A kind of budding Aureobasidium pullulans and method for producing dietary fiber by fermentation

Abstract

The present invention relates to a budding short-stalked mold and a method for fermenting dietary fiber thereof, and belongs to the field of microbial technology. In order to solve the problem that byproducts of potato starch industry are seriously polluted and difficult to handle, the present invention provides a budding short-stalked mold and a method for fermenting dietary fiber thereof, potato residue and potato juice are used as fermentation substrates, budding short-stalked mold is added, fermentation is carried out under the conditions of 26-30°C and ventilation volume of 0.1-0.3vvm, fermentation is carried out for 2-10 days to obtain fermentation liquid, solid-liquid separation of the obtained fermentation liquid is carried out, the supernatant is collected and alcohol precipitation treatment is carried out, and the precipitate is collected to obtain dietary fiber, the obtained dietary fiber has a proliferation effect on lactobacillus and bifidobacterium, can be used for the production of food and health products such as biscuits and meal replacement powder, realizes the resource utilization of pollutants, and has good social and economic benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a budding Aureobasidium pullulans and a method for producing dietary fiber by fermentation thereof. Background Art

[0002] Potatoes have high nutritional and economic value and are widely used in food, starch, feed, medicine and other fields, especially in the starch industry. The production of potato starch is accompanied by the production of by-products such as potato residue and potato juice. The nutritional components of potato residue include starch, hemicellulose, cellulose, pectin, free amino acids, ash, polypeptides, oligopeptides, etc. The rich crude fiber, pectin, starch, etc. contained in it have high recycling value. Potato juice comes from the cell water separated from the potato raw materials during the physical processing process. The solid with the highest relative content in the juice is potato protein. If the potato juice is not recycled, the protein is directly discharged into the wastewater treatment station, which not only increases the wastewater treatment load, but also causes a waste of high-quality protein resources.

[0003] The dietary fiber content in potato residue is as high as 50-60%, which is a high-quality dietary fiber raw material. The process of preparing dietary fiber with potato residue as raw material mainly uses amylase and protease treatment to remove impurities, and then uses cellulase treatment to improve the dietary fiber component or increase the soluble dietary fiber content. However, the moisture and starch content in potato residue are very high, and the starch is tightly wrapped in pectin and cellulose components, making it difficult to effectively remove starch, the main interfering component. How to more efficiently achieve the green treatment and development and utilization of potato starch industrial by-products is still a technical problem to be solved by technicians in this field. Summary of the invention

[0004] The invention provides a budding Aureobasidium pullulans and a method for producing dietary fiber by fermentation.

[0005] The technical solution of the present invention:

[0006] A budding short-stalked mold, the budding short-stalked mold was deposited in the China Center for Type Culture Collection on October 27, 2023, the deposit address is Wuhan University, Wuhan, China, the deposit number is CCTCC NO: M20232055, and the classification name is Aureobasidium pullulans YQ-65.

[0007] A method for producing dietary fiber by fermenting budding Aureobasidium pullulans, comprising: taking potato residue and potato juice as fermentation substrates, inoculating budding Aureobasidium pullulans, fermenting at 26-30 DEG C and with an aeration volume of 0.1-0.3 vvm, fermenting for 2-10 days to obtain a fermentation liquid, separating the obtained fermentation liquid into solid and liquid, collecting the supernatant and performing alcohol precipitation treatment, and collecting the precipitate to obtain the dietary fiber.

[0008] Furthermore, the mass ratio of the potato residue to the potato juice is 1:1 to 1:3.

[0009] Furthermore, the pH of the fermentation system is maintained at 6.0.

[0010] Furthermore, 20% sucrose is added to the fermentation substrate.

[0011] Furthermore, 1% ammonium sulfate is added to the fermentation substrate.

[0012] Furthermore, the inoculation amount of the budding Aureobasidium pullulans is 0.5 to 2‰ of the mass of dry matter in the fermentation substrate.

[0013] Furthermore, the solid-liquid separation is carried out by centrifugal separation, and the centrifugal speed is 3000 rpm.

