CN109181843B - Device and method for on-line heating separation of microbial oil and microbial oil - Google Patents
Device and method for on-line heating separation of microbial oil and microbial oil Download PDFInfo
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- CN109181843B CN109181843B CN201811048702.1A CN201811048702A CN109181843B CN 109181843 B CN109181843 B CN 109181843B CN 201811048702 A CN201811048702 A CN 201811048702A CN 109181843 B CN109181843 B CN 109181843B
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
- C11B1/02—Pretreatment
- C11B1/025—Pretreatment by enzymes or microorganisms, living or dead
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B1/00—Production of fats or fatty oils from raw materials
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
- C11B3/00—Refining fats or fatty oils
- C11B3/16—Refining fats or fatty oils by mechanical means
Abstract
The invention relates to the field of extraction of microbial oil, and discloses a device and a method for on-line heating separation of microbial oil and microbial oil, wherein the device comprises: the system comprises a raw material tank, a heat exchanger, a steam generating device, a three-phase centrifuge and a water supply unit; the heat exchanger is respectively communicated with the raw material tank, the steam generating device, the water supply unit and the three-phase centrifuge. The method comprises the following steps: exchanging heat between water and steam to enable the effluent to have a first preset temperature, and enabling the effluent to enter a three-phase centrifuge to reach a second preset temperature; carrying out heat exchange on the pyrolysis liquid and steam to enable the effluent liquid to reach a third preset temperature; and separating in a preheated three-phase centrifuge to obtain microbial oil; wherein the lysate is a lysate of fermentation broth of oleaginous microorganisms. The invention increases the safety of the microbial oil, improves the yield of the microbial oil to 98-99%, obviously improves the quality such as peroxide value and the like in the crude oil, and reduces the pollution degree of pollutants such as trichloropropanol and the like.
Description
Technical Field
The invention relates to the field of microbial oil separation, in particular to a device for separating microbial oil by online heating, a method for separating microbial oil online and microbial oil extracted by the method.
Background
The microbial oil is prepared by biosynthesis and conversion of yeast, mould, bacteria, algae and other microbes under certain conditions by using carbohydrate, hydrocarbon and common oil as nitrogen source, carbon source, trace elements and the like.
At present, two extraction processes of wet method and dry method are mainly used for extracting the microbial oil. Because the dry extraction process has the problems of large oil oxidation loss, organic solvent extraction, high energy consumption and the like caused by high-temperature dehydration, more and more microbial oil extraction is carried out by adopting a wet extraction process. In the wet process, the primary process task is to break the cell wall by enzymolysis, mechanical crushing, thermal dissolution and other methods, and then separate the water by utilizing the specific gravity difference of oil and water to obtain the crude oil of the microbial oil. During the separation process, various problems have to be solved, such as: the oil emulsification causes the problem of difficult separation; the problem of water-phase grease residue; residue phase grease residue, thereby improving the extraction efficiency.
The conventional demulsification method comprises adding demulsification substances such as salt and other electrolytes, increasing the temperature of feed liquid, adding acid liquid and the like. The addition of electrolyte can cause the generation and pollution of 3-chloropropanol and other hazardous substances, and the generation of the pollutants is aggravated; the method for improving the temperature of the feed liquid is characterized in that a fermentation tank or an enzymolysis tank is generally adopted in the industry for heating, an inner coil pipe and an outer coil pipe are used for preheating to about 70 ℃, then steam is directly introduced for heating, the heating time is very long, and dozens of tons of enzymolysis liquid are integrally heated to the completion of centrifugal treatment for more than 10 hours, so that the oxidation of microbial oil which is dissociated from cell walls is aggravated by the long-time maintenance type heating demulsification mode, and the quality of the oil is poor and the loss is large; the acid added in the acid liquor demulsification is usually citric acid or phosphoric acid, so that the production cost is increased, the peroxide value is increased due to the maintenance of a certain demulsification time, and the oil quality is reduced.
In addition, the existing separation method has the defects that the residual oil content in the separated water phase and the residue phase is high, and the extraction efficiency is low.
Disclosure of Invention
The present invention has been made to overcome the above problems of the prior art, and an object of the present invention is to provide a microbial oil separator having a low degree of oxidation of microbial oil, a low residual oil content in the separated aqueous phase and the residual oil phase, and a high efficiency of oil extraction, a method for separating microbial oil using the same, and microbial oil obtained by the separation.
During the research process, the inventor of the present invention finds that the following heating methods are generally adopted in the prior art for increasing the temperature of the feed liquid: (1) the heating mode is a holding type heating mode, the heating is uneven, and the emulsification phenomenon is easy to generate due to the alternate mixing of cold and hot grease; (2) when the coil is heated, the temperature of the grease close to the coil is locally increased quickly, the temperature of the grease in other areas is low by heat transfer, and the grease in the state of uneven temperature is easy to emulsify under the stirring state; (3) the temperature rising mode of directly jetting high-temperature steam into the cracking liquid ensures that the nonuniformity of the grease temperature is more obvious and the emulsification phenomenon is more easily generated by the high-temperature steam; (4) the fermentation tank or the whole fermentation tank needs to be heated, the time consumption is long, and the oxidation of the grease is accelerated.
In order to accomplish the above objects, according to one aspect of the present invention, there is provided an apparatus for on-line instantaneous heating and separation of microbial oils and fats, the apparatus comprising:
the device comprises a feed liquid tank, a heat exchanger, a steam supply device, a three-phase centrifuge and a water supply unit;
the heat exchanger is respectively communicated with the feed liquid tank, the steam supply device, the water supply unit and the three-phase centrifuge;
wherein, the feed liquid tank is filled with lysate of oleaginous microorganism fermentation liquor.
Preferably, the heat exchanger is a plate heat exchanger or a tube heat exchanger.
The invention provides a separation method for on-line instantaneous heating and separation of microbial oil, which is characterized by comprising the following steps:
(1) preheating a system: the water supply unit continuously supplies water to the heat exchanger, the steam supply device continuously supplies steam to the heat exchanger so as to exchange heat between water flow and the steam in the heat exchanger, maintain the temperature of outlet water flow of the water at a first preset temperature, and then introduce the water into the three-phase centrifuge to preheat the three-phase centrifuge so as to enable the three-phase centrifuge to reach a second preset temperature;
(2) material-water switching: cutting off water supply of the water supply unit, continuously supplying cracking liquid to the heat exchanger by the liquid feed tank, continuously supplying steam to the heat exchanger by the steam supply device, exchanging heat between a cracking liquid material flow and the steam in the heat exchanger, and enabling the temperature of a liquid material flow of the cracking liquid to reach a third preset temperature; then introducing the lysate into a preheated three-phase centrifuge for separation of an oil phase, a water phase and a solid phase to obtain microbial oil;
wherein the lysate is a lysate of fermentation broth of oleaginous microorganisms.
Preferably, the lysate is an enzymatic hydrolysate of a fermentation broth of oleaginous microorganisms, and the preparation method of the enzymatic hydrolysate comprises the following steps: and (3) contacting the fermentation liquor of the oleaginous microorganism with cell wall lyase in an aseptic environment to carry out enzymolysis on the oleaginous microorganism in the fermentation liquor to obtain the enzymolysis liquid.
Preferably, the cell wall-lysing enzyme comprises an alkaline protease and optionally other enzymes, the other enzymes being at least one of cellulase, hemicellulase, pectinase, helicase, chitinase and ligninase.
