Antibacterial Activity of Hydrolized Palm Kernel Oil and Red Palm Super Olein Blend against Gram negative and Gram-positive Bacteria

Antibacterial Activity of Hydrolized Palm Kernel Oil and Red Palm Super Olein Blend against Gram negative and Gram-positive Bacteria | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Antibacterial Activity of Hydrolized Palm Kernel Oil and Red Palm Super Olein Blend against Gram negative and Gram-positive Bacteria Ilmi Fadhilah Rizki, Frisda Rimbun Panjaitan, Manda Edy Mulyono, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6181954/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract This study investigated the antibacterial activity of a novel compound, Hydrolyzed Palm Kernel Oil and Red Palm Super Olein Blend (HPRB), against Gram-negative ( Escherichia coli , Salmonella typhi ) and Gram-positive ( Staphylococcus aureus ) bacteria. HPRB was synthesized through enzymatic hydrolysis with four different ratios of palm kernel oil (PKO) and red palm super olein (RPSO). The antibacterial activity was evaluated using disc diffusion and microdilution methods. The results showed that HPRB-C (60% PKO, 40% RPSO) and HPRB-D (80% PKO, 20% RPSO) exhibited the highest antibacterial activity against all bacteria tested, attributed to the high concentration of 1-monolaurin (17.54% and 24.99%, respectively). The phytonutrient content also contributed to the antibacterial activity of HPRB. HPRB has the potential to be used as a natural antibacterial agent. Biological sciences/Microbiology Biological sciences/Microbiology/Antimicrobials Antibacterial activity Red palm super olein palm kernel oil monolaurin phytonutrients Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction The escalating prevalence of foodborne illnesses and the alarming rise of antibiotic-resistant bacteria pose a significant threat to global health (M et al., 2020). The World Health Organization (WHO) estimates that millions fall ill each year from foodborne pathogens, with the Southeast Asia region alone experiencing a staggering 150 million cases and over 175,000 deaths annually (World Health Organization, 2016 ). The impact of these illnesses is particularly devastating for vulnerable populations such as young children, the elderly, and the immunocompromised, often leading to severe complications and exacerbating malnutrition (Tao et al., 2022 ). The situation is further compounded by the limitations of current treatments, including antibiotics, due to the emergence of multidrug-resistant pathogens (Wu & Zeng, 2024 ). The urgent need for novel and sustainable antibacterial agents has spurred extensive research into natural products, particularly those derived from plants. These plant-based antimicrobials offer several advantages over conventional antibiotics, including a broader spectrum of activity, reduced susceptibility to resistance development, and fewer side effects (Arip et al., 2022 ). The ability of plant-derived compounds to target multiple bacterial components simultaneously, such as cell membranes, protein synthesis, and DNA replication, contributes to their efficacy and reduces the likelihood of resistance emergence (Li et al., 2024 ). Furthermore, Kouitcheu et al. ( 2013 ) observed the synergistic effects when combining plants with antibiotics enhanced antibacterial activity, indicating the potential for plant-derived compounds to complement existing treatment strategies. Cheong et al. ( 2022 ) also emphasized the importance of exploring natural products, including plants, as sources of novel antibacterial agents to address the growing challenge of bacterial resistance. Palm oil and its derivatives have garnered significant attention among the various natural products explored for their antimicrobial potential (Habibiasr et al., 2022 ; Hovorková et al., 2018 ). Palm kernel oil (PKO), a widely available and sustainable resource, is rich in medium-chain fatty acids (MCFAs) and is known for its broad-spectrum antibacterial activity. In their research, Laloučková et al. (2019) found that PKO has potential antibacterial effects against Gram-positive bacteria, including those causing mastitis. Ustadi et al. ( 2022 ) research study showed