CN103864949A - Method for preparing maca polysaccharide and maca alkaloid by use of membrane separation technique - Google Patents
Method for preparing maca polysaccharide and maca alkaloid by use of membrane separation technique Download PDFInfo
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Abstract
The invention provides a method for preparing maca polysaccharide and maca alkaloid by use of a membrane separation technique. The method is used for orderly preparing the maca polysaccharide and the maca alkaloid by using the maca as a raw material through the steps of water (or acidic water) extraction, alcohol extraction, micro-filtration, enzymolysis, nano-filtration, spray drying and the like. The maca polysaccharide and the maca alkaloid are important functional components in the maca, and are efficiently separated out by adopting the membrane separation technique in combination with an enzymolysis technique, the process is simple and easy to implement, the production cost is low and convenience is provided for industrial production. The prepared maca polysaccharide has a remarkable effect of improving the immunity, and the prepared maca alkaloid has the effects of resisting fatigue and increasing the quantity of sperms.
Description
Technical field
The present invention relates to Separation of Natural Products field, relate to particularly the method and the application that utilize membrane technique associating enzymolysis process to prepare agate coffee functional ingredient.
Background technology
Agate coffee (Lepidium meyenii Walp.), has another name called Maca, is the piece root class plant of likeness in form radish, belongs to Cruciferae, originates in 4000 meters of above mountain areas, Andean of Peru's middle part height above sea level.Because Maca is low temperature resistant, strong wind, can under severe ecological condition, grow, become one of local staple crop.Long-term edible experience and research show that Maca is nutritious, there is the people and animals Fertility of raising, improve sexual function, suppress prostate gland increase, antifatigue, antidepressant, the osteoporosis of improving, improve the effects such as memory, cause common people's extensive concern, and progressively entered Chinese market in recent years.
Agate coffee nutritive ingredient is abundant, according to surveying and determination, in the agate coffee dry sample of Yunnan, contain 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 lipid acid, contain 38.64% linolic acid, 26.46% linolenic acid, 17.73% palmitinic acid, be rich in 14.04% amino acid simultaneously.In addition, agate coffee also contains other activeconstituentss as ester glucoside (glucosinolates), the alkaloid etc. of maca polysaccharide, agate coffee acid amides (mecamide), agate coffee alkene (maceaen), sulfur-bearing, there is the body immunity of enhancing, regain one's strength fast, the effect such as Ginseng Extract, in recent years, cause the great attention of international health food, the world of medicine.
Alkaloid (Alkaloids) is the nitrogenous alkaline organic of a class being present in plant materials more, and great majority have the complicated ring texture that comprises nitrogen-atoms, and have significant biological activity, is one of effective constituent important in herbal medicine.Alkaloid is also one of important active effective constituent of agate coffee simultaneously, in the early stage very big concern that has just been subject to numerous scientific researchers of agate coffee research.
Vegetable polysaccharides has diversified bioactive functions, and functional food and being widely used clinically, makes the research of vegetable polysaccharides become focus in recent years.Up to the present, existing more than 300 kind of polysaccharide compound separated from natural product, and the water-soluble polysaccharide wherein extracting from plant is the most important.These polysaccharide have extremely important and special physiologically active, have as immunomodulatory, antitumor, hypoglycemic, reducing blood-fat, radioprotective, antiviral, anti-inflammatory, antifatigue, the 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, provide a kind of technique simple, easy to operate, to production unit strong adaptability, the maca polysaccharide, the alkaloidal preparation method of agate coffee that are easy to suitability for industrialized production, 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, through pulverizing, adds water and fully mixes according to solid-liquid ratio 1:10～1:40, under 20～90 DEG C of conditions of temperature, extracts 1～2h, and 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), extracts the ethanol that slag A and volume fraction are 20～100% and mixes according to solid-liquid ratio 1:5～1:20, under 20～90 DEG C of conditions of temperature, extracts 1～2h, and solid-liquid separation obtains agate coffee extracting solution B and extracts 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 the concentrated removal of reduced vacuum ethanol, adopts millipore filtration to carry out filtering and impurity removing, obtains liquor C;
Step (4), liquor C adds proteolytic enzyme, and enzymolysis 1～5h adopts the nanofiltration membrane that molecular weight ranges is 3000～5000Da to filter enzymolysis solution, obtains filtrate D, and nanofiltration membrane is held back part for maca polysaccharide;
Step (5), filtrate D adds polysaccharide hydrolase, enzymolysis 1～3h, first adopting molecular weight is the nanofiltration membrane filtration enzymolysis solution of 200Da, contains monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts the nanofiltration membrane that molecular weight is 1000Da to filter, and obtains filtrate E;
Step (6), the spray-dried agate coffee alkaloid that obtains of filtrate E.
