CN110734862A - Aspergillus oryzae and method for producing fructo-oligosaccharide by fermenting Aspergillus oryzae - Google Patents

Aspergillus oryzae and method for producing fructo-oligosaccharide by fermenting Aspergillus oryzae Download PDF

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CN110734862A
CN110734862A CN201911022662.8A CN201911022662A CN110734862A CN 110734862 A CN110734862 A CN 110734862A CN 201911022662 A CN201911022662 A CN 201911022662A CN 110734862 A CN110734862 A CN 110734862A
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aspergillus oryzae
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宋建民
王德海
宛荣生
张琴
王颂
黄祥君
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Anhui Minzhen Biological Engineering Co ltd
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Abstract

The invention belongs to the field of fructo-oligosaccharide, and particularly discloses Aspergillus oryzae and a method for producing fructo-oligosaccharide by fermenting Aspergillus oryzae, wherein a new strain is named as Aspergillus oryzae (Aspergillus oryzae) MZ30, the strain preservation number is CGMCC No. 18106.

Description

Aspergillus oryzae and method for producing fructo-oligosaccharide by fermenting Aspergillus oryzae
Technical Field
The invention relates to the field of fructo-oligosaccharide, in particular to kinds of aspergillus oryzae and a method for producing fructo-oligosaccharide by fermenting aspergillus oryzae.
Background
The fructo-oligosaccharide is natural active substances, the sweetness of the fructo-oligosaccharide is 0.3-0.6 times of that of cane sugar, the pure sweet taste of the cane sugar is kept, and the fructo-oligosaccharide is fresher than the sweet taste of the cane sugar, and is known as a new generation additive, namely a growth promoting substance, with the most potential after the age of antibiotics.
Besides the physicochemical properties of functional oligosaccharides, fructo-oligosaccharide has the most remarkable physiological characteristics of obviously improving the microbial population ratio in intestinal tract, being an activated proliferation factor of bifidobacterium in the intestinal tract, reducing and inhibiting the generation of putrefactive substances in the intestinal tract, inhibiting the growth of harmful bacteria, regulating the balance in the intestinal tract, promoting the absorption and utilization of trace elements of iron and calcium to prevent osteoporosis, reducing liver toxins, generating anticancer organic acids in the intestine, having obvious cancer prevention function, pure, sweet and delicious taste, having the fragrance similar to fat and tasty and refreshing smoothness, and being a health-care product market in recent years plus fructo-oligosaccharide products in European, American and other countries.
The inulin is hydrolyzed by endo-inulase to obtain fructo-oligosaccharide with the polymerization degree of 2-8, is also mixed with a small amount of fructan without terminal glucose residue, which inevitably reduces the physiological effect, the nutritional value, the application and the like of the fructo-oligosaccharide, so the preparation of the high-purity fructo-oligosaccharide is more and more concerned by people.
Disclosure of Invention
In view of the above, the present invention aims to provide Aspergillus oryzae strains and a method for producing fructooligosaccharides by fermenting the Aspergillus oryzae strains, wherein the fructooligosaccharides prepared by taking Aspergillus oryzae (Aspergillus oryzae) MZ30 with the strain preservation number of CGMCC No.18106 as a production strain have high yield and purity.
In order to achieve the above purpose, the invention provides the following technical scheme:
Aspergillus oryzae strain, which is separated from natural soybean paste in Huaibei area of Anhui province, China, and is named as Aspergillus oryzae (Aspergillus oryzae) MZ30, and the preservation number of the strain is CGMCC No. 18106.
The invention also provides a method for producing fructo-oligosaccharide by using aspergillus oryzae strains through fermentation, which comprises the following steps:
1) adding water into white sugar to prepare a white sugar solution;
inoculating Aspergillus oryzae (Aspergillus oryzae) MZ30 with the strain preservation number of CGMCC No.18106 to an activation medium under aseptic condition for culturing at the culture temperature of 28 ℃ at the rotation speed of 120r/min of a shaking table for 48 h;
2) adding agar, yeast extract and K into the white sugar solution obtained in the step 1)2HPO4、MgSO4NaCl and NaH2PO4After uniformly mixing, adjusting the pH value of the mixture to 6-7, and sterilizing at the temperature of 115 ℃ for 15min to obtain a fermentation medium; inoculating the strain solution of Aspergillus oryzae (MZ 30) with the strain preservation number of CGMCC No.18106 obtained in the step 1), and fermenting at 25-35 ℃ for 30-50 h to obtain fermentation liquor;
3) removing thalli in the fermentation liquor obtained in the step 2), and adding activated carbon for decoloring to obtain supernatant;
4) sequentially passing the supernatant obtained in the step 3) through an ultrafiltration membrane, a cation exchange resin and an anion exchange resin, and collecting eluent;
5) concentrating and drying the eluent obtained in the step 4) to obtain the fructo-oligosaccharide.
Preferably, in the step 2), the inoculation amount of the Aspergillus oryzae (Aspergillus oryzae) MZ30 bacterial solution in the fermentation medium is 5-10% (v/v).
