Gastrointestinal delivery vehicle for red yeast high activity complex and its use in colonic fermentation

Abstract

The application discloses a gastrointestinal delivery carrier of high-activity compound of Rhodotorula and application thereof in colon fermentation, and belongs to the field of food science, pharmaceutical preparation, nutrition and microbiology research. The application explores the storage stability and digestive tract metabolism of the high-activity compound of Rhodotorula in different carrier forms, namely corn oil loaded with the high-activity compound of Rhodotorula (F-oil), spray-dried powder of corn oil nanoemulsion of the high-activity compound of Rhodotorula (F-EP) and alginate gel of corn oil nanoemulsion of the high-activity compound of Rhodotorula (F-EPA), wherein the storage stability of F-EPA is the best. The three carriers in the application do not release the high-activity compound of Rhodotorula in the oral cavity and stomach stages in vitro, but in the small intestine stage, the digestion rate and bioavailability of F-EP are the highest, and the release of F-EPA is the least. F-EPA can effectively promote the regulation of intestinal microbial community structure and abundance value by Rhodotorula rubin.

Description

TECHNICAL FIELD

[0001] The present application relates to a gastrointestinal delivery carrier of high-activity Monascus compound and its application in colon fermentation, focusing on improving its stability and metabolic release in the gastrointestinal tract, while covering its application in in vitro simulation of continuous colon fermentation process, belonging to the field of food science, pharmaceutical preparation, nutrition and microbiology research. BACKGROUND

[0002] Monascus purpureus is a natural Monascus fermentation product pigment, which is prone to oxidation during storage, has poor stability and low bioavailability in the gastrointestinal tract. Researchers have explored various technologies to prepare Monascus purpureus nanoparticle delivery systems to solve the above problems, for example, for the small intestine part, the existing technology loads Monascus purpureus into liposomes, but the bilayer structure of liposomes is destroyed by intestinal bile salts and enzymes after oral administration, resulting in premature release of embedded macromolecules and reduced residence time.

[0003] For colon targeting, different sustained-release systems have also been developed in the prior art, such as micellar loading, emulsion, nanoemulsion, nanoliposome and hydrogel, etc. But different sustained-release systems also have various problems, such as: micellar loading, a technology that absorbs fat-soluble molecules on the surface of micelles (Qian J, Guo Y, Xu Y, et al. Combination of micelles and liposomes as a promising drug delivery system: a review. Drug Deliv Transl Res. 2023 Nov; 13(11): 2767-2789.), but it has the disadvantages of low loading capacity, premature drug release and poor stability, etc. In addition, some researchers also use oil-in-water nanoemulsion and microemulsion to embed and transport lipophilic substances (Pavoni L, Perinelli DR, Bonacucina G, et al. An Overview of Micro- and Nanoemulsions as Vehicles for Essential Oils: Formulation, Preparation and Stability. Nanomaterials (Basel). 2020 Jan 12; 10(1): 135.), but they also have the characteristics of complex preparation method (most of which involve organic solvents), poor loading capacity and instability during storage, etc. For example, the common nanohydrogel technology, a three-dimensional soft gel formed by cross-linking water-soluble materials (Akram, Muhammad, and Rafaqat Hussain. "Nanohydrogels: History, development, and applications in drug delivery." Nanocellulose and Nanohydrogel Matrices: Biotechnological and Biomedical Applications (2017): 297-330.), although it has great potential in promoting the effective delivery of carotenoids, but also has some limitations including poor loading capacity, premature release of carotenoids and oxidation, etc.

[0004] The release rate of red yeast rubixanthin in the red yeast rubixanthin electrospun microspheres prepared by patent CN115399471B is not high in the small intestine, and is higher in the colon, so it is mainly used for targeted colon delivery; the release rate of red yeast rubixanthin in the bile system of red yeast rubixanthin prepared by patent CN115226898B is also not high in the small intestine, although it has a certain sustained release effect, but in the actual use process, the red yeast rubixanthin takes a long time to achieve sufficient release.

[0005] A study (Li Xingming. Mechanism of red yeast rubixanthin intervention in high-fat obesity [D]. Jiangnan University, 2022.) prepared three kinds of red yeast rubixanthin carrier systems, and explored the storage stability of red yeast rubixanthin in the three carriers and the metabolism in the digestive tract; a study (B. Hu, C. Liu, W. Jiang, et al, Chronic in vitro fermentation and in vivo metabolism: Extracellular polysaccharides from Sporidiobolus pararoseus regulate the intestinal microbiome of humans and mice, International Journal of Biological Macromolecules 192 (2021) 398-406.) found that the red yeast polysaccharide extracted from red yeast had significant intestinal prebiotic properties, but no study has confirmed whether the combination of red yeast polysaccharide and red yeast rubixanthin can synergistically promote the stable release of red yeast rubixanthin after embedding and enhance the function of the intestinal environment. SUMMARY

[0006] In order to solve the above problems, the present application first mixes red yeast rubixanthin and red yeast polysaccharide to obtain a red yeast high-activity compound, and then prepares three different red yeast high-activity compound carriers, namely plant oil loaded with red yeast high-activity compound, red yeast high-activity compound plant oil nanoemulsion spray dried powder, and red yeast high-activity compound plant oil nanoemulsion alginate gel, to improve the stability of red yeast rubixanthin and promote the metabolic function of the gastrointestinal tract. Among them, the plant oil loaded with red yeast high-activity compound is the best in improving the stability of red yeast rubixanthin, and the red yeast high-activity compound plant oil nanoemulsion alginate gel is the best in promoting the metabolic function of the gastrointestinal tract.

[0007] The first object of the present application is to provide a preparation method of a red yeast high-activity compound carrier, which is plant oil loaded with red yeast high-activity compound, comprising the following steps:

[0008] (1) mixing the rhodozyme carotenoid and the rhodozyme polysaccharide to obtain a high-activity rhodozyme compound;

[0009] (2) dissolving the high-activity rhodozyme compound obtained in step (1) in vegetable oil to obtain vegetable oil loaded with the high-activity rhodozyme compound.

[0010] In one embodiment, in step (1), the rhodozyme carotenoid and the rhodozyme polysaccharide are mixed at a mass ratio of (8-12):1.

[0011] In one embodiment, in step (2), the vegetable oil includes but is not limited to corn oil, soybean oil, and sunflower seed oil.

[0012] In one embodiment, in step (2), the mass ratio of the high-activity rhodozyme compound to the vegetable oil is (3-5):1000.

[0013] In one embodiment, in step (1), the method for preparing the rhodozyme carotenoid includes the following steps:

[0014] Sporidiobolus pararoseus JD-2 is inoculated into a culture medium at an inoculation ratio of 2%-5% (volume ratio of bacterial solution to culture medium) and cultured to obtain a rhodozyme fermentation broth;

[0015] The rhodozyme fermentation broth is centrifuged, the supernatant is discarded, and the precipitate is added with water to obtain a yeast suspension;

[0016] The yeast suspension is first homogenized, centrifuged, and the supernatant is then washed, the precipitate is taken, the precipitate is extracted, and vacuum drying is performed to obtain the rhodozyme carotenoid.

[0017] In one embodiment, the culture is carried out at a temperature of 25-30°C and a pH value of 5.5-6.5.

