A composition for increasing enzyme productivity of genus Trichoderma comprising lactose

Abstract

The present invention relates to a composition for inducing enzyme production in Trichoderma genus strains containing lactose. Specifically, it has been confirmed that a composition containing both monosaccharides and disaccharides, and containing glucose and lactose in a certain ratio, exhibits excellent activity in inducing enzyme production from Trichoderma genus strains. Thus, the composition can be usefully utilized for mass production of enzymes from Trichoderma genus strains.

Description

Technology Field

[0001] The present invention relates to a composition for increasing the enzyme productivity of a Trichoderma genus strain containing lactose, and a method for producing an enzyme using said composition. Background Technology

[0003] Cellulose is the most abundant carbon source on Earth, and attention has been focused on producing glucose, used in the production of fuels, food, and chemicals, through the hydrolysis of cellulose. Cellulose can be hydrolyzed by chemical or biological methods; however, the chemical method of acid hydrolysis requires higher temperatures compared to the biological method using enzymes and has the disadvantage of producing a large amount of unwanted byproducts.

[0004] Enzymes that break down fibrous material are obtained from microorganisms such as fungi, bacteria, and actinomycetes. The most commercially effective cellulose-degrading enzymes are from the genus Trichoderma ( Trichoderma It is an enzyme obtained from microorganisms (sp.), and in addition, the genus Aspergillus ( Aspergillus sp.), genus Penicillium ( Penicillium Microorganisms such as sp.) are also known to produce cellulose-degrading enzymes.

[0005] The process of cellulose-degrading enzyme biosynthesis is known to be regulated by induction and catabolite repression. Specifically, sophorose, gentiobiose, or laminaribiose Penicillium purpurogenum It is known that it induces cellulase synthesis in the P-26 strain (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1992, p. 106-110), and trehalose Clostridium papyrosolvensIt is known that cellulase synthesis is induced in the CFR-703 strain (Process Biochemistry 37 (2001) 241-245). In addition, cellulase, whose activity of cellulose-degrading enzymes is also regulated by end-product inhibition, is induced by Cellobiose, and Thermonospora fusca strains and Trichoderma reesei In the case of strains, β-glucosidase is inhibited by glucose. The activity of cellulose-degrading enzymes synthesized as a result of such complex regulatory mechanisms varies depending on the type of microorganism and culture conditions. The problem to be solved

[0007] The object of the present invention is a trichoderma comprising glucose and lactose ( Trichoderma The purpose is to provide a composition for increasing the enzyme productivity of microorganisms in the genus ).

[0008] Another object of the present invention is a trichoderma comprising glucose and lactose ( Trichoderma The invention provides a composition for enhancing the activity of enzymes produced from microorganisms of the genus ).

[0009] Another object of the present invention is a trichoderma comprising glucose and whey permeate ( Trichoderma The purpose is to provide a composition for increasing the enzyme productivity of microorganisms in the genus ).

[0010] Another object of the present invention is a trichoderma comprising glucose and whey permeate ( Trichoderma The invention provides a composition for enhancing the activity of enzymes produced from microorganisms of the genus ).

[0011] Another objective of the present invention is to provide a method for producing an enzyme from a Trichoderma genus strain using a composition for inducing enzyme production of a Trichoderma genus strain containing the disaccharide.

[0012] Another objective of the present invention is to provide a culture medium for Trichoderma microorganisms comprising a composition containing glucose and lactose, or a composition containing glucose and whey permeate.

[0013] Another object of the present invention provides a method for producing an enzyme from a microorganism of the genus Trichoderma, comprising the steps of: preparing a culture medium by adding a composition comprising glucose and lactose, or a composition comprising glucose and whey permeate; and culturing a microorganism of the genus Trichoderma in the culture medium to obtain a culture. means of solving the problem

[0015] Each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention is not to be considered limited by the specific descriptions provided below. Additionally, a person skilled in the art can recognize or identify a number of equivalents to the specific embodiments of the present invention described in this invention using only ordinary experiments. Moreover, such equivalents are intended to be included in the present invention.

[0017] One aspect of the present application is a trichoderma comprising glucose and lactose ( Trichoderma A composition for increasing the enzyme productivity of microorganisms of the genus ) is provided.

[0018] Another aspect of the present application is a trichoderma comprising glucose and whey permeate ( Trichoderma A composition for enhancing the activity of enzymes produced from microorganisms of the genus ) is provided.

[0019] In the present application, the “tricoderm ( Trichoderma The “microorganism of the genus )” may be a microorganism of the genus Trichoderma that produces enzymes, and the microorganism of the genus Trichoderma may be a microorganism that produces cellulase, xylanase, phytase, or glucanase, and may be a microorganism of the genus Trichoderma capable of producing all of the enzymes of cellulase, xylanase, phytase, and glucanase.

[0020] Specifically, the above-mentioned microorganism of the genus Trichoderma is Trichoderma resei ( Trichoderma reesei It can be.

[0021] In the present application, the “enzyme” may be produced from a microorganism of the genus Trichoderma, and specifically, may be one or more enzymes selected from the group consisting of cellulase, xylanase, phytase, and glucanase, and may include all of cellulase, xylanase, phytase, and glucanase.

[0022] In this application, “enzyme productivity increase” may mean increasing the amount of enzyme produced from microorganisms. The “enzyme productivity increase” can be confirmed by measuring the fermentation activity of the produced enzyme, and “enzyme productivity increase” may be interpreted as having the same meaning as “enzyme production induction” or “enzyme activity enhancement.”

