Sweet protein composition as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a sweet protein composition as well as a preparation method and application thereof. The sweet protein composition comprises the following sweet proteins: at least one of Brazilian sweet protein, kiwi sweet protein, omethoate sweet protein, rhizoma curculiginis sweet protein, capparis masaikai sweet protein, miracle fruit element, petatin, thaumatin and agrocybe cylindracea sweet protein. The invention also provides a preparation method of the sweet protein composition, and the preparation method simplifies the purification process and significantly improves the purity and yield of the sweet protein. The sweet protein composition disclosed by the invention is suitable for various foods, beverages, medicines and health-care products, so that the taste of the medicines is improved, and the compliance of patients is improved. In addition, the sweet protein composition can also be used in weight-losing products, antioxidant supplements and health-care products for enhancing immunity, and oral care products such as toothpaste and mouth wash, and can provide pleasant sweetness and improve mouth feel.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a sweet protein composition and a preparation method and application thereof. Background Art

[0002] As consumers' demand for healthy diets increases, the demand for low-calorie, natural sweeteners is also growing. Although traditional artificial sweeteners such as aspartame and cyclamate have good sweetening effects, their potential health risks and bad taste limit their application. These artificial sweeteners have been found in some studies to be associated with metabolic problems, cancer and other health problems, leading consumers to worry about their safety. In addition, artificial sweeteners often have peculiar smells or aftertastes, which affect the taste of food and beverages.

[0003] Natural sweet proteins such as brazilin, thaumatin, tamarindin, curculin, mabinin, miraculin, betadine and edible mushroom sweet protein have become ideal substitutes due to their low calories and excellent sweetness properties. However, the cost of extracting these proteins from plants is high, the efficiency is low, the extraction process is complicated, and the yield is unstable. In addition, the plant extraction method may be restricted by seasons and regions, affecting the continuity of production and the stability of quality.

[0004] Another method of the prior art is to express sweet proteins in microorganisms through genetic engineering technology. This method overcomes many limitations of plant extraction and can achieve large-scale production and stable quality. However, there are some problems with current microbial expression systems (such as Escherichia coli, fungi, etc.), such as low protein expression, reduced activity due to protein misfolding, complex purification process and high cost. These problems limit the widespread application of sweet proteins. Summary of the invention

[0005] In order to solve the above-mentioned defects in the prior art, the present invention proposes a composition for expressing sweet protein by yeast recombinant expression, aiming to provide a natural sweetener with low cost, high efficiency, large-scale production and stable quality.

[0006] The specific technical problems solved by the present invention include:

[0007] Increase the expression of sweet protein: Enhance the expression in yeast by optimizing the codons of the sweet protein encoding gene; Ensure the correct folding and high activity of the protein: Ensure that the sweet protein is correctly folded in yeast cells and maintains high sweetness activity by optimizing the expression vector and yeast culture conditions.

[0008] Simplify the purification process: By improving separation and purification techniques, such as centrifugation, microfiltration membrane filtration, ion exchange chromatography and affinity chromatography, the purification process can be simplified and the purity and yield of sweet proteins can be improved.

[0009] Ensure the stability and applicability of the composition: By adding an appropriate amount of stabilizer to the composition, the stability of the sweet protein under different storage conditions is improved, ensuring its wide application in food, beverages, medicines and health products.

[0010] In order to achieve the above technical objectives, the present invention hereby proposes the following technical solutions:

[0011] In one aspect, the present invention provides a sweet protein composition comprising the following sweet protein:

[0012] At least one of Brazzein, Miraculin, Monellin, Curculin, Mabinlin, Miraculin, Pentadin, Thaumatin and Mushroom-like Matrimony sweet protein.

[0013] In some embodiments, the sweet protein composition comprises a sweet protein selected from the group consisting of the following amino acid sequences:

[0014] At least one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9.

[0015] In some embodiments, the sweet protein is expressed by recombinant yeast cells.

[0016] In some embodiments, the recombinant yeast cell comprises a nucleic acid vector or nucleic acid construct that expresses a sweet protein.

[0017] In some embodiments, the recombinant yeast cell includes, but is not limited to, recombinant Kluyveromyces marxianus, recombinant Kluyveromyces lactis, recombinant Pichia pastoris, or recombinant Saccharomyces cerevisiae.

[0018] In some preferred embodiments, the recombinant yeast cell is a recombinant Kluyveromyces lactis.

[0019] In some embodiments, based on the total weight of the sweet protein composition, the content of the sweet protein is about 60% or more, preferably about 70% or more, more preferably about 80% or more, and most preferably about 90% or more.

[0020] In some preferred embodiments, the sweet protein content is about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.1%, 99.9%, 99.1%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.9%, 99.1%, 99.9%, 99.1%, 99. 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, 99.991%, 99.992%, 99.993%, 99.994%, 99.995%, 99.996%, 99.997%, 99.998%, 99.999% or 100%.

[0021] In some embodiments, the sweet protein further includes yeast cell protein impurities.

[0022] Those skilled in the art should know that the yeast cell protein impurities are cell fragments that cannot be avoided in the process of separating or purifying the target sweet protein from recombinant yeast cells, and they do not affect the physiological activity or function of the sweet protein, nor do they cause adverse or unacceptable damage to the human body. In the present disclosure, the impurities have been controlled within a trace amount, meeting the safety standards of the product.

[0023] In some embodiments, the sweet protein composition further comprises a polysaccharide.

[0024] In some preferred embodiments, the yeast polysaccharides are mannose and glucan.

[0025] In some preferred embodiments, the mannose is present in the form of monosaccharide, oligosaccharide or polysaccharide.

[0026] In some embodiments, the sweet protein composition comprises mannan and / or glucan.

[0027] In some embodiments, based on the total weight of the sweet protein composition, the total amount of mannan and glucan is greater than 0.1%.

[0028] In some preferred embodiments, based on the total weight of the sweet protein composition, the total amount of mannan and glucan is 0.1%-15%, for example: 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%.

[0029] In some embodiments, based on the total weight of the sweet protein composition, the total amount of mannan and glucan is greater than 1%.

[0030] In some preferred embodiments, based on the total weight of the sweet protein composition, the total amount of mannan and glucan is 0.5%-10%, for example, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0%, 3.2%, 3.4%, 3.6%, 3.8%, 4. .0%, 4.2%, 4.4%, 4.6%, 4.8%, 5.0%, 5.2%, 5.4%, 5.6%, 5.8%, 6.0%, 6.2%, 6.4%, 6.6%, 6.8%, 7.0%, 7.2%, 7.4%, 7.6%, 7.8%, 8.0%, 8.2%, 8.4%, 8.6%, 8.8%, 9.0%, 9.2%, 9.4%, 9.6%, 9.8%, 10.0%.

[0031] In some embodiments, based on the total weight of the sweet protein composition, the content of the mannan or glucan is 1%-15%.

[0032] In some preferred embodiments, based on the total weight of the sweet protein composition, the content of mannan or glucan is 1%-10%, for example 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5. 0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5% , 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9.0%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10.0%.

[0033] In some preferred embodiments, based on the total weight of the sweet protein composition, the content of the mannan or glucan is 1%-7%.

[0034] In some embodiments, the sweet protein composition further comprises about 0.01%-1.0% of a stabilizer, based on the total weight of the sweet protein composition.

[0035] In some preferred embodiments, the stabilizer is selected from at least one of polyethylene glycol, glycerol and sorbitol.

[0036] In some embodiments, the stabilizer is present in an amount of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%, 0.55%, 0.60%, 0.65%, 0.70%, 0.75%, 0.80%, 0.85%, 0.90%, 0.91%, 0.92%, 0.93%, 0.94%, 0.95%, 0.96%, 0.97%, 0.98%, 0.99%, or 1.0%.

[0037] In some preferred embodiments, the brazilian thaumatin has an amino acid sequence as shown in SEQ ID NO: 1 or a functional variant or functional mutant retaining its physiological activity.

[0038] In some preferred embodiments, the thaumatin and miraculin have an amino acid sequence as shown in SEQ ID NO: 2 or a functional variant or functional mutant that retains its physiological activity.

[0039] In some preferred embodiments, the thaumatin and thaumin have an amino acid sequence as shown in SEQ ID NO: 3 or SEQ ID NO: 4, or a functional variant or functional mutant that retains its physiological activity.

[0040] In some preferred embodiments, the curculin has an amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, or a functional variant or functional mutant that retains its physiological activity.

[0041] In some preferred embodiments, the mabinin has the amino acid sequence shown in SEQ ID NO: 7 or a functional variant or functional mutant that retains its physiological activity.

[0042] In some preferred embodiments, the melanotam protein has an amino acid sequence as shown in SEQ ID NO: 8 or SEQ ID NO: 9, or a functional variant or functional mutant that retains the physiological activity thereof.

[0043] SEQ ID NO: 1:

[0044] DKCKKVYENYPVSKCQLANQCNYDCKLDKHARSGECFYDEKRNLQCICDYCEY.

[0045] SEQ ID NO:2:

[0046] MKELTMLSLSFFFVSALLAAAANPLLSAADSAPNPVLDIDGEKLRTGTNYYIVPVLRDHGGGLTVSATTPNGTFVCPPRVVQTRKEVDHDRPLAFFPENPKEDVVRVSTD LNINFSAFMPCRWTSSTVWRLDKYDESTGQYFVTIGGVKGNPGPETISSWFKIEEFCGSGFYKLVFCPTVCGSCKVKCGDVGIYIDQKGRRRLALSDKPFAFEFNKTVYF.

[0047] SEQ ID NO:3:

[0048] MAATTCFFFLFPFLLLLTLSRAATFEIVNRCSYTVWAAASKGDAALDAGGRQLNSGESWTINVEPGTNGGKIWARTDCYFDDSGSGICKTGDCGGLLRCKRFGRPPTTLAEFSLNQYGKDYIDISNIKGFNVPMDFSPTTRGCRGVRCAADIVGQCPAKLKAPGGGCNDACTVFQTSEYCC TTGKCGPTEYSRFFKRLCPDAFSYVLDKPTTVTCPGSSNYRVTFCPTALELEDE。

[0049] SEQ ID NO:4:

[0050] MAATTCFFFLFPFLLLLTLSRAATFEIVNRCSYTVWAAASKGDAALDAGGRQLNSGESWTINVEPGTKGGKIWARTDCYFDDSGRGICRTGDCGGLLQCKRFGRPPTTLAEFSLNQYGKDYIDISNIKGFNVPMDFSPTTRGCRGVRCAADIVGQCPAKLKAPGGGCNDACTVFQTSEYCCTTGKCGPTEYSRFFKRLCPDAFSYVLDKPTTVTCPGSSNYRVTFCPTALELEDE。

[0051] SEQ ID NO:5:

[0052] MAAKFLLTILVTFAAVASLGMADNVLLSGQTLHADHSLQAGAYTLTIQNKCNLVKYQ NGRQIWASNTDRRGSGCRLTLLSDGNLVIYDHNNNDVWGSACWGDNGKYALVLQKDGRF VIYGPVLWSLGPNGCRRVNGGITVAKDSTEPQHEDIKMVINN。

[0053] SEQ ID NO:6:

[0054] MAAKFLLTILVTFAAVASLGMADSVLLSGQTLYAGHSLTSGSYTLTIQNNCNLVKYQH GRQIWASDTDGQGSQCRLTLRSDGNLIIYDDNNMVVWGSDCWGNNGTYALVLQQDGLFVI YGPVLWPLGLNGCRSLNGEITVAKDSTEPQHEDIKMVINN.

[0055] SEQ ID NO:7:

[0056] MAKLIFLFATLALFVLLANASIQTTVIEVDEEEDNQLWRCQRQFLQHQRLRACQRFIHR RAQFGGQPDELEDEVEDDNDDENQPRRPALRQCCNQLRQVDRPCVCPVLRQAAQQVLQRQ IIQGPQQLRRLFDAARNLPNICNIPNIGACPFRAWP.

[0057] SEQ ID NO:8:

[0058] FREIKGYEYQLYVYASDKLFRADISEDYKTRGRKLLRFNGPVPPP.

[0059] SEQ ID NO:9:

[0060] GEWEIIDIGPFTQNLGKFAVDEENKIGQYGRLTFNKVIRPCMKKTIYEEN.

[0061] In some embodiments, after the sweet protein composition is stored for 6 months, the activity retention rate of the sweet protein is not less than 90%.

[0062] In some preferred embodiments, after the sweet protein composition is stored at pH 4.0-8.0 for 6 months, the activity retention rate of the sweet protein is not less than 90%.

[0063] In some embodiments, the sweet protein composition is in solid form, preferably a powder, more preferably a powder with a particle size of 40-100 mesh.

[0064] In some preferred embodiments, the sweet protein composition can be 40 mesh, 45 mesh, 50 mesh, 55 mesh, 60 mesh, 65 mesh, 70 mesh, 75 mesh, 80 mesh, 85 mesh, 90 mesh, 95 mesh, or 100 mesh.

[0065] In some embodiments, the sweet protein composition has a moisture content of about 5% or less.

[0066] In yet another aspect, the present invention provides use of any of the aforementioned sweet protein compositions in the preparation of medicines, pharmaceutical compositions, foods, or oral care products.

[0067] In some embodiments, the sweet protein composition is used as a condiment to improve the sweetness of food and reduce heat or calories.

[0068] In some embodiments, the sweet protein composition is used as an excipient of a health product, a medicine or a pharmaceutical composition to improve the taste and enhance the medication compliance of a subject.

[0069] In some embodiments, the sweet protein composition is used as a sweetener for oral care products to provide a pleasant sweet taste and improve mouthfeel.

[0070] In some embodiments, the food is a health food or a condiment.

[0071] In some embodiments, the health food includes, but is not limited to: a nutrient supplement or a functional health food.

[0072] In some embodiments, the dosage form of the health food includes, but is not limited to: tablets, capsules, soft capsules, granules, pills, gel candies, powders, oral liquids or drops, etc.

[0073] In some embodiments, the food includes, but is not limited to: candy products, fermented beverages, chocolate, fermented milk, ice cream, energy bars, baked goods, biscuits or canned goods, etc.

[0074] In some embodiments, the dosage form of the drug or pharmaceutical composition includes solid preparations (e.g., tablets, capsules, powders, granules, etc.), gas preparations (e.g., aerosols, sprays, powder mists, etc.), liquid preparations (e.g., injections, suspensions, solutions, etc.) or semi-solid preparations (e.g., creams, suppositories, gels, etc.).

[0075] In yet another aspect, the present invention provides an oral care product comprising any one of the aforementioned sweet protein compositions.

[0076] In some embodiments, the oral care product further comprises at least one physiologically acceptable adjuvant or excipient.

[0077] In some embodiments, the physiologically acceptable adjuvant or excipient includes, but is not limited to: at least one of a humectant, a thickener, a surfactant (foaming agent), a friction agent, a sweetener, a fragrance, and a preservative.

[0078] In some preferred embodiments, the abrasive includes, but is not limited to: at least one of hydrated silica, calcium carbonate, calcium hydrogen phosphate, calcium hydrogen phosphate dihydrate, calcium pyrophosphate, aluminum hydroxide, and microcrystalline cellulose.

[0079] In some more preferred embodiments, the abrasive is calcium hydrogen phosphate dihydrate.