[0018] In one embodiment, the rhodozyme fermentation broth is centrifuged at 6500-7500 r / min for 15-20 min, the supernatant is discarded, and the precipitate is added with water to obtain a yeast suspension.

[0019] In one embodiment, vacuum drying is performed to obtain the rhodozyme carotenoid in the form of a freeze-dried powder.

[0020] In one embodiment, the yeast suspension is first homogenized at 60-100 MPa for 2-4 times, centrifuged at 4000-6000 r / min for 5-15 min, the supernatant is then washed 2-3 times, the precipitate is taken, the precipitate is extracted for 20-30 min, and vacuum drying is performed to obtain the rhodozyme carotenoid freeze-dried powder.

[0021] In one embodiment, in step (1), the method for preparing the rhodozyme polysaccharide includes the following steps:

[0022] Sporidiobolus pararoseus JD-2 is inoculated into culture medium at an inoculation ratio of 2%-5% (volume ratio of bacterial solution to culture medium) for culture, to obtain a red yeast fermentation liquor;

[0023] The red yeast fermentation liquor is centrifuged to obtain a precipitate;

[0024] The precipitate is extracted, and after centrifugation at the end of the extraction, the supernatant is obtained, and the protein is removed, and the supernatant is obtained by further centrifugation, and the precipitate is obtained by centrifugation after standing, and the precipitate is freeze-dried to obtain red yeast polysaccharide freeze-dried powder.

[0025] In one embodiment, the red yeast fermentation liquor is centrifuged at 6500-7500 r / min for 15-20 min, and the precipitate is obtained.

[0026] In one embodiment, the freeze-dried powder is obtained by freeze-drying the red yeast polysaccharide.

[0027] In one embodiment, the precipitate is extracted, and after centrifugation at 6000-7000 r / min for 5-15 min at the end of the extraction, the supernatant is obtained, and the protein is removed from the supernatant, and the supernatant is obtained by further centrifugation at 6000-7000 r / min for 5-15 min, and the precipitate is obtained by centrifugation at 6000-7000 r / min for 5-15 min after standing, and the precipitate is freeze-dried to obtain red yeast polysaccharide freeze-dried powder.

[0028] A second object of the present application is to provide a preparation method of a red yeast high-activity complex carrier, which is a red yeast high-activity complex plant oil nanoemulsion spray-dried powder, comprising the following steps:

[0029] (1) mixing OSA starch solution with plant oil loaded with red yeast high-activity complex of the present application, homogenizing to obtain red yeast high-activity complex plant oil nanoemulsion;

[0030] (2) spray-drying the red yeast high-activity complex plant oil nanoemulsion obtained in step (1) to obtain red yeast high-activity complex plant oil nanoemulsion spray-dried powder.

[0031] In one embodiment, in step (1), the OSA starch solution and the plant oil loaded with red yeast high-activity complex are mixed at a mass ratio of 1:(8-10).

[0032] In one embodiment, in step (1), the mass fraction of the OSA starch solution is 30-50%.

[0033] In one embodiment, in step (1), the OSA starch solution with a mass fraction of 30-50% is mixed with the plant oil loaded with the high-activity complex of Rhodotorula to obtain an initial emulsion, and the initial emulsion is subjected to temperature-controlled circulation homogenization 4-8 times at 15-25℃ and 90-110 MPa by using a high-pressure homogenizer to obtain the high-activity complex of Rhodotorula plant oil nanoemulsion.

[0034] In one embodiment, in step (2), the operating conditions of the spray drying are as follows: the air inlet temperature is 180-200℃, the air outlet temperature is 80-90℃, the feeding flow rate is 1-3 L / h, the nozzle atomization pressure is 2-4 bar, and the duration of the spray drying process is 1-3 h.

[0035] A third object of the present application is to provide a preparation method of a carrier of the high-activity complex of Rhodotorula, which is a high-activity complex of Rhodotorula plant oil nanoemulsion alginate gel, comprising the following steps:

[0036] The alginate solution is mixed with the high-activity complex of Rhodotorula plant oil nanoemulsion of the present application to obtain a gel, thereby obtaining the high-activity complex of Rhodotorula plant oil nanoemulsion alginate gel.

[0037] In one embodiment, the alginate solution and the high-activity complex of Rhodotorula plant oil nanoemulsion are mixed at a mass ratio of (1-2):(1-2), homogenized, and stirred to form a gel block, thereby obtaining the high-activity complex of Rhodotorula plant oil nanoemulsion alginate gel.

[0038] In one embodiment, the gel block is formed by mixing the alginate solution and the high-activity complex of Rhodotorula plant oil nanoemulsion at a mass ratio of (1-2):(1-2), homogenizing, stirring at a speed of 200-400 rpm for 3-10 minutes, and reacting at 30-40℃ for 5-15 minutes.

[0039] In one embodiment, the alginate solution includes, but is not limited to, a sodium alginate solution, a potassium alginate solution, and a calcium alginate solution, and preferably a 2% sodium alginate solution.

[0040] In one embodiment, a crosslinking agent with a mass concentration of 1-3% is used for gelation to obtain the high-activity complex of Rhodotorula plant oil nanoemulsion alginate gel.

[0041] In one embodiment, the crosslinking agent includes, but is not limited to, CaCl2, propylene diamine, polyethylene glycol, and polypropylene glycol glycidyl ether.

[0042] The fourth object of the present application is the application of the plant oil loaded with the high-activity complex of Rhodotorula, the spray-dried powder of the high-activity complex of Rhodotorula plant oil nanoemulsion, and the alginate gel of the high-activity complex of Rhodotorula plant oil nanoemulsion prepared by the present application in improving the stability of Rhodotorula rubin.

[0043] The fifth object of the present application is the application of the plant oil loaded with the high-activity complex of Rhodotorula, the spray-dried powder of the high-activity complex of Rhodotorula plant oil nanoemulsion, and the alginate gel of the high-activity complex of Rhodotorula plant oil nanoemulsion prepared by the present application in preparing products for promoting the metabolic function of the gastrointestinal tract.

[0044] Beneficial effects

[0045] (1) The present application can significantly improve the stability of Rhodotorula rubin by preparing a new high-activity complex of Rhodotorula and applying it to a carrier packaging delivery system. The corn oil (F-oil) loaded with the high-activity complex of Rhodotorula performs superiorly in terms of storage stability. The high-activity complex of Rhodotorula corn oil nanoemulsion alginate gel (F-EPA) has better sustained-release effect and can effectively delay the release of Rhodotorula rubin, so that it can stably exist in the gastrointestinal tract for a longer time.

[0046] (2) The present application uses alginate as an emulsifier, combines with OSA starch, prepares a nanoemulsion of the high-activity complex of Rhodotorula, and coats it to form a new carrier form (F-EPA) of the high-activity complex of Rhodotorula, which significantly improves the stability of Rhodotorula rubin during storage and the gastrointestinal transit process, and makes the encapsulation process simple and efficient.

[0047] (3) The delivery effect of the high-activity complex of Rhodotorula in the form of F-EPA in the digestive tract is superior to other carriers. It can effectively target the release of Rhodotorula rubin, which is not resistant to gastric acid and has poor stability, to the colon, ensure the stable existence of Rhodotorula rubin in the gastrointestinal tract, and play a health function in the colon through interaction with the microbiota.

