CN116983249A - Production process of molecules in microorganism protoplasm - Google Patents

Production process of molecules in microorganism protoplasm Download PDF

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CN116983249A
CN116983249A CN202310855141.0A CN202310855141A CN116983249A CN 116983249 A CN116983249 A CN 116983249A CN 202310855141 A CN202310855141 A CN 202310855141A CN 116983249 A CN116983249 A CN 116983249A
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substances
nanofiltration
protoplasm
microbial
microorganism
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凌丰
丁文勇
宋彦霞
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Beijing Vitality Blue Crystal Biotechnology Co ltd
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Beijing Vitality Blue Crystal Biotechnology Co ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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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    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
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    • A61K2800/805—Corresponding aspects not provided for by any of codes A61K2800/81 - A61K2800/95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/84—Products or compounds obtained by lyophilisation, freeze-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine

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Abstract

The invention discloses a production process of molecules in microorganism protoplasm, which takes microorganisms as raw materials, adopts a low-temperature wall breaking method to break walls of the microorganisms and simultaneously maintains the activity of substances in the protoplasm, adopts separation to remove solid substances in microorganism wall breaking liquid, adopts microfiltration to remove macromolecular substances in wall breaking liquid separation liquid, adopts nanofiltration to remove color, smell substances and micromolecular substances in microfiltration permeation liquid, adopts a resin adsorption column to remove residual color, smell substances and micromolecular substances in water-washed nanofiltration concentrate diluent, utilizes nanofiltration to concentrate the nanofiltration concentrate diluent after adsorption to obtain colorless, odorless and micromolecular substance-free microorganism protoplasm molecules, and maintains low temperature all the time in the whole production process and maintains the original biological activity of the molecules in the obtained microorganism protoplasm.

Description

Production process of molecules in microorganism protoplasm
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a production process of molecules in microorganism protoplasm.
Background
Protoplasm (Protoplasm) is not a compound, but a complex colloid composed of a plurality of compounds, and the colloid has continuous self-renewal capacity and becomes a system of living substances. In other words, a protoplast is a living substance that constitutes a cell.
Protoplasts are the material basis for cellular structure and vital activity. The chemical composition of the protoplast is extremely complex and constantly changing, but can be divided into two main categories, organic and inorganic.
The protoplasm is a generic term for various structures within the cell wall, and also a morphological structural unit constituting a cell, and various metabolic activities in living cells are performed therein. Protoplasts include cell membranes (cell membranes), cytoplasms (cytoplasms), and nuclei (nuclei), among others. The chemical components of the protoplasm are very complex, the components of the protoplasm also change along with the continuous metabolic activity of cells, the relative component proportion is 85% -90% of water, 7% -10% of protein, 1% -2% of lipid, 1% -1.5% of other organic matters (including nucleic acid) and 1% -1.5% of inorganic matters. Among them, protein (protein) and nucleic acid (nucleic acid) are the most important components related to vital activities.
Cytoplasm is a generic term for all translucent, gelatinous, particulate matter surrounded by the cytoplasmic membrane except for the nuclear region. The water content is about 80%. The major components of the cytoplasm are ribosomes, reservoirs, various enzymes and intermediary metabolites, various nutrients and monomers of macromolecules, etc., and the cytoplasm includes stroma, organelles and inclusions.
The cytoplasmic matrix (cytoplasmic matrix), also known as cytosol, is a homogeneous and translucent colloidal fraction of the cytoplasm, filled between other tangible structures, accounting for about 55% of the total volume of the cell, in which thousands of enzymes are present, most of the intermediary metabolism including glycolysis, gluconeogenesis and synthesis of sugars, fatty acids, nucleotides and amino acids is carried out in the cytoplasmic matrix. About 20% of the cytoplasmic matrix is protein, and the chemical composition of the cytoplasmic matrix can be divided into three classes, small, medium and large, according to their relative molecular mass sizes. Small molecules include water, inorganic salts, etc., medium molecules include lipids, carbohydrates, amino acids, nucleotides and their derivatives, etc., and large molecules include proteins, fats and lipoproteins, polysaccharides and RNAs, etc.
