CN109380063B - Method for producing edible fungus liquid strain by using cordyceps militaris culture medium waste - Google Patents
Method for producing edible fungus liquid strain by using cordyceps militaris culture medium waste Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
- A01G18/40—Cultivation of spawn
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
A method for producing liquid strain of edible fungi by using scarlet caterpiller fungus culture medium waste comprises pulverizing culture medium waste generated in scarlet caterpiller fungus planting, adding water, stirring, and gelatinizing with boiling water; cooling and regulating the pH value, adding alpha-amylase and glucoamylase into the mixture, mixing the mixture and stirring the mixture, performing double enzyme digestion reaction, and terminating the enzymatic reaction; cooling and supplementing water, adjusting the pH value, adding papain, and carrying out enzymatic reaction; carrying out solid-liquid separation and deslagging after enzymatic reaction to obtain a culture medium waste hydrolysate; diluting with water, sterilizing, fermenting, and directly using in edible fungus production. The advantages are that: the cordyceps sinensis extracellular enzyme is decomposed and is suitable for being used as a culture raw material of other large fungi, and the waste is converted into a fermentation industrial culture medium and is successfully used for producing liquid strains of tricholoma giganteum, shiitake mushroom, hericium erinaceus or pleurotus citrinopileatus. The method also has the characteristics of cheap and easily obtained raw materials, simple preparation process, waste recycling and environmental protection idea conformity.
Description
Technical Field
The invention belongs to the field of recycling of agricultural production waste resources, and relates to a method for producing edible fungus liquid strains by using cordyceps militaris culture medium waste, in particular to a method for producing edible fungus liquid strains such as agaric, shiitake mushroom, hericium erinaceus or pleurotus citrinopileatus by using cordyceps militaris culture medium waste, so that the cordyceps militaris culture medium waste is recycled through biotransformation.
Background
Cordyceps militaris (Cordyceppsmilitaris) is a traditional medicine-food dual-purpose fungus and rich in nutrient components. Is an industry with rapid development speed and large scale. Shenyang city in Liaoning province is a discovery place of the cordyceps militaris and a source place of an artificial cultivation and planting technology of the cordyceps militaris, 6000 tons of dry products are produced in Shenyang region every year through more than thirty years of planting development, the yield accounts for about 70% of the national cordyceps militaris yield, and large-scale production is realized in Shenyang region. The economic benefit of cordyceps planting is higher than that of traditional basic agriculture, and the cordyceps planting method becomes a prop industry for partial farmers to delight and enrich in areas around Shenyang. With the improvement and maturity of the planting skills of farmers, the planting area is increased year by year. The method is parallel to the increase of the productivity of the cordyceps militaris, the culture medium waste is rapidly increased, and the generated culture medium waste is 3.5-4 times of the finished product and reaches nearly 24000 tons per year, thereby bringing great pressure and trouble to the production environment. The culture medium waste lacks an effective recycling way, pollutes the natural ecological environment and the reproduction environment, and restricts the sustainable development of the cordyceps industry.
Disclosure of Invention
The invention provides a method for producing liquid strains of edible fungi by using cordyceps militaris culture medium waste, which enables the cordyceps militaris waste to be subjected to biotransformation to change biomolecular substances into carbohydrates, proteins and amino acids which can be utilized by the edible fungi, successfully solves the problem of high-efficiency ecological cyclic utilization of current agricultural waste resources, can reduce resource waste and environmental pollution, can reduce the cost for producing the liquid strains, and supports high-efficiency sustainable development of an edible fungi industrial chain.
