KR20230171667A - An integrated process for conversion of spent coffee grounds into value-added biochemicals and biofuel - Google Patents

Abstract

The present invention relates to a biorefinery process that can produce biofuel and biocompounds from biomass, and more specifically, to produce coffee oil, bioethanol, mannose, manno-oligosaccharides, cafestol and carwell from discarded coffee grounds in one process. It relates to a biorefinery process of coffee grounds that can be produced by, and biocompounds and biofuel produced by the process. According to the present invention, the production yield of biofuel and biocompounds produced from coffee grounds can be significantly improved.

Description

Biorefinery process of coffee grounds, biocompounds and biofuel produced by the process {An integrated process for conversion of spent coffee grounds into value-added biochemicals and biofuel}

The present invention relates to a biorefinery process that can produce biofuel and biocompounds from biomass, and more specifically, to produce coffee oil, bioethanol, mannose, manno-oligosaccharides, cafestol and carwell from discarded coffee grounds in one process. It relates to a biorefinery process of coffee grounds that can be produced by, and biocompounds and biofuel produced by the process.

The growing distribution of biodiesel is causing a rise in the price of vegetable oil, unstable supply and demand, and conflict with food resources. Soybeans, rapeseeds, sunflowers, and palms, which are used as raw materials for domestic biodiesel production, all form a supply and demand balance with the edible market. If biodiesel production increases, the supply and demand balance in the edible oil market will be broken, causing fluctuations in oil grain prices. It will be done. Therefore, research is needed on biomass, a non-food resource that can replace food resources used to produce conventional biofuel.

Meanwhile, as coffee consumption increases worldwide, the amount of coffee grounds disposed of is also increasing every year. About 15g of coffee beans are used to make a cup of Americano, of which 14.97g, or 99.8%, are discarded as coffee grounds. Currently, coffee grounds are discharged as waste in Korea, and their high moisture content causes environmental pollution due to rot. Based on the amount of coffee grounds generated in 2019, it was found that about 4.1 billion won was spent just on the price of garbage bags.

Coffee grounds are organic biomass and contain various useful components such as carbohydrates including glucan and galactomannan, crude fat, crude fiber, crude protein, and ash. In addition, coffee grounds contain various polysaccharides, and among them, the content of mannan, a polymer of mannose, is very high compared to other plants. Enzymatic hydrolysis of mannan can produce not only the monomeric mannose, but also manno-oligosaccharides such as dimers, trimers, and tetramers. It has various effects such as antibacterial, antioxidant, blood sugar reduction, prebiotic, and moisturizing properties in medicine and beauty. It is used in industry. In particular, manno-oligosaccharides are indigestible and cannot be digested in the human intestines, so they have an intestinal digestion effect. In addition to being functional as prebiotics that help the activity of beneficial bacteria in the intestines, they are also effective in reducing human body fat, making them effective in suppressing obesity. It is known that However, currently commercially available manno-oligosachharide (MOS) is a yeast cell wall extract and is not a highly pure composition and does not have a consistent composition.

In this way, coffee grounds have the disadvantage that efficiency can be improved only by applying conversion technology appropriate for their characteristics due to their complex composition, but as a non-food resource, it is an organic resource with high recycling value that can be applied to the biorefinery process.

Moreover, cafestol and kahweol are reported to have functions such as reducing the risk of rectal cancer, preventing obesity, and suppressing the progression of Parkinson's disease. These are fat-soluble compounds known as diterpenes molecules, which are contained in coffee. Although it is known that these useful components have not yet been separated from coffee.

Domestic registered patent number 10-1763064

As a result of research efforts to solve the above-mentioned problems, the present inventors completed the present invention by developing a biorefinery process that can produce not only biofuel but also various biocompounds at high yield from coffee grounds.

In other words, coffee grounds contain not only mannan but also a large amount of phenolic compounds and lipid components, including lignin, making it difficult to hydrolyze them, and have the disadvantage of low purity due to mixing of other components. To solve this problem, manno-oligosaccharides are used. In order to improve the purity of mannose, a technology to remove lignin and lipids is needed, and the development of an enzyme system technology that selectively decomposes only mannose is required.

Therefore, the purpose of the present invention is to develop a new integrated process that can maximize production efficiency by using discarded coffee grounds as a biomass feedstock in the biorefinery process, but solving the disadvantages of low purity due to difficulty in hydrolysis and the inclusion of various components. A biorefinery process for coffee grounds that can produce high value-added substances such as bioethanol as well as various functional biocompounds including coffee oil, D-mannose, MOS, cafestol, and carwell by establishing conditions, and the above process. Providing produced biocompounds and biofuels.

The object of the present invention is not limited to the objects mentioned above, and other objects not mentioned will be clearly understood by those skilled in the art from the description below.

In order to achieve the object of the present invention described above, the present invention includes the steps of extracting coffee oil from coffee grounds; Extracting galactomannan (GM) from the first solid remaining after extracting the coffee oil; A first pretreatment step of removing lignin and lipids from the second solid remaining after the galactomannan extraction; a second pretreatment step of removing phenol compounds from the first pretreated third solid; A first hydrolysis step of first hydrolyzing the second pretreated fourth solid material; and a second hydrolysis step of secondly hydrolyzing the fifth solid material that has not been decomposed after the first hydrolysis.

In a preferred embodiment, the step of extracting the coffee oil includes mixing 2 to 4 parts by volume (w/v) of an organic solvent per 1 part by weight of the coffee grounds and stirring at 50° C. to 150° C. to extract the coffee into the organic solvent layer. An extraction step in which oil is extracted; Separating the reactant obtained in the extraction step into an organic solvent from which the coffee oil was extracted and a first solid; and removing the organic solvent from the organic solvent from which the coffee oil was extracted to obtain coffee oil.

In a preferred embodiment, the organic solvent is at least one selected from the group consisting of hexane (n-hexane), ethyl ether, chloroform, dichloromethane, ethanol, and methanol.