[0014] Furthermore, during the alcohol precipitation treatment, 1 to 2 volumes of anhydrous ethanol are added to the supernatant, and the precipitate is collected after 6 to 12 hours.

[0015] Furthermore, the precipitate collected after the alcohol precipitation treatment is dried and crushed, and the drying is performed at 60° C. overnight.

[0016] Beneficial effects of the present invention:

[0017] The present invention provides a budding short-stalked mold, and uses the budding short-stalked mold to ferment potato juice and potato residue to produce dietary fiber, with a dietary fiber yield of more than 30 g / L, solving the problem that byproducts of the potato starch industry are seriously polluted and difficult to handle. The dietary fiber obtained by fermentation has a proliferation effect on lactobacilli and bifidobacteria, can be used for the production of foods such as biscuits and meal replacement powders, health products, and pharmaceuticals, realizes the resource utilization of pollutants, and has good social and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The curve diagram of the change of dry matter content in the fermentation broth at different fermentation times in Example 2;

[0019] Figure 2 This is a comparison chart of the proliferation effects of dietary fiber obtained by fermentation for 6 days in Example 2 on lactobacilli and bifidobacteria;

[0020] Figure 3 This is a comparison chart of the dietary fiber yields obtained by fermenting for 6 days in Example 2 and in Comparative Example 1 for 6 days. DETAILED DESCRIPTION

[0021] The present invention is further described below in conjunction with the examples. The process equipment or devices not specifically noted in the following examples are all conventional equipment or devices in the art. If not specifically noted, the raw materials used in the examples of the present invention can be obtained commercially. If not specifically noted, the technical means used in the examples of the present invention are all conventional means well known to those skilled in the art.

[0022] Example 1

[0023] This embodiment provides a budding Aureobasidium pullulans and a method for isolating and identifying the same.

[0024] 1. Sample collection and pretreatment

[0025] The humus soil obtained from under the cherry trees in Purple Mountain, Nanjing, China (32.07°N, 118.83°E) was placed in a petri dish and dried in a cool place for 7 days, then ground and set aside for use.

[0026] 2. Isolation and culture of strains

[0027] Accurately weigh 5 g of sample and place it in a conical flask containing 45 mL of sterile water. Add sterile glass beads and shake on a shaker for 30 min. Take 1 mL of the supernatant for gradient dilution and select 10 -4 , 10 -5 , 10 -6 Three concentration gradients were set up, and three parallel experiments were set up. 200 μL of each concentration gradient was applied to the screening medium, and after 1 day of upright culture, it was placed in a 30°C incubator for 3-4 days. The growth of the strain on the medium was observed regularly, and the colonies were picked in time and transferred to the YPD medium without resistance. After multiple passages and purification, a single colony with stable morphology was grown, which was numbered YQ-65. The isolated strain was inoculated into the seed medium for culture. After 3 days of culture, 1 mL was taken and placed in 70% glycerol and stored in a -80°C refrigerator.

[0028] 3. Molecular Biological Identification of Strains

[0029] The genomic DNA of strain YQ-65 was extracted, separated and purified using the Tiangen yeast genomic DNA extraction kit DP307-02. The genomic DNA was used as a template and PCR amplification was performed using primer ITS1 with a nucleotide sequence as shown in SEQ ID No.1 and primer ITS4 with a nucleotide sequence as shown in SEQ ID No.2.

[0030] SEQ ID No.1:ITS1(5′→3′):TCCGTAGGTGAACCTGCGG;

[0031] SEQ ID No. 2: ITS4 (5′→3′): GCTGCGTTCTTCATCGATGC.

[0032] The PCR amplification reaction system is as follows:

[0033] 10.0 μL 2×Premix Taq enzyme, 1.0 μL DNA template, 1.0 μL 10.0 μmol / L ITS1, 1.0 μL 10.0 μmol / L ITS4, 7.0 μL ddH2O, a total of 20 μL.

[0034] The PCR amplification reaction conditions are as follows:

[0035] 94 ℃ for 5 min; 94 ℃ for 30 s, 50 ℃ for 1 min, 72 ℃ for 1 min, 30 cycles; 72 ℃ for 10 min.

