CN117229920A - Multifunctional yeast hydrolysate and preparation method thereof - Google Patents
Multifunctional yeast hydrolysate and preparation method thereof Download PDFInfo
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- CN117229920A CN117229920A CN202310932403.9A CN202310932403A CN117229920A CN 117229920 A CN117229920 A CN 117229920A CN 202310932403 A CN202310932403 A CN 202310932403A CN 117229920 A CN117229920 A CN 117229920A
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention provides a yeast hydrolysate product, wherein the polysaccharide component from the yeast cell wall is less than or equal to 1%, the total amino acid is more than or equal to 50%, the polypeptide or small molecular peptide component is more than or equal to 20%, the nucleic acid is more than or equal to 10%, and the mineral component is more than or equal to 1%, wherein the polysaccharide from the yeast cell wall is mannans and glucans, can form a self-assembled gel system, and can be used in the fields of drug carriers, health care products and cosmetics.
Description
Technical Field
The invention relates to the field of yeast hydrolysates and functional health products, in particular to a multifunctional yeast hydrolysate, a preparation method and application thereof.
Background
Yeast cells are rich in nutrition, have been used in China for thousands of years, contain rich proteins, vitamin ores, polysaccharides and other substances, can promote the growth, development, digestion and absorption of organisms, and the polysaccharides in the yeast cells can promote the proliferation of beneficial bacteria, and are widely used in the fields of food, feed, brewing beverages, medicines, cosmetology and water treatment. For example, CN109423449B discloses a selenium yeast hydrolysate and a preparation method thereof, which is obtained by selecting high selenium yeast for fermentation and performing enzymolysis by combining a plurality of enzymes to obtain the hydrolysate which can be used as a feed product. CN106659217B discloses a low gluten yeast hydrolysate for use in the food field.
The yeast hydrolysate is yeast enzymolysis product containing multiple components, and is obtained by autolysis, chemical treatment, enzymolysis, etc. However, the autolysis method requires longer time and harsh conditions, the chemical treatment brings about organic reagent and causes protein deterioration and denaturation, the mechanical method requires energy consumption, the enzymolysis by using different complex enzyme components is also a biological method, and the products have different enzyme proportions, PH and action time, and the flavor is also different. However, the preparation of yeast hydrolysates generally requires long-term maintenance of pH stability at temperature, and usually requires supplementation of enzymes and buffers during the enzymatic hydrolysis process to maintain the system reaction and stability. Although the polysaccharide and protein components are important nutrients of yeast, the polysaccharide and protein components often cause immune activation and some unnecessary allergic reactions, and have unique nutritional value and flavor when used as food and beverage feeds, the polysaccharide and protein components are difficult to use as functional auxiliary materials, firstly, the polysaccharide and protein components are active, are usually used for balancing intestinal flora, are easy to ferment and are difficult to use in medicines, and secondly, yeast hydrolysate is easy to absorb, inactivate and degrade or is hydrolyzed by in vivo enzymes.
Tumor cell metastasis is the main cause of cancer death, and cancer metastasis refers to that tumor cells of a primary focus enter other tissues through lymph or blood circulation to form new secondary tumors, so that the survival probability of patients is seriously reduced. The radiotherapy and chemotherapy operations can only treat residual cancer cells at the primary part of the tumor to prevent the diffusion of the residual cancer cells, but the radiotherapy and chemotherapy methods cannot be used for a long time, and the cancer is characterized in that the cells grow uncontrollably in the body to cause invasion and death of basic organs. Therefore, the method for effectively controlling the spread metastasis of cancer cells after operation and inhibiting infiltration of the cancer cells to peripheral vascular lymph from the earliest source is a strategy with practical significance.
The development of hydrogels for medical applications can be traced to decades ago, and at present, some special researches on synthesis and characteristics thereof are carried out, and the hydrogels are mostly used for absorbing materials, biomedical engineering or drug carriers (see CN 103494668B), and the hydrogels are usually prepared from crosslinking agents, are used as a polymer system containing a three-dimensional network structure, are easy to be degraded under environmental influence, and are easy to generate toxic side effects when added with organic solvents, so that the application of the hydrogels in biomedical materials is limited.
Disclosure of Invention
The inventor optimizes the formula of the yeast hydrolysis complex enzyme and the process thereof to produce a hydrolysate product which basically does not contain polysaccharide from the cell wall of the yeast and has the health care effect, the hydrolysate is prepared under specific conditions by selecting specific complex enzyme, the hydrolysate product can also have the double functions of health care and carrier through a hydrogel self-assembly system, the filtered residues can be independently packaged as feed, in addition, the hydrolysate product is found to have the protection effect on basal blood vessels in a cell test, the inhibition effect on tumor metastasis and recurrence is shown, and the hydrolysate product has application prospect in functional food and beverage and rehabilitation of cancer patients. In addition, the problem that the system needs complex control to keep the PH stable is solved by the intervention of a high polymer carrier in the enzyme hydrolysis process, the enzymolysis time is longer, the system basically does not contain an organic solvent, and the system has unique fragrance and is easy to accept by consumers.
The specific technical scheme comprises the following steps:
a yeast hydrolysate is provided, characterized in that the polysaccharide component derived from the cell wall of the yeast is not higher than 1%, the total amino acid is not lower than 50%, the polypeptide (or called small molecular peptide) component is not lower than 20% and not higher than 35%, the nucleic acid is not lower than 10%, the mineral component is not lower than 1% by weight, wherein the polysaccharide derived from the cell wall of the yeast is mannans and glucans, the hydrolysate of the invention is substantially free of mannans and glucans, and the hydrolyzed polysaccharide products thereof, in particular, is free of water-soluble beta-glucans. Preferably, the arginine, tryptophan and lysine content of the total amino acids is more than or equal to 20%, further more than or equal to 25%, 30% and 35% by weight. For example, in the case where the total amino acid content in the yeast hydrolysate is 50%, the total of arginine, tryptophan and lysine is 10% or more of the yeast hydrolysate, and 20% or more of the total amino acid content; preferably, the total amino acid is more than or equal to 55 percent, the small molecular polypeptide is more than or equal to 26 percent, and the nucleic acid is more than or equal to 11 percent.