[0023] The above composition contains a specific amount of lactose based on the total weight of the composition, thereby trichoderma ( Trichoderma It can increase the enzyme productivity of microorganisms of the genus ) or enhance the activity of enzymes produced from microorganisms of the genus Trichoderma.

[0024] The content of lactose included in the above composition is 5 to 20 wt%, 7 to 20 wt%, 9 to 20 wt%, 11 to 20 wt%, 13 to 20 wt%, 5 to 18 wt%, 7 to 18 wt%, 9 to 18 wt%, 11 to 18 wt%, 13 to 18 wt%, 5 to 17 wt%, 7 to 17 wt%, 9 to 17 wt%, 11 to 17 wt%, 13 to 17 wt%, 5 to 15 wt%, 7 to 15 wt%, 9 to 15 wt%, 11 to 15 wt%, 13 to 15 wt%, 5 to 14 wt%, 7 to 14 wt%, 9 to 14 wt%, 11 to 14 wt%, or 13 to 14 wt% based on the total weight of the composition. It can be weight %.

[0025] The content of glucose included in the above composition may be 39 to 55 wt%, 41 to 55 wt%, 43 to 55 wt%, 39 to 53 wt%, 41 to 53 wt%, 43 to 53 wt%, 39 to 52 wt%, 41 to 52 wt%, 43 to 52 wt%, 39 to 50 wt%, 41 to 50 wt%, 43 to 50 wt%, 39 to 48 wt%, 41 to 48 wt%, 43 to 48 wt%, 39 to 47 wt%, 41 to 47 wt%, 43 to 47 wt%, 39 to 45 wt%, 41 to 45 wt%, or 43 to 45 wt% based on the total weight of the composition.

[0026] The glucose and lactose included in the above composition may have a specific content ratio (mixing ratio).

[0027] Specifically, the ratio of glucose to lactose content included in the composition may be 7:1 to 2:1, 6:1 to 2:1, 5:1 to 2:1, 4:1 to 2:1, 7:1 to 3:1, 6:1 to 3:1, 5:1 to 3:1, or 4:1 to 3:1.

[0028] The above composition may further include fructose as a monosaccharide.

[0029] The monosaccharide content included in the above composition may be 60% by weight or less, 57% by weight or less, 55% by weight or less, 43 to 55% by weight, 45 to 55% by weight, 47 to 55% by weight, 43 to 53% by weight, 45 to 53% by weight, 47 to 53% by weight, 43 to 52% by weight, 45 to 52% by weight, 47 to 52% by weight, 43 to 50% by weight, 45 to 50% by weight, 47 to 50% by weight, 43 to 48% by weight, 45 to 48% by weight, or 47 to 48% by weight, based on the total weight of the composition.

[0030] The content of fructose included in the above composition may be 1 to 5 weight%, 2 to 5 weight%, 3 to 5 weight%, 1 to 4 weight%, 2 to 4 weight%, or 3 to 4 weight% based on the total weight of the composition.

[0031] The above composition may further include one or more disaccharides selected from the group consisting of trehalose, isomaltose, gentiobiose, cellobiose, and sophorose.

[0032] The disaccharide content included in the above composition is 21 wt% or more, 23 wt% or more, 25 wt% or more, 27 wt% or more, 29 wt% or more, 32 wt% or more, 34 wt% or more, 27 wt% to 40 wt%, 30 wt% to 40 wt%, 33 wt% to 40 wt%, 34 wt% to 40 wt%, 27 wt% to 38 wt%, 30 wt% to 38 wt%, 33 wt% to 38 wt%, 34 wt% to 38 wt%, 27 wt% to 36 wt%, 30 wt% to 36 wt%, 33 wt% to 36 wt%, 34 wt% to 36 wt%, 27 wt% to 35 wt%, 30 wt% to 35 wt%, 33 wt% to 35 wt%, or 34 wt% It may be in the range of 35% by weight.

[0033] The content of trehalose included in the above composition may be 1 to 4 weight%, 2 to 4 weight%, 1 to 3 weight%, or 2 to 3 weight% based on the total weight of the composition.

[0034] The content of isomaltose included in the above composition may be 4.5 to 7 weight%, 5 to 7 weight%, 4.5 to 6 weight%, or 5 to 6 weight% based on the total weight of the composition.

[0035] The content of gentiobiose included in the above composition may be 5 to 10 weight%, 6 to 10 weight%, 7 to 10 weight%, 5 to 10 weight%, 6 to 10 weight%, 7 to 10 weight%, 5 to 9 weight%, 6 to 9 weight%, 7 to 9 weight%, 5 to 8 weight%, 6 to 8 weight%, or 7 to 8 weight% based on the total weight of the composition.

[0036] The content of cellobiose included in the above composition may be 1.0 to 3.0 weight% relative to the total weight of the composition.

[0037] The content of sophorose included in the above composition may be 2 to 6 weight%, 3 to 6 weight%, 4 to 6 weight%, 2 to 5 weight%, 3 to 5 weight%, 4 to 5 weight%, 2 to 4.5 weight%, 3 to 4.5 weight%, or 4 to 4.5 weight% based on the total weight of the composition.

[0038] The above composition may additionally include an oligomer.