The technical solution of the invention is as follows:
a method for producing liquid strains of edible fungi by using cordyceps militaris culture medium waste comprises the following steps:
(1) crushing culture medium waste generated in the planting of the cordyceps militaris to 40-60 meshes, adding water according to the material-water ratio of 1: 6-1: 8g/mL, uniformly stirring, and gelatinizing with boiling water for 20min to obtain a culture medium waste pasty material;
by crushing, the small particles of the culture medium waste are uniformly distributed, which is beneficial to the next treatment. The wet starch is expanded by pasting, which is beneficial to enzyme digestion;
(2) cooling the culture medium waste gelatinized material to 55-60 ℃, adjusting the pH value to 4.0-4.5, adding double enzymes according to the addition amounts of 4U/g of alpha-amylase and 6U/g of saccharifying enzyme, mixing and stirring, wherein the mass ratio of the alpha-amylase to the saccharifying enzyme is 1: 1.5-1: 1.8, carrying out double enzyme digestion at 55-60 ℃, and stopping enzymatic reaction after reacting for 40 min;
(3) cooling to 55 ℃, supplementing water to the original volume of the culture medium waste gelatinized material, adjusting the pH value to 5.0-7.0, adding papain accounting for 0.1-0.2% of the culture medium waste, reacting for 4-6 h at 55-65 ℃, and terminating the enzymatic reaction;
through enzymolysis, the long-chain starch and large protein of the culture medium waste can be digested into usable small molecules;
(4) centrifuging for 5-10 min at the rotating speed of 1200-1500 r/min by using a centrifuge, carrying out solid-liquid separation for deslagging, and then further deslagging by using filtration or Chua's funnel negative pressure suction filtration to obtain a culture medium waste hydrolysate; through detection, the hydrolysis rate of the cordyceps militaris culture medium waste reaches 70-80 percent;
(5) diluting the culture medium waste hydrolysate with 3-5 times of water, adjusting the pH value to 6.0-6.2, and canning according to the volume of a fermentation tank 2/3;
(6) medicine for curing excess syndrome
Sterilizing at 121 deg.C under high pressure for 30min, cooling to 25 deg.C, inoculating strain with an inoculum size of 0.5% -1.5% of the fermentation tank charging volume;
(7) fermenting the mixture
Controlling fermentation conditions of a fermentation tank to ferment, and controlling fermentation tank parameters: the temperature is 25 ℃, the pressure in the tank is 0.02 Mpa-0.06 Mpa, the fermentation time is 6 d-7 d, and the number of liquid strain bacterial balls reaches more than 80 percent of the total volume. Can be directly used for edible fungus production to obtain good cultivation and planting effect.
Further, the enzymatic reaction is terminated in the step (2), and the temperature is raised to 90 ℃ and maintained for 5-10 min to inactivate the enzyme.
Further, step (3) terminates the enzymatic reaction, and the temperature is raised to 100 ℃ for 5min to inactivate the enzyme.
Furthermore, the enzyme activity of the papain is 80 ten thousand U/g.
Further, the strain inoculated in the step (6) is agaricus, shiitake mushroom, hericium erinaceus or pleurotus citrinopileatus.
The invention has the beneficial effects that:
the cordyceps militaris waste contains rich protein and nutrient components which are not completely utilized, and culture medium waste generated in the planting production of the cordyceps militaris is scientifically developed and utilized. The cordyceps sinensis extracellular enzyme is decomposed and is suitable for being used as a culture raw material of other large fungi, and the waste is converted into a fermentation industrial culture medium and is successfully used for producing liquid strains of tricholoma giganteum, shiitake mushroom, hericium erinaceus or pleurotus citrinopileatus. The method also has the characteristics of cheap and easily obtained raw materials, simple preparation process, waste recycling and environmental protection idea conformity. More importantly, the method is convenient for large-scale industrial production, and has the advantages of good stability, low cost, high safety index and the like.
