JP2002045195A - Method for carrying out fermentation production of sophorose lipid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fermentation production method in which residual oil and fat in culture solution after finishing fermentation is slight and sophorose lipid is produced in a high yield, and provide a culturing method capable of utilizing an inexpensive raw material as a culture medium. SOLUTION: In this fermentation production method, an yeast of the genus Candida which has been known as sophorose lipid-producting live germ hitherto is used and a fatty acid is mixed with a vegetable oil or waste edible oil as an oily substrate adding to liquid culture medium. Thereby, sophorose lipid production amount and yield is increased and culturing period is shortened and oil and fat amount left after culturing is remarkably reduced compared with the case when fatty acid is not added. The above method simply enables purifying sophorose lipid in large amount from culture solution and can inexpensively carry out fermentation production by utilizing the waste edible oil.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing sophorose lipid, a kind of biosurfactant, using Candida yeast.

[0002]

2. Description of the Related Art Sophorose lipid, which is one of glycolipid biosurfactants produced by yeasts of the genus Candida such as Candida bombicola or C. apicola, is biodegradable. Has attracted attention as a next-generation surfactant that is highly toxic, low in toxicity and environmentally friendly. However, for actual use,
Due to its high production cost, it is actually limited to those with high cost performance, such as raw materials for moisturizing and wetting agents used in cosmetics (Kozaric Surfa).
ctant science series vol.
48, Biosurfactants pp. 378-
382 (1993)). When Candida bombicola is fed with a high concentration of sugar and an oily substrate at the same time, or when an oily substrate is continuously added to a medium containing a high concentration of sugar and cultured, a high concentration of sophorose lipid accumulates in the medium. Are already known (Asmer et al., J. Am. Oil Ch).
em. Soc. 65: 1460-6 (1988), Ko
Zaric et al. Am. Oil Chem. Soc. 7
2: 67-71 (1992), JP-A-6-6287.
7). As the oily substrate, vegetable and animal fats and oils, fatty acids and fatty acid esters or n-alkanes have been reported. Among them, a high yield is obtained when vegetable oils or fatty acid esters derived from vegetable oils and fats are used. (Davila et al. Appl. M
microbiol. Biotechnol. 38: 6-
11 (1992)). The sophorose lipid accumulated in the medium is first separated from the culture solution by a method such as centrifugation, decantation, or ethyl acetate extraction, and further washed with hexane to remove residual fats and oils that have not been used. However, when vegetable oils are used, the accumulation of sophorose lipid reaches a plateau at a low level depending on the type and quality of the oils and fats, and a phenomenon that a large amount of oils and fats remain in the culture termination solution is often observed (Casas et al. Biosci.Bioeng.88: 488-49.
4 (1999)). This is one of the factors that not only reduces the product yield but also increases the load on the refining process for removing residual fats and oils, thereby increasing the production cost of sophorose lipid.

[0003]

