Method for preparing konjac oligosaccharide by fermentation of konjac powder

Abstract

The invention relates to the technical field of konjak, in particular to a method for preparing konjak oligosaccharide through fermentation of konjak powder. The method comprises the following steps of: inoculating bacillus licheniformis BJ2022 into fermentation liquor containing konjac powder, and fermenting, the bacillus licheniformis BJ2022 is preserved in the China Center for Type Culture Collection on June 7, 2022, and the preservation number of the bacillus licheniformis BJ2022 is CCTCC (China Center for Type Culture Collection) NO: M 2022816. The method has the advantages of high efficiency, environmental protection, low cost, good activity and the like, and is a technology with a wide application prospect.

Description

Technical Field

[0001] The present application relates to the technical field of konjac, and in particular to a method for preparing konjac oligosaccharides by fermenting konjac flour. Background Art

[0002] Konjac (Amorphophallus konjac K.Koch) is a herbaceous plant belonging to the genus Amorphophallus of the Araceae family. It is rich in various nutrients such as konjac polysaccharide, pine oleic acid and lignin. Konjac polysaccharide is a natural high molecular weight (200-2000kDa) water-soluble dietary fiber, and its main chain is composed of D-glucose and D-mannose connected by β-1,4 bonds. Konjac polysaccharide has the characteristics of low calories, difficult to digest and absorb, and is widely used in the field of food additives. Konjac oligosaccharide is the hydrolysis product of konjac polysaccharide, which is mainly composed of 2-10 sugar units connected by glycosidic bonds. Compared with konjac polysaccharide, konjac oligosaccharide has a low molecular weight, high solubility, is more easily absorbed and utilized by the human body, and has better biological activity. Therefore, reducing the degree of polymerization of konjac polysaccharide can not only improve the biological activity of konjac polysaccharide, but also expand the application range of konjac.

[0003] The existing technology mainly prepares konjac oligosaccharides through physical, chemical and enzymatic methods. Traditional physical and chemical methods have a series of problems such as complex process, low extraction efficiency and environmental pollution. Enzymatic method has the advantages of environmental friendliness and high energy efficiency, and is a commonly used method in the preparation of oligosaccharides, but there are still problems such as strict enzymatic reaction conditions and high cost of obtaining high-purity enzymes. Summary of the invention

[0004] Microbial fermentation method, as a new technology, has low pollution, low energy consumption, low cost, and has been applied to the preparation research of multiple active substances. Therefore, the application utilizes Bacillus licheniformis one-step fermentation to prepare konjac oligosaccharides, and obtains konjac flour. The method has the advantages of high efficiency, environmental protection, low cost, good activity, and is a technology with broad application prospects.

[0005] To this end, the embodiments of the present application at least disclose the following technical solutions:

[0006] The embodiment discloses a method for preparing konjac oligosaccharides by fermenting konjac flour. The method includes the steps of inoculating Bacillus licheniformis BJ2022 into a fermentation broth containing konjac flour for fermentation, wherein the Bacillus licheniformis BJ2022 was deposited in the China Center for Type Culture Collection on June 7, 2022, with a deposit number of CCTCC NO: M 2022816.

[0007] The method for preparing konjac oligosaccharides by fermenting konjac flour provided in the embodiment further comprises the step of activating and enriching Bacillus licheniformis BJ2022 to obtain seed liquid.

[0008] In some embodiments, the activation step includes: streaking Bacillus licheniformis BJ2022 on a solid LB medium to obtain an isolated single colony; inoculating the single colony into a liquid LB medium, and enriching and culturing the single colony at 30-37°C and 200rpm until its OD600 value reaches 0.6-0.8, thereby obtaining a seed solution.

[0009] In some embodiments, the fermentation step includes: inoculating the seed liquid into a fermentation medium at an inoculum amount and fermenting for 12 hours. Wherein, the fermentation medium comprises 6% konjac flour, 2g / L (NH4)2SO4, 2g / L KH2PO4, 4g / L NaCl and 0.04g / L MgSO4, and the pH of the fermentation medium is 7.0-7.5. The fermentation conditions include a fermentation temperature of 37°C and a stirring speed of 200rpm.

[0010] The method for preparing konjac oligosaccharides by fermenting konjac flour provided in some embodiments further comprises the step of extracting the konjac oligosaccharides from the fermentation broth. The step comprises treating the fermentation broth by centrifugation at 3000-8000 rpm for 5-10 min; collecting the supernatant and concentrating it by 3-8 times, adding 2-5 times the volume of anhydrous ethanol, and standing overnight to obtain a crude polysaccharide precipitate; and re-dissolving, concentrating, and freeze-drying the crude polysaccharide precipitate to obtain the konjac oligosaccharide.

