Method of purifying distillers solubles and use of the purified matter
专利汇可以提供Method of purifying distillers solubles and use of the purified matter专利检索，专利查询，专利分析的服务。并且Distillers solubles are purified by a centrifugal separation to a degree above 50,000, which is the product of g.times.minute, or a filtration with addition of a filter aid. The clarified matter is purified by a molecular sieve treatment, ultrafiltration, reverse osmosis or an organic solvent precipitation. The clarified matter or purified matter thus obtained is pulverized by spray drying, lyophilic drying or air drying.Said clarified matter, purified matter or pulverized matter is added to a fermentation medium or a feed as an available component.，下面是Method of purifying distillers solubles and use of the purified matter专利的具体信息内容。
What is claimed is:1. A method of purifying distillers solubles unknown growth factor from distillers solubles comprising the steps of clarifying distillers solubles by treatment selected from the group consisting of(1) a centrifugal separation to a degree of above 50,000 (g.times.minute) and(2) a filtration with addition of a filter aid; and collecting the liquid parts of molecular weights of 10.sup.3- 10.sup.5 by subjecting the obtained clarified liquid to a molecular sieve treatment.2. A method in accordance with claim 1, wherein said clarifying step is carried out by centrifugal separation to a degree of at least about 70,000 (g.times.minute).3. A method in accordance with claim 1, wherein said clarifying is carried out by filtration using 1-10% of said filter aid.4. A method in accordance with claim 1, wherein said filter aid is selected from the group consisting of celite, talc, paper pulp, sawdust and short fibers.
FIELD OF INVENTION
The present invention relates to a method of purifying distillers solubles.
An object of the present invention is to remove contaminants from distillers solubles, and to provide a fermentation medium and an available component of feed thus utilizing it. Although the available component obtained and utilized by the present invention is one which has been unknown and not been identified, this component is referred to herein as distillers solubles unknown growth factor. Hereinafter the available component of the present invention is abbreviated simply as DSUGF.
BACKGROUND OF INVENTION
In general, the distillers solubles are generally deformed as for example, distillation residues soluble or suspended in water and from which the rough parts of the particles have been removed, such residues being from stillage (residue of distillation) of whisky and so on, and they may be dried into dried solubles or concentrated or made into semisolid state and added to feed, thus having been broadly utilized.
Also, these distillers solubles have been known for their effects as a growth source of Actinomyces and other various microorganisms or as a growth promoting factor and thereby have been broadly used.
However, even such useful distillers solubles have large drawbacks in use. For example, in the case where the distillers solubles are used as growth source of microorganisms, as they are or in concentrated state, many suspended contaminants and lipid materials may contaminate the microorganisms cells or products; but the removing thereof is very difficult, so that they can not be possibly used for propagation of cells which are adapted for food, such as bakers yeast, starter and so on as they are. Also such distillers solubles have never been utilized for products which are difficult to purify. Moreover, the distillers solubles concentrated and formed into solid state have poor solubility, and in use they are inconvenient, so that they have been difficult to use as a general source of culture.
Heretofore, the distillers solubles sold in market generally are those produced by removing large solids from stillage of whisky or the like, and concentrated, and it has been believed that the distillers solubles themselves constitute an available component as a whole.
SUMMARY OF INVENTION
Studies have now been conducted to modify the properties of such distillers solubles to allow the use thereof, for example, as a substitute for yeast extract, and as a result it was found unexpectedly that the distillers solubles can be rapidly clarified by a centrifugal treatment or a filtration treatment, and the resultant clarified liquid has a large amount of DSUGF and if said liquid is subjected to further purifying process, its available component can be more concentrated.
The present invention relates to fermentation medium and feed which are produced by subjecting distillers solubles or their concentrate to a centrifugal treatment or a filtration treatment, and, if necessary, the resultant supernatant to a further purifying treatment, and allowing them to contain the resultant clarified liquid or its treated matter thus obtained therein as DSUGF.
The distillers solubles used in the present invention may be any one of stillages such as whisky stillage, grain alcohol stillage and so on, and these unconcentrated liquids, partly concentrated concentrates and a marketed matter concentrated to 1/5-1/10 ratio by volume are adapted to the present invention as they are.
According to the present invention, a centrifugal treatment or a filtration treatment is adopted to obtain clarified filtrate.
DETAILED DESCRIPTION OF EMBODIMENTS
In the centrifugal treatment, defatting is not caused, so that if defatting is carried out previously, the clarified liquid can be obtained directly by the centrifugal treatment. To carry out such a previous defatting, these distillers solubles or their relevant matter is contacted with an organic solvent such as ether etc. and. The lipid portions extracted and removed therefrom. This defatting process is not an indispensable one before the centrifugal treatment, but an optional one, and if it is not carried out before the centrifugal treatment, it is sufficiently done after the centrifugal treatment by solvent extraction and filtration etc.
The centrifugal separating treatment of distillers solubles herein is carried out to a degree above 50,000, which is the product of g×minute, preferably to a degree above 70,000 (g×minute).
In general, g is represented by the following formula,
g=S2 r/89500
s: rpm
r: distance (cm) from rotating shaft to the end of liquid portion.
The degree of centrifugal treatment is represented by a product of g multiplied by minute, that is to say, g×minute.
Herein, using a centrifugal separator of r=9.6cm, unconcentrated liquid of distillers solubles was defatted by ether, and subjected to centrifugal treatments of various g, and the resultant clarified liquids were measured of their turbidity by means of a wave length of 660 nm, and thereby the results shown in the following Table 1 were obtained.
TABLE 1______________________________________ Time Degree ofg (minute) g × minute transmission (%)______________________________________ 965 10 9650 0.3001716 10 17160 0.2652681 10 26810 0.0703861 10 38610 0.0605255 10 52550 0.0506864 10 68640 0Unconcentrated liquid ofdistillers solubles 56Defatted liquid of unconcen-trated liquid by ether 13______________________________________
Also, using a constant g (10733g), the same ether defatted unconcentrated liquid of distillers solubles was subjected to centrifugal treatments with various times, and the resultant supernatants were measured of their turbidity by means of a wave length of 660 nm, and thereby the results shown in the following Table 2 were obtained.
TABLE 2______________________________________ Time Degree ofg (minute) g × minute transmission (%)______________________________________10733 3 32199 0.07010733 4 42932 0.06010733 5 53665 0.04010733 6 64398 0.02010733 7 75131 0Unconcentrated liquid ofdistillers solubles 56Defatted liquid of unconcen-trated liquid by ether 13______________________________________
From these results, it is obvious that the distillers solubles can be fairly clarified by centrifugation to a degree above 50,000 (g×minute) and completely clarified by centrifugation to a degree 70,000 (g×minute).
The obtained light fulvous transparent liquid has lipid material suspended therein in the case where defatting has not been carried out before the centrifugal separation treatment, and therefore if it is subjected to a filtration by means of a thick filter paper, for example, Whatman filter paper No.32, the fat can be separated easily.
Also, according to the present invention, in the case the distillers solubles are filter treated, this is carried out under addition of a filter aid to the distillers solubles. As filter aids, celite, talc, paper pulp, saw dust, short fibers and so on are used effectively.
To the distillers solubles or their concentrate, a filter aid is added in an amount of 1-10%, and the mixture is sufficiently stirred and filtered.
The filtration may be of the continuous type or the batch type, however, filtration under pressure or vacuum is preferable in efficiency, and therefore continuous type drum filter machines etc. are most preferable as means of industrial filtering.
The obtained matter by the centrifugal treatment or filtration treatment is light fulvous transparent liquid which is quite different from the distillers solubles of dark brown mud like property which has been heretofore known, and is thought a distinct one therefrom.
The light fulvous transparent liquid contains almost all of DSUGF and therefore is very effective as various kinds of microorganisms fermentation medium and feed. Since this transparent liquid is free of suspended contaminant, it can be broadly used for culture of edible fungi, production of starter and bakers yeast and so on, and therefore becomes very useful.
Thus obtained transparent supernatant is effective as it is, but from the view point of contained components, in a low molecular range and in a high molecular range a fairly large amount of matter is contained in said supernatant, and it is thought that a true available component or a group of available components exist therein. According to the present invention, it is possible that this supernatant is subjected to a treatment such as ultrafiltration etc., if necessary, thereby separating a liquid containing low molecular matter group and a high molecular material group and thus notably concentrating DSUGF.
In order to obtain liquids containing low molecular matters and high molecular matters, any means can be adopted, such as use of molecular sieve, membrane filter and so on, and it is industrially effective to use an ultrafiltration using a cheap membrane filter. For the ultrafiltration, various kinds of membrane filters can be used, and for example, membrane filters made by Amicon Co., XM-100, UM-10, UM-2, UM-05 etc. can be combined to obtain a liquid containing matters of various molecular weights. In this case, if a liquid part passing through UM-05 is separated, low molecular matters containing liquid can be obtained, and if a liquid part not passing through UM-05 is separated, high molecular matters group containing liquid can be obtained, and said high molecular matters containing liquid can be further graduated into respective molecular weight matters group containing liquids of about 103 -104, 104 -105 and above 105 of molecular weight respectively by using UM-2, UM-10 and XM-100 etc.
