CN106667894A - Pineapple ferment and application of pineapple ferment as tyrosinase inhibitor - Google Patents
Pineapple ferment and application of pineapple ferment as tyrosinase inhibitor Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
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Abstract
The invention relates to pineapple ferment and application of the pineapple ferment as a tyrosinase inhibitor. The pineapple ferment is obtained by naturally fermenting wild yeast on the surfaces of the pineapple peels or pineapple leaves or bromelin in the presence of glucose, and has the characteristics that raw materials are cheap and easy to get and waste can be converted into treasure. By adopting the pineapple ferment and the application of the pineapple ferment, not only can the problems that byproducts of the pineapple processing-pineapple peel resources are wasted and the environment is polluted be solved, but also the pineapple ferment with an effect on inhibiting tyrosinase is obtained by virtue of the natural fermenting technology. The pineapple ferment can be used as a whitening agent in the field of cosmetics and is natural and non-irritating.
Description
Technical field
The invention belongs to cosmetic field, and in particular to a kind of bromelain and preparation method thereof and suppression tryrosinase
Purposes.
Background technology
The main melanocyte by human body of melanin (melanin) is produced, and it can reduce injury of the ultraviolet to skin；
But melanic abnormal accumulation can cause hyperpigmentation, easily cause freckle, senile plaque, melasma etc..Researcher is sent out
Existing tryrosinase has significant role in melanic biosynthetic process.During melanin biosynthesis, tryrosinase
Play highly important catalytic action.Tryrosinase can promote L-Tyrosine to be converted into L-3,4 dihydroxyphenylalanine, and L-3,4 dihydroxyphenylalanine is again through oxidation
And DOPA quinone is converted into, DOPA quinone poly generates melanin.
Fructus Ananadis comosi sarcocarp belongs to the Leaf-feeding insects of Fructus Ananadis comosi, containing abundant nutrient substance；But pineapple peel and pineapple leaves are
Inedible part, is the by-product of Fructus Ananadis comosi processing generation, if not being acted upon and utilizing, can be to environment and resource
Waste.The purpose of the present invention is the inhibitory action for studying Fructus Ananadis comosi sarcocarp, peel and leaf to tryrosinase, if there is certain effect,
The wasting of resources would not be caused in productive life from now on, while can also reduce the pollution to environment.
The content of the invention
The present invention provides a kind of bromelain, it is characterised in that the bromelain is prepared by the following method, methods described
Comprise the steps：
The raw material of certain mass is taken, after pretreatment, in being placed in the fermentation tank of Jing aseptic process, raw materials quality is subsequently adding
20%-35% mass glucose after, fermentation tank is sealed, at 15-25 DEG C, natural fermentation, 25-35 days.The raw material
For fresh pineapple peel, Fructus Ananadis comosi sarcocarp or pineapple leaves；The pretreatment comprises the steps：Raw material is rinsed into 2-3 with tap water
It is secondary, then with distilled water flushing 1-2 time, then with aseptic water washing 1-2 time, then with 75% alcohol flushing 2 times, ultra violet lamp
5 minutes, finally shredded.
The bromelain that the present invention is provided is by pineapple peel or wild yeasts bacterium or the bromelain of Fructus Ananadis comosi leaf surface
Enzyme, in the presence of glucose, what natural fermentation was obtained, cheap and easy to get with raw material, the characteristics of turning waste into wealth, the present invention is not only solved
By-product --- the pineapple peel wasting of resources, the problem to environment that Fructus Ananadis comosi of having determined processing is produced, and by certainly
So fermentation technique is also obtained with the bromelain for suppressing tryrosinase effect, can be used for cosmetic field as whitening agent,
Natural no stimulation.
The present invention provides a kind of preparation method of bromelain, it is characterised in that comprise the steps：Take certain mass
Raw material, after pretreatment, in being placed in the fermentation tank of Jing aseptic process, is subsequently adding the Portugal of the 20%-35% mass of raw materials quality
After grape sugar, fermentation tank is sealed, at 15-25 DEG C, natural fermentation, 25-35 days.The raw material is fresh pineapple peel, Fructus Ananadis comosi
Sarcocarp or pineapple leaves；The pretreatment comprises the steps：Raw material is rinsed 2-3 time with tap water, distilled water flushing is then used
1-2 time, then with aseptic water washing 1-2 time, then with 75% alcohol flushing 2 times, ultra violet lamp 5 minutes is finally shredded.
