TWI414596B - Method for production of biomembrane - Google Patents

Abstract

A method and a tray for production of a bio-membrane are disclosed in the present invention. The bio-membrane produced in the method and in the tray of the present invention can be processed into a mask, wherein processes for changing the shape of the bio-membrane are unnecessary. The mask produced from the bio-membrane can be efficient to maintain the skin, and to supply the moisture thereto.

Description

Biofilm manufacturing method

The present invention relates to a method for producing a biofilm and a culture container, and more particularly to a method and a culture container suitable for forming a biofilm by using a microorganism to produce a polymer.

In order to meet the various needs of human skin care, there are a variety of masks on the market that meet the needs. Among them, the common ones are non-woven fabrics, and various types of skin care products and cosmeceutical ingredients are added. Since the non-woven fabric mask is not a fabric woven by a thread, but a textile material and a textile process which is not woven, it has the properties of gas permeability, moisture absorption, durability, chemical resistance, insulation, and the like. . However, due to the excessively ventilating nature, the various active substances added therein are exposed to both sides, causing decomposition or volatilization too fast to make the mask function not fully exerted.

Therefore, the production of high molecular polymers through microorganisms has been developed, collectively referred to as biofilms. After treatment, such biofilms are suitable for various uses, such as dietary fiber for eating, facial mask for skin care, and the like. This kind of biofilm has strong water absorption and can load a large amount of nutritive functional substances. If it is made into a mask, it can provide nutrients on the surface of the skin, and because it has a large number of free functional groups, it is easy to absorb the dirt of the skin. In addition, it has many good effects.

In order to prevent the eyes, nose, mouth and other organs of the user's face from being obscured, it is impossible to see, smell, eat, talk, etc. Whether it is a traditional non-woven fabric or a biofilm to make a mask, a reserved hole is provided to meet The user's aforementioned needs. However, the preparation of the reserved hole must be formed by the secondary knife film, which causes complicated construction, and often causes the edge of the mask or the reserved hole to be damaged, so that the yield of the mask product is reduced, resulting in waste of cost.

In view of the above disadvantages, the main object of the present invention is to provide a culture container for a biofilm, by which the biofilm cultured by the culture container does not need to undergo a more dynamic shape process, and the container can be stacked vertically to save lateral production. The space, and the longitudinally stacked container still has a good ventilation effect.

Another object of the present invention is to provide a method for producing a biofilm, which utilizes a microorganism strain capable of naturally producing a high molecular polymer such as a polysaccharide or a polyglutamic acid to form a specific shaped organism in a container of a specific shape. membrane. Moreover, the biofilm can be made into a mask without increasing the yield by any shape-changing process, and the mask can be an effective mask for skin care and skin replenishment.

In order to achieve the above object, the present invention provides a culture container for a biofilm, comprising a bottom plate; a peripheral wall, the peripheral wall is located on the bottom plate, surrounding the periphery of the bottom plate, and forming an accommodation space with the bottom plate; and at least one A protrusion located at and protruding from the surface of the bottom plate.

In the culture container provided above, a concave edge may be formed on the upper portion of the peripheral wall, and a convex edge may be formed on the lower portion of the peripheral wall. And the upper surface of the peripheral wall may have a plurality of slots, and the lower surface of the peripheral wall of the slots may include a plurality of protruding bolts. If the culture containers are stacked in the longitudinal direction, the lateral culture space can be saved, and the convex plugs are corresponding to the slots, or the concave and convex edges are correspondingly fitted to the upper and lower edges, and the longitudinal direction of the culture container is further The structure of the stack makes it difficult to loosen it. The depth of the slots may be less than or equal to the height of the bumps, and the cross-sectional area of the upper portions of the bumps may be greater than or equal to the cross-sectional area of the slots.

