Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic

Definitions

• the present invention relates to a process for the preparation of primary alcohols containing about 4 to about 20 carbon atoms from organic acids or their derivatives.
• the process is carried out by cultivating a fungus of the genus Mucor in a suitable medium which contains a carboxylic acid or a derivative thereof.
• Hexanol Long-chain primary alcohols, such as octanol and hexanol, have useful organoleptic properties and have long been employed as fragrance and flavor materials.
• Hexanol has important uses in coconut and various berry complexes in fruit flavor compositions, while octanol is used primarily in citrus flavors as well as peach, pineapple, coconut and chocolate flavorants.
• Hexanol occurs naturally in several essential oils such as strawberry, Java citronella, Bourbon geranium, lavender and bitter orange.
• Octanol is found in green tea, grapefruit, and several types of oranges.
• Extracts of various dried mushrooms have also been reported in J. Agr. Food Chem. 21: 955-958 (1973) to contain trace amounts of hexanol and octanol. It has recently been reported in Acta Biotechnol, 2:209-219 (1987) that strains of Zymomonas are capable of producing trace amounts of hexanol. Additional reports have also been made regarding the production of low levels of long-chain alcohols by yeasts during the fermentation of wine and beer. These chemicals are generally considered undesirable in these products.
• Another object of the invention is to provide a microbial process for the production of primary alcohols of variable carbon chain length.
• a microbial process for the production of various long- chain alcohols wherein a Mucor fungal culture, or an enzyme extract thereof, is incubated with a substrate comprising a carboxylic acid having at least four carbon atoms or a derivative thereof, including a salt, an alkyl ester, a mono-, di- or triglyceride or an unsubstituted, monoalkyl or dialkyl amide thereof, to produce the desired alcohol through fermentation.
• the organic carboxylic acid or its derivative used as the substrate for the microbiological process of this invention may be substituted with any functional group that does not interfere with the formation of a terminal hydroxyl group by the reduction of the carboxylic acid moiety.
• a saturated carboxylic acid of from 4 to 20 carbons in length or a derivative thereof, to produce a C. - C 2Q primary alcohol.
• the following invention is directed to a microbial process useful for the production, in unexpectedly high yields, of long-chain primary alcohols from an organic carboxylic acid of at least 4 carbons or a derivative thereof.
• the organic carboxylic acid is preferably a C. - C 2Q alkanoic or alkenoic acid and the corresponding primary alcohols will be the same number of carbon atoms in length.
• organoleptic refers to compounds of the invention which stimulate the sense of smell or taste, and are thus perceived as having a characteristic odor and/or flavor.
• odor e.g., acetyl
• odor e.g., acetyl
• flavor e.g., acetyl
• flavoring e.g., acetyl
• flavorant e.g., acetyl
• alkyl refers to a branched or linear saturated carbon chain containing 1 to 20 carbon atoms
• alkenyl refers to branched or linear carbon chains containing one or more double bonds.
• the long-chain primary alcohols produced by the process of the present invention are organoleptic compounds which possess useful, characteristic flavor and fragrance properties. By including effective amounts of one or more of the alcohols produced according to this invention, it is possible to augment or enhance the flavor or fragrance of consumable materials. These alcohols are particularly useful in certain flavor compositions where wholly natural ingredients are required.
• Primary alcohols of varying molecular weight can be prepared, based upon the fermentative incubation of a fungus of the genus Mucor, in conjunction with an appropriate substrate, to obtain a high yield of the desired alcohol.
• Mucor fungus reduces the carboxylic acid moiety of the substrate to form an alcohol.
• organic carboxylic acid or its derivative used as the substrate for the microbiological process of this invention may be substituted with 1 or more functional groups that do not interfere with the formation of a terminal hydroxyl group.
• Preferred substrates include compounds of the formula:
• Z is -OX, -OCH 2 CHOR 3 CHOR 4 or -NR 5 R 6 where X is hydrogen, alkyl of 1 to 6 carbons, an alkali or alkaline earth metal cation or an ion exchange resin; R 3 and R4 are independently hydrogen, alkyl of 1 to 6 carbons or R 2C(0) with R2 as defined above; and
• R and R independently are hydrogen or alkyl of 1 to 6 carbons.
• alpha carbon is unsubstituted and unbranched.
• Another preferred substrate group is C to C_ straight chain saturated aliphatic carboxylic acids, i.e., R is alkyl and X is hydrogen.
• R 2 represents R 2 ' (CH 2 ) 4
• R 2 ' i hydrogen, C , 15 alkyl, or alkenyl of 1 to 15 carbon atoms.
• the substrate acid may be added to the fungus directly as the free acid or in the form of a salt, e.g., sodium, potassium, calcium, magnesium, ammonium, and the like.
• a salt e.g., sodium, potassium, calcium, magnesium, ammonium, and the like.
• any of the acid derivatives e.g., ester, amide, anhydride, and the like
• the alcohol portion preferably contains up to 6 carbon atoms.
• Glycerol esters can also be employed.
