JP3835848B2 - Method for producing lactic acid - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing lactic acid. More specifically, the present invention is capable of culturing bacterial cells in a very well-disintegrated state, increasing the mass transfer rate of oxygen, substrate, etc. The present invention relates to a method for producing lactic acid capable of improving the speed.
[0002]
[Prior art]
Lactic acid is a substance useful as a food additive, a raw material for brewing, a raw material for pharmaceuticals and agricultural chemicals, a synthetic raw material for chemicals and plastics, and is produced by a chemical synthesis method and a fermentation method.
[0003]
In the latter fermentation method, a method is used in which a filamentous fungus capable of producing lactic acid is inoculated into a liquid medium containing saccharides and the like and cultured and fermented while stirring by aeration. The filamentous fungus is used as a support or carrier. It is also often used by supporting it on the surface.
[0004]
Japanese Patent Application Laid-Open No. 57-144985 describes the use of L-lactic acid-producing cells immobilized on a gel carrier for lactic acid fermentation.
[0005]
Japanese Patent Laid-Open No. 6-253871 discloses that lactic acid can be produced by bringing a lactic acid fermentation broth into contact with an aggregate of fibers carrying lactic acid-producing microorganisms, in particular, a biocompatible non-woven fabric carrying lactic acid-producing microorganisms. Are listed.
[0006]
[Problems to be solved by the invention]
When filamentous fungi are grown in submerged culture (liquid culture), they take various forms from pellets entangled with mycelium to pulp that is uniformly dispersed in the culture solution. Since the mycelial form in the submerged culture affects industrial substance production, it is a key to high-yield fermentation production to induce a highly productive mycelial form.
[0007]
At present, we are trying to control the morphology of bacterial cells by lactic acid production methods such as culture conditions, but it is difficult to obtain the conditions, and even if the expected morphology is obtained, it is easily affected by subtle environmental changes. . Many filamentous fungi, especially molds, form pellets with large and small spheres during deep culture, but depending on the size, diffusion of oxygen or substrate occurs in the cells inside the pellet.
[0008]
When the applicant conducts culture by supporting filamentous fungi on a holmite clay mineral such as sepiolite, the mass transfer rate of oxygen, substrate, etc. is increased, the production rate of the substance is improved, and the separation of the cells is also effective. It has been found that this is done (Japanese Patent Laid-Open No. 7-115961).
[0009]
The present inventors applied this method to a method for producing lactic acid. However, when sepiolite was added, the time during which the cells were kept in a stable dispersed state was slightly extended as compared with the case where sepiolite was not added. However, finally, it was inevitable that a mass of bacterial cells would stick to the wall of the fermentation apparatus and further cause troubles such as a decrease in production speed.
[0010]
Therefore, the object of the present invention is to cultivate the cells in a very well-disintegrated state, and to increase the mass transfer rate of oxygen, substrate, etc., thereby significantly increasing the production rate and yield of the material. Another object of the present invention is to provide a method for producing lactic acid by a fermentation method that can be increased.
Another object of the present invention is to provide a method capable of fermentative production of lactic acid with good workability and productivity without growth inhibition or increase in viscosity with respect to bacterial cells.
[0011]
[Means for Solving the Problems]
According to the present invention, a formicite clay mineral is added to a liquid medium inoculated with a filamentous fungus capable of producing lactic acid or a spore thereof, and cultured, and aggregated when the growth rate of the bacterial cells becomes slow after the start of the culture. There is provided a method for producing lactic acid, characterized in that an agent is added and fermentation is performed while adding an alkaline substance so as not to lower the pH.
[0012]
In the production method of the present invention,
1. The holmite clay mineral is sepiolite having a fiber diameter of 70 to 400 angstroms and a fiber length of 0.2 to 400 μm ;
2. Adding holmite clay mineral to the liquid medium to a concentration of 1 to 50 g / L, particularly 1 to 10 g / L;
3. The flocculant is an anionic, nonionic or amphoteric flocculant;
4). Adding a flocculant to the liquid medium to a concentration of 1 to 20 mg / L, especially 1 to 10 mg / L;
5). Fermenting while maintaining the pH of the medium at 4-8, especially 5-7,
6). Culturing and fermenting while stirring by aeration,
7). The alkaline substance is calcium carbonate,
Is preferred.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
In the method for producing lactic acid according to the present invention, a holmite clay mineral represented by sepiolite was selected as a carrier for filamentous fungi having an ability to produce lactic acid, and the culture was performed in a liquid medium to which the holmite clay mineral was added. It is a remarkable feature that the flocculant is added when the growth rate of the bacterial cells becomes slow after the start of the culture.