" 75% alcohol flushing 2 times, ultra violet lamp 5 minutes " is to kill antibacterial, ferment in preparation method of the present invention
Female bacterium is substantially unaffected.
The present invention provides a kind of purposes of bromelain in whitening agent is prepared, and the bromelain is selected from pineapple peel ferment
Element, Fructus Ananadis comosi sarcocarp ferment or pineapple leaves ferment.
The present invention provides a kind of application of bromelain in terms of tryrosinase is suppressed, and the bromelain is selected from pineapple sarcocarp
Skin ferment, Fructus Ananadis comosi sarcocarp ferment or pineapple leaves ferment.
Description of the drawings
The melanic light absorption values of Fig. 1 and temperature (A-T) figure
The melanic light absorption values of Fig. 2 and response time (A-t) figure
The melanic light absorption values of Fig. 3 and L-Tyrosine substance withdrawl syndrome (A-C) figure
The melanic light absorption values of Fig. 4 and L-Tyrosine consumption (A-V) figure
The melanic light absorption values of Fig. 5 and Rhizoma Solani tuber osi quality/volume of buffer solution figure
The graph of a relation of Fig. 6 pineapple peel ferment concentration and tyrosinase inhibition rate
The graph of a relation of Fig. 7 Fructus Ananadis comosis sarcocarp ferment concentration and tyrosinase inhibition rate
The graph of a relation of Fig. 8 pineapple leaves ferment concentration and tyrosinase inhibition rate
The graph of a relation of Fig. 9 arbutin ferment concentration and tyrosinase inhibition rate
Specific embodiment
The tryrosinase being related in the present invention be can by document method (the good sample of Lee, Lv Haiyan, Dong Jinlong. in Rhizoma Solani tuber osi
The extraction of tryrosinase and its research [J] of activity. spectrographic laboratory, 2008,25 (6)：1040-1043) extract from Rhizoma Solani tuber osi
's；Or extract as follows：Rhizoma Solani tuber osi is cleaned and is peeled, refrigerator freezing layer, freeze overnight are positioned over after stripping and slicing.The used time is needed to take out
Weigh, buffer solution [the Rhizoma Solani tuber osi quality with corresponding proportion：Volume of buffer solution=1：5 (g/mL)] it is co-located in being stirred in blender
Mix；Resulting solution is centrifuged, centrifugation rate is 4000r/min, a length of 5min during centrifugation, it is used to test to take supernatant
(i.e. Rhizoma Solani tuber osi extracting solution).
Buffer solution of the present invention is the mixed phosphate salt buffer solution of pH=6.86.
The present invention extracts ferment from the different parts (peel, sarcocarp, leaf) of Fructus Ananadis comosi, determines it to L-Tyrosine in Rhizoma Solani tuber osi
The inhibitory action of enzymatic activity, by calculating suppression ratio inhibitory action is reflected.With arbutin as positive control, compare IC50's
Size (Ren Hongrong, singly holds Oriolus chinensis diffususs, Jiang Hongfang, etc. perfume Flos Nelumbinis extract suppresses the research [J] of tyrosinase activity. natural product
Research and development, 2011,23 (06)：1122-1126；Ye Xiaozhao, Gong Shengzhao, Liao Guojun, etc. Radix Angelicae Sinensis extract is to tryrosinase
Inhibitory action [J]. daily chemical industry, 2010,40 (02)：98-100).
Suppression ratio computing formula：[1- (T2-T1)/(C2-C1)] × 100%
C1 represents the absorbance of only substrate (mixed solution of Rhizoma Solani tuber osi extracting solution and buffer solution), and C2 is represented the bottom of containing
The absorbance of thing and L-Tyrosine, C2-C1 represents the melanic absorbance generated after the absorbance for deducting substrate.T1
The absorbance containing substrate and sample is represented, T2 represents the absorbance containing substrate, L-Tyrosine and sample, T2-T1 tables
Show the suppression through sample, the remaining melanic absorbance after the absorbance of sample and substrate is deducted.
