WO2023021264A1

WO2023021264A1 - Edible fungus

Info

Publication number: WO2023021264A1
Application number: PCT/GB2022/051898
Authority: WO
Inventors: Robert Johnson
Original assignee: Marlow Foods Limited
Priority date: 2021-08-18
Filing date: 2022-07-21
Publication date: 2023-02-23
Also published as: GB202111820D0; GB2610554A

Abstract

A biomat (1) comprising filamentous fungus is produced by a surface fermentation process. The mat may be transferred from a tray in which it is produced (by hand or using an automated apparatus) onto a belt of a moving belt filter (8). On the belt filter, the biomat may be sprayed from spray head (9) with 65°C water for a period of 15 minutes. The treatment is suitably arranged so the water penetrates and passes through the biomat (1) from top to bottom and then passes through the filter surface of the filter (8). The passage of water will cause removal of RNA from the filamentous fungus of the biomat, thereby producing a biomat and/or filamentous fungus with a reduced level of RNA, compared to the level produced naturally during production of the biomat.

Description

Edible Fungus

This invention relates to edible fungus, for example, comprising particles of a filamentous fungus belonging to an order selected from the group consisting of Mucorales Ustilaginales, Russulales, Polyporales, Agaricales, Pezizales and Hypocreales, wherein, suitably, the filamentous fungus comprises greater than about 40 wt. % protein content and less than about 8 wt. % RNA content. Particularly, although not exclusively, the filamentous fungus may belong to a family selected from the group consisting of Mucoraceae, Ustilaginaceae, Hericiaceae, Polyporaceae, Grifolaceae, Lyophyllaceae, Strophariaceae, Lycoperdaceae, Agaricaceae, Pleurotaceae, Physalacriaceae, Omphalotaceae, Tuberaceae, Morchellaceae, Sparassidaceae, Nectriaceae, Bionectriaceae, and Cordycipitaceae. In preferred embodiments, the filamentous fungus may belong to a species selected from the group consisting of Rhizopus oligosporus, Ustilago esculenta, Hericululm erinaceus, Polyporous squamosus, Grifola fondrosa, Hypsizygus marmoreus, Hypsizygus ulmarius, Calocybe gambosa, Pholiota nameko, Calvatia gigantea, Agaricus bisporus, Stropharia rugosoannulata, Hypholoma lateritium, Pleurotus eryngii, Pleurotus ostreatus, Tuber borchii, Morchella esculenta, Morchella conica, Morchella importuna, Sparassis crispa, Fusarium venenatum, strain MK7 (ATCC Accession Deposit No. PTA- 10698), Disciotis venosa, Clonostachys rosea, Cordyceps militaris, Trametes versicolor, Ganoderma lucidum, Flammulina velutipes, Lentinula edodes, Pleurotus djamor, Pleurotus ostreatus, and Leucoagaricus spp.

It is well-known, for example from W02020/176758 (Sustainable Bioproducts, Inc), to use filamentous fungus in foodstuffs. To produce foodstuffs containing filamentous fungus which are suitable for human consumption, the filamentous fungus should include a nucleic acid content which does not exceed 2% by weight.

W02020/176758, at [182], recognises the need for filamentous fungus to have low RNA content when used in foodstuffs. In preferred embodiments according to W02020/176758, filamentous fungus is selected which has an inherently low RNA level such as in Fusarium oxysporum strain referred to as MK7 (ATCC Accession Deposit No.PTA-10698).

The filamentous fungus described in W02020/176758 is made in trays (by production of so-called “biomats” of filamentous fungus) in a batch process which is relatively slow and/or produces a low yield of product per unit time. It is, therefore, desirable to increase yield of the filamentous fungus and/or to provide at least a partially continuous process. However, it is found that steps taken to increase yield and/or speed of production and/or which provide at least a partially continuous process tend to increase the level of RNA produced in the fungus, even in fungus which is believed to inherently produce relatively low levels of RNA per unit of fungal weight. In addition, manual manipulation of trays and biomats produced therein as described in, for example, W02020/176758 tends to result in breakage of fungal hyphae in the biomats. It is, however, desirable to minimise such breakage because, firstly, the smaller the hyphal lengths, the greater the loss of fungal biomass in downstream processing, such as filtration; and, secondly, in foodstuff comprising filamentous fungus, it is desirable to maintain hyphal lengths thereby to optimise rheological properties of the filamentous fungus in the foodstuff and/or produce optimum texture.

It is an object of the present invention to address the above-described problems.

According to a first aspect of the invention, there is provided a method of treating a biomat comprising:

(i) associating a biomat with a filter membrane;

(ii) contacting said biomat with a treatment fluid;

(iii) moving fluid away from the biomat via the filter membrane.

A biomat may be as described in and/or be produced as described in US10787638 or in THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. XXX1 . No 1 (THE CITRIC ACID FERMENTATION OF ASPERGILLUS NIGER by JAMES N CURRIE), the content of which is incorporated herein by reference.

A said biomat is suitably generated via surface fermentation after inoculation of a desired fungal strain into a growth media where, suitably, no aeration may be required. This method of surface fermentation is applicable to a large variety of fungal species. The media developed generates rapid cell growth, creates high density filamentous fungi biomats with long filaments, produces small waste streams, and allows engineering of the filamentous fungi biomat produced as a function of carbon source, carbon to nitrogen ratio (C:N), and process parameters. The overall effect is one in which high production rates occur with minimal environmental impact as measured by water usage, energy usage, equipment requirements, and carbon footprint.

