CN111549083B - Application of trichoderma pleuroticola ZJ-03 in deep processing of industrial hemp - Google Patents

Application of trichoderma pleuroticola ZJ-03 in deep processing of industrial hemp Download PDF

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CN111549083B
CN111549083B CN202010426590.XA CN202010426590A CN111549083B CN 111549083 B CN111549083 B CN 111549083B CN 202010426590 A CN202010426590 A CN 202010426590A CN 111549083 B CN111549083 B CN 111549083B
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hemp
trichoderma
ion exchange
resin column
exchange resin
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CN111549083A (en
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钱朋智
张梅娟
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Qiqihar University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/04—Disaccharides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase

Abstract

Application of trichoderma pleuroticola ZJ-03 in deep processing of industrial hemp relates to the technical field of food processing. The invention aims to solve the problems of how to induce trichoderma pleuroticola to produce xylanase with high enzyme activity and how to prepare a health syrup product with high purity and main components of xylobiose and xylotriose. The method comprises the following steps: adding hemp scrap powder and hemp seed meal powder into the nutrient solution, stirring, and inoculating trichoderma aureoviride ZJ-03 spore suspension; inoculating, culturing in a fermentation chamber to obtain a trichoderma aureobasicola solid fermentation material, standing and leaching to obtain a leaching solid-liquid mixture, performing coarse filtration on the solid-liquid mixture, centrifuging again, and taking supernatant to obtain a coarse sugar liquid; and sequentially carrying out first evaporation concentration, first ion exchange, second evaporation concentration, second ion exchange and third evaporation concentration on the crude sugar solution to obtain a functional syrup product. The invention can obtain the application of the trichoderma aureoviride ZJ-03 in the deep processing of industrial hemp.

Description

Application of trichoderma pleuroticola ZJ-03 in deep processing of industrial hemp
Technical Field
The invention relates to the technical field of food processing, in particular to application of trichoderma aureobasilicum strain ZJ-03 in fine and further processing of industrial hemp.
Background
Industrial hemp (hemp), a plant of the genus Cannabis of the family Moraceae, is a hemp variety that has been artificially bred and approved (identified or registered) by crop varieties, has a tetrahydrocannabinol content of less than 0.3% (dry matter percentage) at the top leaves and flower ears of the flowering season of the plant population, and has no drug extraction or direct drug utilization value.
The continuous development of the hemp industry becomes a difficult problem to be solved urgently, modern food mechanical equipment integrates deep processing of the hemp into the food industry, a wider development space is opened up for the hemp, and the value of the hemp is improved while the industrial chain is deepened. In the international market, the types of hemp food products reach more than 3000. The hemp food has rich varieties, wide industry prospect in the fields of catering, health-care food and the like, and great market potential.
The hemp functional components are effectively utilized to develop hemp functional food, health food or medicine, which not only plays an important role in the development of hemp industry, but also is beneficial to the health of people. At present, research and development on hemp functional products have become one of hot industries. But functional products with high added value are still to be researched and developed. China hemp industry needs to gradually improve comprehensive utilization rate, realize international layout and enrich market application, which is the key for improving basic capability and industrial chain level of China hemp industry. The key to realize new breakthrough in the fields of food, medicine, new materials and the like is to further enrich high value-added products taking the intensive extraction of the hemp as raw materials. Wherein, the trichoderma pleuroticola fermentation is applied to the fine and further processing of industrial hemp to prepare syrup, which is not reported yet; in addition, how to produce xylanase with high enzyme activity and how to prepare high-purity health syrup products become technical problems in the food processing industry.
Disclosure of Invention
The invention aims to solve the problems of inducing trichoderma aureoviride to produce xylanase with high enzyme activity and preparing a health syrup product with high purity and main components of xylobiose and xylotriose, and provides application of trichoderma aureoviride ZJ-03 in deep processing of industrial hemp.