that waste palm cooking has an in vitro antibacterial effect against Staphylococcus aureus. Furthermore, the latest palm oil derivative, red palm super olein (RPSO), contains concentrated phytonutrients, including carotene and vitamin E, which may contribute to their antimicrobial properties (Mulyono et al., 2023 ). Building upon this foundation, the present study focuses on a novel compound, Hydrolyzed PKO-RPSO Blend (HPRB), created by combining PKO and RPSO through hydrolysis. The unique composition of HPRB, encompassing MCFAs, acylglycerols, and phytonutrients, presents a promising avenue for developing a potent and broad-spectrum antibacterial agent. This research aims to comprehensively analyze the composition of HPRB and evaluate its antibacterial activity against common human pathogens, including Staphylococcus aureus , Escherichia coli , and Salmonella typhi . Furthermore, the study seeks to identify the specific component within HPRB that is responsible for its antibacterial effects, providing valuable insights for future development and applications. By elucidating HPRB's antibacterial potential and key components, this study contributes to the growing body of knowledge on natural antimicrobials. The findings may pave the way for developing novel, effective, and sustainable strategies to combat foodborne illnesses and address the pressing challenge of antibiotic resistance. 2. Materials and Methods 2.1 Chemicals The RPSO was manufactured in the Oleofood Laboratory of the Indonesian Oil Palm Research Institute (IOPRI), Medan (Mulyono et al., 2023 ). First, the PKO was obtained from a local palm oil mill in North Sumatra, Indonesia. The chemicals used in this process include the immobilized lipase (Novozymes (N435), Denmark), Mueller Hinton Agar (MHA), Mueller Hinton Broth (MHB), and 2,3,5-triphenyl tetrazolium chloride (TTC) (Himedia Laboratories PVT, Mumbai, India). The TTC (1.25%) was prepared with sterile distilled water and stored in sterile flasks covered with aluminum paper at 2 to 8 o C. Tween-80 (Sigma-Aldrich, St. Louis, MO, USA) was used as a solvent. 2.2 Microorganisms The antibacterial activity of samples alone and in combination with antibiotics such as vancomycin and penicillin was tested against three bacterial strains, E. coli ( ATCC 25922), S. aureus ( ATCC 25923), and S. typhi ( ATCC 1408), purchased from the Faculty of Medicine Sumatera Utara University (North Sumatra, Indonesia). The inoculum suspension was obtained from the 24 h culture colonies and suspended in 0.9% sodium chloride (NaCl) sterile aqueous solution. The density was adjusted to 0.5 McFarland standard (10 8 colony-forming unit [CFU]/mL) turbidity. 2.3 Production of hydrolyzed PKO and RPSO blend Lipase-catalyzed hydrolysis produced Hydrolized PKO and RPSO Blend (HPRB) (Fig. 1 ). The HPRB formulations were prepared by mixing PKO and RPSO in four different combinations: HPRB - A (20% PKO, 80% RPSO), HPRB - B (40% PKO, 60% RPSO), HPRB - C (60% PKO, 40% RPSO), and HPRB - D (80% PKO, 20% RPSO). The hydrolysis involved blending PKO and RPSO according to the respective ratio with 8% Novozymes 435 and water at 10% (w/w) relative to the oil. The reactants were mixed via magnetic stirring, and the reaction was maintained at 50 o C. After 48 h, the result was filtered to remove the catalysts, and monolaurin-rich HPRPB was obtained. The monolaurin-rich HPRB was then characterized, including fatty acid, acylglycerol, phytonutrient composition, and lipase residue. 2.4 Characterization of HPRB Fatty acids composition The fatty acid composition was determined as fatty acid methyl esters via Gas Chromatography-2010 (Shimadzu, Japan) and a flame ionization detector (FID) equipped with a Shimadzu AOC-20i autosampler (Shimadzu, Japan). The fatty acid composition used DB-23 gas chromatography column (Agilent, USA) with increasing oven temperature from 90 to 208 ◦C (7 ◦C/min) and maintained at 208 ◦C for 5 min. The injector was set to 260 ◦C, and the nitrogen gas column flow was set at 1 mL/min. Acylglycerol composition The acylglycerol composition was determined using the Shimadzu GCMS-QP2010 Plus (Shimadzu, Japan) equipped with a Shimadzu AOC-20i auto-injector (Shimadzu, Japan), an Agilent DB-5HT column (Agilent, USA), and a helium mobile phase at 100 psi. The injector was set at 325°C, while the interface and ion source were at 280°C. The oven temperature was initially set at 100°C for 1 min, increased to 223°C at 30°C/min, and later 1°C/min to 227°C. The final increase in temperature was at a rate of 5°C/min to 360°C for 10 min. Samples were prepared by silyl derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA). Squalene Squalene was analyzed using Shimadzu GCMS-QP2010 Plus (Shimadzu, Japan) equipped with an Agilent HP-5MS column (Agilent, USA). Samples were prepared by saponification in the presence of ascorbic acid. The unsaponifiable fraction was injected into the GC–MS at 1:50 ratio split injection mode. The helium gas was used at a pressure of 100 psi, while the injector, interface, and ion source temperatures were set at 325°C, 280°C, and 250°C, respectively. The oven temperature was initially set at 50°C for 2 minutes and increased at a rate of 10°C/minute to 300°C for 5 minutes. Total carotene The HPRB sample was dissolved in hexane and analyzed using a Shimadzu UV-1700 UV–Visible spectrophotometer (Shimadzu, Japan) at 446 nm. The total carotene was calculated as follows: $$\:Total\:carotene\:\left(ppm\right)=\frac{383\times\:volume\times\:absorbance\:at\:446\:nm}{mass\:of\:sample\times\:100}$$ Vitamin E composition Vitamin E was analyzed with ultra-performance liquid chromatography (UPLC) (Waters 600 system, USA), equipped with an autosampler and ultraviolet (UV) detector. The analysis was performed in the Inertsil ODS-3 column (Inertsil, USA) with methanol as the mobile phase with a 1 mL/min flow rate. The injection volume was 20 µL, and the UV absorbance was measured at 292 nm. 2.5 Disc Diffusion Assay of Antibacterial Activity The anti-bacterial activity for all HPRB combinations was determined using the disc diffusion method, and the best formula against E. coli ( ATCC 25922), S. aureus ( ATCC 25923), and S. typhi ( ATCC 1408) were identified. The HPRB combinations were adjusted to several concentrations, 10% (250 mg/ml), 30% (500 mg/ml), and 50% (1000 mg/ml), using Tween 80. The bacterial suspension (0.1 ml) was placed in a 10 mL agar medium test tube and homogenized to achieve 0.5 McFarland unit turbidity (1.5 x 10 8 CFU/mL). The suspension was then spread over a petri dish containing solidified MHA using a hockey stick and allowed to dry. Sterilized discs (diameter: 6 mm) were dipped into each HPRB formulation and placed on the agar surface inoculated with the respective bacteria. Subsequently, the petri dishes were incubated at 37 o C for 48 h. Tween 80 was used as a negative control, and two standard antibiotics, penicillin (10 mcg/disc) and vancomycin (30 mcg/disc) acted as the positive controls. The HPRB anti-bacterial activity was determined based on the diameter of the inhibition zone around the disc. Each experiment was carried out in triplicates, and the results obtained were subjected to an analysis of variance (ANOVA) test in the Statistical Package for Social Sciences (SPSS) version 25 (IBM, USA). The confidence level was set at p < 0.05 2.6 Determination of Minimum Inhibitory Concentration (MIC) Minimum Inhibitory Concentration (MIC) was determined using a microdilution test in 96-well plates according to the National Committee for Clinical Laboratory Standards with modification. Successive dilutions half ranging of HPRB and antibiotics were prepared by adding 50 µL of Mueller-Hinton broth (MHB) to achieve concentrations between 2 and 1000 mg/ml for HPRB and 0.5 to 250 mg/ml for the antibiotics in the wells. Each well's bacterial suspension density (50 µL) was 10 6 CFU/mL, prepared by diluting 0.5 McFarland suspension 100 times. The 96-well plates were prepared in triplicates, covered, and sealed tightly to prevent evaporation, and incubated at 37°C for 22 h. The bacterial growth was visualized by adding 20 µl of 2,3,5-triphenyl tetrazolium chloride (0.125%) solution to each well and incubated for two hours to observe the color change. 