WO2001087045A1

WO2001087045A1 - Novel mushroom spawn

Info

Publication number: WO2001087045A1
Application number: PCT/GB2001/002230
Authority: WO
Inventors: Peter Hawton
Original assignee: Mycocell Technologies Limited
Priority date: 2000-05-18
Filing date: 2001-05-18
Publication date: 2001-11-22
Also published as: GB0012046D0; AU2001256549A1

Abstract

A granular support suitable for growing mushroom mycelium comprises particles of a base material capable of absorbing or entrapping water and, coated on the surface of the said particles, a layer of powdered phase II compost. The support can be inoculated with mushroom mycelium to form spawn.

Description

NOVEL MUSHROOM SPAWN

The present invention relates to a support suitable for the growth of mushroom mycelium and to a spawn comprising the support..

The production of mushrooms worldwide is a vast business worth hundreds of millions of US dollars per annum. Mushroom cultivation usually involves, as a first step, generating a mushroom spawn which consists of a support material inoculated with mushroom mycelium. The purpose of the spawn is to boost the mycelium such that it can be launched into bulk substrates. The support material serves both as a vehicle for evenly distributing the mycelium and as a nutritional source which can support the expansion of the mycelial mass. The mushroom spawn is then transferred to a suitable substrate for further growth. The substrate must be moistened since water is essential to mushroom growth. Various materials are known for use as supports for mushroom spawn. For a long time compost was a favoured medium, both as a carrier for the mushroom mycelium (compost spawn) and as a bulk substrate for subsequent mushroom growth. The compost used in this context is known as "phase II compost". Phase II compost is compost which has undergone a controlled heat treatment and conditioning and has been made selective for muslirooms over other microorganisms.

Compost spawn was usually made by filing bottles with phase II compost, plugging the bottle with cotton wool which acts as an air/gas filter, and steam sterilising. When the bottles had cooled down a pure culture of mushroom mycelium was added and the bottles were then left to grow through. The grower would receive the compost spawn in the bottles, which would be cracked open to yield the fully-grown compost spawn in one piece. The spawn was then broken into small lumps about the size of a small walnut and the lumps were pushed into bulk phase II compost.

Compost spawn ceased to be the favoured medium for several reasons. One was that the relatively large lumps into which it was broken were unsuitable for use with modern machinery. Another was that, since mushroom mycelium can grow right through compost, the compost spawn tended to form a solid block of mycelium which was difficult to handle.

Cereal grain then superseded compost as the preferred medium for spawn. US-A-1 869 577 discloses a grain spawn and US-A-2 044 861 describes an improved version which is still the standard today for grain spawn production. Grain spawn has specific advantages over compost. These include smaller particle size, which allows more points of inoculation when the spawn is launched into a bulk substrate for mushroom growth. Grain spawn is also more suitable than compost spawn for bulk handling and for use with the modern machinery employed in the mushroom industry.

Grain nonetheless possesses significant disadvantages as a support for mushroom spawn. In order to flourish mushrooms require a variety of nutrients including cellulose, starch or sugar, a nitrogen source and water. To maximise mushroom growth the balance of nutrients must be correct. However, grain possesses a nutrient imbalance in that its starch to cellulose ratio is much too high.

This means that grain spawn can sometimes give a low yield of mushrooms.

Another drawback of grain spawn is that it has a shelf life of only about three months in a cold store. After that time the mycelium starts to break down the grain cellulose and the grain becomes soft. It therefore becomes difficult to handle and may also be dangerous to use because the culture becomes unreliable.

A third drawback relates to the need to sterilise a spawn medium prior to inoculation with the mushroom mycelium. In modern procedures irradiation is one of the new techniques available for sterilisation. However, the density of cereal grain means that a high dose of radiation is required to achieve sterilisation. This is economically undesirable since is significantly increases a mushroom producer's costs. In addition grain is difficult to sterilise consistently by a conventional technique owing to its structure.

In view of these disadvantages there has been a move more recently towards the use of mineral substances as supports for mushroom spawn. FR-A-1 438 817 describes a support for the cultivation of mushroom mycelium comprising a stack of thin mica sheets. However, mica requires a thermal treatment before it can be used which involves heating to 1000°C. This weakens the material and diminishes its ability to support mycelial growth.

