10-HDA对上面发酵小麦啤酒中Pectinatus spp及野生酵母的作用研究
|
摘要
作为蜂王浆标志物的10-HDA,具有抑菌杀菌效果同时还具备抗癌、修复辐射及化学物质损伤、增强免疫力等营养保健功能。上面发酵小麦啤酒为含有活酵母的未经过滤的鲜啤酒,生物稳定性较差。前期实验证明10-HDA对G+啤酒有害菌中短乳杆菌和有害片球菌具有抑菌性,本实验以具有典型性的革兰氏阴性啤酒有害菌Pectinatus.spp及野生酵母为供试菌,研究10-HDA对主要啤酒有害菌的作用影响,开发10-HDA作为上面发酵小麦啤酒的天然啤酒防腐剂。
比较三种有机溶剂乙醚、乙酸乙酯、乙醇,分别采用两种方法提取蜂王浆中的10-HDA,利用高效液相色谱法得出:使用乙醇、采用振荡提取法得到的提取效果最好。通过正交试验优化提取工艺,试验结果表明最佳提取工艺条件为:萃取剂乙醇浓度100%,料液比1:6,萃取时间60min,萃取次数1次,10-HDA提取率为33.893%,纯度为73.5325%。
以酿酒酵母303、糖化酵母、菌膜假丝酵母、Pectinatus cerevisiiphilus和Pectinatus frisingensis为供试菌,对10-HDA乙醇提取物进行抑菌试验。通过牛津杯法检测出10mg/mL浓度范围的10-HDA对酿酒酵母303、糖化酵母不具有抑菌性,对菌膜假丝酵母、Pectinatus cerevisiiphilus及Pectinatus frisingensis具有抑菌作用,通过倍比稀释法得出10-HDA对三株有害菌的最小抑菌浓度为:0.375 mg/mL、0.625 mg/mL、1.25mg/mL。
在此基础上,研究10-HDA乙醇液抑菌活性的稳定性,结果表明,高温处理不利于10-HDA抑菌活性的发挥,但121℃处理30min后,10-HDA仍有抑菌活性;PH对10-HDA活性影响较大,酸性条件更利于抑菌性的发挥,而紫外线对其稳定性几乎没有影响。研究10-HDA对供试菌生长的影响,结果表明10-HDA对供试菌不仅是抑制作用而且是迅速的杀灭作用。
啤酒中异α-酸具有抑菌杀菌性,为验证将10-HDA添加入啤酒后,10-HDA抑菌作用的发挥是否受到异α-酸的协同,对前述供试菌进行异α-酸的酒花抗性实验。结果表明,100mg/L浓度范围内的异α-酸对酿酒酵母303、糖化酵母、Pectinatus cerevisiiphilus及Pectinatus frisingensis不具有抑制作用;随异α-酸乙醇液浓度的增大,异α-酸作用于菌膜假丝酵母的抑菌圈直径逐渐增大,通过稀释法得出异α-酸对菌膜假丝酵母的最低抑菌浓度为62.5mg/L。
本实验中,我们将10-HDA加入到苦味值为20.25mg/L的上面发酵小麦啤酒中,利用NBB和菌种计数法来检测10-HDA在这种啤酒中的抑菌效能,结果显示,10-HDA发挥了良好的抑菌效果,且该效能的发挥与异-α酸无关。
因此,我们有理由得出结论,10-HDA完全可以开发成一种上面发酵小麦啤酒的天然食品添加剂。
As the sign matter of royal jelly, the 10-HDA posses bacteriostatic action and bactericidal ability it also exhibits several nutritional healthy biological activities, including anti-cancer, enhancing immune function and so on. Top-fermented wheat beer with living yeasts has not been filtered, which made it bad biological stability. The antimicrobial effect of 10-HDA on G+ beer-spoilage bacteria Lactobacillu brevis and Pediococcus damnosus was determined in our early experiment stage. In the present study, we chose typical G- beer-spoilage bacteria Pectinatus.spp and wild yeasts to detect the effect of 10-HDA on these different beer-spoilage strains, we will develop the 10-HDA to be a natural preservative of top-fermented wheat beer.
This study used three organic solutions such as ether, ethyl acetate and ethanol as the extractive solution, and also employed two extractive processions. It used the method of high pressure liquid chromatography to compared the extraction efficiency of 10-HDA. The result of the compare is that using the ethanol shake the royal jelly can receive the highest extractive ratio of 10-HDA.The process of extraction of 10-HDA from royal jelly was optimized through the orthogonal experiments. The optimal conditions included: ethanol concentration 100% ,solid/liquid 1∶6 , extraction time was 60 min,the frequency of the extraction was 1. Under these conditions, the yield of 10-HDA in the extraction was 33.893%, purity was 73.5325%.
The antimicrobial effect of 10-HDA on two strains of bacteria and three strains of yeasts was determined by Oxford cup assay and we also determined the minimum inhibitory concentration (MIC) of 10-HDA by double dilution method. Our results showed that 10-HDA could significantly inhibit the Candidamy coderma, Pectinatus cerevisiiphilus, Pectinatus frisingensis in beer. In contrast, no inhibition effect was found while treating top-fermenting yeasts No.303 and Saccharomyces c.diastaticus with 10-HDA. The minimum inhibitory concentration (MIC) of 10-HDA on Candidamy coderma, Pectinatus cerevisiiphilus, Pectinatus frisingensis were as follows: 0.375 mg/mL, 0.625 mg/mL,1.25mg/mL.
