US3909356A - Production of fatty acid esters of fructose - Google Patents

Production of fatty acid esters of fructose Download PDF

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US3909356A
US3909356A US500720A US50072074A US3909356A US 3909356 A US3909356 A US 3909356A US 500720 A US500720 A US 500720A US 50072074 A US50072074 A US 50072074A US 3909356 A US3909356 A US 3909356A
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fatty acid
fructose
atcc
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acid esters
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Takeo Suzuki
Seiga Ito
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KH Neochem Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • C07H13/06—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12P19/00—Preparation of compounds containing saccharide radicals
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    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/06—Arthrobacter
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/29—Micromonospora
    • C12R2001/31—Micromonospora purpurea ; Micromonospora echinospora; Micromonospora rhodorangea
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/32—Mycobacterium
    • C12R2001/34—Mycobacterium smegmatis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/365—Nocardia
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/83—Arthrobacter
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/843—Corynebacterium
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/863—Mycobacterium
    • Y10S435/866—Mycobacterium smegmatis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/867—Micromonospora
    • Y10S435/869—Micromonospora purpurea
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/872—Nocardia

Definitions

  • the present inventionf relates to the production of fatty acid esters of fructose by fermentation. More speci fically, this invention relates to a process for producing fatty acid esters of fructose by culturing a fructoseutilizing microorganism capable of producing fatty acid esters of fructose and belonging tothe genus Arthrobacter, Corynebacterium, Nocardia or Mycobacterium in a medium containing fructose as a carbon source.
  • the fatty acid esters of fructose of the present invention are glycolipids which are used for biochemical experiments.
  • the product has a surface activity and may be used as a surfactant.
  • the present inventors have previously found that a variety of hydrocarbon-utilizing microorganisms can produce a remarkable amount of fatty acid esters of glucose, trehalose or sucrose from n-paraffins (U.S. Pat. No. 3,637,461).
  • n-paraffins U.S. Pat. No. 3,637,461.
  • micro-organisms of the genus Arthrobacter, Corynebacterium, Nocardia or Mycobacterium capable of utilizing fructose are cultured in a medium containing fructose as a carbon source, glycolipids quite different from those obtained by using n-paraffins are produced.
  • the glycolipids are identified with fatty acid esters of fructose.
  • fatty acid esters of fructose are obtained by culturing a fructose-utilizing microorganism belonging tothe genus Arthrobacter, Corynebacterium, Nocardia or Mycobacterium and capable of producing fatty acid esters of fructose in a medium containing fructose as a carbon source, forming the fatty acid esters of fructose intracellularly and recovering the esters from the microbial cells.
  • the fatty acid ester of fructose obtained according to the present process consists of one mol of fructofuranose having the following structure: 3
  • the .microorganisms applicable in the present process are found widely among the microorganisms of the genera Arthrobacter, Corynebacterium, Nocardia and Mycobacterium. Examples thereof are as follows:
  • ammonium salts such as ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate and ammonium phosphate, ammonia and urea may be used.
  • natural substances containing nitrogen such as corn steep liquor, yeast extract, meat extract, peptone, casamino acid, etc. may also be used. These substances may be used either singly or in combinations of two or more.
  • Fermentation is carried out under aerobic conditions at 25f40C. During'the'fermentation, the pH of the fermentation liquor is adjusted to 4-9, preferably, 6-8
  • the microbial cells are separated from the fermentation liquor, for example, by centrifugation.
  • the cells are subjected to extraction with a mixture of chloroform and methanol in a volume ratio ranging from 50:100 to :50.
  • the solvent in the resulting extract is distilled off under reduced pressure, for example, at 20 to 30 mmHg.
  • the resulting residue is subjected to extraction with an appropriate solvent such as chloroform, hexane and ethylacetate.
  • the solvent of the extract is removed off.
  • the residue is again dissolved in a small amount of a solvent such as chloroform, hexane and ethylacetate and subjected to filtration.
  • the filtrate is poured into a silica gel column.
  • non-polar substances such as pigments and free fatty acids are eluted with chloroform.
  • elution is further carried out with a chloroformmethanol mixture, while stepwise varying the volume ratio of chloroform to methanol in the mixture from 99:1 to 95:5.
  • the eluate containing the desired product is recovered and the solvent is removed.
  • the resulting residue is dissolved in warm acetone.
  • the solution is allowed to stand still in a cold place, e.g. at 15C for about 5 to 24 hours.
  • the resulting precipitates are recovered and dried, whereby a white powder of the product is obtained.
  • the white powder is subjected to thin layer chromatography using a mixture of chloroform, methanol and acetic acid.
  • the powder When the powder is found to contain two kinds of products, the powder is dissolved in n-hexane and the resulting solution is poured into a silica gel column. Elution is carried out first with a mixture consisting of chloroform and methanol ina given volume ratio (e.g. 98:2 and 99:1) andthen with a mixture consisting of ,chloroform and methanol in another given volume ratio (e.g. 95:5 and 97:3), whereby eluates respectively containing the product are obtained. Each of the eluates is concentrated and the concentrate is allowed to stand still in a cold place, e.g. at C. The resulting precipitates are recovered and dired. Thus, the two products are isolated.
