Screening and identification of a Bacillus ...

Food Control 78 (2017) 24e32

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Screening and identification of a Bacillus amyloliquefaciens strain for aqueous enzymatic extraction of medium-chain triglycerides Cheng Zeng a, Rongbin Zhao b, c, Xuefang Wen b, c, d, Ping Yu b, c, d, Zheling Zeng b, c, d, Shuguang Deng c, d, e, Deming Gong b, d, f, * a

The First School of Clinical Medicine, Nanchang University, Nanchang, 330031, China State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang, 330047, China School of Resources, Environmental and Chemical Engineering, Nanchang University, Nanchang, 330031, China d Jiangxi Province Key Laboratory of Edible and Medicinal Plant Resources, Nanchang University, Nanchang, 330031, China e School for Engineering of Matter, Transport and Energy, Arizona State University, Tempe, AZ, 85284, United States f New Zealand Institute of Natural Medicine Research, 8 Ha Crescent, Auckland, 2104, New Zealand b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 June 2016 Received in revised form 14 January 2017 Accepted 12 February 2017 Available online 20 February 2017

A strain with a high yield of neutral proteinase and low yield of lipase, resistance to medium chain fatty acid triglycerides is the key to increasing yield and quality of Cinnamomum camphora seed kernel oil (CCSKO) with aqueous enzymatic extraction technology. A bacterial strain, NCU116 isolated from the waste residue produced in CCSKO production through primary screening with plate and secondary screening with shake-flask fermentation. It was found to be suitable for the extraction of CCSKO or other medium-chain triglycerides by using the extraction technology. Its activity of neutral proteinase was 4536.5 U/mL, and only 0.088 U/mL for lipase production. The strain was identified as Bacillus amyloliquefaciens by morphological, physiological, biochemical and 16S rDNA molecular identification. The extracellular enzymes produced by NCU116 included neutral proteinase, pectinase, glucoamylase, cellulase, amylase and lipase. The neutral proteinase had the maximum activity at 50 C, but was unstable. Its optimum temperature and pH value were approximately 40 C and 7.0 respectively. Mn2þ was an activator of neutral proteinase. The glucoamylase had the maximum activity at 45 C, and was activated by Ca2þ, Zn2þ, Fe3þ and Mn2þ. Its optimum temperatures and pH value were 45e50 C and 6.0 respectively. The pectinase had the maximum activity at 40 C, and was activated by Cu2þ, Fe2þ, Mn2þ and Ca2þ. Its optimum temperatures and pH value were 35e40 C and 7.0 respectively. The cellulase had the maximum activity at 35 C, and was activated by Ca2þ and Mn2þ. Its optimum temperatures and pH value were 30e40 C and 7.0 respectively. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Bacillus amyloliquefaciens Screening Identification Enzymatic properties

1. Introduction Cinnamomum camphora (Lauraceae), commonly known as camphor tree and mostly distributed in China, Japan, Vietnam, is a plant with high values (Babu et al., 2003). The oil of its seed kernel

Abbreviations: CCSKO, Cinnamomum camphora seed kernel oil; H/C, the ratio of hydrolytic zone diameter to colony diameter; MCFA, Medium chain fatty acid; MCT, medium chain fatty acid triglyceride. * Corresponding author. State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang, 330047, China. E-mail address: [email protected] (D. Gong). http://dx.doi.org/10.1016/j.foodcont.2017.02.031 0956-7135/© 2017 Elsevier Ltd. All rights reserved.

(CCSKO) contains a high amount of medium chain fatty acid triglyceride (MCT > 94%), which has high nutrition values to human beings. MCT was reported to reduce deposition of body fat, improve blood lipid and glucose levels (Akpan, Bankole, & Adesemowo, 1999; Lavau & Hashim, 1978; Hainer, Kunesova, Stich, Zak, & Parizkova, 1994; Fu et al., 2015), and prevent human obesity (Berning, 1996). It can also be used as a solvent for vitamins, fungicides, hormones, antibiotics and other medical products (Zhao, Liu, & Zhu, 2005). Meanwhile, MCT was found to have a strong inhibitory effect on Gram-positive bacteria, Gram-negative bacteria, mould and enzymes (Luo et al., 2014; Zeng, Zhao, & Luo, 2013; Zhang, Wei, Cui, Zhao, & Feng, 2009). A number of techniques are available for oil extraction, including pressing process, organic solvent extraction and

