Aspergillus oryzae BLCY-006 strain and application thereof in preparation of galactooligosaccharide

Abstract

Provided are an Aspergillus oryzae BLCY-006 strain and an application thereof in the preparation of a galactooligosaccharide. The strain produces β-galactosidase, and the enzyme activity can reach 300 U/ml after culturing and fermentation, which is more than 50% higher than traditional β-galactosidase activity. The enzyme also has lactose and glucose resistance properties.

Claims

1. An Aspergillus oryzae BLCY-006 strain, which has an accession number: CGMCC No. 16965, wherein said accession number was obtained by depositing said strain on Dec. 5, 2018 in the China General Microbiological Culture Collection Center, the Institute of Microbiology, Chinese Academy of Sciences, at the address: No. 1, West Beichen Road, Chaoyang District, Beijing.

2. A method for culturing the Aspergillus oryzae BLCY-006 according to claim 1, characterized by comprising the following steps: (1) inoculating the Aspergillus oryzae BLCY-006 into a solid medium, and performing an activating cultivation at a temperature of from 28° C. to 35° C. for 20 to 30 hours to obtain an activated strain; (2) inoculating the activated strain as obtained in step (1) into a seed culture medium, and performing a proliferating cultivation at a temperature of from 28° C. to 35° C. for 20 to 30 hours to prepare a seed broth; (3) inoculating the seed broth as prepared in step (2) into a fermentation medium at a volume percentage of 2% to 10%, and performing an expanding cultivation at a temperature of from 28° C. to 35° C. for 25 to 35 hours to obtain a bacterial fermentation broth.

3. The method according to claim 2, characterized in that the seed culture medium in step (2) has components as follows, in terms of percentages by weight: ammonium nitrate 0.2%, ammonium sulfate 0.1%, potassium dihydrogen phosphate 0.1%, urea 0.05%, peptone 1%, sucrose 2%, glucose 5%, balance water, pH 4.5 to 6.5.

4. The method according to claim 2, characterized in that the fermentation medium in step (3) has components as follows, in terms of percentages by weight: sucrose 5%, glucose 5%, peptone 1%, ammonium sulfate 0.1%; potassium dihydrogen phosphate 0.1%, balance water.

5. The method according to claim 2, characterized in that the solid medium in step (1) is potato dextrose agar (PDA) solid medium.

6. A method comprising the following steps: (a) culturing Aspergillus oryzae BLCY-006 using the method of claim 2, and collecting bacterial cells by filtration; (b) adding the bacterial cells as collected in step (a) to a pre-cooled phosphate buffer and then having them react with a pre-treated adsorbent for a reaction time of 5 to 25 hours so that the bacterial cells are fixed on a surface of the adsorbent to obtain a β-galactosidase; (c) preparing a lactose solution with a mass concentration of 40% to 60%, and adding the β-galactosidase obtained in step (b) to the lactose solution, and performing an incubating reaction for 12 hours to prepare a crude galactooligosaccharides solution; (d) subjecting the crude galactooligosaccharides solution as prepared in step (c) to decolorization, filtration, ion exchange, chromatographic separation, concentration and drying, and galactooligosaccharides were obtained.

7. The method according to claim 6, characterized in that the filtration in step (a) is carried out by adopting a plate and frame filter-press at a working pressure of 0.3 to 0.5 MPa, and a flow rate of 5 to 10 m.sup.3/h.

8. The method according to claim 6, characterized in that the adsorbent in step (b) is selected from alumina, diatomaceous earth, porous ceramics or cellulose.

9. The method according to claim 6, characterized in that the amount of β-galactosidase added in step (c) is 0.1% to 10% by weight based on the mass of lactose; the temperature of the incubating reaction is 30° C. to 60° C.

10. The method according to claim 6, characterized in that the decolorization in step (d) is carried out by adding an activated carbon in an amount of 0.1 wt %, the decolorization is performed for 1.5 hours; the chromatographic separation is carried out at an operating pressure of 0.2 MPa, a temperature of 60° C., a water consumption ratio of 1:1.2, and a feed rate of 1.8 m.sup.3 per hour.

