Edible carrier as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an edible carrier as well as a preparation method and application thereof. The carrier is prepared by taking the fungal chitosan and the bacterial cellulose as raw materials, the cost is low, the preparation method is simple and efficient, the bacterial cellulose has excellent mechanical properties and biocompatibility, and amino in the fungal chitosan as a cation has a good charge attraction effect on cells; the fungal chitosan and the bacterial cellulose are compounded according to a certain mass ratio, the animal cell culture meat can be prepared without introducing any animal components such as ECM protein, good adhesiveness is shown, cell proliferation can be promoted, and the carrier obtained by taking the fungal chitosan and the bacterial cellulose as raw materials can be subjected to enzymolysis and can be recycled for multiple times.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an edible carrier beneficial to cell culture and a preparation method and application thereof. Background Art

[0002] Animal meat is an important part of the human diet and the main source of high-quality protein. However, the existing animal meat production methods are highly dependent on natural resources and bring a huge burden to the environment. Globally, animal husbandry occupies 39% of habitable land; consumes 50% to 70% of fresh water, which is not conducive to alleviating water shortages; and emits 18% to 30% of greenhouse gases, which exceeds the total emissions of all forms of transportation. Therefore, it is urgent to transform the production methods of animal meat. Cell cultured meat is a fashionable new source of protein, which helps to improve the quality and safety of meat, reduce the addition of hormones and antibiotics, flexibly regulate the nutrition and flavor of meat, reduce resource consumption, accelerate the realization of carbon neutrality, and protect animal welfare. The essence of cell cultured meat is in vitro animal cell culture.

[0003] At present, one of the key technical requirements for large-scale production of cell-cultured meat is high-density culture, which means achieving >10 8 The mainstream reaction system for high-density culture is the stirring reactor, which has a simple structure and is easy to operate, but the stirring process will generate a large shear force, which will cause mechanical damage to the cells. The carrier can provide sufficient anchoring points and growth space for adherent cells, reducing the possible shear force damage to the cells.

[0004] However, existing carrier preparation raw materials or modified materials usually include inedible materials such as polystyrene, polyester, polyethylene, glass, etc. or inedible groups such as DEAE, cationic trimethylammonium, quaternary ammonium salts, triethylamine, etc., such as Eppendof's Fibra-Cel sheet carrier, Cytiva TM Cytodex and Cytopore series of spherical microcarriers, The 2DMicroHex two-dimensional hexagonal carriers of GE Healthcare, the Cytoline series of lens-shaped microcarriers of GE Healthcare, and the DE-52 and DE-53 cylindrical microcarriers of Whatman have hindered the direct application of carriers in the production of cell-cultured meat. Therefore, it is particularly urgent to develop and screen edible, biodegradable, and recyclable carriers for cell-cultured meat. Summary of the invention

[0005] The purpose of the present invention is to provide an edible, low-cost carrier that is beneficial to cell culture and does not introduce any animal components such as ECM proteins, and has good biocompatibility and can promote cell adhesion and proliferation.

[0006] In order to achieve the above object, the present invention provides an edible carrier, which is composed of bacterial cellulose and fungal chitosan cross-linked on the bacterial cellulose; the mass ratio of the bacterial cellulose to the fungal chitosan is 1: (0.00005-1).

[0007] Preferably, the mass ratio of the bacterial cellulose to the fungal chitosan is 1:(0.0005-0.005).

[0008] Preferably, the mass ratio of the bacterial cellulose to the fungal chitosan is 1:(0.05-0.5).

[0009] Preferably, the fungal chitosan comprises mushroom chitosan.

[0010] The present invention also provides a method for preparing the edible carrier described in the above technical solution, comprising the following steps:

[0011] Drying the solution containing bacterial cellulose and fungal chitosan to obtain the edible carrier;

[0012] The dry weight ratio of bacterial cellulose to fungal chitosan in the solution containing bacterial cellulose and fungal chitosan is 1:(0.00005-1).

[0013] Preferably, the drying temperature is 10-88°C.

[0014] Preferably, after the drying process, the method further comprises: cutting the film obtained by the drying process into sheets to obtain sheet-shaped edible carriers.

[0015] Preferably, before the drying treatment, if the solution containing bacterial cellulose and fungal chitosan contains bubbles, the solution containing bacterial cellulose and fungal chitosan is subjected to a degassing treatment.

[0016] Preferably, the degassing treatment comprises ultrasound and / or vacuuming.

[0017] The present invention also provides the use of the edible carrier described in the above technical solution in the preparation of cell cultured meat.

