CN114794338B - Preparation method of composite edible fungus health flavor drink - Google Patents
Preparation method of composite edible fungus health flavor drink Download PDFInfo
- Publication number
- CN114794338B CN114794338B CN202210491347.5A CN202210491347A CN114794338B CN 114794338 B CN114794338 B CN 114794338B CN 202210491347 A CN202210491347 A CN 202210491347A CN 114794338 B CN114794338 B CN 114794338B
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- Prior art keywords
- edible fungus
- polysaccharide
- compound
- folic acid
- liposome
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Classifications
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- A—HUMAN NECESSITIES
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Abstract
The invention relates to a preparation method of a compound edible fungus health flavor drink, belonging to the technical field of drink processing. The preparation process of the invention mainly comprises the following steps: selecting and preprocessing three edible fungus raw materials of lentinus edodes, cordyceps sinensis flowers and tremella, extracting crude polysaccharide of compound edible fungus, preparing compound edible fungus particles, preparing a liposome compound edible fungus polysaccharide delivery system containing folic acid modification, adding auxiliary materials for compound preparation, sterilizing and filling to prepare the compound edible fungus health flavor drink. After the three edible fungus crude polysaccharides are micronized and the liposome containing folic acid modification is encapsulated, the molecular weight of the polysaccharide is reduced, the aggregation of polysaccharide particles is prevented, the polysaccharide particles are easier to be absorbed and utilized by human bodies, and the active targeting of the liposome is improved by utilizing a folic acid receptor-ligand specific homing device. The invention achieves the purpose of regulating and controlling the flavor and rheological property of the composite edible fungi healthy drink by regulating and controlling the particle size, the sugar-acid ratio and the content of ethyl maltol of the composite edible fungi polysaccharide liposome.
Description
Technical Field
The invention belongs to the technical field of beverage preparation, and particularly relates to a preparation method of a compound edible fungus health flavor beverage.
Background
The edible fungus polysaccharide is called as a biological reaction regulator, is an immunostimulant or an immunomodulator, can enhance humoral immunity and cellular immunity, can be used for improving and preventing diseases caused by abnormal immune functions, and has stronger anticancer and cancer preventing effects. Through scientific compatibility, the limitation of the efficacy of single polysaccharide can be overcome by compounding a plurality of edible fungus polysaccharide, and the synergistic effect is achieved. The compound polysaccharide obtained by compounding three edible fungus polysaccharides of lentinan, cordyceps flower (also called as cordyceps militaris or cordyceps militaris) polysaccharide and tremella polysaccharide can play a role in synergistically regulating the immune function on different mechanisms; wherein lentinan has effects of enhancing immunity and enhancing immune response, cordyceps polysaccharide has effects of activating complement system, and Tremella polysaccharide has effects of improving phagocyte ability and regulating immunocyte.
The immunoregulation function and the anti-tumor activity of three edible fungi polysaccharide, namely lentinan, cordyceps polysaccharide and tremella polysaccharide, and the compound fungus polysaccharide are fully verified. Wang Limin (2018) has carried out research on induction of anti-tumor active immune response by combining multifunctional artificial antigen presenting cells with lentinan, and results show that the independent application of lentinan and artificial antigen presenting cells (aAPCs) can obviously inhibit tumor growth and induce corresponding anti-tumor immune response, and the combined application of lentinan and aAPCs not only can non-specifically enhance the in-vivo NK cell tumor killing activity of a treatment mouse, improve the T cell frequency and reduce the proportion of Treg cells, but also can enhance the capability of the aAPCs in induction of specific active immune response. Li Jiang (2017) is subjected to a synergistic interaction and mechanism study of lentinan and antitumor drugs, and the result shows that lentinan and oxaliplatin have inhibition effects on in vitro proliferation of human hepatoma cells HepG2 and mouse hepatoma cells H22 and are dose-dependent. Flow cytometry examined HepG2 apoptotic cells, and found that the apoptosis rates of lentinan (800. Mu.g/mL), oxaliplatin (20. Mu.M) and the combination (800. Mu.g/mL+20. Mu.M) were 16.24%, 20.01% and 30.60%, respectively. Chen Ruizhan and the like (2020) are subjected to researches on physicochemical characteristics, antioxidation and immunocompetence of Cordyceps militaris polysaccharide, and the results show that the Cordyceps militaris polysaccharide can remarkably promote lipopolysaccharide to activate proliferation activity of RAW264.7 cells, and improve phagocytic capacity of the RAW264.7 cells, so that immunity of a human body is enhanced. Hu Ting et al (2005) have carried out the influence of tremella polysaccharides on the activity of mouse spleen lymphocyte protein kinase C, and the result shows that tremella polysaccharides can enhance the activity of mouse spleen lymphocyte Protein Kinase C (PKC) in vitro, and through activating the activity of cell membrane PKC, the intracellular series protein cascade phosphorylation reaction is caused, so that the immune cell regulation function is realized. Shuping (2021) performs the immunoregulatory effect of different edible fungus polysaccharide compounds on RAW264.7 macrophages, and the result shows that 10 polysaccharide compounds obtained by compounding any 3 kinds of polysaccharides from 5 kinds of edible fungus polysaccharides (tremella polysaccharide, pachyman, lentinan, hericium erinaceus polysaccharide and bamboo fungus polysaccharide) can realize the immunoregulatory effect by promoting proliferation of the RAW264.7 macrophages and secretion of TNF-alpha and enhancing phagocytic capacity of the macrophages. The compound polysaccharide 303 based on tremella polysaccharide, pachyman and Dictyophora indusiata polysaccharide has the best effect of promoting macrophage proliferation, the compound polysaccharide 301 based on tremella polysaccharide, pachyman and lentinan and the compound polysaccharide 303 based on tremella polysaccharide, pachyman and Dictyophora indusiata polysaccharide have the best effect of improving TNF-alpha secretion level, and the compound polysaccharide 301 based on tremella polysaccharide, pachyman and lentinan has the strongest effect of enhancing phagocytic capacity of macrophages.