C. Zeng et al. / Food Control 78 (2017) 24e32

supercritical CO2 extraction (Abbasi, Rezaei, & Rashidi, 2008; Panfili, Cinquanta, Fratianni, & Cubadda, 2003). In consideration of the efficiency, safety and cost, an alternative method, aqueous enzymatic technology has attracted attention. Aqueous enzymatic technology not only has mild extraction conditions, a low energy consumption and safe process, but also can get a high quality of vegetable oil and vegetable protein. It reflects a new direction in oil production (Latif & Anwar 2011; Rosenthal, Pyle, & Niranjan, 1996; Tabtabaei & Diosady, 2013; Yusoff, Gordon, Ezeh, & Niranjan, 2016). The oil extraction by aqueous enzymatic method mostly used neutral proteinases from Bacillus subtilis AS1.398, Aspergillus terricola 3942, Actinomycetes 166 and other strains. However, it was found that the yield of CCSKO was less than 83% using these strains, and its acid value was higher than 5 mg KOH/g. This may be due to the antibacterial activity of MCT. Therefore, the key to improving the yield and quality of CCSKO is to identify a strain with high proteinase and low lipase activities, and can grow in the MCTcontaining medium. In the present study, we have for the first time identified a new bacterial strain which can be used for medium-chain triglycerides extraction, and analyzed the properties of its extracellular enzymes. 2. Materials and methods 2.1. Bacteria and reagents Waste residue of CCSKO production was from Xiangzhanglin High-technology Co. (Nanchang, China), restaurant waste water was from Nanchang University Student Canteen (Nanchang, China), sufu blank was from Jiuhong Food Co. (Nanchang, China). Agar and soluble starch were from Beijing Aobox Biotechnology Co. (Beijing, China). Folin phenol was purchased from Lida Biotechnology Co. (Shanghai, China). Carboxymethyl cellulase sodium was obtained from Sinopharm Chemical Reagent Co. (Shanghai, China). Casein was from Damao Chemical Reagent Co. (Tianjin, China). Na2HPO4$12H2O, KH2PO4, NaCl, Na2CO3, NaOH, trichloroacetic acid, glucose, purchased from Xilong Chemical Co. (Guangzhou, China) were of analytical grade.

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2.3. Strain screening 2.3.1. Enrichment culture of the strain Approximately 1 g of strain sample was made into solutions in different dilutions (101-107) by using saline and ten-fold dilution method. Diluted solution (100 mL) was taken from three suitable dilutions and applied onto a plate of C. camphora seed kernels medium. Four parallel experiments were conducted for every dilution. After incubation at 37 C for 24 h, plates with around 10 bacterial colonies were selected. 2.3.2. Screening and selection of bacterial strains with high proteinase and low lipase activities The bacterial colonies obtained above were spot-inoculated onto a plate of skim milk bouillon culture medium by using a sterile tooth pick, and then incubated at 37 C for 24 h. According to the ratio of hydrolytic zone diameter to colony diameter (H/C), strains with proteinase activity were screened out. The bacterial colonies with higher H/C were spot-inoculated onto a plate of CCSKO medium, incubated at 37 C for 24 h. Red transparent circles produced around bacterial colonies were observed. The strain without red transparent circle was selected. The strain obtained from the first screening was inoculated into bouillon culture medium, and then shake-flask cultivated at 37 C, 220 rpm for 24 h. The produced seed liquid was added into fermentation medium with an inoculum of 4% (v/v), and was cultivated at 37 C, 220 rpm for 48 h. The fermentation liquid was then separated by centrifugation at 4250 g for 10 min, and the obtained supernatant contained crude enzymes. The proteinase activity was analysed using the Folin-reagent method by Monnet, Le Bars, Neviani, and Gripon (1987) with a slight modification. One unit of proteinase activity was defined as the amount of enzyme which liberated 1 mg tyrosine per min at 37 C. The lipase activity was analyzed by titration method by Brockman (1981). One unit of lipase activity was defined as the amount of enzyme that produced 1 mmol of fatty acid per minute at 37 C. The strain with the highest proteinase activity and lowest lipase activity was selected. 2.4. Strain identification