FR-A-207 1058 describes a support for the growth of mushroom mycelium comprising expanded perlite and a nutrient medium. The stated purpose of the perlite is to absorb moisture from the nutrient medium. The nutrient medium typically comprises a cereal product such as grain, bran or flour. The cereal may be cooked in water to form a broth into which the perlite is mixed. Alternatively the cereal may be crushed and mixed with the perlite and then moistened with water. In either case the resulting moist substrate consists of beads of perlite with a surface coating of nutrients. It is said that the support should possess a moisture level between 54 and 62%.

EP-A-077 6154 relates to an improvement in the substrate of FR-A-207 1058. It teaches a granular support suitable not only for mycelium growth but also for mushroom (carbophore) development comprising an inert, porous substrate and a nutrient phase. The nutrient phase includes water as an essential component. The porous substrate has a defined porosity which enables it to absorb both the water from the nutrient phase and the nutrients themselves. This creates an environment inside the support particles in which the mushroom mycelium can grow, thereby giving rise to a higher density of mycelial filaments than is possible when growth occurs only at the surface.

The above two disclosures require a nutrient medium to be made up in water and applied to the perlite particles. Care therefore needs to be taken to create the correct recipe of nutrients. Furthermore, once the nutrient medium has been added to the particles the system is moist. This has implications for shelf life and adds considerably to the weight of the material, which affects ease of handling.

The present invention seeks to overcome the drawbacks associated with the known compost spawn, grain spawn and mineral-based spawns described above. Accordingly the invention provides a granular support suitable for growing mushroom mycelium, comprising particles of a base material capable of absorbing or entrapping water and, coated on the surface of the said particles, a layer of powdered phase II compost. The invention essentially involves converting the medium which is most favourable for mushroom growth from a nutritional viewpoint, namely phase II compost, into a uniform particulate or seed-like form that can be conveniently handled and processed in modern equipment. The support of the invention is typically dry and need not then be moistened until it is inoculated with mushroom mycelium. As a result it is relatively light and has an almost indefinite shelf-life. It can be readily sterilised, for example by irradiation, prior to being formed into spawn by inoculation with mushroom mycelium. The support of the invention can also be inoculated after sterilisation with dry mycelium which will lie dormant until contacted with water. This means that dry spawn comprising the support of the invention inoculated with dry mushroom mycelium can be conveniently transported and stored.

The base material may be, for instance, a mineral, ceramics or polymeric substance. Suitable examples include vermiculite and perlite. Vermiculite is a member of the phyllosilicate group of minerals and has the ability to expand to many times its original volume on heating. A typical elemental analysis is as follows:

Element % bv weight

SiO₂ 38-46

Al₂O₃ 10-16

MgO 16-35

CaO 1-5

K₂O 1-6

Fe₂O₃ 6-13

TiO₂ 1-3

H₂O 8-16

Other 0.2-1.2

Perlite is a naturally occurring siliceous rock which, when heated to a suitable temperature in its softening range, expands to from 4 to 20 times its original volume. This expansion is due to the presence of two to six percent combined water in the crude perlite rock. When quickly heated to above 871°C, the crude rock pops in a manner similar to popcorn as the combined water vaporizes and creates countless tiny bubbles that account for the light weight and other physical properties of expanded perlite. Expanded perlite can be manufactured to weigh as little as 32 kgm^"3. Since perlite is a form of natural glass, it is classified as chemically inert and has a pH of approximately 7. A typical elemental analysis is as follows:

Element % bv weight

Silicon 33.8

Aluminium 7.2

Potassium 3.5

Sodium 3.4

Iron 0.6

Calcium 0.6

Magnesium 0.2

Trace 0.2

Oxygen 47.5 (by difference)

Net total 97.0

Bound water 3.0

Total 100.0

When the base material is perlite the weight ratio of the pearlite to the powdered compost is preferably 3 : 1 or less. Typically the weight ratio is from 3 : 1 to 1:1.

Alternatively the particulate base material may be, for example, a material lαiown as a superabsorber which is a water-absorbing polymer such as a cross-linked polyacrylate. Such a material may, for instance, be potassium-based or sodium- based. The support of the invention is typically produced by a process which comprises (a) drying phase II compost and grinding it to a powder, and

(b) combining the base material with the said powder such that the powder adheres to the surface of individual particles of the base material.

The process as defined above typically comprises the further step of sterilising the support thus produced. Preferably sterilisation is carried out by exposing the support to radiation.