[0039] The oligomer content included in the above composition may be 12 to 23 weight%, 14 to 23 weight%, 16 to 23 weight%, 17 to 23 weight%, 12 to 21 weight%, 14 to 21 weight%, 16 to 21 weight%, 17 to 21 weight%, 12 to 19 weight%, 14 to 19 weight%, 16 to 19 weight%, 17 to 19 weight%, 12 to 18 weight%, 14 to 18 weight%, 16 to 18 weight%, or 17 to 18 weight% based on the total weight of the composition.

[0040] In one example of this specification, it was confirmed that when microorganisms of the genus Trichoderma are cultured using a composition containing lactose, the enzyme productivity of the microorganisms of the genus Trichoderma is increased compared to a composition not containing lactose, and the activity of the enzymes produced by the microorganisms of the genus Trichoderma is enhanced. Therefore, a composition containing lactose can be utilized to increase the enzyme productivity of microorganisms of the genus Trichoderma or to enhance the activity of enzymes produced by microorganisms of the genus Trichoderma.

[0042] One aspect of the present application is a trichoderma comprising glucose and whey permeate ( TrichodermaA composition for increasing the enzyme productivity of microorganisms of the genus ) is provided.

[0043] Another aspect of the present application is a trichoderma comprising glucose and whey permeate ( Trichoderma A composition for enhancing the activity of enzymes produced from microorganisms of the genus ) is provided.

[0044] The above-mentioned Trichoderma genus microorganisms, enzymes, for increasing enzyme productivity and enhancing enzyme activity, etc., are as described above.

[0045] In this application, the term “whey permeate” may refer to a whey permeate obtained through a membrane-based treatment process such as ultrafiltration or reverse osmosis, and the whey permeate may be used interchangeably with “a solution of whey permeate powder.” Specifically, the whey permeate may be prepared by adding whey permeate powder to a solution.

[0046] The above composition comprises a specific amount of whey membrane permeate based on the total weight of the composition, thereby including trichoderma ( Trichoderma It can increase the enzyme productivity of microorganisms of the genus ) or enhance the activity of enzymes produced from microorganisms of the genus Trichoderma.

[0047] The content of the whey membrane permeate included in the above composition is 5 to 20 wt%, 7 to 20 wt%, 9 to 20 wt%, 11 to 20 wt%, 13 to 20 wt%, 5 to 18 wt%, 7 to 18 wt%, 9 to 18 wt%, 11 to 18 wt%, 13 to 18 wt%, 5 to 17 wt%, 7 to 17 wt%, 9 to 17 wt%, 11 to 17 wt%, 13 to 17 wt%, 5 to 15 wt%, 7 to 15 wt%, 9 to 15 wt%, 11 to 15 wt%, 13 to 15 wt%, 5 to 14 wt%, 7 to 14 wt%, 9 to 14 wt%, 11 to 14 wt%, or 13 wt% based on the total weight of the composition. It may be up to 14 weight%.

[0048] The content of glucose included in the above composition may be 39 to 55 wt%, 41 to 55 wt%, 43 to 55 wt%, 39 to 53 wt%, 41 to 53 wt%, 43 to 53 wt%, 39 to 52 wt%, 41 to 52 wt%, 43 to 52 wt%, 39 to 50 wt%, 41 to 50 wt%, 43 to 50 wt%, 39 to 48 wt%, 41 to 48 wt%, 43 to 48 wt%, 39 to 47 wt%, 41 to 47 wt%, 43 to 47 wt%, 39 to 45 wt%, 41 to 45 wt%, or 43 to 45 wt% based on the total weight of the composition.

[0049] The glucose and whey membrane permeate included in the above composition may have a specific content ratio (mixing ratio).

[0050] Specifically, the content ratio of glucose to whey membrane permeate included in the composition may be 7:1 to 2:1, 6:1 to 2:1, 5:1 to 2:1, 4:1 to 2:1, 7:1 to 3:1, 6:1 to 3:1, 5:1 to 3:1, or 4:1 to 3:1.

[0051] The above composition may further include fructose as a monosaccharide.

[0052] The monosaccharide content included in the above composition may be 60% by weight or less, 57% by weight or less, 55% by weight or less, 43 to 55% by weight, 45 to 55% by weight, 47 to 55% by weight, 43 to 53% by weight, 45 to 53% by weight, 47 to 53% by weight, 43 to 52% by weight, 45 to 52% by weight, 47 to 52% by weight, 43 to 50% by weight, 45 to 50% by weight, 47 to 50% by weight, 43 to 48% by weight, 45 to 48% by weight, or 47 to 48% by weight, based on the total weight of the composition.

[0053] The content of fructose included in the above composition may be 1 to 5 weight%, 2 to 5 weight%, 3 to 5 weight%, 1 to 4 weight%, 2 to 4 weight%, or 3 to 4 weight% based on the total weight of the composition.

[0054] The above composition may further include one or more disaccharides selected from the group consisting of trehalose, isomaltose, gentiobiose, cellobiose, and sophorose.

[0055] The disaccharide content included in the above composition is 21 wt% or more, 23 wt% or more, 25 wt% or more, 27 wt% or more, 29 wt% or more, 32 wt% or more, 34 wt% or more, 27 wt% to 40 wt%, 30 wt% to 40 wt%, 33 wt% to 40 wt%, 34 wt% to 40 wt%, 27 wt% to 38 wt%, 30 wt% to 38 wt%, 33 wt% to 38 wt%, 34 wt% to 38 wt%, 27 wt% to 36 wt%, 30 wt% to 36 wt%, 33 wt% to 36 wt%, 34 wt% to 36 wt%, 27 wt% to 35 wt%, 30 wt% to 35 wt%, 33 wt% to 35 wt%, or 34 wt% It may be in the range of 35% by weight.

