Morel Mushroom Mycelium Growth in Waste Sulfite Liquors as Source of Protein and Flavouring.**

Morel Mushroom Mycelium Growth in Waste Sulfite Liquors as Source of Protein and Flavouring.~ A. LeDllY, N. Kosaric and .T. E. Zajic Chemical and Biochemical Engineering Faculty of Engineering Science The University of Westem Ontario London, Ontario, Canada

Abstraet Three species of morels (Morchella spp., MorchelIa crassipes and Morchella esculenta) were cultivated in the waste sulfite \iquors (Mg-base and NH 3 -base)-ammonium phosphate dibasiccorn steep \iquor media. Thc spectrum úÍ essential amino acids is comparable to the FAO standard, except for the levels of methionine and isoleucine. The gas-liquid chromatogram shows the presence of aH essential fatty acids induding palmitic, stearic, oleic and linoleic acids. All amino acids and fatty acids present in the fruiting bodies of the meadow mushrooms (Agaricus bispoms) were found in the samples of morel mushroom mycelium (MMM) grown in waste sulfite liquors (WSL). A strong mushroom f1avour (aroma and taste) is also conserved in the freeze-dried powdered samples. The flavour concentrate of Morchella crassipes grown in NH 3 - WSL is recovered and compared with that of the meadow mushroom using gas-liquid chromatography, infrared spectrophotometry and ultra-violet absorption spectrophotometry. The flavour concentrates prepared from the freeze-dried samples lack low boiling compounds, while they are richer in high boiling components. Tentative interpretation of the infrared spectra revealed the presence of ketones, aldehydes, alcohols, phenols and esters in all flavour concentrates.

Résumé Trois especes de morilles (MorcheHa spp., Morchella crassipes et Morchella esculenta) sont cultivées dans le milieu de culture composé de la liqueur noire de sulfite (de la base Mg ou NH 3 ) du phosphate dibasique d'amme,nium, et de la liqueur venant des procédés industriels de mals. Le spectrum des acides aminés essentiels est comparable a la combinaison type F.A.O., sauf pour les niveaux de méthionine et isoleucine. La chromatographie liquide-gazeuse a démontré la présence de tous les acides gras essentiels, ainsi que des acides palmitique, stéarique, oléique et linoléiaue. Tous les acides aminés et acides gras présents dans les champignons communs (Agaricus bisporus) sont trouvés aussi dans le mycélium de morilles (MM) cultivé dans la liqueur noire de sulfite (LNS). Une forte saveur de champignons (l'odeur et le gout) est aussi conservée dans les échantillons séchés a froid. La saveur concentrée du Morchella crassipes cultivé dans la LNS-NH3 est préparée et comparée avec ceHe deschampignons communs, utilisant la chromatographie liquid-gazeuse, la spectrophotométrie en régime infrarouge et ceHe en régime ultraviolet. Les saveurs préparées a partir des échantillons séchés a froid sont dépourvu de substances a basse température d'ébullition, tandis qu'elles sont plus riches en substances a haute température d'ébullition. L'interprétation globale des spectra de la spectrophotométrie en régime infrarouge a révélé la présence des groupes de kétone, aldéhyde, alcool, phénol et ester dans tous les échantillons de la saveur concentrée.

Introduetion Many mushroom eaters considered that the morels, Morchella species, have the finest flavour of aH mushrooms (Groves, 1958). A favourable aroma and taste have also been found in the mycelium of morels (Costantin, 1936). Morel mushroom mycela (MMM) have been cultivated in various substrates by many workers (Brock, 1951; Falanghe et al., 1964; Fron, 1905; Janardhanan et al., 1970; Litchfield et al., 1963a, b; Szuecs, 1956). In a sugar medium, the MMM * based on the paper presented at the 16th Natlonal Conference of the Canadian Instltute of Food Science and Techno!ogy. Vancouver, May 30 - June 1. 1973.

44

have high nutritional value in terms of protein and amino acids (Litchfield et al.) 1963c) and B-vitamin contents (Litchfield, 1964). Due to their potential use as flavouring materials (Litchfield, 1967), the l\fMM have been produced in large scale for commercial use under the trade name Powdered Morel Mushroom Flavouring (Heinemann, 1963; Klis, 1963). However, this production was discontinued (B. Heinemann, MidAmerica Dairymen, Inc., personal communication). Waste sulfite liquors (WSL) have been used as a cheap substrate for the growth of MMM (Cirillo et al.) 1960; Kosaric et al., 1973; Reusser et al., 1958). Because of the abundance and availability of the WSL and because of the disposal problems associated with WSL, it is of particular interest to convert the organic material from these liquors to a valuable source of protein and flavouring. High protein content is recently reported for the samples of MMM grown in various WSL (Kosaric et al., 1973). In this papel', the amino acid and faHy acid como position of the samples of Morchella crassipes, Morchella esculenta and Morchella spp. grown in the Mg-WSL and NH)"vVSL are investigated. The flavour of the MMM in WSL is extracted, analyzed and compared with that of fresh meadow mushrooms.

