Composition for increasing enzyme productivity of trichoderma sp. strain containing lactose

Abstract

The present invention relates to a composition for inducing enzyme production of a Trichoderma sp. strain containing lactose, and specifically, it was confirmed that a composition comprising both monosaccharides and disaccharides and containing glucose and lactose at a predetermined ratio has excellent activity of inducing enzyme production from a Trichoderma sp. strain, and thus the composition can be usefully employed in mass production of enzymes from a Trichoderma sp. strain.

Description

Composition for increasing enzyme productivity of Trichoderma strain containing lactose

[0001] Cross-citation with related application(s)

[0002] This application claims the benefit of priority to Korean Patent Application No. 10-2023-0085743, filed July 3, 2023, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to a composition for increasing enzyme productivity of a Trichoderma genus strain containing lactose and a method for producing enzymes using the composition.

[0004]

[0005] Cellulose is the most abundant carbon source on Earth, and interest has focused on hydrolyzing it to produce glucose, which is used in fuel, food, and chemical production. Cellulose can be hydrolyzed by chemical or biological methods. However, chemical hydrolysis using acid requires higher temperatures than enzymatic biological hydrolysis, and produces a large amount of undesirable byproducts.

[0006] Enzymes that degrade fibrous materials are obtained from microorganisms such as fungi, bacteria, and actinomycetes. The most commercially effective cellulose-degrading enzymes are those obtained from Trichoderma spp., although other microorganisms, such as Aspergillus sp. and Penicillium sp., are also known to produce cellulose-degrading enzymes.

[0007] The process of cellulolytic enzyme biosynthesis is known to be regulated by induction and catabolite repression. Specifically, sophorose, genthiobiose, or laminaribiose are known to induce cellulase synthesis in Penicillium purpurogenum P-26, and trehalose is known to induce cellulase synthesis in Clostridium papyrosolvens CFR-703 (Process Biochemistry 37 (2001) 241-245). In addition, the activity of cellulolytic enzymes is also regulated by end product inhibition. Cellulase is inhibited by cellobiose, and β-glucosidase is inhibited by glucose in Thermonospora fusca and Trichoderma reesei, respectively. As a result of such complex regulatory mechanisms, the activity of the synthesized cellulolytic enzymes differs depending on the type of microorganism and culture conditions.

[0008]

[0009] The purpose of the present invention is to provide a composition for increasing enzyme productivity of microorganisms of the genus Trichoderma, which comprises glucose and lactose.

[0010] Another object of the present invention is to provide a composition for enhancing the activity of an enzyme produced from a microorganism of the genus Trichoderma, which contains glucose and lactose.

[0011] Another object of the present invention is to provide a composition for increasing enzyme productivity of microorganisms of the genus Trichoderma, comprising glucose and whey permeate.

[0012] Another object of the present invention is to provide a composition for enhancing the activity of an enzyme produced from a microorganism of the genus Trichoderma, comprising glucose and whey permeate.

[0013] Another object of the present invention is to provide a method for producing an enzyme from a Trichoderma spp. strain using a composition for inducing enzyme production of a Trichoderma spp. strain containing the disaccharide.

[0014] Another object of the present invention is to provide a medium for culturing microorganisms of the genus Trichoderma, comprising a composition comprising glucose and lactose, or a composition comprising glucose and whey permeate.

[0015] Another object of the present invention is to provide a method for producing an enzyme from a microorganism of the genus Trichoderma, comprising the steps of: preparing a 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 medium to obtain a culture.

[0016] Another object of the present invention is to provide a use for enhancing the activity of an enzyme produced from a microorganism of the genus Trichoderma in a composition comprising glucose and lactose, or a composition comprising glucose and whey permeate, or a use for increasing the enzyme productivity of a microorganism of the genus Trichoderma.

[0017]

[0018] Each description and embodiment disclosed in this invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this invention fall within the scope of this invention. Furthermore, the scope of this invention is not limited by the specific descriptions set forth below. Furthermore, those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments of the invention described herein. Furthermore, such equivalents are intended to be encompassed by this invention.

[0019]

[0020] One aspect of the present application provides a composition for increasing enzyme productivity of a microorganism of the genus Trichoderma, comprising glucose and lactose.

[0021] Another aspect of the present application provides a composition for enhancing the activity of an enzyme produced from a microorganism of the genus Trichoderma, comprising glucose and whey permeate.

[0022] In the present application, the “microorganism of the genus Trichoderma” may be a microorganism of the genus Trichoderma that produces an enzyme, 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 that is capable of producing all of the enzymes cellulase, xylanase, phytase and glucanase.