[0014]
Since chain clay minerals such as sepiolite are fibrous and have a three-dimensional chain structure and voids in the gaps, they are more effective than filamentous fungi compared to ordinary clay minerals and other inorganic carriers. By improving the entanglement and fixing the filamentous fungus firmly and stably, the fixed cell can be made porous, so that the supply of the culture solution and oxygen to the cell can be improved, and the filterability can be improved.
[0015]
In the present invention, by adding a flocculant to the liquid medium at the end of the culturing stage of the bacterial cells, the production rate of lactic acid is remarkably increased as compared to the case where the flocculant is not added, as shown in the examples described later. And the final yield can be increased.
[0016]
It has already been pointed out that when sepiolite alone is added to the liquid medium, the bacterial cells eventually aggregate into large clumps, adhere to the walls of the apparatus, etc., and the production rate also decreases. By adding a flocculant to the medium, the formation of large lumps is suppressed, and the production rate and yield are improved.
[0017]
In general, the flocculant is used to agglomerate suspended matters in a liquid. Therefore, the addition of the flocculant to the medium is expected to promote the aggregation of bacterial cells. The medium used in the present invention Then, surprisingly, the strong flocculation of the cells is prevented, and the loosely dispersed state is maintained until the final stage of fermentation.
[0018]
According to the observations of the present inventors, the addition of a flocculant results in mild floc-like aggregation of filamentous fungus cells fixed on holmite clay mineral, but this floc-like aggregation state is extremely stable and fluid. It is also believed that it has excellent dispersion stability and maintains a high mass transfer rate and mass production rate of oxygen and substrates.
[0019]
The chain clay mineral used in the present invention is a fibrous magnesium silicate clay mineral represented by sepiolite, attapulgite, and palygorskite, etc., but these have a three-dimensional chain structure and are two-dimensional like talc. Unlike the three-dimensional crystal structure, the pores formed in the gaps of the chain structure have a large specific surface area in the range of 100 to 350 m ² / g in the BET method specific surface area, and also have an adsorption action. It is a porous clay mineral.
[0020]
Sepiolite or the like is also characterized by being not swollen in an aqueous system unlike ordinary layered clay minerals represented by montmorillonite, which is also a porous clay mineral.
[0021]
Due to the characteristics of such chain clay minerals such as sepiolite, that is, that it is fibrous and that it is porous, it is less likely to be a filamentous fungus than ordinary clay minerals and other inorganic carriers. By improving the entanglement and fixing the filamentous fungus firmly and stably, the fixed cell can be made porous, so that the supply of the culture solution and oxygen to the cell can be improved, and the filterability can be improved.
[0022]
In the present invention, the chain clay mineral can be used alone, or in combination with a two-layered fibrous mineral such as halloysite or asbestos, or a volcanic fibrous mineral containing Kanuma soil, Akadama soil, or the like. Can do. Further, if necessary, zeolite, or adsorptive clay minerals typified by acidic clay, and rocks such as cristobalite, quartz and feldspar may be used in combination with the fibrous clay mineral.
[0023]
Sepiolite preferably used in the present invention is represented by the formula (1)
(OH ₂ ) ₄ (OH) ₄ Mg ₈ Si ₁₂ O ₃₀ · 6-8H ₂ O (1)
A two-dimensional crystal structure such as talc forms a chain crystal structure in which bricks are alternately stacked.
In addition, it is fibrous due to the pores formed in the chain gaps, but it is also a great feature that it has a large specific surface area and adsorptivity unlike other fibrous minerals.
[0024]
In the present invention, sepiolite having a specific surface area of 100 to 350 m ² / g and an oil absorption of 100 to 300 ml / 100 g is preferably used. If the specific surface area is smaller than this range, the cohesive strength of the sludge is small, while it is larger than this range and no further effect can be obtained.
