JP2009207493A - Method for producing biosurfactant using fermentation product of plant as medium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for efficiently producing a highly functional microbial surface active substance (biosurfactant) by efficiently making use of compositions being byproducts in the processes of producing the liquors, excluding liquors, as unused resource. <P>SOLUTION: The biosurfactant is produced by fermentation at lower cost in a shorter period of time and more efficiently by using a medium containing a composition excluding liquor which is a byproduct in the process of producing the liquor, compared with a process using a conventional production medium, and in addition, the compositions excluding liquors which have been the unused resource of the byproducts in the process of producing the liquor is effectively used. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to a biosurfactant and / or a method for producing a highly functional metabolite other than a biosurfactant using a fermentation product obtained by fermenting a plant obtained in the production process of alcoholic beverages as a medium.
In the present invention, biosurfactant means a highly functional surfactant derived from microorganisms.

  In general, glycolipids are known to be substances in which 1 to 10 monosaccharides are bound to lipids. In addition, since it is involved in the transmission of information between cells in vivo, it has been revealed that it plays an important role in maintaining the functions of the nervous system and immune system. In addition, this glycolipid is called a surfactant because it is an amphiphile having both hydrophilic properties derived from the properties of sugar and lipophilic properties derived from the properties of lipid.

  Biosurfactants that are efficiently produced by some microorganisms are said to have new physiological functions that are highly biodegradable, have low toxicity, and are environmentally friendly. Therefore, the wide application of biosurfactants to the food industry, cosmetics industry, pharmaceutical industry, chemical industry, environmental field, etc. has both the realization of a sustainable society and the provision of highly functional products, and is extremely meaningful.

  This biosurfactant is classified into five types: glycolipid type, acyl peptide type, phospholipid type, fatty acid type and polymer compound type. In particular, glycolipid biosurfactants have been studied the most, and many types of substances produced by bacteria and yeast have been reported.

Rhamnolipid (hereinafter abbreviated as RL) was first discovered from the culture solution of Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) as an antibiotic of Mycobacterium tuberculosis (see Non-Patent Document 1).
In addition, four kinds of homologues have been reported so far from bacteria of the genus Pseudomonas , and the initial production was about several g / L, but now it is possible to produce over 100 g / L. (See Non-Patent Document 2).

Trehalose lipid (hereinafter abbreviated as TL) has been discovered as a cell surface material such as Corynebacterium . Similar substances have also been reported from Mycobacterium , Nocardia and Rodococcus bacteria (see Non-Patent Document 3).
In general, production is low because it is bound to the cell wall, has been reported to 32 g / L produced succinonitrile yl trehalose lipid When Rhodococcus erythropolis the (Rodococcus erythropolis) is cultured under nitrogen limitation (Refer nonpatent literature 4).

Sophorose lipids (also referred to as “sophorose lipids”; hereinafter abbreviated as SL) A. It has been discovered by Gorin et al. In a culture solution of Starmerella ( Candida ) bombicola (see Non-Patent Document 5).
Later, other yeasts such as Candida bogoriensis , Candida magnoliae , Candida gropengisseri , and Candida apicola were also used in the culture. It has been reported that it is produced in a relatively large amount (see Non-Patent Document 6).
Furthermore, production of 300 g / L or more is now possible (see Non-Patent Documents 7 and 8).

Cellobiose lipid (hereinafter abbreviated as CL) is a glycolipid with high antimicrobial activity, also called ustilagic acid or flocculosin.
In addition, CL is produced by Ustilago maydis over 15 g / L (see Non-Patent Documents 9, 10 and 11), Cryptococcus humicola (see Non-Patent Document 12), Pseudozyma flocculusa (see Pseudozyma flocculosa) (see non-Patent Document 13), it has been reported to be produced from such Pseudozyma Fuji folder meter (Pseudozyma fusiformata) (see non-Patent Document 14).
Furthermore, there is also a report example of oligosaccharide lipid produced at 30 g / L by yeast of the genus Tsukamurella (see Non-Patent Document 15).

Sophorose lipid (SL) in the glycolipid biosurfactant described above can obtain a high production amount from a relatively inexpensive raw material. Therefore, it is a kind of typical biosurfactant currently used commercially.
This SL is a glycolipid composed of sophorose or sophorose having a partially acetylated hydroxyl group and a hydroxy fatty acid. Sophorose is a sugar composed of two molecules of glucose linked by β1 → 2, and hydroxy fatty acid is a fatty acid having a hydroxyl group in its structure.
SL is roughly classified into an acid type in which the carboxyl group of hydroxy fatty acid is liberated and a lactone type bonded to sophorose in the molecule (Formula 1). Generally, SL obtained by fermentation production is obtained as a mixture of a lactone type and an acid type, and usually contains 50% or more of a lactone type.

In recent years, SL has been industrially used in a wide range of fields such as detergents, cosmetics, and foods.
For example, use of a sophorolipid derivative as a cosmetic wetting agent (see Non-Patent Document 16) and a gelling agent (see Patent Document 1), and use of a sophorolipid in the form of a mixture in improving the quality of wheat products (see Patent Document 2) In addition, there are reports on detergents containing sophorolipid (see Patent Documents 3 and 4), bleaching agents (see Patent Document 5), and the like.

By the way, in order to use SL in a wide range of applications, it is necessary to efficiently produce by reducing production cost. As a method for efficiently producing SL, a method for continuously providing a high-concentration sugar and an oily substrate (see Non-Patent Document 17 and Patent Document 6), and production of SL by mixing fatty acids with vegetable oil or waste edible oil Examples include a method for increasing the amount and the yield (see Patent Document 7).
However, these methods are mainly solutions relating to the addition of a substrate serving as a carbon source, and no effective solution relating to an auxiliary material in a medium other than the carbon source has been reported.