Because of the large molecular weight of macromolecular polysaccharide, the macromolecular polysaccharide is not easy to be absorbed and utilized by human bodies. The dynamic high-pressure homogenizing technology has the advantages that the macromolecular structure is well crushed, degraded and homogenized through a series of comprehensive effects such as strong shearing, high-speed impact, instantaneous pressure release, high-frequency oscillation, swelling, cavitation and the like on macromolecular substances. The liposome is adopted to encapsulate the dynamic high-pressure homogenized micromolecular polysaccharide, so that aggregation of polysaccharide molecules is avoided, and a liposome drug delivery system is constructed. The liposome is modified by a specific homing device such as an antibody, a sugar residue, a hormone, a receptor ligand and the like through modifying common liposome, so that the liposome can reach specific tissues in a targeted manner, the in vivo selective release can be realized, and the liposome is a hot pharmacological research direction for improving the curative effect and reducing the toxicity. Tumor cells in the dividing phase secrete excess folate receptor compared to normal cells, and folate-modified liposomes can be targeted to tumor cells by receptor-ligand binding. The Yao (2021) carries out preparation of the targeted liposome of the co-carried paclitaxel and itraconazole and in-vitro tumor inhibition effect evaluation, and the result shows that the folic acid modification can improve the targeting capability of the liposome to tumor cells and enhance the uptake effect; the addition of the free folic acid to compete for the folic acid receptor has the effect of weakening the fluorescence intensity of coumarin 6, which indicates that the folic acid modified liposome system enters cells through endocytosis mediated by combining with the folic acid receptor overexpressed on the surfaces of tumor cells. The construction of a folic acid modified amphipathic taxol micelle drug-carrying system and the research on cervical cancer targeting drug delivery are carried out on that (2021) show that the cytotoxicity of taxol-folic acid modified amphipathic cholesterol chitosan micelle is higher than that of a taxol control group under different concentrations, and particularly has higher killing power on Hela cells. The fluorescence microscope shows that the folic acid modified amphipathic cholesterol chitosan micelle has a strong fluorescence signal in Hela cells, and the fluorescence signal in A549 cells is very weak, which indicates that the folic acid modified amphipathic cholesterol chitosan micelle has targeting property on the Hela cells.
In addition, the purpose of regulating and controlling the rheological property of the beverage can be achieved by controlling the particle size of the liposome after encapsulation. In the aspect of beverage flavor regulation, the rheological property of the beverage and the control of the sugar acid ratio and the ethyl maltol content in the compounding process have influence on the overall flavor of the beverage. Therefore, the parameters can be used as a method for regulating and controlling the flavor and the rheology of the composite edible fungi healthy drink.
The invention discloses cordyceps militaris polysaccharide and edible fungus ferment compound drink and a preparation method thereof (bulletin number: CN 106038614B), wherein the compound drink is prepared by taking cordyceps militaris crude polysaccharide and ferment prepared by fermenting compound edible fungi of oyster mushrooms, agrocybe cylindracea, lentinus edodes, flammulina velutipes and pleurotus eryngii as main raw materials, wherein the hydrolysis of macromolecular polysaccharide is accelerated after various edible fungi are fermented, and the bioavailability of the polysaccharide is improved. The invention is different from the method in that dynamic high-pressure homogeneous degradation macromolecular polysaccharide is used for preparing small molecular crude polysaccharide particles, and liposome is used for wrapping the degraded small molecular polysaccharide for micronization to prevent re-agglomeration, so that the bioavailability is improved.