An artificial media suitable for culturing filamentous fungi and enabling their production of a filamentous fungal biomat may comprises at least the following macronutrients: nitrogen (N), phosphorus (P), calcium (Ca), magnesium (Mg), carbon (C), potassium (K), sulfur (S), oxygen (O), hydrogen (H) and the following trace nutrients: iron (Fe), boron (B), copper (Cu), Manganese (Mn), molybdenum (Mo), and zinc (Zn). In some instances, the trace nutrients are augmented with the following additional trace nutrients: chromium (Or), selenium (Se), and vanadium (V). The artificial media has varying C:N ratios which favour production of filamentous fungi biomats having either a high protein:lipid ratio or a high lipid:protein ratio. Filamentous fungi for filamentous fungi biomat production may be as described in the introduction of this specification. Filamentous fungi may be acidophilic, such as species and/or strains of Fusarium, Fusisporium, Pseudofusarium, Gibberella, Sporotrichella, Aspergillus. Penicillium, Triocoderma, species within the Mucorales sp. (e.g., Rhizopus sp.) and the filamentous fungal strain designated as MK7. Depending on the species and/or strain, the pH of the culturing media may range from about 0.68 to about 8.5 and in some cases up to 10.5.

The filamentous fungi biomats produced may arise from anaerobic, microaerobic, aerobic conditions of a combination thereof via surface fermentation. The filamentous fungi biomats comprise the fungal species and/or strain and/or progeny thereof in the form of conidia, microconidia, macroconidia, pycnidia, chlamydospores, hyphae, fragments of hyphae, or any and all combination thereof.

Surface fermentation which is used to produce biomats suitably refers to those fermentations in which the microorganisms employed grow on the surface of the fermentation media without any further support. The media is typically a free-flowing aqueous media. Without being bound by theory, it is thought that filamentous biomats result from some combination of aerobic, microaerobic and/or anaerobic metabolism. For example, the surface of the biomat is thought to rely on aerobic respiration while the bottom of the biomat may be microaerobic to highly anaerobic.

Surface fermentation may be initiated by inoculating artificial media with a suspension of planktonic cells of the desired filamentous fungal species and/or strain(s). Inoculum culture from an inoculum reactor is added to the artificial media at a concentration that will produce a mature biomat in the desired time. Typically, inoculation with 0.5-1 .0 g of cells per litre of growth media will produce a biomat in 3 to 6 days. For example, adding inoculum containing about 10 g/L of cells at 7.5% (volume to volume) of the medium used will produce a biomat in 3 to 6 days. No external oxygen needs be introduced to the artificial media by bubbling or other means; sufficient oxygen can be culled from ambient or near ambient conditions. However, rate of production of biomats may be increased by increasing the oxygen concentration.

Typically, shallow trays containing artificial media are used for surface fermentation under controlled conditions of temperature, humidity, and airflow suitable for the fungal species and/or strains(s) employed. Sterile conditions are maintained for optimal filamentous fungi biomat growth. Sufficient airflow is maintained to remove heat and carbon dioxide produced from microbial respiration and supply oxygen without agitating the surface of the artificial media and disrupting fungal hyphae growth. In general, a “skin” begins to form on the surface of the artificial media on day 2 after inoculation. This “skin” is the initial filamentous fungi biomat which frequently includes aerial hyphae as well as hyphae in contact with the artificial media and which continues to grow and increase in cell density. Typically, three to six days after inoculation, the resultant filamentous fungi biomats are 1 to 30 mm thick and have sufficient tensile strength and structural integrity to be handled without tearing.

In one embodiment, mycelium may be grown in a fermenter and the broth then transferred into a tray. The fungus may then move to the top and filaments of mycelia may knit together. Advantageously, growth time on the tray may be reduced, leading to mat production process which is quicker than if the process does not include the fermentation step.

The filamentous fungi biomats produced have a structure as described which is not seen in nature. First, naturally formed filamentous fungi biomats are not composed of a pure culture/co-culture/substantially pure culture. Typically, the biomats formed in nature contain various types of algae and/or bacteria in addition to at least one filamentous fungal species and form an artificial microecosystem. Thus, preferably, biomats produced as described herein do not include algae and/or bacteria in addition to at least one filamentous fungal species.

Second, the biomats formed using the methods and techniques described herein may have a significantly greater cell density than those found in nature, even taking into account the multiple species found in naturally formed biomats. The produced filamentous fungi biomats tend to be very dense typically, 50-200 grams per litre. Natural and submerged processes for growth of filamentous fungi commonly result in biomass densities of about 15 grams per litre. From the perspective of percent solids, the methods disclosed herein produce filamentous fungi biomats that commonly range from 5-20wt% solids. In contrast, natural and submerged processes for growth of filamentous fungi commonly result in percent solid ranges of less than 1.5wt%. One result of the densities achieved, the filamentous nature, and the extracellular matrix found in these dense biomats is an ability to be maintained as a cohesive mat upon drying. This is in stark contrast to the powdery and/or non-cohesive form normally found with other dried filamentous fungal biomats.