[0025]
Further, the fibrous sepiolite preferably used in the present invention preferably has a fiber length of 70 to 400 angstroms, a fiber length of 0.2 to 400 μm, and an aspect ratio in the range of 5 to 500.
[0026]
Table 1 below shows an example of the general chemical composition of sepiolite (dried product at 110 ° C. for 2 hours).
Table 1 General chemical composition of sepiolite 52.50 (wt%) SiO ₂
MgO 22.8
Al ₂ O ₃ 1.7
Fe ₂ O ₃ 0.8
CaO 0.8
H ₂ O + 10.5
H ₂ O- 11.0
[0027]
The filamentous fungus used in the present invention may be any filamentous fungus capable of producing lactic acid, such as Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and Bifidobacterium. And fungi such as Streptococcus genus, Enterococcus genus, Bacillus genus, Scrotridium genus, Sporolactobacillus genus, molds such as Rhizopus genus, etc., and these can be used alone or in combination of two or more. Preferred is Rhizopus Oryzae .
[0028]
The liquid medium used in the present invention can be used without particular limitation as long as it can grow filamentous fungi capable of producing lactic acid. This liquid medium contains a nutrient component that is a raw material for lactic acid and an inorganic component (mineral component) necessary for the growth of bacterial cells. As the nutrient component, sugars such as glucose and polysaccharides such as starch are used. Is done. Examples of inorganic salts include ammonium nitrate, ammonium sulfate, magnesium sulfate, sodium chloride, zinc sulfate, and potassium dihydrogen phosphate. The carbon source concentration is not particularly limited, but 100 to 250 g / L, preferably about 100 to 150 g / L is suitable.
[0029]
The holmite clay mineral is preferably added to the liquid medium so as to have a concentration of 1 to 50 g / L, particularly 1 to 10 g / L, more preferably 2 to 7 g / L. The effect due to the addition is not recognized, and when the amount is larger than the above range, the production rate is lowered.
[0030]
There is no particular limitation on the method of inoculating the microbial cells or their spores on the liquid medium, and the above-described microbial cells or their spores can be suspended in sterile water or physiological saline and the suspension can be inoculated into the liquid medium. Good.
[0031]
Since the microbial cells used in the present invention are aerobic, it is preferable to culture and ferment while stirring by aeration. That is, it is preferable to supply oxygen-containing gas such as air to the microbial cells, and simultaneously stir the culture medium by rising bubbles to uniformly supply substances such as oxygen and substrate to the microbial cells.
[0032]
For this purpose, an airlift bioreactor can be used, and an antifoaming agent can be added to the system in order to suppress the generation of bubbles accompanying aeration. As the antifoaming agent, a silicone surfactant or the like is used.
[0033]
In FIG. 1 showing an example of an airlift bioreactor, a draft tube 5 is formed in a cylindrical reaction vessel 16 containing a culture fermentation solution so that a gap is formed between both ends and the reaction vessel. Is provided. At the center of the bottom of the reaction vessel 16, a sparger 6 made of, for example, sintered glass is provided for dispersing air in the liquid medium. The cooling jacket 4 is provided on the outer periphery of the reaction vessel 16, and the liquid medium in the vessel can be maintained at a constant temperature by passing cooling water through the jacket.
[0034]
The sparger 6 is connected to an air compressor 8 via a flow meter 7, and a constant flow rate of air is supplied as bubbles from the bottom of the container into the liquid medium. A condenser 12 is provided at the top of the reaction vessel, and excess air in the reaction vessel is discharged to the outside after the liquid is removed by the condenser 12. In addition, a DO electrode 13 that detects dissolved oxygen in the culture medium, an antifoaming sensor 14 that detects the foam level, and a detection sensor 15 that detects the liquid level of the liquid culture medium are provided at the top of the reaction vessel.
[0035]
The antifoaming agent sensor 14 is connected to the antifoaming agent controller 2 and can supply the antifoaming agent from the antifoaming agent storage tank 1 via the pump 3 according to the foam level.
[0036]
Further, the level sensor 15 is connected to the level controller 10 so that sterilized water can be supplied from the sterilized water tank 11 according to the level of the liquid medium so that the liquid level is constant by the pump 3. Yes.
