WO2016107776A1

WO2016107776A1 - A process for preparing cordyceps extract

Info

Publication number: WO2016107776A1
Application number: PCT/EP2015/080757
Authority: WO
Inventors: Rebecca Lian Hwee Peng; Yongcheng Liao; Longfei WANG; Xiaolu GU; Chunling Li; Christoph Hartmann; Jinrui DU
Original assignee: Nestec S.A.
Priority date: 2014-12-31
Filing date: 2015-12-21
Publication date: 2016-07-07
Also published as: CN107105736A; WO2016106704A1

Abstract

A method for preparing cordyceps extract containing adenosine in high concentration is disclosed. The extract contains from 100 mg to 220 mg of adenosine per 100g. The extract may be used for the preparation of a nutritional composition for the treatment or prevention of fatigue, of cardiovascular disease, or of inflammation, or for the improvement of immunity.

Description

A PROCESS FOR PREPARING A CORDYCEPS EXTRACT TECHNICAL FIELD

The invention relates to a method for preparing a cordyceps extract. More particularly, the invention relates to a method for preparing a cordyceps extract containing adenosine in high concentration.

BACKGROUND OF THE INVENTION

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Cordyceps is a genus of ascomycete fungi that grow mainly on insects and other arthropods. Cordyceps is a well-known ingredient in traditional Chinese medicine. Cordyceps is also named DongChongXiaCao in Chinese or "caterpillar fungus" or "winter worm summer grass" in English. When used in traditional Chinese medicine, the ingredient consists in fact of the fungus together with the caterpillar it developed on. In the rest of the specification, the word "cordyceps" will refer to the traditional medicinal ingredient, as used in traditional Chinese medicine. When referring to a fungus specie, the name of the species or the genus will be capitalised and in italics, such as Cordyceps sinensis.

According to traditional Chinese medicine, cordyceps is commonly used as a tonic and for medicinal purposes, such as to replenish the kidney, to soothe the lung, and for the treatment of fatigue. Because of the rarity and outstanding curative effects of Cordyceps sinensis, several strains have been isolated from wild Cordyceps sinensis mycelium. These isolated strains are used in fermentation technology and they are commonly sold as functional food products. Quality control of C. sinensis and its products is very important to ensure their safety and efficacy. In addition, some substitutes such as C. . militaris and C. adulterants also have been used. Cordyceps seems to be a source of interesting chemical constituents such as cordycepin (3'-de-oxyadenosine), adenosine and its derivatives, ergosterol, or polysaccharides.

Adenosine is a purine nucleoside and plays an important role in biochemical processes, such as energy transfer— as adenosine triphosphate (ATP) and adenosine diphosphate (ADP) — as well as in signal transduction as cyclic adenosine monophosphate (cAMP). It is also a neuromodulator to promote sleep and suppress arousal. Adenosine also plays a role in regulation of blood flow to various organs through vasodilation. Adenosine acts through several types of receptors which are located, for instance in the immune, nervous, circulatory, respiratory or urinary systems. It is suggested that adenosine and cordycepin are some of the bio-active compounds which provide the anti-inflammation, anti-oxidant and heart-protective effects of cordyceps.

Cordycepin (3'-deoxyadenosine) and adenosine are generally suggested as quality markers of cordyceps. Fresh cordyceps also contains high amount of water. Therefore, cordyceps must be dried for commercial purposes and long-time storage. Also, cordyceps used for food purposes must comply with regulatory requirements. For instance, in China, cordyceps products must contain at least 50 mg/lOOg of adenosine, and have a total plate count (TPC) below 10000.

Years of research have proven that the characteristic and nutritional components of cultured Cordyceps sinensis are the same as those of wild Cordyceps sinensis, if not higher. Additional studies showed that Cordyceps sinensis can be cultivated to produce spores which are considered as the best part of Cordyceps sinensis.

The traditional method of preparation of cordyceps includes drying, milling, sterilising and packing. However, the cordyceps extract prepared traditionally does not provide the required adenosine content and does not meet the microbiological safety standards. In order to have a high adenosine concentration in the finished products, suppliers have to be very considerate when selecting fresh cordyceps and the processing parameters. However, these operations still do not guarantee the consistency of the product, especially a reproducible batch quality.

In addition, in order to eliminate microbiological risks, suppliers usually apply sterilization and irradiation. However, these operations may cause loss of adenosine or degradation of other active components, which in turn may cause compliance concerns.