[0056] The content of trehalose included in the above composition may be 1 to 4 weight%, 2 to 4 weight%, 1 to 3 weight%, or 2 to 3 weight% based on the total weight of the composition.

[0057] The content of isomaltose included in the above composition may be 4.5 to 7 weight%, 5 to 7 weight%, 4.5 to 6 weight%, or 5 to 6 weight% based on the total weight of the composition.

[0058] The content of gentiobiose included in the above composition may be 5 to 10 weight%, 6 to 10 weight%, 7 to 10 weight%, 5 to 10 weight%, 6 to 10 weight%, 7 to 10 weight%, 5 to 9 weight%, 6 to 9 weight%, 7 to 9 weight%, 5 to 8 weight%, 6 to 8 weight%, or 7 to 8 weight% based on the total weight of the composition.

[0059] The content of cellobiose included in the above composition may be 1.0 to 3.0 weight% relative to the total weight of the composition.

[0060] The content of sophorose included in the above composition may be 2 to 6 weight%, 3 to 6 weight%, 4 to 6 weight%, 2 to 5 weight%, 3 to 5 weight%, 4 to 5 weight%, 2 to 4.5 weight%, 3 to 4.5 weight%, or 4 to 4.5 weight% based on the total weight of the composition.

[0061] The above composition may additionally include an oligomer.

[0062] The oligomer content included in the above composition may be 12 to 23 weight%, 14 to 23 weight%, 16 to 23 weight%, 17 to 23 weight%, 12 to 21 weight%, 14 to 21 weight%, 16 to 21 weight%, 17 to 21 weight%, 12 to 19 weight%, 14 to 19 weight%, 16 to 19 weight%, 17 to 19 weight%, 12 to 18 weight%, 14 to 18 weight%, 16 to 18 weight%, or 17 to 18 weight% based on the total weight of the composition.

[0063] In one example of this specification, it was confirmed that when microorganisms of the genus Trichoderma are cultured using a composition containing whey membrane permeate, the enzyme productivity of the microorganisms of the genus Trichoderma is increased compared to a composition not containing whey membrane permeate, and the activity of the enzymes produced by the microorganisms of the genus Trichoderma is enhanced. Therefore, a composition containing whey membrane permeate can be utilized to increase the enzyme productivity of microorganisms of the genus Trichoderma or to enhance the activity of enzymes produced by microorganisms of the genus Trichoderma.

[0065] One aspect of the present application provides a culture medium for Trichoderma microorganisms comprising a composition comprising glucose and lactose, or a composition comprising glucose and whey permeate.

[0066] Another aspect of the present invention provides a method for producing an enzyme from a microorganism of the genus Trichoderma, comprising the steps of: preparing a culture medium by adding a composition comprising glucose and lactose, or a composition comprising glucose and whey permeate; and culturing a microorganism of the genus Trichoderma in the culture medium to obtain a culture.

[0067] The above-mentioned microorganisms of the genus Trichoderma, enzymes, whey membrane permeate, etc. are as described above.

[0068] The above medium may include a composition containing glucose and lactose, or a composition containing glucose and whey permeate as a carbon source.

[0069] In this application, “culture” may be a fed-batch culture.

[0070] In this application, the term “fed-batch culture” refers to a culture method in which a feed medium is supplied after starting culture with a fermentation medium or a base medium. Fed-batch culture has the advantage of allowing arbitrary control of the substrate concentration in the culture medium, the addition of substrate at an appropriate rate, and the free control of the amount of supplied substrate because there is no outflow.

[0071] In this application, the term “medium” refers to a substance mixed with nutrients as the main component required for culturing microorganisms, and supplies nutrients and growth factors, including water, which is indispensable for survival and growth. Specifically, the medium and other culture conditions used for culturing microorganisms in this application may be used without special limitations as long as they are media commonly used for culturing microorganisms.

[0072] The composition comprising glucose and lactose of the present application, or the composition comprising glucose and whey permeate, may be included in both the fermentation medium and the feed medium used for fed-batch culture. Effects of the invention

[0074] The present invention relates to a composition for inducing enzyme production in Trichoderma genus strains containing lactose. Specifically, it has been confirmed that a composition containing both monosaccharides and disaccharides, and containing glucose and lactose in a certain ratio, exhibits excellent activity in inducing enzyme production from Trichoderma genus strains. Thus, the composition can be usefully utilized for mass production of enzymes from Trichoderma genus strains. Specific details for implementing the invention

[0076] The present invention will be explained in more detail below through examples. However, these examples are intended to illustrate one or more specific embodiments, and the scope of the present invention is not limited to these examples.

[0078] Comparative Example 1. Preparation of a disaccharide composition

[0079] Glucose powder with a purity of 95% or higher (hydrated crystalline glucose, Daejeong Hwakum) was dissolved using steam to prepare 40 kg of a glucose solution with a concentration of 71% w / w. 0.3 kg of β-glucosidase at a concentration of 1,650 U / ml was added to the prepared glucose solution and left to stand at 60°C for 7 to 21 days to prepare a composition containing disaccharides (Treated Glucose Syrup, TGS). Hereinafter, the above composition is referred to as the control group.

