WO2023105878A1 - Porous material and method for producing same - Google Patents

Abstract

The present invention provides a novel porous material that is suitable for the culture of cells. The porous material according to the present invention comprises an edible material and has a specific surface area of 85.0 mm2/mm3 or more per unit volume. The porous material has, for example, edible properties. The method for producing a porous material according to the present invention includes: bringing a gelatinized product comprising an edible material and water and having a porous structure into contact with an alcohol; and drying the gelatinized product that has been contacted with the alcohol.

Description

Porous body and its manufacturing method

The present invention relates to a porous body and its manufacturing method.

In recent years, it is expected that the demand for meat will increase as the world's population increases. In order to meet the future increase in meat demand, it is not enough to simply increase the production efficiency of conventional protein sources, and it is essential to develop new protein sources. New protein sources include vegetable meat produced from plants, meat produced from insects, cultured meat produced by culturing microorganisms or cells themselves, and the like. “Vegetable meat” is a processed food made by adding additives to vegetable protein such as soybeans as a raw material, and is also called “fake meat”. “Cultured meat” means meat produced by culturing muscle cells using regenerative medicine technology, and is also called “cultured meat” or “clean meat”.

One of the benefits of cultured meat is its safety. For example, in the process of meat production and processing, there is always the risk of contamination with pathogens that cause food poisoning. However, since cultured meat is cultivated in almost sterile conditions, the risk of contamination with pathogenic bacteria is low. In addition, cultured meat can not only reduce the cost of the processing process, but it is also attracting attention from an environmental point of view, as research results show that it can reduce greenhouse gases by 96% compared to conventional production methods. At present, minced meat has been reported as cultivated meat.

JP 2014-140381 A

In order to produce chunks of meat of a certain size, such as steak, sashimi, and fillets, it is necessary to use scaffolding materials to three-dimensionally culture cells such as muscle cells. This scaffolding material is preferably a porous body composed of an edible material such as a polysaccharide. An example of a porous material containing polysaccharides is disclosed in Patent Document 1. In the field of cultured meat, new porous bodies suitable for culturing cells are desired.

Therefore, an object of the present invention is to provide a new porous body suitable for culturing cells.

The present invention
Provided is a porous body containing an edible material and having a specific surface area per unit volume of 85.0 mm ² /mm ³ or more.

Furthermore, the present invention provides
Contacting a gelled product containing an edible material and water and having a porous structure with alcohol;
drying the gelled product after contact with the alcohol;
to provide a method for manufacturing a porous body.

According to the present invention, a new porous body suitable for culturing cells can be provided.

3 is a three-dimensional reconstruction image formed from continuous transmission images of the porous body of Example 1. FIG. 1 is a scanning electron microscope (SEM) image showing a cross section of the porous body of Example 1. FIG. 3 is a three-dimensional reconstruction image formed from continuous transmission images of the porous body of Example 2. FIG. 4 is an SEM image showing a cross section of the porous body of Example 2. FIG. 3 is a three-dimensional reconstruction image formed from continuous transmission images of the porous body of Example 3. FIG. 4 is an SEM image showing a cross section of the porous body of Example 3. FIG. 4 is a three-dimensional reconstruction image formed from continuous transmission images of the porous body of Example 4. FIG. 4 is an SEM image showing a cross section of the porous body of Example 4. FIG.

The porous body according to the first aspect of the present invention is
It contains an edible material and has a specific surface area per unit volume of 85.0 mm ² /mm ³ or more.

In the second aspect of the present invention, for example, the porous body according to the first aspect is edible.

In the third aspect of the present invention, for example, the porous body according to the first or second aspect has a porosity of 90% or more.

In the fourth aspect of the present invention, for example, the porous body according to any one of the first to third aspects has an open cell ratio of 90% or more.

In the fifth aspect of the present invention, for example, in the porous body according to any one of the first to fourth aspects, when the cross section of the porous body is observed with a scanning electron microscope, the range is 1800 μm long × 2400 μm wide. The maximum diameter of the pores within is 1-1000 μm.

In the sixth aspect of the present invention, for example, in the porous body according to any one of the first to fifth aspects, the edible material contains polysaccharides.

In the seventh aspect of the present invention, for example, in the porous body according to any one of the first to sixth aspects, the edible material contains alginic acid and/or alginate.

In the eighth aspect of the present invention, for example, in the porous body according to any one of the first to seventh aspects, the edible material contains at least one selected from the group consisting of glucomannan and cellulose derivatives.

In the ninth aspect of the present invention, for example, the porous body according to any one of the first to eighth aspects includes a body portion containing the edible material and an adhesion improver that improves adhesion of cells, and a covering layer that covers the main body.

In the tenth aspect of the present invention, for example, the porous body according to any one of the first to ninth aspects is a foam.

The method for producing a porous body according to the eleventh aspect of the present invention comprises:
Contacting a gelled product containing an edible material and water and having a porous structure with alcohol;
drying the gelled product after contact with the alcohol;
including.

In the twelfth aspect of the present invention, for example, in the production method according to the eleventh aspect, the alcohol is ethanol.

In the thirteenth aspect of the present invention, for example, the production method according to the eleventh or twelfth aspect comprises: foaming a solution containing a foaming agent and water; adding a gelling agent to the foamed solution; creating a compound.

In the 14th aspect of the present invention, for example, in the production method according to the 13th aspect, at least one selected from the group consisting of the foaming agent and the gelling agent contains the edible material.

In the fifteenth aspect of the present invention, for example, in the production method according to the thirteenth or fourteenth aspect, the gelling agent contains an alginate, a compound that generates divalent metal ions, and an acid generator.

In the sixteenth aspect of the present invention, for example, in the production method according to any one of the thirteenth to fifteenth aspects, the foaming agent contains at least one selected from the group consisting of glucomannan and cellulose derivatives.

