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OfflineAnnoA
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: Randoscious_McCray]
    #2128826 - 11/22/03 07:01 AM (20 years, 3 months ago)

Something of interest:

From http://diseyes.lycaeum.org/teo/peele.txt

"Take Psilocybe Cyanescens for example. Most of you might know that
this is one of the most powerful of all psychoactive mushrooms. I have
talked to other people who ate this mushroom. Some, who by the way could
boast about how many Psilocybe cybensis mushrooms they could eat, can't
even look at a mushroom they might see in the grocery store..........I
would advise anybody to go lightly on this one. The point I want to make
is that this mushroom, and Psilocybe pelliculosa, prove the somewhat theory
I have. When these mushrooms are grown under liquid media, they do not
produce any psilocybin or psilocin. This was also found to be true by P.
Catalfomo and V.E. Tyler, Jr.. They published the same findings about
Psilocybe cyanescens and Psilocybe pelliculosa......they do not produce
psilocybing or any other analogs (Catalfomo, P. and V.E. Tyler, Jr. _The
production of psilocybin in submerged culture of Psilocybe cubensis_
LLoydia 27:53-63, 1964). However, once the mycelium of one of these two
is transferred to an agar or grain media, it does produce psilocybin and
psilocin."

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Offlineragadinks
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Re: Growing in Liquid Medium. Using knowledge from Reactor D [Re: Anno]
    #2128926 - 11/22/03 09:19 AM (20 years, 3 months ago)

What species do grow a lot of psylocibin in liquid media ?
(P.azur ? P. cubensis ? )


--------------------
-> Errors Are A Great Source Of Knowledge <-
-> It Is Not Important WHO Is Right But WHAT Is Right <-

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Offlinebonsai
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: Randoscious_McCray]
    #2129538 - 11/22/03 07:13 PM (20 years, 3 months ago)

Randoscious_McCray,
Are you looking to grow the mycelium for extraction?  And are you interested in cubensis only?  I would like to see a few of us try some of these liquid growth medias for medicinal shrooms.  Any takers. 

Any one have any idea how to grow mycelium for maximum efficiency in a liquid media?  Are stirrers and injected air needed.  More info please  :smile:.

bonsai 


--------------------
"Without deviation from the norm, progress is not possible."
--Frank Zappa--

:laugh:

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Offlineragadinks
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Re: Growing in Liquid Medium. Using knowledge from Reactor D [Re: bonsai]
    #2130440 - 11/23/03 11:59 AM (20 years, 3 months ago)

Any one have any idea how to grow mycelium for maximum efficiency in a liquid media? Are stirrers and injected air needed. More info please

There is a section about liquid inoculation techniques in Staments TMC and GGMM. There he describes the usage of magnetic stirrers in order to break up the mycelium and provide oxygen to it. In China they use huge vessels that can be autoclaved. They use aeration stones to oxygenate the broth. According to him almost all wood and straw saprophytic mushrooms are qualifying for liquid medium (There is also a list of the species in the book ). In the book there is also this recipe given as a liquid culture media for Wood Decomposers:

1000 ml water
40 grams barely malt sugar
3-5 grams hardwood sawdust
2 grams yeast
1 gram calcium sulfate

Here is also an article about liquid media for Enoki.
And here is another article about growing a medical mushroom ( Phellinus linteus ) in liquid media.

Edited by ragadinks (12/25/04 05:34 PM)

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Invisiblemicro
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Re: Growing in Liquid Medium. Using knowledge from Reactor D [Re: ragadinks]
    #2130519 - 11/23/03 12:58 PM (20 years, 3 months ago)

Honestly, all you need is for the cultures to be shaking, and a decent amount of surface area to be there and the oxygen will get dissolved. You could use closed mason jars filled up like 20% of the way, or something else.... All you need to do is get them to shake, all you need to do this is make a platform that shakes, for example the same way as a choo-choo chrain works -- get a motor with a rod that moves up and down that moves a platform:



Sorry for the bbad drawing, but whatever....

--
Micro


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InvisibleLeaveMeAlone
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Re: Growing in Liquid Medium. Using knowledge from Reactor D [Re: Anno]
    #2223117 - 01/06/04 12:44 PM (20 years, 2 months ago)

Old link to a cool thread. Zen's posts are the meat and potatoes.

Quote:

This method of harvest is far superior to fruited bodies in relation to alkaloid yields, processing, extraction, and overall time.




--------------------
Love.

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Invisiblewoodrow
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Registered: 03/17/03
Posts: 142
Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: Randoscious_McCray]
    #2227275 - 01/08/04 01:09 AM (20 years, 2 months ago)

A great deal of research on growing shroom mycelium in liquid reactors was done in the fifties by a man named J. Szuecs (pronounced Sooch) and he patented the process for commercial use. Patents 2,693,664 (Sept. 1956) and 2,761,246 (Sept.1958).
The Szeucs reactor is a simple glass jug, stoppered on top, with about a 5 gallon capacity and a glass rod extending to the bottom. Sterile air is bubbled through the glass rod and bubbling alone provides adequate mixing. There is no effluent exchange. Szeucs found that any kind of paddles or stirrers did too much damage to the mycelium. He also found that with rapid aeration the mycelium would grow as hard pellets but slow aeration produced a more mushy product. A good source of information about submerged culture is MICROBIAL TECHNOLOGY by Henry J. Peppler, Reinhold Publishing 1967.

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OfflineMycena
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Re: Growing in Liquid Medium. Using knowledge from Reactor D [Re: blackout]
    #2296711 - 02/03/04 08:10 AM (20 years, 1 month ago)

Dextrose by the Kilo can also be found at beer brewing shops

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Invisiblemycofile
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: woodrow]
    #2300478 - 02/04/04 12:00 PM (20 years, 1 month ago)

Hey woodrow, where did you find those patents? the US patent office doesn't seem to have them (says text not available, view images for full patents, but the images don't show up).

I'm always interested in reading shroom patents, but couldn't seem to pull those up. Any help appreciated.


--------------------
"From a certain point of view"
-Jedi Master Obi Wan Kenobi

PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.

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OfflineAnnoA
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: mycofile]
    #2300641 - 02/04/04 12:43 PM (20 years, 1 month ago)




Sorry, they were that small, I didn't make them any smaller.

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Invisiblemycofile
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: Anno]
    #2304548 - 02/05/04 11:17 AM (20 years, 1 month ago)

Thanks anno, will see how they look under magnification.


--------------------
"From a certain point of view"
-Jedi Master Obi Wan Kenobi

PM me with any cultivation questions.

I just looked at my profile and realized I had a website at one point in time on geocities, it's not there anymore and I have no idea what I had on it. Anybody remember my website from several years aga? PM if so please.

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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: mycofile]
    #2307577 - 02/06/04 02:39 AM (20 years, 1 month ago)

is it worth doing? i.e. is there any chance of them being potent. 6 days is fast. note they mention molasses, could be good for liquid innocs

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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: mycofile]
    #2307642 - 02/06/04 04:19 AM (20 years, 1 month ago)

--------------------------------------------------------------------------------
United States Patent 3,940,883
Kasahara , et al. March 2, 1976
--------------------------------------------------------------------------------
Process for the growth and production of mushroom tissue

Abstract
A process for the growth and production of mushroom tissue which comprises the steps of suspending mushroom tissue in an aqueous nutrient fermentation medium solution, inoculating said solution into a mushroom growth and production bed comprising saw dust, wheat bran, rice bran, peptide for synthetic Japanese sake or liquor, acid potassium phosphate and/or chaff, and contained in a container, adjusting the pH of said bed to pH of 6.0-5.0 with an acid, ageing said mushroom growth and production bed under fermentation conditions, cooling the bed, turning the container upside-down and subjecting said bed to mushroom growth and production conditions.

--------------------------------------------------------------------------------
Inventors: Kasahara; Nobuo (Nagoya, JA); Shiota; Asao (Kasugai, JA); Kitaguchi; Isamu (Nagoya, JA)
Assignee: Sanwa Kagaku Kenkyusho Co., Ltd. (Nagoya, JA)
Appl. No.: 429122
Filed: December 28, 1973

Current U.S. Class: 47/1.1; 71/5
Intern'l Class: A01G 001/04
Field of Search: 47/1.1 71/5
--------------------------------------------------------------------------------
References Cited [Referenced By]
--------------------------------------------------------------------------------
U.S. Patent Documents
1832593 Nov., 1931 Szucs 47/1.
2300983 Nov., 1942 Sleighter 47/1.
2761246 Sep., 1956 Szuecs 47/1.
2928210 Mar., 1960 Cirillo et al. 47/1.
3286399 Nov., 1966 Laniece 47/1.
Foreign Patent Documents
37,047 Nov., 1970 JA.

