"Submerged cultures of mushroom mycelium as sources of protein and flavour compounds"
Dissertation Delft, 1976
F.IJ. Dijkstra

After a brief introduction on the subject and a review of the most important developments as regards the production of mushroom mycelium since Worgan's extensive review [160], the purpose of our own investigations is outlined in Chapter 1.

The nutritional requirements of our strains of Agaricus bisporus and Coprinus comatus are presented in Chapter 2. An incubation temperature of 25 °C and an initial pH between 6.5 and 7.5 were optimal. Several carbohydrates were tested as sources of carbon and energy. For Agaricus bisporus the optimum initial concentration of glucose was 30 g/1 and for Coprinus comatus 60 g/l. Several differences were observed in the ability of our strains and those examined by other workers to utilise carbohydrates. Saccharose could be used after acid hydrolysis. Coprinus comatus could metabolise lactose in the presence of glucose. Several non-carbohydrate compounds were tested as carbon and energy sources. Alcohols, hydrocarbons and salts of organic acids were not suitable. Several lipids could be utilised, especially oleic acid esters. Small amounts of these esters also stimulated mycelial growth when carbohydrates were major sources of carbon and energy.

Agaricus bisporus and Coprinus comatus needed an organic compound as source of nitrogen and one or more amino acids. Several differences were observed between the amino acid requirements of our strain of Agaricus bisporus and different strains investigated by other authors. The carbon to nitrogen ratio resulting in a maximal yield of Coprinus comatus was between 5 and 10. For Agaricus bisporus this ratio was between 2.5 and 10, depending on the nature of the nitrogen source.

Both strains needed thiamine as growth factor. In addition, the growth of Coprinus comatus was promoted by adenine. Maillard compounds, which were formed during heat sterilisation of the culture medium, stimulated the growth of Agaricus bisporus but not that of our strain of Coprinus comatus. A number of other mushroom species were examined for their growth in the presence of Maillard compounds. Several strains were stimulated by Maillard compounds, while no inhibition of the growth of any species was observed.

Various natural media were used as complex sources of nutrients for Agaricus bisporus and Coprinus comatus. Malt extract, skim milk, casein, vegetable oil, an extract of cauliflower leaves, cotton seed flour and single cell protein were suitable for the growth of one or both strains.

The possibilities of using our Agaricus bisporus and Coprinus comatus strains as sources of protein for food purposes are discussed in Chapter 3. In view of the high nutritional requirements and the slow growth rate, the prospects do not seem to be very promising; this was confirmed by amino acid analyses. The balances of almost all essential amino acids were negative; hence the dissimilation of the tested complex media by these mushroom mycelia did not result in any nutritional enrichment.

Concerning the amino acid composition of mycelium of our strains, we came to the same conclusions as other authors working with mushroom mycelium. The mycelial protein of both strains contained sufficient quantities of most amino acids essential to human nutrition. Only the concentrations of the sulphur containing amino acids were too low. At most 24 to 34% of the nitrogen in the mushroom mycelium was represented by non-protein compounds, for instance nucleic acids.

Chapter 4 begins with a review of the literature on mushroom flavours. Because several aroma compounds had already been identified in fruit bodies of Agaricus bisporus, we only determined their concentrations and evaluated their effect on the flavour. The natural (-)-form of 1-octen-3-ol had a stronger flavour than the (+)-form and was the most important aroma component. Nucleotides, amino acids and carbohydrates also contributed significantly. Benzaldehyde, benzyl alcohol, 1-octen-3-one, n-butyric and isovaleric acids as well as low boiling volatiles contributed less to the flavour. Nucleotides did not stimulate the flavour of 1-octen-3-ol nor that of carbohydrates.

In an aqueous extract of fruit bodies of Coprinus comatus 3-octanone, 3-octanol, 1-octen-3-ol, 1-octanol, 2-methyl-2-penten-4-olide, 1 -dodecanol and caprylic acid were identified conclusively and n-butyric and isobutyric acids tentatively. Amino acids, nucleotides and sugars were also determined. A synthetic mixture of 36 compounds found in the extract had a stronger flavour than the natural extract. 3-Octanol, 1-octen-3-ol, 1-octanol and 2-methyl-2-penten-4-olide, were the volatiles with the strongest flavour.