• the nutrient broth used according to the process of the invention includes the usual sources of nitrogen, carbohydrates and minerals.
• Incubative fermentation conditions used according to the process may include any pH, temperature, substrate concentration and substrate feed rate which will maintain the viability of the culture.
• the process of the present invention may be conducted in batch or continuous mode operations. In a batch fermentation, the nutrient medium, culture and substrate are combined and fermented until the alcohol concentration becomes constant. In a continuous process, the substrate or nutrient medium may be continuously recirculated through a fermentation reactor. The substrate is continuously added and product is similarly removed from the recirculating medium.
• a suitable medium is one which contains carbon sources, nitrogen sources, inorganic salts and growth factors.
• Suitable carbon sources include, glucose, fructose, xylose, sucrose, maltose, lactose, mannitol, sorbitol, glycerol, corn syrup and corn syrup solids.
• suitable nitrogen sources include organic and inorganic nitrogen-containing substances such as peptone, corn steep liquor, meat extract, yeast extract, casein, urea, amino acids, ammonium salts, nitrates and mixtures thereof.
• Phosphates, sulfates, magnesium, sodium, calcium, and potassium are examples of inorganic salts which may be used in the aqueous medium of the process of the present invention.
• Suitable growth factors include one or more vitamins of the B group, and one or more trace minerals, such as iron, manganese, cobalt or copper.
• peptone When used as a nitrogen source, peptone is preferably utilized at a concentration of about 0.1 to about 3 weight percent. It is preferable to utilize peptone at a concentration, relative to the total weight of the broth, of about 0.01 to about 1.0 weight percent.
• Yeast extract when used as a vitamin source in the aqueous medium, is preferably utilized at a concentration of about 0.1 to 2 weight percent. It is more preferable to utilize yeast extract at a concentration, relative to the total weight of the broth, of about 0.2 to about 1 weight percent.
• the nutrients may be supplemented with various vitamins and minerals, such as one or more B vitamins and trace minerals, such as iron, manganese, cobalt and copper and the like.
• B vitamins either as a separate supplement or in the form of a yeast extract.
• Dextrose may optionally be included in the nutrient medium, preferably at a concentration, for example, of about 1 to about 20 weight percent, and more preferably from about 2 to about 10 weight percent. The most preferred concentration is from about 2 to 5 weight percent.
• concentration for example, of about 1 to about 20 weight percent, and more preferably from about 2 to about 10 weight percent. The most preferred concentration is from about 2 to 5 weight percent.
• the kind and amount of the above mentioned additives to the aqueous medium may be determined based upon the specie selected and the time and temperature of incubation.
• a Mucor fungus is cultivated in innoculum quantities to produce a mature culture.
• the culture is innoculated into a fermenter and allowed to incubate.
• the substrate is then added and fermentation is carried out continuously until a steady concentration of alcohol is present.
• the cultivation and fermentative incubation of the fungus can be carried out as a stationary culture or as a submerged culture (e.g., shake-flask, fermentor) under anaerobic conditions. Cultivation and incubation may proceed at any suitable pH range, e.g., from about 3 to about 9, more preferably in the range of from about 4 to about 8, and most preferably from about 6 to about 7.
• the pH may be adjusted b the addition of organic or inorganic acids or bases, such as hydrochloric acid, acetic acid, sodium hydroxide, calcium carbonate, ammonia, ion-exchange resins, or by the addition o a buffer such as phosphate, phthalate or Tris ® .
• the incubation temperature is suitably maintained at between about 18°C and about 31°C, with a preferred range of from about 20°C to about 28"C and a particularly preferred range from about 24°C to about 27°C. While anaerotic conditions are preferable, the cultivation and fermentative incubation of the fungus may be carried out in an atmosphere containing up to about 100% dissolved oxygen.
• the substrate is added to the culture medium at the outset under anaerobic conditions.
• the substrate may be added either alone or in combination with another carbon source, such as glucose, during fermentative incubation or after cultivation is complete. It is preferable to add the substrate to the culture medium during the period from about 7 to 24 hours after growth of the culture in the fermentative broth has commenced.
• the substrate may be continuously added during fermentation, after the first 7 to 12 hours.
• a preferred continuous feed rate for this addition is about 0.001 to 2 g/hour/liter of medium.
• a more preferred feed rate is about 0.01 to 1 g per hour per liter, and the most preferred feed rate is 0.1 to 0.3 g per hour per liter.
• the steady state concentration of the substrate in the medium may vary depending on the conditions.
• the concentration of the substrate in the medium may conveniently vary, relative to the total weight of the nutrient broth, from about 0.01% to about 10%, preferably about 0.1% to about 5%, more preferably 0.1 to about 0.5%, consistent with the manner in which it is added to the culture.
• the reaction period will vary according to the specific incubation parameters, such as the strain of microorganism employed, the composition of the culture medium and type of substrate present. In general, shake-flask culture techniques require from between 2 hours and about 10 days to reach steady state depending upon the microbial strain and substrate utilized. When a fermenter is used, however, the fermentation period may be reduced to about 90 hours .or less.