  • a given volume ratio e.g. 98:2 and 99:1
  • a mixture consisting of ,chloroform and methanol in another given volume ratio e.g. 95:5 and 97:3
  • the product is an ester of fructose and a fatty acid having the above mentioned structure.
  • the ester is subjected to the anthrone reaction and periodate oxidation in order to determine the composition ratio of fructose to fatty acid in its molecule. According to the results, the structure of the ester is determined.
  • Fructose g/l (N -M2 4 5 Com steep liquor 3 NaHPO,.12 O 2 KH PO 2 MgS0 ,.7H,O 1 FeSO..7l-l O 0.5 g/l MnSO .4H O 20 mg/l ZnSO .7H O 10 CaC
  • 2.8 liters of the fermentation liquor is subjected to centrifugation to obtain 250 g (wet weight) of the microbial cells.
  • the cells are subjected to extraction at room temperature for three times, each with one liter of a mixture of chloroform and methanol in a volume ratio of 1:1.
  • Three liters of the resulting extract is concentrated in a flash evaporator. 10 g of the residue is subjected to extraction with 100 m1 of chloroform and the solvent in the obtained extract is removed.
  • the residue is dissolved in 100 ml of n-hexane. After concentration and centrifugation, 20 ml of n-hexane solution is obtained.
  • the thus obtained solution is poured into a column having an inner diameter of 4 cm and a height of 20 cm packed with silica gel. Then, chloroform is passed through the column to remove pigments and free fatty acids. Elution is carried out with a mixture of chloroform and methanol while stepwise varying the volume ratio of chloroform to methanol in the mixture from 99:1 to 95:5. The fractions obtained by the elution with a mixture of chloroform and methanol in a volume ratio of 95:5 are collected and the solvent is removed. The resulting residue is dissolved in warm acetone. Upon allowing the solution to stand still at l5C, precipitates are obtained. The precipitates are dried, whereby 2.5 g of a white powder is obtained.
  • Product A is found to be a mono-fatty acid ester of fructose wherein fatty acid of the formula II is bonded to fructose at 6- position.
  • R of the fatty acid is found to be an alkyl group having l0, 12 or 14 carbon atoms and R an alkyl group having 15 or 17 carbon atoms.
  • Product B is found to be a di-fatty acid ester of fructose having two fatty acid residues at land 6-positions. The fatty acid residues are respectively identical with that of Product A.
  • Products A and B are shown below:
  • Product A is powdery when dried; readily absorbs moisture in the air and becomes pasty; has an average molecular weight of 714; is soluble in ether, chloroform and n-hexane at room temperature, soluble in methanol, ethanol and acetone havinga temperature of 4060C and further soluble in hot water; and is light yellow.
  • FIG. 1 is an infrared absorption spectrum of this ester.
  • EXAMPLE 3 In 'this example, Corynebacterium hydrocarboclastus ATCC 12628 is .used. Fermentation is carried out in the same manner as in Example 1 except for using this strain and continuing the fermentation for 30 hours. As the result, 220 g (wet weight) of the microbial cells is obtained. From these cells, 1.7 g of Product A and 0.3 g of Product B are isolated in the same manner as in Example 1.
  • Product A is found to be a mono-fatty acid ester of fructose wherein a fatty acid of the formula II is bonded to fructose at 6- position.
  • R of the fatty acid is found to be an alkyl group having 8, 10 or 12 carbon atoms and R an alkyl group having'33,35 or 37 carbon atoms.
  • Product B is found to be a di-fatty acid ester of fructose having two fatty acid residues at 1- and o-positions.
  • the fatty acid residues are respectively identical with that of Product EXAMPLE 4
  • the same fermentation procedures as those in Example 1 are repeated, except for using Corynebacterium psudodiphtheriticum ATCC 10701 and continuing the fermentation for 48 hours.
  • EXAMPLE 5 In this example, Nocardia paraffinica ATCC 21 198 is used. Fermentation is carried out in the same manner as in Example 1, except for using this strain and continuing the fermentation for 38 hours. As the result, g (wet weight) of the microbial cells are obtained. From these cells, 1.3 g of Product A is isolated in the same manner as in Example 1.
  • Product A is found to be a mono-fatty acid ester of fructose wherein a fatty acid of the formula II is bonded to fructose at 6- position.
  • R of the fatty acid is found to be an alkyl group having 10, 12 or 14 carbon atoms and R" an alkyl group having 33, 35 or 37 carbon atoms.
  • the alkyl groups in the fatty acid residue of Product A have .a large number of carbon atoms as compared with those in the fatty acid residue of Products A and B obtained in Example 1.
  • Such a fatty acid having alkyl groups of a large number of carbon atoms is generally called nocardomycolic acid.
  • Example 6 The same fermentation procedures as those in Example 1 are repeated, except for using Nocardia globerula ATCC"13130 and continuing the fermentation for 53 hours. As the result, 200 g (wet weight) of the microbial cells is obtained. From these cells, Product A and Product B are isolated in the same manner as in Example l in amounts of 0.8 g and 0.2 g, respectively.
  • Product A is found to be a mono-fatty acid ester of fructose wherein a fatty acid of the formula II is bonded to fructose at 6- position.
  • R of the fatty acid is found to be an alkyl group having 20 or 22 carbon atoms and R an alkyl group having 57, 59 or 61 carbon atoms.
  • Product 8. is found to be a di-fatty acid ester of fructose having two fatty acid residues at 1- and 6-positions.
  • the fatty acid residues are respectively identical with that of Product A EXAMPLE 8
  • Mycobacterium smegmatis ATCC 21293 is used. Fermentation is carried out in the same manner as in Example 1, except for using this strain. As the result, 165 g (wet weight) of the microbial cells is obtained. From these cells, 0.8 g of Product A and a trace amount of Product B are obtained.