[0011] The present application discloses a method for preparing konjac oligosaccharides by fermenting konjac flour with Bacillus licheniformis BJ2022 in a one-step manner, thereby achieving bioconversion of konjac flour and improving its biological utilization rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Dynamic monitoring of the components of fermentation liquid of konjac flour fermented by Bacillus licheniformis. A) is the growth curve of Bacillus licheniformis at different fermentation time points; B) is the pH change of fermentation liquid at different fermentation time points; C) is the change of total sugar content of fermentation liquid at different fermentation time points; D) is the change of reducing sugar content of fermentation liquid at different fermentation time points.

[0013] Figure 2 The results of a single factor experiment on the fermentation of konjac flour by Bacillus licheniformis. A) shows the variation of konjac oligosaccharide production with konjac flour concentration; B) shows the variation of konjac oligosaccharide production with Bacillus licheniformis concentration; C) shows the variation of konjac oligosaccharide production with fermentation time; D) shows the variation of konjac oligosaccharide production with buffer salt content.

[0014] Figure 3The molecular weight, degree of polymerization and monosaccharide composition of konjac oligosaccharide prepared by fermentation of Bacillus licheniformis. A) is the average molecular weight of konjac oligosaccharide; B) is the result of the degree of polymerization of konjac oligosaccharide; C) is the standard product of monosaccharide composition; D) is the monosaccharide composition of konjac oligosaccharide. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application is further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described herein are only used to explain the present application and are not intended to limit the present application. The reagents not described separately in detail in the present application are all conventional reagents and can be obtained from commercial channels; the methods not described in detail are all conventional experimental methods and can be obtained from the prior art.

[0016] Source, activation and enrichment culture of Bacillus licheniformis BJ2022

[0017] Embodiments Konjac oligosaccharides were prepared by fermenting konjac flour with Bacillus licheniformis BJ2022 in one step. The strain was named Bacillus licheniformis BJ2022, which has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: M2022816 and a deposit date of June 7, 2022.

[0018] Activation of Bacillus licheniformis: Use a sterile inoculation loop to dip into the frozen Bacillus licheniformis culture liquid, and activate the strain by streaking three zones on LB solid medium. The activated strain is cultured at 37°C until a single clone grows out.

[0019] Enrichment culture of Bacillus licheniformis: Pick a single clone into liquid LB medium and carry out enrichment culture at 37°C and 100-200rpm. 600 The value reaches 0.6-0.8 to be used as seed liquid.

[0020] Konjac flour fermentation

[0021] The embodiment provides a fermentation medium. The fermentation medium contains 6% konjac flour, 2g / L (NH4)2SO4, 2g / L KH2PO4, 4g / L NaCl and 0.04g / L MgSO4, and the pH of the fermentation medium is 7.0-7.5.

[0022] The embodiment provides a step of inoculating Bacillus licheniformis BJ2022 into a fermentation broth containing konjac flour for fermentation.

[0023] Experimental group: This step includes inoculating the above-obtained seed solution into a fermentation medium at an inoculum amount of 6% for fermentation. The fermentation medium comprises 6% konjac flour, 2g / L (NH4)2SO4, 2g / L KH2PO4, 4g / L NaCl and 0.04g / LMgSO4, and the pH of the fermentation medium is 7.0-7.5. The fermentation conditions include a fermentation temperature of 37°C and a stirring speed of 200rpm.

[0024] Control group 1: This step includes inoculating the seed liquid with a 6% inoculum into a fermentation medium containing different concentrations of konjac flour for fermentation. Wherein, the fermentation medium contains 1-10% konjac flour, 2g / L (NH4) 2SO4, 2g / L KH2PO4, 4g / L NaCl and 0.04g / LMgSO4, and the pH of the fermentation medium is 7.0-7.5. The fermentation conditions include a fermentation temperature of 37°C and a stirring speed of 200rpm. After optimizing the conditions by changing the konjac flour content, the konjac oligosaccharide extraction rate in the experimental group is higher than that in the control group.

[0025] Control group 2: This step includes inoculating a seed solution with a concentration of 1%-10% into a fermentation medium for fermentation. Wherein, the fermentation medium contains 6% konjac flour, 2g / L (NH4) 2SO4, 2g / L KH2PO4, 4g / LNaCl and 0.04g / LMgSO4, and the pH of the fermentation medium is 7.0-7.5. The fermentation conditions include a fermentation temperature of 37°C and a stirring speed of 200rpm. After optimizing the conditions by changing the inoculation amount of the seed solution, the konjac oligosaccharide extraction rate of the experimental group was higher than that of the control group.