Third, the biomats formed using the methods and techniques described herein have a high tensile strength compared to naturally occurring biomats, allowing them to be lifted and moved without breakage.

Fourth, the instant biomats have a defined structure comprising, in some instances, a single dense layer comprised of long filaments generally aligned parallel to the airbiomat interface. In some filamentous fungi biomats at least two layers exist: (a) a dense bottom layer and (b) an aerial hyphae layer. In some filamentous fungi biomats at least three structurally different layers are visible: (a) a dense bottom layer, (b) an aerial hyphae layer and (c) a transition zone layer. In systems with aerial hyphae and systems with three layers, the aerial hyphae layer is typically most visibly dominant, followed by the dense bottom layer, while the transition zone layer, if present, is smallest. Each of the layers normally has a characteristic cell density associated with it as compared to the other layer(s). For example, the aerial hyphae layer is significantly less dense than the bottom layer of the biomat. If aerial hyphae are produced, they are predominantly oriented perpendicular to the biomat:air and/or biomat:media interface. For all biomats, the dense layer is comprised of long filaments which are predisposed to be aligned parallel with the biomat:air and/or biomat:media interface. Further, the resulting biomat is comprised of at least a majority of the fungal biomass and in preferred embodiments, comprised of essentially no residual feedstock and is essentially pure fungal biomass.

Biomats are normally harvested between day 3 and day 12 after inoculation, depending on the species/strain(s) used and the desired product, although later harvest times are also possible.

Said biomat may comprise a filamentous fungus, wherein the biomat has a cell density of at least 25 g dry weight /L media and/or the biomat is 1 mm to 30 mm thick. The biomat is preferably produced by a surface fermentation method which may comprise growth on the surface of an aqueous media comprising nutrients in dissolved or submerged form. In preferred embodiments, the biomat has a cell density of at least 50 g dry weight/L media or at least 75 g dry weight /L media or at least 120 g dry weight /L media or at least 180 g dry weight /L media. The biomat may comprise at least two structurally different cell layers in contact with each other, including an aerial hyphae layer and a bottom layer in contact with an artificial medium, wherein the aerial hyphae layer is less dense than the bottom layer. The ratio of thickness of the bottom layer to the thickness of the upper aerial hyphae layer may be at least 0.41 .

Said biomat suitably comprises a filamentous fungus and, preferably, at least 80 wt%, or at least 90 wt% or at least 95 wt% of said biomat comprises filamentous fungus, the aforementioned wt% being on a dry matter basis - i.e. ignoring water associated with the biomat (which may be contained within cells of filamentous fungus or be contained between filaments of said filamentous fungus). Said filamentous fungus is preferably a Fusarium species, for example Fusarium venenatum or Fusarium-oxysporum, for example a strain referred to a MK7.

Said treatment fluid may be arranged to support growth and/or facilitate growth of filamentous fungus of said biomat; or to heat shock the filamentous fungus of said biomat, for example to render said filamentous fungus non-viable; or to wash the filamentous fungus of the biomat. In an embodiment (A), said treatment fluid may comprise culture medium which is suitable arranged to support growth and/or facilitate growth of said filamentous fungus of said biomat. The culture medium (suitably along with other suitable process conditions) is preferably arranged to help to maintain the filamentous fungus in a viable state.

In embodiment (A), said biomat may be associated with a filter membrane, for example, by resting on a surface of said filter membrane and, when so disposed, may be contacted with said treatment fluid (e.g. said culture medium). The culture medium may be sprayed onto said biomat, suitably so it permeates the biomat.

Preferably, the biomat is produced in a receptacle, for example a tray as described, and the method suitably comprises transferring the biomat from said receptacle to said filter membrane, where it may be contacted with said treatment fluid. Said transfer may be automated. For example, said biomat may be conveyed, for example dragged, from said receptacle to a position in which it is associated with said filter membrane.

In an embodiment (B), said treatment fluid may comprise a heated fluid which may comprise steam or heated water. Such a treatment fluid may be arranged to heat shock the filamentous fungus of the biomat. Said treatment fluid may be arranged to raise the temperature of the filamentous fungus of the biomat to a temperature in the range 60-70°C. The treatment is suitably arranged to disrupt cell membranes of the filamentous fungus and/or to facilitate passage of RNA and/or break down products thereof out of the cells of the filamentous fungus. The effect of the treatment, and particularly hyphal disruption, can be observed by use of a suitable straining process.

In an embodiment (C), said treatment fluid may comprise water, for example at ambient temperature and/or at a temperature in the range 10 to 30°C which is suitably arranged to wash filamentous fungus of the biomat. In embodiment (C), said treatment fluid may comprise 99 wt% or more water.

The method preferably includes the wash step of embodiment (C). This may be preceded by the step of embodiment (A) and/or embodiment (B). For example, a biomat may be transferred to said filter membrane, treated as described in embodiment (B) and then washed as described in embodiment (C). Alternatively, a biomat may be transferred to said filter membrane, treated to maintain the filamentous fungus in a viable state as described in embodiment (A), then treated as described in embodiment (B), followed by treatment as described in embodiment (C). Said filter membrane is preferably moveable and/or moves in the process, suitably to convey a biomat thereon from a first position at which a biomat is introduced onto the filter membrane to a second position from which the biomat is withdrawn from the membrane, said second position suitably being downstream of said first position.