[0023] Specifically, the above Trichoderma genus microorganism may be Trichoderma reesei.

[0024] In the present application, the “enzyme” may be produced from a microorganism of the genus Trichoderma, and specifically, may be at least one enzyme selected from the group consisting of cellulase, xylanase, phytase, and glucanase, and may include all of cellulase, xylanase, phytase, and glucanase.

[0025] In this application, "increasing enzyme productivity" may refer to increasing the amount of enzyme produced by a microorganism. This "increasing enzyme productivity" can be confirmed by measuring the fermentation activity of the produced enzyme, and "increasing enzyme productivity" may be interpreted as having the same meaning as "inducing enzyme production" or "enhancing enzyme activity."

[0026] The above composition can increase enzyme productivity of microorganisms in the genus Trichoderma or enhance the activity of enzymes produced from microorganisms in the genus Trichoderma by including a specific amount of lactose based on the total weight of the composition.

[0027] The content of lactose included in the 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 It can be 14% by weight.

[0028] The content of glucose included in the 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.

[0029] Glucose and lactose included in the above composition may have a specific content ratio (mixing ratio).

[0030] Specifically, the content ratio of glucose and lactose 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.

[0031] The above composition may further include fructose as a monosaccharide.

[0032] The monosaccharide content included in the composition may be 60 wt% or less, 57 wt% or less, 55 wt% or less, 43 to 55 wt%, 45 to 55 wt%, 47 to 55 wt%, 43 to 53 wt%, 45 to 53 wt%, 47 to 53 wt%, 43 to 52 wt%, 45 to 52 wt%, 47 to 52 wt%, 43 to 50 wt%, 45 to 50 wt%, 47 to 50 wt%, 43 to 48 wt%, 45 to 48 wt%, or 47 to 48 wt%, based on the total weight of the composition.

[0033] The content of fructose included in the composition may be 1 to 5 wt%, 2 to 5 wt%, 3 to 5 wt%, 1 to 4 wt%, 2 to 4 wt%, or 3 to 4 wt% based on the total weight of the composition.

[0034] The above composition may further include at least one disaccharide selected from the group consisting of trehalose, isomaltose, gentiobiose, cellobiose, and sophorose.

[0035] The disaccharide content included in the 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 It can be 34% to 35% by weight.

[0036] The content of trehalose included in the composition may be 1 to 4 wt%, 2 to 4 wt%, 1 to 3 wt%, or 2 to 3 wt% based on the total weight of the composition.

[0037] The content of isomaltose included in the above composition may be 4.5 to 7 wt%, 5 to 7 wt%, 4.5 to 6 wt%, or 5 to 6 wt% based on the total weight of the composition.

[0038] The content of gentiobiose included in the composition may be 5 to 10 wt%, 6 to 10 wt%, 7 to 10 wt%, 5 to 10 wt%, 6 to 10 wt%, 7 to 10 wt%, 5 to 9 wt%, 6 to 9 wt%, 7 to 9 wt%, 5 to 8 wt%, 6 to 8 wt%, or 7 to 8 wt% based on the total weight of the composition.

[0039] The content of cellobiose included in the above composition may be 1.0 to 3.0 wt% based on the total weight of the composition.

[0040] The content of sophorose included in the composition may be 2 to 6 wt%, 3 to 6 wt%, 4 to 6 wt%, 2 to 5 wt%, 3 to 5 wt%, 4 to 5 wt%, 2 to 4.5 wt%, 3 to 4.5 wt%, or 4 to 4.5 wt% based on the total weight of the composition.

[0041] The above composition may additionally comprise an oligomer.

[0042] The oligomer content included in the composition may be 12 to 23 wt%, 14 to 23 wt%, 16 to 23 wt%, 17 to 23 wt%, 12 to 21 wt%, 14 to 21 wt%, 16 to 21 wt%, 17 to 21 wt%, 12 to 19 wt%, 14 to 19 wt%, 16 to 19 wt%, 17 to 19 wt%, 12 to 18 wt%, 14 to 18 wt%, 16 to 18 wt%, or 17 to 18 wt%, based on the total weight of the composition.

[0043] In one example of the present specification, when culturing a microorganism of the genus Trichoderma using a composition containing lactose, it was confirmed that the enzyme productivity of the microorganism of the genus Trichoderma was increased (increased) and the activity of the enzyme produced from the microorganism of the genus Trichoderma was enhanced compared to a composition not containing lactose. Therefore, the composition containing lactose can be utilized for the purpose of increasing the enzyme productivity of the microorganism of the genus Trichoderma or for the purpose of enhancing the activity of the enzyme produced from the microorganism of the genus Trichoderma.

