酒酒球菌液氮超低溫保存研究

THE STUDY ON LIQUID NITROGEN STORAGE OF OENOCOCCUS OENI

作者：杜立業(yè)　　導(dǎo)師：王　華　李　華

西北農(nóng)林科技大學(xué)　　發(fā)酵工程2011屆碩士

摘　要

目前，已成功采用液氮超低溫保存法對(duì)多種微生物進(jìn)行了長(zhǎng)期保存，如藻類、原生動(dòng)物、酵母菌、乳酸菌、鐵細(xì)菌、硫細(xì)菌等。但關(guān)于酒酒球菌液氮超低溫保存的研究還很少。本文通過(guò)研究酒酒球菌生長(zhǎng)時(shí)間、冷凍方法、解凍溫度、菌密度以及保護(hù)劑等對(duì)酒酒球菌細(xì)胞冷凍存活率的影響，建立最優(yōu)的酒酒球菌液氮超低溫保存方法。此外，還對(duì)液氮超低溫保存前后酒酒球菌生長(zhǎng)性能、釀酒特性和酶活性的變化進(jìn)行了比較。主要結(jié)果如下：

1.　酒酒球菌液氮超低溫保存條件

首先在穩(wěn)定期前期離心收集菌體；再加入保護(hù)劑（20g/L 酵母浸提物，40V/V 甘油，20g/L 蔗糖，30g/L谷氨酸鈉）稀釋菌體，使菌密度為10⁹CFU/mL；然后直接投入液氮冷凍；最后在37℃溫水浴中迅速解凍。

2.　保存時(shí)間對(duì)酒酒球菌細(xì)胞冷凍存活率的影響

保存8個(gè)月后，酒酒球菌細(xì)胞冷凍存活率仍很高，其中22株酒酒球菌的存活率高于98%，sx-1b的細(xì)胞存活率也達(dá)到了97.1%，初步證明此法可用于酒酒球菌的長(zhǎng)期保存。然而使用甘油保存法保存酒酒球菌8個(gè)月后的最低存活率為11.9%，最高存活率也只有26.9%。

3.　液氮超低溫保存對(duì)酒酒球菌生長(zhǎng)的影響

液氮保存前后酒酒球菌的生長(zhǎng)差異不明顯，液氮保存后酒酒球菌的生長(zhǎng)速率比保存前的生長(zhǎng)速率略有提高。pH隨著菌的生長(zhǎng)不斷降低，最低pH達(dá)到3.3左右。液氮保存后，酒酒球菌對(duì)培養(yǎng)基pH變化的影響也不顯著，將液氮保存后的酒酒球菌接種于ATB培養(yǎng)基中，其pH降低比保存前稍快。液氮超低溫保存前，SD-2a 在ATB培養(yǎng)基中培養(yǎng)12 h、36 h、60 h、84 h的OD值和pH值分別為0.100、4.8，1.459、3.85，2.180、3.49，2.240、3.31。液氮超低溫保存后，SD-2a在ATB培養(yǎng)基中培養(yǎng)12 h、36 h、60 h、84 h后的OD值和pH值分別為0.150、4.79，1.573、3.77，2.180、3.41，2.240、3.30。

4.　液氮超低溫保存對(duì)酒酒球菌蘋(píng)果酸分解能力的影響

液氮超低溫保存后，23株酒酒球菌均保持了原有的釀酒活性。酒酒球菌SD-2a在液氮超低溫保存前后的蘋(píng)果酸分解量分別為1.74和1.75；31-DH在液氮超低溫保存前后的蘋(píng)果酸分解量分別為1.36和1.66；其他菌株的蘋(píng)果酸分解量差異也不大。

5.　液氮超低溫保存對(duì)酒酒球菌酶活性的影響

液氮超低溫保存前后酒酒球菌MLE和ATPase活性變化不顯著，菌體仍保持著原來(lái)的活性。液氮超低溫保存前，SD-2a在對(duì)數(shù)期前期、對(duì)數(shù)期中期、穩(wěn)定期前期和穩(wěn)定期中期的MLE活性分別為60.00、170.00、249.00、158.00 μmol.malate.h^－1mg^－1protein。保存后，SD-2a在對(duì)數(shù)期前期、對(duì)數(shù)期中期、穩(wěn)定期前期和穩(wěn)定期中期的MLE活性分別為55.00、171.00、251.00、156.00 μmol.malate.h^－1mg^－1protein。液氮超低溫保存前，SD-2a在對(duì)數(shù)期前期、對(duì)數(shù)期中期、穩(wěn)定期前期和穩(wěn)定期中期的ATPase活性值分別為4.25、12.24、18.21、10.49 μmol.Pi.h^－lmg^－lprotein。保存后，酒酒球菌SD-2a在對(duì)數(shù)期前期、對(duì)數(shù)期中期、穩(wěn)定期前期和穩(wěn)定期中期的ATPase活性值分別為4.13、12.63、17.94、10.95 μmol.Pi.h^－lmg^－lprotein。

關(guān)鍵詞　酒酒球菌　液氮超低溫保存　冷凍存活率　模擬酒發(fā)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