CN103864949B - Membrane separation technique is utilized to prepare maca polysaccharide, the alkaloidal method of agate coffee - Google Patents
Membrane separation technique is utilized to prepare maca polysaccharide, the alkaloidal method of agate coffee Download PDFInfo
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Abstract
The invention provides and utilize membrane separation technique to prepare maca polysaccharide, the alkaloidal method of agate coffee, with agate coffee for raw material, prepare maca polysaccharide and agate coffee alkaloid in turn through steps such as water (or sour water) extraction, ethanol-extracted, micro-filtration, enzymolysis, nanofiltration, spraying dry.Maca polysaccharide and alkaloid are functional ingredient important in agate coffee, and the present invention adopts membrane separation technique to combine zymolysis technique by the two high efficiency separation out, and simple process is easy, and production cost is low, is convenient to suitability for industrialized production.The maca polysaccharide tool preparing gained increases significantly and exempts from power effect, and the agate coffee alkaloid preparing gained has antifatigue, improves the effect of sperm quantity.
Description
Technical field
The present invention relates to Separation of Natural Products field, relate to the method and the application that utilize membrane technique associating enzymolysis process to prepare functional maca composition particularly.
Background technology
Agate coffee (LepidiummeyeniiWalp.), has another name called Maca, for being similar to the block root class plant of radish, belonging to Cruciferae, originating in the mountain area, Andean of height above sea level more than 4000 meters in the middle part of Peru.Because Maca is low temperature resistant, strong wind, can grow under severe ecological condition, become one of local staple crop.Long-term edible experience and research show that Maca is nutritious, have and improve the effect such as people and animals' Fertility, improvement function, the increase of suppression prostate gland, antifatigue, antidepressant, osteoporosis of improving, raising memory, cause common people's extensive concern, and in progressively entering Chinese market in recent years.
Agate coffee rich in nutrition content, according to surveying and determination, containing the multiple nutritional components such as protein 12.31%, robust fibre 32.57%, fat 0.92%, VC314.97mg/kg, calcium 1818mg/kg, iron 81.2mg/kg, zinc 23.8mg/kg, potassium 18.7mg/kg, phosphorus 1895mg/kg in the agate coffee dry sample of Yunnan, containing 38.64% linolic acid, 26.46% linolenic acid, 17.73% palmitinic acid in lipid acid, be rich in 14.04% amino acid simultaneously.In addition, agate coffee also contains other activeconstituentss as the ester glucoside (glucosinolates), alkaloid etc. of maca polysaccharide, macamide (mecamide), agate coffee alkene (maceaen), sulfur-bearing, have strengthen body immunity, regain one's strength fast, the effect such as Ginseng Extract, in recent years, the great attention of international health food, the world of medicine is caused.
Alkaloid (Alkaloids) is the nitrogenous alkaline organic of the class be present in plant materials more, and great majority have the complicated ring texture comprising nitrogen-atoms, and have significant biological activity, is one of effective constituent important in herbal medicine.Simultaneously alkaloid is also one of important active ingredients of agate coffee, in the early stage very big concern just receiving numerous scientific researcher of agate coffee research.
Vegetable polysaccharides has diversified bioactive functions, and functional food and widely using clinically, makes the research of vegetable polysaccharides become focus in recent years.Up to the present, existing more than 300 kind of polysaccharide compound is separated from natural product, and the water-soluble polysaccharide wherein extracted from plant is the most important.These polysaccharide have extremely important with special physiologically active, have as immunomodulatory, antitumor, hypoglycemic, reducing blood-fat, radioprotective, antiviral, anti-inflammatory, antifatigue, anti-ageing bioactive functions of waiting for a long time.
Summary of the invention
Technology of the present invention is dealt with problems: overcome the deficiencies in the prior art, there is provided a kind of technique simple, easy to operate, to production unit strong adaptability, be easy to the maca polysaccharide of suitability for industrialized production, the alkaloidal preparation method of agate coffee, the present invention seeks to be achieved through the following technical solutions:
Utilize membrane separation technique to prepare maca polysaccharide, the alkaloidal method of agate coffee, comprise the following steps:
Step (1), agate coffee is through pulverizing, and add water according to solid-liquid ratio 1:10 ~ 1:40 and fully mix, under temperature 20 ~ 90 DEG C of conditions, extract 1 ~ 2h, solid-liquid separation obtains agate coffee extracting solution A and extracts slag A;
Wherein, extracting mode can take heated and stirred to extract, and also can adopt ultrasonic-assisted extraction;
Step (2), extract slag A and volume fraction be 20 ~ 100% ethanol mix according to solid-liquid ratio 1:5 ~ 1:20, under temperature 20 ~ 90 DEG C of conditions, extract 1 ~ 2h, solid-liquid separation obtains agate coffee extracting solution B and extraction slag B;
Wherein, extracting mode can take heated and stirred to extract, and also can adopt ultrasonic-assisted extraction;
Step (3), extracting solution B merges with extracting solution A after reduced vacuum concentrated removal ethanol, adopts millipore filtration to carry out filtering and impurity removing, obtains liquor C;
Step (4), liquor C adds proteolytic enzyme, enzymolysis 1 ~ 5h, and employing molecular weight ranges is the nanofiltration membrane enzymolysis solution of 3000 ~ 5000Da, obtains filtrate D, and nanofiltration membrane retains part for maca polysaccharide;
Step (5), filtrate D adds polysaccharide hydrolase, enzymolysis 1 ~ 3h, and first employing molecular weight is the nanofiltration membrane enzymolysis solution of 200Da, containing monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts molecular weight to be the nanofiltration membrane of 1000Da, obtains filtrate E;
Step (6), filtrate E is spray-dried obtains agate coffee alkaloid.