Preferably, the conditions of the enzymatic hydrolysis include: pH 8-10, temperature 40-60 deg.C, pressure 0.02-0.05MPa, and ventilation amount 0.2-0.6VVM for 4-15 hr.
In a third aspect, the invention provides a microbial oil prepared by the method described above, wherein the microbial oil is a crude oil having a DHA (docosahexaenoic acid) content of greater than 35 wt% or an ARA (arachidonic acid) content of greater than 35 wt%, 3-chloropropanol < 350 μ g/kg, an anisidine value of less than 25, and a peroxide value of less than 20 meq/kg.
The invention only heats the effluent stream of the cracking solution in an on-line instantaneous heating temperature rise mode so as to meet the temperature requirement of centrifugal separation in the whole process, the heat efficiency of the heat exchanger is very high, the short heating time of the cracking solution is ensured, the cracking solution is heated uniformly, the heating temperature rise of the old whole tank of the cracking solution is avoided, the old whole tank of the cracking solution is kept under the high temperature condition for a long time, the speed and the degree of oxidation of the grease are effectively reduced, the production cost is reduced, and the quality of the finally harvested crude oil is better than that of the grease in the old centrifugal separation heating mode. The final yield reaches 98-99%, the quality indexes of the obtained crude oil are that the peroxide value is less than or equal to 20meq/kg, the anisidine value is less than or equal to 25, and the content of DHA and ARA is also improved. In addition, the separation method does not need to add substances such as a demulsifier, an electrolyte (for example, 3-chloropropanol), acid and the like into the cracking liquid, so that the safety of the product can be better ensured, and the cost is reduced.
Drawings
FIG. 1 is an apparatus for separating microbial oils and fats according to an embodiment of the present invention.
Description of the reference numerals
1 liquid feed tank 2 heat exchanger 3 steam supply device
4-three-phase centrifuge 5 water supply unit 6 first pipeline
7 second pipeline 8 third pipeline 9 temperature monitoring device
10 pressure gauge, 11 pressure pump and 12 visual cup
61 first branch line 62 second branch line
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
In a first aspect, as shown in fig. 1, the present invention provides an apparatus for on-line separation of microbial oils and fats, comprising:
the device comprises a feed liquid tank 1, a heat exchanger 2, a steam supply device 3, a three-phase centrifuge 4 and a water supply unit 5;
wherein the heat exchanger 2 is respectively communicated with the feed liquid tank 1, the steam supply device 3, the water supply unit 5 and the three-phase centrifuge 4;
wherein, the feed liquid tank 1 is a lysate of oleaginous microorganism fermentation liquor.
According to the present invention, the connection mode of the feed liquid tank 1, the heat exchanger 2, the steam supply device 3, the three-phase centrifuge 4, and the water supply unit 5 is not particularly limited in the present invention as long as the heat exchanger 2 can be ensured to communicate with the feed liquid tank 1, the steam supply device 3, the water supply unit 5, and the three-phase centrifuge 4, respectively. According to a preferred embodiment of the present invention, the heat exchanger 2 is communicated with the feed tank 1 and the water supply unit 5 through a first pipeline 6 (i.e. the feed tank 1 and the water supply unit 5 are both connected to a branch pipeline of the first pipeline, preferably, the water supply unit 5 is connected to the first pipeline through a first branch pipeline 61, the feed tank 1 is connected to the first pipeline through a second branch pipeline 62), is communicated with the steam supply device 3 through a second pipeline 7, and is communicated with the three-phase centrifuge 4 through a third pipeline 8. Wherein, in order to facilitate the opening and closing of the material flow and the flow rate of the material flow, valves for controlling the material flow are also arranged on each pipeline, and the material flow is controlled by the opening and closing of the valves. In this preferred manner, the water supply unit 5 can be switched off by first opening the valve on the first branch line 61 and the valve on the second line 7, so that the water provided by the water supply unit 5 exchanges heat with the steam provided by the steam supply device 3 in the heat exchanger 2, after the water flow of the effluent reaches the preset temperature, the valve on the third pipeline 8 is opened, water is introduced into the three-phase centrifuge 4 to preheat the three-phase centrifuge, after the preheating is finished, the valves on the first branch line 61 and the third branch line 8 of the water supply unit 5 are closed, and a valve on a second branch pipeline 62 of the feed liquid tank 1 is opened, the cracking liquid flows into the heat exchanger 2 from the valve to exchange heat with the steam provided by the steam supply device 3, after the discharged material flow of the cracking liquid reaches a preset temperature, and opening the valve on the third pipeline 8 again, and introducing the lysate into the three-phase centrifuge 4 for separating the microbial oil.
According to the present invention, in order to facilitate the switching of the feed water, it is preferable that the first branch conduit 61 and the water supply unit 5 are connected through a distribution station, and the second branch conduit 62 and the feed liquid tank are also connected through the distribution station, and the distribution station distributes water and the lysis solution to the respective conduits.
According to a preferred embodiment of the invention, after the feed water is switched, the method further comprises the step of draining the water in the pipeline through the distribution station so as not to influence the quality of the lysis solution. To facilitate observation of the water emptying, the station is also preferably associated with a sight cup 12, the discharge being stopped when a change in colour of the liquid flowing through the sight cup 12 is observed. Wherein, the discharged water can be directly discharged as sewage and can also be used for other purposes.
According to the present invention, it is preferable that the water pressure of the water supply unit 5 supplying water to the heat exchanger 2 is 0.1 to 0.2 MPa. The water supply unit 5 may be a tap water pipe or a water storage container. When the water supply unit 5 is a tap water pipe, the flow rate of tap water can be controlled by adjusting the opening degree of the valve to control the water pressure, and when the water supply unit is a water storage container, a pressure pump can be arranged on the first branch pipeline 61 to adjust the water pressure.
According to the invention, in order to further effectively control the heating temperature of the cracking liquid, the second branch pipeline 62 is also provided with the pressure pump 11, and the control of the cracking liquid material flow is realized through the adjustment of the pump pressure of the pressure pump 11, so that the accurate control of the temperature is realized. According to a preferred embodiment of the invention, the pump pressure is preferably such that the pressure of the lysate stream entering heat exchanger 2 is between 0.1 and 0.2 MPa. The pressure pump 11 may be a rotary pump.
Thus, preferably, a pressure gauge 10 is also provided on the first pipe 6.
According to a preferred embodiment of the present invention, in order to improve the heat exchange efficiency, the heat exchanger 2 is a plate heat exchanger or a tube heat exchanger.
According to a preferred embodiment of the present invention, in order to facilitate monitoring the temperature of the material flow entering the three-phase centrifuge 4, a temperature monitoring device 9 is further disposed on a pipeline of the heat exchanger 2 communicating with the three-phase centrifuge 4, wherein the temperature monitoring device 9 is disposed near one end of the heat exchanger 2, and more preferably disposed at the material flow outlet of the heat exchanger 2. Wherein the temperature monitoring device 9 may be a thermometer.
According to the invention, the feed liquid tank 1 can be any feed liquid tank filled with lysate of oleaginous microorganism fermentation broth, for example, an enzymolysis tank or a fermentation tank.