The application of the trichoderma pleuroticola ZJ-03 in the fine and further processing of industrial hemp is completed according to the following steps:
inoculating trichoderma aureoviride ZJ-03 spores into a PDA slant culture medium under an aseptic condition, and then placing the culture medium into a mould incubator for culture to obtain activated trichoderma aureoviride ZJ-03 strain spores; taking down the activated spore of Trichoderma aureoviride ZJ-03 strain, scattering and shaking to obtain the product with concentration of 1.5 × 1072.0X 10 to one/mL7each/mL of Trichoderma aureoviride ZJ-03 spore suspension;
secondly, adding hemp seed meal powder and hemp crumb powder into the nutrient solution, fully stirring and sterilizing, and then inoculating the trichoderma aureobasicola ZJ-03 spore suspension obtained in the first step under an aseptic condition, wherein the inoculation amount is 1.5-2%; after inoculation, placing the inoculated product in a sterilized fermentation chamber, firstly culturing for 68-72 h at 28-30 ℃, then culturing for 20-24 h at 33-37 ℃, and then continuously culturing for 22-26 h at 37-40 ℃ to obtain a trichoderma aureobasicola solid fermentation material; leaching the trichoderma aureobasicola solid fermentation material to obtain a leached solid-liquid mixture; carrying out coarse filtration and centrifugation on the solid-liquid mixture obtained by leaching, and taking supernatant to obtain a crude sugar solution;
and thirdly, sequentially carrying out first evaporation concentration, first ion exchange, second evaporation concentration, second ion exchange and third evaporation concentration on the crude sugar liquid obtained in the step two to obtain a functional syrup product.
The invention has the beneficial effects that:
1. the invention realizes that the trichoderma aureoviride ZJ-03 strain takes the hemp chips and the hemp seed meal as main growth substrates for solid state fermentation, and induces the produced xylanase to degrade the hemp chips and the hemp seed meal to be synchronously carried out in the growth period, thereby saving the time and the cost for extracting and refining the xylanase, simplifying the preparation process of the functional syrup product, having feasibility, simultaneously taking the hemp chips and the hemp seed meal as the base material for microbial growth and the acting substrate of the xylanase, fully utilizing the hemp chips and the hemp seed meal, increasing the additional values of the hemp chips and the hemp seed meal, and exploring a new way for the comprehensive utilization of the hemp and the preparation of the functional syrup product.
2. According to the industrial hemp (hemp) functional syrup product, the hemp sawdust powder and the hemp seed meal powder are used as main solid fermentation growth substrates, so that the enzyme activity of xylanase produced by trichoderma pleuroticola is induced to be improved by more than 20% compared with the existing enzyme activity (namely, more functional syrup products with the same purity can be produced by the same amount of xylanase); the impurities such as pigment, protein, monosaccharide and polysaccharide in the functional syrup product are removed by adopting separation and purification technical means such as leaching, coarse filtration and centrifugation, and subsequent multiple evaporation concentration and ion exchange of a coarse sugar solution, so that the purity of the final functional syrup product is as high as 72.80%, and the main components are xylobiose (the purity is as high as 46.00%) and xylotriose (the purity is as high as 26.80%), the main components in the functional syrup product are xylobiose and xylotriose, the products decomposed by digestive enzymes after the xylobiose and the xylotriose are absorbed by a human body are xylose, the xylose is difficult to be decomposed by the digestive enzymes of the human body, saccharides such as glucose and the like which can increase the blood sugar and the blood fat of the human body can not be generated, and the xylose only contains little heat; therefore, the functional syrup product is low-calorie health syrup, and has multiple effects of controlling blood sugar, preventing obesity, improving diabetes symptoms and the like when being added into food.
3. The invention has simple technical route, low cost and good development prospect and market prospect.
The invention can obtain the application of the trichoderma aureoviride ZJ-03 in the deep processing of industrial hemp.
Drawings
FIG. 1 is a flow chart of the process of producing functional syrup products from solid state fermentation of trichoderma pleurorum and separating and purifying functional syrup products according to the embodiment;
FIG. 2 is an activated spore of Trichoderma aureoviride ZJ-03 strain;
FIG. 3 is a schematic diagram of a Trichoderma reesei solid fermentation material;
FIG. 4 is a high performance liquid chromatogram of a functional syrup product.