Detailed Description
Example 1
(1) Crushing culture medium waste generated in the planting of the cordyceps militaris to 40-60 meshes, adding water according to the material-water ratio (the mass-volume ratio of the culture medium waste to the water) of 1:8g/mL, uniformly stirring, and gelatinizing with boiling water for 20min to obtain a culture medium waste gelatinized material;
(2) cooling the culture medium waste gelatinized material to 60 ℃, adjusting the pH value to 4.5 by using dilute hydrochloric acid with the mass fraction of 18%, adding double enzymes according to the addition amounts of 4U/g of alpha-amylase and 6U/g of saccharifying enzyme, mixing and stirring, wherein the mass ratio of the alpha-amylase to the saccharifying enzyme is 1:1.8, carrying out double enzyme digestion at 60 ℃, heating to 90 ℃ after reacting for 40min, maintaining for 10min, and stopping enzymatic reaction;
(3) cooling to 55 ℃, supplementing water to the original volume of the culture medium waste gelatinized material, adjusting the pH value to 7.0 by using dilute hydrochloric acid and NaOH solution with the mass fraction of 18%, adding papain with the enzyme activity of 80 ten thousand U/g accounting for 0.2% of the culture medium waste, reacting for 6 hours at 65 ℃, heating to 100 ℃ and maintaining for 5 minutes, and stopping enzymatic reaction;
(4) centrifuging at 1500r/min for 10min with a centrifuge, performing solid-liquid separation to remove residue, and filtering or vacuum filtering with Chua's funnel to remove residue to obtain culture medium waste hydrolysate; the hydrolysis rate of the culture medium waste of the cordyceps militaris is up to 75 percent calculated by solid residue;
(5) diluting the culture medium waste hydrolysate with 5 times of water, adjusting the pH value to 6.2 by using dilute hydrochloric acid with the mass fraction of 18%, and canning according to the volume of a fermentation tank 2/3;
(6) medicine for curing excess syndrome
Sterilizing at 121 deg.C under high pressure for 30min, cooling to 25 deg.C, inoculating Lentinus Edodes strain with inoculation amount of 1.5% of the fermentation tank;
(7) fermenting the mixture
Controlling fermentation conditions of a fermentation tank to ferment, and controlling fermentation tank parameters: the temperature is 25 ℃, the pressure in the tank is 0.06Mpa, the fermentation time is 7d, the concentration of the liquid strain reaches more than 80 percent, namely the number of the liquid strain bacterial balls reaches more than 80 percent of the total volume, and the method is directly used for producing edible fungi.
The method for producing the mushroom liquid strain has the culture period of 7d in the fermentation tank, which is shortened by 3 d-5 d compared with the culture period of the conventional potato glucose culture medium; the activity of the inoculated three-level strain (the fungus bag) is vigorous, the culture period of the fungus bag is 28 days, and the culture period is shortened by 5 days to 10 days compared with that of the strain produced by a potato glucose culture medium; because the fungus age is greatly shortened, the hypha is thick and strong, the vitality is strong, and the cultivation yield of the mushroom can be stably improved by more than 10 percent.
Example 2
(1) Crushing culture medium waste generated in the planting of the cordyceps militaris to 40-60 meshes, adding water according to the material-water ratio of 1:6g/mL, uniformly stirring, and gelatinizing with boiling water for 20min to obtain culture medium waste pasty material;
(2) cooling the culture medium waste gelatinized material to 55 ℃, adjusting the pH value to 4.0 by using dilute hydrochloric acid with the mass fraction of 18%, adding double enzymes according to the addition amounts of 4U/g of alpha-amylase and 6U/g of saccharifying enzyme, mixing and stirring, wherein the mass ratio of the alpha-amylase to the saccharifying enzyme is 1:1.5, carrying out double enzyme digestion at 55 ℃, heating to 90 ℃ after reacting for 40min, maintaining for 5min, and stopping enzymatic reaction;
(3) cooling to 55 ℃, supplementing water to the original volume of the culture medium waste gelatinized material, adjusting the pH value to 5.0 by using dilute hydrochloric acid with the mass fraction of 18%, adding papain with the enzyme activity of 80 ten thousand U/g accounting for 0.1% of the culture medium waste, reacting for 4 hours at 55 ℃, heating to 100 ℃ and maintaining for 5 minutes, and terminating the enzymatic reaction;
(4) centrifuging at 1200r/min for 5min with a centrifuge, removing residues by solid-liquid separation, and further removing residues by filtration or vacuum filtration with Chua's funnel to obtain culture medium waste hydrolysate; the hydrolysis rate of the culture medium waste of the cordyceps militaris reaches 70 percent calculated by solid residue;
(5) diluting the culture medium waste hydrolysate with 3 times of water, adjusting the pH value to 6.0 by using dilute hydrochloric acid with the mass fraction of 18%, and canning according to the volume of a fermentation tank 2/3;
(6) medicine for curing excess syndrome
Sterilizing at 121 deg.C under high pressure for 30min, cooling to 25 deg.C, inoculating Auricularia auricula-judae shake flask propagation strain with an inoculum size of 0.5% of the fermentation tank charging volume;
(7) fermenting the mixture
Controlling fermentation conditions of a fermentation tank to ferment, and controlling fermentation tank parameters: the temperature is 25 ℃, the pressure in the tank is 0.02Mpa, the fermentation time is 6d, the number of the liquid strain bacterial balls reaches more than 80 percent of the total volume, and the liquid strain bacterial balls are directly used for producing edible fungi.