Materials and Methods Waste sulfite liquors: The eoncentrated Mg·WSL and NH 3 -WSL samples were received from various pulp and papel' milIs. They were prepared and stored as previously reported (Kosaric et al., 1973). Culture maintenance and cultivation method: The maintenance of three species of morels, Morchella c1'assipes NRRL-2369, Morchella esculenta NRRL-2603, and MM'chella spp., the procedure for the preparation of inoculum as well as the composition and conditions for the cultivation of these fungi in a 5-gaHon botUe fermentor were performed as previously described (Kosaric et al., 1973). The MMM growth was filtered by gravity through cotton gauze, then washed twice with 1% NaCl in distilIed water and then freeze-dried. The dry samples were ground into fine powder and stored in closed botUes at room temperature for further analyses. Determination of amino acids: For amino acid determination, the dry mycelium (305.0-315.8 p,g) was hydrolyzed by refluxing with 500 mI of 5.7 N HCI for 24 hours. The hydrolyzate was dried in the rotary evaporator and then redissolved in an appropriate volume of buffer. Norleucine was added as internal standard and aliquot was applied to sample J. Inst. Ca·n. Sel. TechnoJ. Aliment. Vol. 7, No 1. 1974

cartridge for anal,rsis using Acid Analyzer.

Tech~icon

Determination of fatty acids: The fatty acids of MMM on WSL wel'e analyzed by gas-liquid chromatography of their methyl esters. The transmethylation of individual MM:M: fatty acids was performed according to Carroll et al. (1968). Thc chloroform soluble lipid extracts of IMMM (50 to 100 mg dry weight) were mixed with 10 mI of 10% acetyl chloride solution in methanol and then refluxed fol' about 2 hours at 70°C. Methyl esters were extractcd three times with petroleum ether. Combined petrolelllH ether extracted portions were then washed with deionized water until neutral. They were evaporated to dryness under vacuum and redissolved in chloroform prior to injecting into the gas-liquid chromatograph. The gas-liquid chromatography was performed on an F&:M: Model 5750 Research Gas Chromatograph using flame ionization detector. An 8-foot long and % inch ID stainless steel column was packed with 10% EGSSX on Chromosorb P. The carrier gas used was heliull1 at arate of 60 cc/minute. The fatty acid methyl ester standards (National Heart Institute, Bethesda, Md., U.S.A.) were used for identification of MMJt[ methyl ester fatty acids and also for testing the linearity of response in the gas-liquid chromatograph. Quantitation of the fatty acids was based on products of retention time and peak height (Carroll, 1961). Analysis of mushroom flavour: The flavour concentrates were prepared from the following: (i) 10 g freeze-dried powder, and (ii) 100 g fresh pellets of MMM 01' fresh ground fruiting bodies. They were continuously extracted with 250 mI diethyl ether for 24 hours using Soxhlet apparatus. The extracts (solvent phase) were filtrated and reduced to approximately 5 mI under vacuum and stüred in the refrigerator (O°C) for further use. The gas-liquid chromatography analyses of flavou!' concentrates were performed on an F&:M: Model 5750 Research Gas Chromatograph provided with an 8-foot by Va inch OD column packed with 20% Carbowax 20 M on 60-80 mesh Chromosorb "'VAW-DMCS. The operat· ing conditions were: temperature programming from 65° to 250°C at 10°C per minute, then isothel'mal for 40 minutes. Both injection port and detector tempera· tu res were 210°C. The ultraviolet absorption spectrum of the flavour concentrates was determined on the Perkin-Elmer Model 124 Double Beam Grating Spectrophotometer. The infrared spectrum of the flavour concentrates was determined on the Beckman Model IR-20 Double Beam Infrared Spectrophotometer. The diethyl ether (solvent) from the flavour concentrates was removed under vaCUUlll and at 30°C. The residue was dissolved in an excess alllount of anhydrous lllagnesium sulfate treated spectral grade chloroform. For IR spectrophotollletry tl1e solution was treated with anhydrous magnesium sulfate, then filtrated and concentrated under vacuum. The thickness of the potassium bromide cells was 0.1 mm. Can. Inst. Food SeL Teehnol. J. Vol. 7, No. 1, 1974

.