Solid-liquid ratio 1:20 ~ the 1:40 of agate coffee and water in described step (1).
Solid-liquid ratio 1:20 ~ the 1:30 of agate coffee and water in described step (1).
In described step (1), water is the inorganic acid aqueous solution of concentration 0.5-2%, and described mineral acid is the one or more combination in hydrochloric acid, sulfuric acid or phosphoric acid.
Described step (2) middle extraction slag A and volume fraction are that the ethanol of 60-65% mixes according to solid-liquid ratio 1:10 ~ 1:15.
When described step (1) and step (2) middle employing ultrasonic extraction, Extracting temperature is 20 ~ 40 DEG C.
When adopting ultrasonic extraction in described step (1), the ultrasonic power 50 ~ 2000W/h/L(W/h/L i.e. ultrasonic energy of often liter of extracting solution reception per hour), preferred 1000W/h/L; Dutycycle 1:1 ~ 1:5, preferred 1:2; Rotating speed 600 ~ 1500rpm, preferred 1200rpm.
When adopting supersound extraction in described step (2), ultrasonic power 20 ~ 2000W/h/L, preferred 800W/h/L; Dutycycle 1:1 ~ 1:5, preferred 1:2; Rotating speed 500 ~ 1500rpm, preferred 1000rpm.
In described step (3), the aperture of millipore filtration is 0.1 ~ 10 μm, filter membrane is several hybrid films of one in mineral-type, polysulfones, fluoro-containing macromolecule material class, polyamide-based, polyolefins, cellulose family or chitin kind mould material, wherein preferably ceramic film.
In described step (4), nanofiltration membrane retains part for maca polysaccharide, and maca polysaccharide rinses 2 ~ 3 times repeatedly with 80 ~ 85% ethanol, to remove residual monose, pigment and small-molecule substance.
In described step (4), proteolytic enzyme is one or more of trypsinase, Quimotrase (Chymotrypsin), thermolysin, stomach en-, papoid, SP and clostripain.
In described step (5), polysaccharide hydrolase is one or more in amylase, zytase, chitinase, cellulase, N,O-Diacetylmuramidase, polygalacturonase, beta-glucanase, beta-galactosidase enzymes.
In described step (6), spray drying condition is: inlet temperature is 150 ~ 220 DEG C, preferably 180 ~ 200 DEG C, more preferably 180 DEG C; Air outlet temperature is 60 ~ 130 DEG C, preferably 80 ~ 100 DEG C, more preferably 90 DEG C; Velocity of evaporation 1 ~ 200L/h.
The filtrate E of described step (6) gained can obtain the highly purified macamide of 80%-99% through column chromatography purification, and described column chromatography selects anti-phase C18 post or silicagel column.
The above-mentioned maca polysaccharide stated and the alkaloidal purposes of agate coffee, the application of described Maca extract in functional foodstuff, healthcare products, medicine.
The present invention's advantage is compared with prior art: the present invention adopts membrane filtration technique to combine zymolysis technique and is separated maca polysaccharide and agate coffee alkaloid, the two separates by a technique in turn, whole simple process is easy, and production cost is low, is convenient to suitability for industrialized production.The maca polysaccharide tool preparing gained increases significantly power of exempting from, anti-fatigue effect, and the agate coffee alkaloid preparing gained has the effect improving sperm quantity.
Accompanying drawing explanation
Fig. 1 Technology schema of the present invention.
Embodiment
The present invention is introduced in detail below in conjunction with drawings and the specific embodiments.But following embodiment is only limitted to explain the present invention, and protection scope of the present invention should comprise the full content of claim, is not limited only to the present embodiment.
Embodiment 1 utilizes membrane separation technique to prepare maca polysaccharide, the alkaloidal method of agate coffee
(1) agate coffee is through pulverizing, and adding water according to solid-liquid ratio 1:10 fully mixes, and under temperature 90 DEG C of conditions, stirs and extracts 1h, and solid-liquid separation obtains agate coffee extracting solution A and extracts slag A;
(2) extract slag A and volume fraction be 20% ethanol mix according to solid-liquid ratio 1:5, under temperature 90 DEG C of conditions, stir and extract 1h, solid-liquid separation obtains agate coffee extracting solution B and extraction slag B;
(3) extracting solution B merges with extracting solution A after reduced vacuum concentrated removal ethanol, adopts 0.1 μm of millipore filtration to carry out filtering and impurity removing, obtains liquor C; Filter membrane is mineral-type filter membrane;
(4) liquor C adds trypsinase, enzymolysis 1h, and employing molecular weight ranges is the nanofiltration membrane enzymolysis solution of 3000, obtains filtrate D, and nanofiltration membrane retains part for maca polysaccharide; Maca polysaccharide 80 ethanol rinse 2 times repeatedly, to remove residual monose, pigment and small-molecule substance;
(5) filtrate D adds amylase, enzymolysis 1h, and first employing molecular weight is the nanofiltration membrane enzymolysis solution of 200Da, containing monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts molecular weight to be the nanofiltration membrane of 1000Da, obtains filtrate E;
(6) filtrate E is spray-dried obtains agate coffee alkaloid, and spray drying condition is: inlet temperature is 150 DEG C, and air outlet temperature is 60, velocity of evaporation 1L/h.
