CN112244233A

CN112244233A - Processing method of leisure food with cultivated meat as raw material

Info

Publication number: CN112244233A
Application number: CN202011064541.2A
Authority: CN
Inventors: 王守伟; 李莹莹; 李石磊; 李雨爽; 刘文婷; 杨峰; 曲超
Original assignee: China Meat Research Centre
Current assignee: China Meat Research Centre
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2021-01-22

Abstract

The invention belongs to the technical field of animal cell culture and meat product processing, and discloses a processing method of leisure food taking cultured meat as a raw material, which comprises the steps of collecting muscle tissues by using a sterile surgical instrument, cutting the muscle tissues into minced meat, and extracting, culturing, differentiating and collecting myogenic cells; the muscle tissue is pretreated, including pickling, coloring, seasoning and the like; molding and hot processing; and (5) after the sample is cooled, performing packaging treatment to obtain the instant meat product after the cultured meat is processed. The invention improves the efficiency of cell culture, reduces cell death and obtains effective cells to the maximum extent; the use of the cell factory is 5-20 times higher than the collection efficiency of the cells in a normal culture dish, so that the collection of the cells is accelerated, the cell culture efficiency is improved, and the cell factory is a large-size basis for subsequent food processing; by adding various auxiliary materials and additives, the freezing and forming are convenient for batch operation, and more abundant product forms can be obtained.

Description

Processing method of leisure food with cultivated meat as raw material

Technical Field

The invention belongs to the technical field of animal cell culture and meat product processing, and particularly relates to a processing method of a leisure food taking cultivated meat as a raw material.

Background

At present: the cultured meat is also called cultured meat, clean meat and the like, and is a novel meat food which is prepared by controlling the rapid proliferation and the directional differentiation of animal cells in an in vitro culture mode and collecting and processing the animal cells. The traditional meat production mode is at the cost of consuming a large amount of grains and water resources and seriously polluting the environment. With the great increase of economic development, population expansion and middle income crowds, the global meat demand is rapidly increasing, and the production of cultivated meat is taken as a novel efficient, environment-friendly and sustainable meat production mode to meet the meat supply of human beings in the future. The research of meat cultivation belongs to international leading-edge technical research, and European and American countries accelerate research progress and marketization progress of the product. European new food regulations enforced from 2018, 1 month onwards, clearly list cell culture meat as a novel food.

However, at present, the main research on cell culture meat focuses on the aspects of extraction, proliferation, optimization of culture technology, product plasticity and the like of muscle stem cells (the research progress of the 'meat culture' and related patent applications, Chinese inventions and patents, 2019 (7): 71-75.), and the research on the use of the cell culture meat in food is very rare.

The leisure meat product mainly refers to a cooked meat finished product or a semi-finished product which is prepared by taking livestock and poultry meat as a main raw material and seasoning. The leisure meat product as a middle-high-end leisure food has rich nutrition and unique flavor and mouthfeel, is convenient to carry, becomes an important leisure food for home and travel, and is more and more popular with consumers. The cultivated meat is used as a raw material, the production plasticity is strong, the leisure meat product is processed and manufactured through a certain production process, the utilization rate of the raw material is high, the product types and the product forms are more abundant, and the prospect is very wide.

Through the above analysis, the problems and defects of the prior art are as follows:

(1) the research on the direction of meat cultivation by organisms at home and abroad is less and insufficient, the existing mature products are few, and patents on the aspect of meat cultivation and food production at home and abroad are also very lacking;

(2) the current production process of biological cultivation meat is carried out in an aseptic environment, a large amount of culture medium and serum are used, the production cost is higher, and the quantity of obtained products in industrial production is limited, so the using amount and the product form of the products are also limited.

The difficulty in solving the above problems and defects is: the meat raw materials for food cultivation are collected, the moisture content is high, the color is white and tasteless, and the product meeting the requirements of current consumers can be prepared only by properly seasoning and mixing colors. And the production cost of the early raw materials for cultivating the meat is high, and the problem that the maximum use is required is also a project difficulty.

The significance of solving the problems and the defects is as follows: the method for processing the leisure food by taking the cultivated meat as the raw material provides a new thought for commercialization of the cultivated meat, can accelerate industrialization of the cultivated meat, and can produce products meeting the requirements of consumers early.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a processing method of leisure food taking cultivated meat as a raw material.