Said filter membrane is suitably part of a belt filter wherein, suitably, the filter membrane is perforate and is conveyed. Preferably, in the process (for example when treatments are carried out as described in embodiments (A), (B) and/or (C), fluid passes through the biomat and, thereafter, passes through the perforations of the filter membrane, suitably as the belt filter conveys the biomat.

The method preferably comprises isolating the biomat and/or removal of biomat from association with the filter membrane after step (iii).

In an embodiment (D), a receptacle in which a biomat is produced in a surface fermentation process may comprise a moveable filtration membrane. The filtration membrane may be moveable between a first position in which the membrane is below a surface of liquid in the receptacle and may, for example, be relatively close to a base or lower wall of said receptacle; and a second position in which the filtration membrane is closer to a surface of liquid in the receptacle. For example, in the second position, the filtration membrane may contact an underside of the formed biomat and/or it may lift the biomat upwards, thereby to remove the biomat substantially from contact with culture medium in the receptacle in which the biomat is grown. When so disposed, the biomat, present on the filamentous membrane, may be treated as described according to embodiments (A), (B) and/or (C).

Said biomat (e.g. dewatered biomat) may, subsequent to step (iii), include less than 82 wt%, for example less than 80 wt% water; and suitably includes at least 18 wt%, preferably at least 20 wt% of filamentous fungus (on a dry matter basis).

After step (iii), said biomat may include less than 2 wt% of RNA on a dry matter basis.

Said filamentous fungus preferably comprises fungal mycelia and suitably at least 80 wt%, preferably at least 90 wt%, more preferably at least 95 wt% and, especially, at least 99 wt% of the fungal particles in said biomat comprise fungal mycelia. Some filamentous fungi may include both fungal mycelia and fruiting bodies. Said fungal particles preferably comprise a filamentous fungus of a type which does not produce fruiting bodies.

Said filamentous fungus preferably comprise fungus selected from fungi imperfecti. Preferably, filamentous fungus comprise, and preferably consist essentially of, cells of Fusarium species, especially of Fusarium venenatum A3/5 (formerly classified as Fusarium graminearum) (IMI 145425; ATCC PTA-2684 deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA.).

Said filamentous fungus in said biomat of step (i) may comprise filaments having lengths greater than 100pm, preferably greater than 200pm, more preferably greater than 500pm. Preferably, fewer than 5wt%, preferably substantially no, filaments in said biomat have lengths of greater than 20000pm. Preferably, values for the number average of the lengths of said filamentous fungus in said formulation are also as stated above.

Said filamentous fungus in said biomat of step (i) may comprise filaments having diameters of less than 20pm, preferably less than 10pm, more preferably 5pm or less. Said filaments may have diameters greater than 1 pm, preferably greater than 2pm. Preferably, values for the number average of said diameters of said fungal particles in said formulation are also as stated above.

Step (i) of the method is suitably undertaken with said biomat in the presence of its growth medium.

According to a second aspect of the invention, there is provided an assembly comprising a biomat associated with a filter membrane.

The biomat and filter membrane may be as described in the first aspect.

In the assembly, said filter membrane is preferably moveable, suitably to move the biomat between first and second positions. Said filter membrane may be a component of a belt filter as described in the first aspect.

In said assembly, the biomat may include a reduced level of RNA, compared to the level of RNA produced by culturing filamentous fungus to produce the biomat. Filamentous fungus in said biomat may include disrupted cell membranes and/or include cell membranes from which RNA and/or fragments thereof have been removed. The invention extends to an RNA- reduced biomat as described per se.

In the assembly, there may be a spray device for spraying culture medium and/or water onto a biomat associated with said filter membrane. In the assembly, there may be a heater arranged to heat fluid which may then be sprayed onto the biomat associated with the filter membrane. According to a third aspect of the invention, there is provided a method of producing an assembly according to the second aspect, the method comprising providing a biomat on a filter membrane. The method may comprise transferring a biomat from a receptacle, for example in which it is produced onto the membrane.

Biomats as described may be produced in a relatively slow process and/or the yield of filamentous fungus produced may be relatively low. To address this problem, in a fourth aspect, there is provided a method of producing a biomat, suitably as described in any preceding aspect, the method comprising contacting the biomat with culture medium from a position above a biomat. For example, the method may comprise spraying culture medium on top of the biomat. Advantageously, this may facilitate enhanced growth of the biomat due to more ready availability of nutrients. Additionally, the biomat may be subjected to an oxygen-enriched atmosphere which may also advantageously enhance aerobic fermentation of filamentous fungus of the biomat.

According to a fifth aspect of the invention, there is provided a method of producing a biomat which may be as described according to any preceding aspect, the method comprising:

(i) providing a liquid for surface fermentation to produce a biomat; and

(ii) causing the liquid to move, for example substantially linearly (as opposed to liquid being rotated as may occur during mixing). Such movement may facilitate supply of oxygen and/or nutrients to the biomat and increase its rate of growth. The method of the fifth aspect may be carried out in combination with the features described in the fourth aspect.