In a preferred embodiment, the step of extracting the galactomannan involves adjusting a 4.5 to 5.5% (w/v) sodium chloride solution to pH 2.5 to 3.5 with acetic acid and adding 4 to 6 mixed solvents per 1 part by weight of the first solid. Mixing parts by volume (w/v) and then autoclaving at 100°C to 150°C; Separating the reactant obtained after autoclave into a second solid and a solution; And obtaining galactomannan from the separated solution.

In a preferred embodiment, the step of obtaining the galactomannan includes adding 2 to 4 parts by volume of ethanol per 1 part by volume of the separated solution to precipitate the galactomannan; Separating the precipitated galactomannan by centrifugation; and freeze-drying the separated galactomannan.

In a preferred embodiment, a step of producing manno-oligosaccharide (MOS) from the freeze-dried galactomannan is further performed.

In a preferred embodiment, the step of generating MOS includes a first step of mixing the freeze-dried galactomannan with a buffer solution having a pH of 6.5 to 7.5; A second step of adding mannanase to the mixture and reacting at 35°C to 45°C for 30 to 90 minutes; A third step of separating the reactant into a hydrolyzate and a solid is included, and the first to third steps are repeated until the solid is not produced.

In a preferred embodiment, the first pretreatment step includes mixing 5 to 15 parts by volume of a sodium hydroxide solution per 1 part by weight of the second solid and then reacting at 50°C to 100°C; Separating the obtained reaction mixture into a third solid and a reaction solution; Washing the third solid with water until pH reaches 6.5 to 7.5; and drying the washed third solid material.

In a preferred embodiment, the method further includes extracting cafestol and kawal contained in the reaction solution with an organic solvent.

In a preferred embodiment, when the organic solvent is ethyl ether, an extraction step of mixing the reaction solution and ethyl ether in a volume ratio of 0.5 to 1: 2 to 5; Repeating the extraction step two or more times to separate the resulting ethyl ether organic phase and then removing moisture; And removing ethyl ether from the organic phase to obtain cafestol and carwell.

In a preferred embodiment, the second pretreatment step includes mixing 1 to 5 parts by volume of hydrogen peroxide solution per 1 part by weight of the third solid and then reacting at 50°C to 100°C; Separating the fourth solid from the obtained reaction mixture; Washing the fourth solid with water until pH reaches 6.5 to 7.5; and drying the washed fourth solid material.

In a preferred embodiment, the primary hydrolysis is performed by mannanase, and the secondary hydrolysis is performed sequentially by cellulase and mannanase.

In a preferred embodiment, the first hydrolysis step includes mixing the fourth solid with a buffer solution of pH 4.5 to pH 5.5; Adding mannanase to the mixture and then performing a primary hydrolysis reaction at 35°C to 40°C for 36 to 60 hours; and separating the first hydrolyzed liquid containing D-mannose into a fifth solid.

In a preferred embodiment, 0.01 to 0.1 parts by weight of the mannanase is added per 1 part by weight of the fourth solid.

In a preferred embodiment, the second hydrolysis step includes mixing the fifth solid with a buffer solution of pH 4.5 to pH 5.5; Adding cellulase and mannanase to the mixture and then performing a secondary hydrolysis reaction at 45°C to 55°C for 36 to 60 hours; and obtaining a secondary hydrolyzed solution.

In a preferred embodiment, 0.01 to 0.1 parts by weight of mannanase and cellulase are added per 1 part by weight of the fifth solid.

In a preferred embodiment, the step of producing bioethanol from the secondary hydrolysis liquid is further performed.

In a preferred embodiment, coffee oil, galactomannan, MOS, D-mannose, bioethanol, cafestol, and carweol are produced from the coffee grounds.

In addition, the present invention provides coffee oil, which is obtained by performing any of the above-described biorefinery processes, and is obtained in an amount of more than 90 ml per 1 kg of coffee grounds.

In addition, the present invention provides manno-oligosaccharides, which are obtained by performing any of the biorefinery processes described above, and are obtained in an amount of more than 100 g per 1 kg of coffee grounds.

In addition, the present invention provides mannose, which is obtained by performing any of the above-described biorefinery processes, and is obtained in an amount of more than 160g per 1kg of coffee grounds.

In addition, the present invention provides bioethanol, which is obtained by performing any of the biorefinery processes described above, and is characterized in that more than 90 g per 1 kg of coffee grounds is obtained.

According to the above-described present invention, discarded coffee grounds are used as biomass feedstock for the biorefinery process, but the disadvantage that arises when using coffee grounds is that they contain not only mannan but also a large amount of phenolic compounds and lipid components, including lignin, and are hydrolyzed. By establishing new integrated process conditions that can maximize the production efficiency of biomass by overcoming the shortcomings of low purity due to difficult mixing of other ingredients, coffee oil, D-mannose, MOS, cafestol, cowol and bioethanol, etc. High value-added materials can be produced with high efficiency.

This technical effect of the present invention is not limited to the scope mentioned above, and even if not explicitly mentioned, the effect of the invention that can be recognized by a person of ordinary skill in the art from the description of the specific contents for implementing the invention described later. Of course it is included.

Figure 1 is a schematic diagram showing useful products produced from coffee grounds through an integrated biorefinery process according to various embodiments of the present invention.
Figure 2 shows the efficiency of biomass by simultaneously producing high value-added substances such as coffee oil, D-mannose, MOS, cafestol, cowol, and bioethanol using coffee grounds biomass as a feedstock according to various embodiments of the present invention. This is a flowchart of the entire integrated biorefinery process that can maximize .
Figures 3a and 3b show manno-oligosaccharide (MOS) production and The results of examining the degree of polymerization are shown.
Figure 4 compares the MOS production results according to time changes through TLC analysis.
Figure 5 is a graph showing the results of examining the yield after repeated hydrolysis using PP mannnase to increase the MOS production yield.
Figure 6 shows the results of examining the mannose production yield using coffee grounds before and after pretreatment.
Figure 7 shows the results of examining saccharides after secondary hydrolysis using PP mannanse and cellulase to produce bioethanol from the residue remaining after producing mannose using Na and HP pretreated OG-NH-SCG using PP mannanse. .
Figure 8 shows the results of bioethanol production after the SHF fermentation process using the hydrolyzate obtained after secondary enzymatic hydrolysis.
Figure 9 shows the results of extraction of cafestol and Kahweol from the NaOH solution obtained after Na pretreatment and analysis using UV-Vis spectra and 1H NMR.
Figures 10a to 10c show the mass balance results of high value-added materials produced from coffee grounds, comparing existing research and the present invention.

