FUNGAL STRAINS, PRODUCTION AND USES THEREOF

Abstract

This invention relates to novel Penicillium strains, the production of novel Penicillium strains, and the use of novel Penicillium strains in various end applications.

Claims

1. A method of producing strains of Penicillium by sexual reproduction comprising: a. crossing complementary strains of Penicillium of mating type MAT1-1 and MAT1-2; b. inoculating the isolates onto an agar medium; c. incubating the inoculated isolates at a temperature of between about 10-20° C.; and d. obtaining ascospores.

2. Method according to claim 1, further comprising the step of obtaining progeny strains from said ascospores.

3. Method according to claim 1 or 2, wherein said mating type strains are selected from any of Penicillium roqueforti strains 74-130; 74-144; 74-133; 74-146; strain Penicillium roqueforti 5A (74-256) deposited under the Budapest Treaty as CBS 145358, referred to herein as “5A”; strain Penicillium roqueforti 12A (74-257) deposited under the Budapest Treaty as CBS 145359, referred to herein as “12A”; strain Penicillium roqueforti A22 (74-160) deposited under the Budapest Treaty as CBS 145360, referred to herein as “A22”; strain Penicillium roqueforti B20 (74-170) deposited under the Budapest Treaty as CBS 145361, referred to herein as “B20”; a strain obtainable by sexually crossing strains of Penicillium roqueforti 74-130 and 74-144 and having similar characteristics to A22; a strain obtainable by sexually crossing strains of Penicillium roqueforti 74-133 and 74-146 and having similar characteristics to B20; variant strains having at least 99% sequence identity to any of the aforementioned strains.

4. Method according to claim 3, wherein strain 74-130 is sexually crossed with strain 74-144 and optionally produces strain A22 or a strain having similar characteristics to A22.

5. Method according to claim 3, wherein strain 74-133 is sexually crossed with strain 74-146 and optionally produces strain B20 or a strain having similar characteristics to B20.

6. Method according to any preceding claim, wherein said Penicillium is Penicillium roqueforti, Penicillium paneum, Penicillium glaucum or Penicillium camemberti.

7. Ascospores obtainable by a method according to any preceding claim and progeny strains obtainable from said ascospores.

8. A strain of Penicillium selected from: a. strain Penicillium roqueforti 5A (74-256) deposited under the Budapest Treaty as CBS 145358, referred to herein as “5A”; b. strain Penicillium roqueforti 12A (74-257) deposited under the Budapest Treaty as CBS 145359, referred to herein as “12A”; c. strain Penicillium roqueforti A22 (74-160) deposited under the Budapest Treaty as CBS 145360, referred to herein as “A22”; d. strain Penicillium roqueforti B20 (74-170) deposited under the Budapest Treaty as CBS 145361, referred to herein as “B20”; e. a strain obtainable by sexually crossing strains of Penicillium roqueforti 74-130 and 74-144 and having similar characteristics to A22; f. a strain obtainable by sexually crossing strains of Penicillium roqueforti 74-133 and 74-146 and having similar characteristics to B20; g. variant strains having at least 99% sequence identity to the genome sequence of any of the strains defined in (a) to (f).

9. A derivative of a Penicillium strain having an altered pigment development pathway and capable of producing spores of a different colour from corresponding strains having an unaltered pigment development pathway.

10. A derivative strain according to claim 9, wherein said derivative is a derivative of ascospores or progeny strains according to claim 7 or a derivative of a strain according to claim 8.

11. A derivative strain according to claim 9 or 10, wherein the spores are any one or more of the following colours: light olive brown, light brown, olive brown, brown, reddish, reddish-brown, reddish-brown-pink, intense blue to light blue, green and albino.

12. A derivative strain according to any one of claims 9 to 11 having altered (increased or decreased or substantially eliminated) expression of one or more of the following metabolites: heptaketide naphthopyrone; 1,3,6,8-tetrahydroxynaphthalene; scytalone; 1,3,8-trihydroxynaphthalene; vermelone; 1,8-dihydroxynaphthalene, or metabolites produced as a result of the action of genes Alb1, Ayg1, Arp2, Arp1, Abr1, Abr2 from Penicillium species.