2.2. Preparation of media C. camphora seed kernels were mixed with water at a ratio of 1:2 (w/v). The mixture was then grinded for 4 min in colloid mill (50 Hz, 1.1 kw, 220 V, 2810 r/min, JMS50DX, Langtong Machinery Co., Langfang, China), and the resulting white liquid was dried to get C. camphora seed kernels powder (oil content 57.9%, moisture content 2.4%). C. camphora seed kernels medium consisted of 2% (w/v) C. camphora seed kernels powder, 0.7% (w/v) glucose, 0.03% (w/v) KH2PO4, 0.4% (w/v) Na2HPO4$12H2O and 2% (w/v) agar, pH 7.0. CCSKO medium was made up of 1% (w/v) peptone, 0.5% (w/v) beef extract, 0.5% (w/v) NaCl, 1.2% CCSKO, 1.6% natural red and 2% agar, pH 7.0. Bouillon culture medium was made up of 1% (w/v) peptone, 0.3% (w/v) beef extract and 0.5% (w/v) NaCl, pH 7.0. Skim milk powder (1.5%, w/v) was added to the bouillon culture medium to make skim milk bouillon culture medium. Agar culture medium contained 2% (w/v) agar, 1% (w/v) peptone, 0.3% (w/v) beef extract and 0.5% (w/v) NaCl, pH 7.0. Fermentation medium consisted of 4% (w/v) corn flour, 2.5% (w/v) wheat bran, 3% (w/v) soya bean meal, 0.03% (w/v) KH2PO4 and 0.4% (w/v) Na2HPO4$12H2O, pH 7.0. All these media were sterilized for 20 min at 121 C in a portable sterilizer.

2.4.1. Morphological and biochemical identification Upon dilution, the strain was spread onto a nutrient agar medium, and the characteristics of bacterial colony such as shape, color, size and protuberance were recorded after cultivation for 24 h. The strain was gram-stained, and the cellular morphology of the strain was then observed under a microscope. The strain was also identified by a biochemical method (Dong & Cai, 2001). 2.4.2. Molecular identification by 16S rDNA Strain genomic DNA was extracted by using Ezpu pillar bacterial genomic DNA extraction kit (Shanghai Sangon Biotechnology Co., China). A PCR using extracted DNA as a template, bacterial 16S rDNA gene general primer 27F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 -GGTTACCTTGTTACGACTT-30 ) was performed. The reaction mixture (25.0 mL) consisted of 0.5 mL sense primer, 0.5 mL anti-sense primer, 0.5 mL DNA, 3.7 mL Taq mix and 19.8 mL ddH2O. The amplification reaction conditions were as follows: initial denaturation at 95 C for 5 min, 30 cycles of denaturation at 95 C for 30 s, annealing at 55 C for 30 s and extension at 72 C for 1 min, with extension repair at 72 C for 10 min. Amplified PCR products were analysed by agarose gel (1%) electrophoresis. After gel

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Table 1 Results of screening for the strains with high neutral proteinase and low lipase production. Strains

H/C

Oil hydrolysis circle or not

Neutral proteinase activity (U/mL)

Lipase activity (U/mL)

NCU101 NCU102 NCU103 NCU104 NCU105 NCU106 NCU107 NCU108 NCU109 NCU110 NCU111 NCU112 NCU113 NCU114 NCU115 NCU116 NCU117 NCU201 NCU202 NCU203 NCU301 NCU302 NCU303

4.3 3.6 3.8 3.6 4.9 3.7 3.9 3.6 4.5 3.8 4.6 4.2 3.9 4.1 3.9 5.2 4.8 4.2 2.3 3.1 2.1 2.9 2.6