Primary Examiner: Bagwill; Robert E.
Attorney, Agent or Firm: Hammond & Littell
--------------------------------------------------------------------------------
Claims
--------------------------------------------------------------------------------

What is claimed is:

1. A process for the growth and production of mushroom tissue comprising the steps of providing mushroom tissue of wood rotting fungi, suspending said mushroom tissue in an aqueous nutrient fermentation solution, inoculating said mushroom tissue suspending solution into a prepared mushroom growth and production bed contained in a container under sterilized conditions, ageing said nutrient fermentation solution inoculated bed, cooling the contents of said container, turning said container upside down and subjecting the contents of said container to mushroom growth and production conditions.

2. The process for the growth and production of mushroom tissue of claim 1 wherein said mushroom growth and production bed comprises a mixture the pH of which is adjusted to pH 6.0-5.0 with an acid.

3. The process for the growth and production of mushroom of claim 2 wherein said acid is citric acid.

4. The process for the growth and production of mushroom tissue of claim 1 wherein said mushroom growth and production bed comprises a mixture consisting of saw dust, wheat bran, rice bran, peptide for synthetic Japanese rice liquor, acid potassium phosphate and chaff.

5. The process for the growth and production of mushroom tissue of claim 1 wherein said mushroom growth and production bed comprises a mixture consisting of saw dust, rice bran, peptide for synthetic Japanese rice liquor, acid potassium phosphate and chaff.

6. The process for the growth and production of mushroom tissue of claim 1 wherein said mushroom growth and production bed comprises a mixture consisting of saw dust, wheat bran, peptide for synthetic Japanese rice liquor, acid potassium phosphate and chaff.

7. The process for the growth and production of mushroom tissue of claim 1 wherein said mushroom tissue growth and production bed comprises a mixture consisting of saw dust, wheat bran, rice bran, acid potassium phosphate and chaff.

8. The process for the growth and production of mushroom tissue of claim 1 wherein said mushroom tissue comprises a mixture consisting of saw dust, wheat bran, rice bran, peptide for synthetic Japanese rice liquor and chaff.

9. The process for the growth and production of mushroom tissue of claim 1 wherein said mushroom tissue comprises a mixture consisting of saw dust, wheat bran, rice bran, peptide for synthetic Japanese rice liquor and acid potassium phosphate.

10. The process for the growth and production of mushroom tissue of claim 1 wherein said ageing is carried out under the conditions of a temperature of about 25.degree.C, in a humidity range of 75-80 percent and an intensity of illumination of about 100 candela.

11. The process for the growth and production of mushroom of claim 1 wherein said cooling is carried out at 5.degree.C.

12. The process for the growth and production of mushroom tissue of claim 1, wherein said mushroom tissue growth and production conditions comprise a temperature range of 10.degree.-15.degree.C, a humidity range of 70-75 percent and an intensity of illumination of about 200 candela.
--------------------------------------------------------------------------------
Description
--------------------------------------------------------------------------------

BACKGROUND OF THE INVENTION

This invention relates to an improved process for the growth and production of mushroom tissue by the utilization of an artificial mushroom growth and production bed and more particularly, to an improved process for the growth and production of mushroom tissue by the utilization of an artificial mushroom growth and production bed which essentially comprises providing mushroom tissue, suspending the mushroom tissue in an aqueous nutrient fermentation solution, inoculation of the mushroom tissue suspending nutrient fermentation solution into an artificial growth and production bed, ageing the nutrient fermentation inoculated bed and growing the mushroom tissue within the bed under growth conditions.

Edible mushrooms have been highly esteemed as a high class foodstuff because of their refined flavor, but at present the mushrooms are drawing one's attention on account of their nutritive qualities with demand for the foodstuff increasing year by year. Up to date, mushroom tissues have been cultivated and produced by placing the mushroom tissues into suitably cut holes in growth and production tree trunks, stems, stocks or branches cut from a tree selected from the group comprising Quercus glandulifera, Quercus serrata and chestnut trees, covering the holes with covers and suitably arranging and leaving such growth and production tree cuts in a forest or wood until the mushroom tissues will be fully grown. However, such a conventional growth and production process for mushrooms suffer from the disadvantages that areas where the process can be carried out are not readily available, that the process requires a great deal of labor and time and a relatively large space and that the time for harvest for mushrooms is limited to only two seasons of a year. Of the above-mentioned disadvantages inherent in the conventional mushroom growth and production process, the disadvantages in connection with area and space are most serious because the prior art process for growing and producing mushrooms has to be carried out at a mountainous area where trees suitably used for the process are available and the process requires a spacious land area. In order to eliminate such disadvantages inherent in the prior art growth and production process of mushrooms, of late a variety of improved processes for the growth and production of mushrooms which employs the so-called artificial mushroom growth and production bed instead of the conventional tree medium have been proposed and actually employed.

All of such processes for the growth and production of mushrooms employing the artificial growth and production bed have the disadvantages that they require a great deal of labor and a substantially long period of time for the growth and production of mushrooms though they are free of the disadvantages relating to area and space limitations.

SUMMARY OF THE INVENTION

Therefore, one object of the present invention is to provide a novel and improved process for the growth and production of mushroom tissue which can be effectively eliminate the disadvantages inherent in the prior art process for the growth and production of mushroom tissue.

Another object of the present invention is to provide a novel and improved process for the growth and production of mushroom tissue employing an artificial growth and production bed which makes it easier to grow and produce mushroom tissue and shortens the time required for the growth and production of mushroom tissue.

A further object of the present invention is to provide a novel and improved process for the growth and production of mushroom tissue employing an artificial growth and production bed which can be readily applicable to a mass production operation of mushroom tissue.

PREFERRED EMBODIMENT OF THE INVENTION

To describe briefly, according to the present invention, there is provided a process for the growth and production of mushroom tissue which essentially comprises providing mushroom tissue, suspending the mushroom tissue into an aqueous mushroom nutrient fermentation solution, inoculating the mushroom tissue suspending nutrient fermentation solution into a prepared artificial growth and production bed contained in a container, ageing the solution inoculated bed under ageing conditions at a temperature of 25.degree.C, in a humidity of 75-80 percent and the intensity of illumination of about 100 candela until the bed ages sufficiently, cooling the contents of the container at 5.degree.C for 50 hours, turning the container upside-down and subjecting the contents of the container under growth and production conditions at a temperature range of 10.degree.-15.degree.C, in a humidity of 70-75 percent and the intensity of illumination of about 200 candela. The above-mentioned artificial mushroom tissue growth and production bed is prepared by mixing saw dust, wheat bran, rice bran, peptide for synthetic sake (synthetic Japanese rice liquor), acid potassium phosphate and chaff together and adjusting the mixture to pH 6.0-5.0 with a suitable acid such as citric acid.

The process of the present invention will be described in further detail by way of one preferred example in which the process is successfully carried out, but it is to be understood that the present invention is not limited to such an example because the process can be carried in other various ways.

First of all, a nutrient fermentation solution is prepared by mixing together potato extract (an extract solution obtained by extracting 200 g. of potato per 1000ml. of the nutrient fermentation solution in heated water), 0.1 percent by weight of peptide for synthetic Japanese sake or rice liquor "APT" (the trade name of an acid decomposition product of soybean protein by the Ajinomoto K.K.) based on the weight of the nutrient solution and 10 percent by weight of glucose based on the weight of the nutrient solution in a container, mushroom tissue is suspended in the mixture solution, the suspension is adjusted to pH 6.0 and the contents of the container are then shaked at 25.degree.C for 168 hours to thereby complete the prenutrient fermentation of the mushroom tissue. In a separate operation, a growth and production bed is prepared by mixing together 6 l. of saw dust, 1 l. of wheat bran, 1/10000 l. of peptide APT for synthetic Japanese sake or rice liquor, 1/10000 l. of acid potassium phosphate (KH.sub.2 PO.sub.4) and 8 l. of chaff, 6 l. of water is added to the mixture and the resultant mixture is adjusted to pH 6.0-5.0 with citric acid. The thus obtained growth and production bed is then placed into an open top heat-resistance container with about one-fifth of space leaving above the content (based on the capacity of the container), the open top of the container is covered with a cellophane film, for example and thereafter, the content of the container is sterlized at 120.degree.C for 30 minutes to thereby complete the preparation of the growth and production bed.