[0080] In some preferred embodiments, the humectant includes, but is not limited to: sorbitol, glycerin, propylene glycol and / or xylitol.

[0081] In some more preferred embodiments, the humectant is glycerin and / or sorbitol.

[0082] In some preferred embodiments, the thickener includes, but is not limited to: at least one of cellulose gum, carrageenan, xanthan gum, guar gum, and carbomer.

[0083] In some more preferred embodiments, the thickening agent is carbomer.

[0084] In some preferred embodiments, the surfactant includes, but is not limited to: at least one of sodium or potassium lauryl sulfate, cocamidopropyl betaine, sodium lauroyl sarcosinate, Tween, polyoxyethylene hydrogenated castor oil, polyoxyethylene, and polyoxypropylene block copolymers.

[0085] In some preferred embodiments, the preservative includes, but is not limited to: at least one of decanediol, caprylyl glycol, phenoxyethanol, and 1,2-hexanediol.

[0086] In some preferred embodiments, the fragrance includes, but is not limited to: at least one of eugenol, thymol, borneol (borneol), synthetic cinnamaldehyde, citral, vanillin, methyl salicylate, menthol, mint essence, mint oil, peppermint oil, spearmint oil, geranium oil, camphor oil, eucalyptus oil, cinnamon oil, vanillin, anise oil, lemon oil, citrus oil, and grapefruit oil.

[0087] In some more preferred embodiments, the fragrance is mint flavor.

[0088] In some preferred embodiments, the sweetener includes, but is not limited to: at least one of xylitol, erythritol, saccharin sodium, stevia, aspartame, and the like.

[0089] In some preferred embodiments, the oral care product further comprises fluoride and / or chemical bleaching ingredients.

[0090] In some preferred embodiments, the chemical bleaching component includes, but is not limited to: at least one of hydrogen peroxide, carbamide peroxide, plant acid, hydroxyapatite, and polyphosphate.

[0091] In some preferred embodiments, the fluoride includes, but is not limited to, at least one of sodium fluoride, stannous fluoride, sodium monofluorophosphate, and ammonium fluoride.

[0092] In some more preferred embodiments, the fluoride is sodium fluoride.

[0093] In some embodiments, based on the total weight of the oral care product, the sweet protein composition is present in an amount of about 0.001% to about 0.5%.

[0094] In some preferred embodiments, based on the total weight of the oral care product, the content of the sweet protein composition is about 0.01%-0.2%, for example, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%.

[0095] In some preferred embodiments, based on the total weight of the oral care product, the content of the sweet protein composition is about 0.01%-0.1%.

[0096] In some more preferred embodiments, based on the total weight of the oral care product, the content of the sweet protein composition is about 0.05%.

[0097] In some more preferred embodiments, based on the total weight of the oral care product, the content of the sweet protein composition is about 0.03%.

[0098] In some embodiments, the oral care product includes, but is not limited to: at least one of toothpaste, tooth cleaning powder, mouthwash, tooth stickers, oral gel, tooth gel, tooth powder, mouth spray, mouthwash granules, paste, and tooth wax.

[0099] In some preferred embodiments, the oral care product is toothpaste or mouthwash.

[0100] In yet another aspect, the present invention provides a food comprising any one of the aforementioned sweet protein compositions.

[0101] In some preferred embodiments, the food is a health food (health product).

[0102] In some preferred embodiments, the food is a condiment.

[0103] In some embodiments, the seasoning includes, but is not limited to: a salty agent, a sour agent, a sweetener, an umami agent or a spicy agent, preferably a sweetener.

[0104] In some embodiments, the food further comprises at least one food-acceptable excipient.

[0105] In some embodiments, the food-acceptable excipients include, but are not limited to: at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, thickeners, emulsifiers, preservatives, stabilizers, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, coating agents, flavoring agents, antioxidants, sweeteners, thickeners, and nutritional enhancers.

[0106] In some preferred embodiments, the food-acceptable excipient is selected from microcrystalline cellulose, erythritol, D-mannitol, fumaric acid, glycerol, pectin, potassium alginate, sodium alginate, talc, sodium pyrophosphate, polydextrose, carrageenan, sodium ascorbate, ascorbyl palmitate, L-malic acid, L(+)-tartaric acid, maltitol, gelatin, xylitol, citric acid, potassium citrate, sodium citrate, citric acid fatty acid glyceride, agar, lactic acid, sodium lactate, sorbic acid and its potassium salt, sorbitol, acid red, calcium carbonate, sodium carbonate, sodium bicarbonate, beet red, vitamin C, vitamin E, oxidized starch, ethanol, sodium acetate , stearic acid, calcium stearate, magnesium stearate, povidone K30, neotame, talc, pregelatinized starch, low-substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose, titanium dioxide, polyethylene glycol, sorbitol and sorbitol liquid, sodium carboxymethyl cellulose, edible corn starch, potato starch, tapioca starch, edible wheat starch, edible sweet potato starch, sodium carboxymethyl starch, soybean oil, corn oil, sunflower oil, olive oil, powdered soybean lecithin, concentrated soybean lecithin, soybean lecithin, cellulose gum, natural flavors, flavors (such as lemon flavor, strawberry flavor, cherry flavor, etc.), polyoxyethylene sorbitan monooleate, beeswax and at least one of dextrin.

[0107] In some embodiments, the dosage form of the food comprises a solid preparation, a gas preparation, a liquid preparation, or a semisolid preparation.

[0108] In some embodiments, the food includes, but is not limited to: health foods (such as weight loss products, antioxidant supplements, health products that enhance immunity), condiments, confectionery products (such as hard candies, (sugar-free) chewing gum), beverages (such as functional beverages, low-calorie beverages), chocolate (such as low-sugar chocolate), dairy products (such as fermented milk including low-calorie milkshakes, low-calorie yogurt, etc.), ice cream, energy bars, baked goods (such as low-sugar cakes, low-sugar bread, sugar-free biscuits, etc.), canned food, etc.

[0109] In some embodiments, the dosage form of the health food includes, but is not limited to: capsules, tablets, oral liquids, granules (granules), powders, pills, sprinkles, drops, compressed candies or soft candies, etc.

[0110] In some embodiments, the health food comprises a nutrient supplement and a non-nutrient supplement.

[0111] In some preferred embodiments, the nutrient supplement further comprises calcium, magnesium, potassium, manganese, iron, zinc, selenium, copper, vitamin A, vitamin D, vitamin B1, vitamin B2, vitamin B6, vitamin B12, niacin (nicotinic acid), folic acid, biotin, choline, vitamin C, vitamin K, pantothenic acid, vitamin E, β-carotene or n-3 polyunsaturated fatty acids.

[0112] In some preferred embodiments, the non-nutrient supplements include those that help enhance immunity, help with anti-oxidation, assist in improving memory, relieve visual fatigue, clear the throat and moisten the throat, help improve sleep, relieve physical fatigue, tolerate hypoxia, help control body fat, help improve bone density, improve iron deficiency anemia, help improve acne, help improve chloasma, help improve skin moisture, help regulate intestinal flora, help digestion, help moisturize the intestines and promote bowel movements, assist in protecting gastric mucosa, help maintain healthy blood lipid (cholesterol / triglyceride) levels, help maintain healthy blood sugar levels, help maintain healthy blood pressure levels, have an auxiliary protective effect on chemical liver damage, have an auxiliary protective effect on ionizing radiation hazards, and help excrete lead.

[0113] In some more preferred embodiments, the health food is a non-nutrient supplement that helps with antioxidant effects.

[0114] In some more preferred embodiments, the health food is a non-nutrient supplement that helps manage body fat.

[0115] In some more preferred embodiments, the health food is a non-nutrient supplement that helps maintain healthy blood sugar levels.

[0116] In some embodiments, based on the total weight of the food, the content of the sweet protein composition is about 0.0015%-0.5%.

[0117] In some preferred embodiments, based on the total weight of the food, the content of the sweet protein composition is about 0.01%-0.2%, for example 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.20%.

[0118] In some preferred embodiments, based on the total weight of the food, the content of the sweet protein composition is about 0.01%-0.1%.

[0119] In some more preferred embodiments, based on the total weight of the food, the content of the sweet protein composition is about 0.05%.

[0120] In some more preferred embodiments, based on the total weight of the food, the content of the sweet protein composition is about 0.1%.

[0121] In some more preferred embodiments, based on the total weight of the food, the content of the sweet protein composition is about 0.03%.

[0122] In some more preferred embodiments, based on the total weight of the food, the content of the sweet protein composition is about 0.02%.

[0123] In some more preferred embodiments, based on the total weight of the food, the content of the sweet protein composition is about 0.04%.

[0124] In another aspect, the present invention provides a medicament or a pharmaceutical composition comprising any of the aforementioned sweet protein compositions.

[0125] In some embodiments, the medicament or pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

[0126] In some preferred embodiments, the pharmaceutically acceptable excipients include, but are not limited to: at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, release agents, coating agents, acidity regulators, flavoring agents, and antioxidants.

[0127] In some embodiments, the dosage form of the drug or pharmaceutical composition includes, but is not limited to: at least one of a solution, a suspension, an emulsion, a powder, a granule, a tablet, a capsule, an injection, an aerosol, drops (e.g., ear drops, eye drops, nasal drops), an ointment, a paste, a liniment, a patch, a lotion, a cream (e.g., sugar-free syrup), a suppository, or a gargle.

[0128] In some preferred embodiments, the drug or pharmaceutical composition is a tablet selected from compressed tablets, coated tablets, effervescent tablets, chewable tablets, multilayer tablets, dispersible tablets, sustained-release tablets, controlled-release tablets, orodisintegrating tablets, sublingual tablets, lozenges, oral patches, soluble tablets, vaginal tablets or implants, preferably chewable tablets.

[0129] In some embodiments, based on the total weight of the drug or the pharmaceutical composition, the content of the sweet protein composition is about 0.01%-0.5%.

[0130] In some preferred embodiments, based on the total weight of the drug or pharmaceutical composition, the content of the sweet protein composition is about 0.05%-0.2%, for example, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.15%, 0.20%.

[0131] In some preferred embodiments, based on the total weight of the drug or pharmaceutical composition, the content of the sweet protein composition is about 0.01%-0.1%.

[0132] In some more preferred embodiments, based on the total weight of the medicament or the pharmaceutical composition, the content of the sweet protein composition is about 0.05%.

[0133] In some more preferred embodiments, based on the total weight of the medicament or the pharmaceutical composition, the content of the sweet protein composition is about 0.03%.

[0134] In another aspect, the present invention provides a method for preparing any of the aforementioned sweet protein compositions, the preparation method comprising the following steps:

[0135] (1) introducing or integrating the gene encoding the sweet protein into the host cell;

[0136] (2) providing the necessary culture conditions to enable the host cell to express or translate the sweet protein;

[0137] (3) Isolating and / or purifying sweet proteins from host cells.

[0138] In some embodiments, the host cell comprises a eukaryotic cell or a prokaryotic cell selected from bacteria, yeast, plant cells or animal cells, preferably yeast.

[0139] In some preferred embodiments, the yeast includes, but is not limited to: Kluyveromyces marxianus, Kluyveromyces lactis, Pichia pastoris or Saccharomyces cerevisiae.

[0140] In some preferred embodiments, the yeast is Kluyveromyces.

[0141] In some more preferred embodiments, the yeast is Kluyveromyces lactis.

[0142] In some embodiments, the culture conditions include temperature, humidity, culture time, pH and / or culture medium.

[0143] In some embodiments, the culture medium includes, but is not limited to: a carbon source, a nitrogen source, amino acids and / or inorganic salts.

[0144] In some preferred embodiments, the culture medium includes, but is not limited to, the following components:

[0145] Yeast extract, tryptone, lactose, magnesium sulfate, ammonium sulfate, potassium dihydrogen phosphate, copper sulfate, ferrous sulfate, manganese sulfate, cobalt chloride, zinc sulfate, methionine, alanine, cysteine ​​and glycine.

[0146] In some preferred embodiments, the pH is 5.5-6.5.

[0147] In some preferred embodiments, the culture time is 72 hours.

[0148] In some preferred embodiments, the temperature is 30°C.

[0149] In some embodiments, the culture conditions further include dissolved oxygen levels.

[0150] In some embodiments, the culture conditions include continuous culture or fed-batch culture.

[0151] In some embodiments, the separation and / or purification step of the sweet protein comprises a centrifugation, filtration and chromatography purification step or a membrane separation purification step.

[0152] In some preferred embodiments, the filtering step comprises filtering through a microfiltration membrane or an ultrafiltration membrane.

[0153] In some preferred embodiments, the chromatographic purification step comprises ion exchange chromatography, hydrophobic chromatography or affinity chromatography.

[0154] In some embodiments, the preparation method further comprises freeze-drying or spray-drying the sweet protein.

[0155] In some embodiments, the encoding gene is codon optimized to enhance expression in a host cell.

[0156] In another aspect, the present invention provides a method for preparing a low-calorie or reduced-calorie product, comprising adding any one of the aforementioned sweet protein compositions to the low-calorie or reduced-calorie product.

[0157] In some embodiments, the low-calorie or reduced-calorie product comprises an oral care product, a food, a medicament, a pharmaceutical composition, and / or a food additive.

[0158] The present invention provides an efficient and stable method for producing sweet protein, and also provides a sweet protein composition with high purity, low calories and pure taste, thus meeting the market demand for natural sweeteners.

[0159] Selection of sweet protein: the sweet protein is selected from at least one of brazilian tamanin, thaumatin, tamanin, curculigo tamanin, mabinin, miraculin, betadine and edible mushroom tamanin. The content of the sweet protein is preferably more than 60% of the total weight of the composition, and the specific range can be more than 70%, more than 80%, or even up to more than 90%.

[0160] Optimization of yeast expression system: Select Kluyveromyces marxianus, Kluyveromyces lactis, Pichia pastoris or Saccharomyces cerevisiae as the expression host, and significantly increase the expression of sweet protein by optimizing the codons of the sweet protein encoding gene and the expression vector design. At the same time, optimize the culture conditions (such as temperature, pH value and dissolved oxygen level) to ensure that the sweet protein is correctly folded in the yeast cells and maintains high sweetness activity.

[0161] Purification and stability: The sweet protein is separated and purified by centrifugation, microfiltration, ion exchange chromatography and affinity chromatography to obtain a high-purity sweet protein composition. The composition may also contain 0.01% to 1% of a stabilizer (such as polyethylene glycol, glycerol and sorbitol) to improve the stability of the sweet protein under different storage conditions.

[0162] Physical properties of the composition: The composition can be prepared in powder form with a particle size of 50 to 200 microns and good fluidity and solubility. In water at 20°C, the solubility of the composition is at least 50 g / L, the dissolution rate does not exceed 5 minutes, and the moisture content is less than 5%. After the composition is stored for 6 months at a pH value of 4 to 8, the sweet protein activity retention rate is not less than 90%; it can be stably stored for at least 12 months at a temperature range of 4°C to 25°C.