The invention is realized in such a way that a method for processing a leisure food using cultivated meat as a raw material comprises the following steps:

collecting muscle tissue with sterile surgical instrument, and cutting into paste;

adding enzyme solution into the obtained muscle tissue for digestion, adding culture solution, repeatedly blowing and beating with a suction pipe, filtering, centrifuging, sucking supernatant, and obtaining lower-layer sediment as target muscle satellite cells;

resuspending the obtained target muscle satellite cells by using a cell culture medium, inoculating a cell suspension to a plate, adding a cell culture solution, uniformly shaking, and putting the cell suspension into a cell culture box for culture;

when the cell fusion degree reaches more than 90%, digesting with pancreatin, stopping digestion by using a culture medium, and carrying out cell passage by using a cell factory;

after the cells grow over the bottom area of the cell factory, replacing a cell proliferation culture medium with a cell differentiation culture medium, inducing the myosatellite cells to differentiate into the multinucleated myotube cells to form the multinucleated myotube cells, after inducing the differentiation, replacing the culture medium with the cell proliferation culture medium again, and continuing culturing until the cells are completely differentiated to form the multinucleated myotube cells;

rinsing and digesting the cell factory, terminating digestion, immediately centrifuging, sucking supernatant, and precipitating to obtain target collected cells;

collecting multinucleated myotube cells;

pretreatment: adding auxiliary materials into the meat raw materials, uniformly mixing, and pickling;

uniformly spreading the pretreated raw materials in a baking tray, freezing, slightly thawing, cutting into slices or other required shapes, brushing butter or olive oil on the surface, baking in an oven, cooling, and packaging to obtain instant meat product.

Further, the enzyme solution comprises single collagenase, protease, pancreatin or a combination thereof, and the enzyme concentration is 0.15% -0.45%; the obtained muscle tissue was digested by adding 1 to 5mL/g of an enzyme solution, and 4 to 6mL of a culture solution was added.

Further, the digestion is carried out for 20-50min at 37 ℃, the filtration and centrifugation are carried out by using a filter screen, and the filtrate is centrifuged for 3-6min at 1200 rpm.

Further, when the cell suspension was seeded on a plate, 10cm was used²Culture dishes, 106 muscle satellite cells per dish, then 4-8mL cell culture fluidThe cell culture solution comprises a high-sugar DMEM culture medium, penicillin and streptomycin are added, the working concentration of the penicillin is 100-300U/mL, and the working concentration of the streptomycin is 0.05-1 mg/mL; the cell culture chamber is 5% CO at 37 ℃₂A cell culture box.

Further, when cell passaging was performed using a cell factory having a growth surface area of 600-700cm, 1 monolayer cell factory was passaged per 3 dishes of cells²Passage of 1 ten cell factories per 3 monolayer factories; when the cell fusion degree reaches more than 90%, digesting by using 0.15-0.45% of pancreatin, stopping digestion by using 1 time of culture medium, and carrying out cell passage by using a cell factory;

and after the cells grow to fill 50-80% of the area of the bottom of the cell factory, replacing a cell proliferation culture medium with a cell differentiation culture medium, inducing the myosatellite cells to differentiate into the multinucleated myotube cells to form the multinucleated myotube cells, and inducing differentiation for 3-7 days.

Further, when the cell factory is rinsed, digested and immediately centrifuged, the specific steps are adopted:

the cell factory was rinsed with DPBS at a concentration of 8-15 mL/layer, digested with 0.15% -0.45% trypsin at a concentration of 8-15 mL/layer for 4-8min, the digestion was stopped with twice the volume of DPBS and immediately centrifuged at 600-1500rpm for 4-8 min.

Further, the auxiliary materials comprise 0.5-1.5g of table salt, 0.1-0.3g of pepper powder, 8-12mL of vegetable oil, 4-8mL of water, 2-5 drops of monascus red, 0.5-1.5g of carrageenan or xanthan gum, 0.8-2.0g of oyster sauce, 0.15-0.35g of glutamine transaminase and 1-2g of solid honey according to the amount added in 100g of meat raw materials.

Further, the pickling is carried out at 0-4 ℃ for 8-12 h.

Further, the freezing conditions are as follows: freezing at-18 deg.C for 6-12 h; spreading sesame after brushing butter or olive oil on the surface.

The invention improves the efficiency of cell culture, reduces cell death and obtains effective cells to the maximum extent; the use of the cell factory is 5-20 times higher than the collection efficiency of the cells in a normal culture dish, so that the collection of the cells is accelerated, the cell culture efficiency is improved, and the cell factory is a large-size basis for subsequent food processing; by adding various auxiliary materials and additives, the monascus red mainly has a color mixing effect, carrageenan and glutamine transaminase can perform gel curing molding and the like, pepper can remove fishy smell, and solid honey can be used for seasoning and removing fishy smell; the freezing and forming are convenient for batch operation, and more abundant product forms can be obtained.