  • Product A is found to be a mono-fatty acid ester of fructose wherein a fatty acid of the formula II is bonded to fructose at 6- position.
  • R of the fatty acid is found to be an alkyl group having 10, 12 or 14 carbon atoms and R" an alkyl group having 15, 17 or 19 carbon atoms.
  • Product B is found to be a di-fatty acid ester of fructose having two fatty acid residues at land 6-positions.
  • the fatty acid residues are respectively identical with that of Product A EXAMPLE 9
  • the same fermentation procedures as those in Example 1 are repeated, except for using Mycobacterium smegmatis ATCC 607. As the result, 250 g (wet weight) of the microbial cells is obtained. From these cells, one g of Product A and a trace amount of Product B are isolated.
  • Product A is found to be a mono-fatty acid ester of fructose wherein a fatty acid of the formula II is bonded to fructose at 6- position.
  • R of the fatty acid is found to be an alkyl group having l0, 12 or 14 carbon atoms and R" an alkyl group having l5, 17 or 19 carbon atoms.
  • Product B is found to be a di-fatty acid ester of fructose having two fatty acid residues at 1- and 6-positions.
  • the fatty acid residues are respectively identical with that of Product A EXAMPLE 10
  • Nocardia convoluta ATCC 4275 is used. Seed culturing is carried out in the same manner as in Example 1 except the culturing is continued for 48 hours.
  • 1.2 liters of the resulting seed culture is inoculated into 15 liters of a fermentation medium in a 30 liter-jar fermenter.
  • the fermentation medium has the same composition as that of Example 1 except for containing 120 g/l fructose and further containing 3 g/l yeast extract. Fermentation is carried out with stirring at 400 rpm. and an aeration of l l/l/min at 30C for 45 hours. During the fermentation, the pH of the fermentation liquor is automatically adjusted to 6.5-7.0 with ammonia.
  • the fermentation liquor is subjected to centrifugation to obtain 3.0 kg (wet weight) of the microbial cells.
  • the microbial cells are washed with a mixture of ether and ethanol having a volume ratio of 1:1.
  • extraction is car ried out twice, each with 4.5 liters of a mixture of chloroform and methanol in a volume ratio of 1:1 at room temperature.
  • 9 liters of the resulting extract is concentrated in a flash evaporator.
  • the residue is subjected to extraction with 500 ml of ethyl acetate.
  • the solvent in the obtained extract is removed and the residue is dissolved in chloroform. After filtration, 100 ml of chloroform solution is obtained.
  • the thus obtained solution is poured into a silica gel column having an inner diameter of 4 cm and a height of 50 cm. Chloroform is then passed through the column to remove pigments and free fatty acids. Elution is carried out initially with a mixture of chloroform and methanol in a volume ratio of 99:1 while stepwise varying the mixing ratio by volume of chloroform to methanol from 99:1 to :5.
  • the fractions obtained by the elution with a chloroform-methanol mixture having a volume ratio of 99: 1 contain Product D and those obtained by the elution with a chloroform-methanol mixture having a volume ratio of 97:3, contain Product C.
  • Each of the eluates is concentrated and the concentrate is poured into a silica gel column.
  • Product C is found to be a tetra-fatty acid ester of fructose wherein four residues of a fatty acid of the formula II are bonded to fructose.
  • the four fatty acid residues have the same structure.
  • R of the fatty acid residue is found to be an alkyl group having 10, 12, 14 or 16 carbon atoms and R an alkyl group having 45, 47, 49 or 51 carbon atoms.
  • Product D is found to be a penta-fatty acid ester of fructose. The five fatty acid residues are identical with those of Product C.
  • FIG. 2 shows an infrared absorption spectrum of Product C.
  • the infrared absorption spectrum of Product D is almost identical with that of Product C.
  • Example 1 1 In this example, Mycobacterium rubrum ATCC 14346 is used. Fermentation is carried out in the same manner as in Example 10 except for using this strain and continuing the fermentation for 65 hours. As the result, 1.2 kg (wet weight) of the microbial cells is obtained. From these cells, 0.5 g of Product C and 0.15 g of Product D are isolated in the same manner as in Example 10.
  • Product C is found to be a tetra-fatty acid ester of fructose wherein four residues of a fatty acid of the formula 11 are bonded to fructose.
  • the four fatty acid residues are identical.
  • R of the fatty acid residue is found to be an alkyl group having or 22 carbon atoms and R" an alkyl having 53, 55 or 57 carbon atoms.
  • Product D is found to be a penta-fatty acid ester of fructose. The five fatty acid residues are identical with those of Product C.
  • a process for producing fatty acid esters of fructose which comprises culturing a fructose-utilizing microorganism capable of producing fatty acid esters of fructose and belonging to the genus Arthrobacter, Corynebacterium, Nocardia or Mycobacterium in a medium containing fructose as a carbon source, forming the fatty acid esters of fructose intracellularly and recovering the esters from the microbial cells.
  • esters are recovered from the microbial cells by initially separating the microbial cells from the culture liquor, extracting the cells with an organic solvent system, vaporizing the solvent system, further extracting the resulting residue with another solvent and finally effecting elution of the fatty acid esters.
  • fatty acid esters obtained are a reaction product of 1 mole of fructofuranose and 1, 2, 4 or 5 mols of a fatty acid, the fatty acid residue in the esters having the following formula:
  • R is an alkyl group having 8 to 22 carbon atoms and R" is an alkyl group having 15 to 61 carbon atoms.
  • microorganism is selected from the group consisting of:
  • Arthrobacter paraffineus ATCC 15591 Arthrobacter hydrocarboglutamicus ATCC 15583 Cofynebacterium hydrocarboclastus ATCC 21628 Corynebacterium psudodiphtheriticum ATCC 10701 Nocardia repeatfinica ATCC 21198 Nocardia globerula ATCC 13130 Nocardia convoluta ATCC 4275 Mycobacterium rubrum ATCC 14346 Mycobacterium paraflinicum ATCC 12670 Mycobacterium smegmatis ATCC 21293 Mycobacterium smegmatis ATCC 607