[0018] Beneficial effects:

[0019] Fungal chitosan and bacterial cellulose are both derived from microorganisms. Bacterial cellulose is the main component of coconut fruit, and fungal chitosan is allowed to be added to food. The present invention uses fungal chitosan and bacterial cellulose as raw materials to prepare carriers at a low cost. Bacterial cellulose has excellent mechanical properties and biocompatibility. The amino group in fungal chitosan has a good charge attraction effect on cells as a cation. Fungal chitosan, especially mushroom chitosan and bacterial cellulose are compounded according to a certain mass ratio. Without introducing any animal ingredients, it can be used for the preparation of animal cell cultured meat, showing good adhesion, and can promote cell proliferation. The composite sheet carrier of fungal chitosan and bacterial cellulose can be fully degraded by cellulase-lysozyme or cellulase-chitosanase system, and can be recycled many times. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0021] Figure 1 The macroscopic structure of the carrier obtained in Examples 4 and 7 and Comparative Examples 1 to 5, the scale is 3 mm;

[0022] Figure 2 is the thickness of the carrier obtained in Examples 4 and 7 and Comparative Examples 1 to 5;

[0023] Figure 3 This is the state of the carrier obtained in Example 3 after being immersed in the culture medium for 12 hours, and the scale is 2 cm;

[0024] Figure 4 is the C2C12 living cell area ratio on the sheet carrier obtained in Example 7 and Comparative Examples 1, 3 and 5;

[0025] Figure 5 is the C2C12 viable cell area ratio on the sheet carrier obtained in Example 4 and Comparative Examples 1, 2 and 4;

[0026] Figure 6 is the C2C12 viable cell area ratio on the BCMC sheet carrier in the multiple recycling test;

[0027] Figure 7 The enzymatic hydrolysis effect of Application Example 2; wherein (a) is the enzymatic hydrolysis effect in PBS solution, (b) is the enzymatic hydrolysis effect in citric acid solution, and (c) is the enzymatic hydrolysis effect after the enzyme solution is recycled 1 to 4 times after inoculating cells. DETAILED DESCRIPTION

[0028] The invention provides an edible carrier, which consists of bacterial cellulose and fungal chitosan cross-linked on the bacterial cellulose; the mass ratio of the bacterial cellulose to the fungal chitosan is 1:(0.00005-1).

[0029] In the present invention, the mass ratio of bacterial cellulose to fungal chitosan is preferably 1:(0.00005-0.001) or 1:(0.005-1), more preferably 1:(0.00008-0.0008) or 1:(0.008-0.8), and more preferably 1:(0.0001-0.0005) or 1:(0.01-0.5). In the embodiment of the present invention, it is preferred to take any value within the range of 1:(0.00005~1), for example, 1:0.00005, 1:0.00008, 1:0.0001, 1:0.0005, 1:0.0008, 1:0.001, 1:0.005, 1:0.008, 1:0.01, 1:0.05, 1:0.08, 1:0.1, 1:0.5, 1:0.8 or 1:1.

[0030] Bacterial cellulose and fungal chitosan are widely available, and using fungal chitosan and bacterial cellulose as raw materials is low-cost. Moreover, fungal chitosan and bacterial cellulose are both derived from microorganisms. Bacterial cellulose is the main component of coconut fruit. Mushroom chitosan in fungal chitosan comes from natural edible mushrooms and is allowed to be added to food. Compounding fungal chitosan and bacterial cellulose in a certain proportion, without the introduction of any animal ingredients, can have excellent mechanical properties and biocompatibility, promote cell adhesion and proliferation, and can be used for the preparation of animal cell cultured meat; and fungal chitosan and bacterial cellulose can be fully degraded by cellulase-lysozyme or cellulase-chitosanase system, and can be recycled many times.

[0031] In the present invention, the fungal chitosan preferably includes mushroom chitosan; the degree of deacetylation of the mushroom chitosan is preferably ≥70%; the viscosity of the mushroom chitosan is preferably 10-80cps, more preferably 20-60cps, more preferably 20-40cps, and most preferably 20cps.

[0032] In the present invention, the bacterial cellulose is preferably obtained by fermentation culture of bacteria producing bacterial cellulose. The pH value of the fermentation culture of the present invention is preferably 4.0-7.0, more preferably 4.5-6.5, and more preferably 5.0-5.5; the temperature of the fermentation culture is preferably 10-35°C, more preferably 15-30°C, and more preferably 25-30°C. The fermentation culture of the present invention is preferably aerobic fermentation. In the embodiment of the present invention, there are no strict requirements for the culture medium used for the fermentation culture. According to the specific type of bacteria producing bacterial cellulose, the culture medium can be selected conventionally for culture, for example, Hestrin-Schramm culture medium (HS culture medium for short, with ingredients of 20g / L glucose, 5g / L peptone, 5g / L yeast extract, 1.5g / L citric acid monohydrate, and 6.8g / L disodium hydrogen phosphate dodecahydrate). The present invention has no strict requirements for the fermentation culture method, and static or dynamic culture is possible, and dynamic culture is for example cyclotron oscillation culture or reciprocating oscillation culture.

[0033] In the present invention, the bacteria producing bacterial cellulose preferably include Acetobacter, Gluconacetobacter, Gluconobacter, Asaia, Komagataeibacter, Alcaligenes, Sarcina, Rhizobium, Pseudomonas, Azotobacter, r), Agrobacterium, Achromobacter, Aerobacter, Bacillus, Leifsonia, Salmonella, Erwinia, Enterobacter, Shewanella, Zoogloea and Rhodobacter bacteria. The present invention is described in the embodiments by taking Acetobacter xylinum ATCC 700178 and Acetobacter xylinum ATCC 23767 as examples, but it should not be understood as the full scope of the present invention.

