膜醭毕赤酵母真空干燥制剂对热环境耐受力的提高
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摘要
采用热处理、过氧化氢处理以及在酵母培养基中添加氯化钠、氯化钙等预处理方式提高膜醭毕赤酵母(Pichia membranifaciens)对逆境的耐受力,以此提高真空干燥后膜醭毕赤酵母的活性。对膜醭毕赤酵母进行40℃、40 min热处理后,膜醭毕赤酵母细胞内海藻糖含量升高,真空干燥后,经热处理后的膜醭毕赤酵母较对照组活性更高。5 mmol/L过氧化氢对膜醭毕赤酵母处理40 min后,膜醭毕赤酵母细胞内海藻糖含量升高,真空干燥后,经过氧化氢处理后的膜醭毕赤酵母较对照组活性更高。在培养基中添加0.1～0.9 mol/L氯化钠,1～10 mmol/L氯化钙均能诱导膜醭毕赤酵母细胞内海藻糖含量的显著提高,其中添加0.7 mol/L氯化钠后诱导的膜醭毕赤酵母细胞内海藻糖产生量最高,添加0.9 mol/L次之,添加5 mmol/L氯化钙后诱导的膜醭毕赤酵母细胞内海藻糖产生量最高;不添加任何保护剂时,添加0.7 mol/L和0.9 mol/L氯化钠培养后的膜醭毕赤酵母经真空干燥后活性较未添加氯化钠的对照组高;添加5 mmol/L氯化钙培养后的膜醭毕赤酵母经真空干燥后较未添加氯化钙的对照组活性高。在添加保护剂时,添加不同浓度氯化钠处理和添加不同浓度氯化钙处理后的膜醭毕赤酵母干燥后活性与对照组基本没有显著性差异,说明添加氯化钠或氯化钙培养后的膜醭毕赤酵母在真空干燥过程中,保护剂对其活性的影响更大。
We subjected Pichia membranifaciens to thermal and hydrogen peroxide pretreatments and added sodium chloride and calcium chloride to its culture medium in order to enhance its stress tolerance and hence improve the viability of vacuum-dried P. membranifaciens. The trehalose content of heat-adapted P. membranifaciens(40 ℃higher than that of the control; the survival of heat-adapted P. membranifaciens after vacuum-drying was higher than that of the control. The trehalose content of oxidative stress-adapted P. membranifaciens(40 min treatment with 5 mmol/L hydrogen peroxide) was higher than that of the control. Similarly, the survival of oxidative stress-adapted P. membranifaciens after vacuum-drying was higher than that of the control. The addition of 0.1–0.9 mol/L sodium chloride or 1–10 mmol/L calcium chloride to the culture medium induced a marked increase of the trehalose content, and P. membranifaciens cells showed the highest amount of trehalose upon adding 0.7 mol/L sodium chloride, followed by 0.9 mol/L sodium chloride; 5 mmol/L calcium chloride treated P. membranifaciens induced the highest amount of trehalose in yeast cells. The viability of vacuumdried P. membranifaciens without any protectant and with 0.7 or 0.9 mol/L sodium chloride added was higher than that of all other treatments and the control. The viability of vacuum-dried P. membranifaciens with a protectant and sodium chloride or calcium chloride at each concentration was not signi?cantly different from that of the control, indicating that the effect of adding protectant on the viability of vacuum-dried P. membranifaciens is greater than that of pretreating the yeast with sodium chloride or calcium chloride.