Embodiment 2 utilizes membrane separation technique to prepare maca polysaccharide, the alkaloidal method of agate coffee
(1) agate coffee is through pulverizing, and adds sour water fully mix according to solid-liquid ratio 1:10, under temperature 90 DEG C of conditions, stirs and extracts 1h, and solid-liquid separation obtains agate coffee extracting solution A and extracts slag A;
Wherein sour water is the aqueous hydrochloric acid of 0.5%;
(2) extract slag A and volume fraction be 20% ethanol mix according to solid-liquid ratio 1:5, under temperature 90 DEG C of conditions, stir and extract 1h, solid-liquid separation obtains agate coffee extracting solution B and extraction slag B;
(3) extracting solution B merges with extracting solution A after reduced vacuum concentrated removal ethanol, adopts 0.1 μm of millipore filtration to carry out filtering and impurity removing, obtains liquor C; Filter membrane is mineral-type filter membrane;
(4) liquor C adds trypsinase, enzymolysis 1h, and employing molecular weight ranges is the nanofiltration membrane enzymolysis solution of 3000, obtains filtrate D, and nanofiltration membrane retains part for maca polysaccharide; Maca polysaccharide 80 ethanol rinse 2 times repeatedly, to remove residual monose, pigment and small-molecule substance;
(5) filtrate D adds amylase, enzymolysis 1h, and first employing molecular weight is the nanofiltration membrane enzymolysis solution of 200Da, containing monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts molecular weight to be the nanofiltration membrane of 1000Da, obtains filtrate E;
(6) filtrate E is spray-dried obtains agate coffee alkaloid, and spray drying condition is: inlet temperature is 150 DEG C, and air outlet temperature is 60, velocity of evaporation 1L/h.
Embodiment 3 utilizes membrane separation technique to prepare maca polysaccharide, the alkaloidal method of agate coffee
(1) agate coffee is through pulverizing, add water according to solid-liquid ratio 1:40 and fully mix, under temperature 20 DEG C of conditions, supersound extraction 2h, the ultrasonic power 50W/h/L(W/h/L i.e. ultrasonic energy of often liter of extracting solution reception per hour), dutycycle 1:1, rotating speed 600rpm, solid-liquid separation obtains agate coffee extracting solution A and extracts slag A;
(2) extract slag A and volume fraction be 100% ethanol mix according to solid-liquid ratio 1:20, under temperature 20 DEG C of conditions, extraction 2h, ultrasonic power 2000W/h/L, dutycycle 1:5, rotating speed 1500rpm.Solid-liquid separation obtains agate coffee extracting solution B and extracts slag B;
(3) extracting solution B merges with extracting solution A after reduced vacuum concentrated removal ethanol, adopts 10 μm of microporous membranes to carry out filtering and impurity removing, obtains liquor C;
(4) liquor C adds Quimotrase (Chymotrypsin), enzymolysis 5h, and employing molecular weight ranges is the nanofiltration membrane enzymolysis solution of 5000Da, obtains filtrate D, and nanofiltration membrane retains part for maca polysaccharide; Maca polysaccharide 85% ethanol rinses 3 times repeatedly, to remove residual monose, pigment and small-molecule substance;
(5) filtrate D adds zytase, enzymolysis 3h, and first employing molecular weight is the nanofiltration membrane enzymolysis solution of 200Da, containing monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts molecular weight to be the nanofiltration membrane of 1000Da, obtains filtrate E;
(6) filtrate E is spray-dried obtains agate coffee alkaloid, and spray drying condition is: inlet temperature is 220 DEG C, air outlet temperature is 130 DEG C, velocity of evaporation 200L/h.
Embodiment 4 utilizes membrane separation technique to prepare maca polysaccharide, the alkaloidal method of agate coffee
(1) agate coffee is through pulverizing, add sour water according to solid-liquid ratio 1:40 fully to mix, under temperature 20 DEG C of conditions, supersound extraction 2h, the ultrasonic power 50W/h/L(W/h/L i.e. ultrasonic energy of often liter of extracting solution reception per hour), dutycycle 1:1, rotating speed 600rpm, solid-liquid separation obtains agate coffee extracting solution A and extracts slag A;
Wherein sour water is the aqueous sulfuric acid of 2%;
(2) extract slag A and volume fraction be 100% ethanol mix according to solid-liquid ratio 1:20, under temperature 20 DEG C of conditions, extraction 2h, ultrasonic power 2000W/h/L, dutycycle 1:5, rotating speed 1500rpm.Solid-liquid separation obtains agate coffee extracting solution B and extracts slag B;
(3) extracting solution B merges with extracting solution A after reduced vacuum concentrated removal ethanol, adopts 10 μm of microporous membranes to carry out filtering and impurity removing, obtains liquor C;
(4) liquor C adds Quimotrase (Chymotrypsin), enzymolysis 5h, and employing molecular weight ranges is the nanofiltration membrane enzymolysis solution of 5000Da, obtains filtrate D, and nanofiltration membrane retains part for maca polysaccharide; Maca polysaccharide 85% ethanol rinses 3 times repeatedly, to remove residual monose, pigment and small-molecule substance;
(5) filtrate D adds zytase, enzymolysis 3h, and first employing molecular weight is the nanofiltration membrane enzymolysis solution of 200Da, containing monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts molecular weight to be the nanofiltration membrane of 1000Da, obtains filtrate E;
(6) filtrate E is spray-dried obtains agate coffee alkaloid, and spray drying condition is: inlet temperature is 220 DEG C, air outlet temperature is 130 DEG C, velocity of evaporation 200L/h.
