CN101273120A - Method of producing fungal culture - Google Patents

Abstract

It is intended to provide a method of producing a fungal culture obtained by culturing a fungus in a liquid medium, which contains as a culture material at least one member selected from among cereals, beans, potatoes, amaranthus and quinua, wherein the speed of releasing a nutrient in the culture material into the culture system is controlled to thereby control the productivity of an enzyme (in particular, a starch digesting enzyme, a vegetable fiber digesting enzyme or a protease) of the fungal culture. Namely, a method of producing a fungal culture characterized by comprising culturing a fungus by using a liquid medium, which contains as a culture material at least one member selected from among cereals, beans, potatoes, amaranthus and quinua, while controlling the speed of releasing a nutrient in the culture material into the culture system to thereby control the enzyme productivity of the fungal culture.

Description

Produce the method for fungal culture

Technical field

The present invention relates to use liquid nutrient medium to produce the method for fungal culture, particularly, relate to the method for producing fungal culture, wherein when cultivating filamentous fungus, thereby the control nutrition is regulated enzyme productivity in the fungal culture to the culture systems release rate from cultivate starting material.

Background technology

In order to produce leavened food and beverage such as liquor (shochu), use a kind of filamentous fungus, aspergillus.Cultivate aspergillus by solid culture method, aspergillus is grown on the surface of cereal, promptly be called as the solid koji method.Using the method for solid koji is a kind of conventional production methods.Yet described method is a kind of specific cultural method, and promptly therefore solid culture is not suitable for large-scale production.

Culture can easily be controlled as the liquid koji of the aspergillar cultured products that obtains by liquid culture aspergillus in another aspect, and is a kind of cultural method of effectively producing that is fit to.Yet, be well known that liquid koji can not provide produces enough enzymic activitys (seeing non-patent literature 1-4) that leavened food and beverage such as liquor zythepsary need.Therefore, the example that liquid koji is used for actual production is seldom arranged.

Comprising that by the liquid culture filamentous fungus aspergillus produces in the enzyme, be appreciated that, reduce the productivity that nutrition such as the concentration of glucose in culture systems improve enzyme by control.Usually, the concentration of nutrition such as sugar is suppressed by feeding culture process, and wherein nutrition such as sugar add outside culture systems gradually.Yet, expection exploitation simpler method (seeing non-patent literature 5-6).

We have developed the method for the aspergillus cultured products of producing the enzyme that comprises capacity such as glucoamylase and acid acceptance α-Dian Fenmei, it is undertaken by using liquid nutrient medium to cultivate aspergillus, wherein said starting material are covered by skin or shell, and submitted to patent application (to see Japanese patent application No. 2004-350661,2004-352320,2004-352324,2004-378453, the specification sheets of 2005-290651 and 2005-290648).Yet, how still not understand the mechanism of the enzyme of production high yield in production method, how regulate the method for the enzyme productivity in aspergillus cultured products and how to regulate the method for the enzyme productivity of the filamentous fungus except aspergillus based on this mechanism.

On the other hand, proposed a kind of by using the enzyme liquid of cultivating acquisition in the liquid medium within, the method of the starch that do not boil and do not steam liquefies, described liquid nutrient medium comprises the starting material that do not boil and do not steam, mineral substance etc., have that high saccharification is not boiled and the new bacterial strain (seeing patent documentation 1) of the corticium (Corticium) of the starch ability of not steaming.Also proposed to produce the method for sake wine, wherein above-mentioned enzyme liquid and the starting material reactions (seeing patent documentation 2) of not boiling and not steaming.Yet corticium is a kind of bacidiomycetes, and therefore, it significantly is different from the aspergillus that is widely used in production leavened food and beverage.In addition, patent documentation 1 and patent documentation 2 are described aspergillus niger (Aspergillus awamori (Aspergillus awamori)) and Rhizopus (Rhizopus) deficiency on the saccharification ability.Whether have on enough acid acceptance alpha-amylase activities at enzyme liquid and it be unclear that.

Non-patent literature 1:Hata Y. etc.: J.Ferment.Bioeng. (fermenting organism engineering magazine), 84,532-537 (1997)

Non-patent literature 2:Hata Y. etc.: Gene. (gene), 207,127-134 (1998)

Non-patent literature 3:Ishida H. etc.: J.Ferment.Bioeng. (fermenting organism engineering magazine), 86,301-307 (1998)

Non-patent literature 4:Ishida H. etc.: Curr.Genet. (contemporary gene), 37,373-379 (2000)

Non-patent literature 5:Bhargava S. etc.: Biotechnol Bioeng. (biotechnology biotechnology), 82 (1), 111-7 (2003)

Non-patent literature 6:Pedersen H. etc.: Appl Microbiol Biotechnol. (using microbe biotechnology), 53 (3): 272-7 (2000)

Patent documentation 1:JP H05-068237 B

Patent documentation 2:JP H06-053059 B

Summary of the invention

The problem that the present invention is to be solved

An object of the present invention is to provide to comprise and be selected from by cereal by use, beans, stem tuber, at least a method that is adjusted in the productivity of the enzyme in the fungal culture as the raw-material liquid nutrient medium of cultivation of the group that Amaranthus (amaranthus) and Chenopodium (quinoa) plant are formed, described enzyme particularly refers to amylolytic enzyme, vegetable fibre degraded enzyme and proteolytic ferment, wherein when filamentous fungus was cultivated, the control nutrition was cultivated the release rate of raw-material cereal to culture systems from conduct.The method of dealing with problems

The present inventor has carried out broad research to address the above problem, found that when starting material that will be coated with skin during, may control the release rate of glucose in liquid nutrient medium by regulating raw-material shelling ratio as one of nutrition as the starting material of liquid nutrient medium.The starting material that have different shelling ratios by use are cultivated filamentous fungus, found that the enzyme productivity in the fungal culture is to be controlled by the shelling ratio.

The present inventor also finds aspergillus cultivated in the liquid medium within generation and comprises the required glucoamylase of mass production leavened food and beverage and the liquid koji of acid acceptance α-Dian Fenmei, described liquid nutrient medium comprises the starting material that contain starch of having removed shell or shell (husks or hulls), and described starting material are not by gelationization.This may be because not by the starting material of gelationization, also is difficult to be decomposed even without being covered by shell or shell, and sugar wherein, the release in culture systems such as amino acid is suppressed, and as a result of, has obtained the enzymic activity that needs.

Therefore the present inventor has finished the present invention based on these discoveries.

Promptly, according to a first aspect of the invention, provide by using liquid nutrient medium to produce the method for fungal culture, described liquid nutrient medium comprises and is selected from by cereal, beans, stem tuber, starting material are cultivated at least a conduct of the group that Amaranthus and Chenopodium plant are formed, described method comprises cultivates filamentous fungus, thereby controls nutrition simultaneously from cultivating the productivity that the release rate of starting material to culture systems regulated the enzyme in the fungal culture.

According to a second aspect of the invention, provide the method for production fungal culture according to a first aspect of the invention, wherein

Described cultivation starting material are that the cereal of shell covering is at least used on its surface wholly or in part, and

Nutrition from cereal to the release rate of culture systems by the shelling ratio of regulating cereal Be Controlled.

According to a third aspect of the invention we, the method of production fungal culture according to a first aspect of the invention is provided, and wherein nutrition is removed and is not controlled by the cultivation starting material of gelationization by using its shell or shell from cultivating the release rate of starting material to culture systems.

According to a forth aspect of the invention, provide the method for production fungal culture according to a first aspect of the invention, wherein

Described liquid nutrient medium is heat-treated, and

Control nutrition from cultivating the release rate of starting material to culture systems by the inorganic salt concentration of regulating when the thermal treatment in the liquid nutrient medium.

According to a fifth aspect of the invention, provide the method for production fungal culture according to a first aspect of the invention, wherein said nutrition comprises from the sugar of cultivating starch in the starting material and/or from cultivating proteinic amino acid in the starting material.

According to a sixth aspect of the invention, provide the method for production fungal culture according to a first aspect of the invention, wherein said enzyme comprises and is selected from by amylolytic enzyme, the group that vegetable fibre degraded enzyme and proteolytic ferment are formed at least a.

According to a seventh aspect of the invention, provide the method for production fungal culture according to a first aspect of the invention, wherein filamentous fungus comprises and being selected from by aspergillus, the group that Trichoderma (Trichoderma) and white-rot fungi are formed at least a.

According to an eighth aspect of the invention, provide fungal culture, it is by obtaining according to any one method of first to the 7th aspect of the present invention.

According to a ninth aspect of the invention, provide the method for production zymin, described method comprises the fungal culture of use according to eighth aspect present invention.

According to the tenth aspect of the invention, provide a kind of zymin, it obtains by method according to a ninth aspect of the invention.

According to an eleventh aspect of the invention, provide to comprise to be selected from by cereal by use, beans, stem tuber, the group that Amaranthus and Chenopodium plant are formed is at least a produces the method for enzyme as cultivating raw-material liquid nutrient medium, and described method comprises

Cultivate filamentous fungus, thereby control nutrition simultaneously from cultivating the productivity that starting material are regulated the enzyme the fungal culture to the release rate of culture systems.

According to a twelfth aspect of the invention, provide enzyme, it obtains by method according to an eleventh aspect of the invention.

According to a thirteenth aspect of the invention, provide the method for producing leavened food and beverage, described method comprises the fungal culture of use according to eighth aspect present invention.

According to a fourteenth aspect of the invention, provide the method for producing leavened food and beverage, described method comprises use zymin according to the tenth aspect of the invention.

According to a fifteenth aspect of the invention, provide the method for producing leavened food and beverage, described method comprises the enzyme of use the 12 aspect according to the present invention.

According to a sixteenth aspect of the invention, provide leavened food and beverage, it obtains by any the method to the 15 aspect according to a thirteenth aspect of the invention.

The invention effect

According to the present invention, the control nutrition is from cultivating the release rate of starting material to culture systems, wherein nutrition in the culture systems is maintained as sugar and amino acid whose concentration low-level, and the enzyme productivity in the adjusting fungal culture.

According to a second aspect of the invention, by regulating the release rate of shelling ratio control glucose in the substratum of culture systems.The starting material that have different shelling ratios by use are produced aspergillus liquid culture product, found that the enzyme productivity of having controlled amylolytic enzyme and vegetable fibre degraded enzyme by the shelling ratio.Also have been found that the productivity of having controlled the enzyme of producing by the filamentous fungus except aspergillus by identical method.

According to a third aspect of the invention we, produce fungal culture by using the starting material that do not adhere to shell or shell on it, it comprises with balance mode produces leavened food and beverage such as required glucoamylase and the acid acceptance α-Dian Fenmei of liquor.

Therefore, for example, in producing barley liquor etc., make in the step of producing liquid koji and fermentation step and use identical starting material to become possibility, reduced production cost thus.Shell of barley or chaff may influence the quality of alcoholic beverage unfriendly.In addition, starting material have been saved energy without the heating use.

When the fungal culture and the thread bacterial strain production fungal culture that pass through to make up by using different starting material to obtain also use at that time, can easily produce various leavened foods and beverage.

Expect that method of the present invention controlled the many nutraceutical release rate except that glucose that exists in the cereal similarly, as various sugar and amino acid.Therefore, the metabolite that is caused by the sugared concentration in the culture systems or the amino acid concentration enzyme productivity that suppresses to be influenced has obtained regulating widely.

Method of the present invention is applied to produce M-band probably, the promoter region of its applying starch lytic enzyme gene etc.

