CN101904498B - Method for improving vitamin D content in cordyceps sinensis calcium strengthening agent by ultraviolet irradiation - Google Patents
Method for improving vitamin D content in cordyceps sinensis calcium strengthening agent by ultraviolet irradiation Download PDFInfo
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Abstract
The invention discloses a method for improving vitamin D content in a cordyceps sinensis calcium strengthening agent by ultraviolet irradiation, which comprises the following steps of: a, preparing a culture medium: preparing a liquid culture medium from a carbon source, a nitrogen source, mineral elements and the like, and sterilizing the prepared culture medium; b, inoculating cordyceps sinensis fungus into the culture medium, and performing shake culture to prepare liquid cordyceps sinensis fungus; c, inoculating the cordyceps sinensis fungus into the culture medium, performing the shake culture under certain conditions, and taking fermentation liquor out; and d, selecting a UV-B ultraviolet light source with the power of 14 to 60 W and the wavelength of 280 to 325 nm, pouring the cordyceps sinensis fungus fermentation liquor into a large culture dish, placing the ultraviolet light source over the culture dish, irradiating the prepared fermentation liquor, concentrating and drying the fermentation liquor, and breaking wall and grinding the obtained product into a powdery product. The method has the advantages of simple process, convenient operation and wide application to prevention and treatment of rickets of children, acalcerosis of women and osteoporosis of old peoples.
Description
Technical field
The present invention relates to supplementary calcium food and medical product, more particularly relate to a kind of method of improving vitamin D content in cordyceps sinensis calcium strengthening agent by ultraviolet irradiation, concrete grammar is by ultraviolet source irradiation liquid state fermentation aweto, makes the interior ergosterol light of cell be converted into vitamin D
2, can be used for preparation prevention and treatment children, women's calcium deficiency; The medicine of the various illnesss such as old man's osteoporosis or the application in the health food.
Background technology
The patent that Chinese patent discloses a kind of " preparation method of cordyceps biological activity multivitamin intensifying agent " (application number is 03106294.6).Utilize the medicinal fungi aweto to be bio-carrier, inorganic calcium can be changed into the calcium activated of large biological molecule chelating attitude through liquid state fermentation.Product contains multivitamin, thalline immune protein and mycelial polysaccharides, has immunoloregulation function, has fundamentally improved the utilization rate of calcium.Its defective is only to have considered to replenish the calcium, and does not consider to promote body to the absorption of calcium.If calcium supplementing product is vitamin D simultaneously, effect of supplemented calcium is better than simple additional calcium.The existing definite sufficient vitamin D that studies have shown that not only can promote body to the absorptivity of calcium, and can reduce the generation of heart disease, obesity, diabetes, arthritis even cancer.Although edible medicinal fungi is the important sources of vitamin D in the natural food, its content far can not satisfy human body to the demand of vitamin D.Vitamin D in the Creta Preparata also is that the people absorbs for adding with auxiliary calcium in the market, does not have biological homology and balance.Ergosterol is a plant sterols, and this material is the precursor of vitamin D, the domestic industrial production that only is used at present vitamin D.Contain abundant ergosterol among the fungal cell, Singapore scientist research in recent years is found, shines in the growth or the new fresh mushroom of just having plucked with ultraviolet ray, can make and produce a large amount of vitamin Ds in the mushroom
2(Food Chemistry Volume 95, Issue 4, April 2006, Pages 638-643).
Summary of the invention
The object of the present invention is to provide a kind of method of improving vitamin D content in cordyceps sinensis calcium strengthening agent by ultraviolet irradiation, technique of the present invention is simple, easy to operate, can be widely used in prevention and the treatment of children rachitis, women's calcium deficiency, senile osteoporosis disease.It is to carry out the calcium fortified while take the liquid state fermentation aweto as carrier, with ultraviolet source zymocyte liquid is being shone, and by control irradiation temperature and irradiation time, makes intracellular ergosterol be converted into to greatest extent vitamin D
2, vitamin D when replenishing the calcium is to reach the purpose that promotes that body calcium absorbs.By the promotion of vitamin D, body can improve absorptivity and the utilization rate to calcium greatly.