Moreover, it is necessary to add nitrogen sources, such as urea, nitrate, and ammonium salt, phosphate, magnesium salt, etc. to a culture medium as an auxiliary material at the time of generally producing SL. Furthermore, in order to supply trace elements such as vitamins and amino acids, an expensive yeast extract or the like needs to be used (see Patent Document 7).
Therefore, in order to promote the industrial use of SL widely, production costs must be reduced, and cheaper auxiliary materials are required. In particular, it would be extremely effective if a method capable of producing SL in a high yield from an inexpensive medium, such as using waste as a main raw material and auxiliary raw material, could be developed.

As an example, a step of filtering mash subjected to alcoholic fermentation such as sake, beer, a step of squeezing mash of wine, etc., a step of distilling mash of shochu, whiskey, brandy, etc. and a step of centrifuging mash , Liquor by-produced in the production process of liquors such as sake, sake lees, beer, beer lees, wine, wine lees, shochu, shochu distillation residue, whiskey, whiskey distillation residue, brandy, brandy distillation residue, etc. The excluding composition is rich in nutrient sources.
In particular, shochu distillation residue produced as a by-product in the production of shochu is rich in the above-mentioned nutrient sources, and is used as a raw material for fermentation in addition to use in livestock feed (see Patent Document 8) and compost (see Patent Documents 9 and 10). Production of fermentation metabolites such as butanol production (see Patent Literature 11), lactic acid (see Patent Literature 12), dipicolinic acid (see Patent Literature 13), γ-aminobutyric acid (see Patent Literature 14), nisin (see Patent Literature 15), etc. Use for production has been reported.
However, although the composition excluding alcoholic beverages obtained in the alcoholic beverage production process contains abundant nutrients, some of them are subjected to disposal such as incineration. Thus, the current situation is that not all of them can be used effectively, and the development of an effective usage method is desired.

As one of the effective utilization methods of the fermentation residue of a plant, there is a method of producing mannosyl erythritol lipid (MEL) using soy sauce oil as a raw material as a by-product in the soy sauce production process (see Patent Document 16). In this case, only the soy sauce oil that is a small part of the fermented product by-produced in the soy sauce production process is used as a raw material, and it is not a technique for effectively using the entire by-product. Also, from the perspective of BS production medium, this soy sauce oil is only used as a substitute for fatty acids such as oleic acid and ethyl oleate, that is, as a carbon source for microorganisms that produce MEL. In addition, other components such as nitrogen sources, vitamins and inorganic salts necessary for the growth of other microorganisms must be added separately.
As described above, there is no report that biosurfactants were produced in large quantities and with high efficiency by using substantially the entire composition excluding liquors by-produced in the alcohol production process as a medium.

Japanese Patent Publication No. 7-17668 JP-A 61-205449 JP 2003-13093 A JP 2006-83238 A JP 2006-274233 A JP-A-6-62877 JP 2002-45195 A JP-A-10-287485 Japanese Patent No. 3449757 Japanese Patent No. 2784642 JP 2005-328801 A JP 2000-236891 A JP 2004-275075 A JP 2004-290114 A Japanese Patent No. 3672259 JP 2007-101847 A Japanese Patent Application No. 2009-1914

“Journal of the American Chemical Society” (JJ Am. Chem. Soc.), 71, p4124-4126 (1949). “Applied Microbiology and Biotechnology (Appl. Microbiol. Biotechnol.)”, 51, p22-32 (1999). “Biosurfactant and Biotechnology,” (USA), Marshall Decker, New York (1987). "Journal of Biotechnology (J. Biotechnol.)" (UK), Vol. 13, p257-266 (1990). “Canadian Journal of Chemistry”, Vol. 39, p. 848-855 (1961). “Biodegradation”, Vol. 1, p107-119 (1990). “Applied Microbiology and Biotechnology (Appl. Microbiol. Biotechnol.)”, Vol. 47, p. 496-501 (1997). “Biotechnology. Lett.”, Volume 20, p1153-1156 (1998). “Biotechnol. Lett.”, (Netherlands), Kluwer Academic Publishers, Vol. 8, p757-762 (1986). “Applied Microbiology and Biotechnology” (Appl. Microbiol. Biotechnol.), (Germany), Springer-Verlag, 51, p33-39 (1999). “Molecular Biology.”, Blackwell Publishing, 66, p525-533 (2007). “Biochimica Biophysica Acta”, (Netherlands), Elsevier, 1558, p161-170 (2002). “Applied Environment and Microbiology” (Appl. Environ. Microbiol.), (USA), American Society for Microbiology, 69, p2595-2602 (2003). "FEMS Yeast Res." (Netherlands), Elsevier, Vol. 5, p919-923 (2005). "Fett / Lipid.", Volume 101, p389-394 (1999). Yoshimura Kimura, “Oil Chemistry”, 36, p748-753 (1987). “Journal of American Oil Chemistry (J. Am. Oil. Chem.)”, 65, p. 1460-1466 (1988). “Biotechnology Letters.” 28, p253-260 (2006).

Thus, the inventors of the present invention have made diligent efforts and found that such a problem can be solved by using a fermentation product obtained by fermenting a plant obtained in the alcohol production process as a medium for biosurfactant production. It has been completed.
That is, the present invention effectively uses by-products other than liquor produced in the production process of liquor that is an unused resource, and is a microorganism-derived highly functional surfactant (biosurfactant) and / or a highly functional metabolite other than biosurfactant. The purpose is to provide an efficient production method.