According to the present invention, by regulating the speed that nutrition discharges from the starting material that add culture systems in advance, the concentration of nutrition in the substratum such as sugar is suppressed to low-level, even, has also obtained the culture effect identical with feeding culture by than the simpler batch culture of feeding culture.Cultural method of the present invention is a kind of new training method of not reporting in the past.

In addition, compare with solid culture, liquid culture has been subjected to strict control.Therefore, according to the present invention, it has produced the fungal culture with stabilised quality with cheap cost.

The accompanying drawing summary

Fig. 1 has shown by making the barley substrate solution that comprises the barley with different shelling ratios react the temporary transient variation of the glucose concn in the reaction liquid that obtains respectively with the aspergillus culture supernatant.

Fig. 2 shows first glucose release rate of 1 hour of reaction, wherein, makes to comprise the barley substrate solutions with different shelling ratios and react with the aspergillus culture supernatant respectively.

Fig. 3 shows by use to have the barley of different shelling ratios as the enzymic activity in the white aspergillus cultured products of cultivating the starting material acquisition respectively.Fig. 3 (A) shows the enzymic activity of glucoamylase (showing with informal voucher) and α-Dian Fenmei (showing with secret note), and Fig. 3 (B) shows the enzymic activity of acid acceptance α-Dian Fenmei.

Fig. 4 shows by use to have the barley of different shelling ratios as the enzymic activity in the white aspergillus cultured products of cultivating the starting material acquisition respectively.The plain enzymic activity of Fig. 4 (A) display fibers, Fig. 4 (B) shows the enzymic activity of beta-glucosidase enzyme.

Fig. 5 is presented at and uses the barley with different shelling ratios as glucoamylase (GA) activity of cultivating in the raw-material black aspergillus cultured products.

Fig. 6 is presented at and uses the barley with different shelling ratios as α-Dian Fenmei (AA) activity of cultivating in the raw-material black aspergillus cultured products.

Fig. 7 is presented at and uses the barley with different shelling ratios as acid acceptance α-Dian Fenmei (ASAA) activity of cultivating in the raw-material black aspergillus cultured products.

Fig. 8 is presented at and uses the barley with different shelling ratios as glucoamylase (GA) activity of cultivating in the raw-material yellow aspergillus cultured products.

Fig. 9 is presented at and uses the barley with different shelling ratios as α-Dian Fenmei (AA) activity of cultivating in the raw-material yellow aspergillus cultured products.

Figure 10 is presented at and uses the barley with different shelling ratios as cellulase (CEL) activity of cultivating in raw-material viride (Trichoderma viride) cultured products.

Figure 11 is presented at and uses the barley with different shelling ratios as cellulase (CEL) activity of cultivating in the raw-material Trichoderma reesei cultured products.

Figure 12 is presented at and uses the barley with different shelling ratios as zytase (XYL) activity of cultivating in the raw-material Trichoderma reesei cultured products.

Figure 13 is presented at and uses the barley with different shelling ratios as beta-glucosidase enzyme (BGL) activity of cultivating in the raw-material Trichoderma reesei cultured products.

Figure 14 is presented at and uses the barley with different shelling ratios as cellulase (CEL) activity of cultivating in raw-material microorganism Aspergillus aculeatus (Aspergillus aculeatus) cultured products.

Figure 15 is presented at and uses the barley with different shelling ratios as beta-glucosidase enzyme (BGL) activity of cultivating in the raw-material microorganism Aspergillus aculeatus cultured products.

Figure 16 is presented at and uses the barley with different shelling ratios as cellulase (CEL) activity of cultivating in the raw-material white-rot fungi cultured products.

Figure 17 is presented at and uses the barley with different shelling ratios as zytase (XYL) activity of cultivating in the raw-material white-rot fungi cultured products.

Figure 18 is presented at the glucose concn in the reaction liquid, and described reaction liquid has different salt concn when sterilizing by making barley substrate solution obtains with the reaction of aspergillus culture supernatants respectively.

Figure 19 is presented at glucoamylase (GA) activity in the white aspergillus cultured products, the liquid nutrient medium that has different salt concn when wherein using sterilization.

Figure 20 is presented at acid acceptance α-Dian Fenmei (ASAA) activity in the white aspergillus cultured products, the liquid nutrient medium that has different salt concn when wherein using sterilization.

Implement optimum implementation of the present invention

To describe the present invention thereafter.

A first aspect of the present invention relates to the method for producing fungal culture, it is characterized in that comprising and be selected from by cereal, beans, stem tuber, raw-material liquid nutrient medium is cultivated at least a conduct of the group that Amaranthus and Chenopodium plant are formed, when cultivating filamentous fungus, the control nutrition from cultivate starting material to the release rate of culture systems to regulate the productivity of the enzyme the fungal culture.

In the present invention, be controlled at and cultivate the nutrition that exists in the starting material release rate to culture systems, the nutrition that will be included in thus in the culture systems maintains lower level as sugar or amino acid whose concentration, and increases the enzymic activity in the fungal culture.

The example of the mode of the release rate of nutrition in culture systems in the control cultivation starting material comprises the method by the shelling ratio of regulating cereal; by using the attached beans thereon of shell; stem tuber; Amaranthus or Chenopodium plant are as cultivating raw-material method; by using its shell or shell to be removed; but not by the raw-material method of the cultivation of gelationization; when the heat processing liquid substratum, regulate the method for inorganic salt concentration and by the artificial edible film etc. of forming on the cereal surface with the starch component in the protection cereal and the method for protein component.Yet method is not limited to this.Control nutraceutical release rate by suppressing to cultivate raw-material physical decomposition in the cultivation liquid.For example, can have weak stir and the culture apparatus of shearing force suppresses nutraceutical release rate by use.

The example of the enzyme of being produced by filamentous fungus comprises one group of enzyme, and its productivity is subjected in the culture systems influence about the metabolite inhibition of the protein of sugar as glucose and decomposition such as amino acid whose concentration.Its concrete example comprises, but is not necessarily limited to amylolytic enzyme such as glucoamylase, acid acceptance α-Dian Fenmei and α-Dian Fenmei, vegetable fibre degraded enzyme such as cellulase, zytase and beta-glucosidase enzyme and proteolytic ferment such as proteolytic enzyme, peptase and L-Glutamine deaminase.

The raw-material example of used in the present invention cultivation comprises cereal such as barley, paddy rice, wheat, buckwheat, barnyard grass (barnyard millet), grain (foxtail millet), millet, Chinese sorghum and corn, beans such as soybean and red bean, stem tuber such as sweet potato and miscellaneous cereal such as Amaranthus and Chenopodium plant.

Amaranthus is the generic term that belongs to the plant of Amaranthaceae family Amaranthus.In cereal, Amaranthus has high protein content and as the lysine content of one of amino acid, and is identical with content in the soybean.In addition, compare with the paddy rice of shelling, Amaranthus is the high nutrient grain that comprises a large amount of calcium, iron and fiber.Producing the area is south/Sino-U.S. state country, India, the Himalaya and Nepalese concrete zone.

On the other hand, the Chenopodium plant is the annual herb of Agatha family, and it mainly is grown in the plateau as being positioned at the An Disi mountain range in south of Peru and Bolivia western part.Chenopodium plant rich in mineral substances, VITAMIN, protein and food fibre.

Described cultivation starting material can use separately, or its two or more can be used in combination.Raw-material shape is not particularly limited.

To cultivate raw-material any mixes with water with the preparation liquid nutrient medium.

Regulate respectively and cultivate raw-material mixture ratio to tending to the accumulative enzyme by the degree that optionally produces and in fungal culture, accumulate.

For example, for glucoamylase and acid acceptance α-Dian Fenmei, when barley is used as starting material, by preparing liquid nutrient medium in the thick Meccah entry with 1-20% (weight/volume) with balance mode production high yield.When unhulled barley is used as thick wheat, more preferably add 8-10% (weight/volume) preparation liquid nutrient medium.When the barley that will shell as the 95%-of thick wheat is used as starting material, by adding more preferably 1-4% (weight/volume) preparation liquid nutrient medium.

When the amount of used thick wheat surpasses 20% (weight/volume), cultivate the under-supply of the oxygen that viscosity increases and aerobic cultivation filamentous fungus is required of liquid or air.This has reduced the oxygen level in the cultured products, has limited the cultivation process, is not preferred therefore.

Next, when with paddy rice when cultivating starting material, by adding 1-20% (weight/volume), 5-13% (weight/volume) preferably, the or more preferably paddy rice of 8-10% (weight/volume) prepares liquid nutrient medium in water.

When with beans when cultivating starting material, the beans by adding 1-10% (weight/volume) are in water, or preferably, soybean by adding 8-10% (weight/volume) or the red bean of 1-2% (weight/volume) prepare liquid nutrient medium in water.When stem tuber is used as the cultivation starting material, in water, prepare liquid nutrient medium by the stem tuber that adds 1-10% (weight/volume).

When with Amaranthus when cultivating starting material, for example by adding 1.5-15% (weight/volume), 2-10% (weight/volume) preferably, the or more preferably Amaranthus of 2-8% (weight/volume) prepares liquid nutrient medium in water.When with the Chenopodium plant when cultivating starting material, by adding 1.5-7% (weight/volume), 2-6% (weight/volume) preferably, or more preferably the Chenopodium plant of 2-4% (weight/volume) prepares liquid nutrient medium in water.

Can treat blended and cultivate raw-material amount and suitably select because be suitable for most the blended amount according to the enzyme that is intended to use, used raw-material shelling degree, used filamentous fungal strains, variations such as raw-material kind.

Except above-mentioned raw-material any, preferably with organic substance, inorganic substance etc. add liquid nutrient medium as nutrition source.

For example, when with Aspergillus albicans such as Aspergillus kawachii and aspergillus niger such as Aspergillus awamori or aspergillus niger (Aspergillus niger) during as filamentous fungus, preferably be used in combination nitrate and phosphoric acid salt, or more preferably, except that them, unite use vitriol.The example of nitrate comprises SODIUMNITRATE and saltpetre, and saltpetre is particularly preferred.Phosphatic example comprises potassium primary phosphate and ammonium phosphate, and special preferably phosphoric acid potassium dihydrogen.The example of vitriol comprises bitter salt, seven ferric sulfate hydrates and ammonium sulfate, and preferred especially bitter salt and seven ferric sulfate hydrates.Can be used in combination the two or more of those inorganic salt.

White bent and black when bent when using, the concentration of the inorganic salt in the liquid nutrient medium is adjusted to glucoamylase and α-Dian Fenmei respectively and is optionally produced and be accumulated in degree in the aspergillus cultured products.Particularly, the concentration of nitrate is 0.1-2.0%, or 0.2-1.5% preferably, phosphatic concentration is 0.05-1.0%, or 0.1-0.5% preferably, and the concentration of vitriol is 0.01-0.5%, or 0.02-0.1% preferably, condition is that each value is represented with weight/volume.

When with flavus such as aspergillus oryzae (Aspergillus oryzae) or Aspergillus sojae (Aspergillussojae) during as filamentous fungus, liquid nutrient medium preferably makes up and comprises nitrate, phosphoric acid salt and vitriol.The example of nitrate comprises SODIUMNITRATE and saltpetre, and preferred especially SODIUMNITRATE.Phosphatic example comprises potassium primary phosphate and ammonium phosphate, and potassium primary phosphate is particularly preferred.The example of vitriol comprises bitter salt, seven ferric sulfate hydrates and ammonium sulfate, and preferred especially bitter salt and seven ferric sulfate hydrates.Can be used in combination the two or more of those inorganic salt.