The 10-HDA was also studied for its antimicrobial stability to temperature, PH and ultraviolet. The results showed that 10-HDA is very stable to ultraviolet. High temperature was not good for the performance of 10-HDA, but it still have antibacterial effects after dealing with 121℃for 30 minutes; PH has obviously effect on 10-HDA, the higher antibacterial activities were showed at lower PH value. The study of the 10-HDA effect on the growth of microorganism for test, found that the 10-HDA not only antiblastic the growth of microorganism for test, but also kill them.
The hops in beer with its main components as iso-α-acid could inhibit the activity of beer spoilage bacteria. We detected the hops resistance of tested strains to determine if the performance of antibacterial effects was produced by 10-HDA or cooperated with iso-α-acid. The results showed that iso-α-acid could significantly inhibit the Candidamy coderma at the MIC 62.5mg/L. In contrast, no inhibition effect was found while treating Pectinatus.spp and Saccharomyces c .diastaticus with iso-α-acid.
In this study, 10-HDA was added to the top-fermented wheat beer with the bitterness 20.25mg/L. The antimicrobial effect of 10-HDA in this beer was determined by NBB culture medium test and microorganism index. The results showed that the 10-HDA performed good antibacterial effects without conjunction with iso-α-acid.
Therefore,it was confirmed that 10-HDA could be developed as a novel antimicrobial agent for top-fermented wheat beer.
比较三种有机溶剂乙醚、乙酸乙酯、乙醇,分别采用两种方法提取蜂王浆中的10-HDA,利用高效液相色谱法得出:使用乙醇、采用振荡提取法得到的提取效果最好。通过正交试验优化提取工艺,试验结果表明最佳提取工艺条件为:萃取剂乙醇浓度100%,料液比1:6,萃取时间60min,萃取次数1次,10-HDA提取率为33.893%,纯度为73.5325%。
以酿酒酵母303、糖化酵母、菌膜假丝酵母、Pectinatus cerevisiiphilus和Pectinatus frisingensis为供试菌,对10-HDA乙醇提取物进行抑菌试验。通过牛津杯法检测出10mg/mL浓度范围的10-HDA对酿酒酵母303、糖化酵母不具有抑菌性,对菌膜假丝酵母、Pectinatus cerevisiiphilus及Pectinatus frisingensis具有抑菌作用,通过倍比稀释法得出10-HDA对三株有害菌的最小抑菌浓度为:0.375 mg/mL、0.625 mg/mL、1.25mg/mL。
在此基础上,研究10-HDA乙醇液抑菌活性的稳定性,结果表明,高温处理不利于10-HDA抑菌活性的发挥,但121℃处理30min后,10-HDA仍有抑菌活性;PH对10-HDA活性影响较大,酸性条件更利于抑菌性的发挥,而紫外线对其稳定性几乎没有影响。研究10-HDA对供试菌生长的影响,结果表明10-HDA对供试菌不仅是抑制作用而且是迅速的杀灭作用。
啤酒中异α-酸具有抑菌杀菌性,为验证将10-HDA添加入啤酒后,10-HDA抑菌作用的发挥是否受到异α-酸的协同,对前述供试菌进行异α-酸的酒花抗性实验。结果表明,100mg/L浓度范围内的异α-酸对酿酒酵母303、糖化酵母、Pectinatus cerevisiiphilus及Pectinatus frisingensis不具有抑制作用;随异α-酸乙醇液浓度的增大,异α-酸作用于菌膜假丝酵母的抑菌圈直径逐渐增大,通过稀释法得出异α-酸对菌膜假丝酵母的最低抑菌浓度为62.5mg/L。
本实验中,我们将10-HDA加入到苦味值为20.25mg/L的上面发酵小麦啤酒中,利用NBB和菌种计数法来检测10-HDA在这种啤酒中的抑菌效能,结果显示,10-HDA发挥了良好的抑菌效果,且该效能的发挥与异-α酸无关。
因此,我们有理由得出结论,10-HDA完全可以开发成一种上面发酵小麦啤酒的天然食品添加剂。
As the sign matter of royal jelly, the 10-HDA posses bacteriostatic action and bactericidal ability it also exhibits several nutritional healthy biological activities, including anti-cancer, enhancing immune function and so on. Top-fermented wheat beer with living yeasts has not been filtered, which made it bad biological stability. The antimicrobial effect of 10-HDA on G+ beer-spoilage bacteria Lactobacillu brevis and Pediococcus damnosus was determined in our early experiment stage. In the present study, we chose typical G- beer-spoilage bacteria Pectinatus.spp and wild yeasts to detect the effect of 10-HDA on these different beer-spoilage strains, we will develop the 10-HDA to be a natural preservative of top-fermented wheat beer.
This study used three organic solutions such as ether, ethyl acetate and ethanol as the extractive solution, and also employed two extractive processions. It used the method of high pressure liquid chromatography to compared the extraction efficiency of 10-HDA. The result of the compare is that using the ethanol shake the royal jelly can receive the highest extractive ratio of 10-HDA.The process of extraction of 10-HDA from royal jelly was optimized through the orthogonal experiments. The optimal conditions included: ethanol concentration 100% ,solid/liquid 1∶6 , extraction time was 60 min,the frequency of the extraction was 1. Under these conditions, the yield of 10-HDA in the extraction was 33.893%, purity was 73.5325%.
The antimicrobial effect of 10-HDA on two strains of bacteria and three strains of yeasts was determined by Oxford cup assay and we also determined the minimum inhibitory concentration (MIC) of 10-HDA by double dilution method. Our results showed that 10-HDA could significantly inhibit the Candidamy coderma, Pectinatus cerevisiiphilus, Pectinatus frisingensis in beer. In contrast, no inhibition effect was found while treating top-fermenting yeasts No.303 and Saccharomyces c.diastaticus with 10-HDA. The minimum inhibitory concentration (MIC) of 10-HDA on Candidamy coderma, Pectinatus cerevisiiphilus, Pectinatus frisingensis were as follows: 0.375 mg/mL, 0.625 mg/mL,1.25mg/mL.
