• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    基于無性繁殖過程的蝙蝠蛾擬青霉循環(huán)發(fā)酵方法

    2018-11-05 00:49:58SayedHassanRazaShah陸震鳴李碩碩李華祥羅志珊張曉娟史勁松許正宏
    中國釀造 2018年9期
    關(guān)鍵詞:藥學(xué)院無錫江南

    Sayed-Hassan-Raza Shah,陸震鳴,李碩碩,李華祥,羅志珊,張曉娟,史勁松,許正宏*

    (1.江南大學(xué) 藥學(xué)院,江蘇 無錫 214122;2.江南大學(xué) 生物工程學(xué)院 糧食發(fā)酵工藝與技術(shù)國家工程實驗室,江蘇 無錫 214122)

    Ophiocordycepssinensis isawell-known medicinal fungus that has been used as a traditional Chinese medicine for thousand years.Nowadays,the wild O.sinensis is in great market demand due to the presence of potent bioactivities,but it is extremely expensive due to the rarity in nature,and the difficulty of artificial cultivation.Thus,several fermentation products produced from the fungal isolates are used to substitute the wild O.sinensis[1-2].In previous studies,more than 10 fungal specieswereisolated from natural O.sinensis.Among these species,Hirsutella sinensis is an adopted anamorph of O.sinensis[3-6].Other fungal species isolated from O.sinensis,such as Paecilomyces hepiali,Synnematum sinensis,Cephalosporum sinensis,Gliocladium roseum,and Mortierellahepiali,have also been applied in large-scaleproduction and used ascommercial products[7].

    The mycelial powder of P.hepiali has been comprehensively studied and it has been evolved into functional food in China for ages.Studies have showed that P.hepiali has various functions,such as restricting tumor proliferation,invasion,metastasis,and neovascularisation,inducing apoptosis,reversing drug resistance,enhancing immunity as well as protecting hepatic function[8].Adenosine in the mycelial powder of P.hepiali is believed as functional compounds which are beneficial to the human body[9].JinShuiBao capsule,the commercial product of P.hepiali Cs-4,hasbeen used in clinicsthroughout China.The annual output value of JinShuiBao capsulein 2014 ismorethan 2.2 billion RMB[6].

    Submerged fermentation(SmF)isconsidered asthemost efficient way in industrial production of P.hepiali,to satisfy itslargeconsumption demand.However,the SmFof filamen-tous fungus is complex[10]due to many factors,such as strain,inoculum size,mycelium morphology,medium composition,and operation condition,which affect the biosynthesis of metabolites[11].Thus,it is necessary to continue with the ex cellent batch-repeatability,less time consuming,low cost,and lesslabor consuming fermentation method of P.hepiali.

    Usually,mycelia grow on agar plate were used asinoculum for the SmFof P.hepiali.In thisstudy,an asexual reproduction-based repeated batch fermentation(RBF)of P.hepiali was performed with a modified inoculation method.A twenty-cycle RBFwas carried out in this study,and the biomass,sporulation,and metabolites calculation were examined in each batch.Themethodisdesigned tosavecostandimproveefficiency.

    1 Materials and Methods

    1.1 Materialsand Reagents

    1.1.1 Fungal strains

    P.hepiali were provided by theculture bank of National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University.Thestrainswerecultured on potato dextrose agar(PDA)medium.

    1.1.2 Reagents

    Yeastextractwaspurchased from Oxoid Ltd.(Basingstoke,UK).Other chemicalswere purchased from Sinopharm Chemical Reagent Co.,Ltd.(Shanghai,China).Adenosine and oleanolic acid standardswere purchased from Sigma-Aldrich(MO,USA).All the chemicals used were either of analytical gradeor of thehighest purity commercially available.

    1.1.3 Culture medium

    PDA medium:200 g diced potatoes were boiled in 1∶1 of distilled water for 30 min and then were filtered with gauze with four layers;20 g glucose and 20 g agar were added into 1 L of filtrate.The medium wassterilized by autoclaving at 100 kPa121℃for 20 min.

    Seed culture medium:30 g glucose,1 g yeast extract,0.5 g MgSO4,1 g KH2PO4,0.1 g vitamin B1,1 L distilled H2O,pH 6.5.

    Basic sporulation medium:20 g glucose,0.5 g yeast extract,0.5 g MgSO4,3.0 g KH2PO4,1 L distilled H2O,pH 6.5.Themedium wassterilized by autoclaving at 100 kPa,121℃for 20 min.