[0048] (4) In the in vitro simulated continuous colon fermentation system, F-EPA significantly improves the concentrations of propionic acid, butyric acid and valeric acid, and effectively regulates the composition and abundance of intestinal flora. This effect shows that the carrier form can directly or indirectly promote the metabolism of Rhodotorula rubin in the intestinal flora of the human body, increase the generation of beneficial short-chain fatty acids, and improve intestinal health. BRIEF DESCRIPTION OF DRAWINGS

[0049] Figure 1 The flowchart of the overall idea of the present application.

[0050] Figure 2The changes of short-chain fatty acids (SCFAs) in vitro fermentation of the high-activity compound of Rhodotorula corn oil nanoemulsion alginate gel (F-EPA) and the rhodotorula rubin corn oil nanoemulsion alginate gel (T-EPA) in the present application and the effects on the phylum level species and the genus level species in the human intestinal microbiome; wherein (A) and (D) are the changes of SCFAs of F-EPA and T-EPA respectively; (B) and (E) are the effects of F-EPA and T-EPA on the phylum level species in the human intestinal microbiome respectively; (C) and (F) are the effects of F-EPA and T-EPA on the genus level species in the human intestinal microbiome respectively. DETAILED DESCRIPTION

[0051] The present application will be further described below in conjunction with examples, but the embodiments of the present application are not limited thereto.

[0052] 1. Raw materials used in examples and comparative examples

[0053] Corn oil, soybean oil, and sunflower oil were purchased from Wuxi Xuelang District Farmers Market;

[0054] OSA modified starch was provided by Huahao Huafeng Starch Co., Ltd.;

[0055] Lipase, mucin, pepsin, bile extract, and Nile red were purchased from Sigma Company, USA;

[0056] Acetic acid, propionic acid, isobutyric acid, n-butyric acid, iso-valeric acid, and n-valeric acid standard samples were purchased from Sigma-Aldrich Chemical (St. Louis, Missouri, USA); the rest of the reagents were analytical pure and purchased from Tianjin Kaitong Chemical Reagent Co., Ltd.

[0057] The Sporidiobolus pararoseus JD-2 strain involved in the following examples is recorded in the Chinese invention patent with publication number CN102115716B, and the preservation number is CCTCC M 2010326.

[0058] 2. Detection of physical and chemical properties of different carriers

[0059] (1) Measurement of emulsion particle size and zeta potential of different carriers

[0060] The corn oil loaded with high-activity compound of Rhodotorula (F-oil) and the high-activity compound of Rhodotorula corn oil nanoemulsion spray-dried powder (F-EP) were respectively dissolved in ultrapure water at a dilution ratio of 1:1000, and a nanoparticle size and zeta potential instrument was used to measure the particle size and charge (zeta potential) of the emulsion.

[0061] For the gel particles of F-EPA, the diameters of at least 20 particles were measured using a digital micrometer (0-300 mm, EC10, high-precision digital caliper), and the average and standard deviation were calculated.

[0062] The particle size and zeta potential of different carriers of Rhodotorula rubinum were also determined according to the above method.

[0063] (2) The encapsulation yield and encapsulation efficiency of Rhodotorula rubinum in different carriers

[0064] The role of encapsulation yield (EY) and encapsulation efficiency (EE):

[0065] For Rhodotorula rubinum: EY is the ratio of the final amount of Rhodotorula rubinum to the total amount of Rhodotorula rubinum initially added.

[0066] For Rhodotorula rubinum: EY is the ratio of the final amount of Rhodotorula rubinum to the total amount of Rhodotorula rubinum initially added.

[0067] Encapsulation efficiency (EE):

[0068] For Rhodotorula rubinum: EE is the ratio of the total amount of Rhodotorula rubinum in the prepared Rhodotorula rubinum embedding sample to the total amount of Rhodotorula rubinum in the prepared Rhodotorula rubinum embedding sample.

[0069] For Rhodotorula rubinum: EE is the ratio of the total amount of Rhodotorula rubinum in the prepared Rhodotorula rubinum embedding sample to the total amount of Rhodotorula rubinum in the prepared Rhodotorula rubinum embedding sample.

[0070] Encapsulation efficiency (EE):

[0071] For Rhodotorula rubinum: EE is the ratio of the total amount of Rhodotorula rubinum in the prepared Rhodotorula rubinum embedding sample to the total amount of Rhodotorula rubinum in the prepared Rhodotorula rubinum embedding sample.

[0072] For Rhodotorula rubinum: EE is the ratio of the total amount of Rhodotorula rubinum in the prepared Rhodotorula rubinum embedding sample to the total amount of Rhodotorula rubinum in the prepared Rhodotorula rubinum embedding sample.

[0073] (3) Determination of the storage stability of Rhodotorula rubinum in different carriers

[0074] The 2.0 mL of red yeast high-activity complex loaded corn oil (F-oil), 0.15 g of red yeast high-activity complex corn oil nanoemulsion spray-dried powder (F-EP) and 0.25 g of red yeast high-activity complex corn oil nanoemulsion alginate gel (F-EPA) samples were respectively loaded into 10 mL brown bottles, and stored at 50°C, 25°C and 4°C in the dark, while the same amount of samples were placed in transparent glass bottles for light storage. At fixed time intervals, the concentration of the main component (torulene) in the red yeast high-activity complex sample and its change were measured.

[0075] The calculation formula of the retention rate is C t / C1, wherein C1 is the concentration of torulene measured before storage of the red yeast high-activity complex, and C t is the concentration of torulene in the red yeast high-activity complex at the storage time t.

[0076] The storage stability of torulene in different carriers and other carrier forms of the red yeast high-activity complex was also determined according to the above method.

[0077] 3. In vitro simulation of ascending-transverse-descending colon fermentation process to study the metabolism of red yeast high-activity complex in F-EPA and torulene in T-EPA. The specific steps are as follows:

[0078] Fecal sample treatment: Collect the feces of 8 healthy young people, mix with sterile PBS (pH = 6.8) (ratio 1:80), stir and filter to obtain intestinal bacteria liquid, and place in an anaerobic environment.

[0079] Fermentation setting: inoculate the bacteria liquid into a fermentation tank containing 10% medium, adjust the pH to 7.0, and ferment at 37°C, 150 rpm.

[0080] Add samples: add F-EPA, T-EPA (1% mass ratio) to the fermentation tank, supplement 30 mg per day, and continue to ferment for 2 days.

[0081] Sample collection and analysis: collect the fermentation liquid periodically during 0-48 h, and perform metabolic analysis.

[0082] 4. Digestion characteristics and bioaccessibility of red yeast high-activity complex and torulene in different carriers

[0083] (1) In vitro simulation of oral, gastric and small intestinal (GIT) digestion

[0084] The digestion characteristics of the sample were studied. The operation steps are as follows:

[0085] Oral stage: 5 mL sample was mixed with 4 mL simulated oral fluid (1.408 g KCl, 1.428 g NaHCO3, 0.629 g KH2PO4, 0.038 g MgCl2 6H2O, 0.007 g (NH4)2CO3 dissolved in 1 L deionized water) and incubated at 37 °C. 25 μL 0.3 mol / L CaCl2, 0.975 mL water was added and mixed well before incubation in 37 °C water bath for 2 min.