Hydrolysis using the following complex enzymes: candida rugosa lipase, trypsin, arginase, papain, nuclease, glutamine transaminase and beta-glucanase, wherein the proportion of beta-glucanase in the complex enzyme is less than or equal to 10% by weight, more preferably between 2% and 8%; the carrier of the complex enzyme is MOFs (i.e., metal-organic frameworks, also known as porous coordination polymers) selected fromUiO-66, MIL-101 or ZIF-8, preferably UiO-66 (C 48 H 28 O 32 Zr 6 Also known as metal Zr-containing porous coordination polymers); the specific surface area of the metal frame is 100-2000 m g/m, preferably 500-1000 m g/m, most preferably 700-900 m g/m, and the pore diameter is less than or equal to 80 nm, preferably less than or equal to 50nm, more preferably less than or equal to 20nm. The weight ratio of the complex enzyme to the metal frame carrier is 1:0.5-3, preferably 1: the MOFs may be subjected to an activation step, preferably with a buffer or phosphate until the PH is stable within the desired range, and a washing step, the PH being adjusted as desired by phosphate or other buffer, preferably 5-7.5, more preferably 5.5-6.5; the washing solvent is preferably ethanol and/or water, and the MOFs are preferably activated after being formulated as a suspension. It may be placed in an activation bed, column or other vessel and kept at a steady temperature, preferably 20-50 ℃.
The preparation method comprises the following steps of,
1) Culturing yeast: inoculating yeast into a yeast pool containing a culture solution, culturing at room temperature to obtain amplified yeast solution, and drying; wherein the yeast is selected from Saccharomyces cerevisiae, saccharomyces boulardii, preferably Saccharomyces cerevisiae; the culture broth may be conventional (e.g., commercially available medium containing glucose, amino acids, tryptone, etc.), or additional nitrogen source, carbon source, or corresponding nutrient growth factor, etc., according to the desired health function, the amplification may be exponential, the primary seed broth may be followed by the secondary seed amplification, a suitable temperature may be selected, e.g., 20-80 ℃, preferably 30-60 ℃, for 5-72 hours, preferably 12-48 hours, and the fermentation tank may be incubated. Wherein preferably, the water addition amount is estimated so that the concentration of the yeast milk in the prepared yeast pool is 10-15%, and the yeast milk is uniformly stirred. And subsequently dried.
2) Loading a complex enzyme carrier:
cleaning metal frame material (MOF or MOFs, also called MOFs porous material), removing impurities, suspending in ethanol solution or water solution, slowly dripping complex enzyme solution into MOF suspension, stirring overnight, centrifuging, and drying. Methods for removing impurities include, but are not limited to, water rinsing, ultrasonic cleaning, ethanol solution cleaning or post-soaking rinsing. The carrier may be activated at sites by adding a buffer, including but not limited to phosphate (e.g., sodium dihydrogen phosphate), pH preferably ranging from 6 to 7, MOFs suspension preferably using an ethanol solution or an aqueous solution as a solvent, and an aqueous solution may be added with a small amount of NaCl or its electrolyte, ethanol solution preferably having a concentration of 50% or more; the dripping process speed of the complex enzyme is preferably 10ml/min or less, and the complex enzyme is slightly stirred at a certain speed, preferably the stirring speed is lower than 50rpm, so that the enzyme is uniformly distributed at the inner hole sites. The temperature is room temperature or 25-35 ℃; the time is preferably 10-36 hours; the weight ratio of trypsin, arginase, papain, nuclease, glutamine transaminase and beta-glucanase from candida rugosa (Lipase from Candida rugosa) is 2:2:2:2:1:1:1. the beta-glucanase is less than or equal to 10wt%, preferably less than or equal to 5wt%, so that the beta-glucanase can help wall breaking and reduce degradation of macromolecular proteins and polysaccharides, and is convenient for subsequent removal. Wherein the weight ratio of the complex enzyme to the carrier is 1-3:1, preferably 1:1, further, no additional supplementary enzyme and buffer solution are needed in the enzymolysis process, the system can keep stable PH (with electrolyte property) and enzyme reaction sites are uniform and thorough.
3) Obtaining hydrolysate:
adding proper amount of water into saccharomycete to prepare suspension, introducing the suspension into a reactor with MOFs metal frame loaded with compound enzyme, stabilizing pH between 6 and 6.5, heating to hydrolyze for 1 to 24 hours, ultrasonically oscillating and filtering, taking filtrate, heating to inactivate enzyme, concentrating and drying to obtain hydrolysate. Wherein the pH is naturally stable without the need for additional detection or addition of buffer components, and the heating temperature is 35-70deg.C, preferably 40-65deg.C, and the reaction time is greater than 6h, preferably 6-18h, more preferably 8h. A small amount of electrolyte can be added, and the reaction time is preferably 6 hours; the enzyme and saccharomycete are uniformly distributed at inner hole sites, and the enzyme and saccharomycete have the advantages of replacing ions to control PH to be stable, avoiding the adsorption agglomeration of saccharomycete and products thereof and PH movement problem of a reaction system, and ensuring more thorough reaction. Preferably by ultrasonic vibration, accelerating the reaction, more preferably by vacuum drying filtration, vacuum pressurization of 10 mbar to 100 mbar, preferably 20 mbar to 80 mbar, more preferably 40 mbar to 60 mbar, filtration using filtration membranes, including but not limited to Pellicon2Biomax5kDa ultrafiltration membrane package, can also be used in combination or alone to ultrafiltration the separated filtrate. The filtrate is taken to obtain supernatant, and the enzyme is deactivated by heating or chemical process, preferably heating and enzyme deactivation, wherein the heating temperature is 60-100deg.C, preferably 80deg.C, for 10-30min. The drying method may be vacuum drying, freeze-drying or spray-drying.