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.
In described step (2), extracting the ethanol that slag A and volume fraction are 60-65% mixes according to solid-liquid ratio 1:10～1:15.
While adopting ultrasonic extraction in described step (1) and step (2), extracting temperature is 20～40 DEG C.
While adopting ultrasonic extraction in described step (1), ultrasonic power 50～2000W/h/L(W/h/L is the ultrasonic energy of every liter of extracting solution reception per hour), preferably 1000W/h/L; Dutycycle 1:1～1:5, preferably 1:2; Rotating speed 600～1500rpm, preferably 1200rpm.
While adopting supersound extraction in described step (2), ultrasonic power 20～2000W/h/L, preferably 800W/h/L; Dutycycle 1:1～1:5, preferably 1:2; Rotating speed 500～1500rpm, preferably 1000rpm.
In described step (3), the aperture of millipore filtration is 0.1～10 μ m, 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 is held back 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), spraying drying conditions is: inlet temperature is 150～220 DEG C, preferably 180～200 DEG C, and more preferably 180 DEG C; Air outlet temperature is 60～130 DEG C, preferably 80～100 DEG C, and more preferably 90 DEG C; Velocity of evaporation 1～200L/h.
The filtrate E of described step (6) gained can obtain the highly purified agate coffee of 80%-99% acid amides through column chromatography purification, and described column chromatography is selected anti-phase C18 post or silicagel column.
The above-mentioned maca polysaccharide of stating and the alkaloidal purposes of agate coffee, the application of described agate coffee extract in functional foodstuff, healthcare products, medicine.
The present invention's advantage is compared with prior art: the present invention adopts membrane filtration technique associating zymolysis technique to separate maca polysaccharide and agate coffee alkaloid, a technique is separated the two in turn, whole simple process is easily gone, and production cost is low, is convenient to suitability for industrialized production.The maca polysaccharide tool of preparing gained the increase significantly power of exempting from, anti-fatigue effect, the agate coffee alkaloid of preparing gained has the effect that improves sperm quantity.
Brief description of the drawings
Fig. 1 Technology schema of the present invention.
Embodiment
Introduce in detail the present invention below in conjunction with drawings and the specific embodiments.But following embodiment only limits 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, through pulverizing, adds water and fully mixes according to solid-liquid ratio 1:10, under 90 DEG C of conditions of temperature, stirs and extracts 1h, and solid-liquid separation obtains agate coffee extracting solution A and extracts slag A;
(2) extract the ethanol that slag A and volume fraction are 20% and mix according to solid-liquid ratio 1:5, under 90 DEG C of conditions of temperature, stir and extract 1h, solid-liquid separation obtains agate coffee extracting solution B and extracts slag B;
(3) extracting solution B merges with extracting solution A after the concentrated removal of reduced vacuum ethanol, adopts 0.1 μ m millipore filtration to carry out filtering and impurity removing, obtains liquor C; Filter membrane is mineral-type filter membrane;
(4) liquor C adds trypsinase, and enzymolysis 1h adopts the nanofiltration membrane that molecular weight ranges is 3000 to filter enzymolysis solution, obtains filtrate D, and nanofiltration membrane is held back part for maca polysaccharide; Maca polysaccharide rinses 2 times repeatedly with 80 ethanol, to remove residual monose, pigment and small-molecule substance;
(5) filtrate D adds amylase, enzymolysis 1h, and first adopting molecular weight is the nanofiltration membrane filtration enzymolysis solution of 200Da, contains monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts the nanofiltration membrane that molecular weight is 1000Da to filter, and obtains filtrate E;
(6) the spray-dried agate coffee alkaloid that obtains of filtrate E, spraying drying conditions 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, through pulverizing, adds sour water according to solid-liquid ratio 1:10 and fully mixes, and under 90 DEG C of conditions of temperature, stirs and extracts 1h, and solid-liquid separation obtains agate coffee extracting solution A and extracts slag A;
The aqueous hydrochloric acid that wherein sour water is 0.5%;
(2) extract the ethanol that slag A and volume fraction are 20% and mix according to solid-liquid ratio 1:5, under 90 DEG C of conditions of temperature, stir and extract 1h, solid-liquid separation obtains agate coffee extracting solution B and extracts slag B;