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Abstract

A process for producing fatty acid esters of fructose including the steps of (a) culturing a fructose-utilizing microorganism capable of producing fatty acid esters of fructose and belonging to the genus Arthrobacter, Corynebacterium, Nocardia or Mycobacterium in a medium containing fructose as a carbon source, (b) forming the fatty acid esters of fructose intracellularly and (c) recovering the esters from the microbial cells.

Description

United States Patent [191 Suzuki et al.
[451 Sept. 30, 1975 1 PRODUCTION OF FATTY ACID ESTERS OF FRUCTOSE [75] Inventors: Takeo Suzuki, Hofu; Seiga lto,
Machida, both of Japan [73] Assignee: Kyowa Hakko Kogyo Co., Ltd.,
Japan [22] Filed: Aug. 26, 1974 '21 Appl. No.: 500,720
[30] Foreign Application Priority Data Aug. 29, 1973 Japan 48-96175 [52] U.S. Cl 195/47; 195/42 [51] Cl2D 13/02 [58] Field of Search 195/31 R, 31 P, 28 R, 96,
[56] References Cited UNITED STATES PATENTS l/l972 Tawaka et al 195/28 R OTHER. PUBLICATIONS Brennan et a1., Acylglucoses in Corynebacteria,"
Chemical Abstracts, Vol. 71, p. 63, 36500t, (1969).
Winder et al., Turnover of Acylglucose Acyltrehlose and Free Trehalose During the Growth of Mycobacterium Smegmatis on Glucose, Chemical Abstracts, Vol. 78, 26247w, (l973) Brennan et al., Acylglucoses of the Corynebacteria and Mycobacteria."
Primary E.\'anziner-Lionel M. Shapiro Assistant E.ranzinerThomas G. Wiseman Att rney, Agent, or FirmCraig & Antonelli [57] ABSTRACT v 6 Claims, 2 Drawing Figures US, Patent Sept, 30,1975 3,909,356
PRODUCTION OF FATTY ACID ESTERS OF FR-UCTOSE BACKGROUND OF THE INVENTION The present inventionfrelates to the production of fatty acid esters of fructose by fermentation. More speci fically, this invention relates to a process for producing fatty acid esters of fructose by culturing a fructoseutilizing microorganism capable of producing fatty acid esters of fructose and belonging tothe genus Arthrobacter, Corynebacterium, Nocardia or Mycobacterium in a medium containing fructose as a carbon source.
The fatty acid esters of fructose of the present invention are glycolipids which are used for biochemical experiments. The product has a surface activity and may be used as a surfactant.
Heretofore, there has been a report on the production of fatty acid esters of glucose by culturing a bacterium of the genus Mycoplasma, Aerobacter, Pseudomonas, Escherichia, Sreptococcus, Mycobacterium or Corynebacterium in the presence of glucose [1 J. Brennan et al.: Eur. J. Biochem. 13 (1970) 117-123].
The present inventors have previously found that a variety of hydrocarbon-utilizing microorganisms can produce a remarkable amount of fatty acid esters of glucose, trehalose or sucrose from n-paraffins (U.S. Pat. No. 3,637,461). As the result of continued research, it has been found that when micro-organisms of the genus Arthrobacter, Corynebacterium, Nocardia or Mycobacterium capable of utilizing fructose are cultured in a medium containing fructose as a carbon source, glycolipids quite different from those obtained by using n-paraffins are produced. The glycolipids are identified with fatty acid esters of fructose.
BRIEF DESCRIPTION OF THE DRAWINGS SUMMARY OF THE INVENTION In accordance with the present invention, fatty acid esters of fructose, particularly a-branched-B-hydroxy fatty acid esters of fructofuranose, are obtained by culturing a fructose-utilizing microorganism belonging tothe genus Arthrobacter, Corynebacterium, Nocardia or Mycobacterium and capable of producing fatty acid esters of fructose in a medium containing fructose as a carbon source, forming the fatty acid esters of fructose intracellularly and recovering the esters from the microbial cells. 7
DETAILED DESCRIPTION OF THE INVENTION The fatty acid ester of fructose obtained according to the present process consists of one mol of fructofuranose having the following structure: 3
HOHC OH H -CH OH- 2 and 1,2, 4 or 5 mols of an a-branched-B-hydroxy fatty acid represented by the general formula:
(wherein R is an alkyl group having 8 to 22 carbon atoms and R" is an alkyl group having 15 to 6 1 carbon atoms). v
The .microorganisms applicable in the present process are found widely among the microorganisms of the genera Arthrobacter, Corynebacterium, Nocardia and Mycobacterium. Examples thereof are as follows:
' Either a synthetic culture medium or a natural nutrient medium may be employed for the culturing of the microorganisms so long as the medium properly contains a carbon source, a nitrogen source, inorganic materials and other nutrients which may be required by I the specific strains employed.
It is essential that the medium contains fructose, or a crude substance containing fructose, such as molasses, as the carbon source.
As the nitrogen source, ammonium salts such as ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate and ammonium phosphate, ammonia and urea may be used. Further, natural substances containing nitrogen, such as corn steep liquor, yeast extract, meat extract, peptone, casamino acid, etc. may also be used. These substances may be used either singly or in combinations of two or more.
As inorganic materials, potassium phosphate, magnesium sulfate, iron, manganese salts, calcium chloride, sodium chloride and zinc sulfate may be used.
When the microorganism employed has a require ment for some nutrients such as amino acids and vita; mins, the nutrients must, of course, be supplemented to the medium.
Fermentation is carried out under aerobic conditions at 25f40C. During'the'fermentation, the pH of the fermentation liquor is adjusted to 4-9, preferably, 6-8
with a urea solution, an aqueous ammonia or an aque- I ous ammonium carbonate solution. Usually, fermentation is complete in to 7 days. When the yield of the desired product reaches a maximum, fermentation is discontinued. The product is formed predominantly in the microbial cells.