2.7 Bacterial Staining Assay After different treatment from the Minimum Inhibitory Concentration (MIC) procedure, the bacteria suspensions were transferred gently to the microscope slide. The slide is then heated over a gentle flame to help the cell adhesion to the glass slide and prevent the loss of bacteria during rinsing. The gram staining started with an added crystal violet stain over the slide. After 5 minutes, the stain is poured off, and the excess stain is rinsed with tap water. The iodine solution was poured off for 1 minute over the slide and rinsed with tap water. A few drops of decolorizer (mixed solvent of ethanol and acetone) were added to the slide for 10 seconds and rinsed with tap water. For the last step, the slide is counterstained with fuchsin solution for 40–60 seconds and washed off with tap water. The slide was then air-dried and examined under a microscope under oil immersion. 3 Result and Discussion 3.1 Characterization of HPRB 3.1.1 Fatty Acid Composition of HPRB The fatty acid compositions of HPRB formulations, PKO, and RPSO were analyzed using gas chromatography with flame ionization detection (GC/FID) (Table 1 ). PKO, derived from the palm kernel, is rich in medium-chain fatty acids (MCFAs), notably lauric acid (C12:0). In contrast, RPSO is characterized by a high content of long-chain fatty acids (LCFAs), particularly oleic acid (C18:1) and palmitic acid (C16:0). RPSO is derived from CPO, initially made into red palm olein (RPO), and fractionated through dry fractional crystallization to obtain a red palm super olein (RPSO) consisting of high oleic acid and low palmitic acid. Dry fractional crystallization was carried out by temperature adjustment without additives, concentrating the natural phytonutrients contained in CPO, such as carotene, vitamin E, and squalene (Table 3 ) (Mulyono et al., 2023 ). Table 1 Fatty acid composition (wt.%) of HPRB, PKO, and RPSO Composition HPRB - A HPRB - B HPRB - C HPRB - D PKO (E) RPSO (F) C6:0 - - 0.10 ± 0.00 0.15 ± 0,00 0.19 ± 0,00 - C8:0 0.60 ± 0.00 1.17 ± 0.00 1.82 ± 0.01 2.42 ± 0.16 3.16 ± 0.00 - C10:0 0.57 ± 0.00 1.14 ± 0.01 1.77 ± 0.02 2.38 ± 0.13 3.06 ± 0.00 - C12:0 8.95 ± 0.01 18.52 ± 0.02 28.61 ± 0.25 39.06 ± 0.84 49.78 ± 0.07 0.17 ± 0.00 C14:0 3.61 ± 0.00 6.52 ± 0.00 9.55 ± 0.02 12.91 ± 0.06 16.02 ± 0.01 0.89 ± 0.02 C16:0 30.98 ± 0.05 25.78 ± 0.05 20.32 ± 0.15 14.27 ± 0.32 8.21 ± 0.02 35.91 ± 0.15 C16:1 0.14 ± 0.00 0.12 ± 0.01 0.08 ± 0.00 - - 0.17 ± 0.00 C18:0 3.58 ± 0.00 3.27 ± 0.00 2.93 ± 0.01 2.58 ± 0.07 2.15 ± 0.00 3.87 ± 0.01 C18:1 39.64 ± 0.06 33.98 ± 0.00 27.93 ± 0.10 21.72 ± 0.57 15.05 ± 0.02 44.97 ± 0.13 C18:2 11.23 ± 0.01 8.91 ± 0.01 6.41 ± 0.03 4.11 ± 0.10 2.16 ± 0.00 13.23 ± 0.03 C18:3 0,23 ± 0.00 0.17 ± 0.00 0.11 ± 0.00 0.06 ± 0.00 - 0.28 ± 0.00 C20:0 0.28 ± 0.01 0.24 ± 0.00 0.19 ± 0.00 0.15 ± 0.00 0.08 ± 0.00 0.33 ± 0.00 C20:1 0.14 ± 0.00 0.12 ± 0.00 0.10 ± 0.00 0.09 ± 0.00 0.08 ± 0.00 0.14 ± 0.00 ΣSFA 48.59 ± 0.08 56.67 ± 0.00 65.34 ± 0.15 73.95 ± 0.68 82.69 ± 0.02 41.19 ± 0.16 ΣUSFA 51.40 ± 0.08 43.32 ± 0.00 34.65 ± 0.15 25.99 ± 0.68 17.30 ± 0.02 58.80 ± 0.16 ΣMUFA 39.93 ± 0.06 34.22 ± 0.00 28.12 ± 0.11 21.82 ± 0.57 15.14 ± 0.02 45.29 ± 0.13 ΣPUFA 11.46 ± 0.01 9.09 ± 0.01 6.53 ± 0.04 4.17 ± 0.10 2.16 ± 0.00 13.51 ± 0.03 Results are reported as mean ± SD, n = 3 As shown in Table 1 , lauric acid (49.78%) was the predominant fatty acid in PKO, followed by myristic acid (16.02%). In contrast, RPSO contained the highest amount of oleic acid (44.97%), followed by palmitic acid (35.91%). The highest SFA content was observed in PKO (82.69%), followed by HPRB-D (73.95%), HPRB-C (65.34%), HPRB-B (56.67%), HPRB-A (48.59%), and RPSO (41.19%). Conversely, unsaturated fatty acid (USFA) and polyunsaturated fatty acid (PUFA) compositions were most abundant in RPSO and least abundant in PKO. The highest monounsaturated fatty acid (MUFA) content was found in RPSO (45.39%), followed by HPRB-A (39.93%), HPRB-B (34.22%), HPRB-C (28.12%), HPRB-D (21.82%), and PKO (15.14%). The fatty acid composition of the HPRB formulations was influenced by the relative proportions of PKO and RPSO used in their production. As the proportion of PKO increased in HPRB, the levels of lauric (C12:0) and myristic (C14:0) acids also increased, ranging from 8.95–39.06% and 3.61–12.91%, respectively. Conversely, an increase in the RPSO level