CN115678938A - Method for preparing mannan-oligosaccharide by fermenting coffee grounds - Google Patents
Method for preparing mannan-oligosaccharide by fermenting coffee grounds Download PDFInfo
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- CN115678938A CN115678938A CN202211399310.6A CN202211399310A CN115678938A CN 115678938 A CN115678938 A CN 115678938A CN 202211399310 A CN202211399310 A CN 202211399310A CN 115678938 A CN115678938 A CN 115678938A
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Abstract
The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for preparing mannan-oligosaccharide by fermenting coffee grounds, which comprises the following steps: raw material treatment: removing impurities from coffee grounds, then crushing and sieving; activating strains: inoculating the slant mycelia with culture medium from the slant with an inoculating hook into the middle of solid culture medium, and culturing at 28 + -2 deg.C for 3-6 days; preparing a seed solution: mashing the solid culture medium with Ganoderma with inoculating hook, and adding appropriate amount of sterile water to obtain Ganoderma seed solution; fermentation culture: mixing the ganoderma lucidum seed liquid and the coffee grounds raw material powder according to the mass ratio of (2-5) to 1, putting the mixture into a fermentation tank, controlling the oxygen concentration of the space in the fermentation tank to be 0.010-0.025 mol/L under the condition of 28 +/-2 ℃ for 5-10 days; removing impurities: removing impurities in the fermentation liquor by filtration or centrifugation, and collecting the supernatant to obtain a solution containing the mannanoligosaccharide. The method of the invention uses the ganoderma lucidum fermented coffee grounds to obtain the mannanoligosaccharide with higher yield and purity.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a method for preparing mannan-oligosaccharide by fermenting coffee grounds.
Background
Coffee is one of the most popular beverages in the world, and coffee grounds constitute about 2/3 of the quality of the dry coffee beans as a by-product left when producing instant coffee. 30g of coffee grounds are produced for each cup of freshly ground coffee. At present, foreign coffee grounds are mainly used as fertilizers and fuels, and are mostly discarded as wastes at home, so that environmental pollution and resource waste are caused. The main components of the coffee grounds comprise carbohydrate, nitrogen-containing compounds, lipid compounds and some mineral elements, wherein the content of the carbohydrate is about 45 percent, and the content of mannan is as high as about 24 percent, and the coffee grounds are high-quality raw materials for preparing mannan-oligosaccharide. In the paper, "guozping, xuguangwei, wijowen, octoyinjun, shenxueliang, research on preparation of mannooligosaccharides by using an enzymatic method of coffee bean dregs, food research and development, 2017,38 (21): 59-64", the yield of the mannooligosaccharides obtained by enzymatic hydrolysis of coffee bean dregs by using two beta-mannases from different sources is 50% -60%. As early as 2002, the company AGF, japan, succeeded in extracting mannooligosaccharides from coffee grounds by mixing water with ground coffee grounds, then decomposing them at a high temperature of 220 ℃, filtering, decolorizing and deoxidizing to obtain high-purity mannooligosaccharides. Although the two methods obtain the mannase with higher purity, a large amount of impurities are not removed after enzymolysis or high-temperature treatment, so that the utilization rate of coffee grounds is low, a more complex process is required in the process of separating and purifying the mannanoligosaccharide, the generation of waste water and other wastes is increased, the problems of more serious resource waste, environmental pollution and the like still exist, the production cost is high, and the enterprise burden is increased.
Mannan-oligosaccharide, also known as mannan-oligosaccharide and mannan-oligosaccharide, widely exists in konjac flour, guar gum, sesbania gum and cell walls of various microorganisms, is sweet as a bifidobacterium proliferation factor, is often used as a sugar substitute, has the effects of protecting intestinal tracts, improving immunity and the like, and is widely used as a food and feed additive. The method for preparing the mannan-oligosaccharide by hydrolyzing the raw materials such as the konjak, the guar gum, the coconut, the locust bean gum and the like by the enzyme method is a common method for producing nowadays. The preparation of mannooligosaccharides by fermentation has not been reported.
Disclosure of Invention
Aiming at the problem of poor utilization rate of coffee grounds in the prior art, the invention provides a method for preparing mannan-oligosaccharide by fermenting coffee grounds, wherein mannan-oligosaccharide with higher yield and purity is obtained by fermenting coffee grounds with lucid ganoderma.