[0034] In the present invention, the shape of the edible carrier is preferably a sheet, more preferably a disc; the thickness of the sheet or disc is preferably 10 to 20 μm; the diameter of the disc is preferably 6 mm. The present invention limits the shape of the edible carrier, which can further increase the packing density of the carrier in the bioreactor and promote high-density cell culture.

[0035] The present invention also provides a method for preparing the edible carrier described in the above technical solution, comprising the following steps:

[0036] Drying the solution containing bacterial cellulose and chitosan to obtain the edible carrier;

[0037] The dry weight ratio of bacterial cellulose to chitosan in the solution containing bacterial cellulose and chitosan is 1:(0.00005-1).

[0038] In the present invention, the solution containing bacterial cellulose and the solution containing chitosan are preferably mixed to obtain a solution containing bacterial cellulose and chitosan.

[0039] In the present invention, the pH of the solution containing bacterial cellulose is preferably neutral. The method for preparing the solution containing bacterial cellulose of the present invention preferably comprises the following steps: treating bacterial cellulose with a NaOH solution, adjusting the pH to neutral, removing the residual bacteria and endotoxins therein, and obtaining a pretreatment liquid; homogenizing the pretreatment liquid to obtain the dispersion containing bacterial cellulose. The concentration of the NaOH solution of the present invention is preferably 0.1 to 1M, and more preferably 0.5 to 0.8M. The treatment method of the present invention preferably includes boiling or soaking; the boiling time is preferably 10 to 120 minutes, more preferably 20 to 100 minutes, more preferably 30 to 80 minutes, and most preferably 50 to 60 minutes; the soaking time is preferably several days. The present invention uses a NaOH solution to treat bacterial cellulose to remove the residual culture medium, bacteria and endotoxins therein, so that the membrane is milky white and translucent. The speed of the homogenization of the present invention is preferably 4500-20000rpm, more preferably 5000-15000rpm, and more preferably 8000-10000rpm; the homogenization time is preferably 2-30min. In the embodiment of the present invention, a high-speed shearing machine with a blade of 18-60mm is preferably used for the homogenization. The homogenization treatment of the pretreatment liquid of the present invention can make the bacterial cellulose uniformly dispersed in the solution.

[0040] In the present invention, the solution containing chitosan preferably comprises chitosan acetic acid solution. The concentration of chitosan in the chitosan acetic acid solution of the present invention is preferably 2wt%, and the concentration of acetic acid is preferably 1wt%. Chitosan molecules have ionizable amino groups and can be dissolved in acetic acid aqueous solution to form a solution.

[0041] The present invention has no strict requirements on the mixing ratio of the solution containing bacterial cellulose and the solution containing chitosan, and the dry weight ratio of bacterial cellulose to chitosan in the obtained solution containing bacterial cellulose and chitosan is 1:(0.00005-1), preferably 1:(0.00005-0.001) or 1:(0.005-1), further preferably 1:(0.00008-0.0008) or 1:(0.008-0.8), more preferably 1:(0.0001-0.0005) or 1:(0.01-0.5). In the embodiment of the present invention, it is preferred to take any value within the range of 1:(0.00005-1), such as 1:0.00005, 1:0.00008, 1:0.0001, 1:0.0005, 1:0.0008, 1:0.001, 1:0.005, 1:0.008, 1:0.01, 1:0.05, 1:0.08, 1:0.1, 1:0.5, 1:0.8 or 1:1. In the present invention, the solution containing bacterial cellulose and the solution containing chitosan are mixed to obtain a solution containing bacterial cellulose and chitosan. If the solution containing bacterial cellulose and chitosan contains bubbles, the solution containing bacterial cellulose and chitosan is preferably degassed. The degassing treatment method of the present invention preferably includes ultrasound and / or vacuum, and more preferably ultrasound; the power of the ultrasound is preferably 100-1000W, more preferably 200-800W, and more preferably 300-500W; the ultrasound time is preferably 10 minutes. The present invention has no strict requirements on the mixing method, and conventional operations can be used.

[0042] After obtaining the solution containing bacterial cellulose and chitosan, the present invention performs drying treatment on the solution containing bacterial cellulose and chitosan to obtain the edible carrier.

[0043] In the present invention, the temperature of the drying treatment is preferably 10-88°C, more preferably 15-40°C or 45-80°C, more preferably 20-35°C or 50-65°C, and most preferably 25-30°C or 55°C. The drying treatment method of the present invention is preferably vacuum drying or normal pressure drying; the vacuum degree of the vacuum drying is preferably -0.09 to -0.1 MPa. The present invention can form a thin film by drying the solution containing bacterial cellulose and chitosan, and the operation is simple and the processing cost is low.