Embodiment 5 utilizes membrane separation technique to prepare maca polysaccharide, the alkaloidal method of agate coffee
(1) agate coffee is through pulverizing, and adding water according to solid-liquid ratio 1:30 fully mixes, and under temperature 50 C condition, stirs and extracts 1.5h, and solid-liquid separation obtains agate coffee extracting solution A and extracts slag A;
(2) extract slag A and volume fraction be 85% ethanol mix according to solid-liquid ratio 1:10, under temperature 30 DEG C of conditions, supersound extraction 2h, ultrasonic power 1000W/h/L, dutycycle 1:3, rotating speed 1000rpm, solid-liquid separation obtains agate coffee extracting solution B and extraction slag B;
(3) extracting solution B merges with extracting solution A after reduced vacuum concentrated removal ethanol, adopts 0.22 μm of polysulfones millipore filtration to carry out filtering and impurity removing, obtains liquor C;
(4) liquor C adds thermolysin, enzymolysis 3h, and employing molecular weight ranges is the nanofiltration membrane enzymolysis solution of 4000Da, obtains filtrate D, and nanofiltration membrane retains part for maca polysaccharide; Maca polysaccharide 80% ethanol rinses repeatedly, to remove residual monose, pigment and small-molecule substance;
(5) filtrate D adds chitinase and zytase mixed enzyme, enzymolysis 1h, and first employing molecular weight is the nanofiltration membrane enzymolysis solution of 200Da, containing monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts molecular weight to be the nanofiltration membrane of 1000Da, obtains filtrate E;
(6) filtrate E is spray-dried obtains agate coffee alkaloid, and spray drying condition is: inlet temperature is 200 DEG C, and air outlet temperature is 100 DEG C, velocity of evaporation 100L/h.
Embodiment 6 utilizes membrane separation technique to prepare maca polysaccharide, the alkaloidal method of agate coffee
(1) agate coffee is through pulverizing, and adding water according to solid-liquid ratio 1:20 fully mixes, under temperature 40 DEG C of conditions, and supersound extraction 1h, ultrasonic power 100W/h/L, dutycycle 1:4, rotating speed 8500rpm, solid-liquid separation obtains agate coffee extracting solution A and extracts slag A;
(2) extract slag A and volume fraction be 60% ethanol mix according to solid-liquid ratio 1:15, under temperature 80 DEG C of conditions, stir and extract 2h, solid-liquid separation obtains agate coffee extracting solution B and extraction slag B;
(3) extracting solution B merges with extracting solution A after reduced vacuum concentrated removal ethanol, adopts 10 μm of polyamide-based millipore filtrations to carry out filtering and impurity removing, obtains liquor C;
(4) liquor C adds the mixed enzyme of thermolysin, stomach en-and papoid, enzymolysis 3h, and employing molecular weight ranges is the nanofiltration membrane enzymolysis solution of 3000Da, obtains filtrate D, and nanofiltration membrane retains part for maca polysaccharide; Maca polysaccharide 80% ethanol rinses repeatedly, to remove residual monose, pigment and small-molecule substance;
(5) filtrate D adds the mixed enzyme of N,O-Diacetylmuramidase, pectin, enzymolysis 1h, and first employing molecular weight is the nanofiltration membrane enzymolysis solution of 200Da, containing monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts molecular weight to be the nanofiltration membrane of 1000Da, obtains filtrate E;
(6) filtrate E is spray-dried obtains agate coffee alkaloid, and spray drying condition is: inlet temperature is 180 DEG C; Air outlet temperature is 90 DEG C; Velocity of evaporation 10L/h.
Embodiment 7 utilizes membrane separation technique to prepare maca polysaccharide, the alkaloidal method of agate coffee
(1) agate coffee is through pulverizing, and adds sour water fully mix according to solid-liquid ratio 1:20, under temperature 40 DEG C of conditions, and supersound extraction 1h, ultrasonic power 100W/h/L, dutycycle 1:4, rotating speed 8500rpm, solid-liquid separation obtains agate coffee extracting solution A and extracts slag A;
Wherein sour water is the phosphate aqueous solution of 1.5%;
(2) extract slag A and volume fraction be 60% ethanol mix according to solid-liquid ratio 1:15, under temperature 80 DEG C of conditions, stir and extract 2h, solid-liquid separation obtains agate coffee extracting solution B and extraction slag B;
(3) extracting solution B merges with extracting solution A after reduced vacuum concentrated removal ethanol, adopts 10 μm of polyamide-based millipore filtrations to carry out filtering and impurity removing, obtains liquor C;
(4) liquor C adds the mixed enzyme of thermolysin, stomach en-and papoid, enzymolysis 3h, and employing molecular weight ranges is the nanofiltration membrane enzymolysis solution of 3000Da, obtains filtrate D, and nanofiltration membrane retains part for maca polysaccharide; Maca polysaccharide 80% ethanol rinses repeatedly, to remove residual monose, pigment and small-molecule substance;
(5) filtrate D adds the mixed enzyme of N,O-Diacetylmuramidase, pectin, enzymolysis 1h, and first employing molecular weight is the nanofiltration membrane enzymolysis solution of 200Da, containing monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts molecular weight to be the nanofiltration membrane of 1000Da, obtains filtrate E;
(6) filtrate E is spray-dried obtains agate coffee alkaloid, and spray drying condition is: inlet temperature is 180 DEG C; Air outlet temperature is 90 DEG C; Velocity of evaporation 10L/h.