Another object of the present invention is to provide a snack food using cultured meat as a raw material, which is prepared by the method for processing a snack food using cultured meat as a raw material.

By combining all the technical schemes, the invention has the advantages and positive effects that: the invention provides a production process suitable for production and processing of the leisure meat product by using the cultured meat as a raw material and combining the properties of the cultured meat product for the first time.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.

Fig. 1 is a flow chart of a method for processing a snack food using a meat as a raw material according to an embodiment of the present invention.

FIG. 2 is a schematic diagram of immunofluorescence staining of porcine muscle stem cells during differentiation, provided by an embodiment of the present invention; in fig. 2: (a2) a muscle satellite cell; (b2) a stage of transition of the myosatellite cells to multinucleated myotube cells; (c2) multinucleated myotube cells.

FIG. 3 is a schematic diagram of the results of detecting expression levels of proteins MYH and MyoD in the differentiation process of the porcine muscle stem cell provided by the embodiment of the invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In view of the problems in the prior art, the present invention provides a method for processing a snack food using a cultivated meat as a raw material, which is described in detail below with reference to the accompanying drawings.

As shown in fig. 1, the processing method of a snack food using cultivated meat as a raw material according to an embodiment of the present invention includes:

s101, collecting muscle tissues by using a sterile surgical instrument, and shearing the muscle tissues into minced shapes;

s102, adding 1-5mL/g of enzyme solution into the muscle tissue obtained in the S101 for digestion, adding 4-6mL of culture solution, repeatedly blowing and beating by using a suction pipe, filtering, centrifuging, sucking out supernatant, and obtaining lower-layer sediment which is target muscle satellite cells;

s103, resuspending the target muscle satellite cells obtained in the step S102 by using a cell culture medium, inoculating the cell suspension to a plate, adding a cell culture solution, uniformly shaking, and putting the plate into a cell culture box for culture;

s104, when the cell fusion degree in the S103 reaches more than 90%, digesting by using 0.15-0.45% of pancreatin, stopping digestion by using 1 time of culture medium, and carrying out cell passage by using a cell factory;

s105, after the cells grow to fill 50-80% of the area of the bottom of the cell factory, replacing a cell proliferation culture medium with a cell differentiation culture medium, inducing the myosatellite cells to differentiate into the multinucleated myotube cells to form the multinucleated myotube cells, after inducing the differentiation for 3-7 days, replacing the culture medium with a cell proliferation culture medium again, and continuing culturing until the cells are completely differentiated to form the multinucleated myotube cells;

s106, rinsing and digesting the cell factory, stopping digestion, immediately centrifuging, and sucking out supernatant, wherein the precipitate is the target collection cell;

s107, repeating the steps S103-S106 to collect enough multinuclear myotube cells for subsequent processing;

s108, preprocessing: adding auxiliary materials into the meat raw materials, uniformly mixing, and pickling;

s109, heating: and (3) uniformly spreading the raw materials pretreated in the step (S108) into a baking tray, freezing, slightly thawing, cutting into slices or other required shapes, brushing butter or olive oil on the surface, baking in an oven, cooling after baking, and packaging to obtain the instant meat product processed by the cultured meat.

The processing method of the leisure food using the cultivated meat as the raw material according to the present invention can be implemented by those skilled in the art by adopting other steps, and the processing method of the leisure food using the cultivated meat as the raw material according to the present invention shown in fig. 1 is only one specific example.

The technical solution of the present invention will be further described with reference to the following specific examples.

The processing method of the leisure food taking the cultivated meat as the raw material provided by the embodiment of the invention specifically comprises the following steps:

s1, collecting muscle tissue with sterile surgical instrument, and cutting into paste;

s2, adding 1-5mL/g enzyme solution (comprising single collagenase, protease, pancreatin or combination thereof, the enzyme concentration is 0.15% -0.45%) into the tissue obtained in S1 for digestion, digesting for 20-50min at 37 ℃, adding 4-6mL culture solution, repeatedly blowing and beating by using a suction pipe, filtering by using a filter screen, centrifuging the filtrate for 3-6min at 500-;

s3, resuspending the cell pellet obtained in the step S2 in a cell culture medium, and inoculating the cell suspension on a dish, including a culture flask or the like, if 10cm is selected²The culture dish comprises 106 muscle satellite cells per dish, and each of the other ways is converted according to the change, then 4-8mL of cell culture solution (high-sugar DMEM culture medium, penicillin and streptomycin are added, the working concentration of penicillin is 100-₂The cell culture box is used for culturing.