[0044] The present invention preferably places the solution containing bacterial cellulose and chitosan in a mold and then performs a drying process to obtain an edible carrier having a certain shape. The mold of the present invention is preferably a square polystyrene culture dish. The present invention preferably places the solution containing bacterial cellulose and chitosan at a dry weight of 0.2 g / 100 cm 2The ratio of 20:1 to 20:1 is poured into the square polystyrene culture dish. In the embodiment of the present invention, if bubbles are contained after being placed in the mold, the solution containing bacterial cellulose and chitosan is preferably degassed. The degassed treatment method of the present invention preferably includes ultrasound and / or vacuuming, and is further preferably vacuuming; the vacuum degree of the vacuuming is preferably -0.09 to -0.1 MPa, and the time is preferably 1 to 2 days.

[0045] After obtaining the edible carrier, the present invention preferably cuts the edible carrier into sheets to obtain sheet-shaped edible carriers, and the diameter of the sheet-shaped edible carrier is preferably 6 mm.

[0046] The present invention directly mixes a solution containing bacterial cellulose and a solution containing chitosan, and obtains the edible carrier after drying. The preparation process is simple and the cost is low. The obtained edible carrier does not introduce any animal components, can improve the adhesion of cells on bacterial cellulose and its biocompatibility, promote cell proliferation, and can be recycled and enzymatically hydrolyzed multiple times, and can be used to prepare cell-cultured meat. Therefore, the application of the edible carrier in the preparation of cell-cultured meat also belongs to the protection scope of the present invention.

[0047] In order to further illustrate the present invention, an edible carrier provided by the present invention and its preparation method and application are described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Example 1

[0049] Preparation of bacterial cellulose

[0050] Activation of bacteria: Inoculate the cryopreserved Acetobacter xylinum ATCC 700178 into a fresh plate medium (20 g / L glucose, 5 g / L tryptone, 5 g / L yeast extract, 6.8 g / L disodium hydrogen phosphate dodecahydrate, 1.1 g / L anhydrous citric acid and the balance water, pH 6.0), and culture at 30°C until new colonies grow on the plate.

[0051] Expanded culture: After successful activation, use an inoculation loop to pick up a loop of colonies cultured on the plate and inoculate it into the liquid culture medium (the plate culture medium formula removes agar), and culture it at 30°C for 5 days to produce a sufficient amount of bacteria, then place it in a shaker and shake it to fully disperse the bacteria to make a seed suspension for later use.

[0052] Harvesting bacterial cellulose: Inoculate the seed suspension into a fresh liquid culture medium (same formula as above) at a ratio of 10% (v / v), shake well, and culture at a constant temperature of 30°C for several days. Collect a milky white translucent sheet gel with a thickness of ≥1 cm at the air-liquid interface, cut, fully wash, and soak in glacial acetic acid to obtain the bacterial cellulose, which is stored at 4°C.

[0053] Example 2

[0054] Preparation of bacterial cellulose

[0055] Activate bacteria: Inoculate the cryopreserved Acetobacter xylinum ATCC 23767 into fresh plate culture medium (yeast extract 5 g / L, peptone 5 g / L, mannitol 5 g / L, glucose 5 g / L, MgSO4·7H2O 1 g / L, ethanol 5 mL / L and the balance water, pH 6.6-7.0), and culture at 26°C until new colonies grow on the plate.

[0056] Expanded culture: After successful activation, use an inoculation loop to pick up a loop of colonies cultured on the plate and inoculate it into the liquid culture medium (the plate culture medium formula removes the agar), and culture it at 26°C for 5 days to produce a sufficient amount of bacteria, then place it in a shaker and shake it to fully disperse the bacteria to make a seed suspension for later use.

[0057] Harvesting bacterial cellulose: Inoculate the seed suspension into a fresh liquid culture medium (same formula as above) at a ratio of 10% (v / v), shake well, and culture at a constant temperature of 26°C for several days. Collect a milky white translucent sheet gel with a thickness of ≥1 cm at the air-liquid interface, cut, fully wash, and soak in glacial acetic acid to obtain the bacterial cellulose, which is stored at 4°C.

[0058] Example 3

[0059] The preparation method of the edible carrier comprises the following steps:

[0060] 1. Wash the bacterial cellulose (BC) preserved in Example 1 twice with water and boil it in a 0.25M NaOH solution for 30 minutes. Then rinse with water until the pH value is neutral. After preliminary crushing by a wall breaker, homogenize the bacterial cellulose blocks with a high-speed shearing machine at 4500rpm with a 60mm blade to form a uniform bacterial cellulose dispersion. Take a certain amount of the dispersion, dry it completely in an oven at 103±2℃ and weigh it to calculate the moisture content of the bacterial cellulose dispersion;

[0061] 2. Mix mushroom chitosan with a deacetylation degree of ≥70% and a viscosity of 20 cps with an acetic acid aqueous solution, and stir magnetically at room temperature to fully dissolve the mushroom chitosan to obtain a mushroom chitosan acetic acid solution, wherein the concentration of the mushroom chitosan in the mushroom chitosan acetic acid solution is 2wt%, and the concentration of the acetic acid is 1wt%, and insoluble impurities are filtered out with a 70μm filter.