Description

EXAMPLE 1

(1) The process for screening Aspergillus oryzae BLCY-006 was as follows:

(2) (1) Enrichment Culture

(3) The soil near the the Galactooligosaccharides Production Workshop of Bailong Chuangyuan, Dezhou City, Shandong Province, was selected, and the topsoil was removed with a small shovel; about 10 g of the soil was taken in a depth of 5 to 15 cm from the ground, diluted 10 times with sterile water, added to a PDA medium for enrichment culture, in which the medium had composition as follows:

(4) 200 g of potato, 20 g of glucose, 15 g to 20 g of agar, 1000 ml of deionized water, 0.2% ammonium nitrate; 0.1% ammonium sulfate; 0.1% dipotassium hydrogen phosphate; pH 6.5 to 7.0.

(5) The method for preparing the culture medium was as follows:

(6) 200 g of potato was cut into small pieces, added with water and boiled (boiled for 20 to 30 minutes, until the potato could be pierced by a glass rod), filtered with eight layers of gauze, heated, and then added with 1 g to 10 g of agar, 0.2% ammonium nitrate, 0.1% ammonium sulfate, 0.1% dipotassium hydrogen phosphate according to actual experiment needs; continuously heated and stirred to mix evenly. After the agar was completely dissolved, glucose was added, stirred evenly, cooled down and then added with deionized water to reach 1000 ml, subpackaged into test tubes or conical flasks, stoppered, bandaged, and sterilized (121° C.) for about 20 minutes; then the test tubes were taken out and put on a slope or shaken well, and stored for later use after cooling.

(7) (2) Separation of Pure Strain

(8) In this step the streaking method was used. A large test tube containing 5 ml of sterile water was taken, 2 ml of the bacterial solution after the enrichment culture in step (1) was taken and added to the test tube and diluted, shaken thoroughly for dispersion, a loop of the diluted solution was aseptically picked up by using an inoculation loop and subjected to the first parallel streaking of 3 to 4 streaks on one side of a plate medium; then the petri dish was turned about 60 degrees, the remainder on the inoculation loop was burned off. After cooling, the second streaking was carried out by the method same as that of the first streaking; and the third and fourth streaking were carried out in sequence by the same method. After streaking, the petri dish was covered with a lid, turned upside down, and incubated at a temperature of from 28° C. to 35° C. for 30 hours, then single colony was picked up and inoculated on 10 slant culture media to obtain 10 slant seeds, numbered 01 to 10.

(9) The 01 to 10 slant seeds were separately inoculated into shake flask medium and cultured at a temperature of from 28° C. to 35° C. for 30 hours. The β-galactosidase enzyme activities of the 01 to 10 shake flask fermentation broths were measured, in which the enzyme activity of the 08 shake flask was the highest, reaching 105 U/ml.

(10) The composition of the plate medium was as follows:

(11) 200 g of potato, 20 g of glucose, 15 g to 20 g of agar, 1000 ml of deionized water, 0.2% ammonium nitrate; 0.1% ammonium sulfate; 0.1% dipotassium hydrogen phosphate; pH 6.5 to 7.0.

(12) The composition of the shake flask medium was as follows:

(13) 100 ml of leaching juice of soybean cake, added with 2 g of soluble starch, 0.1 g of potassium dihydrogen phosphate, 0.05 g of magnesium sulfate, 0.05 g of ammonium sulfate, and 2 g of agar, natural pH.

(14) The method for preparing the leaching juice of soybean cake comprised: 100 g of soybean cake was added with 500 ml of water, soaked for 4 hours, boiled for 3 to 4 hours, naturally filtered with gauze, and the liquid was taken and adjusted to 5 Baume.

