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1 32 5, Vol.32 No.5, Sep JOURNAL OF NINGBO UNIVERSITY ( NSEE ) 1,2, 2*, 2, 2 1., ; 2., :, (Cephalosporium acremonium). C (CPC),,, (GC-MS). :, mll, CPC, gl, ;. : ; ; ; C : TS254.9 : A : ,.,,, 40 ~50 [1]., [2].,,., [3],, [4],. [5],.,, [6]. [7],,.,,,. [8],, C (CPC). [9], CPC, CPC., CPC, GC-MS, ; ( Ug ) ( Ug ) ; CPC ; N,O- ( ) : , ,,,, : @qq.com * 1980,,,, :. liye@nbu.edu.cn

2 Sigma ; (Acremonium chrysogenum)(40514). 1.2 KYC102C JN9811 ( ); Agilent SB-C18 Agilent 7890A ( Agilent ); M7-80EI ( ) ,, 1: :1, 60 6h, 95, 5 min, rmin 10 min,. 60, 120 rmin, CPC ( 16 gl 5gL 70 gl 30 mll 6gL 14 gl 4gL 5gL, ph) [10], ( mll ) CPC, ( gl )., CPC, Box-Behnken, 1. 1 gl PC HPLC CPC rmin 10 min,., HPLC. HPLC CPC : 300SB-C18(4.6 mm 250 mm, 5μm); 20 mmoll, 80:20; 254 nm; 1.0 mlmin ; 37 ; 20 μl : ( )50mL, 20, 60 h, 5d , 1.0 g,. 15 ml 60 [11], 20 min 8000rmin 10 min,,, mgml / 100 μl, min; 15 mgml ( ) 100 μl, min [12] ;, 0.10 mgml 100 μl [13], rmin 5min. 100 μl GC-MS GC-MS : DB-5MS (30 m mm 0.25 μm, Agilent); 1μL,, 8:1, He, 1.00 mlmin, 8min. 90 3min, 3 min 160, 20 min; 2 min 220, 1 min; 10 min 290, 10 min. : 250, EI, 70 ev, 280, 45~ 550 mz, 60 min GC-MS WILEY NIST, 80, CPC CPC 2. 2, CPC, 30 mll,,

3 5, : 19 (5.342±0.038) gl., CPC /(mgl ) CPC /(gl ) ± ± ± ± ± ± CPC, Box-Behnken, 3. 3 A/(mLL ) B/(gL ) C/(mLL ) CPC / (gl ) 1 1(20) 1(50) 0(30) (30) 1(70) 1(20) (30) 0(60) 0(30) (30) 0(60) 0(30) (40) 0(60) 1(40) (20) 0(60) 1(20) (20) 0(60) 1(20) (30) 1(70) 1(40) (20) 1(70) 0(30) (40) 1(70) 0(30) (30) 0(60) 0(30) (20) 0(60) 1(40) (30) 0(60) 0(30) (30) 0(60) 0(30) (30) 1(50) 1(20) (30) 1(50) 1(40) (40) 1(50) 0(30) : A ; B ; C..,,. 3, Design Expert 8.0, : S CPC = A+0.56B+0.48C+0.22A B 0.17A C 0.23B C 1.03A B C 2, (1) : S CPC CPC, gl ; A B C. (1) CPC. 4. 4, P<0.05, P>0.05, [14]. A B C AB BC A 2 B 2 C 2 (P<0.05). F 49.85, R , [15], 3,. 4 F P < A B C AB AC BC A < B < C (a),,., CPC.,,,, CPC. 1(b),,,, CPC. 1(a) 1(b), CPC.,,, CPC [16].,, CPC. 1(c),, CPC. :. CPC