"O

'rSM-1 Amino

O'"

<"2 ¡,...;s

CIl

C':lC':lC':lCll

CIl

c.:i

-:t
c.:i

"'l'

,....;

C':l

-:t<

en

,....,

'"

2 obt;., Q,.S --O

.~.-=:

o

"':2..c <'-

Col"'l'Colcot-mColCOm"'l'COO ..... t,.., o ..... o C':lCll '-O ..... Col~ ..... O '-O Col

o '-O"'l' ..... mm

'-OCIlt-Col '-Ot- ..... "'l'

O r-: o e-; e-; -:t< e-;e-;If:i O e-;""'; c.:i-:t<" r-i c-i""¡ 1) c.:i-:t
.....


~..$ .-. o¡,.. '"

Qi

j Col"'l'C':l

Col t-omcomColo,-o ..... m'-OCIl O ... C':lCll"'l't-t- ..... COCO"'l''''l'O'''l'

<:ó

COCll~

c
e-;e-;e-;-:t
.....

.....

co C':l

Col C':l

If:i

-:t<

S

'" &

......, '"

,....,

-:t<

c
c
.....

.J:lbll • >:l

::E

cococo ColCOCll

C':l COColC':lCol ..... m'-O"'l'CIlOC':lm '-O ... '-OCO"'l'mo"'l'COCll'-O"'l'OCO

o.E~

Col o

'-O CIl

e-;

e-;

.~

¡::

..... Col'-O m ..... co

t- com ..... m"'l' ..... t-'-OOOColCol CIl ... CIlOCO ..... CIlCOColC':lCllt- ..... t-

=8~ c.>::l",

-:t<

"'
c
c
.....

"'l' co

m CIl

e-;

r-:

...

'"

OCOCO CIlCll"'l'

CIlCOO ..... Col'-O Col "'l' ..... t-t-o Col co I~~'"'"!'"'"!'"'"!'"'"!~~~oq~t-: -:t< ColC':l"'l'mC':l,-oOC':l ..... Colm .....

o

"'O"" f.E~ c;¡bll

-i,....i-:¡

.....

'-O "'l'

o o

e-;

e-;

Id

¡:i.

OCllCllm"'l't-Colt-"'l'C':l .......... OCll CO ..... Colcot-m'-OCIlCol,-oCIlm"'l'm

olf:ioc
.....

::E .~

..... m"'l' m om

o

C'i,....i0

..... CIl"'l'Col mt- ..... '-O

.;;

I

,.::bll

~::E o ¡::

¡::

o

Q,

..... t- "'l' "'l'COCllCol Col ..... CIlmColmC':l ..... (2;'-Oco "'l'C':l C':l Col I ~~~'"'"!~~~~ .t-:'"'"! .. mm"'l' ..... t-Colm 0<:6 "'l''''l'C':l'''l''''l'mO'''l' ..... C':l'-O O e-; c
.....

~

o

--o


:>

.f!,..:¡

;.,

1::

en

-ELS c;¡ I

",bll

::E'" ::E~::E .s --o

..

'-' Col '-O o "'l' '-O co t- ..... m\f)"'l'mmcoo~C':lmco ..... CIl CIl t"'l' ..... I ~~~oq'"'"!~~ .t-:'"'"!~ .. C':l CIl Col 0<:6 "'l''''l'CIlC':l'''l'mom ..... '''l''-O O e-;c
"'''':¡

'-

'Z

~en

::E .~~ f! ~ ::E c;¡::r:

¡:: ¡::

CIl tmco

olf:i

"'l''-OC':l'-OColco':t''''l'co ..... m

I ;~~~~~~;:;~~ ..

CIl Col '-O \f) CO CIl

t-mt-tt-mC':lCO

olf:i,....; .. olf:ie-;e-;

.s

8

' " bll I ...

< ::E~::E .S
<~

'o'" "

d.

t- ..... comOCol"'l' ..... C':lOC':l\f)OCO ~o ..... mC':lt-O\f)Colcot-OCllt-

..... o '-O m C':l0

cot-mm mOC':lm

ocóoe-;e-;lf:ie-;e-;oioe-;,....;c
.....

o

¡::

::l

,....,0

+8

......,'" .-0 8 ¡::

o 1:: '" '-"0 8'~

'"o

.Si'"

,....;

:o E-< '"

::l

¡::

'" "'2

~,..:¡

l:l.en

'A"

....:<

::E .s '~LS

t:

>:l ~

"O

o

:=§

o

¡::

'" ::E

'" '8 <

--o

'O '"

o

S

o o

::E .~

'"::l

::E

..


~,..:¡ "'en :> ~LS
.J:l

Q,

,...., o-:t
"'2

..

..

&
'"
o

¡::

.....