In a second aspect, the present invention provides a method for separating microbial oil by on-line instantaneous heating, the method comprising:
(1) preheating a system: the water supply unit continuously supplies water to the heat exchanger, the steam supply device continuously supplies steam to the heat exchanger so as to exchange heat between water flow and the steam in the heat exchanger, maintain the temperature of outlet water flow of the water at a first preset temperature, and then introduce the water into the three-phase centrifuge to preheat the three-phase centrifuge so as to enable the three-phase centrifuge to reach a second preset temperature;
(2) material-water switching: cutting off water supply of the water supply unit, continuously supplying cracking liquid to the heat exchanger by the liquid feed tank, continuously supplying steam to the heat exchanger by the steam supply device, exchanging heat between a cracking liquid material flow and the steam in the heat exchanger, and enabling the temperature of a liquid material flow of the cracking liquid to reach a third preset temperature; then introducing the lysate into a preheated three-phase centrifuge for separation of an oil phase, a water phase and a solid phase to obtain microbial oil;
wherein the lysate is a lysate of fermentation broth of oleaginous microorganisms.
According to the present invention, the method for on-line separation of microbial oil is carried out in the apparatus for on-line separation of microbial oil as described above. According to the invention, the whole tank of enzymolysis liquid is prevented from being heated by entering the heat exchanger in the form of the enzymolysis liquid material flow to be instantly heated to the preset temperature on line, so that the enzymolysis liquid is exposed to high temperature for too long time, and the enzymolysis liquid is heated in a flowing and variable manner in the form of the material flow, so that the enzymolysis liquid is heated more uniformly. In addition, the heating of demulsifier, electrolyte, acid and other substances is saved, and the finally obtained crude oil is free from pollution. Wherein, in order to further improve the efficiency of heating, the heat exchanger is a plate heat exchanger or a tubular heat exchanger.
Preferably, the first predetermined temperature may be 80-95 ℃.
Preferably, the second predetermined temperature may be 80-95 ℃.
Preferably, the third predetermined temperature may be 80-95 ℃.
According to the invention, the water supply unit supplies water to the heat exchanger continuously by delivering water to the distribution station and through the first branch line 61 and the first line 6; the water supply pressure is preferably 0.1-0.2 MPa;
according to the invention, the pyrolysis liquid from the feed liquid tank enters the replacement tap water from the distribution station, and is continuously supplied to the heat exchanger through the second branch pipeline 62; the hydraulic pressure of the liquid supply of the cracking liquid to the heat exchanger is preferably 0.1-0.2 MPa;
according to the invention, it is preferred that after the water feed has been switched, the method further comprises discharging the water in the distribution station, the first branch line 61 and the first line 6. Because the color phase difference of water and feed liquid is great, can observe whether color change water drains through the sight cup 12 that sets up in distribution station blow off pipe department.
According to the invention, after the feed water is switched, the method also comprises discharging hot water used for preheating in the three-phase centrifuge.
According to the invention, the time of the centrifugation can be 15 to 20 hours.
According to the invention, before the device starts to operate, the device also comprises a water leakage check device for checking the heat exchanger before the heat exchange is effectively carried out, for example, water can be supplied to the heat exchanger through a water supply unit to check whether the heat exchanger leaks water.
According to the present invention, preferably, when the preheated water in step (1) enters the three-phase centrifuge, the three-phase centrifuge is in a full speed starting state, and the rotation speed thereof can be 5000-.
According to the present invention, preferably, when the pyrolysis solution preheated in step (2) enters the three-phase centrifuge, the three-phase centrifuge is in a full speed start state, and the rotation speed thereof can be 8000rpm of 5000-.
According to the present invention, the lysate may be obtained by breaking walls of microorganisms in an oleaginous microorganism fermentation broth by various methods conventionally used in the art, for example, a product of breaking walls by enzymatic hydrolysis, mechanical disruption, thermal dissolution, and the like.
In the process of wall breaking by means of enzymolysis, the inventor of the present invention finds that the main reasons of low microbial oil extraction efficiency, high anisidine value and peroxide value, and low DHA or ARA content are that in the current process, substances such as an enzyme preparation, a pH regulator, etc. need to be introduced into a fermentation concentrated solution, and the substances such as the enzyme preparation, the pH regulator, etc. are usually directly added into the concentrated fermentation solution after preparation, so that pollution of foreign bacteria to the fermentation solution is caused.
Generally, the whole process takes more than 20 hours from the adjustment of pH, the preparation and the addition of an enzyme preparation, the implementation of enzymolysis and the subsequent heating treatment. The propagation speed of the bacterial microorganisms is very high, the bacterial microorganisms generally propagate one generation every 20 minutes, and the bacterial propagation amount in the fermentation liquor rich in the carbonitrogen is very large after more than 20 hours in the whole process of extracting the grease. Meanwhile, although a certain amount of microorganisms can be killed by later-stage heating enzymolysis, bacteria and other microorganisms secrete a large amount of spore exotoxin in the process, the live bodies of the bacteria microorganisms can only be killed by thermal heating, the spore exotoxin is a protein, the action of the spore exotoxin is limited by heat, and the spore exotoxin enters grease as a protein fragment or a peptide and becomes a factor influencing the safety of the grease. In addition, due to the existence of a large amount of foreign bacteria, competition can be formed with the oleaginous microorganisms in the treatment process, so that the treatment effect on the oleaginous microorganisms is weakened, intracellular substances of the foreign bacteria enter the obtained microbial oil in a large amount, the extraction efficiency of the microbial oil is low, and the content of main components such as DHA, ARA and the like is reduced. In addition, because of the infection and mass propagation of bacteria, the fermentation liquor generates foul smell in the treatment process, and the production environment is influenced.
Therefore, preferably, the lysate is an enzymatic hydrolysate of a fermentation broth of an oleaginous microorganism, and the preparation method of the enzymatic hydrolysate comprises: and (3) contacting the fermentation liquor of the oleaginous microorganism with cell wall lyase in an aseptic environment to carry out enzymolysis on the oleaginous microorganism in the fermentation liquor to obtain the enzymolysis liquid.
In the present invention, it should be noted that the sterile environment refers to the environment in which the enzymatic system is sterile except the oleaginous microorganisms contained in the fermentation broth.
According to the invention, the fermentation broth of the oleaginous microorganism is directly obtained after fermentation of the oleaginous microorganism, and does not need any treatment, so that under the preferable condition, compared with the prior art, the method disclosed by the invention also saves the steps of concentration of the fermentation broth, inactivation of the oleaginous microorganism and the like.
According to the present invention, a method for producing a fermentation broth of the oleaginous microorganism is well known to those skilled in the art, for example, an oleaginous microorganism is inoculated into a fermentation sugar solution to perform fermentation, thereby obtaining a fermentation broth of the oleaginous microorganism.
The oleaginous microorganism can be any of various existing oleaginous microorganisms, such as bacteria, molds, yeasts and algae, and preferably any of the molds, yeasts and algae. Among them, examples of the mold may include, but are not limited to, terrae (Asoergullus terreus), Clavicepins purpurea (Claviceps purpurea), Ustilago kawakamii (Tolypospora), Mortierella alpina (Mortierella alpina), and Mortierella pusilla (Mortierella isabellina); examples of the yeast may include, but are not limited to, Cryptococcus albidus (Cryptococcus albicus), Cryptococcus curvatus (Cryptococcus albicus), Lipomyces starchya (Lipomyces), saccharomyces pullulans (trichospirans), Lipomyces oleans (liposlpofer), Rhodotorula mucilaginosa (Rhodotorula glutinis), and Rhodotorula toruloides (Rhodotorula toruloides); examples of the algae may include, but are not limited to, Thraustochytriales (Thraustochytriales), Schizochytrium (Schizochytrium), Crypthecodinium (Crypthecodinium), diatoms (diatom), Spirulina (Spirulina), and wurkinje.
