酒酒球菌液氮超低温保存研究

酒酒球菌液氮超低温保存研究

THE STUDY ON LIQUID NITROGEN STORAGE OF OENOCOCCUS OENI

作者：杜立业　　导师：王　华　李　华

西北农林科技大学　　发酵工程2011届硕士

摘　要

目前，已成功采用液氮超低温保存法对多种微生物进行了长期保存，如藻类、原生动物、酵母菌、乳酸菌、铁细菌、硫细菌等。但关于酒酒球菌液氮超低温保存的研究还很少。本文通过研究酒酒球菌生长时间、冷冻方法、解冻温度、菌密度以及保护剂等对酒酒球菌细胞冷冻存活率的影响，建立最优的酒酒球菌液氮超低温保存方法。此外，还对液氮超低温保存前后酒酒球菌生长性能、酿酒特性和酶活性的变化进行了比较。主要结果如下：

1.　酒酒球菌液氮超低温保存条件

首先在稳定期前期离心收集菌体；再加入保护剂（20g/L 酵母浸提物，40V/V 甘油，20g/L 蔗糖，30g/L谷氨酸钠）稀释菌体，使菌密度为10⁹CFU/mL；然后直接投入液氮冷冻；最后在37℃温水浴中迅速解冻。

2.　保存时间对酒酒球菌细胞冷冻存活率的影响

保存8个月后，酒酒球菌细胞冷冻存活率仍很高，其中22株酒酒球菌的存活率高于98%，sx-1b的细胞存活率也达到了97.1%，初步证明此法可用于酒酒球菌的长期保存。然而使用甘油保存法保存酒酒球菌8个月后的最低存活率为11.9%，最高存活率也只有26.9%。

3.　液氮超低温保存对酒酒球菌生长的影响

液氮保存前后酒酒球菌的生长差异不明显，液氮保存后酒酒球菌的生长速率比保存前的生长速率略有提高。pH随着菌的生长不断降低，最低pH达到3.3左右。液氮保存后，酒酒球菌对培养基pH变化的影响也不显著，将液氮保存后的酒酒球菌接种于ATB培养基中，其pH降低比保存前稍快。液氮超低温保存前，SD-2a 在ATB培养基中培养12 h、36 h、60 h、84 h的OD值和pH值分别为0.100、4.8，1.459、3.85，2.180、3.49，2.240、3.31。液氮超低温保存后，SD-2a在ATB培养基中培养12 h、36 h、60 h、84 h后的OD值和pH值分别为0.150、4.79，1.573、3.77，2.180、3.41，2.240、3.30。

4.　液氮超低温保存对酒酒球菌苹果酸分解能力的影响

液氮超低温保存后，23株酒酒球菌均保持了原有的酿酒活性。酒酒球菌SD-2a在液氮超低温保存前后的苹果酸分解量分别为1.74和1.75；31-DH在液氮超低温保存前后的苹果酸分解量分别为1.36和1.66；其他菌株的苹果酸分解量差异也不大。

5.　液氮超低温保存对酒酒球菌酶活性的影响

液氮超低温保存前后酒酒球菌MLE和ATPase活性变化不显著，菌体仍保持着原来的活性。液氮超低温保存前，SD-2a在对数期前期、对数期中期、稳定期前期和稳定期中期的MLE活性分别为60.00、170.00、249.00、158.00 μmol.malate.h^－1mg^－1protein。保存后，SD-2a在对数期前期、对数期中期、稳定期前期和稳定期中期的MLE活性分别为55.00、171.00、251.00、156.00 μmol.malate.h^－1mg^－1protein。液氮超低温保存前，SD-2a在对数期前期、对数期中期、稳定期前期和稳定期中期的ATPase活性值分别为4.25、12.24、18.21、10.49 μmol.Pi.h^－lmg^－lprotein。保存后，酒酒球菌SD-2a在对数期前期、对数期中期、稳定期前期和稳定期中期的ATPase活性值分别为4.13、12.63、17.94、10.95 μmol.Pi.h^－lmg^－lprotein。

