Flavoring agent from filamentous fungus

Abstract

Flavoring materials may be produced from filamentous fungi by contacting them with water at a temperature sufficient to reduce their nucleic acid content and concentrating or separating solids from the resulting aqueous solution. The materials may be further subjected to a chemical reaction for example with a sulphur containing amino acid.

Description

THIS INVENTION relates to flavouring materials.

It is known to use hydrolysed yeast extracts as flavouring materials. Yeast is non-toxic to humans and is normally cultured at high density (high dry cell weight per litre).

The nucleic acid content of filamentous fungi may be reduced by contacting them with water at high temperatures and separating them from the water, and such a process is described for Fusarium in PCT patent Application WO95/23843. We have discovered that the water from which the fungus is separated contains materials which can be used as or converted to flavouring materials for foods, especially if the fungus is Fusarium, for example Fusarium IMI 145,425.

The current invention comprises a method whereby the soluble components lost from filamentous fungal cells as a result of this heat treatment can be isolated and used as, or converted into, flavouring substances for foods.

This invention comprises a method of processing a filamentous fungus to improve its suitability as food which comprises subjecting it in the presence of water to a temperature sufficient to reduce its nucleic acid content substantially characterised by using materials removed from the fungus in the said method directly or after chemical reaction to flavour food.

The invention also comprises a flavouring material for food which is an aqueous solution which comprises nucleic acids removed from a filamentous fungus by contacting it with water at an elevated temperature in which the concentration of dissolved solids is sufficiently high to render the material stable to storage at a temperature of 20° C. for a period of one month or is a solid comprising such nucleic acids or is a flavouring material comprising a reaction product of such nucleic acids with a sulphur containing amino acid, hydrogen sulphide or ammonium sulphide.

The flavouring materials when in the form of an aqueous solution preferably comprises at least 30% by weight and more preferably 45 to 60% by weight of solids.

Whilst taste is an important factor in food flavours, the odours of flavouring materials are also important

The soluble components are preferably concentrated from the aqueous solution arising from the nucleic acid reduction step by removing water for example by evaporation, distillation (preferably at reduced pressure) reverse osmosis, freeze drying or freezing out the water as ice leaving an aqueous concentrate. It may suitably be removed by evaporation at reduced pressure for example at a temperature of 40 to 70° C.

The dissolved solids may be separated as such or left as a concentrated solution where the Aw (water activity) is reduced sufficiently to ensure biostasis at a range of storage temperatures.

If the nucleic acid content of the filamentous fungus is reduced by raising the temperature of its growth medium the water recovered will contain salts and other nutrients, for example glucose and/or complex nitrogen nutrients in addition to the nucleic acids and other materials derived from the fungus. If the flavour imparted by such materials is required they may be left in the materials, but if not they may either be removed, for example by osmosis or ultrafiltration, or the fungus may be washed before its nucleic acid content is reduced thereby avoiding their presence. In WO95/23843 the removal of nucleic acid from a filamentous fungus in its growing state is described; such a process is an improvement over the treatment of fungus in its resting state, for example in pure water. We have found however that the organism takes a short time to adjust from its growing to its resting state and that providing the nucleic acids are removed soon after it is separated from its growth medium the nucleic acids may be satisfactorily removed according to the procedure of WO95/23843.

We have found that after partial or complete removal of water as aforesaid the concentrate can be used as an alternative to hydrolysed vegetable proteins, yeast autolysates or yeast extract as an additive for food. The materials removed from the fungus of value in the production of savoury flavouring preparations and process flavourings. Because of the savoury nature of the flavour it may be used directly in the flavouring of Snacks, Biscuits, Stocks, Soups, Stews, Sauces and Gravies at inclusion levels of preferably between 0.1 and 15 for example 1 to 10 dry weight %.

We have also found that on heating it produces an attractive roast-type aroma. If desired it may be partially hydrolysed before heating, for example by hydrolysis with acetic acid, to produce modified roast flavours.

It may also be reacted, optionally after at least partial hydrolysis, with sulphur containing amino acids, preferably cysteine or optionally with H 2 S and/or (NH 4 ) 2 S to produce savoury flavours.

The savoury nature of the material may be altered by chemical reaction to provide a different flavour profile in that the meaty/roasted flavour notes are increased. Such “reaction flavourings” may be used in flavouring Meat (beef, chicken, lamb, pork, etc), meat alternatives (e.g. based on soy, wheat, pea protein, myco-protein), prepared meals, snacks and drinks at inclusion levels of preferably between 0.1 and 10 for example 1 to 8 dry weight %.

The flavourings may be produced by reacting materials removed from the fungus as aforesaid with cysteine. This may be carried out in the presence of water if desired; for example a 1.5 to 75 and preferably a 5 to 50 weight % solution of such materials may be reacted with cysteine in quantities of up to 10%, for example 1 to 5% of cysteine by weight based on such materials. The reaction may be carried out at a temperature of for example 110 to 140° C. at a pH of 5.5 to 9. Reaction is suitably continued for 0.5 to 7.5 hours.

It is believed that hydrolysis increases the free ribose content of the concentrate and this may be appropriate if certain flavours are desired. It is desirable to avoid treatment with hydrochloric acid for regulatory reasons (possible production of chloro-propanol derivatives), but hydrolysis with for example acetic acid may be desirable.

In the following descriptions the term “Centrate” is used for the extracellular liquid recovered by the heat shock treatment of a suspension of Fusarium at about 70° C. in the presence of its growth medium after separation of the cellular material. The term FDC means “freeze dried centrate”.

EXPERIMENTAL PROCEDURES

Material Preparation

The liquid centrate was freeze dried in order to:

reduce the water content and therefore inhibit microbial growth;

carry out studies at a high concentration of centrate;

facilitate the handling of the product

All further analyses described in this report deal with the freeze dried centrate abbreviated FDC.

Methods—Compositional Analyses

Moisture

The moisture content was determined by measuring the weight decrease of the FDC, until constant weight, while placed in an oven at 100° C.

Ash

The ash content was determined by placing the FDC in an oven at 600° C. until constant weight was obtained.

Organic Nitrogen

Kjeldahl nitrogen determination was carried out; sucrose was used as blank and glycine as standard. The results are shown in Table 1.

TABLE 1
Moisture, Ash and Organic Nitrogen Content of FDC
	Replicates
	1	2	3	Mean
	Moisture (%)	13	13	13	13
	Ash (%)	18	18	17	18
	Organic Nitrogen (%)	6	6	6	6

Amino Acids

The amino acids determination was done with a 6300 Beckman auto-analyser. The free amino acids present in the FDC were analysed using a 0.06% solution of the FDC. It was possible to measure the total amino acid content by a prior hydrolysis (HCl 6N, 24 h, oven 110° C.) of the FDC. However, the acid is known to hydrolyse tryptophan and the sulphur amino acids. The hydrolysis of the sulphur amino acids can be avoided by a prior oxidation of cysteine into cysteic acid, and methionine into methionine sulfone. This was carried out by treating the FDC with a solution of formic acid/hydrogen peroxide/methanol (48.5/1/0.5) during 4 h at 0° C. in the dark. The results are shown in Table 1a.

TABLE 1a
Amino Acid Content of FDC mean results in g/100 g FDC
		FDC	Hydrolysed FDC
	Amino Acids	(free AA)	(total AA)
	CYSTEIC ACID	0.09	0.32
	ASP	0.14	0.50
	THR	0.02	0.24
	SER	0.12	0.16
	GLU	2.02	1.70
	CYSTEINE	0.00	0.21
	PRO	0.00	0.20
	GLY	0.05	0.24
	ALA	2.05	1.73
	VAL	0.14	0.37
	CYSTINE	0.00	0.01
	METH	0.00	0.13
	ILE	0.00	0.21
	LEU	0.00	0.30
	TYR	0.00	0.06
	PHE	0.00	0.17
	TRYPTOPHAN	0.00	0.00
	NH3	1.49	4.56
	LYS	0.15	0.26
	HIS	0.00	0.08
	ARG	0.71	0.65
	TOTAL	6.98	11.80

Total Carbohydrates

The carbohydrate content of the FDC was assessed by the phenol-sulphuric acid assay method (Carbohydrate analysis: a practical approach, ed. Chaplin, Kennedy, IRL Press). Solutions of FDC and glucose (standard for calibration) were mixed with a solution of phenol in water (5% w/v). Concentrated sulphuric acid (1 ml) was added rapidly and directly to the solution surface without allowing it to touch the sides of the tube. The solutions were left undisturbed for 10 min before shaking vigorously. The absorbencies were read at 490 nm after a further 30 min.