In addition, the upper or lower side of the side wall of the peripheral wall may have a plurality of notches, and the notches do not define the shape thereof. The shape of the peripheral wall is not limited and may be circular, elliptical, polygonal, irregular, or egg-shaped. The protrusion is also not limited, and may be columnar, tapered, or sheet-shaped, and the position of the protrusion in the container is not limited, and may correspond to an eye, a nose, a mouth, and the like of a person. The bottom plate can be planar or non-planar, and the non-planar refers to a curved surface.

In the above culture container, the molding method of the at least one protrusion may be recessed from the outer side of the bottom side to the inner side to form a relative relief, which can effectively save the use of the material; as the material used is not limited, as Transparent or non-transparent organic materials such as PVC (polyvinyl chloride), PP (polypropylene) or PE (polyethylene), or made of anti-oxidation metal. In addition, the surface of the culture dish can be selectively coated with a layer of PEP (rustproof film).

The invention further provides a method for producing a biofilm, comprising the steps of: (a) placing a strain in a first medium, shaking the culture to form a strain; (b) placing the strain in a culture vessel, the culture The container contains a second medium, and the culture container comprises a bottom plate, a peripheral wall, and at least one protrusion, wherein the peripheral wall is located above the bottom plate, surrounds the periphery of the bottom plate, and forms an accommodation space with the bottom plate. And the at least one protrusion is located at and protrudes from a surface of the bottom plate; and (c) forms a biofilm of a predetermined thickness.

In the production method provided by the present invention, the strain is preferably Acetobacter spp., Gluconacetobacter spp., Xanthomonas spp., or Bacillus. ( Bacillus spp.). Preferably, the strain of the genus Acetobacter is A. xylinum ; the strain of the genus Acetobacter is more preferably selected from the group consisting of G. xylinus subsp. Xylinus ATTC 10821, G. xylinus subsp. Xylinus ATTC 700187 and G. Hansenii ATCC A group consisting of 23769 ; the strain of Xanthomonas is more preferably X. campeastris ; and the strain of Bacillus is more preferably B. subtilis var Natto .

In the manufacturing method provided by the present invention, the first medium and the second medium contain carbohydrates, nitrogen-containing compounds, minerals, and auxiliary growth factors. The content of the carbohydrate is preferably from 1% to 30%, the content of the nitrogen-containing compound is preferably from 0.1% to 6%, the content of the mineral is preferably from 0.05% to 3%, and the auxiliary growth factor is The content is preferably from 0.05% to 2%. The carbohydrate is not limited, and is preferably a group consisting of at least one selected from the group consisting of a monosaccharide, a disaccharide or a polysaccharide, a compound having a carboxyl group, and a mixture thereof. The nitrogen-containing compound is not limited, and is preferably at least one selected from the group consisting of yeast extract, peptone, soybean powder, gelatin, and mixtures thereof. The mineral is not limited, and is preferably at least one selected from the group consisting of a chloride salt, an ammonium salt, a sulfur salt, a potassium salt, a phosphate salt, a magnesium salt, a sodium salt, and a mixture thereof. The auxiliary growth factor is not limited, and is preferably at least one selected from the group consisting of vitamins, niacin, citric acid, and mixtures thereof.

In the manufacturing method provided by the present invention, in order to obtain a mass-produced inoculum, the strain and the strain may be cultured at 25 ° C to 37 ° C, and further comprise a step (a1) after the step (a), The inoculum is amplified and cultured, so that after the inoculum is amplified and cultured, a large number of inoculum can be obtained to facilitate the mass production of the subsequent biofilm.

The biofilm produced by the manufacturing method of the present invention can be used as a biofilm for effectively providing skin moisture and maintaining the effect of the skin by using a subsequent process, the membrane, the eye mask, the pleura or the lip membrane. And its subsequent process does not require any processing of its shape, but can prevent damage and yield degradation caused by the process of shape processing.

In the following examples, the strain used in the method for producing a biofilm of the present invention is Acetobacter spp., Gluconacetobacter spp., Xanthomonas spp., or spore. Bacillus spp. In the genus Acetobacter, A. xylinum is mainly used as a strain; in the genus Acetobacter, G. xylinus subsp. Xylinus ATTC 10821, G. xylinus subsp. Xylinus ATTC 700187 and G. Hansenii ATCC 23769 are mainly used as strains; In Xanthomonas, X. campeastris is mainly used as a strain; and in Bacillus, B. subtilis var Natto is mainly used as a strain. Among the above strains, A. xylinum and G. xylinus are different strain names, but they are actually the same strain.