Sun Yijie et al (2020) discloses a tremella polysaccharide high protein drink and a preparation method thereof (bulletin number: CN 111743069A), and the method comprises the following steps: grinding 30-35% of tremella into powder, adding distilled water, pulping, cooling, filtering, adding absolute ethyl alcohol into filtrate, and centrifuging to obtain tremella stock solution; mixing 5-7% of whey protein, 4-6% of polydextrose and 3-5% of xylitol, adding water, uniformly stirring, and homogenizing under high pressure to obtain a mixed solution; adding the tremella stock solution into the mixed solution to obtain a final solution; sterilizing the final solution at high temperature and normal pressure, and packaging. The edible fungus polysaccharide is wrapped by liposome.
Zhou Ping et al (2014) discloses a preparation process of an edible fungus composite polysaccharide (bulletin number: CN 102627789B), which comprises the following steps: the fruiting bodies of the mushrooms, the tremella and the hericium erinaceus are taken as raw materials, and are respectively cleaned, dried to water content of 5% -10%, crushed and sieved to obtain edible fungus powder; mixing edible fungus powder of Lentinus Edodes, tremella and Hericium Erinaceus at a weight ratio of 1:1:1, degreasing, ultrasonic extracting with water, filtering, ultrasonic extracting with water again, concentrating, washing, and drying to obtain edible fungus compound polysaccharide. The edible fungus polysaccharide is wrapped by liposome.
Zheng Baodong (2021) discloses a preparation method of a compound edible fungus polysaccharide compound with an anti-fatigue effect, which is characterized in that polysaccharide is extracted from mushrooms, flammulina velutipes and tremella, lentinan, flammulina velutipes polysaccharide and tremella polysaccharide are compounded for use, 3 edible fungus polysaccharide is taken as glycosyl, bivalent manganese inorganic salt is taken as glycosyl, and a meridian combination reaction is carried out to form the compound edible fungus polysaccharide compound with a stable structure. The edible fungus polysaccharide is wrapped by liposome.
The invention discloses DHA microcapsule powder containing lentinan and a preparation method thereof (bulletin number: CN 107736611A), wherein the DHA microcapsule powder containing lentinan is prepared by mixing and homogenizing and emulsifying the lentinan, the lentinan and water phase wall materials (whey protein, sodium caseinate, soy protein isolate or silkworm chrysalis protein powder) under high pressure and spray drying. The invention is different from the above-mentioned three fungus mushroom polysaccharide which is packed by soybean lecithin/cholesterol liposome and micronized, and uses it as main functional component to make liquid beverage.
Wei Hongjun (2013) discloses a health-care beverage containing lentinan and agaric polysaccharide and a preparation method thereof (bulletin number: CN 102948840A), wherein the invention uses the lentinan: 5-10 parts; agaric polysaccharide: 5-10 parts; jujube: 10-15 parts; honey: 5-15 parts; white sugar: 10-20 parts of a health-care beverage containing lentinan and auricularia auricula polysaccharide is prepared, but no further processing treatment is carried out on the lentinan and the auricularia auricula polysaccharide. The invention takes three edible fungus polysaccharide as raw materials, and the three edible fungus polysaccharide is subjected to dynamic high-pressure homogenization and micronization and then liposome encapsulation, and finally the compound edible fungus drink is prepared.
Jiang Xueping (2021) discloses a compound edible fungus oral liquid and a preparation process thereof (application publication number: CN 112535287A), and the compound edible fungus oral liquid is prepared by taking a mixed fermentation liquid of a boletus, lentinus edodes, grifola frondosa, poria cocos, green mushroom extract, truffle extract, morchella enzymatic hydrolysate, medlar, spina date seed, angelica sinensis, lily, dried orange peel, liquorice and chicory pollen typhae as raw materials. The invention is different from the method in that edible fungus polysaccharide which is widely accepted to have a powerful immunoregulation function is used as a functional component to prepare a composite beverage, and the polysaccharide is treated by using a dynamic high-pressure homogenizing micronization and liposome encapsulation technology to improve the bioavailability.
Liu Qun et al (2012) have invented a double mushroom polysaccharide preparation containing gold needle mushroom polysaccharide and lentinan, and its preparation method and use (bulletin number: CN 101721433B), the invention uses gold needle mushroom polysaccharide and lentinan as main components, and adds ethyl maltol and other auxiliary components to prepare the double mushroom polysaccharide preparation, and the preparation method adopted is simple and the polysaccharide is not processed. The invention is different from the above-mentioned three edible fungi polysaccharide as raw material, firstly making dynamic high-pressure homogenizing and micronizing, then making liposome package, finally adding auxiliary material so as to obtain the invented compound edible fungi beverage.
Zhouqi et al (2020) carried out the research on the processing technology of the lentinan compound oral liquid preparation. The lentinan and the auricularia auricula polysaccharide obtained by ultrasonic synergistic compound enzyme method extraction are used as raw materials to prepare the lentinan oral liquid, and the sensory score value is used as an evaluation index to optimize the optimal formula of the compound oral liquid preparation. The three edible fungi polysaccharide adopted by the invention are subjected to dynamic high-pressure homogenization and micronization, and then are subjected to liposome encapsulation, and finally the beverage is prepared.