The terms used in the present invention are general terms that are currently widely used as much as possible. However, in certain cases, there are terms arbitrarily selected by the applicant, and in this case, the meaning described or used in the detailed description of the invention rather than the simple name of the term is considered. Therefore, its meaning must be understood.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the technical field to which the present invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and unless clearly defined in the present invention, should not be interpreted in an idealized or excessively formal sense. No.

The terms used in the present invention are merely used to describe specific embodiments and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In the present invention, terms such as "comprise" or "have" are intended to designate the presence of features, numbers, steps, operations, components, parts, or combinations thereof described in the description of the invention, but are intended to indicate the presence of one or more other It should be understood that this does not exclude in advance the presence or addition of features, numbers, steps, operations, components, parts, or combinations thereof.

Terms such as first, second, etc. may be used to describe various components, but the components are not limited by the terms. The above terms are used only for the purpose of distinguishing one component from another. For example, a first component may be referred to as a second component, and similarly, the second component may also be referred to as a first component without departing from the scope of the present invention.

When interpreting a component, it is interpreted to include the margin of error even if there is no separate explicit description. In particular, when terms of degree such as "about", "substantially", etc. are used, they may be interpreted as being used at or close to that value when manufacturing and material tolerances inherent in the stated meaning are presented. .

In the case of a description of a temporal relationship, for example, if a temporal relationship is described as ‘~after’, ‘after~’, ‘~next’, ‘before’, etc., ‘immediately’ or ‘directly’ This also includes non-consecutive cases unless used.

Hereinafter, the technical configuration of the present invention will be described in detail with reference to the attached drawings and preferred embodiments.

However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Like reference numerals used to describe the invention throughout the specification represent like elements.

The technical feature of the present invention is to establish new integrated biorefinery process conditions that can maximize biomass production efficiency while using discarded coffee grounds as biomass feedstock for the biorefinery process, thereby producing coffee oil, D-mannose, MOS, It is a biorefinery process from coffee grounds that can produce high value-added substances such as cafestol, cowol, and bioethanol with high efficiency, and biocompounds and biofuel produced through the process.

In other words, coffee grounds contain not only mannan but also a large amount of phenolic compounds and lipid components including lignin, making it difficult to hydrolyze them, and have the disadvantage of low purity due to mixing of other components. Technology to remove lignin and lipids from coffee grounds that can improve purity, as well as enzyme-related technology to selectively decompose only mannan or improve bioethanol yield, and extraction of cafestol and cawell from solutions discarded during the pretreatment process. This is because various technologies suitable for integrated process conditions have been developed that can produce various bio compounds and biofuels with high efficiency at each stage of the coffee grounds biorefinery process, including technologies for

Therefore, the biorefinery process of coffee grounds according to the present invention includes the steps of extracting coffee oil from coffee grounds; Extracting galactomannan (GM) from the first solid remaining after extracting the coffee oil; A first pretreatment step of removing lignin and lipids from the second solid remaining after the galactomannan extraction; a second pretreatment step of removing phenol compounds from the first pretreated third solid; A first hydrolysis step of first hydrolyzing the second pretreated fourth solid material; and a second hydrolysis step of secondly hydrolyzing the fifth solid material that has not been decomposed after the first hydrolysis.

The step of extracting coffee oil includes mixing 2 to 4 parts by volume (w/v) of an organic solvent per 1 part by weight of the coffee grounds and stirring at 50° C. to 150° C. to extract the coffee oil into the organic solvent layer; Separating the reactant obtained in the extraction step into an organic solvent from which the coffee oil was extracted and a first solid; and removing the organic solvent from the organic solvent from which the coffee oil was extracted to obtain coffee oil. Here, the mixing ratio of coffee grounds and organic solvent was determined through experiment. If the mixing ratio (w/v) of coffee grounds: organic solvent is less than 1:2, the coffee oil contained in the coffee grounds is not completely extracted, so the yield of coffee oil is reduced. When this becomes low and exceeds 1:4, too much organic solvent is used, increasing the time and cost required for additional processing. The organic solvent is not limited as long as it can extract coffee oil from coffee grounds, but as an example, it is selected from the group consisting of hexane (n-hexane), ethyl ether, chloroform, dichloromethane, ethanol, and methanol. It can be at least one of them. The step of extracting coffee oil into the organic solvent layer is performed by stirring at 50℃ to 150℃. If it is less than 50℃, the extraction time takes a long time and the yield is low, and if it exceeds 150℃, a lot of energy is used and costs increase. There may be a problem. In the step of separating the organic solvent from which the coffee oil was extracted and the first solid, filter paper was used in the examples described later, where all known techniques for separating the liquid phase and the solid phase can be used. The step of obtaining coffee oil is also not limited as long as the organic solvent can be removed from the organic solvent from which the coffee oil was extracted, but as an example, only coffee oil can be easily obtained by removing the organic solvent using a rotary evaporator.