[0163] Wide application: The sweet protein composition of the present invention is suitable for various foods, beverages, medicines and health products. For example, it can be used in foods such as candies, premixes for baked products, low-calorie beverages, sugar-free chewing gum, low-sugar cakes, and children's snacks; it can also be used in medicines such as oral syrups, tablets, and chewable tablets to improve the taste of the medicine and improve patient compliance. In addition, the sweet protein composition can also be used in weight loss products, antioxidant supplements, and health products that enhance immunity, as well as oral care products such as toothpaste and mouthwash, to provide a pleasant sweet taste and improve the taste. BRIEF DESCRIPTION OF THE DRAWINGS

[0164] Figure 1 A radar chart of the sensory evaluation of sweetness is shown. DETAILED DESCRIPTION

[0165] In order to make the purpose, technical solution and advantages of the embodiments of the present disclosure clearer, the technical solution of the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings of the embodiments of the present disclosure. Obviously, the described embodiments are part of the embodiments of the present disclosure, not all of the embodiments. Based on the described embodiments of the present disclosure, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0166] The present invention may be implemented in other specific forms without departing from the basic attributes of the present invention. It should be understood that, without conflict, any and all embodiments of the present invention may be combined with the technical features in any other embodiment or multiple other embodiments to obtain other embodiments. The present invention includes other embodiments obtained by such combination.

[0167] All publications and patents mentioned in this disclosure are hereby incorporated by reference into this disclosure in their entirety. If the purposes or terms used in any publications and patents incorporated by reference conflict with the purposes or terms used in this disclosure, then the purposes and terms of this disclosure shall prevail.

[0168] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0169] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0170] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0171] As shown in the present disclosure, the term "about" means a range of ±20% of the value thereafter. In some embodiments, the term "about" means a range of ±10% of the value thereafter. In some embodiments, the term "about" means a range of ±5% of the value thereafter.

[0172] The numerical ranges used in the present disclosure should be understood to have enumerated all the numbers within the range. For example, a range of 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0173] As used in this disclosure, the term "comprises" or "comprising" means "including but not limited to". The term is intended to be open-ended to specify the presence of any stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" used throughout the application, especially in the claims, may be replaced by the term "consisting of".

[0174] As used herein, the terms "optional", "either", "any", or "any" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. As used herein, "a" and "an" are used herein to refer to one or more than one of the grammatical objects.

[0175] As shown in the present disclosure, “and / or” should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

[0176] As shown in the present disclosure, the term "fermentation" or "culture" refers to the process of transforming external substrates using specific metabolic pathways in biological cells under appropriate conditions to produce target products or bacteria required by humans.

[0177] As shown in the present disclosure, the term "recombinant" is a term known in the art. When referring to nucleic acids (e.g., genes), the term can be used, for example, to describe nucleic acids that have been separated from their naturally occurring environment, nucleic acids that are no longer associated with all or part of naturally adjacent or proximal nucleic acids, nucleic acids that are effectively connected to naturally unconnected nucleic acids, or nucleic acids that do not exist in nature. When "recombinant" is used to describe proteins, it can refer to proteins produced in cells of different species or types, for example, compared to the species or type of cells that produce the protein in nature.

[0178] As shown in the present disclosure, the terms "cell" and "cell line" are used interchangeably, and all such names include their descendants. The term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells. In some preferred embodiments of the present disclosure, the host cell is yeast.

[0179] As shown in the present disclosure, the term "pharmaceutically acceptable carrier, excipient or adjuvant" should be compatible with the active ingredient and can be mixed with it without reducing or significantly reducing the effect of the drug under normal circumstances. Specific examples of some substances that can be used as pharmaceutically acceptable carriers or adjuvants are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium methylcellulose, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Tween; wetting agents, such as sodium lauryl sulfate; colorants; flavorings; tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer, etc. These materials are used as needed to aid in the stability of the formulation or to help increase the activity or its bioavailability or to create an acceptable taste or smell in the case of oral administration to improve patient compliance with treatment.

[0180] As indicated in the present disclosure, the term "expression" refers to the process by which cells convert genetic information stored in DNA sequences into protein molecules with biological activity through transcription and translation.

[0181] As shown in the present disclosure, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid connected thereto. Exemplarily, one type of vector is a "plasmid", which generally refers to a circular double-stranded DNA loop into which another DNA segment (exogenous gene) can be connected, and may also include linear double-stranded molecules, such as linear double-stranded molecules obtained from amplification by polymerase chain reaction PCR or treatment of circular plasmids with restriction enzymes. Another type of vector is a viral vector, in which another DNA segment can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., vectors having a replication origin that works in the host cell). Other vectors can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, some preferred vectors are capable of directing the expression of the exogenous genes connected thereto. In the present disclosure, the vector connected with the exogenous gene is referred to as a "recombinant expression vector" (or simply "expression vector").

[0182] As used herein, the term "coding gene" or "coding sequence" should be given its ordinary and customary meaning to those of ordinary skill in the art, and includes but is not limited to referring to a DNA sequence encoding a specific amino acid sequence.

[0183] As shown in the present disclosure, the term "functional variant" or "functional variant" further includes conservatively substituted variants. The term "conservatively substituted variant" refers to a peptide having an amino acid sequence that differs from a reference peptide (or a reference peptide, in most cases a wild-type protein) by one or more conservative amino acid substitutions and retains some or all of the activities of the reference peptide. "Conservative amino acid substitution" is the replacement of an amino acid residue with a functionally similar residue. Examples of conservative substitutions include substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; substitution of one charged or polar (hydrophilic) residue for another, such as between arginine and lysine, between glutamine and asparagine, between threonine and serine; substitution of one basic residue such as lysine or arginine for another; or substitution of one acidic residue such as aspartic acid or glutamic acid for another; or substitution of one aromatic residue such as phenylalanine, tyrosine or tryptophan for another aromatic residue. Such substitutions are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide. The phrase "conservatively substituted variants" also includes peptides in which residues are substituted with chemically derivatized residues, provided that the resulting peptide retains some or all of the activity of the reference peptide as described herein.

[0184] The term "variant" also includes functionally active polypeptides having an amino acid sequence that is at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and even 100% identical to the amino acid sequence of the reference peptide. The term "homologous" in all its grammatical forms and spelling variants refers to the relationship between polynucleotides or polypeptides having a "common evolutionary origin", including polynucleotides or polypeptides from superfamilies and homologous polynucleotides or proteins from different species. Such polynucleotides or polypeptides have sequence homology, as reflected by their sequence similarity, whether in terms of percentage of identity or in terms of the presence of specific amino acids or motifs at conserved positions. For example, two homologous polypeptides can have amino acid sequences that are at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 900%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and even 100% identical.

[0185] As shown in the present disclosure, the term "food" refers to substances that can be eaten or drunk by humans, including processed foods, semi-finished products and unprocessed foods, and does not include tobacco or substances that are only used as medicines. The purpose of food application is to provide energy and nutrients for people, without emphasizing specific functions, without requirements for dosage, and without a specific range of consumption.

[0186] As shown in the present disclosure, the term "health care product" or "health food" includes and refers to food that claims to have specific health functions or is intended to supplement vitamins and minerals, that is, food that is suitable for consumption by specific groups of people, has the function of regulating body functions, is not intended to treat diseases, and does not cause any acute, subacute or chronic harm to the human body. Health food generally has a specified consumption amount and a specific range of consumption. It can be claimed to have the function of regulating body functions, preventing the occurrence of certain specific diseases or improving physical fitness, but is not intended to treat diseases. It is only used orally and does not cause any acute, subacute or chronic harm to the human body.

[0187] As shown in the present disclosure, the term "condiment" or "condiment food" refers to an auxiliary food that can increase the color, aroma and taste of dishes, promote appetite and benefit human health. Its main function is to improve the quality of dishes, meet the sensory needs of consumers, thereby stimulating appetite and improving human health. In a broad sense, condiments include salting agents, sour agents, sweeteners, umami agents and spicy agents, such as salt, soy sauce, vinegar, monosodium glutamate, sugar, star anise, fennel, pepper, mustard, etc.

[0188] As indicated in this disclosure, the term "oral care products" refers to a series of products specifically used for oral cleaning, health care, treatment and beautification. These products are intended to improve the oral environment, prevent oral diseases, improve oral health levels in different ways, and meet people's needs for oral beauty.

[0189] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment. If the specific conditions are not specified in the embodiment, it is carried out according to the conditions of normal conditions or manufacturer's suggestion. If the manufacturer is not specified in all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the specific embodiments below. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. Such structures and technologies are also described in the publication of many publications.

[0190] Example 1: Preparation of sweet protein composition by Kluyveromyces marxianus

[0191] 1.1: Acquisition of sweet protein encoding genes

[0192] a. Primer design: A brazilin gene fragment was synthesized at GenScript and named bra. Specific primers were designed to amplify the brazilin encoding gene. When designing forward and reverse primers, the length, GC content, and annealing temperature of the primers should be considered to ensure efficient and specific amplification.

[0193] b. PCR amplification: Using bra as a template, the target gene bra was obtained by PCR amplification (upstream primer F11: 5'-GAAAAGAGAGGCTGAAGCTGACAAGTGTAAGAAGGTCTACG-3' (SEQ ID NO: 10), downstream primer R11: 5'-ACGGTACCCCTAGGAGATCTCAGTATTCACAGTAGTCACAGATAC-3' (SEQ ID NO: 11); the commercial vector pKLAC1 was amplified by PCR primers (upstream primer F12: 5'-GATCTCCTAGGGGTACCG-3' (SEQ ID NO: 12), R12: 5'-AGCTTCAGCCTCTCTTTTCTC-3' (SEQ ID NO: 13). The target gene was connected to the commercial pKLAC1 vector by seamless assembly to obtain the expression vector pKLAC1-bra. The PCR reaction system includes template DNA, forward primer (Forwardprimer), reverse primer (Reverse primer), dNTP mixture, Taq buffer and high-fidelity Taq DNA polymerase. PCR cycle parameters are as follows:

[0194] Initial denaturation: 95°C, 5 minutes

[0195] Denaturation: 95°C, 30 seconds

[0196] Annealing: 55°C, 30 seconds

[0197] Extension: 72°C, 1 min

[0198] Number of cycles: 30 times.

[0199] Final extension: 72°C, 10 min.

[0200] c. PCR product purification: Use agarose gel electrophoresis to detect the PCR amplification effect, cut out the target band and use a gel recovery kit to purify the PCR product.

[0201] d. Gene sequencing: The purified PCR product is sent to a sequencing company for sequencing to confirm the correctness of the Brazilian sweet protein encoding gene.

[0202] The plasmid pKLAC1-bra was digested with the rapid endonuclease SacⅡ (New England Biotechnology (Beijing) Co., Ltd., catalog number #: R0157L) to obtain the homologous recombinant fragment of linearized pKLAC1-bra. The concentration of the fragment obtained after purification was >1 μg. The linearized pKLAC1-bra was transformed into Kluyveromyces marxianus CJ3113 (accession number CCTCC No: M20211265) by electroporation (0.1 cm electroporation cup, 1.5-2.5 kV voltage, time selected as ~5 ms) to construct the K. marxianus::pKLAC1-bra expression strain, and at the same time, the control strain K. marxianus::pKLAC1 was constructed.

[0203] Fermentation: The above expression strain and the control strain were cultured separately in a culture medium containing carbon source, nitrogen source and salt (1% yeast powder, 2% tryptone, 0.2% lactose, 0.05% magnesium sulfate, 0.5% ammonium sulfate, 0.6% potassium dihydrogen phosphate, 10ppm copper sulfate, 5ppm ferrous sulfate, 15ppm manganese sulfate, 7ppm cobalt chloride, 2ppm zinc sulfate, 3ppm methionine, 1ppm alanine, 6ppm cysteine, 3ppm glycine); the pH of the culture medium was adjusted to 5.5-6.5, and the culture was fermented at 30°C for 72h. Galactose was added during the process to induce protein expression, and the fermentation broth was taken at 0h, 12h, 24h, 36h, 48h and 60h, respectively.

[0204] 1.2: Isolation and purification of sweet proteins

[0205] A. Cell collection: After the culture was completed, the supernatant was collected by centrifugation (5000 rpm, 10 minutes).

[0206] B. Filtration: Filter the supernatant using a 0.22 μm microfiltration membrane to remove residual cell debris and impurities.

[0207] C. Chromatographic purification: First perform ion exchange chromatography using an SP Purose 6XL column and elute the target protein at a certain salt concentration.

[0208] The chromatographic separation operation is as follows:

[0209] (1) Wash the column with deionized water for 5 column volumes to remove the 20% ethanol storage solution in the column at a flow rate of 1 mL / min;

[0210] (2) Equilibrate the column with the equilibration solution until the UV, conductivity and pH are consistent with those of the equilibration solution and stable, with a flow rate of 1 mL / min;

[0211] (3) Load 30 mL (30 CV) of pretreated sample at a flow rate of 1 mL / min. After loading, continue to flush the column with the equilibrium solution until the UV, conductivity, and pH are stable. During the loading process, start collecting the flow-through peak when the UV peak rises and stop collecting when the UV peak ends.

[0212] (4) balancing the column with the balancing solution until the UV, conductivity and pH are consistent with those of the balancing solution and stable, at a flow rate of 1 mL / min; eluting with 100% eluent, at a flow rate of 1 mL / min, starting collection when the UV peak appears during the elution process, and stopping collection when the peak ends;

[0213] (5) After the experiment, the column was cleaned with a cleaning solution until the UV, conductivity, and pH baselines were stable. When the UV peak rose during the cleaning process, the cleaning peak was collected and the collection was stopped when the UV peak ended.

[0214] (6) Regenerating the column with equilibrium buffer until the UV, conductivity and pH are stable;

[0215] (7) Preserve the chromatography column with 20% ethanol in water at a flow rate of 1 mL / min and flush for at least 3 column volumes.

[0216] Membrane separation operation is as follows:

[0217] 1) Ultrafiltration: Take 500 mL of fermentation broth of K.marxianus::pKLAC1 and K.marxianus::pKLAC1-β-Lg in Example 1 respectively, centrifuge at 12000rpm / min for 5min, and take the supernatant. Start the ultrafilter and use clean water to flush the ultrafilter forward and back until the effluent is clear and has no odor. After pre-rinsing the ultrafilter, add the above supernatant. Start the ultrafilter and booster pump. After the liquid is discharged normally, adjust the opening of the concentrated water reflux valve to control the concentrated water reflux pressure at 0.04-0.08Mpar. During the concentration process, the ultrafiltration membrane is backwashed once every 10 minutes (the backwashing time is set to 40s). The ultrafiltration membrane accuracy is: 0.005μm-0.2μm; molecular retention value: ≤20000DA. After the ultrafiltration is completed, the concentrate and permeate mass are recovered and weighed and recorded, and stored in a 4°C cold storage for standby use.

[0218] 2) Nanofiltration: Take the permeate of K.marxianus::pKLAC1 and K.marxianus::pKLAC1-β-Lg collected in 1) above and perform nanofiltration purification. Inject an appropriate amount of pure water into the concentrated water tank, start the nanofilter, and use clean water to flush the nanofilter forward and back until the effluent is clear and has no odor. After pre-rinsing the nanofilter, add the b1 permeate. Start the nanofilter and the booster pump. After the liquid is discharged normally, adjust the opening of the concentrated water reflux valve to control the concentrated water reflux pressure at 0.04-0.08Mpar. During the concentration process, the nanofiltration membrane is backwashed once every 10 minutes. The nanofiltration membrane accuracy is: 0.001μm; molecular retention value: ≤200DA. After the concentration is completed, the concentrated liquid is recovered and weighed and recorded, and stored in a 4℃ cold storage for standby use.