(15) (3) Mutagenesis Screening

(16) The 08 strain was subjected to ultraviolet mutagenesis. The ultraviolet mutagenesis was performed by irradiation with a 20 W ultraviolet lamp from a distance of 15 cm, and the irradiation time was 200 s. The obtained high-yield strain was subjected to ion implantation mutagenesis treatment, and finally a high-yield β-galactosidase strain was obtained, named as BLCY-006, which had an enzyme activity of 300 U/ml. The strain was white and yellow at first, then turned yellowish brown to light greenish brown. The conidia heads were radial with a diameter of 150 to 300 μm, and a few were loose columnar. The conidiophore was about 2 mm. The strain was identified as Aspergillus oryzae.

(17) The aforementioned Aspergillus oryzae was named as BLCY-006, deposited under Accession number: CGMCC No. 16965, wherein said Accession number was obtained by depositing said strain on Dec. 5, 2018 in the China General Microbiological Culture Collection Center, the Institute of Microbiology, Chinese Academy of Sciences, at the address: No. 1, West Beichen Road, Chaoyang District, Beijing.

EXAMPLE 2

(18) The method for culturing the Aspergillus oryzae BLCY-006 as described in Example 1 comprised the following steps:

(19) (1) the Aspergillus oryzae BLCY-006 was taken and inoculated into a PDA medium, and an activating cultivation was performed at 30° C. for 30 hours to obtain an activated strain;

(20) (2) the activated strain as obtained in step (1) was taken and inoculated into a seed culture medium, and a proliferating cultivation was performed at 30° C. for 30 hours to prepare a seed broth;

(21) the composition of the seed medium was as follows:

(22) 100 ml of leaching juice of soybean cake, added with 2 g of soluble starch, 0.1 g of potassium dihydrogen phosphate, 0.05 g of magnesium sulfate, 0.05 g of ammonium sulfate, and 2 g of agar, natural pH;

(23) the method for preparing the leaching juice of soybean cake comprised: 100 g of soybean cake was added with 500 ml of water, soaked for 4 hours, boiled for 3 to 4 hours, naturally filtered with gauze, and the liquid was taken and adjusted to 5 Baume;

(24) (3) the seed broth as prepared in step (2) was taken and inoculated into a fermentation medium at a volume percentage of 1%, and an expanding cultivation was performed at 30° C. for 35 hours to obtain a bacterial fermentation broth;

(25) the fermentation medium had components as follows, in terms of percentages by weight:

(26) sucrose 10%, bran 2%, yeast extract 1%, sodium nitrate 0.3%, MgSO.sub.4.7H.sub.2O 0.05%, balance water.

EXAMPLE 3

(27) The method for culturing the Aspergillus oryzae BLCY-006 as described in Example 1 comprised the following steps:

(28) (1) the Aspergillus oryzae BLCY-006 was taken and inoculated into a PDA medium, and an activating cultivation was performed at 35° C. for 20 hours to obtain an activated strain;

(29) (2) the activated strain as obtained in step (1) was taken and inoculated into a seed culture medium, and a proliferating cultivation was performed at 35° C. for 20 hours to prepare a seed broth;

(30) the composition of the seed medium was as follows:

(31) 100 ml of leaching juice of soybean cake, added with 2 g of soluble starch, 0.1 g of potassium dihydrogen phosphate, 0.05 g of magnesium sulfate, 0.05 g of ammonium sulfate, and 2 g of agar, natural pH;

(32) the method for preparing the leaching juice of soybean cake comprised: 100 g of soybean cake was added with 500 ml of water, soaked for 4 hours, boiled for 3 to 4 hours, naturally filtered with gauze, and the liquid was taken and adjusted to 5 Baume;

(33) (3) the seed broth as prepared in step (2) was taken and inoculated into a fermentation medium at a volume percentage of 10%, and an expanding cultivation was performed at 38° C. for 20 hours to obtain a bacterial fermentation broth;

(34) the fermentation medium had components as follows, in terms of percentages by weight:

(35) sucrose 10%, bran 2%, yeast extract 1%, sodium nitrate 0.3%, MgSO.sub.4.7H.sub.2O 0.05%, balance water.