  According to the present invention, a method for fermentative production of a biosurfactant using a microorganism, the fermentation obtained by using a microorganism having a biosurfactant-producing ability as a microorganism, and subjecting a plant to alcohol fermentation as a medium for culturing the microorganism. A method for producing a biosurfactant and / or a highly functional metabolite other than a biosurfactant characterized by using a product can be provided to solve the above-described problems.

  Further, in carrying out the biosurfactant and / or the method for producing a high-functional metabolite other than the biosurfactant of the present invention, a step of filtering the mash from which the fermentation product has been subjected to alcohol fermentation, a step of squeezing the mash, the mash The liquor obtained by going through at least one process selected from the group consisting of the process of distilling the mash and the process of centrifuging the mash, or a by-product other than the liquor is preferred.

  Further, in carrying out the biosurfactant and / or the method for producing a highly functional metabolite other than the biosurfactant according to the present invention, the plant is barley, hato wheat, sweet potato (sweet potato), rice, brown sugar, buckwheat, grape and sesame. It is preferably at least one plant selected from the group consisting of

  Moreover, in carrying out the method for producing a biosurfactant and / or a highly functional metabolite other than the biosurfactant of the present invention, the biosurfactant is preferably a sophorose lipid.

  Moreover, in carrying out the method for producing a biosurfactant and / or a highly functional metabolite other than the biosurfactant of the present invention, it is preferable that the biosurfactant is cellobiose lipid.

In carrying out the method for producing a biosurfactant and / or a high-functional metabolite other than a biosurfactant of the present invention, a microorganism having a biosurfactant-producing ability is selected from Starmerella ( Candida ) bombicola , Candida & Bogorienshisu (Candida bogoriensis), Candida & Magnolie (Candida magnoliae), Candida & Apikora (Candida apicola), Ustilago Maidisu (Ustilago maydis), or in a microorganism belonging Ustilago Esukyurenta (Ustilago esculent a) Is preferred.

  Further, in carrying out the biosurfactant of the present invention and / or the method for producing a high-performance metabolite other than biosurfactant, glucose, sucrose, glycerin, vegetable oil, fatty acid, fatty acid methyl ester, molasses as a carbon source in the medium It is preferable to add at least one selected from the group consisting of waste cooking oil.

According to the present invention, in addition to the carbon source, selected from the group consisting of a step of filtering mash subjected to alcoholic fermentation, a step of squeezing the mash, a step of distilling the mash and a step of centrifuging the mash. A fermentation product obtained by fermenting a plant for the purpose of producing an alcoholic beverage obtained through at least one kind of process or an alcoholic byproduct obtained from the fermentation product and / or an alcoholic by-product produced in the alcoholic beverage production process. It is possible to produce SL, which is a kind of biosurfactant, at a lower cost. In other words, it is cheaper without using nitrogen sources such as urea, inorganic salts such as potassium monohydrogen phosphate, magnesium sulfate, and expensive yeast extracts, which have been generally used as auxiliary materials, as medium components. Thus, highly efficient production of SL is possible from a medium having a simple composition.
Among them, alcoholic beverages and / or alcohol obtained through a process of distilling moromi that has been subjected to alcoholic fermentation using plants such as barley, hato wheat, sweet potato (sweet potato), rice, brown sugar, buckwheat, grapes, sesame and the like as raw materials By-products other than liquor produced in the liquor production process are suitable as a medium for producing SL, which is a kind of biosurfactant, at a lower cost, and a culture solution in which the concentration of the distillation residue is controlled is prepared. By appropriately setting the combination, SL can be produced in a shorter culture time than the conventional production method.
That is, as described in the present invention, a by-product other than liquor produced in the production process of liquor, which is an unused resource as a medium for producing SL, which is a kind of biosurfactant, more preferably a secondary product in the production process of liquor. Since the medium cost can be reduced by using the produced distillation residue, and the fixed cost such as running cost and labor cost can be reduced by shortening the culture time, the SL production cost can be greatly reduced. In addition, the use of SL in a wide range of fields is expected.
At the same time, the present invention provides a method for the treatment and effective use of by-products other than liquors produced in the liquor production process, which has been limited in its use so far. A significant reduction in the quantitative environmental impact of the composition excluding by-product liquors is also expected.

In Example 3, it is a graph which shows the result of the SL production test with respect to SS concentration removal barley shochu distillation residue density | concentration in a culture medium. In Example 5, it is the graph which tracked the result of the culture test performed using the fermenter along culture | cultivation time. In Example 6, it is the graph which followed the result of the fed-batch culture test performed using the fermenter along culture | cultivation time. When a shochu distillation residue was used as a culture medium (Example 8) and when a shochu distillation residue was not used as a culture medium (Comparative Example 2), a fed-batch culture test of Ustilago esculent a MK-1 strain It is the graph which followed the result along culture time.

  Hereinafter, an embodiment according to a method for producing a biosurfactant of the present invention will be specifically described with reference to the drawings as appropriate.

[Microbe used]
The microorganism that can be used in the method for producing SL, which is a kind of biosurfactant according to the present invention, is not particularly limited as long as it has the ability to produce SL. Examples of microorganisms producing SL, e.g. Sutamerera (Candida) Bonbikora (Starmerella (Candida) bombicola), Candida & Bogorienshisu (Candida bogoriensis), Candida & Magnolie (Candida magnoliae), or Candida-Apikora ( Candida apicola ) and the like, and among these, microorganisms belonging to Starmerella (Candida) and Bonbicola are particularly preferred.
These may be strains handed over from a storage organization, or subcultures thereof.