Detailed Description
The first embodiment is as follows: the application of the trichoderma aureobasilicum strain ZJ-03 in the deep processing of industrial hemp in the embodiment is completed according to the following steps:
inoculating trichoderma aureoviride ZJ-03 spores into a PDA slant culture medium under an aseptic condition, and then placing the culture medium into a mould incubator for culture to obtain activated trichoderma aureoviride ZJ-03 strain spores; taking down the activated spore of Trichoderma aureoviride ZJ-03 strain, scattering and shaking to obtain the product with concentration of 1.5 × 1072.0X 10 to one/mL7each/mL of Trichoderma aureoviride ZJ-03 spore suspension;
secondly, adding the hemp seed meal powder and the hemp sawdust powder into the nutrient solution, fully stirring, filling the mixture into a container for sterilization, and then inoculating the trichoderma aureobasicola ZJ-03 spore suspension obtained in the first step under the aseptic condition, wherein the inoculation amount is 1.5-2%; after inoculation, placing the inoculated product in a sterilized fermentation chamber, firstly culturing for 68-72 h at 28-30 ℃, then culturing for 20-24 h at 33-37 ℃, and then continuously culturing for 22-26 h at 37-40 ℃ to obtain a trichoderma aureobasicola solid fermentation material; leaching the trichoderma aureobasicola solid fermentation material to obtain a leached solid-liquid mixture; carrying out coarse filtration and centrifugation on the solid-liquid mixture obtained by leaching, and taking supernatant to obtain a crude sugar solution;
and thirdly, sequentially carrying out first evaporation concentration, first ion exchange, second evaporation concentration, second ion exchange and third evaporation concentration on the crude sugar liquid obtained in the step two to obtain a functional syrup product.
The beneficial effects of the embodiment are as follows:
1. the embodiment realizes that the trichoderma aureoviride ZJ-03 strain takes the hemp chips and the hemp seed meal as main solid fermentation growth substrates, and induces the produced xylanase to degrade the hemp chips and the hemp seed meal to be synchronously carried out in the growth period, thereby saving the time and the cost for extracting and refining the xylanase, simplifying the preparation process of the functional syrup product, having feasibility, simultaneously taking the hemp chips and the hemp seed meal as the base material for microbial growth and the acting substrate of the xylanase, fully utilizing the hemp chips and the hemp seed meal, increasing the additional values of the hemp chips and the hemp seed meal, and exploring a new way for the comprehensive utilization of the hemp and the preparation of the functional syrup product.
2. In the industrial hemp (hemp) functional syrup product of the embodiment, the hemp sawdust powder and the hemp seed meal powder are used as main solid fermentation growth substrates, so that the enzyme activity of the xylanase produced by trichoderma pleuroticola is induced to be improved by more than 20% compared with the prior enzyme activity (namely, more functional syrup products with the same purity can be produced by the same amount of xylanase); the impurities such as pigment, protein, monosaccharide and polysaccharide in the functional syrup product are removed by adopting separation and purification technical means such as leaching, coarse filtration and centrifugation, and subsequent multiple evaporation concentration and ion exchange of the coarse sugar solution, so that the purity of the final functional syrup product is as high as 72.80%, and the main components are xylobiose (the purity is as high as 46.00%) and xylotriose (the purity is as high as 26.80%), the main components in the functional syrup product of the embodiment are xylobiose and xylotriose, the products decomposed by digestive enzymes after the xylobiose and the xylotriose are absorbed by a human body are xylose, the xylose is difficult to be decomposed by the digestive enzymes of the human body, saccharides such as glucose and the like which can increase the blood sugar and the blood fat of the human body can not be generated, and the xylose only contains little heat; therefore, the functional syrup product is low-calorie health syrup, and has multiple effects of controlling blood sugar, preventing obesity, improving diabetes symptoms and the like when being added into food.