13. A derivative strain according to any of claims 9 to 12, wherein said pigment development pathway is altered by any one or more of: U.V., X-ray or chemical mutagenesis, gene editing, gene transformation, for example, to overexpress or downregulate one or more of the following genes: Alb1, Ayg1, Arp2, Arp1, Abr1, Abr2.

14. A strain according to claim 8, or a strain derived from ascospores according to claim 7, or a derivative according to any of claims 9 to 13, having altered protease and/or lipase levels and/or activity and/or altered levels of metabolites relative to either one or both parental strains used in the sexual cross, or relative to known strains used in the manufacture of dairy products.

15. A strain according to claim 8, or a strain derived from ascospores according to claim 7, or a derivative according to any of claims 9 to 13, when used in the production of a food product, produce food products having an altered organoleptic profile relative to either one or both parental strains used in the sexual cross, or relative to known strains used in the manufacture of dairy products.

16. A method for altering the colour of fungal spores, optionally spores of a Penicillium strain, comprising altering the pigment development pathway.

17. Fungal spores obtainable by a method according to claim 16, wherein said spores differ in colour from corresponding strains having an unmodified pigment development pathway.

18. A composition comprising a strain according to claim 8, or ascospores or a strain derived from ascospores according to claim 7, or a derivative according to any of claims 9 to 13, or fungal spores according to claim 17, and an acceptable carrier, such as an agriculturally acceptable carrier, a carrier suitable for nutraceutical administration, a carrier suitable for use in food applications, a carrier suitable for bioremediation applications.

19. Composition according to claim 18, comprising at least one additional bacteria or fungus.

20. Use of a composition according to claim 18 in any one or more of the following: (i) the production of or in a foodstuff, (ii) production of a nutraceutical, (iii) bioremediation, (iv) enzyme productions, (v) fermentation process, e.g. in the production of biofuels, (vi) in the production of or in animal feed.

21. Use according to claim 20, wherein said foodstuff is a cheese starter culture, cheese or a cheese product, such as a cheese sauce, cheese spread, dried cheese ingredient.

22. A cheese comprising a composition according to claim 18, a strain according to claim 8, or ascospores or a strain derived from ascospores according to claim 7, or a derivative according to any of claims 9 to 13, or fungal spores according to claim 17.

23. A cheese according to claim 22 which is a hard cheese or a soft cheese, a blue cheese, whether ripened by a Penicillium internal or external mould, optionally wherein the cheese is a Roquefort, Bleu de Bresse, Bleu du Vercors-Sassenage, Brebiblu, Cabrales, Cambozola (Blue Brie), Cashel Blue, Danish blue, blue Cheddar, Fourme d′Ambert, Fourme de Montbrison, Lanark Blue, Maytag Blue, Strathdon Blue, Blue Murder, Shropshire Blue, Dorset blue vinney, Brighton blue, Moyden's Wrekin blue, picos blue, cabrales, rokpol, dolcelatte, and Stilton, and some varieties of Bleu d′Auvergne, a Gorgonzola, a Brie, a Camembert or any other cheese ripened by a Penicillium internal or external mould.

24. A method of screening for strains having flavour profiles comparable to the strains of any preceding claim, comprising the steps of: a. carrying out GC-MS analysis of strains B20, A22, 5A or 12A, variants or derivatives thereof as defined in any preceding claim; b. carrying out GC-MS analysis of one or more query strains; c. selecting those strains in (b) which group together with strains B20, A22, 5A or 12A, variants or derivatives thereof as shown in the GC-MS readings.

25. Strains obtainable by a screening method according to claim 24.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0103] One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0104] FIG. 1 illustrates the positioning of aliquots of fungal spore suspensions on 9 cm Petri dishes of oatmeal agar (1=isolate of MAT1-1 genotype; 2=isolate of MAT1-2 genotype).