Thereafter, the thus obtained growth and production bed in the container is inoculated with the afore-mentioned mushroom tissue suspending nutrient fermentation solution in the amount of 1/100 l., the container is placed in a growth and production room adjusted to the mushroom tissue growth and production conditions at a temperature of 25.degree.C, in a humidity of 75-80 percent and the intensity of illumination of about 100 candela. The container is left in the room under the afore-mentioned conditions until the artificial growth and production bed ages sufficiently for growth and production of the mushroom tissue. The artificial growth and production initiates its ageing within 60-80 days after the inoculation of the nutrient fermentation solution and ages sufficiently for growth and production of the mushroom tissue within 90-100 days after the inoculation whereupon the bed sets in the mushroom growth stage. Thereafter, the contents in the container are cooled at 5.degree.C for 48 hours to transit from the nutrient growth stage to the reproduction growth stage whereupon the container is turned upside-down to position the now bottom top and the now top bottom whereby the space of one-fifth of the capacity of the reversed container is again provided above the contents. The contents of the container are then again replaced in the growth and production room under the growth and production conditions of the temperature range of 10.degree.-15.degree.C, the humidity range of 70-75 percent and the intensity of illumination of about 200 candela. The container is left in the room for about 2 weeks whereupon the growth of mushroom tissue sets in. In two weeks after the initiation of the reproduction growth stage, a first yield is made possible. After the second yield, mushroom tissue is reproduced and grown with replenishment of only water. The growth and production bed continues to reproduce mushroom tissue for about 2 months without any replenishment of nutriment.

Various mushroom growth and production beds were prepared by modifying the constituent components in various ways and experimentally employed for the growth and production of mushroom tissue. The results of the experiments will be given in the following Table 1. From the results, it will be noted that the rice bran has an important influence on the reproduction of mushroom tissue and the wheat bran, peptide for synthetic Japanese sake or liquor (APT) and chaff accelerate the growth of mushroom tissue and shorten the ageing time of the bed. The saw dust serves as the support for the other constituents of the bed and also as the nutriment for mushroom tissue. Although the effect of the acid potassium phosphate cannot be positively determined, it is considered that the component or constituent may maintain the pH of the nutrient solution within 5-6 and stabilize the growth conditions.

The various mushroom tissue growth and production beds of different compositions having the afore-mentioned nutrient fermentation solution inoculated therein were employed under the same growth and production conditions. Each of the growth and production containers had the capacity of about 1 kg. and 13 containers of the same-type composition were employed, respectively. Each of the growth and production bed compositions comprised the constituent ratio relationship of 6 by volume of saw dust, 1 by volume of wheat bran, 1 by volume of rice bran, 1/1000 by volume of APT, 8 by volume of chaff and/or 1/1000 by volume of KH.sub.2 PO.sub.4.



Since many embodiments may be made of this invention and since many changes may be made in the embodiments described, the foregoing is to be interpreted as illustrative only and the invention is defined by the appended claims.

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OfflineAnnoA
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: Anno]
    #2307653 - 02/06/04 04:29 AM (20 years, 1 month ago)

--------------------------------------------------------------------------------
United States Patent 4,212,947
Torev July 15, 1980
--------------------------------------------------------------------------------
Method for obtaining mycelium from the genus Polyporus

Abstract
The strains, PS 64-103; PS 24-44; and PB 33-48 are mutation products of the perfect fungi Polyporus squamosus and Polyporus brumalis and are characterized by the fact that in a liquid nutrient medium they tend to form hiffs, colonies and secondary spores, subdividing in geometric progression with rapid growth which varies in speed, depending on the seeding material ratio in a period of from 6 to 36 hours. The nutrient medium in which the mycelium is developed is quite simple in composition and comprises three components, i.e., 4-5% of molasses; 0.2% of NH.sub.4 NO.sub.3 ; 0.12% of KH.sub.2 PO.sub.4 and 0.04% of vegetable oil as foam suppressant. The strains assimilate from 50 to 60% of a given amount of saccharoses, accumulating to 1 to 1.2% of dry biological mass. The fungi mycelium can be adapted as a food product and is also used as an additive to various meat and other food products, i.e., sausage, minced meat, soft and smoked cheeses, vegetable canned food, soups, and bread and other bakery products. The waste water stimulates the growth of vegetables.

--------------------------------------------------------------------------------
Inventors: Torev; Atanas K. (Plovdiv, BG)
Assignee: DSO "HRANMASH" (Stara Zagora, BG)
Appl. No.: 878959
Filed: February 17, 1978

Foreign Application Priority Data

--------------------------------------------------------------------------------
May 22, 1975[BG] 30055

Current U.S. Class: 435/256.8; 426/656; 435/254.11; 435/804; 435/911
Intern'l Class: C12N 001/14; C12R 001/645
Field of Search: 195/115,81,1,32 426/656,48,60 47/1.1 435/254,804,911

--------------------------------------------------------------------------------
References Cited [Referenced By]
--------------------------------------------------------------------------------

U.S. Patent Documents
2850841 Sep., 1958 Szuecs 47/1.
3097145 Jul., 1963 Shimazono et al. 195/62.
3151038 Jun., 1964 Gray et al. 195/32.

Other References
Maslova, "Effect of the Different Sources of Nitrogen on the Growth of Wood--rotting Fungi in Culture", Chem. Abstracts, vol. 72, No. 13, p. 115, (1970), abs. No. 63869x.
Robbins et al., "Unidentified Growth Factors for Polyporus schneinitzii", Chem. Abstracts, vol. 72, No. 3, p. 221, (1970), abs. No. 11531n.

Primary Examiner: Wiseman; Thomas G.
Attorney, Agent or Firm: Berry; E. Janet, Rosen; Lawrence

--------------------------------------------------------------------------------
Parent Case Text
--------------------------------------------------------------------------------

This application is a continuation-in-part application of copending application Ser. No. 687,817, filed May 19, 1976, abandoned.
--------------------------------------------------------------------------------

Claims

--------------------------------------------------------------------------------

What is claimed is:

1. A method for obtaining mycelium from a strain of polyporus genus selected from the group consisting of cultivated fungi having essentially all the characteristics of Polyporus squamosus-PS 64-103 and PS 24-44 and Polyporus brumalis PB 33-48, which comprises submerged cultivation of said strain of fungi on a nutrient media containing carbohydrate, nitrogen and phosphorus sources and then harvesting said mycelium.

2. The method of claim 1, wherein said fungi are cultivated at a temperature of 22.degree. to 50.degree. C., a pH from 4.5 to 7.5 and air from 0.6 m.sup.3 to 1.2 m.sup.3 per each 1 m.sup.3 of nutrient media per minute.

3. The method of claim 1 wherein said fungi are cultivated at a volume of 2 m.sup.3 in the course of a twenty-four hour time cycle, at a volume of 20 m.sup.3 in an eighteen hour cycle, and in three growth cycles in progressively greater volumes of nutrient media contained in separate fermentation vessels, with the proviso that the first cycle involves a nutrient media volume of 2 m.sup.3 and a 24 hour time period, the second cycle involves a nutrient media volume of 20 m.sup.3 and a time period of 18 hours, and the third cycle involves a nutrient media volume of 100 m.sup.3 and a time period of 6 hours, and with the further proviso that 50% of the volume of the cultivation media is separated and removed at the end of each cycle and the fungal mycellia being harvested at intervals during the cycles is continuously repeated every six hours, the fungi mycelium being harvested at intervals during said time cycle.

4. A method for obtaining mycelium from a strain of polyporus genus selected from the group consisting of cultivated fungi having essentially all the characteristics of Polyporus squamosus-PS 64-103 and PS 24-44 and Polyporus brumalis PB 33-48, which comprises submerged cultivation of said strain of fungi on a nutrient media containing carbohydrate, nitrogen and phosphorus sources at a temperature of 22.degree. to 50.degree. C., a pH from 4.5 to 7.5, and 1 m.sup.3 of nutrient media per minute, whereby there is a rapid increase in the protein concentration of the fungi mycelium, and afterward isolating said mycelium.

5. The method of claim 4 wherein a semi-continuous method of cultivation is used in which one-tenth of the volume of the initial fermentation is retained as an inoculate and the cultivation is completed in 14 hours.

6. The method of claim 4 wherein said mutant strains are cultivated in a nutrient media containing 3 to 5% molasses, 1.10 to 0.20% ammonium nitrate, 0.08 to 0.15% potassium phosphate (KH.sub.2 PO.sub.4), and 0.02 to 0.06% of a foam supressor.
--------------------------------------------------------------------------------
Description
--------------------------------------------------------------------------------

The present invention relates to a method for obtaining mycelium from fungi (genus Polyporus) by culturing in depth to give products for foodstuffs having high protein content.

In 1938, for the first time, Lambert (USA) developed a project for cultivating mycelium from fungi for use as foodstuff.

Later Humfeld, Block, Jennison and others carried out tests for cultivating mycelium from Agaricus cisporus, Morchela esculenta and other varieties of fungi.

