Optimization of Headspace Solid-phase Microextraction for GC-MS Analysis of Volatile Compounds Produced by Biocontrol Strain <i>Bacillus subtilis</i> CF-3 Using Response Surface Methodology

Abstract

The volatiles of Bacillus subtilis CF-3 showed a significant (p < 0.05) suppression of the mycelial growth of Monilinia fracticola and Colletotrichum gloeosporioides which caused postharvest decay of Peach and Litchi. An 85-μm polyacrylate (PA) extraction fiber was initially selected used for the optimized experiments. For an optimal CF-3 volatile collection, HS-SPME was suggested to be performed with extraction time of 40.8 min and extraction temperature of 38.5℃ and with 41.5 mL LB broth medium and 4.15 mL CF-3 fermentation liquor (1×10 8 CFU/ mL) based on the response surface methodology (RSM). 61 volatile compounds, including fourteen alcohols, fifteen ketones, two aldehydes, eight acids, five phenols, three pyrazines, four esters, three olefins, two amines, two ethers, etc., were extracted using three types of fibers by the optimum method with similarity index (SI)>850 from the database search. Good predictability of the model, satisfactory repeatability for all volatile compounds according to the optimized HS-SPME conditions indicated that the HS-SPME procedure was applicable for the analysis of volatile release, six compounds were selected and assayed for antifungal activity in divided Petri plates, 1-Octanol and 2,4-Di-tert-butylthiophenol can inhibit the mycelial growth of M. fracticola and C. gloeosporioides, which will aid in further research on bio-bacterium volatile metabolites as biocontrol agents against postharvest diseases in fruits and vegetables.

Research on Volatile Organic Compounds From Bacillus subtilis CF-3: Biocontrol Effects on Fruit Fungal Pathogens and Dynamic Changes During Fermentation

“…Bacillus subtilis CF-3 (registered in the China Center for Type Culture Collection, CCTCC M 2016125) was isolated from fermented bean curd and identified by the Laboratory of Food Safety and Quality Control (School of Life Sciences, Shanghai University) (Gao et al, 2016 ). For short-term storage, B. subtilis CF-3 was incubated at 37°C for 24 h on Luria-Bertani (LB) agar (Gao H. Y. et al, 2017 ). All chemicals used were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).…”

“…A. alternata, B. cinerea, M. kuwatsukai , and P. expansum were provided by the College of Horticulture and Landscape, Tianjin Agricultural University; C. gloeosporioides was provided by the Chinese Academy of Tropical Agricultural Sciences Environment and Plant Protection Institute; and M. fructicola was provided by the Laboratory of Agricultural Products Processing and Storage, Food Science and Engineering College, Beijing University of Agriculture. They were plated onto potato dextrose agar (PDA) medium (Gao H. Y. et al, 2017 ) in Petri dishes (Ø90 mm) and incubated at 28°C for 7 days prior to use.…”

“…After 24 h of incubation on LB agar, B. subtilis CF-3 was gently scraped and transferred into 100 mL of LB liquid medium in a conical flask and cultivated at 37°C in a rotary shaker at 150 rpm for 24 h. Subsequently, approximately 1 × 10 8 colony forming units (CFU)/mL were counted using a hemocytometer. Afterwards, 4.15 mL of the prepared B. subtilis CF-3 liquid was transferred into 41.5 mL of fresh culture solution, which was then sealed with a silicone pad and aluminum cap in a custom-made conical flask with side arms bound with eight layers of sterile gauze (Gao H. Y. et al, 2017 ). Eight such conical flasks were prepared and placed in a 150-rpm rotary shaker at 37°C for 12–96 h: the flasks were taken out one by one at 12-h intervals, i.e., at 12, 24, and 36 h, and so forth.…”

“…Subsequently, the fibers were removed from the flasks and immediately inserted into the injection port of the GC-MS apparatus for analysis. The GC-MS conditions and procedures were selected as previously described (Gao H. Y. et al, 2017 ): when one compound was identified by multiple extraction fibers simultaneously, the conditions that yielded the highest probability were adopted. Each fermentation liquid was analyzed in triplicate.…”

“…In a previous study, we isolated, identified, and named the B. subtilis strain CF-3, which exhibited biocontrol effects (Gao et al, 2016 ). Moreover, the VOCs produced by B. subtilis CF-3 were also identified after 48 h of cultivation (Gao H. Y. et al, 2017 ). Considering these findings, the objectives of the present study were: (1) to evaluate the antifungal spectrum and inhibitory effects of VOCs produced by B. subtilis CF-3 on common fungal pathogens in vitro ; (2) to identify the dynamic changes in the levels of VOCs secreted by B. subtilis CF-3 during fermentation using headspace solid-phase micro-extraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS); (3) to discover the correlation between compounds and inhibition rates by evaluating the dynamic inhibition rates of VOCs secreted from B. subtilis CF-3; and (4) to ascertain which compounds were key factors in the inhibition of fungal pathogens both in vivo and in vitro .…”