An alternate solution to improving yield of biomat in a commercially viable, continuous process is described hereinafter in Example 9 which is encompassed by the aspects of the invention which follow.

According to a sixth aspect of the invention, there is provided an assembly comprising a biomat associated with a solid support which supports the biomat.

The support is preferably inert. It is preferably not living. It may comprise a man-made material.

The biomat may be grown on the support. The thickness of the biomat at a first position on the support may be less than at a second position on the support. For example, the ratio of the thickness of the biomat at the second position divided by the thickness at the first position may be at least 2 or at least 5. The thickness at the second position may be the maximum thickness of the biomat on the support. The thickness at the first position may be the minimum thickness of the biomat on the support. The second position may be at or adjacent a region wherein the assembly includes means for disengaging the biomat from the support. The biomat may be of gradually increasing thickness on moving from the first to the second positions.

The biomat may be as described in any preceding aspect. However, preferably, it has a width which is at least 1 m, more preferably at least 2m. The biomat may have a length on the support of at least 2m, preferably at least 4m. The area of a face of the biomat may be at least 3m² or at least 10m².

Said assembly preferably includes culture medium which may be as described in any preceding aspect. Said culture medium is preferably in contact with the support. A mass of culture medium may be in contact with a surface of the support which faces in an opposite direction to the direction in which a surface of the support with which the biomat is associated faces. A mass of culture medium may be in contact with a lower surface of the support and the biomat may be in contact with an upper surface of the support, wherein said upper and lower surfaces suitably face in the opposition directions.

Said culture medium may be provided in a receptacle which is suitably arranged under the biomat. The biomat may overlie the culture medium in the receptacle.

Said support is preferably arranged for passage of fluid, for example, culture medium, from one surface (e.g. the lower surface) of the support to another surface (e.g. the upper surface) of the support. Said support suitably includes openings for passage of fluid. Said support is suitably porous. Said support is preferably arranged to transport fluid by capillary action and/or to wick fluid, suitably so fluid, for example culture medium, can pass through the support.

Said support is preferably movable, suitably to convey the biomat, between a position A and a position B which may correspond to the first and second positions described. The assembly may be arranged for removal of the biomat from the support at position B.

Said support may comprise an endless surface which may pass around a series of rollers which are components of the assembly.

Said support may comprise a filter membrane which may be as described in any preceding aspect.

Said support may comprise a belt filter which may be as described in any preceding aspect. Said assembly may include means for urging said support into culture medium and/or into a volume defined by a receptacle for culture medium.

Said assembly may include means for removing fluid from the biomat, which is suitably at or downstream of said second position and/or at or downstream of position (B). Said means may be arranged to compress the biomat to remove fluid therefrom.

Said assembly may include any feature of the assembly of the second aspect. It may include the spray device of the second aspect.

The invention extends, in the seventh aspect, to a biomat per se having any feature of the biomat as described in the sixth aspect.

According to an eighth aspect of the invention, there is provided a method of producing an assembly according to the sixth aspect, the method comprising growing a biomat on a solid support, suitably having any feature of the support of the sixth aspect.

The method may comprise producing a biomat having any feature of the biomat of the sixth aspect.

The method may comprise the passage of fluid, for example, culture medium from one surface of the support to another surface as described in the sixth aspect. The method may comprise transport of fluid, for example culture medium, by capillary action and/or by wicking through the support.

Said method may comprise moving the support between a position A and position B as described in the sixth aspect.

The method may comprise movement of the support in an endless travel path.

Said method may comprise urging the support into a receptacle containing fluid, for example culture medium.

Said method may comprise removal of fluid from the biomat, suitably after a predetermined thickness of biomat has been produced.

Said method may include any feature(s) of the methods of the third and/or fourth aspects. According to a ninth aspect of the invention, there is provided a method of producing a biomat, the method comprising the method of the eighth aspect, followed by removal of the biomat from the support. The biomat may be as described in any statement therein.

Any feature of any aspect of any invention described herein may be combined with any feature of any other invention or embodiment described herein mutatis mutandis.

Specific embodiments of the invention will now be described, by way of example, with reference to the accompanying figures in which:

Figure 1 is a schematic view of a biomat on a belt filter;

Figure 2 is a schematic representation of a modified apparatus for producing an RNA- reduced biomat;

Figure 3 is a schematic representation of an alternative modified apparatus for producing an RNA-reduced biomat; and

Figure 4 is a schematic representation of a further apparatus for producing a biomat.

In the figures, the same or similar parts are annotated with the same reference numerals.

In the following examples, Examples 1 and 2 describe a culture medium and its inoculation with a filamentous fungus. Example 3 describes preparation of a biomat and Examples 4 to 7 describe processes which include RNA reduction of biomats. Example 8 describes an integrated apparatus for producing a biomat and reducing its RNA using a novel apparatus/method.

Example 1 - Preparation of liquid culture medium

A skilled person in the art is familiar with selection of suitable liquid growth media for growth of filamentous fungus. For example, a culture medium may be as described in WO2017/151684 in Example 2 (MK7-1 or MK7-3 liquid media) or in any of Examples 6, 7, 10, 11 , 12, 13, 14 and 16. The content of the aforementioned examples are incorporated herein by the aforementioned references. In addition, the culture medium may be as described in US5938841 in Example 1.