Sugars

The sugar analysis was performed using a Dionex System of High Pressure Liquid Chromatography (HPLC), in which an eluent of HPLC grade water (1 ml/min) was used with an anion-exchange column (column Dionex PA-1) and a pulsed amperometric detector. Pure compounds were used as standards for retention time determination and quantitation. Free sugars were analysed using a 0.15% solution of FDC after filtering the solution through a 0.45 μm Minisart 25 membrane. Total sugars were also evaluated after a preliminary acid hydrolysis of the FDC (solution 0.15% in HCl 1N, 2 h, oven 110° C.) and filtration through first an Ag filter (precipitate of AgCl) and second a 0.45 μm Minisart 25 filter. The results are shown in Table 2.

TABLE 2
HPLC Analysis of Sugar Content
of FDC g sugar per 100 g of FDC
	FDC	HYDROLYSED FDC
	mean	mean
	arabinose	0.02	0.04
	galactose	0.10	0.10
	glucose	8.62	23.01
	sucrose	0.09	0.23
	xylose	0.00	0.00
	mannose	0.00	0.10
	fructose	0.01	0.00
	ribose	0.00	0.06
	maltose	2.08
	TOTAL SUGARS	12.66	25.10

Nucleic acid Derivatives

A Perkin Elmer Binary HPLC pump 250 equipped with a Spectroflow 757 ABI Analytical Kratos Division was used. Standards and samples were filtered through Acrodisc 0.45 μm Gelman Sciences membranes filters and injected by means of an injector valve equipped with a 20 μl injection loop into a reverse-phase μBondapack C18 (3.9×300 mm) Waters analytical column, protected by a μBondapack C18 guard column. A wavelength of 254 nm was used. A gradient programme with two mobile phases was used: mobile solvent A was a 60/40 methanol/water mixture and mobile solvent B was 0.02M KH 2 PO 4 (pH 5.5) prepared from potassium dihydrogen orthophosphate in distilled water and pH adjusted with IM KOH. All mobile solvents were filtered (Nylaflo 0.2 μm Gelman Sciences membranes filters) and degassed with Helium before use. The total run time was 51 min and the flow rate 1 ml/min which consisted of 100% solvent B during 5 min, followed by a gradient from 0% to 36% solvent A in 36 min and 36% solvent A for 5 min. Then a reverse gradient of 36% to 0% A was set for 5 min and the HPLC was ready for further injection after 15 min equilibrium.

Identification of the compounds was made by comparison with the retention time obtained from standards analysed in the same HPLC conditions. Standards were analysed separately to know their individual retention time and then all together to check any elution over lap that may occur in the sample case. These standards are presented in Table 3.

TABLE 3
HPLC Retention Times of Nucleic Acid Standards
			Hypox
Bases	Cytosine	Uracil	guanine	Xanthine
Ribonucleosides								C	U
2′Deoxy-
ribonucleosides
Ribonucleotides				CMP		UMP			GMP
3′MP
Ribonucleotides	CMP	UMP				GMP	IMP
5′MP
2′Deoxyribo-										DGMP
nucleotides 3′MP
2′Deoxyribo-											DIMP
nucleotides 5′MP
Retention times	3′73	4′29	4′69	5′20	5′62	6′32 + 6′32	6′70	7′52	9′79 + 9′79 +	10′74	11′49 +
									9.79 + 9.79		11′49
	Bases	Purine	Adenine
	Ribonucleosides			G		I			A
	2′Deoxy-						DG	DI		DA
	ribonucleosides
	Ribonucleotides					AMP
	3′MP
	Ribonucleotides	AMP
	5′MP
	2′Deoxyribo-					DAMP
	nucleotides 3′MP
	2′Deoxyribo-	DGMP				DAMP
	nucleotides 5′MP
	Retention times	13′33 +	17′36	21′05	21′56	22′58 + 22′58 +	23′99	26′08	32′90	35′81
		13′33				22′58

Hydrolysis of FDC

Since free ribose is highly reactive in the Maillard reaction, the effect of gentle hydrolysis conditions on FDC and the subsequent effects on flavour generation were investigated. Acid hydrolysis was carried out with sodium acetate 0.01M, pH4 adjusted with acetic acid. Standards (inosine, adenosine 5′mono phosphate-AMP5′, guanosine and guanosine 5′mono phosphate-GMP5′) were prepared at 4000 μM in duplicate and an aliquot of each solution was taken and run under the same HPLC conditions as were adapted for the analysis of nucleic acids derivatives above. The solutions were then subjected to hydrolysis for 7.5 h in an oven 100° C. (GC oven Carlo Erba). The reaction was stopped by placing the tubes in an ice bath and kept in freezer until analysis.

FDC at 2% w/v was subjected to similar hydrolysis conditions.

Flavour Mixture Preparation

As indicated previously, FDC is considered to have the potential of being either a flavouring in its own right, or a precursor in the generation of reaction—product flavours. Therefore a range of reaction mixtures were prepared and are presented in Table 4.