Preferably, each timeThe weight grams of each component in the activation medium are 20-30 of corn starch, 5-8 of yeast extract and KNO31～1.5，K2HPO40.4～0.6，MgCl 0.5～0.7，NaCl 0.5～0.8，FeSO4·7H20.01-0.02% of O and 15-20% of agar.
Preferably, each liter of fermentation medium contains 30-35 g of white sugar, 4-7 g of agar, 10-15 g of yeast extract and K2HPO41～2、MgSO41-2, NaCl 1-2 and NaH2PO40.5～1。
Preferably, in the step 2), an enzyme activator is added while inoculating Aspergillus oryzae (Aspergillus oryzae) MZ30, and the enzyme activator is zinc chloride or glutathione.
Preferably, the mass ratio of the enzyme activator to the white sugar is (0.05-0.1): 100.
preferably, the step 3) is specifically that the fermentation liquor is subjected to tangential flow ultrafiltration, thalli are removed by adopting an ultrafiltration membrane with the aperture of 0.2 mu m in the tangential flow ultrafiltration, and activated carbon is added for decolorization to obtain a supernatant.
Preferably, in the step 4), the cutoff molecular weight of the ultrafiltration membrane is 300-500 Da.
Preferably, in step 4), the anion exchange resin is a D001FD macroporous strongly acidic styrene cation exchange resin, and the cation exchange resin is a D750 macroporous strongly basic acrylic acid anion exchange resin.
The invention provides Aspergillus oryzae strains and a method for producing fructo-oligosaccharide by fermenting the Aspergillus oryzae strains, wherein the new strain is named as Aspergillus oryzae (Aspergillus oryzae) MZ30, the strain preservation number is CGMCC No. 18106. the invention takes white sugar as a raw material, adds Aspergillus oryzae (Aspergillus oryzae) MZ30 to enable the fermentation process to generate fructosyltransferase, catalyzes the white sugar to synthesize fructo-oligosaccharide, and then decolors the fructo-oligosaccharide through active carbon, filters, concentrates and dries through an ultrafiltration membrane, a cation exchange resin and an anion exchange resin.
Detailed Description
Aspergillus oryzae strain is separated from natural soybean paste in Huaibei area of Anhui province, and named as Aspergillus oryzae (Aspergillus oryzae) MZ30, the preservation number of the strain is CGMCC No.18106, the preservation time is 7 months and 10 days in 2019, the preservation unit is the common microorganism center of China Committee for culture Collection of microorganisms, and the address is No. 3 of West Lu No.1 of the sunward area of Beijing city.
Aspergillus oryzae (Aspergillus oryzae) MZ30 produces a fructosyltransferase during fermentation, which catalyzes the synthesis of fructooligosaccharides from white sugar.
The method for producing fructo-oligosaccharide by fermenting the aspergillus oryzae strain comprises the following steps:
1) adding water into white sugar to prepare a white sugar solution;
inoculating Aspergillus oryzae (Aspergillus oryzae) MZ30 with the strain preservation number of CGMCC No.18106 to an activation culture medium under aseptic condition for culturing, wherein the rotating speed of a shaker is 120r/min, the culture temperature is 28 ℃, and the culture time is 48 h;
2) adding agar, yeast extract and K into the white sugar solution obtained in the step 1)2HPO4、MgSO4NaCl and NaH2PO4After uniformly mixing, adjusting the pH value of the mixture to 6-7, and sterilizing at the temperature of 115 ℃ for 15min to obtain a fermentation medium; inoculating the strain solution of Aspergillus oryzae (MZ 30) with the strain preservation number of CGMCC No.18106 obtained in the step 1), and fermenting at 25-35 ℃ for 30-50 h to obtain fermentation liquor;
3) removing thalli in the fermentation liquor obtained in the step 2), and adding activated carbon for decoloring to obtain supernatant;
4) sequentially passing the supernatant obtained in the step 3) through an ultrafiltration membrane, a cation exchange resin and an anion exchange resin, and collecting eluent;
5) concentrating and drying the eluent obtained in the step 4) to obtain the fructo-oligosaccharide.
Adding water into white sugar to prepare white sugar solution. Wherein the white sugar solution is mixed with agar, yeast extract, and K2HPO4、MgSO4NaCl and NaH2PO4After uniformly mixing, adjusting the pH value of the mixture to 6-7, and sterilizing at the temperature of 115 ℃ for 15min to obtain a fermentation medium; each liter of fermentation medium comprises 30-35 g of white sugar, 4-7 g of agar, 10-15 g of yeast extract and K2HPO41～2、MgSO41-2, NaCl 1-2 and NaH2PO40.5～1。
Inoculating Aspergillus oryzae (Aspergillus oryzae) MZ30 with the strain preservation number of CGMCC No.18106 to an activation culture medium under aseptic condition for culturing, wherein the rotating speed of a shaker is 120r/min, the culture temperature is 28 ℃, and the culture time is 48 h; in the embodiment of the invention, each liter of activation medium contains 20-30 grams of corn starch, 5-8 grams of yeast extract and KNO31～1.5，K2HPO40.4～0.6，MgCl 0.5～0.7，NaCl 0.5～0.8，FeSO4·7H20.01-0.02% of O and 15-20% of agar.