When using flavus, the concentration of the inorganic salt in the liquid nutrient medium is adjusted to glucoamylase and α-Dian Fenmei respectively and is optionally produced and be accumulated in degree in the aspergillus cultured products.Particularly, the concentration of nitrate is 0.1-2.0%, or 0.2-1.5% preferably, phosphatic concentration is 0.05-1.0%, or 0.1-0.5% preferably, and the concentration of vitriol is 0.01-0.5%, or 0.02-0.1% preferably, condition is that each value is represented with weight/volume.

Can randomly organic substance except that above-mentioned inorganic salt and inorganic salt be added in the liquid nutrient medium of the present invention as nutrition source.Those additives are not limited especially, as long as they are generally used for cultivating filamentous fungus.The example of organic substance comprises rice bran, wheat bran, corn steep liquor, soya-bean cake and skimmed soy beans.The example of other inorganic salt comprises water-soluble cpds such as ammonium salt, sylvite, calcium salt and magnesium salts.Can use two or more of organic substance and/or inorganic salt simultaneously.Its addition is not limited especially, as long as it has promoted the growth of filamentous fungus.The preferably about 0.1-5% of the addition of organic substance (weight/volume), the preferably about 0.1-1% of the addition of inorganic salt (weight/volume).

The addition of preferred those nutrition sources does not surpass the upper limit, because can suppress the growth of filamentous fungus.Also not preferred addition is less than lower limit, because enzyme can not capacity produce.

Except those nutrition sources, can be randomly with in additive such as microbiotic or the sterilant adding liquid nutrient medium.

As described in a fourth aspect of the present invention, when heat treated liquid nutrient medium being used for when of the present invention, also control nutrition from cultivating the release rate of starting material to culture systems by the inorganic salt concentration of suitably regulating in the liquid nutrient medium when the thermal treatment.

That is, when thermal treatment, suppress nutrition from cultivating the release of starting material to culture systems by the inorganic salt concentration that increases in the liquid nutrient medium.This may be because in the presence of inorganic salt, cultivates starting material by heating and has suppressed to cultivate raw-material physical decomposition.

The example to inorganic salt does not limit especially, can use any inorganic salt of the liquid nutrient medium that is generally used for filamentous fungus as mentioned above.Yet, more preferably nitrate, phosphoric acid salt and vitriol.

The 1-10 that the inorganic salt concentration in the liquid nutrient medium of heat treated can be set in the aforesaid liquid nutrient medium preferred inorganic salt concentration doubly.

Inorganic salt concentration in the liquid nutrient medium in heat treated surpass to be cultivated the filamentous fungus preferred concentration range, can be after heat treated the diluted liquid substratum suitably regulating its inorganic salt concentration, and be then used in cultivation.

Can or preferably carry out above-mentioned thermal treatment under 100 to 121 ℃ the temperature condition at 80 to 130 ℃, carry out 5-120 minute, or preferably 10-30 minute.Particularly, the preferred general condition of the heat sterilization treatment liq substratum that carries out with autoclave etc., that is, 110-121 ℃ temperature was handled 5-20 minute, because carried out the sterilising treatment of substratum simultaneously.

Next, filamentous fungus is inoculated in the liquid nutrient medium.For being used for filamentous fungus of the present invention, can be extensive use of such filamentous fungus, it is produced enzyme and is subjected to suppressing about the metabolite of nutrition as sugar or amino acid whose concentration in culture systems.The example comprises Aspergillus (Aspergillus), Trichoderma (Trichoderma) and white-rot fungi a kind of, that is, and milky white rake bacterium (Irpex lacteus).The specific examples of Aspergillus comprises Aspergillus albicans, is typically Aspergillus kawachii etc., flavus, and aspergillus oryzae typically, Aspergillus sojaes etc., aspergillus niger are Aspergillus awamori typically, aspergillus niger etc., and microorganism Aspergillus aculeatus.The specific examples of Trichoderma comprises viride and Trichoderma reesei, and it is the fungi of cellulase-producing.

Those filamentous funguss can be used for single strain culturing or be used for mixed culture with two or more homologies or allos bacterial strain.Allow spore or mycelium form that use obtains in pre-the cultivation.Yet, preferably use mycelium, because its time that needs for the logarithmic growth stage is still less.The filamentous fungus amount that is inoculated in the liquid nutrient medium is not limited especially, but the amount of spore can be about 1 * 10 ⁴-1 * 10 ⁶In the scope of/ml liquid nutrient medium.With regard to mycelium, the preculture liq of the about 0.1-10% of preferred inoculation.

Preferably 25-45 ℃ of the culture temperature of filamentous fungus, or more preferably 30-40 ℃, but do not limited especially, as long as growth is not by disadvantageous effect.If culture temperature is low, the microbial contamination of the infected property of possibility is because the growth meeting of filamentous fungus is slack-off.Incubation time is preferably in 24-120 hour scope.Culture apparatus can be that those can carry out liquid culture any.Filamentous fungus must aerobic cultivation.Therefore, cultivation should under aerobic conditions be carried out, and wherein oxygen or air is fed in the substratum.In addition, thereby preferred stir culture base makes starting material, and oxygen and filamentous fungus are evenly distributed in the device in culturing process.Agitation condition and ventilation can be arbitrarily, as long as keep aerobic culture environment, and therefore can be according to culture apparatus, the viscosity of substratum etc. is suitably selected.

A second aspect of the present invention, in the method for above-mentioned production fungal culture, use cereal that its surface covers with shell at least wholly or in part as cultivating starting material, and the shelling ratio of regulating cereal with the control nutrition from the release rate of cereal to culture systems.

In the present invention, the surface of cereal need be used shell (husks) covering at least wholly or in part.Can use unhulled raw material or its to have the raw material identical or ratio that more shells (polishing ratio), wherein it shells with such shelling ratio, thereby makes shell be retained at least on the surface of grain.Can also use thick paddy rice, thick wheat etc.For example, when cereal is barley, can use the shelling ratio is 100% unhulled raw material, or condition is that the do not shell shelling ratio of raw material is restricted to 100%, the shelling ratio is no less than the raw material of the value that the shell ratio (normally 7-8%) that deducts barley in the shelling ratio by the raw material that never shells determines, described value is 92-93%.

According to a second aspect of the invention, regulate the shelling ratio of cereal, thereby and in the control culture systems nutraceutical release rate increase enzymic activity in the fungal culture.Therefore, according to the kind of enzyme to be produced, the kind of starting material cereal etc. are selected the shelling ratio of optimization.For example, when when using barley to produce glucoamylase or acid acceptance α-Dian Fenmei as starting material, the ratio that will shell is set at 90-100% or more preferably 98%, and wherein two kinds of enzymes produce with the balance mode high yield.

Term " shelling ratio " refers to the per-cent at the remaining cereal in cereal shelling back.For example, term " 90% shelling ratio " refers to that surface layer part at cereal has shell of 10% etc. to be sloughed.In the present invention, term " thick wheat " comprises that those still are retained in the lip-deep hulled barley of grain from unhulled barley to shell, promptly has 90% or the raw material of more shelling ratios.Term " shell (husk) " refers to cover the exterior portion on cereal-granules surface.

Can before cultivation, at first the starch that exists in the cereal be carried out gelationization.Can basis, but be not limited to ordinary method especially any carry out gelation starch, described ordinary method comprises vaporization method, baking method etc.In step as hereinafter described to the liquid nutrient medium sterilization, by sterilization under high temperature and high pressure starch is heated to gelatinization temperature or higher, carry out the gelationization of starch simultaneously by such processing.

By mixing water, the liquid nutrient medium that above-mentioned cultivation starting material and the preparation of other medium component are used in a second aspect of the present invention.If desired, liquid nutrient medium can be carried out sterilising treatment and the step of such processing do not limited especially.For example, can carry out the high temperature and high pressure sterilising method 121 ℃ temperature and reach 15 minutes.

A third aspect of the present invention, in the method for above-mentioned production fungal culture, use its shell or shell to be removed and not by the raw material of gelationization as cultivating starting material, the control nutrition is from cultivating the release rate of starting material to culture systems.

In a third aspect of the present invention, need to remove raw-material shell of above-mentioned cultivation or shell, and do not have gelationization to cultivate starting material.

For example, when cultivating starting material and be barley, the shelling ratio of described raw material is no less than the value that the shell ratio (normally 7-8%) that deducts barley in the shelling ratio (100%) by the raw material that never shells is determined, promptly about 92-93%.Can also use pearled barley (Pearled barley) (have 65% shelling ratio).

Above-mentioned cultivation starting material are not handled, as heating, by heating, starch wherein will be by gelationization.Yet, as required, above-mentioned cultivation starting material can be carried out such processing such as threshing, shelling, peeling, washing chops up (chopping), extruding and freezing.

In a third aspect of the present invention, the mixture ratio in the above-mentioned cultivation starting material liquid medium within is set to such degree, promptly glucoamylase and acid acceptance α-Dian Fenmei are optionally produced and are accumulated in fungal culture.Particularly, can be with about liquid nutrient medium 1-10% (weight/volume), preferably the amount of 2-6% (weight/volume) adds and cultivates starting material.Yet, because, cultivating starting material etc. according to the kind of used filamentous fungal strains, the mixture ratio of optimizing changes, can suitably select mixture ratio.

Use therein in the situation of aerobic filamentous fungus,, cultivate the oxygen that viscosity increases and aerobic cultivation filamentous fungus is required of liquid or the supply of air and become not enough if the raw-material amount of used cultivation surpasses the upper limit.This has reduced the oxygen level in the cultured products, has limited the cultivation process, is not preferred therefore.On the other hand, when used raw-material amount is less than lower limit, glucoamylase and acid acceptance α-Dian Fenmei can not produce with high yield.

The used liquid nutrient medium of a third aspect of the present invention passes through mixing water, and above-mentioned cultivation starting material prepare with other medium component.In this case, if desired, will mix with water, mixture is sterilized in advance, and then can will do not added wherein in addition by the amyloid starting material of gelationization except the medium component cultivating starting material.Alternatively, can take the raw-material part of cultivation and other medium component and water blended method are sterilized mixture in advance, and then will do not added wherein by the remaining cultivation starting material of gelationization.

Sterilising method is not particularly limited.It for example can be the autoclave sterilization method of under 121 ℃ temperature, carrying out 15 minutes.

Cultivate filamentous fungus to obtain fungal culture by above-mentioned cultural method, wherein the enzyme that is intended to effectively produces and accumulation.Fungal culture of the present invention comprises by cultured products being carried out the cultivation liquid that centrifugation etc. obtains except cultured products itself, its enriched material, and its purified product, or its desciccate etc.

As mentioned above, according to above-mentioned cultural method, can one group of enzyme of High-efficient Production, in culture systems, its productivity is subjected to the influence about the metabolite inhibition of the protein of sugar as glucose or decomposition such as amino acid whose concentration.

Therefore, produce the method for enzyme according to an eleventh aspect of the invention, identical with the method for above-mentioned production fungal culture.