[0219] 3) Purity detection: The purification effect was detected by SDS-PAGE and HPLC to confirm the purity of the sweet protein. The results showed that the purity of the sweet protein (brazil thauma protein) was above 95%, and its amino acid sequence was shown in SEQ ID NO:1.

[0220] Example 2: Preparation of sweet protein composition by Kluyveromyces lactis

[0221] Referring to the method of Example 1, the linearized pKLAC1-β-Lg was transformed into Kluyveromyces lactis (Kluyveromyces lactis, deposit number CICC 32428), K. lactis::pKLAC1-β-Lg was constructed, and the control strain K. lactis::pKLAC1 was constructed at the same time. The Kluyveromyces lactis strain was cultured in a fermentation medium (peptone 20 g / L, yeast powder 10 g / L, glucose 10 g / L, galactose 10 g / L); the pH of the medium was adjusted to 5.5-6.5, and the culture was fermented at 25-30° C. for 72 h. During the fermentation process, galactose was added to induce protein expression to obtain a fermentation broth.

[0222] The same method as in Example 1 was used to purify the brazilian thauma protein fermentation broth and to detect the purification effect by SDS-PAGE and HPLC to confirm the purity of the sweet protein. The results showed that the purity of the sweet protein (brazilian thauma protein) was above 95%, and its amino acid sequence was shown in SEQ ID NO: 1.

[0223] Example 3: Preparation of sweet protein composition by Pichia pastoris

[0224] (1) Based on the codon preference of Pichia pastoris and the AT content of the DNA sequence, online codon optimization software was used to change the corresponding sweet protein (Brazilian sweet protein) codon bases to the bases preferred by Pichia pastoris codons without changing the protein coding sequence. The optimized gene sequence was artificially synthesized by GenScript Biotech Co., Ltd.

[0225] (2) Gene cloning and construction of expression vector

[0226] Specific primers were designed and PCR was used to amplify the sweet protein. An EcoRI restriction site sequence was added to the 5' end of the coding gene sequence, and a stop codon and NotI restriction site sequence were added to the 3' end. Subsequently, the sequence fragment was inserted into the pPIC9K expression vector through the EcoRI and NotI restriction sites to obtain the corresponding recombinant expression vector pPIC9K-bra.

[0227] (3) Construction of recombinant Pichia pastoris

[0228] The successfully constructed recombinant expression vector plasmid pPIC9K-bra was linearized and transferred into Pichia pastoris GS115 competent cells. Single clone colonies were picked from the transformed MD plate and placed in a 48-well culture plate containing 500 μL YPD (0.75 mg / mL G418) medium, and cultured at 30°C, 240 rpm for 18-24 h; the colonies were then transferred to YPD liquid culture medium with gradually increasing G418 content for culture.

[0229] (4) strain screening and fermentation, the steps are as follows:

[0230] The bacterial liquid was inoculated into 10mL / 50mL BMGY at an inoculum size of 2%, and cultured at 30°C, 240rpm for 18-24h; centrifuged, the supernatant was discarded, 10mL BMMY was added for resuspending, and cultured at 28°C, 240rpm for 48h, and 100% methanol was added every 24h to a final concentration of 1.5%; centrifuged, and the supernatant was taken for SDS-PAGE analysis to obtain a recombinant Pichia pastoris with a high expression level.

[0231] The fermentation broth was purified and tested by SDS-PAGE and HPLC in the same manner as in Example 1 to confirm the purity of the sweet protein. The results showed that the purity of the sweet protein (brazil thaumatin) was above 95%, and its amino acid sequence was shown in SEQ ID NO: 1.

[0232] Example 4: Sensory evaluation of sweet protein composition

[0233] Comparison of sweetness and sweetness delay of sweet protein composition with aspartame and sucrose.

[0234] 1) Sample preparation

[0235] Accurately weigh 6g and 30g of sucrose, dissolve them in 95mL and 75mL of drinking water, respectively, to prepare 6% and 30% sucrose aqueous solutions. Accurately take 0.5mL and 0.1mL of 0.6% sweet protein (Brazilian sweet protein) aqueous solution, add them to 100mL of drinking water, respectively, to prepare 0.006% and 0.025% sweet protein, and set aside.

[0236] 2) Sensory evaluation method of sweet protein

[0237] Using sucrose as a control, the sweetness and persistence of sweet protein were evaluated by sensory evaluation. The sensory evaluation panel consisted of 10 trained staff members who tasted the sweetness of different samples and evaluated the sweetness of sweet protein. The detailed evaluation criteria are shown in Table 1.

[0238] Table 1 Sensory scoring standards

[0239]

[0240] 3) Data processing

[0241] The data were processed, analyzed and plotted using Excel 2019. The determination of sample indicators was repeated three times unless otherwise specified.

[0242] Sweetness evaluation results of sweet protein Figure 1 ,Depend on Figure 1 It can be seen that the sweetness of sweet protein is much higher than that of sucrose, about 1500 times sweeter than that of sucrose, and the sweetness is long-lasting. As the concentration increases, the retention time in the mouth will also be prolonged. When the concentration is 0.025%, the sweetness can be retained in the mouth for more than 60 minutes.

[0243] A sweet protein composition containing 0.01% mass concentration of sweet protein (Brazilian sweet protein) was configured. After adding different amounts of mannan (brand Kemike, item number Cl8127397) and / or glucan, the sensory evaluation results are shown in Tables 2 and 3. The addition amount ranged from 0.1% to 7%, and there was no significant effect on the sweetness of the 0.01% sweet protein composition. After adding more than 1% of mannan or glucan alone, the delayed sweetness of the sweet protein composition increased. When glucan and mannan were mixed and added, and the total amount was more than 0.5%, the delayed sweetness of the sweet protein composition increased. The above results show that glucan and / or mannan play an assisting role in the delayed sweetness of the sweet protein composition, and the superimposed application of glucan and / or mannan has a better effect. The microorganisms prepare the sweet protein product, and the residual mannan and / or glucan in the product, or the additionally added mannan and / or glucan, assists the sweetness delay of the sweet protein composition.

[0244] Table 2

[0245]

[0246] Table 3

[0247]

[0248] Example 5: Application of sweet protein composition in oral care products 5.1: Application of sweet protein composition in toothpaste

[0249] 5.1.1: Formulation design

[0250] a.Basic formula:

[0251] Sodium fluoride (anti-caries agent): 0.24%

[0252] Sweet protein composition (Brazilian thauma): 0.1%

[0253] Calcium hydrogen phosphate dihydrate (abrasive): 20%

[0254] Glycerin (humectant): 20%

[0255] Sorbitol (humectant): 10%

[0256] Carbomer (thickener): 1.5%

[0257] Natural flavor (mint flavor): 1%

[0258] Deionized water: 47.21%.

[0259] b. Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.02%, 0.05%, 0.1%, and 0.2%.

[0260] 5.1.2: Preparation of toothpaste

[0261] A. Dissolving the sweet protein: Dissolve the sweet protein composition in deionized water and stir until completely dissolved to ensure that there are no undissolved particles.

[0262] B. Mixing: Add glycerol, sorbitol, carbomer and calcium hydrogen phosphate dihydrate to the sweet protein solution and stir thoroughly to form a homogeneous toothpaste base.

[0263] C. Add flavors and anti-caries agents: Add sodium fluoride and natural flavors and continue stirring until uniform.

[0264] D. Adjust viscosity: Adjust the amount of carbomer as needed to achieve the desired viscosity.

[0265] E. Filling: Fill the prepared toothpaste into the toothpaste tube, seal it and store it at room temperature.

[0266] 5.1.3: Sensory testing

[0267] (1) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score toothpastes with different additive amounts, evaluate their sweetness intensity compared with other toothpastes on the market, and determine the optimal additive amount.

[0268] (2) Taste test: Further sensory testing was conducted on the toothpaste with the optimal amount of addition to evaluate its taste, aftertaste, aroma, overall acceptance and pleasantness. A questionnaire survey was used to record the subjective feelings of the subjects and statistical analysis was performed.

[0269] (3) Dopamine secretion detection: Some subjects were selected for saliva sampling to detect the changes in dopamine levels in saliva before and after using toothpaste. High performance liquid chromatography-mass spectrometry (HPLC-MS) was used for analysis to evaluate the dopamine secretion after toothpaste use.

[0270] Result analysis: According to the feedback data of the subjects and the dopamine test results, the optimal addition amount of the sweet protein composition was determined to be 0.05%. Data analysis showed that the use of toothpaste containing the sweet protein composition can significantly improve the sense of pleasure and dopamine secretion level.

[0271] 5.1.4: Stability testing

[0272] 1) Storage stability: The prepared toothpaste was stored at 4°C and 25°C, and samples were taken at 0, 1, 3, 6, and 12 months to test the activity, appearance, and viscosity of the sweet protein.

[0273] 2) Accelerated aging test: The toothpaste was placed in a 40°C environment for 30 days, and the activity of the sweet protein and the sensory properties of the toothpaste were tested weekly to evaluate its long-term storage stability.

[0274] 3) Result analysis: The sweet protein remained stable during storage, the sweetness intensity and taste did not change significantly, and the appearance and viscosity of the toothpaste remained good.

[0275] 5.2: Application of sweet protein composition in mouthwash

[0276] 5.2.1: Formulation design

[0277] (a) Basic formula:

[0278] Sodium fluoride (anti-caries agent): 0.05%

[0279] Sweet protein composition (Brazilian thauma): 0.05%

[0280] Glycerin (humectant): 10%

[0281] Ethanol (preservative): 5%

[0282] Natural flavors (such as mint flavor): 0.5%

[0283] Citric acid (acidity regulator): 0.2%

[0284] Deionized water: 84.22%.

[0285] (b) Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.01%, 0.03%, 0.05%, and 0.1%.

[0286] 5.2.2: Preparation of mouthwash

[0287] (A) Dissolving the sweet protein: Dissolve the sweet protein composition in deionized water and stir until completely dissolved to ensure that there are no undissolved particles.

[0288] (B) Mixing: Add glycerol, ethanol, citric acid and natural flavors to the sweetened protein solution and mix thoroughly.

[0289] (C) Adding anticaries agent: Add sodium fluoride and continue stirring until uniform.

[0290] (D) Homogenization: Use a homogenizer to homogenize the mouthwash to ensure even distribution of ingredients and a delicate taste.

[0291] (E) Filling: Fill the prepared mouthwash into bottles under a sterile environment, seal them and store them at room temperature.

[0292] 5.2.3: Sensory testing

[0293] (1) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score mouthwashes with different added amounts, evaluate their sweetness intensity compared with other mouthwashes on the market, and determine the optimal addition amount.

[0294] (2) Taste test: Further sensory testing was conducted on the mouthwash with the optimal amount of addition to evaluate its taste, aftertaste, aroma, overall acceptance and pleasantness. A questionnaire survey was used to record the subjective feelings of the subjects and statistical analysis was performed.

[0295] (3) Dopamine secretion detection: Some subjects were selected for saliva sampling to detect the changes in dopamine levels in saliva before and after using mouthwash. High performance liquid chromatography-mass spectrometry (HPLC-MS) was used for analysis to evaluate the dopamine secretion after using mouthwash.

[0296] (4) Result analysis: Based on the feedback data from the subjects and the dopamine test results, the optimal addition amount of the sweet protein composition was determined to be 0.03%. Data analysis showed that the use of mouthwash containing the sweet protein composition can significantly improve the sense of pleasure and dopamine secretion level.

[0297] 5.2.4: Stability testing

[0298] 1) Storage stability: The prepared mouthwash was stored at 4°C and 25°C, and samples were taken at 0, 1, 3, 6, and 12 months to test the activity, pH value, appearance, and microbial indicators of the sweet protein.

[0299] 2) Accelerated aging test: The mouthwash was placed in a 40°C environment for 30 days and the activity of the sweet protein and the sensory properties of the mouthwash were tested weekly to evaluate its long-term storage stability.

[0300] 3) Result analysis: The sweet protein remained stable during storage, and there was no significant change in sweetness intensity and taste. The appearance and microbial indicators of the mouthwash met national standards.

[0301] Example 6: Application of sweet protein composition in food

[0302] 6.1: Application of sweet protein composition in low-calorie (sugar-free) beverages

[0303] 6.1.1: Formulation design

[0304] Basic recipe:

[0305] Water: 95%

[0306] Natural flavor: 1%

[0307] Citric acid (acidity regulator): 0.5%

[0308] Potassium sorbate (preservative): 0.1%

[0309] Sweet protein composition (Brasilan): 0.02%.

[0310] Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.01%, 0.02%, 0.05%, 0.1% and 0.2%.

[0311] 6.1.2: Preparation of low-calorie beverages

[0312] a. Dissolve the sweet protein: Dissolve the sweet protein composition in warm water and stir until completely dissolved to ensure that there are no undissolved particles.

[0313] b. Mixing: Add the dissolved sweet protein composition to the main formula, and then add natural flavors, acidity regulators and preservatives, and stir thoroughly.

[0314] c. Homogenization: Use a high-pressure homogenizer to homogenize the mixture to ensure even distribution of ingredients and a delicate taste.

[0315] d. Sterilization: The homogenized beverage is pasteurized, usually at 85°C for 15 minutes, to ensure the microbiological safety of the beverage.

[0316] e. Filling: Fill the sterilized beverage into PET bottles or glass bottles under a sterile environment, seal them and store them in a refrigerated environment at 4°C.

[0317] 6.1.3: Sensory testing

[0318] a) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score beverages with different added amounts, evaluate their sweetness intensity compared with sucrose solution, and determine the optimal addition amount.

[0319] b) Taste test: The beverages with the optimal addition amount were further subjected to sensory testing to evaluate their taste, aftertaste, aroma and overall acceptance.

[0320] c) Result analysis: According to the feedback data from the subjects, it was determined that the optimal addition amount of the sweet protein composition was 0.02%.

[0321] 6.1.4: Stability testing

[0322] A: Storage stability: Store the prepared low-calorie beverages at 4°C and 25°C, and take samples to test the activity, pH value, appearance and microbial indicators of the sweet protein at 0, 1, 3, 6 and 12 months.

[0323] B: Accelerated aging test: The beverage is placed in a 40°C environment for 30 days, and the activity of the sweet protein and the sensory properties of the beverage are tested weekly to evaluate its long-term storage stability.

[0324] C: Result analysis: The sweet protein remained stable during storage, with no significant changes in sweetness intensity and taste, and the microbial indicators met national standards.

[0325] 6.1.5: Nutritional analysis

[0326] A) Calorie determination: The final beverage was calorie determined, and the result showed that the calorie content of each 100 ml of the beverage did not exceed 10 kcal, which met the standard for low-calorie beverages.

[0327] B) Other ingredient testing: Testing the main nutrients in the beverage, such as protein, carbohydrates, fat, vitamins and minerals, to ensure that they meet relevant food safety standards.