in HPRB resulted in a corresponding increase in oleic (C18:1) and palmitic (C16:0) acids, ranging from 21.72–39.64% and 14.27–30.98%, respectively. The varying fatty acid profiles of these oils are expected to influence the antimicrobial properties of the resulting HPRB formulations. Medium-chain fatty acids (MCFAs), particularly lauric and myristic acids have been shown to possess broad-spectrum antibacterial activity against various bacteria, including human pathogens bacteria (Casillas-Vargas et al., 2021 ; Desbois & Smith, 2010 ; Huang et al., 2012 ; Matsue et al., 2019 ; Okukawa et al., 2021 ). Therefore, in this study, HPRB was hydrolyzed enzymatically, incorporating both PKO and RPSO, to enrich the composition of fatty acids and their corresponding acyglycerols composition, to enhance the potential antimicrobial activity of these formulations. 3.1.2 Acyglycerol Composition of HPRB The HPRB production process resulted in a unique acylglycerol profile that sets it apart from the original raw materials, PKO and RPSO. This distinction in composition significantly impacts the anti-bacterial properties of the various HPRB formulations. The analysis of the acylglycerol composition, as presented in Table 2 , highlights the diversity in the distribution of different acylglycerol classes across the HPRB formulations. Table 2 Acylgycerol composition (wt.%) of HPRB, PKO, and RPSO Acylglycerol species b HPRB A HPRB B HPRB C HPRB D PKO (E) RPSO (F) FFA P 4.42 ± 0.01 7.04 ± 0.19 6.75 ± 0.27 6.11 ± 0.30 ND ND O 9.82 ± 0.44 0.59 ± 0.13 1.52 ± 0.14 3.11 ± 0.06 ND 0.53 ± 0.12 S 0.70 ± 0.02 1.44 ± 0.14 1.36 ± 0.09 ND ND ND Total 14.94 ± 0.47 9.07 ± 0.20 9.63 ± 0.20 9.21 ± 0.36 0 0.53 ± 0.12 MAG -Ca- ND ND ND 0.53 ± 0.06 ND ND Ca-- ND ND ND 2.20 ± 0.59 ND ND -La- ND 0.79 ± 0.06 1.76 ± 0.39 2.30 ± 0.10 ND ND La-- ND 16.95 ± 1.15 17.54 ± 0.72 24.69 ± 1.10 ND ND -M- ND ND ND 0.52 ± 0.03 ND ND M-- 1.15 ± 0.09 2.22 ± 0.02 3.61 ± 0.14 5.91 ± 0.12 ND ND P-- 0.80 ± 0.08 1.36 ± 0.05 1.10 ± 0.07 2.67 ± 0.24 ND ND O-- 1.63 ± 0.14 3.17 ± 0.06 4.70 ± 0.23 5.43 ± 1.18 ND 0.40 ± 0.02 S-- ND 0.29 ± 0.02 0.58 ± 0.03 0.68 ± 0.16 ND ND Total 3.58 ± 0.15 24.79 ± 1.36 30.18 ± 1.57 44.93 ± 3.09 0 0.40 ± 0.02 DAG La-Cp ND ND ND 1.27 ± 1.27 ND ND La-Ca ND 0.61 ± 0.05 0.99 ± 0.05 1.75 ± 0.13 ND ND LaLa- 0.62 ± 0.04 1.52 ± 0.01 2.75 ± 0.13 3.93 ± 0.01 0.15 ± 0.00 ND La-La 1.22 ± 0.01 3.52 ± 0.17 6.81 ± 0.01 8.90 ± 0.41 0.44 ± 0.03 ND LaM- ND 0.49 ± 0.02 1.05 ± 0.01 1.78 ± 0.20 ND ND La-M ND 1.24 ± 0.03 2.33 ± 0.16 3.20 ± 0.61 0.14 ± 0.02 ND La-P ND 0.67 ± 0.01 1.77 ± 0.01 2.46 ± 0.32 0.05 ± 0.05 ND LaO- ND 0.33 ± 0.02 0.71 ± 0.07 1.12 ± 0.10 ND ND P-P ND ND ND ND ND 0.12 ± 0.12 P-O ND ND ND ND ND 1.18 ± 0.11 O-O ND ND ND ND ND 0.85 ± 0.19 Total 1.83 ± 0.06 8.37 ± 0.20 16.39 ± 0.43 24.40 ± 1.94 0.78 ± 0.00 2.14 ± 0.18 TAG LaLaCo ND ND ND ND 0.71 ± 0.06 ND LaLaCp ND 1.11 ± 0.01 2.18 ± 0.00 2.78 ± 0.02 6.61 ± 0.34 ND LaLaCa ND ND ND ND 8.90 ± 0.11 ND LaLaLa 2.28 ± 0.03 2.53 ± 0.15 2.10 ± 0.30 2.57 ± 0.75 27.72 ± 0.35 ND LaLaM 1.02 ± 0.00 1.34 ± 0.04 1.78 ± 0.35 1.83 ± 0.01 20.58 ± 0.15 ND LaLaP ND 0.17 ± 0.17 0.65 ± 0.01 1.07 ± 0.18 10.37 ± 0.02 ND LaLaO ND 0.31 ± 0.05 0.64 ± 0.02 0.47 ± 0.47 2.71 ± 0.04 ND LaMP ND ND ND ND 5.71 ± 0.04 ND LaMO ND ND ND 0.30 ± 0.30 2.97 ± 0.04 ND LaMS ND ND ND ND 2.04 ± 0.00 ND LaOP ND ND ND ND 3.52 ± 0.14 ND PPP 0.50 ± 0.04 0.52 ± 0.06 0.57 ± 0.20 0.26 ± 0.2 4.07 ± 0.25 0.10 ± 0.25 POP 20.19 ± 1.11 12.83 ± 0.33 9.53 ± 0.06 3.68 ± 0.40 1.27 ± 0.04 24.96 ± 1.54 POO 45.11 ± 1.21 32.02 ± 0.56 20.57 ± 1.77 7.90 ± 1.02 1.07 ± 0.05 56.51 ± 1.85 OOO 10.55 ± 0.49 6.95 ± 0.73 4.89 ± 0.09 0.59 ± 0.59 0.97 ± 0.09 14.47 ± 0.03 Total 79.64 ± 0.67 57.77 ± 1.37 43.80 ± 2.21 21.46 ± 0.78 99.23 ± 0.00 96.93 ± 0.04 Co = caproate (C6:0), Cp = caprylate (C8:0), Ca = caprate (C10:0), La = laurate (C12:0), M = myristate (C14:0), P = palmitate (C16:0), O = oleate (C18:1), S = stearate (C18:0), ND not detected. a Mean ± standard deviation ( n = 3) b Some acylglycerols might be undetected due to limit of detection. Table 3 Phytonutrient composition (mg/kg) of HPRB, PKO, and RPSO Composition HPRB – A HPRB - B HPRB - C HPRB - D PKO (E) RPSO (F) Total carotene 604.08 ± 0.74 452.69 ± 