To not only be used to by the fungal culture that the present invention obtains produce leavened food and beverage, also be used for producing sugar, amino acid, its derivative, zymin and medicine digestive pharmaceutical.Can use the aspergillus cultured products to replace solid koji, for example, in the situation of producing sake wine, in the stage of preparation yeast mash or sake wine mash; In the situation of producing liquor, in the stage of wine with dregsization (mashing) liquor mash; In the situation of producing dip, at accumulative phase; In the situation of producing miso, in the stage of wine with dregsization; In the situation that production makes vinegar, in the stage of wine with dregsization; In the situation of producing sweet sake wine, in the stage of wine with dregsization; With in the situation of producing Japanese sweet wine (amazake), in the stage of wine with dregsization.The proferment material (other starting material) that is used to produce those leavened foods and beverage can be carried out gelationization or not carry out gelationization.

Can be with the part of the fungal culture that obtains initiator as the production of subsequently fungal culture.By continuous production fungal culture by this way, obtain stable product and improved production efficiency simultaneously.

As method from fungal culture production zymin of the present invention, with cultured products itself, its filtrate, the supernatant liquors by centrifugation etc. are as liquid enzyme formulation, or it can be dried or fixing to support by conventional preparation method.At this moment, can be wherein with addings such as suitable vehicle.

When food and drink that use above-mentioned fungal culture production fermentation such as alcoholic beverage, can carry out all steps in liquid stage.For example, when producing liquor, with corn, wheat, paddy rice, potato, sugarcane etc. as starting material and then with its in about 80 ℃ of heating to liquefy by dissolving with the heat-proof zymin, above-mentioned aspergillus cultured products and yeast are added wherein so that mash carries out zymamsis, and then it is distilled down in standard atmosphere pressure or decompression etc.

Embodiment

Hereinafter, the present invention will describe in detail by the mode of embodiment.Yet the present invention is not limited to these embodiment.

＜EXPERIMENTAL EXAMPLE 1 〉

Measure the release rate of glucose from barley with different shelling ratios

Make the barley (Stirling (Stirling) originates in Australia) of difference shelling ratio and enzyme reaction, and measure the release rate of glucose from barley from the aspergillus cultured products with 65%-98%.

Particularly, 2 g that weigh respectively are the 65%-hulled barley, the 83%-hulled barley, the 92%-hulled barley, the 95%-hulled barley, the crushing product of 98%-hulled barley and 98%-hulled barley, and every kind of material of weighing and 50ml water placed the 200-ml erlenmeyer flask.It was sterilized 15 minutes at 121 ℃ with autoclave, with preparation " barley substrate solution ".

Subsequently, will cultivate raw-material aspergillus cultured products by the conduct of using barley (Stirling (Stirling) originates in Australia) thereby producing and separate acquisition " aspergillus culture supernatants " by carry out solids liq with filter paper filtering.

Be described below the method for using production aspergillus cultured products in this experiment.

1. pre-incubated method

The barley and the 100ml water of 8g 65%-shelling are placed the baffled erlenmeyer flask of 500-ml, and it is sterilized 15 minutes to obtain pre-incubated substratum with autoclave at 121 ℃.After the cooling, with Aspergillus albicans (Aspergillus kawachii NBRC4308) with 1 * 10 ⁶/ ml is seeded in the pre-incubated substratum, and 37 ℃ and 100rpm wave and culture 24 hours to obtain pre-incubated liquid.

2. main cultured method

With the barley of 98%-shelling add replenished 0.2% (weight/volume) saltpetre and 0.3% (weight/volume) thus making the amount of the barley of 98%-shelling in the water of potassium primary phosphate is that 2.0% (weight/volume) is with the preparation liquid nutrient medium.With 3, the liquid nutrient medium of 000ml preparation places 5, the small-sized fermentation jar of 000-ml is (by (the Marubishi Co. of ball water chestnut Co., Ltd., Ltd) produce) in and with autoclave (121 ℃ 15 minutes) sterilization, use the preculture liq inoculation of the Aspergillus albicans (Aspergillus kawachiiNBRC4308) of 30ml subsequently, in its liquid medium within, cultivate in advance in advance by aforesaid method.Subsequently, in 37 ℃ temperature and the stir speed (S.S.) of 300rpm, the aeration volume of 0.5vvm was cultivated 42 hours, used filter paper (No. 2 Toyo filter paper) filtration product to obtain " aspergillus culture supernatants ".

3. measuring method

The aspergillus culture supernatants that barley substrate solution that 50ml is prepared thus and 50ml prepare thus kept 5 minutes at 37 ℃ respectively, then mixed with initial action.With glucose C-II Test Wako (by watt gram pure chemistry product (the Wako Pure Chemical Industries Co. of Industrial Co., Ltd, Ltd.) produce), measurement behind initial action 1 hour, 2 hours, the glucose concn after 3 hours and 4 hours in the reaction liquid of sampling.

4. result

The temporary transient variation of glucose concn that will be in reaction liquid is shown among Fig. 1.The amount of the glucose that confirm to discharge is according to the shelling degree of the barley that is used for the barley substrate solution and difference.Generally speaking, barley has about 10% shell proportion, and the 65%-hulled barley and the 83%-hulled barley that are used in respectively in this experiment do not have shell in its surface.Relative therewith, the 92%-hulled barley, 95%-hulled barley and 98%-hulled barley still have shell in its surface.

As shown in FIG. 1, used respectively therein during the experiment of the 65%-that do not have shell (husks) in its surface and 83%-hulled barley and 98%-hulled barley crushing product draws, shown high glucose concn.Relative therewith, used 92%-therein respectively, the experiment of 95%-and 98%-hulled barley has confirmed that the shelling ratio is low more in drawing, glucose concn is also low more.Reason has disclosed the existence control of the amount of the glucose that discharges by shell for this reason.

Fig. 2 is presented at the calculated value of glucose release rate in first reaction of 1 hour of this experiment.Confirmed that from Fig. 2 the shelling ratio is low more, glucose release rate Be Controlled gets also low more.

In yet another aspect, used therein during the experiment of the crushing product of 98%-hulled barley draws, the glucose release rate has been increased to the level identical with the 65%-hulled barley.Therefore, the shell that the starch component that covers barley physically is described is the principal element of regulating the glucose release rate.

＜embodiment 1 〉

The barley that use has different shelling ratios produces white bent cultured products

By method as described below, use hulled barley (Stirling (Stirling) originates in Australia) in various degree, produce the Aspergillus albicans cultured products, and measure enzymic activity wherein.

1. pre-incubated method

The barley and the 100ml water of 8g 65%-shelling are placed the baffled erlenmeyer flask of 500-ml, and it is sterilized 15 minutes to obtain pre-incubated substratum with autoclave at 121 ℃.After the cooling, with Aspergillus albicans (Aspergillus kawachii NBRC4308) with 1 * 10 ⁶/ ml is seeded in the pre-incubated substratum, and 37 ℃ and 100rpm wave and culture 24 hours to obtain pre-incubated liquid.

2. main cultured method

With 2g 65%-hulled barley, the 83%-hulled barley, the 92%-hulled barley, any of 95%-hulled barley and 98%-hulled barley, 0.2g saltpetre, 0.3g potassium primary phosphate and 100ml water place the erlenmeyer flask of 500-ml band baffle plate, and it is sterilized 15 minutes with the preparation main medium with autoclave at 121 ℃.After the cooling, main medium is inoculated with the preculture liq of 1ml, and 37 ℃ and 100rpm wave and culture 48 hours.

3. measuring method

After cultivating end, measure glucoamylase (GA), the activity of α-Dian Fenmei (AA) and acid acceptance α-Dian Fenmei (ASAA) as amylolytic enzyme.

By using saccharification underworld differential to quantize test kit (saccharification powerfractionalquantification kit, produce by Kikkoman (Kikkoman) Co., Ltd.) measurement glucoamylase (GA) activity, and by using α-Dian Fenmei to measure test kit (producing by Kikkoman (Kikkoman) Co., Ltd.)) measurement α-Dian Fenmei (AA) activity.In order to measure acid acceptance α-Dian Fenmei (ASAA) activity, will be at Sudo S. etc.: J.Ferment.Bioeng. (fermenting organism engineering magazine), 76,105-110 (1993), Sudo S. etc.: J.Ferment.Bioeng. (fermenting organism engineering magazine), 77,483-489 (1994), with Shigetoshi Sudo etc.: Journal of theBrewing Society of Japan (association's magazine is brewageed by Japan), 89, the method described in the 768-774 (1994) is improved slightly.That is, with the alpha-amylase activity deactivation of acid labile, then measure test kit (producing) and measure the acid acceptance alpha-amylase activity by Kikkoman (Kikkoman) Co., Ltd. with α-Dian Fenmei by using the acid treatment cultured products.More particularly, the 100mM acetate buffer liquid (pH 3) of 9ml is added in the cultivation liquid of 1ml, carried out acid treatment 1 hour, and measure test kit (producing) with α-Dian Fenmei and measure by Kikkoman (Kikkoman) Co., Ltd. at 37 ℃.

Then measure activity as the cellulase (CEL) and the beta-glucosidase enzyme (BGL) of cellulolytic enzyme.The method of the amount by the reducing sugar that quantizes with dinitrosalicylic acid (DNS) method to be produced by the hydrolysis as the carboxymethyl cellulose (CMC) of substrate is measured cellulase (CEL) activity.More specifically, the cultivation liquid of 1ml is added the 1%CMC substrate solution of 1ml (by the low viscosity that the dissolving SIGMA-Ao Er Freundlich (Sigma-Aldrich) is produced in 100mM acetate buffer liquid (pH 5) ^TMAnd enzyme reaction was accurately carried out 10 minutes at 40 ℃ the solution that obtains).Subsequently, will comprise 0.75% dinitrosalicylic acid, 1.2% sodium hydroxide, the 4ml DNS reagent of four hydration sodium-potassium tartrates of 22.5% and a Lactose hydrate of 0.3% adds in the mixture, and thorough mixing is with termination reaction.For after termination reaction, quantize the amount of the reducing sugar in the liquid, the liquid after the termination reaction was accurately heated 15 minutes in the water-bath of boiling.Subsequently, to room temperature, the absorbancy that is determined at 540nm is to quantize the amount of the reducing sugar corresponding with the amount of glucose with liquid cooling.The active expression of the cellulase of a unit (CEL) per minute produces the needed enzyme amount of reducing sugar corresponding to the glucose of 1 μ mol.

Measure the activity of beta-glucosidase enzyme by method as described below.Right-nitrophenyl-β-D-the glucopyranoside (PNPG) that uses 1mM accurately carried out enzyme reaction 10 minutes in 50mM acetate buffer liquid (pH5) at 37 ℃ as substrate.Behind the reaction terminating, the amount of the right-nitrophenols that quantizes to produce by the absorbancy at 410nm is to calculate enzymic activity.Come termination reaction by the 200mM sodium carbonate solution that adds the double amount of reaction liquid therein.With an active unit representation is the activity that discharges the glucose of 1 μ mol by its per minute.

Fig. 3 and 4 shows measuring result.

4. result

As shown in Fig. 3, when the shelling ratio reduced, the productivity of amylolytic enzyme increased.Even for the 98%-hulled barley with low shelling ratio, when barley was crushed, enzyme productivity wherein significantly reduced.In addition, as shown in Fig. 4, observe when the shelling ratio reduces the trend that the productivity of cellulolytic enzyme increases.By this way, the enzyme productivity of observing in the Aspergillus albicans cultured products has the trend that becomes retrocorrelation with the glucose release rate, as as shown in the EXPERIMENTAL EXAMPLE 1, and be disclosed in enzyme productivity in the Aspergillus albicans cultured products by the shelling ratio that changes barley Be Controlled.