The invention is realized by the following technical scheme:
a method of fermenting coffee grounds to make mannanoligosaccharides, the method comprising:
treating raw materials: drying coffee grounds, removing impurities, then crushing and sieving;
activating strains: inoculating the slant mycelium of Ganoderma lucidum with the culture medium cut from the slant into the solid culture medium, and culturing at appropriate temperature for a period of time;
preparing a seed solution: mashing the solid culture medium with the ganoderma lucidum, and then adding a proper amount of sterile water to obtain ganoderma lucidum seed liquid;
fermentation culture: mixing the ganoderma seed liquid and the coffee grounds raw material powder according to a certain proportion, putting the mixture into a fermentation tank, fermenting the mixture for a certain time at a certain temperature, and controlling the oxygen concentration of the space in the fermentation tank within a certain range;
removing impurities: removing impurities in the fermentation liquor by filtration or centrifugation, and collecting supernatant to obtain solution containing mannan-oligosaccharide.
Further, the Ganoderma is one of Ganoderma lucidum (G.lucidum), ganoderma sinensis (G.sinense), and Ganoderma tsugae (G.tsugae Murr); three Ganoderma fungi, ganoderma lucidum (Ganoderma lucidum), ganoderma sinensis (G.sinense) and Ganoderma tsugae (G.tsugae Murr.) are included in the list of fungi for health food published by the Ministry of health in 2001. In order to ensure that the obtained product can be applied to health-care food in the future, the invention finally selects three ganoderma lucidum strains of ganoderma lucidum, ganoderma sinense and ganoderma tsugae.
Further, the strain activation specifically comprises the following steps: cutting Ganoderma with inoculating hook from inclined plane to 0.5-1cm 2 Inoculating the slant mycelium with culture medium into the middle of solid culture medium, and culturing at 28 + -2 deg.C for 3-6 days. Wherein, the solid culture medium is a potato glucose agar culture medium which is sold in accordance with GB 4789.15-2016 food safety national standard food microbiology inspection mould and yeast counting, 0.3-1.2% of lignin is added by mass percent, the lignin is added for inducing the expression and secretion of a lignin degradation enzyme system in ganoderma lucidum, so that the smooth degradation in the liquid fermentation process is ensured, the effective degradation of the lignin is ensured, the lignin cannot be induced by adding less than 0.3%, the growth of ganoderma lucidum is inhibited by adding more than 1.2%, and the failure of the subsequent liquid fermentation is caused.
Further, the preparation steps of the seed liquid are as follows: mashing the solid culture medium with Ganoderma with inoculating hook, adding sterile water with the volume of 2-5 times of the solid culture medium to obtain Ganoderma seed solution.
Further, the fermentation culture step specifically comprises: mixing the ganoderma lucidum seed liquid and the coffee grounds raw material powder according to the mass ratio of (2-5) to 1, putting the mixture into a fermentation tank, culturing for 5 to 10 days at the temperature of 28 +/-2 ℃, and enabling the oxygen concentration of the space in the fermentation tank to be 0.010-0.025 mol/L through stirring rotation speed and ventilation; because the ganoderma lucidum belongs to aerobic microorganisms, if the supply of oxygen is insufficient, the growth of the ganoderma lucidum is slow, the decomposition of cellulose and lignin by the ganoderma lucidum is reduced, the yield of the manna oligosaccharide is low finally, the ganoderma lucidum is easily polluted by other microorganisms, the fermentation failure is caused, and in order to ensure the yield of the manna oligosaccharide, the fermentation condition needs to be strictly controlled. In the fermentation culture process, yeast powder is added into a fermentation culture medium to supplement a nitrogen source, wherein the addition amount of the yeast powder is as follows: 0.5-5.0g of the fermentation medium per liter.
Further, in the step of removing impurities, the mass percent of the mannanoligosaccharide in the obtained solution containing mannanoligosaccharide is 2.5-6.2%.
Further, the method also comprises the steps of filtering the solution containing the manna oligosaccharide, decolorizing with active carbon, concentrating under reduced pressure at the temperature of between 30 and 40 ℃ under the pressure of between 0.001 and 0.01MPa to obtain manna oligosaccharide solid, and refining and drying the manna oligosaccharide solid to obtain manna oligosaccharide with the purity of between 80 and 90 percent.