1.42 303.29 ± 1.24 165.05 ± 2.33 2.18 ± 1.07 761.23 ± 1.64 Total vitamin E 867.54 ± 3.10 608.19 ± 10.29 251.32 ± 2.51 78.61 ± 1.63 15.60 ± 1.65 1118.17 ± 7.24 δ-Tocotrienol 142.64 ± 0.88 131.74 ± 1.70 102.46 ± 2.38 49.36 ± 0.43 15.60 ± 1.65 167.11 ± 2.60 γ-Tocotrienol 438.12 ± 0.86 336.39 ± 4.95 148.85 ± 2.85 29.24 ± 2.06 - 549.16 ± 2.43 α-Tocotrienol 230.57 ± 1.70 115.16 ± 6.78 - - - 313.02 ± 1.43 α-Tocopherol 56.21 ± 0.62 24.88 ± 0.40 - - - 88.87 ± 2.74 Squalene 178.5 ± 3.53 131 ± 2.82 87.5 ± 2.12 51 ± 1.41 23.5 ± 2.12 257.5 ± 17.67 Results are reported as mean ± SD, n = 3 HPRB-A stands out with the highest concentration of free fatty acids (14.94%) and TAGs (79.64%). In contrast, HPRB-D displays a markedly different profile, rich in MAGs (44.93%) and DAGs (24.40%). The abundance of these monoacylglycerols might contribute to enhanced anti-bacterial activity, as certain MAGs are known to disrupt bacterial cell membranes (Jackman et al., 2016 ). HPRB-C also shows a notable presence of MAGs (30.18%) and DAGs (16.39%), suggesting it may share some of the anti-bacterial properties observed in HPRB-D. The free fatty acid composition of HPRB is primarily characterized by the presence of palmitic, oleic, and stearic acids. The MAG fraction in HPRB is mainly composed of 1-monolaurin and 1-monomyristin. Notably, HPRB-D boasts the highest levels of both 1-monolaurin (24.69%) and 1-monomyristin (5.91%), while HPRB-C contains the second-highest amount of 1-monolaurin (17.54%). The prevalence of 1-monolaurin is particularly noteworthy due to its potent anti-bacterial (Ngatirah et al., 2022 ). The formation of 1-monolaurin and 1-monomyristin in HPRB is attributed to the partial hydrolysis of PKO and RPSO during the lipase-catalyzed reaction. This process selectively cleaves ester bonds in triglycerides, leading to the generation of MAGs. The specific composition of MAGs is influenced by the fatty acid profile of the starting materials and the selectivity of the lipase enzyme (Monteiro et al., 2003 ; Subroto, 2020 ; Zeng et al., 2010 ). 3.1.3 Phytonutrient Composition of HPRB The raw materials used to synthesize HPRB have limited nutrients. The PKO lacks phytonutrients, while RPSO has low MCFA. Thus, combining PKO and RPSO potentially increases the composition of MCFA and phytonutrients in the HPRB. The resulting HPRB in this study exhibited changes in the MCFA and phytonutrient contents, and MAG was present as the fatty acid. Table 3 details the phytonutrient composition of each HPRB formulation, PKO, and RPSO, consisting of total carotene, vitamin E and its isomers, and squalene. The RPSO has the highest phytonutrient among the oils: total carotene = 761.23 ppm, total vitamin E = 1118.17 ppm, and squalene = 257.5 ppm. Moreover, RPSO has low palmitic acid (saturated fatty acid) and high oleic acid (unsaturated fatty acid), as shown in Table 2 . The double bond presence is attributed to the high solubility of unsaturated fats. RPSO is the primary source of phytonutrients in HPRB synthesis; thus, phytonutrients composition increases with higher RPSO inclusion in the formulation. The antibacterial activity of carotenoids is often linked to their ability to modulate oxidative stress. The bioactive properties of carotenoids are not limited to their antioxidant effects; they also exhibit direct antibacterial activity. For example, studies have shown that carotenoids can inhibit the growth of both Gram-positive and Gram-negative bacteria, suggesting a broad spectrum of antimicrobial action (Karpiński & Adamczak, 2019 ). In addition to their antioxidant and antibacterial properties, carotenoids have been implicated in various therapeutic applications. The extraction and application of carotenoids in food and pharmaceutical industries are also gaining traction. In this research, the synergy between carotenoids and other bioactive compounds, such as lauric acid in monoacylglycerol composition, can further amplify their health benefits, including enhanced antibacterial effects Another research also indicates that tocotrienols can modulate immune responses, which may enhance the ability to combat bacterial infections. For instance, tocotrienol treatment has been shown