The unconcentrated liquid of distillers solubles was subjected to a centrifugal treatment after defatting process, and the obtained clarified liquid was passed through each membrane filter, and the resultant liquid part and its five times concentrate were measured to determine the amount of nitrogen, and thereby the results shown in Table 3 were obtained.
TABLE 3______________________________________Ultrafiltration of defatted liquid ofdistillers solubles unconcentrated liquid.(By means of membrane filters, XM-100, UM-10,UM-2, UM-05, made by Amicon Co.) Amount of N ofMolecular five times con-weight Amount of N centrate______________________________________Above 105 (XM-100) 0.082mg/ml. 0.412mg/ml104 -105 (UM-10) 0.342 1.712103 -104 (UM-2) 0.056 0.284500-103 (UM-05) 0.020 0.100Below 500 0.137 0.685Distillers solubles 0.7 --unconcentrated liquid______________________________________ Wherein amount of N was analyzed by Kjeldahl method, and each fractionate matter was represented by a conversion into distillers solubles unconcentrated liquid.
For example, the resultant liquids containing matters of UM-05 pass and XM-100 pass - UM-10 no pass respectively are very light yellowish transparent solutions containing a large amount of DSUGF, and effective as various kinds of microorganisms medium.
Also, the distillers solubles clarified liquid can be fractionated into a high molecular matter group and a low molecular matter group by the reverse osmosis method in very short time. In the case of use of the reverse osmosis method, AS-215, AS-230 and AS-290 etc. (all made by Abcor Co.) are used as RO reverse osmosis membranes, and this method is effective to approximately fractionate DSUGF and purify it. The transparent liquids obtained by these purifying methods are free of contaminants, and therefore these becomes very useful for production of foods and biochemical medicines.
The obtained DSUGF containing liquid does not contain contaminants, so that various kinds of treatments such as concentrating and drying etc. can be carried out thereto. In the case of drying, said liquid is often added with various kinds of salts, sugars and other assistant so as to make suitable at the time of resolution, and mixed therewith and then pulverized by spray drying, lyophilic drying and so on. The obtained condensate and powder are convenient to transport, and at the time of use, can immediately dissolve in water thereby reproducing transparent liquid state, and accordingly these can be added to all media as an available component for propagation of microorganism.
As an effective concentration method for DSUGF, there is a precipitation treatment by means of organic solvents such as ethanol, acetone, methanol etc. The available component of distillers solubles progressively precipitates as the concentration of organic solvent becomes higher, so that the resultant precipitate is filter separated, and the separated precipitate can be used as it is or after being subjected to air drying, and pulverized appropriately. For example, in the case of the use of ethanol to precipitate DSUGF, when it is added up to 50-90% of ethanol final concentrations, almost all of the DSUGF precipitates at respective concentrations, and therefore if these precipitates are individually separated appropriately by filtration or centrifugal separation, DSUGF can be obtained in solid state in notably purified condition. Also, since there are available components such as yeast etc. in the soluble section of above 90%, the liquid part can be distillation removed so as to obtain DSUGF in solid state.
The fermentation medium of the present invention is formed by allowing DSUGF to be contained therein. Forming of the medium is effected by mixing the supernatant together with sugars, various kinds of salts and a very small amount of nutritious source previously, and subjecting the resultant mixture to lyophilic or spray drying, or by mixing the dried supernatant as it is. Forming of the medium can be carried out before the culture. In this case, the supernatant, its condensate, its dried matter etc. are previously provided, and these are mixed with sugars, various kinds of salts, a very small amount of nutrious source etc. thereby forming the fermentation medium.
Addition into the fermentation medium can be carried out in a great amount if this supernatant does not particularly obstruct the culture, and also the substitution for all nitrogen compounds is possible. Relating to amount of addition, it is preferable to add above about 21 mg in 100 ml as amount of nitrogen.
DSUGF of the present invention is effective to the growth of the rumen microorganisms isolated from the rumen, and also stimulates the growth of the rumen microorganisms by giving it to cows, and serves for the growth of cows and the increase of body-weight thereof. It is also very effective for the growth and the increase of body-weight of sheep, pigs, minks, hens, fish etc. besides for cows when adding it to their feed.
Various kinds of feeds according to the present invention are produced by adding the centrifugally treated matter or the filter treated matter of distillers solubles or their concentrate or their further treated matter to general feed component in the state of liquid, concentrate or powder, and mixing them together.
The available component containing feed of the present invention serves notably for promoting of the growth and increasing of body-weight by giving it to cows, sheep, pigs, minks, hens and fish etc.
Next, embodiments of the present invention will be shown in relation to experimental examples, production examples, fermentation examples and feed examples, and the light fulvous transparent liquid obtained in Production example 1 will be expressed as "defatted DS" in some cases.
EXPERIMENTAL EXAMPLE 1
Using the available component of distillers solubles obtained in Production example 1 and the precipitate separated at that time, and using Lentinus edodes (IFO 4902) as microorganism, effects to cell propagation of marketed distillers solubles (whisky stillage 1/5 concentrate) and its available component liquid obtained in the method of Production example 1 were examined.
As the basic medium, a medium of the following composition was used:
______________________________________Glucose 10 gYeast extract 2.4 gPolypeptone 2.4 gKH2 PO4 1 gMgSO4 . 7H2 O 0.5 gCaCl2 . 2H2 O 0.5 gFeCl3 . 6H2 O 10 mgMnCl2 . 4H2 O 7.2 mgZnCl2 4.0 mgCuSO4 . 5H2 O 1.0 mgWater 1000 ml(pH = 5.0)______________________________________
Using this basic medium, the following media were made;
1. Only 100 ml of the basic medium.
2. Such one prepared by adding 1.5 g of the distillers solubles to 100 ml of the basic medium.
3. Such one prepared by adding 1.0 g of the supernatant liquid resulting from a centrifugal treatment to a degree of 70,000 (g×minute) of the distillers solubles (this amount corresponds to 1.5 g of the distillers solubles.) to 100 ml of the basic medium.
4. Such one prepared by adding 0.5 g of the precipitate resulting from a centrifugal treatment to a dgreee of 70,000 (g×minute) of the distillers solubles (this amount corresponds to 1.5 g of the distillers solubles.) to the basic medium.
5. Such one prepared by removing the yeast extract and polypeptone from the basic medium and adding 1.5 g of the distillers solubles thereto.
6. Such one prepared by removing the yeast extract and polypeptone from the basic medium and adding 1.0 g of the supernatant liquid resulting from a centrifugal treatment to a degree of 70,000 (g×minute) of the distillers solubles (this amount corresponds to 1.5 g of the distillers solubles.) thereto.
7. Such one prepared by removing the yeast extract and polypeptone from the basic medium and adding 0.5 g of the precipitate resulting from a centrifugal treatment to a degree of 70,000 (g×minute) of the distillers solubles (this amount corresponds to 1.5 g of the distillers solubles.) thereto.
100 ml of each of said prepared medium was put in a 500 ml Sakaguchi flask, and Lentinus edodes (IFO 4902) was inoculated therein and a shaking culture was carried out at 25° C. and 100 rpm (with 7 cm stroke).
After seven days culturing, the produced cells were collected by a filtration, and dried, and amount of cells in each medium was investigated as the dried matter.
The results are shown in the following Table, and it is obvious from the Table that DSUGF exists in large quantities in the liquid part and very slightly in the precipitate.
TABLE______________________________________Medium number Amount of dried cells (g/100ml)______________________________________1 0.362 1.98*3 1.354 0.72*5 1.056 0.947 0.69*______________________________________ *In this case, since it is observed that the precipitated solid matters are taken in the fungi pellets and left therein as they are, true weight of the fungi is considered to be fairly less than this value.
EXPERIMENTAL EXAMPLE 2
Using the available component liquid of distillers solubles obtained in Production example 2 and using Lentinus edodes IFO 4902 as microorganism, effects to cell propagation of commercial distillers solubles (whisky stillage 1/5 concentrate) and of its available component liquid obtained in the method of Production example 2 were compared with those of yeast extract and of polypeptone.
As the basic medium, a medium of the following composition was used.
______________________________________Glucose 10 gYeast extract 2.4 gPolypeptone 2.4 gKH2 PO4 1 gMgSO4 . 7H2 O 0.5 gCaCl2 . 2H2 O 0.5 gFeCl3 . 6H2 O 10 mgMnCl2 . 4H2 O 7.2 mgZnCl2 4.0 mgCuSO4 . 5H2 0 1.0 mgWater 1000 ml(pH = 5.0)______________________________________
Using this basic medium, the following media were prepared;
1. Only 100 ml of the basic medium.
2. Such one prepared by adding 1.5 g of the distillers solubles to 100 ml of the basic medium.
3. Such one prepared by adding 1.0 g of the available component liquid resulting from a filter treatment of the distillers solubles (this amount corresponds to 1.5 g of the distillers solubles.) to 100 ml of the basic medium.
4. Such one prepared by adding 1.5 g of the distillers solubles to the basic medium free from yeast extract and polypeptone.
5. Such one prepared by adding 1.0 g of the DSUGF component liquid resulting from a filter treatment of the distillers solubles (this amount corresponds to 1.5 g of the distillers solubles.) to the basic medium free from yeast extract and polypeptone.