Organelles are structures distributed in the cytoplasm, have a morphology, and play an important role in physiological activities of cells. It comprises the following steps: mitochondria, endoplasmic reticulum, intranet apparatus, golgi apparatus, lysosomes, microfilaments, microtubules, centrosomes, ribosomes, etc., and the chemical components mainly comprise water, proteins and lipids, and in addition, contain small molecules such as coenzymes and nucleic acids in small amounts.
The inclusion is a structure in the cytoplasm that is not itself metabolically active, but has a specific morphology. Some are stored energy substances, such as glycogen particles, lipid droplets; some are cellular products such as secretory granules, melanin granules; the residue may also be considered as inclusion.
The substances in the nucleus are similar to those in the cytoplasm.
As can be seen from the above description of the substances contained in the microorganism's protoplasm, the soluble substances in the protoplasm mainly include proteins, lipids, saccharides, nucleic acids and inorganic substances.
Proteins include macromolecular proteins, small molecular proteins, polypeptides, oligopeptides and amino acids.
Lipids include large and small molecule fats and lipids, glycerol, fatty acids, and the like.
The saccharide includes polysaccharide, oligosaccharide, monosaccharide, etc.
From the above analysis of the soluble matters in the protoplasm, it is known that matters in the protoplasm are similar to the components of the matrix of the cytoplasm, and include macromolecular matters, medium molecular matters and small molecular matters.
The macromolecular substance includes: proteins, fats, lipoproteins, polysaccharides, RNA, and the like.
The medium molecular substances include: including oligopeptides, lipids, oligosaccharides, nucleotides, multivitamins, particularly B vitamins, derivatives thereof, and the like.
Small molecules include: water, inorganic salts, monosaccharides, amino acids, etc.
Their compositions are different and their effects are different, and if they are used to fully exert their effects, they are separated so that they can actually exert their intended effects, and the substances in the protoplasm include color and odor substances and inorganic salts, which affect the quality of cosmetics, they must be removed when applied to the cosmetic field.
The protoplasm molecular substances mainly comprise bioactive soluble middle molecular substances such as oligopeptides, lipids, oligosaccharides, nucleotides, various vitamins, particularly B vitamins and derivatives thereof, and the like, have great benefits on human health, have multiple functions on protecting and repairing human skin so as to treat the human skin, provide bioactive middle molecular nutrient substances for skin cells, promote the skin cells to be in an optimal growth state, ensure vigorous metabolism of the skin cells, and keep the skin in a healthy state, and have the main functions of moisturizing, resisting oxidization, tightening the skin, enhancing skin resistance, inhibiting melanin generation, whitening, improving the skin state, improving skin roughness and the like in cosmetics.
During the fermentation culture process of the microorganism, the microorganism protoplasm can contain yellow or yellow brown substances, substances with special odor and a large amount of inorganic salts, and if the traditional wall breaking technology such as autolysis (autolysis) and enzymolysis (enzymes hydrolysis) is adopted, the biological activity of molecular substances in the microorganism protoplasm can be destroyed.
The color causes pigmentation of skin and the smell causes unpleasant feeling of the produced cosmetics, so the color and smell seriously affect the quality of the produced cosmetics, the skin is stimulated by long-term contact with inorganic salt, especially the salt concentration is too high, the skin is corroded, skin allergy is caused, erythema, pimple, blister, seepage, itching and many other symptoms occur, once the biological activity of molecular substances in microorganism protoplasm is destroyed in the production process, the substances cannot exert the due effect, and the use effect is reduced, thus affecting the quality of the cosmetics, so if the purpose of applying the protoplasm molecules to the cosmetic field and exerting the maximum effect is achieved, the color and smell substances and inorganic salt in the protoplasm molecules must be removed and the biological activity of the molecular substances in the protoplasm is maintained, and the production process for achieving the effect is not mentioned in the existing patent and literature, how to solve the problems become the key of producing the molecular substances in protoplasm suitable for cosmetic application.
Therefore, a set of production process of the microorganism protoplasm molecules is found, which can remove small molecular substances, decolorize and deodorize while maintaining the biological activity of the substances in the microorganism protoplasm molecules, and plays a vital role in the application of the microorganism protoplasm molecules, especially in the application of the microorganism protoplasm molecules as cosmetic raw materials.