In order to realize above-mentioned task, the present invention adopts following solution:
Cordyceps sinensis biological calcium activated hardening agent makes inorganic calcium change into the biological active calcium of large molecule chelating attitude, improved the utilization rate of calcium at body, rely on simultaneously the amino acid that contains in the Cordyceps sinensis, polysaccharide, sterol, vitamin and trace element performance pharmacologically active, strengthen immunity of organisms when replenishing the calcium, regulate the digestive system and respiratory system function, anti-ageing, antitumor, have simultaneously health care and drug therapy effect.
Vitamin D is the conditioning agent of calcium, and it promotes small intestine and kidney to the absorption of calcium by the generation that promotes calbindin and heavily absorbs.In addition, vitamin D can also reduce the incidence of disease of heart disease, immunologic deficiency disease, diabetes, arthritis and some cancer.Contain 7-DHC in the normal human skin, be subjected to the irradiation of daylight middle-ultraviolet lamp namely to change vitamin D into
3, contain ergosterol in the plant, can not be absorbed by the body, but change vitamin D into through the ultraviolet ray irradiation
2Could be absorbed by human.
The present invention confirms UV-irradiation by experiment to the conversion capability of ergosterol in the liquid towards fermentation Cordyceps sinensis bacterial cell, and a kind of method of improving vitamin D content in cordyceps sinensis calcium strengthening agent by ultraviolet irradiation the steps include:
A, be formulated as follows culture medium: carbon source comprises the carbohydrate of 2~8% (weight/volume percent) such as glucose, sucrose, molasses, or starch based such as cereal starch, corn flour, sweet potato powder a kind of or form arbitrarily below six kinds; Nitrogenous source comprises dusty yeast, peptone, analysis for soybean powder, groundnut meal, corn steep liquor a kind of of 1~4% (weight/volume percent) or forms arbitrarily below five kinds; Carbon, nitrogen ratio are 10: 1~40: 1; It is as follows to add mineral element in every 100ml culture medium:
Peptone 0.5-2.0g
Glucose 2.0-10.0g
Dusty yeast 0.1-1.0g
KH
2PO
4 0.05-0.5g
MgSO
4 0.02-0.2g
With FeSO
4Add iron 0.001~0.1g
With ZnSO
4Add zinc 0.001~0.1g
With Na
2SeO
3Add selenium 0.000001~0.0001g
With CrCl
3Add chromium 0.000001~0.0001g
With CaCl
2, CaNO
3Or CaCO
3Add calcium 0.01~1.0g
VB
1 0.001-0.01g
Be settled to 100ml after adding water-solubleization;
With every bottle of 100ml packing 300ml triangular flask, 120 ℃ of sterilizations were cooled to below 30 ℃ after 30 minutes;
B, to the inoculation of medium of above-mentioned steps a in the center preservation of Chinese Typical Representative culture collection, preserving number is the aweto (Cordyceps Sinensis) of CCTCC AF99009, the 150rpm shaking flask was cultivated 80-120 hour under 26~30 ℃ of conditions, obtained the liquid aweto strain;
C, to the liquid aweto strain of the inoculation of medium 2-10% volume of above-mentioned steps a, the 150rpm shaking flask was cultivated 60-120 hour under 26~30 ℃ of conditions, took out zymotic fluid;
D, to select UV-B ultraviolet lamp tube, power 14W, wavelength be 280~325nm.Ultraviolet lamp tube is placed in the superclean bench top apart from sample 15cm place.When room temperature was 25 ℃, the cultured aweto liquid that step c is obtained was poured in the large culture dish, and thickness is 1cm with interior (because ultraviolet penetration power is limited), shines.Irradiation time is 1h, shine complete with zymotic fluid through concentrated, dry, be broken and crushed into powder products, record the thalline yield and be: 2% (2g freeze-dried powder/100ml zymotic fluid).