[0062] 3. Mix the bacterial cellulose dispersion and the mushroom chitosan acetic acid solution evenly, so that the dry weight ratio of bacterial cellulose to mushroom chitosan in the obtained mixture is 1:0.005, and degas for 10 minutes at 100-1000W ultrasonic. Pour the mixture into a 10×10cm 2 The mixture was placed in a square polystyrene dish, vacuumed to remove bubbles, dried in an oven at 45°C to form a film, and cut into sheets with a diameter of 6 mm to obtain a sheet-like edible carrier (denoted as BCMC 1:0.005), which was placed in an autoclave at 121°C for 15 min.

[0063] Example 4

[0064] The preparation method of the edible carrier comprises the following steps:

[0065] 1. Boil the bacterial cellulose (BC) preserved in Example 2 in a 0.1M NaOH solution for 2 hours. Then adjust the pH to neutral with an acetic acid solution. After preliminary crushing with a wall breaker, homogenize the bacterial cellulose blocks with a high-speed shear at 20,000 rpm with an 18 mm blade to form a uniform bacterial cellulose dispersion. Take a certain amount of the dispersion, completely dry it in an oven at 103±2°C and weigh it to calculate the moisture content of the bacterial cellulose dispersion;

[0066] 2. Mix mushroom chitosan with a deacetylation degree of ≥70% and a viscosity of 20 cps with an acetic acid aqueous solution, and stir magnetically at room temperature to fully dissolve the mushroom chitosan to obtain a mushroom chitosan acetic acid solution, wherein the concentration of the mushroom chitosan in the mushroom chitosan acetic acid solution is 2wt%, and the concentration of the acetic acid is 1wt%, and insoluble impurities are filtered out with a 70μm filter.

[0067] 3. Mix the bacterial cellulose dispersion and the mushroom chitosan acetic acid solution evenly, so that the dry weight ratio of bacterial cellulose to mushroom chitosan in the obtained mixture is 1:1, and degas for 10 minutes at 100-1000W ultrasonic. Pour the mixture into a 10×10cm 2 The mixture was placed in a square polystyrene dish, vacuumed to remove bubbles, dried in an oven at 80°C to form a film, and cut into sheets with a diameter of 6 mm to obtain a sheet-like edible carrier (denoted as BCMC 1:1), which was placed in an autoclave at 121°C for 15 min.

[0068] Example 5

[0069] The same as Example 3, except that in step 3, the bacterial cellulose dispersion and the mushroom chitosan acetic acid solution are mixed evenly so that the dry weight ratio of bacterial cellulose to mushroom chitosan in the obtained mixed solution is 1:0.00005, and the obtained sheet-like edible carrier is recorded as BCMC 1:0.00005.

[0070] Example 6

[0071] The same as Example 3, except that in step 3, the bacterial cellulose dispersion and the mushroom chitosan acetic acid solution are mixed evenly so that the dry weight ratio of bacterial cellulose to mushroom chitosan in the obtained mixed solution is 1:0.0005, and the obtained sheet-like edible carrier is recorded as BCMC 1:0.0005.

[0072] Example 7

[0073] The same as Example 3, except that in step 3, the bacterial cellulose dispersion and the mushroom chitosan acetic acid solution are mixed evenly so that the dry weight ratio of bacterial cellulose to mushroom chitosan in the obtained mixed solution is 1:0.5, and the obtained sheet-like edible carrier is recorded as BCMC 1:0.5.

[0074] Example 8

[0075] The same as Example 3, except that in step 3, the bacterial cellulose dispersion and the mushroom chitosan acetic acid solution are evenly mixed so that the dry weight ratio of bacterial cellulose to mushroom chitosan in the obtained mixed solution is 1:0.05, and the obtained sheet-like edible carrier is recorded as BCMC 1:0.05.

[0076] Comparative Example 1

[0077] The same as Example 3, except that step 2 is omitted and step 3 does not prepare a blended liquid. Instead, the bacterial cellulose dispersion is directly subjected to a series of treatments such as degassing, drying, and cutting to obtain a bacterial cellulose sheet carrier (BC).

[0078] Comparative Example 2

[0079] The same as Example 4, the only difference is that a 2 wt % polylysine (PL) aqueous solution is prepared, and the mushroom chitosan acetic acid solution of Example 4 is replaced by a polylysine aqueous solution to obtain a bacterial cellulose-polylysine sheet carrier, wherein the dry weight ratio of bacterial cellulose (BC) to polylysine (PL) is 1:1, recorded as BCPL 1:1.

[0080] Comparative Example 3

[0081] The same as Example 7, the only difference is that a 2 wt % polylysine (PL) aqueous solution is prepared, and the mushroom chitosan acetic acid solution in Example 7 is replaced by a polylysine aqueous solution to obtain a bacterial cellulose-polylysine sheet carrier, wherein the dry weight ratio of bacterial cellulose (BC) to polylysine (PL) is 1:0.5, recorded as BCPL 1:0.5.