[0086] Stomach stage: The mixture from oral stage was mixed with 8 mL simulated gastric fluid (0.643 g KCl, 2.625 g NaHCO3, 0.153 g KH2PO4, 0.031 g MgCl2 6H2O, 0.060 g (NH4)2CO3, 2.808 g NaCl dissolved in 1 L deionized water), 5 μL 0.3 mol / L CaCl2, 4 mg gastric lipase (60 U / mL), 400 mg pepsin (2000 U / mL), 1.895 mL H2O was added and pH was adjusted to 3.0 before incubation in 37 °C water bath.

[0087] Intestinal stage: The mixture from stomach stage was mixed with 8.5 mL simulated intestinal fluid (3.452 g NaCl, 0.634 g KCl, 8.952 g NaHCO3, 0.136 g KH2PO4, 0.084 g MgCl2 6H2O dissolved in 1 L deionized water), 40 μL 0.30 mol / L CaCl2, 16.00 mg trypsin (100 U / mL), 2.67 mg pancreatic lipase (2000 U / mL), 272.38 mg cholic acid (10 mmol / L), 11.26 mL H2O was added and mixed well, pH was adjusted to 7.0 before incubation in 37 °C water bath.

[0088] The free fatty acids (FFAs) released from the digestion of lipids in the sample can be calculated according to the volume of the base solution consumed to neutralize the sample (pH 7.0) during the simulated intestinal digestion process, the calculation formula is as follows:

[0089] Total free fatty acid release (%)= V NaOH(t) x C NaOH x M w,lipid / 2m lipid x 100%

[0090] In the formula, m lipid is the total mass of oil in the simulated digestion solution (g), V NaOH(t) is the volume of NaOH added to the reaction container at time t of simulated intestinal digestion, C NaOH is the concentration of NaOH in the burette (0.25 M), M w,lipid is the average molecular weight of lipids (872 g / mol for corn oil).

[0091] (2) Release rate of rhodoxanthin in different carriers

[0092] After oral and gastric digestion, the digestion products of different carrier forms reach the small intestine. In this experiment, samples were taken at different digestion time points in the small intestine, and the concentration of rhodoxanthin in the dissolution solution was determined by high performance liquid chromatography (HPLC) and ultraviolet-visible spectrophotometry (UV-Vis). Ultraviolet spectroscopy is usually used to determine the content of rhodoxanthin, and rhodoxanthin usually has an absorption peak near 480 nm at a specific wavelength.

[0093] Calculate the release amount at each time point:

[0094] Based on the concentration of rhodoxanthin in the dissolution solution and the sampling volume, the amount of rhodoxanthin released at each time point can be calculated: M(t) = C(t) x V;

[0095] Where M(t) is the mass of rhodoxanthin released at time t (units: mg or pg); C(t) is the concentration of rhodoxanthin in the dissolution solution at time t (units: mg / mL or pg / mL); and V is the volume of each sample (units: mL).

[0096] Calculate the total release rate:

[0097] The total release rate refers to the proportion of the amount of rhodoxanthin released in the dissolution solution at a certain time point or within a certain time interval to the initial total amount. The release rate R(t) at each time point can be calculated as: R(t) = M(t) / M0 x 100% R(t);

[0098] Where R(t) is the release rate at time t (%); M(t) is the mass of rhodoxanthin released at time t; and M0 is the total mass of rhodoxanthin initially input into the sample.

[0099] The release rate of rhodoxanthin in different carriers and other carrier forms of high-activity rhodotorula complex is also determined according to the above method.

[0100] (3) Bioavailability of rhodoxanthin in different carriers

[0101] After the end of the small intestine digestion stage, an equal amount of chyme (10000 g) was collected and centrifuged at 4°C for 40 minutes; then, the centrifuged chyme (0.1 g) was dispersed in deionized water (0.9 mL), and 0.1 mL of the solution was mixed with 0.9 mL of DMSO, and then extracted with 2.0 mL of n-hexane / methylene chloride (4:1) to extract torularhodin; after centrifugation (3000 rpm, 10 min), the content of torularhodin in the organic phase was determined by high-performance liquid chromatography, and the content of torularhodin in the supernatant was detected by the above method. The formula for calculating the bioavailability is:

[0102] Bioavailability (%) = (C Micelle / C Digesta ) x 100%;

[0103] In the formula, C Micelle is the concentration of torularhodin in the digestion supernatant, and C Digesta is the concentration of torularhodin in the chyme at the end of digestion.

[0104] 5. Distribution of torularhodin in F-EPA and T-EPA and its efficacy evaluation in regulating the intestinal environment

[0105] (1) Detection of short-chain fatty acid (SCFA) content

[0106] According to the results of in vivo studies (Liu C, Li X, Li J, et al. Role of the gut microbiota in dietary patterns rich in torularhodin via OSA colon-targeted delivery. Food Funct. 2022 Oct 31; 13(21): 11034-11048.), further detection of SCFA content in F-EPA samples was carried out:

[0107] Put 200 mg of torularhodin high-activity complex corn oil nanoemulsion alginate gel (F-EPA) sample into a 2 mL centrifuge tube, add 0.1 mL of saturated KHSO4 solution and 0.1 mL of 2-ethylbutyric acid internal standard solution (0.1 mg / mL), add 1 mL of dimethyl carbonate (DMC), and shake vigorously for 10 minutes;

[0108] Then centrifuge the mixture at 3800 r / min for 10 minutes to separate the solid phase and aqueous phase from the DMC upper layer, and aspirate the DMC phase into a gas-phase vial;

[0109] Formulate mixed SCFAs standard solution (including formic acid, acetic acid, propionic acid, n-butyric acid, isobutyric acid, n-valeric acid and iso-valeric acid) of 0.1 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL and 3.0 mg / mL, and pretreat according to the above method.

[0110] The SCFAs detection uses the GC-MS method, the ionization mode is EI, the emission current is 1 mA, the electron energy is 70 eV, the interface temperature is 250°C, the source temperature is 210°C, the voltage is 2000 V, the initial temperature is 60°C, the initial time is 1 min, the temperature rising rate is 15°C / min, the final temperature is 240°C, and the holding time is 10 min.

[0111] (2) 16S rRNA gene sequencing

[0112] Extract sample DNA using a DNA extraction kit and perform electrophoresis detection;

[0113] Use primer pairs to perform PCR amplification of the V3-V4 region of sample 16S rRNA. The PCR amplification product is detected by 2% agarose gel electrophoresis, and the PCR product is recovered using the AMPure XT beads kit;

[0114] The purified PCR product is quantitatively detected using Qubit, and the qualified sequencing library is subjected to PE300 sequencing using an Illumina MiSeq sequencer;

[0115] Use QIIME2 software to perform microbial sequencing clustering operation classification unit (OTUs) table screening, and store the results in the Greengene database;

[0116] Use R and other software to analyze the diversity of the microbial composition of the sample.

[0117] Phaffia rhodozyma astaxanthin corn oil nanoemulsion alginate gel (T-EPA) regulates the intestinal environment, and is also determined according to the above method.