[0105] FIG. 2 illustrates the presence of sexual reproductive structures in P. roqueforti. (a-d) Paired cultures of 74-88×74-92 on oatmeal agar with yellow cleistothecia (arrowed) produced along the barrage zones following four weeks incubation at 10° C. Scale bar, 300 μm.

[0106] FIG. 3 provides evidence for meiotic recombination in P. roqueforti. Segregation patterns of a RAPD-PCR amplicon parental isolates (P1, P2) and 12 ascospore progeny (1-12) from the cross 74-92 (P1)×74-88 (P2), using primer OPW10 MM, molecular weight marker; C, water control; kb, kilobase. Arrow indicates the diagnostic RAPD band. Lane headings: MAT1-1 (red) and MAT1-2 (green) genotypes.

[0107] FIG. 4 shows a principle component analysis to illustrate that the sexual offspring from a P. roqueforti cross (labelled 1-30) show production of combinations of flavour volatiles which differ from those of the parental strains (Parent 5 and Parent 18) derived from Gorgonzola and Roquefort cheeses.

[0108] FIG. 5 shows a principle component analysis to illustrate that the sexual offspring from a P. roqueforti cross (labelled E-O) show production of combinations of metabolites including mycotoxins which differ from those of the parental strains (labelled A and B).

[0109] FIG. 6 shows results of a real-time PCR assay to study induction of protease genes when P. roqueforti cultures of both parental isolates (labelled 88 and 92) and sexual offspring (labelled 118-127) were transferred to a milk casein inducing medium.

[0110] FIG. 7 illustrates a dose response ‘kill curve’ when spores of P. roqueforti strain 74-88 were exposed to a UV radiation source for between 0-150 seconds.

[0111] FIG. 8 illustrates some of the colour mutant strains of P. roqueforti produced after UV mutagenesis, including light brown, reddish-brown, green and intense blue colour sporulating mutants.

[0112] FIG. 9 shows the pigment development pathway of Aspergillus and Penicillium (taken from Krijgsheld et al., 2013).

[0113] FIG. 10 shows GC-MS “electronic nose” results for 5A, 12A, A22, B20.

[0114] FIG. 11 shows the lipolytic activity of the novel strains A22, B20, 5A and 12A.

[0115] FIG. 12 shows the proteolytic activity of the novel strains A22, B20, 5A and 12A.

[0116] FIG. 13 shows the growth rate of the novel strains A22, B20, 5A and 12A.

DETAILED DESCRIPTION

Sexual Crossing in Penicillium

[0117] Penicillium roqueforti

[0118] A worldwide strain collection of over 100 P. roqueforti isolates was established from various sources including cheese (for example blue cheeses), cheese ripening cultures, tortillas, bread, hay silage, contaminated foodstuffs and cooking oil, and samples from this collection were used in this study.

Routine Culture Growth and Maintenance

[0119] Cultures of P. roqueforti were grown according to routine methods on potato dextrose agar (PDA—39 g potato dextrose agar power (Oxoid, UK) made up to 1 L with distilled water) or malt extract agar (MEA—20 g Malt Extract powder (Sigma, UK), 1 g Peptone (Oxoid, UK), 20 g Agar (Oxoid, UK) made up to 1 L with distilled water) on 9 cm Petri plates or as slope cultures in Universal tubes, using a swab to transfer mycelia from a pre-existing culture for inoculum as appropriate. Cultures were grown in the light for one week at 28° C., then sealed with Nescofilm and stored at 4° C. Long term stocks were also established under liquid nitrogen or as silica gel stock at 4° C.