100 ml of each of said prepared medium was put in a 500 ml Sakaguchi flask, and a Japanese mushroom (Lentinus edodes IFO 4902) was inoculated and incubated with shaking at 25° C. and 100 rpm (with 7 cm stroke).
After 7 days of culture, the produced cells were collected by a filtration, and dried, and amount of cells in each medium was investigated as the dry matter.
The results are shown in the following Table, and it is obvious from the Table that the available component of the distillers solubles exists in large quantities in the filter part.
TABLE______________________________________ Amount of driedMedium No. cells (g/100ml)______________________________________1 0.362 0.98*3 1.254 0.94*5 0.88______________________________________ *In this case, since it is observed that the precipitated solid matters are taken in the cell pellets and left therein as they are, true weight o the cells is considered to be fairly less than this value.
EXPERIMENTAL EXAMPLE 3
Using the liquid available component of distillers solubles passing through the molecular filter obtained in Production example 3 and each filtrate containing the matter of each molecular weight separated at that time, and using Lentinus edodes IFO 4902 (a species a Japanese mushroom) as microorganism, and, after regulating an amount of nitrogen at the same level, effects to cell propagation in each medium were examined. The uncondensate of distillers solubles and the basic medium were used as controls.
As the basic medium, a medium of the following composition was used:
______________________________________Glucose 10 gYeast extract 2.4 gPolypeptone 2.4 gKH2 PO4 1 gMgSO4 . 7H2 O 0.5 gCaCl2 . 2H2 O 0.5 gFeCl3 . 6H2 O 10 mgMnCl2 . 4H2 O 7.2 mgZnCl2 4.0 mgCuSO4 . 5H2 O 1.0 mgWater 1000 ml(pH = 5.0)______________________________________
Using this basic medium, the following media were prepared;
1. Only 100 ml of the basic medium.
2. Such one prepared by removing the yeast extract from the basic medium and add the uncondensate of distillers solubles in same amount of nitrogen as the yeast extract thereto.
3. Such one prepared by removing the yeast extract from the basic medium and adding the liquid passing through UM-05 in the same amount of nitrogen as the yeast extract thereto.
4. Such one prepared by removing the yeast extract from the basic medium and adding the liquid passing through UM-2-no passing through UM-05 in the same amount of nitrogen as the yeast extract thereto.
5. Such one prepared by removing the yeast extract from the basic medium and adding the liquid passing through UM-10-no passing through UM-2 in the same amount of nitrogen as the yeast extract thereto.
6. Such one prepared by removing the yeast extract from the basic medium and adding the liquid passing through XM-100-no passing through UM-10 thereto in the same amount of nitrogen as the yeast extract.
7. Such one prepared by removing the yeast extract from the basic medium and adding the liquid no passing through XM-100 thereto in the same amount of nitrogen as the yeast extract.
100 ml of each of said prepared medium was put in a 500 ml Sakaguchi flask, and Lentinus edodes IFO 4902 (a species of Japanese mushroom) was inoculated therein and incubated with shaking at 25° C. and 100 rpm (with 7 cm stroke).
After 7 days of culture, the propagated cells were collected by a filtration, and dried, and amount of cells in each medium was investigated as the dry matter.
The results are shown in the following Table, although the available component of the distillers solubles seems to have wide molecular weight distribution, it is obvious from the Table that the available component exists particularly in large quantities in the component passing through XM-100-no passing through UM-10 (molecular weight of about 104 -105).
TABLE______________________________________ Amount ofMedium No. Medium pH cells (g/100ml)______________________________________ 3.34 0.4812 3.77 0.8393 3.49 0.5424 3.35 0.3885 3.23 0.7136 3.31 0.8627 3.26 0.541______________________________________
EXPERIMENTAL EXAMPLE 4
In the same manner as in Production example 11, pulverization treatment was carried out at various temperature in the entrance and exhaust zone, and after investigation of the state of resulting powder the following results were obtained:
______________________________________Temperature (°C.) State ofNo. Entrance Exhaust the powder______________________________________1 290-300 130-120 No Good2 205-210 105-110 Good3 150-160 95-85 Good______________________________________
Using the powder thus obtained (No. 1, 2, 3), Saccharomyces cererisiae IFO 0203 was incubated with shaking for 40 hours at 28° C. and pH 5.5 by using the medium described in Production example 11, and also Bacillus subtilis IFO 3007 was incubated with shaking for 39 hours at 28° C. in the medium described in Production example 12. The results are shown in the following Table, and it is obvious from the Table that an activity decreases only a little by the treatment of pulverization.
TABLE______________________________________ B. subtilis S. cerevisae Specific SpecificNo. Component OD10 660 activity OD10 660 activity______________________________________1 Distillers solubles1 unconcentrated 0.490 1 0.625 1liquid2 Powder 1 0.455 0.93 0.535 0.8563 Powder 2 0.483 0.99 0.535 0.8654 Powder 3 0.488 1 0.570 0.912______________________________________
EXPERIMENTAL EXAMPLE 5
To the light fulvous transparent liquid obtained in Production example 1 ethanol was progressively added from 0% to 95% at ethanol final concentration, and after slow agitation, a precipitation occured at respective ethanol concentration. Each precipitation was isolated by filtration, then the amount of the solid matter and of sugars were determined. The results are shown in the following Table:
______________________________________Ethanol final concentration (%) 0-70 70-80 80-85 85-90 90-9570% 80% 85% 90% 95% 95-unsol- unsol- unsol- unsol- unsol- 95%uble uble uble uble uble solublesection section section section section section______________________________________Solid 10.8 8.4 2.5 2.5 3.5 72.4matter(%)Sugars 9.6 3.0 0.9 1.4 1.5 81.4(%)______________________________________
Powder was respectively obtained by lyophile treatment of each unsoluble and soluble section obtained thus.
The powder above was added in such amount as corresponding to 25% of the defatted DS in 4 ml of the following medium:
______________________________________Glucose 5 gKH2 PO4 0.5 gMgSO4 . 7H2 O 0.2 gPolypeptone 0.4 gadd water to 100 ml______________________________________
and resulting medium containing the powder was adjusted at pH 70.
Bacillus subtilis IFO 3007 was inoculated in each medium thus obtained and incubated at 30° C. After 40 hours of culture, the propagation of cells was investigated.
The results are shown in the following Table, and it is obvious from the Table that a high activity exists in the 80% unsoluble section.
__________________________________________________________________________ Ethanol final concentration 0-70 70-80 80-85 85-90 90-95 more than 95% 70% 80% 85% 90% 95% 95%Control unsoluble unsoluble unsoluble unsoluble unsoluble solubleNo added section section section section section section__________________________________________________________________________Specific1.0 1.11 3.89 1.00 1.05 1.01 1.34Activity1.0 1.10 3.93 1.01 1.02 1.02 1.23__________________________________________________________________________
EXPERIMENTAL EXAMPLE 6
With respect to the light fulvous transparent luquid obtained in Production example 1, the reverse osmosis membrane separation was carried out by using the RO reverse osmosis membrane of AS-215, AS-230 and AS-290 made by Abcor Co.
The characteristic of each RO reverse osmosis membrane used in the Example was as follows:
______________________________________RO reverse osmosis Inhibition ratiomembrane of sucrose______________________________________AS - 215 15 ± 5%AS - 230 30 ± 10%AS - 290 90 ± 5%______________________________________ Using a solution containing 10% of sucrose
Using these reverse osmosis membranes, the liquid obtained in Production example 1 was treated at operation pressure of 20-21 Kg/cm2 and water temperature of 25°-27° C., then liquid passing through the membrane and liquid remaining were respectively obtained. Each remaining percentage is shown in the following Table;
______________________________________ Passing or Amount ofMedium Treated remaining nitrogenNo. liquid percentage (mg/ml)______________________________________1 Liquid no passing through AS-215 6 0.392 Liquid passing through AS-215 94 0.093 Liquid no passing through AS-230 5 0.354 Liquid passing through AS-230 95 0.145 Liquid no passing through AS-290 10 0.386 Liquid passing through AS-290 90 0.157 Untreated liquid 0.54______________________________________
Culture test was carried out by using each liquid of medium number in the Table above described.