    Fermentation medium:20 g glucose,0.5 g yeast extract,0.5g MgSO4,3.0g KH2PO4,1Ldistilled H2O,pH 6.5.Thismedium wassterilized by autoclaving at 100 kPa121℃for 20 min.

    1.2 Instrumentsand Equipments

    BX60 Light microscope:Japan Olympus;SX-40 electron microscope,SX-40 Scanning electronmicroscope,HPLC chromaster:Japan Hitachi Group.

    1.3 Methods

    1.3.1 Inoculum preparation

    The mycelial colony of P.hepiali was picked from the PDA slant to a500 ml Erlenmeyer flask containing 100 ml of seed culture medium.After inoculation,it was cultured by shake-flask cultivation at 150 r/min for 4 d in thecondition of 26℃.The asexual spores in the supernatant of fermentation broth were diluted in fermentation medium,counted using a hemacytometer under a light microscope,and adjusted to adequate concentrations.

    1.3.2 Repeated batch fermentation

    A total of 20 cycles of RBF were performed in 500 ml Erlenmeyer flaskscontaining100 ml of fermentation medium.P.hepiali sporesuspensionwasadded into fermentationmedium with the initial spore concentration of 1×105spores/ml,incubating for 4 d at 26℃with 150 r/min shaking speed.Fermentation broth(2 ml)in theflask wassampled every 24 h for 4 d.An appropriate portion of the supernatant(spore suspension)at 48 h in the cycle 1 wasinoculated into the cycle 2 with inoculum size of 1×105spores/ml.Subsequent cycleswere performed in the same manner asdescribed above.At least three timesparallel testswereperformed.

    1.3.3 Microscopic observations

    During the fermentation process of P.hepiali in flasks,the morphology of spores was observed under a light microscope.Suspensions(2 ml)containing spore aliquotswere taken out from shaking flask at daily intervals and were examined under a microscope.

    (1)Specimenspreparation and microscopic observation

    The fungal spores were produced in liquid culture after 1 d,and then centrifuged at a low speed and washed three times with 0.1 mol/L phosphate-buffered saline(PBS)buffer(adding 3.1 g of NaH2PO4·H2Oand 10.9 g of Na2HPO4to distilled H2O to make a volume of 1 L).Then fixed the acquisi tion in 3%glutaraldehydefor 2 h and in 1%osmic acid for 1 h respectively at 4℃.After washed four times in 0.1 mol/L PBSbuffer,the specimenswere dehydrated through a graded seriesof ethanol,immersed in 100%isoamyl acetate,dried by critical point drying,and coated with gold.Specimens were observed under ascanning electron microscope.

    (2)Sporulation detection

    Spore concentrationswerecalculated by hemacytometer counts.The fungal spores were determined by the sporulation number and the sporulation capacity was determined by the number of fungal sporesper gram of biomass.

    1.3.4 Analytical methods

    (1)Obtaining of mycelia:The fermentation broth was filtered in culture flask through a mesh(pore size,30μm),washed with distilled water.Then the mycelia sediment was obtained after centrifugation at 5 000 r/min for 10 min at 4℃.The mycelia were lyophilized and weighed to calculate the biomass,and stored at 4℃for further analysis[12].

    (2)Determination of crude triterpenoids:Colorimetric method with vanillin-acetic acid system was used to analyze the crude triterpenoids production.The mycelia powders of P.hepiali(0.1 g)were extracted with methanol(5 ml)for 2 h at room temperature.The methanol extract wascentrifuged at 5 000 r/min for 10 min at 4℃,and then 0.2 ml of supernatant(with 0.2 ml methanol asthe blank)was transferred to a tube and heated withboilingwater until thesolventwascompletely evaporated.0.3 ml of 5%freshly prepared vanillin-acetic acid solution and 1.0 ml perchloric acid wereadded into theabove acquisition.The mixture was incubated at 60℃for 20 min,then immediately cooled with icewater.Acetic acid(10.0 ml)was added to each sample,and the absorbance was detected using a spectrophotometer at 550 nm[13].

    Standard curve:Different concentrations of oleanolic acid wereused to obtain the standard curve[14](Y=4.83X-0.04,R2=0.996).X representsthemassof oleanolic acid(0.02-0.18mg),whereas Y representsoptical density at 550 nm.