Inoculating a thallus solution of Aspergillus oryzae (Aspergillus oryzae) MZ30 in an activation culture medium into a fermentation culture medium; wherein the inoculation amount of an Aspergillus oryzae (Aspergillus oryzae) MZ30 thallus solution in the fermentation medium is 5-10% (v/v) of the volume of the fermentation medium.
In the embodiment of the invention, the strain is added and an enzyme activator is also added, and the enzyme activator is zinc chloride or glutathione. The enzyme activator can improve fructosyl transferase activity, and further improve fructo-oligosaccharide yield. In a preferred embodiment, the mass ratio of the enzyme activator to the white sugar is (0.05-0.1): 100.
removing thalli in the fermentation liquor, and adding activated carbon for decolorization to obtain supernatant; in the embodiment of the invention, the fermentation liquor is subjected to tangential flow ultrafiltration, thalli are removed by an ultrafiltration membrane with the aperture of 0.2 mu m adopted in the tangential flow ultrafiltration, and activated carbon is added for decolorization to obtain the supernatant. The method can effectively remove thallus in the fermentation liquor and improve the purity of fructo-oligosaccharide.
Sequentially passing the supernatant through an ultrafiltration membrane, a cation exchange resin and an anion exchange resin, and collecting the eluent; in the embodiment of the invention, the molecular weight cut-off of the ultrafiltration membrane is 300-500 Da; the anion exchange resin is D001FD macroporous strong acidic styrene cation exchange resin, and the cation exchange resin is D750 macroporous strong basic acrylic acid anion exchange resin. The purity of the fructo-oligosaccharide can be obviously improved by the supernatant through an ultrafiltration membrane, cation exchange resin and anion exchange resin.
Concentrating and drying the eluent to obtain fructo-oligosaccharide
According to the fructo-oligosaccharide, white sugar is used as a raw material, Aspergillus oryzae (Aspergillus oryzae) MZ30 is added, fructosyltransferase can be generated in the fermentation process, the white sugar is catalyzed to synthesize fructo-oligosaccharide, and then the fructo-oligosaccharide is decolorized by active carbon, filtered by an ultrafiltration membrane, cation exchange resin and anion exchange resin, concentrated and dried to obtain the high-purity fructo-oligosaccharide. The fructo-oligosaccharide prepared by taking white sugar as a raw material can promote the proliferation of bifidobacteria in intestinal tracts, inhibit the proliferation of enterobacteria and enterococci, and has the effects of regulating intestinal flora and inhibiting enterobacteria and enterococci. The results of the experiments show that the fermentation broth without Aspergillus oryzae (Aspergillus oryzae) MZ30 produced no fructooligosaccharides under the same fermentation conditions.
To further illustrate the present invention at , the Aspergillus oryzae strains and the method for producing fructo-oligosaccharide by fermentation using the Aspergillus oryzae strains provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
In the embodiment, the Aspergillus oryzae strain is separated from natural soybean paste in the northern area of Huaihei of Anhui province in China, and is named as Aspergillus oryzae (Aspergillus oryzae) MZ30, the preservation number of the strain is CGMCC No.18106, the preservation time is 7 months and 10 days in 2019, and the preservation unit is as follows: china general microbiological culture Collection center, address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
Example 1
1) Adding water into white sugar to prepare a white sugar solution;
inoculating Aspergillus oryzae (Aspergillus oryzae) MZ30 with the strain preservation number of CGMCC No.18106 to an activation culture medium under aseptic condition for culturing, wherein the rotating speed of a shaker is 120r/min, the culture temperature is 28 ℃, and the culture time is 48 h; each liter of activation medium comprises 20 grams of corn starch, 5 grams of yeast extract and KNO31，K2HPO40.4，MgCl0.6，NaCl 0.7，FeSO4·7H2O0.015, agar 18;
2) adding agar, yeast extract and K into the white sugar solution obtained in the step 1)2HPO4、MgSO4NaCl and NaH2PO4After being uniformly mixed, the pH value of the mixture is adjusted to 7, and the mixture is sterilized for 15min at the temperature of 115 ℃ to obtain a fermentation medium; inoculating the strain solution of Aspergillus oryzae (MZ 30) with the strain preservation number of CGMCC No.18106 obtained in the step 1), adding zinc chloride, and fermenting at 25 ℃ for 30h to obtain fermentation liquor; the mass ratio of the zinc chloride to the white sugar is 0.05: 100, respectively;
the inoculation amount of an Aspergillus oryzae (Aspergillus oryzae) MZ30 thallus solution in the fermentation medium is 5% of the volume of the fermentation medium;
each liter of fermentation medium contains 32 grams of white sugar, 4 grams of agar, 10 grams of yeast extract and K2HPO41.5、MgSO41.5, NaCl 1.5 and NaH2PO40.8；
3) Performing tangential flow ultrafiltration on the fermentation liquor obtained in the step 2), wherein thalli are removed by using an ultrafiltration membrane with the aperture of 0.2 mu m for the tangential flow ultrafiltration, and then adding activated carbon for decolorization to obtain supernatant;
4) sequentially passing the supernatant obtained in the step 3) through an ultrafiltration membrane with the molecular weight cutoff of 300Da, D001FD macroporous strongly acidic styrene cation exchange resin and D750 macroporous strongly basic acrylic acid anion exchange resin, and collecting eluent;
5) concentrating and drying the eluent obtained in the step 4) to obtain the fructo-oligosaccharide.