(Mode of Culture Test)
Used strain:
Bacillus subtilis IFO 3007, Saccharomyces cerevisiae IFO 0203, Aspergillus oryzae ATCC 128
Basic medium:
Medium for B. subtilis: glucose 5.0%, polypeptone 0.5%, KH2 PO4 0.5%, MgSO4.7H2 O 0.2%, pH7.0
Medium for S. cerevisiae: glucose 5.0%, (NH4)2 SO4 0.5%, KH2 PO4 0.1%, MgSO4.7H2 O 0.05%, CaCl2.2H2 O 0.01%, NaCl 0.01%, vitamin solution, pH5.5
Medium for A. oryzae: glucose 3.0%, K2 HPO4 0.1%, KCl 0.05%, MgSO4.7H2 O 0.05%, FeSO4.7H2 O 0.001%, NaNO3 0.07%, pH5.5
Preparation of medium and culture condition: Both B. subtilis and S. cerevisiae were cultured in the medium so adjusted that the all amount of nitrogen become equal by substituting respectively medium sample of said medium No. 1-7 for nitrogen source of the basic medium in the quantity of 30% (V/V). After 24 and 38 hours of culture with shaking at 28° C., the growth was respectively investigated by measuring OD10 660. With respect to A. oryzae, after 4 days culture under these conditions the growth was respectively investigated by dry cell weight (dcw)
The results of the culture test are shown in the following Table:
__________________________________________________________________________ B. subtilis S. cerevisiae A. oryzae Specific Specific dcw SpecificIncubation OD10 660 activity OD10 660 activity (mg/100ml) activityperiod 24hr (× 10) 38hr (× 20) 24hr 38hr 24hr (× 10) 38hr (× 20) 24hr 38hr 4 days 4__________________________________________________________________________ daysControl 0.212 0.136 0.307 0.1800 1 1 1 1 380.2 1 0.250 0.147 0.265 0.178Medium 0.308 0.195 0.600 0.351No. 1 1.34 1.41 2.05 1.94 523.8 1.38 0.313 0.203 0.570 0.343 0.406 0.255 0.527 0.3102 1.83 1.87 1.87 1.74 318.9 0.84 0.440 0.275 0.542 0.313 0.297 0.183 0.592 0.3603 1.26 1.26 2.08 1.98 612.0 1.61 0.287 0.174 0.595 0.348 0.420 0.315 0.530 0.3154 1.82 2.24 1.90 1.88 308.3 0.81 0.420 0.320 0.555 0.358 0.300 0.170 0.613 0.3925 1.36 1.34 2.14 2.07 608.3 1.60 0.330 0.208 0.610 0.346 0.378 0.292 0.593 0.3656 1.62 2.16 2.05 1.99 303.6 0.80 0.370 0.318 0.580 0.348 0.312 0.270 0.720 0.4757 1.36 2.01 2.45 2.63 631.8 1.66 0.318 0.298 0.682 0.467__________________________________________________________________________
PRODUCTION EXAMPLE 1
100 ml of the distillers solubles unconcentrated liquid was subjected to a centrifugal separating treatment for 10 minutes at 13,000 rpm by means of KR-200A type centrifugal separator (made by Kubota Seisakusho) as it was.
The obtained liquid part was light fulvous and turbid with fine oil droplets.
This liquid part was filtered by means of Whatman No. 32 filter paper (Toyo Filter Paper No. 5C) and thereby 94 ml of light fulvous transparent liquid was obtained. The content of nitrogen thereof was 0.7 mg/ml.
PRODUCTION EXAMPLE 2
100 ml of the distillers solubles unconcentrated liquid was added with 4 g of Celite (Trade name: Supercell) and mixed therewith sufficiently, and then the mixture was filtered by means of a suction filter (Nutsche), thereby obtaining 94 ml of light fulvous transparent liquid.
PRODUCTION EXAMPLE 3
100 ml of the distillers solubles unconcentrated liquid was subjected to a centrifugal separating treatment at 9,000 rpm for 8 minutes (80,000 g×minutes) by means of KR-200A type centrifugal separator (Kubota Seisakusho).
The obtained liquid part was light fulvous and turbid with fine oil droplets.
This liquid part was filtered by means of Whatman No. 32 filter paper, and thereby 94 ml of light fulvous transparent liquid was obtained.
The obtained light fulvous transparent liquid was treated by means of a molecular sieve in which membrane filters, XM-100 and UM-10, made by Amicon Co., were set, and thereby the liquid part which passed through XM-100 and did not pass through UM-10 and thus remained was collected thereby obtaining 9.4 ml of light yellow transparent liquid. Content of nitrogen thereof was 3.4 mg/ml.
PRODUCTION EXAMPLE 4
100 ml of the distillers solubles unconcentrated liquid was subjected to a centrifugal separating treatment at 9,000 rpm for 8 minutes (80,000 g×minutes) by means of KR-200A type centrifugal separator (Kubota Seisakusho).
The obtained liquid part was light fulvous and turbid with fine oil droplets.
This liquid part was filtered by means of Whatman No. 32 filter paper, and thereby 94 ml of light fulvous transparent liquid was obtained.
200 ml of the obtained light fulvous transparent liquid (total nitrogen content 0.74 mg/ml) was treated by means of a molecular sieve in which membrane filter of UM-05, made by Amicon Co., was set, and thereby the liquid part which passed through it was collected, yielding 160 ml (total nitrogen content 0.14 mg/ml) of light fulvous transparent liquid.
PRODUCTION EXAMPLE 5
1,000 ml of the distillers solubles unconcentrated liquid was added with 40 g of Celite (Trade name: Supercell), and mixed therewith sufficiently, and then the mixture was filtered by means of a suction filter (Nutsche), yielding 940 ml of light fulvous transparent liquid.
200 ml (total nitrogen content 0.74 mg/ml) of the obtained light fulvous transparent liquid was treated by means of a molecular sieve in which a membrane filter of UM-05, made by Amicon Co., was set, and thereby the liquid part which passed through it, yielding 160 ml (total nitrogen content 0.15 mg/ml) of light fulvous transparent liquid.
PRODUCTION EXAMPLE 6
30 ml of the light fulvous transparent liquid obtained in Production example 1 was mixed with the mixture consisting of K2 HPO4 0.1 g, KCl 0.05 g, MgSO4.7H2 O 0.05 g, FeSO4.7H2 O 0.001 g, glucose 3 g and NaNO3 0.07 g, adding water to 100 ml, adjusting pH 5.5, and thus a medium for molds being prepared.
PRODUCTION EXAMPLE 7
30 ml of the light fulvous transparent liquid obtained in Production example 1 was mixed with the mixture consisting of NH4 Cl 0.12 g, glucose 5 g, K2 HPO4 0.1 g, MgSO4.7H2 O 0.3 g, and a small amount of vitamins, adding water to 100 ml, adjusting pH 5.5, and thus a medium for yeasts being prepared.
PRODUCTION EXAMPLE 8
30 ml of the light fulvous transparent liquid obtained in Production example 2 was pulverized by lyophile as it was. Thus obtained powder was mixed with the mixture consisting of glucose 5 g, (NH4)2 SO4 0.4 g, KH2 PO4 0.1 g, CaCl2.2H2 O 0.01 g, MgSO4.7H2 O 0.05 g, NaCl 0.01 g and a small amount of vitamins in the dry powder state, and thereby a medium for yeasts being obtained.
This medium is instantly used for culture of yeast by adding water to 100 ml and adjusting pH 5.5 on the spot.
PRODUCTION EXAMPLE 9
6 ml of the light fulvous transparent liquid obtained in Production example 4 was mixed with the mixture consisting of glucose 5 g, KH2 PO4 0.5 g, MgSO4.7H2 O 0.2 g and polypeptone 0.42 g, adding water to 100 ml, adjusting pH 7.0, and thus a medium for Bacillus subtilis.
PRODUCTION EXAMPLE 10
30 ml of the light fulvous transparent liquid obtained in Production example 1 was mixed with the mixture of polypeptone 1.06 g, glucose 2 g, Na-acetate.3H2 O 0.7 g, K2 HPO4 0.04 g, KH2 PO4 0.04 g, MgSO4.7H2 O 0.025 g, NaCl 0.001 g, MnSO4 0.001 g, FeSO4.7H2 O 0.001 g, K-citrate 0.003 g and CaCO3 1 g, adding water to 100 ml, and thus a medium for lactic acid bacteria being prepared.
PRODUCTION EXAMPLE 11
The light fulvous transparent liquid was pulverized as it was by means of spray dryer at 150°-300° C. of entrance temperature and at 85°-130° C. of exhaust temperature.
A medium for yeast was prepared by adding such amount of the resulting powder that corresponds to that of obtained by 30 ml of light fulvous transparent liquid to the following composition:
______________________________________Glucose 5 g(NH4)2 SO4 0.5 gKH2 PO4 0.1 gMgSO4 . 7H2 O 0.05 gCaCl2 . 2H2 O 0.01 gNaCl 0.01 gVitamins small amountWater 100 ml(pH = 5.5)______________________________________
PRODUCTION EXAMPLE 12
A medium for bacteria was prepared by adding such amount of the powder obtained in Production example 11 as corresponds to that of obtained by 30 ml of light fulvous transparent liquid to the following composition:
______________________________________Glucose 5 gPolypeptone 0.5 gKH2 PO4 0.5 gMgSO4 . 7H2 O 0.2 gWater 100 ml(pH = 7.0)______________________________________
PRODUCTION EXAMPLE 13
50 ml of the light fulvous transparent liquid obtained in Production example 1 was added with about 117 ml of ethanol, and the mixture was slowly agitated, thus formed precipitate was removed by filtration. The filtrate was further added with about 83 ml of ethanol, after slow agitation of the mixture, formed precipitate was isolated by filtration and air-dried.
PRODUCTION EXAMPLE 14
100 ml of the light fulvous transparent liquid obtained in Production example 1 was treated by means of RO reverse osmosis membrane "AS-230 (made by Abcor Co.)" at operation pressure of 20-21 Kg/cm2 and water temperature of 25°-27° C., and thereby yielding 95 ml of the passing liquid.
The following examples illustrate culturing examples using the fermentation medium according to this invention. In these culturing examples, the light fulvous transparent liquid obtained in Production example 1 was referred as "defatted DS", that liquid obtained in Production example 3 was referred as "DS 104 -105 ", weight of the dry cells obtained by culturing were represented by "dcw (g)", and when the cells in culture fluids were measured by means of absorption at 660 nm, the amount of cells was represented by "absorbance".
CULTURING EXAMPLE 1
As the strain, Aspergillus oryzae ATCC 128 was used.
______________________________________Glucose 3 gK2 HPO4 0.1 gKCl 0.05 gMgSO4 . 7H2 O 0.05 gFeSO4 . 7H2 O 0.001 gNaNO3 VariationCasamino acid ChangeDefatted DS ChangeDS 104 -105 ChangepH = 5.5______________________________________
The above compositions were become 100 ml by water.
An inoculation of the spore suspension of said strain was made to each of four new 500 ml-Sakaguchi flasks containing the medium of No. 1-4 prepared as following Table by using the basic medium above described. And a shaking culture was carried on for 4 days at 30° C.
______________________________________Medium Casamino DefattedNo. NaNO3 acid DS DS 104 -105 Total-N______________________________________1 0.2g 32.9mg2 0.44g 32.93 0.07 30ml 32.94 0.14 6ml 32.9______________________________________ (in 100ml of medium)
The cells were filter separated from the obtained culture broth, and the weight of cells (dry matters), pH of the filtrate and residual sugar were measured.
The condition of drying the cells was at 110° C. and for 2 hours, and the quantitative determination of sugar was carried out by modified Somogyi's method.
The results were as follows:
TABLE______________________________________Medium N source Residual SpecificNo. g/100ml dcw (g) pH sugar (%) activity______________________________________1 NaNO3 0.2 0.400 4.2 0.45 1 0.416 4.5 0.512 Casamino 0.44 0.474 3.9 0.37 1.05 acid 0.431 3.8 0.44 0.953 NaNO3 0.07 0.769 3.7 0.24 1.70 Defatted DS 30ml 0.745 3.7 0.17 1.654 NaNO3 0.14 0.662 4.8 0.03 1.46 DS 104 -105 6ml 0.636 4.3 0.03 1.40______________________________________
CULTURING EXAMPLE 2
As the strain, Rhizopus delemar IFO 4697 was used. A composition of the basic medium was as follows.
______________________________________Glucose 3 gK2 HPO4 0.1 gKCl 0.05 gMgSO4 . 7H2 O 0.05 gFeSO4 . 7H2 O 0.001 gPolypeptone VariationCasamino acid ChangeDefatted DS ChangeDS 104 -105 ChangeAdjust pH 5.5 and add water to 100ml______________________________________
Said strain was incubated by using each medium of No. 1-4 prepared as following Table as described in Culturing example 1, except that it was incubated for 5 days.
______________________________________Medium poly- Casamino DefattedNo. peptone acid DS DS 104 -105 Total-N______________________________________1 0.25g 32.9mg2 0.44g "3 0.09 30ml "4 0.17 6ml "______________________________________ (in 100ml of medium)
After culture the measurments were performed according to the procedure of Culturing example 1. The results were as follows:
______________________________________Medium N source Residual SpecificNo. (g/100ml) dcw (g) pH sugar (%) activity______________________________________1 Polypeptone 0.231 2.4 -- 0.25 0.223 2.4 -- 12 Casamino acid 0.313 2.5 -- 1.38 0.44 0.314 2.5 -- 1.383 Polypeptone 0.292 2.9 -- 1.29 0.09 defatted DS 0.277 2.9 -- 1.22 30ml4 Polypeptone 0.256 2.6 -- 1.13 0.17 DS 104 -105 0.277 2.6 -- 1.22 6ml______________________________________
CULTURING EXAMPLE 3
As the strain, Saccharomyces cerevisiae IFO 0203 was used. With respect to the medium, the following composition was used as the basic medium:
______________________________________Glucose 5 gK2 HPO4 0.1 gMgSO4 . 7H2 O 0.3 gVitamins small amountWater 100 ml(pH = 5.5)______________________________________
the basic medium above was added with NH4 Cl, defatted DS and/or DS 104 -105 as described in the following Table, and thereby the medium of No. 1-3 being respectively prepared:
______________________________________MediumNo. NH4 Cl defatted DS DS 104 -105 Total-N______________________________________1 0.2g 53mg2 0.12 30ml "3 0.16 6ml "______________________________________ (in 100ml of medium)
By using the medium of No. 1 above mentioned, a preculture was carried out by shaking culture at 30° C. for one night. The strains after preculture were inoculated in medium size test tubes containing 5 ml of medium in such manner that they were finally diluted, a thousand times, and incubated with shaking (180 rpm) at 30° C. for 30 hours.
The growth of cells was measured by measuring absorbance at 660 nm of the culture fluids thus obtained.
The results were shown in the following Table.
TABLE______________________________________Medium Absorb- SpecificNo. N Source ance activity______________________________________1 NH4 Cl 0.2g 3.20 12 NH4 Cl 0.12g, defatted DS 30ml 8.40 2.633 NH4 Cl 0.16g, DS 104 -105 6ml 4.44 1.39______________________________________
CULTURING EXAMPLE 4
Culture and measurement were carried out by using same medium, culture conditions, measuring method etc. as those described in Culturing example 3, except that Candida tropicalis IFO 0006 was used as strain. The results were shown in the following Table:
TABLE______________________________________Medium N source SpecificNo. (g/100 ml) Absorbance activity______________________________________1 NH4 Cl 0.2 6.4 1 6.32 NH4 Cl 0.12 14.6 2.30 defatted DS 30ml 14.8 2.323 NH4 Cl 0.16 8.56 1.34 DS 104 -105 6ml 9.40 1.48______________________________________
CULTURING EXAMPLE 5
As the strain, Pichia membranaefaciens IFO 0128 was used. With respect to the medium, the following composition being used as the basic medium:
______________________________________Glucose 3 gKH2 PO4 0.1 gCaCl2 . 2H2 O 0.01 gMgSO4 . 7H2 O 0.05 gNaCl 0.01 gVitamins small amountWater 100 ml(pH = 5.5)______________________________________
the basic medium above was added with (NH4)2 SO4, defatted DS and/or DS 104 -105 as described in the following Table, and thereby the medium of No. 1-4 being respectively prepared:
______________________________________Medium Casamino defatted Total-No. (NH4)2 SO4 acid DS DS104 -105 N______________________________________1 0.5g 10.6mg2 1.4g "3 0.4 30ml "4 0.46 6ml "______________________________________ (in 100ml of medium)
Culture was carried out as described in Culturing example 4, except that the temperature was maintained at 25° C. Measuring the culture fluids thus obtained, the results are shown in the following Table: (The measuring method is the same as described in Culturing example 3)
TABLE______________________________________Medium N source SpecificNo. (g/100ml) Absorbance activity______________________________________1 (NH4)2 SO4 0.5 3.92 1 3.702 Casamino 1.4 4.21 1.10 acid 4.80 1.263 (NH4)2 SO4 0.4 7.04 1.85 defatted DS 30ml 6.80 1.784 (NH4)2 SO4 0.46 5.90 1.55 DS 104 -105 6ml 5.90 1.55______________________________________
CULTURING EXAMPLE 6
As the strain, Lactobacillus casei IFO 3425 was used. With respect to the medium, the following composition being used as the basic medium:
______________________________________Glucose 2 gNa-acetate . 3H2 O 0.7 gK2 HPO4 0.04 gKH2 PO4 0.04 gMgSO4 . 7H2 O 0.025 gNaCl 0.001 gMnSO4 0.001 gFeSO4 . 7H2 O 0.001 gK-citrate 0.003 gCaCO3 1 gWater 100 ml______________________________________
the basic medium above was added with polypeptone, yeast extract, defatted DS and DS 104 -105 as described in the following Table, and thereby the medium of No. 1-7 being respectively prepared:
______________________________________Medium poly- yeast defattedNo. peptone extract DS DS104 -105 Total-N______________________________________1 1.22g 160.8mg2 1.05 0.2g "3 1.06 30ml "4 1.14 6ml "5 0.8 0.5 "6 0.8 80 "7 1.02 16 "______________________________________ (in 100ml of medium)
10 ml of each medium was respectively transferred in medium size test tube. A preculture was carried out at 30° C. for one night by using the same medium as that of No. 5, and the inoculum size was adjusted as a thousand times dilution was finally obtained.
A culture was accomplished by stationary manner at 30° C. for 48 hours.
The culture broth obtained was respectively added with 1 ml of lactic acid to dissolve calcium carbonate, adequately diluted, and then the absorbance thereof at 660 nm was measured.