And (5) drying filter residues by a roller after discharging, and carrying out additional treatment after crushing by a crusher.
In a preferred technical scheme, the yeast hydrolysate can be industrially produced, and the enzymolysis step can be carried out without adding enzyme, so that the hydrolysis time is shortened, and the PH is stable. And after the carrier is injected, a steam valve is opened, stirring and heating are carried out, the temperature of the feed liquid is controlled, and after enzymolysis is carried out for a certain time, the feed liquid is communicated with a filtering post to be discharged.
The concentration process may be operated as follows: the circulating pump, the discharging pump and the condensing water pump are advanced to cool water, quantitative feed liquid is fed in, the vacuum hydraulic jet pump is started, the vacuum degree is regulated, the air inlet valve is started, and concentration is started. The feeding amount and the steam valve are regulated, the first-effect temperature is controlled to be 85+/-10 ℃, the second-effect temperature is controlled to be 75+/-10 ℃, and the third-effect temperature is controlled to be 65+/-10 ℃. And (3) before the product reaches the technical requirements of the process, a small amount of feed liquid is discharged, the concentration is measured by a Baume meter, and when the concentration reaches 18-25Bx (the concentration of solid matters is 25-45%), the material is discharged and sent to the next drying process.
For the treatment of the filter residues, specifically, the filter residues are driven into a roller, the roller is preheated, and the rotating speed of the roller and the steam consumption are regulated according to the dryness. And naturally cooling the powdery filter residues obtained by roller drying, and filling the cooled filter residues into a woven bag. After a certain amount is collected, drying, crushing, checking and packaging are carried out, wherein the feed is rich in polysaccharide and protein components, and can be used as feed.
The product was detected by HPLC, gel electrophoresis or amino acid analyzer.
The invention also provides a functional hydrogel which can be used as a carrier of health care food or auxiliary medicine, and is characterized by comprising the yeast hydrolysate and hyaluronic acid, wherein the hydrogel is formed by self-assembly, the hydrolysate is prevented from being aggregated and released too fast, the enzyme hydrolysate is prevented from being aggregated and adsorbed, and the salt water (the sodium chloride content is not less than 3.5%) can be further solidified and crosslinked to form a three-dimensional space so as to meet different requirements; wherein hyaluronic acid is dissolved in a buffer solution, and an enzyme hydrolysate is added for crosslinking, preferably a phosphate, most preferably a sodium dihydrogen phosphate solution, preferably, the hydrolysate does not contain glucan and mannans, contains more than or equal to 50% of total amino acids, further, a hydrophilic gel can be formed by self-assembly, a seawater salt solution is added to improve the solidification degree of the hydrogel, preferably, the hydrophilic gel before the salt solution is added can be used as a cosmetic carrier for external use or drinking and can be used as a food additive, can be used as a surgical material, and has viscosity of more than 100mPa.s, more preferably more than 120mPa.s, less than 500mPa.s, most preferably between 100 and 200 mPa.s, and has viscosity of more than 500mPa.s, preferably more than 650 mPa.s, more preferably between 650 and 1000 mPa.s after the salt solution is added.
To maintain a uniform distribution of the yeast hydrolysate, it is preferable to add dropwise the solution to the hyaluronic acid buffer solvent with stirring, and to prepare the yeast hydrolysate as a 1-5% solution,
the preparation method comprises the following steps:
1) Yeast hydrolysate, 2% concentration, water as solvent and proper amount of sodium dihydrogen phosphate solution. Adding hyaluronic acid into phosphate buffer solution to prepare 2% solution with pH of 5.5-6.5.
2) Slowly adding the yeast hydrolysate solution into the hyaluronic acid solution to uniformly distribute the yeast hydrolysate solution, wherein the ratio of the yeast hydrolysate solution to the hyaluronic acid solution is 1:1-3, stirring slightly to form a fluid, if too viscous to form a fluid, may be accelerated or diluted with a small amount of water.
3) If further curing is desired, sea brine with a concentration greater than 3.5% (i.e., naCl content greater than 3.5%) may be added, which may crosslink the fluid hydrogel, cure, increase the hydrogel adhesion, and slowly release the hydrolysate component therein.
4) The viscosity and the sustained-release performance of the hydrogel are detected, the viscosity of the hydrogel before and after adding the sea salt water is measured by using a viscometer, the self-assembled hydrogel containing yeast hydrolysate-hyaluronic acid and the hydrogel after adding the sea salt water are placed in a simulated body fluid buffer solution, the sea salt water is added until the viscosity of the gel is more than 500mPa.s, the gel is incubated at 36 ℃, samples are taken from the simulated body fluid at different time points (1 h to 72 h), and the content (%) of amino acid and polypeptide in the hydrogel is detected, so that the data of the hydrogel release product under the simulated actual condition is obtained.
Further, the invention provides a health care product which comprises the edible hydrogel, wherein the health care product can be a Chinese patent medicine or a nutritional health care product and can be in the forms of beverage, yoghurt, jelly, chewing gum and the like.
Further, the present invention provides a cosmetic comprising the above hydrogel, and other cosmetic ingredients.