The step of extracting galactomannan involves adjusting a 4.5 to 5.5% (w/v) sodium chloride solution to pH 2.5 to 3.5 with acetic acid and adding 4 to 6 parts by volume (w/v) of a mixed solvent per 1 part by weight of the first solid. After mixing, autoclave at 100°C to 150°C; Separating the reactant obtained after autoclave into a second solid and a solution; And obtaining galactomannan from the separated solution. The mixing ratio of the first solid and the mixed solution was determined through experiment. If the mixing ratio (w/v) of the first solid:mixed solution is less than 2, the solid absorbs all of the solvent, making it difficult to separate the solution and solid in the future. If it exceeds 5, there was a problem that the amount of ethanol used increased in the subsequent reaction of adding ethanol. In addition, the autoclave step is performed at 100°C to 150°C in a reactor with a pressure of 121°C or higher. If it is below 100°C, the extraction yield of galactomannan is low, and if it exceeds 150°C, it requires a lot of energy and the first solid is There may be a throwing away problem. In the step of separating the second solid and the solution, filter paper was used in the examples described later, where all known techniques for separating the liquid phase and the solid phase can be used. The step of obtaining galactomannan is not limited as long as it can be separated after precipitating galactomannan from the separated solution, but in one embodiment, 2 to 4 volumes of ethanol are added per volume of the separated solution to precipitate galactomannan. After centrifugation, the precipitated galactomannan can be separated, and the separated galactomannan can be obtained by freeze-drying. A further step of producing manno-oligosaccharide (MOS) can be performed from the obtained freeze-dried galactomannan. The step of generating MOS involves mixing the freeze-dried galactomannan with a buffer solution having pH 6.5 to pH 7.5. Step 1; A second step of adding mannanase to the mixture and reacting at 35°C to 45°C for 30 to 90 minutes; It includes a third step of separating the reactant into hydrolyzate and solid, and the production efficiency of MOS can be maximized by repeating the first to third steps until the solid is not produced.

The first pretreatment step is performed to remove lignin and lipids from the second solid, including mixing 5 to 15 parts by volume of sodium hydroxide solution per 1 part by weight of the second solid and reacting at 50°C to 100°C; Separating the obtained reaction mixture into a third solid and a reaction solution; washing the third solid with water until pH reaches 6.5 to 7.5; and drying the washed third solid material. The mixing ratio of the second solid and the sodium hydroxide solution was determined through experiment. If the mixing ratio (w/v) of the second solid:sodium hydroxide solution is less than 1:5, the second solid absorbs all of the solution, causing a problem of insufficient reaction solvent. There was a problem that if it exceeded 1:15, a large amount of sodium hydroxide was required, resulting in high costs. Additionally, the concentration of the sodium hydroxide solution may be 0.5M to 2M, and lignin and lipids can be effectively removed only when mixed in the above concentration range. It may be performed further by extracting cafestol and carol contained in the reaction solution obtained in the separation step with an organic solvent. At this time, the organic solvent is not limited as long as it can extract cafestol and carwell at the same time. In one embodiment, it may be any one selected from the group consisting of ethyl ether, chloroform, dichloromethane, ethyl acetate, etc. If the organic solvent is ethyl ether, an extraction step of mixing the reaction solution and ethyl ether at a volume ratio of 0.5 to 1: 2 to 5; Repeating the extraction step two or more times to separate the resulting ethyl ether organic phase and then removing moisture; And removing ethyl ether from the organic phase to obtain cafestol and carwell. The mixing ratio of the reaction solution and ethyl ether was determined through experiment. If the mixing ratio (v/v) of reaction solution:ethyl ether is less than 0.5, the yield of cafestol and carwell is low, and if it exceeds 5, a lot of solvent is required and the cost is high. There was a problem.

The second pretreatment step is performed to remove the phenolic compound from the third solid obtained in the first pretreatment step, mixing 1 to 5 parts by volume of hydrogen peroxide solution per 1 part by weight of the third solid and then reacting at 50°C to 100°C. ; Separating the fourth solid from the obtained reaction mixture; Washing the fourth solid with water until pH reaches 6.5 to 7.5; and drying the washed fourth solid material. The mixing ratio of the third solid and the hydrogen peroxide solution was determined through experiment. If the mixing ratio (w/v) of the third solid: hydrogen peroxide solution is less than 1:2, the third solid absorbs the hydrogen peroxide solution and the reaction cannot proceed. There was a problem that if it exceeded 1:10, the amount of hydrogen peroxide solution used increased, resulting in high costs. Additionally, the concentration of the hydrogen peroxide solution may be 1 to 30%, and only when mixed in the 30% concentration range can the reaction time be short and the phenol compound be removed most efficiently.

The present invention sequentially performs two hydrolysis using different types of enzymes to increase the purity of mannose and MOS obtained from coffee grounds and increase the yield of bioethanol. The first hydrolysis is performed by mannanase. And the secondary hydrolysis can be performed by cellulase and mannanase.

The first hydrolysis step is to produce high purity D-mannose from the fourth solid without a separate purification process, including mixing the fourth solid with a buffer solution of pH 4.5 to pH 5.5; Adding mannanase to the mixture and then performing a primary hydrolysis reaction at 35°C to 40°C for 36 to 60 hours; and separating the first hydrolyzed liquid containing D-mannose into a fifth solid. At this time, 0.01 to 0.1 parts by weight of the mannanase is added per 1 part by weight of the fourth solid. If the mannanase is added in less than 0.01 parts by weight, the D-mannose production yield is low, and if it is added in more than 0.1 parts by weight, the mannanase is high. There was a problem that it was required and cost a lot of money. Here, mannanase is not limited as long as it decomposes mannan into D-mannose, but as an example, mannanase (PP mannanase) obtained from Penicillium purpurogenum can be used.

The second hydrolysis step is a process to obtain a saccharification solution for bioethanol production by hydrolyzing some of the mannan and polysaccharides such as cellulose other than the mannan contained in the fifth solid at once, and the fifth solid and pH 4.5 to pH 4.5. Mixing the buffer solution in step 5.5; Adding cellulase and mannanase to the mixture and then performing a secondary hydrolysis reaction at 45°C to 55°C for 36 to 60 hours; and obtaining a secondary hydrolyzed solution. At this time, 0.01 to 0.1 parts by weight of mannanase and cellulase are added per 1 part by weight of the fifth solid. If mannanase is added in less than 0.01 parts by weight, the D-mannose production yield is low, and if added in more than 0.1 parts by weight, There was a problem that a lot of mannanase was required, so it was expensive. If less than 0.01 part by weight of cellulase is added, the glucose production yield is low, and if added more than 0.1 part by weight, a large amount of cellulase is required, resulting in high cost. Here, mannanase is the same as in the first hydrolysis step and cellulase is not limited as long as it decomposes cellulose into glucose, but in the examples described later, RUT-30 cellulase, a self-produced enzyme, was used. A step of producing bioethanol from the secondary hydrolysis liquid is further performed. The step of producing bioethanol is not limited as long as ethanol can be obtained from the secondary hydrolysis liquid, but as an embodiment, SHF (Separate hydrolysis and fermentation) In other words, it can be performed by sequentially performing enzyme saccharification and fermentation. Here, enzymatic saccharification can be performed by adding yeast extract to the secondary hydrolyzation solution, and fermentation is performed by adding 0.05 to 0.15 parts by weight of dry yeast per 100 volumes of enzymatic saccharification solution as an inoculum, then 30 to 35%. It may be performed for 36 to 60 hours while stirring at 150 to 200 rpm at °C.