In the 17th aspect of the present invention, for example, in the manufacturing method according to any one of the 13th to 16th aspects, a foaming agent is further added to the solution.

In the 18th aspect of the present invention, for example, in the production method according to the 17th aspect, the foaming agent is a chemical foaming agent that generates gas upon contact with an acid.

In the 19th aspect of the present invention, for example, in the production method according to any one of the 11th to 18th aspects, the dried product obtained by drying the gelled product is attached to the dried product to improve the adhesiveness of cells. Further comprising applying an enhancer.

Although the details of the present invention will be described below, the following description is not intended to limit the present invention to specific embodiments.

The porous body of this embodiment contains an edible material and has a specific surface area A per unit volume of 85.0 mm ² /mm ³ or more. Edible material means a substance recognized as a food or food additive by the laws and regulations of each country.

The specific surface area A can be specified by the following method. First, a porous body to be evaluated is prepared. This porous body is preferably in a dry state. As used herein, "dry state" means that the water content in the porous body is 10 wt% or less, preferably 1 wt% or less. Next, if necessary, the porous body to be evaluated is cut out to prepare a test piece. The test piece is, for example, a disc with a thickness of 5.5 mm and a diameter of 8 mm.

Next, with the test piece set in the holder, an X-ray CT device is used to take a transmission image of the test piece. Specifically, an arbitrary region inside the test piece (near the center) is photographed. The size of the area is, for example, 2 mm long×2 mm wide×0.6 mm thick. As the X-ray CT apparatus, for example, Xradia 620 Versa manufactured by Zeiss can be used. The transmission image is captured, for example, under the conditions of a tube voltage of 40 kV, a tube current of 73 μA, and a pixel size of 0.3 μm/Pixel. As an example, 1601 transmission images (successive transmission images) are taken in the range of 0° to 360° with respect to an arbitrary reference plane passing through the center of gravity of the area to be imaged. A tomographic image is created by reconstructing all the obtained transmission images, and a three-dimensional reconstructed image (TIFF format stack image) and a reconstructed cross-sectional image (three views) are also created. A three-dimensional reconstructed image can be created using known software (eg, Avizo).

Next, the surface area a of the test piece in the photographed region is calculated from the obtained stereoscopic image (three-dimensional reconstructed image). Calculation of the surface area a can be performed using known software (eg, Avizo). The above surface area a is the area of the surface (hole wall) facing the hole present in the photographed area. Next, the stereoscopic image is binarized to create a binarized image. Using the binarized image, the volume v of the portion (matrix) other than the holes in the photographed area is calculated. Calculation of the matrix volume v can be performed using known software (eg, ImageJ). A value obtained by dividing the surface area a (mm ² ) by the matrix volume v (mm ³ ) can be regarded as the specific surface area A of the porous body.

The specific surface area A of the porous body is 85.0 mm ² /mm ³ or more, preferably 88 mm ² /mm ³ or more, 90 mm ² /mm ³ or more, 100 mm ² /mm ³ or more, 110 mm ² /mm ³ or more. , 120 mm ² /mm ³ or more, 130 mm ² /mm ³ or more, 140 mm ² /mm ³ or more, or even 150 mm ² /mm ³ or more. The larger the specific surface area A of the porous body, the easier it is for the culture solution for culturing the cells to permeate the inside of the porous body and for the cells themselves to reach the inside of the porous body. The upper limit of the specific surface area A of the porous body is not particularly limited, and is, for example, 500 mm ² /mm ³ .

The specific surface area A of the porous body is determined by the shape of the pores included in the porous body. In this embodiment, the porous body has, for example, continuous pores. In other words, the pores included in the porous body are communicated. A continuous pore is a pore that is continuously formed in a three-dimensional shape inside a porous body. The continuous pores may penetrate the porous body. In this embodiment, the continuous pore is formed by, for example, opening a part of the surface (pore wall) facing a specific independent pore and communicating the independent pore with the adjacent independent pore. That is, the continuous hole has, for example, a plurality of holes p1 derived from a plurality of independent holes, and a hole p2 connecting two adjacent holes p1 among the plurality of holes p1. As an example, the continuous hole has a plurality of holes p2, and the plurality of holes p1 communicate with each other through the plurality of holes p2. The diameter of the hole p1 is usually larger than the diameter of the hole p2. The porous body of the present embodiment tends to have a large number of pores p2, and due to this, the specific surface area A tends to be large. The porous body may further have independent pores in addition to the continuous pores.

The porosity of the porous body is, for example, 80% or more, and may be 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, or even 94% or more. The higher the porosity of the porous body, the easier it is to increase the volume ratio of cells to the porous body. By adjusting the volume ratio of cells to a high value, cultured meat with good texture tends to be produced. The upper limit of the porosity of the porous body is not particularly limited, and is, for example, 99%. The porosity means the ratio of the total volume (mm ³ ) of all pores contained in the porous body to the volume (mm ³ ) of the porous body. The porosity of the porous body can be calculated from the binarized image of the specific surface area A described above. Calculation of the porosity can be performed using known software (eg, ImageJ).

The open cell ratio of the porous body is, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or even 100%. good too. The higher the open cell ratio, the easier it is for the culture solution for culturing cells to permeate into the interior of the porous body, and for the cells themselves to reach the interior of the porous body. The open cell ratio means the ratio of the volume (mm ³ ) of continuous pores contained in the porous body to the total value (mm ³ ) of the volume of all pores contained in the porous body. The open-cell ratio can be specified by analyzing the stereoscopic image and the binarized image described above for the specific surface area A.