Furthermore, the invention also provides a pharmaceutical application, namely an application of the yeast hydrolysate in preparing medicines, in particular to a yeast hydrolysate-hyaluronic acid self-assembled hydrogel system. The application shows that the yeast hydrolysate-hyaluronic acid self-assembled hydrogel can be used in operation, before operation and after operation, preferably can be used with chemotherapy drugs in a tandem manner, and can also be used independently. In the yeast hydrolysate-hyaluronic acid self-assembled hydrogel, the mass percentage of the yeast hydrolysate to the hyaluronic acid is 1:1-3, preferably 1:2.
wherein trypsin, arginine protease, papain, nuclease, glutamine transaminase and beta-glucanase in the complex enzyme are all purchased from Guangzhou Weber technologies, inc.;
candida rugosa Lipase, also known as derived from Candida rugosa (Lipase fromCandida rugosa) Lipase, also known as lipase AP6, manufactured by sigma-aldrich, type VII, > 700 unit/mg solid state, CAS number 9001-62-1, available from North solid aspect science company;
MOFs metal frameworks were purchased from DuPont, U.S., where UiO-66 is of formula C 48 H 28 O 32 Zr 6 The remaining compounds were purchased from merck (sigma) and the assay equipment was purchased from mertrer-tolidol instruments limited.
Saccharomyces boulardii YT-35%Saccharomyces boulardii) The strain is preserved in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms (China) on the date of 2014, 07 and 03Center, save number is: CGMCC No.940.
The beer yeast strain is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 13500 in the 12 th month and 26 th month of 2016.
The saccharomyces boulardii is purchased from the wuhan kemicin biomedical technology limited company, the sample is cultured after being dissolved and diluted by sterile normal saline, and DNA detection shows that the saccharomyces boulardii YT-35 with the number of CGMCC No.940Saccharomyces boulardii) The strain has homology and purity higher than 99.9%. The beer yeast is purchased from the Siamiliaxi biotechnology Co-Ltd, the sample is cultured after being dissolved and diluted by sterile normal saline, and DNA detection shows that the beer yeast has homology with the gene sequence of the beer yeast strain with the number of CGMCC No.13500 and the purity is higher than 99.9 percent.
The terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a product or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such product or system. Without further limitation, an element defined by the phrase "comprising … …" does not exclude that an additional identical element is present in a commodity or system comprising the element. The room temperature in the present invention means 15-25 ℃, and all percentages by weight unless otherwise indicated are by weight, and the present solution is an aqueous solution.
The invention has the beneficial effects that:
1) The invention is inspired by the biological adhesion principle of shells, polysaccharide components (mainly glucan and manna) from cell walls in hydrolysate are removed (used as high protein feed), the prepared hydrolysate can form a self-gelling system with hyaluronic acid, can be added into local application or health-care food, has adsorption effect on mucous membrane, has special fragrance, is easy to be tolerated by patients, and provides nutrient substances and health-care components which are used as part of self-assembled carriers.
2) The method improves the yeast hydrolysis process, uses specific complex enzyme to ensure that the cell wall macromolecular polysaccharide is easy to separate, reduces the use of glucokinase, increases the use of amino acid acquisition enzyme, overcomes the problem that saccharomycetes and complex enzyme reaction sites are easy to agglomerate and adsorb by a nano metal frame of a complex enzyme carrier, accelerates the reaction time, can accelerate the reaction time and strengthen the reaction force by conducting electrolyte, ensures that the PH is kept stable in a subsequent hydrolysis system, and has controllable and simplified industrialization process.
3) The hydrolysate can obviously improve vascular heterogeneity, down regulate matrix metalloproteinase MMP2, avoid degradation of surrounding matrix and reduce entry of tumor cells into blood vessels. Has wide prospect in the fields of postoperative rehabilitation and health care of cancer patients, prevention of cancer metastasis and recurrence, and the like.
Drawings
Fig. 1: preparation flow diagram
Fig. 2: yeast hydrolysate preparation process flow chart
Fig. 3: MMP-2 concentration inhibitory effect
Detailed Description
Example 1:
1) Obtaining amplified saccharomycetes through yeast culture:
inoculating Saccharomyces boulardii into culture solution, culturing at room temperature for 48 hr, taking bacterial liquid in exponential growth phase as primary seed liquid, continuously inoculating into secondary seed tank, performing exponential amplification, filtering and drying to obtain yeast liquid;
2) Loading a composite enzyme carrier, immersing MOFs metal frame material (UiO-66 (Zr) nano particles) into ethanol solution (more than or equal to 75 percent), cleaning, removing impurities, and detecting after vacuum drying, wherein the specific surface area is 800 m/g, and the aperture is less than or equal to 20 nanometers. Adding 5% sodium dihydrogen phosphate solution for activation, preparing MOFs suspension, and adding complex enzyme with pH of 6.5-7: MOFs vector = 1:1 (weight) of lipase from candida rugosa (Lipase from Candida rugosa), trypsin, arginase, papain, nuclease, glutamine transaminase and beta-glucanase in a weight ratio of 2:2:2:2:1:1:1, slowly dripping the complex enzyme solution into MOFs suspension, stirring (25 rpm), adding a small amount of electrolyte to form osmotic pressure to promote the adsorption of enzyme in MOFs pore diameter, overnight, centrifuging and drying.
3) Obtaining hydrolysate, adding proper amount of water into amplified saccharomycetes to prepare 15% suspension, introducing the suspension into a reactor loaded with MOFs (UiO-66) metal frame nano particles of complex enzyme, stabilizing the PH between 6 and 6.5, adding electrolyte and electrodes to accelerate the process if necessary, hydrolyzing at a temperature of between 40 and 55 ℃ for at least 6 hours, ultrasonically oscillating and vacuum filtering through 40 mbar pressure, heating the filtrate to above 80 ℃, inactivating enzyme for 20min, concentrating and drying to obtain hydrolysate. And (5) further detecting.