    (3)Determination of adenosine:Adenosine concentration was measured by HPLC,according to the procedure reported by LU et al.with some modification[15].The mycelia powdersof P.hepiali(0.5g)wereextractedfor2hwithmethanol(1.5 ml)at room temperature,and the supernatant was collected by centrifugation(4℃,5000 r/min,10 min).The extractions were concentrated to 1.0 ml through volatilization and filtration through a 0.22μm microporous membrane.Samplesolution(10μl)wasapplied to HPLCanalysis.Separation wasperformed in a Waters XBridge C18reversed-phase analysis column(4.6 mm×250 mm,5μm)at a flow rate of 1.0 ml/min,and the absorbance was determined at 254 nm.Solvent A(water,containing 0.5%acetic acid)and solvent B(100%acetonitrile)were used as the mobile phase.The gra dientconditionswere:0-20 min,5%-20%B;20-40 min,20%-50%B;40-70 min,50%-60%B;70-120 min,60%-80%B;120-150 min,80%-90%B;150-170 min,90%-100%B;170-171 min,100%-5%B;171-180 min,5%B.Content of adenosinewasdetermined by theexternal standard method.

    Standard curve:Different concentrations of adenosine wereused to obtain thestandard curve(Y=65036X-10.5,R2=0.998).X representsthemassof adenosine(0.01-0.05 mg/ml),whereas Y representsoptical density at 254 nm.

    1.3.5 Statistical analysis

    The data in this study were plotted via Origin Pro 8.0 software(http://www.OriginLab.com).Thestatistic significant analysiswasanalyzed by SPSSPASWStatistics16.0 software(SPSSLab Corporation,http://www.spss.com),and the level of significancewasgiven asvaluesof Prob>Flessthan 0.05.

    2 Results and analysis

    2.1 Morphology of P.hepiali myceliaand spores

    Filamentous hyphae of P.hepiali did not aggregate to form pelletsin shaking flask.Asexual sporulation began after 36 h of incubation,and a large number of spores were produced.Morphology of P.hepiali mycelia and spores in submerged culture were observed by scanning electron microscope(Fig.1).The reproduced sporeswere able to germinate when introduced into anutrient-rich environment.

    Fig.1 Morphology of P.hepiali mycelia(A)and asexual spores(B)in submerged culture under scanning electron microscope

    2.2 Timecourseof RBF

    Fig.2 shows the time course of biomass,spore number,and metabolites production in the first three cycles of RBF.Thethreelinesrepresent thefirst three fermentation cyclesof RBF.

    After inoculation,all of thesporesweregerminated within 6 h and filamentous mycelia was grew out of the spores.The biomassof P.hepiali increased quickly to 25 g/L within 2.5 d of incubation(Fig.2A).After 2 d of incubation,P.hepiali in the flasks sporulated asexually,and spore number in the fermentation broth increased to 0.6×108spores/ml(Fig.2B).The sporesin thefermentation broth on day 2 wereused astheinoculum for next batch of RBF.The production of adenosine and crudetriterpenoids(Fig.2Cand 2D)accumulated from day 0 to 4,although the biomass did not increase significantly from day 2.5.After incubated for 4 d,thesporeconcentration of 1.5×108spores/ml,biomassof(25.0±0.3)g/L,adenosine production of(0.93±0.5)mg/L,and crude triterpenoids production of(0.33±0.2)g/L in thefermentation broth wereobtained.

    Fig.2 Time course of biomass,spore number,and metabolites production in the first three cycles of RBF

    2.3 Stability of twenty-cycle RBFof P.hepialid

    To check the stability of asexual sporulation and bioactivecompoundsformation for along-lasting operation,a total twenty-cycle RBFwere performed.Asshown in the Table 1,the biomass of P.hepialid on the 4 th day of fermentation changed between 25.5 g/L and 27.2 g/L,and no significant difference of biomass among different fermentation batches was observed(P>0.05).Spore numbers(0.8×108to 1.4×108spores/ml),adenosine production(49.5-62.3 mg/L),and crude triterpenoids production(126.3-142.7 mg/L)also remained constant throughout the RBFprocess(P>0.05).It indicated that this RBFprocess with P.hepiali exhibited good stability and sustainability.

    Table1 Biomass,sporulation,and triterpenoids and adenosine productions of P.hepiali during the 20 fermentation batches

    3 Conclusion

    In this study,twenty-cycle RBFof P.hepiali was established,by using an asexual spores-based inoculation method.Preparation of mycelial inoculum isnot needed during thefor initiating the fermentation process.The RBFprocess notably reduces cost and labor since it is omitting out inoculums preparation each time,which make the production more expensiveand extravagant.

