixing (3-5) parts of the enzyme preparation with (5000-10000) parts of the water.

(12) According to the embodiment of the invention, prepare the rice alcohol has alcohol range 30%-50% by fermenting a homogeneous mixture twice, then distilling and aging the alcohol using an alcohol aged machine or alcohol aging equipment.

(13) According to the preferred embodiment of the present invention, the rice alcohol has alcohol range 45%-50%.

(14) According to the embodiment of the invention, the homogeneous mixture includes 600 parts of a cooked rice mixture, and (1-2) parts of a wine yeast ingredient.

(15) According to the preferred embodiment of the invention, the cooked rice mixture is cooked from 3 parts glutinous rice with 1 part plain rice, 0.2 part shelled green beans, and 4.5 parts water.

(16) According to the embodiment of the invention, the wine yeast ingredient includes 11 parts of a rice flour, (0.6-0.8) parts of an extracted herbal, and (0.001-0.1) parts of a yeast ingredient.

(17) According to the embodiment of the invention, the wine yeast ingredient includes 11 parts of the rice flour, (0.6-0.8) parts of the extracted herbal, and (0.01-0.1) parts of the yeast ingredient.

(18) According to the preferred embodiment of the invention, the wine yeast ingredient includes 11 parts of the rice flour, 0.7 parts of the extracted herbal, and (0.01-0.1) parts of the yeast ingredient.

(19) According to the embodiment of the invention, the extracted herbal is extracted from a herbal mixture crushed/chopped, and soaked in solvent, or saturated brine solution. The herbal mixture is listed in Table 2 below, including: Myristica fragrans Houtt., Curcuma aromatica Salisb., Atractylodes macrocephala Koidz., Mentha arvensis L., Amomum aromaticum Roxb., Glycyrrhiza uralensis Fisch., Foeniculum vulgare Mill., Illicium verum Hook. f., and Cortex Cinnamomi cassiae.

(20) TABLE-US-00002 TABLE 2 Components of the herbal mixture are used in the embodimentof the present invention No. Species Collection region Used parts 1 Myristica fragrans Houtt. Vietnam seeds 2 Curcuma aromatica Salisb. rhizomes 3 Atractylodes macrocephala Koidz. rhizomes 4 Mentha arvensis L. leaves 5 Amomum aromaticum Roxb. fruits 6 Glycyrrhiza uralensis Fisch. rhizomes 7 Foeniculum vulgare Mill. fruits 8 Illicium verum Hook. f. fruits 9 Cortex Cinnamomi cassiae bark

(21) According to the embodiment of the invention, the yeast ingredient is selected from the group consisting of Saccharomyces cerevisiae NH2, Saccharomyces cerevisiae NT3, Saccharomyces cerevisiae MS42, Saccharomyces cerevisiae CM3.2, Saccharomyces cerevisiae D8, Saccharomyces cerevisiae NM11, and Candida tropicalis NM2; all are listed in Table 3 below.

(22) TABLE-US-00003 TABLE 3 The strains of yeasts present in the yeast ingredient according to the embodiment of the invention No. Type species Storage location Reference 1 Saccharomyces Institute of Bnh, L. N., Thnh, N. V., Th o, H. P., cerevisiae NH2 Biotechnology & Khnh, T. V. (2015). Isolating and 2 Saccharomyces Research and screening strongly active yeast cerevisiae NT3 Development, Can strains from local alcoholic Tho University fermentation starters. CTU Journal of Science and Technology, (39), 18-28. 3 Saccharomyces Institute of Nguy n V n Quyn et al (2018). cerevisiae MS42 Biotechnology and The influence of some factors on the Food Industry, Hanoi production of alcohol whisky by University of Science Saccharomyces cerevisiae MS42 and Technology from barley malt. Journal of Biotechnology 16(3), 525-532. 4 Saccharomyces Institute of on Th Ki u Tin el al (2018). cerevisiae CM3.2 Biotechnology Evaluation of total polyphenol and Research and antioxidant capacity in wine Development, Can fermentation of three-leaf cayratia Tho University from Ca Mau province using Saccharomyces cerevisiae CM3.2. CTU Journal of Science and Technology, 55 (2), 285-291. 5 Saccharomyces Institute of Le Thuong, H. T., Thuy, T. T., & Hao, cerevisiae D8 Biotechnology N. Q. (2018). Improvement of the Research and ethanol production of saccharomyces Development, Tan cerevisiae D8 by the random Trao University mutagenesis. Vietnam Journal of Biotechnology, 16(2), 337-344. 6 Saccharomyces Institute of Th Bch Th y et al (2023). Effect cerevisiae NM11 Biotechnology of some factors on the content of the Research and Compounds in watermelon juice Development, Hue fermented by Saccharomyces University cerevisiae NM11. Journal of Agricultural Science and Technology. 7 Candida tropicalis Institute of Doan Van Thuoc et al (2015). NM2 Biotechnology Characterization of alcohol producing Research and yeast isolated from fermented fruit Development, Hanoi juice of sonneratia caseolaris. National University of Vietnam Journal of Biotechnology, Education 37(1), 69-75.