The mycelium from these types of fungi grows for 3 days to a week. The technology suggested by the above authors employs an interrupted method. This technology and the long period of time required for the mycelium to grow have a number of disadvantages:

(1) The required long period of time under sterile production may cause infection of the media by outside microflora and result in obtaining a product of bad quality.

(2) The prolonged growing period prevents advancing to a chain production method.

(3) The product thus obtained is very expensive.

It is a purpose of this invention to teach a method for obtaining mycelium of high food value and rich in protein by cultivating in depth using a chain method under industrial conditions.

The method according to the invention consists in using strains of non-toxic edible high fungis of the genus Polyporus or its variants and mutants for obtaining fungis mycelium of high protein content. The media wherein the fungi mycelium is grown contains as a base, the carbohydrates, both mono and disaccharides. Dairy-production wastes and hydrolysates can also be used. The already used Polyporus strain can be also used. The Polyporus strain used can be Polyporus squamosus or Polyporus brumalis. Polyporus squamosus Hudo strain PS 64 is preferred. The preferred strains and variants of polyporous are deposited in the State Institute of Treatment Means Control, Bulgaria, Sofia, B1. VI. Zaimov 26, under the numerals PS-64-103; Ps-24-44; and PB-33-48. The following variants can also be used: PS 24-44; PB 33-48. The strain 64-103 is characterized by its very quick growth.

If the quantity of the innoculum is in a ratio 1:1 to the media, the cycle of growth is completed in 7 hours. This strain has the following morphological characteristics: hyphae several millimeters to several centimeters long and 8-10.mu. thick; on solid media it forms fluffy columns, in liquid media it forms columns with budding hyphae and secondary (vegetative) spores, it propagates only vegetatively through germination and cell divisions and by elongating the hyphae and the colonies. Strain PS 24-44 differs from PS 64-103 only in the thickness of the hyphae 6-8.mu. and in its more slightly expressed germination. Strain PB 33-48 differs from PS 64-103 only by its longer hyphae, its relatively poor germination, and its inability to produce secondary spores. Strain PS 64-103 grows on nutrient media having mono- and disaccharides. It grows particularly well on nutrient media having glucose and molasses. From all nitrogen salts it absorbs carbamide, ammonium nitrate and ammonium sulfate. From all phosphorus compounds it absorbs potassium and sodium mono- and dibasic phosphate. The growth conditions are temperature, 26.degree. C..+-.2.degree. C.; pH, 6.5.+-.0.5; air, 0.8.+-.0.2 m.sup.3 to each m.sup.3 nutrient media per minute. Strain PS 24-44 has the same physiological characteristics as strain PS 64-103. Strain PB 33-48 has the same physiological characteristics as strain PS 64-103. Strain PS 64-103 contains 55-58% raw protein, 42-46% protein, 19-21% carbohydrates, 6-8% cellulose-like substances, and 3-4% fats. If nitrogen is lacking, fats up to 50% can be produced in the nutrient media at the expense of the protein. Total ash is 6-7% and there are present vitamins from the B-complex. Strain 24-44 is characterized by its lower content of raw protein (50-52%) and higher content of cellulose-like substance. Strain PB 33-48 also has a low content of raw protein (48-50%) and a higher content of carbohydrates of about 25%.

Nutrient media for growing the fungus mycelium:

______________________________________
Solid Nutrient Media
Ingredient Amount
______________________________________
Glucose 20 g
NH.sub.4 NO.sub.3 3 g
KH.sub.2 PO.sub.4 1 g
MgSO.sub.4 0.5 g
ZnSO.sub.4 0.01 g
CoCl.sub.2 0.01 g
Agar-agar 20 g
water 1 liter
______________________________________
______________________________________
Liquid Nutrient Media for Innoculum
Ingredient Amount
______________________________________
Glucose 20 g
NH.sub.4 NO.sub.3 3 g
KH.sub.2 PO.sub.4 1 g
MgSO.sub.4 0.5 g
ZnSO.sub.4 0.01 g
CoCl.sub.2 0.01 g
water 1 liter
______________________________________
______________________________________
Liquid Nutrient Media for Industrial Production
______________________________________
Beet or red molasses 5%
NH.sub.4 NO.sub.3 0.15% or carbamide 0.1%
KH.sub.2 PO.sub.4 or NaH.sub.2 PO.sub.4 0.1%
Sunflower-seed oil as foam-
suppressor 0.03%
______________________________________

The fungus mycelium is stored on solid substrate as defined for the nutrient media described above. The agar media is poured into Ackerman's test tubes, sterilized at 124.degree. C. for 45 min. and then placed in a tilted position. On agar media the fungus mycelium produces white aerial hyphae of loose structure. It is resown once every six months. It is stored in a refrigerator at +2.degree. C.

The seeding material of liquid substrate is obtained on liquid nutrient media constituted as described above. The media is transferred into Erlenmeyer flasks of 200 ml. each, and the flasks are closed with cotton-lint corks. They are sterilized at 124.degree. C. for 45 min. in an autoclave. They are cooled down to 26.degree. C. and sown in a sterile box, the mycelium having only aerial hyphae or hyphae with part of agar. The test tubes are put in a shaker of 140 revolution per minute in a camera at 26.degree. C. temperature. The period of development is from 72 to 96 hours. The second and third screening takes place from liquid onto liquid media of the same nutrient media and under the same conditions. When sown from liquid onto liquid media for the first time the material takes 60 hours to grow. In the subsequent sowing with sowing material in the ratio of 1:10 the period of growing is about 36 hours.

The liquid nutrient media of the mycelium is stored in a refrigerator at +2.degree. C. for up to 3 months and at 26.degree. C. for up to one week.

Sowing Mycelium in an Innoculator

The innoculator can be 1 or 2 m.sup.3. It should be washed and sterilized while empty. Then it is loaded with the molasses nutrient media described above up to 80% of its overall volume. The nutrient media is prepared in a media-boiling apparatus, sterilized in sterilization columns and cooled in cooling columns. The innoculator, loaded with nutrient media, is sown sterile with 5 sowing test tubes. The technological regime in the apparatus is t, 26.degree. C..+-.1.degree. C.; pH, 6.5.+-.0.5; air 0.8.+-.0.2 m.sup.3 in 1 m.sup.3 nutrient media for 1 minute without agitation. The pressure in the apparatus is 0.5 atm. .+-.0.2 atm. The period of growth of the mycelium is 48 hours.

Intermediate Apparatus Operation

The intermediate apparatus is 15-20 m.sup.3. It is washed and sterilized while empty. Then it is loaded with molasses nutrient media, which has been previously sterilized and cooled. The usable volume of the apparatus is 80% (nutrient media with innoculum). It is sown with mycelium from the innoculator through sterile lines. The technological parameters are the same as those for the innoculator stage. It requires the mycelium 24 hours to grow.

Growth of the Mycelium in Working Apparatus

The working apparatus can have a volume of 50-100 m.sup.3 or more. The apparatus is washed and sterilized while empty whereupon it is loaded with prepared nutrient media (molasses). The quantity of the nutrient media is calculated so as to occupy 80% of the overall volume of the apparatus together with the sowing material from the intermediate apparatus. The sowing is carried out by using the intermediate apparatus, the mycelium being transplanted on previously sterilized lines. The technological parameters maintained in the apparatus are: temperature, 26.degree. C..+-.1.degree. C.; pH 6.5.+-.0.5; air, 0.8 m.sup.3 .+-.0.2 m.sup.3 in 1 m.sup.3 nutrient media for 1 minute without agitation. The pressure is 0.5.+-.0.2 atm. The period of growth of the mycelium depends on the quantity of the innoculum. When its ratio to the nutrient media is 1:10 the growth continues for 24 hours. After full growth of the mycelium is reached, half of the cultivation media is passed to filtration and the other half remains as sowing material. The working apparatus is filled again with prepared nutrient media and operating thus, the quantity of the sowing material to the nutrient media is in a ratio of 1:1. Full growth of the mycelium in this case is reached in 6 hours.+-.1 hour. Then again half of the cultivation media is pumped out for filtration, fresh nutrient media is added and this process is repeated for 3 days.+-.2 days, whereupon everything starts once more from the innoculator and the intermediate apparatus stages. The quantity of fungus mycelium obtained from 1 liter nutrient media is 35 g.+-.5 g; 9 g.+-.1 g dry (28% dry substance).

Separation of the Fungus Mycelium from the Liquid Phase

Separation is carried out by filtration in filter presses, vacuum drum filters or by centrifugation. The mycelium thus separated is washed with water in the same quantity as the cultivation media filtrate.