    猜你喜歡
    藥學(xué)院無錫江南
    無錫一棉
    紡織報告(2024年1期)2024-02-27 06:53:52
    無錫一棉
    China Textile(2022年3期)2022-07-12 05:37:36
    蘭州大學(xué)藥學(xué)院簡介
    無錫確定11月1日為“無錫企業(yè)家日”
    華人時刊(2020年21期)2020-11-17 11:28:32
    小編有話說①
    小編有話說②
    小編有話說①
    無錫公交
    HSCCC-ELSD法分離純化青葙子中的皂苷
    湖北旋覆花化學(xué)成分的研究
    麻豆乱淫一区二区| 久久久久久久大尺度免费视频| 精品人妻1区二区| 亚洲成国产人片在线观看| 视频在线观看一区二区三区| 国产免费福利视频在线观看| 成人黄色视频免费在线看| 久久久精品国产亚洲av高清涩受| 国产国语露脸激情在线看| 国产激情久久老熟女| 精品少妇内射三级| 欧美成人午夜精品| 波多野结衣av一区二区av| 国产不卡av网站在线观看| 午夜福利在线免费观看网站| 日韩av不卡免费在线播放| 欧美精品一区二区免费开放| 欧美精品一区二区免费开放| 最黄视频免费看| 成人黄色视频免费在线看| av福利片在线| 老司机午夜十八禁免费视频| 色婷婷av一区二区三区视频| 国产视频首页在线观看| 一区二区av电影网| 亚洲七黄色美女视频| 亚洲国产精品国产精品| 妹子高潮喷水视频| 在线观看一区二区三区激情| kizo精华| 午夜影院在线不卡| 在线亚洲精品国产二区图片欧美| 一边摸一边抽搐一进一出视频| 亚洲国产精品成人久久小说| 久热爱精品视频在线9| 秋霞在线观看毛片| 黄网站色视频无遮挡免费观看| 婷婷色av中文字幕| 自线自在国产av| a级片在线免费高清观看视频| 亚洲欧美精品自产自拍| 亚洲欧美成人综合另类久久久| 欧美大码av| 女性被躁到高潮视频| 国产深夜福利视频在线观看| 欧美激情 高清一区二区三区| 国产成人精品久久久久久| 亚洲av欧美aⅴ国产| 亚洲av日韩在线播放| 国产欧美亚洲国产| 成年动漫av网址| 91精品三级在线观看| av网站免费在线观看视频| 日本午夜av视频| 精品久久蜜臀av无| 国产免费视频播放在线视频| 亚洲三区欧美一区| 精品视频人人做人人爽| 美女脱内裤让男人舔精品视频| 如日韩欧美国产精品一区二区三区| 免费久久久久久久精品成人欧美视频| 久久精品国产综合久久久| 久久久久网色| 亚洲五月色婷婷综合| 国产精品久久久久久精品古装| 好男人电影高清在线观看| 精品亚洲成a人片在线观看| 日韩 欧美 亚洲 中文字幕| kizo精华| 精品国产一区二区三区久久久樱花| 欧美激情 高清一区二区三区| 午夜影院在线不卡| 亚洲图色成人| 欧美97在线视频| 亚洲九九香蕉| 国产国语露脸激情在线看| 亚洲伊人久久精品综合| 国产男人的电影天堂91| 久久午夜综合久久蜜桃| 高清不卡的av网站| 女人爽到高潮嗷嗷叫在线视频| 欧美日韩av久久| 老司机靠b影院| 午夜福利视频在线观看免费| 赤兔流量卡办理| 国产在线免费精品| 日韩电影二区| 无遮挡黄片免费观看| 精品亚洲成a人片在线观看| 久久精品熟女亚洲av麻豆精品| 99精国产麻豆久久婷婷| 精品熟女少妇八av免费久了| 国产成人欧美在线观看 | √禁漫天堂资源中文www| 在线观看国产h片| 美女福利国产在线| 美女国产高潮福利片在线看| 亚洲成人免费电影在线观看 | 国产熟女午夜一区二区三区| 亚洲一区二区三区欧美精品| 久久精品亚洲av国产电影网| 国产色视频综合| 国产精品偷伦视频观看了| 亚洲熟女精品中文字幕| 