It is well known to those skilled in the art that (1) the extracellular matrix of animals is in a sense, i.e., the cell wall, and its chemical composition is collagen, fibronectin, aminopolysaccharide and proteoglycan. (2) The main component of the bacterial cell wall is peptidoglycan. (3) The main components of the fungal cell wall are chitin, cellulose, glucan, mannan and the like, and the polysaccharides are polymers of monosaccharides. (4) The plant cell wall is mainly cellulose, hemicellulose and pectin, and a large amount of lignin is also contained in the secondary cell wall. Generally, oleaginous microorganisms are bacteria, fungi, yeasts and algae, and thus, enzymatic hydrolysis of the cell wall of oleaginous microorganisms is currently limited to cellulase, hemicellulase, pectinase, helicase, chitinase, ligninase, and the like. However, in the process of breaking the cell wall of the oil-producing microorganism by using the currently adopted enzyme preparation, the wall breaking performance of the enzyme preparation is unstable, and the performance of the enzyme preparation fluctuates greatly no matter the enzyme is liquid or solid, so that the production operation is difficult to control. The inventor of the invention unexpectedly finds that by introducing alkaline protease into an enzyme preparation, not only can excellent enzymolysis efficiency be ensured, but also the enzymolysis process can be stably carried out, so that the extraction efficiency of microbial oil is further improved, the anisidine value and the peroxide value are reduced, and the content of DHA or ARA is improved.
Thus, according to a preferred embodiment of the invention, the cell wall cleaving enzyme comprises an alkaline protease. Further, preferably, the cell wall-lysing enzyme may further include at least one of cellulase, hemicellulase, pectinase, helicase, chitinase, and ligninase.
According to the present invention, the amount of the cell wall-cleaving enzyme may be selected within a wide range as long as the cell wall of the oleaginous microorganism is sufficiently cleaved to release the microbial oil. Preferably, the cell wall-lysing enzyme is used in an amount of 1 to 5g per liter of said fermentation broth.
According to the invention, the condition of enzymolysis can be the condition which is conventionally used for producing the oil microbial cell wall by enzymolysis and cracking, but the inventor of the invention finds that the enzymolysis efficiency can be further improved by carrying out the enzymolysis under the aerobic condition, so that the extraction efficiency of the microbial oil is improved, and the DHA or ARA of the obtained microbial oil is improved. Therefore, preferably, the conditions of the enzymatic hydrolysis include: the pH value is 8-10 (for example, 8, 8.5, 9, 9.5, 10), the temperature is 40-60 ℃ (for example, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃) and the pressure is 0.02-0.05MPa (for example, 0.02MPa, 0.03MPa, 0.04MPa, 0.05MPa), the ventilation amount is 0.2-0.6VVM (the amount of gas introduced per unit volume of fermentation broth per minute is 0.2-0.6 volume) (for example, 0.2VVM, 0.3VVM, 0.4VVM, 0.5VVM, 0.6VVM), and the time is 4-15 hours (for example, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours).
According to the present invention, in order to further improve the contact of the enzyme with the oleaginous microorganism, thereby the efficiency of the enzymatic hydrolysis, the enzymatic hydrolysis is carried out under stirring conditions, and the stirring speed may be 8-30 rpm.
Wherein, the pH value of the enzymolysis system can be controlled between 8 and 10 by adding alkali liquor into the fermentation liquor. The selection of the alkaline solution in the present invention is not particularly limited as long as the pH adjustment can be accomplished and the side reaction with the produced microbial oil does not occur. Examples of the alkali solution may include, but are not limited to, at least one of a sodium oxide solution, a potassium hydroxide solution, a sodium carbonate solution, a sodium bicarbonate solution, a potassium carbonate solution, a potassium bicarbonate solution, and ammonia water. The concentration of the alkali solution is not particularly limited in the present invention as long as the pH adjustment can be achieved, and for example, the concentration of the alkali solution may be 15 to 25 wt%.
Preferably, in order to ensure the safety of the finally obtained microbial oil, the alkali liquor is food-grade alkali liquor.
According to the invention, the enzymatic hydrolysis can be directly carried out in a fermentation tank, in order to ensure the sterile environment of the system, the alkali liquor is preferably sterilized and then conveyed to the fermentation tank through a sterile pipeline, and the cell wall lyase is prepared into a solution with the enzyme activity, and then is sterilized and then conveyed to the fermentation tank through the sterile pipeline.
The method for sterilizing the alkali solution and the pipeline for delivering the alkali solution and the cell wall lytic enzyme can be various sterilization methods known in the art, for example, a filtration method, an ozone sterilization method, and a high-temperature saturated steam method. The invention preferably sterilizes the alkali liquor and the pipeline for conveying the alkali liquor and the cell wall lyase by a high-temperature saturated steam method. The pressure of the high-temperature saturated steam can be 0.1-0.35MPa, the temperature can be 121-145 ℃, and the sterilization time can be 40-80 min.
In order to ensure the activity of the cell wall-cleaving enzyme, the conventional aseptic filtration method of the present invention may be employed for the cell wall-cleaving enzyme, and for example, the filtration sterilization may be performed by using a filtration system, and the pore size of the aseptic liquid filter of the filtration system may be 0.15 to 0.25. mu.m. Preferably, the filtration system further comprises a step of sterilizing the filtration system before use, wherein the sterilization method can be a method of ozone sterilization, a method of high temperature saturated steam, and the like known in the art. The present invention preferably sterilizes the filtration system by means of high temperature saturated steam. The pressure of the high-temperature saturated steam can be 0.1-0.35MPa, the temperature can be 121-145 ℃, and the sterilization time can be 40-80 min.
According to the invention, the enzymolysis can also be carried out in an enzymolysis tank, in order to ensure the sterile environment of the system, the alkali liquor is preferably sterilized and then conveyed to the enzymolysis tank through a sterile pipeline, the cell wall lyase is prepared into a solution with the enzyme activity, then the solution is sterilized through a sterile liquid filter and then conveyed to the enzymolysis tank through a sterile pipeline, and the fermentation liquor is conveyed to the enzymolysis tank through a sterile pipeline.
The method for sterilizing the alkali liquor and the pipeline for conveying the alkali liquor, the cell wall lytic enzyme and the fermentation liquor can be various sterilization methods known in the art, for example, a filtration method, an ozone disinfection method and a high-temperature saturated steam method can be used. The invention preferably sterilizes the alkali liquor and pipelines for conveying the alkali liquor, the cell wall lyase and the fermentation liquor by a high-temperature saturated steam method. The pressure of the high-temperature saturated steam can be 0.1-0.35MPa, the temperature can be 121-145 ℃, and the sterilization time can be 40-80 min.
In order to ensure the activity of the cell wall-cleaving enzyme, the conventional room temperature sterilization method of the present invention may be used for the cell wall-cleaving enzyme, and for example, the filtration sterilization may be performed by using a filtration system, and the pore size of the liquid aseptic filter of the filtration system may be 0.15 to 0.25 μm. Preferably, the filtration system further comprises a step of sterilizing the filtration system before use, wherein the sterilization method can be a method of ozone sterilization, a method of high temperature saturated steam, and the like known in the art. The present invention preferably sterilizes the filtration system by means of high temperature saturated steam. The pressure of the high-temperature saturated steam can be 0.1-0.35MPa, the temperature can be 121-145 ℃, and the sterilization time can be 40-80 min.