Embodiment 8 utilizes membrane separation technique to prepare maca polysaccharide, the alkaloidal method of agate coffee
(1) agate coffee is through pulverizing, and adding water according to solid-liquid ratio 1:30 fully mixes, and under temperature 70 C condition, extracts 2h, and solid-liquid separation obtains agate coffee extracting solution A and extracts slag A;
(2) extract slag A and volume fraction be 65% ethanol mix according to solid-liquid ratio 1:20, under temperature 30 DEG C of conditions, supersound extraction 2h, ultrasonic power 800W/h/L; Dutycycle 1:2; Rotating speed 1000rpm, solid-liquid separation obtains agate coffee extracting solution B and extracts slag B;
(3) extracting solution B merges with extracting solution A after reduced vacuum concentrated removal ethanol, adopts 0.1 μm of cellulose family millipore filtration to carry out filtering and impurity removing, obtains liquor C;
(4) liquor C adds the mixed enzyme of papoid, SP and clostripain, enzymolysis 1 ~ 5h, and employing molecular weight ranges is the nanofiltration membrane enzymolysis solution of 5000Da, obtains filtrate D, and nanofiltration membrane retains part for maca polysaccharide; Maca polysaccharide 80 ethanol rinse 2 ~ 3 times repeatedly, to remove residual monose, pigment and small-molecule substance;
(5) filtrate D adds the mixed enzyme of polygalacturonase, beta-glucanase, beta-galactosidase enzymes, enzymolysis 3h, and first employing molecular weight is the nanofiltration membrane enzymolysis solution of 200Da, containing monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts molecular weight to be the nanofiltration membrane of 1000Da, obtains filtrate E;
(6) filtrate E is spray-dried obtains agate coffee alkaloid, and spray drying condition is: inlet temperature is 190 DEG C, and air outlet temperature is 110 DEG C, velocity of evaporation, 50L/h.
Embodiment 9 utilizes membrane separation technique to prepare maca polysaccharide, the alkaloidal method of agate coffee
(1) agate coffee is through pulverizing, and adding water according to solid-liquid ratio 1:40 fully mixes, under temperature 30 DEG C of conditions, and supersound extraction 1.5h, ultrasonic power 1000W/h/L, dutycycle 1:4, rotating speed 800rpm, solid-liquid separation obtains agate coffee extracting solution A and extracts slag A;
(2) extract slag A and volume fraction be 55% ethanol mix according to solid-liquid ratio 1:10, under temperature 90 DEG C of conditions, stir and extract 2h, solid-liquid separation obtains agate coffee extracting solution B and extraction slag B;
(3) extracting solution B merges with extracting solution A after reduced vacuum concentrated removal ethanol, adopts 5 μm of fluorine macromolecular material class millipore filtrations to carry out filtering and impurity removing, obtains liquor C;
(4) liquor C adds thermolysin, stomach en-, papoid, SP mixed enzyme, enzymolysis 1.5h, and employing molecular weight ranges is that the nanofiltration membrane enzymolysis solution of 3000 ~ 5000Da obtains filtrate D, and nanofiltration membrane retains part for maca polysaccharide; Maca polysaccharide 80% ethanol rinses repeatedly, to remove residual monose, pigment and small-molecule substance;
(5) filtrate D adds cellulase, N,O-Diacetylmuramidase, polygalacturonase mixing polysaccharide lytic enzyme, enzymolysis 2h, and first employing molecular weight is the nanofiltration membrane enzymolysis solution of 200Da, containing monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts molecular weight to be the nanofiltration membrane of 1000Da, obtains filtrate E;
(6) filtrate E is spray-dried obtains agate coffee alkaloid.
Spray drying condition is: inlet temperature is 220 DEG C, and air outlet temperature is 130 DEG C, velocity of evaporation 10L/h.
Embodiment 10 utilizes membrane separation technique to prepare maca polysaccharide, the alkaloidal method of agate coffee
(1) agate coffee is through pulverizing, and adding water according to solid-liquid ratio 1:40 fully mixes, under temperature 50 C condition, and supersound extraction 2h, ultrasonic power 300W/h/L, dutycycle 1:3, rotating speed 700rpm, solid-liquid separation obtains agate coffee extracting solution A and extracts slag A;
(2) extract slag A and volume fraction be 100% ethanol mix according to solid-liquid ratio 1:20, under temperature 90 DEG C of conditions, stir and extract 1h, solid-liquid separation obtains agate coffee extracting solution B and extraction slag B;
(3) extracting solution B merges with extracting solution A after reduced vacuum concentrated removal ethanol, adopts 0.1 μm of ceramic capillary filter membrane to carry out filtering and impurity removing, obtains liquor C;
(4) liquor C adds trypsinase, Quimotrase (Chymotrypsin), thermolysin, stomach en-mixing protease, enzymolysis 1 ~ 5h, employing molecular weight ranges is that the nanofiltration membrane of 3000 ~ 5000Da obtains filtrate D, and nanofiltration membrane retains part for maca polysaccharide; Maca polysaccharide 80 ~ 85% ethanol rinse 2 ~ 3 times repeatedly, to remove residual monose, pigment and small-molecule substance;
(5) filtrate D adds amylase, zytase, chitinase, cellulase mixing polysaccharide lytic enzyme, enzymolysis 1h, and first employing molecular weight is the nanofiltration membrane enzymolysis solution of 200Da, containing monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts molecular weight to be the nanofiltration membrane of 1000Da, obtains filtrate E;
(6) filtrate E is spray-dried obtains agate coffee alkaloid, and spray drying condition is: inlet temperature is 190 DEG C, and air outlet temperature is 100 DEG C, velocity of evaporation 100L/h.
Embodiment 11 is further purified the method preparing macamide
High purity macamide prepared further by alkaloid prepared by the method that one of utilizes embodiment 1-7 any, upper anti-phase C18 post after the filtrate E dissolve with methanol extract of step (6) gained, carries out the degree such as 3 sections with the methanol aqueous solution of three concentration of 8 column volumes, 30%, 8 column volumes 50% and 1 column volume 75% successively and washes assorted; With the anhydrous methanol wash-out of 4 column volumes, elutriant concentrate drying obtains 80% high purity macamide.
Embodiment 12 is further purified the method preparing macamide
High purity macamide prepared further by alkaloid prepared by the method that one of utilizes embodiment 1-7 any, the filtrate E of step (6) gained silicagel column upper after dissolve with methanol extract, carries out the degree such as 3 sections with the methanol aqueous solution of three concentration of 6 column volumes, 30%, 5 column volumes 50% and 2 column volumes 75% successively and washes assorted; With the anhydrous methanol wash-out of 3 column volumes, elutriant concentrate drying obtains 99% high purity macamide.
Embodiment 13 maca polysaccharide and the alkaloidal purposes of agate coffee
Alkaloid prepared by the method that one of utilizes embodiment 1-7 any and the application of maca polysaccharide in exploitation functional maca nourishing drink, by 100 parts of quality, composed as follows: maca polysaccharide 1 part, agate coffee alkaloid 0.5 part, vitamin complex 5 parts, seasonings 0.5 part, compound amino acid powder 5 parts, all the other are 88 parts, water.
The purposes of embodiment 14 macamide
The application of macamide in exploitation functional maca product (tablet) prepared by the method that one of utilizes embodiment 8-9 any, tablet is composed as follows: macamide content 80%, and all the other are hypromellose, Microcrystalline Cellulose, Magnesium Stearate, silicon-dioxide.
The Efficacy experiments of maca polysaccharide
Maca polysaccharide raises Induced by Cyclophosphamide mouse peripheral blood total white blood cells and lowly acts on (table 1): 9 groups: A. blank group is established in experiment; B. endoxan group; Remainder is maca polysaccharide 1.0g/kg group; After modeling and in the 3rd, 6 day tail venous blood sampling, measure total white blood cells.
Table 1 maca polysaccharide raises Induced by Cyclophosphamide mouse peripheral blood total white blood cells and lowly acts on
Note: compare with negative control group, * P<0.01
Result: compare with endoxan group, to after endoxan the 3rd day, maca polysaccharide group total white blood cells all obviously raised (table 1).Show that maca polysaccharide has to raise Immunosuppressant Cyclophosphamide and bring out murine interleukin and lowly act on.
The alkaloidal Efficacy experiments of agate coffee
Selecting healthy Kunming kind mouse 80, body weight 18-22g, being divided into control group by often criticizing animal at random by body weight, agate coffee alkaloid group (embodiment 1-9 group, 0.1g/kg/day), (embodiment 13 groups, 10ml/kg/day), often organize 8, per os gavage, 1 time/d, continuous 30d, after last gives mouse tested material 30min, carry out indices mensuration.
1, Grasping clubglass test
During test, pole-climbing is placed in the basin of water temperature 15 DEG C, dark about 10cm, mouse is placed on perspex bar, muscle is made to be in static(al) Stress, the time that record mouse drops down from perspex bar due to muscle fatigue, termination test when third time is overboard, the time of accumulative three times is as the pole-climbing time.Concrete function is more as shown in table 2.
2, the mensuration of liver starch
Mouse is put into the water tank went swimming 90min that temperature is 30 DEG C after each group of mouse last administration 30min, then it is put to death immediately, get liver to blot with filter paper after physiological saline rinsing, accurately take liver 200mg, add 4mLTCA, often pipe homogenate lmin, pours homogenate into centrifuge tube, with the centrifugal 15min of 3000r/min, supernatant liquor is transferred to another in vitro.In precipitation, add 4mLTCA again, homogenate 1min, recentrifuge 15min, gets supernatant liquor, and the supernatant liquor centrifugal with first time merges, and fully mixes.Get lmL supernatant liquor and put into 10mL centrifuge tube, often pipe adds the ethanol 4mL of 95%, and fully mixing does not leave interface between two kinds of liquid.With clean stopper beyond the Great Wall, test tube is placed on 40 DEG C of water-bath 3h.After precipitation is complete, by test tube in the centrifugal 15min of 3000r/min.Carefully outwell supernatant liquor and test tube is stood upside down and place 10min.Dissolve glycogen with 2ml distilled water, under 620nm wavelength, measure liver starch with anthrone method.Concrete function is more as shown in table 2.