国产欧美日韩综合在线一区二区| av又黄又爽大尺度在线免费看| 一边亲一边摸免费视频| 真人做人爱边吃奶动态| 亚洲欧美激情在线| 乱人伦中国视频| 亚洲国产精品999| 在线看a的网站| 久久久精品国产亚洲av高清涩受| 老司机午夜十八禁免费视频| 欧美国产精品一级二级三级| 男人爽女人下面视频在线观看| 久久人人97超碰香蕉20202| av视频免费观看在线观看| 你懂的网址亚洲精品在线观看| 免费观看人在逋| 精品国产一区二区三区久久久樱花| 日本91视频免费播放| 午夜免费观看性视频| 国产不卡av网站在线观看| videosex国产| 伊人亚洲综合成人网| 欧美精品一区二区免费开放| 极品人妻少妇av视频| 美女扒开内裤让男人捅视频| www.精华液| www.熟女人妻精品国产| 久久青草综合色| 午夜福利视频精品| 精品少妇一区二区三区视频日本电影| 国产视频一区二区在线看| 又紧又爽又黄一区二区| www日本在线高清视频| 亚洲自偷自拍图片 自拍| 国产精品一区二区精品视频观看| 老汉色∧v一级毛片| 嫩草影视91久久| 日韩中文字幕欧美一区二区 | 丁香六月欧美| 激情五月婷婷亚洲| 黑人猛操日本美女一级片| 中国国产av一级| 在线观看免费高清a一片| 国产精品熟女久久久久浪| 免费日韩欧美在线观看| 狂野欧美激情性bbbbbb| 飞空精品影院首页| 国产成人系列免费观看| 深夜精品福利| 国产精品九九99| 亚洲欧美精品自产自拍| 国产免费福利视频在线观看| 视频在线观看一区二区三区| 国产成人av教育| 久久影院123| 人人妻人人添人人爽欧美一区卜| 国产av国产精品国产| 午夜福利视频精品| 久久久久网色| 精品少妇一区二区三区视频日本电影| 国产无遮挡羞羞视频在线观看| www.自偷自拍.com| 人人妻人人爽人人添夜夜欢视频| 久久久久久久久久久久大奶| 亚洲免费av在线视频| 国产精品 欧美亚洲| 欧美精品啪啪一区二区三区 | 亚洲精品国产av成人精品| 极品少妇高潮喷水抽搐| 久久女婷五月综合色啪小说| 观看av在线不卡| 中文字幕最新亚洲高清| 日韩一区二区三区影片| 久久天堂一区二区三区四区| 丝袜人妻中文字幕| 男女国产视频网站| 久久人人爽人人片av| 欧美黑人精品巨大| 一级片免费观看大全| 国产黄色免费在线视频| 国产成人啪精品午夜网站| 高清欧美精品videossex| 亚洲少妇的诱惑av| 人体艺术视频欧美日本| 后天国语完整版免费观看| 午夜久久久在线观看| 成年人午夜在线观看视频| 久久精品久久久久久久性| 欧美黑人精品巨大| 黑丝袜美女国产一区| 欧美人与性动交α欧美精品济南到| 亚洲欧洲国产日韩| 国产男女超爽视频在线观看| 麻豆av在线久日| 国产成人欧美在线观看 | 免费观看a级毛片全部| 久久ye,这里只有精品| 9热在线视频观看99| 在线观看一区二区三区激情| 性高湖久久久久久久久免费观看| 国产在线视频一区二区| 国产精品久久久久久人妻精品电影 | 天天躁夜夜躁狠狠久久av| 国产精品人妻久久久影院| 亚洲欧美一区二区三区久久| 亚洲五月色婷婷综合| 亚洲欧洲国产日韩| 国产精品亚洲av一区麻豆| 男女高潮啪啪啪动态图| 高清黄色对白视频在线免费看| 久久这里只有精品19| 亚洲中文日韩欧美视频| 免费一级毛片在线播放高清视频 | 亚洲色图综合在线观看| 丰满少妇做爰视频| 天天躁夜夜躁狠狠久久av| 少妇猛男粗大的猛烈进出视频| 亚洲av男天堂| 国产精品 欧美亚洲| 手机成人av网站| 考比视频在线观看| 男女边摸边吃奶| 日本猛色少妇xxxxx猛交久久| 黄色视频在线播放观看不卡| 午夜免费观看性视频| 国产成人精品无人区| 精品欧美一区二区三区在线| 