We subjected Pichia membranifaciens to thermal and hydrogen peroxide pretreatments and added sodium chloride and calcium chloride to its culture medium in order to enhance its stress tolerance and hence improve the viability of vacuum-dried P. membranifaciens. The trehalose content of heat-adapted P. membranifaciens(40 ℃higher than that of the control; the survival of heat-adapted P. membranifaciens after vacuum-drying was higher than that of the control. The trehalose content of oxidative stress-adapted P. membranifaciens(40 min treatment with 5 mmol/L hydrogen peroxide) was higher than that of the control. Similarly, the survival of oxidative stress-adapted P. membranifaciens after vacuum-drying was higher than that of the control. The addition of 0.1–0.9 mol/L sodium chloride or 1–10 mmol/L calcium chloride to the culture medium induced a marked increase of the trehalose content, and P. membranifaciens cells showed the highest amount of trehalose upon adding 0.7 mol/L sodium chloride, followed by 0.9 mol/L sodium chloride; 5 mmol/L calcium chloride treated P. membranifaciens induced the highest amount of trehalose in yeast cells. The viability of vacuumdried P. membranifaciens without any protectant and with 0.7 or 0.9 mol/L sodium chloride added was higher than that of all other treatments and the control. The viability of vacuum-dried P. membranifaciens with a protectant and sodium chloride or calcium chloride at each concentration was not signi?cantly different from that of the control, indicating that the effect of adding protectant on the viability of vacuum-dried P. membranifaciens is greater than that of pretreating the yeast with sodium chloride or calcium chloride.
引文
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[14]BONATERRA A,CABREFIGA J,CAMPS J,et al.Increasing survival and efficacy of a bacterial biocontrol agent of fire blight of rosaceous plants by means of osmoadaptation[J].FEMSMicrobiology Ecology,2007,61(1):185-195.DOI:10.1111/j.1574-6941.2007.00313.x.
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[17]NIU X,DENG L,ZHOU Y,et al.Optimization of a protective medium for freeze-dried Pichia membranifaciens and application of this biocontrol agent on citrus fruit[J].Journal of Applied Microbiology,2016,121(1):234-243.DOI:10.1111/jam.13129.
[18]BEKER M J,RAPOPORT A I.Conservation of yeasts by dehydration[J].Advancesin Biochemical Engineering&Biotechnology,1987,35:127-171.DOI:10.1007/BFb0004428.
[19]TAO N,JIA L,ZHOU H.Anti-fungal activity of Citrus reticulata Blanco essential oil against Penicillium italicum and Penicillium digitatum[J].Food Chemistry,2014,153:265-271.DOI:10.1016/j.foodchem.2013.12.070.
[20]LI H,WANG H L,DU J,et al.Trehalose protects wine yeast against oxidation under thermal stress[J].World Journal of Microbiology and Biotechnology,2010,26(6):969-976.DOI:10.1007/s11274-009-0258-1.
[21]骆莹.酿酒酵母胞内海藻糖的提取纯化及近红外检测[D].杨凌:西北农林科技大学,2011.
[22]TEIXIDóN,CA?AMáS T P,USALL J,et al.Accumulation of the compatible solutes,glycine-betaine and ectoine,in osmotic stress adaptation and heat shock cross-protection in the biocontrol agent Pantoea agglomerans CPA-2[J].Letters in Applied Microbiology,2005,41(3):248-252.DOI:10.1111/j.1472-765X.2005.01757.x.
[23]DESMOND C,STANTON C,FITZGERALD G F,et al.Environmental adaptation of probiotic Lactobacilli towards improvement of performance during spray drying[J].International Dairy Journal,2001,11(10):801-808.DOI:10.1016/S0958-6946(01)00121-2.
[24]唐飞.海洋生防酵母Rhodosporidium paludigenum干燥工艺研究[D].杭州:浙江大学,2012:14-23.
[25]WANG Y,WANG P,XIA J,et al.Effect of water activity on stress tolerance and biocontrol activity in antagonistic yeast Rhodosporidium paludigenum[J].International Journal of Food Microbiology,2010,143(3):103-108.DOI:10.1016/j.ijfoodmicro.2010.07.035.
[26]LI C,ZHANG H,YANG Q,et al.Ascorbic acid enhances oxidative stress tolerance and biological control efficacy of Pichia caribbica against postharvest blue mold decay of apples[J].Journal of Agricultural and Food Chemistry,2014,62(30):7612-7621.DOI:10.1021/jf501984n.
[27]TEIXIDóN,VI?AS I,USALL J,et al.Improving ecological fitness and environmental stress tolerance of the biocontrol yeast Candida sake by manipulation of intracellular sugar alcohol and sugar content[J].Mycological Research,1998,102(11):1409-1417.DOI:10.1017/S0953756298006716.