4 宁波大学学报理工版的产量, 使其产量达到 g L. 因此, 考虑在诱变育种的基础上, 用金枪鱼加工副产物酶解液优化培养基, 以期产生更好的效果. 2.3 顶头孢霉菌胞内代谢物的 GC-MS 分析顶头孢霉菌代谢物种类比较通过 GC-MS 在对照组和最优实验组顶头孢霉菌菌体中分别鉴定出 42 种和 40 种化合物, 主要为 (a) 氨基酸(8 种和 19 种) 酸类化合物(18 种和 12 种) 糖类化合物(各 4 种) 醇类化合物(6 种和 3 种) 胺类化合物(3 种和 1 种) 其他化合物(3 种和 1 种). 对照组和最优实验组代谢物的种类组成相似, 其中氨基酸和酸类化合物所占比重较大, 对照组中两者所占比例为 59.81, 最优实验组中为但不同种类代谢物所占比例差异较大(图 2). (b) 对照组图2 最优实验组酶解液对顶头孢霉菌发酵代谢物的影响顶头孢霉菌主要代谢物差异性分析 (c) 图1 图 3 为对照组和最优实验组中氨基酸类代谢交互项因素对 CPC 含量影响的响应面三维图物的质量浓度比较. 氨基酸是微生物胞内代谢活量为: 酶解液 ml L, 玉米浆 g L, 豆动产生的重要初级代谢产物之一, 是微生物生命油 ml L. 在此条件下进行实验, 得到头孢活动不可或缺的重要代谢产物 [19]. 实验中共检测菌素 C 的产量为 g L. 未添加酶解液时产量出 19 种氨基酸, 对照组与实验组中氨基酸含量差为 g L (表 2), 增加了 67.82, 与理论值基异较大的有 7 种, 包括 L-缬氨酸丝氨酸 L-异亮本相符. 氨酸等, 与对照组相比, 实验组中除了赖氨酸含量氮源是菌体细胞的构成物以及合成代谢产物. 氮源的种类质量浓度都会影响发酵产量. 有研 [17] 4+ 减少, 其余 6 种含量均增加. 对氨基酸类化合物的种类及含量分析发现,, 在顶头孢霉菌中, 高浓度铵盐(NH ) 实验组的甘氨酸苏氨酸 L-谷氨酸丙氨酸 L- 会强烈地抑制抗生素的产生, 因此发酵培养基中更适合以 L-天门冬酰胺和 L-精氨酸作为氮源. 玉米浆是顶头孢霉菌发酵过程中常用的氮源, 但玉米浆质量波动较大, 且总氮和氨基态氮含量较低, 往往不能完全满足顶头孢霉菌的菌体生长和合成 CPC 的需要. 用金枪鱼加工副产物制备的酶解液能有效弥补玉米浆的不足. 本研究表明: 在发酵培养基中, 添加一定量的酶解液, 能有效提高 CPC 的产量. 此外, 有研究 [18]通过诱变育种来提高 CPC 缬氨酸和 L-异亮氨酸含量较高. L-谷氨酸在转氨酶究表明的作用下能产生 α-酮戊二酸, 其对氨基酸的代谢具有重要作用[20]. 有研究表明[21], L-谷氨酸在 CPC 发酵过程中含量变化较大, 对 CPC 的合成具有促进作用. L-缬氨酸被认为在 CPC 合成的第二步反应中具有不可或缺的作用, 其与二肽通过加成反应形成三肽, 所以实验组中 L-缬氨酸含量的提高对 CPC 产量具有很大的促进作用[22]. 此外, L-天门冬氨酸 L-苏氨酸和丝氨酸等其他氨基酸对 CPC 的

5 5, : 21,,.,, 9- (Z) ; 2 ; 3 ; 4 ; 5 (Z)-9- ; 6 ; 7 ; 8 ; 9 (Z,Z)-9,12- ; ; 11 (E)-2- ; 12 ; 13 ; ; 15 ; 16 1,2,3- ; 17 10,12- ; 18 ; 19 1,2- - ; ; 21 ; 22. 3, [21].,, , 9-10,12-,. CPC, CPC [23]. (Z,Z)-9,12-, (Z,Z)-9,12- ; (E)-2-1,2, , 2, 3 L-, 4 L-, 5 L-, 6, 7 L-, 8 L-, 9 L-, 10 L-, 11 L-, 12 β-, 13 -L-, 14 DL-, 15 L-, 16 N- -L-, 17, 18 L-, 19 L-. 4, C gl, ,,. CPC,, CPC,. : [1],,,. [J]., 2012, 37(8): [2],,,. [J]., 2012, 33(24): [3],,,. [J]., 2006, 11(2):81. [4],,,. [J]., 2014, 35(20):1-5. [5],,,. [J]., 2010, 26(11): [6],,,. [J]., 2012, 12(7): [7],,,. [J]., 2007, 19(5): [8],. C [J]., 2002, 15(2): [9]. C [D]. :, [10]. C [D]. :, 2011.