In the method of producing a biofilm of the present invention, the medium mainly contains carbohydrates, nitrogen-containing compounds, minerals, and auxiliary growth factors. In the following examples, the content of the carbohydrate ranges from 1% to 30%, the content of the nitrogen-containing compound ranges from 0.1% to 6%, the content of the mineral ranges from 0.05% to 3%, and the auxiliary growth factor The content ranges from 0.05% to 2%. Further, the types of carbohydrates used in the examples are usually monosaccharides, disaccharides or polysaccharides, compounds having a carboxyl group, or a mixture of the above-mentioned carbohydrates; the types of nitrogen-containing compounds are usually yeast extract, peptone ( Peptone), soy flour, gelatin, or a mixture of the above nitrogen-containing compounds; the mineral species are usually chloride, ammonium, sulfur, potassium, phosphate, magnesium, sodium, or the above salts Mixture; Auxiliary growth factor is typically a mixture of vitamins, niacin, citric acid, or a supplemental growth factor as described above.

The shape of the container used in the method for producing a biofilm of the present invention generally depends on the desired shape. If a rectangular shape is required, a rectangular parallelepiped container is used, and if a circular shape is required, a cylindrical container is used. In the following embodiments, the biofilm growth container of the present invention is used as shown in FIGS. 1A to 4 to obtain the desired shape. However, this does not mean that the container of FIG. 1A to FIG. 4 can be used only, and the material thereof can be Made of PVC (polyvinyl chloride), PP (polypropylene) or PE (polyethylene), or made of anti-oxidation metal. The surface of the selectively cultivable container is coated with PEP (anti-rust film), which is a polymer anti-corrosion film, which can be covered by a barrier to prevent penetration of corrosive gas, and penetrates rust in corrosive gas. The membrane will be neutralized, and the corrosive gas that will be coated inside will react to neutralize it, making the air inside the coating no longer corrosive.

Example 1~30

A. xylinum cultured on a slanted surface , inoculated with about 1 to 5 platinum ear to the sterilized liquid medium formula (as shown in Table 1 below), shaken at 37 ° C for 1 to 3 days, and then cultured at 2 to 30 times. The culture is magnified again, and the amplification culture can be carried out in a fermentation tank or a shaking incubator, and the growth-producing inoculum can be produced after 1 to 2 days of growth.

The culture container used in this embodiment is shown in FIG. 1A, and the culture container comprises a bottom plate 11; a peripheral wall 12, the peripheral wall 12 is located above the bottom plate 11, surrounds the periphery of the bottom plate 11, and forms with the bottom plate 11 An accommodation space; and at least one protrusion 13 is located at and protrudes from a surface of the bottom plate 11. The protrusion 13 described above comprises a plurality of different shapes, for example, the first protrusion 132 forms an elliptical cylinder, the second protrusion 133 is tapered, and the third protrusion 133 is a leaf-shaped column or the like, which can be formed according to the desired shape. Biofilm shapes are designed and placed in different relative positions.

Further, as shown in FIG. 1B, about 1% to 50% of the inoculum is mixed with the culture liquid phase of Table 2, and placed in the accommodating space of the culture container, and the biofilm is grown into a shape according to the shape of the model container. 20, and the culture is allowed to stand at a constant temperature until the biofilm 20 is formed to an appropriate thickness to complete the production stage. The biofilm 20 is then removed, and as shown in Fig. 5, the biofilm 20 is formed with a plurality of openings 232, 233, and 234 at the relative positions of the projections 13 shown in Fig. 1A.

After testing with different culture fluids, the results are shown in Fig. 6. According to the weight of biofilm formation, if the culture solution contains 1% yeast extract, 0.1% calcium carbonate and 2.5% glucose are added, which is the best organism. Membrane production.