精品久久久精品久久久| 十八禁高潮呻吟视频| 日韩熟女老妇一区二区性免费视频| 免费观看a级毛片全部| 高潮久久久久久久久久久不卡| 性色av一级| 99re6热这里在线精品视频| 丝袜美腿诱惑在线| 免费在线观看黄色视频的| 国产av国产精品国产| 丝瓜视频免费看黄片| 捣出白浆h1v1| 欧美精品人与动牲交sv欧美| 国产精品免费大片| 国产精品久久久久久精品电影小说| 久久国产精品男人的天堂亚洲| 精品国产乱码久久久久久男人| 日本vs欧美在线观看视频| 热re99久久国产66热| 老熟女久久久| 国产高清不卡午夜福利| 视频区图区小说| 啦啦啦视频在线资源免费观看| 久久久久久久大尺度免费视频| 黄片小视频在线播放| 99热国产这里只有精品6| www日本在线高清视频| 亚洲,欧美,日韩| 首页视频小说图片口味搜索 | 国产高清不卡午夜福利| 久久久精品94久久精品| 午夜精品国产一区二区电影| 永久免费av网站大全| 免费在线观看日本一区| 波多野结衣av一区二区av| 肉色欧美久久久久久久蜜桃| 少妇人妻 视频| 久久精品久久久久久噜噜老黄| 黑人欧美特级aaaaaa片| 一区二区三区激情视频| 99国产综合亚洲精品| 国产视频首页在线观看| 精品人妻1区二区| 电影成人av| 欧美精品av麻豆av| 久久久久久免费高清国产稀缺| 久久精品国产亚洲av涩爱| 制服诱惑二区| 国产成人免费无遮挡视频| 每晚都被弄得嗷嗷叫到高潮| 美女高潮到喷水免费观看| 日本wwww免费看| 9191精品国产免费久久| 午夜免费成人在线视频| 少妇裸体淫交视频免费看高清 | 国产国语露脸激情在线看| 精品福利观看| 色94色欧美一区二区| 激情五月婷婷亚洲| 99久久人妻综合| 丝袜人妻中文字幕| 一区二区三区乱码不卡18| 久久亚洲国产成人精品v| 女人精品久久久久毛片| 免费看av在线观看网站| 男女免费视频国产| 丁香六月欧美| 少妇被粗大的猛进出69影院| 菩萨蛮人人尽说江南好唐韦庄| 黄色视频在线播放观看不卡| 亚洲精品一二三| 如日韩欧美国产精品一区二区三区| 中文字幕av电影在线播放| 精品亚洲乱码少妇综合久久| 少妇裸体淫交视频免费看高清 | 久久精品亚洲熟妇少妇任你| 人妻 亚洲 视频| 精品福利永久在线观看| 亚洲国产中文字幕在线视频| 最近手机中文字幕大全| 国产深夜福利视频在线观看| 性高湖久久久久久久久免费观看| 欧美日韩成人在线一区二区| 亚洲精品国产av成人精品| 亚洲男人天堂网一区| 777米奇影视久久| 亚洲精品国产av蜜桃| 婷婷色麻豆天堂久久| 美国免费a级毛片| 一二三四社区在线视频社区8| 亚洲国产中文字幕在线视频| 亚洲精品一区蜜桃| 香蕉国产在线看| 亚洲午夜精品一区,二区,三区| 人人妻人人澡人人爽人人夜夜| 别揉我奶头~嗯~啊~动态视频 | √禁漫天堂资源中文www| 国产精品二区激情视频| 最黄视频免费看| 久久国产精品影院| 久久人妻福利社区极品人妻图片 | 一二三四社区在线视频社区8| 日本猛色少妇xxxxx猛交久久| 精品人妻1区二区| 国产精品一国产av| 婷婷色综合www| 黄色视频不卡| av天堂久久9| 精品一区二区三卡| 精品一区二区三区四区五区乱码 | 国产男女超爽视频在线观看| 50天的宝宝边吃奶边哭怎么回事| 一区福利在线观看| 性少妇av在线| 亚洲五月色婷婷综合| 午夜福利在线免费观看网站| 日韩av免费高清视频| 天天躁夜夜躁狠狠躁躁| 亚洲欧美一区二区三区黑人| 国产人伦9x9x在线观看| 午夜精品国产一区二区电影| 成年美女黄网站色视频大全免费| 欧美少妇被猛烈插入视频| 黄色 视频免费看| 大香蕉久久网| 亚洲综合色网址| 老汉色∧v一级毛片| 免费观看av网站的网址| 