[0037]
Furthermore, the DO electrode 13 is connected to the DO meter 9 so that the degree of ventilation can be adjusted based on an instruction from the DO meter.
[0038]
In this airlift bioreactor, filamentous fungi or their spores are inoculated into a lactic acid culture and fermentation medium, holmite clay mineral is inoculated into the medium, and air is blown into the medium through a sparger 6.
[0039]
As a result, the oxygen-containing gas such as the blown air becomes bubbles and rises in the draft tube 5, and by this upward flow, a circulating flow is formed that rises in the draft tube 5 and descends outside the draft tube 5. . As a result, sufficient mixing and stirring is performed between the liquid medium and the microbial cells, and supply to the microbial cells such as oxygen and substrate is sufficiently performed. Is done.
[0040]
The production of lactic acid in the present invention is carried out in two stages, ie, a microbial cell culture process in the former stage and a lactic acid fermentation process in the latter stage. In these culturing and fermentation processes, the temperature of the medium is preferably maintained in the range of 20 to 45 ° C, particularly 25 to 40 ° C.
[0041]
The culturing process of the bacterial cells and the fermentation process are not strictly strict, but the end point of the culturing process can be known when the growth of the bacterial cells becomes slow. This time is generally about 40 to 80 hours, particularly about 48 to 72 hours.
[0042]
In the present invention, the flocculant is added to the liquid medium at the time when the bacterial cell growth is slow. As the flocculant, any known flocculant is used, but anionic, nonionic or amphoteric flocculants are preferred.
[0043]
Suitable examples of flocculants include, but are in no way limited to:
Examples of the anionic flocculants include sodium polyacrylate or a copolymer of sodium polyacrylate and polyacrylamide, gelatin, glue, casein, sodium polymethacrylate, sodium alginate, ammonium alginate, carboxymethyl starch, carboxy Methyl cellulose, polyacrylamide partial hydrolyzate, polyacrylonitrile partial hydrolyzate, acrylamide-acrylic acid copolymer, maleic anhydride-vinyl ether copolymer, maleic anhydride-vinyl acetate copolymer, chitosan, sodium styrenesulfonate A copolymer or the like is used.
Nonionic flocculants include polyacrylamide, polyvinyl alcohol (PVA), starch, cyanated starch, guar gum, tragacanth gum, galaya gum, gum arabic, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, bee gum, gelatin, polyethylene glycol, polyoxyethylene, poly Examples thereof include oxypropylene and polyoxyethylene-polyoxypropylene copolymers.
Examples of the amphoteric flocculant include a mixture or copolymer of methacrylic acid ester and polysodium acrylate, a mixture or copolymer of acrylate ester and sodium polyacrylate, and the like.
Of course, these flocculants can be used alone or in combination of two or more.
[0044]
Since the amount of the flocculant used is to form a light floc, it may be a very small amount and is generally 1 to 20 mg / L, particularly 1 to 10 mg / L, more preferably 2 to 8 mg / L in the liquid medium. It is good to add so that it may become a concentration. When the amount of the flocculant used is less than the above range, the formation of large lumps of cells is inevitable, and when the amount is larger than the above range, dense aggregation occurs when the flocculant is added. There is a tendency for the production rate of lactic acid to decrease.
[0045]
Production of lactic acid is started when the growth of the bacterial cells becomes slow. The temperature of the medium is suitably 20 to 45 ° C., particularly 25 to 40 ° C., as in the culture process. With the production of lactic acid, the pH of the medium shifts to the acidic side, so an alkaline substance is added as appropriate, and the pH of the medium is kept in a range suitable for fermentation, generally 4 to 8, particularly 5 to 7. To be.
[0046]
As the alkaline substance, calcium carbonate, ammonia, sodium hydroxide, potassium hydroxide and the like can be used, and calcium carbonate is particularly preferable.
[0047]
The fermentation process is completed when nutrients in the medium are consumed. This process is generally about 48 to 80 hours, although it varies depending on the type of strain and the type or concentration of the raw material.
[0048]
The fermentation broth thus obtained is separated from the cells by filtration or the like, the calcium lactate is neutralized with sulfuric acid, etc., the byproduct gypsum is separated to give crude lactic acid, which is concentrated and purified by means known per se. Product.