The mycelium is divided into solid and liquid phases. The liquid phase (the mother liquor) occupies up to 70% of the cultivation area. The mother liquor is a product having an aqueous base, but also containing substantial quantities of substances from the mycelium metabolism and most important, physiologically active substances having stimulating effects upon plants.

Characteristics of the Final Product

The fungus mycelium can be given two shapes: pressed-fresh and dry.

The pressed mycelium is the fresh mycelium mass containing 28%.+-.1% dry matter. Its color is gray-white to light-cream. It has a specific taste similar to the taste of the sour dough. It is obtained in rectangular shape or in the shape of a parallelepipod weighing 5 kg. It is packed in polyethylene wrap and stored frozen at temperatures of -18.degree. C. to -23.degree. C. The product keeps for 6 months. Dry mycelium is obtained by drying in spray driers, air-heated fan driers or boiling-layer driers at 65.degree. C..+-.5.degree. C. The dry mycelium is a flour of coarse or fine particles according to the method of drying. Its color is white to light-cream. It has a specific taste, similar to that of fungus flour. It is poured into double-bottomed paper bags of 25 kg each and stored in a dry airy area at indoor temperature. The product keeps for 1 year.


______________________________________
Biochemical Characteristics of Strain
PS 64-103 Fungus Mycelium
Ingredient Content
______________________________________
Raw protein 55.8%
Pure protein 45%
Carbohydrates
(without cellulose) 21%
Fats 3.6%
Cellulose-like
substances 6.4%
Mineral composition 7.0%
Thiamin mkg/g 18.9
Riboflavin mkg/g 73.0
Niacin mkg/g 240
Holin mkg/g 6000
Protein assimil-
ation 83%
Caloricity of 100 g
product 340.16 calories
______________________________________



The chemical composition of the fungi mycelium is characterized by its high protein content (50-60%) and a complete set of unique amino acids featuring a high lysine content (8-10%). In addition, there are present mycelium "B"-complex vitamins and a number of other physiologically active substances. The protein digestibility is 83%.

With regard to the essential amino acids and digestibility, (i.e. overall biological value) the fungi mycelium almost equals beef meat in value. Thus, the total content of essential amino acids in beef is 39.9% as compared to 39.0% in the mycelium; digestibility of beef is 85% as compared to 83% for the fungi mycelium.

The fungi mycelium obtained from the aforesaid strains was investigated over a period of three years with respect to its effect if any on cancerogen, theratogen and pathomorphological alterations. All results were found to be negative. The tests carried out on the physical and general health effects of the fungi mycelium as a food product have shown that it raises the stamina of organisms, while simultaneously improving the blood condition.

The amino-acid composition of strain PS 64-103 fungus mycelium as compared to the amino-acid composition of beef, casein, soy-bean flour and standard protein according to FAO is shown in Table 1 below. The data are in % relative to the total content of amino-acids.


TABLE 1
______________________________________
Standard
Fungus Soy Protein
Mycelium Bean According
P-64 Beef Casein Flour to FAO
______________________________________
Lysine 8.5 8.4 8.4 6.4 5.5
Threonine 5.3 4.0 5.0 3.8 4.0
Valine 6.0 5.7 7.4 5.0 5.0
Isoleucine 5.1 5.1 6.2 6.4 4.0
Leucine 7.2 8.4 9.4 6.6 7.0
Tryptophan 1.4 1.1 1.2 1.2 1.0
Methionine 1.9 2.3 2.0 0.7 3.5
Cystine 0.9 1.4 0.3 --
Phenylalenine 3.9 4.0 5.1 4.8
Tyrosine 3.4 4.0 6.4 3.1 6.0
Total 43.6 44.4 51.4 38.0 36.0
Histidine 2.9 2.9 3.2 2.3
Arginine 5.8 6.6 4.2 6.0
Aspargic acid 10.3 8.8 3.7
Serine 4.8 3.8 6.4
Glutaminic acid 16.2 14.2 22.9
Proline 4.0 5.4 10.9
Glycine 4.8 7.1 2.0
Alanine 7.6 6.4 3.3
______________________________________



A BRIEF DESCRIPTION OF THE FIGURE

The technological process flow diagram for obtaining fungus mycelium is shown on FIGURE wherein the numbers have the following meanings: 1 is the media-boiling apparatus; 2--sterilization columns; 3--cooling columns; 4--container for sterilized nutrient media; 5--total air filter; 6--individual air filter; 7--sowing apparatus; 8--intermediate apparatus; 9--working apparatus; 10--container for cultivation media (developed mycelium); 11--filter presses; 12--screw conveyers or conveyor belts; 13--container for cleaning the mycelium; 14--press machine; 15--molding and packing machine; 16--conveyor belt; 17--cooling chamber.

The Biological Value and Toxicity of the Fungus Mycelium from Fungus (genus Polyporus)

For comparison, a group of rats was used. They were fed with protein from casein and with protein from the fungus mycelium. The comparative results are shown in Table 2 below.


TABLE 2
______________________________________
Protein substituted
Protein substituted
with protein from
with protein from
fungus
Biological Indices
casein mycelium
______________________________________
Increase in %
212.6 175.2
Digestibility in %
93.52 82.79
______________________________________



A blood test has been carried out, the liver has been tested, and also the stomach and the small intestines, and the weight coefficient determined among the inner organs. All the indices are followed up to 7 months. Toxicity or deviation from the acceptable norms have not been observed.

Application of the Fungus Mycelium in the Food Industry

It may be used as an addition of 10 to 20% in meat products (sausages, minced meat for meat-balls, kebapcheta, tinned meat) as a source for obtaining synthetic meat, as an addition to processed and smoked cheeses up to 20%, as an enricher for various tinned vegetables with fungus mycelium up to 25%, in preparing various dry soups (meat and vegetable) in up to 20%; and when preparing bread and other dough products in up to 10%. The following Examples illustrate the application of fungus mycelium in the food industry.

EXAMPLE 1


______________________________________
Preparation of Perishable Sausages with Fungus Mycelium
Ingredient Amount
______________________________________
Fat Pork 20%
Semi-fat Pork 65%
Pressed fungi
mycelium 15%
Salt 2.2%
Nitrite 0.01%
Sugar 0.1%
Garlic 0.1%
Pepper 0.2%
______________________________________



They are made according to the technology for perishable sausages.

EXAMPLE 2


______________________________________
Preparation of Non-Perishable Sausages with Fungus Mycelium
Ingredient Amount
______________________________________
Semi-fat pork 40%
Beef 43%
Pressed fungi
mycelium 15%
Salt 2.2%
Pepper 0.3%
______________________________________



They are prepared in the technology for non-perishable sausages.

EXAMPLE 3


______________________________________
Processed Cheese with Fungus Mycelium
Ingredient Amount
______________________________________
Cheese 30%
"Cheddar" cheese 30%
Curds 18%
Pressed fungi
mycelium 15%
Butter 4%
Emulsified salts 3%
Pepper 0.3%
______________________________________



The products were prepared according to the technology for processed cheeses.

EXAMPLE 4


______________________________________
Vegetable Paste with Fungus Mycelium
Ingredient Amount
______________________________________
Pressed fungus
mycelium 20%
Peas 12%
Blanched celery roots
8%
Blanched parsnip 8%
Blanched carrots 10%
Marinated cucumbers
10%
Pepper puree 8%
Onion 2%
Overheated cooking
oil 8%
Vinegar 8%
Salt 1%
Pepper 0.06%
Water 9%
______________________________________



All components are minced, mixed and homogenized and thereafter sterilized.

EXAMPLE 5


______________________________________
Fungus Soup
Ingredient Amount
______________________________________
Fungus flour 10%
Dry fungus
mycelium (flour) 30%
Farina 20%
Flour 16%
Dried carrots 2.8%
Dried parsley 1.2%
Dried Celery 0.8%
Powdered milk 9%
Pepper 0.1%
Monosodium
glutamate 0.2%
Salt 10%
______________________________________



All components were well homogenized, and the mixture packed in suitable bags of 50 g each.

EXAMPLE 6

The mother liquors are diluted 20-30 fold and then plants are watered with them. The watering is best done in a shower, so that the stimulating substances can be absorbed not only by the root system but also by the leaf system. The vegetables watered are mainly tomatoes, peppers, and cucumbers, grown in hot-houses, where the regime of watering can be controlled. The waterings numbered about 15 and start at the budding stage and go on into the mass picking stage.

The effect of stimulating these vegetables with mother waters raises the index of early ripening up to 5% in the first 10 pickings and raises the total output by about 15%.