[0044]

[0045] One aspect of the present application provides a composition for increasing enzyme productivity of a microorganism of the genus Trichoderma, comprising glucose and whey permeate.

[0046] Another aspect of the present application provides a composition for enhancing the activity of an enzyme produced from a microorganism of the genus Trichoderma, comprising glucose and whey permeate.

[0047] The above Trichoderma genus microorganisms, enzymes, enzyme productivity enhancement, and enzyme activity enhancement are as described above.

[0048] In the present 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.

[0049] The above composition can increase enzyme productivity of microorganisms in the genus Trichoderma or enhance the activity of enzymes produced from microorganisms in the genus Trichoderma by including a specific content of whey membrane permeate based on the total weight of the composition.

[0050] The content of the whey membrane permeate included in the 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 It can be 13 to 14 wt%.

[0051] The content of glucose included in the 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.

[0052] The glucose and whey membrane permeate contained in the above composition may have a specific content ratio (mixing ratio).

[0053] Specifically, the content ratio of glucose and 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.

[0054] The above composition may further include fructose as a monosaccharide.

[0055] The monosaccharide content included in the composition may be 60 wt% or less, 57 wt% or less, 55 wt% or less, 43 to 55 wt%, 45 to 55 wt%, 47 to 55 wt%, 43 to 53 wt%, 45 to 53 wt%, 47 to 53 wt%, 43 to 52 wt%, 45 to 52 wt%, 47 to 52 wt%, 43 to 50 wt%, 45 to 50 wt%, 47 to 50 wt%, 43 to 48 wt%, 45 to 48 wt%, or 47 to 48 wt%, based on the total weight of the composition.

[0056] The content of fructose included in the composition may be 1 to 5 wt%, 2 to 5 wt%, 3 to 5 wt%, 1 to 4 wt%, 2 to 4 wt%, or 3 to 4 wt% based on the total weight of the composition.

[0057] The above composition may further include at least one disaccharide selected from the group consisting of trehalose, isomaltose, gentiobiose, cellobiose, and sophorose.

[0058] The disaccharide content included in the 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 It can be 34% to 35% by weight.

[0059] The content of trehalose included in the composition may be 1 to 4 wt%, 2 to 4 wt%, 1 to 3 wt%, or 2 to 3 wt% based on the total weight of the composition.

[0060] The content of isomaltose included in the above composition may be 4.5 to 7 wt%, 5 to 7 wt%, 4.5 to 6 wt%, or 5 to 6 wt% based on the total weight of the composition.

[0061] The content of gentiobiose included in the composition may be 5 to 10 wt%, 6 to 10 wt%, 7 to 10 wt%, 5 to 10 wt%, 6 to 10 wt%, 7 to 10 wt%, 5 to 9 wt%, 6 to 9 wt%, 7 to 9 wt%, 5 to 8 wt%, 6 to 8 wt%, or 7 to 8 wt% based on the total weight of the composition.

[0062] The content of cellobiose included in the above composition may be 1.0 to 3.0 wt% based on the total weight of the composition.

[0063] The content of sophorose included in the composition may be 2 to 6 wt%, 3 to 6 wt%, 4 to 6 wt%, 2 to 5 wt%, 3 to 5 wt%, 4 to 5 wt%, 2 to 4.5 wt%, 3 to 4.5 wt%, or 4 to 4.5 wt% based on the total weight of the composition.

[0064] The above composition may additionally comprise an oligomer.

[0065] The oligomer content included in the composition may be 12 to 23 wt%, 14 to 23 wt%, 16 to 23 wt%, 17 to 23 wt%, 12 to 21 wt%, 14 to 21 wt%, 16 to 21 wt%, 17 to 21 wt%, 12 to 19 wt%, 14 to 19 wt%, 16 to 19 wt%, 17 to 19 wt%, 12 to 18 wt%, 14 to 18 wt%, 16 to 18 wt%, or 17 to 18 wt%, based on the total weight of the composition.

[0066] In one example of the present specification, when culturing a microorganism of the genus Trichoderma using a composition containing a whey membrane permeate, it was confirmed that the enzyme productivity of the microorganism of the genus Trichoderma was increased (increased) and the activity of the enzyme produced from the microorganism of the genus Trichoderma was enhanced compared to a composition not containing a whey membrane permeate. Therefore, a composition containing a whey membrane permeate can be utilized for the purpose of increasing the enzyme productivity of a microorganism of the genus Trichoderma or for the purpose of enhancing the activity of an enzyme produced from a microorganism of the genus Trichoderma.