啦啦啦在线观看免费高清www| 久久人人爽人人片av| 90打野战视频偷拍视频| 18禁黄网站禁片午夜丰满| 99久久99久久久精品蜜桃| 日韩精品免费视频一区二区三区| 国产麻豆69| 国产无遮挡羞羞视频在线观看| av网站免费在线观看视频| 最近手机中文字幕大全| videosex国产| 免费在线观看日本一区| 宅男免费午夜| 日本猛色少妇xxxxx猛交久久| 丝袜喷水一区| 黄色片一级片一级黄色片| 老汉色∧v一级毛片| 十八禁人妻一区二区| a 毛片基地| 久久精品国产亚洲av高清一级| 校园人妻丝袜中文字幕| 天天躁狠狠躁夜夜躁狠狠躁| 嫁个100分男人电影在线观看 | 久久精品国产亚洲av涩爱| 中文字幕色久视频| 在线观看www视频免费| 人体艺术视频欧美日本| av天堂久久9| 亚洲少妇的诱惑av| 日韩大码丰满熟妇| 美国免费a级毛片| 亚洲av美国av| 大片电影免费在线观看免费| 久久精品国产a三级三级三级| 亚洲人成电影观看| 国产精品一区二区精品视频观看| 亚洲七黄色美女视频| 亚洲欧美一区二区三区久久| 女性生殖器流出的白浆| 精品少妇久久久久久888优播| 亚洲精品一二三| 欧美少妇被猛烈插入视频| 久久精品熟女亚洲av麻豆精品| 亚洲精品乱久久久久久| 人人妻,人人澡人人爽秒播 | xxx大片免费视频| 亚洲中文字幕日韩| 国产一区二区三区综合在线观看| 亚洲欧洲国产日韩| 亚洲国产精品成人久久小说| 性色av乱码一区二区三区2| 精品国产国语对白av| 成人亚洲精品一区在线观看| 国精品久久久久久国模美| 十八禁网站网址无遮挡| 黄片播放在线免费| 日韩精品免费视频一区二区三区| 欧美日韩福利视频一区二区| 国产精品一区二区精品视频观看| 黑丝袜美女国产一区| 久久久久久久久免费视频了| 亚洲第一av免费看| 国产欧美日韩综合在线一区二区| 777久久人妻少妇嫩草av网站| 国产免费现黄频在线看| 成人影院久久| 亚洲欧美色中文字幕在线| 中文字幕人妻熟女乱码| 欧美精品亚洲一区二区| 天天添夜夜摸| 国产成人av激情在线播放| 人妻一区二区av| 久久国产精品人妻蜜桃| 熟女av电影| 国产一区二区 视频在线| 国产91精品成人一区二区三区 | 亚洲精品自拍成人| 久热爱精品视频在线9| 伊人亚洲综合成人网| 曰老女人黄片| 国产精品久久久久久人妻精品电影 | 叶爱在线成人免费视频播放| 欧美日韩国产mv在线观看视频| 日韩电影二区| 人人妻人人澡人人看| 999久久久国产精品视频| 在线观看免费午夜福利视频| 国产精品一二三区在线看| 亚洲一区二区三区欧美精品| 国产av国产精品国产| 国产精品国产av在线观看| 婷婷丁香在线五月| 伊人久久大香线蕉亚洲五| 99国产精品一区二区蜜桃av | 国产精品麻豆人妻色哟哟久久| 在线av久久热| 丝袜喷水一区| av又黄又爽大尺度在线免费看| 国产免费又黄又爽又色| 亚洲综合色网址| 国产亚洲av片在线观看秒播厂| 中文欧美无线码| 99热全是精品| 欧美精品人与动牲交sv欧美| 精品久久久精品久久久| 国产欧美亚洲国产| 成人18禁高潮啪啪吃奶动态图| 国产一区有黄有色的免费视频| 久久精品久久久久久噜噜老黄| 建设人人有责人人尽责人人享有的| 男女下面插进去视频免费观看| 天天躁日日躁夜夜躁夜夜| 黄色一级大片看看| 亚洲情色 制服丝袜| 97精品久久久久久久久久精品| 欧美在线一区亚洲| www日本在线高清视频| 人体艺术视频欧美日本| 亚洲精品久久午夜乱码| 久久av网站| 亚洲欧洲日产国产| 成年动漫av网址| 日韩伦理黄色片| 日韩制服丝袜自拍偷拍| 日本vs欧美在线观看视频| 日本91视频免费播放| 久久久久国产一级毛片高清牌| 