(3) extracting solution B merges with extracting solution A after the concentrated removal of reduced vacuum ethanol, adopts 0.1 μ m millipore filtration to carry out filtering and impurity removing, obtains liquor C; Filter membrane is mineral-type filter membrane;
(4) liquor C adds trypsinase, and enzymolysis 1h adopts the nanofiltration membrane that molecular weight ranges is 3000 to filter enzymolysis solution, obtains filtrate D, and nanofiltration membrane is held back part for maca polysaccharide; Maca polysaccharide rinses 2 times repeatedly with 80 ethanol, to remove residual monose, pigment and small-molecule substance;
(5) filtrate D adds amylase, enzymolysis 1h, and first adopting molecular weight is the nanofiltration membrane filtration enzymolysis solution of 200Da, contains monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts the nanofiltration membrane that molecular weight is 1000Da to filter, and obtains filtrate E;
(6) the spray-dried agate coffee alkaloid that obtains of filtrate E, spraying drying conditions 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 and fully mix according to solid-liquid ratio 1:40, under 20 DEG C of conditions of temperature, supersound extraction 2h, ultrasonic power 50W/h/L(W/h/L is the ultrasonic energy of every 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 the ethanol that slag A and volume fraction are 100% and mix according to solid-liquid ratio 1:20, under 20 DEG C of conditions of temperature, extract 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 the concentrated removal of reduced vacuum ethanol, adopts 10 μ m microporous membranes to carry out filtering and impurity removing, obtains liquor C;
(4) liquor C adds Quimotrase (Chymotrypsin), and enzymolysis 5h adopts the nanofiltration membrane that molecular weight ranges is 5000Da to filter enzymolysis solution, obtains filtrate D, and nanofiltration membrane is held back part for maca polysaccharide; Maca polysaccharide rinses 3 times repeatedly with 85% ethanol, to remove residual monose, pigment and small-molecule substance;
(5) filtrate D adds zytase, enzymolysis 3h, and first adopting molecular weight is the nanofiltration membrane filtration enzymolysis solution of 200Da, contains monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts the nanofiltration membrane that molecular weight is 1000Da to filter, and obtains filtrate E;
(6) the spray-dried agate coffee alkaloid that obtains of filtrate E, spraying drying conditions 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, adding sour water according to solid-liquid ratio 1:40 fully mixes, under 20 DEG C of conditions of temperature, supersound extraction 2h, ultrasonic power 50W/h/L(W/h/L is the ultrasonic energy of every 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;
The aqueous sulfuric acid that wherein sour water is 2%;
(2) extract the ethanol that slag A and volume fraction are 100% and mix according to solid-liquid ratio 1:20, under 20 DEG C of conditions of temperature, extract 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 the concentrated removal of reduced vacuum ethanol, adopts 10 μ m microporous membranes to carry out filtering and impurity removing, obtains liquor C;
(4) liquor C adds Quimotrase (Chymotrypsin), and enzymolysis 5h adopts the nanofiltration membrane that molecular weight ranges is 5000Da to filter enzymolysis solution, obtains filtrate D, and nanofiltration membrane is held back part for maca polysaccharide; Maca polysaccharide rinses 3 times repeatedly with 85% ethanol, to remove residual monose, pigment and small-molecule substance;
(5) filtrate D adds zytase, enzymolysis 3h, and first adopting molecular weight is the nanofiltration membrane filtration enzymolysis solution of 200Da, contains monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts the nanofiltration membrane that molecular weight is 1000Da to filter, and obtains filtrate E;
(6) the spray-dried agate coffee alkaloid that obtains of filtrate E, spraying drying conditions 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, through pulverizing, adds water and fully mixes according to solid-liquid ratio 1:30, 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 the ethanol that slag A and volume fraction are 85% and mix according to solid-liquid ratio 1:10, under 30 DEG C of conditions of temperature, supersound extraction 2h, ultrasonic power 1000W/h/L, dutycycle 1:3, 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 the concentrated removal of reduced vacuum ethanol, adopts 0.22 μ m polysulfones millipore filtration to carry out filtering and impurity removing, obtains liquor C;
(4) liquor C adds thermolysin, and enzymolysis 3h adopts the nanofiltration membrane that molecular weight ranges is 4000Da to filter enzymolysis solution, obtains filtrate D, and nanofiltration membrane is held back part for maca polysaccharide; Maca polysaccharide rinses repeatedly with 80% ethanol, to remove residual monose, pigment and small-molecule substance;