[0118] Example 1: Preparation of corn oil loaded with high-activity phaffia rhodozyma complex (F-oil)

[0119] The specific embodiment includes the following steps:

[0120] 1. Preparation of raw materials

[0121] (1) Preparation of phaffia rhodozyma astaxanthin freeze-dried powder:

[0122] Red yeast rubin (95%, HPLC), which was separated and purified from the extract of Sporidiobolus pararoseus JD-2 (which has been disclosed in the Chinese invention patent CN102115716B) in the laboratory, and the specific preparation method is as follows:

[0123] Sporidiobolus pararoseus JD-2 was inoculated into the medium containing glucose, yeast extract and minerals at an inoculation ratio of 3% (volume of bacterial solution / volume of medium), and then was subjected to shake flask culture under the conditions of temperature of 28℃, pH value of 6 and 12 hours light / 12 hours dark cycle, to obtain red yeast fermentation liquor;

[0124] The red yeast fermentation liquor was centrifuged at 6500r / min for 15min by using an ultracentrifuge, and the supernatant was discarded, and the precipitate was made into a yeast suspension with a concentration of 500g / L by using deionized water;

[0125] Then the yeast suspension was added into a high-pressure homogenizer, and was homogenized for 3 times under the pressure of 80MPa, and then was centrifuged for 10min (5000r / min) by using a low-speed centrifuge, and the supernatant was taken and washed twice with deionized water, and the precipitate was obtained, and 100mL of a mixture of ethanol and n-hexane (volume ratio of 1:1) was added into the precipitate to extract for 25min in the dark, to obtain a red yeast rubin extraction liquor, and the red yeast rubin extraction liquor was vacuum freeze-dried to obtain a red yeast rubin freeze-dried powder.

[0126] (2) Preparation of red yeast polysaccharide:

[0127] Red yeast polysaccharide (purity of 83%), which was also separated and purified from the fermentation liquor of Sporidiobolus pararoseus JD-2, and the specific preparation method is as follows:

[0128] The red yeast fermentation liquor was prepared according to the method in step (1), and then was centrifuged at 6500r / min for 15min by using an ultracentrifuge, and the supernatant was discarded, and the precipitate was taken;

[0129] The precipitate was subjected to hot water extraction (the hot water extraction conditions were set as follows: extraction time of 3h, temperature of 80℃, and solid-liquid ratio of precipitate and hot water of 1:20), and after the extraction was completed, the precipitate was centrifuged at 6500r / min for 10min, and the supernatant was taken;

[0130] After the supernatant was subjected to zinc acetate-potassium ferrocyanide method for protein removal, the supernatant was centrifuged at 6500r / min for 10min, and the supernatant was taken, and 4 times the volume of anhydrous ethanol was added into the supernatant and was allowed to stand for 24h, and after the standing was completed, the supernatant was centrifuged at 6500r / min for 10min, and the precipitate was collected, and the collected precipitate was subjected to freeze-drying, to obtain a red yeast polysaccharide freeze-dried powder.

[0131] 2. Preparation of corn oil loaded with high-activity complex of Rhodotorula (F-oil):

[0132] The lyophilized powder of Rhodotorula rubinum and the lyophilized powder of Rhodotorula polysaccharide obtained in step 1 were mixed in a mass ratio of 10:1, i.e. 10 parts of the lyophilized powder of Rhodotorula rubinum and 1 part of the lyophilized powder of Rhodotorula polysaccharide were mixed to obtain a mixture (i.e. high-activity complex of Rhodotorula), and 0.3 g of the mixture was added to 75 mL of corn oil to obtain corn oil loaded with high-activity complex of Rhodotorula (F-oil) with a mass fraction of 0.4%.

[0133] Example 2: Preparation of spray-dried powder of corn oil nanoemulsion of high-activity complex of Rhodotorula (F-EP)

[0134] The specific implementation includes the following steps:

[0135] (1) Disperse OSA starch in ultrapure water, stir at 80°C at 12000 rpm for 30 minutes until dissolved to obtain an OSA starch solution with a mass fraction of 40%;

[0136] (2) Mix the F-oil obtained in Example 1 with the OSA starch solution obtained in step (1) in a mass ratio of 1:9, stir at 15000 rpm for 3 minutes to obtain an initial emulsion, and use a high-pressure homogenizer to homogenize the obtained initial emulsion at 20°C at 100 MPa for 6 cycles to obtain a corn oil nanoemulsion of high-activity complex of Rhodotorula;

[0137] (3) Spray-dry the corn oil nanoemulsion of high-activity complex of Rhodotorula obtained in step (2) using a Mobile Minor spray dryer, control the drying chamber size to be 800x620mm, 60° cone, equipped with a counter-flow nozzle atomizer, air inlet temperature 190°C, outlet temperature 85°C, feed flow rate 2L / h, nozzle atomization pressure 3bar, and the duration of the spray-drying process is 2h to obtain a spray-dried powder of corn oil nanoemulsion of high-activity complex of Rhodotorula (F-EP), and store the F-EP in a brown glass bottle, and store it at 4°C in the dark for standby use. TM Spray-dry the corn oil nanoemulsion of high-activity complex of Rhodotorula obtained in step (2) using a Mobile Minor spray dryer, control the drying chamber size to be 800x620mm, 60° cone, equipped with a counter-flow nozzle atomizer, air inlet temperature 190°C, outlet temperature 85°C, feed flow rate 2L / h, nozzle atomization pressure 3bar, and the duration of the spray-drying process is 2h to obtain a spray-dried powder of corn oil nanoemulsion of high-activity complex of Rhodotorula (F-EP), and store the F-EP in a brown glass bottle, and store it at 4°C in the dark for standby use.

[0138] Example 3: Preparation of alginate gel of corn oil nanoemulsion of high-activity complex of Rhodotorula (F-EPA)

[0139] The specific implementation includes the following steps:

[0140] (1) Preparation of 2% sodium alginate:

[0141] Deionized water was heated to room temperature, stirred at 300 rpm for 1.5 hours to ensure complete dissolution of sodium alginate, and then adjusted to pH 6.5 using 0.1M dilute hydrochloric acid. The solution was filtered through a 0.45μm filter membrane to obtain a 2% sodium alginate solution by mass concentration;

[0142] (2) The 2% sodium alginate solution by mass fraction obtained in step (1) was mixed with the high-activity compound of red yeast and corn oil nanoemulsion in Example 2 at a mass ratio of 1:1. A 2% CaCl2 solution by mass concentration was used as a crosslinking agent to gel the alginate (the stirring speed was maintained at 300 rpm for 5 minutes, the reaction was carried out at 30°C for 10 minutes, and a gel block with a size of about 5cm×5cm×1cm was formed). A high-activity compound of red yeast and corn oil nanoemulsion alginate gel (F-EPA) was obtained, which was freeze-dried for 48 hours and then stored for use.

[0143] Comparative Example 1: Red yeast rubixanthin was used alone to prepare red yeast rubixanthin-loaded corn oil (T-oil), red yeast rubixanthin corn oil nanoemulsion spray-dried powder (T-EP), and red yeast rubixanthin corn oil nanoemulsion alginate gel (T-EPA)

[0144] According to the preparation methods of Examples 1-3, the difference is that 1 part of red yeast rubixanthin freeze-dried powder is used instead of 1 part of red yeast polysaccharide freeze-dried powder (red yeast rubixanthin freeze-dried powder accounts for 11 parts, i.e. 0.3g of red yeast rubixanthin freeze-dried powder). Three different carriers of red yeast rubixanthin were prepared, namely red yeast rubixanthin-loaded corn oil (T-oil), red yeast rubixanthin corn oil nanoemulsion spray-dried powder (T-EP), and red yeast rubixanthin corn oil nanoemulsion alginate gel (T-EPA).

