【摘要】：共軛亞油酸(conjugated linoleic acid, CLA)是一系列含共軛雙鍵的亞油酸的位置(8,10-、9,11-、10,12-、11,13-)和幾何(cis, cis; cis, trans; trans, cis; trans, trans)異構(gòu)體的混合物的總稱。CLA具有抗癌、抗腫瘤、提高免疫力、減少脂肪沉積等生理功能。在CLA的多種異構(gòu)體中,9c,11t-CLA和10t,12c-CLA均具有獨(dú)特的生理活性。天然CLA主要存在于反芻動(dòng)物的肉和乳制品中且含量很少,因此,利用生物轉(zhuǎn)化法制備CLA是一個(gè)很好的途徑。 以本實(shí)驗(yàn)室分離保存的一株高產(chǎn)9c,11t-CLA的瑞士乳桿菌L7(L. helveticusL7)為材料,首先通過(guò)發(fā)酵條件優(yōu)化,提高亞油酸(LA)在發(fā)酵液中的生物轉(zhuǎn)化率,增加9c,11t-CLA的產(chǎn)量,進(jìn)而對(duì)其細(xì)胞進(jìn)行透性化處理以提高細(xì)胞膜的通透性,并對(duì)透性化處理細(xì)胞進(jìn)行固定化,研究了固定化細(xì)胞的理化性質(zhì)和轉(zhuǎn)化特性。主要結(jié)果如下： 1)優(yōu)化了菌種發(fā)酵產(chǎn)9c,11t-CLA的最佳條件。確定L. helveticus L7發(fā)酵的最適菌體濃度為5×1010CFU/mL左右。在發(fā)酵進(jìn)行到36h時(shí)發(fā)酵液中的9c,11t-CLA達(dá)到最高值。底物L(fēng)A的最適添加濃度為1mg/mL。 2)對(duì)L. helveticus L7細(xì)胞透性化條件進(jìn)行了研究。分別使用十六烷基三甲基溴化銨(CTAB)、乙醚、乙醇、CTAB、甲苯以及溶菌酶對(duì)L. helveticus L7進(jìn)行透性化處理,結(jié)果表明：透性化處理的效果最好的是20%的乙醇,室溫下處理30min,制得的透性化細(xì)胞的產(chǎn)率達(dá)到468μg/mL;對(duì)超聲波細(xì)胞破碎法制得的亞油酸異構(gòu)酶粗酶液的操作穩(wěn)定性和貯藏穩(wěn)定性進(jìn)行研究,結(jié)果表明：粗酶液的酶活操作穩(wěn)定性較差；保存于4℃時(shí)酶活性逐步降低,120h后酶活降至初始活性的35%左右。經(jīng)溶菌酶處理的透性化細(xì)胞轉(zhuǎn)化率也較高。透性化細(xì)胞的酶活穩(wěn)定性較粗酶液有較大提高。 3)應(yīng)用響應(yīng)面方法對(duì)透性化細(xì)胞的最佳固定化的條件進(jìn)行了優(yōu)化。結(jié)果表明海藻酸鈣膠珠最佳固定化條件為：海藻酸鈉濃度1.78%、氯化鈣濃度3.13%、固定化時(shí)間60.30min,利用在此條件下制備所得的固定化膠珠進(jìn)行發(fā)酵,9c,11t-CLA的產(chǎn)量達(dá)到561μg/mL。與未固定細(xì)胞相比,固定化細(xì)胞單次9c,11t-CLA產(chǎn)量沒(méi)有明顯的變化,但固定化細(xì)胞可多次重復(fù)利用,重復(fù)利用5次后活力沒(méi)有明顯降低,9c,11t-CLA產(chǎn)量保持在549μg/mL。于4℃貯存120h后酶活無(wú)明顯下降,保留在90%以上,具有較好的貯藏穩(wěn)定性。電鏡觀察發(fā)現(xiàn),固定化膠珠表面及內(nèi)部為錯(cuò)綜復(fù)雜的網(wǎng)狀結(jié)構(gòu),能夠很好的滿足底物和產(chǎn)物的傳遞。實(shí)驗(yàn)結(jié)果表明：經(jīng)海藻酸鈣固定,可以有效提高菌體的重復(fù)利用率,從而降低9c,11t-CLA的生產(chǎn)成本。
[Abstract]:Conjugated linoleic acid (conjugated linoleic acid, CLA) is a mixture of a series of linoleic acids containing conjugated double bonds (810 ~ (-10) ~ (9) C ~ (11) ~ (10) ~ (10) ~ (10) and geometric (cis, cis; cis, trans; trans, cis; trans, trans) isomers. CLA has the physiological functions of anti-cancer, anti-tumor, improving immunity, reducing fat deposition and so on. Among the various isomers of CLA, both 9ct 11t-CLA and 10tl 12c-CLA have unique physiological activities. Natural CLA mainly exists in ruminant meat and dairy products and the content is very small. Therefore, bioconversion is a good way to prepare CLA. A strain of Lactobacillus helittidis L7 (L7) with high yield of 9cnc-11t-CLA was isolated and preserved in our laboratory. The fermentation conditions were optimized to improve the bioconversion of linoleic acid (LA) in the fermentation broth and to increase the yield of 9cnc-11t-CLA. In order to improve the permeability of the cell membrane and immobilize the cells, the physicochemical properties and transformation characteristics of the immobilized cells were studied. The main results are as follows: 1) the optimum fermentation conditions of 9 cu 11 t CLA were optimized. The optimum concentration of L. helveticus L7 was about 5 脳 1010CFU/mL. When fermentation lasted for 36 h, the maximum value of 9cnc-11t-CLA in the fermentation broth was reached. The optimal concentration of substrate LA was 1 mg / mL.2) the condition of L. helveticus L7 cell permeability was studied. (CTAB), ether, ethanol, CTAB, toluene and lysozyme were used to treat L. helveticus L7 with hexadecyltrimethylammonium bromide, respectively. The results showed that 20% ethanol was the best permeation treatment. When treated at room temperature for 30 min, the yield of permeable cells reached 468 渭 g / mL. The operating stability and storage stability of the crude linoleic acid isomerase solution prepared by ultrasonic cell fragmentation were studied. The results showed that the enzyme activity stability of the crude enzyme solution was poor. The enzyme activity decreased gradually at 4 鈩，

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