EXAMPLE 4

(36) The method for culturing the Aspergillus oryzae BLCY-006 as described in Example 1 comprised the following steps:

(37) (1) the Aspergillus oryzae BLCY-006 was taken and inoculated into a PDA medium, and an activating cultivation was performed at 32° C. for 25 hours to obtain an activated strain;

(38) (2) the activated strain as obtained in step (1) was taken and inoculated into a seed culture medium, and a proliferating cultivation was performed at 32° C. for 25 hours to prepare a seed broth;

(39) the composition of the seed medium was as follows:

(40) 100 ml of leaching juice of soybean cake, added with 2 g of soluble starch, 0.1 g of potassium dihydrogen phosphate, 0.05 g of magnesium sulfate, 0.05 g of ammonium sulfate, and 2 g of agar, natural pH;

(41) the method for preparing the leaching juice of soybean cake comprised: 100 g of soybean cake was added with 500 ml of water, soaked for 4 hours, boiled for 3 to 4 hours, naturally filtered with gauze, and the liquid was taken and adjusted to 5 Baume;

(42) (3) the seed broth as prepared in step (2) was taken and inoculated into a fermentation medium at a volume percentage of 10%, and an expanding cultivation was performed at 38° C. for 20 hours to obtain a bacterial fermentation broth;

(43) the fermentation medium had components as follows, in terms of percentages by weight:

(44) sucrose 10%, bran 2%, yeast extract 1%, sodium nitrate 0.3%, MgSO.sub.4.7H.sub.2O 0.05%, balance water.

COMPARATIVE EXAMPLE 1

(45) The original strain of Aspergillus oryzae obtained from the soil, but not yet subjected to mutagenesis and screening, was cultured, and the culture conditions were the same as those in Example 2.

COMPARATIVE EXAMPLE 2

(46) Chinese patent application CN101691538A (application number: 200910018452.1) disclosed a method for preparing high-purity galactooligosaccharides. The Aspergillus oryzae BLB-21 (Accession number: CGMCC No. 2951) disclosed in the patent application was taken and cultured, and the culture conditions were the same as those in Example 2.

EXPERIMENTAL EXAMPLE 1

(47) The bacterial fermentation broths obtained by culturing in Examples 2 to 4 and Comparative Examples 1 to 2 were taken, and the enzyme activities of β-galactosidase in these fermentation broths were detected. The results were shown in Table 1:

(48) TABLE-US-00001 TABLE 1 Enzyme activity of bacterial fermentation broth Group Enzyme activity of β-galactosidase Example 2 305 U/mL Example 3 311 U/mL Example 4 315 U/mL Comparative Example 1 162 U/mL Comparative Example 2 189 U/mL

(49) It can be seen from the data in Table 1, when Examples 2 to 4 were compared with Comparative Examples 1 to 2, the β-galactosidase enzyme activities in the bacterial fermentation broths prepared by Aspergillus oryzae BLCY-006 provided by the present invention were significantly improved.

EXPERIMENTAL EXAMPLE 2

(50) The use of Aspergillus oryzae BLCY-006 in the production of β-galactosidase comprised the following steps:

(51) (a) the bacterial fermentation broths prepared in Examples 2 to 4 and Comparative Examples 1 to 2 were taken and filtered by a plate and frame filter-press at a working pressure of 0.3 to 0.5 MPa, and a flow rate of 5 to 10 m.sup.3/h, and bacterial cells were collected;

(52) (b) the bacterial cells as collected in step (a) were added to a pre-cooled phosphate buffer and then reacted with a pre-treated adsorbent for a reaction time of 15 hours so that the bacterial cells were fixed on a surface of the adsorbent to obtain β-galactosidases;

(53) (c) lactose solutions with mass concentrations of 40% and 60% were prepared, respectively, the β-galactosidases obtained in step (b) were added to the lactose solutions at an addition amount of 5 wt % based on the mass of lactose, and an incubating reaction was performed for 12 hours to prepare crude galactooligosaccharide;

(54) (d) the crude galactooligosaccharides solution as prepared in step (c) were subjected to decolorization, filtration, ion exchange, chromatographic separation, concentration and drying, and galactooligosaccharides were obtained.