Q, '"
'"'" ~.=: ~bll ~ o ¡::

.;

...o

..c::l

'" '" '" =8

8o

"'2 o

¡::

.~

--o
~

'"Q,'"

::E•

J 'o" "'3


bll

,....,

...¿

=s .S .~

.&o .&
...o

d
g

...i:f

"O


'-O .....

..

o

'-

l:l o ~o""

'bll

2

J

P-.::E .~

'2

";

...

I

~ '" o o::l:>

~

..... ......,

--o

::E '" --oSbt ~ 8-S
~

o C':l '-O CIl

~

O s¡.enl:i ...

+

~

<

.=: o~~< u:>::E;:::; ~"; ~ ~ ~~ e- a ~::r: te ,..:¡ E-< ;>-'if::r: E-< o ...¡ Z <

E-< <::E E-< JS ü.t ü



e


~Z'

,..:¡'"

45

Results and Discussion Pl'oximate _.1nalysis: The proximate analysis of the MMM biomass was earlier reported (Kosaric et al., 1973). The freeze-dried samples of MMM in 1VSL contained,
chromato~rams

was the same for a-amino-n-butyric acid and cysteine, and these two acids were not resolved on the column. Hatanaka et al., (1968) reported the presence of ,B-alanine and y-aminobutyric acid and 4 unidentified ninhydrin positive substances in the fruiting body of Morchella esculenta. Hatanaka (1969) identified one of these compounds as a new amino acid having the structure cis-3-amino-L-proline. This amino acid was also found in the fruiting body of Morchella conica and Morchella crassipes, and in the mycelium of Morchella esculenta and Morchella conica. Litchfield et al. (1963c) reported that the trace amounts of cysteic acid, ethanolamine, methionine sulfoxide, methionine sulfoxamine, homocitrulline and a-aminon-butyric acid were detected in Morchella escnlenta, Morchella hortensis and the Powdered Morel Mushroom Flavouring, but not in Morchella crassípes. The mycelia of Morchella crassipes, Morchella csculenta and }Iorche71a hortensis contained 1.15, 2.00 and 2.00% respectively (dry-weight basis) of a-aminoisobutyric acid, whereas the Powdered Morel Mushroom Flavouring did not contain this compound in detectable concentration. A trace of 2,4-diaminobutyric acid was found in an four samples tested by these authors. Seven unidentified ninhydrin positive substances were also found in various samples of MMM (Table 2). Compounds No. 3 and No. 4 are present in large alllounts in an salllples tested, including the fresh llleadow lllushrooms. They might be responsible for the typical mushr.aolll flavour (taste and/or aroma). The cOlllpounds No. 6 and No. 7 were also comlllon in an samples tested, but usuany in trace amounts. Compound No. 1 is present in trace amounts in an samples except in Morchella spp. grown in a glucose llledium. Compound No. 2 was found in an samples except in Morchella esculenta in iMg-WSL. Oompound No. 5 was not detected in an samples includin~ the fresh meadow lllushrooms, but it was found in substantial alllounts in the species of Morchella crassipes. No attempt was made to further identify these substances. Fatty acids: Table 3 shows the quantitative results of fatÍ\' acids as compared to values obtained frolll oth~r sources. The chromatograms of fatty acids of MMM show 18 to 20 detectable substances as compared with 16 for the llleadow mushrooms. Five saturated (myristic, pentadecanoic, palmitic, heptadecanoic and stearic acids) and four unsaturated (palmitoleic, oleic, linoleic, linolenic acids) fatty acids were identified in all samples tested. The rest of the peaks might represent various isolllers, branched·chain 01' the hiaher molecular weight fatty acids. h The essential fatty acids (palmitic, oleic, 1¡I:olci~' and linolenic acids) are present in a large amount in an samples of MMM in WSL. The levels of palmitic and oleic acids are superior in the MM'M as compared to the meadow mushrooms. 'l'he concentration of linolenic acid is also higher in the samples of Morchella crassipes in Mg-WSL and NH¡-WSL. As compared to Ivanov et al., (1967) tbe linoleic acid content was J. Inst. Can. Sel. Teehnol. Aliment. Vol. 7. No 1, 1974

Table 2.

Unidentified Ninhydrin Positive Compounds of MMM and Meadow Mushrooms.