[28]ABADIAS M,TEIXIDóN,USALL J,et al.Survival of the postharvest biocontrol yeast Candida sake CPA-1 after dehydration by spray-drying[J].Biocontrol Science and Technology,2005,15(8):835-846.DOI:10.1080/09583150500187041.
[29]LI B Q,TIAN S P.Effect of intracellular trehalose in Cryptococcus laurentii and exogenous lyoprotectants on its viability and biocontrol efficacy on Penicillium expansum in apple fruit[J].Letters in Applied Microbiology,2007,44(4):437-442.DOI:10.1111/j.1472-765X.2006.02080.x.
[2]ZHOU Y,DENG L,ZENG K.Enhancement of biocontrol efficacy of Pichia membranaefaciens by hot water treatment in postharvest diseases of citrus fruit[J].Crop Protection,2014,63:89-96.DOI:10.1016/j.cropro.2014.05.015.
[3]ABADIAS M,BENABARRE A,TEIXIDO N,et al.Effect of freeze drying and protectants on viability of the biocontrol yeast Candida sake[J].International Journal of Food Microbiology,2001,65(3):173-182.DOI:10.1016/S0168-1605(00)00513-4.
[4]FU N,CHEN X D.Towards a maximal cell survival in convective thermal drying processes[J].Food Research International,2011,44(5):1127-1149.DOI:10.1016/j.foodres.2011.03.053.
[5]GECIOVA J,BURY D,JELEN P.Methods for disruption of microbial cells for potential use in the dairy industry:a review[J].International Dairy Journal,2002,12(6):541-553.DOI:10.1016/S0958-6946(02)0038-9.
[6]DONSìF,FERRARI G,LENZA E,et al.Main factors regulating microbial inactivation by high-pressure homogenization:operating parameters and scale of operation[J].Chemical Engineering Science,2009,64(3):520-532.DOI:10.1016/j.ces.2008.10.002.
[7]COX C S.Roles of water molecules in bacteria and viruses[J].Origins of Life and Evolution of the Biosphere,1993,23(1):29-36.DOI:10.1007/BF01581988.
[8]SANTIVARANGKNA C,KULOZIK U,FOERST P.Inactivation mechanisms of lactic acid starter cultures preserved by drying processes[J].Journal of Applied Microbiology,2008,105(1):1-13.DOI:10.1111/j.1365-2672.2008.03744.x.
[9]ABEE T,WOUTERS J A.Microbial stress response in minimal processing[J].International Journal of Food Microbiology,1999,50(1/2):65-91.DOI:10.1016/S0168-1605(99)00078-1.
[10]LIU J,WISNIEWSKI M,DROBY S,et al.Effect of heat shock treatment on stress tolerance and biocontrol efficacy of Metschnikowia fructicola[J].FEMS Microbiology Ecology,2011,76(1):145-155.DOI:10.1111/j.1574-6941.2010.01037.x.
[11]CHENG Z,CHI M,LI G,et al.Heat shock improves stress tolerance and biocontrol performance of Rhodotorula mucilaginosa[J].Biological Control,2016,95:49-56.DOI:10.1016/j.biocontrol.2016.01.001.
[12]LIU J,WISNIEWSKI M,DROBY S,et al.Increase in antioxidant gene transcripts,stress tolerance and biocontrol efficacy of Candida oleophila following sublethal oxidative stress exposure[J].FEMSMicrobiology Ecology,2012,80(3):578-590.DOI:10.1111/j.1574-6941.2012.01324.x.
[13]TEIXIDóN,VINAS I,USALL J,et al.Ecophysiological responses of the biocontrol yeast Candida sake to water,temperature and pHstress[J].Journal of Applied Microbiology,1998,84(2):192-200.DOI:10.1046/j.1365-2672.1998.00320.x.
[14]BONATERRA A,CABREFIGA J,CAMPS J,et al.Increasing survival and efficacy of a bacterial biocontrol agent of fire blight of rosaceous plants by means of osmoadaptation[J].FEMSMicrobiology Ecology,2007,61(1):185-195.DOI:10.1111/j.1574-6941.2007.00313.x.
