• <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 ?

    Optimizing of Liquid Medium Formula about Medicinal Fungus Phellinus igniarius

    2015-01-16 05:48:18XUQian
    微生物學(xué)雜志 2015年4期
    關(guān)鍵詞:桑黃菏澤菌絲體

    XU Qian

    (Dept. of Life Sci., Heze Uni., Heze 274015)

    ?

    Optimizing of Liquid Medium Formula about Medicinal Fungus Phellinus igniarius

    XU Qian

    (Dept. of Life Sci., Heze Uni., Heze 274015)

    The influence of liquid medium formula on biomass ofPhellinusigniariusmycelium and the yield of exocellular polysaccharide were studied adopting four factors and three levels orthogonal experiment. The suitable formula for mycelium biomass and exocellular polysaccharide yield ofP.igniariuswas A3B1C3D2(wheat bran 10%, peptone 0.5%, KH2PO40.35%, MgSO40.15%), pH 6.The total yield of mycelium biomass and exocellular polysaccharide yield was 1.007 7 g/100 mL. The optimized formula can be used in industrial setting in order to get more yield ofP.igniariusmycelium biomass and exocellular polysaccharide.

    liquid medium formula; mycelium biomass; exocellular polysaccharide yield; total yield; orthogonal test;Phellinusigniarius

    Phellinusigniarius(L. ex Fr.) Quél. called asSanghuangin Chinese, belongs to the genus pfPhellinusQuél, Hymenochaetaceae, Aphyllophorales, Hymenomycetes, Basidiomycotina. It has been widely used in Asian countries for many years[1-4]. It is a kind of precious mushroom with important medical values. Its natural production is limited and it is called as "woods gold". It is rich in polysaccharide, triterpenoids, flavone etc., these life active substance are used to treat the diseases of cardiovascular, liver, nervous system, kidney and have bioactivities of anticancer, anti-liver fibrosis, anti-lipid peroxidation, immunity enhancement, hypoglycemic action, anti-pneumonia, weight control etc.[5-12]. During the past decades, polysaccharides isolated fromPhellinusspecies have been revealed as some pharmacological activities such as inhibiting tumor growth and metastasis, and low toxicity[13-22].

    Traditionally, polysaccharide ofPhellinusigniariuswas extracted from fruit body. It will spend quite a long time in gaining fruit body in nature and artificial cultivation due to its special and complex physiological ecology[23], therefore, liquid culture is employed to developP.igniarius[24]. Liquid culture is a time-saving and high-yield method and mycelium gained through liquid culture has nutrients including polysaccharide similar to fruit body[25-26]. At the same time, quite a lot exocellular polysaccharide yield was got through liquid culture. Therefore, it is necessary to optimize liquid medium formula ofP.igniariusby using the total yield (mycelium biomass and exocellular polysaccharide yield) as an index in industry. Wang et al[27]had studied the influence of nutrients onP.igniariusmycelium biomass. He picked ten activated round fungus blocks in outer circle with hole puncher (diameter 0.6 cm), inoculated the ten round fungus blocks to liquid medium including different carbon source or nitrogen source, the biggest mycelium biomass was that 100 mL liquid medium and 10 round fungus blocks could get 1.52 g mycelium, that is to say, 100 mL liquid medium and 2 round fungus blocks (diameter 1 cm) could get about 0.844 4 g mycelium. Li et al[28]had studied liquid fermentation medium ofP.igniariusand got the biggest mycelium biomass (3.56 g/L) by using 10% inoculation quantity. These two studies both got limited mycelium biomass by using a large amount of spawn. This study paid more attention to simplify cultivation process and get more total yield (mycelium biomass and exocellular polysaccharide yield) by using a small amount of spawn.

    1 Materials and Methods

    1.1 Experimental materials

    1.1.1 SpawnP.igniarius(Ph001) was provided by Edible Fungus Institute of Huazhong Agricultural University.

    1.1.2 Culture medium PDA culture medium: peeled potatoes 200 g, glucose 20 g, agar 15 g, and water 1 000 mL. Liquid medium (designed by orthogonal test): carbon sources (cornmeal, cotton seed hulls, and wheat bran), peptone, KH2PO4, MgSO4.

    1.1.3 Instruments Electronic analytical balance (model: FA1604, Shanghai balance instrument plant), Air temperature oscillator (model: HZQ-C, Harbin city DongLian electronic technology development Co., LTD, China), Automatic control vertical electric steam sterilizer (model: YX40011, Sanshen, Shanghai), Electric drying oven with forced convection (model: 101-2, Beijing Ever Bright Medical Instrument Factory), MJX intelligent mold incubator (Ningbo Jiangnan Instrument Factory), Pointer type electrothermal constant temperature water-bath water pot, Hole puncher (diameter 1 cm), Conical flask (250 mL).

    1.1.4 Reagents Glucose (analytical purity, Tianjin Hongyan Reagent Factory), KH2PO4(analytical purity, West Long Chemical Co., LTD), MgSO4(analytical purity, Tianjin Hongyan Reagent Factory), ethanol (95%) (Anhui Antell Biological Chemical Co., LTD), agar (biochemical reagent, Beijing Spiritualizing Star Biological Technology Co., LTD).