TABLE 4
Flavoured mixtures analysed by sniffing panel
Samle Name	Aqueous Centrate	Heated Aqueous Centrate
Sample Composition	1.7% (w/v, solids of centrate/water)	1.7% (w/v, solids
		of centrate/water)
		0.5 h 140° C.
Sample Name	Heated Buffered (pH 5.5) Centrate	Heated Hydrolysed Buffered	Heated Hydrolysed Buffered (pH
		(pH 5.5 Centrate	5.5) Centracte + C
Sample Composition	1.7% (w/v, solids of	1.7% (w/v, solids of	1.7% (w/v, solids
	centrate/sodium acetate 0.01M)	centrate/sodium	of centrate/sodium
	0.5 h, 140° C.	acetate 0.01M)	acetate 0.01M)
		7.5 h, 110° C.	1 g cysteine/17 g solids
		0.5 h, 140° C.	of centrate
			7.5 h, 110° C.
			0.5 h, 140° C.
Sample Name	Heated Aqueous Centrate 12% pH	Heated Aqueous Centrate 20% pH	Heated Aqueous
	5.5	5.5	Centrate 30% pH
			5.5
Sample Composition	12% (w/v, solids of centrate/water)	20% (w/v solids of centrate/water)	30% (w/v, solids
	0.5 h, 140° C.	0.5 h, 140° C.	of centrate/water)
			0.5 h, 140° C.
Sample Name	Heated Aqueous Centrate 50% pH	Heated Aqueous Centrate 75% pH	Heated Aqueous
	5.5	5.5	Centrate 87% pH
			5.5
Sample Composition	50% (w/v, solids of centrate/water)	75% (w/v, solids of centrate/water)	87%(w/v, solids
	0.5 h, 140° C.	0.5, 140° C.	of centrate/water)
			0.5 h, 140° C.
Sample Name	Heated Aqueous Centrate 20% pH	Heated Aqueous Centrate 30% pH
	7.5	7.5
Sample Composition	20% (w/v, solids of centrate/water)	30% (w/v, solids of centrate/water)
	0.5 h, 140° C.	0.5 h, 140° C.
Sample Name	Heated Aqueous Centrate 20% pH	Heated Aqueous Centrate 30% pH
	9	9
Sample Composition	20% (w/v, solids of centrate/water)	30% (w/v, solids of centrate/water)
	0.5 h, 140° C.	0.5 h, 140° C.
Sample Name	Heated Aqueous Centrate pH 5.5 +	Heated Aqueous Centreate pH	Heated Aqueous
	C {fraction (1/20)}	5.5 + C{fraction (1/10)}	Centrate pH 5.5 +
			C⅕
Sample Composition	20% (w/v, solids of centrate/water)	20% (w/v, solids	20% (w/v, solids
	1 g cysteine/20 g solids of centrate	of centrate/water)	of centrate/water)
	0.5 h, 140° C.	1 g cysteine/10 g solids	1 g cysteine/5 g solids
		of centrate	of centrate
		0.5 h, 140° C.	0.5 h, 140° C.
Sample Name	Heated Aqueous Centrate pH 9 + C	Heated Aqueous Centrate pH 9 + C	Heated Aqueous Centrate pH 9 + C
	{fraction (1/20)}	{fraction (1/10)}	⅕
Sample Composition	20% (w/v, solids of centrate/water)	20% (w/v, solids	20% (w/v, solids
	1 g cysteine/20 g solids of centrate	of centrate/water)	of centrate/water)
	0.5 h, 140° C.	1 g cysteine/10 g solids	1 g cysteine/5 g solids
		of centrate	of centrate
		0.5 h, 140° C.	0.5 h, 140° C.
Sample Name	175° C. Heated Aqueous Centrate	100° C. 1 h Heated Aqueous	100° C. 1.5 h Heated Aqueous
	pH 5.5 + C {fraction (1/20)}	Centrate pH 5.5 + C {fraction (1/20)}	Centrate pH 5.5 + C {fraction (1/20)}
Sample Composition	20% (w/v, solids of centrate/water)	20% (w/v, solids of centrate/water)	20% (w/v, solids
	1 g cysteine/20 g solids of centrate	1 g cysteine/20 g solids of centrate	of centrate/water)
	5 min, 175° C.	1 h, 100° C.	1 g cysteine/20 g solids
			of centrate
			1.5 h, 100° C.
Sample Name	175° C. Heated Aqueous	100° C. 1 h Heated Aqueous	100° C. 1.5 h
	Centrate pH 9 + C ⅕	Centrate pH 9 + C ⅕	Heated Aqueous
			Centrate pH 9 + C ⅕
Sample Composition	20% (w/v, solids of centrate/water)	20% (w/v, solids of centrate/water)	20% (w/v, solids
	1 g cysteine/5 g solids of centrate	1 g cysteine/5 g solids of centrate	of centrate/water)
	5 min, 175° C.	1 h, 100° C.	1 g cysteine/5 g solids
			of centrate
			1.5 h, 100° C.
Selected mixtures for GS-MS analysis are underlined
C = Cysteine

Reaction mixtures (2 ml) were prepared by mixing appropriate quantities of stock solutions in glass tubes and then transferring to 20 ml Kimble ampules that were sealed in hot flame. The ampules were then placed in a metal cover and heated in a Carlo Erba 4200 gas chromatograph oven.

The reaction mixtures were stored in the freezer at −20° C. before analysis. The ampules were broken for analysis after bringing the reaction mixtures to room temperature.

Sensory Evaluation of Aroma Volatiles

An informal panel of 6 persons (3 females, 3 males) experienced in flavour evaluation was recruited.

For sensory evaluation, 1 ml aliquots of the samples under investigation were transferred into brown screw-cap bottles and diluted 10 times (except for the concentration study where no dilution was applied). The coded samples were presented to one panellist at one time at room temperature and the panellists were asked to describe the aromas using their own terms.

Instrumental Evaluation of Aroma Volatiles Determination

A dynamic headspace collection procedure was used. Each sample (1.7 ml of reaction mixture so that it was equivalent to 0.4 g of FDC) was placed in a 250 ml conical flask fitted with a Drechsel head. Distilled water was added to a final volume of 10 ml and the mixture shaken gently. Oxygen-free nitrogen was passed over the sample for 1 h at a rate of 40 ml/min. The volatiles were swept onto a preconditioned glass-lined stainless-steel trap (105 mm×3 mm i.d.) packed with 85 mg Tenax GC (CHIS system. SGE Limited). Throughout the collection, the sample was maintained at 37° C. using a water bath. The internal standard was 1,2-dichlorobenzene in ether (130 μl/ml) and 1 μl was injected onto the trap at the end of the collection time, the trap was then flushed with nitrogen for 10 min.

A Hewlett-Packard (HP) 5890/5972 gas chromatrograph-mass spectrometer (GC-MS), fitted with a 50 m×0.32 mm i.d. fused-silica capillary column coated with BPX-5) SGE Limited) at 0.5 μm film thickness, was used to analyse the collected volatiles. These were thermally desorbed at 250° C. in the CHIS injection port (SGE Limited) and cryofocused directly onto the front of the GC column, while the oven was held at 0° C. for 5 min. The oven temperature was then raised to 40° C. over 1 min and held for 5 min before raising the temperature to 250° C. at a rate of 4° C./min and holding for a further 10 min. The helium carrier gas flow rate was 1.5 ml/min. Mass spectra were recorded in the electron impact mode at an ionisation voltage of 70 eV and source temperature of 200° C. A scan range of 29-400 m/z and a scan time of 0.69 s were used. The date were controlled and stored by the HP G1034C Chemstation data system.

Volatiles were identified by comparison of their mass spectra with the spectra from authentic compounds in the Reading Laboratory or in the NIST/EPA/MSDC Mass Spectral Database or other published spectra. The linear retention index (LRI) was calculated for each component using the retention times of a homologous series of C 6 -C 22 n-alkanes.

Nucleic Acid Derivatives

The nucleic acid composition of the centrate was determined from 3 replicates and is presented in Table 5. It explains most of the HPLC eluted peaks. A number of compounds co-eluted, but it was not possible to investigate alternative analysis conditions and, therefore, for co-eluting compounds it was not possible to determine which of the compounds contributed to the peak obtained in the centrate analysis.

As expected, there were few deoxyribonucleic acid derived compounds compared with the ribonucleic acid derived compounds, which are more abundant in nature. The major nucleic acid components are cytosine 5′ monophosphate (26% of the total nucleic acid content), uridine 3′ monophosphate and/or guanosine 5′ monophosphate (18%), adenosine 5′ monophosphate and/or deoxyribo guanosine 5′ monophosphate (16%). All of them are potential sources of ribose and ribose phosphate which are good reactive precursors the Maillard reaction. Excluding the bases, the potential source of ribose or ribose phosphate represents 96% of the nucleic acid content of the centrate, which is equivalent to 202 ppm of the content of the centrate.

TABLE 5
Centrate Nucleic Acids HPLC Analysis Results
							Hypox +	AMP5′ +		AMP5′ +
							guanine +	I +		I +
Nucleic			Ura-	UMP3′ +		Cyti-	uridine +	DAMP3′ +		DAMP3′ +			Adeno-
Acid Type	CMP′5	UMP5′	cil′	GMP5′	IMP5′	dine	GMP3′	DAMP5′	Purine	DAMP5′	DG	DI	sine	Total
nucleic acid	54	23	1	38	5	5	7	34	2	6	2	15	19	212
(ppm in
centrate
nucleic acid	26	11	0	18	3	2	3	16	1	3	1	7	9	100
(% of total)
Standard	3	1	0	1	0	1	0	2	0	0	0	1	2
deviation
(ppm in
centrate)
hypox = hypoxanthine
I = inosine
C = cytidine
U = Uridine
G = Guanosine
A = Adenosine
MO = mono phosphate
D = deoxyribose

Effects of Acid Hydrolysis on Nucleic Acid Derivatives

Table 6 presents the results of hydrolysis of solutions of inosine, guanosine and their respective 5′ phosphate ribonucleotides. The method is based on that used by Matoba et al (J. Food Science, vol. 53, n.4, 1988, p1156). The last column gives an indication of quantity recovery and it can be seen that the results of the hydrolysis on the guanosine showed a significant loss, which suggests that the guanine molecule is unstable.