Moreover, if it has the capability to produce CL as a microorganism which can be used for the manufacturing method of CL which is 1 type of the biosurfactant which concerns on this invention, it will not specifically limit. Examples of microorganisms that produce CL include microorganisms belonging to, for example, Ustilago maydis , Ustilago esculenta, and the like.
These may be strains handed over from a storage organization, or subcultures thereof.
As a microorganism belonging to the above-mentioned Ustilago esculenta ( Ustilago esculenta ), the strain Ustylago esculenta MK-1 is exemplified as a preferred strain (see Patent Document 17).
Ustilago esculenta strain MK-1 is a strain isolated from a plant collected in Japan (Makomo), and is 25 ° C on YM agar medium, PDB agar medium and 5% malt extract medium. After culturing for 7 days, corrugated, semi-lens, moist and creamy colonies are formed. The formation of mycelia with a width of about 4-5 μm is confirmed after culturing at 25 ° C. for 3 days in YM liquid medium and PD liquid medium. The formation of hyphae with septa and pseudohyphae was also observed after culturing at 25 ° C. for 7 days on the YM agar medium, PDB agar medium and 5% malt extract medium plate medium. In either case, no clear sexual genital formation is observed.
Ustilago esculent a MK-1 strain determines the nucleotide sequence (rDNA sequence) of the 26S rDNA-D1 / D2 region of the ribosomal RNA gene, accesses the DNA database (DDBJ), and accesses the FASTA program (http: / The homology search using /www.ddbj.nig.ac.jp/search/fasta-j.html) was 100% identical to the rDNA sequence of Ustilago esculent a. Together with the results of the physiological property test of MK-1, it was found that this strain belongs to Ustylago esculenta, a kind of Basidiomycota, and it was named as Ustilago esculenta (Kustilago esculenta ) MK-1. The results of the physiological property test are as shown in Table 1. This strain has been deposited on November 17, 2008 at the National Institute of Advanced Industrial Science and Technology Patent Microorganism Depositary Center (IPOD) (1-1-3 Higashi 1-1-3, Tsukuba, Ibaraki) under the accession number FERM P-21725 .
+: Positive reaction within several days after the start of the test,-: Negative response, S: Gradual positive reaction was observed from 2 to 3 weeks after the start of the test, L: Rapid positive after 2 weeks of the start of the test Reaction was observed

[Medium and culture conditions]
In the present invention, a fermented product obtained by fermenting a plant obtained in the alcohol production process is used as the medium, but it is more preferable to add a carbon raw material as a main raw material to the medium in order to efficiently perform BS production.
The above-mentioned liquors include a step of filtering mash that has undergone alcoholic fermentation such as sake and beer, a step of squeezing mash of wine, etc., a step of distilling mash of shochu, whiskey, brandy, etc. and a step of centrifuging mash. Alcohol obtained by passing through is not particularly limited, but liquor obtained through a step of distilling mash is preferred.

The plant to be subjected to the fermentation is not particularly limited, such as barley, hato barley, sweet potato (sweet potato), rice, brown sugar, buckwheat, grapes, sesame, but cereals such as barley, hato barley, and rice are preferred.
The fermented products mentioned above include alcohol fermented products such as moromi fermented plants for the purpose of producing alcoholic beverages, alcoholic products obtained from the fermented products and / or by-products other than alcoholic beverages produced in the alcoholic beverage production process. Although it does not restrict | limit in particular, By-products other than liquor produced in the manufacturing process of liquor are preferable from the point that it is cheap and can be obtained.

By-products other than liquor produced in the liquor production process include sake lees produced as a by-product in the sake production process, beer lees produced as a by-product in the beer production process, wine lees produced as a by-product in the wine production process, and shochu production processes. Shochu distillation residue produced as a by-product in the process, whiskey distillation residue produced as a by-product in the whiskey production process, brandy distillation residue produced as a by-product in the brandy production process are not particularly limited, but a shochu distillation residue produced as a by-product in the shochu production process is preferred. .
In addition, when using the fermentation product obtained by fermenting the plant obtained in the above-described liquor production process as a medium, the entire fermentation product may be used as a medium, or a water-insoluble fermentation residue or the like (SS content). It may be removed.

In addition, methods for removing water-insoluble fermentation residues (SS) from fermentation products include screw presses, filter presses, Iwata-type moromi squeezing equipment, F-type moromi squeezing equipment, ceramic filtration equipment, Although it does not restrict | limit especially filtration operation, such as a filtration apparatus, and / or a decanter continuous horizontal centrifuge, a decone continuous centrifuge, It is preferable to use the fermentation product obtained by the combination of a screw press and a ceramic filtration apparatus.
Furthermore, the non-adsorbed fraction when treated with the synthetic adsorbent, the non-adsorbed fraction when treated with the ion exchange resin and / or the carbon filtration treatment of the fermentation product from which the water-insoluble fermentation residue (SS content) was removed. The non-adsorbed fraction can also be used.

  As the carbon raw material, it is preferable to use sugars and fats and oils. The saccharide is not particularly limited as long as the yeast can assimilate. For example, monosaccharides such as glucose, galactose and fructose, and disaccharides such as sucrose and maltose are used, and glucose is preferred. The initial culture concentration is 20-150 g / L, preferably 50-120 g / L. Moreover, as fats and oils to be used, although it does not specifically limit, it is preferable to use vegetable oil. Furthermore, fatty acids or esters thereof may be used.