The tryrosinase of embodiment 1 promotes L-Tyrosine to generate the determination of melanic optimum activity condition
(1) impact of the temperature to tryrosinase optimum activity
The brown volumetric flask 8 that 10mL is totally dried is taken, is divided into 4 groups, 2 per group, be respectively labeled as C1 and C2.According to table
1 adds each test solution：
The system of table 1 is constituted and each reagent dosage
According to upper table the buffer solution and L-Tyrosine solution of corresponding amount are added as the bottom of reaction system to 4 pool-size bottles
Thing, is subsequently adding the Rhizoma Solani tuber osi extracting solution of corresponding amount, by 4 groups of solution be respectively placed in 0 DEG C, 10 DEG C, 30 DEG C, at 40 DEG C, react 35min
Afterwards, the measurement (it is best that measurement wavelength is 475nm) of absorbance is carried out immediately.Such as Fig. 1.
(2) impact of the system response time to tryrosinase optimum activity
The brown volumetric flask two that 100mL is totally dried is taken, C1 and C2 is labeled as, according to table 2 each test solution is added：
The system of table 2 is constituted and each reagent dosage
According to upper table buffer solution and L-Tyrosine solution that 2 volumetric flasks add corresponding amount are given as the bottom of reaction system
Thing, is subsequently adding the Rhizoma Solani tuber osi extracting solution of corresponding amount, and volumetric flask is placed in into 10 DEG C of water-baths, measures an absorbance every 5min, instead
Total duration is answered for 90min (measurement wavelength is 475nm).Such as Fig. 2.
(3) impact of the L-Tyrosine substance withdrawl syndrome to tryrosinase optimum activity
The L-Tyrosine powder of 0.0181g is accurately weighed with electronic balance, constant volume is dissolved in the appearance of 100ml with buffer solution
In measuring bottle, the L-Tyrosine solution of 1mmol/L can be obtained.
Take the volumetric flask that 6 10mL are totally dried, be separately added into above-mentioned solution 1.0mL, 2.0mL, 3.0mL, 4.0mL,
5.0mL, 6.0mL, with buffer solution graduation mark is settled to, obtain final product substance withdrawl syndrome be respectively 0.1mmol/L, 0.2mmol/L,
The L-Tyrosine solution of 0.3mmol/L, 0.4mmol/L, 0.5mmol/L, 0.6mmol/L.
The brown volumetric flask 12 that 10mL is totally dried separately is taken, is divided into 6 groups, 2 per group, be respectively labeled as C1 and C2.Press
Each test solution is added according to table 3：
The system of table 3 is constituted and each reagent dosage
Add the L-Tyrosine of the amount concentration of the buffer solution and different material of corresponding amount molten to 4 pool-size bottles according to upper table
Liquid is subsequently adding the Rhizoma Solani tuber osi extracting solution of corresponding amount as the substrate of reaction system.Volumetric flask is placed in into 10 DEG C of water-baths, is reacted
After 35min, absorbance is measured immediately (measurement wavelength is 475nm).Such as Fig. 3.
(4) impact of the L-Tyrosine consumption to tryrosinase optimum activity
The volumetric flask that 10 10mL are totally dried is taken, is divided into 5 groups, 2 per group, be respectively labeled as C1 and C2.Add according to table 4
Enter each test solution：
The system of table 4 is constituted and each reagent dosage
In table x be 1.0mL, 1.5mL, 2.0mL, 2.5mL, 3.0mL, correspondence y be 8.0mL, 7.5mL, 7.0mL, 6.5mL,
6.0mL, to 5 pool-size bottles the buffer solution and different amounts of L-Tyrosine solution (concentration is 0.3mmol/L) of corresponding amount are added
As the substrate of reaction system, the Rhizoma Solani tuber osi extracting solution of corresponding amount is subsequently adding.Volumetric flask is placed in into 10 DEG C of water-baths, 35min is reacted
Afterwards, absorbance is measured immediately (measurement wavelength is 475nm).Such as Fig. 4.
(5) Rhizoma Solani tuber osi quality and volume of buffer solution compare the impact of tryrosinase optimum activity
The volumetric flask that 10 10mL are totally dried is taken, is divided into 5 groups, 2 per group, be respectively labeled as C1 and C2.Add according to table 4
Enter each test solution：
The system of table 5 is constituted and each reagent dosage
Rhizoma Solani tuber osi quality is respectively 1 with volume of buffer solution ratio (g/mL) in Rhizoma Solani tuber osi extracting solution:1、1:2、1:3、1:4、1:5.