[0081] Example 1. Preparation of disaccharide compositions containing lactose in various proportions

[0082] A mixed composition with a final concentration of 70-75% by adding lactose instead of glucose to the composition containing the disaccharide prepared in Comparative Example 1, such that the content of lactose (purity 95% by weight or higher, Daejeong Hwakum) was 6.86% by weight (hereinafter, composition of Example 1-1), 10.29% by weight (hereinafter, composition of Example 1-2), 13.72% by weight (hereinafter, composition of Example 1-3), or 17.14% by weight (hereinafter, composition of Example 1-4) based on the total weight of the composition.

[0084] Example 2. Preparation of a disaccharide composition containing whey membrane permeate

[0085] A mixed composition with a final concentration of 70-75% by adding whey permeate powder instead of glucose to the disaccharide-containing composition prepared in Comparative Example 1, such that the content of the whey membrane permeate (purity 88% by weight or higher) was 13.72% by weight based on the total weight of the composition. The composition of the whey permeate powder is shown in Table 1 below.

[0087] Composition of whey permeable powder NO Composition (dry weight basis) Whey permeate powder 1 DM (%) 24 2 CP (%) 2.6 3 Nonprotein N (%) 0.36 4 Ash (%) 7.63 5 Crude fat (%) 0.03 6 Lactose (%) 88.1 7 Total sugars (%) 88.7 8 Organic acids (%) 4.1 9 Calcium (%) 0.67 10 Phosphorus (%) 1.12 11 Magnesium (%) 0.17 12 Potassium (%) 2 13 Chloride (%) 1.12 14 Sodium (%) 0.67 15 Iron (mg / kg) 45.81 16 Copper (mg / kg) 4.16 17 Zinc (mg / kg) 20.82 18 pH 5.76 19 Density (kg / L) 1.11 20 Viscosity1 (cP) 3.5

[0088] Experimental Example 1. Analysis of sugar content of a composition containing lactose or whey permeated powder

[0089] The following experiment was performed to analyze the sugar content of the compositions containing disaccharides prepared in Comparative Example 1, Example 1, and Example 2, the mixed composition further containing lactose as a disaccharide, and the mixed composition further containing whey permeate powder as a disaccharide.

[0090] Specifically, the concentrations of monosaccharides and disaccharides included in each composition Measurements were performed using a Bio-LC system (Dionex ICS-3000, Sunnyvale, CA, United States) equipped with an electrochemical detector and a CarboPac PAI column. The column was eluted with 0.1M NaOH (0–5 min) at 30°C, followed by a linear gradient of sodium acetate (0–0.2M) at 1 mL / min for 5–35 minutes. The content (%) of the monosaccharides glucose and fructose, and the disaccharides trehalose, isomaltose, maltose, cellobiose, gentiobiose, sophorose, and lactose in the monosaccharide and disaccharide mixed compositions prepared in Comparative Example 1, Example 1, and Example 2 was measured and is shown in Tables 2 and 3 below.

[0091] Analysis results of sugar content of a composition containing lactose division Content (%) etc Degree of polymerization Sugars Comparative example Example 1-1 Examples 1-2 Examples 1-3 Examples 1-4 DP1 Glucose 57.77 50.92 47.49 44.06 40.63 C-source Fructose 3.66 3.66 3.66 3.66 3.66 sum 61.44 54.58 51.15 47.72 44.29 DP2 Trehalose 2.67 2.67 2.67 2.67 2.67 α(1→1) Isomaltose 5.31 5.31 5.31 5.31 5.31 α(1→6) Gentiobiose 7.42 7.42 7.42 7.42 7.42 β(1→6) Cellobiose 1.3 1.3 1.3 1.3 1.3 β(1→4) sophorose 4.03 4.03 4.03 4.03 4.03 β(1→2) Lactose 0 6.86 10.29 13.72 17.14 β(gla1→glu 4) sum 20.73 27.59 31.02 34.45 37.88 DP3 or higher oligomer 17.83 17.83 17.83 17.83 17.83 Total 100 100 100 100 100

[0092] division Content (%) etc Degree of polymerization Sugars Comparative example Example 2 DP1 Glucose 57.77 44.06 C-source Fructose 3.66 3.66 sum 61.44 47.72 　 DP2 Trehalose 2.67 2.67 α(1→1) Isomaltose 5.31 5.31 α(1→6) Gentiobiose 7.42 7.42 β(1→6) Cellobiose 1.3 1.3 β(1→4) sophorose 4.03 4.03 β(1→2) whey permeate 0 13.72 β(gla1→glu 4)

[0093] Experimental Example 1. Confirmation of the effect of inducing enzyme production according to the lactose content included in the composition

[0094] To confirm the difference in the effect of inducing enzyme production in Trichoderma genus strains according to the lactose content included in the composition, the fermentation activity of the enzymes was verified using a fed-batch culture method with a composition without added lactose and a composition with added lactose.

[0096] Experimental Example 1-1. Preparation of enzyme-producing strains

[0097] As an enzyme-producing strain, the Trichoderma lessei QM6a strain (ATCC13631), capable of producing acidic cellulase, neutral xylanase, phytase, and glucanase, was prepared. The Trichoderma lessei QM6a strain was grown on potato dextrose agar plates at 30°C for 5 days to form spores. After spore formation, the spores of each strain were resuspended in sterile NaCl solution (9 g / L), and 30% sterile glycerol was added. This mixture was stored in a 1.8 ml tube at -80°C.