[0036] After the PCR amplification results were verified by gel electrophoresis, the target fragment was recovered, connected with the pMD18T vector and transferred into the competent cells of Escherichia coli (DH5α), the positive clones were picked, the plasmids were extracted and verified, and then the ITS sequence was determined. The nucleotide sequence of the ITS sequence of strain YQ-65 was obtained by sequencing as shown in SEQ ID No.3.

[0037] The ITS sequence obtained by sequencing was compared with the known sequences in the Genebank database, and it was determined that the isolated and purified strain YQ-65 belonged to the Ascomycetes, Docomycetes, and Chrysobasidiaceae in the Fungi kingdom. Aureobasidium The genus is Aureobasidium, which is classified as Aureobasidium pullulans YQ-65 was deposited in the China Center for Type Culture Collection on October 27, 2023, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M20232055.

[0038] Example 2

[0039] This embodiment provides a method for producing dietary fiber by fermenting Aureobasidium pullulans in Example 1, and the specific steps are as follows:

[0040] Step 1: Press the budding mold in Example 1 for 10 8 cfu / mL was inoculated into seed culture medium, and the seed solution was obtained after culturing at 28°C and 180 rpm for 1 day. The seed culture medium of Aureobasidium pullulans was PDB medium.

[0041] Step 2: potato residue and potato juice with a mass ratio of 1:2 were used as fermentation substrates, added into a 5L airlift fermenter, sterilized at 121°C for 15 min, and after cooling to room temperature, the budding short-stalked mold seed liquid was inoculated into the fermenter with a peristaltic pump. The inoculation amount was 1‰ of the dry matter mass in the fermentation substrate. During this period, the pH of the fermentation system was controlled at 6.0, and the fermentation was carried out under the fermentation conditions of a temperature of 28°C and a ventilation volume of 0.2 vvm for 10 days. 100 mL of fermentation broth was taken out at 2, 4, 6, 8 and 10 days, and the solid-liquid separation of the obtained fermentation broth was carried out at a centrifugal speed of 3000 rpm, the amount of dry matter was determined, and the cell protein was used after drying. The protein content in the dry matter was detected by the Kjeldahl method; the centrifuged supernatant was taken, 1 volume of anhydrous ethanol was added to the supernatant, and the precipitate was collected after 12 hours, dried at 60°C overnight, and crushed to obtain dietary fiber, and the content of water-soluble dietary fiber and water-insoluble dietary fiber was detected.

[0042] Figure 1 It is a curve diagram of the change of dry matter in the fermented liquid at different fermentation times in this embodiment; the dry matter at the beginning of fermentation is 35g of potato residue added, and the rapid decline of dry matter content at 2 days is due to the consumption of the growth and reproduction of budding short stalk mold in the early stage of fermentation. With the increase of fermentation days, the potato residue content gradually decreases, but the biomass of budding short stalk mold gradually increases. When the fermentation is 6d, the dry matter of the fermented liquid is about 19 g / L, and then it remains basically unchanged with the increase of days.

[0043] The results of the protein content test in the fermentation broth at different fermentation times in this example are shown in Table 1.

[0044] Table 1

[0045]

[0046] As shown in Table 1, the protein content on day 0 of fermentation is the protein in potato residue. As the fermentation days increase, the protein content also increases, reaching a maximum value of 23.2% on day 10.

[0047] The yields of dietary fiber in the fermentation broth at different fermentation times in this example are shown in Table 2.

[0048] Table 2

[0049]

[0050] As can be seen from Table 2, the dietary fiber content reached the highest yield at 6 days of fermentation, reaching 30.93 g / L, of which the water-soluble dietary fiber content was 24.16 g / L and the water-insoluble dietary fiber content was 6.76 g / L.

[0051] The dietary fiber obtained by fermentation for 6 days was used as substrate to examine the proliferation of lactobacilli and bifidobacteria.

[0052] The lactobacillus used was purchased from the China Industrial Microbiological Culture Collection Center, product number CICC23941;

[0053] The bifidobacterium used was purchased from China Industrial Microbiological Culture Collection Center with product number CICC 6070.