(23) The term homogeneous/homogeneously is understood to mean the uniform distribution, or complete dissolution of, substances present in a solution/mixture.

(24) At step 120, creating a chitin ingredient from shrimp shells comprising performing in a specific order from (a) to (c): (a) collecting a molting shell of shrimp, then washing to remove impurities, and soaking with HCl solution in a ratio of 1: (3-5) (w/v) for 15-20 days, then filtering to remove liquid, washing twice with the rice alcohol has alcohol range 30%-50 to obtain a first temporary mixture; in which HCl solution obtained by diluting a solution concentrated HCl contains 40% (concentrated grade) with water to 5%-18% HCl solution; (b) treating the first temporary mixture to obtain a basic solution including: dissolving a quicklime (CaO) ingredient in solution concentrated HCl contains 40% (concentrated grade) with combined stirring at 50 rpm for 5 minutes to obtain a solution 1; admixing the first temporary mixture to the solution 1 with combined stirring at 50 rpm for 5 minutes, then stop stirring and let stand for 7-10 days at 28 C.-40 C. to obtain the basic solution; wherein a ratio of the first temporary mixture and the quicklime (CaO) ingredient is 1: (2-5) (w/w); wherein a ratio of the quicklime (CaO) ingredient and solution concentrated HCl contains 40% (concentrated grade) is 1: (2-5) (w/v); (c) admixing 1 part of the enzyme solution at step 110 into 10 parts of the basic solution, then stop stirring and let stand for 12-18 hours to obtain the chitin ingredient from shrimp shells.

(25) According to the priority embodiment of the invention, at step (b) the ratio of the first temporary mixture and the quicklime (CaO) ingredient is 1:3 (w/w).

(26) According to the embodiment of the invention, the molting shell of shrimp is selected from the group consisting of litopenaeus vannamei (Penaeus vannamei), Penaeus monodon, Penaeus merguiensis, Macrobrachium rosenbergii, Metapenaeus ensis, Macrobrachium lanchesteri, Fenneropenaeus merguiensis, Penaeus semisulcatus, and a combination thereof; all are listed in Table 4 below.