According to the biorefinery process of coffee grounds according to the present invention having the above-described configuration, coffee oil, galactomannan, MOS, D-mannose, bioethanol, cafestol, and carol are produced as shown in Figure 10c through each step of the process. You can see that all of these are produced.

In particular, the coffee oil, MOS, D-mannose, and bioethanol of the present invention are produced by the biorefinery process of coffee grounds as described above. As shown in Figure 1, more than 90 ml of coffee oil can be obtained per 1 kg of coffee grounds. More than 100g of MOS can be obtained per 1Kg of coffee grounds, more than 160g of mannose can be obtained per 1Kg of coffee grounds, and more than 90g of bioethanol can be obtained per 1Kg of coffee grounds.

Example

Each step shown in Figure 2 was performed as follows to produce coffee oil, galactomannan, MOS, cafestol and cowol, D-mannose, and bioethanol.

1. Steps to extract coffee oil

Coffee grounds (SCG) and hexane (n-hexane) were mixed at a ratio of 1:3 (w/v) and stirred at 80°C for 3 hours to react and extract coffee oil. After extracting the coffee oil, the hexane from which the coffee oil was extracted is separated from the first solid (O-SCG), which is the residue of the coffee grounds, using filter paper, and the hexane solvent is removed from the coffee oil extracted in the hexane layer using a rotary evaporator. I got coffee oil.

2. Step to extract galactomannan (GM)

To extract galactomannan from the first solid (O-SCG), a 5% (w/v) sodium chloride mixed solution adjusted to pH 3 using the first solid (O-SCG) and acetic acid was mixed 1:5 (w). /v) and autoclaved at 121°C for 15 minutes. After autoclaving, the obtained reaction product was separated into a second solid (OG-SCG) and a solution using filter paper, and ethanol was added to the separated solution in a 1:3 ratio and left overnight at 4°C to precipitate galactomannan. Afterwards, the precipitated galactomannan was separated by centrifuge at 3500 rpm for 20 minutes and freeze-dried to obtain galactomannan (GM).

The steps for generating manno-oligosaccharide (MOS) from lyophilized galactomannan (GM) were performed as follows to obtain MOS. MOS was produced by hydrolyzing GM with PP mannanase. The first hydrolysis was performed at pH 7 and 40°C for 1 hour with 2% (w/v) GM in a total volume of 10 mL. The remaining solid that did not react with the hydrolysate produced after the initial hydrolysis was separated using a centrifuge. The remaining solid was hydrolyzed again for 1 hour under the same conditions. This process was repeated five times until there were no solids remaining.

The PP mannanase used at this time was produced using a strain called Penicillium purpurogenum as follows. P. purpurogenum KACC 47037 was pre-cultured in 100 mL of YPD (yeast extract-pepton-dextrose) medium and then cultured in 1 L Czapex-dox medium (5g/L ammonium sulfate, 2g/L) using 1% coffee grounds (SCG) as a carbon source. L Potassium phosphate, 0.3 g/L Calcium chloride, 0.3 g/L Magnesium sulfate, 2.0 % corn steep, 0.2 % tween 80, 0.2% trace elements solution, g/L Magnesium chloride, 2 g/L Magnesium chloride, 0.2% (v /v) containing trace elements solution) and cultured. Protein concentration was measured using the Bradford protein assay at 595 nm. Mannanase activity was measured using dinitrosalicylic acid (DNS) assay with 1.0% galactomannan as substrate.

3. First pretreatment step to remove lignin and lipids

The first pretreatment, that is, Na pretreatment, was performed to remove lignin and lipid from the second solid (OG-SCG) obtained after galactomannan extraction. 50 mL of 1 M NaOH solution was added per 5 g of the dried second solid (OG-SCG) and reacted at 80°C for 1 hour. For the reaction, a 250 mL Erlenmeyer flask and water bath were used. The obtained reaction mixture was filtered and separated into a third solid (OG-N-SCG) and a reaction solution, and washed with water until the pH of the third solid (OG-N-SCG) reached 7. Afterwards, it was dried at 70°C for 48 hours and used.

The separated sodium hydroxide reaction solution was extracted three times for 1 hour each with 200 mL of ethyl ether. After extraction, the resulting organic layer was separated and moisture was removed using Na ₂ SO ₄ . Afterwards, ethyl ether was removed using a vacuum rotary evaporator, and as a result, cafestol and carwell were obtained.

4. Second pretreatment step to remove phenol compounds

5g of Na-pretreated coffee grounds, that is, the third solid (OG-N-SCG), was reacted at 80°C for 2 hours after adding 50mL of 30% H ₂ O ₂ . For the reaction, a 250 mL Erlenmeyer flask and water bath were used. The obtained reaction mixture was filtered to separate the fourth solid (OG-NH-SCG), and the separated fourth solid (OG-Nh-SCG) was washed with water until the pH reached 7. The separated fourth solid (OG-Nh-SCG) was dried at 70°C for 48 h.

5. First hydrolysis step

Using the fourth solid (OG-NH-SCG) as a substrate, first hydrolysis was performed using PP mannanase as follows. The first hydrolysis reaction was performed with 5.0% substrate (w/v) in 50 mM sodium citrate buffer (pH 5.0) at 37°C for 48 hours. 50 mg of PP mannanase was added per 1g of the fourth solid (OG-NH-SCG). After the first hydrolysis, it was centrifuged at 13,000 rpm to separate it into the first hydrolyzate and the fifth solid. The first hydrolysis solution contains only D-mannose as a monosaccharide.