[0049]
Embodiments of the present invention will be described below by way of examples, but these are merely examples and do not limit the present invention in any way. In addition, glucose and lactic acid in Examples were quantified by the following method.
[0050]
(1) Glucose determination method The culture broth was diluted 2000 times.
Take 1 ml and add 0.5 ml of 80% phenol aqueous solution.
Add 5 ml of concentrated sulfuric acid.
Leave for 30-40 minutes. Colorimetric determination at 500 nm.
(2) Quantification method of lactic acid Trichloroacetic acid was added to 1 ml of a sample and centrifuged at 3000 rpm for 10 minutes, and the supernatant was collected and diluted. 1 ml of the solution was added to a 3% copper sulfate solution, calcium hydroxide was added, and the mixture was allowed to stand at room temperature. Then, it centrifuges, the supernatant liquid is extract | collected, and a copper sulfate and a sulfuric acid are added. Next, after heating in a boiling bath for 5 minutes and cooling, p-hydroxyphenyl was added, left in a 30 ° C. water bath for 30 minutes, further placed in a boiling bath for 90 seconds and then cooled and colorimetrically determined at a wavelength of 560 nm. It was.
[0051]
(Example 1)
<Fermental production of lactic acid in a stirring culture tank>
L (+) - a Rhizopus oryzae NRRL395 a lactic acid-producing strain was suspended in physiological saline to a concentration of 10 ⁷ spores / ml, it was inoculated with 1% of the culture liquid amount in the following culture land. Glucose 120 g / L, ammonium sulfate 3.02 g / L, potassium dihydrogen phosphate 0.25 g / L, zinc sulfate heptahydrate 0.04 g / L, magnesium sulfate heptahydrate 0.15 g / L, sepiolite 3 g / L L.
The culture was performed at 35 ° C. and a stirring speed of 300 rpm. Polyethylene oxide was added as a flocculant at a concentration of 5 mg / L in the vicinity of the slow growth rate of the cells after the start of culture, and then calcium carbonate was added so that the pH was not lowered by the produced lactic acid. .
The progress of the culture is shown in FIG. Compared with the case of no addition (Comparative Example 1), the production rate of lactic acid is very fast, and the final production amount is 69.6 g / L in the case of no addition, whereas sepiolite and flocculant are added. Was 115.1 g / L. Accordingly, the lactic acid concentration increased to about 1.67 times that in the case of no addition.
When sepiolite and flocculant were added, the cells maintained a loose shape until the end of the culture.
[0052]
(Example 2)
In Example 1, it carried out similarly by changing the ratio of sepiolite and a flocculant (polyethylene oxide). The result is shown in FIG.
[0053]
(Comparative Example 1)
In Example 1, the same procedure was performed without adding sepiolite and a flocculant (polyethylene oxide). The result is shown in FIG.
As a result, the microbial cells became large lumps, and the amount of lactic acid produced was low, and the microbial cells adhered to the baffle plates and the stirring shaft in the reactor, giving a burden to the apparatus.
[0054]
(Comparative Example 2)
In Example 1, it carried out similarly without adding a flocculant (polyethylene oxide). The result is shown in FIG.
As a result, the microbial cells became large lump, and the production amount of lactic acid was between Example 1 and Comparative Example 1 as apparent from FIG. In addition, although the amount of bacterial cells attached to the baffle plate and the stirring shaft in the reactor was smaller than that in Comparative Example 1, the load was imposed on the apparatus.
[0055]
Example 3
<Lactic acid fermentation production in an airlift bioreactor>
Cultivation was attempted in an airlift bioreactor (FIG. 1) , which is advantageous when the viscosity is low, because the viscosity of the culture medium during culture can be kept almost the same as that of water by adding Ade Plus. The method was the same as in Example 1. The results are shown in FIG. When sepiolite and polyethylene oxide as a flocculant were added to a concentration of 5 mg / L, a lactic acid production amount of 116.6 g / L was exhibited after 56 hours.
[0056]
Example 4
<When the flocculant is changed (air lift)>
L (+) - a Rhizopus oryzae NRRL395 a lactic acid-producing strain was suspended in physiological saline to a concentration of 10 ⁷ spores / ml, it was inoculated with 1% of the culture liquid amount in the culture ground. Component of the culture ground is the same as in Example 1, the amount of sepiolite was 5 g / L.