Sample

Unidentified Ninhydrin Positive Compound 1

M. crassipes in Mg-WSL M. crassipes in NH 3 -WSL M. esculenta in Mg-WSL MorcheIla spp. in Mg-WSL Morchella spp. in glucose Fruiting Body of A. bisporus

2

3

4

5

6

'"'

'"'

'"'

'"'

'"'

'"'

'"'

'"'

'"'

t

O

'"'

'"'

O

t

'"'

'"'

'"'

O

'"'

'"'

'"'

O

'"'

'"'

O

O

'"'

7

t

t

t

Legend: (t) trace amount; ('"') present in wbstantial amount; (O) not detected.

particularly high in our samples representing Morchella spp. in Mg-"'\VSL and in the fruiting bodies of Agaricus bisporus. The MorcheZla crassipes in Mg-WSL and NH3'WSL contained a lower amount of linoleic acid, about one-half of the aboye two samples. The distribution of essential fatty acids is somehow more uniform in samples of Morchella cra8sipes in Mg-WSL and NH3'vVSL and MorcheZla esculenta (Ivanov et al., 1967) while the heavy weight was confined to the linoleic acid alone as found in the samples of ~1 orchella spp. in Mg-WSL and the meadow mushrooms. The abundance of unsaturated and essential fatty acids indicates a good nutritional quality of this biomass. In this respect MMM seems also to be better than the commercial meadow mushrooms. The characteristics and pleasant flayour of MMM j:?;rown on WSL might also be partly attributed to this specific fatty acid spectrum. Cha1'acteristics of MMM flavour: Three main catej:?;ories of MMM flavours have been observed during experiments in our laboratory: a) Highly volatile components with pleasant flower-like odour. These were detected around the fermentor near the exhaust air stream and also in the filtered MM1\1 and even in the frozen sample. This flayour was only preserved if the sal11ple was stored in a frozen state. b) Less volatile cOl11ponents which smelI like ordinary mushrooms. This flavour characteristic is not lost even when the sal11ple is stored in a closed flask at room temperature. Table 3.

c)

Non-volatile components which particularly represent the characteristic mushrüom taste which is preserved in the air-dried sample. However, samples dried at 75°0 had a strong peanut 01' similar taste. Strong mushroom flavour is conseryed in the freeze-dried powder samples. It was found that Mm'cheZla crassipes in Mg-WSL have a stronger flavour than that of M orcheZla spp. in Mg-WSL, and Morchellct esculenta in Mg-WSL have a more pleasant flavour than Morchella spp. grown in j:?;lucose medium. GlIbert (1960) characterized the odour of the effluent air from submerged cultures of MMM as follows: MorcheZla hortensis and MorcheZla rimosipes, aromatic 01' estery, Morchella esculenta, earthy, Morchella an,qusticeps, slippery elm, and other species, farinaceous. He further stated that Morchella spp. develop their characteristic flavour in tIle mycelium as well as in the sporocarp. He suggested that the flavour appears to be a geneticalIy controlled characteristic in MorcheZla and the characteristic flavour of each species has been obtained in cultures of that Fpecies regardless of the medium. The flavour of the fresh MMM has also been reported by Gilbert (1960). According to taste panel evaluations, the flavour
Fatty Acids of MM},! and Meadow Mushrooms (% of total methyl esters).

Fatty Acid

- - - --

- - ._--

M. crassipes M. crassipes MorcheIla spp. M. esculenta A. bisporus (a) fruiting body bMg-WSL in NH 3-WSL in Mg-WSL

Myristic (14:0) Pentadecanoic (15:0) Palmitic (16:0) Palmitoleic (16:1) Heptadecanoic (17:0) Stearic (18:0) Oleie (18:1) Linoleic (18:2) Linolenic (18:3)

0.22 0.46 11.79 1.12 0.40 1.58 28.30 47.40+ 4.25

0.27 0.21 12.92 1.15 0.99 6.01 17.14 34.75+ 8.50

0.16 0.39 9.69 0043 0.51 1.47 5.04 72.02+ 1.20

14.1 0.4 5.4 23.6 52.8

0.24 0.76 8.69 0.33 0040 2.25 1.18 80.04 2.88

Legend: (-) not reported (+) contains alw an unknown partially resolved component (a) From Ivanov et al. (1967) Can. Inst. Food SeL Teehnol. J. Vol. 7, No. 1, 1974

47

B M CRASSlFB FhEE2E - CRIEO R:JMX B~,FlE2E-DAEDI'OIoOO'l

e

A

8IS~,

B

"'-

C

\.J

o

"-

FRESH SAMPLE

\,\L_Jv-.J>AJ----J"--"~_~"---c_A!l6PC!M __ , _l're5><_SAMf'lE

_

o ~,FREEZE -CRIED PCM1:ER 65'C

(a)

A

(b )

Gas-liquid chromatograms of the ether extracts of in NH3 -WSL; (B) freeze-dried powder of M. crassipes dried powder of A. bisporus; (a) low attenuation;