[0099] Experimental Example 1-2. Flask culture

[0100] Trichoderma lessei QM6a strains were cultured on potato dextrose agar plates for 5 to 7 days, and the mycelia were inoculated into 50 ml of PDB (potato dextrose broth) and pre-cultured for 72 hours at 30°C with shaking at 200 rpm. For the main culture, a liquid medium composed of 30 g / L lactose, 10 g / L ammonium sulfate, 20 g / L yeast extract, 5 g / L potassium phosphate (KH2PO4), and 0.5 g / L magnesium sulfate was used. 5% (v / v) of the pre-culture solution was inoculated into 200 ml of the liquid medium, and the culture solution (enzyme solution) was collected while incubating at 28°C with 200 rpm for 5 to 7 days. During this process, the culture temperature was maintained in the range of 28°C to 32°C, and the pH of the liquid medium was maintained in the range of 4.0 to 6.0.

[0102] Experimental Example 1-3. Obtaining culture medium after fed-batch culture of enzyme-producing strains

[0103] The Trichoderma resei QM6a strain, which produces the enzyme of Experimental Example 1-1, was cultured using a fed-batch culture method as follows, and a culture medium for measuring enzyme activity was obtained.

[0104] Specifically, fed-batch culture was carried out using a fermentation medium containing 20.0 g / L of carbohydrates, 5.0 g / L of ammonium sulfate, 1.5 g / L of magnesium sulfate, 0.5 g / L of calcium chloride, 5.0 g / L of potassium dihydrogen phosphate, 10.0 g / L of yeast extract, 10.0 mg / L of ferric sulfate, 4.0 mg / L of cobalt chloride, 2.0 mg / L of sodium molybdate, 0.4 mg / L of boric acid, and 1.0 g / L of Tween 80. Using DO as an indicator, the supply rate of the carbon source in the feed medium (Comparative Example 1 or the compositions of Examples 1-1 to 1-4, which are lactose-added compositions) was supplied at a constant rate of 5.0 to 9.0 g / L per hour at the point when DO increased. The supply of the feed medium was performed using a peristatic pump. The feed medium containing the compositions of Examples 1-1 to 1-4, which are the control group or lactose-added compositions, was composed of 460.0 g / L of carbohydrates, 6.0 g / L of yeast extract, and 1.0 g / L of sodium chloride, with a weight ratio of carbon source to yeast extract of 76:1. Meanwhile, the fermentation conditions were maintained at a culture temperature of 28°C, and the pH was maintained at 4.0 to 6.0 using ammonia water. The stirring speed was started at 600 rpm and increased to 1,000 rpm to prevent DO (dissolved oxygen) limitation. Feed medium was added at a rate of 0.84 kg to 1.51 kg during fed-batch culture at pH 4 and 28°C for 168 hours. For inoculum culture, 1 ml of frozen spore mixture was inoculated into 150 ml of medium in a 500 ml Erlenmeyer flask and pre-cultured in a shaker at 28°C and 200 rpm for 72 hours, with 10% (v / v) of the culture medium being inoculated. After the culture was completed, the supernatant of the culture medium was collected and used to measure enzyme activity to confirm the effect of inducing enzyme production.

[0106] Experimental Example 1-4. Analysis of Acidic Cellulase Activity

[0107] The fermentation liquid of each Trichoderma lessei QM6a strain cultured in a fermentation medium and a feed medium containing the compositions of Comparative Example 1, Example 1-1, Example 1-2, Example 1-3, or 1-4 (which are lactose-added compositions) as a carbon source (carbohydrate) obtained in Experimental Example 1-3 above was centrifuged. T. reesei Cells and other solid materials were removed. The culture supernatant was appropriately diluted for enzyme analysis. All enzyme activities were expressed as specific activity using International Units (IU) per mL of supernatant. 1 IU was defined as the amount of enzyme required to release 1 μmol of D-glucose per minute under standard analytical conditions (5 mg / mL CMC, pH 4.8, 50℃).

[0108] Cellulase hydrolyzes cellulose to produce monosaccharides and oligosaccharides under specific temperature and pH conditions. Oligosaccharides with reducing ends and monosaccharides with reducing groups undergo a color reaction with DNS reagent under high temperature conditions, and the color intensity of the reaction solution is proportional to the amount of reducing sugars produced by enzymatic hydrolysis and the amount of reaction that reduces sugar production. Cellulase activity was calculated by measuring the absorbance of the reaction solution by spectrophotometry, and the results are shown in Table 4 below.

[0109] Comparison of the effect of inducing acidic cellulase production according to the lactose content included in the composition experimental conditions Cell concentration (g / L) Enzyme activity (IU / mL) Relative Activity Comparison (%) Comparative Example 1 (Control Group) 201 2604 0 Example 1-1 (containing 6.86 wt% lactose) 197 2776 6.6 Examples 1-2 (containing 10.29 wt% lactose) 200 2873 10.4 Examples 1-3 (containing 13.72 wt% lactose) 219 3207 23.2 Examples 1-4 (containing 17.14 wt% lactose) 210 2704 3.8

[0110] As a result, as shown in Table 4, when cultured in a fermentation medium and a feed medium containing the composition of Examples 1-3 with a lactose content of 13.72 wt%, the enzymatic activity of acid cellulase produced from the Trichoderma genus strain increased by 23.2%, exhibiting maximum activity. It was confirmed that when a composition containing a lower glucose content and lactose as a substitute for glucose was added as a carbon source to the fermentation medium and feed medium for culture, the acid cellulase produced from the Trichoderma genus strain showed higher activity than when a composition not containing lactose (control group) was added as a carbon source to the fermentation medium and feed medium for culture.