[0067]

[0068] One aspect of the present application provides a medium for culturing microorganisms of the genus Trichoderma, comprising a composition comprising glucose and lactose, or a composition comprising glucose and whey permeate.

[0069] 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 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 medium to obtain a culture.

[0070] The above Trichoderma genus microorganisms, enzymes, whey membrane permeate, etc. are as described above.

[0071] The medium may comprise a composition comprising glucose and lactose, or a composition comprising glucose and whey permeate as a carbon source.

[0072] In this application, “culture” may be a fed-batch culture.

[0073] 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 basal medium. Fed-batch culture offers the advantages of freely controlling the substrate concentration in the culture medium, adding the substrate at an appropriate rate, and controlling the amount of substrate supplied without runoff.

[0074] As used herein, the term "medium" refers to a substance containing nutrients essential for culturing microorganisms, primarily water, and other nutrients and growth factors. Specifically, the medium and other culture conditions used for culturing microorganisms in this application may be used without special restrictions, as long as they are conventional media used for culturing microorganisms.

[0075] 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 in fed-batch culture.

[0076]

[0077] One aspect of the present application provides a use for enhancing the activity of an enzyme produced from a microorganism of the genus Trichoderma in a composition comprising glucose and lactose, or a composition comprising glucose and whey permeate, or a use for increasing enzyme productivity of a microorganism of the genus Trichoderma.

[0078] The above lactose, whey membrane permeate, Trichoderma genus microorganisms, enzymes, etc. are as described above.

[0079]

[0080] The present invention relates to a composition for inducing enzyme production from a Trichoderma spp. strain containing lactose, and specifically, it has been confirmed that a composition containing both monosaccharides and disaccharides and containing glucose and lactose in a certain ratio has excellent activity in inducing enzyme production from a Trichoderma spp. strain, and thus the composition can be usefully utilized for mass production of enzymes from a Trichoderma spp. strain.

[0081]

[0082] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify one or more specific examples, and the scope of the present invention is not limited to these examples.

[0083]

[0084] Comparative Example 1. Preparation of disaccharide composition

[0085] Glucose powder (hydrated crystalline glucose, Daejung Chemical Co., Ltd.) with a purity of 95% or more 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 the solution was allowed to stand at 60°C for 7 to 21 days to prepare a composition containing disaccharides (Treated Glucose Syrup, TGS). Hereinafter, the composition is referred to as the control group.

[0086]

[0087] Example 1. Preparation of disaccharide compositions containing lactose in various proportions

[0088] In the composition containing the disaccharide prepared in Comparative Example 1, lactose (purity 95 wt% or more, Daejung Chemical) was added instead of glucose so that the content of lactose (hereinafter, Example 1-1 composition), 10.29 wt% (hereinafter, Example 1-2 composition), 13.72 wt% (hereinafter, Example 1-3 composition), or 17.14 wt% (hereinafter, Example 1-4 composition) based on the total weight of the composition, thereby preparing a mixed composition having a final concentration of 70 to 75 wt%.

[0089]

[0090] Example 2. Preparation of a disaccharide composition containing whey membrane permeate

[0091] In Comparative Example 1, whey permeate powder was added instead of glucose to the composition containing disaccharides so that the content of whey permeate (purity 88 wt% or more) was 13.72 wt% based on the total weight of the composition, thereby preparing a mixed composition having a final concentration of 70 to 75 wt%. The composition of the whey permeate powder is shown in Table 1 below.

[0092]

[0093] Composition of whey permeate powder NO Composition (dry weight basis) Whey permeate powder 1 DM (%) 242 CP (%) 2.63 Nonprotein N (%) 0.364 Ash (%) 7.635 Crude fat (%) 0.036 Lactose (%) 88.17 Total sugars (%) 88.78 Organic acids (%) 4.19 Calcium (%) 0.67 10 Phosphorus (%) 1.12 11 Magnesium (%) 0.17 12 Potassium (%) 213 Chloride (%) 1.12 14 Sodium (%) 0.67 15 Iron (mg / kg) 45.8 116 Copper (mg / kg) 4.16 17 Zinc (mg / kg) 20.8 2 18 pH 5.7 6 19 Density (kg / L) 1.11 20 Viscosity 1 (cP) 3.5

[0094]

[0095] Experimental Example 1. Analysis of sugar content in a composition containing lactose or whey permeate powder

[0096] The following experiments were performed to analyze the sugar content contained in the compositions containing each disaccharide prepared in Comparative Example 1, Example 1, and Example 2, the mixed compositions further containing lactose as the disaccharide, and the mixed compositions further containing whey permeate powder as the disaccharide.