Ge Yingliang et al (2012) developed lentinan health drinks. The lentinan beverage is prepared from Lentinus edodes by extracting Lentinus edodes with hot water, breaking wall with ultrasonic wave, extracting lentinan, and preparing into lentinan beverage. The three edible fungi polysaccharide adopted by the invention are subjected to dynamic high-pressure homogenization and micronization, and then are subjected to liposome encapsulation, and finally the beverage is prepared.
Wei Shaowen (2020) has been studied and developed for lentinan oral liquid. Extracting lentinan by hot water extraction, adding sucrose, citric acid, xylitol and vanillin, and flavoring to obtain the final product. But the lentinan is not degraded and further processed. The three edible fungus polysaccharides adopted by the invention are subjected to dynamic high-pressure homogenization and micronization, and then are subjected to liposome encapsulation, and finally the compound edible fungus flavor drink is prepared.
Disclosure of Invention
The invention aims to provide a preparation method of a compound edible fungus health flavor drink. According to the invention, a certain amount of folic acid-polyethylene glycol-distearoyl phosphatidylethanolamine (Folate-PEG-DSPE) or folic acid-polyethylene glycol-Cholesterol (Folate-PEG-cholestenol) is added into liposome raw materials to construct a folic acid-modified liposome composite edible fungus polysaccharide delivery system, and an anti-tumor active targeting of the liposome drug delivery system is improved through a folic acid receptor-ligand specific homing device; the purposes of regulating and controlling the rheological property and the flavor of the composite edible fungi healthy drink are achieved by regulating and controlling the particle size, the sugar-acid ratio and the content of ethyl maltol of the composite edible fungi polysaccharide liposome.
The technical scheme of the invention is as follows:
a preparation method of a compound edible fungus health flavor drink mainly comprises the following preparation processes:
(1) Selecting and preprocessing edible fungus raw materials: selecting three edible fungi, namely mushroom, cordyceps flower and tremella, drying, crushing and sieving with a 60-mesh sieve to obtain three edible fungus raw material dry powders respectively;
(2) Extracting the compound edible fungus crude polysaccharide: mixing the three edible fungus raw materials of the mushrooms, the cordyceps sinensis flowers and the tremella in the step (1) according to a proportion, putting the three edible fungus raw materials into an extraction container, adding water, heating to above 80 ℃ to extract for 1-3 hours, filtering the extract to obtain filtrate, concentrating the filtrate, slowly adding ethanol into the filtrate to enable the ethanol concentration to reach 70% -90%, refrigerating for 8 hours at 4 ℃, centrifuging, and obtaining a precipitate which is the compound edible fungus crude polysaccharide;
(3) Preparing composite edible fungus particles: completely dissolving the crude polysaccharide of the compound edible fungi obtained in the step (2) by using water to obtain crude polysaccharide extraction raw pulp, and dynamically homogenizing the crude polysaccharide extraction raw pulp at high pressure by using a dynamic high-pressure homogenizer to obtain the crude polysaccharide particles of the compound edible fungi;
(4) Preparation of a composite edible fungus polysaccharide delivery system: preparing a folic acid modified liposome-containing composite edible fungus polysaccharide delivery system by using a pre-synthesized lipophilic folic acid derivative;
(5) Adding auxiliary materials: adding water into the liposome composite edible fungus polysaccharide delivery system containing folic acid modification prepared in the step (4) to prepare a certain concentration, adding sucrose, citric acid and ethyl maltol according to a certain sugar acid ratio for compounding, and controlling the pH of the beverage to be below 3.6 to obtain a composite edible fungus healthy beverage;
(6) Sterilizing and filling: filling after pasteurization, and sealing;
(7) Cooling and storing at normal temperature.
The mixing ratio of the three edible fungus raw material dry powders of the mushroom, the cordyceps sinensis flower and the tremella in the step (2) is 3:1:1.
The preparation method of the folic acid modified liposome composite edible fungus polysaccharide delivery system in the step (4) comprises the steps of preparing folic acid-polyethylene glycol-distearoyl phosphatidylethanolamine and folic acid-polyethylene glycol-cholesterol as raw materials; adding folic acid-polyethylene glycol-distearoyl phosphatidylethanolamine or folic acid-polyethylene glycol-cholesterol as a trace lipid component into hydrogenated soybean phospholipid or cholesterol to obtain a lipid mixture, fully dissolving the lipid mixture and the edible fungus crude polysaccharide particles in the step (3) in absolute ethyl alcohol, quickly injecting the absolute ethyl alcohol into 0.01mol/L phosphoric acid buffer solution, performing ultrasonic treatment for 15min to form a homogeneous system, evaporating the ethanol under reduced pressure at 60 ℃, and extruding and passing through a film by using a polycarbonate film to obtain a liposome composite edible fungus polysaccharide delivery system containing folic acid modification, wherein the particle size of the liposome composite edible fungus polysaccharide delivery system is uniform;
the addition amount of the folic acid-polyethylene glycol-distearoyl phosphatidylethanolamine or folic acid-polyethylene glycol-cholesterol in the hydrogenated soybean phospholipid or cholesterol is 0.1-1mol%.
