Optimization of culture conditions for maximum viable count of Brevibacillus laterosporus based on MTT colorimetry and response surface methodology

Authors

SONG Peng, College of Agriculture, Henan University of Science and Technology, Luoyang, Henan 471000, ChinaFollow
LI Lixiang, College of Agriculture, Henan University of Science and Technology, Luoyang, Henan 471000, China
ZHAO Biao, College of Agriculture, Henan University of Science and Technology, Luoyang, Henan 471000, China
WANG Zele, College of Agriculture, Henan University of Science and Technology, Luoyang, Henan 471000, China
SUN Xingxin, College of Agriculture, Henan University of Science and Technology, Luoyang, Henan 471000, China

Abstract

Objective: This study aimed to explore the optimal medium composition and culture conditions for obtaining the maximum viable count of Brevibacillus laterosporus. Methods: Based on establishing the correlation regression equation between the MTT colorimetric method and plate counting method, the optimal medium composition (carbon source, nitrogen source, inorganic salt) and fermentation conditions (initial pH, temperature, inoculation amount, KH₂PO₄) for obtaining the maximum viable count of B. laterosporus were optimized. Results: A significant linear correlation for viable count determination between MTT colorimetry and plate counting (R²>0.999) was found; Maltose, CaCl₂, initial pH and KH₂PO₄ were the significant factors. The optimal fermentation condition was 8.75 g/L maltose, 0.17 g/L CaCl₂, 7.07 initial pH and 3.73 g/L KH₂PO₄. Under the control of these conditions, the number of viable counts was 8.12×10⁸ CFU/mL. However, no significant difference from the theoretical number (8.25×10⁸ CFU/mL) was found. Conclusion: Based on MTT colorimetry and response surface methodology, the culture conditions for the maximum viable count of B. laterosporus were optimized and the number of viable counts was higher than before optimization increased by 3.02 times.

Publication Date

3-27-2024

First Page

28

Last Page

35,62

DOI

10.13652/j.spjx.1003.5788.2023.80635

Recommended Citation

Peng, SONG; Lixiang, LI; Biao, ZHAO; Zele, WANG; and Xingxin, SUN (2024) "Optimization of culture conditions for maximum viable count of Brevibacillus laterosporus based on MTT colorimetry and response surface methodology," Food and Machinery: Vol. 40: Iss. 2, Article 4.
DOI: 10.13652/j.spjx.1003.5788.2023.80635
Available at: https://www.ifoodmm.cn/journal/vol40/iss2/4