    1.2 Methods

    1.2.1 Preparation of solid medium PDA culture medium (300 mL).

    1.2.2 Activating spawnP.igniariusspawn was activated twice on PDA culture medium. The first activation was to pick a little bit of spawn on the slant culture spawn to a PDA plate, and the plate was put in incubator (28 ℃) for 15 days. The second activation was to pick a round fungus block (diameter 1 cm with a hole puncher) from the first activated spawn to the center of another PDA plate and it was put in incubator (28 ℃) for another 15 days.

    1.2.3 Formulas of liquid medium The four factors and three levels orthogonal test were used[29]. The four factors were carbon source, peptone, KH2PO4and MgSO4, and each factor had three levels (Table 1). There were nine formulas of the L9(34) orthogonal test (Table 2).

    Table 1 Factors and levels of the orthogonal test

    1.2.4 Confecting liquid mediums Corn flour, cotton seed hulls, wheat bran were smashed and screened with a 20 mesh screen, and then put into the right amount of water and boiled for 30 min. Flowing filtered by four layers of gauze, other reagents were added into the filtrate. Mediums with carbon source, peptone, KH2PO4and MgSO4were confected according to the formulas designed in Table 2. Each formula was repeated three times with a 150 mL volume of medium, and the medium was placed in 250 mL conical flask and sterilized at 121 ℃ for 30 min.

    1.2.5 Liquid culture of spawn Two activated strains bacteria breads in outer circle were picked with hole puncher (diameter 1 cm) and inoculated to liquid medium, and then they were cultured in shake under the condition of 28 ℃, 180 r/min for 15 days. The best medium formula with the highest total yield (g/100 mL) (mycelium biomass and exocellular polysaccharide yield) were determined actually or theoretically.

    1.2.6 Mycelium biomass assay The mycelium in the conical flask was filtered with eight layers of gauze, and the mycelium was washed with distilled water for three times. They were dried to constant weight in 60 ℃ electric drying oven with forced convection, mycelia dry weight was weighed with electronic analytical balance after being cooled.

    Mycelium biomass (g/100 mL) = Mycelia dry weight / Medium volume

    1.2.7 Exocellular polysaccharide yield assay 20 mL of filtrate and 60 mL of 95% ethanol were mixed uniformly and placed in 4 ℃ refrigerator for one day (each formula was repeated three times), then the mixture was filtered, the filter residue was exocellular polysaccharide[27], the exocellular polysaccharide was dried to constant weight in 60 ℃ electric drying oven with forced convection and weighed with electronic analytical balance.

    Exocellular polysaccharide yield (g/100 mL)=Exocellular polysaccharide dry weight/Medium volume

    1.2.8 Total yield assay

    Total yield (g/100 mL)=Mycelium biomass+Exocellular polysaccharide yield

    1.2.9 Verification Test Corresponding verification tests were done on the base of the orthogonal test’s results. As to the formula which took the total yield as the index, verification test were done with formula 7 (A3B1C3D2) which gained the highest total yield selected through the orthogonal test and the optimal formula theoretically (A3B3C3D2).

    2 Results

    2.1 Some stages of spawn activation and liquid culture

    Fig.1 Pictures of spawn activation and liquid culture

    P.igniariusspawn was activated twice on PDA culture medium for 15 days, and then cultured in liquid medium under the condition of 28 ℃, 180 r/min for 15 days

    A: the spawn activated on the 6th day;B: was the spawn cultured on the 11th day

    Figure 1 showed some stages of spawn activation and liquid culture. While activating spawn, the mycelia were white at the beginning, then became yellow; during the period of liquid culture, the color of mycelia pellet changed from white to yellow too.

    2.2 Results of the orthogonal test

    The results of the total yield of each formula gained through the orthogonal test were in Table 2.

    Table 2 Results of the L9 (34) orthogonal test

    Table 2 showed the formula 7 gained the highest total yield, formula 4 gained the lowest total yield, the prominence order of the factors was A (carbon source) > C (KH2PO4) > B (peptone) > D (MgSO4), as to the formula which took total yield as the index, the optimal formula theoretically was A3B3C3D2: wheat bran 10%, Peptone 1.5%, KH2PO40.35%, MgSO40.15%.

    Table 3 Analysis of variance of orthogonality of medium formula’s influence on the total yield

    SourcesofvariationSSdfMSFSignificancelevelA2.07140521.0357033957.36??B0.05350220.026751102.2136??C0.10218720.051094195.2256??D0.01900420.00950236.30706??e1000.000262e20.00471118

    Note: * indicates significant influence, ** indicates very significant influence, blank indicates no significant influence

    F1-0.05(2, 18): 3.54, F1-0.01(2, 18): 6.01

    Table 3 showed the four factors of medium formula all had significantly higher influence on the total yield ofP.igniarius[29], the prominence order of the factors was A (carbon source) > C (KH2PO4) > B (peptone) > D (MgSO4), and A (carbon source) had the biggest influence on the total yield ofP.igniarius.

    2.3 Medium formula’s influence on the total yield

    Figure 2 showed the relationship between the total yield and the factors, the optimal formula theoretically was assured: wheat bran 10%, peptone 1.5%, KH2PO40.35%, MgSO40.15% while taking the total yield as the index. The prominence order of the carbon sources three levels was wheat bran>cornmeal>cotton seed hull, the influence of peptone trend on mycelium biomass was polarized, the percentage of KH2PO4still had rising space.