[0328] 6.2: Application of sweet protein composition in sugar-free chewing gum

[0329] 6.2.1: Formulation design

[0330] Basic recipe:

[0331] Gum base: 50%

[0332] Polyethylene glycol (PEG): 15%

[0333] Glycerin: 5%

[0334] Sweet protein composition (Brazilian thauma): 0.05%

[0335] Mint flavor (natural flavor): 2%

[0336] Citric acid: 0.2%

[0337] Potassium sorbate (preservative): 0.1%

[0338] Other ingredients (such as thickeners, antioxidants): 27.6%.

[0339] Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.05%, 0.1%, 0.2%, and 0.5%.

[0340] 6.2.2: Preparation of sugar-free chewing gum

[0341] a. Melting of the matrix: Heat the gum matrix to about 60°C until it is completely melted, keeping stirring to avoid sticking of the matrix.

[0342] b. Dissolving the sweet protein composition: Dissolve the sweet protein composition in the glycerol and PEG mixture and stir until completely dissolved to ensure that there are no undissolved particles.

[0343] c. Mixing: Add the sweet protein mixture, natural flavors, citric acid and preservatives to the melted gum base and mix thoroughly.

[0344] d. Molding: Pour the mixture into a pre-prepared mold, cool to room temperature, and remove from the mold after solidification.

[0345] e. Packaging: Cut and package the formed chewing gum, seal it and store it in a dry environment.

[0346] 6.2.3: Sensory testing

[0347] a) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score the chewing gums with different added amounts, evaluate their sweetness intensity compared with traditional chewing gums, and determine the optimal added amount.

[0348] b) Taste test: The chewing gum with the optimal addition amount was further subjected to sensory testing to evaluate its taste, aftertaste, aroma and overall acceptance.

[0349] c) Result analysis: According to the feedback data from the subjects, it was determined that the optimal addition amount of the sweet protein composition was 0.1%.

[0350] 6.2.4: Stability testing

[0351] A: Storage stability: Store the prepared sugar-free chewing gum at 4°C and 25°C, and take samples at 0, 1, 3, 6, and 12 months to test the activity, appearance, and microbial indicators of the sweet protein.

[0352] B: Accelerated aging test: The chewing gum is placed in an environment of 40°C for 30 days and the activity of the sweet protein and the sensory properties of the chewing gum are tested every week to evaluate its long-term storage stability.

[0353] C: Result analysis: The sweet protein remained stable during storage, with no significant changes in sweetness intensity and taste, and the microbial indicators met national standards.

[0354] 6.3: Application of sweet protein composition in children's snacks

[0355] 6.3.1: Formulation design

[0356] Basic recipe:

[0357] Wheat flour: 40%

[0358] Milk powder: 20%

[0359] Vegetable oil: 10%

[0360] Sweet protein composition (Brazilian thauma): 0.03%

[0361] Vanilla extract (natural flavor): 1%

[0362] Sodium bicarbonate (leavening agent): 0.5%

[0363] Other ingredients (such as thickeners, antioxidants): 28.47%.

[0364] Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.01%, 0.02%, 0.03%, and 0.05%.

[0365] 6.3.2: Preparing snacks for children

[0366] a. Mix dry ingredients: Mix wheat flour, milk powder, starter and other dry ingredients well.

[0367] b. Dissolving the sweet protein: Dissolve the sweet protein composition in an appropriate amount of water and stir until completely dissolved to ensure that there are no undissolved particles.

[0368] c. Mix liquid ingredients: Add sweetened egg white solution, vegetable oil and natural flavors to dry ingredients and mix thoroughly until a homogeneous dough forms.

[0369] d. Shaping and baking: Roll out the dough and cut into small pieces. Bake in a preheated oven at 180°C for 15 minutes until golden brown.

[0370] e. Cooling and packaging: After the baked children's snacks are cooled to room temperature, they are packaged, sealed and stored in a dry environment.

[0371] 6.3.3: Sensory testing

[0372] a) Sweetness intensity test: Invite 20 children and parents to conduct a blind test to rate snacks with different added amounts, evaluate their sweetness intensity compared with traditional sugary snacks, and determine the optimal added amount.

[0373] b) Taste test: Further sensory testing was conducted on the children’s snacks with the optimal addition amount to evaluate their taste, aftertaste, aroma and overall acceptability.

[0374] c) Result analysis: According to the feedback data from children and parents, it was determined that the optimal addition amount of the sweet protein composition was 0.03%.

[0375] 6.3.4: Nutritional analysis

[0376] A: Calorie measurement: The calorie content of the final children's snacks was measured, and the results showed that the calories per 100 grams of snacks did not exceed 300 kcal, which met the standards for low-calorie snacks.

[0377] B: Other ingredient testing: Test the main nutrients in snacks, such as protein, carbohydrates, fat, vitamins and minerals, to ensure that they meet relevant food safety standards.

[0378] 6.3.5: Stability testing

[0379] A) Storage stability: The prepared children's snacks were stored at 4°C and 25°C, and samples were taken at 0, 1, 3, 6, and 12 months to test the activity, appearance, and microbiological indicators of the sweet protein.

[0380] B) Accelerated aging test: The snacks were placed in an environment of 40°C for 30 days for accelerated aging test, and the activity of the sweet protein and the sensory properties of the snacks were tested weekly to evaluate their long-term storage stability.

[0381] C) Result analysis: The sweet protein remained stable during storage, with no significant changes in sweetness intensity and taste, and the microbial indicators met national standards.

[0382] Example 7: Application of sweet protein composition in health (food) products

[0383] 7.1: Application of sweet protein composition in weight loss products

[0384] 7.1.1: Formulation design

[0385] Basic recipe:

[0386] Green tea extract, L-carnitine: 10%

[0387] Sweet protein composition (Brazilian thauma): 0.02%

[0388] Microcrystalline cellulose (filler): 20%

[0389] Dextrin (carrier): 10%

[0390] Cellulose gum (thickener): 5%

[0391] Magnesium stearate (lubricant): 1%

[0392] Lemon flavor (natural flavor): 1%

[0393] Other ingredients (such as antioxidants, stabilizers): 52.98%.

[0394] Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.01%, 0.02%, 0.03%, and 0.05%.

[0395] 7.1.2: Formulating weight loss products

[0396] a. Mix dry ingredients: Mix the active ingredients, sweet protein composition, microcrystalline cellulose, dextrin, cellulose gum, natural flavors and other dry ingredients evenly.

[0397] b. Tableting: Compress the mixture into tablets to control the weight and hardness of each tablet.

[0398] c. Coating: The tablets are film-coated to improve taste and appearance.

[0399] d. Packaging: Package the coated tablets, seal them and store them in a dry environment.

[0400] 7.1.3: Sensory testing

[0401] a) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score the weight loss tablets with different added amounts, evaluate their sweetness intensity compared with traditional weight loss tablets, and determine the optimal added amount.

[0402] b) Taste test: The weight loss tablets with the optimal addition amount were further subjected to sensory testing to evaluate their taste, aftertaste, aroma and overall acceptance.

[0403] c) Result analysis: According to the feedback data from the subjects, it was determined that the optimal addition amount of the sweet protein composition was 0.02%.

[0404] 7.1.4: Stability Testing

[0405] A: Storage stability: The formulated weight loss tablets were stored at 4°C and 25°C, and samples were taken at 0, 1, 3, 6, and 12 months to test the activity, appearance, and hardness of the sweet protein.

[0406] B: Accelerated aging test: The tablets were placed in an environment of 40°C for 30 days for accelerated aging test. The activity of the sweet protein and the sensory properties of the tablets were tested weekly to evaluate their long-term storage stability.

[0407] C: Result analysis: The results showed that the sweet protein remained stable during storage, with no significant changes in sweetness intensity and taste, and the tablet hardness and appearance remained good.

[0408] 7.2: Application of sweet protein compositions in antioxidant supplements

[0409] 7.2.1: Formulation design

[0410] Vitamin C, Vitamin E: 20%

[0411] Sweet protein composition (Brazilian thauma): 0.03%

[0412] Plant cellulose: 10%

[0413] Orange flavor (natural flavor): 0.5%

[0414] Citric acid (acidity regulator): 0.2%

[0415] Purified water: 69.27%.

[0416] Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.02%, 0.03%, 0.04%, and 0.05%.

[0417] 7.2.2: Preparation of antioxidant beverages

[0418] a. Dissolving the sweet protein: Dissolve the sweet protein composition in part of the purified water and stir until completely dissolved to ensure that there are no undissolved particles.

[0419] b. Mixing: Add active ingredients, plant cellulose, citric acid and natural flavors into the sweet protein solution and stir evenly.

[0420] c. Replenish water: Add the remaining purified water to the mixture and stir well.

[0421] d. Homogenization: Use a high-pressure homogenizer to homogenize the beverage to ensure even distribution of ingredients and a delicate taste.

[0422] e. Sterilization: The homogenized beverage is pasteurized, usually at 85°C for 15 minutes, to ensure the microbiological safety of the product.

[0423] f. Filling: Fill the sterilized beverage into PET bottles or glass bottles under a sterile environment, seal them and store them in a refrigerated environment at 4°C.

[0424] 7.2.3: Sensory testing

[0425] a) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score antioxidant drinks with different added amounts, evaluate their sweetness intensity compared with other products on the market, and determine the optimal addition amount.

[0426] b) Taste test: The antioxidant beverages with the optimal addition amount were further subjected to sensory testing to evaluate their taste, aftertaste, aroma and overall acceptability.

[0427] c) Result analysis: According to the feedback data from the subjects, it was determined that the optimal addition amount of the sweet protein composition was 0.03%.

[0428] 7.2.4: Stability testing

[0429] A: Storage stability: The prepared antioxidant beverage was stored at 4°C and 25°C, and samples were taken at 0, 1, 3, 6, and 12 months to test the activity, pH value, appearance, and microbial indicators of the sweet protein.

[0430] B: Accelerated aging test: The beverage is placed in a 40°C environment for 30 days, and the activity of the sweet protein and the sensory properties of the beverage are tested weekly to evaluate its long-term storage stability.

[0431] C: Result analysis: The sweet protein remained stable during storage, with no significant changes in sweetness intensity and taste, and the microbial indicators met national standards.

[0432] 7.3: Application of sweet protein composition in health products for enhancing immunity

[0433] 7.3.1: Formulation design

[0434] Basic recipe:

[0435] β-Glucan, Vitamin D: 15%

[0436] Sweet protein composition (Brazilian thauma): 0.04%

[0437] Plant cellulose: 10%

[0438] Apple flavor (natural flavor): 0.5%

[0439] Citric acid (acidity regulator): 0.3%

[0440] Purified water: 74.16%.

[0441] Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.03%, 0.04%, 0.05%, and 0.06%.

[0442] 7.3.2: Preparation of immunity-enhancing beverages

[0443] a. Dissolving the sweet protein: Dissolve the sweet protein composition in part of the purified water and stir until completely dissolved to ensure that there are no undissolved particles.

[0444] b. Mixing: Add active ingredients, plant cellulose, citric acid and natural flavors into the sweet protein solution and stir evenly.

[0445] c. Replenish water: Add the remaining purified water to the mixture and stir well.

[0446] d. Homogenization: Use a high-pressure homogenizer to homogenize the beverage to ensure even distribution of ingredients and a delicate taste.

[0447] e. Sterilization: The homogenized beverage is pasteurized, usually at 85°C for 15 minutes, to ensure the microbiological safety of the product.

[0448] f. Filling: Fill the sterilized beverage into PET bottles or glass bottles under a sterile environment, seal them and store them in a refrigerated environment at 4°C.

[0449] 7.3.3: Sensory testing

[0450] a) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score the immunity-enhancing drinks with different added amounts, evaluate their sweetness intensity compared with other products on the market, and determine the optimal addition amount.

[0451] b) Taste test: The immunity-enhancing beverage with the optimal addition amount was further subjected to sensory testing to evaluate its taste, aftertaste, aroma and overall acceptance.

[0452] c) Result analysis: According to the feedback data from the subjects, it was determined that the optimal addition amount of the sweet protein composition was 0.04%.

[0453] 7.3.4: Stability testing

[0454] A: Storage stability: Store the formulated immunity-enhancing beverage at 4°C and 25°C, and take samples at 0, 1, 3, 6, and 12 months to test the activity, pH value, appearance, and microbial indicators of the sweet protein.

[0455] B: Accelerated aging test: The beverage is placed in a 40°C environment for 30 days, and the activity of the sweet protein and the sensory properties of the beverage are tested weekly to evaluate its long-term storage stability.

[0456] C: Result analysis: The sweet protein remained stable during storage, with no significant changes in sweetness intensity and taste, and the microbial indicators met national standards.

[0457] Example 8: Application of sweet protein composition in dairy products

[0458] 8.1: Application of sweet protein composition in low-calorie yogurt

[0459] 8.1.1: Formulation design

[0460] Basic recipe:

[0461] Skim milk powder: 10%

[0462] Sweet protein composition (Brazilian thauma): 0.02%

[0463] Lactic acid bacteria starter: 0.1%

[0464] Pectin (thickener): 0.5%

[0465] Vanilla extract (natural flavor): 0.1%

[0466] Purified water: 89.28%.

[0467] Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.01%, 0.02%, 0.03%, and 0.05%.

[0468] 8.1.2: Preparation of low-calorie yogurt

[0469] a. Mix ingredients: Dissolve skimmed milk powder in purified water, add pectin and natural flavors, and stir well.

[0470] b. Heating and homogenization: Heat the mixture to 85°C and keep it for 10 minutes for pasteurization, followed by homogenization.

[0471] c. Cooling and fermentation: Cool the pasteurized mixed solution to 42°C, add the lactic acid bacteria starter and the sweet protein composition, and stir evenly. Put the mixed solution into a sterile fermentation tank and ferment at 42°C for 6-8 hours until the desired acidity and consistency are reached.

[0472] d. Refrigeration and packaging: After fermentation is completed, the yogurt is cooled to 4°C, divided into sterile packaging containers, sealed and stored in a refrigerated environment.

[0473] 8.1.3: Sensory testing

[0474] a) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score yogurts with different added amounts, evaluate their sweetness intensity compared with other low-calorie yogurts on the market, and determine the optimal addition amount.

[0475] b) Taste test: The yogurt with the optimal addition amount was further subjected to sensory testing to evaluate its taste, consistency, aroma and overall acceptance.

[0476] c) Result analysis: According to the feedback data from the subjects, it was determined that the optimal addition amount of the sweet protein composition was 0.02%.

[0477] 8.1.4: Stability Testing

[0478] A: Storage stability: Store the prepared low-calorie yogurt at 4°C, and take samples at 0, 1, 3, and 6 months to test the activity, pH value, appearance, and microbial indicators of the sweet protein.

[0479] B: Result analysis: The sweet protein remained stable during the storage of yogurt, the sweetness intensity and taste did not change significantly, and the microbial indicators met the national standards.

[0480] 8.2: Application of sweet protein composition in low-calorie milkshake

[0481] 8.2.1: Formulation design

[0482] Basic recipe:

[0483] Skim milk: 60%

[0484] Sweet protein composition (Brazilian thauma): 0.03%

[0485] Pea protein (plant protein powder): 5%

[0486] Oat fiber (thickener): 1%

[0487] Chocolate flavor (natural flavor): 1%

[0488] Ice cubes: 33%.

[0489] Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.01%, 0.02%, 0.03%, and 0.05%.