.After. the completion of fermentation, the microbial cells are separated from the fermentation liquor, for example, by centrifugation. The cells are subjected to extraction with a mixture of chloroform and methanol in a volume ratio ranging from 50:100 to :50. The solvent in the resulting extract is distilled off under reduced pressure, for example, at 20 to 30 mmHg. The resulting residue is subjected to extraction with an appropriate solvent such as chloroform, hexane and ethylacetate. The solvent of the extract is removed off. The residue is again dissolved in a small amount of a solvent such as chloroform, hexane and ethylacetate and subjected to filtration. The filtrate is poured into a silica gel column. First, non-polar substances such as pigments and free fatty acids are eluted with chloroform. Then, elution is further carried out with a chloroformmethanol mixture, while stepwise varying the volume ratio of chloroform to methanol in the mixture from 99:1 to 95:5. The eluate containing the desired product is recovered and the solvent is removed. The resulting residue is dissolved in warm acetone. The solution is allowed to stand still in a cold place, e.g. at 15C for about 5 to 24 hours. The resulting precipitates are recovered and dried, whereby a white powder of the product is obtained.
The white powder is subjected to thin layer chromatography using a mixture of chloroform, methanol and acetic acid.
When the powder is found to contain two kinds of products, the powder is dissolved in n-hexane and the resulting solution is poured into a silica gel column. Elution is carried out first with a mixture consisting of chloroform and methanol ina given volume ratio (e.g. 98:2 and 99:1) andthen with a mixture consisting of ,chloroform and methanol in another given volume ratio (e.g. 95:5 and 97:3), whereby eluates respectively containing the product are obtained. Each of the eluates is concentrated and the concentrate is allowed to stand still in a cold place, e.g. at C. The resulting precipitates are recovered and dired. Thus, the two products are isolated.
The obtained product is subjected to infrared absorption spectrometry. The product exhibits an absorption at 1,7O0 crn' characteristic to an ester linkage.
The product is subjected to gas chromatography. As the result, it is determined that the product has in its molecule'one mol fructose represented by the formula 6 o noa i/ 012i 7 I a a cn on The product is reacted with 0.5N NaOH. The resultingreaction mixture is subjected to paper chromatog- -raphy and it is confirmed that in the mixture there ex- (wherein R is an alkyl group having 8 to 22 carbon atoms and R" is an alkyl group having 15 to 61 carbon atoms).
In the reaction of the product with 0.5N NaOl-l, the fatty acid is readily liberated.
From the foregoing, it is evident that the product is an ester of fructose and a fatty acid having the above mentioned structure.
The ester is subjected to the anthrone reaction and periodate oxidation in order to determine the composition ratio of fructose to fatty acid in its molecule. According to the results, the structure of the ester is determined.
The present invention is further illustrated by the following representative examples.
EXAMPLE 1 Seed Culture Medium:
Fructose 30 g/l Meat extract [0 Peptone 10 NaCl 3 [pH 7.2 before sterilization] Culturing is carried out with shaking at 30C for 24 hours. The resulting seed culture liquor is poured into 3 liters of a medium having the following composition in a 5-liter-jar fermenter.
Main Fermentation Medium:
Fructose g/l (N -M2 4 5 Com steep liquor 3 NaHPO,.12 O 2 KH PO 2 MgS0 ,.7H,O 1 FeSO..7l-l O 0.5 g/l MnSO .4H O 20 mg/l ZnSO .7H O 10 CaC|.2H O 1 Fermentation is carried out with stirring at 600 r.p.m. an aeration of one liter of air/liter of medium/min at 30C for 45 hours. During the fermentation, the pH of the fermentation liquor is adjusted automatically to 6.8-7.2 with ammonia.
After the completion of fermentation, 2.8 liters of the fermentation liquor is subjected to centrifugation to obtain 250 g (wet weight) of the microbial cells. The cells are subjected to extraction at room temperature for three times, each with one liter of a mixture of chloroform and methanol in a volume ratio of 1:1. Three liters of the resulting extract is concentrated in a flash evaporator. 10 g of the residue is subjected to extraction with 100 m1 of chloroform and the solvent in the obtained extract is removed. The residue is dissolved in 100 ml of n-hexane. After concentration and centrifugation, 20 ml of n-hexane solution is obtained.
The thus obtained solution is poured into a column having an inner diameter of 4 cm and a height of 20 cm packed with silica gel. Then, chloroform is passed through the column to remove pigments and free fatty acids. Elution is carried out with a mixture of chloroform and methanol while stepwise varying the volume ratio of chloroform to methanol in the mixture from 99:1 to 95:5. The fractions obtained by the elution with a mixture of chloroform and methanol in a volume ratio of 95:5 are collected and the solvent is removed. The resulting residue is dissolved in warm acetone. Upon allowing the solution to stand still at l5C, precipitates are obtained. The precipitates are dried, whereby 2.5 g of a white powder is obtained.
Further, in thin layer chromatography using as a developer a mixture of chloroform, methanol and acetic acid in a volume ratio of 90:10:5, it is found that two kinds of products are contained in the powder. The powder is dissolved in n-hexane and the solution is poured into a silica gel column having an inner diameter of 4 cm and a height of 30 cm. Elution is carried out first with a mixture of chloroform and methanol in a volume ratio of 98:2. The resulting eluate is concentrated and allowed to stand still at l 5C. The resulting precipitates are collected and dried to obtain 1.2 g of Product A. Further elution of the above column is carried out with a mixture of chloroform and methanol in a volume ratio of 95:5. The eluate is treated in the same manner as above to obtain 0.5 g of Product B.
After various identification tests, Product A is found to be a mono-fatty acid ester of fructose wherein fatty acid of the formula II is bonded to fructose at 6- position. R of the fatty acid is found to be an alkyl group having l0, 12 or 14 carbon atoms and R an alkyl group having 15 or 17 carbon atoms. Product B is found to be a di-fatty acid ester of fructose having two fatty acid residues at land 6-positions. The fatty acid residues are respectively identical with that of Product A.
Properties of Products A and B are shown below: Product A: is powdery when dried; readily absorbs moisture in the air and becomes pasty; has an average molecular weight of 714; is soluble in ether, chloroform and n-hexane at room temperature, soluble in methanol, ethanol and acetone havinga temperature of 4060C and further soluble in hot water; and is light yellow. FIG. 1 is an infrared absorption spectrum of this ester. Elementary analysis: C 69.6 percent, H 1 1.5 percent, 0 19.9 percent Product Bi exists in the form of a paste; has an average molecular weight of 1,240; is soluble in ether, chloroform and n-hexane- 'at room temperature and also in methanol, ethanol and acetone having a temperature of 40-60C;-and is light yellow. The infrared absorption spectrum of Product B is almost identical with that of Product A. Elementary analysis: C 74.7 percent, H= 12.6 percent. 0 12.7