The most interesting model systems are the ribonucleotides since they are major components in the centrate. They were hydrolysed by half or less, producing their respective nucleosides which were further hydrolysed into their bases. Although it is possible to hydrolyse the ribonucleotides into their bases and consequently produce ribose and/or ribose phosphate, relatively low yields of bases were obtained and an optimisation of this process should be carried out.

TABLE 6
Acid hydrolysis results of model systems
	Quantity (%)	After hydrolysis
	initial	hypoxanthine	inosine	IMP5′	total
inosine	100	13	85		98
IMP5′	100	6	15	71	92
		guanine	guanosine	GMP5′
guanosine	100	2	42		44
GMP5′	100	2	35	51	88

Conclusion

The nucleic acid composition of the FDC has been characterised. It comprises mainly ribonucleotides with relatively small amounts of deoxyribonucleotides. Hydrolysis of nucleotides releases free ribose or ribose phosphate only occurs to a relatively small extent in acetate buffer at pH 4.

Results—Sensory Evaluations of Aroma Volatiles

It was decided to present the individual results of each panellist and not to group them under specific common descriptors because of the too large diversity of the terms described.

Tables 7 and 8 present the effect of heating and the impact of the hydrolysis, with or without the addition of cysteine.

TABLE 7
Aroma panel results on centrate - Study of the effect of cooking
Sample name	Aqueous Centrate	Heated Aqueous Centrate
Sample	1.7%(w/v, solids of	1/7%(w/v, solids of centrate/water)
Composition	centrate/water	0.5, 140° C.
Panellist 1	scrumpy, glucose, syrup, molasses	molasses, caramel
Panellist 2	caramel, sweet	burnt
Panellist 3	whey, old yoghurt, sharp, creamy	raw celery, braised celery, weird smell
Panellist 4	wet cloth/ironing/scorching	fermenting cereal, ironing/wet cloth,
	auto-claving media,	cotton/wool, treacle, golden syrup
	slightly acrid
Panellist 5	honey, urine	caramel, fatty
Panellist 6	caramel, slightly fruity	slightly fruity caramel, burnt, sharp

TABLE 8
Aroma panel results on heated buffered centrate - Study of the effect of hydrolysis and of addition of cysteine
		Heated Hydrolysed Buffered (pH 5.5)	Heated Hydrolysed Buffered (pH 5.5)
Sample	Heated Buffered (pH 5.5) Centrate	Centrate	Centrate + Cysteine
Sample	1.7% (w/v solids of centrate/sodium	1.7% (w/v, solids of centrate/sodium	1.7% (w/v, solids of centrate/soldium
Composition	acetate 0.01M)	acetate 0.01M)	acetate 0.01M)
	0.5 h, 140° C.	7.5 h, 110° C.	1 g cysteine/17 g solids of centrate
		0.5 h, 140° C.	7.5 h, 110° C.
			0.5 h, 140° C.
Panellist 1	burnt, caramel, toffee, acrid, acid	resinous, burnt, acrid	burning paper/plastic/hair, putrid, acrid
Panellist 2	burnt, caramel, celery, slightly sweet	stale shall, burnt, acidic	burnt, celery, acid
Panellist 3	braised celery, marmite, jammy,	varnish, paint, biscuit	sweet, grassy, herbal, raw onion,
	cooked apple		vinegar
Panellist 4	rotting vegetable, sulfur, autoclaving	wet cloth, autoclaving, fermenting	onion rotting, hard boiled egg,
	malted barley, burnt, nutty, treacle	cereal, nose catching/sharp	malty/barley/fermenting cereal
Panellist 5	treacle, honey, fatty	nicotine	burning tires
Panellist 6	caramel, toffee, slight fruity, slightly	chemical, slightly burnt rubbery	caramel, toffee, sweet, floral
	burnt

The study of the effect of concentration of centrate involved the range of concentrations likely to be reached in commercial practice, i.e., within the range 12% and 30% of solids. These were compared with the non diluted freeze dried centrate powder (87% solids). The other preparation conditions were kept constant. viz. pH 5.5 and heating at 140° C. for 30 min. The results are presented in Table 9. The odours were very strong and the reproducibility of the results within 2 replicates for each panellist was fairly poor. However, there was a noticeable trend within the sample set from low to high concentration: at 12%, the odours were mainly sweet, vegetable and molasses. These notes became associated with burnt and sharp as well as savoury at 20 and 30% solids. At 50%, the sample had roasted and paint smells that became dominant at 75%. Some extra metallic, burnt rubbery, and sulphur notes were detected with the 87% sample. The 20 and 30% solid samples seemed the most interesting because of their meaty savour smells and therefore they were selected for further analysis it was also decided to dilute the original flavoured reaction mixtures before sniffing further reaction mixtures.

TABLE 9
Aroma panel results on heated centrate pH 5.5 - Study of the effect of concentration
	Heated Aqueous	Heated Aqueous	Heated Aqueous	Heated Aqueous	Heated Aqueous	Heated Aqueous
Sample	Centrate 12% pH	Centrate 20% pH	Centrate 30% pH	Centrate 50% pH	Centrate 7.5% pH	Centrate 87% pH
Name	5.5	5.5	5.5	5.5	5.5	5.5
Sample	12% (w/v, solids of	20% (w/v, solids of	30% (w/v, solids of	50% (w/v, solids of	75% (w/v, solids of	87% (w/v, solids of
Composition	centrate/water)	centrate/water)	centrate/water)	centrate/water)	centrate/water)	centrate/water)
	0.5 h, 140° C.	0.5 h, 140° C.	0.5 h, 140° C.	0.5 h, 140° C.	0.5 h, 140° C.	0.5 h, 140° C.
Panellist 1	burnt, caramel,	sweet,	soja sauce,	burnt, caramel,	burnt, very	burnt sugar
	slightly sweet	slightly meaty,	caramel, toffee,	bitter chocolate	strong,
		nutty	burnt, sweet		chocolate
Panellist 2	sickly molasses	sharp, acrid,	molasses, black	molasses, black	burnt, solvent,	savoury, burnt,
		cloying, burnt paper	treacle	treacle, solvent	sickly	burnt flesh/skin
Panellist 3	celery,	marmite	marmite, emulsion	paint, marmite,	burnt	slightly sweet,
	burnt roast coffee,		paint	slight celery		burnt, marmite
	bovril
Panellist 4	honey, caramel,	honey, caramel,	caramelised	raw vegetables,	caramel, roasted	soy sauce
	stir fry	stir fry, vegetable	vegetables	caramel	slight treacle
Panellist 5	celery, rancid,	sharp, marmite,	sharp, bovril,	strange top note	very burnt sugar +	burnt rubber,
	maggi, bovril	yeasty, bovril +	vinegary top note	of sharp green/	top note	sulphur,
		celery		fruity note/		very burnt
		undertones		household		sugar
				or dry smell
Panellist 6	malty, brewery,	honey, malty	autoclaving, honey,	green banana,	burnt milk, burnt	dry, dusty
	virol (malt extract),		digestive biscuits	honey, slight	sugar	sensation, like
	v. concentrate			autoclaving,	(not caramel -	something burnt
	digestive biscuit,			slight virol	really burnt)	black in oven,
	black treacle,					sharp,
	autoclaving					faint marmite,
						black treacle,
						slightly stale,
						metallic,
						rusty steel
	REPLICATE	REPLICATE	REPLICATE	REPLICATE	REPLICATE	REPLICATE
Panellist 1	sweet, caramel,	burnt, toffee,	soja sauce,	burnt, caramel	burnt, slightly	burnt sugar
	soja sauce	acrid	caramel,		sweet, caramel
			burnt, sweet
Panellist 2	burnt, caramel,	sharp, acrid,	molasses, resinous	burnt, black	very strong	molasses, burnt,
	burnt skin/hair	cloying,		treacle, slightly	marmite, meat	savoury
		burnt paper		acrid	extract, burnt,
					caramel
Panellist 3	malt	marmite, celery	marmite, celery,	paint, marmite,	marmite, burnt	burnt, marmite
			slight	cereal
			roast coffee
Panellist 4	honey, caramel,	honey, earthy,	soy sauce,	vegetable,	damp wood,	vegetable
	stir fry	uncooked potato	wood smoke	roasted,	syrup, honey
				woodland
Panellist 5	celery, meaty	burnt celery	bovril, strange	strong strange	strong strange	marmite, yeasty,
			top note	top note, bovril	top note,	bovril, very
					burnt sugar	concentrated
Panellist 6	black treacle,	concentrated	biscuits, honey,	black treacle,	fresh sensation,	same as other
	virol, autoclaving	honey,	green/fruity,	acrid/burnt	honey, malt	replicate
		biscuity	green bananas	honey,
		digestives,		virol/malt
		autoclaving

The pH effect was studied on the centrate at 3 different values: 5.5, 7.5 and 9, with the heating conditions kept at 140° C. for 30 min. The results are in Table 10. There was not much difference in the results between the 20% and the 30% solids samples. The results at pH 7.5 were similar to those at pH 5.5 and the smells were mainly autoclave and caramel. The odour became burnt with pH 9. Therefore it was decided to carry on the sniffing experiments by selecting the two extreme pHs and to keep only one concentration (20% solids).

TABLE 10
Aroma panel results on heated centrate study of the effect of pH
	Heated aqueous centrate	Heated aqueous centrate
Sample Name	20% pH 5.5	30% pH 5.5
Sample composition	20%(w/v, solids of	30% (w/v, solids of
	centrate/water) 0.5 h 140° C.	centrate/water) 0.5 h 140° C.
Dilution before sniffing	50 for panellist 1	50 for panellist 1
	10 for panellist 2	10 for panellist 2
Panellist 1	1-slight autoclaving	1-slightly autoclave,
	2-diacetyl then going to	“catching” in nose.
	caramel, butter scotch,	2-then quite a lot of caramel
	slightly nutty
Panellist 2	caramel, butter-like	caramel, sweet
	Heated aqueous centrate	Heated aqueous centrate
Sample Name	20% pH 7.5	30% pH 7.5
Sample composition	20%(w/v, solids of	30%(w/v, solids of
	centrate/water 0.5 h 140° C.	centrate/water) 0.5 h 140° C.
Dilution before sniffing	50	50
Panellist 1	similar to sample 20% pH 5.5	1-slightly autoclave
	in the way it changes, bit more	2-caramel
	autoclaving, caramel but not
	really butterscotch
	Heated aqueous centrate	Heated aqueous centrate
Sample Name	20% pH 9	30% pH9
Sample composition	20% (w/v, solids of	30% (w/v, solids of
	centrate/water) 0.5 h 140° C.	centrate/water) 0.5 h 140° C.
Dilution before sniffing	10	10
Panellist 2	burnt, baked, roasted, cereals	slightly burnt, caramel sweet

The effect of addition of cysteine was studied on the centrate in solution at 20% solids at pH 5.5 and 9. There were three concentrations of cysteine tested: ratios 1/20, 1/10 and 1/5 of cysteine (g)/centrate solids (g). The heating conditions were kept the same as previously (104° C. for 30 min). The results are presented in Table 11. Within the pH 5.5 sample series, the low concentration of cysteine sample lead to a somewhat pleasant odour of sweet, greasy, meaty sauce, that was progressively replaced by roasted and rubber notes as the cysteine concentration increased. At pH 9, the burnt roasted cereals notes already mentioned in the previous experiment were present again with cysteine at low concentration. When the cysteine content increased, the odour became strong and more nutty and then close to savoury, meaty stock. It was therefore decided to select pH 5.5 with cysteine 1/20 sample and pH9 with cysteine 1/5 sample for the next set of experiments.

TABLE 11
Aroma panel results on heated centrate pH 5.5 and pH 9 - Study of the effect of addition of cysteine
	Heated Aqueous	Heated Aqueous	Heated Aqueous	Heated Aqueous	Heated Aqueous	Heated Aqueous
	Centrate pH 5.5 +	Centrate pH 5.5 +	Centrate pH 5.5 +	Centrate pH 9 +	Centrate pH 9 +	Centrate pH 9 +
Sample Name	cysteine {fraction (1/20)}	cysteine {fraction (1/10)}	cysteine ⅕	cysteine {fraction (1/20)}	cysteine {fraction (1/10)}	cysteine ⅕
Sample	20% (w/v, solids	20% (w/v, solids	20% (w/v, solids	20% (w.v, solids	20% (w/v, solids	20% (w/v, solids
Composition	of centrate/water)	of centrate/water)	of centrate/water)	of centrate/water)	of centrate/water)	of centrate/water)
	1 g cysteine/20 g	1 g cysteine/10 g	1 g cysteine/5 g	1 g cysteine/20 g	1 g cysteine/10 g	1 g cysteine/5 g solids
	solids of centrate	solids of centrate	solids of centrate	solids of centrate	solids of centrate	of centrate
	0.5 h, 140° C.	0.5 h, 140° C.	0.5 h, 140° C.	0.5 h, 140° C.	0.5 h, 140° C.	0.5 h, 140° C.
Dilution	10	10	10	10	10	10
before
Sniffing
Panellist 1	celery, cooked	bacon fat,	sweet, caramel,	burnt, roasted,	sweet caramel,	meaty, pork
	vegetables,	crispy duck	roasted	nutty	roasted, nutty,	crackling, nutty
	burnt wool,	(Chinese style),			burnt
	sweaty socks	sl. sweaty,
		roasted
Panellist 2	celery (strong),	puffed wheat,	celery,	sweet (strong),	celery (strong),	meaty (strong)
	sl. burnt coffee,	sweet + sour	sweet + sour,	toffee, biscuity,	sl. sweet,
	treacle toffee		puffed wheat	marzipan	biscuity
	(sl.)		(strong)
Panellist 3	strong chicken	burning rubber,	weak rubbery,	savoury,	burning rubber,	strong chicken
	stock, greasy,	savoury, marmite,	acrid	burnt skin,	strong savoury,	stock, chicken
	acidic, rubber	malt extract		stock	malt extract	fat, sl. acrid
Panellist 4	strong celery	medicinal smell,	(sweet and sour),	v. strong weird	fruity, sweet,	sulphury, weak
	(main odour) +	also watery	rubber, magi	note, stale	burnt sugar,	stock, nasty note,
	other notes -	stock (weak)		biscuit crumbs,	vinegar	burnt meat?
	meaty, pork	faint celery/		Farleys rusks,
	and apple,	sweet'n sour		faint
	sweet and sour			sweet'n sour
	sauces			note
Panellist 5	black treacle,	buttery, golden	golden syrup,	burnt,	same as sample	meaty (beef gravy?),
	buttery/caramel,	syrup, something	caramel,	scorched/burnt	{fraction (1/20)} but stronger	burnt, burnt sugar,
	burnt sugar,	burnt? (wet	toasted nuts	wet wool,	burnt, less	plus caramel
	burnt fried	wood or toasted	(not burnt)	golden syrup	golden syrup
	boiled cabbage,	nuts, almond,	sl. sulphur.
	marsala, sweet,	hazelnut)
	nutty

The results of the temperature/duration heating conditions study are presented in Table 12. They involve the two selected samples previously described which were then cooked at a lower temperature and longer time: 100° C. for 60 min and 90 min, and at a higher temperature but for a shorter time: 175° C. for 5 min. Compared to the original heating conditions, similar results were obtained for the pH 5.5 cysteine 1/20 sample by heating 100° C. for 60 min. Longer time of heating at 100° C. resulted in more roasted burnt notes and a similar type of odour was obtained after 5 min at 175° C. Regarding the pH 9 cysteine 1/5 sample, the meaty notes obtained by 30 min at 140° C. were reached by the treatment 5 min at 175° C. At 100° C., some very strong odours of urine and boiled eggs were described.