When the support material is to be exposed to radiation to effect sterilisation it is dried and is usually then placed within a lining material which resists the ingress of microorganisms, for instance inside a plastic bag. Such a bag may be made of any polymeric material which is stable towards radiation, for instance polyethylene. The bags are usually chosen so that their volume is several times bigger than the volume of the contents, so that the bag can be folded and sealed in such a way as to avoid post-sterilisation contamination. If desired, one or more bags may then be placed for additional protection inside a second bag, which is also folded and sealed. The or each bag is then typically placed in a cardboard box. The cardboard box is closed and may then be exposed to radiation.

Any form of radiation capable of effecting sterilisation may be used. Typically the radiation is electron beam or gamma radiation, but in practice gamma rays are preferred because they achieve a higher degree of penetration of the substrate material. A preferred source of gamma rays is Co⁶⁰. The dose of radiation is typically from 5 kGy to 100 kGy, for example from 8 kGy to 100 kGy, more preferably from 10 kGy to 100 kGy. In one preferred aspect of the invention the upper limit is 60 kGy.

The process thus described yield a sterile dry support in a sealed bag, which is very convenient for further handling.

Alternatively the support may be sterilised by exposure to heat or by chemical treatment. When the support is to be sterilised by exposure to heat it is placed in an autoclave or an oven. The temperature in the autoclave is typically of the order of 120°C. The time required for the heating will depend on the nature of the support, for instance its density and its bio-count. Typically the time period is from 5 to 20 hours, more usually from 10 to 20 hours, it being understood that the specific time period must be individually selected depending on not only the nature of the support material but also the equipment being used for heating.

When the support is to be submitted to chemical sterilisation, it may be treated with a chemical such as sodium hypochlorite (bleach) or formaldehyde and subsequently dried. Alternatively the substrate may be treated with a gas or with a mixture of gases. Suitable examples of gases include formalin, ozone and methyl bromide.

As used herein the term "sterile" means substantially free, more typically totally free, of all living organisms. It may also mean substantially free of competitor microorganisms. Preferably it means that the probability that the support material contains even one surviving microorganism is infinitesimally small.

Inoculation of the sterile support is typically carried out in a laminar flow cabinet, which is a conventional structure that allows an operator to work in a sterile environment. The box is opened and each of the bags inside it is opened. A measured amount of mushroom mycelium is then injected into the sterile support and shaken.

A mushroom spawn may be produced by inoculating the support of the invention with mushroom mycelium. The support may be sterilised prior to inoculation with the mycelium. Accordingly the invention further provides a mushroom spawn comprising a support of the invention as defined above and mushroom mycelium. Dry mushroom mycelium is suitable for this. Alternatively the mycelium may be inoculated into the support of the invention via a liquid culture medium. When dry, for instance having a moisture content of 10% by weight or less, the spawn may be subsequently rehydrated. This typically takes place after storage or after transportation to a remote location. The spawn may be launched into a bulk substrate, for instance prior to rehydration.

In one aspect a mushroom spawn of the invention as described above is dried in a low temperature oven. Preferably the temperature within the spawn does not exceed 25°C. A dry, sterile bulk substrate is then prepared, for example by a process as described in PCT/GB00/00340. This process comprises exposing to radation or heat, or submitting to chemical sterilisation, a substrate which has a moisture content of 30% by weight or less and which comprises a particulate base material capable of absorbing or entrapping water. Typically the moisture content is 25% by weight or less, preferably 20% by weight or less, more preferably 12% by weight or less. It is frequently preferable for the moisture content to be uniform throughout the body of the substrate.

In this context the base material may be wood fibre, for instance in the form of sawdust or wood shavings. Alternatively, it is suitably selected from the materials referred to above for use as the base material for the granular support of the present invention. Likewise, the step of sterilizating the base material of the bulk substrate by radiation, heat or chemical sterilization is typically carried out as described above for the sterilisation of the granular support of the present invention.

The dry, sterile bulk substrate is preferably sealed within a lining material that resists the ingress of microorganisms, for example a plastic bag of the type discussed above. The lining material is opened under sterile conditions and innoculated with the mushroom spawn of the invention, dried as described above. It is then re-sealed. Typically a dry bulk substrate is inoculated with from 1 to 10% by weight of dry spawn, preferably from 1 to 2% by weight.

In general the end user would take a bag containing the substrate inoculated with spawn, open the bag in a sterile environment and add sufficient water to bring the contents to a moisture content of about 50% by weight, the exact amount depending on the particular culture. This rehydration step will stimulate the mushroom mycelium, which has remained dormant whilst dehydrated, into growth.