关键词　酒酒球菌　液氮超低温保存　冷冻存活率　模拟酒发酵　酶活

Abstract

At present,liquid nitrogen cryopreservation have been used to conserve multiple microbes for long time successfully,such as alga,protozoa,yeast,lactic acid bacteria,iron bacteria,sulphur bacteria,et al. But there are few researches on the Oenococcus oeni storaging in liquid nitrogen. In this paper,we describe a cryopreservation protocol for the cultures of Oenococcus oeni. We investigated the influence of the age,rate of freezing,the temperature of thawing,their concentration in the protective medium,and the protective medium on the number of viable cells after cryopreservation. Besides,the growth characteristics,vinification characteristics and enzyme activity were also studied. The main results are listed as follows:

1.　Cryopreservation conditions for Oenococcus oeni

We developed an efficient cryopreservation technology for Oenococcus oeni. We got the highest number of viable cells after cryopreservation by collecting cells in the early stationary growth phase,keeping their concentration in the protective medium at 10⁹ CFU/mL,transferring cells into liquid nitrogen directly and thawing at 37℃ in the water bath.

2.　The influence of cryopreservation period to the ratio of viable cells

The ratio of viable cells was above 99% for 23 of the Oenococcus oeni after storaging in liquid nitrogen for 8 months,and the ratio of viable cells for sx-1b also reached 97.1%. But the ratio of viable cells was 11.9% for Oenococcus oeni after storagingin glycerin,the highest ratio of viable cells was only 26.9%.

3.　The influence of cryopreservation to the growth rate of Oenococcus oeni

After storaging in liquid nitrogen,the growth rate of Oenococcus oeni had no obvious change,the cell grow rate increased a little after cryopreservating in liquid nitrogen than before cryopreservation. The pH decreased as the cell grew,and the lowest pH was about 3.3. Oenococcus oeni SD-2a after storaging decreased the medium pH faster than before storaging. When Oenococcus oeni SD-2a before storaging cultured in ATB for 12,36,60,84 hours,the OD and medium pH were 0.100，4.80；1.459，3.85；2.180，3.49；2.240，3.31 respectively. And Oenococcus oeni SD-2a after storaging cultured in ATB for 12,36,60,84 hours,the OD and medium pH were 0.150，4.79；1.573，3.77；2.180，3.41；2.240，3.30 respectively.

4.　The influence of cryopreservation to the ability of consuming malic acid of Oenococcus oeni

After cryopreservation，all of 23 Oenococcus oeni retain the initial vinification characteristics. Oenococcus oeni SD-2a before and after cryopreservation consumed 1.74g/L and1.75g/L malic acid respectively;Oenococcus oeni 31-DH consumed 1.36g/L and 1.66g/L malic acid respectively;and malic acid consumption by the others before and after cryopreservation was also similar. After cryopreservation，volatile acid content of the wine were under 1.2 g/L acetic acid,so the wine were healthy.

5.　The influence of cryopreservation to the enzyme activity of Oenococcus oeni

After storaging in liquid nitrogen,Oenococcus oeni SD-2a intracellular MLE activity and H⁺-ATPase activity were as high as before storaging. Before cryopreservation,the MLE activities of SD-2a at early logarithmic phase,middle logarithmic phase,early stationary phase and middle stationary phase were 60.00,170.00,249.00,158.00μmol.malate.h^－1mg^－1protein. And after cryopreservation,the MLE activities of SD-2a at early logarithmic phase,middle logarithmic phase,early stationary phase and middle stationary phase were 55.00,171.00,251.00,156.00μmol.malate.h^－1mg^－1protein respectively. Before cryopreservation,the H⁺-ATPase activity of SD-2a at early logarithmic phase,middle logarithmic phase,early stationary phase and middle stationary phase were 4.25,12.24,18.21,10.49μmol.Pi.h^－lmg^－lprotein respectively;after cryopreservation,the H⁺-ATPase activity of SD-2a at early logarithmic phase,middle logarithmic phase,early stationary phase and middle stationary phase were 4.13,12.63,17.94,10.95μmol.Pi.h^－lmg^－lprotein respectively.

Key words　Oenococcus oeni　Liquid nitrogen cryopreservation　Viable cells after cryopreservation Model wine fermentation　Enzyme activity