TABLE 12
Aroma panel results on heated centrate pH 5.5 and pH 9 + Cysteine
Study of the effect of temperature and duration of the cooking
				175° C.	100° C. 1 h
			100° C. 1.5 h	Heated	Heated
			Heated	Aqueos	Aqueous	100° C. 1.5 h
	175° C. Heated	100° C. 1 h Heated	Aqueous Centrate	Centrate	Centrate	Heated Aqueous
	Aqueous Centrate	Aqueous Centrate	pH 5.5 +	pH 9 +	pH 9 +	Centrate pH 9
Sample Name	pH 5.5 + cysteine {fraction (1/20)}	pH 5.5 + cysteine {fraction (1/20)}	cysteine {fraction (1/20)}	cysteine ⅕	cysteine ⅕	+ cysteine ⅕
Sample	20% (w/v, solids of	20% (w/v, solids of	20% (w/v,	20% (w/v,	20% (w/v,	20% (w/v, solids
Composition	centrate/water)	centrate/water)	solids of	solids of	solids	of centrate/water)
	1 g cysteine/20 g solids	1 g cysteine/20 g	centrate/water)	centrate/	of centrate/	1 g cysteine/5 g
	of centrate)	solids of centrate	1 g cysteine/	water)	water)	solids of centrate
	5 min 175° C.	1 h, 100° C.	20 g	1 g cysteine/	1 g cysteine/	1.5 h, 100° C.
			solids of	5 g	5 g solids
			centrate	solids	of centrate
			1.5 h, 100° C.	of centrate	1 h, 100° C.
				5 min,
				175° C.
Dilution before	10	10	10	10	10	10
sniffing
Panellist 1	burnt-strong,	fruity-moderate,	celery-moderate,	crackers-	yeast	yeast, bread
	caramel-moderate,	nutty-slight	roasted-light	moderate,	extract-	dough-moderate,
	roasted-moderate			stale-	weak,	vegetables-weak,
				slight,	vegetables-	nutty-background
				baked	moderate,	after other
				bread-	wet	odours decrease
				moderate	washing-
					moderate
Panellist 2	puffed wheat-strong,	celery-strong	celery-medium,	sulphury-	hard	egg-strong
	marmite-medium,		cereal-slight	slight,	boiled
	celery-light			meaty-	egg-
				medium,	strong
				burnt-
				medium
Panellist 3	rancid-v.strong,	chicken fat-medium,	burnt rubber-	burnt	wet wall	state, wet
	acrid-strong,	rubber-medium,	weak,	paper-	paper-	wallpaper, musty
	burnt fat/skin-medium,	burnt rubber-medium	molasses-	weak,	medium,
	molasses-medium/weak		weak	chicken	urine
			medium,	stock-
			burnt/cold	weak/
			wood-weak	medium
				cereal/
				malty-weak

Results—Instrumental Analysis CF Aroma Volatiles by GC/MS

A selection of the reaction mixtures described above were further studied by GC-MS analysis of the headspace volatiles by the method described previously. These mixtures are underlined in Table 2. A sample of autolysed yeast was analysed under the same conditions for comparison of the volatiles. The results of the study are presented in detail in Table 13.