References

[1] HERMANT Y, PALPAL-LATOC D, KOVALENKO N, et al. The total chemical synthesis and biological evaluation of the cationic antimicrobial peptides, laterocidine and brevicidine[J]. Journal of Natural Products, 2021, 84（8）: 2 165-2 174.
[2] 李馨宇, 邹德勇, 杜春梅. 侧孢短芽孢杆菌的研究进展[J]. 中国农学通报, 2018, 34（14）: 28-34. LI X Y, ZOU D Y, DU C M. Research advance in Brevibacillus laterosporus[J]. Chinese Agricultural Science Bulletin, 2018, 34（14）: 28-34.
[3] WANG X N, ZHANG J C, WANG X F, et al. The growth-promoting mechanism of Brevibacillus laterosporus AMCC100017 on apple rootstock Malus robusta[J]. Horticultural Plant Journal, 2022, 8（1）: 22-34.
[4] ZHAO Z, ZHAO C, DO H, et al. Brevibacillus laterosporus ZN5 induces different carbonate precipitations of lead in ammonification and nitrate assimilation processes[J]. Geomicrobiology Journal, 2020, 37（8）: 764-773.
[5] 李理想, 江厚龙, 张艳, 等. 施用侧孢短芽孢杆菌对烟草根际微生态及产量和品质的影响[J]. 湖南农业大学学报（自然科学版）, 2023, 49（2）: 159-164. LI L X, JIANG H L, ZHANG Y, et al. Effects of application of Brevibacillus laterosporus on rhizosphere microecology, yield and quality of tobacco[J]. Journal of Hunan Agricultural University （Natural Sciences）, 2023, 49（2）: 159-164.
[6] WU Y, ZHOU L, LU F, et al. Discovery of a novel antimicrobial lipopeptide, brevibacillin V, from Brevibacillus laterosporus fmb70 and its application on the preservation of skim milk[J]. Journal of Agricultural and Food Chemistry, 2019, 67（45）: 12 452-12 460.
[7] 杨培洁, 李腾, 陈晓红, 等. MTT法测定瑞士乳杆菌MB2-1活菌数[J]. 食品科学, 2013, 34（20）: 99-102. YANG P J, LI T, CHEN X H, et al. MTT colorimetric method for Lactobacillus helveticus MB 2-1 viable cell counting[J]. Food Science, 2013, 34（20）: 99-102.
[8] 周大祥, 熊书, 谢桂香. 利用MTT建立一种快速检测青枯菌活菌的方法[J]. 生物学杂志, 2019, 36（1）: 103-106. ZHOU D X, XIONG S, XIE G X. Establishment of a method for rapid detecting only viable cells of Ralstonia Solanacearum by MTT[J]. Journal of Biology, 2019, 36（1）: 103-106.
[9] STOCKERT J C, HOROBIN R W, COLOMBO L L, et al. Tetrazolium salts and formazan products in cell biology: Viability assessment, fluorescence imaging, and labeling perspectives[J]. Acta Histochemica, 2018, 120（3）: 159-167.
[10] 王丹, 黄紫藤, 马越, 等. 蓝莓花色苷对鸡胚成纤维细胞系生长的影响[J]. 食品与机械, 2016, 32（8）: 13-16. WANG D, HUANG Z T, MA Y, et al. Effect of anthocyanins from blueberry on primary chicken embryonic fibroblast[J]. Food & Machinery, 2016, 32（8）: 13-16.
[11] YAP L S, LEE W L, TING A S Y. Optimization of L-asparaginase production from endophytic Fusarium proliferatum using OFAT and RSM and its cytotoxic evaluation[J]. Journal of Microbiological Methods, 2021, 191: 106358.
[12] 解万翠, 杨亚东, 杨锡洪, 等. 蜡状芽孢杆菌产中性蛋白酶发酵条件的优化[J]. 食品与机械, 2016, 32（1）: 26-30, 129. XIE W C, YANG Y D, YANG X H,et al. Response surface methodology optimization of fermentation conditions for neutral protease production by Bacillus cereus[J]. Food & Machinery, 2016, 32（1）: 26-30, 129.
[13] 游佳欣, 应文俊, 杨倩倩, 等. 杨木酶水解液培养枯草芽孢杆菌试验[J]. 林业工程学报, 2021, 6（4）: 94-100. YOU J X, YING W J, YANG Q Q, et al. The production of Bacillus subtilis by fermentation from enzymatic hydrolysate of poplar[J]. Journal of Forestry Engineering, 2021, 6（4）: 94-100.
[14] 吴窈画, 谈书华, 范超超, 等. MTT法检测细菌细胞数的主要影响因素分析[J]. 微生物学杂志, 2011, 31（3）: 67-72. WU Y H, TAN S H, FAN C C, et al. Analysis of influencing factors on the bacterial cell counting with the MTT method[J]. Journal of Microbiology, 2011, 31（3）: 67-72.
[15] 马林玉, 胡家银, 林煊, 等. MTT法检测奶牛乳房炎无乳链球菌活菌数的研究[J]. 中国动物传染病学报, 2021, 29（6）: 100-104. MA L Y, HU J Y, LIN X, et al. Development of MTT method for viable count of Streptococcus agalactia causing bovine mastitis[J]. Chinese Journal of Animal Infectious Diseases, 2021, 29（6）: 100-104.
[16] 高琼. 大肠杆菌在土壤中的迁移特性实验研究[D]. 天津: 天津理工大学, 2011: 16-18. GAO Q. Experimental study on migration characteristics of Escherichia coli in soils[D]. Tianjin: Tianjin University of Technology, 2011: 16-18.
[17] 吴丽媛. 甜瓜细菌性果斑病生防菌的筛选及其抑菌机理的初步研究[D]. 呼和浩特: 内蒙古农业大学, 2013: 18-21. WU L Y. Screening of biocontrol agent against to Acidovorax citrulli and its antibacterial mechanism[D]. Hohhot: Inner Mongolia Agricultural University, 2013: 18-21.
[18] 陈丹霞, 居晨玉, 郑永标, 等. 培养基组分对细脚拟青霉液体发酵生物量与有机粗提物量的影响[J]. 食用菌, 2012（2）: 23-24. CHEN D X, JU C Y, ZHENG Y B, et al. Effects of culture medium components on liquid fermentation biomass and organic crude extracts of Paecilomyces tenuipes[J]. Edible Fungi, 2012（2）: 23-24.
[19] 李茂琳, 谭军, 王红英, 等. 一株产胶原蛋白酶细菌的鉴定及产酶条件优化[J]. 食品工业科技, 2019, 40（14）: 118-126. LI M L, TAN J, WANG H Y, et al. Identification of a collagenase-producing bacterium and optimization of enzyme producing conditions[J]. Science and Technologr of Food Industry, 2019, 40（14）: 118-126.
[20] 范海延, 梁胜贤, 王栋, 等. 利用甘薯淀粉生产废水培养侧孢短芽孢杆菌的研究[J]. 沈阳农业大学学报, 2015（3）: 352-356. FAN H Y, LIANG S X, WANG D, et al. Production of Brevibacillus laterosporus using sweet potato processing wastewater[J]. Journal of Shenyang Agricultural University, 2015（3）: 352-356.
[21] 杨旭, 武天宁, 荀明强, 等. 1株地衣芽孢杆菌发酵条件研究及其微生物制剂的制备[J]. 饲料研究, 2022, 45（24）: 91-96. YANG X, WU T N, XUN M Q, et al. Study on fermentation conditions of Bacillus licheniformis and preparation of microbial agents[J]. Feed Research, 2022, 45（24）: 91-96.
[22] 刘蒲临, 程德勇, 缪礼鸿. 产几丁质酶侧孢短芽孢杆菌的筛选及其酶学性质研究[J]. 生物技术通报, 2016, 32（6）: 174-180. LIU P L, CHENG D Y, MIAO L H. Isolation of a chitinase-producing strain Brevibacillus laterosporus and its enzymatic properties[J]. Biotechnology Bulletin, 2016, 32（6）: 174-180.
[23] 卜雯丽, 李凤伟, 王杰, 等. 出芽短梗霉固态发酵啤酒糟制备阿魏酰低聚糖和膳食纤维工艺研究[J]. 中国酿造, 2019, 38（5）: 38-43. BU W L, LI Y W, WANG J, et al. Preparation process of feruloyl oligosaccharides and dietary fiber from brewer's spent grain by solid-state fermentation of Aureobasidium pullulans[J]. China Brewing, 2019, 38（5）: 38-43.