CN104824574A - Cordyceps sinensis broad bean - Google Patents

Abstract

With broad beans as a culture medium, cordyceps sinensis and cordyceps militaris are subjected to double-bacterial mixed solid fermentation. The invention belongs to the technical field of biological engineering. The broad beans prepared through the double-bacterial bio-conversion have functions of health caring and are excellent in application prospect.

Description

Chinese caterpillar fungus broad bean

Technical field

Adopting Cordyceps sinensis (Ophiocordyceps sinensis) and Cordyceps militaris (Cordyceps militaris), take broad bean as culture medium, carries out two bacterium mixed solid fermentation, belongs to technical field of bioengineering.Utilizing bacterial strain Cordyceps militaris preserving number to be CICC 14013, bacterial strain Ophiocordyceps sinensis preserving number is CICC 14088, is produced obtain nutrition and health care broad bean by two bacterium mixed solid fermentation.

Background technology

Cordyceps (Cordyceps) is under the jurisdiction of mycota (Fungi), Ascomycota (Ascomycota), Ascomycetes (Ascomycetes), excrement shell bacterium subclass (Sordariomycetidae), Hypocreales (Hypocreales), Clavicipitaceae (Clavicipitaceae).Cordyceps sinensis fungus great majority are medicinal fungis, wherein Cordyceps sinensis (Ophiocordyceps sinensis) is generally well known, applicating history is also remote, and the application study of Cordyceps militaris (Cordyceps militaris) is the most deep.

Cordyceps sinensis is recorded in 710 years Christian eras " moon king medicine is examined " the earliest according to textual research, records first appeared in Qing Dynasty Wu Yi Lip river " new compilation of materia medica " (1757 Christian eras) with medicine, thinks its " sweet flat; to protect lung kidney-nourishing; hemostasis and phlegm, phthisical cough, control diaphragm disease all good ".Cordyceps sinensis is China's tradition rare Chinese medicine, and it and ginseng, pilose antler are described as Chinese medicine Triratna together.It has mend and not high, temperature and not fiery, grow and oiliness pharmacodynamic profile, have more negative and positive with the unique effects of mending, make it become nourishing panacea, be described as " kings of hundred medicines " by physician.

Cordyceps militaris is one of Chinese medicinal material of being of great rarity of China, and its medical value is on the books very early, and Li Shizhen (1518-1593 A.D.) (1518) is pointed out in Compendium of Material Medica, and cicada fungus can cure mainly " children's hangs in sky frightened epilepsy, the morbid night crying of babies, palpitaition ".Cordyceps militaris is easy to large-scale artificial and cultivates, and its active component and pharmacological action are similar to Cordyceps sinensis again.

The current grain raw material that Cordyceps sinensis or Cordyceps militaris is applied to ferments by wide coverage.Such as: patent 200610117997.4 adopts Cordyceps militaris industrialization to cultivate discarded object and makes rice flour product, Chinese caterpillar fungus is pulverized rear mixing with the careless highland barley fried and makes health products by patent 200710050135.9, patent 200710166278.6 adopts wheat to cultivate fruiting bodies of cordyceps militaris, patent 200810068734.8 adopts adopting red yeast rice synergistic to ferment thus improves the output of fruiting bodies of cordyceps militaris and cordycepin, patent 200910218363.1 discloses a kind of method being improved primary medicinal component by Poria cocos and Cordyceps militaris solid co-fermentation, patent 201110055425.9 utilizes solid state fermentation to produce Cordyceps militaris fermentation rice, patent 201110348239.4 take lotus seeds as matrix, carries out the solid state fermentation of Cordyceps militaris or winter grass summer grass, patent 201110064459.4 discloses a kind of preparation method of Cordyceps militaris paddy food, patent 201310150267.4 and 201310151228.6 respectively with soya bean and peanut for matrix, carry out solid state fermentation after inoculation Cordyceps militaris, but need in process to add insect enzymolysis liquid, cause cost and difficult quality to control, patent 201210064068.7 discloses a kind of with glossy ganoderma, Phellinus etc. can the mushroom entity of efficient cellulase-producing as culture medium major ingredient, grain raw material is auxiliary material, inoculate into Cordyceps militaris, Cordyceps sinensis, mushroom, Hericium erinaceus, the bacterial classifications such as acupuncture needle aunt carry out Mixed culture, make health products or additive, but this complex process, edible and medical fungi fructification is expensive, and after autoclaving, destroy the active ingredient of edible and medical fungi fructification, complicated component and uncontrollable after other strain fermentation again, the strain combination of various complexity also will cause the instability of end product quality.