[0112] Experimental Example 1-5. Analysis of Neutral Xylanase Activity

[0113] The fermentation liquid of each Trichoderma lessei QM6a strain cultured in a fermentation medium and a feed medium containing the compositions of Comparative Example 1, Example 1-1, Example 1-2, Example 1-3, or 1-4 (which are lactose-added compositions) as a carbon source (carbohydrate) obtained in Experimental Example 1-3 above was centrifuged. T. reesei Cells and other solid materials were removed. The culture supernatant was appropriately diluted for enzyme analysis. All enzyme activities were expressed as specific activity using International Units (IU) per mL of supernatant. 1 IU was defined as the amount of enzyme required to release 1 μmol of D-xylose per minute under standard analytical conditions (1% xylan, pH 6.5, 50°C).

[0114] Under specific temperature and pH conditions, xylanase breaks down xylan into oligosaccharides and monosaccharides. The terminally reduced oligosaccharides and monosaccharides with reducing groups undergo a color reaction with DNS reagent. The color intensity of the reaction solution is proportional to the amount of reducing sugars produced by enzymatic hydrolysis, and since the amount of reducing sugars produced is proportional to the activity of xylanase in the reaction solution, the activity of xylanase can be calculated by spectrophotometry. The results of measuring xylanase activity derived from Trichoderma resei strains cultured in a feed medium containing each of the above compositions are shown in Table 5 below.

[0115] Confirmation of the effect of inducing neutral xylanase production according to the lactose content included in the composition experimental conditions Cell concentration (g / L) Enzyme activity (IU / mL) Relative Activity Comparison (%) Comparative Example 1 (Control Group) 290 111744 0 Example 1-1 (containing 6.86 wt% lactose) 278 125160 12.01 Examples 1-2 (containing 10.29 wt% lactose) 280 122160 9.32 Examples 1-3 (containing 13.72 wt% lactose) 253 153800 37.64 Examples 1-4 (containing 17.14 wt% lactose) 225 136168 21.86

[0116] As a result, as shown in Table 5, when cultured in a fermentation medium and feed medium containing the composition of Examples 1-3 with a lactose content of 13.72 wt%, the enzymatic activity of xylanase produced from the Trichoderma strain increased by 37.6%, exhibiting maximum activity. It was confirmed that when a composition with a lower glucose content and lactose replacing glucose was added as a carbon source to the fermentation medium and feed medium for culture, the neutral xylanase produced from the Trichoderma strain showed higher activity than when a composition without lactose (control group) was added as a carbon source to the fermentation medium and feed medium for culture. This is the same effect as acidic cellulase.

[0118] Experimental Example 1-6. Phytase Activity Analysis

[0119] The fermentation liquid of each Trichoderma lessei QM6a strain cultured in a fermentation medium and a feed medium containing the compositions of Comparative Example 1, Example 1-1, Example 1-2, Example 1-3, or 1-4 (which are lactose-added compositions) as a carbon source (carbohydrate) obtained in Experimental Example 1-3 above was centrifuged. T. reeseiCells and other solid materials were removed. The culture supernatant was appropriately diluted for enzyme analysis. All enzyme activities were expressed as specific activity using International Units (IU) per mL of supernatant. 1 IU was defined as the amount of enzyme required to release 1 μmol of inorganic phosphorus per minute in a sodium phytate solution at a concentration of 5.0 mmol / L per minute, which is the phytase activity unit, under conditions of 37°C and pH=5.5.

[0120] Under specific temperature and pH conditions, phytase completely hydrolyzes the substrate sodium phytate to produce orthophosphate and inositol derivatives. In acidic solutions, it can form a yellow compound with ammonium vanadium molybdate, which can be measured colorimetrically at a wavelength of 415 nm. The results of measuring phytase activity derived from Trichoderma lessei strains cultured in a feed medium containing each of the above compositions are shown in Table 6 below.

[0121] Confirmation of the effect of inducing phytase production according to the lactose content included in the composition experimental conditions Cell concentration (g / L) Enzyme activity (IU / mL) Relative Activity Comparison (%) Comparative Example 1 (Control Group) 280 24176 0 Example 1-1 (containing 6.86 wt% lactose) 260 19946 -17.49 Examples 1-2 (containing 10.29 wt% lactose) 210 24426 1.03 Examples 1-3 (containing 13.72 wt% lactose) 180 25050 3.61 Examples 1-4 (containing 17.14 wt% lactose) 260 21445 -11.3

[0122] As a result, as shown in Table 6, when cultured in a fermentation medium and a feed medium containing the composition of Examples 1-3 with a lactose content of 13.72 wt%, the enzyme activity of phytase produced from Trichoderma genus strains increased by 3.61%, showing the highest activity, and the improvement effect was low compared to acidic cellulase and neutral xylanase. In addition, while the improvement effect was observed under conditions where the lactose content in the composition was 10.29 wt% to 13.72 wt%, it was confirmed that under other conditions, the enzyme activity was lower than the control group, inhibiting the induction of enzyme production.

[0124] Experimental Example 1-7. Analysis of Glucanase Activity

[0125] The fermentation liquid of each Trichoderma lessei QM6a strain cultured in a fermentation medium and a feed medium containing the compositions of Comparative Example 1, Example 1-1, Example 1-2, Example 1-3, or 1-4 (which are lactose-added compositions) as a carbon source (carbohydrate) obtained in Experimental Example 1-3 above was centrifuged. T. reesei Cells and other solid materials were removed. The culture supernatant was appropriately diluted for enzyme analysis. All enzyme activities were expressed as specific activity using International Units (IU) per mL of supernatant. 1 IU was defined as the amount of enzyme required to release 1 μmol of D-glucose per minute under standard analytical conditions (1 mg / mL dextran, pH 4.8, 50°C).