Disclosure of Invention
The invention aims to provide a production process for removing molecules in microorganism protoplasm for removing micromolecular substances, decoloring and deodorizing while maintaining the biological activity of substances in the microorganism protoplasm.
The invention aims at realizing the following technical scheme:
the production process of the molecules in the microorganism protoplasm specifically comprises the following steps:
(1) Adding water into the used microbial raw materials, uniformly mixing to obtain microbial raw material diluent, and then separating the obtained microbial raw material diluent by using a separator to remove a liquid part to obtain microbial solids;
(2) Repeating the step (1) to obtain a clean microbial raw material;
(3) Adding water into the clean microorganism raw materials, and uniformly mixing to obtain uniform clean microorganism mixed solution;
(4) Breaking the wall of the clean microorganism mixed solution to obtain a wall-broken solution of the microorganism mixed solution;
(5) Separating the wall-broken liquid by using a separator to remove solid parts, thereby obtaining wall-broken liquid separation liquid;
(6) Micro-filtering the obtained wall-broken liquid separation liquid by utilizing a micro-filtration membrane to remove macromolecular substances in the wall-broken liquid separation liquid, thereby obtaining micro-filtration permeation liquid;
(7) Removing color, odor substances and micromolecular substances in the microfiltration permeate by nanofiltration to obtain nanofiltration concentrated solution;
(8) Diluting the nanofiltration concentrated solution by adding water to obtain nanofiltration concentrated solution diluent;
(9) Filtering and concentrating the nanofiltration concentrated solution diluent again by nanofiltration;
(10) Repeating the steps (8) and (9), and further removing color, smell substances and micromolecular substances in the nanofiltration concentrated solution to obtain water-washed nanofiltration concentrated solution;
(11) Diluting the water-washed nanofiltration concentrated solution by adding water to obtain water-washed nanofiltration concentrated solution diluent;
(12) Removing residual color, odor substances and micromolecular substances in the water-washed nanofiltration concentrated solution diluent by adopting a resin adsorption column to obtain the nanofiltration concentrated solution diluent after adsorption;
(13) Concentrating the dilution of the nanofiltration concentrated solution after adsorption by nanofiltration to obtain colorless, odorless and micromolecular substance-free microorganism protoplasm medium molecules.
The microbial feedstock in step (1) includes a single-cell eukaryotic microorganism or a single-cell prokaryotic microorganism.
The dry matter content in the microbial solids described in step (1) is not less than 30% (w/w), calculated as weight percent of dry matter contained in the microbial solids.
The separators in the steps (1) and (5) can separate solid and liquid phases in the separated liquid, and the separators comprise a horizontal decanter centrifuge, a disc separator, a plate-and-frame filter press and the like.
The water adding amount in the step (3) is 0.5-5 times (w/w) of the microbial raw material.
The wall breaking process in the step (4) is a process performed at a low temperature, such as wall breaking using a homogenizer, or the like.
The aperture of the microfiltration membrane in the step (6) is 0.05-1 μm.
The macromolecular substances in the step (6) include proteins, lipoproteins, polysaccharides, RNA and the like.
The molecular weight cut-off of the nanofiltration membrane in the step (7) and the step (13) is 150Da to 1000Da.
The nanofiltration membrane in step (7) and step (13) may be replaced by an ultrafiltration membrane having a molecular weight cut-off of 1000-300000Da.
The small molecule substance in the step (7) includes inorganic salts and the like.
The resin in the step (12) is nonpolar adsorption resin, medium-polarity adsorption resin or polar adsorption resin which can remove color, odor substances and small molecular substances in the nanofiltration concentrated solution.
The whole process described in step (1) to step (13) is free from the addition of enzymes, chemicals, etc. which disrupt the biological activity of substances in the molecules in the protoplasts of microorganisms, and the temperature is maintained at not more than 45 ℃ in the whole process.
The microorganism protoplasm in the step (13) can be a product or can produce a plurality of products according to the molecular weight range.
The microorganism protoplasm molecule in the step (13) can be in the form of concentrated solution or freeze-dried powder as the final product.
The invention has the advantages and beneficial effects that:
1. the product (molecule in the microorganism protoplasm) produced by the invention is a brand new product, and no existing patent or literature describes the same product as the product produced by the invention.