DNA Extraction

[0120] For extraction of genomic DNA from Penicillium species, mycelium was produced from liquid cultures. A spore solution was prepared by adding 1.5 ml 0.05% (v/v) tween 80 to a slope of a culture, which was agitated with a swab and recovered. The spore solution was then added directly to 250 ml conical flasks containing 50 ml of yeast extract glucose liquid growth medium (YEG: 8 g yeast extract, 40 g glucose/per litre). Cultures were grown at 28° C. on a rotary shaker at 150 rpm for 5 days. The resulting mycelium was collected by filtration through a double layer of Miracloth (Calbiochem, USA) and rinsed thoroughly with 0.1M potassium phosphate buffer (pH 7.0). The harvested mycelium was freeze-dried and stored at −80° C. until DNA extraction was performed. DNA was extracted using a DNeasy Plant Mini kit (Qiagen Ltd, UK) in accordance with the manufacturer's instructions. Concentrations of DNA were estimated by comparisons with lambda DNA quantity markers (Promega, USA) on 0.8% agarose gels (SeaKem® LE Agarose, Lonza Group Ltd, USA).

Screening of P. roqueforti for Mating-Type Genes

[0121] A multiplex PCR diagnostic test was employed to amplify mating-type genes and therefore determine the mating type of individual isolates. DNA was obtained from four isolates of P. roqueforti (74-9, 74-10, 74-41, and 74-42) which had previously been confirmed to be P. roqueforti, based on sequencing of the b-tubulin gene.

Sexual Assay of P. roqueforti

Sexual Crosses

[0122] Various crosses were set up to study the potential for sexual reproduction of P. roqueforti. Six isolates of P. roqueforti used in cheese production comprising one MAT1-1 strains and five MAT1-2 strains were crossed in all possible pair wise combinations (n=5). In addition, crosses were set up between four isolates from field sources comprising two MAT1-1 and two MAT1-2 strains.

[0123] Spore suspensions of each isolate (5×10.sup.5 conidia ml.sup.−1) were prepared as follows. Conidia were collected from two month old cultures grown at room temperature on slopes of MEA or PDA. A sterile 10 μl inoculating loop (Greiner) was gently rubbed across the surface of the sporulating colony on the slope. One loop full of conidia was transferred to 5 ml of a 0.05% (v/v) tween 80 solution containing 0.05% agar. The purpose of addition of the agar was to increase the viscosity of the medium in order to prevent the formation of stray colonies during subsequent inoculation. The resulting suspension was homogenized by using a vortex mixer and quantified using a Haemocytometer to estimate spore density and diluted to a target concentration of approximately 5×10.sup.5 conidia ml.sup.−1. The spore suspension was then used immediately to avoid any change in viability due to storage.

[0124] 1 μl aliquots of each spore suspension was separately inoculated on to an oatmeal agar surface (Robert et al. 2007. CBS Yeasts Database, The Netherlands, Centraalbureau voor Schimmelcultures, Utrecht) ca. 4 cm apart and perpendicular to aliquots of conidia of the opposite mating type (O'Gorman et al 2009; Nature, 457, 471-4). This arrangement created four interaction or ‘barrage’ zones as colonies grew (FIG. 1). Petri dishes were sealed with one layer of Parafilm, incubated inverted in continuous darkness at 10° C. or 20° C., and examined periodically for cleistothecia over different lengths of time according to the particular experimental cross.

Preparation of Single Ascospore Cultures

[0125] Mature cleistothecia from P. roqueforti cross 74-88×78-92 were removed and cleaned by rolling on agar and an ascospore suspension prepared by crushing of the cleistothecia.

[0126] More specifically, ascospore suspensions were prepared according to O'Gorman et al Nature, 457, 471-4. Mature cleistothecia were picked off from surrounding hyphae using a flame sterilised needle, observed under a Nikon-SMZ-2B dissecting microscope, taking care to avoid conidia where possible. 5-10 cleistothecia were transferred to a 4% (w/v) water agar plate and gently rolled across the agar surface using a sterilised needle tip to remove any adhering conidia, taking care to keep the cleistothecium intact. Next, 20-50 μl of pH 6 sterile Tween 80 (BDH)(0.05%) was pipetted into a sterile 1.5 ml Eppendorf tube and cleistothecia ruptured against the side of the tube in this small droplet, releasing a macerate of peridium, asci, ascospores and contaminating conidia. This was made up to a final volume of 500 μl in pH 6 sterile Tween 80 (0.05%) washing the cleistothecia contents from the side of the tube with final vortex-mixing for 1 min to release the ascospores. The concentration of the resulting ascospore suspension was determined using a haemocytometer and were diluted as appropriate.