E, vitamin D
2Content with high effective liquid chromatography for measuring (Drying Technology, 2003,21,1093-1101.), chromatographic condition: C18 post (250 * 4.6mm); Mobile phase: acetonitrile-methyl alcohol (75: 25); Flow velocity: 1ml/min; Detect wavelength: 282nm.Sample will be processed through saponification first, get aweto freeze-dried powder 1g, add sodium ascorbate solution (the 17.5g ascorbic acid is dissolved in 100ml 1M NaOH) 4ml, ethanol 50ml (95%), 50%KOH solution 10ml is behind 85 ℃ of water-bath saponification 1h, be cooled to rapidly room temperature (20-25 ℃, below identical), move into separatory funnel.Add deionized water 15ml, ethanol 15ml is with three times (50ml, 50ml, 20ml) of pentane extraction.Keep organic layer, 50ml washes three times with KOH-ethanolic solution (3%KOH, 5% ethanol, solid reagent are weight/volume percent, liquid reagent is volume/volume percentage), is washed till neutrality with deionized water again.Organic layer is moved to round-bottomed flask, at 40 ℃ of lower concentrated evaporates to dryness, add rapidly the dissolving of 5ml ethanol with rotary evaporator.Cross the organic filter membrane of 0.45 μ m, get on the filtrate 20 μ l high efficiency liquid phase and analyze.By internal standard method and external standard method qualitative analysis vitamin D
2, quantize vitamin D in the calculation sample by peak area
2Content.
F, result: vitamin D in the aweto
2Content is brought up to postradiation 488 μ g/g (freeze-dried powder) from 43 μ g/g (freeze-dried powder) of pre-irradiation.
The above results explanation: aweto mycelium is through the UV-B ultra violet lamp after 1 hour, vitamin D in the cell
2Content is significantly increased.To be the ultraviolet ray irradiation radiation that makes the ergosterol absorbing ultraviolet change into first the intermediate of vitamin D by photochemical reaction to mechanism, changes at last vitamin D again
2
According to above-mentioned design and experimental result, realize that the object of the invention adopts following technical scheme:
The microorganism of using:
Produce the various bacterial classifications of the Ascomycetes Clavicipitaceae Cordyceps (Cordyceps (Fr.) Link) of medicinal ingredient.They neither binding neither be exclusive.
Concrete bacterial strain can be:
In the center preservation of Chinese Typical Representative culture collection, preserving number is Cordyceps sinensis (Cordyceps Sinensis) bacterial strain of CCTCC AF99009.The related properties of above-mentioned bacterial classification can be consulted Chinese Typical Representative culture collection center (CCTCC) bacterial classification catalogue, and the present invention repeats no more.Anyone can directly buy this bacterial classification to this preservation center.
The light source of selecting:
It is different that the radiation source of different wave length transforms impact to ergosterol light.The light that is shorter than 280nm is easy to generate tachysterol, and the light of being longer than 284nm generates lumisterol easily.Specifically selecting wavelength is the UV-B ultraviolet source of 280~325nm, can reduce the generation of accessory substance, and power is 14-60W, and the type ultraviolet source all has sale on the market.
The step that improves vitamin D in the cordyceps sinensis biological calcium activated hardening agent is:
A, preparation culture medium: with preparation fluid nutrient mediums such as carbon source, nitrogenous source, mineral elements, add following composition (weight/volume percent) in every 100ml culture medium:
Carbon source in the culture medium comprises the carbohydrate of 2~10% volumes such as glucose, sucrose, molasses, or starch based such as cereal starch, corn flour, sweet potato powder a kind of or form below six kinds;
Nitrogenous source comprises dusty yeast, peptone, analysis for soybean powder, groundnut meal, corn steep liquor a kind of of 1~4% volume or forms arbitrarily below five kinds;
Carbon, nitrogen ratio are 10: 1~40: 1;
Mineral element is the materials such as potassium, magnesium, phosphorus, sodium, sulphur:
KH
2PO
4 0.05-0.5g
MgSO
4 0.02-0.2g
With FeSO
4Add iron 0.001~0.1g
With ZnSO
4Add zinc 0.001~0.1g
With Na
2SeO
3Add selenium 0.000001~0.0001g
With CrCl
3Add chromium 0.000001~0.0001g
With CaCl
2, Ca (NO
3)
2Or CaCO
3Add calcium 0.01~1.0g
VB
1 0.001-0.01g
Be settled to 100ml after adding water-solubleization;
With the medium sterilization for preparing, 120 ℃ of sterilizations were cooled to below 30 ℃ after 30 minutes;
B, to the inoculation of medium of above-mentioned steps a in the center preservation of Chinese Typical Representative culture collection, preserving number is the aweto (Cordyceps Sinensis) of CCTCC AF99009, the 100-150rpm shaking flask was cultivated 80-120 hour under 26~30 ℃ of conditions, obtained the liquid aweto strain;
C, to the liquid aweto strain of inoculation of medium 2~10% volumes of above-mentioned steps a, the 100-150rpm shaking flask was cultivated 60-120 hour under 26~30 ℃ of conditions, took out zymotic fluid;
D, to select UV-B ultraviolet source, power 14-60W, wavelength be 280~325nm.Cordyceps militeris fungus fermentation broth is poured in the large culture dish, because ultraviolet penetration power is limited, bacterium liquid thickness is 0.2~2cm, ultraviolet source is placed in apart from 10~30cm place directly over the culture dish, under 15-30 ℃ room temperature, the zymotic fluid that step (c) is obtained shines, and irradiation time is determined at 20min~60min.Shine complete with zymotic fluid through concentrated, dry, be broken and crushed into powder products.