The 10-HDA was also studied for its antimicrobial stability to temperature, PH and ultraviolet. The results showed that 10-HDA is very stable to ultraviolet. High temperature was not good for the performance of 10-HDA, but it still have antibacterial effects after dealing with 121℃for 30 minutes; PH has obviously effect on 10-HDA, the higher antibacterial activities were showed at lower PH value. The study of the 10-HDA effect on the growth of microorganism for test, found that the 10-HDA not only antiblastic the growth of microorganism for test, but also kill them.
The hops in beer with its main components as iso-α-acid could inhibit the activity of beer spoilage bacteria. We detected the hops resistance of tested strains to determine if the performance of antibacterial effects was produced by 10-HDA or cooperated with iso-α-acid. The results showed that iso-α-acid could significantly inhibit the Candidamy coderma at the MIC 62.5mg/L. In contrast, no inhibition effect was found while treating Pectinatus.spp and Saccharomyces c .diastaticus with iso-α-acid.
In this study, 10-HDA was added to the top-fermented wheat beer with the bitterness 20.25mg/L. The antimicrobial effect of 10-HDA in this beer was determined by NBB culture medium test and microorganism index. The results showed that the 10-HDA performed good antibacterial effects without conjunction with iso-α-acid.
Therefore,it was confirmed that 10-HDA could be developed as a novel antimicrobial agent for top-fermented wheat beer.
引文
[1]王腾飞,王瑞明.10-HDA抗菌实验的初步研究[J].山东轻工业学院报,2009, 3:17-24
[2] J.L.Shimwell. On the relation between the staining properties of bacteria and their reaction towards hop antiseptic. Part I,Ⅱ[J]. J. Inst. Brew, 1937, 43, 111-118
[3] W. J. Simpson. Ionophoric action of trans-isohumulone on Lactobacil-Ius brevis [J]. Journal of General Microbiology, 1993, 139:1041-1045
[4] Kanta Sakamoto., Wil N. Konings. Beer spoilage bacteria and hop resistance. International [J]. Journal of Food Microbiology, 2003, (89):105-124
[5]吴文芳,张成刚,吕安国等.啤酒酿造中野生酵母的研究[J].酿酒,1994,(2):57-63
[6] Shanshan Li., Guangtian Zhou. 10-Hydroxy-2-decenoic Acid as an Antimicrobial Agent in Draft Keg-conditioned Wheat Beer [J]. American Society of Brewing Chemists, 2010, 2, 114-118
[7]郭芳彬.浅析蜂王浆在蜂箱中为何不变质[J].蜜蜂杂志,2003,(8):25-26
[8] Takenaka T. Chemical compositions of royal jelly [J]. Honeybee Science, 1982, 3(1): 69-74
[9] Emanvilovi, Velchevap, etal. Studies on the chemical composition and antibacterial and antitoxic properties of royal jelly[J]. Izv Mikrobiol Inst (Slfiia), 1963, 15: 89-95
[10] Eshraghi S., Seifollajhi F. Antibacterial effects of royal jelly on different strains of bacterial [J]. Iranian JPublHealth, 2003, 32(1):25-30
[12]杨远帆,叶兴乾,倪辉等.蜂王浆抗菌作用的研究进展[J].食品科学, 2007, 11:611-614
[13]黄文诚.蜂王浆肌动蛋白是蜂王浆质量的一种可能的标记物[J].中国养蜂,2003,54(6):41-42
[14]倪辉,杨远帆,彭莺.蜂王浆中10-HDA的研究进展[J].中国蜂业, 2006, 57(3):9-10
[15]徐友第.蜂王浆抗菌活性的研究[J].中国养蜂,1989,3(6):28-29
[16]刘晓雯,刘克武,喻东.蜂王浆的有效成分与人类健康[J].蜜蜂杂志,1999,9:6-8
[17]赵家平,刘继勇,蔡文贵.蜂王浆的药理作用[J].特种经济动植物,1998,5:29-30
[18]李利君,孙亮先,彭莺.蜂王浆对三种革兰氏阴性菌的抑菌特性[J].泉州师范学院学报(自然科学).2008,3:96-99
[19]刘克武,杨守忠,葛绍荣.蜂王浆的抗菌活性研究[J].蜜蜂杂志(月刊), 2001, (2):5-6
[20]倪辉,吉挺,杨远帆.蜂王浆抗菌作用的初步研究[J].蜜蜂杂志(月刊). 2003, (2):9-10
[21]孙亮先,陈朝阳,张昌松等.蜂王浆抗菌作用的研究[J].蜜蜂杂志(月刊). 2008, (2):3-4
[22]苏晔,敬璞,丁晓雯.蜂王浆的化学成分、生理活性及应用.蜜蜂杂志(月刊). 2000(9):23-24
[23]金茜.蜂王浆的生理功能[J].化学教育,2004(9):1-2
[24]王国燕,林志彬. 10-羟基- 2-癸稀酸对小鼠T淋巴细胞及其亚型和白介素2产生的影响[J] .中国药理学与毒理学, 1996 (10): 53- 55
[25]耿纯.蜂王浆的降血脂作用[J] .中国养蜂,2000, 52( 1) : 36
[26]刘晓华,孙文基.王浆酸的研究进展概况[J].中国药品标准.2004(5):9-10
[27]汪多仁.王浆酸的开发与应用进展[J].养蜂科技, 2004(4): 6-10
[28]刘洪云.蜂王酸的制备.四川化工.1996(4):39-40
[29] Kulkarni S.M. A short synthesis of royal jelly acid [10-hydroxy-(E)-2-decenoic acid][J]. Indian J. Chem. Sect.B23B, 1984 (5): 460
[30] FujisavaT. A novel synthesis of royal jelly acids and queen Substance by the five carbon homologation using ropiolactone [J]. Chem, Lett, 1982(2): 219-220
[31] Corey E J, William Suggs J, Pyridinium chlorochromate. An efficient reagent for oxidation of primary and secondary alcohols to carbonyl compounds [J].Tertahedron Letters, 1975(1): 2647-2650.