6 [11] Meyer H, Liebeke M, Lalk M. A protocol for the investigation of the intracellular Staphylococcus aureus metabolome[j]. Analytical Biochemistry, 2010, 401(2): [12] Chen M M, Li A L, Sun M C, et al. Optimization of the quenching method for metabolomics analysis of Lactobacillus bulgaricus[j]. Journal of Zhejiang University (Science B), 2014, 15(4): [13] Huang X, Gong Y F, Yu K, et al. Gas chromatographymass spectrometry based on metabonomics study of carbon tetrachloride-induced acute liver injury in mice[j]. Chinese Journal of Analytical Chemistry, 2007, 35(12): [14],,,. [J]., 2010, 32(3): [15],,,. [J]., 2006, 27(1): [16]. C [D]. :, [17],. C [J]., 2016, 56(3): [18],,,. C [J]., 2014, 34(8): [19] Kunji E R S, Mierau I, Hagting A, et al. The proteolytic systems of lactic acid bacteria[j]. Antonie Van Leeuwenhoek, 1996, 70(2/4): [20] Kieronczyk A, Skeie S, Olsen K, et al. Metabolism of amino acids by resting cells of non-starter lactobacilli in relation to flavour development in cheese[j]. International Dairy Journal, 2001, 11(S4/7): [21]. β- [D]. :, [22] Park H J, Khang Y H. Production of cephalosporin C by immobilized cephalosporin acremonium in polyethylenemodified barium alginate[j]. Enzyme and Microbial Technology, 1995, 17(5): [23],,,. C [J]. ( ), 2014, 28(2): Effects of hydrolysates of tuna processing by-products on the fermentation products of Cephalosporium acremonium WANG Huan 1,2, LI Ye 2*, DAI Juan 2, SU Xiurong 2 ( 1.College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo , China; 2.School of Marine Sciences, Ningbo University, Ningbo , China ) Abstract: The by-products of tuna were used as raw materials to study the effects of the hydrolysates on the fermentation products of Cephalosporium acremonium. Firstly, based on the yield of cephalosporin C (CPC), the effects of different amounts of hydrolysates on the production of antibiotics by C. acremonium were studied by orthogonal experiment and response surface method. The results showed that in the fermentation medium with the amount of hydrolysates being mll, the production of CPC reached a maximum of gl. It was higher than that without the hydrolysates. Further, the effect of the hydrolysates on the metabolites of C. acremonium was qualitatively and quantitatively analyzed by GC-MS. It was found that the addition of the hydrolysates significantly changed the species and content of the metabolites of C. acremonium. It was of great significance to improve the production of CPC and the comprehensive economic and environmental benefits of tuna processing that uses the resources of tuna processing by-products to develop fermentation industrial medium. Key words: tuna by-products; hydrolysates; Cephalosporium acremonium; cephalosporin C

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g

H 2 SO ml ml 1. 0 ml C 4. 0 ml - 30 min 490 nm 0 ~ 100 μg /ml Zhao = VρN 100% 1 m V ml ρ g 16 6 2018 11 Chinese Journal of Bioprocess Engineering Vol. 16 No. 6 Nov. 2018 doi 10. 3969 /j. issn. 1672-3678. 2018. 06. 004 1 2 1 1 1. 330200 2. 330006 4 Box-Behnken 1 25 g /ml 121 92 min 2 6. 284%