The support of the present invention is particularly applicable to the cultivation of compost-loving mushrooms such as white button mushroom (Agaricus).

The support of the invention possesses significant advantages. First, as indicated above, compost is a favourable medium for mushroom growth. This is because its nutrient profile corresponds closely to the optimum balance of nutrients required for the growth of the mycelium and carbophores of mushrooms, especially Agaricus mushrooms.

Second, it is selective for mushrooms over other microorganisms and can thus resist invasion by predator fungi.

Third, when mycelium grows on a particular medium it produces enzymes which convert nutrients in the medium into an assimilable form. The enzymes required for one medium, for instance grain, will differ from those required for another, for instance compost. Usually the profile of nutrients in mushroom spawn is different from that of the bulk substrate into which the spawn is subsequently launched. The mycelium must therefore manufacture two sets of enzymes. In contrast, when spawn is made from the support of the present invention it will already possess the full spectrum of enzymes which are required when the spawn is launched into phase II compost (as the bulk substrate). Mushroom growth in the bulk substrate should therefore be faster.

Fourth, the individual particles of the base material are uniform and are not prone to splitting or cracking, as grain is.

Fifth, since the support is dry it can be stored indefinitely, either by itself or inoculated with dry (dormant) mycelium. It is not susceptible to fermentation.

Sixth, when a mineral substance such as perlite is used as the base material the support is very light due to the air trapped within the perlite structure. Owing to their small particle sizes perlite and vermiculite are also able to provide more points of inoculation than the individual grains of most grain spawns. Yet another advantage of the support of the invention is that spawn made from it can be used in the casing layer. When mycelium reaches an advanced level of development it requires a casing layer to continue its growth into mushrooms. In the natural environment this function is performed by topsoil. Soil (for instance peat) is added to the surface of the spawn-run bulk substrate as a so-called "casing" layer. This retains moisture and prevents the bulk substrate from drying out. The casing layer is thus a physical barrier, not a further source of nutrients. CACing, which stands for Compost Added to Casing, is typically added to the casing soil, for instance in a proportion of 2% by weight.

A widely used commercially available CACing product comprises 75% vermiculite and 25% peat, which is sterilised and inoculated with spawn. CACing is mixed with the casing soil and applied to the bulk substrate, typically phase II compost. Its presence encourages even growth of the mycelium to the surface of the casing layer and shortens the time to the appearance of the mushrooms. Without CACing the mycelium must grow through the casing layer to the surface before mushrooms will appear. In CACing the mycelium is only growing on the cellulose of the peat since other nutrients are lacking. The poor nutrient profile also discourages the growth of competitor moulds.

Grain spawn cannot be used as CACing because it is too vulnerable to attack by competitor moulds in the hostile environment of the casing layer. In contrast, spawn produced from the support of the present invention is ideally suited to use as CACing because its high selectivity for mushroom mycelium means it can resist infestation by adventitious microorganisms.

The present invention will be described in the following Examples:

Example 1: Preparation of granular support using perlite

Phase II compost was dried from 68% moisture to 6/5 % and ground to a fine powder. 100 grams of the powder was then added to 300 grams of perlite and 50 grams of gypsum. The mix was placed in a standard spawn bag with filters and sealed.

Example 2: Preparation of compost spawn

The bag containing the support prepared in Example 1 was sterilized by irradiation. After sterilization the bag was opened in a laminair flow cabinet to avoid contamination and inoculated with a pure culture of agaricus. 0.75 litres of sterile water was added. The bag was then sealed, shaken and placed on a rack. After ten days all the contents of the bag had been colonised by the mycelium of the agaricus culture. The bag was then shaken to break - up the particles of perlite, a small sample was placed on an agar plate (twenty one particles) and incubated. After four days the plate was checked and all the particles were growing mycelium. Example 3: Mushroom growth in bulk substrate

One percent by volume of the spawn produced in Example 2 was added, after the ten day period referred to in that Example, to a bag of phase II compost. After four days it was observed that each particle was growing and after ten days the compost was fully colonised by the culture.

Example 4: Preparation of sterilised bulk substrate inoculated with dried compost spawn

a) Bulk substrate preparation

Sawdust and bran (a source of nitrogen) were mixed together, and the resulting mixture was checked to see that its moisture content was 10% by weight. If not, the mixture was dried in an oven until its weight loss on further drying was 10% or less. The dry mixture was divided into 5 kg samples. Each sample was placed in a plastic bag having a volume approximately five times that of the sample.