When observing the cross section of the porous body with a scanning electron microscope (SEM), the maximum diameter of the pores within the range of 1800 μm long×2400 μm wide is, for example, 1 to 1000 μm. The maximum pore diameter can be determined by the following method. First, the porous body is cut and the cross section is observed with an SEM. The enlargement magnification at this time is, for example, 50 times. From the SEM image, the holes p1 and p2 described above can usually be confirmed. In the SEM image, pores present within a range of 1800 μm long×2400 μm wide are specified. For each identified hole, identify the diameter (diameter of the smallest circle that can enclose the hole). Among the specified diameters, the largest value can be regarded as the maximum diameter of the hole.

As described above, the porous body contains an edible material. Edible materials include, for example, polysaccharides; proteins such as gelatin and collagen; lipids such as beeswax, phospholipids and fatty acids. The edible material is preferably a polymeric compound, ie an edible polymer. The weight average molecular weight of the edible polymer is not particularly limited, and is, for example, 1000 or more.

The edible material preferably contains polysaccharides. The edible material preferably contains alginic acid and/or alginate as polysaccharide. In other words, the porous body preferably contains alginic acid and/or alginate as the polysaccharide. Alginic acid is a polysaccharide contained in seaweeds and the like, and has a structural unit (M block) derived from β-D-mannuronic acid and a structural unit (G block) derived from α-L-guluronic acid. In alginic acid, each structural unit is linked via a 1,4-glycosidic bond. The content of G blocks in alginic acid is not particularly limited, and is, for example, 30 mol % or more, preferably 40 mol % or more, and more preferably 50 mol % or more. The upper limit of the G block content may be 90 mol % or 80 mol %. The content of G blocks may be from 31 mol % to 63 mol %.

The alginate contained in the porous body is, for example, a salt of alginic acid and a divalent metal ion. For example, in alginate, at least one G block contained in alginic acid forms an ionic bond with a divalent metal ion. In other words, in the alginate contained in the porous body, alginic acid partially forms a salt with divalent metal ions. Alginates, for example, have a crosslinked structure via divalent metal ions. Examples of divalent metal ions include calcium ions, barium ions, iron ions, zinc ions and copper ions, with calcium ions being preferred.

The total value of the alginic acid content and the alginate content in the porous body is not particularly limited, and is, for example, 10 wt% or more, preferably 20 wt% or more, more preferably 30 wt% or more, and still more preferably. is 40 wt% or more. The upper limit of this total value is not particularly limited, and is, for example, 80 wt%, preferably 70 wt%, more preferably 60 wt%. In the present specification, the "content rate in the porous body" means the content rate based on the dry porous body, unless otherwise specified.

The porous body may contain polysaccharides P other than alginic acid and alginate together with alginic acid and/or alginate, or instead of alginic acid and/or alginate. It may contain more than In the porous body, two or more other polysaccharides P may be associated with each other. Other polysaccharides P include glucomannan (konnyakumannan) and cellulose derivatives. In other words, the porous body contains, as polysaccharides, at least one selected from the group consisting of glucomannan and cellulose derivatives, preferably both glucomannan and cellulose derivatives.

Glucomannan is a polysaccharide contained in konjac yam and the like, and has a structural unit derived from glucose (glucose unit) and a structural unit derived from mannose (mannose unit). In glucomannan, each structural unit is linked via 1,4-glycosidic bonds. In glucomannan, the molar ratio of mannose unit to glucose unit is not particularly limited, and is, for example, 0.5 to 2, and may be 0.5 to 1.6.

A cellulose derivative has a structure in which a substituent is introduced into cellulose. This substituent preferably functions as a hydrophobic group in the cellulose derivative. Cellulose derivatives include, for example, cellulose ethers. Cellulose ethers include, for example, alkylcelluloses such as methylcellulose (MC); hydroxyalkylcelluloses such as hydroxypropylcellulose (HPC) and hydroxyethylcellulose (HEC); hydroxyalkylalkylcelluloses such as hydroxypropylmethylcellulose (HPMC); carboxymethylcellulose ( CMC) and other carboxyalkyl celluloses. Preferably, the cellulose derivative comprises hydroxypropylmethylcellulose.

The content of the other polysaccharide P in the porous body is not particularly limited, and is, for example, 0.5 wt% or more, preferably 5 wt% or more, more preferably 10 wt% or more, and still more preferably 20 wt% or more. is. The upper limit of the content of the other polysaccharide P is not particularly limited, and is, for example, 90 wt%, preferably 80 wt%, more preferably 70 wt%.

When the porous body contains glucomannan, the content of glucomannan in the porous body is not particularly limited, and is, for example, 0.5 wt% to 5.0 wt%. When the porous body contains a cellulose derivative, the content of the cellulose derivative in the porous body is not particularly limited, and is, for example, 20 wt % to 80 wt %. The weight ratio of glucomannan and cellulose derivative in the porous material is not particularly limited, but is preferably 0.1:99.9 to 9.9:90.1. In addition, the porous body may contain almost no other polysaccharides P, particularly cellulose derivatives.

The porous body may further contain a compound C that generates divalent metal ions. Compound C can generate divalent metal ions, for example, by contact with an acid. Divalent metal ions include those described above for alginates. Compound C is, for example, a salt containing a divalent metal ion. The salts include carbonates such as calcium carbonate. Compound C, especially calcium carbonate, is a component suitable for adjusting the hardness of the porous body. The compound C may exist as a residue in a salt state in the porous body, or may be consumed by a cross-linking reaction of alginic acid molecules, which will be described later. Compound C may remain in the porous body in a solid state.

The content of compound C, particularly calcium carbonate, in the porous body is not particularly limited, and is, for example, 20 wt% or less, preferably 18 wt% or less, more preferably 15 wt% or less, and 12 wt% or less. good too. The lower limit of the content of compound C is not particularly limited, and is, for example, 0.1 wt%. The content of compound C is, for example, 3.5 wt % to 15 wt %. By adjusting the content of compound C within the above range, there is a tendency that the hardness of the porous body can be appropriately adjusted. However, the porous body does not have to contain compound C.