The method for producing black fungus liquid strain has a fermentation tank culture period of 6d, which is shortened by 3d compared with conventional potato glucose culture medium culture period; the culture period of the inoculated black fungus culture bag is 25 days, which is shortened by 5 days compared with the production of the strain by a potato glucose culture medium.
Example 3
(1) Crushing culture medium waste generated in the planting of the cordyceps militaris to 40-60 meshes, adding 7mL of water into each gram of the culture medium waste, namely adding water according to the material-water ratio of 1:7g/mL, uniformly stirring, and gelatinizing with boiling water for 20min to obtain a culture medium waste gelatinized material;
(2) cooling the culture medium waste gelatinized material to 58 ℃, adjusting the pH value to 4.2, adding double enzymes according to the addition amounts of 4U/g of alpha-amylase and 6U/g of saccharifying enzyme, mixing and stirring, wherein the mass ratio of the alpha-amylase to the saccharifying enzyme is 1:1.7, carrying out double enzyme digestion at 58 ℃, heating to 90 ℃ after reacting for 40min, maintaining for 8min, and terminating the enzymatic reaction;
(3) cooling to 55 ℃, supplementing water to the original volume of the culture medium waste gelatinized material, adjusting the pH value to 6, adding papain accounting for 0.15 percent of the culture medium waste, reacting at 60 ℃ for 5 hours, heating to 100 ℃, maintaining for 5min, and stopping enzymatic reaction;
(4) centrifuging for 8min at the rotation speed of 1300r/min by using a centrifuge, carrying out solid-liquid separation for deslagging, then further deslagging by using filtration or Chua's funnel negative pressure suction filtration to obtain culture medium waste hydrolysate, wherein the hydrolysis rate of the culture medium waste of the cordyceps militaris reaches 80% calculated on the basis of insoluble solid residues;
(5) diluting the culture medium waste hydrolysate with 4 times of water, adjusting the pH value to 6.1, and canning according to the volume of a fermentation tank 2/3;
(6) medicine for curing excess syndrome
Sterilizing at 121 deg.C under high pressure for 30min, cooling to 25 deg.C, inoculating Hericium Erinaceus strain in shake flask with an inoculum size of 1% of the fermentation tank charge volume;
(7) fermenting the mixture
Controlling fermentation conditions of a fermentation tank to ferment, and controlling fermentation tank parameters: the temperature is 25 ℃, the pressure in the tank is 0.04Mpa, the fermentation time is 6d, the concentration of the liquid strain reaches more than 80 percent, the quantity of the liquid strain bacteria balls reaches more than 80 percent of the total volume, and the method is directly used for producing edible fungi.
The method for producing Hericium erinaceus liquid strain has a fermentation tank culture period of 6d, which is shortened by 4d compared with conventional potato glucose culture medium; the culture period of the inoculated hericium erinaceus culture bag is 28 days, which is shortened by 5-10 days compared with that of the production strains of the potato glucose culture medium; the cultivation yield of the hericium erinaceus can be stably improved by more than 15%.