    Fig.2 Relationship between factors and the total yield 9 formulas designed with orthogonal test L9 (34) were used to culture P. igniarius by inoculating 2 bacteria breads in each of them for 15 days, 4 factors had different influence on the total yield

    2.4 Results of the verification test

    Verification test was done with formula 7 (A3B1C3D2) which had the highest total yield gained through the orthogonal test and the optimal formula theoretically (A3B3C3D2), results showed that there were differences in the total yield gained based on the two formulas (Table 4), the optimal formula theoretically gained the largest total yield and there were no significant differences between the influence of two formulas on it (Table 5).

    Table 4 Results of the verification test

    Table 5 Analysis of variance of formula 7 and the best formula’s influence on the total yield

    SourcesofvariationSSdfMSFSignificancelevelA0.00384110.0038410.62555error0.02455840.006139sum0.0283985

    Note: * indicates significant influence, ** indicates very significant influence, blank indicates no significant influence

    F1-0.05(1,4)=6.94 , F1-0.01(1,4)=7.71

    3 Discussion

    The influence ofP.igniariusliquid medium formula onP.igniariustotal yield was studied through the four factors and three levels orthogonal test. The levels were determined through the former single factor experiment before the orthogonal test. The three levels were the first level in gaining the most total yield and two other levels designed according to the first level (one is higher, another is lower).

    The study found that the four factors all had highly significant influence on the total yield and got the best liquid medium formula forP.igniariustotal yield: wheat bran 10%, peptone 1.5%, KH2PO40.35%, MgSO40.15%, pH 6, 1.007 7 g/100 mL total yield could be got by using this formula and 2 round fungus blocks (diameter 1 cm). Verification test proved that there were no significant differences between the influences of the two formulas (formula 7 and the optimal formula theoretically) on the total yield, so in order to save the cost, formula 7 (wheat bran 10%, peptone 0.5%, KH2PO40.35%, MgSO40.15%, pH 6) can be used in industrial settings.

    [1] Zou X, Sun M, Guo X. Quantitative response of cell growth and polysaccharide biosynthesis by the medicinal mushroomPhellinuslinteustoNaCl in the medium[J]. World J Microbiol Biotechnol, 2006, 22(11): 1129-1133.

    [2] Yang Y, Zhang JS, Liu YF, et al. Structural elucidation of a 3-O-methyl-D-galactose-containing neutral polysaccharide from the fruiting bodies ofPhellinusigniarius[J]. Carbohydr. Res, 2007, 342(8): 1063-1070.

    [3] Dong W, Li N, Lu WD, et al. Tumor-inhibitory and liver-protective effects ofPhellinusigniariusextracellular polysaccharides[J]. World J Microbiol Biotechnol, 2009, 25(4): 633-638.

    [4] Guo X, Zou X, Sun M. Optimization of a chemically defined medium for mycelial growth and polysaccharide production by medicinal mushroomPhellinusigniarius[J]. World J Microbiol Biotechnol, 2009, 25(12): 2187-2193.

    [5] Chi JH, Ha TM, Kim YH, et al. Studies on the main factors affecting the mycelial growth ofPhellinuslinteus[J]. Ko. J Mycol, 1996, 24: 214-222.

    [6] Kim DH, Yang BK, Jeong SC, et al. Production of a hypoglycemic, extracellular polysaccharide from the submerged culture of the mushroom [J]. Phellinus linteus. Biotechnology letters, 2001, 23 (7): 513-517.

    [7] Hwang HJ, Kim SW, Yun JW. Modern Biotechnology ofPhellinusbaumii-From Fermentation to Proteomics[J]. Food Technol Biotechnol, 2007, 45(3): 306-318.

    [8] Zhu T, Kim SH, Chen CY. A medicinal mushroom:Phellinuslinteus[J]. Current Medicinal Chemistry, 2008, 15(13): 1330-1335.

    [9] Dai HY, Wang P, Feng LY, et al. The molecular mechanisms of traditional Chinese medicine ZHENG syndromes on pancreatic tumor growth[J]. Integr Cancer Ther, 2010, 9(3): 291-297.

    [10]Noh JR, Lee IK, Ly SY, et al. APhellinusbaumiiextract reduces obesity in high-fat diet-fed mice and absorption of triglyceride in lipid-loaded mice[J]. Journal of Medicinal Food, 2011, 14(3): 209-218.

    [11]Li L, Wu G, Choi BY, et al. A mushroom extract Piwep fromPhellinusigniariusameliorates experimental autoimmune encephalomyelitis by inhibiting immune cell infiltration in the spinal cord[J]. Biomed Res Int., 2014, 2014(1): Article ID 218274. Doi.org/10.1155/2014/218274.

    [12]Zhou C1, Jiang SS, Wang CY, et al. Different immunology mechanisms ofPhellinusigniariusin inhibiting growth of liver cancer and melanoma cells[J]. Asian Pac J Cancer Prev, 2014, 15(8):3659-3965.

    [13]Ohtsuka S, Uneo S, Yoshikumi C, et al.Polysaccharides and method for producing same(4051314)[P]. Washington: United States Patent and Trademark Office, 1977: 1-4.

    [14]Kim HM, Han SB, Oh GT, et al. Stimulation of humoral and cell mediated immunity by polysaccharide from mushroomPhellinuslinteus[J]. Int J Immunopharmacol, 1996, 18(5): 295-304.

    [15]Han SB, Lee CW, Jeon YJ, et al. The inhibitory effect of polysaccharides isolated fromPhellinuslinteuson tumor growth and metastasis[J]. Immunopharmacology, 1999, 41(2): 157-164.