To 5 pool-size bottles the buffer solution and L-Tyrosine solution (concentration is 0.3mmol/L) of corresponding amount are added as reaction system
Substrate, is subsequently adding the Rhizoma Solani tuber osi extracting solution of corresponding amount.Volumetric flask is placed in into 10 DEG C of water-baths, after reaction 35min, extinction is measured immediately
Degree (measurement wavelength is 475nm).Such as Fig. 5.
Jing determines that tryrosinase promotes L-Tyrosine to generate melanic optimum activity condition：10 DEG C of temperature, response time
35min, L-Tyrosine substance withdrawl syndrome is 0.3mmol/L, and the volumetric usage of L-Tyrosine is 2.5mL, prepares Rhizoma Solani tuber osi extraction
Rhizoma Solani tuber osi quality and the volume ratio of buffer solution are 1 during liquid：4.
Embodiment 2
Fresh pineapple peel 2.0kg is weighed, is rinsed 2 times with tap water, then with distilled water flushing 1 time, then use sterilized water
Rinse 2 times, then with 75% alcohol flushing 2 times, ultra violet lamp 5 minutes is finally shredded, and is placed in sending out for Jing aseptic process
In fermentation tank, after being subsequently adding 400g glucoses, fermentation tank is sealed, at 15-25 DEG C, after natural fermentation 25 days, by fermentation liquid
Take out, in being placed in centrifuge tube, Jing after centrifugation, take supernatant, carry out concentrating under reduced pressure, obtain solid sample 10.83g, as Fructus Ananadis comosi
Peel ferment.
Embodiment 3
Fresh pineapple peel 1.0kg is weighed, is rinsed 2 times with tap water, then with distilled water flushing 2 times, then use sterilized water
Rinse 1 time, then with 75% alcohol flushing 2 times, ultra violet lamp 5 minutes is finally shredded, and is placed in sending out for Jing aseptic process
In fermentation tank, after being subsequently adding 350g glucoses, fermentation tank is sealed, at 15-25 DEG C, after natural fermentation 35 days, by fermentation liquid
Take out, in being placed in centrifuge tube, Jing after centrifugation, take supernatant, carry out concentrating under reduced pressure, obtain solid sample 6.65g, as pineapple sarcocarp
Skin ferment.
Embodiment 4
Fresh pineapple sarcocarp 1.0kg is weighed, is rinsed 2 times with tap water, then with distilled water flushing 2 times, then use sterilized water
Rinse 1 time, then with 75% alcohol flushing 2 times, ultra violet lamp 5 minutes is finally shredded, and is placed in sending out for Jing aseptic process
In fermentation tank, after being subsequently adding 350g glucoses, fermentation tank is sealed, at 15-25 DEG C, after natural fermentation 35 days, by fermentation liquid
Take out, in being placed in centrifuge tube, Jing after centrifugation, take supernatant, carry out concentrating under reduced pressure, obtain solid sample 7.23g, as pineapple sarcocarp
Meat ferment.
Embodiment 5
Fresh pineapple leaf 2.0kg is weighed, is rinsed 2 times with tap water, then with distilled water flushing 1 time, then rushed with sterilized water
Wash 2 times, then with 75% alcohol flushing 2 times, ultra violet lamp 5 minutes is finally shredded, and is placed in the fermentation of Jing aseptic process
In tank, after being subsequently adding 400g glucoses, fermentation tank is sealed, at 15-25 DEG C, after natural fermentation 25 days, fermentation liquid is taken
Go out, in being placed in centrifuge tube, Jing after centrifugation, take supernatant, carry out concentrating under reduced pressure, obtain solid sample 9.76g, as pineapple leaves ferment
Element.
Jing HPLC are analyzed, and embodiment 2 is consistent with the HPLC figure appearance situations of the pineapple peel ferment that embodiment 3 is prepared
, 95%, it is poor with the appearance concordance of embodiment 4, and similarity is only 47% for property, with the appearance concordance of embodiment 5 more
Difference, similarity is only 36%.
Embodiment 6
Under the conditions of the tryrosinase that embodiment 1 determines promotes the melanic optimum activity of L-Tyrosine generation, survey respectively
Pineapple peel ferment, Fructus Ananadis comosi sarcocarp ferment, the pineapple leaves ferment that examination embodiment 2, embodiment 4, embodiment 5 are prepared is to cheese ammonia
The impact of phytase activity, using arbutin as positive control.