[0097] Specifically, the concentrations of monosaccharides and disaccharides contained in each composition were measured using a Bio-LC system (Dionex ICS-3000, Sunnyvale, CA, United States) equipped with an electrochemical detector and a CarboPac PAI column, and the column was eluted with 0.1 M NaOH (0 to 5 min) at 30°C, followed by a linear gradient of sodium acetate (0 to 0.2 M) at 1 mL / min for 5 to 35 min. The contents (%) of monosaccharides glucose and fructose, and disaccharides trehalose, isomaltose, maltose, cellobiose, gentiobiose, sophorose, and lactose in the monosaccharide and disaccharide mixture compositions manufactured in Comparative Example 1, Example 1, and Example 2 were measured and shown in Tables 2 and 3 below.

[0098] Analysis results of sugar content of compositions containing lactose Classification Content (%) Other degree of polymerization Sugars Comparative examples Example 1-1 Example 1-2 Example 1-3 Example 1-4 DP1 Glucose 5 7.77 5 0.92 47.49 44.06 40.63 C-source Fructose 3.66 3.66 3.66 3.66 3.66 3.66 sum 6 1.44 5 4.58 5 1.15 47.72 44.29 DP2 Trehalose 2.67 2.67 2.67 2.67 2.67 2.67 α(1→1) Isomaltose 5.3 1 5.31 5.315.315.31α(1→6)Gentiobiose7.427.427.427.427.42β(1→6)Cellobiose1.31.31.31.31. 3β(1→4)sophorose4.034.034.034.034.03β(1→2)Lactose06.8610.2913.7217.14β(gla1→glu 4)sum20.7327.5931.0234.4537.88DP3 or more oligomer17.8317.8317.8317.8317.83Total100100100100100

[0099] Classification Content (%)Other degree of polymerizationSaccharidesComparative exampleExample 2DP1Glucose57.7744.06C-sourceFructose3.663.66sum61.4447.72 DP2Trehalose2.672.67α(1→1)Isomaltose5.315.31α(1→6)Gentiobiose7.427.42β(1→6)Cellobiose1.31.3β(1→4)sophorose4.034.03β(1→2)whey permeate013.72β(gla1→glu 4)

[0100]

[0101] Experimental Example 1. Confirmation of the enzyme production induction effect according to the lactose content included in the composition.

[0102] In order to confirm the difference in the enzyme production induction effect of Trichoderma spp. strains according to the lactose content included in the composition, the enzyme fermentation activity was confirmed by a fed-batch culture method using a composition without added lactose and a composition with added lactose.

[0103]

[0104] Experimental Example 1-1. Preparation of enzyme-producing strains

[0105] Trichoderma reesei QM6a strain (ATCC13631), capable of producing acid cellulase, neutral xylanase, phytase, and glucanase, was prepared as an enzyme-producing strain. Trichoderma reesei QM6a strain was grown on potato dextrose agar plates at 30°C for 5 days to form sporulation. After sporulation, 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.

[0106]

[0107] Experimental Example 1-2. Flask Culture

[0108] Trichoderma reesei QM6a strain was 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. The main culture was performed using a liquid medium composed of 30 g / L lactose, 10 g / L ammonium sulfate, 20 g / L yeast extract, 5 g / L potassium phosphate (KH2P04), and 0.5 g / L magnesium sulfate. 5% (v / v) of the pre-culture was inoculated into 200 ml of the above liquid medium and cultured at 28°C with 200 rpm for 5 to 7 days, and the culture solution (enzyme solution) was harvested. At this time, the culture temperature was in the range of 28℃ to 32℃, and the pH of the liquid medium was in the range of 4.0 to 6.0.

[0109]

[0110] Experimental Example 1-3. Obtaining culture solution after fed-batch cultivation of enzyme-producing strain

[0111] The Trichoderma reesei QM6a strain producing the enzyme of Experimental Example 1-1 was cultured using the fed-batch method as follows, and then a culture solution for measuring enzyme activity was obtained.