(27) TABLE-US-00004 TABLE 4 Ingredients of shrimp species present in the shrimp shells according to the embodiment of the invention Time to raise fully No. Species Collection region mature shrimp 1 litopenaeus Mekong Delta region: Soc Trang, Bac Lieu, 180 days vannamei Ca Mau, Kien Giang, Tra Vinh provinces; (Penaeus Central region: Binh Dinh, Phu Yen, Khanh vannamei) Hoa, Ninh Thuan, Binh Thuan provinces; Southeast region: Ba Ria-Vung Tau, Tien Giang, Long An provinces; Northern region: Quang Ninh, Thai Binh, Nam Dinh provinces. 2 Penaeus Mekong Delta region: Ca Mau, Bac Lieu, 4 months monodon Kien Giang, Tra Vinh, Soc Trang, An Giang, Tien Giang provinces; Central Coast region: Binh Dinh, Phu Yen, Ninh Thuan, Binh Thuan provinces; North Central region: Thanh Hoa, Nghe An, Ha Tinh provinces; Southeast region: Ba Ria-Vung Tau, Dong Nai provinces; Northern region: Thai Binh, Nam Dinh, Quang Ninh provinces. 3 Penaeus Mekong Delta region: Ca Mau, Bac Lieu, 3-4 months Merguiensis Tra Vinh, Soc Trang, An Giang, Kien Giang provinces; Central Coast region: Binh Dinh, Phu Yen, Khanh Hoa, Ninh Thuan, Binh Thuan provinces; Southeast region: Ba Ria-Vung Tau, Tien Giang, Long An provinces. 4 Macrobrachium Ca Mau, Kien Giang, Can Tho, Long An, 100-135 days rosenbergii Dong Nai, Ben Tre, Soc Trang, An Giang provinces. 5 Metapenaeus Mekong Delta region: Ca Mau, Bac Lieu, 3-4 months ensis Tra Vinh, Soc Trang, An Giang, Kien Giang provinces; Central Coast region: Binh Dinh, Phu Yen, Khanh Hoa, Ninh Thuan, Binh Thuan provinces; Southeast region: Ba Ria-Vung Tau, Tien Giang, Long An provinces. 6 Macrobrachium North: 3-4 months lanchesteri Red River Basin (Hanoi, Hai Phong, Hai Duong provinces); Thai Binh river basin (Thai Binh, Nam Dinh provinces); Cau River basin (Bac Giang, Bac Ninh provinces); Central region: Ma River basin (Thanh Hoa province); Gianh river basin (Quang Binh province); Vu Gia-Thu Bon river basin (Quang Nam, Da Nang province); Southern: Dong Nai River Basin (Dong Nai province, Ho Chi Minh City); Tien and Hau river basins (Mekong Delta); Coastal lagoon area (Ca Mau, Kien Giang province). 7 Fenneropenaeus Mekong Delta region: Ca Mau, Bac Lieu, 3-4 months Merguiensis Tra Vinh, Soc Trang, An Giang, Kien Giang provinces; Central Coast region: Binh Dinh, Phu Yen, Khanh Hoa, Ninh Thuan, Binh Thuan provinces; Southeast region: Ba Ria-Vung Tau, Tien Giang, Long An provinces. 8 Penaeus Mekong Delta region: Ca Mau, Bac Lieu, 3-4 months Semisulcatus Tra Vinh, Soc Trang, An Giang, Kien Giang provinces; Central Coast region: Binh Dinh, Phu Yen, Khanh Hoa, Ninh Thuan, Binh Thuan provinces; Southeast region: Ba Ria-Vung Tau, Tien Giang, Long An provinces.

(28) According to the embodiment of the invention, the molting shell of shrimp is a molting shell of shrimp from 60-180 days old shrimp.

(29) According to the embodiment of the invention, the molting shell of shrimp is a molting shell of shrimp from 61-90 days old shrimp.

(30) According to the embodiment of the invention, the molting shell of shrimp is a molting shell of shrimp from 71-80 days old shrimp.

(31) Refer to the survey results of chitin, mineral and protein content analyzed from molting shrimp shells at different age stages (01-06), all listed in Table 5 below.

(32) TABLE-US-00005 TABLE 5 Comparison of chitin, mineral and protein content analyzed from molting shrimp shells at different age stages (O1-O6) according to the invention's embodiment Species Stages Age (days) Minerals (%) Protein (%) Chitin (%) litopenaeus vannamei O1 31-60 54.95 2.6 12.45 1.1 20.1 0.8 (Penaeus vannamei) O2 61-70 60.8 1.9 10.4 0.7 23.4 0.5 O3 71-80 53.5 0.5 12.6 0.6 23.7 0.2 O4 81-90 55.1 0.8 12.5 0.8 23.5 0.7 O5 >90 55.8 1.9 12.7 0.7 23.6 0.5 Penaeus monodon O1 31-60 53.66 2.2 11.75 1.3 19.5 0.6 O2 61-70 62.8 1.4 9.84 0.5 22.7 0.8 O3 71-80 52.2 0.7 11.6 0.6 22.5 0.3 O4 81-90 54.5 0.6 11.8 0.4 22.1 0.2 O5 >90 54.7 1.3 11.95 0.7 22.3 0.6 Penaeus Merguiensis O1 31-60 55.45 1.7 12.55 1.2 20.8 0.5 O2 61-70 61.9 1.1 10.3 0.9 24.1 0.3 O3 71-80 52.7 0.6 13.2 0.4 24.9 0.2 O4 81-90 56.2 0.1 13.4 0.5 24.6 0.3 O5 >90 56.5 1.5 13.6 0.2 24.5 0.7 Macrobrachium O1 31-60 54.12 1.9 11.97 1.5 20.5 0.2 rosenbergii O2 61-70 63.4 1.8 9.76 0.8 23.5 0.4 O3 71-80 53.5 0.6 11.8 0.5 23.8 0.7 O4 81-90 54.8 0.7 11.7 0.8 23.4 0.4 O5 >90 54.9 1.1 11.9 0.3 23.3 0.8 Metapenaeus ensis O1 31-60 56.1 1.2 13.11 1.3 21.6 0.7 O2 61-70 61.3 1.4 11.3 0.4 23.1 0.4 O3 71-80 53.9 0.5 13.8 0.7 23.7 0.9 O4 81-90 55.7 0.7 13.5 0.3 23.4 0.7 O5 >90 55.9 1.4 13.7 0.6 23.3 0.3 Macrobrachium O1 31-60 55.84 1.6 12.45 1.6 21.2 0.7 lanchesteri O2 61-70 64.5 1.2 10.63 0.7 24.2 0.3 O3 71-80 54.1 0.4 12.6 0.8 24.9 0.4 O4 81-90 55.2 0.5 12.4 0.6 24.6 0.7 O5 >90 54.4 1.3 12.7 0.5 24.7 0.5 Fenneropenaeus O1 31-60 54.77 2.0 12.25 1.3 20.3 0.7 Merguiensis O2 61-70 60.5 1.5 10.1 0.5 23.2 0.7 O3 71-80 53.8 0.4 12.9 0.3 23.8 0.6 O4 81-90 55.3 0.9 12.4 0.6 23.4 0.6 O5 >90 55.7 1.7 12.6 0.8 23.5 0.4 Penaeus Semisulcatus O1 31-60 55.5 1.8 12.65 1.6 20.8 0.5 O2 61-70 61.7 1.1 10.5 0.6 22.6 0.2 O3 71-80 54.4 0.3 12.7 0.8 22.9 0.8 O4 81-90 56.2 0.8 12.2 0.5 22.5 0.6 O5 >90 56.5 1.4 12.4 0.7 22.7 0.4

(33) At step 130, creating a chitin ingredient from oyster mushroom comprising performing in a specific order from (a) to (d): (a) collecting oyster mushrooms, then washing to remove impurities, and soaking with the fruit vinegar at step 110 in a ratio of 1: (3-5) (w/v) for 15-20 days, then filtering to remove liquid, and washing twice with the rice alcohol has alcohol range 30%-50% to obtain a second temporary mixture; wherein the fruit vinegar has a concentration of 35%-55%; (b) treating the second temporary mixture to obtain a basic temporary solution including: dissolving the quicklime (CaO) ingredient in solution concentrated HCl contains 40% (concentrated grade) with combined stirring at 50 rpm for 5 minutes to obtain the solution 1; admixing the second temporary mixture to the solution 1 with combined stirring at 50 rpm for 5 minutes, then stop stirring and let stand for 7-10 days at 28 C.-40 C. to obtain the basic temporary solution; wherein a ratio of the second temporary mixture and the quicklime (CaO) ingredient is 1: (3-5) (w/w); wherein a ratio of the quicklime (CaO) ingredient and solution concentrated HCl contains 40% (concentrated grade) is 1: (2-5) (w/v); (c) admixing the microorganism solution at step 110 into the basic temporary solution at a ratio of (1-2): 10 (w/v) to obtain a foundation temporary solution; and (d) adjusting pH of the foundation temporary solution reached 6.8-7.2, then fermenting at 30 C.-40 C. for 125-135 hours to obtain the chitin temporary mixture from oyster mushroom.

(34) According to the priority embodiment of the invention, at step (b) the ratio of the second temporary mixture and the quicklime (CaO) ingredient is 1:4 (w/w).

(35) According to another embodiment of the invention, replacing the quicklime (CaO) ingredient with a solution lime for betel chewing. The solution lime for betel chewing is obtained by dissolving 1 part of a powdered lime for betel chewing with 0.5-5 parts of the water; wherein the powdered lime for betel chewing is obtained by calcining (burning) shells, coral, or limestone. The powdered lime for betel chewing comprising chemical components: calcium hydroxide Ca(OH).sub.2, and calcium carbonate CaCO.sub.3.

(36) According to the embodiment of the invention, oyster mushrooms listed in Table 6 below, including: Pleurotus pulmonarius, Pleurotus cf. floridanus, Pleurotus ostreatus, Pleurotus citrinopileutus, and a combination thereof.

(37) TABLE-US-00006 TABLE 6 Ingredients of species present in the oyster mushrooms according to the embodiment of the invention No. Species References 1 Pleurotus Pham Van Loc et al (2023). Identification of pulmonarius Oyster Mushroom (Pleurotus spp.) Strains in 2 Pleurotus cf. the South Vietnam based on Morphological floridanus Characteristics and ITS Sequencing. Vietnam J. 3 Pleurotus Agri. Sci, 21(12): 1569-1580 ostreatus 4 Pleurotus Nguyen Thi Thom et al (2018). Study of citrinopileatus cultivating golden oyster mushroom Pleurotus citrinopileatus utilization of agricultural waste. Journal of Science. University of Education, Hue University, 01(45): 138-148

(38) At step 140, creating a chitin mixture by homogenously mixing the chitin ingredient from shrimp shells at step 120 with the chitin ingredient from oyster mushroom at step 130; wherein a ratio of the chitin ingredient from shrimp shells and the chitin ingredient from oyster mushroom is (1-10): 1 (w/w).

(39) According to the embodiment of the invention, the ratio of the chitin ingredient from shrimp shells and the chitin ingredient from oyster mushroom is (1-7): 1 (w/w).

(40) According to the embodiment of the invention, the ratio of the chitin ingredient from shrimp shells and the chitin ingredient from oyster mushroom is (1-5): 1 (w/w).

(41) According to the embodiment of the invention, the ratio of the chitin ingredient from shrimp shells and the chitin ingredient from oyster mushroom is (1-3): 1 (w/w).

(42) At step 150, loading the chitin mixture into a centrifugal spray drying device having predetermined specifications to create the chitosan composition; in which predetermined specifications include: a rotate speed of 1200 rpm for 3-5 hours at temperature 20 C.-28 C. Step 150 can be done with the centrifugal spray drying device and other similar devices. The centrifugal spray drying device has been known in previous art so the description of the structure and its operating principle will not be described in detail in the invention.

(43) According to the embodiment of the invention, at step 150, the chitosan composition has a pH of 7-8.5.

(44) According to the embodiment of the invention, at step 150, the chitosan composition has a pH of 8-8.5.

(45) According to the embodiment of the invention, at step 150, time is 4-5 hours.

(46) According to the embodiment of the invention, at step 150, time is 5 hours.

(47) According to the embodiment of the invention, at step 150, temperature is 23 C.-26 C.

(48) According to the embodiment of the invention, at step 150, temperature is 25 C.

(49) Finally, at step 160, packaging the chitosan composition at step 150 and preservation.

(50) According to other embodiments of the invention, the method 100 further comprising step drying the chitosan composition at 70 C. for 5-10 minutes before the step 160 packaging and preservation.

(51) According to the invention, the chitosan composition obtained by method 100 is edible and soluble in saliva secreted in the oral cavity by more than 95%, preferably more than 97%, preferably more than 98%.

(52) It should be noted that the term admixed/mixed/admixing/mixing as used in the present invention is understood to mean adding, or reacting, or dissolving homogeneously, or evenly, components in the same solution/mixture.

(53) Now referring to FIG. 2, the process for preparing the microorganism solution 200 (process 200) based on the above principle in accordance with an exemplary embodiment of the present invention. In particular, process 200 includes the following steps:

(54) At step 201, creating a first suspension solution comprising performing in a specific order from (a1) to (c1): (a1) activating Bacillus sp. TV11 on the Nutrient agar medium, incubating at 37 C. for a period of 36-48 hours to obtain an activated Bacillus sp. TV11; (b1) inoculating a single colony of the activated Bacillus sp. TV11 into a test tube containing 10 ml of Nutrient broth medium, shaking at 200 rpm for 24 hours at 37 C. to obtain an increased first biomass solution; and (c1) inoculating the increased first biomass solution into Nutrient broth medium at a ratio of (1-3): 100, shaking at 200 rpm for 24 hours at 37 C. to obtain the first suspension solution.

(55) At step 202, creating a second suspension solution comprising performing in a specific order from (a2) to (c2): (a2) activating Lactobacillus sp. T432 on the Man Rogosa Sharpe (MRS) agar medium, incubating at 37 C. for a period of 36-48 hours to obtain an activated Lactobacillus sp. T432; (b2) inoculating a single colony of the activated Lactobacillus sp. T432 into a test tube containing 10 ml of MRS broth medium, shaking at 200 rpm for 24 hours at 37 C. to obtain an increased second biomass solution; and (c2) inoculating the increased second biomass solution into MRS broth medium at a ratio of (1-3): 100, shaking at 200 rpm for 24 hours at 37 C. to obtain the second suspension solution.

(56) At step 203, creating a third suspension solution comprising performing in a specific order from (a3) to (c3): (a3) activating Lactobacillus plantarum VTCC 431 on the MRS agar medium, incubating at 37 C. for a period of 36-48 hours to obtain an activated Lactobacillus plantarum VTCC 431; (b3) inoculating a single colony of the activated Lactobacillus plantarum VTCC 431 into a test tube containing 10 ml of MRS broth medium, shaking at 200 rpm for 24 hours at 37 C. to obtain an increased third biomass solution; and (c3) inoculating the increased third biomass solution into MRS broth medium at a ratio of (1-3): 100, shaking at 200 rpm for 24 hours at 37 C. to obtain the third suspension solution.

(57) At step 204, creating a fourth suspension solution comprising performing in a specific order from (a4) to (c4): (a3) activating Lactobacillus plantarum VTCC 431 on the MRS agar medium, incubating at 37 C. for a period of 36-48 hours to obtain an activated Lactobacillus plantarum VTCC 431; (b3) inoculating a single colony of the activated Lactobacillus plantarum VTCC 431 into a test tube containing 10 ml of MRS broth medium, shaking at 200 rpm for 24 hours at 37 C. to obtain an increased third biomass solution; and (c3) inoculating the increased third biomass solution into MRS broth medium at a ratio of (1-3): 100, shaking at 200 rpm for 24 hours at 37 C. to obtain the third suspension solution.

(58) Finally, at step 205, mixing (1-2) parts of the first suspension solution at step 201 with (1-2) parts of the second suspension solution at step 202, (1-2) parts of the third suspension solution at step 203, and (1-2) parts of the fourth suspension solution at step 204 to obtain the microorganism solution.

(59) According to the embodiment of the present invention, the microorganism solution contains strains of microorganisms including Bacillus sp. TV11, Lactobacillus sp. T432, Lactobacillus plantarum VTCC 431, and Lactobacillus bulgaricus VTCC 703, all listed in Table 7 below.

(60) TABLE-US-00007 TABLE 7 The strains of microorganisms present in the microorganism solution according to the embodiment of the invention Genus Type species Storage location Reference Bacillus Bacillus sp. Institute of Phong, H. X., Linh, L. ., Nam, TV11 Biotechnology P. H., Thanh, N. N., & Long, B. Lactobacillus Lactobacillus Research and H. . (2020). Production of sp. T432 Development, Can chitin from shrimp shells Tho University (Penaeus monodon) using Bacillus sp. TV11 and Lactobacillus sp. T342. TNU Journal of Science and Technology, 225(08), 230-238. Lactobacillus Vietnam Type Boi, V. N., Trang, N. T. M., plantarum Culture Collection- Thao, N., T., P., Chung, L., P., VTCC 431 Vietnam National Yen, H., T., B. (2016). Study on Lactobacillus University, Hanoi using lactic acid bacteria for bulgaricus demineralization and VTCC 703 deproteinization of heads and shells of shrimp in chitosan production. Journal of Fisheries Science and Technology (01), 11-19.

(61) Referring to FIG. 3, the process for preparing the fruit vinegar 300 (process 300) based on the above principle in accordance with an exemplary embodiment of the present invention. In particular, process 300 includes the following steps:

(62) At step 301, selecting and preparing fruits by a predetermined quality guideline, includes selecting said fruits that have a Brix level of at highest 2.

(63) According to the embodiment of the invention, said fruits is selected from the group consisting of ambarella (Spondias dulcis), plum (Prunus salicina), apricot (Prunus armeniaca (L.)), crocodile fruit (Dracontomelon duperreanum), star gooseberry (Phyllanthus acidus (L.)), and a combination thereof, all listed in Table 8 below.

(64) TABLE-US-00008 TABLE 8 The fruits ingredients for creating the fruit vinegar according to the embodiment of the present invention Fruits Scientific name Collection region Used part Ambarella Spondias dulcis Central Vietnam Fruit Plum Prunus salicina Southern Vietnam Apricot Prunus armeniaca (L.) Northern Vietnam Crocodile fruit/ Dracontomelon Northern Vietnam Dracontomelon duperreanum Star gooseberry Phyllanthus acidus (L.) Northern Vietnam

(65) At step 302, performing visual inspection to select undamaged fruits.

(66) At step 303, washing fruits and cold-squeezing fruits to obtain a fruit juice.

(67) At step 304, creating a foundation solution by mixing the fruit juice at step 303 and the coconut water in a ratio of 1:1.

(68) At step 305, pasteurizing the foundation solution at step 304 at 85 C. for 4 minutes, then let it cool, and adjusting the Brix to 6 to obtain a pasteurized solution.

(69) At step 306, adding ethanol 95% at a ratio of 3% (v/v) to the pasteurized solution at step 305, adjusting the pH to 5 by acetic acid 1.5%, then adding Acetobacter aceti bacteria in ratio of 1, 2% (v/v), and fermenting for 7 days to obtain a solution after fermentation.

(70) Finally, at step 307, filtering the solution after fermentation at step 306 to obtain the fruit vinegar.

(71) In the present invention and the specification of the present application, it should also be noted that the steps washing by distilled water according to method 100, or according to process 200, or according to process 300, all characterized in that the washing temperature of the distilled water is 55 C.-60 C. According to the preferred embodiment of the present invention, washing temperature of the distilled water is 60 C.

(72) According to another embodiment of the invention, the chitosan composition created by method 100 is mixed with a turkey tail mushroom extract ingredient to obtain a complex composition of turkey tail mushroom extract-chitosan.

(73) According to another embodiment of the invention, the chitosan composition created by method 100 is mixed with a turkey tail mushroom extract ingredient, a carboxymethyl cellulose solution 0.375%, a alginate solution 0.375%, a potassium citrate solution, a AgNO.sub.3 solution 0.37%, a Cu(NO.sub.3) 2 solution 0.723%, a Zn(NO.sub.3) 2 solution 3.085%, Co(NO.sub.3) 2 solution 0.746%, a Fe(NO.sub.3) 3 solution 2.5%, a glycerin ingredient, a vitamin E, a tartrazine ingredient 1%, a K.sub.2HPO.sub.4 solution 15%, a mixture extracts/essential oils, and an other ingredient to obtain a composition for prevention and treatment of Early Mortality Syndrome (EMS) shrimp.