The porous body may further contain an acid generator and/or a decomposition product of the acid generator. The acid generator is an undecomposed residue present in the porous body. An acid generator is, for example, a compound that upon hydrolysis forms an acidic group such as a carboxyl group. A specific example of an acid generator is glucono delta lactone (GDL). A specific example of the decomposition product of the acid generator is gluconic acid. The total value of the content of the acid generator and the content of the decomposed product of the acid generator in the porous body is, for example, 50 wt % or less, preferably 30 wt % or less. The porous body may not contain an acid generator, and may not contain a decomposition product of the acid generator.

The porous body may further contain a foaming agent and/or a decomposition product of the foaming agent. The blowing agent is an unreacted residue present within the porous body. The blowing agent is preferably a chemical type blowing agent that generates gas upon contact with acid. Examples of such chemical blowing agents include sodium hydrogen carbonate. When the blowing agent is a chemical type blowing agent, the decomposition product of the blowing agent may form a salt, such as sodium gluconate, with the decomposition product of the acid generator. The total value of the content of the foaming agent and the content of the decomposed product of the foaming agent in the porous body is, for example, 10 wt % or less, preferably 5 wt % or less. The porous body may not contain a foaming agent, and may not contain a decomposition product of the foaming agent. When the decomposition product of the foaming agent forms a salt with the decomposition product of the acid generator, most of the salt is removed from the porous body by the production method described later, particularly the step of bringing the gelled product into contact with alcohol. tend to be removed.

The porous body may contain other components than those mentioned above, but it is preferred that it does not substantially contain them. Other components include, for example, plasticizers (softeners) and surfactants containing low-molecular-weight compounds. The content of other components in the porous body is, for example, 10 wt% or less, preferably 5 wt% or less, more preferably 1 wt% or less.

A porous body is typically edible. In the present specification, "the porous body is edible" means that the porous body is composed only of substances approved as foods or food additives according to the laws and regulations of each country.

The porous body is typically a foam. The shape of the porous body is not particularly limited, and can be appropriately adjusted according to the shape of cultured meat to be produced. As an example, the porous body may be sheet-shaped, cube-shaped or disc-shaped with a thickness of 1 to 30 mm.

The porous body may have a main body containing an edible material and a coating layer covering the main body. The main body is composed of, for example, the above-described components such as polysaccharides. The main body has a porous structure and includes, for example, continuous pores. The coating layer may cover the entire surface (outer surface and pore walls) of the main body, or may partially cover the surface of the main body. The coating layer contains, for example, an adhesion improver that improves the adhesiveness of cells, and is preferably composed substantially only of the adhesion improver.

The adhesion improver is preferably edible. The adhesion improver contains, for example, at least one selected from the group consisting of edible plant-derived components and edible animal-derived components. The adhesion improver may consist essentially of edible plant-derived components, or may consist essentially of edible animal-derived components.

As used herein, the term "edible plant-derived component" means an edible component made from a plant. The edible plant-derived component is not particularly limited, and is derived, for example, from plant seeds, roots, stems, leaves, and the like. The edible plant-derived component is preferably a seed-derived component.

The raw material of the edible plant-derived component is not particularly limited, and examples include leguminosae, grape family, gramineous family, asteraceae, palm family, cotton family, brassicaceae, poppy family, sesame family, rose family, oleaceae, mallow. plants belonging to the family, Pinaceae, Polygonaceae, Ericaceae, Currantaceae, Zingiberaceae, etc., preferably leguminous plants or grape family plants, more preferably leguminous plants. The raw material of the edible plant-derived component is preferably a leguminous plant seed, more preferably a soybean seed (soybean).

Edible plant-derived ingredients are, for example, ingredients processed from soybeans. The component obtained by processing soybeans is not particularly limited. Soy protein.

Defatted soybeans are soybeans from which oil has been removed. For example, they contain 50% or more protein, 35% or more carbohydrates, and 19% or less lipids by weight.

Defatted soymilk is a water-extracted fraction of defatted soybeans, and the dried fraction contains, for example, 59.0% or more protein, 26.9% or more carbohydrate, and 0.9% fat by weight. Including 2% or less.

Isolated soy protein is a protein separated from defatted soy milk by isoelectric precipitation or heating. %, containing 0.2-31% lipids.

Whey is a fraction obtained by removing separated soy protein from skimmed soy milk, and contains oligosaccharides and minerals.

Soybean meat is a product obtained by processing the water-insoluble components of defatted soymilk. Contains 5 to 2.8%.

Tofu cheese is defatted soymilk agglomerated with calcium ions and the like. .9%.

As used herein, the term "edible animal-derived ingredient" means an edible ingredient made from an animal. The edible animal-derived ingredients preferably include non-mortal animal-derived ingredients. "Non-lethal animal-derived ingredient" means an animal-sourced ingredient that can be obtained without slaughtering an animal. Raw materials for non-lethal animal-derived components include, for example, animal milk, eggs, blood, crop milk and the like, preferably animal milk or eggs.

Animal milk is not particularly limited, and examples include cow, goat, sheep, buffalo, camel, donkey, horse, reindeer, and yak milk, preferably cow milk (milk). The edible animal-derived component derived from milk is not particularly limited, and examples thereof include casein, whey, milk fat, lactose, vitamins, minerals, etc., preferably casein or whey. Casein is, for example, sodium caseinate.

The animal eggs are not particularly limited, and examples include chicken, quail, duck, ostrich, and pigeon eggs, preferably chicken eggs (chicken eggs). An egg is, for example, an unfertilized egg. Edible animal-derived components derived from eggs are not particularly limited, and examples thereof include egg yolk, egg white, ovalbumin, egg yolk lecithin, eggshell membrane, and the like.

The coating layer may or may not be gelled. A gelled coating layer can constitute a strong film. Ungelled coating layers tend to be relatively brittle.