to influence the expression of pro-inflammatory cytokines in macrophages, suggesting a role in the regulation of inflammatory responses that are critical during bacterial infections (Qureshi et al., 2010 ). This immunomodulatory effect may be particularly beneficial in managing infections caused by multi-drug resistant pathogens, as tocotrienols have demonstrated the ability to inhibit the growth of both Gram-positive and Gram-negative bacteria, including Staphylococcus aureus (Hartmann et al., 2020 ). This suggests that tocotrienols may not only support the immune system but also exert direct antimicrobial effects, making them a promising candidate for the development of natural antibacterial agents. Tocotrienols have been suggested to work synergistically with carotenoids enhancing their antibacterial effects and providing a multi-faceted approach to infection management. Furthermore, their antioxidant properties may help to mitigate oxidative stress associated with bacterial infections, thereby protecting host tissues and improving overall health outcomes (Shrum et al., 2023 ) 3.1.4 Anti-Bacterial Activity of HPRB The antibacterial activities of the HPRB formulations were evaluated by measuring the inhibition zone diameters (Table 4 ). The most effective formulations were HPRB-C and HPRB-D, which exhibited the largest inhibition zones against all three bacterial strains. Specifically, HPRB-C (500 mg/ml) displayed inhibition zones of 20.4 mm, 10.85 mm, and 14.52 mm against S. aureus , E. coli , and S. typhi . Similarly, HPRB-D (250 mg/ml) showed inhibition zones of 19.47 mm, 14.72 mm, and 13.87 mm against the same strains. The minimum inhibitory concentrations (MICs) for HPRB-C and HPRB-D were also lower than those for HPRB-A (1000 mg/ml; 14.15 mm, 13.57 mm, and 10.77 mm) and HPRB-B (1000 mg/ml; 16.4 mm, 9.12 mm, and 13.7 mm), further supporting their superior antibacterial activity. The raw materials, PKO (palm kernel oil) and RPSO (refined, bleached, and deodorized palm stearin oil), showed either no inhibition (NI) or significantly smaller inhibition zones (PKO: 8.65 mm, 8.22 mm, 12.1 mm; RPSO: 8 mm, 8.55 mm, 9.85 mm) compared to the HPRB formulations, highlighting the enhanced antibacterial properties achieved through the formulation process. Table 4 Inhibition zone diameter and minimum inhibitory concentration of HPRB Sample Staphylococcus aureus Escherichia coli Salmonella typhi Inhibition diameter (mm) MIC (mg/ml) Inhibition diameter (mm) MIC (mg/ml) Inhibition diameter (mm) MIC (mg/ml) HPRB – A 14.15 ± 0.01 c 1000 13.57 ± 0.14 ab 1000 10.77 ± 0.05 ab 1000 HPRB – B 16.4 ± 0.015 b 1000 9.12 ± 0.03 ab 500 13.7 ± 0.00 ab 1000 HPRB – C 20.4 ± 0.065 a 500 10.85 ± 0.03 ab 1000 14.52 ± 0.21 a 500 HPRB – D 19.47 ± 0.05 a 250 14.72 ± 0.47 a 500 13.87 ± 0.01 a 500 PKO (E) 8.65 ± 0.05 d NI* 8.22 ± 0.11 b NI* 12.1 ± 0.26 ab NI* RPSO (F) 8 ± 0.1 d 1000 8.55 ± 0.02 b 1000 9.85 ± 0.00 b 100 The inhibition zone includes the diameter of the disk (6 mm). Values of inhibition diameter are given as mean ± SD, n = 3. Different letters are significantly different in each column by Tukey’s test (P < 0.05). (*NI: No inhibition) Figure 2 , visually demonstrates the antibacterial activity of HPRB-C and HPRB-D against S. aureus , E. coli , and S. typhi . The clear zones of inhibition surrounding the HPRB-treated discs indicate the effective suppression of bacterial growth. The varying diameters of these zones reflect differences in susceptibility among the bacterial strains and potentially concentration-dependent effects of the HPRB formulations. The inclusion of positive (penicillin and vancomycin) and negative (Tween 80) controls further validates the observed antibacterial activity and ensures the reliability of the assay. Furthermore, Fig. 3 illustrates the determination of the minimum inhibitory concentration (MIC) of HPRB against three bacterial strains: Staphylococcus aureus , Escherichia coli , and Salmonella typhi , using a 96-well plate setup with decreasing HPRB concentrations and the colorimetric indicator TTC. The red color, indicative of bacterial growth, diminishes with increasing HPRB concentration, allowing visual identification of the MIC as the