(74) The purpose of the following example is to prove that the technical solution in this invention has been successfully researched and tested by the author. Specifically, according to the method 100 applied to create the chitosan composition including the following specific steps: (Step 1) creating the chitin ingredient from shrimp shells is similar to step 120, characteristic in that: at step (a), the soaking ratio between the molting shell of shrimp and HCl solution is 1:5 for 20 days; at step (b): the ratio of the quicklime (CaO) ingredient and the solution concentrated HCl contains 40% (concentrated grade) is 1:2 (w/v); the soaking ratio between the first temporary mixture and the quicklime (CaO) ingredient is 1:3 (w/v); and at step (c), time for let stand is 15 hours; and (Step 2) creating the chitin ingredient from oyster mushroom is similar to step 130, characteristic in that: at step (a), the soaking ratio between oyster mushroom and the fruit vinegar is 1:5 for 15 days; at step (b): the ratio of the quicklime (CaO) ingredient and the solution concentrated HCl contains 40% (concentrated grade) is 1:2 (w/v); the soaking ratio between the second temporary mixture and the quicklime (CaO) ingredient is 1:4 (w/v); and at step (c), the mixing ratio between the microorganism solution and the second temporary mushroom powder is 1.5:10 (w/v); and at step (d), fermenting time is 130 hours; (Step 3) creating the chitin mixture by homogenously mixing the chitin ingredient from shrimp shells with the chitin ingredient from oyster mushroom is similar to step 140, including five formulas listed in Table 9 below; (Step 4) creating the chitosan composition is similar to step 150, characteristic in that predetermined specifications including: a rotate speed of 1200 rpm for 5 hours at temperature 25 C.

(75) TABLE-US-00009 TABLE 9 Ratio of mixing ingredients for creating the chitin mixture in five formulas according to method 100 of the present invention Ratio (w/w) The chitin ingredient The chitin ingredient Formula from shrimp shells from oyster mushroom 1 1 1 2 3 1 3 5 1 4 7 1 5 10 1

(76) The present invention is further described in detail with reference to the following the ratio (w/w or v/v) of mixing ingredients to create the microorganism solution according to process 200 including fifteen formulas listed in Table 10 below.

(77) TABLE-US-00010 TABLE 10 Ratio of mixing ingredients for creating the microorganism solution in fifteen formulas according to process 200 of the present invention Ratio (w/w or v/v) The first The second The third The fourth suspension suspension suspension suspension Formula solution solution solution solution 1 1 1 1 1 2 1 1 1 2 3 1 1 2 1 4 1 2 1 1 5 2 1 1 1 6 2 1 1 2 7 2 1 2 1 8 2 2 1 1 9 1 1 2 2 10 1 2 1 2 11 1 2 2 1 12 2 2 2 1 13 2 2 1 2 14 2 1 2 2 15 1 2 2 2

(78) The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms a, an and the are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms includes and/or including, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, element components, and/or groups thereof.

(79) While the preferred embodiment to the invention had been described, it will be understood that those skilled in the art, both now and in the future, may make various improvements and enhancements which fall within the scope of the claims which follow. These claims should be construed to maintain the proper protection for the invention first described.

(80) The description of the present invention has been presented for purposes of illustration and description but is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. The embodiment was chosen and described in order to best explain the principles of the invention and the practical application and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.

(81) The flow diagrams depicted herein are just one example. There may be many variations to this diagram or the steps (or operations) described therein without departing from the spirit of the invention. For instance, the steps may be performed in a differing order, or steps may be added, deleted, or modified. All of these variations are considered a part of the claimed invention.

(82) While the preferred embodiment to the invention had been described, it will be understood that those skilled in the art, both now and in the future, may make various improvements and enhancements which fall within the scope of the claims which follow. These claims should be construed to maintain the proper protection for the invention first described.

(83) The foregoing description details certain embodiments of the invention. It will be appreciated, however, that no matter how detailed the foregoing appears in text, the invention can be practiced in many ways. As is also stated above, it should be noted that the use of particular terminology when describing certain features or aspects of the invention should not be taken to imply that the terminology is being re-defined herein to be restricted to including any specific characteristics of the features or aspects of the invention with which that terminology is associated. The scope of the invention should therefore be construed in accordance with the appended claims and any equivalents thereof.