[0145] The different carriers of Examples 1-3 and Comparative Example 1 were detected. The results are as follows.

[0146] 1. Physicochemical property detection

[0147] Table 1. Emulsion particle size, zeta potential, encapsulation efficiency (EE), and encapsulation yield of different carriers of Examples 1-3 and Comparative Example 1

[0148]

[0149] 2. Stability and preservation characteristics detection of red yeast rubixanthin

[0150] The retention rate of red yeast rubixanthin in different carriers is shown in Tables 2 and 3:

[0151] Table 2. Average retention rate of red yeast rubixanthin in F-oil, F-EP, and F-EPA of Examples 1-3 under light and in the dark for 7 days (%)

[0152]

[0153]

[0154] Table 3 Average retention rate (%) of T-oil, T-EP and T-EPA of Comparative Example 1 under light and darkness for 7 days

[0155]

[0156] It can be seen that the retention rate of the high-activity compound group of rhodoerythrin is relatively higher than that of the rhodoerythrin group as a whole.

[0157] 3. Digestion characteristics and bioavailability of rhodoerythrin in different carriers

[0158] (1) In vitro simulation of oral, gastric and small intestinal (GIT) digestion

[0159] The stability of the high-activity compound of rhodoerythrin in the gastrointestinal tract is significantly affected by the digestion characteristics of its oil phase. As a substance dissolved in the oil phase, the digestion rate and extent of the high-activity compound of rhodoerythrin can reflect its stability during gastrointestinal transport, because the digestion of the oil phase will lead to the release of rhodoerythrin therein.

[0160] In vitro simulation of digestion of the oral, gastric and small intestinal (GIT) stages in test tubes showed that the digestion of the oil phase in the oral and gastric stages was less affected, and the main digestion occurred in the small intestine.

[0161] (2) Release rate of FFA and release rate of rhodoerythrin in different carriers after 2 hours of small intestinal digestion

[0162] After the entire simulated GIT model, the release rate (%) of FFA and the release rate (%) of rhodoerythrin in different carriers after 2 hours of small intestinal digestion were determined, and the results are shown in Table 4:

[0163] Table 4 Release rate (%) of FFA and release rate (%) of rhodoerythrin in different carriers of Example 1 to Example 3 and Comparative Example 1 after 2 hours of small intestinal digestion

[0164]

[0165] The release rate of free fatty acids (FFAs) during in vitro simulation of small intestinal lipid digestion showed that:

[0166] In the T-oil sample, FFAs were almost completely released within 2 h, indicating that the oil phase was completely lipolyzed in a short time, thus completely releasing the torulene in the small intestine; in the T-EP sample, the release of FFAs was significantly slower, and about 60% of the FFAs were released after 2 h; T-oil released 40% of the FFAs after 2 h. In comparison, the release rates of FFAs of F-oil, F-EP and F-EPA after 2 h of small intestine digestion were 82.3%, 71.6% and 32.9%, respectively.

[0167] The release rates of torulene in the samples prepared by different embedding methods after 2 h of small intestine digestion were simulated in vitro, and the results showed that:

[0168] The release rates of torulene of F-EPA, F-EP and F-oil were 62.9%, 92.7% and 72.3%, respectively, which were significantly lower than those of T-EPA, T-EP and T-oil, i.e., 67.2%, 96.5% and 84.3%, respectively.

[0169] Based on the experimental results of the above FFA, it was shown that the high-activity complex of Rhodotorula coated with different carriers had stronger colon delivery capacity, and the high-activity complex of Rhodotorula in the form of F-EPA carrier could significantly reduce the loss in the gastrointestinal tract and enhance the colon-targeted delivery capacity.

[0170] (3) Bioavailability of torulene in different carriers after 2 h of small intestine digestion

[0171] Bioavailability refers to the proportion of the high-activity complex of Rhodotorula that is absorbed in the gastrointestinal tract, reflecting its bioavailability. Since the high-activity complex of Rhodotorula is mainly composed of torulene, the bioavailability thereof was evaluated based on torulene. During the hydrolysis of the oil phase, torulene is released and forms micelles containing bile salts, fatty acids and phospholipids, which are absorbed by the small intestine. Therefore, the bioavailability can be measured by the concentration of torulene in the micelles during in vitro digestion.

[0172] After passing through the entire simulated GIT model, the bioavailability of torulene in different carriers after 2 h of small intestine digestion was determined, and the results are shown in Table 5:

[0173] Table 5 Bioavailability of torulene in different carriers of Example 1 to Example 3 and Comparative Example 1 after 2 h of small intestine digestion (%)

[0174]

[0175] Wherein, the bioaccessibility of F-EPA, F-EP and F-oil were all lower than T-EPA, T-EP and T-oil, indicating that the high activity complex of Rhodotorula rubra was more conducive to its function in the colon. The lower bioavailability of the high activity complex of Rhodotorula rubra rubin in F-form than in T-form proved its stability and potential for colon health.

[0176] 4. Test of the influence of different carriers (F-EPA, T-EPA) on the regulation of the intestinal environment

[0177] According to the results of previous in vivo studies, further detection of the SCFAs content of F-EPA and T-EPA samples and the influence of the two samples on intestinal flora:

[0178] (1) Changes in the content of short-chain fatty acids

[0179] Intestinal microbiota can produce short-chain fatty acids (SCFAs) by encoding various endogenous enzymes and fermenting various compounds. Studies have found that SCFAs can act on multiple targets in the host's local and systemic processes, playing an important role in the host's metabolic and immune processes.

[0180] a. Influence of high activity complex of Rhodotorula rubra corn oil nanoemulsion alginate gel (F-EPA) on the content of short-chain fatty acids

[0181] From Figure 2 (A), we can see that:

[0182] The high activity complex of Rhodotorula rubra in F-EPA increased the total SCFAs concentration from 14.53 mM (0 h) to 53.24 mM (48 h), although it was lower than the content of inulin, the positive control group, 61.06 mM (48 h), but significantly higher than the blank control group 16.79 mM (48 h) (p < 0.05).

[0183] Acetic acid, propionic acid and butyric acid are the main fermentation products of F-EPA.

[0184] Compared with the blank control group 3.77 mM, the concentration of acetic acid was significantly increased to 36.23 mM (p > 0.05) after 48 h of enzymolysis of the high activity complex of Rhodotorula rubra;

[0185] The propionic acid content of F-EPA reached 9.76 mM at 48 h, which was significantly higher than that of the control group 3.12 mM (48 h);

[0186] Compared with the blank control group, the concentration of butyric acid in the high activity complex of Rhodotorula rubra group was also significantly increased; while the content of valeric acid showed a slow growth trend, which may be related to the metabolic activity of microorganisms.