[32]刘放,黄卫平,戴惠芳.蜂王浆及制剂中10-羟基-2-癸烯酸测定方法的进展[J].中国养蜂, 1999,(5): 19-21
[33]张小军,郝彦玲,寇晓红.啤酒花及其抗菌机理研究进展[J].酿酒科技, 2007(4):109-111
[34]郝秋娟,董翠英,李崎.啤酒腐败细菌与酒花抗性[J].酿酒科技,2005,9:65-68
[35] K.suzuki., M.koyanagi. H.Yamashita[J]. Journal of Applied Microbiology, 2004, 96: 946-953
[36] Manabu samin, Yamashita H., Kadokura H [J]. J.Am.Soc.Brew, 1997, 55(4): 137-140
[37]朱林江,郑飞云,李崎,顾国贤.酒花苦味物质的抗菌活性[J].啤酒科技, 2007, 2:5-7
[38]何士雯,马永.酒花苦味酸对乳酸菌的抑制作用.啤酒科技,2004(9):7-9
[39] W.J.Simpson, Studies on the sensitivity of lacticacid bacteria to hop bitter acids [J]. J. Inst.Brew, 1993, 99: 405-411
[40]周广田,董小雷,崔云前.啤酒酵母与工厂卫生[M].北京:化学工业出版社,2008:45-47
[41]王树庆.啤酒酿造中的微生物污染[J].酿酒,2008(1):50-53
[42] Back W. Brauwelt online 24/25, 2003
[43] Methner F.J. Monograph 32 Sanitary Engineering&HACCP[R]. Fachverlag Hans Carl: European Brewery Convention, 2003
[44]林先军,李崎,顾国贤.乳酸菌对制麦和啤酒酿造的影响[J].啤酒科技, 2005(7):21-23
[45]赵敏,杨开,孙培龙.啤酒中乳酸杆菌的分离纯化和鉴定[J].酿酒.2005(1):58-61
[46]周广田.现代啤酒工艺技术[M].北京:化学工业出版社,2007:11-12
[47]陈敬春.浅论啤酒有害细菌及其检出[J].啤酒科技,2005(2):5-4
[48]周广田,聂聪,崔云前等.啤酒酿造技术[M].济南:山东大学出版社,2004:23-25
[49]李晓霞,刘新社.纯生啤酒生产中微生物污染的危害及控制技术[J].商丘职业技术学院学报, 2009(2):105-107
[50]王海明.野生酵母与啤酒酿造.酿酒科技,2003(2):59-61
[51]闫洪伟.啤酒生产中主要有害菌的来源及控制.科技信息,2009(13):744-745
[52]管建良.野生酵母对啤酒生产的影响及控制.啤酒科技.2009(2):47-48
[53]詹东.啤酒的稳定性[J].啤酒科技, 2006(10):20-23
[54]王喜萍,李长生.影响啤酒稳定性的因素及预防措施[J].酿酒科技, 2000(4):59-60
[55]顾建国.影响啤酒稳定性的因素及预防[J].张家口农专学报,1996(12):37-39
[56]张全斌,李艳琴.啤酒腐败菌分子检测技术的研究进展[J].食品与药品, 2007(9):55-57
[57]张少兰.啤酒腐败菌种果胶杆菌和巨型球菌的检测[J].广东轻工职业技术学院学报(综合版),2002(1):34-35
[58] Hakalehto E, Finne J. Identification by immunoblot analysis of major antigenic determinants of the anaerobic beer spoilage bacterium genus Pectinatus [J]. FEMS Microbiol Lett, 1990(67):307-312.
[59]陈江,梁风华.API系统对常见啤酒有害菌进行分类鉴定的研究[J].啤酒科技,2005(11):24-25
[60] Sami M, Yamashita H, Kadokura H, et al. A new and rapid method fordetermination of beer-spoilage ability of lactobacilli[J]. J Am Soc Brew Chem, 1997, 55(4): 137-140
[61] HAYASHI N, ITO M, HORIIKE S, etal. Molecular cloning of a putative divalent-cation transporter gene as a new genetic marker for the identification of Lactobacillusbrevis strains capable of growing in beer [J]. Appl Microbiol Biotechnol, 2001, 55: 596-603
[62] ING D, BRANDL A. Demonstration of PCR based methods in brewery quality control[R]. Hartwall Brewery, Lahti Finland: EBC Brewing Science Group-Microbial Contaminants Subgroup, 2004.
[63] Barszczewski W, Robak M. Differentiation of contaminating yeasts in brewery by PCR-based techniques [J]. Food Microbiol, 2004, 21(2): 227-231.
[64] TAKAOMI YASUHARA, Kyoko Takahashi, Yasuo Motoyama, Toshifumi Yuuki, Kazunori Shibata et al. Rapid detection, identification and enumeration of beer-spoilage bacteria by FISH method. Brewing Research, Development Laboratory, Asahi Breweries Lid.,1-1-21 Midori, Moriya-machi, Ibaraki 302-0106, Japan.