3, the impact of mice serum MDA value
MDA is one of primary product of the lipid peroxidation that free radical causes, and its content indirectly can embody antioxidant ability of organism and remove the ability of oxidation products.Exhausted movemeat causes peroxidatic reaction of lipid strengthen and produce more free radical, the microbial film such as sarcolemma and mitochondrial membrane integrity is caused to be lost and damage, thus cause a series of cellular metabolism disorder, the infringement of cell popularity and pathological change, muscular work ability is declined, produces tired.MDA in lipid peroxide degraded product can with thiobarbituricacidα-condensation, formed red product, 532nm place mensuration absorbancy.Concrete function is more as shown in table 2.
Table 2 agate coffee alkaloid is on the impact of mouse pole-climbing time, liver starch, serum MDA
| Sample | Swimming time (min) | Liver starch (mg/g hepatic tissue) | Serum MDA (mmol/L) |
| Contrast | 4.22±1.05 | 14.57±3.12 | 33.26±7.26 |
| Embodiment 1 | 5.73±1.41 | 29.04±2.21* | 25.16±7.01* |
| Embodiment 2 | 13.21±1.43* | 51.33±2.78* | 24.15±3.12* |
| Embodiment 3 | 9.34±1.72* | 63.52±1.88* | 25.89±5.42* |
| Embodiment 4 | 9.24±0.87* | 45.76±2.09* | 24.67±6.20* |
| Embodiment 5 | 8.38±2.02* | 46.32±1.09* | 24.23±6.31* |
| Embodiment 6 | 8.12±1.03* | 55.22±1.06* | 21.98±6.96* |
| Embodiment 7 | 9.32±1.58* | 62.79±2.37* | 22.34±6.89* |
| Embodiment 8 | 8.24±1.12* | 54.12±1.25* | 22.91±6.17* |
| Embodiment 9 | 8.31±1.28* | 67.69±2.22* | 21.14±6.34* |
| Embodiment 13 | 7.68±2.11* | 78.02±1.46* | 24.16±4.31* |
Note: compare with negative control group, * P<0.01
From experimental result, agate coffee alkaloid 10 experimental examples and functional maca nourishing drink (embodiment 13) obviously increase the pole-climbing time of mouse, obviously increase the liver glycogen reserves of mouse, obviously reduce the effect of Content of MDA.Therefore, the change of above-mentioned several index has absolutely proved that agate coffee alkaloid of the present invention has the effect of antifatigue, remains the functional ingredient of agate coffee to greatest extent.
The Efficacy experiments of macamide
Impact on SD rat spermatozoa occurs: three monthly age SD rats 30, be divided into control group (physiological saline) at random, A group (comparative example, 0.05g/10mL), B group (the macamide that embodiment 11 obtains, 0.005g/10mL), C group (macamide that embodiment 12 obtains, 0.005g/10mL), D group (the macamide tablet that embodiment 14 obtains, 0.005g/10mL), every day gavage 1 time, gavage volume is 10mL/kg body weight, each group successive administration 7 days, freely take food and drink water, after last administration, 24h ether is put to death.Measure its day sperm production, epididymal sperm number respectively, the results are shown in Table 3: different Maca extract is on impact (X ± SEM) (n=6) of rat spermatozoa output and quality.
The different Maca extract of table 3 is on the impact of rat spermatozoa output and quality
Note: compare with blank group, * * P<0.01.
Experimental result shows that the Maca extract that embodiment 11,12 obtains and tablet prepared by embodiment 14 obviously increase SD rat day sperm production and epididymal sperm number.
The above; be only part embodiment of the present invention, but protection scope of the present invention is not limited thereto, any those skilled in the art are in the technical scope that the present invention discloses; the change that can expect easily or replacement, all should be encompassed within protection scope of the present invention.
Claims (10)
1. utilize membrane separation technique to prepare maca polysaccharide, the alkaloidal method of agate coffee, it is characterized in that: described method comprises the following steps:
Step (1), agate coffee is through pulverizing, according to solid-liquid ratio 1:10 ~ 1:40, to add water or inorganic acid aqueous solution fully mixes, then adopt heated and stirred or ultrasonic extraction, carry out solid-liquid separation, Extracting temperature 20 ~ 90 DEG C, extraction time 1 ~ 2h, solid-liquid separation obtains agate coffee extracting solution A and extracts slag A;
Step (2), extract slag A and volume fraction be 20 ~ 100% ethanol mix according to solid-liquid ratio 1:5 ~ 1:20, then heated and stirred or ultrasonic extraction is adopted to carry out solid-liquid separation, Extracting temperature 20 ~ 90 DEG C, extraction time 1 ~ 2h, solid-liquid separation obtains agate coffee extracting solution B and extracts slag B;
Step (3), extracting solution B merges with extracting solution A after reduced vacuum concentrated removal ethanol, adopts millipore filtration to carry out filtering and impurity removing, obtains liquor C;
Step (4), liquor C adds protease hydrolyzed 1 ~ 5h, and employing molecular weight ranges is the nanofiltration membrane enzymolysis solution of 3000 ~ 5000Da, obtains filtrate D, and nanofiltration membrane retains part for maca polysaccharide;
Step (5), filtrate D adds polysaccharide hydrolase, enzymolysis 1 ~ 3h, and first employing molecular weight is the nanofiltration membrane enzymolysis solution of 200Da, containing monosaccharide and disaccharide, amino acid in filtered liquid; Then adopt molecular weight to be the nanofiltration membrane of 1000Da the trapped fluid of gained, obtain filtrate E;
Step (6), filtrate E is spray-dried obtains agate coffee alkaloid.