* * * * *
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: Anno]
    #2307655 - 02/06/04 04:31 AM (20 years, 1 month ago)

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United States Patent 6,490,824
Maekawa , et al. December 10, 2002
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Method for culturing a basidiomycetous fungus in a liquid culture medium

Abstract
Disclosed is an efficient method for culturing an edible basidiomycetous fungus such as Mushroom Agaricus Blazei Murill in a liquid culture medium to give fungus aggregates of several centimeter size. Characteristically, the liquid culture medium is formulated with sucrose as a carbon source in the form of crude cane sugar in combination with a water-insoluble growth-supporting material in the form of a fine powder to serve as the core of the fungus aggregates as selected from crushed sugarcane, sugarcane bagasse, pine trees and wheat bran. Further characteristically, the culturing procedure is carried out under an oxygen-enriched condition by blowing oxygen-enriched air of at least 30% by volume oxygen into the culture medium under pressurization at 0.12 to 0.5 MPa (absolute) in a specified blowing rate.

--------------------------------------------------------------------------------
Inventors: Maekawa; Takaaki (Ibaraki, JP); Intabon; Keo (Tsukuba, JP)
Assignee: Tsukuba Biosystems, Ltd. (Ibaraki, JP)
Appl. No.: 958664
Filed: October 12, 2001
PCT Filed: April 20, 2000
PCT NO: PCT/JP00/02595
PCT PUB.NO.: WO00/65029
PCT PUB. Date: November 2, 2000

Foreign Application Priority Data

--------------------------------------------------------------------------------
Apr 23, 1999[JP] 11-116188

Current U.S. Class: 47/1.1; 47/1.4
Intern'l Class: A01G 001/04
Field of Search: 47/1.1,1.4

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References Cited [Referenced By]
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U.S. Patent Documents
2693665 Nov., 1954 Humfeld.
2761246 Sep., 1956 Szuecs.
2850841 Sep., 1958 Szuecs.
3828470 Aug., 1974 Stoller 47/1.
4071973 Feb., 1978 Izuka et al. 47/1.
4127965 Dec., 1978 Mee 47/1.
4420319 Dec., 1983 Holtz 71/5.
4873195 Oct., 1989 Kubo et al. 435/254.
5186731 Feb., 1993 Parker 71/5.
5888803 Mar., 1999 Starkey 435/254.
5934012 Aug., 1999 Holtz et al. 47/1.
Foreign Patent Documents
2108151 May., 1983 GB.
60-54324 Mar., 1985 JP.

Primary Examiner: Jordan; Charles T.
Assistant Examiner: Hayes; Bret
Attorney, Agent or Firm: Wenderoth, Lind & Ponack, L.L.P.

--------------------------------------------------------------------------------

Claims

--------------------------------------------------------------------------------

What is claimed is:

1. A method for culturing a basidiomycetous fungus in an aqueous liquid culture medium which comprises the steps of:

(a) inoculating a liquid culture medium containing inorganic nutrient salts for nitrogen, phosphate and potassium with a body of the fungus;

(b) admixing the liquid culture medium with crude cane sugar in an amount in the range from 50 g to 70 g calculated as sucrose per liter of the liquid culture medium;

(c) admixing the liquid culture medium with a water-insoluble growth-supporting material selected from the group consisting of crushed sugarcane, sugarcane bagasse, pine tree-tissue and wheat bran in an amount in the range from 0.2 g to 15 g as dry per liter of the liquid culture medium;

(d) keeping the liquid culture medium under agitation at a temperature in the range from 20 to 30 .degree. C.; and

(e) blowing, into the liquid culture medium, oxygen-enriched air containing at least 30% by volume of oxygen under a pressure in the range from 0.12 to 0.5 MPa (absolute) at a rate of at least 0.01 liter/minute per liter of the liquid culture medium.

2. The method for culturing a basidiomycetous fungus in an aqueous liquid culture medium according to claim 1 in which the water-insoluble growth-supporting material added to the liquid culture medium in step (c) is in the form of a powder having a particle size to pass a 100 mesh screen.

3. The method for culturing a basidiomycetous fungus in an aqueous liquid culture medium according to claim 2 in which the water-insoluble growth-supporting material added to the liquid culture medium in step (c) is in the form of a powder having a particle size to pass a 200 mesh screen.

4. The method for culturing a basidiomycetous fungus in an aqueous liquid culture medium according to claim 1 in which the oxygen-enriched air blown into the liquid culture medium in step (e) contains from 30% to 90% by volume of oxygen.

5. The method for culturing a basidiomycetous fungus in an aqueous liquid culture medium according to claim 1 in which the blowing rate of the oxygen-enriched air into the liquid culture medium in step (e) is in the range from 0.01 to 1.0 liter/minute per liter of the liquid culture medium.

6. The method for culturing a basidiomycetous fungus in an aqueous liquid culture medium according to claim 1 which further comprises the step of:

(f) controlling the concentration of carbon dioxide in the gaseous phase over the liquid culture medium.
--------------------------------------------------------------------------------

Description

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TECHNICAL FIELD

The present invention relates to a novel and efficient method for culturing a basidiomycetous fungus in an aqueous liquid culture medium or, more particularly, to a method for culturing an edible basidiomycetous fungus such as Mushroom Agaricus Blazei Murill, Cortinellus shuitake, Lyophyllum aggregatum, Pleurotus ostreatus and the like in an aqueous liquid culture medium to obtain aggregates of the fungus body having a several centimeter size as well as to a bioreactor for practicing the culturing method.

BACKGROUND ART

Methods for culturing a basidiomycetous fungus in a liquid culture medium are known as disclosed, for example, in U.S. Pat. Nos. 2,693,665, 2,761,246 and 2,850,841 and elsewhere. A typical liquid culture medium used in the prior art contains 50 g of sucrose, 10 g of ammonium nitrate, 5 g of sodium phosphate, 2.5 g of magnesium sulfate and 0.2 g of iron (II) sulfate each per liter of the liquid culture medium. The liquid culture medium inoculated with the fungus body such as mycelia is gently agitated with a stirrer rotating at a relatively low revolution in air for several days to effect growth of the mycelia into aggregates of globular or polyhedral granules having a diameter of 3 to 40 mm. It is accepted that such an aggregate of mycelia is formed by virtue of the viscous polysaccharide material formed on the surface of the mycelium to act like an adhesive. The productivity of these prior art methods, however, is very low due to the low growth rate of the fungus and a difficulty encountered in the recovery of the fungus body as grown from the culture medium.

The present invention accordingly has an object to provide a novel and efficient industrial method for culturing a basidiomycetous fungus such as Mushroom Agaricus Blazei Murill, referred to as Agaricus fungus hereinafter, and the like in a liquid culture medium. A secondary object of the invention is to provide a novel bioreactor suitable for practicing the above mentioned culturing method in a liquid culture medium for the fungus.

DISCLOSURE OF INVENTION

Thus, the method of the present invention for culturing a basidiomycetous fungus in an aqueous liquid culture medium comprises the steps of:

(a) inoculating a liquid culture medium containing inorganic nutrient salts for nitrogen, phosphate and potassium with a body of the fungus such as mycelia;

(b) admixing the liquid culture medium with crude cane sugar in an amount in the range from 50 g to 70 g calculated as sucrose per liter of the liquid culture medium;

(c) admixing the liquid culture medium with a water-insoluble growth-supporting material selected from the group consisting of crushed sugarcane, sugarcane bagasse and wheat bran in an amount in the range from 0.2 g to 15 g as dry per liter of the liquid culture medium;

(d) keeping the liquid culture medium under agitation at a temperature in the range from 20 to 30 .degree. C.; and

(e) blowing, into the liquid culture medium, oxygen-enriched air containing at least 30% by volume or, preferably, from 60 to 90% by volume of oxygen under a pressure in the range from 0.12 to 0.5 MPa (absolute) at a rate of at least 0.01 liter/minute per liter of the liquid culture medium.

The bioreactor for practicing the above described inventive culturing method comprises:

(A) a pressurizable vessel for containing a liquid culture medium;

(B) a gas inlet tube capable of blowing oxygen-enriched air into the liquid culture medium under a superatmospheric pressure in the range from 0.12 to 0.5 MPa (absolute);

(C) gas outlet tube having a means for regulating the pressure inside of the pressurizable vessel at a pressure in the range from 0.12 to 0.5 MPa (absolute); and

(D) a means for agitating the liquid culture medium contained in the pressurizable vessel.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic cross sectional view of a typical bioreactor system for practicing the method of the invention.

FIG. 2 is a schematic cross sectional view of another bioreactor system for practicing the method of the invention.

FIG. 3 is a schematic cross sectional view of a further different bioreactor system for practicing the method of the invention.

FIG. 4 is a schematic cross sectional view of a bioreactor system for practicing the method of the invention as a continuous process.