TABLE 13
Volatile compounds analysed by headspace concentration
	Approximate quantities (ng/0.4 g of Freeze Dried Centrate, or ng/45.7 g of Centrate
				pH 5.5 +	pH 5.5 +	ph 9 +	pH 9 +	pH 5.5 +	pH 9 +	pH 5.5 +	pH 9 +
		pH 5.5	ph 9	C 1/20	C 1/5	C 1/20	C 1/5	C 1/20	C 1/5	C 1/20	C 1/5
Identified Compounds	LRI	140° C.	140° C.	140° C.	140° C.	140° C.	140° C.	175° C.	175° C.	100° C.	100° C.	Yeast
Pyrazines
pyrazine	762	—	648	—	—	1108	—	—	—	—	—	—
methylpyrazine	845	78	4607	—	37	2521	813	102	132	—	—	—
2-6-	928	—	—	—	—	465	590	—	—	—	3	—
dimethylpyrazine
2,5-	936	104	3859	—	—	382	500	—	—	—	17	4
dimethylpyrazine
2,3-	940	—	966	—	—	30	—	—	157	—	—	—
dimethylpyrazine
ethenylpyrazine	950	44	239	—	—	—	—	—	—	—	—	—
2-ethylpyrazine	956	—	38	—	—	118	186	—	167	—	—	—
2-ethyl-	1014	—	984	—	—	301	343	—	—	—	—	—
6-methylpyrazine
trimethylpyrazine	1018	256	1758	—	63	1453	1758	—	518	—	98	—
propylpyrazine	1025	—	21	—	—	—	—	—	—		—	—
6-methyl-2-	1034	20	160	—	—	—	—	—	—	—	—	—
ethenylpyrazine
tetra-	1098	—	—	—	46	1009	1121	—	660	—	647	—
methylpyrazine
dimethylethyl-	1099	416	120	51	—	85	111	74	—	—	9	—
pyrazine
dimethylethyl-	1101	—	1593	—	—	—	35	—	—	—	—	—
pyrazine
methylpropyl-	1101	26	—	—	—	—	—	—	—	—	—	—
pyrazine
3,5-diethyl-2-	1168	—	137	—	—	—	—	18	—	—	—	4
methylpyrazine
trimethylethyl-	1168	51	—	—	—	120	195	—	75	—	105	—
pyrazine
dimethylpropyl-	1170	12	63	—	—	98	186	—	51	—	15	—
pyrazine
dimethylpropyl-	1179	32	130	—	—	81	146	—	40	—	9	—
pyrazine
dimethylbutyl-	1321	—	—	—	—	—	—	—	—	—	16	2
pyrazine
trimethylpropyl-	1247	75	168	—	23	166	352	36	150	—	265	—
pyrazine
trimethylpropyl-	1260	16	99	—	—	27	—	—	—	—	—	—
pyrazine
2-(2-methylpropyl)-		—	—	—	—	—	—	—	—	—	2	—
3-(1-methylethyl)-
pyrazine
methyldiethyl-		—	—	—	—	—	—	—	—	—	7	—
pyrazine
2,5-dimethyl-		—	—	—	—	—	—	—	—	—	5	—
3,6-dipropyl-
pyrazine
Furans
2-ethylfuran	707	—	—	29	398	32	2037	—	—	—	—	—
dimethylfuran	711	—	—	—	22	—	—	112	—	—	—	—
2,4-	723	111	—	104	111	43	85	—	—	—	—	—
dimethylfuran
dihydro-2methyl-	831	71	69	—	—	—	—	—	—	—	—	—
3(2H)-furanone
2-furancarbox-	857	668	—	253	109	—	—	165	—	—	—	—
aldehyde
2-furanmethanol	881	85	298	153	85	33	—	121	11	—	—	7
2-furan-	926	—	—	—	249	—	—	—	—	—	—	—
methanethiol
2-acetylfuran	924	—	—	—	—	—	—	—	406	—	—	—
1-(2-furanyl)-	929	84	—	629	1193	—	391	494	—	—	7	—
ethanone
2-pentylfuran	997	113	62	?	—	35	47	—	—	—	—	—
1-(2-furanyl)-	1025	21		—	—	—	—	—	—	—	—	—
1-propanone
Pyrans
3,4-dihydro-	809	—	—	—	20	—	—	—	—	—	—	—
2H-pyran
tetrahydro-6-	879	—	—	—	—	—	—	—	—	—	8	—
methyl-2H-
pyran- 2-one
tetrahydro-	884	155	—	—	—	—	—	—	—	—	—	—
2H-pyran-2-oll
Thiophenes
thiophene	677	—	—	—	179	—	—	—	—	—	—	—
methylthiophene	778	—	—	66	63	47	478	62	—	—	—	—
ethylthiophene	875	—	—	—	28	—	58	—	—	—	—	—
2,5 dimethyl	897	—	—	—	19	—	36	38	—	—	—	—
thiophene
thiophenethiol	995	—	—	—	205	—	—	—	—	—	—	—
2-methyltetra-	1014	693	—	1366	—	—	—	—	—	28	—	—
hydrothiophen-
3-one
2-thiophene-	1026	20	—	99	—	104	—	—	—	—	—	—
carboxaldehyde
2-thiophene-	1077	—	—	—	27	—	—	—	—	—	—	—
methanethiol
3-(methylthio)	1103	—	—	—	37	145	—	—	—	—	—	—
thiophene
methylthiophene-	1143	—	—	43	5	—	—	52	97	—	—	—
carboxaldehyde
methylthiophene-	1148	—	—	526	89	134	41	329	—	—	—	—
carboxaldehyde
thienothiophene	1233	—	—	35	49	—	32	16	24	—	—	—
thienothiophene	1276	—	—	—	30	—	—	—	36	—	—	—
Thiazoles
thiazole	734	—	—	46	51	—	1165	219	870	—	—	—
methylthiazole	821	—	—	—	62	72	2	71	134	—	6	—
5-methylthiazole	857	—	—	—	106	57	112	779	82	—	—	—
methylthiazole	866	—	—	—	39	—	—	—	—	—	—	—
dimethylthiazole	947	—	—	—	29	88	289	—	95	—	—	—
2-ethylthiazole	959	—	—	—	22	—	42	26	78	—	—	—
2,4,5-tri-	1016	—	—	—	1499	59	262	—	153	—	—	—
methylthiazole
methyl-	1028	—	—	—	140	—	68	123	290	—	—	—
ethylthiazole
2-acetylthiazole	1041	—	—	—	—	647	361	—	109	—		—
4-methyl-5-	1043	57	67	—	—	—	59	43	—	—	20	—
ethenylthiazole
2-isobutyl-	1065	31	—	—	—	—	—	—	—	—	—	—
thiazole
methylpropyl-	1128	—	—	—	—	36	—	—	—	—	—	—
thiazole
dimethyliso-	1157	—	—	—	24	29	87	—	55	—	—	—
propylthiazole
dimethyliso-	1152	—	—	—	—	—	52	—	37	—	—	—
propylthiazole
Aliphatic compounds
1-propanol	616	65	—	—	101	—	—	—	—	1319	—	—
2-butanone	628	1449	—	—	2021	—	3074	—	3755	—	1164	107
ethylacetate	631	—	144	74	92	112	—	97	47	82	110	13
2,3-butane-	637	1843	1092	2001	—	—	—	1548	—	664	—	—
dione
3-methylbutanal	655	84	3297	1232	—	903	—	1213	752	—	—	1116
pentanal	686	2217	—	—	—	—	—	—	—	—	—	—
2-methylbutanal	691	621	703	1277	—	—	—	1242	151	—	—	582
2-methyl-1-	705	1133	2412	1238	2430	1614	1997	948	1791	1923	3992	17
propanol
3-methyl-2-	672	—	—	—	143	—	2205	—	—	42	62	—
butanone
2-pentenal	698	—	—	48	22	—	—	34	—	—	—	—
2-pentanone	708	108	—	72	279	93	1190	85	445	34	91	—
3-pentanone	708	—	—	—	72	—	502	—	163	—	—	—
2,3-	721	708	487	567	—	240	—	525	309	66	—	—
pentanedione
1-butanol	719	—	132	—	70	100	—	—	56	28	77	—
1-methoxy-2-	723	—	—	—	—	67	—	—	58	—	—	—
propanone
3-hydroxy-2-	728	—	—	—	1377	332	—	1694	—	142	—	—
butanone
2-methyl-2-	753	—	76	—	—	—	—	—	—	—	—	—
butenal
3-methyl-3-	761	511	—	1794	—	—	578	—	—	98	294	—
buten-1-ol
3-methylbutanol	766	682	2221	954	730	254	1278	1375	564	355	796	94
2-butennitrile	770	—	—	—	—	—	249	—	—	—	—	—
heptanol	770	433	—	—	—	—	—	—	—	—	—	—
4-methyl-2,3,-	801	60	607	—	—	—	—	—	—	—	—	—
pentanedione
2,3-	811	1686	137	1791	—	434	—	1404	—	591	—	—
hexanedione
hexanal	818	91	—	38	—	144	—	171	57	—	—	—
3-hexanone	822	36	—	—	203	—	1654	—	244	—	58	—
2-hexanone	803	—	—	—	391	—	1502	—	—	—	80	—
3-hydroxy-2-	831	—	—	75	59	—	—	125	—	—	—	—
pentanone
2-heptanone	903	99	122	106	111	113	206	161	83	57	71	7
heptanal	910	—	—	—	—	—	—	—	—	—	—	138
2-hydroxy-3-	913	138	78	323	234	127	65	432	135	—	10	—
hexanone
6-methyl-2-	967	594	722	9465	15413	1397	4781	7347	—	—	3512	—
heptanone
1,3-cyclo-	973	26	24	—	—	—	—	—	—	—	—	—
pentanedione
5-methyl-	979	31	34	555	684	91	205	572	399	248	247	—
2-heptanol
methylheptanone	984	—	—	—	149	11	234	—	2995	4909	110	19
octenol	988	—	—	—	—	—	—	—	—	—	5	40
6-methyl-5-	997	—	—	114	104	—	—	—	30	25	20	23
hepten-2-one
2-heptenal	1036	—	—	—	—	—	—	—	—	—	15	24
2-nonenal	1113	—	—	—	—	—	—	—	70	25	7	81
2-octenal	1069	—	—	—	—	—	—	—	—	—	—	25
1-octanol	1078	—	—	—	—	—	—	—	—	—	—	25
nonanal	1116	37	—	56	—	—	—	48	—	—	—	—
decanal	1217	28	—	—	—	—	—	—	—	—	6	—
2-undecanone	1298	—	—	—	—	—	—	—	—	—	4	—
butanoic acid	1380	—	—	—	—	—	—	—	—	—	—	3
butyl ester
dodecanal	1417	—	—	—	—	—	—	—	—	—	—	3
unsaturated	1459	—	—	—	9	—	—	—	—	—	—	8
ketone
1-tetradecanol	1480	—	—	—	—	—	—	—	—	—	11
1-hexadecanol	1581	—	—	—	—	—	—	—	—	—	7
Pyrroles/Benropyrroles
pyrrole	765	—	—	—	—	102	238	—	199	—	0	—
2-meth-1H-pyrrole	830	—	—	—	—	—	12	—	—	—	—	—
tetrahydro-6-	879	—	—	—	—	—	—	—	—	—	8	—
methyl-2H-
pyran-2-one
2,4-dimethyl-	1055	—	—	—	—	—	—	—	—	—	22	—
3-ethyl-1H-
pyrrole
indole	1313	—	—	—	—	—	—	—	—	—	5	—
Phenyls
phenol	727	—	—	—	11	—	—	—	—	—	—	—
benzaldehyde	983	223	126	152	60	175	—	106	—	—	17	267
methylethylbenzene	1025	—	—	—	—	—	—	—	—	—	—	20
benzenaceatal-	1059	—	—	—	—	—	—	—	—	—	—	38
dehyde
2-methylphenol	1065	—	—	—	—	—	—	—	—	—	—	5
methyl-	1088	—	—	—	—	—	—	—	—	—	—	6
chlorophenol
acetophenenone	1080	—	—	—	—	—	—	—	—	—	6	—
2-methylthiophenol	1157	—	—	—	—	—	45	—	226	—	—	—
ethoxybenzal-	1244	—	—	228	10	—	—	—	—	—	—	—
dehyde
Pyridines
methylpyridine	833	—	—	—	—	—	96	—	82	—	5	—
2-methylpyridamine	848	52	—	—	—	—	—	—	—	—	—	—
6-methyl-4-(1H)-	937	—	—	253	252	46	—	339	190	84	—	—
pyrimidinone
Terpenes
3,6,6-trimethyl-	934	—	—	—	—	—	—	—	—	—	—	8
bicyclo-
(3.1.1.)hept-2-ene
1,5-dimethyl-	1034	—	—	—	—	—	—	—	—	—	—	22
1-5-cyclo-
octadiene
limonene	1037	—	—	—	—	—	41	—	—	—	15	—
cymene	1031	—	—	—	—	—	—	—	—	—	—	10
eucalyptol	1039	—	—	—	—	—	—	—	—	—	—	13
verbenene	1063	—	—	—	—	—	—	—	—	—	—	4
3-7-dimethyl-	1105	—	—	—	—	—	—	—	—	—	—	19
1,6-octadien-3-Ol
borneol	1184	—	—	—	—	—	—	—	—	—	—	2
terpin-4-ol	1191	—	—	—	—	—	—	—	—	—	—	15
trimethylbicyclo-	1236	—	—	—	38	—	—	—	—	—	—	14
(2.2.1)-
heptan-2-one
bornyl acetate	1292	—	—		—	—	—	—	—	—	—	2
safrole	1305	—	—	—	—	—	—	—	—	—	—	3
terpine	1339	—	—	—	—	—	—	—	—	—	—	3
a sesquiterpene	1383	—	—	—	—	—	—	—	—	—	—	5
a sesquiterpene	1435	—	—	—	5	—	—	—	—	16	12	2
a sesquiterpene	1494	20	—	—	36	—	27	24	15	—	14	14
(cadinene?)
a sesquiterpene	1499	—	—	—	—	—	17	50	—	—	9	2
Oxazoles
4,5-	771	—	—	—	105	—	—	—	—	—	—	—
dimethyloxazole
trimethyloxazole	865	649	540	580	321	174	122	—	68	161	29	—
2,5-dimethyl-4-	932	256	—	—	—	—	—	—	—	48	—	—
ethyloxazole
4,4-dimethyl-	946	10	—	—	—	—	—	—	—	—	—	—
2-ethyloxazole
4,5-dimethyl-	986	237	158	—	147	—	68	160	22	47	—	—
2-iso-
propyloxazole
4,5-dimethyl-2-	1014	—	—	—	—	—	—	1463	—	369	65	—
propyloxazole
2,4-dimethyl-5-	1020	697	—	1448	—	—	—	1555	—	513	—	—
propyloxazole
4-methyl-5-ethyl-2-	1044	—	—	—	20	—	—	48	—	—	—	—
isopropyloxazole
Aliphatic sulfides
dimethyldi-	754	295	389	280	—	311	—	—	—	—	—	81
sulfide
dimethyltri-	987	—	—	402	—	92	—	—	—	62	—	87
sulfide
2-pentanethiol	841	—	—	—	2403	—	—	102	—	—	—	—
1(elhylthio)-	921	—	—	—	51	—	—	—	—	—	—	—
2-propanone
2,2-dithio-	1280	—	—	—	16	—	—	—	—	—	—	—
bis-ethanol
Cyclic polysulfides
3,5-dimethyl-1,2,4-	1280	—	—	—	—	21	91	—	36	—	—	—
trithiolane
Dioxanes
4-methyl-1,	737	321	46	256	—	—	—	142	—	—	—	—
3-dioxane
TOTAL		18678	29633	28779	33228	16478	32517	25890	17369	11936	12195	2982