3. The implementation method has the advantages of simple technical route, low cost, and good development prospect and market prospect.
The second embodiment is as follows: the present embodiment differs from the present embodiment in that: the hemp scrap powder in the step two is prepared according to the following steps: crushing hemp scraps, sieving with a 40-mesh sieve, and taking undersize products to obtain hemp scrap powder; the hemp seed meal powder is prepared by the following steps: crushing hemp seed meal, sieving with a 40-mesh sieve, and taking undersize to obtain hemp seed meal powder; the nutrient solution is prepared by the following steps: adding the glucose mother liquor and the threonine mother liquor into distilled water, and uniformly mixing to obtain the nutrient solution, wherein the ratio of the mass of the glucose mother liquor to the mass of the threonine mother liquor to the volume of the distilled water is (0.5 g-1 g): (0.5 g-1 g): 100 mL.
Other steps are the same as those in the first embodiment.
The third concrete implementation mode: the first or second differences from the present embodiment are as follows: culturing in the step one: culturing at 28-30 ℃ until the spores of trichoderma pleuroticola ZJ-03 are mature; and (2) sterilization in the second step: stirring thoroughly, placing into container, and sterilizing at 121 deg.C for 2 hr; leaching in the second step: adding the trichoderma pleuroticola solid fermentation material into pure water, and statically leaching for 6-8 h at the temperature of 35-37 ℃; centrifuging in the step two: centrifuging the solid-liquid mixture for 10-15 min under 3500-4000 r/min.
The other steps are the same as those in the first or second embodiment.
The fourth concrete implementation mode: the difference between this embodiment and one of the first to third embodiments is as follows: the mass ratio of the hemp seed meal to the hemp scurf powder in the second step is (19-49): 1, and the volume ratio of the total mass of the hemp seed meal and the hemp scurf powder to the volume of the nutrient solution is (1.5-1.8); the ratio of the mass of the trichoderma pleuroticola solid fermentation material to the volume of pure water is 1 g: (8 mL-10 mL).
The other steps are the same as those in the first to third embodiments.
The fifth concrete implementation mode: the difference between this embodiment and one of the first to fourth embodiments is: the first evaporation and concentration in the third step is carried out according to the following steps: and (3) adding the crude sugar solution obtained in the step two into a rotary evaporator, and evaporating and concentrating at the temperature of 75-85 ℃ and at the rotating speed of 25-35 r/min until the concentration of soluble solids in the crude sugar solution is 8-12 g/100mL to obtain a first concentrated solution.
The other steps are the same as those in the first to fourth embodiments.
The sixth specific implementation mode: the difference between this embodiment and one of the first to fifth embodiments is as follows: the first ion exchange in step three is carried out according to the following steps: and (3) passing the first concentrated solution through a first anion exchange resin column, a first cation exchange resin column and a second anion exchange resin column in sequence to obtain a first ion exchange solution.
The other steps are the same as those in the first to fifth embodiments.
The seventh embodiment: the difference between this embodiment and one of the first to sixth embodiments is: the second evaporation and concentration in the third step is carried out according to the following steps: and adding the first ion exchange liquid into a rotary evaporator, and evaporating and concentrating at the temperature of 75-85 ℃ and at the rotating speed of 25-30 r/min until the concentration of soluble solids in the first ion exchange liquid is 10-20 g/100mL to obtain a second concentrated liquid.
The other steps are the same as those in the first to sixth embodiments.
The specific implementation mode is eight: the difference between this embodiment and one of the first to seventh embodiments is: the second ion exchange in step three is carried out according to the following steps: and (3) passing the second concentrated solution through a second cation exchange resin column, a third anion exchange resin column and a third cation exchange resin column in sequence to obtain a second ion exchange solution.
The other steps are the same as those in the first to seventh embodiments.
The specific implementation method nine: the difference between this embodiment and the first to eighth embodiments is: the third evaporation concentration in the third step is carried out according to the following steps: and adding the second ion exchange liquid into a rotary evaporator, and evaporating and concentrating at the temperature of 75-85 ℃ and at the rotating speed of 10-20 r/min until the concentration of soluble solids in the second ion exchange liquid is 65-75 g/100mL, thereby obtaining the functional syrup product.