Examples of vegetable oils to be used include soybean oil, rapeseed oil, corn oil, cottonseed oil, sunflower oil, kapok oil, sesame oil, rice oil, peanut oil, safflower oil, olive oil, linseed oil, gill oil, castor oil, palm oil, palm Examples thereof include nuclear oil, coconut oil, and mixtures thereof.
In addition, as the fatty acid and fatty acid ester to be used, a fatty acid or fatty acid ester having a carbon chain of 10-24, preferably a carbon chain of 16-18 can be used. These fatty acids or fatty acid esters may contain 1-3 unsaturated bonds in the molecule. For example, saturated fatty acids such as decanoic 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 or the like, or tofu Acids, Linderic acid, Tuzic acid, Myristoleic acid, Palmitoleic acid, Petrothelic acid, Oleic acid, Elaidic acid, Vaccenoic acid, Erucic acid, Sorbic acid, Linoleic acid, Linoleic acid, Gamma-linolenic acid, Linolenic acid, Arachidone Examples thereof include unsaturated fatty acids such as acids or esters thereof.

The concentration of the fats and oils added to the medium is in the range of 20-200 g / L, preferably 50-150 g / L. When adopting a fed-batch culture mode, it is added continuously or intermittently during the culture period so that the concentration in the medium falls within the above range.
The culture form is batch culture using a liquid medium or fed-batch culture in which fat and oil and / or shochu distillation residue is continuously added to the culture system, and aeration and agitation are preferred. The initial pH of the medium is preferably adjusted to 4.0-6.0, but the pH need not be adjusted. Moreover, the temperature range suitable for culture is 20-35 ° C, more preferably 28-30 ° C.

[Sophorose lipid recovery and purification method]
Extraction and purification of SL from the culture solution is performed as follows according to the method of Ashby et al. Described in the following literature.
First, after adding an amount of ethyl acetate equal to the culture solution, SL and hydrophobic components are extracted, and the ethyl acetate phase is separated. Then, by removing ethyl acetate with an evaporator or the like, a fraction containing SL and a hydrophobic component is obtained. Furthermore, the obtained fraction is washed with hexane to remove hydrophobic components other than SL, and the residue is dried to obtain an SL preparation.
Also for the hexane washing solution, by removing hexane with an evaporator or the like, the amount of the hydrophobic component remaining in the medium is obtained by collecting the hydrophobic component other than SL and measuring the weight.
Ashby et al .: See Non-Patent Document 18.
[Recovery and purification method of cellobiose lipid]
Extraction and purification of CL from the culture can also be performed in the same manner as the above SL operation.

  EXAMPLES The present invention will be described in detail below with reference to examples. However, these examples show one example of the present invention and do not limit the present invention in any way, and are arbitrary within the scope of the present invention. It is possible to change to

(Adjustment method and component composition of by-products other than liquor produced in the liquor production process)
<Adjustment of distillation residue by-produced in the manufacturing process of shochu>
An evaluation test was performed using a composition (so-called barley shochu distillation residue) excluding alcoholic beverages obtained by passing barley as a plant raw material and subjecting the moromi subjected to alcoholic fermentation to distillation. As a raw material, 70% polished barley was used. For the production of koji, 40% (W / W) of barley was absorbed and steamed for 40 minutes, and then allowed to cool to 40 ° C. Then, after inoculating 1 kg of seed meal (white bacterium) per ton of barley, it was performed at 38 ° C. and RH 5% for 24 hours, and at 32 ° C. and RH 92% for 20 hours. Steamed barley was absorbed with 40% (W / W) of barley, steamed for 40 minutes, allowed to cool to 40 ° C., and added to the primary charge. In the first preparation, 3.6 kiloliters of water and 1 kg (wet weight) of cultured cells of shochu yeast are added to the barley koji (3 tons as barley) produced by the method described above to obtain primary mash. The obtained primary moromi was subjected to fermentation (first stage fermentation) for 5 days.
Next, in the secondary charging, 11.4 kiloliters of water and steamed barley produced by the above-mentioned method (7 tons as barley) are added to the primary mash after the first stage fermentation for 11 days of fermentation ( 2nd stage fermentation). The fermentation temperature was 25 ° C. for both the first charge and the second charge. The secondary mash after the second stage fermentation was subjected to simple distillation by a conventional method to obtain 10 kiloliters of barley shochu and 15 kiloliters of barley shochu distillation residue. Further, 5 kiloliters of the barley shochu distillation residue obtained was treated with a screw press and a ceramic filtration device to remove SS. As a result, the obtained 4 kiloliters of SS-removed barley shochu distillation residue was used in the following examples.
The component composition analysis values of the obtained SS-removed barley shochu distillation residue are shown in Table 2.

(Productivity comparison test of BS when various microorganisms are grown using by-products other than liquor in the liquor production process as a medium)
<Test strain>
・Starmerella bombicola NBRC10243
・Candida bogoriensis NBRC1966
・Candida magnoliae NBRC0705
・Candida apicola NBRC10261

<Seed culture>
Starmerella ( Candida ) bombicola NBRC10243 strain, Candida bogoriensis NBRC1966 strain, Candida magnoliae NBRC0705 strain frozen and stored in a deep freezer (-80 ° C) And 0.5 mL of Candida apicola NBRC10261 strain in a 30 mL YM medium (glucose 10 g / L, yeast extract 3 g / L, malt extract 3 g / L, peptone 5 g / L) And shaking culture at 25 ° C. for 1 day.