percent EXAMPLE 2 The same fermentation procedures as those in'Example l are repeated, except for using zlrthrobacter hydrocarboglutamicus ATCC 15583. As the result, 220 g (wet weight) of the microbial cells is obtained. From these cells, two products are isolated in the same manner as in Example 1 in amounts of 1.0 g and 0.4 g, respectively. As the result of various identification tests, the products are found to have the same chemical structures as those of Products A and B obtained in Example 1, respectively.
EXAMPLE 3 In 'this example, Corynebacterium hydrocarboclastus ATCC 12628 is .used. Fermentation is carried out in the same manner as in Example 1 except for using this strain and continuing the fermentation for 30 hours. As the result, 220 g (wet weight) of the microbial cells is obtained. From these cells, 1.7 g of Product A and 0.3 g of Product B are isolated in the same manner as in Example 1.
After various identification tests, Product A is found to be a mono-fatty acid ester of fructose wherein a fatty acid of the formula II is bonded to fructose at 6- position. R of the fatty acid is found to be an alkyl group having 8, 10 or 12 carbon atoms and R an alkyl group having'33,35 or 37 carbon atoms. Product B is found to be a di-fatty acid ester of fructose having two fatty acid residues at 1- and o-positions. The fatty acid residues are respectively identical with that of Product EXAMPLE 4 The same fermentation procedures as those in Example 1 are repeated, except for using Corynebacterium psudodiphtheriticum ATCC 10701 and continuing the fermentation for 48 hours. As the result, 260 g (wet weight) of the microbial cells are obtained. From these cells, two products are isolated in the same manner as in Example 1 in amounts of 0.7 g and 0.2 g, respectively. As the result of various identification tests, the products are found to have the same chemical structures as those of Products A and B obtained in Example 1 respectively.
EXAMPLE 5 In this example, Nocardia paraffinica ATCC 21 198 is used. Fermentation is carried out in the same manner as in Example 1, except for using this strain and continuing the fermentation for 38 hours. As the result, g (wet weight) of the microbial cells are obtained. From these cells, 1.3 g of Product A is isolated in the same manner as in Example 1.
After various identification tests, Product A is found to be a mono-fatty acid ester of fructose wherein a fatty acid of the formula II is bonded to fructose at 6- position. R of the fatty acid is found to be an alkyl group having 10, 12 or 14 carbon atoms and R" an alkyl group having 33, 35 or 37 carbon atoms.
. The alkyl groups in the fatty acid residue of Product A have .a large number of carbon atoms as compared with those in the fatty acid residue of Products A and B obtained in Example 1. Such a fatty acid having alkyl groups of a large number of carbon atoms is generally called nocardomycolic acid.
' EXAMPLE 6 The same fermentation procedures as those in Example 1 are repeated, except for using Nocardia globerula ATCC"13130 and continuing the fermentation for 53 hours. As the result, 200 g (wet weight) of the microbial cells is obtained. From these cells, Product A and Product B are isolated in the same manner as in Example l in amounts of 0.8 g and 0.2 g, respectively.
After various identification tests, Product A is found to be a mono-fatty acid ester of fructose wherein a fatty acid of formula II is bonded to fructose at 6-position. R of the fatty acid is found to be an alkyl group having 10, 12 or 14 carbon atoms and R" an alkyl group having 35, 37 or 39 carbon atoms. Product 8:, is founded to be a di-fatty acid ester of fructose having two fatty acid residues at land 6-positions. The fatty acid residues are respectively identical with that of Product A EXAMPLE 7 In this example, Mycobacterium paraffinicum ATCC 12670 is used. Fermentation is carried out in the same manner as in Example 1, except for using this strain and continuing the fermentation for 40 hours. As the result, 190 g (wet weight) of the microbial cells is obtained. From these cells, 2.7 g of Product A., and a trace amount of Product 8., are isolated in the same manner as in Example l.
After various identification tests, Product A., is found to be a mono-fatty acid ester of fructose wherein a fatty acid of the formula II is bonded to fructose at 6- position. R of the fatty acid is found to be an alkyl group having 20 or 22 carbon atoms and R an alkyl group having 57, 59 or 61 carbon atoms. Product 8., is found to be a di-fatty acid ester of fructose having two fatty acid residues at 1- and 6-positions. The fatty acid residues are respectively identical with that of Product A EXAMPLE 8 In this example, Mycobacterium smegmatis ATCC 21293 is used. Fermentation is carried out in the same manner as in Example 1, except for using this strain. As the result, 165 g (wet weight) of the microbial cells is obtained. From these cells, 0.8 g of Product A and a trace amount of Product B are obtained.
After various identification tests, Product A is found to be a mono-fatty acid ester of fructose wherein a fatty acid of the formula II is bonded to fructose at 6- position. R of the fatty acid is found to be an alkyl group having 10, 12 or 14 carbon atoms and R" an alkyl group having 15, 17 or 19 carbon atoms. Product B is found to be a di-fatty acid ester of fructose having two fatty acid residues at land 6-positions. The fatty acid residues are respectively identical with that of Product A EXAMPLE 9 The same fermentation procedures as those in Example 1 are repeated, except for using Mycobacterium smegmatis ATCC 607. As the result, 250 g (wet weight) of the microbial cells is obtained. From these cells, one g of Product A and a trace amount of Product B are isolated.