And S4, when the cell fusion degree in S3 reaches more than 90%, digesting by using 0.15-0.45% of pancreatin, stopping digestion by using 1-time culture medium, and carrying out cell passage. Passage of 1 monolayer of cell factory per 3 dishes of cells (growth surface area 600-²) Passage of 1 ten layers of fine cells per 3 monolayer cell factoriesA cell factory.

S5, after the cells grow to fill 50-80% of the area of the bottom of the cell factory, replacing a cell proliferation culture medium with a cell differentiation culture medium, inducing the differentiation of the porcine muscle satellite cells to the multinucleated myotube cells to form the multinucleated myotube cells, after inducing the differentiation for 3-7 days, replacing the culture medium with the cell proliferation culture medium again, and continuing culturing until the cells are completely differentiated to form the multinucleated myotube cells.

S6, rinsing the cell factory with DPBS at a concentration of 8-15 mL/layer, and digesting with 0.15% -0.45% pancreatin at a concentration of 8-15 mL/layer for 4-8 min. Digestion was stopped with two volumes of DPBS and centrifugation was immediately performed at 600-1500rpm for 4-8 min. And (4) sucking off the supernatant, and obtaining the precipitate as the target collection cell.

S7, repeating the steps S3-S6 to collect enough multinucleated myotube cells for subsequent processing.

S8, preprocessing: adding 0.5-1.5g of table salt, 0.1-0.3g of pepper powder, 8-12mL of vegetable oil, 4-8mL of water, 2-5 drops of monascus red, 0.5-1.5g of carrageenan or xanthan gum, 0.8-2.0g of oyster sauce, 0.15-0.35g of glutamine transaminase and 1-2g of solid honey into 100g of meat raw materials, rolling or uniformly mixing in other modes, and pickling at the temperature of 0-4 ℃ for 8-12 h;

s9, heating: spreading S8 uniformly on baking tray, freezing at-18 deg.C for 6-12h, thawing slightly, cutting into sheet or other desired shape, spreading butter or olive oil on the surface, optionally spreading semen Sesami (optional step), and baking at 200 deg.C in oven for 10-25 min.

The technical solution of the present invention is further described with reference to the following examples.

The implementation case is as follows:

1. extraction of porcine cells

Using CO for piglets₂Air-washing, soaking in 75% ethanol for sterilization, dissecting thigh skeletal muscle, removing fascia, cutting skeletal muscle tissue into tissue blocks of about 0.5 g/block, cutting with ophthalmic scissors into paste with particle size of about 1mm³，

Washing with PBS, adding 2.5mL/g trypsin solution for digestion at 37 deg.C for 25min, adding 5mL culture solution, repeatedly beating with a pipette,filtering with filter screen, centrifuging the filtrate at 800rpm for 5min, sucking supernatant to obtain lower layer precipitate as target muscle satellite cell, placing the precipitate in high glucose DMEM proliferation culture medium containing 20% fetal calf serum, standing at 37 deg.C and 5% CO₂And (4) carrying out aseptic culture in a cell culture box under the condition.

Culturing for 2-3 days, pouring out supernatant and the block, washing with DBPS once, adding 1mL of pancreatin (0.25%) for digestion, adding 1mL of culture medium containing 10% FBS and DMEM after the cells begin to shed, gently blowing by using a pipette until the cells completely shed, transferring the cell suspension into a centrifuge tube for centrifugation, centrifuging at 800rpm for 5min, sucking out the supernatant, and suspending the precipitate in high-sugar DMEM culture medium containing 20% FBS for continuous culture. After the cells are expanded to cover 80% of the bottom area of the culture dish, subculture is carried out.

After digestion of the cells in the culture dish, the cells were digested at 1X 10⁷Seeding concentration of cells/layer dispersed cells were seeded in the cell factory. Normally, the fusion degree of 3 cells reaches more than 80 percent and is 10cm²The culture dish is digested and inoculated in 1 monolayer cell factory, and 3 monolayer cells factory cells with 80% cell fusion degree are digested and inoculated in 1 ten layer cell factory. The culture conditions were 37 ℃ and 5% CO₂. As shown in fig. 2 and 3.