2. The bioactive medium molecular substances such as oligopeptides, lipids, oligosaccharides, nucleotides, multivitamins, especially B vitamins and derivatives thereof and the like have various benefits on human health, have various functions on the protection and repair of human skin so as to treat the human skin, provide bioactive medium molecular nutrients for the skin cells, enable the skin cells to be in an optimal growth state, improve the vitality of the skin cells and ensure the vigorous metabolism of the skin cells, so that the skin can be kept in a healthy state.
3. The method adopts the steps (1) and (2), and achieves the purpose of thoroughly removing impurities carried in the microbial raw material by a method of repeatedly diluting and separating the microbial raw material, thereby obtaining clean microbial raw material and laying a good foundation for finally obtaining the molecules in clean microbial protoplasm.
4. The wall breaking process in the step (4) is a wall breaking process which can be performed at a low temperature, such as wall breaking by using a homogenizer, etc., and the low temperature wall breaking adopted by the invention does not damage the biological activity of the substances in the molecules in the protoplasm, thereby ensuring the biological activity of the substances in the molecules in the produced protoplasm, and only ensuring the activity of the substances in the protoplasm, thereby fully playing the functions of the substances in the protoplasm.
5. The method has the advantages that the enzyme which damages the biological activity of the substances in the molecules in the microbial protoplasm is not added in the whole process, a large amount of substances with biological activity are contained in the molecules in the microbial protoplasm, the biological activity of the substances in the molecules in the microbial protoplasm can be guaranteed only by keeping the biological activity of the substances in the molecules, the substances can play a role in application, and the biological activity of the substances in the molecules in the microbial protoplasm can be damaged by high temperature and addition of decomposition enzymes and chemical substances such as strong acid, strong alkali and the like.
6. The aperture of the microfiltration membrane in the step (6) is 0.05-1 mu m, so that the flexibility of a molecular product in the produced microorganism protoplasm is ensured, the size of the aperture of the microfiltration membrane determines the size of the molecular weight of the permeated microorganism protoplasm, and the molecular in the microorganism protoplasm with different molecular weights is produced by adjusting the aperture of the microfiltration membrane according to the requirement.
7. In the step (12), color, smell substances and small molecular substances which cannot be removed by a cleaning method in the dilution of the water-washed nanofiltration concentrated solution can be effectively removed by adopting a resin adsorption column, so that colorless, odorless and salt-free small molecular substances which affect the quality of cosmetics in the final product are ensured.
8. According to the invention, through the nanofiltration membranes in the step (7) and the step (13), in practical application, the nanofiltration membranes can be replaced by ultrafiltration membranes, so that all products covering the molecular weight in the microorganism protoplasm are produced, and the effects in cosmetics are different due to the fact that substances in the products are different, so that the respective effects of the products in different molecular weight ranges are fully exerted.
9. The method thoroughly removes the micromolecular substances, color and smell substances in the molecules in the protoplasm by the methods of the steps (7) - (13), thereby obtaining the pure, colorless, odorless and microorganism protoplasm molecules which have no micromolecular substances and keep the original biological activity.
Drawings
In order that the present invention may be more readily understood, a further detailed description of the invention is provided below with reference to the accompanying drawings, wherein FIG. 1 is a process flow diagram of the present invention.
Detailed Description
The invention will be further described by means of specific examples.
Example 1
A3 liter full-automatic fermenter with automatic control of temperature, pH and dissolved oxygen values (stirring speed and air intake) was used, with a 6-way automatic feed control system.
(1) 70g/L of sterilized sugar, 1g/L of peptone, 2g/L of yeast powder, 1.3g/L of urea, 0.0003g/L of cupric sulfate pentahydrate, 0.01g/L of zinc sulfate heptahydrate, 1.5g/L of dipotassium hydrogen phosphate, 3g/L of potassium dihydrogen phosphate and 0.3g/L of magnesium sulfate heptahydrate are added into a fermentation tank, and then ultrapure water is added to 2.5 liters to obtain a basic culture medium.
(2) Saccharomyces cerevisiae (dried fresh yeast) was used, and the inoculum size of the yeast was 10g/L.
(3) The fermentation tank was set to have an air intake of 1vvm (air intake per minute per volume of the biosynthesis tank), a stirring speed of 200 rpm, a temperature of 30℃and a pH of 6.5, and biosynthesis of yeast was started.