Means for Solving the Problems In order to realize cheaper sophorose lipid production, the present inventors have promoted the use of an oily substrate added to a culture medium and increased the yield and the yield of sophorose lipid. It was investigated. As a result, they have found that these problems can be solved by mixing and giving free fatty acids to vegetable oils conventionally used as an oily substrate added to the medium. More specifically, a culture method in which Candida yeast having a sophorose lipid-producing ability is mostly made of triglyceride and mixed with a fatty acid such as an edible vegetable oil to give a fatty acid is used as an oleaginous substrate. The yield and yield of sophorose lipid were increased and the time to reach maximum product concentration was reduced.
Further, at this time, consumption of the whole fats and oils was promoted, and almost no residue in the culture termination liquid was recognized. Furthermore, in this cultivation method, it became clear that used food waste oil can be used as an oily substrate as well as unused one. When cooking oil is used for cooking, the quality gradually decreases due to coloring and generation of unpleasant odor, and the cooking oil becomes unsuitable for edible use. An acid value is used as one index of the quality deterioration. Here, the waste oil whose acid value has risen to 3.0 or more is defined as waste edible oil. By providing this waste food oil with a fatty acid as a medium component, sophorose lipid is produced in high yield and high yield, and the cultivation is continued. The present inventors have found that the amount of residual fats and oils in the finished liquid is very small, and completed the present invention. The features of the present invention, namely, 1. 1. Significant reduction of residual fat after culture. 2. Increase product yield and yield. All three aspects of the use of used fats and oils reduce the production cost of sophorose lipid. Thus, the present invention provides sophorose lipids at a lower cost on the market, thereby expanding the range of applications from current limited uses to wider fields including cleaning. At the same time, the present invention provides a method for effectively utilizing fats and oils that are no longer edible and used for cooking or the like as resources. Hereinafter, a method of manufacturing the sophorose lipid according to the present invention will be described. The yeast to be cultured belongs to the genus Candida which has sophorose lipid-producing ability, and is preferably Candida bombicola. These may be bacterial strains distributed from a preservation organization or subcultures thereof. The culture mode is batch culture using a liquid medium, or fed-batch culture in which fats and oils are continuously added to the culture system, and it is desirable to perform aeration and stirring. The initial pH of the medium is 4.0-5.5.
The pH is adjusted within the range described above, but pH adjustment during culture may not be performed. The temperature range suitable for culture is 20-35 ° C, more preferably 28-30 ° C. Hereinafter, the composition of the liquid culture medium used for sophorose lipid fermentation production will be described. Fats and oils and sugars are used as carbon sources. Fats and oils are used by mixing fatty acids with vegetable oils or used waste oils, and the total concentration of the fats and oils is 50-200 g / L, preferably 100-200 g / L.
It is in the range of 150 g / L. In the case of continuous supply, an amount of the fat or oil mixture corresponding to the above concentration is added to the culture system at a uniform rate during the culture period. The proportion of the fatty acid in the whole fat or oil is 10% or more and less than 100% by weight, preferably 2% by weight.
It is in the range of 0-50%. The types of vegetable oils used are soybean oil, rapeseed oil, cottonseed oil, sunflower oil, kapok oil,
Sesame oil, corn oil, rice oil, peanut oil, safflower oil, olive oil, linseed oil, drill oil, castor oil, palm oil, palm kernel oil, coconut oil, etc., or a mixture thereof, but more preferably soybean oil, Rapeseed oil, sunflower oil, safflower oil or a mixture thereof. As waste cooking oil, vegetable oils and fats in general used as cooking oils, that is, soybean oil, rapeseed oil, cottonseed oil, sunflower oil, kapok oil, sesame oil, corn oil, rice oil, peanut oil, safflower oil, olive oil, palm oil and the like Deteriorated quality can be used by using the mixed one for cooking or the like, but more preferred waste cooking oil is soybean oil,
Waste cooking oil produced from rapeseed oil, sunflower oil, safflower oil or a mixture thereof. In addition, these waste cooking oils may contain fats and oils derived from cooked foods, and their decomposed / modified products. The fatty acids used have 6-24 carbon atoms
And each molecule may further contain 0-3 unsaturated bonds. Examples include caproic acid, caprylic acid, capric acid, undecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, nonadecanoic acid, arachidic acid, behenic acid, lignoceric acid, Succinic acid, succinic acid, lindelic acid, tunic acid, myristoleic acid, palmitoleic acid, petroselinic acid, oleic acid, elaidic acid, vaccenic acid, erucic acid, sorbic acid, linoleic acid, linoleic acid, γ-
Unsaturated fatty acids such as linolenic acid, linolenic acid and arachidonic acid and mixtures thereof, preferably having 1 carbon atom
4-20 saturated or unsaturated fatty acids and mixtures thereof, more preferably oleic acid or a mixture of oleic acid-based fatty acids. As the saccharides, monosaccharides such as glucose, fructose and galactose or disaccharides such as sucrose and maltose are used,
It is preferably glucose, and the initial culture concentration is 30-
150 g / L, more preferably 90-120 g / L
The range is suitable. 2-5 g / L as a nitrogen source
Of yeast extract, 0.5-2 g / L of urea, etc., are not limited thereto. Further, appropriate amounts of various organic substances and inorganic salts such as phosphates and magnesium salts necessary for the growth of yeast may be added. Representative culture conditions and medium compositions are described below. Culture conditions Culture type: Batch culture method Initial pH: 4.5, pH is not adjusted during culture Culture temperature: 30 ± 1 ° C, aeration and stirring speed: 0.6 vvm, 600 rpm Medium composition (per 1 L) Safflower oil 50 g Industrial Oleic acid for use 50 g Glucose 100 g Yeast extract 2.5 g Urea 1.0 g Magnesium sulfate 5.0 g Sodium chloride 1.0 g Potassium dihydrogen phosphate 10 g The sugars and fats and oils in the culture medium are removed during the culture process. Used at the same time,
The concentration decreases with the passage of time, of which the saccharides are completely consumed first. The production of sophorose lipid stops when all the fats and oils in the medium have been consumed. The oils and fats remaining in the medium are separated into an oil layer by heating the culture solution in a hot water bath and centrifuging. Therefore, the end point of the generation of sophorose lipid can be easily known by removing a part of the culture solution during the culture and performing the above operation to confirm the disappearance of the oil layer. Extraction and purification of sophorose lipid from the culture solution was performed as follows according to the method of Kozaric et al. Since the sophorose lipid precipitates as a viscous liquid substance when the culture solution is allowed to stand, the upper layer of the culture solution is removed by decantation, and distilled water and n-hexane are added to the residue containing the liquid precipitate and mixed well. . The sophorose lipid is precipitated by standing or centrifugation, and the hexane and aqueous solution layers are removed. Add ethyl acetate to the residue and mix well. Since sophorose lipid is dissolved in ethyl acetate to form two layers, the ethyl acetate layer is recovered, and the solvent and water are degassed and removed to obtain a sample of sophorose lipid.