[0082] Comparative Example 4

[0083] The same as Example 4, the only difference is that a 2 wt % gelatin (GT) aqueous solution is prepared, the mushroom chitosan acetic acid solution in Example 4 is replaced by a gelatin aqueous solution, and the mushroom chitosan is replaced by gelatin to obtain a bacterial cellulose-gelatin sheet carrier (BCGT), wherein the dry weight ratio of bacterial cellulose (BC) to polylysine (GT) is 1:1, recorded as BCGT 1:1.

[0084] Comparative Example 5

[0085] The same as Example 7, the only difference is that a 2 wt % gelatin (GT) aqueous solution is prepared, the mushroom chitosan acetic acid solution in Example 7 is replaced by a gelatin aqueous solution, and the mushroom chitosan is replaced by gelatin to obtain a bacterial cellulose-gelatin sheet carrier (BCGT), wherein the dry weight ratio of bacterial cellulose (BC) to polylysine (GT) is 1:0.5, recorded as BCGT 1:0.5.

[0086] Test Example 1

[0087] The carriers obtained in Examples 4 and 7 and Comparative Examples 1 to 5 were used as experimental samples and the following tests were performed:

[0088] (1) Observe the macrostructure of different samples by taking photos with a digital camera; the results are as follows Figure 1 shown.

[0089] according to Figure 1 It can be seen that each group of flaky carriers obtained has relatively good uniformity, among which BC, BCPL, and BCGT carriers are white and translucent, and BCMC carrier is light yellow and translucent.

[0090] (2) The thickness of different samples was measured by a micrometer. The results are shown in Table 1 and Figure 2 shown.

[0091] Table 1 Thickness of different samples

[0092]

[0093] According to Table 1 and Figure 2 It can be seen that the thickness of the obtained sheet carrier is about 20 μm, which is lower than the commercially available sheet carrier, and helps to increase the packing density of the carrier in the bioreactor.

[0094] (3) The carriers obtained in Example 4 and Comparative Example 1 were immersed in culture medium (DMEM + 10% FBS) respectively and observed after immersion for 12 hours. The results are as follows: Figure 3 shown.

[0095] according to Figure 3 It can be seen that BC( Figure 3 Left) and BCMC ( Figure 3Right) The sheet carrier can sink to the bottom of the culture medium, which meets commercial standards.

[0096] Application Example 1 Application of sheet carriers in cell culture

[0097] 1. Test method:

[0098] C2C12 mouse myoblasts with a passage number of less than 10 generations were inoculated into a culture medium (DMEM + 10% FBS) and cultured in an incubator (37°C, 5% CO2). The cultured cells were taken and digested with 0.25% trypsin solution (Shanghai Biyuntian Biotechnology) in an incubator for 2 minutes. The cells were collected by centrifugation at 1000 rpm for 4 minutes and processed as follows:

[0099] Treatment group 1 (BC): The sterilized sheet carriers from comparative example 1 were added to the culture medium (DMEM + 10% FBS) at a concentration of 1×10 4 The cells were inoculated onto a sheet carrier at a density of 10 cells / sheet and cultured in an ultra-low adsorption 96-well plate at 37°C and 5% CO2. 100 μL of culture medium was added to each well and replaced once a day.

[0100] Treatment group 2 (BC:MC=1:1): Same as treatment group 1, except that the sterilized sheet carrier of Example 4 was added to the culture medium.

[0101] Treatment group 3 (BC:MC=1:0.5): Same as treatment group 1, except that the sterilized sheet carrier of Example 7 was added to the culture medium.

[0102] Treatment group 4 (BC:PL=1:1): Same as treatment group 1, except that the sterilized sheet carrier of comparative example 2 was added to the culture medium;

[0103] Treatment group 5 (BC:PL=1:0.5): Same as treatment group 1, except that the sterilized sheet carrier of comparative example 3 was added to the culture medium;

[0104] Treatment group 6 (BC:GT=1:1): Same as treatment group 1, except that the sterilized sheet carrier of comparative example 4 was added to the culture medium;

[0105] Treatment group 7 (BC:GT=1:0.5): Same as treatment group 1, except that the sterilized sheet carrier of comparative example 5 was added to the culture medium.

[0106] 2. Use the Calcein / PI live-dead staining detection kit to perform cell viability and cytotoxicity assays on the 1st, 3rd, 5th, and 7th days after inoculation of cells in treatment groups 1 to 8. In brief, transfer the vector to a new well (24-well plate) and wash once with 300 μL / well PBS to reduce the effects of serum and phenol red on CalceinAM staining. Add 300 μL / well detection working solution (prepared with CalceinAM (1000X), PI (1000X), and detection buffer at a volume ratio of 1:1:1000) and place in an incubator for incubation in the dark for 30 minutes. Subsequently, observe and photograph using a fluorescence microscope, with 3 parallel samples set for each group of samples. Use ImageJ software to analyze the fluorescence area ratio of live and dead cells in the photos at 40 times, and plot the relevant data into a graph. The results are shown in Tables 2 to 3 and Figures 4-5 shown.