• the fermentative incubation of the fungus is carried out under anaerobic conditions, wherein the dissolved oxygen content in the incubation broth is maintained at 0% by sparging nitrogen gas into the vessel.
• the substrate is preferably maintained in continuous contact with the aqueous phase and the microorganism. Generally, vigorous stirring or shaking is satisfactory, but a surface active agent, such as Tween 80, can be added to aid in the dispersion of the substrate if desired.
• Conventional antifoam agents such as silicone oils, polyalkylene glycol derivatives, or soya oil can be used to control foaming.
• the fermentation may be carried out using cells isolated from the culture solution, or with an enzyme extract isolated from the cells in a manner known in the art.
• the reaction can be conveniently carried out in aqueous solution.
• aqueous solutions include buffer solutions and water.
• the isolated cells or an enzyme extract thereof may be immobilized on a solid support and the desired transformation conducted separately. It is convenient to employ the immobilized form of the enzyme extract in a continuous process.
• the fermentation of the substrate may also be effected by mutants of the fungus.
• the progress of the fermentative production of the alcohol can be monitored by assaying for alcohol concentrations using standard analytical techniques such as chromotography (gas-liquid, thin layer or high pressure liquid) and spectroscopy such as IR and NMR.
• Fermentation can also be followed by measuring the consumption of substrate, e.g., fatty acid or derivative thereof, or carbohydrate, e.g., glucose, or by measuring pH changes.
• substrate e.g., fatty acid or derivative thereof, or carbohydrate, e.g., glucose
• the fermentative production of the alcohol is generally terminated when all of the substrate has been consumed, or when no further increase in the alcohol concentration is observed.
• the final products containing four to twenty carbon atoms can be isolated and purified by solvent extraction, distillation, chromatographic separation, high pressure liquid chromatography and the like.
• the present invention produces a generally high yield of such primary alcohols, e.g., from about 50 to 100 mgs. to about 2 to 3 grams of primary alcohol per liter of medium.
• Conventional fermentation methods result in alcohol yields at best on the order of only several milligrams per liter.
• Ten ml of a stationary culture of Mucor circinelloides can be inoculated into a two liter fermentor containing IL of a sterile broth composed of 1% peptone, 0.5% yeast extract, and 5% dextrose dissolved in IL of water (herein after referred to as PYE-5 broth) .
• PYE-5 broth a sterile broth composed of 1% peptone, 0.5% yeast extract, and 5% dextrose dissolved in IL of water
• PYE-5 broth 2-methylbutyl hexanoate
• 2MBH 2-methylbutyl hexanoate
• the extract weighs 8g and the major components are 15% hexanol, 15% methyl hexanoate, 15% hexanoic acid and 40% 2MBH. This represents a hexanol yield of 1.2 g/L broth.
• M. circinelloides (ATCC no. 20983) was grown in 10 ml of PYE broth containing 10% dextrose and 2.5% ethyl octanoate at 27°C and 250 rpm on a rotary shaker. The pH wa periodically adjusted to 7.0 over the course of the next 4 d after which the broth was extracted. The extract weighed 1. and contained 60% ethyl octanoate, 20% octanol, 2% methyl caproate and 4% octanoic acid.
• EXAMPLE 3 FERMENTATION WITH MUCOR CIRCINELLOIDES FOR OCTANOL PRODUCTI 15L of PYE-5 may be inoculated with 100 ml of a stationary culture of M. circinelloides (ATCC no. 20983) . T pH should be maintained at 7.0 and the temperature at 27°C. gases are added to the reactor. After 24 hours, ethyl octanoate is pumped into the culture at a rate of 2 mL/h for hours (total of 48 ml ester added) . The broth is extracted with organic solvent after a total of 36 hours of incubation The extract weighs 35 g and contains 55% ethyl octanoate, 23 octanoic acid and 3% octanol.
• EXAMPLE 4 FERMENTATION OF VARIOUS STRAINS OF MUCOR FOR HEXANOL PRODUCT M. circinelloides, ATCC Deposit Nos. 27649, 8540 a 42258, were grown in PYE-2 containing 1.5% malt extract as described in Example 1. After a 24 hour preincubation perio 0.5% 2MBH was added to each culture. After 3 days, the cultures were extracted, and the extracts contained 2 to 3% hexanol, which represented a yield of less than O.lg hexanol broth.
• strains tested include M. hiemalis, ATCC No.
• 16L of PYE-10 may be inoculated with 300 ml of a 2 hour stationary culture of M. circinelloides (ATCC no. 20983)
• ethyl hexanoate is pumped into the reactor at rate of 1 ml/h, which is increased to 2 ml/h after 17 hours.
• the ester is fed for 72 hours (total ester added was 161 ml) .
• the total volume of broth was then extracted with methylene chloride.
• the resulting extract weighs 27 g and contains 64 25 hexanol (17.4 g) representing a yield of 1.1 g/L.

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• 1991
  • 1991-03-08 WO PCT/US1991/001598 patent/WO1991013997A1/en not_active Application Discontinuation
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