[15]AN B,LI B,QIN G,et al.Exogenous calcium improves viability of biocontrol yeasts under heat stress by reducing ROS accumulation and oxidative damage of cellular protein[J].Current Microbiology,2012,65(2):122-127.DOI:10.1007/s00284-012-0133-4.
[16]CERRUTTI P,DE HUERGO M S,GALVAGNO M,et al.Commercial baker’s yeast stability as affected by intracellular content of trehalose,dehydration procedure and the physical properties of external matrices[J].Applied Microbiology and Biotechnology,2000,54(4):575-580.DOI:10.1007/s002530000428.
[17]NIU X,DENG L,ZHOU Y,et al.Optimization of a protective medium for freeze-dried Pichia membranifaciens and application of this biocontrol agent on citrus fruit[J].Journal of Applied Microbiology,2016,121(1):234-243.DOI:10.1111/jam.13129.
[18]BEKER M J,RAPOPORT A I.Conservation of yeasts by dehydration[J].Advancesin Biochemical Engineering&Biotechnology,1987,35:127-171.DOI:10.1007/BFb0004428.
[19]TAO N,JIA L,ZHOU H.Anti-fungal activity of Citrus reticulata Blanco essential oil against Penicillium italicum and Penicillium digitatum[J].Food Chemistry,2014,153:265-271.DOI:10.1016/j.foodchem.2013.12.070.
[20]LI H,WANG H L,DU J,et al.Trehalose protects wine yeast against oxidation under thermal stress[J].World Journal of Microbiology and Biotechnology,2010,26(6):969-976.DOI:10.1007/s11274-009-0258-1.
[21]骆莹.酿酒酵母胞内海藻糖的提取纯化及近红外检测[D].杨凌:西北农林科技大学,2011.
[22]TEIXIDóN,CA?AMáS T P,USALL J,et al.Accumulation of the compatible solutes,glycine-betaine and ectoine,in osmotic stress adaptation and heat shock cross-protection in the biocontrol agent Pantoea agglomerans CPA-2[J].Letters in Applied Microbiology,2005,41(3):248-252.DOI:10.1111/j.1472-765X.2005.01757.x.
[23]DESMOND C,STANTON C,FITZGERALD G F,et al.Environmental adaptation of probiotic Lactobacilli towards improvement of performance during spray drying[J].International Dairy Journal,2001,11(10):801-808.DOI:10.1016/S0958-6946(01)00121-2.
[24]唐飞.海洋生防酵母Rhodosporidium paludigenum干燥工艺研究[D].杭州:浙江大学,2012:14-23.
[25]WANG Y,WANG P,XIA J,et al.Effect of water activity on stress tolerance and biocontrol activity in antagonistic yeast Rhodosporidium paludigenum[J].International Journal of Food Microbiology,2010,143(3):103-108.DOI:10.1016/j.ijfoodmicro.2010.07.035.
[26]LI C,ZHANG H,YANG Q,et al.Ascorbic acid enhances oxidative stress tolerance and biological control efficacy of Pichia caribbica against postharvest blue mold decay of apples[J].Journal of Agricultural and Food Chemistry,2014,62(30):7612-7621.DOI:10.1021/jf501984n.
[27]TEIXIDóN,VI?AS I,USALL J,et al.Improving ecological fitness and environmental stress tolerance of the biocontrol yeast Candida sake by manipulation of intracellular sugar alcohol and sugar content[J].Mycological Research,1998,102(11):1409-1417.DOI:10.1017/S0953756298006716.
[28]ABADIAS M,TEIXIDóN,USALL J,et al.Survival of the postharvest biocontrol yeast Candida sake CPA-1 after dehydration by spray-drying[J].Biocontrol Science and Technology,2005,15(8):835-846.DOI:10.1080/09583150500187041.
[29]LI B Q,TIAN S P.Effect of intracellular trehalose in Cryptococcus laurentii and exogenous lyoprotectants on its viability and biocontrol efficacy on Penicillium expansum in apple fruit[J].Letters in Applied Microbiology,2007,44(4):437-442.DOI:10.1111/j.1472-765X.2006.02080.x.