响应面法优化含活性肽CC34毕赤酵母重组菌冻干营养保护剂的研究

doi:10.16431/j.cnki.1671-7236.2022.10.011

Abstract

Abstract: 【Objective】 Response surface method was used to optimize the formulation of lyophilized nutritional protectants for Pichia pastoris recombinant bacteria containing active peptide CC34.【Method】 The optimum range of skimmed milk powder,trehalose,sorbitol and monosodium glutamate to improve the survival rate of recombinant Pichia pastoris was screened by single factor test. The regression equation of the survival rate of recombinant Pichia pastoris was established by Box-Behnken method,and the effect relationship of three protectant combinations on the survival rate was analyzed.【Result】 When the concentration of skimmed milk powder was 10%,the freeze-drying survival rate of recombinant Pichia pastoris was the highest,reaching 51.23%. When the concentration of trehalose was 15%,the freeze-drying survival rate was the highest,reaching 67.50%. When the concentration of sorbitol was 20%,the freeze-drying survival rate was the highest,reaching 52.19%.And when the concentration of sodium glutamate was 3%,the freeze-drying survival rate was the highest,reaching 36.92%.The concentrations of skimmed milk powder,trehalose and sorbitol in the freeze-drying protectant combination of Pichia pastoris recombinant strain obtained by response surface were 10.75%,15.70% and 20.19%,respectively. The survival rate of recombinant Pichia pastoris was predicted to be 77.00% by the optimal model.The freeze-drying test with the optimal combination of protectants showed that the survival rate of the cell was 78.98%.【Conclusion】 In this experiment, the formulation of compound freeze-drying protectant for recombinant Pichia pastoris was optimized by response surface methodology. Under the conditions of skim milk powder concentration of 10.75%, trehalose concentration of 15.70% and sorbitol concentration of 20.19%, a higher survival rate (78.98%) of recombinant Pichia pastoris could be obtained. The compound freeze-drying protectant could be used for the preservation of recombinant Pichia pastoris containing active peptide CC34 and subsequent experimental research.

Key words: response surface; freeze drying; lyoprotectant; recombinant Pichia pastoris

CLC Number:

S816.3

LI Shuang, DU Jiahua, BAI Zhihui, QU Ke, SU Miao, JIANG Ning, ZHANG Aizhong. Optimization of Lyophilized Combination for Pichia pastoris Containing Active Peptide CC34 Recombinant with Response Surface Methodology[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 3791-3799.