It is desirable to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. In particular, it is desirable to provide a method to prepare a cordyceps extract with consistent batch quality, with sufficient adenosine content, and with a low microbiological contamination. It is also desirable that the cordyceps extract can be used easily in the preparation of food products, or beverages, such as functional food products.

SUMMARY OF THE INVENTION

The object of the invention is achieved by the subject matter of the independent claims. The dependent claims further develop the idea of the present invention.

Accordingly, in a first aspect, the invention provides a method for preparing a cordyceps extract comprising the steps of: a) grinding cordyceps, b) blanching said cordyceps, c) drying said blanched cordyceps, wherein blanching comprises a treatment of said cordyceps at a temperature of from 27°C to 90°C during 20 to 240 minutes in water.

In a second aspect, the invention provides a cordyceps extract prepared from a method according to the first aspect of the invention, wherein said cordyceps extract contains from 100 mg to 220 mg of adenosine per 100 g of extract.

In a third aspect, the invention provides a nutrition composition comprising a cordyceps extract obtainable by a method according to the first aspect of the invention, or a cordyceps extract according to the second aspect of the invention. The nutrition composition may be a food product, a drink, a functional food, a nutraceutical, a food additive, a nutritional formula, and a pet food product. In a fourth aspect, the invention provides the use of a cordyceps extract according to the second aspect of the invention, or prepared by a method according to the first aspect of the invention, for the preparation of a nutritional connposition for the treatment or prevention of fatigue, of cardiovascular disease, or of inflammation, or for the improvement of immunity.

These and other aspects, features and advantages of the invention will become more apparent to those skilled in the art from the detailed description of embodiments of the invention, in connection with the attached drawings. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a flow chart showing the traditional preparation of cordyceps, as explained in example 1 (prior art).

Figures 2 to 5 are flow charts showing embodiments of a method according to the invention, of preparing the cordyceps extract as explained in example 2 to 5.

Figure 6 is a chromatogram of cordyceps extract by UPLC-PDA, as further explained in Experimental example 6.

DETAILED DESCRIPTION OF THE INVENTION

Throughout the specification, the words "comprise", "comprising" and the like are to be construed in an inclusive sense, that is to say, in the sense of "including, but not limited to", as opposed to an exclusive or exhaustive sense. As used in the specification, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

Unless noted otherwise, all percentages in the specification refer to weight percent.

Unless defined otherwise, all technical and scientific terms have and should be given the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Cordyceps used herein refers to all Cordyceps species, including C. sinensis, C. militaris, C. capita, C. pruinosa and C. ophioglossoides. In an embodiment, Cordyceps species are selected from the group consisting of C. sinensis, C. militaris, C. capita, C. pruinosa and C. ophioglossoides, preferably from C. sinensis, C. militaris and C. ophioglossoides.

When preparing the cordyceps extract according to the invention, a mix of cordyceps may be used, with different Cordyceps species, such as a mix of cordyceps which contains both Cordyceps sinensis and Cordyceps ophioglossoides. It is preferable that cordyceps is fresh. This way, the quality of the cordyceps extract is improved over dried cordyceps. However, it is also possible to prepare the cordyceps extract based on dried cordyceps.

Examples of methods for preparing a cordyceps extract are illustrated in Figures

2 to 5. In Figures 2 to 5, optional steps are indicated in dashed boxes. Grey boxes correspond to the blanching steps, as explained below.

In common to all the embodiments of the method of preparing a cordyceps extract, cordyceps is grinded into powder. Grinding can be performed in water, to obtain a cordyceps slurry direclty. Grinding increases the surface/volume ratio of the cordyceps particle, which improves the efficiency of extraction. Grinding can be achieved by chopping, cutting, or other methods. Preferably, the cordyceps is grinded to a particles size of 0.5mm to 5mm, more preferably of 1mm to 3mm. Preferably, fresh cordyceps is used, although dried cordyceps can also be used.