[65] H.Stender, A. Sage, K. Oliveira, A. J. Broomer, B. Young and J. Coull. Combination of ATP- bioluminescence and PNA probes allows rapid total counts and identification of specific microorganisms in mixed opulations [J]. Microbiol Methods, 2001, 46:69-70
[66]孙培龙,徐巧林.啤酒中污染菌检测与鉴定的研究进展.酿酒2006(6):65-69
[67] GRACIAS K S, MCKILLIP J L. A review of conventional detection and enumeration methods for pathogenic bacteria in food[J]. Can J Microbiol, 2004, 50: 883-890.
[68] TAKAHASHI T, NAKAKITA Y, et al. Application of a bioluminescence method for the beer industry: sensitivity of MicroStarTM-RMDS for detecting beer-spoilage bcteria, biosci[J]. Biotechnol Biochem, 2000, 64(5): 1032-1037.
[69] TAKAHASHI T, NAKAITA Y, etal. A new rapid technique for detection of microorganisms using bioluminescence and fluorescence microscope method[J]. Journal of Bioscience and Bioengineering, 2000, 89(5): 509-513
[70]顾国贤等.啤酒无菌酿造[J].酿酒, 2000(3):49-53
[71]王树庆.啤酒生产中污染微生物的快速检测与鉴定技术.酿酒科技, 2009(7):109-113
[72] Foster, A.[J].Journal of the American Society of Brewing Chemists,1996,54-76.
[73]吴红.啤酒的微生物检验技术[J].酿酒,2000(5):34-39
[74]赵现方,陈林海,李宗义.流式细胞术及其在微生物学中的应用[J].河南农业科学, 2005(6):5-8
[75] Malacrino P, Zapparoli G, Torriani S. Dellaglio rapid detection of viable yeasts and bacteria in wine by flow cytometry [J]. J Microbiol Methods, 2001, 45(2):127-l34.
[76] Day A, Meyling J C. Practical experience with rapid methods of detecting yeasts in breweries [J]. J Inst Brew, 1990, 89: 204-206.
[77]肖安风,周祥山,周利,等.应用流式细胞术检测毕赤酵母的细胞活性[J].微生物学通报, 2006, 33(6): 22-26
[78]李靖,李成斌,顿文涛.流式细胞术( FCM)在生物学研究中的应用[J].中国农学通报, 2008(6):107-111
[79]李庆,马筱玲,翟志敏等.流式细胞术检测药物抗菌效应方法学的建立[J].安徽医学,2003(24): 8-10
[80] Novo J D, Perlmutter N G, Hunt R H, et al. Multiparameter flow cytometr ic analysis of ntibiotic effects on membrane potential, membrane permeability , and bacterial counts of Staphy lococcus aureus and Micrococcus luteus. Antimicrob Agents Chemother, 2000, 44(4): 827-834
[81]李庆,陈多炎,翟志敏.流式细胞术用于药物抗真菌效应的检测[J].临床输血与检验,2007(9):213-216
[82]王文风,徐玲,王腾飞等.从蜂王浆中提取蜂王酸的研究.食品与药, 2008(10):30-32
[83]刘媛,康秀文.王浆渣中反-10-羟基-2-癸稀酸的研究.天津化工.1995,2:4-5
[84]徐响,孙丽萍,董捷.王浆渣中有效成分萃取及分析的研究[J].中国蜂业, 2009, 3:10-12
[85]肖小年,吴凌伟,范青生等.酒花浸膏及其异构化衍生物抗食品腐败菌的初步研究.天然产物研究与开发(NATURAL PRODUCT RESEARCH AND DEVELOPMENT), 2000,1(13):47-50
[86] GB/T 4928-2008,啤酒分析方法[S].北京:中国标准出版社,2008
[87]金侃华,陈小龙.含顺丁烯二酸酐结构化合物抑制黑曲霉性能的研究[J].安徽农业科学, 2009,37(21):9857-9858
[88]徐淑云,卞如濂,陈修.药理实验学方法[M].第三版.北京:人民卫生出版社, 2001:1657-1660
[89]袁景环,贡汉生,孟祥晨.苯乳酸的抗菌作用及其抗菌机理的初步研究[J].食品工业,2009 (5):14-17
[90]邱如意,许军,徐伟等.杏香兔耳风体外抑菌作用[J].中国药业, 2009(11):13-15
[91] J.L Tholozan. Effects of culture conditions on Pectinatus cerevisiiphilus and Pectinatus frisingensis metabolism: a physiological and statistical approach [J]. Journal of Applied Bacteriology, 1996, 80:418-424
[92]陈红漫,谢建飞,杨玉红.白色链霉菌聚赖氨酸抑菌作用的研究[J].中国酿造,2009(4):77-79
[93]张玲玲,张明.肠球菌E4及其抑菌产物特性的研究[J].中国微生态学杂志, 2009(6): 485-487
[94]张其标,杨远帆,倪辉.蜂王浆抗革兰氏阳性菌的特性研究[J].中国蜂业, 2009(10):9-11
[95]赵淑艳,呼世斌,吴焕利.山茱萸提取物稳定性的研究[J].食品科学, 2008(12):98-101
[96]潘凌子,那杰,邢卓等.蜂毒肽对农作物病原菌的抑杀作用[J].科学通报, 2007(3):291-296
[97] Methner, F.J. 2003 European Brewery Convention. Monograph 32 Sanitary Engineering&HACCP. Fachverlag Hans Carl
[98]洪梅,史伯伦.蜂王浆与10-羟基癸烯酸(10-HDA)[J].蜜蜂杂志(月刊), 2001(6):22-23
[99] Eshrachi S., Seifcllahi F. Antibacterial effects of royal jelly on different strains of bacterial [J].Iranian JPubl Health, 2003, 32(1):25-30
[100]康宏.常见啤酒有害菌的主要污染途径及检测[J].啤酒科技.2001(6):8-10
[101]张波,王军厚,卞杰.啤酒厌氧菌的检测[J].中国酿造,2004(4):32-35
[102]孙培龙,徐巧林,赵敏.啤酒中污染菌检测与鉴定的研究进展[J].酿酒, 2006(6):65-69
[103] Sakamoto, Konings W. N. Beer spoilage bacteria and hop resistance[J]. International Journal of Food Microbiology, 2003, 89:105-124
[104]郭秀清.厌氧菌中NBB的使用情况[J].啤酒科技.2008(3):5-6
[105]刘冬梅,李理,杨晓泉.用牛津杯法测定益生菌的抑菌活力[J].食品研究与开发,2006(12):110-111
[106]崔云前等.蜂王浆养生啤酒生产工艺[J].酿酒,2004(6):59-61
[107]赵春杰,李正英.保健型枸杞纯生啤酒酿造方法的研究[J].中国酿造, 2009(5):178-180
[2] J.L.Shimwell. On the relation between the staining properties of bacteria and their reaction towards hop antiseptic. Part I,Ⅱ[J]. J. Inst. Brew, 1937, 43, 111-118
[3] W. J. Simpson. Ionophoric action of trans-isohumulone on Lactobacil-Ius brevis [J]. Journal of General Microbiology, 1993, 139:1041-1045
[4] Kanta Sakamoto., Wil N. Konings. Beer spoilage bacteria and hop resistance. International [J]. Journal of Food Microbiology, 2003, (89):105-124
[5]吴文芳,张成刚,吕安国等.啤酒酿造中野生酵母的研究[J].酿酒,1994,(2):57-63
[6] Shanshan Li., Guangtian Zhou. 10-Hydroxy-2-decenoic Acid as an Antimicrobial Agent in Draft Keg-conditioned Wheat Beer [J]. American Society of Brewing Chemists, 2010, 2, 114-118
[7]郭芳彬.浅析蜂王浆在蜂箱中为何不变质[J].蜜蜂杂志,2003,(8):25-26
[8] Takenaka T. Chemical compositions of royal jelly [J]. Honeybee Science, 1982, 3(1): 69-74
[9] Emanvilovi, Velchevap, etal. Studies on the chemical composition and antibacterial and antitoxic properties of royal jelly[J]. Izv Mikrobiol Inst (Slfiia), 1963, 15: 89-95
[10] Eshraghi S., Seifollajhi F. Antibacterial effects of royal jelly on different strains of bacterial [J]. Iranian JPublHealth, 2003, 32(1):25-30
[12]杨远帆,叶兴乾,倪辉等.蜂王浆抗菌作用的研究进展[J].食品科学, 2007, 11:611-614
[13]黄文诚.蜂王浆肌动蛋白是蜂王浆质量的一种可能的标记物[J].中国养蜂,2003,54(6):41-42
[14]倪辉,杨远帆,彭莺.蜂王浆中10-HDA的研究进展[J].中国蜂业, 2006, 57(3):9-10
[15]徐友第.蜂王浆抗菌活性的研究[J].中国养蜂,1989,3(6):28-29
[16]刘晓雯,刘克武,喻东.蜂王浆的有效成分与人类健康[J].蜜蜂杂志,1999,9:6-8
[17]赵家平,刘继勇,蔡文贵.蜂王浆的药理作用[J].特种经济动植物,1998,5:29-30
[18]李利君,孙亮先,彭莺.蜂王浆对三种革兰氏阴性菌的抑菌特性[J].泉州师范学院学报(自然科学).2008,3:96-99
[19]刘克武,杨守忠,葛绍荣.蜂王浆的抗菌活性研究[J].蜜蜂杂志(月刊), 2001, (2):5-6
[20]倪辉,吉挺,杨远帆.蜂王浆抗菌作用的初步研究[J].蜜蜂杂志(月刊). 2003, (2):9-10
[21]孙亮先,陈朝阳,张昌松等.蜂王浆抗菌作用的研究[J].蜜蜂杂志(月刊). 2008, (2):3-4
[22]苏晔,敬璞,丁晓雯.蜂王浆的化学成分、生理活性及应用.蜜蜂杂志(月刊). 2000(9):23-24
[23]金茜.蜂王浆的生理功能[J].化学教育,2004(9):1-2
[24]王国燕,林志彬. 10-羟基- 2-癸稀酸对小鼠T淋巴细胞及其亚型和白介素2产生的影响[J] .中国药理学与毒理学, 1996 (10): 53- 55
[25]耿纯.蜂王浆的降血脂作用[J] .中国养蜂,2000, 52( 1) : 36
[26]刘晓华,孙文基.王浆酸的研究进展概况[J].中国药品标准.2004(5):9-10
[27]汪多仁.王浆酸的开发与应用进展[J].养蜂科技, 2004(4): 6-10
[28]刘洪云.蜂王酸的制备.四川化工.1996(4):39-40
[29] Kulkarni S.M. A short synthesis of royal jelly acid [10-hydroxy-(E)-2-decenoic acid][J]. Indian J. Chem. Sect.B23B, 1984 (5): 460
[30] FujisavaT. A novel synthesis of royal jelly acids and queen Substance by the five carbon homologation using ropiolactone [J]. Chem, Lett, 1982(2): 219-220
[31] Corey E J, William Suggs J, Pyridinium chlorochromate. An efficient reagent for oxidation of primary and secondary alcohols to carbonyl compounds [J].Tertahedron Letters, 1975(1): 2647-2650.