6. Second hydrolysis step of secondary hydrolysis

A secondary hydrolysis reaction was performed using the residue, that is, the fifth solid, still remaining after the primary hydrolysis treatment, as follows, to produce a saccharification solution for bioethanol production. The secondary hydrolysis reaction was performed with 5.0% substrate (w/v) in 50 mM sodium citrate buffer (pH 5.0) at 50°C for 48 hours to obtain a secondary hydrolysis solution. At this time, 50 mg of two self-produced enzymes, namely RUT-30 cellulase and PP mannanase, were added per 1 g of the fifth solid.

The step of producing bioethanol from the secondary hydrolysis solution was performed using SHF (Separate hydrolysis and fermentation) as follows.

Enzymatic saccharification was performed in an Erlenmeyer flask with a total working volume of 150 mL with 0.1% (w/v) yeast extract, 0.2% (w/v) peptone, and 0.05 M citrate buffer (pH 5.0) as 1% (w/v) substrate. did. For fermentation, 0.1 g dry yeast was added as inoculum to 100 mL of hydrolyzate. Fermentation was performed at 32°C for 48 hours while stirring at 180 rpm to produce bioethanol.

Experimental Example 1

In the example, the fatty acids of coffee oil extracted from coffee grounds were analyzed using a hexane solvent, and the results are shown in Table 1. The fatty acid content of the oil was analyzed using GC.

Fatty acid Wt% Palmitic acid (C16:0) 19.31 Heptadecanoic acid (C17:0) 0.29 Stearic acid (C18:0) 5.54 Arachidic acid (C20:0) 47.09 Behenic acid (C22:0) 0.63 Lignoceric acid (C24:0) 0.40 Saturated 73.26 Oleic acid (C18:1n9c) 6.24 cis-11-Eicosenoic acid (C20:1) 0.38 Monounsaturated 6.620 Linoleic acid (C18:2n6c) 17.82 cis-11,14-Eicosadienoic acid (C20:2) 0.10 cis-13,16-Docosadienoic acid (C22:2) 0.18 Linolenic acid (C18:3n3) 1.19 Arachidonic acid (C20:4n6) 0.10 cis-5,8,11,14,17-Eicosapentaenoic acid (C20:5n3) 0.73 Polyunsaturated 20.12

As shown in Table 1, as a result of coffee oil extraction using hexane, it was possible to extract a yield of about 9.67 wt% compared to the raw material (coffee grounds), and saturated fatty acids and unsaturated fatty acids were 73.26% and 26.74%, respectively. Arachidic acid (C20:0), linoleic acid (C18:2), and palmitic acid (C16:0) account for the majority, and among these, arachidic acid has the highest content at 47.09%. showed. Diesel fuel consists of hydrocarbons with chain lengths between 8 and 21 carbon atoms. The bio coffee oil extracted in the present invention contains a carbon chain distribution in a range similar to biodiesel (C16-C20, 98.69%). These results show that coffee oil extracted from coffee grounds of the present invention can be used as biodiesel, an eco-friendly renewable energy.

Experimental Example 2

Monosaccharide components were analyzed before and after NaHP pretreatment (first and second pretreatment steps) of the second solid material (i.e., residue obtained after extraction of coffee oil and GM from coffee grounds), and the results are shown in Table 2. .

From Table 2, it can be seen that the amount of mannose after NaHP pretreatment is 30.38%, which is 1.34 times more than before the first pretreatment, and the total sugar content also increases from 49.22% to 552.84%. It appears that the mannose content was relatively increased as lignin and fat were removed through NaHP pretreatment. In addition, it was found that the glucose content also increased after NaHP pretreatment, making it possible to improve bioethanol productivity.

As such, the NaHP pretreatment of the present invention, that is, the first pretreatment step and the second pretreatment step, was newly developed to remove lipids and lignin contained in large amounts in coffee grounds in order to efficiently produce high value-added substances from coffee grounds through enzymatic hydrolysis. It was confirmed that the color of coffee grounds changed from dark brown to light yellow after NaHP pretreatment, which is a new pretreatment technology. This appears to be the result of fat and lignin components being removed.

Experimental Example 3

The production and degree of polymerization of manno-oligosaccharide (MOS) produced by varying the amount of PP mannanase enzyme used in the freeze-dried galactomannan (GM) obtained in the example in the range of 50 mg/g substrate to 200 mg/g substrate were investigated, and the results were investigated. It is shown in Figures 3a and 3b. The MOS content after enzymatic hydrolysis was investigated by high-performance liquid chromatography (HPLC). In the drawing, M1 means mannose, M2 means mannobiose, M3 means mannotriose, M4 means mannotetraose, M5 means mannopentaose, and M6 means mannohexaose.

As can be seen in Figures 3a and 3b, the production of MOS increased as the amount of enzyme used increased, and M3, M4, M5, and M6 were mainly produced. The most MOS could be produced when 200 mg of PP mannanase was treated for 60 minutes. In addition, these results showed that PP mannanase is an endo-beta-mannanase as it cuts mannan randomly, and that the degree of polymerization of MOS produced can be controlled by changing time.

Experimental Example 4

MOS production results over time were compared for M1 to M6 obtained in Experimental Example 3 through TLC analysis, and the results are shown in FIG. 4. TLC used a mobile phase of C ₄ H ₁₀ OH/CH ₃ COOH/ H ₂ O (2:1:1, v/v).

From Figure 4, it can be seen that the yield of MOS was the highest when mannanase was treated for 30 to 60 minutes, and the MOS produced at this time had degrees of polymerization 2 (M2) and 3 (M3) the most.

Experimental Example 5

To increase the MOS production yield, the yield was investigated after repeated hydrolysis using PP mannnase, and the results are shown in Figure 5. The concentration of MOS was analyzed using HPLC and TLC. Sugar content was detected using anisidine phthalate reagent containing 1.23 g p-anisidine and 1.66 g phthalic acid in 100 mL 95% ethanol.