FIG. 5 shows growth curves of Agaricus fungus in various liquid culture media.

FIG. 6 shows the mycelium concentration of Agaricus fungus in the mist carried off from a bioreactor equipped (solid-line curve) or not equipped (broken-line curve) with a gas cyclone.

BEST MODE FOR CARRYING OUT THE INVENTION

As is described above, the inventive method for culturing a basidiomycetous fungus or, typically, the Agaricus fungus is characterized in both of the constituents in the liquid culture medium and the running conditions of the culturing process in such a liquid culture medium. When the culturing process is conducted under most desirable conditions, the aggregates of the fungus body granules show rapid growth to give an aggregate having a diameter of 10 to 20 mm if the nutrients are sufficiently provided in the culture medium although the core portion of the aggregates sometimes exhibits a dark color presumably as a consequence of local deficiency in the dissolved oxygen in the liquid culture medium. This problem can be solved by increasing the concentration of the dissolved oxygen in the culture medium up to 15 to 30 mg/liter so that the aggregates of the fungus body granules can be grown to have an approximately 40 mm diameter without blackening in the core portion by a culturing run continued for 3 to 4 days.

The kinds and concentration of inorganic nutrient salts are not particularly limitative but can be conventional including 10 to 15 g/liter of ammonium nitrate as a nitrogen source, 5 to 7 g/liter of sodium phosphate as a phosphate source, 3 to 7 g/liter of potassium sulfate or dipotassium hydrogenphosphate as a potassium source, 2.5 to 3.5 g/liter of magnesium sulfate and 0.2 to 0.3 g/liter of iron(II) sulfate.

Characteristically, the liquid culture medium used in the inventive culturing method is admixed with sucrose as a carbon source which is not in the form of a purified sugar but in the form of a crude cane sugar. The initial amount of the crude cane sugar in the liquid culture medium is in the range from 40 to 200 g/liter calculated as pure sucrose. It is essential that the sucrose is added to the medium as a crude cane sugar and not as a crude beat sugar. Although the reason for this limitation to crude cane sugar is not well understood, it is presumable that, while crude sugars, which may be from sugarcanes or from beats, always contain a substantial amount of impurities besides sucrose, the kinds of the crude sugar impurities are different between crude cane sugar and crude beat sugar and the impurities contained in the crude cane sugar including certain metallic elements such as manganese, zinc and cobalt as well as other unidentified trace elements are particularly effective for promoting growth of the Agaricus fungus.

Further characteristically, the liquid culture medium used in the inventive method is admixed with a water-insoluble growth-supporting material selected from the group consisting of crushed sugarcane, sugarcane bagasse and wheat bran as well as crushed pine tree tissues including woody parts, leaves and fruits in an amount in the range from 0.2 to 15 g/liter calculated as dried material. The growth-supporting material added to the culture medium should be in the form of fine particles of fineness passing a screen of 100 mesh or, preferably, 200 mesh fineness in the Tyler standard. The particles of these growth-supporting materials serve as a core on which the fungus body aggregates grow with improved stability.

The inventors of course have conducted extensive screening tests for uncovering other growth-supporting materials derived from various plants including stalks and leaves of Indian corns, rice bran, barley grains and leaves and other parts of mulberry trees each in the form of fine particles to reach a conclusion that the growth-promoting and --stabilizing effect is specific to crushed sugarcane, sugarcane bagasse, wheat bran and pine tree tissues.

In conducting the inventive method for culturing a basidiomycetous fungus such as Agaricus fungus in the above described liquid culture medium, the first step is to inoculate the liquid culture medium containing the above described various ingredients with the fungus body which is preferably the mycelium of the fungus.

The most unexpected discovery leading to the present invention is that growth of the Agaricus fungus is greatly promoted with stability by conducting the culturing process under an oxygen-enriched condition which can be accomplished by blowing, into the above described liquid culture medium, oxygen-enriched air under pressurization so as to obtain a dissolved oxygen concentration of at least 7 mg O.sub.2 per liter of the liquid culture medium. In order to accomplish this concentration of the dissolved oxygen, the oxygen-enriched air blown into the liquid medium contains at least 30% by volume of oxygen or, preferably, from 30 to 90% by volume of oxygen and the oxygen-enriched air is pressurized to have a pressure in the range from 0.12 to 0.5 MPa (absolute). The blowing rate of the oxygen-enriched air is of course of some importance and should be at least 0.01 liter/minute per liter of the liquid culture medium. The blowing rate has no particular upper limit but the blowing rate is preferably in the-range from 0.01 to 1.0 liter/minute per liter of the liquid medium in consideration of the increase in the mist dissipation carried off by the exhaust out of the gas outlet tube of the bioreactor. Needless to say, the oxygen-enriched air blown into the liquid culture medium must be sterilized, for example, by passing a suitable filter in order to minimize the risk of contamination of the liquid culture medium with various microorganisms which might be inhibitive against stable growth of the desired basidiomycetous fungus.

The temperature at which the culturing process of the Agaricus fungus is carried out is of course very important in order to obtain a best result of culturing. The temperature should be in the range from 20 to 30 .degree. C. for the basidiomycetous fungi in general and must be selected within this range depending on the particular species of the fungus under culturing for the optimum temperature. When the temperature is too low or too high, the growth rate of the fungus is disadvantageously decreased. When the requirements for the above described various factors are satisfied, the culturing process of the Agaricus fungus in a batch process is completed within 2 to 3 days.

It should be noted here that, as a consequence of the rapid growth of the fungus, a large volume of carbon dioxide gas is produced by the assimilation of the carbon source nutrient by the fungus body. Unless the carbon dioxide in the gaseous phase is adequately removed, growth of the fungus body is inhibited due to a decrease in the pH value of the liquid culture medium sometimes down to 3.5 or even lower as a result of the acidic external secretion from the fungus mycelia along with a decrease in the dissolved oxygen concentration therein by the decrease in the oxygen partial pressure in the gaseous phase above the liquid medium.

In the following, the culturing process of the invention and a bioreactor used therefor are illustrated by making reference to the accompanying drawing.

FIG. 1 schematically illustrates a bioreactor system including a culturing vessel consisting of a pressurizable cylindrical body 1 having a gas inlet tube 5 with a gas diffuser 5A at the lower part of the vessel 1, a gas outlet tube 6 connected to the upper part of the vessel 1 and a stirrer 7 with paddle blades to gently agitate the liquid culture medium 2 as driven by the motor M at the top. Oxygen-enriched air is blown into the liquid culture medium 2 through the gas inlet tube 5 as pressurized by a compressor 3 and sterilized by passing a sterilizing filter 4 under a specified pressure regulated by means of the pressure controller 8. The pressure of the gaseous phase over the liquid culture medium 2 in the vessel 1 is monitored and controlled by means of a constant-pressure valve under control of the exhaust pressure regulator 6A connected to the gas outlet tube 6. It is optional that a part of the exhaust air is returned to the air-feed pipeline to be mixed with the fresh air feed.

FIG. 2 is a schematic illustration of another bioreactor system to practice the inventive method, which is equipped with a carbon dioxide concentration controller 9 to discharge excess of carbon dioxide by means of a blower 12 and an oxygen modifier 10 combined with an oxygen concentration controller 11. The oxygen-enriched air inside of the vessel 1 over the liquid culture medium 2 discharged through the gas outlet tube 6 is circulated to the gas inlet tube 5 through the blower 12 along with removal of a part of carbon dioxide gas in the controller 9.

FIG. 3 is a schematic illustration of a further different bioreactor system for practicing the inventive method. When a basidiomycetous fungus is cultured in a culturing system by inoculating the liquid culture medium 2 with mycelia of the fungus, it is sometimes the case that the mist of the liquid culture medium 2 produced by blowing of oxygen-enriched air and containing the mycelia of the fungus under culturing is carried off by the exhaust air to cause deposition of the mycelia onto various parts of the system including the sterilizing filter 4, air discharge valves, inner wall of the pipelines and so on resulting in clogging of these parts so that the culturing run must be interrupted. In order to avoid such troubles, the bioreactor system is provided with a wet cyclone 13 and the mycelia separated in the cyclone 13 and deposited within the cyclone 13 are washed down in the conical part of the cyclone 13 with the liquid sent thereto. The liquid washing containing the mycelia thus washed down is discharged out of the bottom of the cyclone 13 and received in the receiver tank 14 connected to the bottom of the cyclone 13, from which the liquid is returned,to the reactor vessel 1 by means of the pump P.sub.1 under a pressure controlled by means of the pressure controller 16. When adequately designed and operated, the culturing process of a basidiomycetous fungus can be continued without interruption for 4 days or even longer.