MMM and meadow mushrooms: (A) fresh pellets of M. crassipes in NH3 - WSL; (C) fresh fruiting body of A. bisporus; (D) freeze(b) high attenuation.

vnlgaris and Morchella rimosipes was acceptable but not as well liked as that of the first four. The two strains of Morchella semilibera tasted quite blando Intensity of flavour was determined by defrosting the frozen mycelium, squeezing it. and noting the persistence of the odour. In regard to intensity of flavour, the Szuecs strain was not surpassed. The flavour of this extract, as well as that in the freeze-dried samples of MMM was also conserved for a long period of time. Litchfield et al. (1963a) reported that the dry, powdered mycelia of Morchella esctllenta, Morchella crc[ssipes and Morchella hortensis grown in glucose, maltose and lactose media have been subjected alone and in taste panel evaluation. It was found that the flavour of Morchella esculenta was the mildest and Mo1-chella crassipes the strongest. A comparison has also been made between the cooked flavour of fresh sporocarp and freshly cultured Morchella escnlenta mycelium (Gilbert, 1960). The products were fried in butter in the same manner and submitted to a taste panel. The panel found the The relationship between protein as ",ell as nonprotein nitrogenous compounds of both mushroom mycelium and sporophores and flavour of various fungi has been reviewed by Litchfield (1967). However, there is a lack of comparative data on the amino acid contents of sporophores and myceliull1 and their extracts of the Morchella species. The taste of individual amino acids was characterized (Kirimura et al., 1969; Solms, 1969) as being sweet, salty, sour, bitter, monosodium glutamate-like, sulfuric, 01' tasteless. The üorrelation between amino acids and taste is a very difficult skill. It was found that sorne amino acids contribute to the inherent taste of foodstuffs themselves; sorne specific patterns of amino acid mixtures intensify the taste of foodstuffs and in crease the mouthfulness without loosing their inherent taste. The buffer action of amino acids can also contribute to the taste of foodstuffs.

Solms (1969) proposed an integrated flavour profile which is composed of four groups of substances, with close inter-relationships, each of which should occur in a specific balance to give an overall flavour sensation. Amino acids, peptides, and proteins are located at the base of this scheme, providing taste and tactile effects; certain compounds act as flavour potentiators and synergists, and act as a bridge between the nonvolatile and volatile fractions. Of the three main classes of foodstuffs, proteins, carbohydrates and lipids, it might be thought that lipids are the most important as sources of flavour compounds but the least important for their own taste and aroma. Lipids of low volatility are tasteless, partly because they are insoluble in water. Fatty acids of low volatility (above C IO ) do not taste acid 01' sour, nor do they have much aroma. Their flavour has been described as candlelike. Glycerol results from hydrolysis of lipids and has a sweet taste. The role of lipids as flavour precursors for their aldehyde, ketone, lactone and alcohol derivatives has been reviewed by Forss (1969).

Figure 1.

48

The lipid fractions of mushrooms are also oE interest in connection with taste and aroma developmento The typical flavour of mushroOll1S may well depend upon some products ,of autooxidation of unsaturated fatty acids such as palmitoleic, oleic, linoleic and linolenic acids. The essential oil and related constituents of various mushroom sporophores have been isolated and reviewed by Litchfield (1967). The carbohydrate constituents of the fruiting bodies of various mushrooms have been reported and reviewed (Litchfield, 1967). It included various reducing sugars, amino sugars, sugar alcohols, 3ugar acids, methylated sugars, methylated sugar acids, glycogen and hemicellulose. These compounds wuuld most likely contribute to a sweet flavour and probably are not major constituents of typical fresh mushroom flavour. J. Inst. Can. bef. Technol. Aliment. Vol. 7. No 1, 1974

cllella crassiZJes in NH 3-WSL as compared with the fresh pellet growth. The freeze-dried sample of meadow lIlushrooms contained less components in the lower temperature region (below 250°0) but more compounds in the higher temperature region (250°0 isothermal) . It is suggested that the freeze-dried samples contain most of the high boiling compounds while the fresh samples contain most of the low boiling compounds. The low boiling compounds which are not present in the freeze-dried samples are probably lost during the freeze-drying process. The fact that more compounds were found in the freeze-dried samples of both mushrooms, particularly in the high temperature region of the gas-liquid chromatograms might be explained as follows: three categories of less-volatile and non·volatile flavour compounds were present in the mushrooms: (i) soluble in water only, (ii) soluble in diethyl ether only, and (iii) soluble in both water and diethyl ether. The compounds in category (i) were not shown in all of the chromatograms; those in category (ii) were shown in the chromatograms of the fresh samples; and those in category (iii) were shown in the chromato-

WAVELENGTH IN MICRONS

80

60 40 20

A - M. CRASSIPES , FRESH PELLETS

O

60 40 ~

20

~

Z O

O

~

100

iB

B - M. CRASSIPES, FREEZE-DRIED PO'NDER

~

Ci!