After the initial enzymatic hydrolysis using PP mannnase, the total MOS concentration was found to be 3.95 mg/mL, resulting in a hydrolysis efficiency of 43.3%. Therefore, the remaining residues were repeatedly hydrolyzed with PP mannnase to increase MOS yield. The hydrolysis procedure was repeated until no further increase in MOS yield was observed.

As shown in Figure 5, after the first hydrolysis, the main products were M3, M4, M5, and M6, with trace amounts of M2 and M1. After the second hydrolysis, the major products M3 (0.56 mg/mL) and M2 (0.52 mg/mL) were produced, with a trace amount of M1 (0.06 mg/mL). After the third hydrolysis, small amounts of M2 (0.37 mg/mL), M3 (0.09 mg/mL), and M1 (0.06 mg/mL) were confirmed to be produced, and after the fourth hydrolysis, M2 (0.05 mg/mL) mL) was produced. The total sugar content recovered after four repetitions was 5.66 mg/mL (yield 62.0%). The prepared MOS was M6 (0.70 mg/mL), M5 (0.97 mg/mL), M4 (1.08 mg/mL), M3 (1.67 mg/mL), M2 (1.07 mg/mL), and M1 (0.17 mg/mL). ) contained. These results showed that repeated application of PP mannnase under optimal conditions increased the production of MOS, especially the MOS of DP 2-6.

Experimental Example 6

Mannose production yield was investigated for coffee grounds (SCG) and the first solid (O-SCG) to fourth solid (OG-NH-SCG) obtained in each step performed in the examples, and the results are shown in Figure 6. It was.

As shown in Figure 6, the highest mannose yield was obtained from OG-NH-SCG, with a higher yield of OG-N-SCG (4.03 mg/mL, 10.7% yield) compared to SCG without NaHP pretreatment (1.62 mg/mL, 10.7% yield). The mannose content of OG-NH-SCG (26.5%) and OG-NH-SCG (9.28 mg/mL, yield 61.1%) increased by 2.48 times and 5.73 times, respectively. Therefore, it can be seen that NaHP pretreatment, that is, Na pretreatment (first pretreatment) and HP pretreatment (second pretreatment), are necessary processes for optimal production of D-mannose.

Experimental Example 7

After producing mannose through primary hydrolysis of NaHP-pretreated OG-NH-SCG using PP mannanse, the saccharide obtained after secondary hydrolysis using PP mannanse and cellulase was used to produce bioethanol from the residue remaining. The investigation was conducted and the results are shown in Figure 7. The concentration of sugar produced after hydrolysis was analyzed using HPLC.

As a result of analyzing the chemical composition of the fifth solid using GC after the first hydrolysis, it was found that the residue still contained mannose (45.1%) and glucose (13.9%). Therefore, in the present invention, we attempted to further convert bioethanol from the fifth solid using a hydrolysis and fermentation (SHF) process.

As shown in Figure 7, in order to increase the bioethanol yield, the fifth solid was subjected to secondary hydrolysis using self-produced cellulase and PP mannanse as described above in the Example. As a result, not only mannose but also glucose was high. It can be seen that it was produced with high yield.

Experimental Example 8

Bioethanol was produced after an SHF fermentation process using the secondary hydrolyzate, which is a hydrolyzate obtained after secondary hydrolysis, and the results are shown in Figure 8. Glucose consumption and ethanol content were analyzed using HPLC. Ethanol yield was calculated based on the total glucose content in the material and dividing the amount of ethanol produced by the total amount of glucose.

As shown in Figure 8, the ethanol yield from the fourth solid (OG-NH-SCG) was 72.1% of the theoretical maximum after 9 hours, indicating that bioethanol could be produced at a high yield. This shows the potential of coffee grounds as a biomass source for bioethanol production. Additionally, using inexpensive coffee grounds as a feedstock is expected to lower the cost of ethanol production.

Experimental Example 9

Cafestol and Kahweol were extracted from the reaction solution (NaOH solution) obtained after the first pretreatment (Na pretreatment) and analyzed using UV-Vis spectra and 1H NMR, and the results are shown in Figure 9.

As shown in Figure 9, the peaks appearing at 227 nm and 287 nm in the UV-Vis spectrum are peaks showing cafestol and kahweol. In addition, as a result of 1H NMR analysis, the peaks of cafestol and Kawell were confirmed at δ 7.24 (H19 of cafestol) and δ 7.27 (H19 of Kaewol), and the two olefin protons H2 and H1 of Kaewol were doubled at δ 6.21 and δ 5.89, respectively. It appeared.

Experimental Example 10

The mass balance of high value-added materials produced from coffee grounds and the biorefinery process of the present invention were compared with existing studies, and the results are shown in Figures 10A to 10C.

As shown in Figure 10c, according to the biorefinery process of the present invention, about 97 mL of coffee oil, about 164 g of D-mannose, 102 g of MOS, and about 99 g of bioethanol could be successfully produced from 1 kg of dry SCG. . In addition, 78.2% of the initial carbohydrates in SCG, which is coffee grounds, were converted into high value-added substances (35.2% mannose, 21.9% MOS, 21.2% ethanol), and based on 1 kg of total coffee grounds, they were converted into value-added substances with a high yield of 46.2%. I was able to confirm that it was converted.

This product yield value is not only a significantly higher yield compared to the values reported in previous studies shown in Figures 10a and 10b, but also, unlike previous studies, according to the present invention, more diverse and high-value functional biocompounds are produced in coffee. It can be seen that it can be produced from waste.

As such, the biorefinery process of coffee grounds according to the present invention was effective in recovering value-added substances from coffee grounds, and also produced coffee oil, bioethanol, mannose, MOS, cafestol, and carwell in the integrated biorefinery process. It was very effective.

Although the present invention has been illustrated and described by way of preferred embodiments as described above, it is not limited to the above-described embodiments and is intended to be used by those skilled in the art without departing from the spirit of the invention. Various changes and modifications will be possible.