＜embodiment 2 〉

The barley that use has different shelling ratios produces the aspergillus niger cultured products

By using hulled barley (Stirling (Stirling) originates in Australia) in various degree, utilize method as described below to produce the aspergillus niger cultured products, and measure enzymic activity wherein.

1. pre-incubated method

The barley and the 100ml water of 8g 65%-shelling are placed the baffled erlenmeyer flask of 500-ml, and it is sterilized 15 minutes to obtain pre-incubated substratum with autoclave at 121 ℃.After the cooling, with aspergillus niger (Aspergillus awamori NBRC4388) with 1 * 10 ⁶/ ml is seeded in the pre-incubated substratum, and 37 ℃ and 100rpm wave and culture 24 hours to obtain pre-incubated liquid.

2. main cultured method

With 2g 65%-hulled barley, the 83%-hulled barley, the 92%-hulled barley, any of 95%-hulled barley and 98%-hulled barley, 0.2g saltpetre, 0.3g potassium primary phosphate and 100ml water place the erlenmeyer flask of 500-ml band baffle plate, and it is sterilized 15 minutes with the preparation main medium with autoclave at 121 ℃.After the cooling, main medium is inoculated with the preculture liq of 1ml, and 37 ℃ and 100rpm wave and culture 48 hours.

3. measuring method

After cultivating end, measure glucoamylase (GA), the activity of α-Dian Fenmei (AA) and acid acceptance α-Dian Fenmei (ASAA) as amylolytic enzyme.

Quantize test kit (producing) measurement glucoamylase (GA) activity by use saccharification underworld differential, and pass through to use α-Dian Fenmei to measure test kit (producing) by Kikkoman (Kikkoman) Co., Ltd. by Kikkoman (Kikkoman) Co., Ltd.) measurement α-Dian Fenmei (AA) activity.In order to measure acid acceptance α-Dian Fenmei (ASAA) activity, will be at Sudo S. etc.: J.Ferment.Bioeng. (fermenting organism engineering magazine), 76,105-110 (1993), Sudo S. etc.: J.Ferment.Bioeng. (fermenting organism engineering magazine), 77,483-489 (1994), with Shigetoshi Sudo etc.: Journal oftheBrewing Society of Japan (association's magazine is brewageed by Japan), 89, the method described in the 768-774 (1994) is improved slightly.That is, with the alpha-amylase activity deactivation of acid labile, then measure test kit (producing) and measure the acid acceptance alpha-amylase activity by Kikkoman (Kikkoman) Co., Ltd. with α-Dian Fenmei by using the acid treatment cultured products.More particularly, the 100mM acetate buffer liquid (pH 3) of 9ml is added in the cultivation liquid of 1ml, carried out acid treatment 1 hour, and measure test kit (producing) with α-Dian Fenmei and measure by Kikkoman (Kikkoman) Co., Ltd. at 37 ℃.

Fig. 5-7 shows measuring result.

4. result

As shown in Fig. 5-7, when the shelling ratio reduced, the productivity of amylolytic enzyme increased.Even for the 98%-hulled barley with low shelling ratio, when barley was crushed, enzyme productivity wherein significantly reduced.By this way, the enzyme productivity of observing in the aspergillus niger cultured products has the trend that becomes retrocorrelation with the glucose release rate, as as shown in the EXPERIMENTAL EXAMPLE 1, and be disclosed in enzyme productivity in the aspergillus niger cultured products by the shelling ratio that changes barley Be Controlled.

＜embodiment 3 〉

The barley that use has different shelling ratios produces the flavus cultured products

By method as described below, use hulled barley (Stirling (Stirling) originates in Australia) in various degree, produce the flavus cultured products, and measure enzymic activity wherein.

1. cultural method

The 100ml substratum is placed the erlenmeyer flask of the band baffle plate of 500-ml, and with autoclave 121 ℃ the sterilization 15 minutes with the preparation substratum, described substratum comprises 2g 65%-hulled barley, the 83%-hulled barley, the 92%-hulled barley, any of 95%-hulled barley and 98%-hulled barley, the SODIUMNITRATE of 1.2% (weight/volume), the Repone K of 0.8% (weight/volume), the potassium primary phosphate of 0.4% (weight/volume), the bitter salt of 0.2% (weight/volume), seven ferric sulfate hydrates and the water of 0.08% (weight/volume).After the cooling, with flavus (aspergillus oryzae RIB40) with 1 * 10 ⁶/ ml is inoculated in the pre-incubated substratum and 30 ℃ and 100rpm wave and culture 72 hours.

2. measuring method

After cultivating end, measure as the glucoamylase (GA) of amylolytic enzyme and the activity of α-Dian Fenmei (AA).

Quantize test kit (producing) measurement glucoamylase (GA) activity by use saccharification underworld differential, and pass through to use α-Dian Fenmei to measure test kit (producing) by Kikkoman (Kikkoman) Co., Ltd. by Kikkoman (Kikkoman) Co., Ltd.) measurement α-Dian Fenmei (AA) activity.

Fig. 8 and 9 shows measuring result.

3. result

As shown in Fig. 8 and 9, when the shelling ratio reduced, the productivity of amylolytic enzyme increased.Particularly, when using the 98%-hulled barley, the active of glucoamylase significantly increases.Even about having the 98%-hulled barley of low shelling ratio, when using the barley of its crushing, enzyme productivity wherein significantly reduces.As mentioned above, the enzyme productivity that has disclosed in the flavus cultured products is influenced greatly by the glucose release rate, as shown in EXPERIMENTAL EXAMPLE 1, and the enzyme productivity of the shelling ratio control flavus cultured products by changing barley.

＜embodiment 4 〉

The barley that has different shelling ratios by use produces filamentous fungus (viride) cultured products

By method described below, use hulled barley (Stirling (Stirling) originates in Australia) in various degree, production can produce the cultured products of the filamentous fungus (viride) of cellulolytic enzyme, and measures enzymic activity wherein.

1. pre-cultural method

The substratum of 100ml is placed the erlenmeyer flask of 500-ml band baffle plate, and with its with autoclave 121 ℃ the sterilization 15 minutes to prepare pre-culture medium, comprise 2% glucose in the described substratum, 0.5% yeast extract, 0.1% saltpetre, 0.1% potassium primary phosphate, 0.07% ammonium sulfate, 0.03% bitter salt, 0.02% calcium chloride (each % represents with weight/volume) and water.After the cooling, with viride (viride NBRC31137) with 1 * 10 ⁶/ ml is inoculated in the pre-incubated substratum, and 30 ℃ and 100rpm wave and culture 24 hours to obtain pre-incubated liquid.

2. main cultured method

The substratum of 100ml is placed the erlenmeyer flask of 500-ml band baffle plate, and with autoclave 121 ℃ of sterilizations 15 minutes to obtain main medium, described substratum comprises 2% hulled barley, 0.08% tryptone, 0.25% ammonium sulfate, 0.1% ammonium phosphate, 0.03% calcium chloride, 0.03% bitter salt, 0.12% saltpetre (each % represents with weight/volume) and water.

With the 65%-hulled barley, the 83%-hulled barley, the 92%-hulled barley, the 95%-hulled barley, 98%-hulled barley or 98%-hulled barley crushing product are as above-mentioned hulled barley.

After the cooling, with main medium with the inoculation of the preculture liq of 10ml, and 30 ℃ and 100rpm wave and culture 90 hours.

3. measuring method

After cultivating end, measure activity as the cellulase (CEL) of cellulolytic enzyme.The method of the amount by the reducing sugar that quantizes with dinitrosalicylic acid (DNS) method to be produced by the hydrolysis as the carboxymethyl cellulose (CMC) of substrate is measured cellulase (CEL) activity.More specifically, the cultivation liquid of 1ml is added the 1%CMC substrate solution of 1ml (by the low viscosity that the dissolving SIGMA-Ao Er Freundlich (Sigma-Aldrich) is produced in 100mM acetate buffer liquid (pH 5) ^TMAnd enzyme reaction was accurately carried out 10 minutes at 40 ℃ the solution that obtains).Subsequently, will comprise 0.75% dinitrosalicylic acid, 1.2% sodium hydroxide, the 4ml DNS reagent of four hydration sodium-potassium tartrates of 22.5% and a Lactose hydrate of 0.3% adds in the mixture, and thorough mixing is with termination reaction.For after termination reaction, quantize the amount of the reducing sugar in the liquid, the liquid after the termination reaction was accurately heated 15 minutes in the water-bath of boiling.Subsequently, to room temperature, the absorbancy that is determined at 540nm is to quantize the amount of the reducing sugar corresponding with the amount of glucose with liquid cooling.The active expression of the cellulase of a unit (CEL) per minute produces the needed enzyme amount of reducing sugar corresponding to the glucose of 1 μ mol.

4. result

Figure 10 shows measuring result.It has confirmed that in viride cellulase (cellulose) productivity is also different according to the shelling ratio, and described viride is the filamentous fungus except aspergillus.The 98%-hulled barley provides the highest enzyme productivity, and its crushing product significantly reduces on enzymic activity.Therefore, illustrate that the nutrition release restraining effect of barley shell helps the high yield of cellulase.

＜embodiment 5 〉

The barley that has different shelling ratios by use produces filamentous fungus (Trichoderma reesei) cultured products

By method described below, use hulled barley (Stirling (Stirling) in various degree, originate in Australia), production can produce the cultured products of the filamentous fungus (Trichoderma reesei) of cellulolytic enzyme, and measures enzymic activity wherein.

1. cultural method

(1) pre-cultural method

The substratum of 100ml is placed the erlenmeyer flask of 500-ml band baffle plate, and with its with autoclave 121 ℃ the sterilization 15 minutes to prepare pre-culture medium, comprise 2% glucose in the described substratum, 0.5% yeast extract, 0.1% saltpetre, 0.1% potassium primary phosphate, 0.07% ammonium sulfate, 0.03% bitter salt, 0.02% calcium chloride (each % represents with weight/volume) and water.After the cooling, with Trichoderma reesei (Trichoderma reesei NBRC31326) with 1 * 10 ⁶/ ml is inoculated in the pre-incubated substratum, and 30 ℃ and 100rpm wave and culture 72 hours to obtain pre-incubated liquid.

(2). main cultured method

The substratum of 100ml is placed the erlenmeyer flask of 500-ml band baffle plate, and with autoclave 121 ℃ of sterilizations 15 minutes to obtain main medium, described substratum comprises 2% hulled barley, 0.08% tryptone, 0.25% ammonium sulfate, 0.1% ammonium phosphate, 0.03% calcium chloride, 0.03% bitter salt, 0.12% saltpetre (each % represents with weight/volume) and water.

With the 65%-hulled barley, the 83%-hulled barley, the 95%-hulled barley, 98%-hulled barley or 98%-hulled barley crushing product are as above-mentioned hulled barley.

After the cooling, with main medium with the inoculation of the preculture liq of 10ml, and 30 ℃ and 100rpm wave and culture 96 hours.

2. measure the method for enzymic activity

After cultivating end, culture fluid is centrifugal with the collection supernatant liquor, and the activity of the vegetable fibre degraded enzyme in the measurement supernatant liquor.