lowest concentration without red color. The figure enables comparison of MICs across strains, revealing varying susceptibilities and the potential of HPRB as an antibacterial agent. The microscopy images presented in Fig. 4 provide a visual representation of the antibacterial efficacy of HPRB-C and HPRB-D against S. aureus , E. coli , and S. typhi . The untreated bacteria in the positive control groups (Fig. 4 A, 4 D, 4 G) exhibit robust growth, forming dense and confluent bacterial colonies that stain darkly with crystal violet, indicating intact cell walls. In contrast, the treated samples reveal varying degrees of bacterial inhibition that correspond to the minimum inhibitory concentrations (MICs) determined through quantitative assays. The images show that HPRB-D at 500 mg/mL (Fig. 4 C) completely inhibits S. aureus growth, resulting in a clear background devoid of any visible bacterial colonies. In contrast, HPRB-C at the same concentration (Fig. 4 B) shows only partial inhibition, with a few scattered and faintly stained bacterial cells visible. This observation aligns with the MIC data, indicating that a higher concentration of HPRB-C is needed to achieve complete inhibition of S. aureus . For E. coli , both HPRB-C and HPRB-D at 250 mg/mL (Fig. 4 E and 4 F, respectively) appear to significantly reduce bacterial growth compared to the untreated control. However, HPRB-D treatment results in a clearer background with fewer and fainter bacterial cells, suggesting a stronger inhibitory effect compared to HPRB-C at the same concentration. This observation is consistent with the MIC data, which shows that HPRB-D has a lower MIC against E. coli than HPRB-C. In the case of S. typhi , both HPRB-C and HPRB-D at 500 mg/mL (Fig. 4 H and 4 I, respectively) completely inhibit bacterial growth, resulting in a clear background with no visible bacterial colonies. This observation is in agreement with the MIC data, which shows that both compounds have the same MIC against S. typhi . The primary action of HPRB against Gram-positive bacteria is from monolaurin ability to integrate into and disrupt the lipid bilayer of the bacterial cell membrane. This disruption is facilitated by the lipophilic nature of monolaurin, allowing it to interact with the lipid components of the membrane (Farhanghi et al., 2022 ). When monolaurin penetrates the membrane, it alters the membrane's integrity, leading to increased permeability and eventual cell lysis. This mechanism is particularly effective against Gram-positive bacteria, which possess a thick peptidoglycan layer that is more susceptible to such disruptions compared to the outer membrane of Gram-negative bacteria (Dai et al., 2017 ). In addition to direct membrane disruption, monolaurin has been shown to inhibit the growth of bacteria by interfering with their metabolic processes. For instance, it can inhibit the synthesis of essential cellular components, which is vital for bacterial growth and replication. This dual action—disrupting the membrane while also affecting metabolic pathways—enhances the overall antibacterial efficacy of monolaurin. Gram-negative bacteria are composed of a lipid bilayer and a layer of lipopolysaccharides (LPS). This outer membrane serves as a protective barrier, making Gram-negatve bacteria inherently more rresistant to many antimicrobial agents, including monolaurin (Sedlaříková et al., 2021 ). While monolaurin is effective against Gram-positive bacteria, its activity against Gram-negative bacteria is typically lower, as the LPS layer can impede the penetration of monolaurin into the bacterial cell (Buňková et al., 2011 ). Researche indicates that monolaurin can interact with the lipid components of the Gram-negative bacterial membrane, leading to increased permeability and potential cell lysis. However, the effectiveness of monolaurin against Gram-negative bacteria often requires higher concentrations compared to Gram-positive bacteria (Ukachukwu et al., 2022 ). For instance, studies have shown that monolaurin can exhibit antibacterial activity against E. coli, but the concentration required for significant effects is typically higher than that needed for Gram-positive organisms (Mueller & