# US 12,109,509 B2 · Ultrasonic composite acidic water extraction method for cordyceps polysaccharide and cordycepin in Cordyceps militaris

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- Number: US 12,109,509 B2
- Document ID: US12109509B2
- Status: Granted
- Kind: B2
- Application number: 17/419,718
- Filed: 2019-12-06
- Published: 2022-03-17
- Granted: 2024-10-08
- Earliest claimed: 2019-03-01
- Assignees: Jiangnan University
- Applicants: Jiangnan University
- Inventors: Min Zhang; Hao Shi; Sifu Yi; Xiaoling Guo; Lihua Hou; Wuxiong Yang
- Web page: https://patentgrep.com/patent/US12109509B2

## CPC

- B01D 11/0265: {Applying ultrasound}
- B01D 11/0288: {Applications, solvents}
- A01G 18/70: Harvesting
- A23L 31/00: Edible extracts or preparations of fungi; Preparation or treatment thereof
- B01D 11/0211: {in combination with an electric or magnetic field}
- C07H 1/08: from natural products
- C07H 19/16: Purine radicals
- C08B 37/00: Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof (cellulose D21; {microbiological processes C12P})
- C08B 37/0003: {General processes for their isolation or fractionation, e.g. purification or extraction from biomass}
- C08L 5/00: Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
- B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28)
- A01G: HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING (picking of fruits, vegetables, hops or the like A01D46/00; propagating unicellular algae C12N1/12)
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)
- C08B: POLYSACCHARIDES; DERIVATIVES THEREOF (polysaccharides containing less than six saccharide radicals attached to each other by glycosidic linkages C07H; fermentation or enzyme-using processes C12P19/00; sugar industry C13; production of cellulose D21)
- C08L: COMPOSITIONS OF MACROMOLECULAR COMPOUNDS (compositions based on polymerisable monomers C08F, C08G; artificial filaments or fibres D01F; textile treating compositions D06)

## Abstract

Disclosed is an ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris, which falls within the technical field of food processing. The method comprises: picking, washing, drying, pulverizing and degreasing Cordyceps militaris fruiting bodies to obtain a Cordyceps militaris dry powder; then immersing same in a prepared diluted hydrochloric acid solution to perform three cycles of ultra-low temperature freezing and microwave defrosting, and at the same time using low-frequency ultrasonic waves to carry out assisted stirring and extraction; then subjecting an extract to evaporation and concentration treatments; and finally, performing freeze-drying on the extract to collect a dry powder of a water extraction product. The amount of an additive used in the method meets domestic and foreign usage requirements of food additives, and the operation is simple, practical and uses a combination of chemical and physical methods, thereby saving time and being highly efficient.

## Claims

### Claim 1

An ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris, comprising the following specific steps:
(1) culturing and harvesting: culturing Cordyceps militaris fruiting bodies in a solid medium, harvesting after 40-45 days of the culturing, and storing the harvested Cordyceps militaris fruiting bodies under refrigeration at 4° C.;
(2) washing and drying: drying the harvested Cordyceps militaris fruiting bodies for 5 hours in a hot-air drying oven at 60-65° C. during which the Cordyceps militaris fruiting bodies are arranged evenly and kept ventilated; and collecting and storing same in a cool and dry place after drying to a constant weight;
(3) pulverizing: crushing a dry sample of Cordyceps militaris, and passing through a 60-mesh sieve;
(4) degreasing: placing the pulverized sample in a reflux device, performing reflux extraction with 95% ethanol at a material-liquid ratio of 1:5 (w:v) in a boiling water bath for 3 hours, centrifugation, collecting the precipitate, and drying at 60-65° C. to a constant weight, to obtain a degreased cordyceps powder;
(5) ultra-low temperature freezing-microwave defrosting cycle: adding distilled water into the degreased cordyceps powder at a material-liquid ratio of 1:40 (w:v), to obtain an extract; stirring and titrating the extract with 1 mol/L of a diluted acid solution to obtain a pH of 4.8±0.2; freezing the extract for 2 hours at −80° C.; taking out the extract, and defrosting for 2-3 min using microwaves with a power of 300-400 W; and repeating the freezing and the defrosting operations three times, to obtain a crude extraction mixture of Cordyceps militaris;
(6) ultrasonic-assisted extraction: after obtaining the crude extraction mixture of Cordyceps militaris, extracting for 35-40 min at 65-70° C. with the assistance of ultrasonic waves having a frequency of 20-30 KHZ and a power of 300-400 W; and after the ultrasonic-assisted extraction is complete, centrifuging for 15 min at 4,000 r/min, and filtering the supernatant to obtain a crude water extract of the Cordyceps militaris fruiting bodies;
(7) evaporation and concentration: concentrating, by rotary evaporation at 60-65° C. and at 0.095 MPa, the crude water extract of the Cordyceps militaris fruiting bodies to 1% of the original volume, until it finally becomes a viscous and thick slurry; collecting the concentrated crude water extract of the Cordyceps militaris fruiting bodies, and freezing for 24 hours at −70° C.; and
(8) freeze-drying to prepare powder: performing freeze-drying on the frozen concentrated crude water extract of the Cordyceps militaris fruiting bodies for 3 hours in a cold trap at −55° C., to obtain a dry powder of the crude water extract of the Cordyceps militaris fruiting bodies.