(4) After 2 hours of biosynthesis, 7ml/h of glucose solution (glucose content: 50%) and 0.6ml/h of yeast powder solution (yeast powder content: 20%) were fed, and 0.4ml/h of urea solution (urea content: 20%) were fed and stopped until 45 hours.
(5) The biosynthesis was completed for 50 hours, and 2.6L of a biosynthetic solution was obtained.
(6) 2.6L of biosynthetic liquid after the biosynthesis is finished is taken, the taken biosynthetic liquid is separated by a separator, the separator is set to 6000 revolutions per minute, the separation factor is 6439, the separation time is 20 minutes, 541g of wet yeast is obtained through separation, and the content of the wet yeast in the biosynthetic liquid is calculated to be 20.8% (w/v).
(7) 400g of wet yeast (dry matter content: 33%) was taken, 1200g of water was added, the separator was set to 4500 rpm, at which time the separation factor was 3622, and the supernatant was removed for 10 minutes to obtain a yeast solid at the bottom;
(8) Adding 1200g of water into yeast solid, stirring uniformly, setting a separator to 4500 r/min, separating for 10 min with a separation factor of 3622, and removing supernatant to obtain yeast solid at bottom;
(9) Repeating step (8) for 4 times to obtain 392g (dry matter content 32%) of clean yeast raw material;
(10) Adding 1000g of water into a clean yeast raw material, uniformly stirring to obtain a clean yeast mixed solution, then breaking the wall of the clean yeast mixed solution by using a homogenizer, wherein the pressure of the homogenizer is set to 120MPa, the flow is set to 48ml/min, and the circulation times are 5 times to obtain a yeast wall breaking solution;
(11) Separating the wall-broken liquid of the yeast, wherein the rotating speed of the separator is set at 5000 revolutions per minute, the separating factor is 4472, the separation is carried out for 10 minutes, and the solid part is removed, so as to obtain the wall-broken liquid separating liquid containing the protoplasm of the yeast;
(12) Microfiltration is carried out on the obtained wall-broken liquid separation liquid containing yeast protoplasm substances by utilizing a microfiltration membrane, the aperture of the microfiltration membrane is 0.2 mu m, and macromolecular substances in the wall-broken liquid separation liquid are removed by microfiltration to obtain 1234g of microfiltration permeate (the dry matter content is 7.7 percent);
(13) Nanofiltration is carried out on the microfiltration permeate by utilizing a nanofiltration membrane, the interception molecular weight of the nanofiltration membrane is 300Da, and color, odor substances and small molecular substances in the microfiltration permeate are removed by nanofiltration, so that 474g of nanofiltration concentrate (the dry matter content is 18.1 percent) is obtained;
(14) Diluting the nanofiltration concentrated solution by adding 1700g of water to obtain nanofiltration concentrated solution diluent;
(15) Filtering and concentrating the nanofiltration concentrated solution diluent again by using a nanofiltration membrane;
(16) Repeating the steps (14) and (15) to further remove color, odor substances and small molecular substances in the nanofiltration concentrated solution to obtain 461g (dry matter content is 17.8%) of water-washed nanofiltration concentrated solution;
(17) Diluting the water-washed nanofiltration concentrated solution by adding 800g of water to obtain water-washed nanofiltration concentrated solution diluent;
(18) Removing residual color, odor substances and small molecular substances in the water-washed nanofiltration concentrated solution diluent by using a resin adsorption column (macroporous adsorption resin is adopted for resin), so as to obtain 1231g (dry matter content is 6.5%) of the nanofiltration concentrated solution diluent after adsorption;
(19) The nanofiltration concentrate dilution after adsorption was concentrated by nanofiltration with a nanofiltration membrane cut-off of 300Da to obtain 428g (dry matter content of 18.2%) of colorless, odorless and small molecular mass free yeast protoplasm.
Molecular detection in yeast protoplasm:
measurement of chromaticity: the color tristimulus value and the chromatic aberration delta E of the general test method for cosmetics are adopted by the light industry standard QB/T2789-2006 of the people's republic of China * Measurement of (3), measurement results: average reading ΔE of two sample preparation measurements * 4.4, almost colorless.