In the porous body, the weight ratio of the adhesion improver to the weight of the main body is not particularly limited, and is, for example, 0.01 wt% to 50 wt%, preferably 0.1 wt% to 30 wt%, more preferably 1 wt%. % to 10 wt%. The adhesion promoter allows cells to easily adhere to the porous body. During culturing of cells on the porous material, some or all of the adhesion promoter may flow out or elute into the culture medium of the cells.

(Manufacturing method of porous body)
The method for manufacturing the porous body of the present embodiment includes:
Contacting a gelled material G containing an edible material and water and having a porous structure with alcohol;
drying the gelled material G after contact with the alcohol;
including.

The above gelled material G can be produced, for example, by the following method. First, a solution S containing a foaming agent and water is prepared, and the solution S is foamed (foamed). The foaming agent contains, for example, polysaccharides P other than alginic acid and alginate, and preferably contains two or more kinds of other polysaccharides P. As other polysaccharides P, those mentioned above can be used. That is, the foaming agent preferably contains at least one selected from the group consisting of glucomannan and cellulose derivatives, and more preferably contains both glucomannan and cellulose derivatives (especially hydroxypropylmethylcellulose). When the foaming agent contains glucomannan and a cellulose derivative, in solution S the mannose units of glucomannan tend to interact with substituents, especially hydrophobic groups, contained in the cellulose derivative. This interaction brings the glucomannan and the cellulose derivative together. When glucomannan and a cellulose derivative are associated, not only is the solution S easily foamed, but the elasticity of the generated foam tends to be improved. Furthermore, the shape of the foam tends to be maintained for a long period of time.

The concentration of the foaming agent in the solution S is not particularly limited, and is, for example, 0.05 wt% to 10 wt%, and may be 0.05 wt% to 5 wt%. As an example, the concentration of glucomannan in solution S is, for example, 0.05 wt % to 0.5 wt %, preferably 0.1 wt % to 0.3 wt %. The concentration of the cellulose derivative in solution S is, for example, 0.5 wt % to 10 wt %, preferably 1 wt % to 9 wt %. The concentration of the cellulose derivative may optionally be from 0.5 wt% to 5 wt%, or from 1 wt% to 3 wt%.

The method for foaming the solution S is not particularly limited, and known methods can be used. For example, the solution S may be foamed by stirring the solution S using a commercially available homogenizer. The stirring speed, stirring time, and the like of the solution S can be appropriately set according to the viscosity and composition of the solution S. The operation of bubbling the solution S is performed at room temperature (22±3° C.), for example. In this operation, it is preferable to foam the solution S as a whole. Note that the solution S may be foamed while the solution S is being prepared by adding a foaming agent while stirring water.

Next, by adding a gelling agent to the foamed solution S, gelation of the solution S progresses and a gelled product G can be produced. The gelled material G is a hydrogel containing water derived from the solution S. The gelled product G has a porous structure resulting from bubbles generated in the solution S. Most of the pores contained in the gelled product G are independent pores. As described above, the production method of the present embodiment further comprises foaming the solution S containing a foaming agent and water, and adding a gelling agent to the foamed solution S to produce the gelled product G. include.

The gelling agent for producing gelled material G includes, for example, alginate, compound C that generates divalent metal ions, and an acid generator. Thus, at least one selected from the group consisting of a foaming agent and a gelling agent comprises an edible material (particularly a polysaccharide), and typically both the foaming agent and the gelling agent comprise edible materials. contains. The operation of adding the gelling agent to the solution S can be performed, for example, by adding the alginate, the compound C and the acid generator to the solution S in this order.

Examples of alginates added to the solution S include salts of alginic acid and alkali metal ions such as sodium ions and potassium ions. Preferably, the alginate is sodium alginate. The alginate added to solution S is typically substantially free of divalent metal ions.

The alginate is preferably added to the solution S while the solution S is being stirred. Addition of alginate to solution S increases the viscosity of solution S. Thereby, the shape of the bubbles generated in the solution S is easily maintained.

In the solution S to which alginate is added, the alginate concentration is, for example, 1 wt% to 3 wt%, preferably 1 wt% to 2 wt%.

As the compound C and the acid generator added to the solution S, those described above can be used. The compound C and the acid generator are preferably added to the solution S while the solution S is being stirred.

When the acid generator is added to the solution S, acid is generated from the acid generator. Specifically, the acid generator is hydrolyzed in solution S to form acidic groups. By forming an acidic group, the hydrolyzed acid generator functions as an acid. When this acid reacts with compound C, divalent metal ions are generated from compound C.

The divalent metal ions generated from compound C form an ionic bond with the alginate G block. Specifically, metal ions (alkali metal ions) contained in the alginate are exchanged for divalent metal ions. As a result, multiple alginic acid molecules are crosslinked via divalent metal ions. Due to this cross-linking reaction, gelation of the solution S proceeds, and a gelled product G is obtained.

The cross-linking reaction of multiple alginic acid molecules via divalent metal ions is usually an irreversible reaction. That is, the gelled material G obtained by the above method is difficult to return to the solution S. Therefore, the porous body produced from this gelled material G tends to be excellent in heat resistance. When the porous body is excellent in heat resistance, cultured meat produced using this porous body as a scaffolding material easily maintains its shape even when cooked. Thus, a porous body with excellent heat resistance is particularly suitable as a scaffolding material for cultured meat for cooking with heat.

Even when other polysaccharides (for example, chitosan) are used as the gelling agent instead of alginate, gelation of the solution S may progress. However, when other polysaccharides are used, it is necessary to heat the solution S and then cool it in order to accelerate the gelation of the solution S. On the other hand, the gelation of the solution S due to the cross-linking reaction of a plurality of alginic acid molecules has the advantage of proceeding relatively quickly without heating the solution S. In addition, as described above, the porous body produced from the gelled product G obtained by the cross-linking reaction of a plurality of alginic acid molecules also has the advantage of being excellent in heat resistance compared to the case of using other polysaccharides. .

