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THIS ARTICLE IS ABOUT A SHIPMENT OF BREAD THAT WAS OVERFILLED WITH STREPTOMYCES GRISEUS CAUSING THE TASTE AND SMELL OF THE BREAD SMELL AND TASTE LIKE SOIL AND DIRT. 

A bulk shipment of wheat flour (from Australian hard-grain wheat supplied to a miller in Tasmania by the Australian Wheat Board) delivered to a bakery in southern Austalian was observed to possess an abnormal 'earthy-musty' adour and the bread made from it had an intense 'earthy' off-flavour. Coincident with these observations was a report that mill offal, presumably prepared from the same batch of grain, possessed a strong 'musty' odour. A sensory examination of samples of the flour by an experienced panel concluded that the likely cause of the off-flavour was the microbial metabolite geosmin (E-1,10-dimethyl-E-9-decalol) and possibly 2-methylisoborneol (1,2,7,7-tetramethyl-exo-bicyclo[2.2.1]heptan-2-ol).

The aroma of geosmin is intensely earthy, [1] whereas that of 2-methylisoborneol is described as muddy in dilute solutions but camphor-like in high concentrations. [2] In dilute aqueous solutions geosmin has an odour threshold concentration of 7ng [1.sup.-1] [3] and that of 2-methylisoborneol is 29ng [1.sup.-1]. [4] Both compounds are produced separately or together by several species of actinomycetes [5,6] and cyanobacteria. [7-9] Geosmin and 2-methylisoborneol have been reported to cause objectionable tastes in drinking water, [6,10] and have been reported to impart earthy or muddy off-flavours to fish from areas that are commonly affected by infections of cyanobacteria or actinomycetes. [11,12] More recently, both compounds were found in naturally infected wheat grains cultured in glass jars. [13]

Geosmin and 2-methylisoborneol were identified as follows in the sample of off-flavoured flour: the flour (250g) was placed in a Soxhlet apparatus and was extracted continuously with redistilled pentane ([500cm.sup.3]) for 48h. The extract was transferred to a distillation flask and 90 per cent of the pentane was removed by fractional distillation. The residue (about [50cm.sup.3]) was added to a flask containing purified water (11) which was connected to a modified Liken-Nickerson apparatus. [14] The mixture was warmed gently until the pentane had distilled into the solvent flask and U-tube. The residual lipid extract was the extracted by continuous steam distillation-solvent extraction for 3h with the pentane as solvent. Small amounts of water were removed from the volatile extract by cooling to -20 [degrees]C and decanting the organic solvent. The concentrate was further dried (anhydrous [Na.sub.2.SO.sub.4]) and then passed through a silica gel Sep-Pak cartridge. The Sep-Pak cartridge was extracted with 10 per cent methanol in ether (2 [cm.sup.3]) and the extract was concentrated by distillation of the ether through a wet-wall microfractionating column.

Known quantities (1[microgram]) of three chloroalkane standards [15] (1-chlorooctane, 1-chloridecane and 1-chlorododecane) were added to the concentrate. An aliquot (3[microliter]) was injected into a split introducer of a Varian 1440 gas chromatograph fitted with an SGE BP1 column (12 m x 0.3 mm id) and coupled of a Varian-MAT 311A mass spectrometer. Under the control of a Finnigan-MAT INCOS 2200 data system, the magnet was set to transmit ions at m/z 90 and the accelerating voltage was switched to monitor the following ions: m/z 91, 93 and 105 for the chlroalkanes, m/z 95, 107 and 108 for 2-methylisoborneol, and m/z 111, 112 and 125 for geosmin. The five compounds were identified by the presence of peaks in the three ions traces at the correct retention times. On the BP1 column 2-methylisoborneol and geosmin had linear retention indexes of 1158 and 1379, respectively. One ion trace for each compound was used for quantification: m/z 91 for the chloroalkanes, m/z 95 for 2-methylisoborneol and m/z 112 for geosmin. The GC-MS system was calibrated by analysing a solution of geosmin, 2-methylisoborneol and the chloroalkane standards at a known concentration of [1[microgram]/cm.sup.3]. Subsequent quantifications were calculated from the response factors obtained with respect to 1-chlorodecane.