The other steps are the same as those in the first to eighth embodiments.
The detailed implementation mode is ten: the difference between this embodiment and one of the first to ninth embodiments is as follows: the type of the resin in the first anion exchange resin column is D301, the type of the resin in the first cation exchange resin column is 001, the type of the resin in the second anion exchange resin column is D201, the type of the resin in the second cation exchange resin column is 001 multiplied by 7, the type of the resin in the third anion exchange resin column is D301, and the type of the resin in the third cation exchange resin column is 001 multiplied by 7.
The other steps are the same as those in the first to ninth embodiments.
The following examples were used to demonstrate the beneficial effects of the present invention:
the first embodiment is as follows: the application of the trichoderma pleuroticola ZJ-03 in the fine and further processing of industrial hemp is completed according to the following steps:
picking up restored and cultured trichoderma aureoviride ZJ-03 spores by using an inoculating shovel under the aseptic condition, inoculating the trichoderma aureoviride ZJ-03 spores into a PDA slant culture medium, then placing the culture medium into a mould incubator, and culturing at 28 ℃ until the trichoderma aureoviride ZJ-03 spores are mature to obtain activated trichoderma aureoviride ZJ-03 strain spores; washing activated spore of Trichoderma aureoviride ZJ-03 strain on PDA slant culture medium with appropriate amount of sterile water, taking off, scattering with sterilized glass beads, shaking, and counting with blood corpuscle plate to obtain 1.5 × 107Per mL of Trichoderma aureoviride ZJ-03 sporesSuspending the solution; FIG. 2 is an activated spore of Trichoderma aureoviride ZJ-03 strain; the Trichoderma pleuroticola ZJ-03 is separated from a Pleurotus ostreatus bag, is preserved in Guangdong province microorganism strain collection center, and has a preservation number of GDMCC NO. 60689.
Secondly, preprocessing industrial hemp (hemp) scraps: crushing hemp scraps, sieving the crushed hemp scraps by a 40-mesh sieve, and taking undersize products to obtain the hemp scrap powder, wherein the hemp scraps are purchased from Heilongjiang Han Zhenghuma technology Limited company.
Pretreatment of industrial hemp (hemp) seed meal: crushing hemp seed meal, sieving with a 40-mesh sieve, and taking undersize products to obtain hemp seed meal powder.
Preparing a nutrient solution: adding 0.5g of glucose mother liquor and 0.5g of threonine mother liquor into 100mL of distilled water, and uniformly mixing to obtain the nutrient solution.
Adding the hemp sawdust powder and the hemp seed meal powder (the dry basis mass ratio of the hemp sawdust powder to the hemp seed meal is 49:1) into a nutrient solution, fully and uniformly stirring, then filling into a polypropylene cuboid tray (52cm multiplied by 36cm multiplied by 7cm), sterilizing for 2 hours at 121 ℃, and then inoculating the trichoderma pleuroticola ZJ-03 spore suspension obtained in the first step under the aseptic condition, wherein the inoculation amount is 2% (v/m, namely the mass ratio of the volume of the trichoderma pleuroticola ZJ-03 spore suspension to the dry basis of the solid culture material; after inoculation, placing the inoculated strain on a sterilized culture rack of a fermentation chamber with the relative humidity of 80% for culture, wherein the fermentation chamber is provided with a cooling and heating air conditioner, a humidifier and a full-automatic hygrothermograph, culturing the strain at 28 ℃ for 72 hours, then culturing the strain at 37 ℃ for 24 hours, and then continuously culturing the strain at 37 ℃ for 24 hours to obtain a trichoderma aureobasicola solid fermented material, and FIG. 3 is a schematic diagram of the trichoderma aureobasicola solid fermented material; after fermentation is finished, adding the trichoderma aureobasicola solid fermentation material into pure water, and standing and leaching for 6 hours at 37 ℃ to obtain a leaching solid-liquid mixture; coarse filtering the solid-liquid mixture by using 6 layers of gauze to remove spores and solid matters, centrifuging for 10min under the condition of 4000r/min, and taking supernatant to obtain a crude sugar solution; the ratio of the total mass of the hemp seed meal powder and the hemp scrap powder to the volume of the nutrient solution is 1: 1.5; the ratio of the mass of the trichoderma pleuroticola solid fermentation material to the volume of pure water is 1 g: 8 mL;