Claims (22)

Extracting coffee oil from coffee grounds;
Extracting galactomannan (GM) from the first solid remaining after extracting the coffee oil;
A first pretreatment step of removing lignin and lipids from the second solid remaining after the galactomannan extraction;
a second pretreatment step of removing phenol compounds from the first pretreated third solid;
A first hydrolysis step of first hydrolyzing the second pretreated fourth solid material; and
A biorefinery process of coffee grounds comprising a second hydrolysis step of secondary hydrolysis of the fifth solid material that has not been decomposed after the first hydrolysis.
According to claim 1,
The step of extracting the coffee oil is an extraction step in which coffee oil is extracted into the organic solvent layer by mixing 2 to 4 parts by weight (w/v) of an organic solvent per 1 part by weight of the coffee grounds and stirring at 50 ℃ to 150 ℃. ; Separating the reactant obtained in the extraction step into an organic solvent from which the coffee oil was extracted and a first solid; and obtaining coffee oil by removing the organic solvent from the organic solvent from which the coffee oil was extracted. According to claim 2,
A biorefinery process for coffee grounds, wherein the organic solvent is at least one selected from the group consisting of hexane (n-hexane), ethyl ether, chloroform, dichloromethane, ethanol, and methanol.
According to claim 1,
In the step of extracting the galactomannan, a 4.5 to 5.5% (w/v) sodium chloride solution is adjusted to pH 2.5 to 3.5 with acetic acid and mixed with 4 to 6 parts by volume (w/v) of a mixed solvent per 1 part by weight of the first solid. ) and then autoclave at 100°C to 150°C; Separating the reactant obtained after autoclave into a second solid and a solution; And obtaining galactomannan from the separated solution. A biorefinery process of coffee grounds, characterized in that it is performed including.
According to claim 4,
Obtaining the galactomannan includes adding 2 to 4 parts by volume of ethanol per 1 part by volume of the separated solution to precipitate the galactomannan; Separating the precipitated galactomannan by centrifugation; And a step of freeze-drying the separated galactomannan. A biorefinery process of coffee grounds, characterized in that it is performed including.
According to claim 5,
A biorefinery process for coffee grounds, characterized in that the step of generating manno-oligosaccharide (MOS) from the freeze-dried galactomannan is further performed.
According to claim 6,
The step of generating the MOS includes a first step of mixing the freeze-dried galactomannan and a buffer solution of pH 6.5 to pH 7.5; A second step of adding mannanase to the mixture and reacting at 35°C to 45°C for 30 to 90 minutes; A third step of separating the reactants into hydrolysates and solids, and repeating the first to third steps until no solids are produced.
According to claim 1,
The first pretreatment step includes mixing 5 to 15 parts by volume of sodium hydroxide solution per 1 part by weight of the second solid and then reacting at 50°C to 100°C; Separating the obtained reaction mixture into a third solid and a reaction solution; washing the third solid with water until pH reaches 6.5 to 7.5; And a step of drying the washed third solid material. A biorefinery process for coffee grounds, comprising:
According to claim 8,
A biorefinery process for coffee grounds, further comprising extracting cafestol and carwell contained in the reaction solution with an organic solvent.
According to clause 9,
If the organic solvent is ethyl ether, an extraction step of mixing the reaction solution and ethyl ether in a volume ratio of 0.5 to 1: 2 to 5; Repeating the extraction step two or more times to separate the resulting ethyl ether organic phase and then removing moisture; and removing ethyl ether from the organic phase to obtain cafestol and carwell.
According to claim 1,
The second pretreatment step includes mixing 1 to 5 parts by volume of hydrogen peroxide solution per 1 part by weight of the third solid and then reacting at 50°C to 100°C; Separating the fourth solid from the obtained reaction mixture; Washing the fourth solid with water until pH reaches 6.5 to 7.5; And a step of drying the washed fourth solid material. A biorefinery process for coffee grounds, comprising:
According to claim 1,
A biorefinery process of coffee grounds, characterized in that the first hydrolysis is performed by mannanase, and the second hydrolysis is performed sequentially by cellulase and mannanase.
According to claim 1,
The first hydrolysis step includes mixing the fourth solid with a buffer solution of pH 4.5 to pH 5.5; Adding mannanase to the mixture and then performing a primary hydrolysis reaction at 35°C to 40°C for 36 to 60 hours; And separating the first hydrolyzate containing D-mannose and the fifth solid. A biorefinery process of coffee grounds, characterized in that it is performed including.
According to claim 13,
A biorefinery process of coffee grounds, characterized in that 0.01 to 0.1 parts by weight of the mannanase is added per 1 part by weight of the fourth solid.
According to claim 1,
The second hydrolysis step includes mixing the fifth solid with a buffer solution of pH 4.5 to pH 5.5; Adding cellulase and mannanase to the mixture and then performing a secondary hydrolysis reaction at 45°C to 55°C for 36 to 60 hours; and obtaining a secondary hydrolyzate. A biorefinery using coffee grounds.
According to claim 15,
A biorefinery process for coffee grounds, characterized in that 0.01 to 0.1 parts by weight of the mannanase and cellulase are added per 1 part by weight of the fifth solid.
According to claim 15,
A biorefinery process using coffee grounds, further comprising the step of generating bioethanol from the secondary hydrolyzate.
The method according to any one of claims 1 to 17,
A biorefinery process using coffee grounds, characterized in that coffee oil, galactomannan, MOS, D-mannose, bioethanol, cafestol, and carwell are produced from the coffee grounds.
Coffee oil obtained by performing the biorefinery process of any one of claims 1 to 17, wherein more than 90 ml of coffee oil is obtained per 1 kg of coffee grounds.
A manno-oligosaccharide obtained by performing the biorefinery process of any one of claims 1 to 17, and obtained in an amount of more than 100 g per 1 kg of coffee grounds.
Mannose is obtained by performing the biorefinery process of any one of claims 1 to 17, and is obtained in an amount of 160 g or more per 1 kg of coffee grounds. Bioethanol obtained by performing the biorefinery process of any one of claims 1 to 17, and characterized in that more than 90 g per 1 kg of coffee grounds is obtained.