Two of the bags containing a 5 kg sample were then put inside a second bag and a dosimeter (which is an instrument that changes colour on exposure to radiation, the degree of colour change being related to the radiation dose) was placed in the centre. The bag was sealed and placed inside a cardboard carton. The carton was then exposed to a Co⁶⁰ source of gamma-radiation. The carton was retrieved and the dosimeter checked to determine the radiation level at the centre of the box.

The data obtained from this procedure were as follows:

Dose Range Specification: Min 10.0 kGy Max 60.0 kGy

Dimensions of Carton: 430x420x260 mm

Weight of Carton: 11.2 kg Density: 0.24 g per cc

Dwell Time: 223 Sees

Current Cobalt Loading: 1.742651 Mega Curies Standard Plant Dwell Time: 223 Sees Dosimetrv Results

Minimum Dose Reading: 20.2 kGy

Maximum Dose Reading: 29.0 kGy Routine Dosimeter Reading: 25.3 kGy

Dose Required at Routine Dosimeter 12.5 kGy To achieve Min 10.0 kGy

Dose Required at Routine Dosimeter 52.3 kGy To achieve Max 60.0 kGy

b) Dried spawn preparation The support prepared as described in Example 1 was inoculated in a spawn bag with agaricus culture. Following colonisation of the support by mycelium the bag was shaken to break up the particles. The spawn was then dried in a low temperature oven such that the temperature in the spawn did not exceed 25°C.

c) Launch of spawn into substrate

Under sterile conditions the bag of sterile substrate prepared as described under (a) above was opened and inoculated with 1-2% by weight or the dried spawn prepared as described under (b) above. The bag was then re-sealed.

Example 5: Rehydration of inoculated bulk substrate

The substrate containing dried spawn, prepared as described in step (c) of Example 4 above, was opened under sterile conditions. Water was added to the bag until the moisture content was above 50% by weight. After about 4 weeks the substrate was grown over with mushroom mycelium.

Example 6: Use of vermiculite support

The procedures of Examples 1, 2, 3, 4 and 5 were repeated, but using vermiculite in the support in place of perlite. Colonisation of the support by the mycelium as described in Example 2, and colonisation of the bulk substrate as described in Examples 3 and 5, was observed.

Claims

1. A granular support suitable for growing mushroom mycelium, comprising particles of a base material capable of absorbing or entrapping water and, coated on the surface of the said particles, a layer of powdered phase II compost.

2. A support according to claim 1 wherein the base material is a mineral, ceramics or polymeric substance.

3. A support according to claim 1 or 2 wherein the base material is perlite or vermiculite.

4. A support according to any one of the preceding claims wherein the base material is perlite and the weight ratio of the perlite to the compost is 3:1 or less.

A support according to claim 4 wherein the weight ratio is from 3:1 to 1 :1.

6. A process for producing a support as defined in any one of the preceding claims which comprises (a) drying phase II compost and grinding it into a powder; and

7. A process according to claim 6 which further comprises sterilising the support.

8. A mushroom spawn comprising a support as defined in any one of claims 1 to 5 and mushroom mycelium.

9. A spawn according to claim 8 which has a moisture content of 10% by weight or less.

10. A spawn according to claim 9 which has been rehydrated by the addition of water.

11. A process for producing a mushroom spawn, which process comprises inoculating a support as claimed in any one of claims 1 to 5 with mushroom mycelium.

12. A process according to claim 11 which includes the step of sterilising the support prior to inoculation with the mycelium.

13. A process according to claim 11 or 12 which includes the step of drying the resulting spawn in an oven at a temperature no greater than 25°C.

14. A sterile substrate having a moisture content of 30% by weight or less and comprising a particulate base material capable of absorbing or entrapping water, which substrate further comprises a spawn as claimed in claim 8 or 9.

15. A substrate according to claim 14 which comprises from 1 to 10% by weight of the spawn.

16. A substrate according to claim 15 which comprises 1 to 2% by weight of the spawn.

17. A substrate according to any one of claims 14 to 16 which is sealed within a lining material which resists the ingress of microorganisms.

18. A support according to any one of claims 1 to 5 which is sealed within a lining material that resists the ingress of microorganisms.

19. A spawn according to claim 8 or 9 which is sealed within a lining material that resists the ingress of microorganisms.