O~-------------..

1-

~

w

á?

~

20 o~

e-A. BISPORUS, FRESH SAMPLE

W U

z

100

~

40

'-,

t'/',-,

~

CJ)

~

40

I roJI

60 / /

D

-A.BISPORUS,

OL.-_ _---JI V

4000

3000

FREEZE-DRIED PO'N~

L.---'-_-'----'-_-'-_L.---'-_..L....J

2000

WAVENUMBER CMFigure 2.

600 I

Infrared spectra of the ether extracts of MMM an<;! meadow mushrooms: (A) freshpel1ets of M. crassipes in NH3-WSL; (B) freeze-dried powder of M. crassipes in NH3-WSL; (C) fresh fruiting body of A. bisporus; (D) freeze-dried powder of A. bisporus.

The B·vitamin content of Morchella Spp. have been determined (Litchfield, 1964; Szuecs, 1956). However, there is no evidence to indicate that these constituents have any major role as mushroom flavour constituents. Analysis 01 Ilavour extracts: The diethyl ether extractsof both fresh and freezedried samples of Morchella crassipes grown in NH 3 WSL and meadow mushrooms were analyzed by the gas-liquid chromatography, infrared spectrophotometry and uItraviolet absorption spectrophotometry. The gas-liquid chromatograms (Figure 1) showed more compounds in the freeze·dried sample of MorCan. Inst. Food Sci. Technol. J. Vol. 7. No. 1, 1974

~

~

/

I I

,,

/

/ /

/ /

~"

," .

~

~

O ~

_ .... " "

20

I

/

1 1 1

"

I

I

W

40

1 1

100

o~

1

/

80

O

1

/. /

~

20

,,

\

I I I

zU

g ~

60

80

i

~

~

400 Figure 3.

U)travio)et absorption spectra of the ether extracts of MMM and meadow mushrooms: (A) fresh pel1ets of M. crassipes in NH 3-WSL; (B) freeze-dried powder of M. crassipes in NH 3-WSL; (C) fresh fruiting body of A. bisporus; (D) freeze-dried powder of A. bisporus.

49

grallls of the freeze-dried samples. At the end of the Soxhlet extraction, two phases (solvent and water) were obtained from the fresh salllples, while only one phase (solvent) was obtained from the freeze-dried samples. In the latter case, aH compounds in the categories (ii) and (iii) would be expected to be present in the same chrolllatogralll. The gas-liquid cbrolllatograms of MMM and meadow mushrooms showed many similar compounds. The distinguished flavours of both fungi might be due to their non-similar compounds and/or relative proportions of the similar compounds. It is to be noted that during the ether extraction, tbe volatile compounds of the flavour were lost, tbe remaining are 1ll0Stly non-volatile cOlllpounds wbicb are responsible mainly for tbe taste rather than the aroma of lllushrooms. The taste of lllushrooms might play a superior r'Úle to the aroma in practical meal preparations. The infrared spectra (Figure 2) of the ether extracts of MMM and meadow l11ushrooms were similar in the functional group region (above 1300 cm-l ). Eowever the finger print regions (909-1300 cm-l ) were different for two fungi and also depend upon the preparation of the fresh 01' fl'eeze-dried sample before extraction. The difference was also found in the ultraviolet absorption spectl'a (Figure 3) of four samples tested. Tentative intel'pl'etation ol the infral'ed spectrogl'al11S of the ether extracts of mushroom flavours reveals the presence of these functional groups: ketone, aldehyde, alcohol, phenol and estero The str'Úng absorption band at 1725 Cl11-1 (A,B), 1720 Cl11-1 (C) and 1700 cm-l (D) may be attributed to the C=O stretching vibration for ketones, aldehydes and estel's. The C=O stretchin¡.!; vibration for aldebyde may be tbe absorption band in the 1685-1740 cm-l region; the same for alcohols, phenols and esters in the 1000-1260 cm-l region. Tbe absorption band at 2850 Cll1- 1 might confine to the C-E stretchin~ vibration of aldehydes. The O-E bendin¡.!; vibration of alcohol and phenol might be identified by the sharp absorption band at 1370 cm-l and a broad absorption band at tbe 650-769 Cll1-1 region. The ll1ultiple bands of llloderate absorption in the 1100-1300 Cll1-1 might result from the C-C-C O stretching and bending vibrations in the group 11 of ketones. C-C-C