[0112] Specifically, fed-batch culture was performed using a fermentation medium containing 20.0 g / L of carbohydrate, 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 carbon source in the feed medium (the composition of Comparative Example 1 or the compositions of Examples 1-1 to 1-4, which are lactose-added compositions) was supplied at a constant supply rate of 5.0 to 9.0 g / L per hour 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 control or lactose-added compositions, was composed of 460.0 g / L of carbohydrates with a weight ratio of carbon source to yeast extract of 76:1, 6.0 g / L of yeast extract, and 1.0 g / L of sodium chloride. Meanwhile, the fermentation conditions were maintained at a culture temperature of 28°C and a pH of 4.0 to 6.0 using ammonia water. The stirring speed was started at 600 rpm and increased to 1,000 rpm so as not to cause DO (dissolved oxygen) limitation. Fed-batch culture was performed at 28℃ and pH 4 for 168 hours, adding 0.84 kg to 1.51 kg of the feed medium. For the spawn culture, 1 ml of the frozen spore mixture was inoculated into 150 ml of the medium in a 500 ml Erlenmeyer flask, and 10% (v / v) of the culture solution was inoculated by pre-cultivating in a shaker at 28℃ and 200 rpm for 72 hours. After completion of the culture, the supernatant of the culture solution was obtained and used to measure enzyme activity to confirm the effect of inducing enzyme production.

[0113]

[0114] Experimental Example 1-4. Acid Cellulase Activity Analysis

[0115] Each fermented broth of Trichoderma reesei QM6a strain cultured in a fermentation medium and a feed medium containing the composition of Comparative Example 1, lactose-added composition of Example 1-1, Example 1-2, Example 1-3, or 1-4 obtained in Experimental Example 1-3 as a carbon source (carbohydrate) was centrifuged to remove T. reesei cells and other solid materials. The culture supernatant was appropriately diluted for enzyme analysis. All enzyme activities were expressed as specific activities using international units (IU) per mL of supernatant. 1 IU was defined as the amount of enzyme required to liberate 1 μmol of D-glucose per minute under standard assay conditions (5 mg / mL CMC, pH 4.8, 50°C).

[0116] 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 the DNS reagent under high-temperature conditions, and the color intensity of the reaction solution is proportional to the amount of reducing sugar produced by enzymatic hydrolysis and the amount and reaction rate of reducing sugar production. Cellulase activity was calculated by measuring the absorbance of the reaction solution using spectrophotometry, and the results are shown in Table 4 below.

[0117] Comparison of the effect of inducing acid 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) 20126040 Example 1-1 (containing 6.86 wt% of lactose) 19727766.6 Example 1-2 (containing 10.29 wt% of lactose) 200287310.4 Example 1-3 (containing 13.72 wt% of lactose) 219320723.2 Example 1-4 (containing 17.14 wt% of lactose) 21027043.8

[0118] As a result, as shown in Table 4, when cultured with a fermentation medium and a feed medium containing the composition of Example 1-3 having a lactose content of 13.72 wt%, the enzyme activity of the acid cellulase produced from the strain of Trichoderma increased by 23.2%, showing the highest activity. In addition, when the glucose content was lowered and a composition containing more lactose in place of glucose was added as a carbon source to the fermentation medium and the feed medium, it was confirmed that the acid cellulase produced from the strain of Trichoderma showed higher activity than when cultured with a composition not containing lactose (control) added as a carbon source to the fermentation medium and the feed medium.

[0119]

[0120] Experimental Example 1-5. Neutral xylanase activity analysis

[0121] Each fermented broth of Trichoderma reesei QM6a strain cultured in a fermentation medium and a feed medium containing the composition of Comparative Example 1, lactose-added composition of Example 1-1, Example 1-2, Example 1-3, or 1-4 obtained in Experimental Example 1-3 as a carbon source (carbohydrate) was centrifuged to remove T. reesei cells and other solid materials. The culture supernatant was appropriately diluted for enzyme analysis. All enzyme activities were expressed as specific activities using international units (IU) per mL of supernatant. 1 IU was defined as the amount of enzyme required to liberate 1 μmol of D-xylose per minute under standard assay conditions (1% xylan, pH 6.5, 50°C).

[0122] Under specific temperature and pH conditions, xylanase decomposes xylan into oligosaccharides and monosaccharides. The oligosaccharides with reduced terminals and monosaccharides with reducing groups undergo a color reaction with the DNS reagent. The color intensity of the reaction solution is proportional to the amount of reducing sugar produced by enzymatic hydrolysis, and the amount of reducing sugar produced is proportional to the activity of xylanase in the reaction solution, so the activity of xylanase can be calculated spectrophotometrically. The results of measuring the activity of xylanase from Trichoderma reesei strains cultured in a feed medium containing each of the above compositions are shown in Table 5 below.