(1) method of measurement cellulase activity

The method of the amount by the reducing sugar that quantizes with dinitrosalicylic acid (DNS) method to be produced by the hydrolysis as the carboxymethyl cellulose (CMC) of substrate is measured cellulase (CEL) activity.More specifically, the cultivation liquid of 1ml is added the 1%CMC substrate solution of 1ml (by the low viscosity that the dissolving SIGMA-Ao Er Freundlich (Sigma-Aldrich) is produced in 100mM acetate buffer liquid (pH 5) ^TMAnd enzyme reaction was accurately carried out 10 minutes at 40 ℃ the solution that obtains).Subsequently, will comprise 0.75% dinitrosalicylic acid, 1.2% sodium hydroxide, the 4ml DNS reagent of four hydration sodium-potassium tartrates of 22.5% and a Lactose hydrate of 0.3% adds in the mixture, and thorough mixing is with termination reaction.For after termination reaction, quantize the amount of the reducing sugar in the liquid, the liquid after the termination reaction was accurately heated 15 minutes in the water-bath of boiling.Subsequently, to room temperature, the absorbancy that is determined at 540nm is to quantize the amount of the reducing sugar corresponding with the amount of glucose with liquid cooling.The active expression of the cellulase of a unit (CEL) per minute produces the needed enzyme amount of reducing sugar corresponding to the glucose of 1 μ mol.

(2) method of measurement xylanase activity

Next, by making reducing sugar and DNS reaction and quantification measure zytase (XYL) activity in the increase of the absorbancy of 540nm, described reducing sugar is by producing as the substrate enzymically hydrolyse from the xylan of oat-spelt (oat spelts).More specifically, the 1% xylan substrate solution (xylan that the culture fluid of 0.1ml is added 1.9ml, from the oat-spelt that is dissolved in 200mM acetate buffer body (pH 4.5) by SIGMA-Ao Er Freundlich (Sigma-Aldrich) product), and enzymatic reaction was accurately carried out 10 minutes at 40 ℃.Subsequently, will comprise 0.75% dinitrosalicylic acid, 1.2% sodium hydroxide, the 4ml DNS reagent of four hydration sodium-potassium tartrates of 22.5% and a Lactose hydrate of 0.3% adds in the mixture, and thorough mixing is with termination reaction.For after termination reaction, quantize the amount of the reducing sugar in the liquid, after termination reaction, liquid was accurately heated 15 minutes in the water-bath of boiling.Subsequently, to room temperature, the absorbancy that is determined at 540nm is with the amount of quantification corresponding to the reducing sugar of the amount of wood sugar with liquid cooling.The xylanase activity of a unit is illustrated under 40 ℃ and the 10 minutes reaction conditionss, and per minute production is corresponding to the needed enzyme amount of the reducing sugar of the wood sugar of 1 μ mol.

(3) method of measurement beta-glucosidase activity

Measure beta-glucosidase enzyme (BGL) activity by method as described below.In 50mM acetate buffer liquid (pH 5), use 1mM right-nitrophenyl-β-D-glucopyranoside (PNPG) is as substrate, enzymatic reaction was accurately carried out 10 minutes at 37 ℃.Behind the reaction terminating, the amount that quantizes the right-nitrophenols of generation by the absorbancy at 410nm is calculated enzymic activity.The sodium carbonate solution of 200mM by adding 2 times of amounts of reaction liquid comes termination reaction.The activity of a unit is represented to make per minute that the d/d activity of glucose of 1 μ mol be arranged by it.

3. result

Figure 11-13 shows measuring result.Also with the shelling ratio vary, described Trichoderma reesei is the filamentous fungus except aspergillus to the productivity of its confirmation vegetable fibre degraded enzyme in Trichodermareesei.When using 95%-or 98%-hulled barley, obtained high enzyme productivity.Yet when using 98%-hulled barley crushing product, enzymic activity significantly reduces.Therefore, its explanation barley shell discharges the high yield that restraining effect helps vegetable fibre degraded enzyme to the nutrition of nutrition release.

＜embodiment 6 〉

The barley that use has different shelling ratios produces filamentous fungus (microorganism Aspergillus aculeatus) cultured products

By method described below, use hulled barley (Stirling (Stirling) originates in Australia) in various degree, production can produce filamentous fungus (microorganism Aspergillus aculeatus) cultured products of cellulolytic enzyme, and measures enzymatic activity wherein.

1. cultural method

(1) pre-cultural method

The substratum of 100ml is placed the erlenmeyer flask of 500-ml band baffle plate, and with its with autoclave 121 ℃ the sterilization 15 minutes to prepare pre-culture medium, comprise 2% glucose in the described substratum, 0.5% yeast extract, 0.1% saltpetre, 0.1% potassium primary phosphate, 0.07% ammonium sulfate, 0.03% bitter salt, 0.02% calcium chloride (each % represents with weight/volume) and water.After the cooling, with microorganism Aspergillus aculeatus (microorganism Aspergillus aculeatus NBRC3530) with 1 * 10 ⁶/ ml is inoculated in the pre-incubated substratum, and 30 ℃ and 100rpm wave and culture 72 hours to obtain pre-incubated liquid.

(2) main cultured method

The substratum of 100ml is placed the erlenmeyer flask of 500-ml band baffle plate, and with autoclave 121 ℃ of sterilizations 15 minutes to obtain main medium, described substratum comprises 2% hulled barley, 0.08% tryptone, 0.25% ammonium sulfate, 0.1% ammonium phosphate, 0.03% calcium chloride, 0.03% bitter salt, 0.12% saltpetre (each % represents with weight/volume) and water.

With the 65%-hulled barley, the 83%-hulled barley, the 95%-hulled barley, 98%-hulled barley or 98%-hulled barley crushing product are as above-mentioned hulled barley.

After the cooling, with main medium with the inoculation of the preculture liq of 10ml, and 30 ℃ and 100rpm wave and culture 96 hours.

2. measure the method for enzymic activity

After cultivating end, culture fluid is centrifugal with the collection supernatant liquor, and the activity of the vegetable fibre degraded enzyme in the measurement supernatant liquor.

(1) method of measurement cellulase activity

The method of the amount by the reducing sugar that quantizes with dinitrosalicylic acid (DNS) method to be produced by the hydrolysis as the carboxymethyl cellulose (CMC) of substrate is measured cellulase (CEL) activity.More specifically, the cultivation liquid of 1ml is added the 1%CMC substrate solution of 1ml (by the low viscosity that the dissolving SIGMA-Ao Er Freundlich (Sigma-Aldrich) is produced in 100mM acetate buffer liquid (pH 5) ^TMAnd enzyme reaction was accurately carried out 10 minutes at 40 ℃ the solution that obtains).Subsequently, will comprise 0.75% dinitrosalicylic acid, 1.2% sodium hydroxide, the 4ml DNS reagent of four hydration sodium-potassium tartrates of 22.5% and a Lactose hydrate of 0.3% adds in the mixture, and thorough mixing is with termination reaction.For after termination reaction, quantize the amount of the reducing sugar in the liquid, the liquid after the termination reaction was accurately heated 15 minutes in the water-bath of boiling.Subsequently, to room temperature, the absorbancy that is determined at 540nm is to quantize the amount of the reducing sugar corresponding with the amount of glucose with liquid cooling.The active expression of the cellulase of a unit (CEL) per minute produces the needed enzyme amount of reducing sugar corresponding to the glucose of 1 μ mol.

(2) method of measurement beta-glucosidase activity

Measure beta-glucosidase enzyme (BGL) activity by method as described below.In 50mM acetate buffer liquid (pH 5), use 1mM right-nitrophenyl-β-D-glucopyranoside (PNPG) is as substrate, enzymatic reaction was accurately carried out 10 minutes at 37 ℃.Behind the reaction terminating, the amount that quantizes the right-nitrophenols of generation by the absorbancy at 410nm is calculated enzymic activity.The sodium carbonate solution of 200mM by adding 2 times of amounts of reaction liquid comes termination reaction.The activity of a unit represents have the glucose of 1 μ mol to be released by described active per minute.

3. result

Figure 14 and 15 shows measuring result.Also with the shelling ratio vary, described microorganism Aspergillus aculeatus is the filamentous fungus except aspergillus to the productivity of its confirmation vegetable fibre degraded enzyme in microorganism Aspergillus aculeatus.When using the 98%-hulled barley, obtained high enzyme productivity.Yet when using its crushing product, CEL and BGL are active to be reduced.Therefore, its explanation barley shell discharges the high yield that restraining effect helps those enzymes to the nutrition that nutrition discharges.

＜embodiment 7 〉

The barley that use has different shelling ratios produces the white-rot fungi cultured products

By using hulled barley (Stirling (Stirling) originates in Australia) in various degree, can produce the cultured products of the white-rot fungi of cellulolytic enzyme by method production described below, and measure enzymic activity wherein.

1. cultural method

(1) pre-cultural method

The substratum of 100ml is placed the erlenmeyer flask of 500-ml band baffle plate, and with its with autoclave 121 ℃ the sterilization 15 minutes to prepare pre-culture medium, comprise 2% glucose in the described substratum, 0.5% yeast extract, 0.1% saltpetre, 0.1% potassium primary phosphate, 0.07% ammonium sulfate, 0.03% bitter salt, 0.02% calcium chloride (each % represents with weight/volume) and water.After the cooling, with pre-culture medium with the milky white of 30 5mm * 5mm size rake bacterium (milky white rake bacterium NBRC5367) mycelia clump inoculation, and 28 ℃ and 120rpm wave and culture 96 hours to obtain pre-incubated liquid.

(2) main cultured method

The substratum of 100ml is placed the erlenmeyer flask of 500-ml band baffle plate, and with autoclave 121 ℃ of sterilizations 15 minutes to obtain main medium, described substratum comprises 2% hulled barley, 0.1% poly-peptone, 0.14% ammonium sulfate, 0.2% potassium primary phosphate, 0.03% urea, 0.03% bitter salt, 0.03% calcium chloride, 0.1% tween 80 (each % represents with weight/volume) and water.

With the 65%-hulled barley, the 83%-hulled barley, 98%-hulled barley or 98%-hulled barley crushing product are as above-mentioned hulled barley.

After the cooling, with main medium with the inoculation of the preculture liq of 10ml, and 28 ℃ and 120rpm wave and culture 96 hours.

2. measure the method for enzymic activity

After cultivating end, culture fluid is centrifugal with the collection supernatant liquor, and the activity of the vegetable fibre degraded enzyme in the measurement supernatant liquor.

(1) method of measurement cellulase activity

The method of the amount by the reducing sugar that quantizes with dinitrosalicylic acid (DNS) method to be produced by the hydrolysis as the carboxymethyl cellulose (CMC) of substrate is measured cellulase (CEL) activity.More specifically, the cultivation liquid of 1ml is added the 1%CMC substrate solution of 1ml (by the low viscosity that the dissolving SIGMA-Ao Er Freundlich (Sigma-Aldrich) is produced in 100mM acetate buffer liquid (pH 5) ^TMAnd enzyme reaction was accurately carried out 10 minutes at 40 ℃ the solution that obtains).Subsequently, will comprise 0.75% dinitrosalicylic acid, 1.2% sodium hydroxide, the 4ml DNS reagent of four hydration sodium-potassium tartrates of 22.5% and a Lactose hydrate of 0.3% adds in the mixture, and thorough mixing is with termination reaction.For after termination reaction, quantize the amount of the reducing sugar in the liquid, the liquid after the termination reaction was accurately heated 15 minutes in the water-bath of boiling.Subsequently, to room temperature, the absorbancy that is determined at 540nm is to quantize the amount of the reducing sugar corresponding with the amount of glucose with liquid cooling.The active expression of the cellulase of a unit (CEL) per minute produces the needed enzyme amount of reducing sugar corresponding to the glucose of 1 μ mol.