(5) filtrate D adds chitinase and zytase mixed enzyme, enzymolysis 1h, and first adopting molecular weight is the nanofiltration membrane filtration enzymolysis solution of 200Da, contains monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts the nanofiltration membrane that molecular weight is 1000Da to filter, and obtains filtrate E;
(6) the spray-dried agate coffee alkaloid that obtains of filtrate E, spraying drying conditions 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, through pulverizing, adds water and fully mixes according to solid-liquid ratio 1:20, under 40 DEG C of conditions of temperature, 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 the ethanol that slag A and volume fraction are 60% and mix according to solid-liquid ratio 1:15, under 80 DEG C of conditions of temperature, stir and extract 2h, solid-liquid separation obtains agate coffee extracting solution B and extracts slag B;
(3) extracting solution B merges with extracting solution A after the concentrated removal of reduced vacuum ethanol, adopts the polyamide-based millipore filtration of 10 μ m to carry out filtering and impurity removing, obtains liquor C;
(4) liquor C adds the mixed enzyme of thermolysin, stomach en-and papoid, and enzymolysis 3h adopts the nanofiltration membrane that molecular weight ranges is 3000Da to filter enzymolysis solution, obtains filtrate D, and nanofiltration membrane is held back part for maca polysaccharide; Maca polysaccharide rinses repeatedly with 80% ethanol, 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 adopting molecular weight is the nanofiltration membrane filtration enzymolysis solution of 200Da, contains monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts the nanofiltration membrane that molecular weight is 1000Da to filter, and obtains filtrate E;
(6) the spray-dried agate coffee alkaloid that obtains of filtrate E, spraying drying conditions 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, through pulverizing, adds sour water according to solid-liquid ratio 1:20 and fully mixes, under 40 DEG C of conditions of temperature, 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;
The phosphate aqueous solution that wherein sour water is 1.5%;
(2) extract the ethanol that slag A and volume fraction are 60% and mix according to solid-liquid ratio 1:15, under 80 DEG C of conditions of temperature, stir and extract 2h, solid-liquid separation obtains agate coffee extracting solution B and extracts slag B;
(3) extracting solution B merges with extracting solution A after the concentrated removal of reduced vacuum ethanol, adopts the polyamide-based millipore filtration of 10 μ m to carry out filtering and impurity removing, obtains liquor C;
(4) liquor C adds the mixed enzyme of thermolysin, stomach en-and papoid, and enzymolysis 3h adopts the nanofiltration membrane that molecular weight ranges is 3000Da to filter enzymolysis solution, obtains filtrate D, and nanofiltration membrane is held back part for maca polysaccharide; Maca polysaccharide rinses repeatedly with 80% ethanol, 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 adopting molecular weight is the nanofiltration membrane filtration enzymolysis solution of 200Da, contains monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts the nanofiltration membrane that molecular weight is 1000Da to filter, and obtains filtrate E;
(6) the spray-dried agate coffee alkaloid that obtains of filtrate E, spraying drying conditions 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, through pulverizing, adds water and fully mixes according to solid-liquid ratio 1:30, under temperature 70 C condition, extracts 2h, and solid-liquid separation obtains agate coffee extracting solution A and extracts slag A;
(2) extract the ethanol that slag A and volume fraction are 65% and mix according to solid-liquid ratio 1:20, under 30 DEG C of conditions of temperature, 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 the concentrated removal of reduced vacuum ethanol, adopts 0.1 μ m 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, and enzymolysis 1～5h adopts the nanofiltration membrane that molecular weight ranges is 5000Da to filter enzymolysis solution, obtains filtrate D, and nanofiltration membrane is held back part for maca polysaccharide; Maca polysaccharide rinses 2～3 times repeatedly with 80 ethanol, 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 adopting molecular weight is the nanofiltration membrane filtration enzymolysis solution of 200Da, contains monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts the nanofiltration membrane that molecular weight is 1000Da to filter, and obtains filtrate E;