fourthly, adding the crude sugar solution obtained in the third step into a rotary evaporator, and carrying out evaporation concentration at the temperature of 80 ℃ and the rotating speed of 30r/min until the concentration of soluble solids in the crude sugar solution is 10g/100mL to obtain a first concentrated solution; sequentially passing the first concentrated solution through a first anion exchange resin column (the model of resin in the column is D301), a first cation exchange resin column (the model of resin in the column is 001) and a second anion exchange resin column (the model of resin in the column is D201), and performing decolorization, deacidification and metal ion removal to achieve the aim of further purification to obtain a first ion exchange solution; adding the first ion exchange liquid into a rotary evaporator, and carrying out evaporation concentration at the temperature of 80 ℃ and the rotating speed of 30r/min until the concentration of soluble solids in the first ion exchange liquid is 15g/100mL to obtain a second concentrated liquid; passing the second concentrated solution through a second cation exchange resin column (the type of the resin in the column is 001 × 7), a third anion exchange resin column (the type of the resin in the column is D301) and a third cation exchange resin column (the type of the resin in the column is 001 × 7) in sequence, and performing decolorization, deacidification and metal ion removal to achieve the purpose of further purification to obtain a second ion exchange solution; adding the second ion exchange liquid into a rotary evaporator, and evaporating and concentrating at 85 ℃ and at a rotating speed of 15r/min until the concentration of soluble solids in the second ion exchange liquid is 70g/100mL to obtain a functional syrup product; the ion exchange resin columns were purchased from Zhejiang Ushu Kogyo Co.
The yield of the third evaporated concentrated solution (i.e. the functional syrup product) weight relative to the dry basis weight of the industrial hemp (hemp) scraps and the industrial hemp (hemp) seed meal is 8%, i.e. 98 tons of industrial hemp (hemp) scraps (dry basis) and 2 tons of industrial hemp (hemp) seed meal are subjected to solid state fermentation, and finally 8 tons of high-purity functional syrup products can be produced, i.e. 12.25 tons of industrial hemp (hemp) scraps (dry basis) and 0.25 tons of industrial hemp (hemp) seed meal are required for producing 1 ton of functional syrup products.
The detection and calculation method comprises the following steps: the method comprises the steps of detecting the concentration (g/100mL) of total soluble solids in sugar liquid by adopting a handheld refractometer method, detecting the concentration (g/100mL) of sugar in the sugar liquid and the concentration (g/100mL) of total reducing sugar in the sugar liquid by adopting a film reagent method, detecting the light transmittance (%) of the sugar liquid by adopting a spectrophotometer method, detecting the conductivity (mu s/cm) of the sugar liquid by adopting a conductometer method, detecting the pH value of the sugar liquid by adopting a pH meter method, and determining the yield (%) (% of a syrup product relative to the dry basis of industrial hemp (hemp) scraps, namely the mass (g) ÷ 100% of the syrup product relative to the dry basis of the industrial hemp (hemp) scraps.
Fourthly, detecting functional syrup products:
chromatographic conditions:
a chromatographic column: COSMOSIL sugar-D column (4.6 mm. times.250 mm, 5 μm); flow rate: 1 mL/min; a detector: a difference detector; column temperature: 30 ℃; mobile phase: acetonitrile: 75 parts of water: 25; sample introduction amount: 20 mu L of the solution; high performance liquid chromatography instrument: high performance liquid chromatography Primaide (Hitachi).