(1) inhibitory action of the pineapple peel ferment to tyrosinase activity
Take the volumetric flask that 1 25mL is totally dried, the pineapple peel ferment solid sample of accurate weighing 0.25g, with buffering
Solution dissolves constant volume in volumetric flask, can obtain the sample solution that concentration is 10mg/mL.
Separately take the volumetric flask that 9 10mL are totally dried, be separately added in each volumetric flask above-mentioned solution 0.5mL, 0.8mL,
1.0mL, 1.5mL, 2.0mL, 3.0mL, 3.5mL, 4.0mL, 4.5mL, with buffer solution graduation mark is settled to, and can obtain mass concentration
9 bottles of respectively 0.5mg/mL, 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 3.5mg/mL, 4.0mg/mL, 4.5mg/mL are molten
Liquid, labelled labelling.
The volumetric flask that 36 10mL are totally dried is taken again, is divided into 9 groups, 4 per group, be respectively labeled as C1、C2、T1、T2.Press
Each test solution is added according to table 6：
The system of table 6 is constituted and each reagent dosage
According to upper table to 9 pool-size bottles add corresponding amount buffer solution and L-Tyrosine solution (concentration is 0.3mmol/
L) as the substrate of reaction system, (Rhizoma Solani tuber osi quality and volume of buffer solution ratio are 1 to be subsequently adding the Rhizoma Solani tuber osi extracting solution of corresponding amount:
4) sample solution of the equivalent of variable concentrations, is added in different groups in T1 and T2.Volumetric flask is placed in into 10 DEG C of water-baths, is reacted
After 35min, absorbance is measured immediately (measurement wavelength is 475nm).Such as Fig. 6, under tyrosinase activity optimum condition, work as concentration
During between 0.5mg/mL~3.0mg/mL, sample increases the suppression ratio of enzymatic activity with the increase of concentration, and is in concentration
Maximum is reached during 3.0mg/mL, suppression ratio now is 76.11%；When concentration is between 3.0mg/mL~4.5mg/mL,
Suppression ratio reduces with the increase of concentration.In suppression ratio rising part, half-inhibition concentration is 0.98mg/mL, i.e. IC50=
0.98mg/mL。
(2) inhibitory action of the Fructus Ananadis comosi sarcocarp ferment to tyrosinase activity
Take the volumetric flask that 1 25mL is totally dried, the perfume Fructus Ananadis comosi sarcocarp solid sample of accurate weighing 0.25g, with buffering
Solution dissolves constant volume in volumetric flask, can obtain the sample solution that concentration is 10mg/mL.
Separately take the volumetric flask that 8 10mL are totally dried, be separately added in each volumetric flask above-mentioned solution 0.5mL, 1.0mL,
1.5mL, 1.8mL, 2.0mL, 2.5mL, 3.0mL, 4.0mL, with buffer solution graduation mark is settled to, and can be obtained mass concentration and is respectively
8 bottles of solution of 1.0mg/mL, 1.5mg/mL, 2.0mg/mL, 2.5mg/mL, 3.0mg/mL, 4.0mg/mL, labelled labelling.
The volumetric flask that 32 10mL are totally dried is taken again, is divided into 8 groups, 4 per group, be respectively labeled as C1、C2、T1、T2.Press
Each test solution is added according to table 7：
The system of table 7 is constituted and each reagent dosage
According to upper table to 7 pool-size bottles add corresponding amount buffer solution and L-Tyrosine solution (concentration is 0.3mmol/
L) as the substrate of reaction system, (Rhizoma Solani tuber osi quality and volume of buffer solution ratio are 1 to be subsequently adding the Rhizoma Solani tuber osi extracting solution of corresponding amount:
4), T in different groups1And T2The sample solution of the equivalent of middle addition variable concentrations.Volumetric flask is placed in into 10 DEG C of water-baths, 35min is reacted
Afterwards, absorbance is measured immediately (measurement wavelength is 475nm).Such as Fig. 7, under tyrosinase activity optimum condition, when concentration is
When between 0.5mg/mL~2.0mg/mL, sample increases the suppression ratio of enzymatic activity with the increase of concentration, and is in concentration
Maximum is reached during 2.0mg/mL, suppression ratio now is 75.00%；When concentration is between 2.0mg/mL~4.0mg/mL,
Suppression ratio reduces with the increase of concentration.In suppression ratio rising part, half-inhibition concentration is 1.66mg/mL, i.e. IC50=
1.66mg/mL。
(3) inhibitory action of the pineapple leaves ferment to tyrosinase activity
The volumetric flask that 1 25mL is totally dried is taken, the perfume pineapple leaves solid sample of accurate weighing 0.25g is molten with buffering
Liquid dissolves constant volume in volumetric flask, can obtain the sample solution that concentration is 10mg/mL.