The invention has the beneficial technical effects that:
ganoderma (Ganoderma) is an important dual-purpose fungus for medicine and food in Basidiomycetes, polyporaceae and Ganoderma, and is one of traditional and rare medicinal materials in China. Three Ganoderma fungi, ganoderma lucidum (Ganoderma lucidum), ganoderma sinensis (G.sinense) and Ganoderma tsugae (G.tsugae Murr.) are included in the list of fungi for health food published by the Ministry of health in 2001. During the deep fermentation process of the ganoderma lucidum, rich extracellular enzyme systems, such as cellulase, hemicellulase, amylase, pectinase and the like can be generated, and the extracellular enzyme systems can carry out various extracellular biochemical reactions to effectively destroy covalent bonds, hydrogen bonds, ester bonds and the like among cellulose, hemicellulose and lignin in coffee grounds, so that mannan is released and further degraded into mannan-oligosaccharide. Meanwhile, during the growth process of the ganoderma lucidum, a large amount of cellulose and other substances are consumed, so that the separation and purification of the mannanoligosaccharide are simplified. Therefore, the coffee grounds fermented by the lucid ganoderma can be used for obtaining the mannooligosaccharides with higher yield and purity, each kilogram of the coffee grounds can obtain 160-210 g of the mannooligosaccharides, and the yield of the mannooligosaccharides prepared by the coffee grounds hydrolyzed by the prior enzyme method is about 50-60 percent, namely about 120-145 g. The method provided by the invention can remove impurities and ganoderma lucidum through centrifugation, and can obtain the mannan oligosaccharide with the purity of 80-90% after concentration and purification by a reverse osmosis membrane. The invention not only provides a new color idea for high-value utilization of coffee grounds, but also opens up a new method for preparing the mannan-oligosaccharide by a fermentation method.
The method has the advantages of simple operation, mild production conditions, no environmental pollution, low energy consumption and accordance with energy conservation and emission reduction. The method for producing the manna oligosaccharide can greatly reduce the production cost and the environmental pollution, provides a new color idea for high-value utilization of coffee grounds, and develops a new method for preparing the manna oligosaccharide by a fermentation method.
Mannan, a precursor of mannan oligosaccharide, is mainly present in hemicellulose, and the hemicellulose is usually combined with cellulose and lignin in the nature, so that the hemicellulose must be separated from the cellulose and the lignin for high-efficiency utilization of the hemicellulose, which is a main problem for extracting mannan oligosaccharide from coffee residues at present. The cellulose and lignin are degraded by the lucid ganoderma, so that the problem of separating the hemicellulose from the cellulose and lignin is solved, the cellulose and the lignin are utilized and decomposed by the lucid ganoderma in the growth and metabolism processes, the difficulty of subsequent separation and purification is reduced, the quantity of waste residues is further reduced, the utilization rate of coffee grounds is improved, and the risk of environmental pollution is reduced.
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FIG. 1 is a flow chart of a method for preparing mannanoligosaccharide by fermenting coffee grounds in an embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are intended to illustrate the present invention and are not intended to limit the scope of the present invention. Modifications or substitutions to the steps or conditions of the present invention may be made without departing from the spirit and scope of the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The ganoderma lucidum used in the following examples is a strain of the following species: ganoderma lucidum (Ganoderma lucidum), ganoderma sinense (G.sinense), and Ganoderma tsugae (G.tsugae Murr.). Each strain was purchased from China general microbiological culture Collection center (CGMCC).
Example 1
The present embodiment provides a method for preparing mannooligosaccharides by fermenting coffee grounds, the method comprising:
(1) Treating raw materials: drying coffee grounds in an oven at 80 ℃, removing impurities, crushing and sieving by a 20-40 mesh sieve.
(2) Activating strains: cutting Ganoderma (G.lucidum) with inoculating hook to 1cm from the inclined plane 2 Inoculating the slant mycelium with culture medium into the middle of potato glucose agar culture medium containing 0.6 wt% lignin, and culturing at 28 + -2 deg.C for 3-5 days.
(3) Preparing a seed solution: mashing the solid culture medium with Ganoderma lucidum with an inoculating hook, and adding appropriate amount of sterile water to obtain Ganoderma lucidum seed solution; in this example, the amount of sterile water was 2 times the volume of the solid medium;
(4) Fermentation culture: mixing the ganoderma lucidum seed liquid and the coffee grounds raw material powder according to the mass ratio of 5.