久久国产精品男人的天堂亚洲| 国产激情久久老熟女| 欧美变态另类bdsm刘玥| 久久午夜综合久久蜜桃| 中文字幕人妻丝袜一区二区| 国产99久久九九免费精品| 黄色一级大片看看| 美女扒开内裤让男人捅视频| 国产精品久久久av美女十八| 亚洲精品乱久久久久久| 新久久久久国产一级毛片| 亚洲国产av影院在线观看| 国产成人欧美| 日韩大码丰满熟妇| 97精品久久久久久久久久精品| 国产精品国产三级专区第一集| 蜜桃国产av成人99| 日韩伦理黄色片| 国产亚洲精品第一综合不卡| 日韩中文字幕视频在线看片| 久久狼人影院| 国产人伦9x9x在线观看| 亚洲国产精品999| 亚洲精品国产色婷婷电影| 国产av一区二区精品久久| 丰满人妻熟妇乱又伦精品不卡| 精品一区二区三卡| 欧美黄色淫秽网站| 精品亚洲成a人片在线观看| 18禁黄网站禁片午夜丰满| 国产高清视频在线播放一区 | 青草久久国产| 国产精品免费视频内射| 伊人亚洲综合成人网| 亚洲欧美中文字幕日韩二区| 亚洲一码二码三码区别大吗| 手机成人av网站| 日韩人妻精品一区2区三区| 一边摸一边抽搐一进一出视频| 国产av精品麻豆| 一级毛片我不卡| 高清视频免费观看一区二区| 亚洲欧洲日产国产| 久久狼人影院| 欧美少妇被猛烈插入视频| 国产精品二区激情视频| 中文字幕高清在线视频| 香蕉丝袜av| 久久久久国产精品人妻一区二区| 国产精品偷伦视频观看了| 黄网站色视频无遮挡免费观看| 又大又爽又粗| 亚洲精品在线美女| 精品熟女少妇八av免费久了| 三上悠亚av全集在线观看| 在线观看免费午夜福利视频| 中文字幕制服av| 18禁黄网站禁片午夜丰满| 伊人久久大香线蕉亚洲五| 国产精品秋霞免费鲁丝片| 人人妻,人人澡人人爽秒播 | 女人爽到高潮嗷嗷叫在线视频| 精品国产一区二区三区久久久樱花| 久久精品国产综合久久久| 丝袜美腿诱惑在线| 日本欧美国产在线视频| 18在线观看网站| 91字幕亚洲| 777米奇影视久久| 国产伦理片在线播放av一区| 99国产精品99久久久久| 欧美xxⅹ黑人| 侵犯人妻中文字幕一二三四区| 男的添女的下面高潮视频| 日本欧美国产在线视频| 中文字幕人妻丝袜制服| 91九色精品人成在线观看| 天天添夜夜摸| 一本久久精品| 大话2 男鬼变身卡| 亚洲欧美精品综合一区二区三区| 制服诱惑二区| 女人高潮潮喷娇喘18禁视频| 男女边吃奶边做爰视频| 日本黄色日本黄色录像| av电影中文网址| 久久精品成人免费网站| 天天躁夜夜躁狠狠躁躁| 啦啦啦在线观看免费高清www| 欧美黄色片欧美黄色片| 亚洲国产欧美网| 大型av网站在线播放| 欧美日韩黄片免| 国产精品亚洲av一区麻豆| 亚洲视频免费观看视频| 91精品伊人久久大香线蕉| 久久精品国产亚洲av高清一级| 曰老女人黄片| 超色免费av| 亚洲精品国产av蜜桃| 首页视频小说图片口味搜索 | 国产成人欧美在线观看 | 99精国产麻豆久久婷婷| 免费在线观看视频国产中文字幕亚洲 | videos熟女内射| 又粗又硬又长又爽又黄的视频| 日韩欧美一区视频在线观看| 午夜免费鲁丝| 亚洲成色77777| 一本大道久久a久久精品| 欧美xxⅹ黑人| 国产片特级美女逼逼视频| 亚洲黑人精品在线| 麻豆乱淫一区二区| 免费高清在线观看视频在线观看| 精品少妇一区二区三区视频日本电影| 99精品久久久久人妻精品| 国产真人三级小视频在线观看| 黄色a级毛片大全视频| 狠狠婷婷综合久久久久久88av| 精品少妇一区二区三区视频日本电影| 人人妻人人澡人人看| 久热这里只有精品99| 真人做人爱边吃奶动态| 美女大奶头黄色视频| 亚洲专区中文字幕在线| 午夜久久久在线观看| 亚洲中文av在线| 国产精品一区二区精品视频观看|