In sum, the solid ferment process of current Cordyceps militaris or Cordyceps sinensis all can add inorganic salts, insect hydrolyzate or other medicinal ingredient, to improving Chinese caterpillar fungus thalline or cordycepin output and drug effect.Different additives can cause complicated component and import external pollution sources, and is not easy to set up quality index system.In addition, the solid ferment process of Cordyceps militaris often using cordycepin (Cordycepin) as important quality index.Cordycepin, is also referred to as 3'-Deoxyadenosine (3-deoxyadenosine), and since self-discovery, its pharmacological action is constantly made known.Personnel study discovery after deliberation, and cordycepin has immunological regulation, antitumor, anti-leucocyte, the multiple pharmacological effect such as antimycotic, antiviral.What is interesting is, cordycepin is mainly stored in Cordyceps militaris, and content in Cordyceps sinensis is atomic, but the disappearance of cordycepin does not affect the remarkable efficacy of Cordyceps sinensis.Therefore, these two kinds of effects of Cordyceps sinensis and Cordyceps militaris are similar, but the incomplete same bacterial classification of composition has complementarity.

Summary of the invention

Based on current situation, the present invention take broad bean as culture medium, when not adding other additive any, only broad bean is carried out expanding treatment, after sterilizing, Simultaneous vaccination Cordyceps sinensis and Cordyceps militaris carry out Mixed culture, finally obtain the health care broad bean of high cordycepin content and high bioactivity.The method is implemented simple, quality controllable, has wide application and development prospect.

The present invention's Cordyceps strain Ophiocordyceps sinensis preserving number used is CICC14088, and cordyceps militaris link bacterial strain Cordyceps militari preserving number is CICC 14013.

Technical scheme of the present invention is as follows:

(1) part broad bean adopts the twin-screw extruder containing three sections to process.1st section is preheating zone, and serviceability temperature is 40-60 DEG C, and the 2nd district is traffic zone, and temperature is 120-160 DEG C, and the 3rd district is slaking district, and temperature is 140-180 DEG C.The water content of expanded front broad bean is 10%-20%.

(2) part broad bean mechanical crushing is 0.1-2mm to particle diameter.

(3) mixed with certain proportion with the broad bean of mechanical crushing by the broad bean of extrusion, the broad bean proportion of extrusion is 1-100%.Keep the skin wet after mixing and make water content reach 20%-70%, 121 DEG C of insulations sterilizing in 20 minutes.After cooling, access Cordyceps sinensis, inoculum concentration is 1-10%.Meanwhile, access Cordyceps militaris, inoculum concentration is 1-10%.Initial pH and sweat pH is nature, and fermentation temperature is 15-28 DEG C, and envionmental humidity is 60%-100%, and fermentation period is 5-25 days.?

The content of whole cordycepin reaches as high as 8.2%.

The Cordyceps sinensis that the present invention is used and Cordyceps militaris spawn preservation slant medium formula are: glucose 20g/l, potato juice 200g/l, agar 20g/l, pH nature.

The seed culture based formulas of Cordyceps sinensis of the present invention and Cordyceps militaris is: glucose 20g/l, potato juice 200g/l, pH nature.After slant strains is inoculated into the triangular flask that seed culture medium is housed, the incubation time of seed is 96 hours, and the cultivation temperature of seed is 25 DEG C.

Cordycepin content adopts liquid chromatography for measuring.Assay method refers to: Wei Huiping, Ye little Li, Zhang Huaying, Li Xuegang, Zhong Yunjun, with the research of Cordyceps militaris solid fermentation method High-efficient Production cordycepin, and CHINA JOURNAL OF CHINESE MATERIA MEDICA, 1998,33 (19), 2159-2162.

Beneficial effect of the present invention: by expanded, makes Faba bean starch gelatinization and Broad Bean Protein sex change at short notice.Expanded broad bean ferments after mixing with mechanical crushing broad bean, the nutriment making thalline to utilize puffing process to produce fast on the one hand, on the other hand mechanical crushing broad bean is the equal of that solid state fermentation carrier makes fermentation substrate keep loose favourable thalli growth, and when thalli growth is vigorous, thalline produces a large amount of ectoenzyme and can decompose further again and utilize mechanical crushing broad bean.This process, without the need to adding other material, ensures that the quality height of fermented product is controlled.The common fermentation of Cordyceps sinensis and Cordyceps militaris two kinds of similar bacterial classifications of character, on the one hand the tunning of similar quality bacterial classification can not change overall pharmacology pharmacy proterties, can be in harmonious proportion two kinds of strain fermentation products defect separately on the other hand.The method simple possible, is easy to accomplish scale production, is with a wide range of applications.