[32]刘放,黄卫平,戴惠芳.蜂王浆及制剂中10-羟基-2-癸烯酸测定方法的进展[J].中国养蜂, 1999,(5): 19-21
[33]张小军,郝彦玲,寇晓红.啤酒花及其抗菌机理研究进展[J].酿酒科技, 2007(4):109-111
[34]郝秋娟,董翠英,李崎.啤酒腐败细菌与酒花抗性[J].酿酒科技,2005,9:65-68
[35] K.suzuki., M.koyanagi. H.Yamashita[J]. Journal of Applied Microbiology, 2004, 96: 946-953
[36] Manabu samin, Yamashita H., Kadokura H [J]. J.Am.Soc.Brew, 1997, 55(4): 137-140
[37]朱林江,郑飞云,李崎,顾国贤.酒花苦味物质的抗菌活性[J].啤酒科技, 2007, 2:5-7
[38]何士雯,马永.酒花苦味酸对乳酸菌的抑制作用.啤酒科技,2004(9):7-9
[39] W.J.Simpson, Studies on the sensitivity of lacticacid bacteria to hop bitter acids [J]. J. Inst.Brew, 1993, 99: 405-411
[40]周广田,董小雷,崔云前.啤酒酵母与工厂卫生[M].北京:化学工业出版社,2008:45-47
[41]王树庆.啤酒酿造中的微生物污染[J].酿酒,2008(1):50-53
[42] Back W. Brauwelt online 24/25, 2003
[43] Methner F.J. Monograph 32 Sanitary Engineering&HACCP[R]. Fachverlag Hans Carl: European Brewery Convention, 2003
[44]林先军,李崎,顾国贤.乳酸菌对制麦和啤酒酿造的影响[J].啤酒科技, 2005(7):21-23
[45]赵敏,杨开,孙培龙.啤酒中乳酸杆菌的分离纯化和鉴定[J].酿酒.2005(1):58-61
[46]周广田.现代啤酒工艺技术[M].北京:化学工业出版社,2007:11-12
[47]陈敬春.浅论啤酒有害细菌及其检出[J].啤酒科技,2005(2):5-4
[48]周广田,聂聪,崔云前等.啤酒酿造技术[M].济南:山东大学出版社,2004:23-25
[49]李晓霞,刘新社.纯生啤酒生产中微生物污染的危害及控制技术[J].商丘职业技术学院学报, 2009(2):105-107
[50]王海明.野生酵母与啤酒酿造.酿酒科技,2003(2):59-61
[51]闫洪伟.啤酒生产中主要有害菌的来源及控制.科技信息,2009(13):744-745
[52]管建良.野生酵母对啤酒生产的影响及控制.啤酒科技.2009(2):47-48
[53]詹东.啤酒的稳定性[J].啤酒科技, 2006(10):20-23
[54]王喜萍,李长生.影响啤酒稳定性的因素及预防措施[J].酿酒科技, 2000(4):59-60
[55]顾建国.影响啤酒稳定性的因素及预防[J].张家口农专学报,1996(12):37-39
[56]张全斌,李艳琴.啤酒腐败菌分子检测技术的研究进展[J].食品与药品, 2007(9):55-57
[57]张少兰.啤酒腐败菌种果胶杆菌和巨型球菌的检测[J].广东轻工职业技术学院学报(综合版),2002(1):34-35
[58] Hakalehto E, Finne J. Identification by immunoblot analysis of major antigenic determinants of the anaerobic beer spoilage bacterium genus Pectinatus [J]. FEMS Microbiol Lett, 1990(67):307-312.
[59]陈江,梁风华.API系统对常见啤酒有害菌进行分类鉴定的研究[J].啤酒科技,2005(11):24-25
[60] Sami M, Yamashita H, Kadokura H, et al. A new and rapid method fordetermination of beer-spoilage ability of lactobacilli[J]. J Am Soc Brew Chem, 1997, 55(4): 137-140
[61] HAYASHI N, ITO M, HORIIKE S, etal. Molecular cloning of a putative divalent-cation transporter gene as a new genetic marker for the identification of Lactobacillusbrevis strains capable of growing in beer [J]. Appl Microbiol Biotechnol, 2001, 55: 596-603
[62] ING D, BRANDL A. Demonstration of PCR based methods in brewery quality control[R]. Hartwall Brewery, Lahti Finland: EBC Brewing Science Group-Microbial Contaminants Subgroup, 2004.
[63] Barszczewski W, Robak M. Differentiation of contaminating yeasts in brewery by PCR-based techniques [J]. Food Microbiol, 2004, 21(2): 227-231.
[64] TAKAOMI YASUHARA, Kyoko Takahashi, Yasuo Motoyama, Toshifumi Yuuki, Kazunori Shibata et al. Rapid detection, identification and enumeration of beer-spoilage bacteria by FISH method. Brewing Research, Development Laboratory, Asahi Breweries Lid.,1-1-21 Midori, Moriya-machi, Ibaraki 302-0106, Japan.
[65] H.Stender, A. Sage, K. Oliveira, A. J. Broomer, B. Young and J. Coull. Combination of ATP- bioluminescence and PNA probes allows rapid total counts and identification of specific microorganisms in mixed opulations [J]. Microbiol Methods, 2001, 46:69-70
[66]孙培龙,徐巧林.啤酒中污染菌检测与鉴定的研究进展.酿酒2006(6):65-69
[67] GRACIAS K S, MCKILLIP J L. A review of conventional detection and enumeration methods for pathogenic bacteria in food[J]. Can J Microbiol, 2004, 50: 883-890.
[68] TAKAHASHI T, NAKAKITA Y, et al. Application of a bioluminescence method for the beer industry: sensitivity of MicroStarTM-RMDS for detecting beer-spoilage bcteria, biosci[J]. Biotechnol Biochem, 2000, 64(5): 1032-1037.