Examples 31 to 60

A. xylinum cultured on a slant surface , take about 1 to 5 platinum ear, inoculate it into a sterilized liquid medium formula (as in Table 1 of Examples 1 to 30), and grow at 37 ° C for 1 to 3 days with shaking culture. 2~30 times of the culture solution is re-amplified and cultured. The scale-up culture can be carried out in a fermentation tank or a shaking incubator. After 1 to 2 days of growth, the inoculum can be produced in a mass production scale.

The culture container used in this embodiment is as shown in FIG. 2, and the culture container is substantially the same as the container shown in FIG. 1A, except that the culture container additionally has a plurality of slots 123 on the upper surface of the peripheral wall 12, and The lower surface of the peripheral wall 12 has a plurality of protruding bolts 124, and the protruding bolts 124 correspond to the slots 123.

The culture solution is mixed with the culture liquid phase of Table 2 and placed in the accommodating space of the above-mentioned culture container, and the culture container can be vertically stacked and permeated through the culture container as shown in FIG. The protruding bolts 124 and the slots 123 are configured to be more stably longitudinally stacked, so that the culture containers stacked on each other are loosened and tripped, thereby saving lateral space during production. According to the shape of the model container, the biofilm 20 of a certain shape is grown, and the culture is allowed to stand at a constant temperature until the biofilm 20 is formed to an appropriate thickness to complete the production stage. Then, the biofilm 20 is taken out to obtain a biofilm 20 having a shape as shown in FIG.

After testing with different culture fluids, the results are shown in Fig. 7. According to the weight of biofilm formation, if the culture solution contains 0.5% yeast extract, 2% calcium carbonate and 5% glucose are added, which is the best organism. Membrane production.

Examples 61 to 63

G. xylinus subsp. Xylinus ATTC 10821, G. xylinus subsp. Xylinus ATTC 700187, and G. Hansenii ATCC 23769, which were cultured in slant, were each inoculated with about 1 to 5 platinum ear, and inoculated into a sterilized liquid medium formulation (like Table 1) of Examples 1 to 30, grown in shaking culture at 30 ° C for 1 to 2 days, and then re-amplified with 2 to 30 times of culture medium. This amplification culture can be carried out in a fermentation tank or a shaking incubator, and grow 1 to 2 After the day, it can be a mass production scale.

The culture container used in this embodiment is as shown in FIG. 3, and the culture container is substantially the same as the container shown in FIG. 2, but differs in that the upper portion of the inner peripheral wall 12 has a concave edge 121 and a lower portion has a convex edge. 122, and the diameter of the upper portion of the protruding bolts 124 is larger than the diameter of the slots 123.

The amount of the inoculum is mixed with the culture liquid of Table 2 and placed in the accommodating space of the culture container, and the concave layer rim 121 and the convex edge 122 are correspondingly fitted, and the culture can be vertically stacked. container. Although the concave edge 121 and the convex edge 122 are only formed on the inner side of the peripheral wall 12, the concave edge 121 and the convex edge 122 may be formed on the outer side of the peripheral wall 12.

In addition, the longitudinally stacking the culture container may also be vertically stacked corresponding to the slots 123 through the protruding bolts 124 as shown in FIG. 3, and the longitudinally adjacent culture containers may be separated by a certain height. Therefore, air can flow through this height, so ventilation can be increased to promote biofilm growth. According to the shape of the model container, the biofilm 20 of a certain shape is grown, and the culture is allowed to stand at a constant temperature until the biofilm 20 is formed to an appropriate thickness to complete the production stage. Then, the biofilm 20 is taken out to obtain a biofilm 20 having a shape as shown in FIG.

Example 64

X. campeastris cultured in slant , inoculate about 1~5 platinum ear, to the sterilized liquid medium formula (as shown in Table 5 below), shake culture at 30 °C for 1~2 days, 2~30 times culture solution The amplification culture is carried out again, and the amplification culture can be carried out in a fermentation tank or a shaking incubator, and the growth-producing inoculum can be produced after 1 to 2 days of growth.