2. the membrane separation technique that utilizes according to claim 1 prepares maca polysaccharide, the alkaloidal method of agate coffee, it is characterized in that: in described step (1), inorganic acid aqueous solution is the inorganic acid aqueous solution of concentration 0.5-2%, and described mineral acid is the one or more combination in hydrochloric acid, sulfuric acid or phosphoric acid.
3. the membrane separation technique that utilizes according to claim 1 prepares maca polysaccharide, the alkaloidal method of agate coffee, it is characterized in that: described step (2) middle extraction slag A and volume fraction are that the ethanol of 60-65% mixes according to solid-liquid ratio 1:10 ~ 1:15.
4. prepare maca polysaccharide, the alkaloidal method of agate coffee according to one of any described membrane separation technique that utilizes of claim 1-3, it is characterized in that: when adopting ultrasonic extraction in described step (1), Extracting temperature is 20 ~ 40 DEG C, ultrasonic power 50 ~ 2000W/h/L; Dutycycle 1:1 ~ 1:5; Rotating speed 600 ~ 1500rpm; When adopting supersound extraction in described step (2), Extracting temperature is 20 ~ 40 DEG C, ultrasonic power 20 ~ 2000W/h/L; Dutycycle 1:1 ~ 1:5; Rotating speed 500 ~ 1500rpm.
5. prepare maca polysaccharide, the alkaloidal method of agate coffee according to one of any described membrane separation technique that utilizes of claim 1-3, it is characterized in that: the aperture of millipore filtration is 0.1 ~ 10 μm in described step (3), filter membrane is one or more hybrid films in mineral-type, polysulfones, fluoro-containing macromolecule material class, polyamide-based, polyolefins, cellulose family or chitin kind mould material.
6. prepare maca polysaccharide, the alkaloidal method of agate coffee according to one of any described membrane separation technique that utilizes of claim 1-3, it is characterized in that: in described step (4), nanofiltration membrane retains part for maca polysaccharide, maca polysaccharide 80 ~ 85% ethanol rinse 2 ~ 3 times repeatedly, to remove residual monose, pigment and small-molecule substance.
7. prepare maca polysaccharide, the alkaloidal method of agate coffee according to one of any described membrane separation technique that utilizes of claim 1-3, it is characterized in that: in described step (4) proteolytic enzyme be trypsinase, Quimotrase and Chymotrypsin, thermolysin, stomach en-, papoid, SP and clostripain one or more.
8. prepare maca polysaccharide, the alkaloidal method of agate coffee according to one of any described membrane separation technique that utilizes of claim 1-3, it is characterized in that: in step (5), polysaccharide hydrolase is one or more in amylase, zytase, chitinase, cellulase, N,O-Diacetylmuramidase, polygalacturonase, beta-glucanase, beta-galactosidase enzymes.
9. prepare maca polysaccharide, the alkaloidal method of agate coffee according to one of any described membrane separation technique that utilizes of claim 1-3, it is characterized in that: in described step (6), spray drying condition is: inlet temperature is 150 ~ 220 DEG C; Air outlet temperature is 60 ~ 130 DEG C; Velocity of evaporation 1 ~ 200L/h.
10. alkaloid prepared by the described method that one of utilizes claim 1-3 any is further purified the method preparing macamide, it is characterized in that: the filtrate E of described step (5) gained can obtain through column chromatography purification the macamide that purity is 80%-99%, and described column chromatography selects anti-phase C18 post or silicagel column.
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| CN105395592A (en) * | 2014-09-11 | 2016-03-16 | 宁波润沃生物科技有限公司 | Method for preparing maea extract through ultrasonic wave countercurrent |
| CN105558528B (en) * | 2014-10-11 | 2018-09-07 | 华中科技大学 | A kind of antifatigue Maca extract and the drinks including the extract |
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| CN105981876A (en) * | 2015-02-04 | 2016-10-05 | 玛卡龙酒业有限公司 | Maca coffee and preparation method thereof |
| CN104872656B (en) * | 2015-03-06 | 2019-01-15 | 江中（天津）健康科技发展有限公司 | A kind of composition with health care function and preparation method thereof |
| CN105367677A (en) * | 2015-12-01 | 2016-03-02 | 中国药科大学 | Maca polysaccharide of immunoregulatory activity and preparation method therefor |
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Inventor after: Wang Qiang Inventor after: Zhao Qingsheng Inventor after: Zhao Bing Inventor before: Wang Qiang Inventor before: Zhao Qingsheng Inventor before: Zhao Bing Inventor before: Yang Yongwu Inventor before: Chen Kaicai |