[0127] Where appropriate, to inactivate any contaminating conidia, the suspension was then heat treated for different periods of time and at different temperatures to kill any remaining conidia whilst at the same time retaining viable ascospores e.g. heat treatment at 69° C. for 10 min (NB. in some species the ascospores and conidia were killed at the same temperature so this was not possible).

[0128] To isolate individual ascospores, 100 μl of a 5×10.sup.5 ascospore ml.sup.−1 suspension (either with or without heat treatment) was spread inoculated on three defined areas of an ACM plate. Triplicate plates were prepared and incubated at 37° C. for 14 h. Single-spore cultures were established on ACM by transferring individual germinating ascospores with a LaRue lens cutter attached to a Nikon-Optiphot microscope or by picking up individual germinating ascospores with a flame sterilised platinum wire.

Evidence of Recombination

[0129] The possible occurrence of recombination in the ascospore offspring of a sexual reproduction between P. roqueforti was assessed by examining the segregation of RAPD-PCR markers and the mating-type genotype in progeny. An initial screening of nine RAPD primers revealed five primers that were suitable for genotyping of P. roqueforti (OPAX16, UBC90, OPW10, OPAJ05, OPA11).

Results of Sexual Crosses in Penicillium roqueforti

[0130] The availability of the PCR-based MAT diagnostic tests allowed directed crosses to be set up between P. roqueforti isolates of known MAT1-1 or MAT1-2 identity. Results of crossing efforts are described below.

Production of Cleistothecia

[0131] Crosses were set up involving five P. roqueforti isolates used in cheese manufacture, comprising one MAT1-1 isolate (74-88) crossed in all combinations to four MAT1-2 isolates (74-89, 74-90, 74-91 and 74-92) (Table 2). Crosses were incubated at 10° C., 15° C., and 20° C. to assess any capacity to undergo teleomorph stage development. Significantly, after four weeks incubation at all three temperatures, cleistothecia were observed and all were found to contain ascospores when squashed. Similar number of cleistothecia was produced at all three temperatures, however slightly higher numbers were apparent at 10° C. (Table 2). The crosses were then re-incubated for a further month and re-examined for the presence of cleistothecia. However, the longer incubation period yielded no apparent increase in the number of cleistothecia. The pairing of 74-88×78-90 produced the highest number of cleistothecia at all temperatures, and was very consistent with the formation of over 1,000 cleistothecia per plate (Table 2). Developing cleistothecia were initially white and soft when young, and then matured becoming pale orange-brown to bright yellow after 1-2 months of incubation. The cleistothecia formed along the barrage zones between isolates of opposite mating type (FIG. 2(a-d)).

[0132] Cleistothecia were also formed in some crosses between the four P. roqueforti isolates from field sources (Table 3).

TABLE-US-00006 TABLE 2 Number of cleistothecia produced by P. roqueforti crosses on oatmeal agar medium at 10° C. in the dark after one month. Number of cleistothecia MAT1-2 Crosses 74-89 74-90 74-91 74-92 MAT1-1 74-88 > >> > > Ratings indicate the mean number of cleistothecia produced from three replicate crosses on oatmeal agar in 9 cm diameter Petri dishes after incubating in the dark for 1 month at 10° C.: >more than 100 cleistothecia, >>more than 1,000 cleistothecia.

TABLE-US-00007 TABLE 3 Number of cleistothecia produced by P. roqueforti crosses on oatmeal agar medium at 10° C. in the dark after one month. Number of cleistothecia MAT1-2 Crosses 74-4 MAT1-1 74-84 > MAT1-1 74-86 > Ratings indicate the mean number of cleistothecia produced from three replicate crosses on oatmeal agar in 9 cm diameter Petri dishes after incubating in the dark for 1 month at 10° C.: >more than 100 cleistothecia but less than 1,000.