The pyrazines, thiazoles and thiophenes content was very much affected by the reaction conditions. The highest levels of pyrazines were found at pH9, as expected, since the formation of N-heterocyclic compounds in the Maillard reaction is favoured by high pH. With a few exceptions, sulphur compounds were formed only in the presence of cysteine, confirming that the content of sulphur amino acids in the freeze dried centrate was very low.

The yeast autolysate aroma volatiles were dominated by terpenes and its composition was very different from the volatiles obtained from the centrate.

Conclusion

The range of flavours was generated from the centrate, showing its potential as a flavouring ingredient or as a source of precursors for reaction product flavourings. The variables that were applied in this study were the centrate concentration, the pH, the presence of added cysteine, and the temperature/duration of the heating conditions. An addition of cysteine was necessary to generate meaty aromas which derive from sulphur-containing volatiles.

Claims

1. A flavoring material for food which:(a) is an aqueous solution which comprises materials removed from filamentous fungus by contacting filamentous fungal cells in their growing state and/or in the presence of its growth medium with water at an elevated temperature sufficient to reduce the nucleic acid content of the filamentous fungal cells in which the concentration of dissolved solids is sufficiently high to render the solution stable to storage at a temperature of 20° C. for a period of one month; or (b) is a solid comprising materials so removed from the filamentous flingal cells.

2. A flavoring material according to claim 1, which is an aqueous solution comprising at least 30% by weight of solids.

3. A flavoring material as claims in claim 1, wherein said filamentous fungus is Fusarium.

4. A process of producing a flavoring material for food which comprises the step of improving the suitability of filamentous fungus for food by subjecting filamentous fungal cells(a) in their growing state and/or in the presence of its growth medium; and (b) in the presence of water; to a temperature sufficient to reduce its nucleic acid content thereby producing an aqueous solution and concentrating the aqueous solution.

5. A process according to claim 4, wherein said aqueous solution after concentration comprises at least 30% by weight of solids.

6. A process as claimed in claim 4, in which water is removed from the aqueous solution by evaporation, distillation, reverse osmosis, freeze drying or freezing out the water as ice leaving an aqueous concentrate.

7. A process as claimed in claim 4, in which the soluble components are concentrated by removing water at reduced pressure and at a temperature of 40 to 70° C.

8. A process of producing a flavoring material for food according to claim 4, in which nucleic acid is removed from the filamentous fungal cells while the filamentous fungus is in its growing state.

9. A process as claimed in claim 8, in which the filamentous fungus in its growing state is washed to remove growth medium and immediately thereafter heated in the presence of water to remove nucleic acid.

10. A process as claimed in claim 4, in which a solution of 1.5 to 75% of the materials removed from the fungus in water is reacted with cysteine in amounts up to 10% of cysteine by weight based on the said materials at a pH of 5.5 to 9.

11. A process as claimed in claim 10, in which the temperature of the reaction is 110 to 140° C.

12. A process as claimed in claim 4, wherein said filamentous fungus is Fusarium.

13. A food which comprises 0.1 to 15% by weight based on the solids content of a flavoring material as claimed in claim 1.

14. A food according to claim 13, which is a snack, biscuit, stock, soup, stew, sauce, gravy or a drink.

15. A food which comprises 0.1 to 15% by weight based on the solids content of a flavoring material produced by a process as claimed in claim 4.

16. A food according to claim 15, which is a snack, biscuit, stock, soup, stew, sauce, gravy or a drink.

17. A process of flavoring food which comprises the step of improving the suitability of a filamentous fungus as food by subjecting filamentous fungal cells of the filamentous fungus(a) in their growing state and/or in the presence of their growth medium; and (b) in the presence of water; to a temperature sufficient to reduce the nucleic acid content of the cells substantially and flavoring food with materials removed in the said step directly or after chemical reaction.

18. A method of preparing a flavoring material for food which comprises subjecting a filamentous fungus in the presence of water to a temperature sufficient to remove materials therefrom and thereby reduce nucleic acid content of the filamentous fungus; and subjecting materials removed therefrom to a chemical reaction thereby to prepare a flavoring material for food.

19. A method of processing filamentous fungus to improve its suitability as food which comprises subjecting the cells of the filamentous fungus, in the presence of water, to a temperature sufficient to remove materials therefrom and thereby reduce its nucleic acid content; and subjecting materials removed from the fungus to a chemical reaction thereby to prepare a flavoring material for food.