Example 2
1) Adding water into white sugar to prepare a white sugar solution;
collecting strain with preservation number of CGMCC NO.oryzae (Aspergillus oryzae) MZ30 of 18106 was inoculated into an activated medium under sterile conditions for culturing at 28 ℃ with shaker rotation speed of 120r/min for 48 h; each liter of activation medium contains 30 grams of corn starch, 8 grams of yeast extract and KNO31.5，K2HPO40.6，MgCl 0.7，NaCl 0.8，FeSO4·7H2O0.02, agar 20;
2) adding agar, yeast extract and K into the white sugar solution obtained in the step 1)2HPO4、MgSO4NaCl and NaH2PO4After being uniformly mixed, the pH value of the mixture is adjusted to 6, and the mixture is sterilized for 15min at the temperature of 115 ℃ to obtain a fermentation medium; inoculating a thallus solution of Aspergillus oryzae (MZ 30) with the strain preservation number of CGMCC No.18106 obtained in the step 1), adding glutathione, and fermenting at 25 ℃ for 30h to obtain a fermentation liquid; the mass ratio of the glutathione to the white sugar is 0.08: 100, respectively;
the inoculation amount of an Aspergillus oryzae (Aspergillus oryzae) MZ30 thallus solution in the fermentation medium is 10% of the volume of the fermentation medium;
each liter of fermentation medium contains 35 grams of white sugar, 7 grams of agar, 15 grams of yeast extract and K2HPO42、MgSO42. NaCl 2 and NaH2PO41; the mass ratio of the enzyme activator to the white sugar is 0.05: 100, respectively;
3) performing tangential flow ultrafiltration on the fermentation liquor obtained in the step 2), wherein the aperture of an ultrafiltration membrane adopted by the tangential flow ultrafiltration is 0.2 mu m, removing thalli, and adding activated carbon for decolorization to obtain supernatant;
4) sequentially passing the supernatant obtained in the step 3) through an ultrafiltration membrane with the molecular weight cutoff of 500Da, D001FD macroporous strongly acidic styrene cation exchange resin and D750 macroporous strongly basic acrylic acid anion exchange resin, and collecting eluent;
5) concentrating and drying the eluent obtained in the step 4) to obtain the fructo-oligosaccharide.
Example 3
1) Adding water into white sugar to prepare a white sugar solution;
the preservation number of the strain is CGMCC No.18106 Aspergillus oryzae (Aspergillus oryzae) MZ30 is inoculated into an activated medium under aseptic conditions for culturing at a shaker rotation speed of 120r/min at a culture temperature of 28 ℃ for 48 h; each liter of activation medium contains 25 grams of corn starch, 6 grams of yeast extract and KNO31.2，K2HPO40.5，MgCl 0.5，NaCl 0.5，FeSO4·7H2O0.01, agar 20;
2) adding agar, yeast extract and K into the white sugar solution obtained in the step 1)2HPO4、MgSO4NaCl and NaH2PO4After being uniformly mixed, the pH value of the mixture is adjusted to 6, and the mixture is sterilized for 15min at the temperature of 115 ℃ to obtain a fermentation medium; inoculating a thallus solution of Aspergillus oryzae (MZ 30) with the strain preservation number of CGMCC No.18106 obtained in the step 1), adding glutathione, and fermenting at 25 ℃ for 30h to obtain a fermentation liquid; the mass ratio of the glutathione to the white sugar is 0.1: 100, respectively;
the inoculation amount of an Aspergillus oryzae (Aspergillus oryzae) MZ30 thallus solution in the fermentation medium is 8% of the volume of the fermentation medium;
each liter of fermentation medium contains 30 grams of white sugar, 5 grams of agar, 12 grams of yeast extract and K2HPO41、MgSO41. NaCl 1 and NaH2PO 1; the mass ratio of the glutathione to the white sugar is 0.075: 100, respectively;
3) performing tangential flow ultrafiltration on the fermentation liquor obtained in the step 2), wherein the aperture of an ultrafiltration membrane adopted by the tangential flow ultrafiltration is 0.2 mu m, removing thalli, and adding activated carbon for decolorization to obtain supernatant;
4) sequentially passing the supernatant obtained in the step 3) through an ultrafiltration membrane with the molecular weight cutoff of 400Da, D001FD macroporous strongly acidic styrene cation exchange resin and D750 macroporous strongly basic acrylic acid anion exchange resin, and collecting eluent;
5) concentrating and drying the eluent obtained in the step 4) to obtain the fructo-oligosaccharide.