FIG. 4 is a schematic illustration of a bioreactor system suitable for practicing the method of the invention as a continuous process, in which the carbon dioxide gas produced by culturing of the fungus is removed by operating the carbon dioxide absorber 18 and the air having a decreased concentration of carbon dioxide is returned to the culture system through a carbon dioxide concentration controller 9. The air inlet tube 5 is surrounded by a jacket tube 19 which enables upward flowing of the liquid medium within the jacket tube so as to enhance the growth rate of the fungus aggregates even in the absence of a mechanical agitating means such as a stirrer. The liquid culture medium 2, which is prepared in the preparation tank 22 with supplemental addition of the nutrient ingredients through the pipe line 23, is circulated through microfilters or ultrafiltration membranes 20, 21 to remove the suspended fungus mycelia.

In the following, the method of the present invention is described in more detail by way of Examples in which the Agaricus fungus was taken as a typical species of the basidiomycetous fungi.

EXAMPLE 1

A pressurizable cylindrical stainless steel bioreactor vessel of 1 liter capacity was charged with 1 liter of a liquid culture medium containing 60 g of pure sucrose, 10 g of ammonium nitrate, 5 g of sodium phosphate, 2.5 g of magnesium sulfate heptahydrate MgSO.sub.4.7H.sub.2 O, 5 g of dipotassium hydrogenphosphate K.sub.2 HPO.sub.4 and 0.2 g of iron (II) sulfate heptahydrate FeSO.sub.4.7H.sub.2 O each per liter of the liquid medium.

The thus prepared liquid culture medium was inoculated with 3 mg as dry of the mycelia of the Agaricus fungus contaminated with actinomyces and gently agitated at 25 .degree. C. for 3 days to obtain 25 g as dry of granules of the fungus body.

Two fungus granules free from coloration inherent in true bacteria by visual inspection were picked up from the thus obtained mass of granules and triturated in a clean bench. A 1 liter volume of another liquid culture medium containing 60 g of crude cane sugar corresponding to 58 g of sucrose, 10 g of a dried powder of crushed sugarcane having a particle fineness to pass a 200 mesh screen, 12 g of ammonium nitrate, 4 g of sodium phosphate, 2.5 g of magnesium sulfate heptahydrate, 0.2 g of iron (II) sulfate heptahydrate and 5 g of dipotassium hydrogenphosphate dissolved or dispersed each per liter of the liquid medium was inoculated with the triturated fungus granules and culturing was conducted in a batch process. After repeating again this batch process culturing under the same conditions, the fungus mycelia were cultured on a conventional nutrient agar culture medium to obtain fungus body granules free from contamination.

Similar uncontaminated Agaricus granules could be obtained by conducting culturing in the same liquid culture medium inoculated with the initial granules contaminated with actinomyces from which the colored portions contaminated with actinomyces had been shaved off by using a knife.

EXAMPLE 2

A pressurizable stainless steel bioreactor vessel connected to a cyclone as illustrated in FIG. 3 of 1 liter capacity was charged with 1 liter volume of a first liquid culture medium, referred to as the culture medium A hereinafter, containing 60 g of purified sucrose, 10 g of ammonium nitrate, 5 g of sodium phosphate, 2.5 g of magnesium sulfate heptahydrate, 0.2 g of iron (II) sulfate heptahydrate and 7 g of dipotassium hydrogenphosphate dissolved therein each per liter of the liquid medium.

A further pressurizable stainless steel bioreactor vessel of 1 liter capacity was charged with 1 liter of a second liquid culture medium, referred to as the culture medium. B hereinafter, containing 50 g of crude cane sugar, 15 g as dried of a sugarcane bagasse powder having a particle fineness to pass a 200 mesh screen, 12 g of ammonium nitrate, 4 g of sodium phosphate, 2.5 g of magnesium sulfate heptahydrate, 0.2 g of iron (II) sulfate heptahydrate and 6 g of dipotassium hydrogenphosphate dissolved or dispersed therein each per liter of the liquid medium.

Each of the cultured media A and B was inoculated with the uncontaminated Agaricus fungus granules obtained in Example 1 and culturing of the fungus was conducted at 25 .degree. C. under gentle agitation of the liquid medium while oxygen-enriched air containing 30 to 35% by volume of oxygen was blown into the liquid medium at a rate of 0.8 liter/minute. The pressure of the oxygen-enriched air blown into the liquid medium was controlled in such a way that the upstream-side pressure was kept at 0.12 to 0.15 MPa (absolute) for the first 24 hours and at 0.15 to 0.30 MPa (absolute) for the second to fourth days while the downstream-side pressure was kept always at 0.10 to 0.13 MPa (absolute).

The amounts of the fungus aggregates (dry basis) thus obtained by culturing for up to 5 days are shown by the curves A and B in FIG. 5 for the liquid culture media (A) and (B), respectively. The curve A' in FIG. 5 shows the results obtained in the liquid culture medium (A) without agitation with the stirrer driven. The curve C in FIG. 5 shows the results obtained in culturing in a conventional liquid culture medium which was the same as the liquid culture medium (A) excepting for the replacement of sucrose with the same amount of glucose.

EXAMPLE 3

The same culturing process of the Agaricus fungus as in Example 2 was repeated by using the liquid culture medium (B). After 24 hours of running, the liquid surface was covered with a substantial volume of foams which were skimmed up and reserved. Another run of Agaricus culturing was undertaken in the same manner as in Example 2 with a fresh portion of the liquid culture medium (B) and, after 3 hours of running, the liquid culture medium was admixed with the foams reserved above in a volume of about 10% of the liquid medium to continue further running of culturing up to 5 days.

The results of culturing obtained in this run were substantially the same as those shown by the curve B in FIG. 5 indicating that the foams could exhibit antimicrobial-activity against eubacteria and eumycetes.

EXAMPLE 4

The same culturing procedure of the Agaricus fungus was conducted over 4 days in the same manner as in Example 1 in a bioreactor with a cyclone as is illustrated in FIG. 3 or in the same reactor excepting for omission of the cyclone. The liquid discharged from the bioreactor as being carried by the exhaust air was analyzed for the content of the Agaricus mycelia getting out as accompanying the mist to give the results shown in FIG. 6 by the solid line curve and the broken line curve for the runs with and without the cyclone, respectively. As is indicated in this figure, the content of the Agaricus mycelia in the mist could be kept not to exceed 0.1 g as dry per liter of the liquid when the cyclone was equipped while the mycelia content reached 0.4 g as dry per liter of the liquid when no cyclone was equipped.

* * * * *
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OfflineAnnoA
Experimenter
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: Randoscious_McCray]
    #3214731 - 10/04/04 04:21 PM (19 years, 5 months ago)

It would be interesting to know if Randoscious_McCray ever used that reactor.....

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Offline4hodmt
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Registered: 04/06/04
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: Anno]
    #3224510 - 10/06/04 07:50 PM (19 years, 5 months ago)

i was thinking earlier.....

what if u had the liquid KARO shit, and then put a seriese of fine screens on top of each other, going all the way to just out of the water? the screens, if given perhaps a foot or two, could probably grow sum mycelinium. i was thinking bubbling air up through to give them that, and if u did this, u could make sure that the substrate is always full of high amounts of nutrients. u may get infinite flushes?


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OfflineMorbes
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Registered: 11/20/04
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Last seen: 10 years, 4 months
Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: 4hodmt]
    #3435748 - 12/02/04 12:27 AM (19 years, 3 months ago)

I've been curious about the same possibility. Although contamination would be a serious concern (whilst switching up the "balance" of essential nutrients etc) could the mycellium be tricked this way into fruiting forever?

I realise the only real way to know is to do an experiment, but until I have the time/resources for this particular endeavor - anyone have any response to this possibility? Is there a reason why it will not work?

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Offlineragadinks
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Re: Growing in Liquid Medium. Using knowledge from Reactor Des [Re: Morbes]
    #3542115 - 12/25/04 12:30 PM (19 years, 2 months ago)

Just have found a picture of a huge bio reactor that produces mushroom mycelium at this website:



Wonder how this one works exactly ...

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Offlineticktock
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Registered: 10/02/04
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Re: Growing in Liquid Medium. Using knowledge from Reactor D [Re: ragadinks]
    #3542141 - 12/25/04 01:06 PM (19 years, 2 months ago)

Quote:

ragadinks said:

1.00 ml water
40 grams barely malt sugar
3-5 grams hardwood sawdust
2 grams yeast
1 gram calcium sulfate





That can't be right. 1 gram of water and 46-48 grams of dry stuff. Pretty dry for a liquid culture. I'll bet it was 1 liter of water.


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Don't panic!

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