(II) treating the functional syrup subjected to chromatography:
the syrup having a soluble solid concentration of 70g/100mL was diluted with ultrapure water to a soluble solid concentration of 1g/100 mL. Sterilizing and removing impurities by a 0.22-micron needle head type filter, and performing ultrasonic degassing for later use.
(III) detecting the functional syrup product by high performance liquid chromatography:
FIG. 4 is a high performance liquid chromatogram of a functional syrup product showing a peak at 3.007 minutes, the component of which is the solvent peak, acetonitrile;
table 1 shows the hplc component percentages of the functional syrup product:
TABLE 1
Figure BDA0002498943570000071
High performance liquid chromatography detection shows that the purity of the functional syrup product is 72.80%, the functional syrup product mainly contains two effective components of xylobiose and xylotriose, the xylobiose component accounts for 46.00%, and the xylotriose component accounts for 26.80%; in the embodiment, the hemp sawdust powder and the hemp seed meal powder are used as main growth substrates for solid state fermentation, so that the enzyme activity of the xylanase produced by trichoderma pleuroticola is induced to be improved by more than 20% compared with the prior enzyme activity (namely, more functional syrup products with the same purity can be produced by the same amount of xylanase); through adopting the separation and purification technical means of extraction, coarse filtration and centrifugation, and subsequent multiple evaporation concentration and ion exchange of the coarse sugar solution, impurities such as pigment, protein, monosaccharide, polysaccharide and the like in the functional syrup product are removed, so that the purity of the final functional syrup product is as high as 72.80%, and the main components are xylobiose (the purity is as high as 46.00%) and xylotriose (the purity is as high as 26.80%), and as the main components in the functional syrup product are the xylobiose and the xylotriose, the products decomposed by digestive enzymes after the xylobiose and the xylotriose are absorbed by a human body are xylose which is difficult to be decomposed by the digestive enzymes of the human body, glucose and saccharides which can increase blood sugar and blood fat of the human body can not be generated, and the xylose only contains very little heat; therefore, the functional syrup product is low-calorie health syrup, and has multiple effects of controlling blood sugar, preventing obesity, improving diabetes symptoms and the like when being added into food.

Claims (3)

1. The application of trichoderma pleuroticola ZJ-03 in the deep processing of industrial hemp is characterized by comprising the following steps:
inoculating trichoderma aureoviride ZJ-03 spores into a PDA slant culture medium under an aseptic condition, and then placing the culture medium into a mould incubator for culture to obtain activated trichoderma aureoviride ZJ-03 strain spores; taking down the activated spore of Trichoderma aureoviride ZJ-03 strain, scattering and shaking to obtain the product with concentration of 1.5 × 1072.0X 10 to one/mL7each/mL of Trichoderma aureoviride ZJ-03 spore suspension; the Trichoderma pleuroticola strain ZJ-03 is Trichoderma pleuroticola (Trichoderma pleuroticola) ZJ-03 which is preserved in Guangdong province microorganism strain collection center with the preservation number of GDMCC NO. 60689;
secondly, adding hemp seed meal powder and hemp crumb powder into the nutrient solution, fully stirring and sterilizing, and then inoculating the trichoderma aureobasicola ZJ-03 spore suspension obtained in the first step under an aseptic condition, wherein the inoculation amount is 1.5-2%; after inoculation, placing the inoculated product in a sterilized fermentation chamber, firstly culturing for 68-72 h at 28-30 ℃, then culturing for 20-24 h at 33-37 ℃, and then continuously culturing for 22-26 h at 37-40 ℃ to obtain a trichoderma aureobasicola solid fermentation material; leaching the trichoderma aureobasicola solid fermentation material to obtain a leached solid-liquid mixture; carrying out coarse filtration and centrifugation on the solid-liquid mixture obtained by leaching, and taking supernatant to obtain a crude sugar solution; the nutrient solution is prepared by the following steps: adding the glucose mother liquor and the threonine mother liquor into distilled water, and uniformly mixing to obtain the nutrient solution, wherein the ratio of the mass of the glucose mother liquor to the mass of the threonine mother liquor to the volume of the distilled water is (0.5 g-1 g): (0.5 g-1 g): 100 mL; the leaching is as follows: adding the trichoderma pleuroticola solid fermentation material into pure water, and statically leaching for 6-8 h at the temperature of 35-37 ℃; the mass ratio of the hemp seed meal powder to the hemp scurf powder is (19-49): 1, and the volume ratio of the total mass of the hemp scurf powder and the hemp seed meal powder to the volume of the nutrient solution is (1.5-1.8); the ratio of the mass of the trichoderma pleuroticola solid fermentation material to the volume of pure water is 1 g: (8 mL-10 mL);