(5) Removing impurities: removing impurities of the wall-broken homogenate by a filtering or centrifuging mode, collecting supernatant to obtain a mannooligosaccharide solution, and detecting the content of the mannooligosaccharide to be 3.52% by referring to a method of plum daemon and the like (plum daemon, jiangqiang, wei 36191, lirit, nissan Shuanggong. The enzymatic preparation, separation and crystallization of oligomeric mannose [ J ]. Food science, 2005 (S1): 58-60.).
(6) Concentrating and drying: the solution of the manna oligosaccharide is decolorized by active carbon, then decompressed and concentrated at the temperature of 30-40 ℃ under the pressure of 0.001-0.01MPa, and then refined and dried to obtain 198.8 g of manna oligosaccharide solid. Referring to "guozhong, xuguangwei, wijowen, octoyinjun, shenxueliang", research on the preparation of mannooligosaccharides by using coffee bean dregs enzyme method, food research and development, 2017,38 (21): 59-64 ", the mean degree of polymerization DP of the mannooligosaccharides was measured to be 2.83, and the purity was 83.3%.
Example 2
The embodiment provides a method for preparing mannan-oligosaccharide by fermenting coffee grounds, which comprises the following steps:
(1) Raw material treatment: drying coffee grounds in an oven at 80 ℃, removing impurities, crushing and sieving with a 20-40 mesh sieve.
(2) Activating strains: cutting Ganoderma sinensis (G.sinense) with inoculating hook to 1cm from slant 2 Inoculating the slant mycelium with culture medium into the middle of potato glucose agar solid culture medium added with 0.8% lignin by mass, and culturing at 28 + -2 deg.C for 4-5 days.
(3) Preparing a seed solution: mashing the solid culture medium with Ganoderma sinensis with an inoculating hook, and adding appropriate amount of sterile water to obtain Ganoderma sinensis seed solution; in this example, the amount of sterile water was 4 times the volume of the solid medium;
(4) Fermentation culture: mixing the Ganoderma sinensis seed solution and coffee grounds raw material powder according to the mass ratio of 2 to 1, placing into a fermentation tank, culturing for 7 days at 28 + -2 deg.C, and controlling the oxygen concentration of the space in the fermentation tank to be 0.02mol/L.
(5) Removing impurities: removing impurities of the wall-broken homogenate by a filtering or centrifuging mode, collecting supernatant to obtain a mannooligosaccharide solution, and detecting the content of the mannooligosaccharide to be 8.12% by referring to a method of plum sense and the like (plum sense, jiangqiang, wei 36191, lirit, nissan Shuanggong. The enzymatic preparation, separation and crystallization of mannan [ J ]. Food science, 2005 (S1): 58-60.).
(6) Concentrating and drying: the solution of the manna oligosaccharide is decolorized by active carbon, then decompressed and concentrated at the temperature of between 30 and 40 ℃ under the pressure of between 0.001 and 0.01MPa, and then refined and dried to obtain 201.7 g of manna oligosaccharide solid. The average polymerization degree DP value of the mannanoligosaccharide is determined to be 3.22 and the purity is 84.7% by referring to ' Guojipin, xuanguwei, wewawinu, zygunjun, shenxuliang ', research on the preparation of the mannanoligosaccharide by using a coffee bean dreg enzyme method, food research and development, 2017,38 (21): 59-64 '.
Example 3
The embodiment provides a method for preparing mannan-oligosaccharide by fermenting coffee grounds, which comprises the following steps:
(1) Treating raw materials: drying coffee grounds in an oven at 80 ℃, removing impurities, crushing and sieving with a 20-40 mesh sieve.
(2) Activating strains: cutting Ganoderma tsugae (G.tsugae Murr.) with inoculating hook 1cm from the slant 2 Band cultureInoculating the slant mycelium of the culture medium into the middle of a potato glucose agar solid culture medium added with 0.9 mass percent of lignin, and culturing for 4-5 days at 28 +/-2 ℃.
(3) Preparing a seed solution: mashing the solid culture medium with Ganoderma tsugae with an inoculation hook, and adding appropriate amount of sterile water to obtain Ganoderma tsugae seed solution; in this example, the amount of sterile water was 5 times the volume of the solid medium;
(4) Fermentation culture: mixing the ganoderma tsugae seed liquid and the coffee grounds raw material powder according to the mass ratio of 3 to 1, putting the mixture into a fermentation tank, culturing for 8 days at the temperature of 28 +/-2 ℃, and controlling the oxygen concentration of the space in the fermentation tank to be 0.022mol/L.