After various identification tests, Product A is found to be a mono-fatty acid ester of fructose wherein a fatty acid of the formula II is bonded to fructose at 6- position. R of the fatty acid is found to be an alkyl group having l0, 12 or 14 carbon atoms and R" an alkyl group having l5, 17 or 19 carbon atoms. Product B is found to be a di-fatty acid ester of fructose having two fatty acid residues at 1- and 6-positions. The fatty acid residues are respectively identical with that of Product A EXAMPLE 10 In this Example, Nocardia convoluta ATCC 4275 is used. Seed culturing is carried out in the same manner as in Example 1 except the culturing is continued for 48 hours. 1.2 liters of the resulting seed culture is inoculated into 15 liters of a fermentation medium in a 30 liter-jar fermenter. The fermentation medium has the same composition as that of Example 1 except for containing 120 g/l fructose and further containing 3 g/l yeast extract. Fermentation is carried out with stirring at 400 rpm. and an aeration of l l/l/min at 30C for 45 hours. During the fermentation, the pH of the fermentation liquor is automatically adjusted to 6.5-7.0 with ammonia.
After the completion of fermentation, the fermentation liquor is subjected to centrifugation to obtain 3.0 kg (wet weight) of the microbial cells. The microbial cells are washed with a mixture of ether and ethanol having a volume ratio of 1:1. Then, extraction is car ried out twice, each with 4.5 liters of a mixture of chloroform and methanol in a volume ratio of 1:1 at room temperature. 9 liters of the resulting extract is concentrated in a flash evaporator. The residue is subjected to extraction with 500 ml of ethyl acetate. The solvent in the obtained extract is removed and the residue is dissolved in chloroform. After filtration, 100 ml of chloroform solution is obtained.
The thus obtained solution is poured into a silica gel column having an inner diameter of 4 cm and a height of 50 cm. Chloroform is then passed through the column to remove pigments and free fatty acids. Elution is carried out initially with a mixture of chloroform and methanol in a volume ratio of 99:1 while stepwise varying the mixing ratio by volume of chloroform to methanol from 99:1 to :5. The fractions obtained by the elution with a chloroform-methanol mixture having a volume ratio of 99: 1 contain Product D and those obtained by the elution with a chloroform-methanol mixture having a volume ratio of 97:3, contain Product C. Each of the eluates is concentrated and the concentrate is poured into a silica gel column. Elution is carried out with the solvent with which each eluate is obtained. After removing the solvents in the resulting eluates, 1.5 g of a residue containing Product C and 0.5 g of a residue containing Product D are obtained. Each of the residues is dissolved in warm acetone and allowed to stand still at l5C. The resulting precipitates are dried. Thus, 1.0 g of Product C and 0.2 g of Product D are obtained each as a colorless paste.
After various identification tests, Product C is found to be a tetra-fatty acid ester of fructose wherein four residues of a fatty acid of the formula II are bonded to fructose. The four fatty acid residues have the same structure. R of the fatty acid residue is found to be an alkyl group having 10, 12, 14 or 16 carbon atoms and R an alkyl group having 45, 47, 49 or 51 carbon atoms. Product D is found to be a penta-fatty acid ester of fructose. The five fatty acid residues are identical with those of Product C.
Both of Products C and D are soluble in ether, chloroform, ethyl acetate, n-hexane, ethanol, methanol and acetone at room temperature but are insoluble in water. FIG. 2 shows an infrared absorption spectrum of Product C. The infrared absorption spectrum of Product D is almost identical with that of Product C.
Elementary analysis:
Product C: C 80.7%, H 12.9%, 6.4% Product D: C=8l.l%,H=13.0%.O=5.9%
Example 1 1 In this example, Mycobacterium rubrum ATCC 14346 is used. Fermentation is carried out in the same manner as in Example 10 except for using this strain and continuing the fermentation for 65 hours. As the result, 1.2 kg (wet weight) of the microbial cells is obtained. From these cells, 0.5 g of Product C and 0.15 g of Product D are isolated in the same manner as in Example 10.
After various identification tests, Product C is found to be a tetra-fatty acid ester of fructose wherein four residues of a fatty acid of the formula 11 are bonded to fructose. The four fatty acid residues are identical. R of the fatty acid residue is found to be an alkyl group having or 22 carbon atoms and R" an alkyl having 53, 55 or 57 carbon atoms. Product D is found to be a penta-fatty acid ester of fructose. The five fatty acid residues are identical with those of Product C.
What is claimed is:
1. A process for producing fatty acid esters of fructose, which comprises culturing a fructose-utilizing microorganism capable of producing fatty acid esters of fructose and belonging to the genus Arthrobacter, Corynebacterium, Nocardia or Mycobacterium in a medium containing fructose as a carbon source, forming the fatty acid esters of fructose intracellularly and recovering the esters from the microbial cells.
2. The process of claim 1, wherein the culturing is carried out under aerobic conditions at 20C to 40C at a pH of from 4 to 9.
3. The process of claim 1, wherein the esters are recovered from the microbial cells by initially separating the microbial cells from the culture liquor, extracting the cells with an organic solvent system, vaporizing the solvent system, further extracting the resulting residue with another solvent and finally effecting elution of the fatty acid esters.
4. The process of claim 3, wherein elution is effected with a mixture of a chlorinated hydrocarbon and a lower aliphatic alcohol, the mixing ratio by volume of the hydrocarbon to the alcohol being stepwise varied from 99:1 to :5.
5. The process of claim 1, wherein the fatty acid esters obtained are a reaction product of 1 mole of fructofuranose and 1, 2, 4 or 5 mols of a fatty acid, the fatty acid residue in the esters having the following formula:
ll 1 O R" wherein R is an alkyl group having 8 to 22 carbon atoms and R" is an alkyl group having 15 to 61 carbon atoms.
6. The process of claim 1, wherein the microorganism is selected from the group consisting of:
Arthrobacter paraffineus ATCC 15591 Arthrobacter hydrocarboglutamicus ATCC 15583 Cofynebacterium hydrocarboclastus ATCC 21628 Corynebacterium psudodiphtheriticum ATCC 10701 Nocardia praffinica ATCC 21198 Nocardia globerula ATCC 13130 Nocardia convoluta ATCC 4275 Mycobacterium rubrum ATCC 14346 Mycobacterium paraflinicum ATCC 12670 Mycobacterium smegmatis ATCC 21293 Mycobacterium smegmatis ATCC 607