Detailed description of the invention

Embodiment 1

Adopt the DS56-III type twin-screw extruder process broad bean that Jinan Ju Xian plant equipment Co., Ltd produces, broad bean used is commercially available, and measuring its moisture content through moisture teller is 12.1%.Setting charging rate is 120kg/h.Table 1 lists temperature combination and the puffed degree of three sections.When the temperature in preheating zone, traffic zone, slaking district is respectively 60,160,190 DEG C, the puffed degree of broad bean is the highest as can be seen from Table 1, under this temperature conditions, in broad bean, supplement a small amount of sterilized water further or broad bean is dried further, investigating puffed degree during different moisture content.As can be seen from Table 2, moisture content is that expansion effect is good when about 12-18%, therefore can directly carry out expanded with commercially available broad bean.

Table 1: the different each section different temperatures combination of extruder obtains the puffed degree of expanded broad bean

Table 2: the puffed degree after different water cut broad bean is expanded

Moisture content %	10.2	12.1	14.3	16.1	17.3	18.2	19.8
								Puffed degree	1.6	1.8	1.8	1.8	1.7	1.7	1.5

Embodiment 2

Be inoculated into by aweto strain and be equipped with in the 250ml triangular flask of 50ml culture medium, culture medium consists of: glucose 20g/l, and potato juice 200g/l, pH nature, 25 DEG C, shaking speed 150 turns/min, cultivates 96 hours, as inoculation seed.

Be inoculated into by Cordyceps militaris spawn and be equipped with in the 250ml triangular flask of 50ml culture medium, culture medium consists of: glucose 20g/l, and potato juice 200g/l, pH nature, 25 DEG C, shaking speed 150 turns/min, cultivates 96 hours, as inoculation seed.

Get puffed degree be 1.8 expanded broad bean and mechanical crushing degree be 1mm broad bean mixes, amount to 50g, adding distil water 40g again, put into 500ml triangular flask, 121 DEG C of sterilizing 20min, inoculate after cooling into Cordyceps sinensis and each 5ml of Cordyceps militaris seed liquor, control environment humidity 90% in constant temperature and humidity incubator, temperature is 25 DEG C, ferments and measures cordycepin content after 15 days.Cordycepin output under different condition combination is as shown in table 3.When expanded broad bean and mechanical crushing broad bean ratio are 3:2, cordycepin produces the highest as can be seen from Table 3, can reach 6.3%.

Table 3: solid state fermentation cordycepin content under expanded broad bean and mechanical crushing degree different proportion

Expanded broad bean weight (g)	Mechanical crushing broad bean weight (g)	Cordycepin content (%)
			50	0	2.7
1	49	3.4
			25	25	4.5
10	40	3.6
			40	10	3.2
20	30	5.7
			30	20	6.3

Embodiment 3

Get puffed degree be 1.8 expanded broad bean 30g and mechanical crushing degree be 2mm broad bean 20g mixes, adding distil water 40g again, put into 500ml triangular flask, 121 DEG C of sterilizing 20min, inoculate into cultured Cordyceps sinensis and each 5ml of Cordyceps militaris seed liquor after cooling, control environment humidity 80% in constant temperature and humidity incubator, and temperature is 15 DEG C, ferment 20 days afterwards measure cordycepin content be 5.5%.

Embodiment 4

Get puffed degree be 1.8 expanded broad bean 30g and mechanical crushing degree be 0.5mm broad bean 20g mixes, adding distil water 50g again, put into 500ml triangular flask, 121 DEG C of sterilizing 20min, inoculate into cultured Cordyceps sinensis and each 6ml of Cordyceps militaris seed liquor after cooling, control environment humidity 90% in constant temperature and humidity incubator, and temperature is 20 DEG C, ferment 5 days afterwards measure cordycepin content be 2.3%.