As shown on the process diagrams of Figures 2 to 5, water is added to the grinded cordyceps, to obtain a cordyceps slurry. As mentioned before, water can be also added to te cordyceps before grinding. There is no fixed weight ratio of cordyceps to water. Usually the cordyceps to water weight ratio ranges from 0.5:1 to 2:1, and preferably from 1:1 to 1.5:1. If less water is used, then the extraction may be less efficient. If too much water is used with respect to the cordyceps, then it will be necessary to evaporate the excess water, which will require a higher energy input. Water can be distilled water or tap water. Preferably, distilled water is used to reduce the risk of microbiological contamination, especially when the cordyceps extract is not sterilized. The cordyceps slurry is then blanched in water. Blanching comprises a treatment of said cordyceps at a temperature of from 27°C to 90°C during 20 to 240 minutes in water. The inventors have found that blanching the cordyceps slurry improves the adenosine content of the cordyceps extract. The duration of the blanching step depends on the blanching temperature.

In some embodiments, blanching is performed in one step. For instance, the cordyceps slurry is blanched at a temperature of from 30°C to 85°C during 30 minutes to 200 minutes, preferably at a temperature of from 30°C to 45 °C during 120 minutes to 200 minutes. In an embodiment, the cordyceps slurry is blanched by soaking at a temperature of from 30°C to 40°C during 150 minutes to 200 minutes. In another embodiment, the cordyceps slurry is blanched at a temperature of from 70°C to 90 °C during 30 minutes to 60 minutes.

In other preferred embodiments, blanching is divided into two steps: a first step is a mild heat treatment, followed by a second step which is a stronger heat treatment, in water. For instance, the cordyceps slurry is first soaked at a temperature of 30°C to 45°C for 120 minutes to 200 minutes in water, then the slurry is heated to a temperature of from 70°C to 90 °C for 30 to 60 more minutes in water. In another preferred embodiment, blanching of the cordyceps slurry performed by soaking the cordyceps slurry at a first temperature of from 30°C to 45°C for 120 minutes to 200 minutes in water, then increasing the temperature up to a second temperature of 70°C to 90°C within 5 to 15 minutes, and maintaining the second temperature for 30 to 45 minutes.

As will be shown in the examples, the inventors have found that soaking the cordyceps slurry for 180 min at 30°C (mild heat treatment), and then heating the cordyceps slurry 85°C for 30 minutes (stronger heat treatment), allows the preparation of an extract with the highest adenosine content.

Preferably, the blanched cordyceps slurry is sterilised before drying. Sterilization reduces potential contaminations and allows for an longer shelf-life of the product. The blanched cordyceps slurry is sterilized at a temperature of from 98°C to 101°C for 10 to 15 minutes, more preferably for about 15 minutes. After sterilisation, the cordyceps slurry is dried, for instance by freeze-drying, oven-drying, or vacuum-drying. The dried cordyceps may be milled into a powder from 30 to 200 mesh size, and more preferably in 50 to 100 mesh size.

When cordyceps extract is prepared by the method described above, a cordyceps extract containing a high concentration of adenosine can be obtained, for instance a concentration of 100 mg to 220 mg of adenosine per lOOg of cordyceps extract. This is much higher than the concentration of 25 mg/lOOg according to the previous methods of preparation (Figure 1 and example 1 below). In some embodiments, a concentration of adenosine higher than 110 mg/lOOg, and even higher than 150 mg/lOOg can be reached. The final concentration of adenosine depends on the blanching conditions, including the blanching time and temperature, as well as the blanching steps.

An aspect of the invention relates to a nutrition composition comprising a cordyceps extract obtainable by the method described above. The nutrition composition may be a food product, a drink, a functional food, a nutraceutical, a food additive, a nutritional formula, and a pet food product. The nutrition composition may be prepared by mixing the cordyceps extract into a standard nutritional product. When dry nutritional products are considered, the dry cordyceps extract can be dry-mixed into the dry nutritional product. For instance, the dry cordyceps extract can be mixed into milk powder. When wet nutritional products are considered, it may be desirable to mix the blanched cordyceps slurry, maybe concentrated and sterilised, into the wet nutritional product, before sterilisation or heat treatment of the wet nutritional product.

In a fourth aspect, the invention provides the use of a cordyceps extract which contains an amount of adenosine ranging from 100 mg/100 g to 220 mg/100 g, for the preparation of a nutritional composition for the treatment or prevention of fatigue, of cardiovascular disease, or of inflammation, or for the improvement of immunity. The nutrition composition may be a food product, a drink, a functional food, a nutraceutical, a food additive, a nutritional formula, and a pet food product. EXAMPLES

Example 1 - Traditional preparation of cordyceps

As shown on Figure 1, 50 g of fresh cordyceps fruiting body was collected and it was pasteurized at 101°C for 10 minutes. Then the pasteurised cordyceps was freeze- dried at -50°C for 12 hours. The freeze-dried cordyceps was collected and milled. A brown powder was obtained.