N N N N N N N N N N N N N N N N N N N N N N N

480.9 ± 20.1b 663.0 ± 21.5d 950.2 ± 22.1g 571.6 ± 20.2c 89.0 ± 10.5a 732.1 ± 22.3e 1012.7 ± 21.5g 994.2 ± 56.5g 2751.6 ± 20.2l 1556.3 ± 21.7i 1007.7 ± 16.1g 146.5 ± 18.7a 776.4 ± 20.2e 2247.0 ± 20.1k 1657.2 ± 17.8j 4536.5 ± 20.2n 3511.5 ± 19.8m 1014.4 ± 18.7g 563.1 ± 14.4c 1415.1 ± 16.5h 622.0 ± 6.3cd 880.0 ± 9.5f 598.0 ± 10.2cd

e e e e e e e e 0.091 ± 0.015a e e e e e e 0.088 ± 0.006a 0.102 ± 0.008a e e e e e e

N indicates that no oil hydrolysis circle was observed. Strains NCU101-NCU117 were isolated from the waste residue produced in CCSKO production, strains NCU201-NCU203 were isolated from restaurant waste water, strains NCU301-NCU303 were isolated from sufu blank. Values represent the mean ± SD of three replicates. The different letters mean significant differences. aenSignificant difference at p < 0.05.

extraction, the products were purified by SanPrep pillar PCR purification kit, and sent to Shanghai Sangon Biotechnology Co. for DNA sequencing. 2.5. Effect of fermentation time on the activities of extracellular enzymes When the cultivation time was between 38 h and 48 h, it was sampled every 2 h. The fermentation liquid was centrifuged at 4250 g for 10 min, the supernatant obtained at different fermentation times contained the fermentation enzymes. The activities of the extracellular enzymes were measured at 40 C. The extracellular enzymes activities were determined under neutral conditions (pH ¼ 7), which was applicable to the aqueous enzymatic extraction technology. Then, the optimum fermentation times for the extracellular enzymes were determined. Pectinase activity was determined according to QB 1502-92 (1992). One unit of pectinase activity was defined as the amount of enzyme needed to produce 1 mg of galacturonic acid per hour under assay conditions. Glucoamylase activity was determined according to GB 8276-2006 (2006). One unit of glucoamylase activity was defined as the amount of enzyme produced 1 mg of glucose per hour at 40 C under assay conditions. Cellulase activity was determined using the method by Wood and Bha (1988) with a few modifications. One unit of cellulase activity was expressed as 1 mmol of glucose liberated per minute under assay conditions. Amylase activity was determined using the method of GB 8275-2009 (2009). One unit of amylase activity was defined as 1 g soluble starch liquefied per hour under assay conditions. 2.6. Characterization of extracellular enzymes Extracellular enzymes activities were measured under different pH values (3e11), different temperatures (35e65 C) and different incubation times (30e120 min). In addition, the activities of the extracellular enzymes in the presence of 0.01 mol/L metal ions

(Mn2þ, Mg2þ, Ca2þ, Cu2þ, Zn2þ, Fe2þ and Fe3þ) were measured. 2.7. Data analysis Data were expressed as the mean ± standard deviation (SD). Data were analysed using one way analysis of variance (ANOVA), followed by independent-sample t-test (Statistics programming software SPSS 19.0, Chicago, U.S.A.). A p value < 0.05 was considered to be statistically significant. 3. Results and discussion 3.1. Strains screening As shown in Table 1, 17 strains, designated as NCU101-NCU117, were selected from the waste residue of CCSKO production with Table 2 Physiological and biochemical characteristics of the strain NCU116. Biophysical and biochemical tests

NCU116

Bacillus amyloliquefaciens

Catalase Oxidase V.P Test Indole test Xylose L-Arabinose Mannitol Glucose gas Glucose produced acid Using citric acid Nitrite-reducing Amylolysis Gelatin liquefaction 7%NaCl development 50 C development

þ þ þ þ þ þ e þ þ

þ þ þ þ þ þ e v þ þ

“þ” indicates positive reaction, “” indicates negative reaction, “v” indicates unstable reaction between strains.


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H/C values higher than 3.5 and no oil hydrolysis circle. Three strains, designated as NCU201-NCU203, were chosen from restaurant waste water with H/C values greater than 2.0 and no oil hydrolysis circle. Three strains, designated as NCU301NCU303, were obtained from the sufu blank with H/C values greater than 2.0 and no oil hydrolysis circle. Three strains (NCU109, NCU116, NCU117) with high proteinase activities (>2500.0 U/mL) and low lipase activities (