Example 2 - Inoculation process Cultures for inoculation and a process for inoculation may be as described in WO2017/151684 in Example 3 or in Example 1 (column 4, lines 52 to column 5, line 7) of US5938841. The content of the aforementioned examples is incorporated by reference.

Example 3 - Preparation of mats comprising filamentous fungus (also referred to as “biomats”) (“Method PI”)

In general terms, biomats comprising filamentous fungus may be prepared as described in Example 4 of WO2017/051684 and the content of the aforementioned example is incorporated herein by reference. Biomats may be produced as described to produce structures which are 3 to 10mm thick with enough tensile strength and structural integrity so that they can be handled without tearing.

Example 4 - Treatment of biomats to reduce level of RNA (“Method RNA1 ”)

Referring to Figure 1 , a biomat 1 produced as described in Example 3 may be transferred from the tray (by hand or using an automated apparatus) onto a belt of a moving belt filter 8. On the belt filter, the biomat may be sprayed from spray head 9 with 65°C water for a period of 15 minutes. The treatment is suitably arranged so the water penetrates and passes through the biomat 1 from top to bottom and then passes through the filter surface of the filter 8. The passage of water will cause removal of RNA from the filamentous fungus of the biomat, thereby producing a biomat and/or filamentous fungus with a reduced level of RNA, compared to the level produced naturally during production of the biomat.

Optionally, in the method, after treatment with water, the biomat may be directed from the belt filter through one or more groups of paired rollers to sgueeze liguid from the biomat.

Example 5 - Treatment of biomat to reduce level of RNA (“Method RNA2”)

A biomat produced as described in Example 3 may be transferred from the tray into a receptacle containing water at 65°C and maintained in the receptacle for a period of at least 15 minutes. Thereafter, the treated (RNA reduced) biomat may be transferred onto a belt filter and washed with water (by forcing water through the biomat, to wash out RNA breakdown products). Optionally, in the method, the biomat may pass from the belt filter through one or more groups of paired rollers to sgueeze liguid from the biomat, thereby to produce a biomat with a reduced level of RNA.

In a modification of the method, represented in Figure 3, a belt filter 8a, travelling in the direction of arrow 12, carries a biomat 1 a. The biomat 1 a is transferred onto a belt 8b which transports the biomat into a receptacle 14 containing water at 65°C. The biomat is conveyed from belt 8b to a belt 8c (the biomat is numbered 1 c on the belt 8c), A lid or other means may be provided to force the immersion of the biomat. The biomat 1 c is then conveyed from belt 8c to belt 8d and subsequently passes from the receptacle onto belt 8e, the biomat being represented as biomat 1d. On belt 8e, the biomat may be washed to produce a biomat with a reduced level of RNA.

Example 6 - Treatment of biomat to reduce level of RNA (“Method RNA3”)

As an alternative to RNA reduction processes which utilise heated water, RNA reduction may comprise heating by means other than using water. For example, a biomat produced as described in Example 3 may be treated whilst present in a tray in which it is produced, or after removal from such a tray, with means to produce a radiative heating effect on the filamentous fungus of the biomat, thereby to cause cells of the fungus to rupture. The radiative heating effect may be produced, for example, by microwaves, infrared or hot air.

Alternatively, RNA reduction may comprise pulse electric field processing or electroporation of a biomat.

The aforementioned RNA reduction processes may be undertaken on a biomat when still present on a tray. In this case, the treatment may, advantageously, be undertaken whilst the filamentous fungus is in a growing state which is found to enhance RNA reduction/removal. Alternatively, the biomat may be transferred from a tray onto a belt filter. If the process is undertaken with the biomat present on the tray, after treatment, the biomat is suitably washed by spraying water onto the biomat on a belt filter in a manner analogous to that described in Example 4 (although the water sprayed need not be at a temperature of 65°C). Alternatively, the biomat may be washed in a receptacle and treated as described in Example 5 (and, again, the water used need not be at a temperature of 65°C).

Example 7 - Maintaining filamentous fungus in growing state prior to RNA reduction

In a variation of Example 4, the biomat may be transferred from the tray onto the moving belt filter whilst in its growing state and may be maintained in its growing state on the belt filter by spraying fresh culture medium onto the biomat. Then, whilst in the growing state, the biomat may be subjected to a heat shock. The heat shock may comprise spraying the biomat with 65°C water as described in Example 4 or by heating by one of the methods described in Example 6. A further alternative means of delivering a heat shock may comprise directing pressurized steam at the biomat to shock it and raise its temperature to 60-70°C. Heat shock as described in this example is intended to cause rupture of cells of the filamentous fungus and leaking from the cells of RNA and/or products of RNA enzymatic digestion. The RNA and/or products may then be washed from the biomat to produce a biomat comprising filamentous fungus with a reduced RNA level.

Example 8 - Modified belt filter for biomat production and RNA reduction

As an alternative to culturing filamentous fungus in a tray as described in Example 3, an integrated tray/belt filter apparatus may be used as represented in Figure 2. The apparatus 2 includes a tray 4 which contains culture medium 6 in which filamentous fungus may be cultured as described in Example 3. In addition, however, tray 4 incorporates a belt filter apparatus 8 which is positioned towards the bottom of the tray during initial growth of a biomat as described in Example 3. However, after a biomat of appropriate dimensions/density has been produced, the belt filter 8 may be raised (e.g. it may be arranged to be lifted by hydraulic or other means) so that the filter surface of the belt filter contacts the underside of the biomat and lifts it from the surface of the culture medium. When so disposed, the biomat may be treated to reduce the level of RNA and/or washed as described in Examples 4 to 7.