Because in Agaricus bisporus and Coprinus comatus 1-octen-3-ol, 5'-GMP and glutamic acid were important flavour compounds, the concentrations of these substances were compared in 8 fresh, 3 canned and 5 dried mushrooms. The highest amounts of 1-octen-3-oi and 5'-GMP were found in the fresh mushrooms. Agaricus bitorquis, Pleurotus ostreatus and Pholiota squarrosa contained 5 to 7 times as much 1-octen-3-ol as Agaricus bisporus and Calvatia gigantea 58 times as much. Coprinus comatus and Pleurotus ostreatus contained much 5'-GMP. Little 1-octen-3-ol and 5'-GMP were found in most dried and canned mushrooms. Glutamic acid was present in most samples in sufficient quantities to have an important influence on the flavour.

We concluded that 1-octen-3-ol can be used as a test substance for the presence of a mushroom flavour, while the presence of 5'-GMP and glutamic acid may provide further confirmation.

The results of our studies on the production of flavour compounds by mushroom mycelium in submerged culture are reported in Chapter 5. Twelve strains of mushroom mycelium and three strains of Beauveria tenella (originally isolated as Agaricus campestris) were examined for their production of 1-octen-3-ol and 5'-GMP in shaken flasks. Agaricus bisporus, Calvatia gigantea, one strain of Coprinus comatus, Morchella esculenta and Morchella hortensis produced both flavour compounds in varying amounts. Agaricus bitorquis, Armillaria mellea, Beauveria tenella, one strain of Coprinus comatus, Lentinus edodes, Pleurotus ostreatus and Volvariella volvacea produced little or no 1-octen-3-ol and varying amounts of 5'-GMP.

In most cases 1-octen-3-ol was found in the mycelium, whereas 5'-GMP was often released to a large extent into the culture medium. The quantities of these flavour compounds produced by the mycelium in submerged culture were compared with the amounts found in fruit bodies. 1-Octen-3-ol was formed and accumulated by Agaricus bisporus and Coprinus comatus in early stages of the growth, after which the concentration decreased. Contrarily the concentration of 5'-GMP was maximal in later stages of the growth. The production of flavour compounds could be influenced by modification of the culture medium. The original medium contained malt extract and casein: this was modified by the addition of olive oil or Tween 80, and by substitution of glucose plus meat extract, yeast extract, or vegetable extract for malt extract.

For a more detailed investigation and identification of the volatiles present in the mycelium, Agaricus bisporus and Coprinus comatus were grown in 10 litre fermentors. The influence of aeration and agitation on the maximum yield of mycelium, growth rate, crude protein content of the mycelium, carbohydrate consumption, pellet diameter and production of aroma compounds were studied. The development of Agaricus bisporus could be observed indirectly on the basis of the laccase activity.

An aroma concentrate of Agaricus bisporus mycelium from the fermentor under conditions resulting in the highest yield showed the presence of benzaldehyde, benzyl alcohol, 3-octanol, 3-octanone and 1-octen-3-ol. These compounds have all been found in fruit bodies. In the Coprinus comatus mycelium we identified 3-octanone, 2-methyl-2-penten-4-olide and 1-octene-3-ol, which have been found in fruit bodies too, as well as 2-octen-l-ol, which has not yet been identified in the corresponding fruit bodies.

The mycelial pellets of Agaricus bisporus and Coprinus comatus smelled and tasted like mushrooms, but the flavours were not identical. The flavour of Coprinus comatus mycelium was much stronger than that of Agaricus bisporus mycelium.

After the growth of the mycelium the flavour of the culture broth, which contained most of the 5'-GMP, was not very good, necessitating thorough elimination of the medium from the mycelium.

We may conclude, that mushroom mycelium with a good flavour can be grown within one week. With the assay techniques used in the present investigations, it seems possible to screen a great number of strains or mutants and select those with a high flavour production. If strains can be found combining this property with simple nutritional requirements, it may become possible to produce mushroom mycelium economically, for the purpose of improving the aroma properties of foods by addition of such flavour-rich mycelium.