[0490] 8.2.2: Preparing Low-Calorie Milkshakes

[0491] a. Mix ingredients: Add skim milk, plant protein powder, oat fiber and sweetened protein composition into a blender and mix well.

[0492] b. Add flavors: Add natural flavors and ice cubes and continue blending until it becomes a smooth milkshake.

[0493] c. Filling: Pour the evenly mixed milkshake into cups, seal them and store them in a refrigerated environment.

[0494] 8.2.3: Sensory testing

[0495] a) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score milkshakes with different added amounts, evaluate their sweetness intensity compared with other low-calorie milkshakes on the market, and determine the optimal added amount.

[0496] b) Taste test: The milkshakes with the optimal addition amount were further subjected to sensory testing to evaluate their taste, consistency, aroma and overall acceptability.

[0497] c) Result analysis: According to the feedback data from the subjects, it was determined that the optimal addition amount of the sweet protein composition was 0.03%.

[0498] 8.2.4: Stability testing

[0499] A: Storage stability: Store the prepared low-calorie milkshake at 4°C and take samples at 0, 1, 3, and 6 months to test the activity, pH value, appearance, and microbial indicators of the sweet protein.

[0500] B: Result analysis: The sweet protein remained stable during the storage of the milkshake, with no significant changes in sweetness intensity and taste, and the microbial indicators met national standards.

[0501] Example 9: Application of sweet protein composition in baked products

[0502] 9.1: Application of sweet protein composition in low-sugar cakes

[0503] 9.1.1: Formulation design

[0504] Basic recipe:

[0505] Cake flour: 40%

[0506] Sweet protein composition (Brazilian thauma): 0.05%

[0507] Eggs: 20%

[0508] Butter: 10%

[0509] Milk: 15%

[0510] Baking powder: 1%

[0511] Vanilla extract: 0.5%

[0512] Salt: 0.5%

[0513] Water: 13.95%.

[0514] b. Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.02%, 0.05%, 0.1%, and 0.2%.

[0515] 9.1.2: Preparing low-sugar cakes

[0516] a. Mix dry ingredients: Sift together cake flour, baking powder and salt, mix well and set aside.

[0517] b. Mix wet ingredients: Dissolve the sweet protein composition in water, add eggs, butter, milk and vanilla extract, and mix well.

[0518] c. Prepare the cake batter: Gradually add the dry ingredients to the wet ingredients and mix with an electric mixer until smooth, making sure there are no lumps.

[0519] d. Bake: Pour the cake batter into a pre-greased and floured cake pan, place in an oven preheated to 180°C, and bake for 25-30 minutes, until the surface is golden and a toothpick inserted comes out clean.

[0520] e. Cooling and packaging: Take the baked cake out of the mold and place it on a cooling rack to cool to room temperature. After cooling, package it, seal it and store it in a dry environment.

[0521] 9.1.3: Sensory testing

[0522] a) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score cakes with different amounts of added sweetener, evaluate their sweetness intensity compared with traditional cakes, and determine the optimal amount of added sweetener.

[0523] b) Taste test: The cakes with the optimal addition amount were further subjected to sensory testing to evaluate their taste, consistency, aroma and overall acceptability.

[0524] c) Result analysis: According to the feedback data from the subjects, it was determined that the optimal addition amount of the sweet protein composition was 0.05%.

[0525] 9.1.4: Stability Testing

[0526] A: Storage stability: Store the prepared low-sugar cakes at 4°C and 25°C, and take samples at 0, 1, 3, and 6 months to test the activity, pH value, appearance, and microbial indicators of the sweet protein.

[0527] B: Result analysis: The sweet protein remained stable during the storage of the cake, with no significant changes in sweetness intensity and taste, and the microbial indicators met national standards.

[0528] 9.2: Application of sweet protein composition in low-sugar cakes

[0529] 9.2.1: Formulation design

[0530] Basic recipe:

[0531] Cake flour: 50%

[0532] Sweet protein composition (Brazilian thauma): 0.04%

[0533] Butter: 15%

[0534] Eggs: 10%

[0535] Baking soda: 1%

[0536] Vanilla extract: 0.5%

[0537] Salt: 0.5%

[0538] Water: 22.96%.

[0539] Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.02%, 0.04%, 0.06%, and 0.08%.

[0540] 9.2.2: Preparation of sugar-free cookies

[0541] a. Mix dry ingredients: Sift together cake flour, baking soda and salt and set aside.

[0542] b. Mix wet ingredients: Dissolve the sweet protein composition in water, add melted butter, eggs and vanilla extract, and stir well.

[0543] c. Prepare cookie dough: Gradually add dry ingredients to wet ingredients and mix using electric mixer until smooth to form cookie dough.

[0544] d. Shaping and baking: Roll out the dough to about 0.5 cm thick, cut out cookie shapes using a cookie cutter, and place on a baking tray lined with baking paper. Place the baking tray in an oven preheated to 175°C and bake for 12-15 minutes until the cookie surface is golden brown.

[0545] e. Cooling and packaging: Take the baked cookies out of the baking tray and place them on a cooling rack to cool to room temperature. After cooling, package them, seal them and store them in a dry environment.

[0546] 9.2.3: Sensory testing

[0547] a) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score the cookies with different added amounts, evaluate their sweetness intensity compared with traditional cookies, and determine the optimal amount of addition.

[0548] b) Taste test: The biscuits with the best addition amount were further subjected to sensory testing to evaluate their taste, crispness, aroma and overall acceptance.

[0549] c) Result analysis: According to the feedback data from the subjects, it was determined that the optimal addition amount of the sweet protein composition was 0.04%.

[0550] 9.2.4: Stability Testing

[0551] A: Storage stability: Store the prepared sugar-free biscuits at 4°C and 25°C, and take samples at 0, 1, 3, and 6 months to test the activity, pH value, appearance, and microbial indicators of the sweet protein.

[0552] B: Result analysis: The sweet protein remained stable during the storage of biscuits, the sweetness intensity and taste did not change significantly, and the microbial indicators met the national standards.

[0553] 9.3: pH stability test

[0554] 9.3.1: Experimental Design

[0555] Sample preparation: The prepared low-sugar cake and sugar-free biscuit samples were placed in buffer solutions with different pH values ​​(pH 4, pH 6, pH 8).

[0556] Testing time points: sampling for testing at 0, 1, 3, and 6 months.

[0557] 9.3.2: Detection indicators

[0558] A) Sweet protein activity: High performance liquid chromatography (HPLC) was used to detect the content of sweet protein and evaluate its stability under different pH conditions.

[0559] B) Sensory properties: The appearance, smell and taste of the samples are evaluated through sensory evaluation to ensure that the product does not change significantly under different pH conditions.

[0560] C) pH determination: At each testing time point, the pH value of the sample was re-measured to monitor its changes.

[0561] D) Microbiological indicators: Through the detection of total colony count, coliform group and mold and yeast, ensure that the samples are free of microbial contamination under different pH conditions.

[0562] 9.3.3: Result Analysis

[0563] (i) Sweet protein activity: The results showed that the activity of sweet protein was well maintained in the range of pH 4 to pH 8, with the retention rate being above 85% within 6 months.

[0564] (ii) Sensory properties: The sensory properties of all samples remained good at all testing time points, with no obvious changes in appearance, odor and taste.

[0565] (iii) pH value determination: The pH value of the samples remained stable at each testing time point without significant changes.

[0566] (iv) Microbiological indicators: The microbiological indicators of all samples at each testing time point met the national standards and there was no microbial contamination.

[0567] 9.4: Accelerated aging test

[0568] 9.4.1: Experimental Design

[0569] i) Sample preparation: The prepared low-sugar cake and sugar-free biscuit samples were placed in a 40°C environment for accelerated aging testing.

[0570] ⅱ) Testing time points: sampling and testing were carried out at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 weeks.

[0571] 9.4.2: Detection indicators

[0572] Sweet protein activity: The sweet protein content was detected using high performance liquid chromatography (HPLC) and its stability under accelerated aging conditions was evaluated.

[0573] Sensory properties: The appearance, smell and taste of the samples are evaluated through sensory evaluation to ensure that there are no significant changes in the product during accelerated aging.

[0574] pH: Use a pH meter to measure the pH of the samples to ensure that it remains stable during accelerated aging.

[0575] Microbiological indicators: Through the total colony count, coliform bacteria and mold and yeast tests, ensure that the product is free of microbial contamination during accelerated aging.

[0576] 9.4.3: Result Analysis

[0577] (I) Sweet protein activity: The results showed that the activity of the sweet protein was well maintained under accelerated aging conditions, with a retention rate of more than 85% within 8 weeks and a retention rate of more than 75% within 12 weeks.

[0578] (II) Sensory properties: The sensory properties of all samples remained good at each testing time point, with no obvious changes in appearance, odor and taste.

[0579] (III) pH value: The pH value of the samples remained stable under accelerated aging conditions without significant changes.

[0580] (IV) Microbiological indicators: The microbiological indicators of all samples at each testing time point met the national standards and there was no microbial contamination.

[0581] 9.5: Data aggregation and analysis

[0582] 9.5.1: Storage stability

[0583] The results showed that the sweet protein composition had the best storage stability at 4°C and 25°C, and its activity and sensory properties were well maintained, making it suitable for long-term storage. At 40°C, the activity of the sweet protein decreased, but it still maintained a certain stability.

[0584] Recommendation: Store the sweet protein composition at 4°C and 25°C to ensure its optimal storage stability and product quality.

[0585] 9.5.2: pH stability

[0586] The results showed that the sweet protein composition had good stability in the range of pH 4 to pH 8 and was suitable for products in various acidic and alkaline environments.

[0587] Recommendation: Sweet protein compositions should be kept in the pH 4 to pH 8 range during application to ensure their optimal stability.

[0588] 9.5.3: Accelerated Aging

[0589] Summary: The results of the accelerated aging test showed that the sweet protein composition had good stability under high temperature conditions, and its long-term storage stability could be evaluated in a short time.

[0590] Recommendation: Use accelerated aging testing as a rapid method for stability assessment of sweet protein compositions to quickly assess their stability during new product development.

[0591] Through the above stability test, the stability of the sweet protein composition under various storage conditions and pH environments is verified, ensuring its good stability and reliability in application in different baked products.

[0592] 9.6: Nutritional analysis of low-sugar cakes and sugar-free biscuits

[0593] 9.6.1: Experimental Design

[0594] Sample preparation: Low-sugar cake and sugar-free biscuit samples were prepared separately for nutritional analysis.

[0595] Detection indicators: The main nutritional components of the test samples, including protein, fat, carbohydrates, cellulose, vitamins and minerals, etc.

[0596] 9.6.2: Result Analysis

[0597] Protein content: The results showed that the protein content in low-sugar cakes and sugar-free biscuits was 8% and 10% respectively, which met the nutritional requirements.

[0598] Fat Content: The fat content in low sugar cakes and sugar-free biscuits is 12% and 15% respectively, providing good taste and texture to the products.

[0599] Carbohydrate content: The carbohydrate content in low-sugar cakes and sugar-free biscuits is 30% and 40% respectively, which mainly comes from low-gluten flour and other ingredients.

[0600] Fiber content: The fiber content in low-sugar cakes and sugar-free biscuits is 2% and 3% respectively, which helps to improve the health attributes of the products.

[0601] Vitamins and minerals: Low-sugar cakes and sugar-free biscuits contain a certain amount of nutrients such as vitamin B, vitamin E, calcium and iron, which enhance the nutritional value of the product.

[0602] Example 10: Application of sweet protein composition in condiments

[0603] 10.1: Application of sweet protein composition in low-calorie ketchup

[0604] 10.1.1: Formulation Design

[0605] Basic recipe:

[0606] Tomato paste concentrate: 50%

[0607] Sweet protein composition (Brazilian thauma): 0.02%

[0608] White vinegar: 5%

[0609] Salt: 2%

[0610] Onion powder: 1%

[0611] Garlic powder: 0.5%

[0612] Paprika: 0.3%

[0613] Water: 41.18%.

[0614] Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.01%, 0.02%, 0.03%, and 0.05%.

[0615] 10.1.2: Preparation of low-calorie tomato sauce

[0616] a. Mix ingredients: Mix tomato paste concentrate, white vinegar, salt, onion powder, garlic powder and chili powder.

[0617] b. Dissolving the sweet protein: Dissolve the sweet protein composition in water and stir until completely dissolved to ensure that there are no undissolved particles.

[0618] c. Mixing and homogenization: Add the dissolved sweet protein liquid to the ketchup mixture and homogenize using a high shear homogenizer to ensure even distribution of the ingredients.

[0619] d. Pasteurization: The homogenized ketchup is pasteurized at 85°C for 15 minutes to ensure the microbiological safety of the product.

[0620] e. Cooling and packaging: Cool the pasteurized tomato paste to room temperature, divide it into sterile packaging bottles, seal them and store them at room temperature.

[0621] 10.1.3: Sensory testing

[0622] a) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score the tomato sauce with different addition amounts, evaluate its sweetness intensity compared with traditional tomato sauce, and determine the optimal addition amount.

[0623] b) Taste test: The ketchup with the optimal amount of addition was further subjected to sensory testing to evaluate its taste, consistency, aroma and overall acceptability.

[0624] c) Result analysis: According to the feedback data from the subjects, it was determined that the optimal addition amount of the sweet protein composition was 0.02%.

[0625] 10.1.4: Stability Testing

[0626] A: Storage stability: Store the prepared low-calorie ketchup at 4°C and 25°C, and take samples at 0, 1, 3, and 6 months to test the activity, pH value, appearance, and microbial indicators of the sweet protein.

[0627] B: Result analysis: The sweet protein remained stable during the storage of tomato sauce, the sweetness intensity and taste did not change significantly, and the microbial indicators met the national standards.

[0628] 10.2: Application of sweet protein composition in low-calorie ketchup

[0629] 10.2.1: Formulation Design

[0630] Mayonnaise base: 30%

[0631] Sweet protein composition (Brazilian thauma): 0.03%

[0632] White vinegar: 5%

[0633] Lemon juice: 2%

[0634] Salt: 1%

[0635] Garlic paste: 0.5%

[0636] Olive oil: 10%

[0637] Water: 51.47%.

[0638] Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.02%, 0.03%, 0.04%, and 0.05%.

[0639] 10.2.2: Preparation of sugar-free salad dressing

[0640] a. Mix ingredients: Mix mayonnaise base, white vinegar, lemon juice, salt and mashed garlic.

[0641] b. Dissolving the sweet protein: Dissolve the sweet protein composition in water and stir until completely dissolved to ensure that there are no undissolved particles.

[0642] c. Mixing and homogenization: Gradually add the dissolved sweetened protein liquid and olive oil to the salad dressing mixture and homogenize using a high shear homogenizer to ensure even distribution of the ingredients.

[0643] d. Pasteurization: The homogenized salad dressing is pasteurized at 85°C for 10 minutes to ensure the microbiological safety of the product.

[0644] e. Cooling and packaging: Cool the pasteurized salad dressing to room temperature, divide it into sterile packaging bottles, seal them and store them at room temperature.

[0645] 10.2.3: Sensory testing

[0646] a) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score salad dressings with different addition amounts, evaluate their sweetness intensity compared with traditional salad dressings, and determine the optimal addition amount.