### Claim 2 (depends on 1)

The ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris according to claim 1, wherein in the step (1), components of the solid medium comprise rice, wheat, glucose, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, vitamin B1, and vitamin B2.

### Claim 3 (depends on 1)

The ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris according to claim 1, wherein in the step (1), a height of the harvested Cordyceps militaris fruiting bodies is greater than or equal to 7 cm.

### Claim 4 (depends on 1)

The ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris according to claim 1, wherein in the step (3), the dry sample of Cordyceps militaris is cut into segments of 3-4 cm for crushing, and then is pulverized.

### Claim 5 (depends on 2)

The ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris according to claim 2, wherein in the step (3), the dry sample of Cordyceps militaris is cut into segments of 3-4 cm for crushing, and then is pulverized.

### Claim 6 (depends on 1)

The ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris according to claim 1, wherein in the step (4), a speed of the centrifugation is 4,000 r/min, and a time of the centrifugation is 20 min.

### Claim 7 (depends on 2)

The ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris according to claim 2, wherein in the step (4), a speed of the centrifugation is 4,000 r/min, and a time of the centrifugation is 20 min.

### Claim 8 (depends on 3)

The ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris according to claim 3, wherein in the step (4), a speed of the centrifugation is 4,000 r/min, and a time of the centrifugation is 20 min.

### Claim 9 (depends on 1)

The ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris according to claim 1, wherein in the step (5), the diluted acid solution is a diluted hydrochloric acid solution, a citric acid solution or an acetic acid solution.

### Claim 10 (depends on 2)

The ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris according to claim 2, characterized in that, wherein in the step (5), the diluted acid solution is a diluted hydrochloric acid solution, a citric acid solution or an acetic acid solution.

### Claim 11 (depends on 2)

The ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris according to claim 2, wherein in the step (1), a height of the harvested Cordyceps militaris fruiting bodies is greater than or equal to 7 cm.

### Claim 12 (depends on 3)

The ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris according to claim 3, wherein in the step (3), the dry sample of Cordyceps militaris is cut into segments of 3-4 cm for crushing, and then is pulverized.

### Claim 13 (depends on 3)

The ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris according to claim 3, wherein in the step (5), the diluted acid solution is a diluted hydrochloric acid solution, a citric acid solution or an acetic acid solution.

### Claim 14 (depends on 4)

The ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris according to claim 4, wherein in the step (4), a speed of the centrifugation is 4,000 r/min, and a time of the centrifugation is 20 min.

### Claim 15 (depends on 4)

The ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris according to claim 4, wherein in the step (5), the diluted acid solution is a diluted hydrochloric acid solution, a citric acid solution or an acetic acid solution.

### Claim 16 (depends on 6)

The ultrasonic composite acidic water extraction method for a Cordyceps polysaccharide and cordycepin in Cordyceps militaris according to claim 6, wherein in the step (5), the diluted acid solution is a diluted hydrochloric acid solution, a citric acid solution or an acetic acid solution.