    [16]Li G, Kim D, Kim T, et al. Protein-bound polysaccharide fromPhellinuslinteusinduces 2/M phase arrest and apoptosis in SW480 human colon cancer cells[J]. Cancer Lett, 2004, 216(2): 175-181.

    [17]Kim GY, Lee JY, Lee JO, et al. Partial characterization and immunostimulatory effect of a novel polysaccharide-protein complex extracted fromPhellinuslinteus[J].Biosci Biotechnol Biochem, 2006, 70(5): 1218-1226.

    [18]Sliva D, Kawasaki J, Stanley G, et al.Phellinuslinteusinhibits growth and invasive behavior of breast cancer cells through the suppression of Akt signaling[J]. FASEB J, 2006, 20: 559-560.

    [19]Moradali MF, Mostafavi H, Ghods S, et al. Immunonodulating and anticancer agents in the realm of macromycetes fungi (macrofungi)[J]. Int Immunopharmacol, 2007, 7(6): 701-724.

    [20]Zhang M, Cui S W, Cheung P C K, et al. Antitumor polysaccharides from mushrooms: a review on their isolation process, structural characteristics and antitumor activity[J]. Trends Food Sci Technol, 2007, 18(1): 4-19.

    [21]Yang Y, Ye LB, ZHang JS, et al. Structural analysis of a bioactive polysaccharide Structural Analysis of a Bioactive Polysaccharide, PISP1, from the Medicinal MushroomPhellinusigniarius[J]. Biosci Biotechnol Biochem, 2009, 73(1): 134-139.

    [22]Chen L, Pan J, Li X, et al. Endo-polysaccharide ofPhellinusigniariusexhibited anti-tumor effect through enhancement of cell mediated immunity[J]. Int Immunopharmacol, 2011, 11(2):255-259.

    [23]Li GJ, Wu GY, Cui JC, et al.Phelinuslinteushyphae training and applications[J]. Edible Fungi China, 1998, 17(5): 27-28. (in Chinese)

    [24]Song L, Sun PL, Guo BB, et al. The research progress ofPhellinusIgniarius[J]. Edible Fungi China, 2005, 24(3): 7-10. (in Chinese)

    [25]Xiao JH, Chen DX, Wan WH,et al. Enhanced simultaneous production of mycelia and intracellular polysaccharide in submerged cultivation of Cordyceps jiangxiensis using desirability functions[J]. Process Biochem, 2006, 41(8): 1887-1893.

    [26]Hu GQ. The production of edible fungi liquid spawn and application[J]. Edible Fungi,1998, 20(1): 17-18. (in Chinese)

    [27]Wang ZH, Wu ZW, Zhao XF. Nutrients’influence onPhellinusigniariusmycelium biomass and exocellular polysaccharide yield[J]. Chinese Wild Plant Res, 2009, 28(1): 37-41. (in Chinese)

    [28]Li CC,Wei YX,Guo LZ. Preliminary study on liquid fermentation medium ofPhellinusigniarius[J]. Edible Fungi China , 2009, 28(2):46-48. (in Chinese)

    [29]Zhuang CQ, He CX. Applied Mathematical Statistical Basis[M].Guangzhou: South China University of Technology Press, 2009. (in Chinese)

    藥用真菌桑黃液體培養(yǎng)基配方的優(yōu)化

    許 謙

    (菏澤學(xué)院 生命科學(xué)系,山東 菏澤 274015)

    研究液體培養(yǎng)基配方對(duì)桑黃菌絲體生物量和胞外多糖產(chǎn)量的影響。采用四因素三水平的正交試驗(yàn)方法。適宜于菌絲體生物量和胞外多糖產(chǎn)量的配方為A3B1C3D2(麥麩 10%, 蛋白胨 0.5%, KH2PO40.35%, MgSO40.15%), pH 6,菌絲體生物量和胞外多糖總產(chǎn)達(dá)到1.007 7 g /100 mL。優(yōu)化的配方可以用于工業(yè)生產(chǎn)獲得更多的菌絲體生物量和胞外多糖。

    液體培養(yǎng)基配方;菌絲體生物量;胞外多糖產(chǎn)量;總產(chǎn)量;正交試驗(yàn);桑黃

    Q939.11

    A

    1005-7021(2015)04-0029-06

    10.3969/j.issn.1005-7021.2015.04.006

    Found Projecd:Supported by Projects of Heze City Science and Technology Development (2012N002)

    Brief introduction to the writer: XU Qian, female, master, associate professor. Research Area: Development and utilization of