[0054] Lactobacillus or Bifidobacterium were 10 8 cfu / mL was inoculated into LB seed medium and cultured at 28°C, 180 rpm for 1 day to obtain the seed solution.

[0055] Using MRS medium, the dietary fiber obtained by fermentation for 6 days in this example was added to the fermentation substrate at 0, 0.2, 0.4, 0.6, 0.8 and 1.0%, respectively, and sterilized at 121° C. for 15 min. After cooling to room temperature, the lactobacillus or bifidobacterium seed liquid was inoculated into the fermentation tank using a peristaltic pump, and the inoculation amount was 1%. During the fermentation, the pH of the fermentation system was controlled at 6.0, and the fermentation was carried out under the fermentation conditions of a temperature of 28° C. and a ventilation volume of 0.2 vvm for 6 days.

[0056] Figure 2 The figure is a comparison of the proliferation effects of the dietary fiber obtained by fermentation for 6 days on lactobacillus and bifidobacterium in this embodiment. It can be found from the figure that under the condition of not adding dietary fiber, the logCFU of lactobacillus and bifidobacterium are 5.6 and 5.1 respectively, but with the addition of dietary fiber, lactobacillus and bifidobacterium have good proliferation effects, and the best is when the addition amount is 0.8%, at which time the logCFU of lactobacillus and bifidobacterium reaches 11.5 and 10.3 respectively. It shows that the dietary fiber obtained by fermentation has a prebiotic effect.

[0057] Example 3

[0058] This embodiment provides a method for producing dietary fiber by fermenting Aureobasidium pullulans in Example 1, and the specific steps are as follows:

[0059] Step 1: Press the budding mold in Example 1 for 10 8 cfu / mL was inoculated into seed culture medium, and the seed solution was obtained after culturing at 28°C and 180 rpm for 1 day. The seed culture medium of Aureobasidium pullulans was PDB medium.

[0060] Step 2: potato residue and potato juice in a mass ratio of 1:1 are used as fermentation substrates, added into a 5L airlift fermenter with a liquid volume of 2‰, sterilized at 121°C for 15min, and after cooling to room temperature, the budding short-stalked mold seed liquid is inoculated into the fermenter with a peristaltic pump, and the inoculation amount is 1‰ of the dry matter mass in the fermentation substrate. During this period, the pH of the fermentation system is controlled at 6.0, and the fermentation is carried out under the fermentation conditions of a temperature of 28°C and a ventilation volume of 0.2vvm. The fermentation is carried out for 6 days, and the obtained fermentation liquid is separated into solid and liquid at a centrifugal speed of 3000rpm, and dried as cell protein; the centrifuged supernatant is taken, 1 volume of anhydrous ethanol is added to the supernatant, the precipitate is collected after 12h, and it is dried at 60°C overnight and crushed to obtain dietary fiber.

[0061] Example 4

[0062] This embodiment provides a method for producing dietary fiber by fermenting Aureobasidium pullulans in Example 1, and the specific steps are as follows:

[0063] Step 1: Press the budding mold in Example 1 for 10 8 cfu / mL was inoculated into seed culture medium, and the seed solution was obtained after culturing at 28°C and 180 rpm for 1 day. The seed culture medium of Aureobasidium pullulans was PDB medium.

[0064] Step 2: potato residue and potato juice in a mass ratio of 1:3 are used as fermentation substrates, added into a 5L airlift fermenter, sterilized at 121°C for 15 minutes, and after cooling to room temperature, the budding Aureobasidium pullulans seed liquid is inoculated into the fermenter with a peristaltic pump, and the inoculation amount is 1‰ of the dry matter mass in the fermentation substrate. During this period, the pH of the fermentation system is controlled at 6.0, and the fermentation is carried out under the fermentation conditions of a temperature of 28°C and a ventilation volume of 0.2vvm. The fermentation is carried out for 6 days, and the obtained fermentation liquid is separated into solid and liquid at a centrifugal speed of 3000rpm, and dried as cell protein; the centrifuged supernatant is taken, 1 volume of anhydrous ethanol is added to the supernatant, the precipitate is collected after 12 hours, and it is dried at 60°C overnight and crushed to obtain dietary fiber.