[0126] Glucanase hydrolyzes dextran to produce monosaccharides and oligosaccharides under specific temperature and pH conditions. Oligosaccharides with reducing ends and monosaccharides with reducing groups undergo a color reaction with DNS reagent under high temperature conditions, and the color intensity of the reaction solution is proportional to the amount of reducing sugars produced by enzymatic hydrolysis, the amount of sugar production reduced, and the reaction. Glucanase activity was calculated by measuring the absorbance of the reaction solution by spectrophotometry, and the results are shown in Table 7 below.

[0127] Confirmation of the effect of inducing glucanase production according to the lactose content included in the composition experimental conditions Cell concentration (g / L) Enzyme activity (IU / mL) Relative Activity Comparison (%) Comparative Example 1 (Control Group) 320 7475 0 Example 1-1 (containing 6.86 wt% lactose) 240 8363 11.9 Examples 1-2 (containing 10.29 wt% lactose) 280 8792 17.6 Examples 1-3 (containing 13.72 wt% lactose) 250 11205 49.9 Examples 1-4 (containing 17.14 wt% lactose) 230 7967 6.6

[0128] As a result, as shown in Table 7, when cultured in a fermentation medium and a feed medium containing the composition of Examples 1-3 with a lactose content of 13.72 wt%, the enzyme activity of glucanase produced from the Trichoderma genus strain increased by 49.9%, exhibiting the highest activity and showing the highest enzyme production induction effect among the four types of enzymes. On the other hand, it was confirmed that when the lactose content included in the composition was higher than 13.72%, the enzyme production induction effect tended to decrease rapidly.

[0130] Experimental Example 2. Confirmation of the enzyme production-inducing effect of a composition containing whey membrane permeate

[0131] To confirm the effect of a composition containing whey permeate on enzyme production in Trichoderma strains, the fermentation activity of the enzymes was verified using a fed-batch culture method with a composition without whey permeate and a composition with whey permeate.

[0132] Specifically, except that a composition without whey membrane permeate (composition of Comparative Example 1) and a composition with whey membrane permeate (composition of Example 2) were used as carbon sources in the fermentation medium and feed medium, Trichoderma strains were cultured in the same manner as in Experimental Example 1, and the activities of acidic cellulase, neutral xylanase, phytase, and glucanase were measured using the culture medium, and the results are shown in Table 8 below.

[0133] Confirmation of the effect of inducing enzyme production depending on the presence or absence of whey membrane permeate addition enzyme experimental conditions Cell concentration (g / L) Enzyme activity (IU / mL) Relative Activity Comparison (%) acidic cellulase Comparative Example 1 (Control Group) 314 8678 0 Example 2 (addition of whey permeate) 294 13200 52.11 Neutron ylanase Comparative Example 1 (Control Group) 240 106281 0 Example 2 (addition of whey permeate) 300 148399 39.63 phytase Comparative Example 1 (Control Group) 170 23658 0 Example 2 (addition of whey permeate) 190 24389 3.09 glucanase Comparative Example 1 (Control Group) 314 8678 0 Example 2 (addition of whey permeate) 294 13200 52.11

[0134] As a result, as shown in Table 8, it was confirmed that acidic cellulase activity was improved by 52.0% compared to the control group when cultured in a fermentation medium and a feed medium containing a composition with added whey permeate. In addition, the activities of neutral xylanase, phytase, and glucanase were improved by 39.63%, 3.09%, and 52.11%, respectively, compared to the control group, confirming that the production of acidic cellulase, neutral xylanase, phytase, and glucanase can be induced from Trichoderma strains using a composition containing whey permeate.

[0136] From the foregoing description, those skilled in the art to which this application pertains will understand that this application may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of this application should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 delete Claim 7 Trichoderma (comprising glucose and whey permeate, wherein the whey permeate is 5 to 20 weight% based on the total weight of the composition) Trichoderma Composition for increasing enzyme productivity of microorganisms of the genus ). Claim 8 In paragraph 7, the above-mentioned microorganism of the genus Trichoderma is Trichoderma resei ( Trichoderma reesei )-, composition. Claim 9 A composition according to claim 7, wherein the enzyme is one or more enzymes selected from the group consisting of cellulase, xylanase, phytase, and glucanase. Claim 10 delete Claim 11 A composition according to claim 7, wherein the content of the whey membrane permeate included in the composition is 11 to 17 weight%. Claim 12 A composition according to claim 7, wherein the ratio of glucose and whey membrane permeate included in the composition is 7:1 to 2:

1. Claim 13 A culture medium for Trichoderma genus microorganisms comprising the composition of claim 7. Claim 14 A method for producing an enzyme from a microorganism of the genus Trichoderma, comprising: a step of preparing a culture medium by adding the composition of claim 7; and a step of culturing a microorganism of the genus Trichoderma in the culture medium to obtain a culture. Claim 15 In paragraph 14, the above-mentioned microorganism of the genus Trichoderma is Trichoderma resei ( Trichoderma reesei )person, method. Claim 16 A method according to claim 14, wherein the enzyme is one or more enzymes selected from the group consisting of cellulase, xylanase, phytase, and glucanase.