The polycarbonate film described in the above step (4) has a pore size of 0.6 μm or 1. Mu.m.
The concentration of the water added and blended in the step (5) is 0.2% -2%.
The sugar-acid ratio in the step (5) is (13-15): 1.
The added ethyl maltol in the step (5) accounts for 0.03-0.05% of the weight of the compounded beverage.
The pasteurization in step (6) above is carried out at 71.7℃for 15 seconds.
The invention has the beneficial effects that:
(1) The method selects three edible fungus polysaccharides of lentinus edodes, cordyceps sinensis flowers and tremella as main functional components, overcomes the limitation of single polysaccharide efficacy by compounding multiple edible fungus polysaccharides, and has the synergistic effect.
(2) The invention adopts dynamic high-pressure homogenization to micronize and crush macromolecular polysaccharide, and encapsulates micronized micromolecular polysaccharide through liposome, thereby avoiding aggregation of micronized polysaccharide, and simultaneously the micromolecular polysaccharide is easy to be absorbed and utilized by human body; a certain amount of Folate-PEG-DSPE or Folate-PEG-cholestol is added into a liposome raw material to construct a folic acid modified liposome composite edible fungus polysaccharide delivery system, and the anti-tumor active targeting of the liposome drug delivery system is improved through a folic acid receptor-ligand specific homing device.
(3) According to the invention, the rheological property of the composite edible fungus healthy drink is regulated and controlled by controlling the particle size of the liposome, so that the viscosity and shearing stress of the composite edible fungus healthy drink are reduced, the liposome wraps polysaccharide particles, and the flavor property of the drink is regulated and controlled by the sugar-acid ratio and the addition amount of ethyl maltol, so that the too intense special fishy smell of the drink is alleviated, and the sweet and sour flavor is endowed.
Drawings
FIG. 1 is a molecular structural formula of folic acid-polyethylene glycol-distearoyl phosphatidylethanolamine (Folate-PEG-DSPE), folic acid-polyethylene glycol-Cholesterol (Folate-PEG-Cholesterol).
Fig. 2 is a schematic diagram of a folic acid modified liposome composite edible fungus polysaccharide delivery system.
Fig. 3 is a flavor radar chart of the electronic nose evaluation of comparative example and example 1.
Electronic nose sensor description:
the sensitive compounds of 18 sensors of the electronic nose are respectively S1 (alkane, smog), S2 (alcohols, aldehydes, short-chain alkanes), S3 (ozone), S4 (sulfide), S5 (organic amine), S6 (benzene ketone, aldol, aromatic compound), S7 (short-chain alkanes: methane), S8 (short-chain alkanes: propane), S9 (aromatic compound, aldol), S10 (hydrogen-containing gas: hydrogen), S11 (alkanes, alkenes), S12 (short-chain alkanes: liquefied gas), S13 (combustible gases: methane), S14 (combustible gases: combustible gases), S15 (alkanes, organic gases), S16 (sulfide), S17 (nitride), S18 (ketones, alcohols).
Fig. 4 is a graph of rheological properties of a composite edible fungus health drink of comparative example.
Fig. 5 is a graph of rheological properties of a composite edible fungus health drink containing a large particle size folic acid modified liposomal polysaccharide delivery system of example 1.
FIG. 6 shows the results of in vitro HepG2 cell uptake fluorescence analysis by confocal laser scanning microscopy: the non-targeted edible fungus crude polysaccharide liposome (A) and the folic acid modified liposome-containing composite edible fungus polysaccharide delivery system (B) are not added with the Folate-PEG-DSPE.
Fig. 7 is a graph of rheological properties of a composite edible fungus health drink containing a small particle size folic acid modified liposomal polysaccharide delivery system of example 2.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The technical scheme of the invention will be further described with reference to specific embodiments.
Comparative example: preparation method of composite edible fungus healthy drink
Three edible fungi, namely mushroom, cordyceps flower and tremella, are selected, dried, crushed and sieved by a 60-mesh sieve, and three edible fungus raw material powders are obtained. Respectively weighing 50g of mushroom, cordyceps flower and tremella powder and 50g of compound mushroom powder (30 g of mushroom, 10g of cordyceps flower and 10g of tremella), respectively placing into an extraction container, respectively adding 1500mL of water, heating to 95 ℃ for extraction for 2 hours, filtering the extract to obtain filtrate, concentrating the filtrate to 200mL, slowly adding 600mL of 95% ethanol into the filtrate, refrigerating for 8 hours at 4 ℃, centrifuging, and obtaining precipitate which is tremella crude polysaccharide, cordyceps flower crude polysaccharide, tremella crude polysaccharide and compound edible fungus crude polysaccharide, and freeze-drying.