Example 4
(1) Crushing culture medium waste generated in the planting of the cordyceps militaris to 40-60 meshes, adding water according to the material-water ratio of 1:6.8g/mL, uniformly stirring, and gelatinizing with boiling water for 20min to obtain culture medium waste pasty material;
(2) cooling the culture medium waste gelatinized material to 56 ℃, adjusting the pH value to 4.1, adding double enzymes according to the addition amounts of 4U/g of alpha-amylase and 6U/g of saccharifying enzyme, mixing and stirring, wherein the mass ratio of the alpha-amylase to the saccharifying enzyme is 1:1.7, carrying out double enzyme digestion at 56 ℃, heating to 90 ℃ after reacting for 40min, maintaining for 7min, and terminating the enzymatic reaction;
(3) cooling to 55 ℃, supplementing water to the original volume of the culture medium waste gelatinized material, adjusting the pH value to 5.5, adding papain accounting for 0.17 percent of the culture medium waste, wherein the enzyme activity of the papain is 80 ten thousand U/g, reacting for 5.5h at 56 ℃, heating to 100 ℃, maintaining for 5min, and stopping enzymatic reaction;
(4) centrifuging for 7min at the rotation speed of 1400r/min by using a centrifuge, carrying out solid-liquid separation and deslagging, and then further deslagging by using filtration or Chua's funnel negative pressure suction filtration to obtain a culture medium waste hydrolysate; the hydrolysis rate of the culture medium waste of the cordyceps militaris is up to 78 percent calculated by solid residue;
(5) diluting the culture medium waste hydrolysate with 3.5 times of water, adjusting the pH to 6.1, and canning according to the volume of a fermentation tank 2/3;
(6) medicine for curing excess syndrome
Sterilizing at 121 deg.C under high pressure for 30min, cooling to 25 deg.C, inoculating Pleurotus Citrinopileatus Sing strain, wherein the inoculation amount is 1.2% of the charging volume of the fermentation tank;
(7) fermenting the mixture
Controlling fermentation conditions of a fermentation tank to ferment, and controlling fermentation tank parameters: the temperature is 25 ℃, the pressure in the tank is 0.03Mpa, the fermentation time is 5d, the concentration of the liquid strain reaches more than 80 percent, the quantity of the liquid strain bacteria balls reaches more than 80 percent of the total volume, and the method is directly used for producing edible fungi.
The method for producing Pleurotus citrinopileatus liquid strain has a fermentation tank culture period of 6d, which is shortened by 3d compared with conventional potato glucose culture medium culture period; the culture period of the inoculated pleurotus citrinopileatus culture bag is 25 days, which is 5 days shorter than that of the potato glucose culture medium production strain.
The above description is only exemplary of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. A method for producing liquid strains of edible fungi by using cordyceps militaris culture medium waste is characterized by comprising the following steps: the method comprises the following steps:
(1) crushing culture medium waste generated in the planting of the cordyceps militaris to 40-60 meshes, adding water according to the material-water ratio of 1: 6-1: 8g/mL, uniformly stirring, and gelatinizing with boiling water for 20min to obtain a culture medium waste pasty material;
(2) cooling the culture medium waste gelatinized material to 55-60 ℃, adjusting the pH value to 4.0-4.5, adding double enzymes according to the addition amounts of 4U/g of alpha-amylase and 6U/g of saccharifying enzyme, mixing and stirring, wherein the mass ratio of the alpha-amylase to the saccharifying enzyme is 1: 1.5-1: 1.8, carrying out double enzyme digestion at 55-60 ℃, and stopping enzymatic reaction after reacting for 40 min;
(3) cooling to 55 ℃, supplementing water to the original volume of the culture medium waste gelatinized material, adjusting the pH value to 5.0-7.0, adding papain accounting for 0.1-0.2% of the culture medium waste, reacting for 4-6 h at 55-65 ℃, and terminating the enzymatic reaction;
(4) centrifuging the culture medium waste pasty material subjected to the termination of the enzymatic reaction in the step (3) for 5-10 min at the rotating speed of 1200-1500 r/min by using a centrifugal machine, carrying out solid-liquid separation for deslagging, and then further deslagging by using filtration or Chua's funnel negative pressure suction filtration to obtain culture medium waste hydrolysate;
(5) diluting the culture medium waste hydrolysate with 3-5 times of water, adjusting the pH value to 6.0-6.2, and canning according to the volume of a fermentation tank 2/3;
(6) medicine for curing excess syndrome
Sterilizing the diluted culture medium waste hydrolysate with the adjusted pH value in the step (5) at the high pressure at the temperature of 121 ℃ for 30min, cooling to 25 ℃, and inoculating strains, wherein the inoculation amount is 0.5-1.5% of the charging volume of a fermentation tank;
(7) fermenting the mixture
Controlling fermentation conditions of a fermentation tank to ferment, and controlling fermentation tank parameters: the temperature is 25 ℃, the pressure in the tank is 0.02 Mpa-0.06 Mpa, the fermentation time is 6 d-7 d, the number of liquid strain bacterial balls reaches more than 80 percent of the total volume, and the method is directly used for producing edible fungi.