The procedures of the preceding examples may be used to produce biomats which may be further processed to produce foodstuffs. For example, biomats may be size-reduced and a mass of filamentous fungus incorporated into foodstuffs as described in, for example, WO2017/151684, WO2019/046480, W02020/176758 or WO2020243431 , assigned to The Fynder Group, Inc.

Example 9 - Biomat production on belt filter

As an alternative to the culturing methods described, a biomat may be produced on an upper surface of a filter as represented in Figure 4.

Referring to Figure 4, a belt filter apparatus 8 is shown which comprises an endless filtration belt 50 which is arranged to be driven by rollers 52, 54 in the direction of arrow 56. A tray 58 is positioned immediately below an inner surface of the belt, wherein the tray is substantially full of culture medium 60. The apparatus may include a roller (or other guide) (not shown) at or adjacent a region 62 which may function to urge the belt 50 downwards so it contacts the medium 60. The apparatus is thereby arranged so that the belt may wick or absorb medium 60 which may pass from its inner surface to its outer surface, suitably so the belt is saturated with medium. The belt is inoculated and conditions selected so that a biomat 64 gradually grows on the outer surface of the belt. Figure 4 shows how the biomat may become thicker as it is conveyed rightwardly (as shown in Figure 4). During growth of the biomat, culture medium may be continuously introduced into tray 60 to replenish it and allow for continuous wicking or absorption of culture medium into the belt 50 to keep it saturated. Adjacent end 66 of the apparatus, there may be provided a knife or other suitable device for removing the formed biomat 64 continuously from the belt and depositing it in a suitable location. Downstream of end 66, the belt may be treated to clean and sterilise it, with excess fluid being removed by one or more pairs of nip rollers (not shown). Then the portion of the belt from which biomat has been removed may, in due course, pass back around to end 68 whereafter it may again be urged at position 62 into the medium 60 for subsequent growth of biomat.

Thus, the apparatus of Figure 4 may be used continuously to grow and isolate a continuous, long length of biomat.

The apparatus described may be of any suitable size. For example, tray 60 may have a surface area of at least 5 m² or at least 10 m². The tray may, for example, be 3 to 4 m wide and 2 to 4 m long. Thus, relatively large amounts of biomat may be produced in a readily automated continuous process.

The biomat of Figure 4 may, optionally, be treated to reduce RNA as described in any of Examples 4 to 8. Such treatment may be carried out subsequent to biomat growth as described with reference to Figure 4. It may be undertaken on the same belt 50 on which the biomat is grown or biomat may be transferred onto another belt or into another apparatus to reduce the level of RNA using the methods described.

The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Claims

1 A method of treating a biomat comprising:

(i) associating a biomat with a filter membrane;

(ii) contacting said biomat with a treatment fluid;

(iii) moving fluid away from the biomat via the filter membrane.

2 A method according to claim 1 , wherein said biomat is generated via surface fermentation.

3 A method according to claim 1 or claim 2, wherein said biomat comprises a filamentous fungus, wherein the biomat has a cell density of at least 25 g dry weight ZL media and/or the biomat is 1 mm to 30 mm thick.

4 A method according to any preceding claim, wherein said biomass comprises filamentous fungi which, optionally, are acidophilic, such as species and/or strains of Fusarium, Fusisporium, Pseudofusarium, Gibberella, Sporotrichella, Aspergillus. Penicillium, Triocoderma, species within the Mucorales sp. (e.g., Rhizopus sp.) and the filamentous fungal strain designated as MK7.

5 A method according to any preceding claim, wherein said treatment fluid is arranged to support growth and/or facilitate growth of filamentous fungus of said biomat; or to heat shock the filamentous fungus of said biomat, for example to render said filamentous fungus non-viable; or to wash the filamentous fungus of the biomat.

6 A method according to any preceding claim, wherein the biomat is produced in a receptacle, for example a tray, and the method comprises transferring the biomat from said receptacle to said filter membrane, where it is contacted with said treatment fluid.

7 A method according to any preceding claim, wherein, in an embodiment (A), said treatment fluid comprises culture medium which is arranged to support growth and/or facilitate growth of said filamentous fungus of said biomat.

8 A method according to claim 7, wherein said biomat is associated with a filter membrane, by resting on a surface of said filter membrane and, when so disposed, is contacted with said treatment fluid which comprises said culture medium.

9 A method according to claim 7 or claim 8, wherein said culture medium is sprayed onto said biomat, suitably so it permeates the biomat. 10 A method according to any of claims 1 to 6, wherein, in an embodiment (B), said treatment fluid comprises a heated fluid which comprises steam or heated water.

11 A method according to claim 10, wherein said treatment fluid is arranged to raise the temperature of the filamentous fungus of the biomat to a temperature in the range 60-70°C.

