Antibacterial Activity of Free Fatty Acids from Hydrolyzed Virgin Coconut Oil Using Lipase from Candida rugosa

Hindawi Journal of Lipids Volume 2017, Article ID 7170162, 7 pages https://doi.org/10.1155/2017/7170162 Research Article Antibacterial Activity of Free Fatty Acids from Hydrolyzed Virgin Coconut Oil Using Lipase from Candida rugosa Van Thi Ai Nguyen,1,2 Truong Dang Le,1 Hoa Ngoc Phan,2 and Lam Bich Tran2 1 Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City, Ho Chi Minh City, Vietnam Department of Food Technology, Faculty of Chemical Engineering, Ho Chi Minh City University of Technology, Ho Chi Minh City, Vietnam 2 Correspondence should be addressed to Van Thi Ai Nguyen; Received 1 August 2017; Revised 20 September 2017; Accepted 2 October 2017; Published 13 November 2017 Academic Editor: Maurizio Averna Copyright © 2017 Van Thi Ai Nguyen et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Free fatty acids (FFAs) were obtained from hydrolyzed virgin coconut oil (VCO) by Candida rugosa lipase (CRL). Four factors’ influence on hydrolysis degree (HD) was examined. The best hydrolysis conditions in order to get the highest HD value were determined at VCO to buffer ratio 1 : 5 (w/w), CRL concentration 1.5% (w/w oil), pH 7, and temperature 40∘ C. After 16 hours’ reaction, the HD value achieved 79.64%. FFAs and residual hydrolyzed virgin coconut oil (HVCO) were isolated from the hydrolysis products. They were tested for their antibacterial activity against Gram-negative and Gram-positive bacteria, which can be found in contaminated food and cause food poisoning. FFAs showed their inhibition against Bacillus subtilis (ATCC 11774), Escherichia coli (ATCC 25922), Salmonella enteritidis (ATCC 13076), and Staphylococcus aureus (ATCC 25923) at minimum inhibitory concentration (MIC) of 50%, 60%, 20%, and 40%, respectively. However, VCO and HVCO did not show their antibacterial activity against these tested bacteria. 1. Introduction VCO is extracted from fresh kernel by using either cold press or centrifuge process. It does not go through refined, bleached, and deodorized process (RBD). Therefore, its physical properties as flavor, color, and so forth are less changed than RBD oil. VCO has many advantages in skin care, promotes the growth of hair, and enhances the beauty. Antioxidant activity and phenolic compounds in VCO also was conducted by some studies; it was suggested that the consumption of food containing phenolic compound will have a positive contribution in health [1]. Aside from benefits above, VCO is also good in health promotion and prevents some diseases because of the presence of FFAs in VCO. FFAs in VCO are rich in medium chain fatty acids (MCFAs) in which lauric acid takes the highest percentage about 46–48%. MCFAs in VCO are easily digested and absorbed, but fat is harder because it contains long chain fatty acids which need going through circulatory system before absorbing, so VCO can be used to replace cooking oil in daily meal to improve digestion. Moreover, MCFAs are also good for obese people because they increase energy expenditure more than usual. And MCFAs are directly absorbed from the intestine and burned in the liver; this makes them have a feeling which is always early satiety, and weight is decreased [2]. And also absorbing and burning directly in liver make MCFAs not take part in biosynthesis and transport of cholesterol. Thus, MCFAs in VCO have cardioprotective ability [3]. MCFAs also showed antifungal activity; Shino and coworkers (2016) exhibited a comparison of antimicrobial activity of chlorhexidine, coconut oil, probiotics, and ketoconazole on Candida albicans isolated in children with early childhood caries [4]. Parfene and coworkers (2013) gave a result about antifungal activity against Yarrowia lipolytica of MCFAs from crude coconut oil [5]. MCFAs have effective ability to inhibit some species of virus by breaking their membranes [3]. And antibacterial activity of MCFAs was also conducted by some previous studies; Kim and Rhee (2016) presented that MCFAs 2 Journal of Lipids were antibacterial agents against Escherichia coli [6]. Shilling and coworkers (2013) also studied antimicrobial effect of VCO and MCFAs against Clostridium difficile [7]. MCFAs are antibacterial agents; this was demonstrated by previous studies, but MCFAs used in their studies were in form of pure chemical. Therefore, the aim of this study was to use FFAs extracted from hydrolyzed VCO and evaluate their antibacterial against Bacillus subtilis (ATCC 11774), Escherichia coli (ATCC 25922), Salmonella enteritidis (ATCC 13076), and Staphylococcus aureus (ATCC 25923), which can be found in food and cause food poisoning. At the same time, the resistance of VCO and HVCO against these tested bacteria was also evaluated. 2. Materials and Methods 2.1. Materials. VCO was sponsored by Luong Quoi Coconut Co., Ltd. (Ben Tre Province, Vietnam). Candida Rugosa lipase (CRL) (Type VII, ≥700 unit/mg solid) was purchased from Sigma-Aldrich Co. (Canada). Chemicals used in this study were KOH, n-hexane, and iso-octane and all other chemicals from Merck (Germany) and China were analyzed with purification more than 95%. Mediums used in antibacterial test were Nutrient Broth (NB) (Italy), Mueller Hinton Agar (MHA) from HiMedia Laboratories Pvt. Ltd (India), and Mueller Hinton Broth (MHB) from Titan Biotech Ltd (India). And four types of bacteria used in this study were Bacillus subtilis (ATCC 11774), Escherichia coli (ATCC 25922), Salmonella enteritidis (ATCC 13076), and Staphylococcus aureus (ATCC 25923) provided by Microbiologics Co. (USA). Devices used in this study were high speed homogenizer (IKA T25 digital ULTRA-TURRAX, Germany), overhead stirrer (OS20, USA), orbital shaker incubator (LM-2575RD) from Yihder Technology Co. (Taiwan), evaporator (IKA RV digital V) from Germany, and GC-FID SHIMADZU 2010 Plus (Japan). 2.2. Hydrolysis of VCO. VCO dissolved in iso-octane (VCO to solvent ratio 1 : 1 (w/w)) and phosphate buffer solution to adjust pH condition was placed in a 250 mL Erlenmeyer flask [8]. Emulsification of the mixture was carried out by using a stirrer at speed of 10000 rpm in 15 minutes; then the appropriate amount of lipase was added and dissolved by stirring at speed of 350 rpm in 5 minutes. The reaction was conducted in orbital shaker incubator at speed 150 rpm for 2 hours. To stop the reaction, add 1 ml ethanol 99.5% into the erlenmeyer flask. The hydrolysis degree (HD) was calculated as the following formula [9]: HD = 𝑉KOH ∗ 𝑀KOH ∗ 𝑀FFAs (%) , 10 ∗ 𝑚 (1) where 𝑉KOH is the volume of potassium hydroxide (KOH) titrated (mL), 𝑀KOH is the molarity of KOH solution (mol/L), 𝑀FFAs is the average molecular weight of free fatty acids, and 𝑚 is mass of VCO (g). 2.3. Obtaining FFAs. The process was carried out according to the method of Shimada and coworkers (1998) [10]. (...truncated)