The filter residue is discharged and then is dried by a roller, and the filter residue can be used as feed for additional treatment after being crushed by a crusher.
The detection method comprises the following steps:
the hydrolysate was analyzed by High Pressure Liquid Chromatography (HPLC) using an HP1100 instrument run by ChemStation software, the conditions of the HPLC were as follows:
separation column: asahipak HPLCcolumn GS-320 (30 ℃ C., 7.5 mm. Times.300 mm)
Mobile phase: 0.1M sodium phosphate buffer (pH 7.0)
Flow rate: 1.0mL/min
Detection: ultraviolet ray detector (260 nm)
Protein properties of the hydrolysate were detected by electrophoresis using polyacrylamide gel (Invitrogen), protein migration was performed using NuPAGEMES migration buffer, seeBlueLelus 2 was used as a molecular weight marker, protein staining was performed using Coomassie blue R-250, and high molecular proteins, dextran and mannan components were detected in the filtration residue, and the above components were 0.05% in the hydrolyzed filtrate.
Nucleic acid reference spectrophotometry for determination of total phosphorus in feed (GB/T6437).
Peptide content the total amino acids and free amino acids were determined using an amino acid analyzer (Hitachi high speed amino acid analyzer L-8900), and the value obtained by subtracting the free amino acids from the total amino acids was set as the peptide content.
The detection results are mainly amino acids, polypeptides, nucleic acids and mineral components, and the amino acids are analyzed by HPLC to maximize tryptophan, arginine and lysine content. The content of each component is as follows (by weight): 55% of total amino acids, 28% of polypeptides, 12% of nucleic acids and 4% of minerals. Wherein the tryptophan content is 3%, the arginine content is 15%, and the lysine content is 15%.
Example 2:
1) Culturing yeast:
inoculating beer yeast into the culture solution, culturing at room temperature for 48h, taking the bacterial liquid in the exponential growth phase as a first-stage seed liquid, continuously inoculating into a second-stage seed tank, realizing exponential amplification, and filtering the obtained yeast liquid;
2) Loading a composite enzyme carrier, immersing MOFs metal frame material (UiO-66 (Zr) nano particles) into ethanol solution (more than or equal to 75 percent), cleaning, removing impurities, and detecting after vacuum drying, wherein the specific surface area is 800 m/g, and the aperture is less than or equal to 20 nanometers. Adding 10% sodium dihydrogen phosphate solution for activation, preparing MOFs suspension, and adding complex enzyme with pH of 6.5-7: MOFs vector = 1:1 (weight) derived from candida rugosa (Lipase fromCandida rugosa) The weight ratio of trypsin, arginine protease, papain, nuclease, glutamine transaminase and beta-glucanase is 2:1:2:2:2:1:1, slowly dripping the complex enzyme solution into MOF suspension, opening a steam valve, stirring (25 rpm), adding a small amount of sodium chloride to form osmotic pressure to promote the adsorption of enzyme in MOFs aperture, overnight, centrifuging, and drying.
3) Obtaining hydrolysate, adding proper amount of water into amplified saccharomycete to prepare 10% suspension, introducing into a reactor loaded with MOFs (UiO-66) metal frame nano particles of complex enzyme, stabilizing pH between 6 and 6.5, adding electrolyte and electrode to accelerate the process if necessary, stirring and heating, hydrolyzing at 40-55deg.C for at least 6h, carrying out ultrasonic vibration, notifying discharge with filtration post, rough filtering to remove carrier, heating supernatant filtrate to above 80deg.C, inactivating enzyme for 20min, ultrafiltering the separated filtrate with Pellicon2 (Biomax 5 kDa) ultrafiltration membrane package (P2B 005A05, purchased from Shanghai denning technology Co.), removing high molecular weight substances to purify hydrolysate, concentrating supernatant filtrate, and drying. And adding the discharged residues into a roller for crushing, and then treating the feed. The hydrolysate was further tested and the test method was the same as in example 1, wherein total amino acids were 54%, small molecule polypeptides 21%, nucleic acids 19% and minerals 4%. Wherein the tryptophan content is 13%, the arginine content is 10%, the lysine content is 11%, and the polysaccharide and high molecular weight protein content is 0%.
The concentration process may be operated as follows: the circulating pump, the discharging pump and the condensing water pump are advanced to cool water, a certain amount of feed liquid enters, the vacuum hydraulic jet pump is started, the vacuum degree is regulated, the air inlet valve is started, and concentration is started. The feeding amount and the steam valve are regulated, the first-effect temperature is controlled to be 85+/-10 ℃, the second-effect temperature is controlled to be 75+/-10 ℃, and the third-effect temperature is controlled to be 65+/-10 ℃. And (3) before the product reaches the technical requirements of the process, a small amount of feed liquid is discharged, the concentration is measured by a Baume meter, and when the concentration reaches 18-25Bx (the concentration of solid matters is 25-45%), the material is discharged and sent to the next drying process.
For the treatment of the filter residues, specifically, the filter residues are driven into a roller, the roller is preheated, and the rotating speed of the roller and the steam consumption are regulated according to the dryness. And naturally cooling the powdery filter residues obtained by roller drying, and filling the cooled filter residues into a woven bag. After a certain amount is collected, drying, crushing, checking and packaging are carried out, wherein the feed is rich in polysaccharide and protein components, and can be used as feed.
Example 3:
preparation of hydrogels
1) The yeast hydrolysate obtained in example 1 was prepared to have a concentration of 2%, the solvent was water, and a proper amount of sodium dihydrogen phosphate solution was added. Adding hyaluronic acid into phosphate buffer solution to prepare 2% solution with pH of 5.5-6.5.