    edible-medicinal fungi, microbial genetics and breeding.E-mail: xq710301@163.com

    Draft accepted date:2015-05-03;Revision returned date:2015-05-16

    猜你喜歡
    桑黃菏澤菌絲體
    桑黃——古老中藥放新彩
    鄉(xiāng)村振興的“菏澤路徑”
    金橋(2021年4期)2021-05-21 08:19:10
    桑黃黃酮的研究進(jìn)展
    2019年底前山東菏澤境內(nèi)三條高速可通車(chē)
    石油瀝青(2019年2期)2019-02-13 17:24:47
    菏澤牡丹,花開(kāi)全新產(chǎn)業(yè)鏈——第27屆菏澤牡丹文化旅游節(jié)盛大開(kāi)幕
    金橋(2018年5期)2018-09-22 02:16:54
    桑黃纖孔菌發(fā)酵液化學(xué)成分的研究
    中成藥(2018年1期)2018-02-02 07:20:03
    Leadership Change: a Perspective from China
    桑黃液體發(fā)酵茶飲料工藝研究
    葡萄糖酸鈉發(fā)酵廢棄菌絲體提取殼聚糖的研究
    新型環(huán)保吸聲材料——菌絲體膠合秸稈
    安全(2015年7期)2016-01-19 06:19:39
    涩涩av久久男人的天堂| 亚洲欧美成人综合另类久久久| 亚洲欧洲日产国产| a级毛片黄视频| 涩涩av久久男人的天堂| 叶爱在线成人免费视频播放| 久久久精品国产亚洲av高清涩受| 国产精品一区二区在线不卡| 午夜激情av网站| 日本五十路高清| 老汉色av国产亚洲站长工具| 丝袜人妻中文字幕| 亚洲一卡2卡3卡4卡5卡精品中文| 欧美黄色淫秽网站| 国产精品欧美亚洲77777| 黄色a级毛片大全视频| 五月天丁香电影| 丰满饥渴人妻一区二区三| 丝袜喷水一区| 亚洲,欧美精品.| 热99久久久久精品小说推荐| 国产一区二区激情短视频 | 精品久久久久久久毛片微露脸 | 精品久久久久久电影网| 丁香六月欧美| 亚洲专区中文字幕在线| 国产男女超爽视频在线观看| 亚洲伊人久久精品综合| 91精品国产国语对白视频| 蜜桃国产av成人99| 美女高潮到喷水免费观看| 午夜av观看不卡| 欧美大码av| 九色亚洲精品在线播放| 蜜桃在线观看..| 一级片免费观看大全| 国产男人的电影天堂91| 777久久人妻少妇嫩草av网站| 国产一区有黄有色的免费视频| 色94色欧美一区二区| 一区在线观看完整版| 丰满迷人的少妇在线观看| 久热爱精品视频在线9| 男女无遮挡免费网站观看| av国产久精品久网站免费入址| 国产高清视频在线播放一区 | 婷婷色麻豆天堂久久| 国产激情久久老熟女| 日日摸夜夜添夜夜爱| 亚洲中文字幕日韩| 日本黄色日本黄色录像| 国产成人影院久久av| 激情五月婷婷亚洲| 999精品在线视频| 亚洲男人天堂网一区| 男女边摸边吃奶| 91麻豆av在线| 曰老女人黄片| 少妇被粗大的猛进出69影院| 如日韩欧美国产精品一区二区三区| 男女国产视频网站| 老汉色∧v一级毛片| 深夜精品福利| bbb黄色大片| 黄片播放在线免费| 成年av动漫网址| 大香蕉久久网| 91麻豆av在线| 亚洲成人免费电影在线观看 | 男男h啪啪无遮挡| 亚洲精品国产av蜜桃| 国产在线免费精品| 久久久久久亚洲精品国产蜜桃av| 国产老妇伦熟女老妇高清| 欧美97在线视频| 国产成人精品久久二区二区91| 啦啦啦在线观看免费高清www| 国产在视频线精品| 午夜福利乱码中文字幕| 亚洲精品久久久久久婷婷小说| 亚洲精品国产色婷婷电影| 一级黄色大片毛片| 久久久久精品人妻al黑| av片东京热男人的天堂| 在现免费观看毛片| 赤兔流量卡办理| 亚洲精品日韩在线中文字幕| 久久ye,这里只有精品| 亚洲成人免费电影在线观看 | 咕卡用的链子| 男人添女人高潮全过程视频| cao死你这个sao货| 黄片播放在线免费| 精品亚洲成a人片在线观看| 国产精品av久久久久免费| 操美女的视频在线观看| 老司机在亚洲福利影院| 欧美激情 高清一区二区三区| 日本av免费视频播放| 久久青草综合色| 中文字幕制服av| 日韩熟女老妇一区二区性免费视频| 亚洲欧洲国产日韩| 亚洲欧美中文字幕日韩二区| 精品一品国产午夜福利视频| 亚洲色图 男人天堂 中文字幕| av在线播放精品| 日韩制服丝袜自拍偷拍| 国产福利在线免费观看视频| 人人妻人人添人人爽欧美一区卜| 蜜桃国产av成人99| 免费高清在线观看日韩| 国产片内射在线| 久久久久国产精品人妻一区二区| 97在线人人人人妻| 