The culture container used in this embodiment is as shown in FIG. 4, and the culture container is substantially the same as the container shown in FIG. 2. The only difference is that the culture container has a notch 125 on the side of the peripheral wall 12, and the concave The port 125 is not limited to four sides of the peripheral wall 12 as shown in FIG. 4, and may be a single side, an opposite side, or three sides.

The amount of the inoculum is mixed with the culture liquid of Table 2 and placed in the accommodating space of the culture container, and the culture container can pass through the protruding bolts 124 and the grooves as shown in FIG. The holes 123 are vertically stacked, and the air is circulated by the notches 125 to increase ventilation, which is advantageous for biofilm growth. According to the shape of the model container, the biofilm 20 of a certain shape is grown, and the culture is allowed to stand at a constant temperature until the biofilm 20 is formed to an appropriate thickness to complete the production stage. Then, the biofilm 20 is taken out to obtain a biofilm 20 having a shape as shown in FIG.

Example 65

B. subtilis var Natto cultured on a slant surface , take about 1 to 5 platinum ear, inoculate the sterilized liquid medium formula (as shown in Table 5 of Example 64), and grow at 37 ° C for 1 to 3 days with shaking culture. 2~30 times of the culture solution is re-amplified and cultured. The scale-up culture can be carried out in a fermentation tank or a shaking incubator. After 1 to 2 days of growth, the inoculum can be produced in a mass production scale.

In the present embodiment, the container shown in Figs. 1 to 4 above can be used. About 1% to 50% of the inoculum was mixed with the culture liquid phase of Table 2, and placed in the accommodation space of the above culture vessel. According to the shape of the model container, the biofilm 20 of a certain shape is grown, and the culture is allowed to stand at a constant temperature until the biofilm 20 is formed to an appropriate thickness to complete the production stage. Then, the biofilm 20 is taken out to obtain a biofilm 20 having a shape as shown in FIG.

The biofilm prepared in the above Examples 1 to 65 is pretreated with an alkaline solution, and then treated with an acidic solution, and then processed and rinsed and rinsed by a conventional method in the art, and then effectively sterilized and added. After the mask process of ingredients, packaging, and labeling, a biological mask with an effective composition is formed. Therefore, the biofilm provided by the present invention does not need to undergo a process of additionally changing its shape, such as press forming, or cutting, in the process of forming a biological mask, and has a desired shape after being formed, thereby avoiding After the edge of the forming process is damaged, the yield of the mask product can be greatly improved, thereby saving cost and preventing waste.

The above-mentioned embodiments are merely examples for convenience of description, and the scope of the claims is intended to be limited to the above embodiments. Other advantages and utilities of the present invention will be readily apparent to those skilled in the art from this disclosure. The present invention may be embodied or applied in various other specific embodiments, and various modifications and changes can be made without departing from the spirit and scope of the invention.

11. . . Bottom plate

12. . . Zhou wall

121. . . Concave edge

122. . . Convex edge

123. . . Slot

124. . . Bolt

125. . . Notch

13. . . obstructive

132. . . First protrusion

133. . . Second protrusion

134‧‧‧ Third protrusion

232~234‧‧‧ openings

20‧‧‧Biofilm

1A is a schematic view of a biofilm culture container according to an embodiment of the present invention.

Figure 1B is a schematic illustration of the cultivation of a biofilm in accordance with an embodiment of the present invention.

2 is a schematic view showing the culture of a biofilm according to an embodiment of the present invention.

Figure 3 is a schematic illustration of the cultivation of a biofilm in accordance with an embodiment of the present invention.

Figure 4 is a schematic illustration of the cultivation of a biofilm in accordance with an embodiment of the present invention.

Figure 5 is a schematic diagram of a biofilm produced by an embodiment of the present invention.

Fig. 6 is a graph showing the comparison of the amount of biomass cellulose produced in Examples 1 to 30 of the present invention.