Embodiment 5

Get puffed degree be 1.8 expanded broad bean 30g and mechanical crushing degree be 1mm broad bean 20g mixes, adding distil water 45g again, put into 500ml triangular flask, 121 DEG C of sterilizing 20min, inoculate into cultured Cordyceps sinensis seed liquor 5ml after cooling, control environment humidity 90% in constant temperature and humidity incubator, and temperature is 20 DEG C, ferment 25 days afterwards measure cordycepin content be 0.03%.The fermentate that this process obtains is designated as sample 1.

Get puffed degree be 1.8 expanded broad bean 30g and mechanical crushing degree be 1mm broad bean 20g mixes, adding distil water 45g again, put into 500ml triangular flask, 121 DEG C of sterilizing 20min, inoculate into cultured Cordyceps militaris seed liquor 5ml after cooling, control environment humidity 90% in constant temperature and humidity incubator, and temperature is 20 DEG C, ferment 25 days afterwards measure cordycepin content be 7.9%.The fermentate that this process obtains is designated as sample 2.

Get puffed degree be 1.8 expanded broad bean 30g and mechanical crushing degree be 1mm broad bean 20g mixes, adding distil water 40g again, put into 500ml triangular flask, 121 DEG C of sterilizing 20min, inoculate into cultured Cordyceps sinensis and Cordyceps militaris seed liquor 5ml after cooling, control environment humidity 90% in constant temperature and humidity incubator, and temperature is 20 DEG C, ferment 25 days afterwards measure cordycepin content be 8.2%.The fermentate that this process obtains is designated as sample 3.

Get the Kunming mouse 40 purchased from Shanghai Slac Experimental Animal Co., Ltd., Individual Quality 18-22g, male and female half and half.Be divided into 4 groups at random, often organize 10, be respectively sample 1,2,3 administration group and blank group.In first 7 days of experiment, every day, gavage gave different solid state fermentation samples, and the sample size of every mouse gavage is 0.5g.Blank group gavage to be unfermentable puffed degree be 1.8 0.5g broad bean.7th day, every mouse peritoneal injected 2% starch 2ml, after 24h, every mouse peritoneal injects 2% chicken red blood cell 1ml, after 30min, broken end, Intraperitoneal injection 0.2ml heparin, get peritoneal fluid smear, Rui Shi-Ji's nurse Sa dyeing 1min, salt solution gradient washes, oily sem observation phagocytic percentage, phagocytic index, get spleen, thymus gland is weighed, calculate spleen index, thymus index.

Spleen index=spleen average quality (mg)/average body quality (g).

Thymus index=thymus gland average quality (mg)/average body quality (g).

The cell number of the quantity of phagocytic index=engulf/engulf.

The phagocyte number of the cell number of phagocytic rate=engulf/total.

Spleen index is listed, thymus index, phagocytic index and phagocytic rate in table 4.

Table 4: different fermentations sample is on the impact of mouse spleen, thymic factor D injection, Peritoneal Macrophage Phagocytosis

Group	Thymus index	Spleen index	Phagocytic rate (%)	Phagocytic index
					Blank group	1.74±0.25	2.91±0.25	48.2±8.53	1.7±0.1
1 group, sample	2.42±0.21	4.01±0.22	59.3±10.2	2.1±0.3
					2 groups, sample	2.23±0.22	3.52±0.21	66.4±10.2	2.2±0.2
3 groups, sample	3.31±0.31	5.81±0.32	85.2±10.3	3.2±0.4

Index and spleen index and thymus index are the important parameters directly reflecting Immunologic States, and sample 1-3 group all significantly can promote the growth of mouse spleen and thymus gland relative to blank group as can be seen from Table 4, the immune level effect of being significantly improved.The Be very effective that worm winter, worm summer and Cordyceps militaris carry out 3 groups, the sample of mixed culture fermentation is better than 1 group, the sample of Cordyceps sinensis list strain fermentation and 2 groups, the sample of the single strain fermentation of Cordyceps militaris.

1 group, sample fermenting compared to single bacterium of worm winter, worm summer and Cordyceps militaris 3 groups, sample carrying out mixed culture fermentation and 2 groups, sample significantly can promote the phagocytic function of Turnover of Mouse Peritoneal Macrophages as can be seen from Table 4, the anti-infective of body and anti-tumor capacity can be improved, strengthen the immunologic function of body, stable state of keeping fit.

Claims (2)

1. the present invention adopts broad bean to be matrix, carries out Cordyceps sinensis and Cordyceps militaris mixed solid fermentation.

2. mixed solid fermentation according to claim 1, is characterized in that part broad bean need carry out expanding treatment.