References

[1] AGEITOS J M,SANCHEZ-PEREZ A,CALO-MATA P,et al.Antimicrobial peptides(AMPs):Ancient compounds that represent novel weapons in the fight against bacteria[J]. Biochemical Pharmacology,2017,133:117-138.
[2] 卫旭彪,武如娟,郑召君,等.抗菌肽的免疫调节功能及其作用机制[J].饲料工业,2015,22:55-58. WEI X B,WU R J,ZHENG Z J,et al.Immunomodulatory function and action mechanism of antimicrobial peptides[J].Feed Industry,2015,22:55-58.(in Chinese)
[3] WEBB K E,MATTHEWS J C,DIRIENZO D B.Peptide absorption:A review of current concenpts and future perspectives[J].Journal of Animal Science,1992,70(10):3248.
[4] 郭玲玲.微生物菌种保藏方法及关键技术[J].微生物学杂志,2019,39(3):105-108. GUO L L.Preservation method and preservation key technology of microbial strain seed[J].Journal of Microbiology,2019,39(3):105-108.(in Chinese)
[5] 张楠驰,李娟,魏勇.羊源解淀粉芽孢杆菌对断奶金堂黑山羊生长性能、屠宰性能、血清生化和免疫指标以及肌肉发育相关基因表达的影响[J].动物营养学报,2022,34(3):1743-1753. ZHANG N C,LI J,WEI Y,et al.Effect of Bacillus amyloliquefaciens isolated from goats on growth performance,slaughter performance,serum biochemical and immune indexes,and muscle development-related gene expression of weanling jintang black goat[J].Chinese Journal of Animal Nutrition,2022,34(3):1743-1753.(in Chinese)
[6] WANG G Q,PU J,YU X Q,et al.Influence of freezing temperature before freeze-drying on the viability of various Lactobacillus plantarum strains[J].Journal of Dairy Science,2020,103(4):3066-3075.
[7] STEFANWLLO R F,NABESHIMA E H,IAMANAKA B T,et al.Survival and stability of Lactobacillus fermentum and Wickerhamomyces anomalus strains upon lyophilisation with different cryoprotectant agents[J]. Food Research International,2019,115:90-94.
[8] BYUN S Y,KANG J S,CHANG S.Analysis of primary drying of poly-γ-glutamic acid during vacuum freeze drying[J].Journal of Mechanical Science and Technology,2020,34:1-10.
[9] 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.
[10] ABADIAS M,TEIXID N,USALL J,et al.Viability,efficacy,and storage stability of freeze-dried biocontrol agent Candida sake using different protective and rehydration media[J].Journal of Food Protection,2001,64:856-61.
[11] 张菊,苏成文,亓鹏,等.植物乳杆菌冻干保护剂的筛选及冻干工艺的研究[J].中国饲料,2020,17:53-57. ZHANG J,SU C W,QI P,et al.The research of protectants and freeze-drying process for Lactobacillus plantarum[J].China Feed,2020,17:53-57.(in Chinese)
[12] 陈合,张建华,秦涛,等.山梨醇、甘露醇及甜菜碱等对双歧杆菌冻干的影响[J].食品科技,2014,39(1):19-23. CHEN H,ZHANG J H,QIN T,et al.Effect of sorbitol,mannitol and betaine on the freeze-drying of Bifidobacterium bifidum[J].Food Science and Technology,2014,39(1):19-23.(in Chinese)
[13] 蔡鹏,姜宁,张爱忠,等.毕赤酵母表达系统应用于抗菌肽表达的研究进展[J].黑龙江畜牧兽医,2015,11:56-60. CAI P,JIANG N,ZHANG A Z,et al.Research advance in the Pichia pastoris expression system applied to the antibacterial peptides[J].Heilongjiang Animal Science and Veterinary Medicine,2015,11:56-60.(in Chinese)
[14] 徐颖,贺黎,吕嘉枥,等.富硒鼠李糖乳杆菌稳定性及其冻干保护剂研究[J].中国食品学报,2020,20(9):102-108. XU Y,HE L,LYU J L,et al.Studies on Stability of selenium-enriched Lactobacillus rhamnosus and its lyophilized protectant[J]. Journal of Chinese Institute of Food Science and Technology,2020,20(9):102-108.(in Chinese)
[15] 陈颜红,王修俊,张二康,等.植物乳杆菌LP-7501S冻干保护剂优化研究[J].扬州大学学报(农业与生命科学版),2021,42(2):99-104. CHEN Y H,WANG X J,ZHANG E K,et al.Optimization of freeze-drying protectant for Lactobacillus plantarum LP-7501S[J].Journal of Yangzhou University(Agricultural and Life Science Edition),2021,42(2):99-104.(in Chinese)
[16] 盛洁.解淀粉芽孢杆菌fmbj37产γ-聚谷氨酸发酵工艺的优化及应用[D].南京:南京农业大学,2019. SHENG J.Medium optimization of γ-polyglutamic acid produced by Bacillus amyloliquefaciens fmbj37 and application[D].Nanjing:Nanjing Agricultural University,2019.(in Chinese)
[17] 赵禹彤.罗伊氏乳杆菌冻干保护剂的筛选及在发酵乳中的应用[D].长春:吉林大学,2020. ZHAO Y T.Screening of lyophilized protective agent for Lactobacillus reuteri and its application in fermented milk[D].Changchun:Jilin University,2020.(in Chinese)
[18] 李大鹏,高玉荣.益生菌植物乳杆菌G1-28复合冻干发酵剂制备及保藏条件研究[J].食品工业科技,2021,42(10):100-104. LI D P,GAO Y R.Preparation and preservation technology of probiotic Lactobacillus plantarum G1-28 composite lyophilized starter[J].Science and Technology of Food Industry,2021,42(10):100-104.(in Chinese)
[19] 张瑶.生菜发酵过程中微生物多样性研究及发酵菌株冻干发酵剂的优化[D].北京:北京农学院,2021. ZHANG Y.Study on microbial diversity in lettuce fermentation and optimization of freeze drying protectant for fermentation strain[D].Beijing:Beijing University of Agriculture,2021.(in Chinese)
[20] 侯佳玲.添加冻干保护剂制备纤维素纳米纤维气凝胶及其应用[D].天津:天津科技大学,2018. HOU J L.Preparation of cellulose nanofibers aerogels by adding cryoprotectant and its application[D].Tianjin:Tianjin University of Science and Technology,2018.(in Chinese)
[21] 谭莎莎,马方励,崔树茂,等.罗伊氏乳杆菌冻干保护剂的优选及高密度冻干工艺优化[J].食品与发酵工业,2020,46(4):1-6. TAN S S,MA F L,CUI S M,et al.Optimization of lyoprotectants and high-density freeze-drying process for Lactobacillus reuteri[J].Food and Fermentation Industries,2020,46(4):1-6.(in Chinese)
[22] 公丕民.保加利亚乳杆菌喷雾干燥过程中损伤机制及保护方法研究[D].哈尔滨:哈尔滨工业大学,2019. GONG P M.Injury mechanism and protection methods of spray-dried lactobacillus bulgaricus[D].Harbin:Harbin Institute of Technology,2019.(in Chinese)