[0187] F-EPA is metabolized by gut microbiota to produce short-chain fatty acids, such as acetic acid, propionic acid, and butyric acid. These fatty acids can protect the intestinal barrier, regulate immunity, inhibit the growth of harmful microorganisms, and promote liver fatty acid oxidation and regulate liver immune cell activity through the gut-liver axis.

[0188] Therefore, F-EPA has the potential to regulate the intestinal environment.

[0189] b. Effect of red yeast erythrin corn oil nanoemulsion alginate gel (T-EPA) on short-chain fatty acid content

[0190] Depend on Figure 2 (D) It can be concluded that:

[0191] After 48 hours of fermentation, the erythropoietin in T-EPA increased the concentration of total SCFAs from 11.44 mM to 41.99 mM, the concentration of acetic acid from 6.56 mM to 28.91 mM, the concentration of propionic acid from 2.52 mM to 7.35 mM, and the concentration of butyric acid from 1.34 mM to 3.52 mM.

[0192] Therefore, the F-EPA group showed a better effect in producing short-chain fatty acids compared to the T-EPA group.

[0193] (2) Changes in gut microbiota

[0194] The human gut contains a diverse symbiotic microbiota, among which Proteobacteria, Bacteroidetes, and Firmicutes are key groups for degrading complex macromolecules. Firmicutes / Bacteroidetes are associated with diseases such as obesity, while Bacteroidetes metabolites are not only utilized by other microbial groups but also play an important role in improving immune system disorders.

[0195] a. Effects of red yeast highly active complex corn oil nanoemulsion alginate gel (F-EPA) on phylum and genus-level species in the human gut microbiome.

[0196] Figure 2 (B) Display:

[0197] At the phylum level, after 48 hours of fermentation, the dominant bacterial groups in the red yeast high-activity complex group, the positive control inulin group, and the blank control OR group were all Bacteroidetes, Firmicutes, and Proteobacteria.

[0198] Compared with the blank control OR and the positive control inulin group, the number of Proteobacteria was significantly reduced and the number of Bacteroidetes was significantly increased in the red yeast high-activity complex group (F-EPA);

[0199] Compared with the OR group, F-EPA fermentation caused a significant decrease in the ratio of Firmicutes / Bacteroidetes, indicating that the high-activity complex of R. rubrum has the potential to alleviate obesity caused by the imbalance between Firmicutes and Bacteroidetes.

[0200] F-EPA significantly reduced the number of Shigella coli and inhibited the proliferation of harmful bacteria, which helps to prevent intestinal infections. At the same time, F-EPA promoted the proliferation of Bacteroides and improved the fermentation process Figure 2 (C)); while the beneficial bacteria Dialister slightly decreased, which may be related to its selectivity for carbohydrates.

[0201] b. Effects of R. rubrum astaxanthin corn oil nanoemulsion alginate gel (T-EPA) on phylum-level species and genus-level species in human intestinal microbiome

[0202] As shown in Figure 2 (E), Figure 2 (F), the microbial community of the T-EPA fermentation group mainly includes Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria, accounting for 90% of the microbial community. With the extension of fermentation time, the ratio of Firmicutes to Bacteroidetes in the T-EPA fermentation group increased significantly, and the abundance of Proteobacteria also increased significantly, indicating that R. rubrum astaxanthin effectively regulates the composition of intestinal microbiome. At the genus level, the main microorganisms in the T-EPA group include Escherichia-Shigella, Streptococcus, Bacteroides, Bifidobacterium, and Megamonas, etc.

[0203] Compared with each other, the high-activity complex of R. rubrum corn oil nanoemulsion alginate gel (F-EPA) exhibits more significant regulation effect on the composition and abundance of intestinal flora than the R. rubrum astaxanthin corn oil nanoemulsion alginate gel (T-EPA), and can effectively inhibit the proliferation of harmful bacteria, thereby showing potential prebiotic potential.

[0204] Comparative Example 2: Preparation and performance test of high-activity complex of R. rubrum in other carrier forms (liposomes, vesicles, bile bodies, microspheres)

[0205] 1. Preparation of high-activity complex of R. rubrum in other carrier forms

[0206] According to the preparation method in Example 1, R. rubrum astaxanthin freeze-dried powder and R. rubrum polysaccharide freeze-dried powder were first prepared, and then the R. rubrum astaxanthin freeze-dried powder and the R. rubrum polysaccharide freeze-dried powder were mixed in a mass ratio of 10:1 to obtain the high-activity complex of R. rubrum.

[0207] The high-activity complex of R. rubrum was prepared into the following other carrier forms:

[0208] (1) Preparation of high-activity complex of R. rubrum liposomes:

[0209] Dissolve 100 mg of soybean phospholipid in 10 mL of chloroform to obtain a phospholipid chloroform solution with a mass concentration of 10%, and remove the solvent using a rotary evaporator at 40°C to form a phospholipid film; then add 10 mg of high-activity complex of Rhodotorula to the film and perform ultrasonic treatment for 15 min to form liposomes. Finally, remove the supernatant by centrifugation (3000 rpm, 10 min) to remove the uncoated complex and excess phospholipid, and resuspend the collected liposome complex with an appropriate amount of PBS buffer for subsequent detection.

[0210] (2) Preparation of high-activity complex vesicles of Rhodotorula:

[0211] Dissolve polyvinyl alcohol (PVA) in deionized water to obtain a PVA aqueous solution with a concentration of 4%, and add the prepared high-activity complex of Rhodotorula 10 mg to the PVA solution and stir at 1000 r / min for 30 min; use a spray drying method (temperature controlled at 60-80°C) to convert the mixture into solid vesicles, and use a 60-mesh sieve to remove large particles to obtain vesicle complexes for subsequent experiments.

[0212] (3) Preparation of high-activity complex bile bodies of Rhodotorula:

[0213] Mix phospholipid and cholate (taurocholic acid) in a mass ratio of 1:1 and dissolve in deionized water, then gradually add the high-activity complex of Rhodotorula (50 mg) to the mixture and mix uniformly under high shear stirring; use ultrasonic treatment for 20 min to promote the formation of bile bodies, then centrifuge at 12000 r / min for 30 min to remove uncoated components, collect the supernatant, and obtain bile body complexes for subsequent analysis.

[0214] (4) Preparation of high-activity complex microspheres of Rhodotorula:

[0215] Dissolve polylactic acid-glycolic acid copolymer (PLGA) in an organic solvent dichloromethane to form a PLGA solution with a concentration of 10%, add the high-activity complex of Rhodotorula 50 mg to the PLGA solution, and stir at 1000 r / min for 30 min to form an emulsion; slowly add ionized water to the PLGA solution to obtain a precursor of emulsion microspheres, remove the dichloromethane by freeze-drying treatment, and finally obtain microspheres, which are sieved using an 80-μm sieve and separated by centrifugation at 10000 r / min for 20 min, and finally, collect microspheres with appropriate particle sizes for subsequent research.

[0216] 2. Performance test of high-activity complex of Rhodotorula in other carrier forms

[0217] The red yeast high activity complex was prepared into various carrier forms, and their stability and release characteristics were evaluated respectively.

[0218] (1) Stability test

[0219] The stability test was carried out at 4°C, 25°C and 50°C (dark condition), and the 7-day average retention rate of the red yeast rubixanthin in different carriers of Example 3 was shown in Table 6.