The obtained results are shown in the following Table:
TABLE______________________________________Medium N source SpecificNo. (g/100ml) Absorbance activity______________________________________1 polypeptone 1.22 0.88 1 0.852 polypeptone 1.05 1.41 1.63 yeast extract 0.2 1.35 1.563 polypeptone 1.06 1.69 4.53 defatted DS 30ml 1.71 4.094 polypeptone 1.14 1.91 2.87 104 -105 DS 6ml 1.51 3.035 polypeptone 0.8 1.53 3.73 yeast extract 0.5 1.43 3.636 polypeptone 0.8 2.13 5.64 defatted DS 80ml 1.98 5.347 polypeptone 1.02 1.75 3.80 104 -105 DS 16ml 1.88 3.93______________________________________
CULTURING EXAMPLE 7
Culture and measurement were carried out by using same medium, culture conditions, measuring method etc. as those described in Culturing example 6, except that Leuconostoc mesenteroides IFO 3832 was used.
The obtained results are shown in the following Table:
TABLE______________________________________Medium N source SpecificNo. (g/100ml) Absorbance activity______________________________________1 polypeptone 1.22 0.58 1 0.652 polypeptone 1.05 1.69 6.39 yeast extract 0.2 1.70 5.603 polypeptone 1.06 2.20 6.97 defatted DS 30ml 1.95 6.794 polypeptone 1.14 1.78 6.11 DS 104 -105 6ml 1.74 5.895 polypeptone 0.8 1.55 6.63 yeast extract 0.5 1.77 6.456 polypeptone 0.8 2.50 8.94 defatted DS 80ml 2.25 8.377 polypeptone 1.02 2.35 7.65 DS 104 -105 16ml 2.25 7.31______________________________________
CULTURING EXAMPLE 8
As the strain, Streptomyces griseus IFO 13189 was used. The medium was prepared as described in Culturing example 1, except that pH was adjusted to 7.0.
A preculture was respectively carried out in a 500 ml-Sakaguchi flask containing 100 ml of each medium of No. 1-4 at 30° C. for 5 days by using Czapek-Dox medium. After inoculation of 5 ml of each preculture, a shaking culture was carried out at 30° C. for 5 days (stroke: 7 cm, 114 rpm).
Thus obtained culture broth was subjected to a centrifugal separating treatment at 10,000 rpm for 10 minutes, and that, precipitate part was dried at 110° C. for 2 hours to be measured the weight of dry cells, and on the other hand as to that liquid part, the value of pH and residual sugar was respectively measured. The quantitative determination of sugar was carried out by modified Somogyi's method.
The results are as follows:
TABLE______________________________________Medium N source Residual SpecificNo. (g/100ml) dcw(g) pH sugar(%) activity______________________________________1 NaNO3 0.2 0.120 7.8 1.76 1 0.124 7.8 1.542 Casamino 0.44 0.134 7.2 0.04 1.10 acid 0.133 7.3 0.04 1.093 NaNO3 0.07 0.174 7.5 -- 1.43 defatted DS 30ml 0.171 7.4 -- 1.414 NaNO3 0.14 0.186 7.6 -- 1.53 DS 104 -105 6ml 0.186 7.7 -- 1.53______________________________________
CULTURING EXAMPLE 9
Culture and measurement were carried out by using same medium, culture conditions, measuring method etc. as those described in Culturing example 8, except that Streptomyces aureofaciens IFO 12594 was used. The obtained results are shown in the following Table:
TABLE______________________________________Medium N source Residual SpecificNo. (g/100ml) dcw(g) pH sugar(%) activity______________________________________1 NaNO3 0.2 0.601 2.2 -- 1 0.648 1.9 --2 Casamino 0.44 0.699 1.9 -- 1.12 acid 0.735 2.2 -- 1.183 NaNO3 0.07 0.825 2.1 -- 1.32 defatted DS 30ml 0.784 2.1 -- 1.264 NaNO3 0.14 0.777 2.0 -- 1.24 DS 104 -105 6ml 0.758 2.0 -- 1.21______________________________________
CULTURING EXAMPLE 10
Using the low molecular available component (liquid passing through UM-05) of distillers solubles obtained in Production example 4 and using such five strains as:
a. Saccharomyces cerevisiae K7 IFO 0203
b. Candida tropicalis IFO 0006
c. Bacillus subtilis IFO 3007
d. Pichia membranaefaciens IFO 0128
e. Leuconostoc mesenteroides IFO 3832
each strain was respectively incubated in each medium. Distillers solubles unconcentrated liquid and each basic medium were used as control, and in case of culture an amount of nitrogen was so adjusted that it always becomes same.
With respect to the yeasts, the following composition was used as a basic medium:
______________________________________Glucose 5%(NH4)2 SO4 0.5%KH2 PO4 0.1%MgSO4 . 7H2 O 0.05%CaCl2 . 2H2 O 0.01%NaCl 0.01%Vitamins small amount______________________________________ (pH = 5.5)
With respect to the culture of yeasts, Pichia membranaefaciens was incubated with shaking at 25° C. for 40 hours and the other yeasts were incubated with shaking at 28° C. for 36 hours.
With respect to the bacteria, the following composition was used as a basic medium:
______________________________________ Glucose 5% Polypeptone 0.5% KH2 PO4 0.5% MgSO4 . 7H2 O 0.2%______________________________________ (pH = 7.0)
The following composition was used as a basic medium for Leuconostoc mesenteroides:
______________________________________Glucose 2.0% Polypeptone 0.8%Yeast 0.5% Sodium acetate 0.5%extractKH2 PO4 0.04% K2 HPO4 0.04%MgSO4 . 7H2 O 0.025% NaCl 0.001%MnSO4 0.001% FeSO4 . 7H2 O 0.001%FeSO4 . 7H2 O 0.001% Potassium 0.003% citrateCaCO3 1.0% pH 6.5______________________________________
The bacteria were incubated with or without shaking at 28° C. for 39 hours.
Culturing method was as follows: The each basic medium above was added with the filtrate containing the low molecular available component of distillers solubles in various amount as described in the following Table, removing the nitrogen source from the basic medium in such amount as corresponding to the nitrogen content of the filtrate which was added to the basic medium (in case of Leuconostoc mesenteroides, yeast extract being removed as the nitrogen source), and thereby the medium always containing same amount of nitrogen was respectively prepared. For comparison, the two media were further prepared, one being that of added, in various amount, with the transparent defatted and unconcentrated liquid of the distillers solubles as it is, and the other being that of using only the basic medium as nitrogen sources without adding anything. On these media each microorganism of from a to e was incubated, and after the culture fluids obtained being diluted ten times by volume, the quantities of cells propagated in each of media above mentioned were comparatively determined by turbidity.
The results obtained are given in Table below, and it is obvious from the Table that the low molecular available component of distillers solubles is an excellent factor for propagation of each microorganism:
TABLE__________________________________________________________________________ low molecularDefatted, availableunconcentrated component Pichia memb- Leuconostocliquid of (liquid passing S. cerevisiae C. tropicalis B. subtilis ranaefaciens mesenteroidesdistillers through specific specific specific specific specificsolubles UM-05) OD10 660 activity OD10 660 activity OD10 660 activity OD10 660 activity OD10 660 activity__________________________________________________________________________1 0.84 1.00 1.15 1.00 0.38 1.00 0.230 1.00 0.428 1.00 0.92 1.26 0.40 0.260 0.420 1.82 1.64 1.37 0.464 0.4422 30 ml/100 ml 2.06 1.37 3.55 1.84 1.04 (N = 21.0 mg) 1.81 1.66 1.40 0.440 0.440 1.96 1.87 1.31 0.420 0.4603 50 ml/100 ml 2.23 1.47 3.35 1.78 1.10 (N = 35.0 mg) 1.97 1.68 1.30 0.450 0.472 2.09 1.93 1.13 0.503 0.5034 70 ml/100mg 2.35 1.62 2.85 1.99 1.17 (N = 49.0 mg) 2.05 1.98 1.09 0.470 0.488 1.34 1.27 1.41 0.325 0.3275 30 ml/100 ml 1.51 1.02 3.65 1.30 0.79 (N = 4.1 mg) 1.31 1.18 1.44 0.310 0.340 1.44 1.40 1.39 0.365 0.3986 50 ml/100 ml 1.63 1.16 3.69 1.50 0.95 (N = 6.9 mg) 1.42 1.39 1.49 0.372 0.407 1.56 1.52 1.40 0.395 0.4587 70 ml/100 ml 1.78 1.23 3.63 1.58 1.05 (N = 9.6 mg) 1.58 1.45 1.43 0.378 0.433__________________________________________________________________________ Note; OD10 660 : measured value of the culture liquids diluted 1 times by volume
CULTURING EXAMPLE 11
Using the defatted DS the bakers yeast was cultured.
Mode of Incubation:
The N-sources of the following media (1) and (2) were replaced with the defatted DS such that total content of nitrogen become equal with each other.
Culture medium (1): for preculture
As total sugar in molasses 1%, (NH4)2 SO4 0.16%, KH2 PO4 0.014%, MgSO4.7H2 O 0.009%, K-citrate 0.05%, citric acid 0.01%, pH 5.6 (T.N=34 mg/100 ml)
Culture medium (2): for main culture
Add 4.52 ml of the liquid containing 0.655% of (NH4)2 SO4 and 1.22% of (NH2)2 CO to 100 ml of the liquid containing 1% of total sugar in molasses and 0.014% of KH2 PO4 and adjust pH 5.6 (T.N=32 mg/100 ml).