Separately take the volumetric flask that 8 10mL are totally dried, be separately added in each volumetric flask above-mentioned solution 1.0mL, 2.0mL,
3.0mL, 3.5mL, 4.0mL, 4.5mL, 5.0mL, 5.5mL, with buffer solution graduation mark is settled to, and can be obtained mass concentration and is respectively
8 bottles of 1.0mg/mL, 2.0mg/mL, 3.0mg/mL, 3.5mg/mL, 4.0mg/mL, 4.5mg/mL, 5.0mg/mL, 5.5mg/mL
Solution, labelled labelling.
The volumetric flask that 32 10mL are totally dried is taken again, is divided into 8 groups, 4 per group, be respectively labeled as C1, C2, T1, T2.Press
Each test solution is added according to table 8：
The system of table 8 is constituted and each reagent dosage
According to upper table to 8 pool-size bottles add corresponding amount buffer solution and L-Tyrosine solution (concentration is 0.3mmol/
L) as the substrate of reaction system, (Rhizoma Solani tuber osi quality and volume of buffer solution ratio are 1 to be subsequently adding the Rhizoma Solani tuber osi extracting solution of corresponding amount:
4) sample solution of the equivalent of variable concentrations, is added in different groups in T1 and T2.Volumetric flask is placed in into 10 DEG C of water-baths, is reacted
After 35min, absorbance is measured immediately (measurement wavelength is 475nm).Such as Fig. 8, under tyrosinase activity optimum condition, work as concentration
During between 1.0mg/mL~4.0mg/mL, sample increases the suppression ratio of enzymatic activity with the increase of concentration, and is in concentration
Maximum is reached during 4.0mg/mL, suppression ratio now is 81.44%；When concentration is between 4.0mg/mL~5.5mg/mL,
Suppression ratio reduces with the increase of concentration.In suppression ratio rising part, half-inhibition concentration is 1.97mg/mL, i.e. IC50=
1.97mg/mL。
(4) inhibitory action of the arbutin to tyrosinase activity
The volumetric flask that 1 25mL is totally dried is taken, the arbutin solid sample of accurate weighing 0.25g is molten with buffer solution
Solution constant volume can obtain the sample solution that concentration is 10mg/mL in volumetric flask.
Separately take the volumetric flask that 9 10mL are totally dried, be separately added in each volumetric flask above-mentioned solution 0.5mL, 0.8mL,
1.0mL, 1.5mL, 1.8mL, 2.0mL, 2.5mL, 3.0mL, 3.5mL, with buffer solution graduation mark is settled to, and can obtain mass concentration
Respectively 0.5mg/mL, 0.8mg/mL, 1.0mg/mL, 1.5mg/mL, 1.8mg/mL, 2.0mg/mL, 2.5mg/mL, 3.0mg/
9 bottles of solution of mL, 3.5mg/mL, labelled labelling.
The volumetric flask that 36 10mL are totally dried is taken again, is divided into 9 groups, 4 per group, be respectively labeled as C1, C2, T1, T2.Press
Each test solution is added according to table 9：
The system of table 9 is constituted and each reagent dosage
According to upper table to 9 pool-size bottles add corresponding amount buffer solution and L-Tyrosine solution (concentration is 0.3mmol/
L) as the substrate of reaction system, (Rhizoma Solani tuber osi quality and volume of buffer solution ratio are 1 to be subsequently adding the Rhizoma Solani tuber osi extracting solution of corresponding amount:
4) sample solution of the equivalent of variable concentrations, is added in different groups in T1 and T2.Volumetric flask is placed in into 10 DEG C of water-baths, is reacted
After 35min, absorbance is measured immediately (measurement wavelength is 475nm).Such as Fig. 9, under tyrosinase activity optimum condition, work as concentration
During between 0.5mg/mL~2.0mg/mL, sample increases the suppression ratio of enzymatic activity with the increase of concentration, and is in concentration
Maximum is reached during 2.0mg/mL, suppression ratio now is 61.68%；When concentration is between 2.0mg/mL~3.5mg/mL, suppression
Rate processed reduces with the increase of concentration.In suppression ratio rising part, half-inhibition concentration is 1.55mg/mL, i.e. IC50=
1.55mg/mL。
By the test result (Fig. 6-Fig. 9) of the present embodiment (1)-(4), it can be seen that pineapple peel ferment is to tryrosinase
Inhibitory activity preferably (IC50=0.98mg/mL), better than the work of Fructus Ananadis comosi sarcocarp ferment and pineapple leaves ferment and positive control arbutin
Property.Analysis is probably pineapple peel by its surface wild yeasts bacterium in the presence of glucose, and natural fermentation is generated to cheese ammonia
Sour enzyme has the secondary metabolite of higher inhibitory activity, by its HPLC collection of illustrative plates it can also be seen that pineapple peel ferment and Fructus Ananadis comosi
Sarcocarp ferment and pineapple leaves ferment go out peak position have it is dramatically different.