Determination of smell: the sensory test shows that there is almost no special smell generated by yeast during fermentation culture.
Determination of inorganic salts: the detection result is 0.018S/m by using a conductivity meter (Lei Ci desk type conductivity meter DDS-307A), and the salt content of the molecule in the yeast protoplasm is less than 85ppm.
Example 2
Steps (1) - (6) of example 1 were repeated to obtain 553g of wet yeast.
(7) 400g of wet yeast (dry matter content: 33%) was taken, 1200g of water was added, the separator was set to 4500 rpm, at which time the separation factor was 3622, and the supernatant was removed for 10 minutes to obtain a yeast solid at the bottom;
(8) Adding 1200g of water into yeast solid, stirring uniformly, setting a separator to 4500 r/min, separating for 10 min with a separation factor of 3622, and removing supernatant to obtain yeast solid at bottom;
(9) Repeating step (8) for 4 times to obtain 394g (dry matter content 32%) of clean yeast raw material;
(10) Adding 1200g of water into a clean yeast raw material, uniformly stirring to obtain a clean yeast mixed solution, then breaking the wall of the clean yeast mixed solution by using a homogenizer, setting the pressure of the homogenizer to 125MPa, setting the flow to 48ml/min, and setting the circulation time to 5 times to obtain a yeast wall-broken solution;
(11) Separating the wall-broken liquid of the yeast, wherein the rotating speed of the separator is set at 5000 revolutions per minute, the separating factor is 4472, the separation is carried out for 10 minutes, and the solid part is removed, so as to obtain the wall-broken liquid separating liquid containing the protoplasm of the yeast;
(12) Microfiltration is carried out on the obtained wall-broken liquid separation liquid containing yeast protoplasm substances by utilizing a microfiltration membrane, the aperture of the microfiltration membrane is 0.5 mu m, and macromolecular substances in the wall-broken liquid separation liquid are removed by microfiltration to obtain 1471g (dry matter content is 7.0%) of microfiltration permeate;
(13) Nanofiltration is carried out on the microfiltration permeate by utilizing a nanofiltration membrane, the interception molecular weight of the nanofiltration membrane is 200Da, and color, odor substances and small molecular substances in the microfiltration permeate are removed by nanofiltration, so that 528g of nanofiltration concentrate (the dry matter content is 18.3%) is obtained;
(14) Adding 1300g of water to dilute the nanofiltration concentrated solution to obtain nanofiltration concentrated solution diluent;
(15) Filtering and concentrating the nanofiltration concentrated solution diluent again by using a nanofiltration membrane;
(16) Repeating the steps (14) and (15) to further remove color, odor substances and small molecular substances in the nanofiltration concentrated solution to obtain 514g (dry matter content of 18.2%) of water-washed nanofiltration concentrated solution;
(17) Adding 1200g of water to dilute the water-washed nanofiltration concentrated solution to obtain a water-washed nanofiltration concentrated solution diluent;
(18) Removing residual color, odor substances and small molecular substances in the water-washed nanofiltration concentrated solution diluent by using a resin adsorption column (macroporous adsorption resin is adopted for resin), so as to obtain 1684g (dry matter content is 5.4%) of the nanofiltration concentrated solution diluent after adsorption;
(19) The nanofiltration concentrate dilution after adsorption is concentrated by nanofiltration, the nanofiltration membrane has a molecular weight cut-off of 200Da, and 494g of molecular weight 494g (dry matter content of 18.0%) in yeast protoplasm which is colorless, odorless and free of small molecular substances is obtained.
Molecular detection in yeast protoplasm:
measurement of chromaticity: the color tristimulus value and the chromatic aberration delta E of the general test method for cosmetics are adopted by the light industry standard QB/T2789-2006 of the people's republic of China * Measurement of (3), measurement results: average reading ΔE of two sample preparation measurements * 4.8, almost colorless.
Determination of smell: the sensory test shows that there is almost no special smell generated by yeast during fermentation culture.
Determination of inorganic salts: the detection result is 0.02S/m by adopting a conductivity meter (Lei Ci desk type conductivity meter DDS-307A), and the salt content in the molecule in the yeast protoplasm is less than 95ppm.