2) Slowly adding the yeast hydrolysate solution into the hyaluronic acid solution to uniformly distribute the yeast hydrolysate solution, wherein the ratio of the yeast hydrolysate solution to the hyaluronic acid solution is 1:1-3, stirring slightly to form a fluid, if too viscous to form a fluid, may be accelerated or diluted with a small amount of water.
3) The addition of sea brine with a concentration greater than 3.5% (i.e., naCl content greater than 3.5%) can crosslink and solidify the fluid hydrogel, increase the adhesiveness of the hydrogel, and slowly release the hydrolysate component therein.
4) The viscosity and sustained release performance of the hydrogels were measured using a viscometer to measure the viscosity of the hydrogels before and after adding the sea salt water, which were 121mpa.s and 876mpa.s, respectively, the self-assembled hydrogels containing yeast hydrolysate-hyaluronic acid and the hydrogels after adding the sea salt water were placed in a simulated body fluid buffer, the sea salt water was added until the viscosity of the gels was greater than 500mpa.s, incubated at 36 ℃, sampled from the simulated body fluid at different time points (1 h to 72 h), and the content (%) of amino acids and polypeptides was measured to obtain simulated actual hydrogel release product data. The results are shown in Table 1, wherein the hydrogel before addition of the seawater is self-assembled with hyaluronic acid and yeast hydrolysate, and the viscosity increases after addition of the seawater, expressed as hydrogel (front) (back), respectively.
TABLE 1
| T （h/%） | 1h | 10h | 24h | 36h | 48h | 60h | 72h |
| Hydrogel (front) | 45 | 86 | 94 | 95 | 97 | 99 | 99 |
| Hydrogel (rear) | 10 3 | 19 | 32 | 50 3 | 67 | 86 | 97 |
Comparative example 1
1) Obtaining amplified saccharomycetes through yeast culture:
inoculating Saccharomyces boulardii into culture solution, culturing at room temperature for 48 hr, taking bacterial liquid in exponential growth phase as primary seed liquid, continuously inoculating into secondary seed tank, performing exponential amplification, filtering and drying to obtain yeast liquid;
2) Loading a composite enzyme carrier, immersing MOF metal frame materials (UiO-66 (Zr) nano particles) into ethanol solution (more than or equal to 75 percent), cleaning, removing impurities, and detecting after vacuum drying, wherein the specific surface area is 800 m/g, and the aperture is less than or equal to 20 nanometers. Adding sodium dihydrogen phosphate solution for activation, preparing MOF suspension with pH of 6.5-7, adding complex enzyme, and compounding enzyme: MOF vector = 1:1 (weight) adding beta-glucanase, mannanase, saccharifying enzyme, endoenzyme, exonuclease and phosphodiesterase, wherein the components and parts by weight are as follows: 1 part of beta-glucanase, 10 parts of mannase, 20 parts of saccharifying enzyme, 15 parts of endonuclease, 1 part of exonuclease and 10 parts of phosphodiesterase. Slowly dripping the complex enzyme solution into the MOF suspension, stirring (25 rpm), adding a small amount of salt to form osmotic pressure to promote the adsorption of the enzyme in the MOF pore diameter, standing overnight, centrifuging, and drying.
3) Obtaining hydrolysate, adding proper amount of water into amplified saccharomycetes to prepare suspension, introducing the suspension into a reactor loaded with MOFs (UiO-66) metal frame nano particles of complex enzyme, stabilizing the PH between 6 and 6.5, adding electrolyte and electrodes to accelerate the process if necessary, hydrolyzing at the temperature of 40-55 ℃ for at least 6 hours, ultrasonically oscillating and vacuum filtering through the pressure of 40 mbar, heating the filtrate to above 80 ℃, inactivating enzyme for 20min, concentrating and drying to obtain hydrolysate. And (5) further detecting.
The detection method is the same as that of the example 1, and the content of each component is as follows (weight): 35% of total amino acid, 16% of small molecule polypeptide, 11% of nucleic acid, 4% of mineral, 15% of glucan and 16% of mannan.
Comparative example 2
1) Culturing yeast:
inoculating beer yeast into the culture solution, culturing at room temperature for 48h, taking the bacterial liquid in the exponential growth phase as a first-stage seed liquid, continuously inoculating into a second-stage seed tank, realizing exponential amplification, and filtering the obtained yeast liquid;
2) Filtering the fermentation liquor, performing ultrasonic treatment for 40min, heating to 45 ℃, preserving heat for 4h, and heating and boiling for 15min to obtain the autofermentation liquor. Cooling to 50 ℃, regulating the pH (using phosphate buffer solution) to 5.5-6, slowly adding 1 part of compound papain, 1 part of nuclease, 1 part of glutamine transaminase, 2 parts of alkaline protease, 1 part of neutral protease and 2 parts of dipeptidase, and performing enzymolysis for 35 hours at 50 ℃ or more, wherein the pH is regulated to 5.5-6 by adding the buffer solution for multiple times, and continuously adding the compound enzyme components. Heating the hydrolysate to 80 ℃, performing enzyme deactivation treatment for 15min, and performing centrifugal separation to obtain supernatant. Evaporating supernatant to concentrate into concentrated solution with solid content of above 40%, spray drying the concentrated solution to obtain powdery product with water content of not higher than 6%,
and (5) further detecting. The detection method is the same as in example 1, wherein the protein content is 13wt%, the nucleic acid content is 18wt%, the yeast cell wall polysaccharide is 21wt%, the polypeptide content is 19wt%, the ash mineral content is 4wt%, and the total amino acid content is 21%.