The adenosine content was analysed by UPLC, as explained in Experimental example 6. The cordyceps powder contained 25 mg of adenosine per 100 g of powder. Example 2 - Preparation of a cordyceps extract

As shown on Figure 2, 50 g of fresh cordyceps fruiting body was collected and chopped in a miller to obtain a cordyceps slurry. 500g of water was added to the cordyceps slurry. The blanching was performed in two steps (indicated in grey boxes on Figure 2): the cordyceps slurry was then treated for 3 hours (180 minutes) at 30°C (soaking), and then, the cordyceps slurry was further treated at 85°C for 30 minutes. Finally, the cordyceps slurry was sterilized at 101°C for 10 minutes, and freeze-dried at -50°C overnight. The freeze-dried cordyceps was collected and milled as a brown powder.

The adenosine content was analysed by UPLC, as explained in Experimental example 6. The cordyceps powder contained 176 mg of adenosine per lOOg of powder.

Example 3 - Preparation of a cordyceps extract

As shown on Figure 3, 50 g of fresh cordyceps fruiting body was collected and chopped in a miller to obtain a cordyceps slurry. 500g of water was added to the cordyceps slurry. The blanching was performed in two steps (indicated in grey boxes on Figure 3): the cordyceps was then treated for 3 hours (180 minutes) at 30°C (soaking), and then, the cordyceps slurry was further treated at 85°C for 30 minutes. Finally, the cordyceps slurry was freeze-dried at -50°C overnight. The freeze-dried cordyceps was collected and milled as a brown powder. The adenosine content was analysed by UPLC, as explained in Experimental example 6. The cordyceps powder contained 205 mg of adenosine per lOOg of powder. In Example 3, the blanched cordyceps slurry was not sterilised, contrary to Example 2. It seems that sterilisation has a negative impact on the adenosine content.

Example 4 - Preparation of a cordyceps extract

As shown on Figure 4, 50 g of fresh cordyceps fruiting body was collected and chopped in a miller to obtain a cordyceps slurry. 500g of water was added to the cordyceps slurry. The blanching was performed in one step (indicated in a grey box on Figure 4): the cordyceps slurry was treated for 30 minutes at 85°C. Finally, the cordyceps slurry was sterilized at 101°C for 10 minutes, and freeze-dried at -50°C overnight. The freeze-dried cordyceps was collected and milled as a brown powder.

The adenosine content was analysed by UPLC, as explained in Experimental example 6. The cordyceps powder contained 109 mg of adenosine per lOOg of powder. In Example 4, a one-step blanching was used, contrary to Examples 2 and 3. It seems that a one-step blanching yields a lower adenosine content than a two-step blanching.

Example 5 - Preparation of a cordyceps extract

As shown on Figure 5, 50 g of fresh cordyceps fruiting body was collected and chopped in a miller to obtain a cordyceps slurry. 500g of water was added to the cordyceps slurry. The blanching was performed in one step (indicated in a grey box on Figure 5): the cordyceps slurry was treated for 180 minutes at 30°C. Finally, the cordyceps slurry was sterilized at 101°C for 10 minutes, and freeze-dried at -50°C overnight. The freeze-dried cordyceps was collected and milled as a brown powder.

The adenosine content was analysed by UPLC, as explained in Experimental example 6. The cordyceps powder contained 158 mg of adenosine per lOOg of powder. In Example 5, a one-step mild blanching was used, contrary to Example 4. It seems that a mild blanching yields a higher adenosine content than a stronger blanching. The table below summarises the main processing steps of examples 1-5 and compares the adenosine content of the milled cordyceps extract.

Experimental example 6 - Method of analysis of the adenosine content

Referring to the method from Chinese pharmacopeia, fine cordyceps extract powder was extracted by methanol : water (1:1, v/v) under ultrasonic condition for 30 minutes and quantified by UPLC-PDA with a C18 column (for instance, Acquity BEH C18, 2.1*100 mm, 1.7 urn). The analysis was performed with isocratic method at room temperature at a flow speed of 1.0 ml/minutes. The mobile phase was water : methanol (95 : 5, v/v). The absorption wavelength for quantification was 259.4 nm.