thirdly, sequentially carrying out first evaporation concentration, first ion exchange, second evaporation concentration, second ion exchange and third evaporation concentration on the crude sugar solution obtained in the second step to obtain a functional syrup product;
the first evaporation and concentration is carried out according to the following steps: adding the crude sugar solution obtained in the step two into a rotary evaporator, and carrying out evaporation concentration at the temperature of 75-85 ℃ and at the rotating speed of 25-35 r/min until the concentration of soluble solids in the crude sugar solution is 8-12 g/100mL to obtain a first concentrated solution; the first ion exchange is carried out according to the following steps: sequentially passing the first concentrated solution through a first anion exchange resin column, a first cation exchange resin column and a second anion exchange resin column to obtain a first ion exchange solution; the second evaporation concentration is carried out according to the following steps: adding the first ion exchange liquid into a rotary evaporator, and evaporating and concentrating at the temperature of 75-85 ℃ and at the rotating speed of 25-30 r/min until the concentration of soluble solids in the first ion exchange liquid is 10-20 g/100mL to obtain a second concentrated liquid; the second ion exchange is carried out according to the following steps: passing the second concentrated solution through a second cation exchange resin column, a third anion exchange resin column and a third cation exchange resin column in sequence to obtain a second ion exchange solution; the third evaporation concentration is carried out according to the following steps: adding the second ion exchange liquid into a rotary evaporator, and evaporating and concentrating at the temperature of 75-85 ℃ and at the rotating speed of 10-20 r/min until the concentration of soluble solids in the second ion exchange liquid is 65-75 g/100mL, thereby obtaining a functional syrup product; the type of the resin in the first anion exchange resin column is D301, the type of the resin in the first cation exchange resin column is 001, the type of the resin in the second anion exchange resin column is D201, the type of the resin in the second cation exchange resin column is 001 multiplied by 7, the type of the resin in the third anion exchange resin column is D301, and the type of the resin in the third cation exchange resin column is 001 multiplied by 7.
2. The application of trichoderma pleuroticum strain ZJ-03 in the deep processing of industrial cannabis sativa according to claim 1, wherein the hemp sawdust powder in the second step is prepared by the following steps: crushing hemp scraps, sieving with a 40-mesh sieve, and taking undersize products to obtain hemp scrap powder; the hemp seed meal powder is prepared by the following steps: crushing hemp seed meal, sieving with a 40-mesh sieve, and taking undersize products to obtain hemp seed meal powder.
3. The use of Trichoderma aureoviride strain ZJ-03 in the intensive processing of industrial cannabis according to claim 1, wherein the cultivation in step one is as follows: culturing at 28-30 ℃ until the spores of trichoderma pleuroticola ZJ-03 are mature; and (2) sterilization in the second step: stirring thoroughly, placing into container, and sterilizing at 121 deg.C for 2 hr; centrifuging in the step two: centrifuging the solid-liquid mixture for 10-15 min under 3500-4000 r/min.
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CN103757036A (en) * 2007-10-03 2014-04-30 维莱尼姆公司 Xylanases, nucleic acids encoding them and methods for making and using them
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CN103757036A (en) * 2007-10-03 2014-04-30 维莱尼姆公司 Xylanases, nucleic acids encoding them and methods for making and using them
CN110747135A (en) * 2019-12-11 2020-02-04 齐齐哈尔大学 Trichoderma aureoviride and application thereof

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