Evidence of Recombination

[0133] A recombination analysis of the ascospore offspring using molecular markers was conducted to confirm that meiosis had taken place. It was found that a heat shock of 70° C. for 10 min was sufficient to break the ascospore dormancy and activate germination, whilst killing any contaminating conidia. Distinct segregation patterns were clearly observed when six RAPD markers and the MAT genotype were scored in 12 ascospore progeny (FIG. 3 and Table 4). Unique genotypes were found in 83% of the progeny, with only two of the offspring identical to its parent (based on the markers examined (Table 4)). These results provide convincing data that P. roqueforti exhibits a heterothallic sexual breeding system.

TABLE-US-00008 TABLE 4 Genotypes.sup.a in the parental isolates and 12 ascospore progeny of a cross between P. roqueforti isolates 74-88 × 74-92 Mating RAPD band.sup.b Isolate type OPAJ05 UBC 90 OPAX 16 OPW 10 OPA 11 Genotype.sup.c 74-88 MAT1-1 − + − − + P1 74-92 MAT1-2 + − + + − P2 88-92-1 MAT1-1 + + − + + A 88-92-2 MAT1-2 + − − − − B 88-92-3 MAT1-2 − − − − − C 88-92-4 MAT1-2 − + − − − D 88-92-5 MAT1-2 + − − + − E 88-92-6 MAT1-1 + − + − − F 88-92-7 MAT1-1 + + + − − G 88-92-8 MAT1-1 − + + − − H 88-92-9 MAT1-2 + + − − − I 88-92-11 MAT1-1 − − − − − C 88-92-13 MAT1-2 − + + − + K 88-92-18 MAT1-1 − + − + − L P- value(2- tailed).sup.d 1.00 1.00 1.00 0.545 1.00 Contingency χ.sup.ef 0.454(1) .sup.aGenotypic characterization mating- type and RAPD-PCR bands. .sup.bRAPD-PCR bands amplified using operon primers OMT1 or R108. ‘+’ and ‘−’ denotes presence or absence, respectively, of particular amplicon. .sup.cThe genotype of each progeny isolate, defined by unique combinations of mating-type and RAPD markers as distinct from the parental isolates (designated P1 and P2), is identified by a different letter of the alphabet .sup.dFisher's exact test for deviation from the null hypothesis of independent assortment of mating-type and RAPD markers in the progeny (i.e. a 1:1:1:MAT1-1+:MAT1-1−:MATt1-2+:MAT1-2− ratio for each RAPD marker). Fisher's exact test was used instead of the χ.sup.2 test because the expected frequencies were <5 .sup.eTo test for deviation from the null hypothesis of independent assortment of mating-type and RAPD markers in the progeny (i.e. an overall 1:1:1:1 MAT1-1+:MAT1-1−:MAT1-2+:MAT1-2−ratio for the sum of the RAPD markers). .sup.fNumber in parenthesis indicates the degree of freedom.

[0134] The data above clearly demonstrates the ability of Penicillium roqueforti, used in the production of blue-veined cheeses, e.g. Roquefort, Danish blue, and Gorgonzola (Nichol, 2000) to sexually reproduce and produce progeny strains with different genotypes to either parent.

Production of Cleistothecia

[0135] Crosses were set up involving five P. roqueforti isolates used in cheese manufacture, comprising one MAT1-1 isolate (74-88) crossed in all combinations to four MAT1-2 isolates (74-89, 74-90, 74-91 and 74-92) (Table 5). Crosses were incubated at 10° C., 15° C., and 20° C. to assess any capacity to undergo teleomorph stage development. Significantly, after four weeks incubation at all three temperatures, cleistothecia were observed and all were found to contain ascospores when squashed. Similar number of cleistothecia was produced at all three temperatures, however slightly higher numbers were apparent at 10° C. (Table 5). The crosses were then re-incubated for a further month and re-examined for the presence of cleistothecia. However, the longer incubation period yielded no apparent increase in the number of cleistothecia. The pairing of 74-88×78-90 produced the highest number of cleistothecia at all temperatures, and was very consistent with the formation of over 1,000 cleistothecia per plate (Table 5). Developing cleistothecia were initially white and soft when young, and then matured becoming pale orange-brown to bright yellow after 1-2 months of incubation. The cleistothecia formed along the barrage zones between isolates of opposite mating type (FIG. 2(a-d)).