(5) Removing impurities: removing impurities of the wall-broken homogenate by a filtering or centrifuging mode, collecting supernatant to obtain a mannooligosaccharide solution, and detecting the content of the mannooligosaccharide to be 3.81% by referring to a method of plum sense and the like (plum sense, jiangqiang, wei 36191, lirit, nissan Shuanggong. The enzymatic preparation, separation and crystallization of mannan [ J ]. Food science, 2005 (S1): 58-60.).
(6) Concentrating and drying: the solution of the manna oligosaccharide is decolorized by active carbon, then decompressed and concentrated at the temperature of 30-40 ℃ under the pressure of 0.001-0.01MPa, and then refined and dried to obtain 177.3 g of manna oligosaccharide solid. Referring to "guozhong, xuguangwei, wijowen, octoyinjun, shenxueliang", research on the preparation of mannooligosaccharides by using coffee bean dregs enzyme method, food research and development, 2017,38 (21): 59-64 ", the mean degree of polymerization DP of the mannooligosaccharides was determined to be 3.45 and the purity was 86.2%.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (8)
1. A method for preparing mannanoligosaccharide by fermenting coffee grounds, the method comprising:
treating raw materials: drying coffee grounds, removing impurities, then crushing and sieving;
activating strains: inoculating the slant mycelium of Ganoderma lucidum with the culture medium cut from the slant into the solid culture medium, and culturing at appropriate temperature for a period of time;
preparing a seed solution: mashing the solid culture medium with the ganoderma lucidum, and then adding a proper amount of sterile water to obtain ganoderma lucidum seed liquid;
fermentation culture: mixing the ganoderma seed liquid and the coffee grounds raw material powder according to a certain proportion, putting the mixture into a fermentation tank, fermenting the mixture for a certain time at a certain temperature, and controlling the oxygen concentration of the space in the fermentation tank within a certain range;
removing impurities: removing impurities in the fermentation liquor by filtration or centrifugation, and collecting the supernatant to obtain a solution containing the mannanoligosaccharide.
2. The method for preparing mannan-oligosaccharide from fermented coffee grounds according to claim 1, wherein the adopted ganoderma lucidum is one of ganoderma lucidum, ganoderma sinensis and ganoderma tsugae.
3. The method for preparing mannan-oligosaccharide from fermented coffee grounds according to claim 1, wherein the strain activation step comprises the following specific steps: cutting Ganoderma with inoculating hook from inclined plane to 0.5-1cm 2 Inoculating slant mycelia with a culture medium into the middle of a solid culture medium, and culturing at 28 +/-2 ℃ for 3-6 days, wherein the solid culture medium is a potato glucose agar culture medium added with lignin, and the mass percent of the lignin added in the potato glucose agar culture medium is 0.3-1.2%.
4. The method for preparing mannan-oligosaccharide from fermented coffee grounds as claimed in claim 1, wherein the seed liquid preparation step comprises: mashing the solid culture medium with Ganoderma with inoculating hook, adding sterile water with the amount of 2-5 times of the solid culture medium volume to obtain Ganoderma seed solution.
5. The method for preparing mannan-oligosaccharide from fermented coffee grounds according to claim 1, wherein the fermentation culture step comprises the following specific steps: mixing the ganoderma seed liquid and the coffee grounds raw material powder according to the mass ratio of (2-5) to 1, putting the mixture into a fermentation tank, fermenting for 5 to 10 days at the temperature of 28 +/-2 ℃, and controlling the oxygen concentration of the space in the fermentation tank to be 0.010-0.025 mol/L.
6. The method for preparing mannan-oligosaccharide from fermented coffee grounds as claimed in claim 1, wherein in the fermentation culture process, yeast powder is added into the fermentation culture medium to supplement nitrogen source, and the addition amount of the yeast powder is as follows: 0.5-5.0g of the fermentation medium per liter.
7. The method for preparing mannan-oligosaccharide from fermented coffee grounds as claimed in claim 1, wherein the mannan-oligosaccharide contained in the solution containing mannan-oligosaccharide obtained in the step of removing impurities is 2.0-5.0% by mass.
8. The method for preparing mannan-oligosaccharide from fermented coffee grounds according to claim 1, further comprising concentrating the mannan-oligosaccharide-containing solution under reduced pressure at 0.001-0.01MPa and 30-40 ℃, and drying to obtain mannan-oligosaccharide solid.
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