And after the cells grow over 80% of the bottom of the cell factory, replacing the cell proliferation culture medium with a cell differentiation culture medium, inducing the pig muscle satellite cells to differentiate into the multinucleated myotube cells to form the multinucleated myotube cells, after inducing the differentiation for 4 days, replacing the culture medium with the cell proliferation culture medium again, and continuing culturing until the cells are completely differentiated to form the multinucleated myotube cells.

The cell factory was rinsed with DPBS at a concentration of 10 mL/layer and then digested with 0.125% trypsin at a concentration of 10 mL/layer for 4-8 min. Digestion was stopped with two volumes of DPBS and centrifugation was immediately performed at 800rpm for 5 min. And (4) sucking off the supernatant, and obtaining the precipitate as the target collection cell.

The steps are repeated to collect sufficient multinucleated myotube cells for subsequent processing.

Pretreatment: adding 1.0g of salt, 0.2g of pepper, 10mL of vegetable oil, 5mL of water, 4 drops of monascus red, 0.8g of carrageenan or xanthan gum, 1.0g of oyster sauce, 0.25g of glutamine transaminase and 1g of solid honey into 100g of meat raw materials, rolling or uniformly mixing in other modes, and pickling for 6 hours at 0-4 ℃;

s9, heating: spreading S8 uniformly on baking tray, freezing at-18 deg.C for 10 hr, thawing slightly, cutting into 2mm pieces, brushing butter or olive oil on the surface, optionally spreading semen Sesami, placing in oven, and baking at 175 deg.C for 10 min.

The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, and any modifications, equivalents and improvements made by those skilled in the art within the spirit and principle of the present invention are intended to be covered by the present invention.

Claims

1. A method for processing a snack food using meat as a raw material, comprising:

collecting multinucleated myotube cells;

2. A method of processing a leisure food using cultured meat as a raw material according to claim 1, wherein the enzyme solution includes collagenase, protease, pancreatin or a combination thereof alone at an enzyme concentration of 0.15% to 0.45%; the obtained muscle tissue was digested by adding 1 to 5mL/g of an enzyme solution, and 4 to 6mL of a culture solution was added.

3. The method as claimed in claim 1, wherein the digestion is carried out at 37 ℃ for 20-50min, the filtration and centrifugation are carried out by using a filter screen, and the filtrate is centrifuged at 500-.

4. The method for processing a leisure food using meat as a raw material according to claim 1, wherein the cell suspension is seeded on a dish at a length of 10cm²Culture dishes, each dish plate 106 muscle satellite cells, then 4-8mL of cell culture solution is added, the cell culture solution comprises high-sugar DMEM culture medium, penicillin and streptomycin are added, the working concentration of the penicillin is 100-300U/mL, and the working concentration of the streptomycin is 0.05-1 mg/mL; the cell culture chamber is 5% CO at 37 ℃₂A cell culture box.

5. A method of processing a meat-based snack food according to claim 1, wherein the cell factory is used for cell passaging, wherein 1 monolayer of cell factory is passaged every 3 dishes of cells, and the growth table of said cell factory is shownThe area is 600-700cm²Passage of 1 ten cell factories per 3 monolayer factories; when the cell fusion degree reaches more than 90%, digesting by using 0.15-0.45% of pancreatin, stopping digestion by using 1 time of culture medium, and carrying out cell passage by using a cell factory;

6. The method for processing a leisure food using cultured meat as a raw material according to claim 1, wherein the steps of rinsing, digesting, terminating digestion and immediately centrifuging the cell factory are as follows:

7. The method for processing the leisure food using the cultivated meat as the raw material according to claim 1, wherein the auxiliary materials comprise 0.5 to 1.5g of table salt, 0.1 to 0.3g of pepper, 8 to 12mL of vegetable oil, 4 to 8mL of water, 2 to 5 drops of monascus red, 0.5 to 1.5g of carrageenan or xanthan gum, 0.8 to 2.0g of oyster sauce, 0.15 to 0.35g of glutamine transaminase and 1 to 2g of solid honey based on the amount added to 100g of the meat raw material.

8. The method for processing a snack food using a cultured meat as a raw material according to claim 1, wherein the pickling is carried out at 0 to 4 ℃ for 8 to 12 hours.

9. The method for processing a leisure food using meat as a raw material according to claim 1, wherein the freezing is performed under the following conditions: freezing at-18 deg.C for 6-12 h; spreading sesame after brushing butter or olive oil on the surface.

10. A snack food made from meat as claimed in any one of claims 1 to 9, which is produced by the method for processing a snack food made from meat as claimed in any one of claims 1 to 9.