(2) method of measurement xylanase activity

Next, measure zytase (XYL) activity by making reducing sugar and DNS reaction and quantification in the increase of the absorbancy of 540nm, described reducing sugar is by producing as the substrate enzymically hydrolyse from the xylan of oat-spelt.More specifically, the 1% xylan substrate solution (xylan that the culture fluid of 0.1ml is added 1.9ml, from the oat-spelt that is dissolved in 200mM acetate buffer body (pH 4.5) by SIGMA-Ao Er Freundlich (Sigma-Aldrich) product), and enzymatic reaction was accurately carried out 10 minutes at 40 ℃.Subsequently, will comprise 0.75% dinitrosalicylic acid, 1.2% sodium hydroxide, the 4ml DNS reagent of four hydration sodium-potassium tartrates of 22.5% and a Lactose hydrate of 0.3% adds in the mixture, and thorough mixing is with termination reaction.For after termination reaction, quantize the amount of the reducing sugar in the liquid, after termination reaction, liquid was accurately heated 15 minutes in the water-bath of boiling.Subsequently, to room temperature, the absorbancy that is determined at 540nm is with the amount of quantification corresponding to the reducing sugar of the amount of wood sugar with liquid cooling.The xylanase activity of a unit is illustrated under 40 ℃ and the 10 minutes reaction conditionss, and per minute production is corresponding to the needed enzyme amount of the reducing sugar of the wood sugar of 1 μ mol.

3. result

Figure 16 and 17 shows measuring result.It has confirmed that in white-rot fungi the productivity of vegetable fibre degraded enzyme is also with the shelling ratio vary.When using the 98%-hulled barley, obtained the highest enzyme productivity.Yet when using its crushing product, enzymic activity significantly reduces.Therefore, illustrate that the nutrition that the barley shell discharges nutrition discharges the high yield that restraining effect helps vegetable fibre degraded enzyme.

＜embodiment 8 〉

Do not use and produced the Aspergillus albicans cultured products by the pearled barley of gelationization

(1) pre-incubated method:

8g pearled barley (Stirling (Stirling) originates in Australia) and 100ml water are placed the erlenmeyer flask of 500-ml band baffle plate, and sterilize 15 minutes to obtain pre-culture medium at 121 ℃ with autoclave.With Aspergillus albicans (Aspergillus kawachii NBRC4308) with 1 * 10 ⁶/ ml is inoculated in the pre-culture medium, and 37 ℃ and 100rpm wave and culture 24 hours to obtain pre-incubated liquid.

(2) main cultural method:

KNO with 0.2g ₃, the KH of 0.3g ₂PO ₄And 100ml water places the erlenmeyer flask of 500-ml band baffle plate, and sterilizes 15 minutes at 121 ℃ with autoclave.After the cooling, with paraxin (watt gram pure chemistry product (the Wako Pure Chemical Industries Co. of Industrial Co., Ltd, Ltd.)) adding the product obtain, to make its concentration be 50 μ g/ml, and will be not do not add wherein to obtain main medium through the 2g pearled barley of heat treated.With the preculture liq of 1ml inoculation main medium, and 37 ℃ and 100rpm wave and culture 72 hours to obtain the aspergillus cultured products.

Produce the aspergillus cultured products in contrast by the cultivation starting material that use gelationization.That is, with the 2g pearled barley, the KNO of 0.2g ₃, the KH of 0.3g ₂PO ₄And 100ml water places the erlenmeyer flask of 500-ml band baffle plate, and sterilizes 15 minutes at 121 ℃ with autoclave.After the cooling, paraxin is added the product obtain, and to make its concentration be 50 μ g/ml, to obtain main medium.With the preculture liq of 1ml inoculation main medium, and 37 ℃ and 100rpm wave and culture 72 hours to obtain the aspergillus cultured products.

(3) measuring method

The glucoamylase in the aspergillus cultured products that measurement obtains in each experimental design, the activity of acid acceptance α-Dian Fenmei and α-Dian Fenmei.That is, measure glucoamylase activity by using saccharification underworld differential to quantize test kit (producing) by Kikkoman Co., Ltd..For measuring the acid acceptance alpha-amylase activity, will be at J.Ferment.Bioeng. (fermenting organism engineering magazine) such as Sudo S., 76,105-110 (1993), Sudo S. etc.: J.Ferment.Bioeng. (fermenting organism engineering magazine), 77,483-489 (1994), with Shigetoshi Sudo etc.: Journal of the Brewing Society ofJapan (association's magazine is brewageed by Japan), 89, the method described in the 768-774 (1994) is revised slightly.That is,, then measure test kit (producing) and measure the acid acceptance alpha-amylase activity by Kikkoman Co., Ltd. with α-Dian Fenmei by come deactivation acid labile alpha-amylase activity with the acid treatment cultured products.More specifically, 9ml 100mM acetate buffer liquid (pH 3) is added the cultivation liquid of 1ml, and carried out acid treatment 1 hour, and measure test kit (producing) with α-Dian Fenmei and measure by Kikkoman Co., Ltd. at 37 ℃.Measure alpha-amylase activity by using α-Dian Fenmei to measure test kit (producing) by Kikkoman Co., Ltd..The result is presented in the table 1.

(4) result

In the former wheat of pearl of the present invention (pearled raw barley) aspergillus cultured products, produce glucoamylase and acid acceptance α-Dian Fenmei with good and equilibrated mode.And the output of α-Dian Fenmei is low slightly.

In contrast, produce a large amount of relatively acid acceptance α-Dian Fenmei and α-Dian Fenmei.Although this may be because used the starting material of gelationization, because KNO ₃And KH ₂PO ₄Existence, the nutrition condition maintains suitable scope.

Therefore, the aspergillus cultured products that has disclosed produced according to the invention to can be used for producing leavened food and beverage.

Table 1

＜embodiment 9 〉

By using, produce the barley liquor with the Aspergillus albicans cultured products not by the pearled barley of gelationization

(1) pre-cultural method:

8g pearled barley (Stirling (Stirling) originates in Australia) and 100ml water are placed the erlenmeyer flask of 500-ml band baffle plate, and sterilize 15 minutes to obtain pre-culture medium at 121 ℃ with autoclave.With Aspergillus albicans (Aspergillus kawachii NBRC4308) with 1 * 10 ⁶/ ml is inoculated in the pre-culture medium, and 37 ℃ and 100rpm wave and culture 24 hours to obtain pre-incubated liquid.

(2) main cultured method:

With the pearled barley of 0.5g, the KNO of 0.2g ₃, the KH of 0.3g ₂PO ₄Place the erlenmeyer flask of 500-ml band baffle plate with the water of 100ml, and with its with autoclave 121 ℃ of sterilizations 15 minutes to obtain main medium.With main medium with the inoculation of the preculture liq of 1ml, and 37 ℃ and 100rpm wave and culture 24 hours.Subsequently, the 1.5g pearled barley that will not be heated processing adds wherein, and 37 ℃ and 100rpm again wave and culture 48 hours to obtain the aspergillus cultured products.

(3) yeast:

Deer island (Kagoshima) yeast is spent the night at the 100rpm wave and culture in the YPD of 1ml substratum, centrifugal with collecting cell, and with aqua sterilisa washing 2 times.

(4) wine with dregsization:

The wine with dregs combination is presented in the table 2.For additional barley, use pearled barley, it is washed and carried out submergence subsequently 60 minutes, draining 30 minutes and steaming 40 minutes.Use above-mentioned zymic total amount.

Table 2

	One-level	Secondary	Total amount
				Additional barley (g)	310	615	925
Wine with dregs water (ml)	300	650	950
				Aspergillus cultured products (ml)	350	0	350
90% lactic acid (ml)	2	0	2

(5) fermentation condition: will ferment and carry out 20 days at 25 ℃.After the one-level wine with dregsization, carried out the secondary wine with dregsization in 3 days.

(6) distillation: under the decompression of-650mmHg, distill.

(7) result

Ferment continuously, the mash after the fermentation ends has 17.5% ethanol content.

The group that is made up of the expert of 6 alcoholic beverage carries out the sensory evaluation to the sample that distills the back and obtain.As a result, the sample of acquisition is spoken highly of and is the alcoholic beverage prime quality.

From this result, disclose the method according to this invention and produced barley liquor with the quality of having no time.

＜embodiment 10 〉

Do not use and produced the flavus cultured products by the pearled barley of gelationization

(1) pre-incubated method:

8g pearled barley (Stirling (Stirling) originates in Australia) and 100ml water are placed the erlenmeyer flask of 500-ml band baffle plate, and sterilize 15 minutes to obtain pre-culture medium at 121 ℃ with autoclave.With flavus (aspergillus oryzae NRIB40) with 1 * 10 ⁶/ ml is inoculated in the pre-culture medium, and 37 ℃ and 100rpm wave and culture 24 hours to obtain pre-incubated liquid.

(2) main cultural method:

KNO with 0.8g ₃, the KH of 1.2g ₂PO ₄, the MgSO of 0.2g ₄Place the erlenmeyer flask of 500-ml band baffle plate with 100ml water, and sterilized 15 minutes at 121 ℃ with autoclave.After the cooling, (it is 50 μ g/ml that watt product that gram pure chemistry product Industrial Co., Ltd (Wako Pure Chemical IndustriesCo., Ltd.)) adding obtains makes its concentration, and the 2g pearled barley is added wherein to obtain main medium with paraxin.With the preculture liq of 1ml inoculation main medium, and 37 ℃ and 100rpm wave and culture 72 hours to produce the aspergillus cultured products.

For positive control, produce the aspergillus cultured products by method according to a second aspect of the invention.That is, with 2g 95%-hulled barley (Stirling (Stirling) originates in Australia), the KNO of 0.8g ₃, the KH of 1.2g ₂PO ₄, the MgSO of 0.2g ₄Place the erlenmeyer flask of 500-ml band baffle plate with the water of 100ml, and at 121 ℃ with autoclave sterilization 15 minutes.After the cooling, paraxin added to make its concentration in the product obtain be that 50 μ g/ml are to obtain main medium.With main medium with the inoculation of the preculture liq of 1ml, and with its 37 ℃ and 100rpm wave and culture 72 hours to produce the aspergillus cultured products.

(3) result

Measure the glucoamylase in the aspergillus cultured products that in each experimental design, obtains and the activity of α-Dian Fenmei in the mode identical with embodiment 8.Table 3 display result.

In the mould cultured products of the former wheat koji of pearl, produce glucoamylase in good mode, and alpha-amylase activity is low slightly.The activity of two kinds of enzymes is all inferior to those enzymic activitys in the positive control.Yet two kinds of enzymes are all with balance mode production.Therefore, disclose, the aspergillus cultured products that can be used for producing leavened food and beverage is provided.

Table 3

＜EXPERIMENTAL EXAMPLE 2 〉

Measurement the time has glucose release rate in the barley substrate solution of various inorganic salt concentrations in sterilization

Obtain the barley substrate solution by the inorganic salt solution of the various salt concn that comprise barley being sterilized with autoclave.Make every kind of barley substrate solution with from the enzyme reaction of aspergillus cultured products, and measure from the glucose release rate in the barley.

1. the method for preparing the barley substrate solution

The first, the 98%-hulled barley of the 2g that weighs (Stirling (Stirling) originates in Australia) then places the 500-ml erlenmeyer flask with it with 50ml water.With its with autoclave sterilization (121 ℃ 15 minutes) with preparation barley substrate solution, then it is expressed as " No. 1: control design ".