The cordyceps sinensis biological calcium activated hardening agent that said method obtains, vitamin D content is 10~50mg/100g, reach before strengthening more than ten times.
Product can be with beverage, oral liquid, chewable tablets, candy, capsule or other formulation for the preparation for the treatment of and prevention children rachitis, children's calcium deficiency; Women's calcium deficiency; The medicine of the various illnesss such as the decline of old man's immunity, osteoporosis or the application in the health food.
Contain other components (weight portion) in every 100g hardening agent product:
Bacterium protein 15.0~45.0
Total reducing sugar (comprising mycelial polysaccharides) 2.0~10.0
Carbohydrate 20.0~40.0
Total fat 2.0~10.0
Mineral matter 10.0~25.0
Moisture 2.0~10.0
Calcium 1.0~30.0
Iron 0.05~5.0
Zinc 0.05~5.0
Selenium 0.00005~0.005
Chromium 0.00005~0.005
Vitamin A 0.001~0.04
Vitamin B1 0.02~0.1
Vitamin B2 0.02~0.1
Vitamin B5 0.2~0.8
Vitamin B6 0.01~0.2
Vitamin B1 20.0005~0.001
Vitamin C 0.5~5.0
Vitamin E 0.00001~0.0001
Folic acid 0.0001~0.005.
The present invention compared with prior art has the following advantages and effect:
Production technology is simple, utilizes aweto to contain to enrich the characteristic of vitamin D Precursors, adopts cheap ultraviolet source, makes it be converted into vitamin D in cell
2, be equipped with the calcium of large biological molecule chelating attitude, be conducive to improve the bioavilability of product, realized biological active calcium and vitamin D truly.It is active to add the distinctive immunological regulation pharmacology of Cordyceps sinensis, makes product possess multi-efficiency, can be widely used in preparation treatment and prevention children rachitis, children's calcium deficiency; Women's calcium deficiency; The medicine of the various illnesss such as the decline of old man's immunity, osteoporosis or the application in the health food.The absorptivity of calcium reaches 61.2-78.7%, and utilization rate reaches 42.3-55.5%.
The specific embodiment
Embodiment 1:
The bacterial classification that adopts: the Cordyceps sinensis of CCTCC AF99009 (Cordyceps Sinensis) bacterial strain.
A, obtaining liq culture medium, add following composition (weight/volume percent) in every 100ml culture medium:
Sucrose 3.0g
Corn flour 3.0g
Analysis for soybean powder 1.5g
Dusty yeast 0.1g
KH
2PO
4 0.05g
MgSO
4 0.02g
With FeSO
4Add iron 0.01g
With ZnSO
4Add zinc 0.01g
With Na
2SeO
3Add selenium 0.00002g
With CrCl
3Add chromium 0.00002g
With CaCO
3Add calcium 0.2g
VB
1 0.002g
Be settled to 100ml after adding water-solubleization;
120 ℃ of sterilizations were cooled to 30 ℃ after 30 minutes;
B, to the inoculation of medium of above-mentioned steps a in the center preservation of Chinese Typical Representative culture collection, preserving number is the aweto (Cordyceps Sinensis) of CCTCC AF99009, the 120rpm shaking flask was cultivated 100 hours under 26~30 ℃ of conditions, obtained the liquid aweto strain;
C, to the liquid aweto strain of inoculation of medium 3% volume of above-mentioned steps a, the 120rpm shaking flask was cultivated 80 hours under 26 ℃ of conditions, obtained the liquid aweto strain;
D, the Cordyceps fermented liquid that step c is obtained are poured large culture dish into, and thickness is 0.5cm, and under 15 ℃ room temperature, (wavelength is 280~325nm) irradiations, and irradiation distance is 15cm, and irradiation time is 20min with the UV-B ultraviolet source of power 14W.Shine complete with zymotic fluid through concentrated, dry, be broken and crushed into powder products, vitamin D content reaches 13mg/100g in the product.