[0220] Table 6 7-day average retention rate (%) of red yeast rubixanthin in different carriers of Example 3 and Comparative Example 2 under dark condition

[0221]

[0222] The red yeast high activity complex corn oil nanoemulsion alginate gel (F-EPA) of Example 3 exhibited higher stability, and could more effectively protect the red yeast rubixanthin in the red yeast high activity complex, avoiding the degradation of the active ingredient.

[0223] (2) Release characteristic test

[0224] In addition, the release test results were shown in Table 7.

[0225] Table 7 Release rate (%) of red yeast rubixanthin in different carriers of Example 3 and Comparative Example 4 after 2 hours of small intestine digestion

[0226]

[0227] Although the liposomes, vesicles, bile bodies, microspheres and microcapsules all successfully encapsulated the red yeast high activity complex, the red yeast high activity complex corn oil nanoemulsion alginate gel (F-EPA) of Example 3 released more slowly, and under the simulated physiological conditions (PBS buffer at pH 7.4), the red yeast rubixanthin in the encapsulated high activity complex could safely reach the colon, showing the best effect.

[0228] Example 4: Preparation of soybean oil (D-oil) loaded with red yeast high activity complex, red yeast high activity complex soybean oil nanoemulsion spray dried powder (D-EP), and red yeast high activity complex soybean oil nanoemulsion alginate gel (D-EPA)

[0229] According to the preparation methods of Examples 1-3, the difference was that soybean oil was used instead of corn oil, and three kinds of carriers were prepared, which were soybean oil (D-oil) loaded with red yeast high activity complex, red yeast high activity complex soybean oil nanoemulsion spray dried powder (D-EP), and red yeast high activity complex soybean oil nanoemulsion alginate gel (D-EPA).

[0230] Stability test was carried out at 4℃, 25℃, 50℃ (dark condition), and the results are shown in Table 8.

[0231] Table 8 Average retention rate (%) of rhodoxanthin in different carriers of Example 7 in dark for 7 days

[0232]

[0233] In addition, release test was also carried out, and the release rates of rhodoxanthin in D-oil, D-EP and D-EPA were 53.2%, 67.8% and 73.5% respectively after 2 hours of small intestine period digestion. It is shown that the carrier prepared by soybean oil can also be used as a potential colon delivery system to some extent.

[0234] Example 5: Preparation of sunflower oil loaded with high-activity compound of Rhodotorula (K-oil), spray-dried powder of sunflower oil nanoemulsion loaded with high-activity compound of Rhodotorula (K-EP) and sunflower oil nanoemulsion alginate gel loaded with high-activity compound of Rhodotorula (K-EPA)

[0235] According to the preparation method of Examples 1-3, the difference is that sunflower oil is used instead of corn oil to prepare three carriers, which are sunflower oil loaded with high-activity compound of Rhodotorula (K-oil), spray-dried powder of sunflower oil nanoemulsion loaded with high-activity compound of Rhodotorula (K-EP) and sunflower oil nanoemulsion alginate gel loaded with high-activity compound of Rhodotorula (K-EPA).

[0236] Stability test was carried out at 4℃, 25℃, 50℃ (dark condition), and the results are shown in Table 9.

[0237] Table 9 Average retention rate (%) of rhodoxanthin in different carriers of Example 8 in dark for 7 days

[0238]

[0239] In addition, release test was also carried out, and the release rates of rhodoxanthin in K-oil, K-EP and K-EPA were 51.3%, 62.7% and 77.6% respectively after 2 hours of small intestine period digestion. It is shown that the carrier prepared by sunflower oil can also be used as a potential colon delivery system to some extent.

[0240] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for preparing a highly active complex carrier of red yeast, characterized in that, The carrier is a plant oil loaded with a highly active red yeast complex, and the process includes the following steps: (1) A high-activity complex of red yeast was obtained by mixing red yeast erythrin and red yeast polysaccharide; (2) Dissolve the red yeast high-activity complex obtained in step (1) in vegetable oil to obtain vegetable oil loaded with red yeast high-activity complex.

2. The preparation method according to claim 1, characterized in that, In step (1), red yeast erythrin and red yeast polysaccharide are mixed in a mass ratio of (8-12):

1.

3. The preparation method according to claim 1, characterized in that, In step (2), the vegetable oil includes, but is not limited to, corn oil, soybean oil, and sunflower seed oil.

4. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of the red yeast high-activity complex to the vegetable oil is (3-5):1000.

5. A method for preparing a highly active complex carrier of red yeast, characterized in that, The carrier is a spray-dried powder of a highly active red yeast complex plant oil nanoemulsion, comprising the following steps: (1) A high-activity complex of red yeast was obtained by mixing red yeast erythrin and red yeast polysaccharide; (2) Dissolve the red yeast high-activity complex obtained in step (1) in vegetable oil to obtain vegetable oil loaded with red yeast high-activity complex; (3) OSA starch solution was mixed with vegetable oil loaded with red yeast high-activity complex and homogenized to obtain red yeast high-activity complex vegetable oil nanoemulsion. (4) Spray dry the red yeast high-activity complex plant oil nanoemulsion to obtain red yeast high-activity complex plant oil nanoemulsion spray-dried powder.

6. The preparation method according to claim 5, characterized in that, OSA starch solution and vegetable oil loaded with highly active red yeast complex were mixed at a mass ratio of 1:(8-10).

7. A method for preparing a highly active complex carrier of red yeast, characterized in that, The carrier is a red yeast highly active complex plant oil nanoemulsion alginate gel, comprising the following steps: (1) A high-activity complex of red yeast was obtained by mixing red yeast erythrin and red yeast polysaccharide; (2) Dissolve the red yeast high-activity complex obtained in step (1) in vegetable oil to obtain vegetable oil loaded with red yeast high-activity complex; (3) OSA starch solution was mixed with vegetable oil loaded with red yeast high-activity complex and homogenized to obtain red yeast high-activity complex vegetable oil nanoemulsion. (4) The alginate solution was mixed with the red yeast high-activity complex plant oil nanoemulsion and gelled to obtain red yeast high-activity complex plant oil nanoemulsion alginate gel.

8. The preparation method according to claim 7, characterized in that, Alginate solution and red yeast high-activity complex plant oil nanoemulsion are mixed at a mass ratio of (1-2):(1-2), homogenized, and stirred to form a gel block, thus obtaining red yeast high-activity complex plant oil nanoemulsion alginate gel.

9. The application of the vegetable oil loaded with the highly active red yeast complex prepared by the preparation method according to any one of claims 1 to 4, the spray-dried powder of the vegetable oil nanoemulsion of the highly active red yeast complex prepared by the preparation method according to claim 5 or 6, and the alginate gel of the vegetable oil nanoemulsion of the highly active red yeast complex prepared by the preparation method according to claim 7 or 8 in improving the stability of red yeast lycopene.

10. The application of the vegetable oil loaded with highly active red yeast complex prepared by any of the preparation methods of claims 1 to 4, the spray-dried powder of vegetable oil nanoemulsion of highly active red yeast complex prepared by the preparation method of claim 5 or 6, and the alginate gel of vegetable oil nanoemulsion of highly active red yeast complex prepared by the preparation method of claim 7 or 8 in the preparation of products that promote gastrointestinal metabolic function.

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