Comparative example 3
1) Culturing yeast:
inoculating beer yeast into the culture solution, culturing at room temperature for 48h, taking the bacterial liquid in the exponential growth phase as a first-stage seed liquid, continuously inoculating into a second-stage seed tank, realizing exponential amplification, and filtering the obtained yeast liquid;
2) Filtering the fermentation liquor, performing ultrasonic treatment for 40min, heating to 45 ℃, preserving heat for 4h, and heating and boiling for 15min to obtain the autofermentation liquor. Cooling to 50deg.C, adjusting pH to 5.5-6 (using phosphate buffer), and slowly adding complex enzyme (such as lipase derived from candida rugosa (Lipase from Candida rugosa), trypsin, arginase, papain, nuclease, glutamine transaminase and beta-glucanase) at a weight ratio of 2:1:2:2:2:1:1, overnight, centrifuging and drying. Detecting pH instability during enzymolysis at above 50 ℃, supplementing buffer solution every 1h to maintain pH stability, aggregating enzyme groups, reducing reaction rate after time extension, increasing stirring force, affecting stability of a reaction system, detecting sample liquid after 15h, continuously supplementing complex enzyme for reaction overnight (12 h), heating hydrolysate to 80 ℃, inactivating enzyme for 15min, centrifuging and separating to obtain supernatant. The separated filtrate was ultrafiltered using a Pellicon2Biomax5kDa ultrafiltration membrane pack to remove high molecular weight material to purify the hydrolysate. Evaporating supernatant to concentrate into concentrated solution with solid content of above 40%, spray drying the concentrated solution to obtain powdery product with water content of not higher than 6%,
and (5) further detecting. The detection method is the same as in example 1, wherein the protein content is 2.3wt%, the nucleic acid content is 12wt%, the yeast cell wall polysaccharide is 1.8wt%, the polypeptide content is 19wt%, the ash mineral content is 4wt%, the total amino acid content is 49%, the arginine content is 9.5%, the tryptophan content is 9%, and the lysine content is 8.9%.
Because the embodiment does not adopt an enzyme carrier, the reaction process is prolonged, the enzyme agglomeration phenomenon is realized, the reaction sites are uneven, the system stability is poor, and the PH regulation and the supplementation of complex enzyme are required.
Comparative example 4
1) Obtaining amplified saccharomycetes through yeast culture:
inoculating Saccharomyces boulardii into culture solution, culturing at room temperature for 48 hr, taking bacterial liquid in exponential growth phase as primary seed liquid, continuously inoculating into secondary seed tank, performing exponential amplification, filtering and drying to obtain yeast liquid;
2) Loading a composite enzyme carrier, immersing MOF metal frame materials (UiO-66 (Zr) nano particles) into ethanol solution (more than or equal to 75 percent), cleaning, removing impurities, and detecting after vacuum drying, wherein the specific surface area is 800 m/g, and the aperture is less than or equal to 20 nanometers. Preparing MOF suspension, adding complex enzyme, and compounding enzyme: MOF vector = 1:1 (weight) of trypsin, arginine protease, papain, nuclease, glutamine transaminase and beta-glucanase in a weight ratio of 2:2:2:1:1: and 0.5, slowly dripping the complex enzyme solution into the MOF suspension, stirring (25 rpm), adding a small amount of salt to form osmotic pressure to promote the adsorption of the enzyme in the aperture of the MOF, standing overnight, centrifuging, separating and drying to obtain the complex enzyme.
3) Obtaining hydrolysate, adding proper amount of water into amplified saccharomycetes to prepare suspension, introducing the suspension into a reactor loaded with MOFs (UiO-66) metal frame nano particles of complex enzyme, stabilizing the PH between 6 and 6.5, adding electrolyte and electrodes to accelerate the process if necessary, hydrolyzing at the temperature of 40-55 ℃ for at least 6 hours, ultrasonically oscillating and vacuum filtering through the pressure of 40 mbar, heating the filtrate to above 80 ℃, inactivating enzyme for 20min, concentrating and drying to obtain hydrolysate. And (5) further detecting.
The detection method is the same as in example 1, and the content of each component is as follows (weight): total amino acid 45%, small molecule polypeptide 29%, nucleic acid 24%, mineral 1% and polysaccharide 0.5%.
The hydrogel system prepared in accordance with the method of example 3 was not self-assembled since the fluid viscosity of comparative examples 1-2 was too low to be crosslinked by adding NaCl. Related to the fact that it contains a large amount of polysaccharide components and the amino acid composition is different. Comparative examples 3 to 4, although forming a hydrogel system, released faster and crosslinked with sea water insufficiently, the released product was not stable enough even after curing, and the detection method was the same as in example 3. The amino acid and polypeptide indexes are detected after sea brine is crosslinked, and the substitution release effect is poor, and the data are shown in the following table 2:
TABLE 2
| T （h/%） | 1h | 10h | 24h | 36h | 48h | 60h | 72h |
| Comparative example 3 (front) | 49 | 97 | 99 | 99 | 100 | 100 | 100 |
| Comparative example 3 (rear) | 25 | 76 | 87 | 89 | 97 | 99 | 99 |
| Comparative example 4 (front) | 46 | 89 | 98 | 99 | 99 | 100 | 100 |
| Comparative example 4 (rear) | 29 | 48 | 54 | 66 | 87 | 99 | 99 |
Blank group: the blank set in the following test is the use of a commercially available yeast hydrolysate product (e.g., purchased from Angel Yeast Co.).
And (3) testing:
1) Inhibition of tumor cell migration
Human nasopharyngeal carcinoma cells HONE-1 are adopted, HONE-1 cell suspension is added on a Transwell pore plate, culture inducing factors are added below the Transwell pore plate, cell migration is induced, the pore plate is placed in an incubator for incubation, and cells can migrate downwards from an upper pore through a microporous membrane. After 15d incubation the well plate was removed, the cells were fixed and stained and the number of cells migrating through the well membrane was counted for observation. Parallel experiments were performed by setting 9 groups of example 1+ cisplatin, example 2+ cisplatin, example 1, chemotherapeutic group (cisplatin), comparative examples 1-4 and blank group (comparative example 3 because the number of cells was not statistically significant due to agglomeration and adsorption occurring several days after the instillation of the hydrolysate). The results show that the example 1-2+ cisplatin group, the chemotherapeutic drug and the example 1 group have significant inhibition effects.