(6) the spray-dried agate coffee alkaloid that obtains of filtrate E, spraying drying conditions 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, through pulverizing, adds water and fully mixes according to solid-liquid ratio 1:40, under 30 DEG C of conditions of temperature, 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 the ethanol that slag A and volume fraction are 55% and mix according to solid-liquid ratio 1:10, under 90 DEG C of conditions of temperature, stir and extract 2h, solid-liquid separation obtains agate coffee extracting solution B and extracts slag B;
(3) extracting solution B merges with extracting solution A after the concentrated removal of reduced vacuum ethanol, adopts 5 μ m 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, and enzymolysis 1.5h adopts the nanofiltration membrane filtration enzymolysis solution that molecular weight ranges is 3000～5000Da to obtain filtrate D, and nanofiltration membrane is held back part for maca polysaccharide; Maca polysaccharide rinses repeatedly with 80% ethanol, 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 adopting molecular weight is the nanofiltration membrane filtration enzymolysis solution of 200Da, contains monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts the nanofiltration membrane that molecular weight is 1000Da to filter, and obtains filtrate E;
(6) the spray-dried agate coffee alkaloid that obtains of filtrate E.
Spraying drying conditions 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, through pulverizing, adds water and fully mixes according to solid-liquid ratio 1:40, 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 the ethanol that slag A and volume fraction are 100% and mix according to solid-liquid ratio 1:20, under 90 DEG C of conditions of temperature, stir and extract 1h, solid-liquid separation obtains agate coffee extracting solution B and extracts slag B;
(3) extracting solution B merges with extracting solution A after the concentrated removal of reduced vacuum ethanol, adopts 0.1 μ m 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, nanofiltration membrane that to adopt molecular weight ranges be 3000～5000Da the D that filters to get filtrate, nanofiltration membrane is held back part for maca polysaccharide; Maca polysaccharide rinses 2～3 times repeatedly with 80～85% ethanol, 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 adopting molecular weight is the nanofiltration membrane filtration enzymolysis solution of 200Da, contains monosaccharide and disaccharide, amino acid etc. in filtered liquid; Then the trapped fluid of gained adopts the nanofiltration membrane that molecular weight is 1000Da to filter, and obtains filtrate E;
(6) the spray-dried agate coffee alkaloid that obtains of filtrate E, spraying drying conditions 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 for preparing agate coffee acid amides
The alkaloid that utilizes one of any method of embodiment 1-7 to prepare is further prepared high purity agate coffee acid amides, upper anti-phase C18 post after dissolve with methanol extract for the filtrate E 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 agate coffee acid amides.
Embodiment 12 is further purified the method for preparing agate coffee acid amides
The alkaloid that utilizes one of any method of embodiment 1-7 to prepare is further prepared high purity agate coffee acid amides, upper silicagel column after dissolve with methanol extract for the filtrate E of step (6) gained, 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 agate coffee acid amides.
Embodiment 13 maca polysaccharides and the alkaloidal purposes of agate coffee
The alkaloid and the application of maca polysaccharide in exploitation agate coffee functional health-care beverage that utilize one of any method of embodiment 1-7 to prepare, by 100 parts of quality, composed as follows: 1 part of maca polysaccharide, 0.5 part of agate coffee alkaloid, 5 parts of vitamin complexes, 0.5 part of seasonings, 5 parts of compound amino acid powders, all the other are 88 parts, water.
The purposes of embodiment 14 agate coffee acid amides
Utilize agate coffee acid amides prepared by one of any method of the embodiment 8-9 application in exploitation agate coffee functional product (tablet), tablet is composed as follows: agate coffee amide content 80%, all the other are hypromellose, Microcrystalline Cellulose, Magnesium Stearate, silicon-dioxide.
Effect experiment of maca polysaccharide
Maca polysaccharide rising Induced by Cyclophosphamide mouse peripheral blood total white blood cells lowly acts on (table 1): experiment is established 9 groups: A. blank group; B. endoxan group; Remainder is maca polysaccharide 1.0g/kg group; After modeling and in the 3rd, 6 days tail venous blood samplings, measure total white blood cells.