Example 3
Steps (1) to (6) of example 1 were repeated to obtain 547g of wet yeast.
(7) 400g of wet yeast (dry matter content: 33%) was taken, 1200g of water was added, the separator was set to 4500 rpm, at which time the separation factor was 3622, and the supernatant was removed for 10 minutes to obtain a yeast solid at the bottom;
(8) Adding 1200g of water into yeast solid, stirring uniformly, setting a separator to 4500 r/min, separating for 10 min with a separation factor of 3622, and removing supernatant to obtain yeast solid at bottom;
(9) Repeating step (8) for 4 times to obtain 391g (dry matter content of 32%) of clean yeast raw material;
(10) 1600g of water is added into clean yeast raw materials, the mixture is stirred uniformly to obtain clean yeast mixed solution, then a homogenizer is used for breaking the wall of the clean yeast mixed solution, the pressure of the homogenizer is set to 125MPa, the flow is set to 48ml/min, and the circulation times are 5 times to obtain yeast wall breaking solution;
(11) Separating the wall-broken liquid of the yeast, wherein the rotating speed of the separator is set at 5000 revolutions per minute, the separating factor is 4472, the separation is carried out for 10 minutes, and the solid part is removed, so as to obtain the wall-broken liquid separating liquid containing the protoplasm of the yeast;
(12) Microfiltration is carried out on the obtained wall-broken liquid separation liquid containing yeast protoplasm substances by utilizing a microfiltration membrane, the aperture of the microfiltration membrane is 0.05 mu m, and macromolecular substances in the wall-broken liquid separation liquid are removed by microfiltration to obtain 1797g (dry matter content is 4.9 percent) of microfiltration permeate;
(13) Nanofiltration is carried out on the microfiltration permeate by utilizing a nanofiltration membrane, the interception molecular weight of the nanofiltration membrane is 500Da, and color, odor substances and small molecular substances in the microfiltration permeate are removed by nanofiltration, so that 405g of nanofiltration concentrate (the dry matter content is 17.8 percent) is obtained;
(14) Diluting the nanofiltration concentrated solution by adding 1600g of water to obtain nanofiltration concentrated solution diluent;
(15) Filtering and concentrating the nanofiltration concentrated solution diluent again by using a nanofiltration membrane;
(16) Repeating the steps (14) and (15) to further remove color, odor substances and small molecular substances in the nanofiltration concentrated solution to obtain 389g (dry matter content is 17.6%) of water-washed nanofiltration concentrated solution;
(17) Adding 1000g of water to dilute the water-washed nanofiltration concentrated solution to obtain a water-washed nanofiltration concentrated solution diluent;
(18) Removing residual color, odor substances and small molecular substances in the water-washed nanofiltration concentrated solution diluent by using a resin adsorption column (macroporous adsorption resin is adopted for resin), so as to obtain 1359g (dry matter content is 4.9%) of the nanofiltration concentrated solution diluent after adsorption;
(19) The nanofiltration concentrate dilution after adsorption is concentrated by nanofiltration, the nanofiltration membrane has a molecular weight cut-off of 500Da, and 364g (dry matter content of 17.9%) of colorless, odorless and micromolecular substance-free yeast protoplasm molecules are obtained.
Molecular detection in yeast protoplasm:
measurement of chromaticity: the color tristimulus value and the chromatic aberration delta E of the general test method for cosmetics are adopted by the light industry standard QB/T2789-2006 of the people's republic of China * Measurement of (3), measurement results: average reading ΔE of two sample preparation measurements * 3.8, almost colorless.
Determination of smell: the sensory test shows that there is almost no special smell generated by yeast during fermentation culture.
Determination of inorganic salts: the detection result is 0.017S/m by adopting a conductivity meter (Lei Ci desk type conductivity meter DDS-307A), and the salt content in the molecules in the yeast protoplasm is less than 80ppm.