In order to appropriately control the gelation speed of the solution S, the solution S may be cooled while the acid generator is being added to the solution S and/or after the acid generator is added. When the gelation speed of the solution S is appropriately controlled, the gelation tends to proceed uniformly. In addition, when the gelation progresses unevenly, the elastic modulus and apparent density of the porous body formed from the obtained gelled material G tend to increase. The gelation rate can be evaluated based on the time from the addition of the acid generator until the fluidity of the solution S is lost (gelling start time). As an example, the gelation start time is preferably 5 seconds or longer, more preferably 10 seconds or longer. Note that "the fluidity of the solution S is lost" means that the change in the shape of the solution S cannot be visually confirmed when the container holding the solution S is tilted at 45°.

In solution S to which compound C is added, the concentration of compound C is, for example, 0.1 wt% to 2.0 wt%, and in some cases 0.2 wt% to 1.5 wt%. In the solution S to which the acid generator is added, the concentration of the acid generator is, for example, 0.1 wt % to 5.0 wt %, and in some cases 1.0 wt % to 4.0 wt %.

In the production method of the present embodiment, the weight ratio R1 of the amount of compound C added to the amount of alginate added to solution S, and the weight of the amount of acid generator added to the amount of compound C added to solution S Ratio R2 tends to affect the rate of gelation of solution S. The weight ratios R1 and R2 tend to affect the elastic modulus of the obtained porous body and the pH of the culture solution when the porous body is immersed in the culture solution for culturing cells.

The weight ratio R1 is, for example, 0.05 or more, preferably 0.08 or more, more preferably 0.1 or more, and still more preferably 0.2 or more. The weight ratio R1 is, for example, 1.0 or less, preferably 0.8 or less, more preferably 0.7 or less, and even more preferably 0.6 or less. The weight ratio R1 is preferably 0.05 to 1.0, more preferably 0.08 to 0.8, even more preferably 0.1 to 0.7, and 0.2 to 0.6 is particularly preferred.

The weight ratio R2 is, for example, 0.5 or more, preferably 0.7 or more, and more preferably 0.9 or more. The weight ratio R2 is, for example, 10 or less, 8.0 or less, 6.0 or less, 5.0 or less, 4.0 or less, 3.8 or less, 3.6 or less, and further 3.4 or less. good too. The weight ratio R2 is preferably 0.5-10.

A foaming agent may be added to the solution S together with the gelling agent. As the foaming agent, those mentioned above can be used. The blowing agent is preferably added to the solution S before the acid generator is added to the solution S. As an example, the effervescent agent is added to solution S after alginate is added to solution S and before compound C is added to solution S. By using a foaming agent, the solution S can be further subjected to foaming treatment, which tends to further increase the specific surface area A of the porous body. As an example, when a chemical foaming agent capable of generating gas upon contact with an acid is used, the acid generated from the acid generator causes the solution S to undergo gelation and foaming. It can be carried out.

In the solution S to which the foaming agent is added, the concentration of the foaming agent is, for example, 0.1 wt% to 10 wt%.

As described above, the manufacturing method of the present embodiment includes contacting the gelled material G with alcohol and drying the gelled material G after contacting with the alcohol. The method of bringing the gelled product G into contact with alcohol is not particularly limited. As an example, the gelled material G may be brought into contact with alcohol by immersing the gelled material G in alcohol. The temperature of the alcohol brought into contact with the gelled product G is, for example, room temperature. The time for which the gelled product G is brought into contact with the alcohol is not particularly limited, and is, for example, 1 minute to 24 hours, and may be 1 minute to 1 hour. As the alcohol, for example, a lower alcohol having 5 or less carbon atoms can be used, preferably ethanol.

When the gelled product G is brought into contact with alcohol, water contained in the gelled product G is replaced with alcohol. As a result, among the components contained in the gelled product G, components with low solubility in alcohol are partially precipitated and removed from the gelled product G. As an example, components such as polysaccharides (particularly cellulose derivatives such as HPMC) and salts of decomposition products of foaming agents and acid generators (for example, sodium gluconate) are partially precipitated, resulting in gelled substances. tend to be removed from G. Removal of some of the components from the gelled material G tends to reduce the thickness of the pore walls facing the pores (closed pores) contained in the gelled material G.

The drying of the gelled product G may be performed at room temperature or in a heated environment. When the gelled product G is dried by heating, the drying temperature of the gelled product G is not particularly limited, and is, for example, 40°C to 100°C. The drying time of the gelled material G is not particularly limited, and is, for example, 1 minute to 24 hours. By drying the gelled material G, the solvent (alcohol or water) is removed from the gelled material G to obtain a dried material. The solvent content in the dried product is, for example, 10 wt % or less, and may be 1 wt % or less.

In the production method of the present embodiment, for example, the gelled material G whose pore wall thickness has been reduced by contact with alcohol is subjected to a drying treatment. Therefore, the pore walls of the gelled material G shrink during drying, thereby easily forming openings in the pore walls. By forming the openings, in the gelled material G, adjacent independent pores communicate with each other to form continuous pores. Due to the formation of many openings in the pore walls, the dried product obtained from the gelled product G tends to have a large specific surface area per unit volume. Thereby, a porous body having a sufficiently large specific surface area can be produced.

In addition, Patent Document 1 describes that freeze-drying may be performed in the process of producing a porous scaffold. However, freeze-drying is not only a cumbersome process, but also tends to be costly. According to the production method of the present embodiment, a porous body having a large specific surface area can be easily produced by a simple method without using freeze-drying.