2. The method for producing liquid strains of edible fungi from cordyceps militaris culture medium waste as claimed in claim 1, wherein the method comprises the following steps: and (2) stopping the enzymatic reaction, heating to 90 ℃ and maintaining for 5-10 min to inactivate the enzyme.
3. The method for producing liquid strains of edible fungi from cordyceps militaris culture medium waste as claimed in claim 1, wherein the method comprises the following steps: and (3) stopping the enzymatic reaction, heating to 100 ℃ and maintaining for 5min to inactivate the enzyme.
4. The method for producing liquid strains of edible fungi from cordyceps militaris culture medium waste as claimed in claim 1, wherein the method comprises the following steps: the enzyme activity of the papain is 80 ten thousand U/g.
5. The method for producing liquid strains of edible fungi from cordyceps militaris culture medium waste as claimed in claim 1, wherein the method comprises the following steps: the inoculated strain in the step (6) is agaricus, shiitake mushroom, hericium erinaceus or pleurotus citrinopileatus.
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| JPS61183194A (en) * | 1985-02-06 | 1986-08-15 | 金子農機株式会社 | Use of wheat straw or like |
| CN103858676A (en) * | 2014-03-24 | 2014-06-18 | 山东晨阳菌业有限公司 | Method for cultivating liquid cultures of cordyceps militaris |
| CN104987156A (en) * | 2015-07-09 | 2015-10-21 | 辽宁省农业科学院蔬菜研究所 | Lyophyllum fumosurn culture medium using fermented bran and method for cultivating lyophyllum fumosurn |
| CN105684727A (en) * | 2016-01-25 | 2016-06-22 | 赵建荣 | Culture medium and cultivation method for cultivating black termitomyces albuminosus with enzyme bacterium residues as main raw material |
| CN108467292A (en) * | 2018-05-28 | 2018-08-31 | 贵州湄潭兰馨茶业有限公司 | It is a kind of using tealeaves waste as the culture medium of edible fungus of primary raw material and preparation method |
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2018
- 2018-09-27 CN CN201811146372.XA patent/CN109380063B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61183194A (en) * | 1985-02-06 | 1986-08-15 | 金子農機株式会社 | Use of wheat straw or like |
| CN103858676A (en) * | 2014-03-24 | 2014-06-18 | 山东晨阳菌业有限公司 | Method for cultivating liquid cultures of cordyceps militaris |
| CN104987156A (en) * | 2015-07-09 | 2015-10-21 | 辽宁省农业科学院蔬菜研究所 | Lyophyllum fumosurn culture medium using fermented bran and method for cultivating lyophyllum fumosurn |
| CN105684727A (en) * | 2016-01-25 | 2016-06-22 | 赵建荣 | Culture medium and cultivation method for cultivating black termitomyces albuminosus with enzyme bacterium residues as main raw material |
| CN108467292A (en) * | 2018-05-28 | 2018-08-31 | 贵州湄潭兰馨茶业有限公司 | It is a kind of using tealeaves waste as the culture medium of edible fungus of primary raw material and preparation method |
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Address after: 113113 Hou An Zhen Nan Zhang Dang Cun, Fushun County, Fushun City, Liaoning Province Patentee after: Liaoning Sanyou Agricultural Biotechnology Group Co.,Ltd. Address before: 113113 Hou An Zhen Nan Zhang Dang Cun, Fushun County, Fushun City, Liaoning Province Patentee before: LIAONING SANYOU AGRICULTURAL BIOTECHNOLOGY Co.,Ltd. |