[0065] Example 5

[0066] This embodiment provides a method for producing dietary fiber by fermenting Aureobasidium pullulans in Example 1, and the specific steps are as follows:

[0067] Step 1: Press the budding mold in Example 1 for 10 8 cfu / mL was inoculated into seed culture medium, and the seed solution was obtained after culturing at 28°C and 180 rpm for 1 day. The seed culture medium of Aureobasidium pullulans was PDB medium.

[0068] Step 2: potato residue and potato juice in a mass ratio of 1:2 are used as fermentation substrates, added into a 5L airlift fermenter, sterilized at 121°C for 15min, and after cooling to room temperature, the budding Aureobasidium pullulans seed liquid is inoculated into the fermenter with a peristaltic pump, and the inoculation amount is 0.5‰ of the dry matter mass in the fermentation substrate. During this period, the pH of the fermentation system is controlled at 6.0, and the fermentation is carried out under the fermentation conditions of a temperature of 28°C and a ventilation volume of 0.2vvm. The fermentation is carried out for 6 days, and the obtained fermentation liquid is separated into solid and liquid at a centrifugal speed of 3000rpm, and dried as cell protein; the centrifuged supernatant is taken, 1 volume of anhydrous ethanol is added to the supernatant, the precipitate is collected after 12h, and it is dried at 60°C overnight and crushed to obtain dietary fiber.

[0069] Example 6

[0070] This embodiment provides a method for producing dietary fiber by fermenting Aureobasidium pullulans in Example 1, and the specific steps are as follows:

[0071] Step 1: Press the budding mold in Example 1 for 10 8 cfu / mL was inoculated into seed culture medium, and the seed solution was obtained after culturing at 28°C and 180 rpm for 1 day. The seed culture medium of Aureobasidium pullulans was PDB medium.

[0072] Step 2: Using potato residue and potato juice in a mass ratio of 1:2 as fermentation substrates, add them into a 5L airlift fermenter, sterilize at 121°C for 15 minutes, and after cooling to room temperature, inoculate the budding Aureobasidium pullulans seed liquid into the fermenter with a peristaltic pump, and the inoculation amount is 2‰ of the dry matter mass in the fermentation substrate. During this period, the pH of the fermentation system is controlled at 6.0, and the fermentation is carried out under the fermentation conditions of a temperature of 28°C and a ventilation volume of 0.2vvm. The fermentation is carried out for 6 days, and the obtained fermentation liquid is separated into solid and liquid at a centrifugal speed of 3000rpm, and dried as cell protein; taking the centrifugal supernatant, adding 1 volume of anhydrous ethanol to the supernatant, collecting the precipitate after 12 hours, drying at 60°C overnight, and crushing to obtain dietary fiber.

[0073] Example 7

[0074] This embodiment provides a method for producing dietary fiber by fermenting Aureobasidium pullulans in Example 1, and the specific steps are as follows:

[0075] Step 1: Press the budding mold in Example 1 for 10 8 cfu / mL was inoculated into seed culture medium, and the seed solution was obtained after culturing at 28°C and 180 rpm for 1 day. The seed culture medium of Aureobasidium pullulans was PDB medium.

[0076] Step 2: potato residue and potato juice in a mass ratio of 1:2 are used as fermentation substrates, added into a 5L airlift fermenter, sterilized at 121°C for 15min, and after cooling to room temperature, the budding Aureobasidium pullulans seed liquid is inoculated into the fermenter with a peristaltic pump, and the inoculation amount is 1‰ of the dry matter mass in the fermentation substrate. During this period, the pH of the fermentation system is controlled at 6.0, and the fermentation is carried out under the fermentation conditions of a temperature of 28°C and a ventilation volume of 0.2vvm. The fermentation is carried out for 6 days, and the obtained fermentation liquid is separated into solid and liquid at a centrifugal speed of 3000rpm, and dried as cell protein; the centrifuged supernatant is taken, 2 times the volume of anhydrous ethanol is added to the supernatant, and the precipitate is collected after 6 hours, dried at 60°C overnight, and crushed to obtain dietary fiber.