Comparative example 1
1) Adding water into white sugar to prepare a white sugar solution;
taking Aspergillus oryzae (As) with the strain preservation number of CGMCC No.18106Inoculating pergillus oryzae) MZ30 to an activation culture medium under aseptic condition for culturing, wherein the rotating speed of a shaking table is 120r/min, the culture temperature is 28 ℃, and the culture time is 48 h; each liter of activation medium contains 25 grams of corn starch, 6 grams of yeast extract and KNO31.2，K2HPO40.5，MgCl 0.5，NaCl 0.5，FeSO4·7H2O0.01, agar 20;
2) adding agar, yeast extract and K into the white sugar solution obtained in the step 1)2HPO4、MgSO4NaCl and NaH2PO4After being uniformly mixed, the pH value of the mixture is adjusted to 6, and the mixture is sterilized for 15min at the temperature of 115 ℃ to obtain a fermentation medium; inoculating the strain solution of Aspergillus oryzae (MZ 30) with the strain preservation number of CGMCC No.18106 obtained in the step 1), and fermenting for 30h at 25 ℃ to obtain fermentation liquor;
the inoculation amount of an Aspergillus oryzae (Aspergillus oryzae) MZ30 thallus solution in the fermentation medium is 8% of the volume of the fermentation medium;
each liter of fermentation medium contains 30 grams of white sugar, 5 grams of agar, 12 grams of yeast extract and K2HPO41、MgSO41. NaCl 1 and NaH2PO 1; the mass ratio of the glutathione to the white sugar is 0.075: 100, respectively;
3) performing tangential flow ultrafiltration on the fermentation liquor obtained in the step 2), wherein the aperture of an ultrafiltration membrane adopted by the tangential flow ultrafiltration is 0.2 mu m, removing thalli, and adding activated carbon for decolorization to obtain supernatant;
4) sequentially passing the supernatant obtained in the step 3) through an ultrafiltration membrane with the molecular weight cutoff of 400Da, D001FD macroporous strongly acidic styrene cation exchange resin and D750 macroporous strongly basic acrylic acid anion exchange resin, and collecting eluent;
5) concentrating and drying the eluent obtained in the step 4) to obtain the fructo-oligosaccharide.
Comparative example 2
1) Adding water into white sugar to prepare a white sugar solution;
culturing the activated culture medium at a shaking table rotation speed of 120r/min at a temperature of 28 ℃ for 48 h; the mass grams of each component in each liter of activation medium is jadeRice starch 25, yeast extract 6, KNO31.2，K2HPO40.5，MgCl 0.5，NaCl0.5，FeSO4·7H2O0.01, agar 20;
2) adding agar, yeast extract and K into the white sugar solution obtained in the step 1)2HPO4、MgSO4NaCl and NaH2PO4After being uniformly mixed, the pH value of the mixture is adjusted to 6, and the mixture is sterilized for 15min at the temperature of 115 ℃ to obtain a fermentation medium; inoculating the solution obtained in the step 1), adding glutathione, and fermenting at 25 ℃ for 30h to obtain fermentation liquor; the mass ratio of the glutathione to the white sugar is 0.1: 100, respectively;
each liter of fermentation medium contains 30 grams of white sugar, 5 grams of agar, 12 grams of yeast extract and K2HPO41、MgSO41. NaCl 1 and NaH2PO 1; the mass ratio of the glutathione to the white sugar is 0.075: 100, respectively;
3) performing tangential flow ultrafiltration on the fermentation liquor obtained in the step 2), wherein the aperture of an ultrafiltration membrane adopted by the tangential flow ultrafiltration is 0.2 mu m, removing thalli, and adding activated carbon for decolorization to obtain supernatant;
4) sequentially passing the supernatant obtained in the step 3) through an ultrafiltration membrane with the molecular weight cutoff of 400Da, D001FD macroporous strongly acidic styrene cation exchange resin and D750 macroporous strongly basic acrylic acid anion exchange resin, and collecting eluent;
5) concentrating and drying the eluent obtained in the step 4) to obtain the fructo-oligosaccharide.