[0123] 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 11 17 440 Example 1-1 (containing 6.86 wt% of lactose) 278 12 5 16 0 12.01 Example 1-2 (containing 10.29 wt% of lactose) 280 12 2 16 0 9.32 Example 1-3 (containing 13.72 wt% of lactose) 253 15 38 00 37.64 Example 1-4 (containing 17.14 wt% of lactose) 225 136 168 21.86

[0124] As a result, as shown in Table 5, when cultured with a fermentation medium and a feed medium containing the composition of Example 1-3 having a lactose content of 13.72 wt%, the enzyme activity of the xylanase produced from the strain of Trichoderma increased by 37.6%, showing the highest activity. In addition, when cultured by lowering the glucose content and adding a composition containing more lactose as a carbon source to the fermentation medium and the feed medium in place of glucose, it was confirmed that the neutral xylanase produced from the strain of Trichoderma showed higher activity than when cultured by adding a composition not containing lactose (control) as a carbon source to the fermentation medium and the feed medium. This is the same effect as acid cellulase.

[0125]

[0126] Experimental Example 1-6. Phytase Activity Analysis

[0127] Each fermented broth of Trichoderma reesei QM6a strain cultured in a fermentation medium and a feed medium containing the composition of Comparative Example 1, lactose-added composition of Example 1-1, Example 1-2, Example 1-3, or 1-4 obtained in Experimental Example 1-3 as a carbon source (carbohydrate) was centrifuged to remove T. reesei cells and other solid materials. The culture supernatant was appropriately diluted for enzyme analysis. All enzyme activities were expressed as specific activities using international units (IU) per mL of supernatant. 1 IU was defined as the amount of enzyme required to liberate 1 μmol of inorganic phosphorus per minute in a sodium phytate solution at a concentration of 5.0 mmol / L per minute, which is a phytase activity unit, under the conditions of 37°C and pH = 5.5.

[0128] Under specific temperature and pH conditions, phytase completely hydrolyzes its substrate, sodium phytate, producing orthophosphate and an inositol derivative. 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 from Trichoderma reesei strains cultured in a feed medium containing each of the above compositions are shown in Table 6 below.

[0129] 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) 280 241 760 Example 1-1 (containing 6.86 wt% of lactose) 260 199 46-17.49 Example 1-2 (containing 10.29 wt% of lactose) 210 244 261.03 Example 1-3 (containing 13.72 wt% of lactose) 180 250 503.61 Example 1-4 (containing 17.14 wt% of lactose) 260 214 45-11.3

[0130] As a result, as shown in Table 6, when cultured with the fermentation medium and feed medium containing the composition of Example 1-3 having a lactose content of 13.72 wt%, the enzyme activity of the phytase produced from the strain of Trichoderma spp. increased by 3.61%, showing the highest activity, and the improvement effect was low compared to acid cellulase and neutral xylanase. In addition, although the lactose content contained in the composition showed an improvement effect under the concentration conditions of 10.29 wt% to 13.72 wt%, it was confirmed that the enzyme activity was lower than that of the control group under other conditions, inhibiting the induction of enzyme production.

[0131]

[0132] Experimental Example 1-7. Glucanase Activity Analysis

[0133] Each fermentation broth of Trichoderma reesei QM6a strain cultured in a fermentation medium and a feed medium containing the composition of Comparative Example 1, lactose-added composition of Example 1-1, Example 1-2, Example 1-3, or 1-4 obtained in Experimental Example 1-3 as a carbon source (carbohydrate) was centrifuged to remove T. reesei cells and other solid materials. The culture supernatant was appropriately diluted for enzyme analysis. All enzyme activities were expressed as specific activities using international units (IU) per mL of supernatant. All enzyme activities were expressed as specific activities using international units (IU) per mL of supernatant. 1 IU was defined as the amount of enzyme required to liberate 1 μmol of D-glucose per minute under standard assay conditions (1 mg / mL dextran, pH 4.8, 50°C).

[0134] 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 the DNS reagent under high-temperature conditions, and the color intensity of the reaction solution is proportional to the amount of reducing sugar produced by enzymatic hydrolysis and the amount and reaction rate of reducing sugar production. Glucanase activity was calculated by measuring the absorbance of the reaction solution using spectrophotometry, and the results are shown in Table 7 below.

[0135] 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) 3 207 4750 Example 1-1 (containing 6.86 wt% of lactose) 2 408 363 11.9 Example 1-2 (containing 10.29 wt% of lactose) 2 808 792 17.6 Example 1-3 (containing 13.72 wt% of lactose) 2 501 120 549.9 Example 1-4 (containing 17.14 wt% of lactose) 2 307 967 6.6

[0136] As a result, as shown in Table 7, when cultured with the fermentation medium and feed medium including the composition of Example 1-3 having a lactose content of 13.72 wt%, the enzyme activity of the glucanase produced from the strain of Trichoderma spp. increased by 49.9%, showing the highest activity, and exhibiting the highest enzyme production induction effect among the four 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.