Table 1 maca polysaccharide rising Induced by Cyclophosphamide mouse peripheral blood total white blood cells lowly acts on
Note: with negative control group comparison, * P<0.01
Result: with the comparison of endoxan group, give after endoxan the 3rd day, maca polysaccharide group total white blood cells all obviously raise (table 1).Show that maca polysaccharide has rising Immunosuppressant Cyclophosphamide and brings out murine interleukin and lowly act on.
The alkaloidal effect experiment of agate coffee
Select 80 of healthy Kunming kind mouse, body weight 18-22g, is divided into control group by every batch of animal at random by body weight, agate coffee alkaloid group (embodiment 1-9 group, 0.1g/kg/day), (13 groups of embodiment, 10ml/kg/day), every group 8, per os gavage, 1 time/d, 30d continuously, give, after mouse tested material 30min, to carry out indices mensuration in last.
1, pole-climbing experiment
When test, pole-climbing is placed in the basin of 15 DEG C of water temperatures, dark about 10cm, mouse is placed on perspex bar, make muscle in static(al) Stress, record the time that mouse drops down from perspex bar due to muscle fatigue, termination test when overboard for the third time, the time that accumulative total is three times is as the pole-climbing time.Concrete function is more as shown in table 2.
2, the mensuration of liver starch
After each group mouse last administration 30min, mouse is put into temperature and is the water tank went swimming 90min of 30 DEG C, then it is put to death immediately, get liver blots with filter paper after physiological saline rinsing, accurately take liver 200mg, add 4mL TCA, every pipe homogenate l min, pours homogenate into centrifuge tube, with the centrifugal 15min of 3000r/min, supernatant liquor is transferred to another in vitro.In precipitation, add again 4mL TCA, homogenate 1min, recentrifuge 15min, gets supernatant liquor, and merges with centrifugal for the first time supernatant liquor, fully mixes.Get l mL supernatant liquor and put into 10mL centrifuge tube, every pipe adds 95% ethanol 4mL, fully mixes between two kinds of liquid and does not leave interface.With clean stopper beyond the Great Wall, test tube is placed on to 40 DEG C of water-bath 3h.Precipitation completely after, 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 causes of free radical, and its content can indirectly embody antioxidant ability of organism and remove the ability of oxidation products.Power exhausts motion and causes peroxidatic reaction of lipid reinforcement and produce more free radical, cause the microbial film such as sarcolemma and mitochondrial membrane integrity to be lost and damage, thereby cause a series of cellular metabolism disorders, the infringement of cell popularity and pathological change, muscular work ability is declined, produce tired.MDA in lipid peroxide degraded product can with thiobarbituricacidα-condensation, form red product, in 532nm place mensuration absorbancy.Concrete function is more as shown in table 2.
The impact of table 2 agate coffee alkaloid on 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: with negative control group comparison, * P<0.01
From experimental result, 10 experimental examples of agate coffee alkaloid and agate coffee functional health-care beverage (embodiment 13) obviously increase the pole-climbing time of mouse, obviously increase the liver starch deposit of mouse, obviously reduce the effect of Content of MDA.Therefore, the variation of above-mentioned several indexs has absolutely proved that agate coffee alkaloid of the present invention has the effect of antifatigue, has retained the functional ingredient of agate coffee to greatest extent.
Effect experiment of agate coffee acid amides
The impact that SD rat spermatozoa is occurred: three 30 of monthly age SD rats, be divided at random control group (physiological saline), A group (comparative example, 0.05g/10mL), B group (the agate coffee acid amides that embodiment 11 obtains, 0.005g/10mL), C group (the agate coffee acid amides that embodiment 12 obtains, 0.005g/10mL), D group (the agate coffee acid amides tablet that embodiment 14 obtains, 0.005g/10mL), every day gavage 1 time, gavage volume is 10mL/kg body weight, respectively organizes successive administration 7 days, freely take food and drink water, after last administration, 24h ether is put to death.Measure respectively its day sperm production, epididymal sperm number, the results are shown in Table 3: the impact (X ± SEM) of different agate coffee extracts on rat spermatozoa output and quality (n=6).