Claims (16)

1. A process for the production of a molecule in a microbial plasma, characterized in that it comprises the following steps:
(1) Adding water into the used microbial raw materials, uniformly mixing to obtain microbial raw material diluent, and then separating the obtained microbial raw material diluent by using a separator to remove a liquid part to obtain microbial solids;
(2) Repeating the step (1) to obtain a clean microbial raw material;
(3) Adding water into the clean microorganism raw materials, and uniformly mixing to obtain uniform clean microorganism mixed solution;
(4) Breaking the wall of the clean microorganism mixed solution to obtain a wall-broken solution of the microorganism mixed solution;
(5) Separating the wall-broken liquid by using a separator to remove solid parts, thereby obtaining wall-broken liquid separation liquid;
(6) Micro-filtering the obtained wall-broken liquid separation liquid by utilizing a micro-filtration membrane to remove macromolecular substances in the wall-broken liquid separation liquid, thereby obtaining micro-filtration permeation liquid;
(7) Removing color, odor substances and micromolecular substances in the microfiltration permeate by nanofiltration to obtain nanofiltration concentrated solution;
(8) Diluting the nanofiltration concentrated solution by adding water to obtain nanofiltration concentrated solution diluent;
(9) Filtering and concentrating the nanofiltration concentrated solution diluent again by nanofiltration;
(10) Repeating the steps (8) and (9), and further removing color, smell substances and micromolecular substances in the nanofiltration concentrated solution to obtain water-washed nanofiltration concentrated solution;
(11) Diluting the water-washed nanofiltration concentrated solution by adding water to obtain water-washed nanofiltration concentrated solution diluent;
(12) Removing residual color, odor substances and micromolecular substances in the water-washed nanofiltration concentrated solution diluent by adopting a resin adsorption column to obtain the nanofiltration concentrated solution diluent after adsorption;
(13) Concentrating the dilution of the nanofiltration concentrated solution after adsorption by nanofiltration to obtain colorless, odorless and micromolecular substance-free microorganism protoplasm medium molecules.
2. The microorganism of step (1) of claim 1, wherein the microbial feedstock comprises a single-cell eukaryotic microorganism or a single-cell prokaryotic microorganism.
3. The microbial solid according to claim 1, wherein the dry matter content in the microbial solid is not less than 30% (w/w), calculated on the weight percentage of dry matter contained in the microbial solid.
4. The separator according to claim 1, wherein the separator is capable of separating solid and liquid phases in the separated liquid, and comprises a decanter centrifuge, a disc separator, a plate and frame filter press, and the like.
5. The method of claim 1, wherein the water is added to the clean microbial feedstock in step (3) in an amount of 0.5 to 5 times (w/w) the microbial feedstock.
6. The wall breaking process according to claim 1, wherein the wall breaking process is a process performed at a low temperature, such as wall breaking with a homogenizer, or the like.
7. The microfiltration according to claim 1, wherein the microfiltration membrane has a pore size of 0.05-1 μm.
8. The macromolecular substance according to claim 1, wherein said macromolecular substance comprises proteins, lipoproteins, polysaccharides, RNAs and the like.
9. The nanofiltration of step (7) and step (13) of claim 1, wherein the nanofiltration membrane has a molecular weight cut-off of 150Da to 1000Da.
10. Nanofiltration according to step (7) and step (13) of claim 1, wherein the nanofiltration membrane is replaced by an ultrafiltration membrane.
11. The ultrafiltration membrane of claim 10, wherein the ultrafiltration membrane has a molecular weight cut-off of 1000 to 300000Da.
12. The small molecule substance of claim 1, wherein said small molecule substance comprises an inorganic salt or the like.
13. The resin adsorption column according to claim 1, wherein the resin is a nonpolar adsorption resin, a medium-polar adsorption resin, a polar adsorption resin, or the like capable of removing color, odor substances, and small molecular substances from the nanofiltration concentrate.
14. The process according to claim 1, wherein the whole process is free from the addition of enzymes, chemicals, etc. which disrupt the biological activity of the substances in the molecules in the microorganism protoplasm and the temperature is maintained at no more than 45 ℃ in the whole process.
15. The microbial protoplasm molecule according to claim 1, wherein the microbial protoplasm molecule can be a product or can be produced according to different molecular weight intervals.
16. The microbial protoplasm molecule according to claim 1, wherein the microbial protoplasm molecule can be in a concentrated solution form as a final product, or can be freeze-dried to form a freeze-dried powder form as a final product.
CN202310855141.0A 2023-07-13 2023-07-13 Production process of molecules in microorganism protoplasm Pending CN116983249A (en)

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