[0137]

[0138] Experimental Example 2. Confirmation of the enzyme production-inducing effect of a composition containing whey membrane permeate.

[0139] In order to confirm the enzyme production inducing effect of a Trichoderma spp. strain of a composition containing whey permeate, the fermentation activity of the enzyme was confirmed by a fed-batch culture method using a composition without whey permeate and a composition with whey permeate added.

[0140] Specifically, except that a composition without whey membrane permeate added as a carbon source in the fermentation medium and the feed medium (composition of Comparative Example 1) and a composition with whey membrane permeate added (composition of Example 2) were used, a Trichoderma spp. strain was cultured in the same manner as in Experimental Example 1, and the activities of acid cellulase, neutral xylanase, phytase, and glucanase were measured using the culture solution. The results are shown in Table 8 below.

[0141] Confirmation of enzyme production induction effect according to the presence or absence of whey membrane permeate additionEnzyme experimental conditionsCellular concentration (g / L)Enzyme activity (IU / mL)Relative activity comparison (%)Acid cellulaseComparative example 1 (control group)31486780Example 2 (whey permeate added)2941320052.11Neutral xylanaseComparative example 1 (control group)2401062810Example 2 (whey permeate added)30014839939.63PhytaseComparative example 1 (control group)170236580Example 2 (whey permeate added)190243893.09GlucanaseComparative example 1 (control group)31486780Example 2 (whey permeate added) Added)2941320052.11

[0142] As a result, as shown in Table 8, it was confirmed that the acid cellulase activity was improved by 52.0% compared to the enzyme activity of the control group when cultured with the fermentation medium and feed medium containing the composition to which whey permeate was added. In addition, the activities of neutral xylanase, phytase, and glucanase were also improved by 39.63%, 3.09%, and 52.11%, respectively, compared to the control group, confirming that the production of acid cellulase, neutral xylanase, phytase, and glucanase can be induced from a Trichoderma spp. strain using the composition containing whey permeate.

[0143]

[0144] From the above description, those skilled in the art will understand that the present application can be implemented in other specific forms without altering the technical concept or essential features thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of this application should be interpreted to include all changes or modifications derived from the meaning and scope of the following claims and their equivalents, rather than the detailed description above.

Claims

1. A composition for increasing enzyme productivity of a microorganism of the genus Trichoderma, containing glucose and lactose.

2. A composition according to claim 1, wherein the microorganism of the genus Trichoderma is Trichoderma reesei.

3. A composition according to claim 1, wherein the enzyme is at least one enzyme selected from the group consisting of cellulase, xylanase, phytase, and glucanase.

4. A composition according to claim 1, wherein the content of lactose included in the composition is 5 to 20 wt%.

5. A composition according to claim 1, wherein the content of lactose included in the composition is 11 to 17 wt%.

6. A composition according to claim 1, wherein the content ratio of glucose and lactose included in the composition is 7:1 to 2:

1.

7. A composition for increasing enzyme productivity of microorganisms of the genus Trichoderma, comprising glucose and whey permeate.

8. A composition according to claim 7, wherein the microorganism of the genus Trichoderma is Trichoderma reesei.

9. A composition according to claim 7, wherein the enzyme is at least one enzyme selected from the group consisting of cellulase, xylanase, phytase, and glucanase.

10. A composition according to claim 7, wherein the content of the whey membrane permeate contained in the composition is 5 to 20 wt%.

11. A composition according to claim 7, wherein the content of the whey membrane permeate contained in the composition is 11 to 17 wt%.

12. A composition according to claim 7, wherein the content ratio of glucose and whey membrane permeate contained in the composition is 7:1 to 2:

1.

13. A medium for culturing microorganisms of the genus Trichoderma comprising the composition of claim 1 or claim 7.

14. A step of preparing a medium by adding the composition of paragraph 1 or paragraph 7; and A method for producing an enzyme from a microorganism of the genus Trichoderma, comprising: a step of culturing a microorganism of the genus Trichoderma in the above medium to obtain a culture; 15. A method according to claim 14, wherein the microorganism of the genus Trichoderma is Trichoderma reesei.

16. A method according to claim 14, wherein the enzyme is at least one enzyme selected from the group consisting of cellulase, xylanase, phytase, and glucanase.