Comparative example 3
1) Adding water into white sugar to prepare a white sugar solution;
inoculating Aspergillus oryzae (Aspergillus oryzae) MZ30 with the strain preservation number of CGMCC No.18106 to an activation culture medium under aseptic condition for culturing, wherein the rotating speed of a shaker is 120r/min, the culture temperature is 28 ℃, and the culture time is 48 h; each liter of activation medium contains 25 grams of corn starch, 6 grams of yeast extract and KNO31.2，K2HPO40.5，MgCl 0.5，NaCl 0.5，FeSO4·7H2O0.01, agar 20;
2) adding agar into the white sugar solution obtained in the step 1)Lipid, yeast extract, K2HPO4、MgSO4NaCl and NaH2PO4After being uniformly mixed, the pH value of the mixture is adjusted to 6, and the mixture is sterilized for 15min at the temperature of 115 ℃ to obtain a fermentation medium; inoculating a thallus solution of Aspergillus oryzae (MZ 30) with the strain preservation number of CGMCC No.18106 obtained in the step 1), adding glutathione, and fermenting at 25 ℃ for 30h to obtain a fermentation liquid; the mass ratio of the glutathione to the white sugar is 0.1: 100, respectively;
the inoculation amount of an Aspergillus oryzae (Aspergillus oryzae) MZ30 thallus solution in the fermentation medium is 8% of the volume of the fermentation medium;
each liter of fermentation medium contains 30 grams of white sugar, 5 grams of agar, 12 grams of yeast extract and K2HPO41、MgSO41. NaCl 1 and NaH2PO 1; the mass ratio of the glutathione to the white sugar is 0.075: 100, respectively;
3) sequentially passing the fermentation liquor obtained in the step 2) through an ultrafiltration membrane with the molecular weight cutoff of 400Da, D001FD macroporous strongly acidic styrene cation exchange resin and D750 macroporous strongly basic acrylic acid anion exchange resin, and collecting eluent;
4) concentrating and drying the eluent obtained in the step 3) to obtain the fructo-oligosaccharide.
Comparative example 4
1) Adding water into white sugar to prepare a white sugar solution;
inoculating Aspergillus oryzae (Aspergillus oryzae) MZ30 with the strain preservation number of CGMCC No.18106 to an activation culture medium under aseptic condition for culturing, wherein the rotating speed of a shaker is 120r/min, the culture temperature is 28 ℃, and the culture time is 48 h; each liter of activation medium contains 25 grams of corn starch, 6 grams of yeast extract and KNO31.2，K2HPO40.5，MgCl 0.5，NaCl 0.5，FeSO4·7H2O0.01, agar 20;
2) adding agar, yeast extract and K into the white sugar solution obtained in the step 1)2HPO4、MgSO4NaCl and NaH2PO4After being uniformly mixed, the pH value of the mixture is adjusted to 6, and the mixture is sterilized for 15min at the temperature of 115 ℃ to obtain a fermentation medium;inoculating a thallus solution of Aspergillus oryzae (MZ 30) with the strain preservation number of CGMCC No.18106 obtained in the step 1), adding glutathione, and fermenting at 25 ℃ for 30h to obtain a fermentation liquid; the mass ratio of the glutathione to the white sugar is 0.1: 100, respectively;
the inoculation amount of an Aspergillus oryzae (Aspergillus oryzae) MZ30 thallus solution in the fermentation medium is 8% of the volume of the fermentation medium;
each liter of fermentation medium contains 30 grams of white sugar, 5 grams of agar, 12 grams of yeast extract and K2HPO41、MgSO41. NaCl 1 and NaH2PO 1; the mass ratio of the glutathione to the white sugar is 0.075: 100, respectively;
3) performing tangential flow ultrafiltration on the fermentation liquor obtained in the step 2), wherein the aperture of an ultrafiltration membrane adopted by the tangential flow ultrafiltration is 0.2 mu m, removing thalli, and adding activated carbon for decolorization to obtain supernatant;
4) sequentially passing the supernatant obtained in the step 3) through an ultrafiltration membrane with the molecular weight cutoff of 400Da and D750 macroporous strong basic acrylic acid anion exchange resin, and collecting eluent;
5) concentrating and drying the eluent obtained in the step 4) to obtain the fructo-oligosaccharide.
Comparative example 5
1) Adding water into white sugar to prepare a white sugar solution;
inoculating Aspergillus oryzae (Aspergillus oryzae) MZ30 with the strain preservation number of CGMCC No.18106 to an activation culture medium under aseptic condition for culturing, wherein the rotating speed of a shaker is 120r/min, the culture temperature is 28 ℃, and the culture time is 48 h; each liter of activation medium contains 25 grams of corn starch, 6 grams of yeast extract and KNO31.2，K2HPO40.5，MgCl 0.5，NaCl 0.5，FeSO4·7H2O0.01, agar 20;
2) adding agar, yeast extract and K into the white sugar solution obtained in the step 1)2HPO4、MgSO4NaCl and NaH2PO4After being uniformly mixed, the pH value of the mixture is adjusted to 6, and the mixture is sterilized for 15min at the temperature of 115 ℃ to obtain a fermentation medium; inoculating Aspergillus oryzae (CGMCC No. 18106) with the strain preservation number of CGMCC No.18106 obtained in the step 1)Aspergillus oryzae) MZ30 thallus solution, and adding glutathione, fermenting at 25 deg.C for 30h to obtain fermentation liquor; the mass ratio of the glutathione to the white sugar is 0.1: 100, respectively;
the inoculation amount of an Aspergillus oryzae (Aspergillus oryzae) MZ30 thallus solution in the fermentation medium is 8% of the volume of the fermentation medium;
each liter of fermentation medium contains 30 grams of white sugar, 5 grams of agar, 12 grams of yeast extract and K2HPO41、MgSO41. NaCl 1 and NaH2PO 1; the mass ratio of the glutathione to the white sugar is 0.075: 100, respectively;
3) performing tangential flow ultrafiltration on the fermentation liquor obtained in the step 2), wherein the aperture of an ultrafiltration membrane adopted by the tangential flow ultrafiltration is 0.2 mu m, removing thalli, and adding activated carbon for decolorization to obtain supernatant;
4) sequentially passing the supernatant obtained in the step 3) through an ultrafiltration membrane with the molecular weight cutoff of 400Da and D001FD macroporous strong-acid styrene cation exchange resin, and collecting eluent;
5) concentrating and drying the eluent obtained in the step 4) to obtain the fructo-oligosaccharide.