[0136] Cleistothecia were also formed in some crosses between the four P. roqueforti isolates from field sources (Table 6). However, isolate 74-64 (MAT1-2) failed to form cleistothecia in any of the attempted crosses.

TABLE-US-00009 TABLE 5 Number of cleistothecia produced by P. roqueforti crosses on oatmeal agar medium at 10° C. in the dark after one month. Number of cleistothecia MAT1-2 Crosses 74-89 74-90 74-91 74-92 MAT1-1 74-88 > >> > > Ratings indicate the mean number of cleistothecia produced from three replicate crosses on oatmeal agar in 9 cm diameter Petri dishes after incubating in the dark for 1 month at 10° C.: >more than 100 cleistothecia, >>more than 1,000 cleistothecia.

TABLE-US-00010 TABLE 6 Number of cleistothecia produced by P. roqueforti crosses on oatmeal agar medium at 10° C. in the dark after one month. Number of cleistothecia MAT1-2 Crosses 74-4 74-34 MAT1-1 74-84 > — MAT1-1 74-86 > — Ratings indicate the mean number of cleistothecia produced from three replicate crosses on oatmeal agar in 9 cm diameter Petri dishes after incubating in the dark for 1 month at 10 ° C.: —none, >more than 100 cleistothecia but less than 1,000.

Proteolytic Activity, Lipolytic Activity and Growth Rate

[0137] Table 7 below and FIGS. 11, 12 and 13 show the proteolytic activity, lipolytic activity and growth rate for the novel strains A22, B20, 5A and 12A. “Low” defined as below 25% percentile. “Med/Low” up to median. “Med/high” up to 75% percentile. “High” above 75% percentile. Penicillium roqueforti strains 74-88, P2, A7 and F11 may be found at the Nottingham University BDUN Collection.

TABLE-US-00011 TABLE 7 Proteolytic activity, Lipolytic activity and Growth Rate of various strains Proteolytic Lipolytic Isolate Activity Activity Growth Rate 74-88 Med/High Med/High Med/Low P2 Med/High High High A7  Low Low Low A22 High Med/High Med B20 Med/Low Med/Low Low  5A Med/High Low High 12A Low Med/Low Med/High F11 Med/Low Med/High Med/Low

Phenotypic Variations in the Properties of Penicillium Strains Produced by Sexual Crossing

Differences in Flavour Properties

[0138] For example, a cross was set up between strains of Penicillium roqueforti derived from Roquefort and Gorgonzola type-blue cheeses. Thirty sexual progeny were recovered from this cross. The GC-MS technique was then used to determine levels of production of 16 volatiles linked to flavour production in an artificial cheese system based on growth in UHT milk in laboratory shake culture. Data for individual flavour volatiles were then pooled using principal component analysis (FIG. 4). This revealed that the parents (labelled Parent 5 and Parent 18 in bold font) occupied certain flavour spaces corresponding to the relative production of a variety of flavour volatiles. By contrast, the majority of the sexual offspring (labelled 1 to 30) occupied different flavour spaces on the principal component analysis, with some isolates (e.g. 2, 4, 9, 3, 18, 21) exhibiting quite different volatile flavour production from the parents, while others (e.g. 28, 8, 1) exhibited intermediate volatile flavour production between the two parents (FIG. 4). Thus, there is evidence of the production of different relative levels of flavour volatiles in the sexual offspring, likely to correlate with different flavour properties in cheese.