<Main culture>
Into a 300 mL Erlenmeyer flask, Brix. 30 mL of the SS-removed barley shochu distillation residue described in paragraph [0039] adjusted to a concentration of 2% was added. In addition, a medium added with 50 g / L glucose and 50 g / L soybean oil was prepared, and a medium sterilized by autoclaving at 121 ° C. for 20 minutes was prepared. Furthermore, 1.5 mL of each seed culture solution described in Paragraph [0042] was inoculated, and cultured with shaking at 25 ° C. for 7 days. Then, the SL preparation is purified from the culture solution after completion of the culture using the recovery / purification method described in paragraph [0036], and the SL production per medium volume is calculated by measuring the weight of the SL preparation. did.

As shown in Table 3, when the SS-removed barley shochu distillation residue, which is a by-product other than liquor produced in the alcohol production process, was used as the culture medium, production of BS was confirmed in all of the strains used in the test. That is, it was confirmed that by-products other than liquor produced in the liquor production process are good substrates for microbial culture and BS production.
It was also confirmed that a particularly large amount of BS was produced when Stamella bombicola NBRC10243 strain was used. Therefore, the Stamella bombicola NBRC10243 strain was used for evaluation of BS productivity using by-products other than liquors produced in the subsequent liquor production process as a medium.

(Production of sophorose lipids using by-products other than liquor produced in the production process of alcoholic beverages as a medium 1) Effect of shochu distillation residue concentration on SL production in the medium)

[Seed culture]
Starmerella ( Candida ) bombicola NBRC10243 strain stored frozen in a deep freezer (-80 ° C) in 30 mL of YM medium (glucose 10 g / L, yeast extract 3 g / L, malt extract (3 g / L, peptone 5 g / L) was inoculated with 0.5 mL, and cultured with shaking at 28 ° C. for 1 day.

[Main culture]
Into a 300 mL Erlenmeyer flask, Brix. 30 mL of the SS-removed barley shochu distillation residue described in paragraph [0039] adjusted to a concentration of 0.5 to 16% was added. Furthermore, a medium added with 50 g / L glucose and 50 g / L olive oil was prepared, and a medium sterilized by autoclaving at 121 ° C. for 20 minutes was prepared. 1.5 mL of the seed culture solution described in paragraph [0047] was inoculated and cultured at 28 ° C. for 3 days with shaking. SL preparation was refine | purified from the culture solution after completion | finish of culture | cultivation using the collection | recovery and refinement | purification method as described in paragraph [0036], and the SL production amount per culture medium volume was computed by measuring the weight of SL preparation. In addition, the cells contained in the culture solution extracted with ethyl acetate were washed with methanol and water and then dried at 105 ° C. overnight. Thereafter, the amount of cells was measured by measuring the weight.

As shown in FIG. 1, the SS content-removed barley shochu distillation residue of Brix. When the concentration was 1 to 2%, the production of SL was good and the output was about 50 g / L. Also, SS-removed barley shochu distillation residue is Brix. In the concentration range of 4% or more, the SL production decreased. From this, in the SL production in the batch culture method using an Erlenmeyer flask using 50 g / L of olive oil as the carbon source, the medium concentration of the SS-removed barley shochu distillation residue is Brix. 1 to 2% has been shown to be suitable.
Furthermore, Brix. As the cell growth increased greatly with increasing concentration, it was confirmed that SS-removed barley shochu distillation residue was a good substrate for microbial culture.

(Production of Sophorose Lipid Using Shochu Distillation Residue as Medium 2) Effect of Carbon Raw Material Type on SL Production)
In a 300 mL Erlenmeyer flask, Brix. Add 30 mL of the SS-removed barley shochu distillation residue described in 0018 adjusted to a concentration of 2.0%, and add 50 g / L glucose and 50 g / L vegetable oil (olive oil, soybean oil, palm oil) A medium sterilized by autoclaving at 121 ° C. for 20 minutes was prepared. 1.5 mL of the seed culture solution prepared in the same manner as in Example 1 was inoculated, and cultured for 2.5 days under the same conditions as in Example 1 to produce SL.
[Comparative Example 1]

(Production of sophorose lipids using conventional media)
On the other hand, as a comparative example, SL production was performed using a conventional medium according to Patent Document 6 in which shochu distillation residue was not used and inorganic salts and yeast extract were added as auxiliary materials. More specifically, a culture test was conducted with the following medium composition.

  In a 300 mL Erlenmeyer flask, 50 g / L glucose, 1.0 g / L urea, 5.0 g / L magnesium sulfate, 1.0 g / L sodium chloride, 10 g / L phosphoric acid Prepare a medium in which 50 g / L vegetable oil (olive oil, soybean oil, palm oil) is added to 30 mL of medium A containing potassium dihydrogen and 2.5 g / L yeast extract, and sterilized by autoclaving at 121 ° C for 20 minutes A prepared medium was prepared. 1.5 mL of the seed culture solution prepared in the same manner as in Example 3 was inoculated, and cultured for 2.5 days under the same conditions as in Example 3 to produce SL.

  Table 4 summarizes the results of comparing the production amounts of SL produced in Example 4 and Comparative Example 1. The amount of SL produced by the medium (Example 4) using the SS-removed barley shochu distillation residue is approximately twice that of the SL produced by the conventional medium (Comparative Example 1), regardless of the oil used. Increasingly, the effectiveness of using shochu distillation residue as a SL production medium has been demonstrated.

In Example 4 and Comparative Example 1, it is the result of the SL production test which performed various vegetable oils as a carbon raw material in a different culture medium.