Comparative example 6
1) Adding water into white sugar to prepare a white sugar solution;
inoculating Aspergillus oryzae (Aspergillus oryzae) MZ30 with the strain preservation number of CGMCC No.18106 to an activation culture medium under aseptic condition for culturing, wherein the rotating speed of a shaker is 120r/min, the culture temperature is 28 ℃, and the culture time is 48 h; each liter of activation medium contains 25 grams of corn starch, 6 grams of yeast extract and KNO31.2，K2HPO40.5，MgCl 0.5，NaCl 0.5，FeSO4·7H2O0.01, agar 20;
2) adding agar, yeast extract and K into the white sugar solution obtained in the step 1)2HPO4、MgSO4NaCl and NaH2PO4After being uniformly mixed, the pH value of the mixture is adjusted to 6, and the mixture is sterilized for 15min at the temperature of 115 ℃ to obtain a fermentation medium; inoculating the thallus solution of Aspergillus oryzae (MZ 30) with the strain preservation number of CGMCC No.18106 obtained in the step 1), adding glutathione, and adding 2Fermenting at 5 deg.C for 30h to obtain fermentation liquid; the mass ratio of the glutathione to the white sugar is 0.1: 100, respectively;
the inoculation amount of an Aspergillus oryzae (Aspergillus oryzae) MZ30 thallus solution in the fermentation medium is 8% of the volume of the fermentation medium;
each liter of fermentation medium contains 30 grams of white sugar, 5 grams of agar, 12 grams of yeast extract and K2HPO41、MgSO41. NaCl 1 and NaH2PO 1; the mass ratio of the glutathione to the white sugar is 0.075: 100, respectively;
3) performing tangential flow ultrafiltration on the fermentation liquor obtained in the step 2), wherein the aperture of an ultrafiltration membrane adopted by the tangential flow ultrafiltration is 0.2 mu m, removing thalli, and adding activated carbon for decolorization to obtain supernatant;
4) sequentially passing the supernatant obtained in the step 3) through D001FD macroporous strong-acid styrene cation exchange resin and D750 macroporous strong-base acrylic acid anion exchange resin, and collecting eluent;
5) concentrating and drying the eluent obtained in the step 4) to obtain the fructo-oligosaccharide.
The yields and purities of fructooligosaccharides obtained in examples 1 to 3 and comparative examples 1 to 6 were measured, and the results are shown in Table 1.
TABLE 1 Experimental results for examples 1 to 3 and comparative examples 1 to 6
Yield (%) Purity (%)
Example 1 68.84 96.87
Example 2 67.59 97.13
Example 3 69.12 96.59
Comparative example 1 46.37 95.67
Comparative example 2 —— ——
Comparative example 3 66.59 85.19
Comparative example 4 67.59 84.36
Comparative example 5 65.89 83.69
Comparative example 6 66.26 85.97
As can be seen from Table 1, compared with comparative examples 1 to 6, the fructooligosaccharides prepared by the method provided by the invention in examples 1 to 3 have high yield and purity; compared with the comparative example 1, the yield of fructo-oligosaccharide can be obviously improved by adding the glutathione according to the example 3; example 3 compared with comparative example 2, with Aspergillus oryzae (Aspergillus oryzae) MZ30 with the strain preservation number of cgmccno.18106 provided by the present invention, fructooligosaccharide can be prepared, and no fructooligosaccharide is generated without adding Aspergillus oryzae (Aspergillus oryzae) MZ 30; example 3 compared with comparative example 3, the step of removing the thallus can obviously improve the purity of the fructo-oligosaccharide; example 3 compared to comparative example 4, the elimination of D001FD macroporous strongly acidic styrene cation exchange resin in the filtration step affects the yield of fructooligosaccharides; example 3 compared to comparative example 5, the elimination of D750 macroporous strongly basic acrylic acid anion exchange resin in the filtration step affects the yield of fructooligosaccharides; example 3 compared to comparative example 6, the omission of a 400Da ultrafiltration membrane from the filtration step affects the yield of fructooligosaccharides.
Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention.

Claims (10)

1, Aspergillus oryzae strain, which is separated from natural soybean paste in the northern area of Huaihei of Anhui province in China, and is named as Aspergillus oryzae (Aspergillus oryzae) MZ30, and the preservation number of the strain is CGMCC No. 18106.
2, A method for producing fructo-oligosaccharide by Aspergillus oryzae fermentation, which is characterized by comprising the following steps:
1) adding water into white sugar to prepare a white sugar solution;
inoculating Aspergillus oryzae (Aspergillus oryzae) MZ30 with the strain preservation number of CGMCC No.18106 to an activation culture medium under aseptic condition for culturing, wherein the rotating speed of a shaker is 120r/min, the culture temperature is 28 ℃, and the culture time is 48 h;
2) adding agar, yeast extract and K into the white sugar solution obtained in the step 1)2HPO4、MgSO4NaCl and NaH2PO4After uniformly mixing, adjusting the pH value of the mixture to 6-7, and sterilizing at the temperature of 115 ℃ for 15min to obtain a fermentation medium; inoculating the strain solution of Aspergillus oryzae (MZ 30) with the strain preservation number of CGMCC No.18106 obtained in the step 1), and fermenting at 25-35 ℃ for 30-50 h to obtain fermentation liquor;
3) removing thalli in the fermentation liquor obtained in the step 2), and adding activated carbon for decoloring to obtain supernatant;
4) sequentially passing the supernatant obtained in the step 3) through an ultrafiltration membrane, a cation exchange resin and an anion exchange resin, and collecting eluent;
5) concentrating and drying the eluent obtained in the step 4) to obtain the fructo-oligosaccharide.
3. The method for producing fructooligosaccharides by fermentation of Aspergillus oryzae according to claim 2, wherein in step 2), the inoculum amount of the bacterial solution of Aspergillus oryzae (Aspergillus oryzae) MZ30 in the fermentation medium is 5-10% by volume of the fermentation medium.
4. The method for producing fructooligosaccharides by Aspergillus oryzae fermentation as claimed in claim 2, wherein the mass grams of each component in each liter of activation medium is 20-30% of corn starch, 5-8% of yeast extract, and KNO31～1.5，K2HPO40.4～0.6，MgCl 0.5～0.7，NaCl 0.5～0.8，FeSO4·7H20.01-0.02% of O and 15-20% of agar.
5. The method for producing fructooligosaccharides by Aspergillus oryzae fermentation as claimed in claim 2, wherein the mass grams of each component in each liter of fermentation medium is 30-35 g of white sugar, 4-7 g of agar, 10-15 g of yeast extract, K2HPO41～2、MgSO41-2, NaCl 1-2 and NaH2PO40.5～1。
6. The method for producing fructooligosaccharides by fermentation of Aspergillus oryzae according to claim 2, wherein an enzyme activator, which is zinc chloride or glutathione, is added at the same time of inoculating Aspergillus oryzae (Aspergillus oryzae) MZ30 in step 2).
7. The method for producing fructooligosaccharides by fermentation of aspergillus oryzae according to claim 6, wherein the mass ratio of the enzyme activator to the white sugar is (0.05-0.1): 100.
8. the method for producing fructooligosaccharides by fermentation of aspergillus oryzae according to claim 2, wherein step 3) is specifically performed by subjecting the fermentation broth to tangential flow ultrafiltration, removing thalli from the fermentation broth with an ultrafiltration membrane having a pore size of 0.2 μm, and adding activated carbon for decolorization to obtain a supernatant.
9. The method for producing fructooligosaccharides by fermentation of aspergillus oryzae according to claim 2, wherein in the step 4), the ultrafiltration membrane has a molecular weight cutoff of 300-500 Da.
10. The method for producing fructooligosaccharides by fermentation of aspergillus oryzae according to claim 2, wherein in step 4), the anion exchange resin is D001FD macroporous strongly acidic styrene cation exchange resin, and the cation exchange resin is D750 macroporous strongly basic acrylic acid anion exchange resin.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022058460A1 (en) * 2020-09-17 2022-03-24 Beneo-Orafti Sa Method for producing a stable fructooligosaccharide composition, fructooligosaccharide composition, and use thereof
CN114231573A (en) * 2021-12-27 2022-03-25 南京中医药大学 Method for producing essence of heaven by using aspergillus oryzae genetic engineering bacteria

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022058460A1 (en) * 2020-09-17 2022-03-24 Beneo-Orafti Sa Method for producing a stable fructooligosaccharide composition, fructooligosaccharide composition, and use thereof
CN114231573A (en) * 2021-12-27 2022-03-25 南京中医药大学 Method for producing essence of heaven by using aspergillus oryzae genetic engineering bacteria
CN114231573B (en) * 2021-12-27 2024-04-09 南京中医药大学 Method for producing Tianjing by using aspergillus oryzae genetically engineered bacteria

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