50

References Brock. T. D. 1951. Studies on the Nutrition 01 Morchella eseulenta Fries. Mycologia. 43 :402. Carroll, K. K. 1961. Quantitative Estimation of Peak Areas in GasLiquid Chromatography. Nature. 191:377. Carroll, K. K .• Cutts, J. H., and Murray, E. G. D. 1968. The Lipids of Llsteria monoeytogenes. Can. J. Biochem. 46:899. . Cirillo, V. P., Hardwick, W. A., and Seeley, R. D. 1960. FermentatlOn Process for Producing Edible Mushroom Mycellum. U.S. Patent 2, 928, 210. Costantin, J. 1936. La Culture de la Morille et sa Forme Conidienne. Annales des Seienees Naturelles, Botanique et Biologle Végétales. 18:111. Falanghe, R, Smlth, A. K., and Raekis, J. J. 1964. Produetlon of Fungal Myeelial Proteln In Submerged Culture of Soybean Whey. Appl. Mierobiol. 12 :330. Forss, D. A. 1969. Role of Llpids in Flavours. J. Agr. Food Chem. 17 :681. Fron, G. 1905. Sur les Conditlons de Développement du Mycéllum de la Morille. Académie des Seiences á Paris, Compte Rendus Hebdomadaire des Seéanees. 140:1187. Gllbert, F. A. 1960. The Submerged Culture of Morehella. Mycologia. 52 :201. Groves, J. W. 1958. Mushroom Colleeting for Beginners. Canada Department of Agrlculture, Ottawa, 32 pp. Hatanaka, S. l. 1969. A New Amino Aeid Isolated from Morehella esculenta and Related Specles. Phytochemistry. 8 :1305. Hatanaka, S. I. and Terakawa, H. 1968. Bioehemieal Studles on Nitrogen Compounds of Fungi l. Distribution of Some Nonprotein Amlno Aeids 1. Bot. Mag. Tokyo. 81 :259. Heinemann, B. 1963. Proeess and Composition for Growlng Mushroom Mycellum Submerged Fermentation. U.S. Patent 3, 086, 320. Ivanov, S. A. and Bllznakova, L. 1967. Uber die Fettsaure Zusammensetzung des Lipoid-Konzentrats der industriell hergestellten Pllzmizellen von MoreheJla eseulenta Pers. und Cuprinus eomatus Fr.. Natura Plovdlv. 1 :39. Janardhanan, K. K., Kaul, T. N. and Husaln, A. 1970. Use of Vegetable Wastes for the Produetlon of Fungal Proteln from MorehelIa species. J. Food Se!. Technol. 7:197. Klrimura, J., Shimizu, A., Kimizuka, A., Nlnomlya, T. and Katsuya, N. 1969. The Contribution of Peptldes and Amino Aeids to the Taste of Foodstuffs. J. Agr. Food Chem. 17:689. Klis, J. B. 1963. Real Mushrooms In Powder Form. Food Processing. 24:99. Kosarie, N., LeDuy, A. and Zajie, J. E. 1973. Submerged Culture Growth of Edlble Mushrooms on Waste Sulphite Liquors. Can. J. Chem. Eng. 51 :186. Litchfield, J. R 1964. Nutrient Content of Morel Mushroom Mycellum: B-Vitamin Compositlon. J. Food Se!. 29:690. Lltehfleld, J. H. 1967. Morel Mushroom MyeeIlum as a FoodFlavourlng Material. Biotechnol. Bloeng. 9:289. Lltehfleld, J. H., Overbeek, R. C. and Davidson, R. S. 1963a. Faetors Affeetlng the Growth of Morel Mushroom Myeelium in Submerged Culture. Agrie. Food Chem. 11 :158. Lltehfleld, J. H. and Overbeck, R. C. 1963b. Submerged Culture Growth of Morchella Species in Food Processing Waste Substrates. Proe. 1st Intern. Congo Food Se!. TeehnoI., London, Gordon and Breeeh, N.Y., B-6, Part 11, p. 511. Lltehfleld, J. H., Vely, V. G. and Overbeek, R. C. 1963c. Nutrient Content of Morel Mushroom MyeeIlum: Amino Aeid Composltlon of the Protein. J. Food Sel. 28 :741. Reusser, F., Speneer, J. F. T., and Sallans, H. R. 1958. Protein and Fat Content of Some Mushrooms Grown in Submerged Culture. AppI. MierobioI. 6:1. 8olms, J. 1969. The Taste of Amino Aeids, Peptides, and Protelns. J. Agr. Food Chem. 17:686. Szuecs, J. 1956. Mushroom Culture. U.S. Patent 2, 761, 246. Reeelved May 15, 1973.

J. Inst. Can. Sel. TeehnoI. Aliment. Vol. 7, No 1, 1971