The cordyceps extract is analysed by Acquity UPLC-PDA (Waters, USA). Detailed parameters of UPLC are shown in the table below: Column: Waters Acquity BEH C18, 2.1*100mm_/ 1.7 μηη

Mobile phase A: Water

Mobile phase B: Methanol

Flow ratio: 0.4 mL/min

Gradient elution Time (min) B (%)

0.00 5

4.00 5

8.00 10

9.00 10

9.01 5

12.00 5

Injection volume: 2 μΙ_

Column temperature: 30 C

PDA Wavelength: 210nm-400nm_/ Scanning, resolution 1.2 nm

This method was used to analyse the adenosine content in the cordyceps extract described in Examples 1 to 5. Figure 6 is an example of a chromatogram of cordyceps extract by UPLC-PDA

The concentration of adenosine was calculated according to the standard curve created by injecting different level of standards. For example, the standards listed below were injected to get the calibration curve.

Standard name Concentration (X value) Adenosine Response (Y value)

1 μg/mL mix standard 1 μg/mL 16415

2 μg/mL mix standard 2 μg/mL 29055

4 μg/mL mix standard 4 μg/mL 64063

8 μg/mL mix standard 8 μg/mL 130661

15 μg/mL mix standard 15 μg/mL 246860

30 μg/mL mix standard 30 μg/mL 476448

50 μg/mL mix standard 50 μg/mL 810322 Based on results above, a calibration equation for adenosine was obtained, such as Y=aX+b. Then unknown sannples were injected to get response Y value and calculate X value in μg/mL. Finally the adenosine content A in the sannple can be expressed in mg/lOOg according to the formula below:

X * 50 * 100

A =

m * 1000

X adenosine concentration ^g/mL) as calculated with the calibration equation m sample weight (g)

50 dilution volume (mL)

1000 conversion factor from μg to mg

Claims

1. A method for preparing a cordyceps extract comprising the steps of: a) grinding cordyceps,

b) blanching said cordyceps,

c) drying said blanched cordyceps,

wherein blanching comprises a treatment of said cordyceps at a temperature of from 27°C to 90°C during 20 to 240 minutes in water.

2. A method according to claim 1, wherein blanching is performed at a temperature of from 30°C to 85°C during 30 minutes to 200 minutes.

3. A method according to claim 1 or 2, wherein blanching is performed by soaking said cordyceps at a temperature of from 30°C to 45 °C during 120 minutes to 200 minutes.

4. A method according to claim 1, wherein blanching is performed at a temperature of from 70°C to 90 °C during 30 minutes to 60 minutes.

5. A method according to claim 1, wherein blanching is performed by soaking the cordyceps at a temperature of 30°C to 45°C for 120 minutes to 200 minutes in water, then heating the cordyceps to a temperature from 70°C to 90 °C for 30 to 60 minutes in water.

6. A method according to claim 1, wherein blanching is performed by soaking the cordyceps at a first temperature of 30°C to 45°C for 120 minutes to 200 minutes in water, then increasing the temperature up to a second temperature of 70°C to 90°C within 5 to 15 minutes, and maintaining the second temperature for 30 to 45 minutes.

7. A method according to any one of claims 1 to 6, wherein said water and cordyceps are in a weight ratio of from 0.5:1 to 2:1, preferably from 1:1 to 1.5:1.

8. A method according to any one of claims 1 to 7, which comprises a step of sterilising at a temperature of 98°C to 101°C for 10 to 15 minutes between the blanching step and the drying step.

9. A method according to any one of claims 1 to 8, wherein the drying step is performed by freeze-drying, oven-drying, or vacuum-drying.

10. A method according to any one of claims 1 to 9, which comprises a further step of milling after the drying step.

11. A method according to any one of claims 1 to 10, wherein said cordyceps is Cordyceps militaris or Cordyceps sinensis.

12. A cordyceps extract prepared from a method according to any one of claims 1 to 11, wherein said cordyceps extract contains from 100 mg to 220 mg of adenosine per 100 g of extract.

13. A nutrition composition comprising a cordyceps extract obtainable according to any one of claims 1 to 11 or a cordyceps extract according to claim 12.

14. A nutrition composition according to claim 13, wherein said nutrition composition is selected from the group consisting of a food product, a drink, a functional food, a nutraceutical, a food additive, a nutritional formula, and a pet food product. 11 or a cordyceps extract according to claim 12, for the preparation of a nutritional composition for the treatment or prevention of fatigue, of cardiovascular disease, or of inflammation, or for the improvement of immunity.