Geosmin was shown to be present in the off-flavoured flour in a concentration of not less than 1.2[microgram] [kg.sup.-1] and that of 2-methylisoborneol not less than 0.1[microgram] [kg.sup.-1]. The concentration of geosmin was therefore at least 170 times its odour threshold in water and that of 2-methylisoborneol at least three times. These results indicate that geosmin was the principal cause of the off-flavour in the contaminated flour.

The off0flavoured flour was examined for microbiological contamination. Samples were plated onto a variety of microbiological growth media and low numbers of moulds and bacteria were isolated from the flour. An occasional white 'fluffy' colony approximately 2-3 mm in diameter was present on the Dextrose Tryptone Agar plates which also exhibited a strong odour of geosmin. Colony and cell morphology was typical of an actinomycete. Samples of the fluffy colony were transferred to Plate Count Agar and the organism was purified by sub-culturing on this medium. The intense characteristic odour of geosmin was noticeable at each stage of the purification.

The organism was identified by electron microscopy, chemical analysis and by its carbohydrate utilisation patterns [16] as a strain of Streptomyces griseus. [17] Its identification was confirmed by application of the Gordon tests [18,19] and by a comparison of its reactions in these tests with those of 123 strains of the S. griseus taxon. [17] Cultures of this organism prepared in nutrient agar, a mixture of wheat grains and agar (4:1), and sterilised wheat with 50 per cent moisture all gave the intense aroma of geosmin after only two days incubation at 30 [degrees]C.

At least 12 species of Streptomyces are known to produce geosmin: eight species produce 2-methylisoborneol and a proportion of these species produce both compounds. [1,20] The inoculation of 50 per cent whole grain bread (45 to 50 per cent moisture) with S. griseus strain 10137 has been shown to produce high concentrations of geosmin (20[microgram] [kg.sup.-1]) and 2-methylisoborneol (280[microgram] [kg.sup.-1]) after storage at 25 [degrees]C for between 7 and 14 days. [21] Furthermore, these compounds have also been detected in relatively high concentrations when wheat grains infected with unindentified species of actinomycetes were cultured at 16.8 and 18.2 percent moisture at 30 [degrees]C. [13] In these experiments the yield of geosmin was 17.1 and 3.5[micrograms] [kg.sup.-1] and that 2-methylisoborneol 1.4 and 0.3[microgram] [kg.sup.-1]. The lower figures (18.2 per cent moisture) are comparable with those found in the sample of off-flavoured flour. Geosmin has also been identified in some samples of corn kernels, but its occurrence was not related to any particular off-flavour problem, nor was the organism responsible for its production identified. [22] The current study would appear, therefore, to be the first report of the occurrence of geosmin and 2-methylisoborneol in wheat grains or flour where an organism capable of producing these compounds has been identified.

Although it is not possible to identify the sequence of events that caused the off-flavour problem it would appear that the moisture content of the wheat would have been in excess of that needed to support the growth of S. griseus, which would presumably be greater than 16.8 per cent. [13] At the time of the incident, mill staff reported that the wheat was so damp that it had stuck to the sides of the conditioning bin. Excess of water could have been accidentally introduced into the conditioning bin; instead of raising the moisture content from 11 to 16 per cent, which is normal for some types of wheat, the final moisture content may have been in excess of 25 per cent. As the presence of actinomycetes in wheat and other cereal grains can be accepted as a common occurrence, careful control of the moisture content would appear, therefore, to be the only practical method of preventing the occurrence of off-flavours caused by geosmin and 2-methylisoborneol in these materials.

CITATION:

Whitfield, Frank B., Kevin J. Shaw, Angela M. Gibson, and David C. Mugford. "An earthy off-flavour in wheat-flour: geosmin produced by 'streptomyces griseus.'." Chemistry and Industry n22 (Nov 18, 1991 n22): 841(2). General OneFile. Gale. St Andrews Episcopal School - Austin. 9 Dec. 2007 

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