A specific embodiment of the present invention will now be described in detail with reference to fig. 1, in particular,
(1) preparing alkali liquor: adding a certain amount of water into an alkali tank, weighing NaOH according to the concentration of 15-25 wt% and putting the NaOH into the alkali tank for dissolving, placing the prepared alkali liquor into high-temperature saturated steam for sterilization for 1h, then introducing sterile compressed air into the alkali tank to ensure that the tank pressure of the alkali tank is 0.02-0.05MPa, and introducing cooling water into a jacket of the alkali tank to cool the alkali liquor to 35-45 ℃ for later use.
(2) Cell wall lysate preparation: mixing the cell wall lysate with a quantity of water, according to the enzyme: preparing water according to the proportion of 1 to (5-10), sterilizing the sterile filtration system by using high-temperature saturated steam for 1 hour, and then maintaining the pressure by using sterile compressed air for standby.
(3) Adding an enzymolysis liquid and an alkali liquor: the temperature of the fermentation liquor is raised to 35-55 ℃, and then the alkali liquor is pressed into the fermentation tank through a sterile pipeline, so that the pH value of the fermentation liquor is 8-10; the enzymolysis liquid is conveyed into a sterile filtration system through a diaphragm pump for filtration, and then conveyed into a fermentation tank through a sterile pipeline for enzymolysis of the fermentation liquid, wherein the whole enzymolysis time is 4-15 h.
(4) Preheating preparation of a plate heat exchanger: firstly, supplying water into the plate heat exchanger 2 by a water supply unit 5 to ensure that the sealing condition of plates of the plate heat exchanger 2 is good, then slowly introducing steam into the plate heat exchanger by a steam supply device 3 to exchange heat with water, so that the temperature of outlet water of the heat exchanger 2 is slowly increased, then gradually increasing the supply amount of the steam, and ensuring that the temperature of the outlet water is 80-95 ℃ after heat exchange;
(5) preheating preparation of a centrifuge: introducing hot water of 80-95 ℃ into a three-phase centrifuge 4 to ensure that the temperature of a rotary drum body of the centrifuge reaches 80-95 ℃;
(6) feeding and centrifugal treatment: and (3) switching the water in the feed pipeline of the plate heat exchanger 2 into the enzymolysis liquid, and when the temperature of the enzymolysis liquid reaches 80-95 ℃, conveying the enzymolysis liquid to a three-phase centrifuge 4 to start centrifugal treatment, wherein the centrifugation process is performed.
In a second aspect, the invention provides a microbial oil prepared by the above method, wherein the microbial oil is crude oil, and the crude oil has a DHA content of more than 35 wt% or an ARA content of more than 35 wt%, 3-chloropropanol of less than 350 μ g/kg, an anisidine value of less than 25, and a peroxide value of less than 20 meq/kg.
The present invention will be described in detail below by way of examples. In the following examples of the present invention,
alkaline protease was purchased from danisc, PD 216661-7.0 CHN;
pectinase was purchased from bioscience, ltd, donnghenhuadao, P128776;
the cellulase is purchased from Jiangsu Hao Zeng Biotechnology Co., Ltd, with the trade name of 232-;
the oleaginous microorganism fermentation liquor 1 is fermentation liquor obtained by fermenting alpine saturated mold, and mainly contains ARA;
the oil-producing microorganism fermentation liquor 2 is fermentation liquor obtained by fermenting schizochytrium and mainly contains DHA;
determining the DHA content of the obtained crude oil by gas chromatography according to a GB26400-2011 method;
the EPA content is determined by gas chromatography according to the method of GB 5009.168-2016;
the ARA content is measured by gas chromatography according to the GB26401-2011 method;
measuring the content of 3-chloropropanol by high performance liquid chromatography;
the anisidine value was determined by the method GB/T24304-2009;
measuring the peroxide value by an ultraviolet spectrophotometer according to a GB/T24304-2009 method;
the yield of the microbial oil is measured by a rotary evaporator extraction weighing method;
the residual oil rate in the solid phase residue is determined by a rotary evaporator extraction weighing method.
An apparatus for separating microbial oils and fats as shown in fig. 1, comprising: a feed liquid tank 1 (fermentation tank), a heat exchanger 2 (plate heat exchanger), a steam supply device 3, a three-phase centrifuge 4 (purchased from Jiangsu Yixing Huading vessel grain and oil machinery Co., Ltd., a cargo number BTSD95), and a water supply unit 5 (tap water pipe); the heat exchanger 2 is respectively communicated with the feed liquid tank 1 and the water supply unit 5 through a first pipeline 6, communicated with the steam supply device 3 through a second pipeline 7 and communicated with the three-phase centrifuge 4 through a third pipeline 8; a return pipeline 9 is also arranged between the feed liquid tank 1 and the heat exchanger 2; a temperature monitoring device 10 is arranged at the discharge port of the heat exchanger 2. The water supply unit 5 is connected to the first line 6 through a distribution station and a first branch line 61; the tank 1 is connected to the first line (6) by the distribution station and a second branch line 62; the second branch pipeline 62 is provided with a pressure pump 11; a pressure gauge 10 is arranged on the first pipeline 6; a sight cup 12 is also connected to the station.
Example 1
This example illustrates the microbial oils and methods of preparation thereof provided by the present invention
(1) Preparing alkali liquor: adding 200L of softened water into an alkali tank, weighing food-grade NaOH according to the concentration of 20 wt%, putting the food-grade NaOH into the alkali tank for full dissolution, placing the prepared alkali liquor into saturated steam with the pressure of 0.14MPa and the temperature of 145 ℃ for sterilization for 1h, introducing sterile compressed air into the alkali tank to ensure that the pressure of the alkali tank is 0.02-0.05MPa, and introducing cooling water into a jacket of the alkali tank to cool the alkali liquor to 35-45 ℃ for later use;
(2) cell wall lysate preparation: sterilizing the serially connected two-stage liquid sterile filtration system with saturated steam at the pressure of 0.14MPa and the temperature of 145 ℃ for 1h, and then maintaining the pressure with sterile compressed air for later use; the filter specification of the sterile filtration system is aperture 0.2 μm, and the sterile filtration system can resist temperature of 150 ℃; weighing alkaline protease, adding softened water according to the weight ratio of the alkaline protease to the alkaline protease: preparing the enzymolysis liquid according to the water ratio of 1: 10.
(3) Adding an enzymolysis liquid and an alkali liquor: the temperature of the oil-producing microorganism fermentation liquor 1 is raised to 35-55 ℃, then the alkali liquor is pressed into a fermentation tank by using compressed air through an aseptic alkali liquor pipeline, the pH of the fermentation liquor is measured by using a pH probe of 'Mettler', and the addition of the alkali liquor is stopped until the pH of the fermentation liquor is 9; the enzymolysis liquid is conveyed into a secondary liquid sterile filtering system which is connected in series through a diaphragm pump for filtering, and then conveyed into a fermentation tank through a sterile pipeline, wherein the addition amount of the enzyme is 3g relative to each liter of fermentation liquid, and the enzymolysis of the fermentation liquid is carried out; in the enzymolysis process, the temperature is controlled to be 55 ℃, the pH is controlled to be 9, the stirring speed is controlled to be 20rpm, the aeration quantity is controlled to be 0.45vvm, the tank pressure is controlled to be 0.03MPa, and the whole enzymolysis time is 8 hours.