[0107] Table 2 C2C12 living cell area ratio on carriers of treatment groups 2, 4, 6 and 8

[0108]

[0109] Table 3 C2C12 living cell area ratio on carriers of treatment groups 1, 3, 5 and 7

[0110]

[0111] Note: In Tables 2 and 3, "bright" means the end of the sheet carrier that contacts the mold during the preparation process, i.e., the lower surface; "rough" means the end of the sheet carrier that does not contact the mold during the preparation process, i.e., the upper surface.

[0112] According to Tables 2-3 and Figures 4-5It can be seen that the area of ​​live cells on the BC carrier did not exceed 25% within 7 days. At a ratio of 1:0.5, the effect of the BCPL carrier was better than that of the BC carrier, but the area of ​​live cells did not exceed 60% within 7 days. The BCGT carrier had a similar effect to the BC carrier, and the area of ​​live cells did not exceed 10% within 7 days. Among the sheet carriers, only the area of ​​live cells on the BCMC carrier reached more than 90%, that is, the cells basically covered the entire carrier. This peak of live cell area appeared on the third day, which was more than three times that of the first day. The earlier peak means that a shorter cell recovery cycle can be adopted when applied in a bioreactor, and a higher cell culture meat production efficiency can be achieved. After the third day, the area of ​​live cells on the BCMC carrier decreased significantly, and the area of ​​dead cells remained basically unchanged. In the experiment, it was also observed that live cells fell off in sheets at the corresponding time, which was mainly caused by excessive cell density. After some cells fell off, the surface of the carrier restored some growth space, so the area of ​​live cells increased again after the fifth day. Since the results of the rough and smooth surfaces of each carrier are limited and have no obvious rules, and do not affect the overall effect comparison of different modified products, no further discussion is made. The living cell area ratio of various carriers at a ratio of 1:1 is consistent with that at a ratio of 1:0.5, which shows that the sheet carrier (BCMC) provided by the present invention can effectively improve the cell adhesion of bacterial cellulose and promote cell proliferation.

[0113] Application Example 2: Recycling Characteristics of Sheet Carriers in Cell Culture

[0114] 1. Test method:

[0115] (1) C2C12 mouse myoblasts with a passage number of less than 10 generations were inoculated into a culture medium (DMEM + 10% FBS) and cultured in an incubator (37°C, 5% CO2). The cultured cells were taken and digested with 0.25% trypsin solution (Shanghai Biyuntian Biotechnology) in an incubator for 2 minutes, and the cells were collected by centrifugation at 1000 rpm for 4 minutes;

[0116] (2) The sterilized carrier (BC:MC=1:0.5) of Example 7 was added to the culture medium (DMEM+10% FBS), and the cells in step (1) were cultured at 1×10 4 The density of cells / sheet was inoculated on the sheet carrier;

[0117] The sterilized carrier (BC:MC=1:0.05) of Example 8 was added to the culture medium (DMEM+10% FBS), and the cells in step (1) were cultured at 1×10 4 The density of cells / sheet was inoculated on the sheet carrier;

[0118] After inoculation, multiple rounds of cell culture were performed, each round of cell culture cycle was 7 days. After each round of cell culture, the cells on the carrier were fully digested and removed with 0.25% trypsin solution, washed with PBS, soaked with culture medium, and then re-inoculated with cells to start the next round of cell culture. During multiple rounds of cell culture, the living cell area at each culture time was as shown in Table 4 and Figure 6 shown.

[0119] Table 4 C2C12 living cell area ratio on BCMC sheet carrier in recycling test

[0120]

[0121] According to Table 4 and Figure 6 It can be seen that living cells still grow on more than 20% of the area on the carrier, indicating that the sheet-like carrier obtained by the present invention can be recycled.

[0122] Application Example 3 Enzymatic hydrolysis characteristics of sheet carrier

[0123] For the BCMC flake carrier component, a cellulase-lysozyme composite system was used for enzymatic hydrolysis:

[0124] (1) Enzymatic hydrolysis in PBS solution

[0125] The sheet carriers of Examples 3, 5, 6 and 8 and the BC sheet carrier without MC (i.e., the sheet carrier of Comparative Example 1) were placed in a 24-well plate, 300 mL of PBS solution (pH 7.0-7.4) was added to each well, and cellulase and lysozyme were added to start enzymolysis. The cellulase concentration was 1×10 5 FPU / g substrate, lysozyme concentration is 1×10 8 FPU / g substrate. Oscillate the reaction at 37°C and 150 rpm for 3 hours and observe the enzymatic hydrolysis effect. The results are as follows Figure 7 As shown in (a).