[0026] Control group 3: This step includes inoculating the above-obtained seed liquid into the fermentation medium at a 6% inoculation amount and fermenting for different times. Wherein, the fermentation time is 6-72h. The fermentation medium contains 6% konjac flour, 2g / L (NH4) 2SO4, 2g / L KH2PO4, 4g / LNaCl and 0.04g / L MgSO4, and the pH of the fermentation medium is 7.0-7.5. The fermentation conditions include a fermentation temperature of 37°C and a stirring speed of 200rpm. After optimizing the conditions by changing the fermentation time, the konjac oligosaccharide extraction rate of the experimental group was higher than that of the control group.

[0027] Control group 4: This step includes inoculating the above-obtained seed solution into a fermentation medium at a 6% inoculation rate for fermentation. Wherein, the original concentration of the four buffer salt ions in the fermentation medium is 10%-100%, and the pH of the fermentation medium is 7.0-7.5. The fermentation conditions include a fermentation temperature of 37°C and a stirring speed of 200rpm. After optimizing the conditions by changing the buffer salt content, there was no significant difference in the change of konjac oligosaccharide extraction rate.

[0028] Control group 5: Use a sterile inoculation loop to dip the frozen bacterial solution of Lactobacillus acidophilus AS1.2686 (B312098, Mingzhoubio), and activate the strain by three-zone streaking on LB solid medium. The activated strain is cultured at 37°C until a single clone grows. Pick a single clone and add it to liquid LB medium, and enrich it at 37°C and 100-200rpm. Enrich it at 30-37°C and 200rpm until its OD 600 The value reaches 0.6-0.8 to serve as seed liquid. The obtained seed liquid is inoculated into the fermentation medium with an inoculum amount of 6% for fermentation. Wherein, the fermentation medium comprises 6% konjac flour, 2g / L (NH4) 2SO4, 2g / L KH2PO4, 4g / LNaCl and 0.04g / L MgSO4, and the pH of the fermentation medium is 4.0-4.5. The fermentation conditions include a fermentation temperature of 37°C and a stirring speed of 200rpm. After the fermentation is completed, the fermentation broth is collected, graded alcohol precipitation is performed, redissolved, and freeze-dried. The extraction rate of konjac oligosaccharides obtained is lower than that of konjac oligosaccharides prepared by fermentation of Bacillus licheniformis BJ2022 in the experimental group.

[0029] The embodiment provides a step for determining the structure of the prepared konjac oligosaccharide. The molecular weight and degree of polymerization of konjac oligosaccharide are determined by HPGPC; the monosaccharide composition is determined by PMP monosaccharide derivatization. The prepared oligosaccharide has a low molecular weight and a degree of polymerization mainly distributed in 1-8. Konjac oligosaccharide is mainly composed of mannose and glucose in a ratio of 3:1.

[0030] The above is only a preferred specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed in the present application should be covered within the protection scope of the present application.

Claims

1. A method for preparing konjac oligosaccharides by fermenting konjac flour, comprising the step of inoculating Bacillus licheniformis BJ2022 into a fermentation broth containing konjac flour for fermentation, wherein the Bacillus licheniformis BJ2022 was deposited in the China Center for Type Culture Collection on June 7, 2022, with a deposit number of CCTCC NO: M2022816.

2. The method according to claim 1, further comprising the step of activating and enriching Bacillus licheniformis BJ2022 to obtain seed liquid.

3. The method according to claim 2, wherein the activation step comprises: Bacillus licheniformis BJ2022 was streaked on solid LB medium to obtain isolated single colonies; A single colony was inoculated into liquid LB medium, and enriched and cultured at 30-37°C and 200 rpm until its OD600 value reached 0.6-0.8, thereby obtaining seed solution.

4. The method according to claim 1, wherein the fermentation step comprises: The seed liquid is inoculated into a fermentation medium at an inoculation rate of 6% and fermented for 12 hours, wherein the fermentation medium contains 6% konjac flour, 2g / L (NH4)2SO4, 2g / L KH2PO4, 4g / L NaCl and 0.04g / LMgSO4, and the pH of the fermentation medium is 7.0-7.5; the fermentation conditions include a fermentation temperature of 37°C and a stirring speed of 200rpm.

5. The method according to any one of claims 1 to 4, further comprising the step of extracting the konjac oligosaccharide from the fermentation broth.

6. The method according to claim 5, wherein the step of extracting the konjac oligosaccharide from the fermentation broth comprises: The fermentation broth was treated by centrifugation at 3000-8000 rpm for 5-10 min; The supernatant was collected and concentrated 3 to 8 times, and 2 to 5 times the volume of anhydrous ethanol was added, and after standing overnight, a crude polysaccharide precipitate was obtained; The crude polysaccharide precipitate is redissolved, concentrated and freeze-dried to obtain the konjac oligosaccharide.

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