Fig. 7 is a graph showing the comparison of the amount of biomass cellulose produced in Examples 31 to 60 of the present invention.

12. . . Zhou wall

121. . . Concave edge

122. . . Convex edge

123. . . Slot

124. . . Bolt

13. . . obstructive

132. . . First protrusion

133. . . Second protrusion

134. . . Third protrusion

232~234. . . Opening

20. . . Biofilm

Claims (15)

A method for directly forming a biofilm comprises the steps of: (a) placing a strain in a first medium and shakingly culturing to form a bacterium, wherein the strain is Acetobacter spp., Acetobacter glucomannii Genus ( Gluconacetobacter spp.), Xanthomonas spp., or Bacillus spp.; (b) placing the inoculum in a culture vessel containing a second medium And the culture container comprises a bottom plate, a peripheral wall, and at least one protrusion, wherein the peripheral wall is located above the bottom plate, surrounds and connects the circumference of the bottom plate, and forms an accommodation space with the bottom plate, and the peripheral wall has a plurality of a slot and a plurality of latches, the slots are located on the upper surface of the peripheral wall, the latches corresponding to the slots and located at a lower surface of the peripheral wall; and the at least one protrusion is connected to and protrudes from the bottom plate a surface in which two culture containers are correspondingly stacked by inserting the plugs of one of the culture containers into the slots of the other culture container, and the culture containers have a predetermined distance between them to facilitate the respective culture containers. And (c) forming a biofilm having a predetermined thickness and a predetermined shape; wherein the strain of Acetobacter is A.xylinum , and the strain of Acetobacter is G.xylinus subsp. Xylinus Or G. Hansenii , the strain of Xanthomonas is X. campeastris , and the strain of the genus Bacillus is B. subtilis var Natto . The manufacturing method of claim 1, wherein the first medium and the second medium comprise carbohydrates, nitrogen-containing compounds, minerals, and auxiliary growth factors. The manufacturing method according to claim 2, wherein the content of the carbohydrate is 1% to 30%, the content of the nitrogen-containing compound is 0.1% to 6%, and the content of the mineral is 0.05% to 3%. And the content of the auxiliary growth factor is 0.05% to 2%. The method of manufacture of claim 2, wherein the carbohydrate is at least one selected from the group consisting of a monosaccharide, a disaccharide or a polysaccharide, a compound having a carboxyl group, and a mixture thereof. The manufacturing method according to claim 2, wherein the nitrogen-containing compound is at least one selected from the group consisting of yeast extract, peptone, soybean powder, gelatin, and a mixture thereof. Group of. The manufacturing method according to claim 2, wherein the mineral is at least one selected from the group consisting of a chloride salt, an ammonium salt, a sulfur salt, a potassium salt, a phosphate salt, a magnesium salt, a sodium salt, and a mixture thereof. group. The manufacturing method according to claim 2, wherein the auxiliary growth factor is at least one selected from the group consisting of vitamins, niacin, citric acid, and mixtures thereof. The manufacturing method according to claim 1, wherein after the step (a), the step (a1) is further included, and the inoculum is expanded and cultured to obtain a mass-produced inoculum. The manufacturing method of claim 1, wherein the upper portion of the peripheral wall has a concave edge and the lower portion of the peripheral wall has a convex edge. The manufacturing method of claim 1, wherein the slots have a depth less than or equal to a height of the protruding bolts. The manufacturing method of claim 1, wherein the cross-sectional area of the upper portions of the protruding bolts is greater than or equal to the cross-sectional area of the slots. The manufacturing method according to claim 1, wherein the upper portion or the lower portion of the side wall of the peripheral wall has a plurality of notches. The manufacturing method according to claim 1, wherein the protrusion is columnar, tapered, or sheet-shaped. The manufacturing method according to claim 1, wherein the peripheral wall is surrounded by a circular shape, an elliptical shape, a polygonal shape, an irregular shape, or an egg shape. The manufacturing method of claim 1, wherein the container is stacked through the plurality of slots through the plurality of slots.