Embodiment 7
Method according to document (Chinese herbal medicine, the 7th the 949-954 page of phase of volume 46) obtains the ethanol of pineapple leaves 95% and extracts
Thing and pineapple leaves acetone extract is obtained according to the method for the A of patent CN 104523463, according to the side in the embodiment of the present invention 6
Method, carries out test tyrosinase inhibitory activity, as a result finds the ethanol extraction and pineapple leaves acetone extract of pineapple leaves 95%
When concentration is 5mg/mL, it is to tyrosinase inhibition rate less than 15%.
Claims (7)
1. a kind of bromelain, it is characterised in that the bromelain is prepared by the following method, and methods described comprises the steps：
A certain amount of raw material is taken, after pretreatment, in being placed in the fermentation tank of Jing aseptic process, raw materials quality is subsequently adding
After the glucose of 20%-35% mass, fermentation tank is sealed, at 15-25 DEG C, natural fermentation, 25-35 days.
2. the bromelain described in claim 1, it is characterised in that the raw material is fresh pineapple peel, Fructus Ananadis comosi sarcocarp or Fructus Ananadis comosi
Leaf.
3. the bromelain described in any one of claim 1-2, it is characterised in that the pretreatment comprises the steps：By raw material
Rinsed 2-3 time with tap water, then with distilled water flushing 1-2 time, then with aseptic water washing 1-2 time, then with 75% ethanol punching
Wash 2 times, ultra violet lamp 5 minutes is finally shredded.
4. purposes of the bromelain described in any one of claim 1-3 in whitening agent is prepared.
5. the purposes described in claim 4, it is characterised in that the bromelain is selected from pineapple peel ferment.
6. application of the bromelain described in any one of claim 1-3 in terms of tryrosinase is suppressed.
7. the application described in claim 6, it is characterised in that the bromelain is selected from pineapple peel ferment.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112386547A (en) * | 2019-08-14 | 2021-02-23 | 百岳特生物技术（上海）有限公司 | Skin health care application of pineapple extract |
| CN113499298A (en) * | 2021-06-24 | 2021-10-15 | 青岛黛优佳生物科技有限公司 | Cosmetic with whitening and freckle removing synergistic effect and application |
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| CN105567480A (en) * | 2016-02-25 | 2016-05-11 | 青岛洁百氏科技有限公司 | Enzyme soap and preparation method thereof |
| CN106333894A (en) * | 2016-10-10 | 2017-01-18 | 辽宁石油化工大学 | Method for preparing natural sunscreen and skin whitening components in cosmetics by utilizing fruits |
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| CN105567480A (en) * | 2016-02-25 | 2016-05-11 | 青岛洁百氏科技有限公司 | Enzyme soap and preparation method thereof |
| CN106333894A (en) * | 2016-10-10 | 2017-01-18 | 辽宁石油化工大学 | Method for preparing natural sunscreen and skin whitening components in cosmetics by utilizing fruits |
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| CN112386547A (en) * | 2019-08-14 | 2021-02-23 | 百岳特生物技术（上海）有限公司 | Skin health care application of pineapple extract |
| CN113499298A (en) * | 2021-06-24 | 2021-10-15 | 青岛黛优佳生物科技有限公司 | Cosmetic with whitening and freckle removing synergistic effect and application |
| CN113499298B (en) * | 2021-06-24 | 2023-01-24 | 青岛黛优佳生物科技有限公司 | Cosmetic with whitening and freckle removing synergistic effect and application |
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