Weighing a certain amount of tremella polysaccharide, cordyceps polysaccharide, tremella polysaccharide and compound fungus polysaccharide dry powder, and preparing 160, 80, 40, 20 and 10 mug/mL of standby working solution by using a DMEM (medium-electron microscope) culture medium added with 10% fetal calf serum. The control group was added with medium, and the positive group was added with 1. Mu.g/mL lipopolysaccharide medium solution. The mouse macrophage cell line RAW264.7 was cultured in DMEM complete medium, and cells in the logarithmic growth phase were taken for subsequent experiments. RAW264.7 cells in logarithmic growth phase are inoculated into an ELISA plate according to 5X 105 cells/mL each hole, wall adhesion is carried out for 4 hours, 500 mu L of standby working solution is respectively added into each component, the culture is carried out for 12 hours, and supernatant fluid is collected for measuring tumor necrosis factor-alpha (tumor necrosis factor-alpha, TNF-alpha). The secretion level of TNF-alpha was determined according to the instructions of the ELISA kit. The results are shown in Table 1.
According to the results in Table 1, it was shown that all three polysaccharides and the complex polysaccharide showed an increase in TNF-alpha factor secretion by macrophage RAW264.7 compared to the control group. Lentinan appears to be significantly dose dependent on the secretion of TNF-alpha factor. Compared with the positive group added with lipopolysaccharide, the cordyceps polysaccharide obviously promotes the secretion of TNF-alpha factor, but the polysaccharide concentration has no obvious influence on the secretion of TNF-alpha factor. The tremella polysaccharide has no obvious effect on improving the secretion of TNF-alpha factor of macrophage RAW264.7, and does not show the effect of promoting the secretion of TNF-alpha factor with the increase of tremella polysaccharide concentration. The compound edible fungus polysaccharide shows better effect of promoting the secretion of TNF-alpha factor than the three polysaccharides when being singly used. This is due to the mutual supplementation of the compound edible fungi polysaccharide and the synergistic promoting effect.
TABLE 1 Effect of different Compound edible fungi polysaccharide on the secretion of TNF-alpha factor of RAW264.7 macrophages
Taking the obtained compound edible fungus crude polysaccharide, adding a certain amount of water according to the mass fraction of the compound edible fungus crude polysaccharide with the concentration of 1%, completely dissolving the compound edible fungus crude polysaccharide, adding sucrose and citric acid according to the sugar-acid ratio of 15:1, controlling the pH of the beverage to be 3.5, and adding 0.04% of ethyl maltol for compounding to obtain the compound edible fungus healthy beverage. Flavor evaluation was performed using an electronic nose flavor profile (fig. 3), and rheological properties (fig. 4) were evaluated using a rheometer. As can be seen from fig. 3, the beverage in this comparative example has a strong smell compared to the beverage after liposome encapsulation. From the rheological properties of fig. 4, it can be seen that the polysaccharide has a larger particle size and the overall product stability is normal, since no high pressure homogenization is performed. Pasteurizing, maintaining at 71.7deg.C for 15 seconds, filling, sealing, cooling, and storing at normal temperature. The compound edible fungus healthy drink has the advantages of strong fungus mushroom flavor, good sour and sweet taste and good taste, and the total sensory quality score reaches 80 minutes.
Example 1: preparation method of composite edible fungus healthy drink containing large-particle-size folic acid modified liposome polysaccharide delivery system
Three edible fungi, namely mushroom, cordyceps flower and tremella, are selected, dried, crushed and sieved by a 60-mesh sieve, and three edible fungus raw material powders are obtained. Respectively weighing 30g,10g and 10g of three kinds of mushroom, cordyceps flower and tremella powder, putting the three kinds of mushroom, cordyceps flower and tremella powder into an extraction container, adding 1500mL of water, heating to 95 ℃ for extraction for 2 hours, filtering the extract to obtain filtrate, concentrating the filtrate to 200mL, slowly adding 600mL of 95% ethanol into the filtrate, refrigerating at 4 ℃ for 8 hours, centrifuging, and obtaining precipitate as the compound edible fungus crude polysaccharide. And (3) completely dissolving the compound edible fungus crude polysaccharide with water, carrying out dynamic high-pressure homogenization by using a dynamic high-pressure homogenizer to obtain compound edible fungus crude polysaccharide particles, and carrying out spray drying granulation. Folate-PEG-DSPE was added as a minor lipid component to hydrogenated soybean phospholipid at 1mol%. Fully dissolving the components and edible fungus polysaccharide particles in absolute ethyl alcohol, quickly injecting into 0.01mol/L phosphoric acid buffer solution, performing ultrasonic treatment for 15min to form a homogeneous system, performing reduced pressure evaporation at 60 ℃ to remove the ethanol, and extruding through a polycarbonate film with the thickness of 1 mu m to obtain the folic acid-modified liposome-containing composite edible fungus polysaccharide delivery system with the particle size within the range of 1.0 mu m. And (3) adding water into the prepared liposome composite edible fungus polysaccharide delivery system containing the folic acid modification to dilute to 1%, adding sucrose and citric acid according to a sugar-acid ratio of 15:1, controlling the pH of the beverage to be 3.5, and adding 0.04% ethyl maltol for compounding to obtain the composite edible fungus healthy beverage containing the liposome polysaccharide delivery system containing the folic acid modification with large particle size. Flavor evaluation was performed using an electronic nose flavor profile (fig. 3), and rheological properties (fig. 5) were evaluated using a rheometer. As can be seen from fig. 3, the specific smell of the beverage in this example was significantly relaxed compared to the beverage of the comparative example. From the rheological characteristics of fig. 5, it can be seen that the large-particle-size liposome composite edible fungus polysaccharide drink prepared by high-pressure homogenization and passing through a 1 μm polycarbonate film has smaller particle size of the polysaccharide liposome and good overall product stability. Pasteurizing, maintaining at 71.7deg.C for 15 seconds, filling, sealing, cooling, and storing at normal temperature. The compound edible fungus healthy drink is rich in flavor, sweet and sour, good in taste and high in overall sensory quality score of 96 minutes.
Example 2: preparation method of composite edible fungus healthy drink containing small-particle-size folic acid modified liposome polysaccharide delivery system
Three edible fungi, namely mushroom, cordyceps flower and tremella, are selected, dried, crushed and sieved by a 60-mesh sieve, and three edible fungus raw material powders are obtained. Respectively weighing 30g,10g and 10g of three kinds of mushroom, cordyceps flower and tremella powder, putting the three kinds of mushroom, cordyceps flower and tremella powder into an extraction container, adding 1500mL of water, heating to 95 ℃ for extraction for 2 hours, filtering the extract to obtain filtrate, concentrating the filtrate to 200mL, slowly adding 600mL of 95% ethanol into the filtrate, refrigerating at 4 ℃ for 8 hours, centrifuging, and obtaining precipitate as the compound edible fungus crude polysaccharide. And (3) completely dissolving the compound edible fungus crude polysaccharide with water, carrying out dynamic high-pressure homogenization by using a dynamic high-pressure homogenizer to obtain compound edible fungus crude polysaccharide particles, and carrying out spray drying granulation. Folate-PEG-DSPE was added as a minor lipid component to hydrogenated soybean phospholipid at 1mol%. Fully dissolving the components and edible fungus polysaccharide particles in absolute ethyl alcohol, quickly injecting into 0.01mol/L phosphoric acid buffer solution, performing ultrasonic treatment for 15min to form a homogeneous system, performing reduced pressure evaporation at 60 ℃ to remove the ethanol, and extruding through a polycarbonate film with the thickness of 0.6 mu m to obtain the folic acid modified liposome-containing composite edible fungus polysaccharide delivery system with the particle size within the range of 0.6 mu m.
The targeting delivery efficiency of the folic acid modified liposome composite edible fungus polysaccharide delivery system is evaluated through an in vitro cell uptake experiment. Culturing human liver cancer cell HepG2 cells with a vaneless RPMI1640 culture medium containing 1% penicillin streptomycin and 10% fetal bovine serum, taking HepG2 cells in a logarithmic growth phase for in vitro cell uptake experiments, transferring into a sterile centrifuge tube after being digested into a suspension state by pancreatin, centrifuging at 1000r/min for 5min, discarding supernatant, adding fresh culture medium, and sub-packaging into 4 sterile EP tubes. The blank group is not treated by adding medicine, and the non-targeted edible fungus crude polysaccharide liposome without adding Folate-PEG-DSPE and the folic acid modified liposome-containing composite edible fungus polysaccharide delivery system are respectively added into the medicine adding groups, and incubated for 1h at 37 ℃. After the incubation was completed, the cells were washed 3 times with cold PBS to remove unbound liposomes. The fluorescence intensity in HepG2 cells was examined using a laser confocal scanning microscope, as shown in fig. 6. FIG. 6-A shows weak fluorescence of non-targeted edible fungus crude polysaccharide lipid without Folate-PEG-DSPE, which shows that the intake efficiency is lower in HepG2 cells; fig. 6-B shows the uptake of the folic acid modified liposome-containing composite edible fungus polysaccharide delivery system in HepG2 cells, and the result shows that the HepG2 cells have obvious fluorescence and the intensity is higher than that of non-targeted edible fungus crude polysaccharide liposomes without Folate-PEG-DSPE. The result shows that the folic acid modified liposome composite edible fungus polysaccharide delivery system can improve the uptake efficiency of HepG2 cells, which proves that the liposome composite edible fungus polysaccharide delivery system has better targeting property.