[0126] according to Figure 7 As can be seen in (a), the structure of the mushroom chitosan-modified sheet carrier changes to varying degrees under the combined action of cellulase-lysozyme, and the carrier obtained in Example 5 (BC:MC=1:0.00005) can be completely degraded after 3 hours of enzymatic hydrolysis.

[0127] (2) Enzymatic hydrolysis in citric acid solution

[0128] Since the selected cellulase and lysozyme react better in an acidic environment, the sheet carrier of Example 8 was placed in a 24-well plate, 300 mL of citric acid solution (pH = 4.8) was added to each well, and the plates were randomly divided into three treatment groups:

[0129] Treatment group 1: Cellulase and lysozyme were added to start enzymatic hydrolysis. The concentration of cellulase was 2×10 4 FPU / g substrate, lysozyme concentration is 1×10 8 FPU / g substrate, shaken at 37°C, 150 rpm for 2 h;

[0130] Treatment group 2: Cellulase and lysozyme were added to start enzymatic hydrolysis. The concentration of cellulase was 4×10 4 FPU / g substrate, lysozyme concentration is 1×10 8 FPU / g substrate, shaken at 37°C, 150 rpm for 2 h;

[0131] Treatment group 3: Cellulase and lysozyme were added to start enzymatic hydrolysis. The concentration of cellulase was 1×10 5 FPU / g substrate, lysozyme concentration is 1×10 8 FPU / g substrate, shaken at 37°C, 150 rpm for 2 h;

[0132] The enzymatic hydrolysis effect of each treatment group was observed. Figure 7 As shown in (b).

[0133] according to Figure 7 As can be seen from (b), the flake carrier (BC:MC=1:0.05) obtained in Example 8 is 1×10 5 (FPU / g substrate) can be almost completely degraded after 2 hours of reaction. Because the cells in the final product of cell-cultured meat will also die after processing, there is no need to worry about the effect of pH on cell activity during enzymatic hydrolysis of the carrier.

[0134] (3) Enzymatic hydrolysis after cell inoculation

[0135] C2C12s were inoculated onto the sheet carrier (BC:MC=1:0.05) obtained in Example 8. After 7 days, the carrier with cells was transferred to a 12-well plate and washed with PBS, and then degraded with a cellulase-lysozyme system. 1 mL of enzyme solution was added to each well, and the enzyme was oscillated at pH 4.8, 37°C, and 150 rpm for 2 h. The cellulase concentration was 1×10 5 FPU / g substrate, lysozyme concentration is 1×10 8 After the enzymatic hydrolysis is completed, the enzyme solution is collected by centrifugation, and a new carrier is taken. The above enzymatic hydrolysis process is repeated using the recovered enzyme solution, and the degradation effect of the enzyme solution on the carrier is observed for the first, second, third, and fourth times. The results are as follows: Figure 7 As shown in (c).

[0136] according to Figure 7As can be seen in (c), the sheet carrier (BC:MC=1:0.05) obtained in Example 8 can be completely degraded in the cellulase-lysozyme system within 2 hours after cell culture. The enzyme solution still has the ability to degrade the carrier when it is recycled for the fourth time.

[0137] It can be seen from the above content that the edible carrier provided by the present invention has good biocompatibility, increases the adhesion of bacterial cellulose, promotes cell proliferation, and can be enzymatically hydrolyzed and recycled.

[0138] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An edible carrier, characterized in that: It consists of bacterial cellulose and fungal chitosan cross-linked on the bacterial cellulose; the mass ratio of the bacterial cellulose to the fungal chitosan is 1:(0.00005-1).

2. The edible carrier according to claim 1, characterized in that The mass ratio of the bacterial cellulose to the fungal chitosan is 1:(0.0005-0.005).

3. The edible carrier according to claim 2, characterized in that The mass ratio of the bacterial cellulose to the fungal chitosan is 1:(0.05-0.5).

4. The edible carrier according to any one of claims 1 to 3, characterized in that: The fungal chitosan includes mushroom chitosan.

5. The method for preparing the edible carrier according to any one of claims 1 to 4, characterized in that: The steps include: Drying the solution containing bacterial cellulose and fungal chitosan to obtain the edible carrier; The dry weight ratio of bacterial cellulose to fungal chitosan in the solution containing bacterial cellulose and fungal chitosan is 1:(0.00005-1).

6. The preparation method according to claim 5, characterized in that: The temperature of the drying process is 10-88°C.

7. The preparation method according to claim 5, characterized in that: After the drying process, the method further comprises: cutting the film obtained by the drying process into sheets to obtain sheet-shaped edible carriers.

8. The preparation method according to claim 5, characterized in that: Before the drying treatment, if the solution containing bacterial cellulose and fungal chitosan contains bubbles, the solution containing bacterial cellulose and fungal chitosan is subjected to a degassing treatment.

9. The preparation method according to claim 8, characterized in that: The degassing treatment method includes ultrasound and / or vacuum.

10. Use of the edible carrier according to any one of claims 1 to 4 or the edible carrier obtained by the preparation method according to any one of claims 5 to 9 in the preparation of cell-cultured meat.

Images