[0647] b) Taste test: The salad dressing with the optimal amount of addition was further subjected to sensory testing to evaluate its taste, consistency, aroma and overall acceptability.

[0648] c) Result analysis: According to the feedback data from the subjects, it was determined that the optimal addition amount of the sweet protein composition was 0.03%.

[0649] 10.2.4: Stability Testing

[0650] A: Storage stability: Store the prepared sugar-free salad dressing at 4°C and 25°C, and take samples at 0, 1, 3, and 6 months to test the activity, pH value, appearance, and microbial indicators of the sweet protein.

[0651] B: Result analysis: The sweet protein remained stable during the storage of the salad dressing, and there was no obvious change in the sweetness intensity and taste. The microbial indicators met the national standards.

[0652] 10.3: pH stability test

[0653] 10.3.1: Experimental Design

[0654] Sample preparation: The prepared low-calorie ketchup and sugar-free salad dressing samples were placed in buffer solutions with different pH values ​​(pH 4, pH 6, pH 8).

[0655] Testing time points: sampling for testing at 0, 1, 3, and 6 months.

[0656] 10.3.2: Detection indicators

[0657] A) Sweet protein activity: High performance liquid chromatography (HPLC) was used to detect the content of sweet protein and evaluate its stability under different pH conditions.

[0658] B) Sensory properties: The appearance, smell and taste of the samples are evaluated through sensory evaluation to ensure that the product does not change significantly under different pH conditions.

[0659] C) pH determination: At each testing time point, the pH value of the sample was re-measured to monitor its changes.

[0660] D) Microbiological indicators: Through the detection of total colony count, coliform group and mold and yeast, ensure that the samples are free of microbial contamination under different pH conditions.

[0661] 10.3.3: Result Analysis

[0662] Sweet protein activity: The results showed that the activity of sweet protein was well maintained in the range of pH 4 to pH 8, with the retention rate being above 85% within 6 months.

[0663] Sensory properties: The sensory properties of all samples remained good at each testing time point, with no obvious changes in appearance, odor and taste.

[0664] pH determination: The pH value of the sample remained stable at each testing time point without significant changes.

[0665] Microbiological indicators: The microbiological indicators of all samples at each testing time point meet the national standards and there is no microbial contamination.

[0666] 10.4: Accelerated Aging Test

[0667] 10.4.1: Experimental Design

[0668] Sample preparation: The prepared low-calorie ketchup and sugar-free salad dressing samples were placed in a 40°C environment for accelerated aging testing.

[0669] Testing time points: samples were taken for testing at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 weeks.

[0670] 10.4.2: Detection indicators

[0671] Sweet protein activity: The sweet protein content was detected using high performance liquid chromatography (HPLC) and its stability under accelerated aging conditions was evaluated.

[0672] Sensory properties: The appearance, smell and taste of the samples are evaluated through sensory evaluation to ensure that there are no significant changes in the product during accelerated aging.

[0673] pH: Use a pH meter to measure the pH of the samples to ensure that it remains stable during accelerated aging.

[0674] Microbiological indicators: Through the total colony count, coliform bacteria and mold and yeast tests, ensure that the product is free of microbial contamination during accelerated aging.

[0675] 10.4.3: Result Analysis

[0676] Sweet protein activity: The results showed that the activity of sweet protein was well maintained under accelerated aging conditions, with a retention rate of more than 85% within 8 weeks and more than 75% within 12 weeks.

[0677] Sensory properties: The sensory properties of all samples remained good at each testing time point, with no obvious changes in appearance, odor and taste.

[0678] pH value: The pH value of the samples remained stable under accelerated aging conditions without significant changes.

[0679] Microbiological indicators: The microbiological indicators of all samples at each testing time point meet the national standards and there is no microbial contamination.

[0680] 10.5: Data aggregation and analysis

[0681] 10.5.1: Storage stability

[0682] The results showed that the sweet protein composition had the best storage stability at 4°C and 25°C, and its activity and sensory properties were well maintained, making it suitable for long-term storage. At 40°C, the activity of the sweet protein decreased, but it still maintained a certain stability.

[0683] 10.6: Nutritional analysis of low-calorie ketchup and sugar-free salad dressing

[0684] 10.6.1: Experimental Design

[0685] Sample preparation: Low-calorie ketchup and sugar-free salad dressing samples were prepared separately for nutrient analysis.

[0686] Detection indicators: The main nutritional components of the test samples, including protein, fat, carbohydrates, cellulose, vitamins and minerals, etc.

[0687] 10.6.2: Result Analysis

[0688] Protein content: The results showed that the protein content in low-calorie ketchup and sugar-free salad dressing was 1.5% and 1% respectively, which met the nutritional requirements.

[0689] Fat Content: The fat content in low-calorie ketchup and sugar-free salad dressing is 0.5% and 11% respectively, providing good taste and texture to the products.

[0690] Carbohydrate content: The carbohydrate content in low-calorie ketchup and sugar-free salad dressing is 10% and 5%, respectively, mainly from ketchup concentrate and mayonnaise base.

[0691] Fiber content: The fiber content in low-calorie ketchup and sugar-free salad dressing is 2% and 1% respectively, which helps to improve the health attributes of the products.

[0692] Vitamins and minerals: Low-calorie ketchup and sugar-free salad dressing contain a certain amount of nutrients such as vitamin C, vitamin E, calcium and iron, which enhance the nutritional value of the product.

[0693] 10.6.3: Summary

[0694] The above nutritional analysis verifies the superiority of low-calorie ketchup and sugar-free salad dressing in nutritional value, ensuring that they provide healthier choices while meeting consumers' taste needs.

[0695] Example 11: Application of sweet protein composition in beverages

[0696] 11.1: Application of sweet protein composition in low-calorie carbonated beverages

[0697] 11.1.1: Formulation Design

[0698] Basic recipe:

[0699] Carbonated water: 90%

[0700] Sweet protein composition (Brazilian thauma): 0.05%

[0701] Citric acid: 0.2%

[0702] Lemon flavor (natural flavor): 0.3%

[0703] Salt: 0.02%

[0704] Potassium sorbate (preservative): 0.1%

[0705] Water: 9.33%.

[0706] Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.01%, 0.05%, 0.1%, and 0.2%.

[0707] 11.1.2: Preparation of Low-Calorie Carbonated Beverages

[0708] a. Dissolving the sweet protein: Dissolve the sweet protein composition in water and stir until completely dissolved to ensure that there are no undissolved particles.

[0709] b. Mix ingredients: Add citric acid, natural flavors, salt and preservatives to the dissolved sweet protein solution and stir well.

[0710] c. Carbonation: Inject the mixed liquid into the carbonation equipment for carbonation treatment, and control the carbon dioxide gas pressure to ensure the carbonation effect.

[0711] d. Filling and packaging: Fill the carbonated beverage into sterile bottles, seal them and store them in a refrigerated environment at 4°C.

[0712] 11.1.3: Sensory testing

[0713] a) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score carbonated beverages with different added amounts, evaluate their sweetness intensity compared with other low-calorie carbonated beverages on the market, and determine the optimal addition amount.

[0714] b) Taste test: Further sensory testing was conducted on the carbonated beverages with the optimal addition amount to evaluate their taste, bubble sensation, aroma and overall acceptance.

[0715] c) Result analysis: According to the feedback data from the subjects, it was determined that the optimal addition amount of the sweet protein composition was 0.05%.

[0716] 11.1.4: Stability Testing

[0717] A: Storage stability: The prepared low-calorie carbonated beverages were stored at 4°C and 25°C, and samples were taken at 0, 1, 3, and 6 months to test the activity, pH value, appearance, and bubble stability of the sweet protein.

[0718] B: Result analysis: The sweet protein remained stable during the storage of carbonated beverages, with no significant changes in sweetness intensity and taste, and good bubble stability.

[0719] 11.2: Application of sweet protein composition in sugar-free juice beverages

[0720] 11.2.1: Formulation Design

[0721] Basic recipe:

[0722] Pure juice: 50%

[0723] Sweet protein composition (Brazilian thauma): 0.03%

[0724] Citric acid: 0.2%

[0725] Orange flavor (natural flavor): 0.2%

[0726] Water: 49.57%.

[0727] Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.02%, 0.03%, 0.04%, and 0.05%.

[0728] 11.2.2: Preparation of sugar-free juice beverages

[0729] a. Dissolving the sweet protein: Dissolve the sweet protein composition in part of the water and stir until completely dissolved to ensure that there are no undissolved particles.

[0730] b. Mix ingredients: Add citric acid, natural flavors and remaining water to the dissolved sweetened protein solution and stir well.

[0731] c. Mixed juice: Mix pure juice with sweetened protein mixed liquid and stir well.

[0732] d. Pasteurization: The mixed liquid is pasteurized at 85°C for 15 minutes to ensure the microbiological safety of the product.

[0733] e. Filling and packaging: Fill the pasteurized juice beverage into sterile bottles, seal them and store them in a refrigerated environment at 4°C.

[0734] 11.2.3: Sensory testing

[0735] a) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score fruit juice drinks with different added amounts, evaluate their sweetness intensity compared with other sugar-free juices on the market, and determine the optimal addition amount.

[0736] b) Taste test: The fruit juice beverages with the optimal addition amount were further subjected to sensory testing to evaluate their taste, aroma and overall acceptance.

[0737] c) Result analysis: According to the feedback data from the subjects, it was determined that the optimal addition amount of the sweet protein composition was 0.03%.

[0738] 11.2.4: Stability Testing

[0739] A: Storage stability: Store the prepared sugar-free juice beverage at 4°C and 25°C, and take samples at 0, 1, 3, and 6 months to test the activity, pH value, appearance, and microbial indicators of the sweet protein.

[0740] B: Result analysis: The sweet protein remained stable during the storage of the juice beverage, the sweetness intensity and taste did not change significantly, and the microbial indicators met the national standards.

[0741] Example 12: Application of sweet protein composition in medicine

[0742] 12.1: Application of sweet protein composition in oral syrup

[0743] 12.1.1: Formulation Design

[0744] Basic recipe:

[0745] Antibiotics, cough medicine: 5%

[0746] Sweet protein composition (Brazilian thauma): 0.03%

[0747] Glycerin: 10%

[0748] Citric acid (acidity regulator): 0.2%

[0749] Potassium sorbate (preservative): 0.1%

[0750] Strawberry, cherry flavor: 1%

[0751] Purified water: 83.67%.

[0752] Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.01%, 0.02%, 0.03%, and 0.05%.

[0753] 12.1.2: Preparation of oral syrups

[0754] a. Dissolving the sweet protein: Dissolve the sweet protein composition in part of the purified water and stir until completely dissolved to ensure that there are no undissolved particles.

[0755] b. Mixing: Add active ingredients, glycerin, citric acid and flavors into the sweet protein solution and stir evenly.

[0756] c. Replenish water: Add the remaining purified water to the mixture and stir well.

[0757] d. Preservative treatment: Potassium sorbate is added to ensure that the syrup is not contaminated by microorganisms during storage.

[0758] e. Homogenization: Use a high-pressure homogenizer to homogenize the syrup to ensure even distribution of ingredients and a delicate taste.

[0759] f. Sterilization: The homogenized syrup is pasteurized, usually at 85°C for 15 minutes, to ensure the microbiological safety of the drug.

[0760] g. Filling: Fill the sterilized syrup into glass bottles or plastic bottles under a sterile environment, seal them and store them in a refrigerated environment at 4°C.

[0761] 12.1.3: Sensory testing

[0762] a) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score the oral syrups with different addition amounts, evaluate their sweetness intensity compared with traditional syrups, and determine the optimal addition amount.

[0763] b) Taste test: The oral syrup with the optimal addition amount was further subjected to sensory testing to evaluate its taste, aftertaste, aroma and overall acceptability.

[0764] c) Result analysis: According to the feedback data from the subjects, it was determined that the optimal addition amount of the sweet protein composition was 0.03%.

[0765] 12.1.4: Stability Testing

[0766] A: Storage stability: Store the prepared oral syrup at 4°C and 25°C, and take samples at 0, 1, 3, 6, and 12 months to test the activity, pH value, appearance, and microbial indicators of the sweet protein.

[0767] B: Accelerated aging test: The syrup was placed in an environment of 40°C for 30 days for accelerated aging test. The activity of sweet protein and the sensory properties of the syrup were tested weekly to evaluate its long-term storage stability.

[0768] C: Result analysis: The sweet protein remained stable during storage, with no significant changes in sweetness intensity and taste, and the microbial indicators met national standards.

[0769] 12.2: Application of sweet protein compositions in tablets and chewable tablets

[0770] 12.2.1: Formulation Design

[0771] Basic recipe:

[0772] Vitamins, minerals: 30%

[0773] Sweet protein composition (Brazilian thauma): 0.05%

[0774] Microcrystalline cellulose (filler): 35%

[0775] Sodium carboxymethyl cellulose (binder): 5%

[0776] Sorbitol (sweetener): 20%

[0777] Magnesium stearate (lubricant): 1%

[0778] Mint flavor (natural flavor): 1%

[0779] Other ingredients (such as thickeners, antioxidants): 8.95%.

[0780] Optimization of the amount of sweet protein composition added: In order to obtain the best sweet effect, a series of tests are required to determine the optimal amount of addition. The initial addition amount is set at 0.01%, 0.02%, 0.05%, and 0.1%.

[0781] 12.2.2: Formulation of tablets and chewable tablets

[0782] a. Dry mixing: Evenly mix the sweet protein composition with microcrystalline cellulose, sodium carboxymethyl cellulose, sorbitol, natural flavors and other auxiliary materials.

[0783] b. Wet granulation: The dry mixture is moistened and then granulated, and uniform granules are prepared by passing through a sieve.

[0784] c. Drying: Dry the wet granules to a moisture content of less than 5%.

[0785] d. Tabletting: Mix the dry granules with magnesium stearate and then tablet them to control the weight and hardness of each tablet.

[0786] e. Coating: For chewable tablets, you can choose a coating process to cover the tablet surface with a film coating material to improve the taste and appearance.

[0787] 12.2.3: Sensory testing

[0788] a) Sweetness intensity test: Invite 20 subjects to conduct a blind test to score tablets and chewable tablets with different added amounts, evaluate their sweetness intensity compared with traditional tablets, and determine the optimal added amount.

[0789] b) Taste test: Tablets and chewable tablets with the best addition amount were further subjected to sensory testing to evaluate their taste, aftertaste, aroma and overall acceptance.

[0790] c) Result analysis: According to the feedback data from the subjects, it was determined that the optimal addition amount of the sweet protein composition was 0.05%.

[0791] 12.2.4: Stability Testing

[0792] A) Storage stability: The formulated tablets and chewable tablets were stored at 4°C and 25°C, and samples were taken at 0, 1, 3, 6, and 12 months to test the activity, appearance, and hardness of the sweet protein.

[0793] B) Accelerated aging test: The tablets and chewable tablets were placed in an environment of 40°C for 30 days, and the activity of the sweet protein and the sensory properties of the tablets were tested weekly to evaluate their long-term storage stability.

[0794] C) Result analysis: The sweet protein remained stable during storage, with no significant changes in sweetness intensity and taste, and good hardness and appearance.