The production method of the present embodiment includes, for example, cutting the gelled material G into a predetermined shape before drying the gelled material G, and/or cutting the dried material into a predetermined shape after drying the gelled material G. It may further include a cutting step of cutting into . The cutting step is preferably performed on the gelled material G from the viewpoint of ease of cutting. According to the cutting step, a porous body having a shape suitable for producing cultivated meat can be produced. Examples of the predetermined shape include sheet-like, cube-like, disc-like, and the like.

A dense layer called a skin layer may be formed on the surface of the dried product produced by the production method of the present embodiment. A dried product having a skin layer may be difficult to penetrate with a culture solution for culturing cells. Therefore, in the cutting step, it is preferable to cut the dried material so that the skin layer is removed. The manufacturing method of the present embodiment may not include the cutting step, and the dried product itself obtained by drying the gelled product G may be regarded as a porous body.

The production method of the present embodiment may further include applying an adhesion improver that improves the adhesiveness of cells to the dried product obtained by drying the gelled product G. The coating layer described above can be formed by applying an adhesion improver to the dried product. As the adhesion improver, those described above can be used.

The adhesion improver can be applied, for example, by immersing the dried product in an aqueous solution containing the adhesion improver. The time for immersing the dried product in the aqueous solution is not particularly limited, and is, for example, 1 minute or longer, typically 30 minutes. The temperature of the aqueous solution is, for example, room temperature.

After applying the adhesion improver, you may wipe off excess water and heat dry. The drying temperature is not particularly limited, and is, for example, 50°C or higher, typically 90°C. The drying time is not particularly limited, and is, for example, 30 minutes or more, typically 1 hour.

(Characteristics and uses of porous material)
As described above, the porous body of this embodiment has a specific surface area A per unit volume of 85.0 mm ² /mm ³ or more. A porous body having such a large specific surface area A is suitable for culturing cells because the culture solution for culturing cells can easily penetrate into the inside and the cells themselves can easily reach the inside. . Furthermore, the porous body of the present embodiment tends to swell greatly when the culture solution permeates inside. A highly swollen porous body can be used as a bulky culture substrate.

The porous body of this embodiment is particularly suitable for use as a scaffold material for cultured meat. However, the porous body of the present embodiment can also be used for applications other than scaffolding materials for cultured meat, such as foods other than cultured meat, chemical products, and medicines.

The present invention will be described in more detail below with examples and comparative examples, but the present invention is not limited to these.

(Example 1)
First, 62 g of distilled water was added to a 200 mL disposable cup (square type) and stirred with a homogenizer. The rotation speed of the homogenizer was set at approximately 8000 rpm. Next, 1.5 g of powdered hydroxypropyl methylcellulose (HPMC: manufactured by Dow, METHOCEL® E19) was added little by little to the cup and stirred for 3 minutes at a rotation speed of about 8000 rpm to obtain a solution containing HPMC. got The solution was foamy with stirring and foaming was occurring.

Next, 0.1 g of powdered glucomannan (GM: Rheolex (registered trademark) LM manufactured by Shimizu Chemical Co., Ltd.) was added little by little to the cup and stirred for 3 minutes at a rotation speed of about 8000 rpm. This resulted in a solution containing HPMC and GM and in which bubbles were generated.

Next, after increasing the rotation speed of the homogenizer to about 18000 rpm, 1.2 g of powdered sodium alginate (ALG: I-3G manufactured by Kimika Co., Ltd.) was added little by little to the solution. The solution was stirred for an additional 3 minutes while maintaining the rotation speed of the homogenizer. This increased the viscosity of the solution.

Next, while the rotation speed of the homogenizer was maintained at about 18000 rpm, 0.5 g of powdered sodium hydrogen carbonate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., reagent special grade) was added little by little to the solution and stirred for 1 minute.

Next, while maintaining the rotation speed of the homogenizer at about 18000 rpm, 0.36 g of powdered calcium carbonate (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., special reagent grade) was added little by little to the solution and stirred for 1 minute.

Next, 1.9 g of powdered glucono delta lactone (GDL: Fuji Glucon (registered trademark) manufactured by Fuso Chemical Industry Co., Ltd.) was added little by little to the solution while the rotation speed of the homogenizer was maintained at about 18000 rpm. At this time, a food coloring was also added in order to visually confirm the stirring state. The solution was stirred for an additional 5 minutes while maintaining the rotation speed of the homogenizer while ensuring that the coloring of the solution with the food coloring was uniform. At the stage when GDL was added to the solution, foaming by sodium hydrogencarbonate started and gelation of the solution started. Evaporation with sodium bicarbonate further increased the volume of the solution.

Next, the stirring by the homogenizer was stopped, and the solution was allowed to stand still in the disposable cup. In the solution, a cross-linking reaction of a plurality of alginic acid molecules proceeded via divalent calcium ions to form a gelled product having a porous structure.

Next, I took out the gel from the disposable cup. The gelled product was cut with a cutter knife and a punch to obtain 40 disk-shaped gelled products having a thickness of 5.5 mm and a diameter of 8 mm.

Next, 100 mL of ethanol (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd., Wako first grade) was added to a 150 mL disposable cup. 40 disk-shaped gelled products were immersed in this ethanol, and the gelled products were brought into contact with the ethanol. At this time, water contained in the gelled product was replaced with ethanol, and components with low solubility in ethanol partially precipitated, making the ethanol turbid. After the disk-shaped gelled product was immersed in ethanol for 30 minutes, the gelled product was removed from the ethanol.

Next, the disk-shaped gelled product was arranged in an aluminum cup, heated to 90°C and dried for 1 hour to obtain a dried product. The skin layer formed on the surface of this dried product was scraped off with a cutter to obtain a porous body of Example 1.

(Example 2)
A porous body of Example 2 was obtained in the same manner as in Example 1, except that the gelled product was not immersed in ethanol.