(Production of sophorose lipid using fermenter)
[Seed culture]
Three 30 mL YM media prepared in a 300 mL Erlenmeyer flask were inoculated with frozen stocks of Starmerella ( Candida ) bombicola NBRC10243 strain and cultured with shaking at 28 ° C for 1 day. Prepared.
[Main culture]
In a 5 L fermentor (Maruhishi Bioengineer), Brix. 2 L of a medium containing SS-removed barley shochu distillation residue described in paragraph [0039] having a concentration of 4%, 100 g / L glucose, 50 g / L olive oil, and 50 g / L oleic acid were prepared. Components other than glucose were sterilized by autoclave after being put into the fermenter. In order to avoid alteration of the glucose due to the Maillard reaction, a 50% (w / v) aqueous solution was prepared, autoclaved separately from the other components, cooled to room temperature, and then charged into the fermenter. The aeration rate was set to 1 vvm (2 L / min), the stirring speed was set to 600 rpm, and the culture temperature was 28 ° C., and the seed culture solution was seeded in whole. Furthermore, sampling was performed every other day, and SL production, hydrophobic group mass, glucose amount, and dry cell weight were measured.
[Quantitative determination of glucose]
The culture solution was centrifuged and the aqueous phase was separated. The amount of glucose contained in the aqueous solution phase was measured using a commercially available glucose detection kit (Glucose Test Wako C-II, Wako Pure Chemical Industries).

  As shown in FIG. 2, on the second day from the start of the main culture, glucose and fat as carbon sources were completely consumed, and SL synthesis was completed. At that time, the SL production amount reached 80 g / L. When the SL productivity per volume of the medium is calculated, it is 40 g / L / day, which is shorter than the 15.5 g / L / day calculated from the conventional report (see Example 2 of Patent Document 6). It was shown that SL can be produced efficiently.

(SL production by fed-batch culture)
[Seed culture]
The same method as in Example 5 was used.
[Main culture]
Culturing was carried out in the same manner as in Example 5. During the culturing period, an amount of feed medium corresponding to 50 g / L glucose, 50 g / L olive oil, and 50 g / L oleic acid was added in a timely manner. Moreover, sampling was performed in a timely manner, and the SL production amount and the dry cell weight were measured by the above-described methods.

  As shown in FIG. 3, it was confirmed that 184 g / L of SL could be produced in 6 days of culture by adding a fed-batch medium at an appropriate time (added at the timing indicated by the arrow in FIG. 3).

(Production of cellobiose lipid)
[Seed culture]
A potato dextrose medium (PDB medium) prepared in a 300 mL Erlenmeyer flask was inoculated with the cryopreserved Ustilago maydis NBRC6907 strain and cultured with shaking at 28 ° C. for 2 days.
[Main culture]
Into a 300 mL Erlenmeyer flask, Brix. 30 mL of the SS-removed barley shochu distillation residue described in paragraph [0039] adjusted to a concentration of 1% was added, and a medium supplemented with 50 g / L of glucose was prepared. 1.5 mL of the above seed culture solution was inoculated and cultured for 4 days under the same conditions as in Example 1 to produce cellobiose lipid (CL).
[Quantification of CL]
After adjusting the culture solution to pH 3 with dilute hydrochloric acid, an equivalent amount of ethyl acetate was added to extract the CL component, and the ethyl acetate was removed with a rotary evaporator to obtain an ethyl acetate extract fraction. The ethyl acetate extract fraction was washed with hexane to remove residual oil. Furthermore, after removing hexane with a vacuum drier, ethanol was added to the residue, and the glucose equivalent in the ethyl acetate extract fraction was measured using the anthrone sulfuric acid method. The CL concentration was calculated by multiplying the glucose equivalent by the molecular weight.

  Using the medium containing the SS-removed barley shochu distillation residue, it was possible to produce 1.4 ± 0.2 g / L of CL. That is, it was confirmed that the BS production method using the medium containing the SS-removed barley shochu distillation residue can also be applied to CL production.

<Production of Cellobiose Lipit Using Fermenter with Shochu Distillation Residue as Medium-Effect of Medium Type on CL Productivity per Medium Volume>
(Production of cellobiose lipids using culture media using shochu distillation residue)
In the production of shochu, CL fermentation production was performed using shochu distillation residue (fermented barley extract) produced as a by-product.
[Seed culture]
Ustilago esculenta MK-1 strain frozen in a deep freezer (−80 ° C.) was added to 30 mL of YM medium (glucose 10 g / L, yeast extract 3 g / L, malt extract 3 g / L, peptone 5 g / L) was inoculated in 0.5 mL, and shake culture was performed at 28 ° C. for 1 day to prepare a first stage seed culture solution. Prepare 30 ml of fermenter CL production medium in 3 300 mL Erlenmeyer flasks with a glucose concentration of 50 g / L as shown in Table 5 [Composition of fermenter CL production medium] and autoclave at 121 ° C. for 20 minutes. Sterilized. 30 ml of the CL production medium for fermenters was seeded with 1.5 mL of the first stage seed culture solution and cultured for 2 days to obtain a second stage seed culture solution.
[Main culture]
Using a hand-held refractometer on a 5 L jar fermenter, 1.5 L of fermented barley extract having a concentration adjusted to 5% Brix was prepared and sterilized at 121 ° C. for 20 minutes in an autoclave. Separately, 400 ml of a 50% (w / v) glucose solution sterilized by autoclaving was added, the seed culture solution of the second stage was seeded on a jar fermenter, the culture temperature was 25 ° C., the stirring rotation speed was 500 rpm, and the aeration speed was 2 L. The cells were cultured under the conditions of / min (1 VVM). After 65 hours of culture, 400 mL of an autoclaved 50% (w / v) glucose solution was added. A sample was extracted during the culture, and the CL concentration, bacterial cell concentration, and glucose concentration in the medium were measured.
The CL concentration was measured by the method described in Example 5 above, and the glucose concentration was measured by the method described in Example 7 above.