Claims (6)

1. A PROCESS FOR PRODUCING FATTY ACID ESTERS OF FRUCTOSE, WHCH COMPRISES CULTURING A FRUCTOSE-UTILIZING MICROORGANISM CAPABLE OF PRODUCING FATTY ACID ESTERS OF FRUCTOSE AND BELONGING TO THE GENUS ARTHROBACTER, CORYNBACTERIUM, NACARDIA OR MYCOBACTERIUM IN A MEDIUM CONTAINING FRUCTOSE INTRACELLULARLY SOURCE, FORMING THE FATTY ACID ESTERS OF FRUCTOSE INTRACELLULARLY AND RECOVERING THE ESTERS FROM THE MICROBIAL CELLS.
2. The process of claim 1, wherein the culturing is carried out under aerobic conditions at 20*C to 40*C at a pH of from 4 to 9.
3. The process of claim 1, wherein the esters are recovered from the microbial cells by initially separating the microbial cells from the culture liquor, extracting the cells with an organic solvent system, vaporizing the solvent system, further extracting the resulting residue with another solvent and finally effecting elution of the fatty acid esters.
4. The process of claim 3, wherein elution is effected with a mixture of a chlorinated hydrocarbon and a lower aliphatic alcohol, the mixing ratio by volume of the hydrocarbon to the alcohol being stepwise varied from 99:1 to 95:5.
5. The process of claim 1, wherein the fatty acid esters obtained are a reaction product of 1 mole of fructofuranose and 1, 2, 4 or 5 mols of a fatty acid, the fatty acid residue in the esters having the following formula:
6. The process of claim 1, wherein the microorganism is selected from the group consisting of: Arthrobacter paraffineus ATCC 15591 Arthrobacter hydrocarboglutamicus ATCC 15583 Corynebacterium hydrocarboclastus ATCC 21628 Corynebacterium psudodiphtheriticum ATCC 10701 Nocardia praffinica ATCC 21198 Nocardia globerula ATCC 13130 Nocardia convoluta ATCC 4275 Mycobacterium rubrum ATCC 14346 Mycobacterium paraffinicum ATCC 12670 Mycobacterium smegmatis ATCC 21293 Mycobacterium smegmatis ATCC 607
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Cited By (4)

* Cited by examiner, † Cited by third party
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US4284509A (en) * 1979-03-21 1981-08-18 Gesellschaft Fur Biotechnologische Forschung Process for removing oils or petroleum from the surface of the sea
US4363763A (en) * 1980-02-25 1982-12-14 The Procter & Gamble Company Polyol esters of alpha-hydroxy carboxylic acids
US4614718A (en) * 1983-08-23 1986-09-30 Dai-Ichio Kogyo Seiyaku Co., Ltd. Synthesis of sugar or sugar-alcohol fatty acid esters
US5191071A (en) * 1987-08-21 1993-03-02 Novo Nordisk A/S Monoesters of glycosides and a process for enzymatic preparation thereof

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JPS533514A (en) * 1976-06-25 1978-01-13 Ichirou Azuma Adjuvant containing sugarlipid as effective component
DE3312166A1 (en) * 1983-04-02 1984-10-11 Hoechst Ag, 6230 Frankfurt LIPOTENSIDE, METHOD FOR INSULATION AND THEIR USE
DK157308C (en) * 1985-02-27 1990-05-07 Novo Nordisk As PROCEDURE FOR THE PREPARATION OF ACETAL OR CATAL ESTERS OF POLYOLES OR MONOESTERS OF MONO OR DISACCHARIDES OR MONOGLYCERIDES

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US3637461A (en) * 1968-02-01 1972-01-25 Kyowa Hakko Kogyo Kk Process for producing fatty acid esters of sugars

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* Cited by examiner, † Cited by third party
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US3637461A (en) * 1968-02-01 1972-01-25 Kyowa Hakko Kogyo Kk Process for producing fatty acid esters of sugars

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4284509A (en) * 1979-03-21 1981-08-18 Gesellschaft Fur Biotechnologische Forschung Process for removing oils or petroleum from the surface of the sea
US4363763A (en) * 1980-02-25 1982-12-14 The Procter & Gamble Company Polyol esters of alpha-hydroxy carboxylic acids
US4614718A (en) * 1983-08-23 1986-09-30 Dai-Ichio Kogyo Seiyaku Co., Ltd. Synthesis of sugar or sugar-alcohol fatty acid esters
US5191071A (en) * 1987-08-21 1993-03-02 Novo Nordisk A/S Monoesters of glycosides and a process for enzymatic preparation thereof

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