(4) Preheating preparation of the plate heat exchanger 5: opening a valve between a tap water pipe and the plate heat exchanger, wherein the tap water pipe supplies water to the plate heat exchanger through a distribution station, the water supply pressure is 0.1-0.2MPa, the sealing condition of a plate of the plate heat exchanger is checked through whether water leakage exists, after the sealing condition of the plate is determined to be good, the valve between the plate heat exchanger and a steam supply device is slowly opened, steam is supplied to the plate heat exchanger through the steam supply device, heat exchange is carried out between the steam and water in the plate heat exchanger, the temperature of the water is detected through a temperature monitoring device, and the water temperature is;
(5) preheating preparation of a three-phase centrifuge: starting the three-phase centrifuge to cool water, ensuring smooth water flow at a water outlet, simultaneously starting the three-phase centrifuge 13, ensuring that the starting current is 80A, starting a valve between the plate heat exchanger and the three-phase centrifuge after the speed of a full-speed indicator panel is 6700rpm, and allowing hot water at 90 ℃ to enter the three-phase centrifuge to ensure that the temperature of a rotary drum of the three-phase centrifuge reaches 90 ℃;
(6) feeding and centrifugal treatment: and (3) opening a valve between the fermentation tank and the plate heat exchanger, closing the valve between the plate heat exchanger and a tap water pipe, switching tap water in the first pipeline into the enzymolysis liquid obtained in the step (4) through the distribution station, opening a drain valve of the distribution station to drain the tap water in the pipeline, stopping draining when a color change is observed through a visual cup, conveying the enzymolysis liquid reaching 90 ℃ to a three-phase centrifuge when the temperature of the enzymolysis liquid entering the heat exchanger for heat exchange reaches 90 ℃, performing centrifugal treatment for 18h to obtain microbial oil serving as crude oil, wherein the extraction efficiency of the oil, the residual oil rate in solid-phase residue, the ARA content, the EPA content, the 3 chloropropanol content, the anisidine value and the peroxide value in the oil are shown in table 1.
Example 2
This example illustrates the microbial oils and methods of preparation thereof provided by the present invention
(1) Preparing alkali liquor: adding 200L of softened water into an alkali tank, weighing food-grade sodium carbonate according to the concentration of 15 wt%, putting the food-grade sodium carbonate into the alkali tank for full dissolution, placing the prepared alkali liquor into saturated steam with the pressure of 0.14MPa and the temperature of 145 ℃ for sterilization for 1h, introducing sterile compressed air into the alkali tank to ensure that the tank pressure of the alkali tank is 0.02-0.05MPa, and introducing cooling water into a jacket of the alkali tank to cool the alkali liquor to 35-45 ℃ for later use;
(2) cell wall lysate preparation: sterilizing the serially connected two-stage liquid sterile filtration system with saturated steam at the pressure of 0.14MPa and the temperature of 145 ℃ for 1h, and then maintaining the pressure with sterile compressed air for later use; the filter specification of the sterile filtration system is aperture 0.2 μm, and the sterile filtration system can resist temperature of 150 ℃; weighing alkaline protease, pectinase, cellulase and helicase, and adding softened water according to the weight ratio of enzyme: preparing an enzymolysis liquid according to the water ratio of 1:10, wherein the dosage of enzyme in each liter of fermentation liquid is as follows: 2g of alkaline protease, 1g of pectinase, 1.5g of cellulase and 0.5g of snailase.
(3) Adding an enzymolysis liquid and an alkali liquor: the temperature of the oil-producing microorganism fermentation liquor 1 is raised to 35-55 ℃, then the alkali liquor is pressed into a fermentation tank by using compressed air through an aseptic alkali liquor pipeline, the pH of the fermentation liquor is measured by using a pH probe of a Metler, and the addition of the alkali liquor is stopped until the pH of the fermentation liquor is 8; the enzymolysis liquid is conveyed into a secondary liquid sterile filtering system which is connected in series through a diaphragm pump for filtering, and then conveyed into a fermentation tank through a sterile pipeline for enzymolysis of the fermentation liquid; in the enzymolysis process, the temperature is controlled to be 50 ℃, the pH is controlled to be 8, the stirring speed is controlled to be 30rpm, the aeration quantity is controlled to be 0.2vvm, the tank pressure is controlled to be 0.02MPa, and the whole enzymolysis time is 15 h.
(4) Preheating preparation of the plate heat exchanger 5: opening a valve between a tap water pipe and the plate heat exchanger, wherein the tap water pipe supplies water to the plate heat exchanger through a distribution station, the water supply pressure is 0.1-0.2MPa, the sealing condition of a plate of the plate heat exchanger is checked through whether water leakage exists, after the sealing condition of the plate is determined to be good, the valve between the plate heat exchanger and a steam supply device is slowly opened, steam is supplied to the plate heat exchanger through the steam supply device, heat exchange is carried out between the steam and water in the plate heat exchanger, the temperature of the water is detected through a temperature monitoring device, and the water temperature is;
(5) preheating preparation of a three-phase centrifuge: starting a three-phase centrifuge to cool water, ensuring smooth water flow at a water outlet, simultaneously starting the three-phase centrifuge 13, ensuring that the starting current is 80A, starting a valve between a plate heat exchanger and the three-phase centrifuge after the speed of a full-speed indicator panel is 6000rpm, and enabling hot water at 95 ℃ to enter the three-phase centrifuge to ensure that the temperature of a rotary drum of the three-phase centrifuge reaches 95 ℃;
(6) feeding and centrifugal treatment: and (3) opening a valve between the fermentation tank and the plate heat exchanger, closing the valve between the plate heat exchanger and a tap water pipe, switching tap water in the first pipeline into the enzymolysis liquid obtained in the step (4) through the distribution station, opening a drain valve of the distribution station to drain the tap water in the pipeline, stopping draining when a color change is observed by a visual cup, stopping backflow of the enzymolysis liquid when the temperature of the enzymolysis liquid entering the heat exchanger for heat exchange reaches 95 ℃, conveying the enzymolysis liquid reaching 95 ℃ to a three-phase centrifuge, and performing centrifugal treatment for 15 hours to obtain microbial oil serving as crude oil, wherein the extraction efficiency of the oil, the residual oil rate in solid-phase residues, the ARA content, the EPA content, the 3-chloropropanol content, the anisidine value and the peroxide value are shown in table 1.
Example 3
This example illustrates the microbial oils and methods of preparation thereof provided by the present invention
(1) Preparing alkali liquor: adding 200L of softened water into an alkali tank, weighing food-grade sodium bicarbonate according to the concentration of 25 wt%, putting the food-grade sodium bicarbonate into the alkali tank for full dissolution, placing the prepared alkali liquor into saturated steam with the pressure of 0.14MPa and the temperature of 145 ℃ for sterilization for 1h, introducing sterile compressed air into the alkali tank to ensure that the tank pressure of the alkali tank is 0.02-0.05MPa, and introducing cooling water into a jacket of the alkali tank to cool the alkali liquor to 35-45 ℃ for later use;
(2) cell wall lysate preparation: sterilizing the serially connected two-stage liquid sterile filtration system with saturated steam at the pressure of 0.14MPa and the temperature of 145 ℃ for 1h, and then maintaining the pressure with sterile compressed air for later use; the filter specification of the sterile filtration system is aperture 0.2 μm, and the sterile filtration system can resist temperature of 150 ℃; weighing alkaline protease, adding softened water according to the weight ratio of the alkaline protease to the alkaline protease: preparing the enzymolysis liquid with the ratio of water to 1: 10.