[0004]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 In a 300 ml Erlenmeyer flask, 100 ml of a liquid medium containing 100 g / L glucose, 100 g / L edible safflower oil and 2.5 g / L yeast extract (pH 4.5)
Was prepared and steam sterilized with a breathable lid. A loopful of one subculture of Candida bombicola IFO 10243T was inoculated here, and stirred at 30 ° C. with a stirrer.
For 3-5 days. This is used as a preculture. 5L
100 g / L glucose, 50
g / L safflower oil, 50 g / L oleic acid (NAA-34 manufactured by NOF Corporation), 2.5 g / L yeast extract, 10
Prepare 2.5 L of a liquid medium consisting of g / L potassium dihydrogen phosphate, 5 g / L magnesium sulfate, 1 g / L sodium chloride and 1 g / L urea, and adjust the pH to about 4.8 using hydrochloric acid. And then steam sterilized. However, to avoid the formation of insolubles, the phosphate was steam sterilized as a separate solution and mixed after cooling. The pH of the mixed medium was adjusted to 4.5 using hydrochloric acid, aerated for 1 hour or more, and then 100 ml of the above preculture was added. Culture temperature is 30
C., and aeration and agitation were performed at 1.5 L / min and 600 rpm. The culture solution after culturing for 7 days is 130 g per liter
Sophorose lipid preparations were included. Further, the concentration of residual fats and oils was 0.2 g / L as a hexane extract.
No residual glucose was observed. At this time, the sophorose lipid yield based on the consumed carbon source (the weight of sophorose lipid produced / the weight of glucose and fat consumed) was 0.65. As a result, the yield of sophorose lipid was improved about 3 times and the yield was improved about 2 times as compared with Comparative Example 1a described later.

Comparative Example 1 a) The same operation as in Example 1 was carried out, except that the
Only 0 g / L safflower oil was used. After culturing for 7 days, the culture solution contained 45 g of sophorose lipid preparation per liter. The residual oil and fat concentration was 51% as hexane extract.
g / L and the residual concentration of glucose were 9 g / L. At this time, the sophorose lipid yield was 0.32. b) When the culture was further continued under the same conditions, 18
Product formation hardly progressed even after culturing for one day, and the sophorose lipid preparation contained in the culture solution was 48 g / L.
Met. Further, the concentration of residual fats and oils was reduced to 1.6 g / L as a hexane extract, and no residual glucose was observed. At this time, the sophorose lipid yield was 0.24.
Met.