The results are given in the Table below and this shows that the yeast was best propagated in case of adding the defatted DS in an amount of 45%:
______________________________________Adding ratio Medium (1) Medium (2)of the defatted Final FinalDS (%) OD10 660 (X10) pH OD10 660 (X10) pH______________________________________0 0.317 3.56 0.296 4.2315 0.368 3.98 0.371 5.2730 0.416 4.44 0.418 5.5845 0.430 5.14 0.423 5.6060 0.390 5.59 0.368 5.49______________________________________ Concentration of N in the defatted DS:0.53 mg/ml
CULTURING EXAMPLE 12
Using the defatted DS and simultaneously the sulfite waste liquor (SWL), the yeast was incubated.
______________________________________1. Used strain: Candida utilis IFO 06192. Basic medium: (NH4)2 HPO4 0.089 g (NH4)2 SO4 0.307 g KCl 0.048 g MgSO4 . 7H2 O 0.078 g SWL (total 80 ml sugar 3.0%) Add tap water to 100ml and adjust pH 5.5.______________________________________
The five times concentrated liquid of the defatted DS was respectively added to the basic medium in an amount of 20, 30, 50 and 70% expressed as the ratio of the unconcentrated liquid of the defatted DS, and at the same time the amount of ammonium sulfate was respectively reduced according to the amount of the defatted DS added thereto, and then each medium containing same amount of nitrogen was respectively prepared. The yeast was transferred to a 500 ml Sakaguchi flask containing 100 ml of each medium above. The flask was incubated with shaking at 30° C. for 30-40 hours.
The results are shown in the following Table:
______________________________________Amount of thedefatted DS Number of cells × 109 /ml Residualadded thereto After 22 hrs After 40 hrs sugar(%) of culture of culture (%)______________________________________1 -- 0.47 0.74 0.862 20 0.72 1.30 0.463 30 0.61 1.40 0.494 50 0.61 1.40 0.485 70 0.52 1.10 0.47______________________________________
CULTURING EXAMPLE 13
Using the defatted DS, a n-paraffin assimilable yeast was incubated.
1. Used strain: Candida lipolytica
2. Basic medium: n-paraffin 5%, NH4 Cl 0.5%, KH2 PO4 0.02%, MgSO4.7H2 O 0.02%, FeSO4.7H2 O 10 mg/l, MnSO4.4H2 O 2 mg/l, ZnSO4.4H2 O 2 mg/l, CaCO3 1%, yeast extract 0.1%
3. Culture: The yeast extract was removed from the basic medium above, and an amount of nitrogen was replaced in certain ratio with the original liquid of the defatted DS. The medium thus obtained was used for culture. (T.N=130 mg/100 ml)
4. Culturing conditions: Shaking culture (150 rpm) at 30° C. for 60 hours. Using a 500 ml Sakaguchi flask containing 30 ml of the medium. Inoculum size 105 /ml.
The results of culture are shown in the following Table, and it shows that the defatted DS replaces 0.1% of the yeast extract or it is much more effective in propagation of the yeast.
______________________________________ Amount of the defatted DSCul- added thereto Amount of cellsture (%) OD10 660 (X100)______________________________________1 20 0.312 30 0.323 45 0.344 60 0.365 75 0.386 90 0.38Control yeast extract 0.32 0.1% added______________________________________
CULTURING EXAMPLE 14
The defatted DS was concentrated to 1/3 volume under reduced pressure. To 50 ml of the concentrated liquid thus obtained, changing pH conditions, 50 ml of organic solvent was added, then 50% unsoluble fraction was isolated. To the residual liquid part 25 ml of organic solvent was further added, then 60% unsoluble fraction was isolated. To the residual liquid part 42 ml of organic solvent was added, then 70% unsoluble fraction was isolated, and similarly adding 83 ml of organic solvent to the liquid part then 80% unsoluble fraction was isolated, and further adding 250 ml of organic solvent to the liquid part the 90% unsoluble fraction was isolated. Thus remained liquid part was 90% soluble fraction.
The following strains were used:
______________________________________Bacterium: Bacillus subtilis IFO 3007Yeast: Saccharomyces cerevisiae IFO 0203Mold: Penicillium chrysogenum IFO 4826Actinomyces: Streptomyces griseus(Ray fungus) IFO 13189______________________________________
The basic medium and culture conditions were as follows:
______________________________________Basic Medium Culture Conditions______________________________________Bacter- Glucose 5% Inoculate a precultureium Polypeptone 0.5% (carried out at 30° C. for KH2 PO4 0.5% one night by using the basic MgSO4 . 7H2 O 0.2% medium) in such inoculum (pH 7.0) size that a thousand times dilution is finally obtained, and incubate with shaking at 30° C. for 24 hrs.Yeast Glucose 5% Inoculate a preculture NH4 Cl 0.2% (carried out at 30° C. for one K2 HPO4 0.1% night by using the basic MgSO4 . 7H2 O 0.3% medium) in such inoculation (pH 5.5) size that a thousand times dilution is finally obtained, and incubate with shaking at 30° C. for 24 hrs.Mold Glucose 3% Inoculate a spore suspens- NaNO3 0.2% ion of the used strain, KCl 0.05% and incubate with shaking MgSO4 . 7H2 O 0.05% at 30° C. for 4 days FeSO4 . 7H2 O 0.001% (pH 5.5)Actino- Glucose 3% Inoculate respective 5mlmyces NaNO3 0.2% of a preculture carried KCl 0.05% out for 5 days by using the MgSO4 . 7H2 O 0.05% basic medium, and incubate FeSO4 . 7H2 O 0.001% with shaking for 5 days. (pH 7.0)______________________________________
By using such one prepared by adding the defatted DS to the basic medium above mentioned in an amount of 30%, such one prepared by adding the fraction by organic solvent to the basic medium above mentioned in such amount of that corresponding to 30% of the defatted DS, and by using the basic medium as a control, an activity was expressed as specific activity. Each specific activity was respectively symbolized as follows:
______________________________________ MoreSpecific Less than thanactivity 1.0 1.0-1.2 1.2-1.5 1.5-2.0 2.0-2.5 2.5______________________________________symbol - ± + ++ +++ ++++______________________________________
With respect to each strain above, under these conditions a culture test was carried out with changing the fraction conditions.
The results shown in the following Tables were obtained:
a. Fractionation conditions
Organic solvent: Ethanol
pH at fractionation: 3.8
Temperature at fractionation: 25° C.
______________________________________strainsFractionated P.chryso-samples B.subtilis S. cerevisiae A. oryzae genum St.griseus______________________________________0-50% +++ - ± ± +50-60 +++ - ± ± -60-70 +++ - ± - -70-80 +++ - - - -80-90 - - - - -morethan - +++ ± + ++90Defatted +++ +++ + + ++DS______________________________________
b. Fractionation conditions
Organic solvent: Ethanol
pH at fractionation: 7.0
Temperature at fractionation: 25° C.
______________________________________ StrainsFraction- S. A. P. St.ated samples B.subtilis cerevisiae oryzae genum griseus______________________________________Precipitate ++++ - ± - -occurred bypH adjustingand cooling0-50% ++++ - ± ± +50-60 - - - - -60-70 - - - -70-80 - - - - -80-90 - - - - -more than 90 - +++ ± + ++Defatted DS ++++ +++ + + ++______________________________________
c. Fractionation conditions
Organic solvent: Ethanol
pH at fractionation: 3.8
Temperature at fractionation: 5° C.
______________________________________ StrainsFraction- B. S. A. P. chryso- St.ated samples subtilis cerevisiae orzyae genum griseus______________________________________Precipitate ++++ - ± - -occurred bypH adjustingand cooling0-50% + - ± ± +50-60 - - - - -60-70 - - - - -70-80 - - - - -80-90 - - - - -more than 90 - +++ ± ± ++Defatted DS ++++ +++ + + ++______________________________________
d. Fractionation conditions
Organic solvent: Ethanol
pH at fractionation: 3.8
Temperature at fractionation: 5° C.
______________________________________ StrainsFraction- B. S. A. P.chryso- St.ated samples subtilis cerevisiae oryzae genum griseus______________________________________Precipitate +++ - - - -occurred bypH adjustingand cooling0-50% +++ - + + +50-60 +++ - - - -60-70 +++ - - - -70-80 ++ - - - -80-90 - - - - -more than 90 ` +++ ++ + ++Defatted DS +++ +++ ++ + ++______________________________________
e. Fractionation conditions
Organic solvent: Methanol
pH at fractionation: 3.8
Temperature at fractionation: 25° C.
______________________________________ StrainsFractionated B. S. A. P. chryso- St.samples subtilis cerevisiae oryzae genum griseus______________________________________0-50% ++++ - ± ± +50-60 +++ - - - -60-70 +++ - - - -70-80 +++ - - - -80-90 ++ - - - -more than 90 ++ +++ ± + ++Defatted DS ++++ +++ + + ++______________________________________
f. Fractionation conditions
Organic solvent: Acetone
pH at fractionation: 3.8
Temperature at fractionation: 2.5° C.