(3) Adding an enzymolysis liquid and an alkali liquor: the temperature of the oil-producing microorganism fermentation liquor 1 is raised to 35-55 ℃, then the alkali liquor is pressed into a fermentation tank by using compressed air through an aseptic alkali liquor pipeline, the pH of the fermentation liquor is measured by using a pH probe of 'Mettler', and the addition of the alkali liquor is stopped until the pH of the fermentation liquor is 10; the enzymolysis liquid is conveyed into a secondary liquid sterile filtering system which is connected in series through a diaphragm pump for filtering, and then conveyed into a fermentation tank through a sterile pipeline, and the addition amount of enzyme is 5g relative to each liter of fermentation liquid for enzymolysis of the fermentation liquid; in the enzymolysis process, the temperature is controlled to be 50 ℃, the pH is controlled to be 10, the stirring speed is controlled to be 8rpm, the aeration quantity is controlled to be 0.6vvm, the tank pressure is controlled to be 0.05MPa, and the whole enzymolysis time is 4 h.
(4) Preheating preparation of the plate heat exchanger 5: opening a valve between a tap water pipe and the plate heat exchanger, wherein the tap water pipe supplies water to the plate heat exchanger through a distribution station, the water supply pressure is 0.1-0.2MPa, the sealing condition of a plate of the plate heat exchanger is checked through whether water leakage exists, after the sealing condition of the plate is determined to be good, the valve between the plate heat exchanger and a steam supply device is slowly opened, steam is supplied to the plate heat exchanger through the steam supply device, heat exchange is carried out between the steam and water in the plate heat exchanger, the temperature of the water is detected through a temperature monitoring device, and the water temperature is;
(5) preheating preparation of a three-phase centrifuge: starting a three-phase centrifuge to cool water, ensuring smooth water flow at a water outlet, simultaneously starting the three-phase centrifuge 13, ensuring that the starting current is 80A, starting a valve between a plate heat exchanger and the three-phase centrifuge after the speed of a full-speed indicator panel is 7500rpm, and allowing hot water at 85 ℃ to enter the three-phase centrifuge, so that the temperature of a rotary drum of the three-phase centrifuge reaches 85 ℃;
(6) feeding and centrifugal treatment: and (3) opening a valve between the fermentation tank and the plate heat exchanger, closing the valve between the plate heat exchanger and a tap water pipe, switching tap water in the first pipeline into the enzymolysis liquid obtained in the step (4) through the distribution station, opening a drain valve of the distribution station to drain the tap water in the pipeline, stopping draining when a color change is observed by a visual cup, stopping backflow of the enzymolysis liquid when the temperature of the enzymolysis liquid entering the heat exchanger for heat exchange reaches 85 ℃, conveying the enzymolysis liquid reaching 85 ℃ to a three-phase centrifuge, and performing centrifugal treatment for 20 hours to obtain microbial oil serving as crude oil, wherein the extraction efficiency of the oil, the residual oil rate in solid-phase residues, the ARA content, the EPA content, the 3-chloropropanol content, the anisidine value and the peroxide value are shown in table 1.
Example 4
The production of microbial oils was carried out in the same manner as in example 1 except that the oleaginous microorganism fermentation broth 1 in step 3 was replaced with the oleaginous microorganism fermentation broth 2. The extraction efficiency of the oil, the residual oil content in the solid phase residue, the DHA content, the EPA content, the 3-chloropropanol content, the anisidine value and the peroxide value in the oil are shown in Table 1.
Example 5
The production of microbial oils was carried out in the same manner as in example 2, except that the oleaginous microorganism fermentation broth 1 in step 3 was replaced with the oleaginous microorganism fermentation broth 2. The extraction efficiency of the oil, the residual oil content in the solid phase residue, the DHA content, the EPA content, the 3-chloropropanol content, the anisidine value and the peroxide value in the oil are shown in Table 1.
Example 6
The production of microbial oils was carried out in the same manner as in example 3, except that the oleaginous microorganism fermentation broth 1 in step 3 was replaced with the oleaginous microorganism fermentation broth 2. The extraction efficiency of the oil, the residual oil content in the solid phase residue, the DHA content, the EPA content, the 3-chloropropanol content, the anisidine value and the peroxide value in the oil are shown in Table 1.
Example 7
This example illustrates the microbial oils and methods of preparation thereof provided by the present invention
Microbial oil preparation was carried out as in example 1, except that the enzymes used for the enzymatic hydrolysis were replaced by helicase, cellulase and pectinase, and the amounts of enzymes used per liter of fermentation broth were: 2.5g of cellulase, 1.5g of pectinase and 1g of helicase, and the results are shown in Table 1.
Example 8
Comparative example microbial oil to illustrate reference and preparation method thereof
The microbial oils were prepared according to the method of example 1, except that the alkaline solution, the enzymatic hydrolysate, and the piping for transporting the alkaline solution and the enzymatic hydrolysate were not sterilized, and the results are shown in Table 1.
Example 9
Comparative example microbial oil to illustrate reference and preparation method thereof
The microbial oils were prepared according to the method of example 4, except that the alkaline solution, the enzymatic hydrolysate and the piping for transporting the alkaline solution and the enzymatic hydrolysate were not sterilized, and the results are shown in Table 1.
Comparative example 1
The preparation of microbial oils was carried out in the same manner as in example 1, except that the enzymolysis solution was entirely heated to 90 ℃ in the fermenter and then centrifuged, and the results are shown in Table 1.
Comparative example 2
Comparative example microbial oil to illustrate reference and preparation method thereof
The preparation of microbial oils was carried out in the same manner as in example 4, except that the enzymolysis solution was entirely heated to 90 ℃ in the fermenter and then centrifuged, and the results are shown in Table 1.
TABLE 1
As can be seen from Table 1, the method for separating microbial oil does not add any demulsifier, has high safety, and can improve the yield of the microbial oil as crude oil to 98-99%, which is obviously improved compared with a control; in addition, the DHA content or the ARA content in the crude oil is obviously improved, and the trichloropropanol content, the anisidine value and the peroxide value are obviously reduced; in addition, the whole process is controlled to operate in a sterile environment, so that the mass propagation of bacteria in the microbial oil extraction process can be reduced, the safety of the microbial oil, the yield of the microbial oil, the DHA content or the ARA content in the crude oil can be further increased, and the anisidine value and the peroxide value can be further reduced. In addition, the effect can be further improved by preferably using alkaline protease and preferably performing enzymolysis.
Test example
The degree of enzymatic hydrolysis is typically detected by two methods:
(a) contour morphology method for microscopic observation of algal cells: after enzymolysis, sampling 200mL of the sampling opening of the fermentation tank by using a conical flask, picking 2-4-ring enzymolysis fermentation liquid by using an inoculating ring, spreading the fermentation liquid on a cover glass, covering an observation glass slide, dripping 1-2 drops of cedar oil, and observing by using a 100-time oil mirror. See if there are intact algal cells; whether or not there are large pieces of cell debris is found, and the results are shown in Table 2.
(b) Centrifugal observation method: after enzymolysis, sampling 200mL of fermentation liquid by using a conical flask at a sampling port of a fermentation tank, taking 50mL of fermentation liquid by using a 50mL centrifugal test tube, carrying out centrifugal treatment for 3min by using a 6000-plus 10000rpm laboratory centrifuge, observing the separation condition of an oil phase and a water phase, judging whether a layering interface is clear, grading by using a 1-10 grading system, wherein the higher the grade is, the clearer the result is, and the result is shown in Table 2.