一区二区日韩欧美中文字幕| 777久久人妻少妇嫩草av网站| 在线亚洲精品国产二区图片欧美| 91成人精品电影| 中文字幕人妻丝袜一区二区| 午夜福利在线免费观看网站| 日韩伦理黄色片| 69精品国产乱码久久久| 啦啦啦视频在线资源免费观看| 亚洲欧美日韩另类电影网站| 波多野结衣av一区二区av| av有码第一页| 中文欧美无线码| 中文字幕最新亚洲高清| 不卡av一区二区三区| 好男人电影高清在线观看| 宅男免费午夜| 亚洲一码二码三码区别大吗| 亚洲国产av新网站| 50天的宝宝边吃奶边哭怎么回事| 欧美日韩av久久| 丁香六月天网| 大香蕉久久网| 亚洲色图 男人天堂 中文字幕| 亚洲精品国产色婷婷电影| 男人爽女人下面视频在线观看| av天堂在线播放| 深夜精品福利| 亚洲人成电影观看| 国产激情久久老熟女| 亚洲精品国产色婷婷电影| 十分钟在线观看高清视频www| 亚洲av片天天在线观看| 国产精品99久久99久久久不卡| 十八禁高潮呻吟视频| 国产不卡av网站在线观看| 大话2 男鬼变身卡| 日本午夜av视频| 又粗又硬又长又爽又黄的视频| 热re99久久国产66热| 亚洲成人免费电影在线观看 | 在线观看免费日韩欧美大片| 国产男人的电影天堂91| 美国免费a级毛片| 国产一级毛片在线| 熟女av电影| 男的添女的下面高潮视频| 别揉我奶头~嗯~啊~动态视频 | 一级a爱视频在线免费观看| 精品视频人人做人人爽| 欧美日韩一级在线毛片| 99国产精品一区二区三区| 欧美97在线视频| 午夜av观看不卡| 婷婷色综合www| 人妻 亚洲 视频| 大香蕉久久成人网| 一区二区三区乱码不卡18| 青春草视频在线免费观看| 嫁个100分男人电影在线观看 | 男女之事视频高清在线观看 | 国产野战对白在线观看| 亚洲av日韩精品久久久久久密 | 国产成人欧美在线观看 | 老司机在亚洲福利影院| 国产精品香港三级国产av潘金莲 | 免费在线观看日本一区| 亚洲精品av麻豆狂野| 亚洲自偷自拍图片 自拍| 免费在线观看影片大全网站 | 国产成人91sexporn| 高潮久久久久久久久久久不卡| 国产精品99久久99久久久不卡| 亚洲国产毛片av蜜桃av| 国产男女超爽视频在线观看| 免费在线观看黄色视频的| 国产精品一区二区免费欧美 | 午夜视频精品福利| 欧美老熟妇乱子伦牲交| 久久精品成人免费网站| 亚洲精品国产色婷婷电影| 99热全是精品| 久久av网站| 国产成人精品久久久久久| 成人国语在线视频| 精品少妇内射三级| 老司机靠b影院| 91精品三级在线观看| 亚洲天堂av无毛| 亚洲综合色网址| 免费在线观看视频国产中文字幕亚洲 | 美女福利国产在线| 国产高清视频在线播放一区 | 久久人妻熟女aⅴ| 亚洲综合色网址| 在现免费观看毛片| 亚洲国产看品久久| 亚洲黑人精品在线| 国产视频首页在线观看| 亚洲欧美精品综合一区二区三区| 国产精品.久久久| av网站在线播放免费| 午夜视频精品福利| 一区二区三区精品91| 午夜91福利影院| 国产精品久久久人人做人人爽| 别揉我奶头~嗯~啊~动态视频 | 亚洲精品久久成人aⅴ小说| 久9热在线精品视频| 老司机亚洲免费影院| 在线精品无人区一区二区三| 国产亚洲午夜精品一区二区久久| 精品亚洲成a人片在线观看| 国产精品成人在线| 久久久久国产精品人妻一区二区| 大话2 男鬼变身卡| 99久久综合免费| 国产午夜精品一二区理论片| 自拍欧美九色日韩亚洲蝌蚪91| 老汉色∧v一级毛片| 永久免费av网站大全| av在线播放精品| 欧美黑人精品巨大| 两人在一起打扑克的视频| 亚洲熟女毛片儿| 免费观看av网站的网址| 免费在线观看日本一区| 欧美黄色片欧美黄色片| 精品一区在线观看国产| 一区福利在线观看| 久久久久视频综合| 99久久人妻综合| 又大又爽又粗| 中文字幕精品免费在线观看视频| 久久精品人人爽人人爽视色| av在线播放精品| 精品人妻在线不人妻| 菩萨蛮人人尽说江南好唐韦庄| 狂野欧美激情性bbbbbb| 男女免费视频国产| 91精品伊人久久大香线蕉| 午夜福利一区二区在线看| 老司机午夜十八禁免费视频| 18禁观看日本| 婷婷色麻豆天堂久久| 超碰成人久久| 国产精品av久久久久免费| 久久久久久久精品精品| 国产不卡av网站在线观看| 国产一区亚洲一区在线观看| 黑人巨大精品欧美一区二区蜜桃| 成人黄色视频免费在线看| 777久久人妻少妇嫩草av网站| 大陆偷拍与自拍| 青春草亚洲视频在线观看| 欧美在线黄色| 狂野欧美激情性xxxx| 悠悠久久av| 亚洲精品自拍成人| 免费日韩欧美在线观看| 国产男女超爽视频在线观看| 男男h啪啪无遮挡| 深夜精品福利| 两个人看的免费小视频| 国产不卡av网站在线观看| 国产男人的电影天堂91| 日本91视频免费播放| 亚洲美女黄色视频免费看| 久久精品亚洲熟妇少妇任你| 