Measure other content of material in the product, contain following component (weight portion composition) in every 100g Creta Preparata:
Table 1. general analysis
Composition ± 25%
Protein 15
Total reducing sugar (comprising mycelial polysaccharides) 6
Total fat 4
Carbohydrate 30
Mineral matter 20
Moisture 6
Table 2. mineral matter
Composition ± 25%
Calcium 10
Iron 0.5
Zinc 0.4
Selenium 0.001
Chromium 0.001
Table 3. vitamin
Composition ± 25%)
Vitamin A 0.02
Vitamin B1 0.075
Vitamin B2 0.08
Vitamin B5 1.0
Vitamin B6 0.1
Cobalamin 0.0008
Vitamin C 2.0
Vitamin E 0.00008
Folic acid 0.003
Embodiment 2: vitamin D content reaches 35mg/100g in the product
The bacterial classification that adopts: the Cordyceps sinensis of CCTCC AF99009 (Cordyceps Sinensis) bacterial strain.
A, obtaining liq culture medium: add following composition (weight/volume percent) in every 100ml culture medium:
Sucrose 4.0g
Corn flour 4.0g
Analysis for soybean powder 2.0g
Dusty yeast 0.2g
KH
2PO
4 0.1g
MgSO
4 0.02g
With FeSO
4Add iron 0.01g
With ZnSO
4Add zinc 0.01g
With Na
2SeO
3Add selenium 0.00002g
With CrCl
3Add chromium 0.00002g
With CaCl
2Add calcium 0.3g
VB
1 0.003g
Be settled to 100ml after adding water-solubleization;
120 ℃ of sterilizations were cooled to 30 ℃ after 30 minutes;
B, to the inoculation of medium of above-mentioned steps a in the center preservation of Chinese Typical Representative culture collection, preserving number is the aweto (Cordyceps Sinensis) of CCTCC AF99009, the 150rpm shaking flask was cultivated 100 hours under 30 ℃ of conditions, obtained the liquid aweto strain;
C, add the liquid aweto strain of 5% volume in the culture medium of above-mentioned steps a; Air agitation was cultivated 100 hours under 30 ℃ of conditions, obtained Cordyceps fermented liquid;
D, the Cordyceps fermented liquid that step c is obtained are poured large culture dish into, and thickness is 1.0cm, and under 25 ℃ room temperature, (wavelength is 280~325nm) irradiations, and irradiation distance is 20cm, and irradiation time is 40min with the UV-B ultraviolet source of power 30W.Shine complete with zymotic fluid through concentrated, dry, be broken and crushed into powder products, vitamin D content reaches 33mg/100g in the product.
Measure other content of material in the product, contain following component (weight portion composition) in every 100g Creta Preparata:
Table 1. general analysis
Composition ± 25%
Protein 20
Total reducing sugar (comprising mycelial polysaccharides) 7
Total fat 4
Carbohydrate 30
Mineral matter 20
Moisture 6
Table 2. mineral matter
Composition ± 25%
Calcium 15
Iron 0.5
Zinc 0.4
Selenium 0.001
Chromium 0.001
Table 3. vitamin
Composition ± 25%)
Vitamin A 0.02
Vitamin B1 0.075
Vitamin B2 0.08
Vitamin B5 1.0
Vitamin B6 0.1
Cobalamin 0.0008
Vitamin C 3.0
Vitamin E 0.00008
Folic acid 0.003
Embodiment 3:
The bacterial classification that adopts: the Cordyceps sinensis of CCTCC AF99009 (Cordyceps Sinensis) bacterial strain.