TABLE 2
As can be seen from table 2, the effect can be further improved under the preferred use of alkaline protease and the preferred enzymatic conditions.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (16)
1. An apparatus for on-line instantaneous heating and separation of microbial oils and fats, comprising:
the device comprises a feed liquid tank (1), a heat exchanger (2), a steam supply device (3), a three-phase centrifuge (4) and a water supply unit (5);
wherein the heat exchanger (2) is respectively communicated with the feed liquid tank (1), the steam supply device (3), the water supply unit (5) and the three-phase centrifuge (4);
wherein, the feed liquid tank (1) is filled with lysate of oleaginous microorganism fermentation liquor;
the heat exchanger (2) is respectively communicated with the feed liquid tank (1) and the water supply unit (5) through a first pipeline (6), communicated with the steam supply device (3) through a second pipeline (7), and communicated with the three-phase centrifuge (4) through a third pipeline (8), water reaching a preset temperature is introduced into the three-phase centrifuge (4) to preheat the three-phase centrifuge (4), and after preheating is finished, pyrolysis liquid reaching the preset temperature is introduced into the three-phase centrifuge (4) to separate microbial oil.
2. The device according to claim 1, wherein the water supply unit (5) is connected to the first line (6) through a distribution station and a first branch line (61); the feed tank (1) is connected to the first line (6) by the distribution station and a second branch line (62).
3. The device according to claim 2, wherein a pressure pump (11) is further arranged on the second branch line (62).
4. The device according to claim 2, wherein a pressure gauge (10) is also provided on the first line (6).
5. The device according to claim 2, wherein a visual cup (12) is also connected to the dispensing station.
6. The device according to any one of claims 1-5, wherein the heat exchanger (2) is a plate heat exchanger or a tube heat exchanger.
7. The device according to any one of claims 1-5, wherein a temperature monitoring device (9) is further arranged on a pipeline of the heat exchanger (2) communicated with the three-phase centrifuge (4).
8. A separation method for on-line instantaneous heating and separation of microbial oil is characterized by comprising the following steps:
(1) preheating a system: the water supply unit continuously supplies water to the heat exchanger, the steam supply device continuously supplies steam to the heat exchanger so as to exchange heat between water flow and the steam in the heat exchanger, maintain the temperature of outlet water flow of the water at a first preset temperature, and then introduce the water into the three-phase centrifuge to preheat the three-phase centrifuge so as to enable the three-phase centrifuge to reach a second preset temperature;
(2) material-water switching: cutting off water supply of the water supply unit, continuously supplying cracking liquid to the heat exchanger by the liquid feed tank, continuously supplying steam to the heat exchanger by the steam supply device, exchanging heat between a cracking liquid material flow and the steam in the heat exchanger, and enabling the temperature of a liquid material flow of the cracking liquid to reach a third preset temperature; then introducing the lysate into a preheated three-phase centrifuge for separation of an oil phase, a water phase and a solid phase to obtain microbial oil;
wherein the lysate is a lysate of fermentation broth of oleaginous microorganisms.
9. The method of claim 8, wherein the first, second, and third predetermined temperatures are each independently 80-95 ℃.
10. The method according to claim 8 or 9, wherein in step (1), the water supply unit continuously supplies water to the heat exchanger by delivering water to the distribution station and through the first branch line (61) and the first line (6); the water supply pressure is 0.1-0.2 MPa;
in the step (2), pyrolysis liquid from a feed liquid tank enters the alternative tap water from the distribution station, and is continuously supplied to the heat exchanger through a second branch pipeline (62); the hydraulic pressure of the liquid supply of the cracking liquid to the heat exchanger is 0.1-0.2 MPa.
11. A method according to claim 10, wherein after the water switch, the method further comprises draining the water in the distribution station, the first branch line (61) and the first line (6).
12. The method of claim 8, wherein the lysate is an enzymatic hydrolysate of a fermentation broth of oleaginous microorganisms, and the enzymatic hydrolysate is prepared by a method comprising: and (3) contacting the fermentation liquor of the oleaginous microorganism with cell wall lyase in an aseptic environment to carry out enzymolysis on the oleaginous microorganism in the fermentation liquor to obtain the enzymolysis liquid.
13. The method of claim 12, wherein the cell wall-lysing enzyme comprises alkaline protease and optionally other enzymes, the other enzymes being at least one of cellulase, hemicellulase, pectinase, helicase, chitinase, and ligninase.
14. The method of claim 12, wherein the conditions of enzymatic digestion comprise: pH 8-10, temperature 40-60 deg.C, pressure 0.02-0.05MPa, and ventilation amount 0.2-0.6VVM for 4-15 hr.
15. The process according to claim 14, wherein the enzymatic hydrolysis is carried out under stirring at a speed of 8-30 rpm.
16. A microbial oil produced according to any one of claims 8 to 15 which is a crude oil having a DHA content of greater than 35 wt% or an ARA content of greater than 35 wt% and which has a 3-chloropropanol < 350 μ g/kg, an anisidine value of less than 25, and a peroxide value of less than 20 meq/kg.
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| CN102433215A (en) * | 2011-09-22 | 2012-05-02 | 厦门汇盛生物有限公司 | Method of extracting grease from fungi or algae through physical wall-breaking |
| AU2013100882A4 (en) * | 2012-10-18 | 2013-07-25 | Transpacific Industries Group Ltd | Process and apparatus for treating waste |
| TWI646189B (en) * | 2013-12-20 | 2019-01-01 | 荷蘭商Ｄｓｍ智慧財產有限公司 | Method for obtaining microbial oil from microbial cells (5) |
| WO2015095694A1 (en) * | 2013-12-20 | 2015-06-25 | Dsm Ip Assets B.V. | Processes for obtaining microbial oil from microbial cells |
| WO2015092546A1 (en) * | 2013-12-20 | 2015-06-25 | MARA Renewables Corporation | Methods of recovering oil from microorganisms |
| CA2996526A1 (en) * | 2015-08-25 | 2017-03-02 | Dsm Ip Assets B.V. | Refined oil compositions and methods for making |
| AU2017297752B2 (en) * | 2016-07-13 | 2021-09-23 | Dsm Ip Assets B.V. | Method for isolating lipids from lipid-containing cells |
| WO2018011286A1 (en) * | 2016-07-13 | 2018-01-18 | Evonik Degussa Gmbh | Method of separating lipids from a lysed lipids containing biomass |
| BR102016017229B1 (en) * | 2016-07-25 | 2022-10-04 | Daniel Francisco De Souza | EXTRACTION AND SEPARATION PROCESS AND CHARACTERIZATION OF A FRACTION OF URUCUM SEEDS WITH MOSQUITO REPELLENCE ACTIVITY SUCH AS AEDES AEGYPTI |
| CN206345769U (en) * | 2016-12-30 | 2017-07-21 | 杰瑞环保科技有限公司 | Greasy filth piece-rate system |
| CN207016744U (en) * | 2017-05-28 | 2018-02-16 | 天津大学 | The microalgae grease extracting system that vapor recompression and heat exchange integrate |
| CN207193229U (en) * | 2017-09-15 | 2018-04-06 | 山西农业大学 | A kind of device for extracting microalgae wet algae oil |
| CN109181843B (en) * | 2018-08-20 | 2020-08-11 | 梁云 | Device and method for on-line heating separation of microbial oil and microbial oil |
-
2018
- 2018-09-10 CN CN201811048702.1A patent/CN109181843B/en active Active
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2019
- 2019-08-16 WO PCT/CN2019/101030 patent/WO2020038295A1/en active Application Filing
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| Publication number | Publication date |
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| CN109181843A (en) | 2019-01-11 |
| WO2020038295A1 (en) | 2020-02-27 |
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