(55) Wherein, in step (d), the decolorization was carried out by adding an activated carbon in an amount of 0.1 wt % and the decolorization was performed for 1.5 hours; the chromatographic separation was carried out at an operating pressure of 0.2 MPa, a temperature of 60° C., a water consumption ratio of 1:1.2, and a feed rate of 1.8 m.sup.3 per hour.

(56) The crude galactooligosaccharides solution prepared in step (c) were taken and tested for glucose content, lactose content, galactose content, and Galactooligosaccharides content; the galactooligosaccharides prepared in step (d) were taken and tested for the yield of galactooligosaccharides; and the results were shown in Tables 2 to 3.

(57) TABLE-US-00002 TABLE 2 Various indexes of production of Galactooligosaccharides from 40% lactose solution Crude galactooligosaccharides solution Galactooligosaccharides Galacto- Galacto- Galacto- Galactose Glucose Lactose oligosaccharides oligosaccharides oligosaccharides content/% content/% content/% content/% purity/% yield/% Example 2 5.48 21.36 11.64 61.52 76.71 92.95 Example 3 5.76 20.33 11.62 62.29 86.59 93.12 Example 4 6.07 21.44 11.16 61.33 86.22 93.01 Comparative 7.93 32.36 19.13 40.24 70.39 75.32 Example 1 Comparative 9.69 28.75 16.01 45.55 72.47 77.25 Example 2

(58) TABLE-US-00003 TABLE 3 Various indexes of production of Galactooligosaccharides from 60% lactose solution Crude galactooligosaccharides solution Galactooligosaccharide Galacto- Galacto- Galacto- Galactose Glucose Lactose oligosaccharide oligosaccharide oligosaccharide content/% content/% content/% content/% purity/% yield/% Example 2 4.54 22.56 11.59 61.31 86.32 92.83 Example 3 5.07 21.14 11.44 62.35 86.45 93.26 Example 4 5.70 21.29 11.26 61.75 86.17 93.14 Comparative 7.93 32.36 19.13 40.58 70.25 75.28 Example 1 Comparative 11.02 28.95 15.31 44.72 72.36 77.39 Example 2

(59) It can be seen from the above data that by using the bacterial fermentation broths prepared by Aspergillus oryzae BLCY-006 provided by the present invention, the degree of utilization of lactose in the crude galactooligosaccharides solution prepared in Examples 2 to 4 were not lower than 88%, the Galactooligosaccharides contents reached more than 61%; while the degree of utilization of lactose in Comparative Examples 1 to 2 were only about 81%, and the Galactooligosaccharides contents in the crude galactooligosaccharides solution were only about 40%. It could be concluded from the comparison that the using Aspergillus oryzae provided by the present invention for preparing galactooligosaccharides could significantly improve the degree of utilization of lactose, and the content of galacto-oligosaccharides in the produced crude galacto-oligosaccharide solution was also significantly increased.

(60) From the analysis of the final galactooligosaccharides product as obtained, the galactooligosaccharides prepared in Examples 2 to 4 all had a purity of above 85%, and the galactooligosaccharides prepared in Comparative Examples 1 to 2 had a purity of not exceeding 75%. The galactooligosaccharides prepared using the bacterial fermentation broths of Examples 2 to 4 had a yield of about 92%, while the galactooligosaccharides prepared using the bacterial fermentation broths of Comparative Examples 1 to 2 had a yield of only about 75%. As compared to Comparative Examples 1 to 2, Examples 2 to 4 had significantly improved purity and yield of galactooligosaccharides.