2) Down-regulating effect of metalloproteinase MMP-2.
Preparing agarose gel matrix porous plates, adding QXLtm520 fluorogenic substrate and MMP-2, preparing a hydrogel system (because comparative example 2 cannot form a gel system and has high protein components, and is difficult to control and sterile and is removed) by using the method of example 3 for the 9 groups of samples to be tested respectively, adding seawater to moderately crosslink, dripping the seawater into the matrix porous plates, incubating the seawater into an incubator under the condition of 36 ℃ and 40% humidity, detecting fluorescent signals generated by degradation of the substrates by using a fluorescence analyzer after 1,3,7day, and analyzing the influence effect of each group on the down regulation of MMP2 activity according to the fluorescent signal intensity.
The results are shown in FIG. 3.
According to the above experimental results, examples 1-2, taken alone or in combination with a chemotherapeutic agent, have remarkable effects in inhibiting tumor cell migration and inducing metastasis to peripheral stroma degradation, probably related to amino acid or polypeptide components contained therein, tumor cells reduce tumor vessel-related formation in an oxygen-deficient environment, migration of tumor cells is accompanied by epithelial cell switching, while matrix metalloproteinase MMP and the like are down-regulated, tumor cells often degrade stroma around cancer cells by using released metalloproteinase MMP, thereby promoting tumor cells to enter blood vessels or lymph, and migrate to new tissue sites along with the circulatory system and form secondary tumors, the rapid proliferation of which requires vascular participation, and the results of the present invention indicate that hydrolysates can remarkably influence migration of tumor cells, improve heterogeneity of tumor blood vessels, and chemotherapeutic agents, while being effective in inhibiting tumor cell metastasis, cannot prevent release of peripheral MMP factors and degrade stroma to promote the release of cancer cells into blood vessels. The recurrence of metastases at the distal end is therefore difficult to control. The hydrolysate has special composition components, has the effects of resisting tumor migration and recurrence, and particularly has better combined use effect with chemotherapeutic drugs, and the hydrolysate is also effective under the independent test condition.
In addition, the zymolyte of the invention has higher amino acid content and extremely low polysaccharide content, thus self-assembled gel can be formed with hyaluronic acid, but the product hydrolyzed by a conventional way and other conventional complex enzymes can not realize the effect, even if one enzyme is changed, even if gel can be formed, the slow release effect is not obvious, and compared with the embodiment 1 of the invention, the anti-tumor migration and transfer capability of the comparative example are poor; the MOF carrier is used in the preparation process, so that adsorption aggregation of enzyme substrates and hydrolysate is effectively solved, the sites are dispersed uniformly, the enzyme hydrolysis efficiency and degree are guaranteed, the PH is stabilized, the reaction time is shortened, the stability of a self-assembled gel system is also beneficial, and the MOF carrier has special fragrance for enzymolysis products and has excellent use tolerance.
The above embodiments do not limit the scope of the present invention, and those skilled in the art can make various changes and applications of the present invention according to the above description.
Claims (7)
1. A yeast hydrolysate is characterized in that polysaccharide components from yeast cell walls are less than or equal to 1%, total amino acids are more than or equal to 50%, polypeptide components are more than or equal to 35% and are more than or equal to 20%, nucleic acids are more than or equal to 10%, mineral components are more than or equal to 1%, and polysaccharide components from yeast cell walls are mannose and glucan.
2. The yeast hydrolysate of claim 1, wherein the total content of arginine, tryptophan and lysine in the total amino acids is not less than 20% by weight.
3. The yeast hydrolysate of claim 1, characterized in that it is obtained by hydrolysis using a complex enzyme comprising: candida rugosa lipase, trypsin, arginase, papain, nuclease, glutamine transaminase and beta-glucanase, wherein the specific gravity of the beta-glucanase in the complex enzyme is less than or equal to 10 weight percent; the carrier of the complex enzyme is MOFs, and the MOFs metal framework is selected from UiO-66, MIL-101 or ZIF-8.
4. A method for producing a yeast hydrolysate according to claim 1 to 3, comprising the steps of:
1) Culturing yeast:
feeding yeast into a yeast pool rich in culture solution, culturing at room temperature to obtain amplified yeast solution, and drying;
2) Loading a complex enzyme carrier:
washing MOFs metal frame material, removing impurities, suspending in ethanol solution or aqueous solution, dripping complex enzyme solution into MOFs suspension, stirring overnight, centrifuging, and drying to obtain the final product;
3) Obtaining hydrolysate:
adding proper amount of water into the saccharomycete obtained in the step 1) to prepare suspension with concentration of 10-15%, introducing the suspension into a yeast hydrolysis tank with MOFs metal frame loaded with compound enzyme, stabilizing the pH value between 6 and 6.5, heating to above 50 ℃, carrying out enzymolysis for 1-24 hours, carrying out ultrasonic vibration and filtration, heating filtrate to deactivate enzyme, concentrating and drying to obtain hydrolysate, discharging filter residues, drying by a roller, crushing by a crusher, and carrying out additional treatment.
5. Use of the yeast hydrolysate of claim 1 in the preparation of a health product.
6. Use of the yeast hydrolysate of claim 1 for the preparation of a pharmaceutical carrier.
7. A functional hydrogel comprising the yeast hydrolysate of any one of claims 1-3 and hyaluronic acid.
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