______________________________________ strainsFraction- B. S. A. P. chryso- St.ated samples subtilis cerevisiae oryzae genum griseus______________________________________0-70% +++ - + + +70-80 +++ - - - +80-90 - - - - +more than - ++ + + +90Defatted DS +++ +++ + + ++______________________________________
CULTURING EXAMPLE 15
The microorganisms used in this culturing example were isolated and identified from the rumen of cattle by the Second Bacteria Research Section, the National Institute of Animal Health, the Ministry of Agriculture and Forestry, and they are stocked therein. These organisms are not deposited anywhere, however, they are validly stocked in said Research Section with each strain number, and they may be available without restraint.
The name of used strains and its strain numbers in this Example were as follows:
Selenomonas Sp. 4-1-25
Selenomonas sp. 4-3-28
Selenomonas sp. P-3-7
Streptococcus bovis 3-cattle-13
Streptococcus bovis 9809
Bacteroides ruminicola Br-19189
Bifidbacterium 31
Bacteroides brevis 7S-6
Bacteroides brevis 7-2-2
Butyrivibrio fibrisolvens 2-40
Butyrivibrio fibrisolvens 7S-29
Butyrivibrio fibrisolvens 7-33
Butyrivibrio fibrisolvens 7-19
Eubacterium sp. 7S-27
Bacteroides oralis 7-4
Ruminococcus flavefaciens A17
Negasphela elsdenii 4-3-32
Buthyrivibrio sp. 7-7
Buthyrivibrio sp. 7S-30
Buthyrivibrio alactoacidigens 7S-11
The used media were as follows:
______________________________________(1) Basic mediumSugar (Glucose or Maltose) 10 gPolypeptone 10 gYeast extract 10 gCysteine-HC1 0.5 g0.1% Resazulin solution 1 ml*Salts solution A 6 mlSalts solution B 6 mladd water to 1.l andadjust pH 6.9*Salts solution A0.6% K2 HPO4Salts solution BKH2 PO4 0.6%NaCl 1.2(NH4)2 SO4 1.2MgSO4 . 7H2 O 0.12CaCl2 . 2H2 O 0.12(2) Media for experimentationA. Control mediumUse the basic medium above as it wasB. Medium added with the available componentof distillers solubles______________________________________
The transparent light fulvous liquid obtained in Production example 1 (hereinafter designated as "defatted DS") was added in such amount that the content of defatted DS in the resulting liquid medium was equal to 30%, and supplying polypeptone to the same level as the medium, thus the following medium was prepared:
______________________________________Sugar (Glucose or Maltose) 10 gPolypeptone 17.1 gCysteine-HCl 0.5 g0.1% Resazulin solution 1 mlSalts solution A 6 mlSalts solution B 6 mlDefatted DS 300 mlAdjust pH 6.9 and add water to 1l.______________________________________
The mode of experimentation by using the strains and media above was as follows:
Mode of Experimentation
(1) Preculture
Use such one culturing on a slant culture containing rumen extract at 37° C for 2 days.
(2) Inoculation
0.6ml of the salts solution being respectively added to the preculture slant, thus a cell suspension of strain was prepared. An inoculation was carried out by adding a drop of the suspension into each test tube by means of same pipette.
Such operation was always carried out under gas (CO2) jet conditions in order to keep anaerobic conditions.
(3) Culturing
Stationary culture at 37° C.
(4) Measurement
The growth of cells were measured by measuring the OD 660 nm after both 24 hours and 48 hours of culture.
The results obtained from this experiment are as follows:
______________________________________(Results in the experiment) OD60 660Species . Strain number Medium 24 hrs 48 hrs______________________________________ Selenomonas sp. 4-1-25 A 0.688 0.648 B 0.745 0.675Selenomonas sp. 4-3-28 A 0.313 0.698 B 0.176 0.691Selenomonas sp. P-3-7 A 0.684 0.674 B 0.725 0.715Streptococcus bovis 3-cattle-13 A 0.414 0.669 B 0.333 0.708Streptococous bovis 9809 A 0.485 0.545 B 0.440 0.540Bacteroides ruminicola Br-19189 A 0.011 0.059 B 0.056 0.371Bifidacterium sp. 31 A 0.441 0.526 B 0.690 0.620Bacteroides brevis 7S-6 A 0.185 0.085 B 0.520 0.480Bacteroides brevis 7-2-2 A 0.010 0.015 B 0.145 0.235Butyrivibrio fibrisolvens 2-40 A 0.014 0.020 B 0.00 0.105Butyrivibrio fibrisolvens 7S-29 A 0.00 0.00 B 0.158 0.107Butyrivibrio fibrisolvens 7-33 A 0.017 0.009 B 0.063 0.088Butyrlvibrio fibrisolvens 7-19 A 0.00 0.004 B 0.161 0.114Eubacterium sp. 7S-27 A 0.015 0.291 B 0.046 0.707Bacteroldes oralis 7-4 A 0.078 0.495 B 0.075 0.601Ruminococcus flavefaciens A-17 A 0.025 0.397 B 0.001 0.522Negasphela elsdenii 4-3-32 A 0.011 0.009 B 0.030 0.555Butyrivibrio sp. 7-7 A 0.034 0.020 B 0.082 0.200Butyrivibrio sp. 7S-30 A 0.00 0.00 B 0.162 0.113______________________________________
The following facts became clear from the results obtained by the experiments above described:
(1) The strains which grew only in the medium B (defatted DS added) were as follows:
Negasphela elsdenii 4-3-32
Butyrivibrio fibrisolvens 7S-29
Butyrivibrio fibrisolvens 7-33
Butyrivibrio fibrisolvens 2-40
Butyrivibrio fibrisolvens 7-19
Butyrivibrio sp. 7S-30
Butyrivibrio alactoacidigens 7S-11
Bacteroides brevis 7-2-2
(2) The strains which grew much more in the medium B (defatted DS added) than in the medium A (defatted DS no added) were as follows:
Bacteroides ruminicola ATCC-19189
Bacteroides brevis 7S-6
Eubacterium sp. 7S-27
Bifidbacterium thermophilum 31
Ruminococcus flavefaciens A-17
Bacteroides oralis 7-4
(3) The strains which grew well both in the medium A and in the medium B (there being not appreciable difference between those media) were as follows:
Selenomonas sp. 4-1-25
Selenomonas sp. 4-3-28
Selenomonas sp. P-3-7
Streptococcus bovis 3-cattle-13
Streptococcus bovis 9809
The feed examples according to the present invention are as follows:
FEED EXAMPLE 1
A basic composition used was as follows:
______________________________________Yellow corn 570 KgTallow 25Soybean cake 255Fish meal (crudeprotein content:60%) 50Gluten meal 20Alfalfa meal 20Calcium phosphate 15Calcium carbonate 7.5Sodium Chloride 4.5Magnesium sulfate 0.3Zinc oxide 0.1Methionine 0.3Vitamins 5______________________________________
The composition above was added with 800 l of the transparent light fulvous liquid and all amount of the powder respectively obtained in Production examples 1 and 6, mixing them well, then a feed for broiler was prepared.
FEED EXAMPLE 2
A basic composition used was as follows:
______________________________________Soybean cake (crudeprotein content: 44%) 250 KgAlfalfa meal 70Molasses 70Ground bones 25Sodium chloride 8Vitamin A (10,000 I.U./g) 0.8Vitamin D2 (1,500 I.U./g) 0.3______________________________________
The composition above was added with 2,800 l of the transparent light fulvous liquid and all amount of the powder respectively obtained in Production examples 2 and 7, mixing them well, them a feed for beef cattle was prepared.
FEED EXAMPLE 3
A basic composition used was as follows:
______________________________________Yellow corn 410 KgMilo 480Soybean cake (crudeprotein content: 44%) 40Fish solubles (crudeprotein content: 33%) 10Calcium carbonate 7.5Calcium phosphate 10Sodium chloride 3Minerals 2Indian millet 480______________________________________
The composition above was added with 1,600 l of the transparent light fulvous liquid obtained in Production example 2, mixing them well, drying and then a feed for pig was prepared.
FEED EXAMPLE 4
A basic compositon used was as follows:
______________________________________Corn 440 KgSoybean cake (crudeprotein content: 44%) 75Powdered meat 180Powdered embryo 85Tallow 50Pressed wheat 25Alfalfa meal 15Grated cheese 15Powdered fish 15Powdered liver 7.5Ground bones 10Sodium chloride 10vitamin A (4,000 I.U./g) 1.5Vitamin D2 (1,500 I.U./g) 1______________________________________
The composition above was added with 2,300 l of the transparent light fulvous liquid and all amount of the passing liquid respectively obtained in Production example 1 and 8, mixing them well, drying, and then a dog food was prepared.
FEED EXAMPLE 5
A basic compositon used was as follows:
______________________________________Wheat flour 50 KgWhite powdered fish 150Oat 110Powdered meat and Groundbones (crude protein 225content: 505)Soybean cake 50Alfalfa meal 50Cotton seed cake 50Powdered embryo 50Powdered liver 25Skim milk powder 70Sodium chloride 20______________________________________
The composition above was added with 5,000 l of the transparent light fulvous liquid obtained in Production example 2, mixing them well, granulating and then a bait for trout was prepared.
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