[0077] Comparative Example 1

[0078] In this comparative example, dietary fiber was prepared by fermenting potato industrial byproducts with Bacillus subtilis. The Bacillus subtilis used in this comparative example was purchased from China General Microbiological Culture Collection Center with a product number of CGMCC 1.15792.

[0079] Bacillus subtilis 10 8 cfu / mL was inoculated into the seed culture medium, and the seed solution was obtained after culturing at 28°C and 180 rpm for 1 day. The seed culture medium of Bacillus subtilis was LB medium.

[0080] The method for producing dietary fiber by fermentation with Bacillus subtilis is as follows:

[0081] Potato residue and potato juice with a mass ratio of 1:2 were used as fermentation substrates, added into a 5L airlift fermenter, sterilized at 121°C for 15 min, and after being cooled to room temperature, Bacillus subtilis seed liquid was inoculated into the fermenter with a peristaltic pump, and the inoculation amount was 1‰ of the dry matter mass in the fermentation substrate. During this period, the pH of the fermentation system was controlled at 6.0, and fermentation was carried out under the fermentation conditions of a temperature of 28°C and a ventilation volume of 0.2vvm. The fermentation lasted for 6 days, and the obtained fermentation liquid was separated into solid and liquid at a centrifugal speed of 3000rpm, and dried as cell protein; the centrifuged supernatant was taken, 2 times the volume of anhydrous ethanol was added to the supernatant, and the precipitate was collected after 6 hours, dried at 60°C overnight, and crushed to obtain dietary fiber.

[0082] Figure 3 This is a comparison chart of the yield of dietary fiber obtained by fermenting for 6 days in Example 2 and fermenting for 6 days in Comparative Example 1; it can be found that the total amount of dietary fiber produced by budding Aureobasidium pullulans after 6 days of fermentation is 30.9 g / L, which is much greater than the 21.8 g / L produced by Bacillus subtilis, and the content of water-soluble dietary fiber and water-insoluble dietary fiber produced by budding Aureobasidium pullulans is also higher than that of Bacillus subtilis.

Claims

1. A budding Aureobasidium pullulans, characterized in that: The budding short-stalked mold was deposited in the China Center for Type Culture Collection on October 27, 2023, with the deposit address being Wuhan University, Wuhan, China, with the deposit number being CCTCC NO: M20232055, and the classification name being Aureobasidium pullulans YQ-65.

2. A method for producing dietary fiber by fermenting Aureobasidium pullulans according to claim 1, characterized in that: Potato residue and potato juice are used as fermentation substrates, budding Aureobasidium pullulans is inoculated, and fermentation is carried out at 26-30°C and aeration volume of 0.1-0.3 vvm. Fermentation liquid is obtained after 2-10 days of fermentation, and the obtained fermentation liquid is separated into solid and liquid, the supernatant is collected and subjected to alcohol precipitation treatment, and the precipitate is collected to obtain dietary fiber.

3. The method according to claim 2, characterized in that The mass ratio of the potato residue to the potato juice is 1:1 to 1:

3.

4. The method according to claim 2 or 3, characterized in that: The pH of the fermentation system was maintained at 6.

0.

5. The method according to claim 4, characterized in that The inoculation amount of the budding Aureobasidium pullulans is 0.5-2‰ of the mass of dry matter in the fermentation substrate.

6. The method according to claim 5, characterized in that The solid-liquid separation adopts centrifugal separation method, and the centrifugal speed is 3000rpm.

7. The method according to claim 6, characterized in that During the alcohol precipitation treatment, 1 to 2 volumes of anhydrous ethanol are added to the supernatant, and the precipitate is collected after 6 to 12 hours.

8. The method according to claim 7, characterized in that The precipitate collected after the alcohol precipitation treatment is dried and crushed, and the drying is performed at 60° C. overnight.

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