Optimization of Lyophilized Combination for Pichia pastoris Containing Active Peptide CC34 Recombinant with Response Surface Methodology

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 13

Recommended Articles

Metrics

Comments

[1]	LI Weihao, SUN Yuanyuan, LIU Juxiang. Preparation of Oregano Oil Macleaya cordata Oral Solution and Its Antibacterial Activity in vitro [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 718-730.
[2]	SUN Yuanyuan, LIANG Chuncai, ZHANG Hailong, LIU Juxiang. Preparation of Hydrogel Drug Carrier for Chicks [J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(10): 4075-4084.
[3]	XU Baichang, FANG Ziyi, MENG Jinyan, CUI Qinghan, LI Hongman, SI Hongbin. Optimization of Extraction Process and Antioxidant Activity of Polyphenols from Leaves of Broussonetia papyrifera [J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(3): 1046-1053.
[4]	WU Xinyu, YAN Lin, WU Hong. Optimizing Conditions Analysis of Levan by Whole-cell Biocatalysis with Corynebacterium glutamicum [J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3474-3483.
[5]	CHEN Keyuan, HAO Baocheng, DING Ziqiang, LIANG Jianping. Optimization of Liquid Fermentation Process for Danggui Buxue Tang by Response Surface Methodology [J]. China Animal Husbandry and Veterinary Medicine, 2019, 46(10): 2946-2954.
[6]	YAN Lin, ZHONG Hongmei, ZUO Jie, CHEN Zhihui, LUO Youcai, CHEN Qiyue, WU Hong. Optimization of Culture Condition in Trichoderma reesei Tu6-VHb Which Transferred VHb Gene Cellulase Production by Surface Method [J]. , 2018, 45(3): 663-672.
[7]	BIAN Ya-bin, ZHANG Jing-yan, HOU Yan-hua, WANG Xu-rong, ZHANG Kai, LI Jian-xi. Optimization of Polysaccharides Extraction from Astragalus membranaceus Fermented by FGM Strain with Response Surface Method [J]. , 2017, 44(2): 440-446.
[8]	MO Hai-fei, CHEN Lian-hong, HUANG Lin, WU Hong. Optimization of Culture Conditionsin in Trichoderma reesei Solid-state Fermentation Producing α-galactosidase using Response Surface Method [J]. , 2016, 43(8): 2038-2046.
[9]	LIANG Kong-xian, LV Xiao-jun, DING Dan, XU Qi, ZHOU Jie, LIU Wei. Preparation of Adjuvant Diluent for Live Vaccine against Classical Swine Fever and Evaluation of its Immune Effects [J]. , 2015, 42(9): 2467-2473.
[10]	DING Dan, SHENG Xiao-li, LIANG Kong-xian, XU Qi, LIU Wei. Study on Ivermectin Nanoemulsion for Transdermal Drug Delivery [J]. , 2015, 42(2): 401-407.
[11]	ZHANG Dai-bao, WU Zhi-ming, ZHAO Ming-jun, ZHANG Lin-hai, YAN Ruo-qian. Research Progress on Interferon Freeze-drying Technology [J]. , 2012, 39(10): 57-60.
[12]	NING Yu-chang;YANG Xiang-ke. Preparation of Microcapsules and Characteristic Research of Lactobacillus from Chicken [J]. , 2011, 38(2): 220-223.
[13]	WANG Jing;JI Hai-feng;WANG Si-xin;ZHANG Dong-yan;WANG Ya-min. Advances on Lactobacillus Preparation Research andApplication in Pig Production [J]. , 2010, 37(3): 38-40.