A, obtaining liq culture medium: add following composition (weight/volume percent) in every 100ml culture medium:
Sucrose 6.0g
Corn flour 4.0g
Analysis for soybean powder 2.0g
Dusty yeast 0.3g
KH
2PO
4 0.1g
MgSO
4 0.02g
With FeSO
4Add iron 0.01g
With ZnSO
4Add zinc 0.01g
With Na
2SeO
3Add selenium 0.00002g
With CrCl
3Add chromium 0.00002g
With Ca (NO
3)
2Add calcium 0.4g
VB
1 0.003g
Be settled to 100ml after adding water-solubleization;
120 ℃ of sterilizations were cooled to 30 ℃ after 30 minutes;
B, to the inoculation of medium of above-mentioned steps a in the center preservation of Chinese Typical Representative culture collection, preserving number is the aweto (Cordyceps Sinensis) of CCTCC AF99009, the 150rpm shaking flask was cultivated 120 hours under 28 ℃ of conditions, obtained the liquid aweto strain;
C, to above-mentioned steps a culture medium in add the liquid aweto strain of 8% volume; Air agitation was cultivated 120 hours under 28 ℃ of conditions, obtained Cordyceps fermented liquid;
D, the Cordyceps fermented liquid that step c is obtained are poured large culture dish into, and thickness is 1.0cm, and under 30 ℃ room temperature, (wavelength is 280~325nm) irradiations, and irradiation distance is 20cm, and irradiation time is 60min with the UV-B ultraviolet source of power 30W.Shine complete with zymotic fluid through concentrated, dry, be broken and crushed into powder products, vitamin D content reaches 48mg/100g in the product.
Measure other content of material in the product, contain following component (weight portion composition) in every 100g Creta Preparata:
Table 1. general analysis
Composition ± 25%
Protein 24
Total reducing sugar (comprising mycelial polysaccharides) 8
Total fat 4
Carbohydrate 30
Mineral matter 20
Moisture 6
Table 2. mineral matter
Composition ± 25%
Calcium 18
Iron 0.5
Zinc 0.4
Selenium 0.001
Chromium 0.001
Table 3. vitamin
Composition ± 25%)
Vitamin A 0.02
Vitamin B1 0.075
Vitamin B2 0.08
Vitamin B5 1.0
Vitamin B6 0.1
Cobalamin 0.0008
Vitamin C 4.0
Vitamin E 0.00008
Folic acid 0.003.
Claims (1)
1. the method for an improving vitamin D content in cordyceps sinensis calcium strengthening agent by ultraviolet irradiation the steps include:
A, preparation culture medium: prepare fluid nutrient medium with carbon source, nitrogenous source, mineral element, add following composition in every 100ml culture medium: the carbon source in the culture medium is glucose, sucrose, the molasses of 2~10% weight/volume percent, or cereal starch, corn flour, sweet potato powder a kind of or form below six kinds;
Nitrogenous source is dusty yeast, peptone, analysis for soybean powder, groundnut meal, corn steep liquor a kind of of 1~4% weight/volume percent or forms below five kinds;
Carbon, nitrogen ratio are 10: 1~40: 1;
Mineral element is potassium, magnesium, phosphorus, iron, zinc, sodium, sulphur, selenium, chromium, calcium:
Be settled to 100ml after adding water-solubleization;
With the medium sterilization for preparing, 120 ℃ of sterilizations were cooled to below 30 ℃ after 30 minutes;
B, to above-mentioned inoculation of medium in the center preservation of Chinese Typical Representative culture collection, preserving number is the aweto Cordyceps Sinensis of CCTCC AF99009, the 100-150rpm shaking flask was cultivated 80-120 hour under 26~30 ℃ of conditions, obtained the liquid aweto strain;
C, to the liquid aweto strain of inoculation of medium 2~10% volumes of above-mentioned steps (a), the 100-150rpm shaking flask was cultivated 60-120 hour under 26~30 ℃ of conditions, took out zymotic fluid;
D, select the UV-B ultraviolet source, power 14-60W, wavelength is 280~325nm, and cordyceps militeris fungus fermentation broth is poured in the large culture dish, and bacterium liquid thickness is 0.2~2cm, ultraviolet source is placed in apart from 10~30cm place directly over the culture dish, under 15-30 ℃ room temperature, the zymotic fluid that step (c) is obtained shines, and irradiation time is determined at 20min~60min, shine complete with zymotic fluid through concentrated, dry, be broken and crushed into powder products.
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