(Example 3)
Same as Example 1 except that the amount of sodium hydrogen carbonate added was changed to 0.25 g, the amount of calcium carbonate added was changed to 0.6 g, and the amount of GDL added was changed to 1.8 g. A porous body of Example 3 was obtained by the method.

(Example 4)
By the same method as in Example 1, except that the amount of HPMC added was changed to 3.0 g, the amount of calcium carbonate added was changed to 0.6 g, and the amount of GDL added was changed to 1.8 g. A porous body of Example 4 was obtained.

[Imaging with an X-ray CT device]
The produced porous body was photographed with an X-ray CT apparatus by the method described above for the specific surface area A. Specifically, using the prepared disk-shaped porous body as a test piece, an area of 2 mm long×2 mm wide×0.6 mm thick near the center of the test piece was photographed. Xradia 620 Versa manufactured by Zeiss was used as the X-ray CT apparatus. A tomographic image was created by reconstructing a transmission image obtained by imaging, and a three-dimensional reconstructed image and a reconstructed cross-sectional image were also created. The obtained stereoscopic image (three-dimensional reconstructed image) was binarized to create a binarized image. The three-dimensional reconstruction image obtained from the porous body of Example 1 is shown in FIG. 1A, the three-dimensional reconstruction image obtained from the porous body of Example 2 is shown in FIG. The original reconstructed image is shown in FIG. 3A, and the three-dimensional reconstructed image obtained from the porous body of Example 4 is shown in FIG. 4A.

[Continuous cell rate]
Using ImageJ, the total volume of all pores included in the photographed region was calculated from the above binarized image. Furthermore, Avizo was used to calculate the volume of the pore having the largest volume from the three-dimensional image, and the obtained calculated value was regarded as the volume of the continuous pore included in the photographed area. The ratio of the volume (mm ³ ) of continuous pores included in the above region to the total value (mm ³ ) of the volume of all pores included in the above region is calculated, and the obtained calculated value is the continuity of the porous body. It was specified as the foam rate.

[Porosity]
Using ImageJ, the volume ratio of the holes in the imaged region and the volume ratio of the portion (matrix) other than the holes in the region were calculated from the binarized image. The calculated pore volume ratio can be regarded as the porosity of the porous body.

[Specific surface area]
Using Avizo, the surface area a of the porous body in the photographed region was calculated from the three-dimensional image. Furthermore, using ImageJ, the volume v of the matrix in the photographed region was calculated from the binarized image. A value obtained by dividing the surface area a (mm ² ) by the matrix volume v (mm ³ ) was regarded as the specific surface area A of the porous body.

[Observation with a scanning electron microscope (SEM)]
The cross section of the produced porous body was observed by SEM. The SEM image of the porous body of Example 1 is shown in FIG. 1B, the SEM image of the porous body of Example 2 is shown in FIG. 2B, the SEM image of the porous body of Example 3 is shown in FIG. is shown in FIG. 4B. From the SEM images, it can be seen that in these porous bodies, a part of the surface facing the independent pore is open, and the independent pore and the adjacent independent pore communicate with each other. As can be seen from the SEM images, in the porous bodies of Examples 1 and 3 to 4, the number of holes (above-mentioned holes p2) communicating between independent pores was larger than that of the porous body of Example 2.

As can be seen from Table 1, the porous bodies of Examples 1, 3 and 4 produced by contacting the gelled product with alcohol had a larger specific surface area A than the porous body of Example 2. A porous body having a specific surface area A as large as about 85.0 mm ² /mm ³ or more easily penetrates the culture solution for culturing cells into the inside, and the cells themselves easily reach the inside. suitable for doing

The porous body of this embodiment is suitable as a scaffolding material for producing cultured meat. The porous body of the present embodiment can also be used for applications other than scaffolding materials for cultured meat, such as foods other than cultured meat, chemical products, and medicines.

Claims (19)

A porous body containing an edible material and having a specific surface area per unit volume of 85.0 mm ² /mm ³ or more. The porous body according to claim 1, which is edible. The porous body according to claim 1, which has a porosity of 90% or more. The porous body according to claim 1, wherein the open cell rate is 90% or more. The porous body according to claim 1, wherein the maximum diameter of pores within a range of 1800 µm long x 2400 µm wide is 1 to 1000 µm when the cross section of the porous body is observed with a scanning electron microscope. The porous body according to claim 1, wherein the edible material contains polysaccharides. The porous body according to claim 1, wherein the edible material contains alginic acid and/or alginate. The porous body according to claim 1, wherein the edible material contains at least one selected from the group consisting of glucomannan and cellulose derivatives. a body portion containing the edible material;
a coating layer that includes an adhesion improver that improves cell adhesion and that covers the main body;
The porous body according to claim 1, having The porous body according to claim 1, which is a foam. Contacting a gelled product containing an edible material and water and having a porous structure with alcohol;
drying the gelled product after contact with the alcohol;
A method for producing a porous body, comprising: The production method according to claim 11, wherein the alcohol is ethanol. frothing a solution comprising a foaming agent and water;
adding a gelling agent to the foamed solution to produce the gelled product;
12. The manufacturing method of claim 11, further comprising: The manufacturing method according to claim 13, wherein at least one selected from the group consisting of the foaming agent and the gelling agent contains the edible material. The production method according to claim 13, wherein the gelling agent includes an alginate, a compound that generates divalent metal ions, and an acid generator. The production method according to claim 13, wherein the foaming agent contains at least one selected from the group consisting of glucomannan and cellulose derivatives. The manufacturing method according to claim 13, further comprising adding a foaming agent to the solution. The manufacturing method according to claim 17, wherein the foaming agent is a chemical foaming agent that generates gas upon contact with an acid. 12. The production method according to claim 11, further comprising applying an adhesion improver for improving adhesion of cells to the dried product obtained by drying the gelled product.

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