Example 2 The same procedure as in Example 1 was carried out except that the amount of the fat added to the medium was 50 g /
L soybean oil and 50 g / L oleic acid (same as in Example 1). After 6 days of culture, the culture solution is 140
g of sophorose lipid preparation. The residual oil and fat concentration was 3.3 g / L as a hexane extract, and the residual glucose concentration was 2 g / L. At this time, the sophorose lipid yield was 0.72. This result was about 5% lower in sophorose lipid yield than Comparative Example 2a described later.
And the yield was about three times higher.

Example 3 The same procedure as in Example 1 was carried out except that the amount of the fat added to the medium was 80 g / g.
L soybean oil and 20 g / L oleic acid (same as in Example 1). After culture for 6 days, the culture solution is 110
g of sophorose lipid preparation. The concentration of residual fats and oils was 12 g / L as a hexane extract, and no residual glucose was observed. At this time, the sophorose lipid yield was 0.59. As a result, the yield of sophorose lipid was improved about 4 times and the yield was improved about 2 times as compared with Comparative Example 2a described later.

Example 4 The same procedure as in Example 1 was carried out, except that the fat and oil added to the medium was 90 g / g.
L soybean oil and 10 g / L oleic acid (same as in Example 1). After 9 days of culture, the culture solution was 102
g of sophorose lipid preparation. The residual fat concentration was 49 g / L as a hexane extract, and the residual concentration of glucose was 6 g / L. At this time, the sophorose lipid yield was 0.70. As a result, the yield of sophorose lipid was about three times higher than that of Comparative Example 2b in which cultivation was performed for a longer period, and the yield was about three times that of Comparative Examples 2a and 2b.

Comparative Example 2 a) The same procedure as in Example 1 was carried out, except that the
Only 0 g / L soybean oil was used. The culture solution after culturing for 6 days contained 27 g of sophorose lipid preparation per liter. The residual fat concentration was 93 g / hexane extract.
The residual concentration of L and glucose was 1 g / L. At this time, the sophorose lipid yield was 0.25.
b) When the culture was further continued under the same conditions, the culture solution after 11 days of culture contained 31 g of sophorose lipid preparation per liter. Further, the concentration of residual fats and oils was 73 g / L as a hexane extract, and no residual glucose was observed. At this time, the sophorose lipid yield was 0.2.
It was 4.

Example 5 The same procedure as in Example 1 was carried out, except that the amount of the fat added to the medium was 50 g / g.
L waste cooking oil (soy oil, acid value 3.7) and 50 g / L oleic acid (same as in Example 1). This waste food oil is obtained by collecting what was actually used as frying oil in a fryer of a meat store and removing food residues such as dregs. The culture solution after culturing for 9 days contained 141 g of sophorose lipid preparation per liter. The residual oil and fat concentration was 1.7 g / L as a hexane extract, and the residual glucose concentration was 9.7 g / L. At this time, the sophorose lipid yield was 0.74. This result shows that the sophorose lipid yield was 3 compared to Comparative Example 3 described below.
More than twice, and the yield was improved about twice.

Comparative Example 3 The same procedure as in Example 1 was carried out, except that 100 g of fat or oil was added to the medium.
/ L waste cooking oil (soy oil, acid value 3.7) only. The culture solution after culturing for 9 days contained 41 g of sophorose lipid preparation per liter. The residual oil concentration was 84 g / L as a hexane extract, and no residual glucose was observed. At this time, the sophorose lipid yield was 0.35. The following pages show Examples and Comparative Examples in a table.

Claims (4)

[Claims] 1. A method for producing fermented sophorose lipid by liquid culture using yeast of the genus Candida, comprising the step of mixing free fatty acids with lipids added to a culture medium. 2. The sophorose lipid fermentation production method according to claim 1, wherein components other than free fatty acids among lipids added to the culture medium are waste oils. 3. The free fatty acid added to the medium has a carbon number of 6
3. The method according to claim 1, wherein said range is -24.
Sophorose lipid fermentation production method. 4. The amount of free fatty acid added to the medium is 0 in the molecule.
3. The composition according to claim 3, wherein the composition contains three unsaturated bonds.
Sophorose lipid fermentation production method.