大陆偷拍与自拍| 亚洲色图综合在线观看| 人人妻人人爽人人添夜夜欢视频| 一边亲一边摸免费视频| 91成人精品电影| 99精品久久久久人妻精品| 一级a爱视频在线免费观看| 久久中文字幕一级| 国产av国产精品国产| 国产午夜精品一二区理论片| 黑人猛操日本美女一级片| 午夜影院在线不卡| 日韩大片免费观看网站| 久久久久久久国产电影| 久久免费观看电影| 不卡av一区二区三区| 中文字幕人妻丝袜一区二区| 两个人看的免费小视频| 成人国产一区最新在线观看 | 少妇人妻久久综合中文| 成年动漫av网址| 亚洲国产毛片av蜜桃av| 亚洲欧美精品自产自拍| 午夜激情久久久久久久| 少妇 在线观看| 亚洲欧美精品自产自拍| 中国美女看黄片| 50天的宝宝边吃奶边哭怎么回事| 久久精品国产a三级三级三级| 少妇裸体淫交视频免费看高清 | 97人妻天天添夜夜摸| 国产三级黄色录像| 午夜老司机福利片| 一边摸一边抽搐一进一出视频| 亚洲成av片中文字幕在线观看| 国产精品亚洲av一区麻豆| 老熟女久久久| 极品人妻少妇av视频| 999久久久国产精品视频| 青草久久国产| 国产精品一二三区在线看| 日韩av在线免费看完整版不卡| 我要看黄色一级片免费的| 国产免费现黄频在线看| 国产免费一区二区三区四区乱码| 人人妻人人澡人人爽人人夜夜| av国产精品久久久久影院| 欧美黑人欧美精品刺激| 黄片播放在线免费| 老汉色av国产亚洲站长工具| av国产精品久久久久影院| 国产精品二区激情视频| 欧美久久黑人一区二区| 欧美激情 高清一区二区三区| av一本久久久久| 精品人妻在线不人妻| 一边亲一边摸免费视频| 日韩欧美一区视频在线观看| av又黄又爽大尺度在线免费看| 午夜福利视频在线观看免费| 亚洲av成人精品一二三区| 国产高清视频在线播放一区 | 亚洲欧美成人综合另类久久久| 亚洲伊人久久精品综合| 国产国语露脸激情在线看| 欧美精品av麻豆av| 我要看黄色一级片免费的| 国产成人一区二区在线| 少妇的丰满在线观看| 亚洲成人免费电影在线观看 | 国产精品一区二区免费欧美 | 国产精品久久久人人做人人爽| 午夜老司机福利片| 亚洲五月色婷婷综合| 久久精品人人爽人人爽视色| 久久久久精品国产欧美久久久 | 婷婷色av中文字幕| 精品免费久久久久久久清纯 | 久久久欧美国产精品| 大码成人一级视频| 搡老岳熟女国产| xxx大片免费视频| 国产在线观看jvid| 国产亚洲欧美精品永久| 在线观看免费视频网站a站| 大片电影免费在线观看免费| 精品一区二区三卡| 又粗又硬又长又爽又黄的视频| 国产免费又黄又爽又色| 日韩av免费高清视频| 国产免费又黄又爽又色| 欧美少妇被猛烈插入视频| 欧美黑人精品巨大| 亚洲国产精品一区三区| 亚洲久久久国产精品| 亚洲精品久久午夜乱码| 久久久国产精品麻豆| 亚洲成人手机| 丝袜美腿诱惑在线| 美女国产高潮福利片在线看| 婷婷色麻豆天堂久久| 亚洲中文字幕日韩| 亚洲一卡2卡3卡4卡5卡精品中文| 亚洲图色成人| av片东京热男人的天堂| av在线老鸭窝| 国产精品人妻久久久影院| 午夜影院在线不卡| 一级毛片电影观看| 中文字幕人妻丝袜一区二区| 久久 成人 亚洲| 波野结衣二区三区在线| 欧美性长视频在线观看| 一级片免费观看大全| 亚洲精品成人av观看孕妇| 视频在线观看一区二区三区| 老汉色∧v一级毛片| 操出白浆在线播放| 午夜福利视频在线观看免费| www.精华液| 国产av一区二区精品久久| 久久久精品94久久精品| 国产精品久久久人人做人人爽| 国产日韩一区二区三区精品不卡| 亚洲av美国av| 久久久久久久久免费视频了| 九草在线视频观看| 啦啦啦视频在线资源免费观看| 90打野战视频偷拍视频| 久久精品亚洲熟妇少妇任你| 亚洲av国产av综合av卡| 少妇粗大呻吟视频| 99热网站在线观看| 精品一品国产午夜福利视频| 久久人人爽av亚洲精品天堂| 岛国毛片在线播放| 一区二区三区乱码不卡18| 在线看a的网站| 97精品久久久久久久久久精品| 成人手机av| 久久久久久久国产电影| 成人国语在线视频| 另类亚洲欧美激情| 久久99热这里只频精品6学生| 亚洲国产日韩一区二区| 亚洲精品国产av成人精品| 国产女主播在线喷水免费视频网站| 久久久久国产精品人妻一区二区| 一区在线观看完整版| av线在线观看网站| 中文字幕色久视频| 午夜福利一区二区在线看| 久久久久久久久免费视频了| 精品久久久久久久毛片微露脸 | 亚洲精品在线美女| 国产一区二区在线观看av| 日本91视频免费播放| 免费久久久久久久精品成人欧美视频| 18禁国产床啪视频网站| 99国产精品99久久久久| 久久99一区二区三区| 久久人人爽人人片av| 麻豆国产av国片精品| 精品人妻1区二区| 久久久国产一区二区| 久久毛片免费看一区二区三区| 午夜免费鲁丝| 国产有黄有色有爽视频| 又紧又爽又黄一区二区| 国产欧美日韩综合在线一区二区| 日本91视频免费播放| 欧美日韩视频精品一区| 97精品久久久久久久久久精品| 亚洲色图 男人天堂 中文字幕| av线在线观看网站| 啦啦啦在线观看免费高清www| 91麻豆精品激情在线观看国产 | 丝袜在线中文字幕| 99热网站在线观看| 国产精品久久久人人做人人爽| xxxhd国产人妻xxx| 久久女婷五月综合色啪小说| 男人爽女人下面视频在线观看| 只有这里有精品99| 久久久久久免费高清国产稀缺| 亚洲一卡2卡3卡4卡5卡精品中文| 欧美av亚洲av综合av国产av| av一本久久久久| 国产在线免费精品| 中文乱码字字幕精品一区二区三区| 欧美日韩成人在线一区二区| 久久人人97超碰香蕉20202| 一区二区三区四区激情视频| 色网站视频免费| 亚洲精品国产av成人精品| 亚洲七黄色美女视频| 国产精品国产三级国产专区5o| 又大又黄又爽视频免费| 国产又爽黄色视频| 午夜久久久在线观看| 十八禁网站网址无遮挡| 欧美 亚洲 国产 日韩一| 日韩大码丰满熟妇| www.自偷自拍.com| 国产精品欧美亚洲77777| 欧美黄色片欧美黄色片| 亚洲中文日韩欧美视频| 男女下面插进去视频免费观看| 午夜两性在线视频| 国产麻豆69| 美女午夜性视频免费| 精品第一国产精品| 一区二区日韩欧美中文字幕| 一本色道久久久久久精品综合| 国产精品久久久人人做人人爽| 看免费av毛片| 在线看a的网站| 超碰97精品在线观看| e午夜精品久久久久久久| 亚洲人成网站在线观看播放| 精品少妇内射三级| 亚洲精品自拍成人| 99久久人妻综合| 欧美另类一区| 久久女婷五月综合色啪小说| 国产精品99久久99久久久不卡| 99香蕉大伊视频| 国产一区有黄有色的免费视频| 亚洲国产精品一区三区| 国产熟女欧美一区二区| 欧美成人午夜精品| 桃花免费在线播放| 亚洲精品一二三| 久久精品国产综合久久久| 1024视频免费在线观看| 亚洲精品一区蜜桃| 国产精品一区二区在线不卡| 大陆偷拍与自拍| 亚洲欧美激情在线| 99久久精品国产亚洲精品| 久久久欧美国产精品| 精品卡一卡二卡四卡免费| 亚洲激情五月婷婷啪啪| 精品亚洲乱码少妇综合久久| 制服人妻中文乱码| 人体艺术视频欧美日本| 侵犯人妻中文字幕一二三四区| 国产极品粉嫩免费观看在线| 人妻 亚洲 视频| 热re99久久国产66热| 亚洲精品自拍成人| 国产亚洲一区二区精品| 男人操女人黄网站| 纯流量卡能插随身wifi吗| 久9热在线精品视频| 蜜桃国产av成人99| 久久九九热精品免费| 建设人人有责人人尽责人人享有的| 免费看十八禁软件| 另类精品久久| 波多野结衣一区麻豆| 狠狠精品人妻久久久久久综合| 亚洲国产av新网站| 欧美av亚洲av综合av国产av| 美女扒开内裤让男人捅视频| h视频一区二区三区| 伦理电影免费视频| 十八禁网站网址无遮挡| 黑人欧美特级aaaaaa片| 欧美激情极品国产一区二区三区| 99久久综合免费| 精品人妻1区二区| a级毛片在线看网站| 97人妻天天添夜夜摸| 国产黄色免费在线视频| 久久久精品区二区三区| 日本av免费视频播放| 在线亚洲精品国产二区图片欧美| 女警被强在线播放| av线在线观看网站| 国产成人欧美在线观看 | 在线精品无人区一区二区三| 一区二区三区四区激情视频| 亚洲av电影在线进入| 日本欧美国产在线视频| 久久免费观看电影| 欧美日韩黄片免| 精品一区二区三区四区五区乱码 | 日韩人妻精品一区2区三区| 又紧又爽又黄一区二区| 别揉我奶头~嗯~啊~动态视频 | 日韩,欧美,国产一区二区三区| 亚洲国产欧美网| 狠狠婷婷综合久久久久久88av| 老汉色∧v一级毛片| 国产精品三级大全| 美女脱内裤让男人舔精品视频| 建设人人有责人人尽责人人享有的| 国产一区亚洲一区在线观看| 美女高潮到喷水免费观看| 一级黄片播放器| 最新在线观看一区二区三区 | 国产精品一区二区在线不卡| 成年美女黄网站色视频大全免费| 三上悠亚av全集在线观看| 亚洲精品国产色婷婷电影| 狠狠婷婷综合久久久久久88av| 国产高清视频在线播放一区 | 97精品久久久久久久久久精品| 桃花免费在线播放| 在线天堂中文资源库| 水蜜桃什么品种好| 少妇人妻久久综合中文| 国产精品二区激情视频| 97人妻天天添夜夜摸| 十分钟在线观看高清视频www| 国产一区二区三区综合在线观看| a 毛片基地| 成人国产av品久久久| 乱人伦中国视频| 黄色a级毛片大全视频| 亚洲国产欧美一区二区综合| 亚洲成人免费av在线播放| 高清不卡的av网站| 亚洲人成电影观看| 男人添女人高潮全过程视频| av国产久精品久网站免费入址| 一区在线观看完整版| 久久久欧美国产精品| 老司机亚洲免费影院| 极品人妻少妇av视频|