The impact of the different agate coffee of table 3 extract on rat spermatozoa output and quality
Note: with the comparison of blank group, * * P<0.01.
Experimental result shows that tablet prepared by agate coffee extract that embodiment 11,12 obtains and embodiment 14 obviously increases 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 to this, in the technical scope that any those skilled in the art disclose in the present invention; the variation that can expect easily or replacement, within all should being encompassed in 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, preferably 1:20～1:40 or 1:20～1:30, add water and fully mix, then adopt heated and stirred or ultrasonic extraction to carry out solid-liquid separation, extract 20～90 DEG C of temperature, extraction time 1～2h, solid-liquid separation obtains agate coffee extracting solution A and extracts slag A;
Step (2), extract the ethanol that slag A and volume fraction are 20～100% and mix according to solid-liquid ratio 1:5～1:20, then adopt heated and stirred or ultrasonic extraction to carry out solid-liquid separation, extract 20～90 DEG C of temperature, 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 the concentrated removal of reduced vacuum ethanol, adopts millipore filtration to carry out filtering and impurity removing, obtains liquor C;
Step (4), liquor C adds protease hydrolyzed 1～5h, adopts the nanofiltration membrane that molecular weight ranges is 3000～5000Da to filter enzymolysis solution, obtains filtrate D, and nanofiltration membrane is held back part for maca polysaccharide;
Step (5), filtrate D adds polysaccharide hydrolase, enzymolysis 1～3h, first adopting molecular weight is the nanofiltration membrane filtration enzymolysis solution of 200Da, contains monosaccharide and disaccharide, amino acid in filtered liquid; Then the trapped fluid of gained is adopted the nanofiltration membrane that molecular weight is 1000Da to filter, obtain filtrate E;
Step (6), the spray-dried agate coffee alkaloid that obtains of filtrate E.
2. the membrane separation technique of utilizing according to claim 1 is prepared maca polysaccharide, the alkaloidal method of agate coffee, it is characterized in that: 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.
3. the membrane separation technique of utilizing according to claim 1 is prepared maca polysaccharide, the alkaloidal method of agate coffee, it is characterized in that: in described step (2), extract the ethanol that slag A and volume fraction are 60-65% and mix 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 of utilizing of claim 1-3, it is characterized in that: while adopting ultrasonic extraction in described step (1), extracting temperature is 20～40 DEG C, ultrasonic power 50～2000W/h/L(W/h/L is the ultrasonic energy of every liter of extracting solution reception per hour), preferably 1000W/h/L; Dutycycle 1:1～1:5, preferably 1:2; Rotating speed 600～1500rpm, preferably 1200rpm; While adopting supersound extraction in described step (2), extracting temperature is 20～40 DEG C, ultrasonic power 20～2000W/h/L, preferably 800W/h/L; Dutycycle 1:1～1:5, preferably 1:2; Rotating speed 500～1500rpm, preferably 1000rpm.
5. prepare maca polysaccharide, the alkaloidal method of agate coffee according to one of any described membrane separation technique of utilizing of claim 1-3, it is characterized in that: in described step (3), the aperture of millipore filtration is 0.1～10 μ m, 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.
6. prepare maca polysaccharide, the alkaloidal method of agate coffee according to one of any described membrane separation technique of utilizing of claim 1-3, it is characterized in that: in described step (4), nanofiltration membrane is held back part for maca polysaccharide, maca polysaccharide rinses 2～3 times repeatedly with 80～85% ethanol, 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 of utilizing of claim 1-3, it is characterized in that: in described step (4), proteolytic enzyme is that trypsinase, Quimotrase are one or more of Chymotrypsin, thermolysin, stomach en-, papoid, SP and clostripain.
8. prepare maca polysaccharide, the alkaloidal method of agate coffee according to one of any described membrane separation technique of utilizing 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 of utilizing of claim 1-3, it is characterized in that: in described step (6), spraying drying conditions 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, and more preferably 90 DEG C; Velocity of evaporation 1～200L/h.
10. the alkaloid that utilizes one of any described method of claim 1-3 to prepare is further purified the method for preparing agate coffee acid amides, it is characterized in that: the filtrate E of described step (6) gained can obtain the agate coffee acid amides of high purity 80%-99% through column chromatography purification, described column chromatography is selected anti-phase C18 post or silicagel column.
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