In " No. 2: use the design of salt ", carry out the autoclave sterilization in the mode identical, except the inorganic salt solution of the potassium primary phosphate of the saltpetre that comprises 0.1g that uses 50ml and 0.15g replaces water with " No. 1: control design ".Salt concn when in other words, sterilizing with autoclave is 0.2% saltpetre and 0.3% potassium primary phosphate.

In " No. 3: use the design of the salt of high density during sterilization ", carry out the autoclave sterilization in the mode identical with " No. 1: control design ", the 10ml inorganic salt solution that comprises the potassium primary phosphate of the saltpetre of 0.1g and 0.15g except use replaces water, and the aqua sterilisa of 40ml is added wherein.In other words, the salt concn during sterilization is 1.0% saltpetre and 1.5% potassium primary phosphate, and it is those 5 times in No. 2, the experimental design.Yet the salt concn after regulating sterilization and adding water to be obtaining 0.2% saltpetre and 0.3% potassium primary phosphate, and it is identical with in No. 2, the experimental design those.

2. the method for preparing the aspergillus culture supernatants

Subsequently, separate by carry out solid-liquid with filter paper filtering as the aspergillus cultured products of cultivating starting material production by using barley (Stirling (Stirling) originates in Australia), to obtain " aspergillus culture supernatants ".

Produce and use the method for aspergillus cultured products in this experiment as described below.

(1) pre-incubated method

8g 65%-hulled barley and 100ml water are placed the erlenmeyer flask of 500-ml band baffle plate and sterilize 15 minutes to obtain pre-culture medium with autoclave at 121 ℃.After the cooling, with Aspergillus albicans (Aspergillus kawachii NBRC4308) with 1 * 10 ⁶/ ml be seeded in the pre-incubated substratum and 37 ℃ and 100rpm wave and culture 24 hours to obtain preculture liq.

(2) main cultured method

The 98%-hulled barley is added in the water of potassium primary phosphate of the saltpetre and 0.3% (weight/volume) replenished 0.2% (weight/volume) so that the amount of 98%-hulled barley is that 2.0% (weight/volume) is with the preparation liquid nutrient medium.With 3, the preparation liquid nutrient medium of 000ml places 5, the small-sized fermentation jar of 000-ml is (by (the Marubishi Co. of ball water chestnut Co., Ltd., Ltd) produce) in, and with autoclave (at 121 ℃, 15 minutes) sterilization, use the preculture liq inoculation of the Aspergillus albicans (Aspergillus kawachiiNBRC4308) of 30ml subsequently, described Aspergillus albicans is with cultivating in advance in advance in the aforesaid method liquid medium within.Subsequently, in 37 ℃ temperature and the stir speed (S.S.) of 300rpm, and the aeration volume of 0.5vvm, cultivated 42 hours, filter the product that obtains with filter paper (No. 2 Toyo filter paper) to obtain " aspergillus culture supernatants ".

3. measure the method for glucose release rate

The aspergillus culture supernatant of the barley substrate solution of 50ml and 50ml is remained on 37 ℃ respectively reach 5 minutes, and then mix with initial action.With glucose C-II Test Wako (producing) by a watt gram pure chemistry product Industrial Co., Ltd measure initial back 3 hours of reaction respectively the glucose concn in the reaction liquid of sampling with the release rate of calculating glucose.

4. result

By as shown in Figure 18, the result who measures the glucose concn in the reaction liquid calculates the glucose release rate.Confirmed that the glucose release rate changes with salt concn on preparation barley substrate solution.In EXPERIMENTAL EXAMPLE 1, shown that the glucose release rate regulates with the shelling ratio of barley.EXPERIMENTAL EXAMPLE 2 also discloses, with as the thermal treatment of autoclave sterilization after, exist the glucose release rate slack-off owing to salt.This may be that the existence of salt has suppressed the physical decomposition of barley because when the thermal treatment barley.

＜embodiment 11 〉

The barley that has various salt concn when using sterilization produces the Aspergillus albicans cultured products

Use hulled barley (Stirling (Stirling) originates in Australia) in various degree, produce the Aspergillus albicans cultured products by described method below, and measure enzymic activity wherein.

1. pre-cultural method

8g 65%-hulled barley and 100ml water are placed the erlenmeyer flask of 500-ml band baffle plate and sterilize 15 minutes to obtain pre-culture medium with autoclave at 121 ℃.After the cooling, with Aspergillus albicans (Aspergillus kawachii NBRC4308) with 1 * 10 ⁶/ ml be seeded in the pre-incubated substratum and 37 ℃ and 100rpm wave and culture 24 hours to obtain preculture liq.

2. main cultural method

The weigh 98%-hulled barley (Stirling (Stirling) originates in Australia) of 2g, and it is placed the erlenmeyer flask of 500-ml band baffle plate with 100ml water.With autoclave it is sterilized 15 minutes with the preparation main medium at 121 ℃, then it is expressed as " No. 1: control design ".

In " No. 2: use the design of salt ", carry out the autoclave sterilization in the mode identical, except the inorganic salt solution of the potassium primary phosphate of the saltpetre that comprises 0.2g that uses 100ml and 0.3g replaces water with " No. 1: control design ".

In " No. 3: use the design of the salt of high density during sterilization ", carry out the autoclave sterilization in the mode identical with " No. 1: control design ", the 20ml inorganic salt solution that comprises the potassium primary phosphate of the saltpetre of 0.2g and 0.3g except use replaces water, and the aqua sterilisa of 80ml is added wherein.In other words, salt concn is respectively 1.0% saltpetre and 1.5% potassium primary phosphate during sterilization, and it is those 5 times in No. 2, the experimental design.Yet the salt concn after regulating sterilization and adding water to be obtaining 0.2% saltpetre and 0.3% potassium primary phosphate, and it is identical with in No. 2, the experimental design those.

After the cooling, with the pre-culture medium inoculation of the main culture medium of preparation thus with 1ml, and 37 ℃ and 100rpm wave and culture 48 hours.

3. measuring method

After cultivating end, measure as the glucoamylase (GA) of amylolytic enzyme and the activity of acid acceptance α-Dian Fenmei (ASAA).

Measure glucoamylase (GA) activity by using saccharification power fractional to quantize test kit (producing) by Kikkoman Co., Ltd..

In order to measure acid acceptance α-Dian Fenmei (ASAA) activity, will be at Sudo S. etc.: J.Ferment.Bioeng. (fermenting organism engineering magazine), 76,105-110 (1993), Sudo S. etc.: J.Ferment.Bioeng. (fermenting organism engineering magazine), 77,483-489 (1994), with ShigetoshiSudo etc.: Journal ofthe Brewing Society ofJapan (association's magazine is brewageed by Japan), 89, the method described in the 768-774 (1994) is improved slightly.That is, with the alpha-amylase activity deactivation of acid labile, then measure test kit (producing) and measure the acid acceptance alpha-amylase activity by Kikkoman Co., Ltd. with α-Dian Fenmei by using the acid treatment cultured products.More particularly, the 100mM acetate buffer liquid (pH 3) of 9ml is added in the culture fluid of 1ml, carried out acid treatment 1 hour, and measure test kit (producing) with α-Dian Fenmei and measure by Kikkoman Co., Ltd. at 37 ℃.

4. result

Figure 19 shows the measuring result of glucoamylase activity, and Figure 20 shows the measuring result of acid acceptance alpha-amylase activity.

As shown in Figure 19 and 20, after cultivation, compare with the enzyme productivity in No. 2 designs that wherein have identical salt concn, improved the enzyme productivity in No. 3 designs that salt concn is higher when sterilizing therein.In EXPERIMENTAL EXAMPLE 2, confirmed when the inorganic salt concentration in the barley substrate solution uprises during in thermal treatment, from the sugared release rate step-down of starting material barley.Therefore, illustrated that the productivity of enzyme also is subjected to the influence of sugared release rate except the effect of salt pair aspergillus growth.No. 1 design does not comprise salt, thinks that therefore the aspergillar growth is suppressed and enzyme productivity wherein is because high sugared release rate by remarkable the inhibition.

Up to date, think that the inorganic salt that add only relate to the aspergillar growth in the culturing process when the preparation substratum.Yet,, illustrated that the enzyme production in the aspergillar liquid culture is also discharged the promoted possibility of restraining effect by the sugar from the starting material barley according to this embodiment.

Industrial applicability

The method according to this invention, the control of the nutrient concentrations in the culture systems is lower, therefore obtain to add the training mode that cultivation equates with stream by simple batch culture, add cultivation and need not to carry out wherein outside culture systems, to add gradually nutraceutical stream.

According to the present invention, the method of stably producing fungal culture with cheapness is provided, described fungal culture is used for removing fermented food and the beverage of producing for the production of by described fungal culture, in the various industry outside enzyme and the enzyme preparation.

In addition, expection applies the present invention to M-band production, the promoter region of its use amylolytic enzyme gene etc.

Therefore, the present invention can be applicable to comprise food-processing industry in the widely industry, fermentation industry, pharmaceutical industries etc.

Claims (16)

1. one kind comprises by use and to be selected from by cereal, beans, and stem tuber, at least a of the group that Amaranthus and Chenopodium plant are formed produces the method for fungal culture as cultivating raw-material liquid nutrient medium, and described method comprises,

When cultivating filamentous fungus, thus the productivity that the control nutrition is regulated the enzyme in the fungal culture from the release rate of described cultivation starting material to culture systems.

2. produce the method for fungal culture according to claim 1, wherein

The cereal of described cultivation starting material to be its surfaces be coated with wholly or in part shell at least and

By regulating the shelling ratio of described cereal, the control nutrition is from the release rate of cereal to described culture systems.

3. produce the method for fungal culture according to claim 1, wherein by using its shell or shell to be removed and not controlled nutrition from the release rate of described cultivation starting material to described culture systems by the cultivation starting material of gelationization.

4. produce the method for fungal culture according to claim 1, wherein

Described liquid nutrient medium is heat-treated, and

Control nutrition from the release rate of described cultivation starting material to described culture systems by the inorganic salt concentration of regulating in the liquid nutrient medium when the thermal treatment.

5. produce the method for fungal culture according to claim 1, wherein said nutrition comprises from the sugar of starch in the described cultivation starting material and/or from proteinic amino acid in the described cultivation starting material.

6. produce the method for fungal culture according to claim 1, wherein said enzyme comprises and is selected from by amylolytic enzyme, the group that vegetable fibre degraded enzyme and proteolytic ferment are formed at least a.

7. produce the method for fungal culture according to claim 1, wherein said filamentous fungus comprises and being selected from by Aspergillus, the group of Trichoderma (Trichoderma) and white-rot fungi composition at least a.

8. fungal culture, it is by obtaining according to each method of claim 1-7.

9. method of producing zymin, described method comprises use fungal culture according to Claim 8.

10. zymin, it obtains by the method according to claim 9.

11. a method of producing enzyme, it comprises by use and is selected from by cereal, beans, and stem tuber, at least a of the group that Amaranthus and Chenopodium plant are formed carries out as cultivating raw-material liquid nutrient medium, and described method comprises

Cultivate filamentous fungus, thereby control the productivity that nutrition is regulated the enzyme the described fungal culture from described cultivation starting material to the release rate of described culture systems simultaneously.

12. enzyme, it obtains by the method according to claim 11.

13. produce the method for leavened food and beverage, described method comprises use fungal culture according to Claim 8.

14. produce the method for leavened food and beverage, described method comprises the zymin of use according to claim 10.

15. produce the method for leavened food and beverage, described method comprises the enzyme of use according to claim 12.

16. leavened food and beverage, it obtains by each the method according to claim 13-15.

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