And (3) adding water into the prepared liposome composite edible fungus polysaccharide delivery system containing the folic acid modification to dilute to 1%, adding sucrose and citric acid according to a sugar-acid ratio of 15:1, controlling the pH of the beverage to be 3.5, and adding 0.04% ethyl maltol for compounding to obtain the composite edible fungus healthy beverage containing the liposome polysaccharide delivery system containing the folic acid modification with small particle size. Flavor evaluation was performed using an electronic nose flavor profile (fig. 3), and rheological properties (fig. 7) were evaluated using a rheometer. As can be seen from fig. 3, the specific smell of the beverage in this example was further alleviated compared to the beverage of example 1. From the rheological characteristics of fig. 7, it can be seen that the small-particle-size liposome composite edible fungus polysaccharide drink prepared by high-pressure homogenization and passing through a 0.6 μm polycarbonate film has small particle size of polysaccharide liposome and good overall product stability. Pasteurizing, maintaining at 71.7deg.C for 15 seconds, filling, sealing, cooling, and storing at normal temperature. The compound edible fungus healthy drink is rich in flavor, sweet and sour, good in taste and high in overall sensory quality score of 93 minutes.
Claims (7)
1. A preparation method of a compound edible fungus health flavor drink is characterized in that the preparation steps are mainly as follows
The following are provided:
(1) Selecting and preprocessing edible fungus raw materials: three edible fungi of mushroom, cordyceps flower and tremella are selected,
drying, crushing and sieving with a 60-mesh sieve to obtain three edible fungus raw material dry powders respectively; the lentinus edodes and the insect
The mixing ratio of the dry powder of the three edible fungus raw materials of the grass flower and the tremella is 3:1:1;
(2) Extracting the compound edible fungus crude polysaccharide: taking three kinds of mushrooms, cordyceps sinensis flowers and tremella prepared in the step (1)
Mixing edible fungus dry powder at a certain proportion, placing into an extraction container, adding water, and heating to 80deg.C
Extracting for 1-3 hr, filtering the extractive solution to obtain filtrate, concentrating the filtrate, and slowly adding ethanol to obtain filtrate
In the liquid, the concentration of ethanol reaches 70% -90%, the mixture is refrigerated at 4 ℃ for 8 hours and centrifuged, and the precipitate is composite food
Using fungus crude polysaccharide;
(3) Preparing composite edible fungus particles: finishing the compound edible fungus crude polysaccharide obtained in the step (2) by using water
Fully dissolving to obtain crude polysaccharide extract, and dynamically homogenizing the crude polysaccharide extract with dynamic high-pressure homogenizer
Homogenizing under high pressure to obtain coarse polysaccharide particles of the composite edible fungi;
(4) Preparation of a folic acid modified liposome composite edible fungus polysaccharide delivery system: folic acid-polyethylene glycol
Alcohol-distearoyl phosphatidylethanolamine or folic acid-polyethylene glycol-cholesterol as trace lipid component
Obtaining a lipid mixture by hydrogenation of soybean phospholipids or cholesterol, and feeding the lipid mixture to step (3)
Fully dissolving the fungus crude polysaccharide particles in absolute ethyl alcohol, quickly injecting into 0.01mol/L phosphoric acid buffer solution,
ultrasonic treating for 15min to form homogeneous system, evaporating under reduced pressure at 60deg.C to remove ethanol, and extruding with polycarbonate film
The liposome composite edible fungus polysaccharide delivery system containing folic acid modification and having uniform particle size is obtained through membrane passing;
(5) Adding auxiliary materials: delivering the folic acid modified liposome-containing composite edible fungus polysaccharide prepared in the step (4)
Adding water into the system to prepare a certain concentration, adding sucrose, citric acid and ethyl maltol according to a certain sugar acid ratio
Compounding and controlling the pH of the beverage to be below 3.6 to obtain the composite edible fungus healthy beverage;
(6) Sterilizing and filling: pasteurizing, filling and sealing;
(7) Cooling and storing at normal temperature.
2. The method for preparing a compound edible fungus health-care flavor drink according to claim 1, which is characterized in that
Characterized in that the folic acid-polyethylene glycol-distearoyl phosphatidylethanolamine or folic acid-polyethylene glycol-cholesterin
The addition amount of alcohol in hydrogenated soybean phospholipid or cholesterol is 0.1-1mol%.
3. The method for preparing a compound edible fungus health-care flavor drink according to claim 1, which is characterized in that
Characterized in that the pore size of the polycarbonate membrane is 0.6 μm or 1 μm.
4. The method for preparing a compound edible fungus health-care flavor drink according to claim 1, which is characterized in that
Characterized in that the concentration of the water added and blended in the step (5) is 0.2% -2%.
5. The method for preparing a compound edible fungus health-care flavor drink according to claim 1, which is characterized in that
Characterized in that the sugar-acid ratio in the step (5) is (13-15): 1.
6. The method for preparing a compound edible fungus health-care flavor drink according to claim 1, which is characterized in that
Characterized in that the mass of the ethyl maltol added in the step (5) accounts for 0.03-0.05% of the beverage after being compounded.
7. The method for preparing a compound edible fungus health-care flavor drink according to claim 1, which is characterized in that
Characterized in that the pasteurization in step (6) is carried out at 71.7 ℃ for 15 seconds.
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