[0795] Example 13: Stability test of sweet protein composition

[0796] 13.1: Formulation and sample preparation

[0797] The products prepared in Examples 5-12 include low-calorie beverages, sugar-free chewing gum, children's snacks, oral syrups, tablets and chewable tablets, diet beverages, antioxidant beverages, immunity-enhancing beverages, toothpaste and mouthwash. The content of the sweet protein composition and related ingredients in each product formula are prepared according to the optimal addition amount and process to ensure the quality and consistency of each sample.

[0798] 13.2: Storage stability test

[0799] 13.2.1: Experimental Design

[0800] a. Sample preparation: The prepared sweet protein composition samples were stored in 4° C., 25° C. and 40° C. environments, respectively. Three groups of samples were prepared for each product so as to be tested at different time points.

[0801] b. Testing time points: sampling and testing at 0, 1, 3, 6, 9 and 12 months.

[0802] 13.2.2: Detection indicators

[0803] A: Sweet protein activity: The content of sweet protein was detected using high performance liquid chromatography (HPLC) to evaluate its stability under different storage conditions.

[0804] B: Sensory properties: The appearance, smell and taste of the samples are evaluated through sensory evaluation to ensure that there is no significant change in the product during storage.

[0805] C: pH: Use a pH meter to measure the pH of the sample to ensure it remains stable during storage.

[0806] D: Microbiological indicators: Through the detection of total colony count, coliform group and mold and yeast, ensure that the product is free of microbial contamination during storage.

[0807] E: Physical and chemical properties: Additional physical and chemical properties testing such as viscosity, solubility, etc. is performed on specific products (such as toothpaste, mouthwash).

[0808] 13.2.3: Result Analysis

[0809] (1) Sweet protein activity: The results showed that the activity of sweet protein was well maintained at 4°C and 25°C, with a retention rate of more than 90% within 12 months. In a 40°C environment, the activity of sweet protein decreased after 3 months, but the retention rate was still more than 80% within 6 months.

[0810] (2) Sensory properties: The sensory properties of all samples remained good at each testing time point, with no obvious changes in appearance, odor and taste.

[0811] (3) pH value: The pH value of the samples remained stable under all storage conditions without significant changes.

[0812] (4) Microbiological indicators: The microbiological indicators of all samples at each testing time point met the national standards and there was no microbial contamination.

[0813] (5) Physical and chemical properties: The viscosity of toothpaste and the solubility of mouthwash remain stable under all storage conditions without significant changes.

[0814] 13.3: pH stability test

[0815] 13.3.1: Experimental Design

[0816] a) Sample preparation: The sweet protein composition solution was adjusted to different pH values ​​(4, 5, 6, 7, 8), and three groups of samples were prepared for each pH value and stored in 4° C., 25° C., and 40° C. environments, respectively.

[0817] b) Testing time points: sampling and testing at 0, 1, 3, 6, 9 and 12 months.

[0818] 13.3.2: Detection indicators

[0819] A) Sweet protein activity: High performance liquid chromatography (HPLC) was used to detect the content of sweet protein under different pH conditions and to evaluate its stability under different storage conditions.

[0820] B) pH change: The pH change of samples was tested regularly to evaluate the stability of the sweet protein composition under different pH conditions.

[0821] C) Sensory properties: The appearance, smell and taste of the samples are evaluated through sensory evaluation to ensure that the product does not change significantly under different pH conditions.

[0822] D) Microbiological indicators: Through the total colony count, coliform group and mold and yeast tests, ensure that the product is free of microbial contamination under different pH conditions.

[0823] 13.3.3: Result Analysis

[0824] (a) Sweet protein activity: The results showed that the activity of sweet protein was well maintained within the pH range of 4 to 8, with a retention rate of more than 85% within 12 months. The activity of sweet protein was most stable at pH 6 and 7.

[0825] (b) pH value change: The pH value of the samples did not change significantly during storage, indicating good pH stability.

[0826] (c) Sensory properties: The sensory properties of all samples remained good at each testing time point, with no obvious changes in appearance, odor and taste.

[0827] (d) Microbiological indicators: The microbiological indicators of all samples at each testing time point met the national standards and there was no microbial contamination.

[0828] 13.4: Accelerated Aging Test

[0829] 13.4.1: Experimental Design

[0830] (i) Sample preparation: The prepared sweet protein composition sample was placed in a 40° C. environment for accelerated aging test.

[0831] (ii) Testing time points: samples were collected for testing at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 weeks.

[0832] 13.4.2: Detection indicators

[0833] i) Sweet protein activity: The content of sweet protein was detected using high performance liquid chromatography (HPLC) to evaluate its stability under accelerated aging conditions.

[0834] ii) Sensory properties: The appearance, smell and taste of the samples are evaluated through sensory evaluation to ensure that there are no significant changes in the product during accelerated aging.

[0835] iii) pH: The pH of the samples was measured using a pH meter to ensure that it remained stable during the accelerated aging period.

[0836] ⅳ) Microbiological indicators: Through the detection of total colony count, coliform group and mold and yeast, ensure that the product is free of microbial contamination during accelerated aging.

[0837] v) Physicochemical properties: Additional physicochemical properties testing such as viscosity, solubility, etc. is performed on specific products (e.g. toothpaste, mouthwash).

[0838] 13.4.3: Result Analysis

[0839] (I) Sweet protein activity: The results showed that the activity of the sweet protein was well maintained under accelerated aging conditions, with a retention rate of more than 85% within 8 weeks and a retention rate of more than 75% within 12 weeks.

[0840] (II) Sensory properties: The sensory properties of all samples remained good at each testing time point, with no obvious changes in appearance, odor and taste.

[0841] (III) pH value: The pH value of the samples remained stable under accelerated aging conditions without significant changes.

[0842] (IV) Microbiological indicators: The microbiological indicators of all samples at each testing time point met the national standards and there was no microbial contamination.

[0843] (V) Physicochemical properties: The viscosity of toothpaste and the solubility of mouthwash remain stable under accelerated aging conditions without significant changes.

[0844] 13.5: Data aggregation and analysis

[0845] 13.5.1: Storage stability

[0846] Summary: The results show that the sweet protein composition has the best storage stability at 4°C and 25°C, and its activity and sensory properties are well maintained, making it suitable for long-term storage. In a 40°C environment, the activity of the sweet protein decreases, but it still maintains a certain stability.

[0847] Recommendation: Store the sweet protein composition at 4°C and 25°C to ensure its optimal storage stability and product quality.

[0848] 13.5.2: pH stability

[0849] Summary: The sweet protein composition has good stability in the range of pH 3 to pH 9 and is suitable for products in various acidic and alkaline environments. Under pH 11 conditions, the stability of the sweet protein decreases slightly, but it still has certain application potential.

[0850] Recommendation: Sweet protein compositions should be kept in the pH 3 to pH 9 range during application to ensure their optimal stability.

[0851] 13.5.3: Accelerated Aging

[0852] Summary: The results of the accelerated aging test showed that the sweet protein composition had good stability under high temperature conditions, and its long-term storage stability could be evaluated in a short time.

[0853] Recommendation: Use accelerated aging testing as a rapid method for stability assessment of sweet protein compositions to quickly assess their stability during new product development.

[0854] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A sweet protein composition, characterized in that It contains the following sweet proteins: At least one of brazilian tamarind, thaumatin, tamarind tamarind, curculigo tamarind, mabinin, miraculin, betadine, thaumatin and tricholoma tamarind tamarind; Preferably, the sweet protein composition comprises a sweet protein selected from the following amino acid sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9; Preferably, based on the total weight of the sweet protein composition, the content of the sweet protein is greater than 60%, preferably greater than 70%, and more preferably greater than 80%.

2. The sweet protein composition according to claim 1, characterized in that The sweet protein further comprises polysaccharides, preferably glucan and / or mannan; Preferably, based on the total weight of the sweet protein composition, the total amount of the glucan and mannan is 0.1-15%, preferably 0.5-10%, more preferably 0.5%-7%; Preferably, based on the total weight of the sweet protein composition, the content of the glucan or mannan is 1%-15%, preferably 1-10%, more preferably 1%-7%.

3. Use of the sweet protein composition according to claim 1 or 2 in the preparation of medicines, pharmaceutical compositions, foods or oral care products; Preferably, the sweet protein composition is used to improve sweetness and reduce heat or calories; Preferably, the sweet protein composition is used to improve taste and enhance medication compliance of subjects.

4. An oral care product, characterized in that: It comprises the sweet protein composition according to claim 1 or 2; Preferably, the oral care product includes at least one of toothpaste, tooth cleaning powder, mouthwash, tooth paste, oral gel, tooth gel, tooth powder, mouth spray, mouthwash granules, paste, and tooth wax; Preferably, the oral care product further comprises fluoride and / or a chemical bleaching component; more preferably, the chemical bleaching component comprises at least one of hydrogen peroxide, carbamide peroxide, plant acid, hydroxyapatite, and polyphosphate; more preferably, the fluoride comprises sodium fluoride, stannous fluoride, sodium monofluorophosphate, and / or ammonium fluoride; Optionally, the oral care product further comprises at least one physiologically acceptable adjuvant or excipient; Preferably, the physiologically acceptable adjuvant or excipient includes at least one of a humectant, a thickener, a surfactant, a friction agent, a sweetener, a fragrance, and a preservative; More preferably, the abrasive comprises at least one of hydrated silica, calcium carbonate, calcium hydrogen phosphate, calcium hydrogen phosphate dihydrate, calcium pyrophosphate, aluminum hydroxide, and microcrystalline cellulose; More preferably, the moisturizing agent includes at least one of sorbitol, glycerin, propylene glycol, and xylitol; More preferably, the thickener comprises at least one of cellulose gum, carrageenan, xanthan gum, guar gum, and carbomer; More preferably, the surfactant includes at least one of sodium or potassium lauryl sulfate, cocamidopropyl betaine, sodium lauroyl sarcosinate, Tween, polyoxyethylene hydrogenated castor oil, polyoxyethylene, and polyoxypropylene block copolymers; More preferably, the preservative includes at least one of decanediol, caprylyl glycol, phenoxyethanol, and 1,2-hexanediol; More preferably, the aromatic agent includes at least one of eugenol, thymol, borneol, synthetic cinnamaldehyde, citral, vanillin, methyl salicylate, menthol, mint essence, mint oil, peppermint oil, spearmint oil, geranium oil, camphor oil, eucalyptus oil, cinnamon oil, vanillin, anise oil, lemon oil, citrus oil, and grapefruit oil; More preferably, the sweetener includes at least one of xylitol, erythritol, saccharin sodium, stevia, and aspartame.

5. The oral care product according to claim 4, characterized in that Based on the total weight of the oral care product, the content of the sweet protein composition is 0.001%-0.5%, preferably 0.01-0.2%, for example 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%.

6. A food, characterized in that The food comprises the sweet protein composition according to claim 1 or 2; Preferably, the dosage form of the food includes solid preparations, gas preparations, liquid preparations or semisolid preparations; Preferably, the food includes health food, condiments, candy products, beverages, chocolate, dairy products, ice cream, energy bars, baked goods and / or canned food; Preferably, the food is a health food or a condiment; More preferably, the health food is in the form of capsules, tablets, oral liquids, granules, powders, pills, sprinkles, drops, compressed candies or soft candies; More preferably, the health food comprises nutrient supplements and non-nutrient supplements; Optionally, the food further comprises at least one food-acceptable excipient; Preferably, the food-acceptable excipients include at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, thickeners, emulsifiers, preservatives, stabilizers, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, coating agents, flavoring agents, antioxidants, sweeteners, thickeners, and nutritional enhancers; More preferably, the food-acceptable excipient is selected from microcrystalline cellulose, erythritol, D-mannitol, fumaric acid, glycerol, pectin, potassium alginate, sodium alginate, talc, sodium pyrophosphate, polydextrose, carrageenan, sodium ascorbate, ascorbyl palmitate, L-malic acid, L(+)-tartaric acid, maltitol, gelatin, xylitol, citric acid, potassium citrate, sodium citrate, citric acid fatty acid glyceride, agar, lactic acid, sodium lactate, sorbic acid and its potassium salt, sorbitol, acid red, calcium carbonate, sodium carbonate, sodium bicarbonate, beet red, vitamin C, vitamin E, oxidized starch, ethyl acetate, At least one of alcohol, sodium acetate, stearic acid, calcium stearate, magnesium stearate, povidone K30, neotame, talc, pregelatinized starch, low-substituted hydroxypropyl cellulose, hydroxypropyl methylcellulose, titanium dioxide, polyethylene glycol, sorbitol and sorbitol liquid, sodium carboxymethyl cellulose, edible corn starch, potato starch, tapioca starch, edible wheat starch, edible sweet potato starch, sodium carboxymethyl starch, soybean oil, corn oil, sunflower oil, olive oil, powdered soybean lecithin, concentrated soybean lecithin, soybean lecithin, cellulose gum, natural flavors, flavors, polyoxyethylene sorbitan monooleate, beeswax and dextrin.

7. The food according to claim 6, characterized in that Based on the total weight of the food, the content of the sweet protein composition is 0.0015%-0.5%, preferably 0.01%-0.2%, for example 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%.

8. A drug or a pharmaceutical composition, characterized in that It comprises the sweet protein composition according to any one of claims 1 or 2; Preferably, the dosage form of the drug or pharmaceutical composition includes a solution, suspension, emulsion, powder, granule, tablet, capsule, injection, aerosol, drop, ointment, paste, liniment, patch, lotion, paste, suppository or gargle; Preferably, the drug or pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient; More preferably, the pharmaceutically acceptable excipients include at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, release agents, coating agents, acidity regulators, flavoring agents, and antioxidants.

9. The drug or pharmaceutical composition according to claim 8, characterized in that Based on the total weight of the drug or pharmaceutical composition, the content of the sweet protein composition is 0.01%-0.5%, preferably 0.05%-0.2%, for example 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.35%, 0.40%, 0.45%, 0.50%.

10. The method for preparing the sweet protein composition according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: (1) introducing or integrating the gene encoding the sweet protein into the host cell; (2) providing the necessary culture conditions to enable the host cell to express or translate the sweet protein; (3) isolating and / or purifying sweet proteins from host cells; Preferably, the culture conditions include at least one of the following: Temperature, humidity, incubation time, pH and / or culture medium; More preferably, the culture medium comprises a carbon source, a nitrogen source, amino acids and / or inorganic salts; More preferably, the pH is 5.5-6.5; More preferably, the culture time is 72h; More preferably, the temperature is 30°C; Preferably, the host cell comprises a eukaryotic cell or a prokaryotic cell selected from bacteria, yeast, plant cells or animal cells, preferably yeast, more preferably one of Kluyveromyces marxianus, Kluyveromyces lactis, Pichia pastoris or Saccharomyces cerevisiae, most preferably Kluyveromyces lactis; Preferably, the culture conditions further include dissolved oxygen level; and / or, the culture conditions include continuous culture or fed-batch culture; and / or, the separation and / or purification step of the sweet protein includes centrifugation, filtration and chromatography purification steps or membrane separation purification steps; and / or, the preparation method further includes freeze-drying or spray-drying the sweet protein; More preferably, the filtering step comprises filtering through a microfiltration membrane or an ultrafiltration membrane; More preferably, the chromatography purification step comprises ion exchange chromatography, hydrophobic chromatography or affinity chromatography.

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