12 A method according to any preceding claim, wherein said filter membrane is moveable and/or moves in the process, suitably to convey a biomat thereon from a first position at which a biomat is introduced onto the filter membrane to a second position from which the biomat is withdrawn from the membrane, said second position suitably being downstream of said first position.

13 A method according to any preceding claim, wherein said filter membrane is part of a belt filter wherein, suitably, the filter membrane is perforate and is conveyed.

14 A method according to any preceding claim, wherein, in the process, fluid passes through the biomat and, thereafter, passes through the perforations of a or said filter membrane.

15 A method according to any preceding claim, wherein a receptacle in which a biomat is produced in a surface fermentation process comprises a moveable filtration membrane which is moveable between a first position in which the membrane is below a surface of liquid in the receptacle and a second position in which the filtration membrane is closer to a surface of liquid in the receptacle.

16 A method according to any preceding claim, wherein said biomat, subsequent to step (iii), includes less than 82 wt% water; and includes at least 18 wt% of filamentous fungus (on a dry matter basis).

17 A method according to any preceding claim, wherein said biomat, subsequent to step (iii), includes less than 2 wt% of RNA on a dry matter basis.

18 A method according to any preceding claim, wherein step (i) of the method is undertaken with said biomat in the presence of its growth medium.

19 An assembly comprising a biomat associated with a filter membrane, wherein the biomat and filter membrane are as described in any preceding claim. 19

20 An assembly according to claim 19, wherein said filter membrane is moveable to move the biomat between first and second positions and/or said filter membrane is a component of a belt filter.

21 An assembly according to claim 19 or claim 20, wherein, in said assembly, the biomat includes a reduced level of RNA, compared to the level of RNA produced by culturing filamentous fungus to produce the biomat.

22 An assembly according to any of claims 19 to 21 , wherein said assembly includes a spray device for spraying culture medium and/or water onto a biomat associated with said filter membrane.

23 A method of producing an assembly according to any of claims 19 to 22, the method comprising transferring a biomat from a receptacle, for example in which it is produced onto said filter membrane.

24 A method of producing a biomat, suitably as described in any preceding claim, the method comprising contacting the biomat with culture medium from a position above a biomat, wherein, optionally, the method comprises spraying culture medium on top of the biomat.

25 An assembly comprising a biomat associated with a solid support which supports the biomat.

26 An assembly according to claim 25, wherein the thickness of the biomat at a first position on the support is be less than at a second position on the support, wherein optionally, the biomat is of gradually increasing thickness on moving from the first to the second positions.

27 An assembly according to claim 25 or claim 26, wherein the biomat has a width which is at least 1 m, more preferably at least 2m; and/or the biomat has a length on the support of at least 2m, preferably at least 4m; and/or the area of a face of the biomat is at least 3m² or at least 10m².

28 An assembly according to any preceding claim, wherein said assembly includes culture medium (which may be as described in any preceding claim), wherein said culture medium is in contact with the support.

29 An assembly according to claim 28, wherein said culture medium is provided in a receptacle which is arranged under the biomat. 20

30 An assembly according to any preceding claim, wherein said support includes openings for passage of fluid and, optionally, said support is arranged to transport fluid by capillary action and/or to wick fluid.

31 An assembly according to any preceding claim, wherein said support is movable, suitably to convey the biomat, between a position A and a position B and, optionally, the assembly is arranged for removal of the biomat from the support at position B.

32 An assembly according to any preceding claim, wherein said support comprises an endless surface.

33 An assembly according to any preceding claim, wherein said support comprises a filter membrane which may be as described in any preceding claim and, optionally, said support comprises a belt filter which is as described in any preceding claim.

34 An assembly according to any preceding claim, wherein said assembly include means for urging said support into culture medium and/or into a volume defined by a receptacle for culture medium.

35 An assembly according to any preceding claim, wherein said assembly includes means for removing fluid from the biomat, which is, optionally, arranged to compress the biomat to remove fluid therefrom.

36 An assembly according to any preceding claim, wherein said assembly include any feature of the assembly of any of claims 19 to 22.

37 A method of producing an assembly according to any of claims 25 to 36, the method comprising growing a biomat on a solid support having any feature of the support of any of claims 25 to 36.

38 A method according to claim 37, wherein the method comprises producing a biomat having any feature of the biomat of any of claims 25 to 36.

39 A method according to claim 37 or claim 38, wherein the method comprises the passage of fluid, for example, culture medium from one surface of the support to another surface as described in any of claims 25 to 36; and/or the method comprises transport of fluid, for example culture medium, by capillary action and/or by wicking through the support. 21

40 A method according to any of claims 37 to 39, wherein said method comprises moving the support between a position A and position B as described in claim 31 and any claim dependent thereon.

41 A method according to any of claims 37 to 40, wherein the method comprises movement of the support in an endless travel path.

42 A method according to any of claims 37 to 41 , wherein said method comprises urging the support into a receptacle containing fluid, for example culture medium.

43 A method according to any of claims 37 to 42, wherein said method comprises removal of fluid from the biomat, suitably after a predetermined thickness of biomat has been produced.

44 A method according to any of claims 37 to 43, wherein said method includes any feature(s) of the methods of any preceding claim.

45 A method of producing a biomat, the method comprising the method of any of claims 37 to 44 followed by removal of the biomat from the support, wherein, optionally, the biomat may be as described in any preceding claim.