Culture ground temperature, 35 ℃, ventilation was carried out at 0.5~1.0vvm. To culture locations flocculant (sodium polyphosphate) in the vicinity of the start of culture after the cell growth rate became gentle, it was added to a concentration of 7 mg / L, so that pH does not drop by then produced lactic acid Calcium carbonate was added.
The final lactic acid production was 98.3 g / L, slightly inferior to that when polyethylene oxide was added, but the cells remained loose.
[0057]
(Comparative Example 3)
In Example 3, the same procedure was performed without adding sepiolite and a flocculant (polyethylene oxide). The result is shown in FIG.
In the case of no addition, it adhered to the vent and grew in a lump, which prevented the substrate and oxygen from being supplied to the cells, and the production rate also decreased.
[0058]
【The invention's effect】
According to the present invention, by adding a holmite clay mineral to a culture solution to grow a filamentous fungus having a lactic acid-producing ability, and adding a flocculant to this prior to fermentation, a state in which the cells are loosened very well It became possible to perform culture and fermentation.
[0059]
Since the holmite clay mineral and the flocculant to be added are very small amounts, there are no adverse effects such as growth inhibition on the cells and increase in viscosity.
By making the cells loose, the mass transfer rate of oxygen, substrate, etc. is increased compared with the case where only the surface cells take the form of a lump that is involved in production, so the substance production rate is improved. .
If the culture was performed in this way, cells since forming the loosely aggregated floc contained therein the hormite type clay mineral, it is easy to separate the bacteria and culture.
Therefore, it can be applied to all culture methods such as batch, semi-batch, and continuous.
If continuous culture becomes possible, the cells that have been disposed of up to now can be reused.
Not only the productivity is improved, but also the cell separation property is improved in the recovery and purification of the fermentation product, so that the cost required for the recovery and purification can be minimized.
In order to suppress the viscosity of the culture solution itself, it is effective in improving the physical properties of the culture solution in the cultivation of filamentous fungi in which the mycelia are intertwined in a complicated manner and the viscosity of the culture solution is inevitably high.
It becomes possible to control the form of the fungus body, and it is possible to use an airlift bioreactor with less damage due to the shearing of the fungus body, and the productivity can be further improved.
[Brief description of the drawings]
FIG. 1 is a layout view showing an example of an airlift bioreactor used in Examples.
FIG. 2 is a diagram showing a change with time of lactic acid production by a stirring bioreactor.
FIG. 3 is a graph showing the relationship between the ratio of sepiolite and flocculant and the amount of lactic acid produced.
FIG. 4 is a diagram showing a change over time in lactic acid production in an airlift bioreactor.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Defoamer storage tank 2 Defoamer controller 3 Pump 4 Cooling jacket 5 Draft tube 6 Sparger 7 Flow meter 8 Air compressor 9 DO meter 10 Level controller 11 Sterile water tank 12 Condenser 13 DO electrode 14 Defoamer sensor 15 level Sensor 16 Reaction vessel

Claims (8)

A liquid medium inoculated with filamentous fungi having the ability to produce lactic acid or its spores is added and cultured, and after the start of the culture, when the growth rate of the cells becomes slow, a flocculant is added, and the pH is increased. A method for producing lactic acid, wherein fermentation is carried out while adding an alkaline substance so that the temperature does not decrease. The method according to claim 1, wherein the holmite clay mineral is sepiolite having a fiber diameter of 70 to 400 angstroms and a fiber length of 0.2 to 400 µm. The production method according to claim 1 or 2, wherein the holmite clay mineral is added to the liquid medium so as to have a concentration of 1 to 50 g / L. The production method according to claim 1, wherein the flocculant is an anionic or nonionic flocculant. The production method according to any one of claims 1 to 4, wherein the flocculant is added to the liquid medium so as to have a concentration of 1 to 20 mg / L. The production method according to any one of claims 1 to 5, wherein fermentation is performed while maintaining the pH of the medium at 4 to 8. The production method according to any one of claims 1 to 6, wherein the culture and fermentation are performed while stirring by aeration. The production method according to claim 1, wherein the alkaline substance is calcium carbonate.

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