Differences in Mycotoxin Production

[0139] For example, a cross was set up between two blue cheese production strains of Penicillium roqueforti (A and B) and sexual progeny (E-O) were recovered from this cross. The HPLC technique was then used to determine levels of production of over 25 metabolites including known mycotoxins from representative offspring when grown in laboratory liquid culture media. Data for individual metabolites and mycotoxins were then pooled using principal component analysis (PCA) (FIG. 5). This revealed that the parents (labelled A and B in blue font) occupied certain areas of the PCA spaces corresponding to the relative production of the variety of metabolites including mycotoxins. The majority of the sexual offspring (also labelled in blue) exhibited intermediate metabolite and mycotoxin production between the two parents (e.g. K, O, M). However, some offspring showed quite different levels of metabolite including mycotoxin production (e.g. G, J, L). Thus, there is evidence of the production of different relative levels of metabolites including mycotoxins in the sexual offspring, offering the opportunity to select for offspring with lowered levels of mycotoxin production.

Differences in Protease Profile

[0140] A cross was set up between two blue cheese production strains of Penicillium roqueforti (88 and 92) and six sexual progeny (118, 119, 121, 124, 126 and 127) were recovered from this cross. Both the parental isolates and the sexual offspring were then first grown on a non-milk based nutrient agar. They were then transferred to media containing milk protein (casein) and levels of induction of protease enzymes were determined using real-time PCR methodology, measuring induction of protease genes (FIG. 6). This analysis revealed differences in the protease profiles of the parental isolates (88 and 92), and that the sexual progeny exhibited levels of protease production which differed considerably from parent 88, but which were mostly similar to parent 92 although there were some more intermediate values (118) and some offspring showed protease production lower than either parent (119, 124) (FIG. 6). This small sample indicates that differences in protease production will be present in the sexual offspring relative to the parents, offering the opportunity to select for offspring with desirable levels of protease production.

Mutagenesis of Penicillium Strains to Produce Different Coloured Spores

[0141] To produce mutants of Penicillium roqueforti by UV mutagenesis, a UV cabinet was switched on and allowed to warm up for 30 minutes (this also helps sterilize the UV box). A dose response curve was then performed to determine a suitable time to expose fungal material to result in a suitable decrease in viability. Fungal spores were produced on PDA and spore suspensions were made in sterile water aseptically and diluted to a concentration of 200,000 spores/ml, and cooled in a fridge for at least an hour. Spores were then transferred to a sterile 5 cm Petri dish and were continuously mixed using a sterilized magnetic stirrer in a 10 ml volume of spore suspension. The spores were then exposed to the UV radiation source at a certain distance from the lamp (standard between 30-40 cm below lamp) and aliquots of the spore suspension removed at suitable intervals (for example, between 30 seconds to 10 minutes) and stored in a fridge for 1 hour, using 3 technical replicates per time point. Spores were then transferred to 12 cm square plates containing PDA and then surviving spore numbers counted approximately 30 to 36 hours later.

[0142] In the case of P. roqueforti strain 74-88, it was found that a suitable 5-10% survival rate resulted after exposure of spores for 60-90 seconds (FIG. 7). The resulting germinating fungal colonies that were exposed to UV radiation for 60-90 seconds were allowed to grow and sporulate. At this point mutants showing differences in colour from the wild type were then selected, such as light brown, reddish-brown, pink, green and intense blue mutants (FIG. 8).

Production of a Blue Cheese Using a Penicillium Strain of the Invention.

[0143] In small-scale production trials, approximately 40 litres of whole, pasteurised milk was placed in hard plastic buckets. Milk was added at just over 34° C. to allow for the small volumes and the culture room was held at around 24° C. A mixed mesophilic starter culture from Christian Hansen and a gas giver, leuconostoc, from Danisco were added together with 5 ml of calcium chloride per bucket. Chymax rennet from Christian Hansen, 10 ml per bucket, diluted with 4 parts water was also added. The buckets were incubated, resulting in curd and whey formation. The curds were cut using an 11 mm square cutter and removed with a colander, then during moulding and turning further whey was removed by gravity, resulting in cheese wheels of perhaps 2.8-3 kg in size. The cheese was then turned a number of further times over 3-4 days. After one further day firming up is performed at around 16° C. followed by first and second salting in successive days then ripening at 12° C. Cheeses are then pierced during week 2 and by week 3 mould development should be apparent. Cheeses are then allowed to ripen for a suitable time between 2 and 3 months.