[69] TAKAHASHI T, NAKAITA Y, etal. A new rapid technique for detection of microorganisms using bioluminescence and fluorescence microscope method[J]. Journal of Bioscience and Bioengineering, 2000, 89(5): 509-513
[70]顾国贤等.啤酒无菌酿造[J].酿酒, 2000(3):49-53
[71]王树庆.啤酒生产中污染微生物的快速检测与鉴定技术.酿酒科技, 2009(7):109-113
[72] Foster, A.[J].Journal of the American Society of Brewing Chemists,1996,54-76.
[73]吴红.啤酒的微生物检验技术[J].酿酒,2000(5):34-39
[74]赵现方,陈林海,李宗义.流式细胞术及其在微生物学中的应用[J].河南农业科学, 2005(6):5-8
[75] Malacrino P, Zapparoli G, Torriani S. Dellaglio rapid detection of viable yeasts and bacteria in wine by flow cytometry [J]. J Microbiol Methods, 2001, 45(2):127-l34.
[76] Day A, Meyling J C. Practical experience with rapid methods of detecting yeasts in breweries [J]. J Inst Brew, 1990, 89: 204-206.
[77]肖安风,周祥山,周利,等.应用流式细胞术检测毕赤酵母的细胞活性[J].微生物学通报, 2006, 33(6): 22-26
[78]李靖,李成斌,顿文涛.流式细胞术( FCM)在生物学研究中的应用[J].中国农学通报, 2008(6):107-111
[79]李庆,马筱玲,翟志敏等.流式细胞术检测药物抗菌效应方法学的建立[J].安徽医学,2003(24): 8-10
[80] Novo J D, Perlmutter N G, Hunt R H, et al. Multiparameter flow cytometr ic analysis of ntibiotic effects on membrane potential, membrane permeability , and bacterial counts of Staphy lococcus aureus and Micrococcus luteus. Antimicrob Agents Chemother, 2000, 44(4): 827-834
[81]李庆,陈多炎,翟志敏.流式细胞术用于药物抗真菌效应的检测[J].临床输血与检验,2007(9):213-216
[82]王文风,徐玲,王腾飞等.从蜂王浆中提取蜂王酸的研究.食品与药, 2008(10):30-32
[83]刘媛,康秀文.王浆渣中反-10-羟基-2-癸稀酸的研究.天津化工.1995,2:4-5
[84]徐响,孙丽萍,董捷.王浆渣中有效成分萃取及分析的研究[J].中国蜂业, 2009, 3:10-12
[85]肖小年,吴凌伟,范青生等.酒花浸膏及其异构化衍生物抗食品腐败菌的初步研究.天然产物研究与开发(NATURAL PRODUCT RESEARCH AND DEVELOPMENT), 2000,1(13):47-50
[86] GB/T 4928-2008,啤酒分析方法[S].北京:中国标准出版社,2008
[87]金侃华,陈小龙.含顺丁烯二酸酐结构化合物抑制黑曲霉性能的研究[J].安徽农业科学, 2009,37(21):9857-9858
[88]徐淑云,卞如濂,陈修.药理实验学方法[M].第三版.北京:人民卫生出版社, 2001:1657-1660
[89]袁景环,贡汉生,孟祥晨.苯乳酸的抗菌作用及其抗菌机理的初步研究[J].食品工业,2009 (5):14-17
[90]邱如意,许军,徐伟等.杏香兔耳风体外抑菌作用[J].中国药业, 2009(11):13-15
[91] J.L Tholozan. Effects of culture conditions on Pectinatus cerevisiiphilus and Pectinatus frisingensis metabolism: a physiological and statistical approach [J]. Journal of Applied Bacteriology, 1996, 80:418-424
[92]陈红漫,谢建飞,杨玉红.白色链霉菌聚赖氨酸抑菌作用的研究[J].中国酿造,2009(4):77-79
[93]张玲玲,张明.肠球菌E4及其抑菌产物特性的研究[J].中国微生态学杂志, 2009(6): 485-487
[94]张其标,杨远帆,倪辉.蜂王浆抗革兰氏阳性菌的特性研究[J].中国蜂业, 2009(10):9-11
[95]赵淑艳,呼世斌,吴焕利.山茱萸提取物稳定性的研究[J].食品科学, 2008(12):98-101
[96]潘凌子,那杰,邢卓等.蜂毒肽对农作物病原菌的抑杀作用[J].科学通报, 2007(3):291-296
[97] Methner, F.J. 2003 European Brewery Convention. Monograph 32 Sanitary Engineering&HACCP. Fachverlag Hans Carl
[98]洪梅,史伯伦.蜂王浆与10-羟基癸烯酸(10-HDA)[J].蜜蜂杂志(月刊), 2001(6):22-23
[99] Eshrachi S., Seifcllahi F. Antibacterial effects of royal jelly on different strains of bacterial [J].Iranian JPubl Health, 2003, 32(1):25-30
[100]康宏.常见啤酒有害菌的主要污染途径及检测[J].啤酒科技.2001(6):8-10
[101]张波,王军厚,卞杰.啤酒厌氧菌的检测[J].中国酿造,2004(4):32-35
[102]孙培龙,徐巧林,赵敏.啤酒中污染菌检测与鉴定的研究进展[J].酿酒, 2006(6):65-69
[103] Sakamoto, Konings W. N. Beer spoilage bacteria and hop resistance[J]. International Journal of Food Microbiology, 2003, 89:105-124
[104]郭秀清.厌氧菌中NBB的使用情况[J].啤酒科技.2008(3):5-6
[105]刘冬梅,李理,杨晓泉.用牛津杯法测定益生菌的抑菌活力[J].食品研究与开发,2006(12):110-111
[106]崔云前等.蜂王浆养生啤酒生产工艺[J].酿酒,2004(6):59-61
[107]赵春杰,李正英.保健型枸杞纯生啤酒酿造方法的研究[J].中国酿造, 2009(5):178-180