As shown in Fig. 4, when CL is produced using a fermentor in a medium using shochu distillation residue using Ustilago esculent a MK-1 strain isolated from Makomo which can be used for food In 7 days, 36,000 mg / L of CL accumulated in the medium. On the seventh day, the CL productivity per medium volume was 5,100 mg / L / day. As a result of TLC and NMR, biosurfactants other than CL such as MEL were not detected. In previous reports, 30 g / L glycolipid can be produced in 7 days by culturing with Ustilago maydis (Appl. Microbiol. Biotechnol., 51, 33-39 (1999) In this case, the resulting glycolipid is a mixture of MEL and CL, and the content ratio is MEL / CL = 9/1. In other words, the production of pure CL is less than about 3 g / L. Recently, it has been reported that 15 g / L of floculosin can be produced in 7 days, the highest value so far, by culturing with yeast of another genus, Pseudozyma flocculosa (Appl. Microbiol. Biotechnol., 80, 307-315 (2008)), this technology can easily produce only CL at a production amount more than twice that of the prior art. That is, it was confirmed that CL can be efficiently produced in a natural medium using fermented barley extract. Further, when CL was recovered from the fermentation broth after completion of the culture using a mixed solvent of ethyl acetate and 2-propanol (mixing ratio 4: 1), 90.1 g of CL could be recovered.
[Comparative Example 2]

(Production of cellobiose lipids using medium without shochu distillation residue)
On the other hand, as a comparative example, CL production was performed using a medium that does not use shochu distillation residue using MK-1 strain. That is, a 2 L portion of the CL production medium for fermenter was prepared in a 5 L jar fermenter as shown in Table 5 [Composition of CL production medium for fermenter], and sterilized by autoclaving at 121 ° C. for 20 minutes. The second stage seed culture described in Example 8 was seeded on a jar fermenter, and cultured under conditions of a culture temperature of 25 ° C., a stirring rotation speed of 500 rpm, and an aeration speed of 2 L / min (1 VVM). After 55 hours of culture, 400 ml of an autoclaved 50% (w / v) glucose solution was added. A sample was extracted during the culture, and the CL concentration, bacterial cell concentration, and glucose concentration in the medium were measured. The CL concentration and glucose concentration were measured in the same manner as in Example 8 above.

As shown in FIG. 4, when CL was produced in a medium not using shochu distillation residue using Ustilago esculent a MK-1 strain isolated from Makomo, which can be used for food, in 5 days, 13,000 mg / L of CL was accumulated in the medium. On the fifth day, the CL productivity per medium volume was 2,580 mg / L / day. As a result of TLC and NMR, biosurfactants other than CL such as MEL were not detected.

  Table 6 summarizes the results of comparison of CL productivity per medium volume in Example 8 and Comparative Example 2. The CL productivity (5,100 mg / L / day) per volume of the culture medium (Example 8) using the SS-removed barley shochu distillation residue was determined based on the culture medium using no shochu distillation residue (Comparative Example 2). Compared with the CL productivity per volume (2,580 mg / L / day), it increased about twice, indicating the effectiveness of using shochu distillation residue as a CL production medium.

It is possible to provide an effective method of using the composition excluding alcoholic beverages obtained in the production process of alcoholic beverages, and a significant reduction in the quantitative environmental burden due to the composition is also expected.
Since the production cost of sophorose lipid (SL) can be greatly reduced, the use of SL in a wide range of fields is expected. The same applies to cellobiose lipid (CL).

  FERM P-21725

Claims (7)

  A biosurfactant (except Mannosylerythritol lipid; MEL) characterized by using a microorganism having an ability to produce biosurfactant and using a fermentation product obtained by subjecting a plant to alcohol fermentation as a medium for culturing the microorganism. Production method.   At least one step selected from the group consisting of a step of filtering the mash obtained through alcoholic fermentation, a step of pressing the mash, a step of distilling the mash and a step of centrifuging the mash. The method for producing a biosurfactant (Mannosylerythritol lipid; excluding MEL) according to claim 1, wherein the alcohol is a liquor obtained by passing through or a by-product other than the liquor.   The biosurfactant (Mannosylerythritol lipid) according to claim 1 or 2, wherein the plant is at least one plant selected from the group consisting of barley, wheat, sweet potato (sweet potato), rice, brown sugar, buckwheat, grape and sesame. Production method of excluding MEL.   The method for producing a biosurfactant (Mannosylerythritol lipid; excluding MEL) according to claim 1, 2 or 3, wherein the biosurfactant is a sophorose lipid.   The method for producing a biosurfactant (Mannosylerythritol lipid; excluding MEL) according to claim 1, 2 or 3, wherein the biosurfactant is cellobiose lipid. The microorganisms capable of producing biosurfactants are Starmerella ( Candida ) bombicola , Candida bogoriensis , Candida magnoliae , Candida apicola. ), Ustilago maydis , or Ustilago esculenta , the production of the biosurfactant (except Mannosylerythritol lipid; MEL) according to any one of claims 1 to 5. Method.   The carbon source of the medium is added with at least one selected from the group consisting of glucose, sucrose, glycerin, vegetable oil, fatty acid, fatty acid methyl ester, molasses and waste edible oil. 7. A method for producing a biosurfactant (Mannosylerythritol lipid; excluding MEL) according to any one of 6 above.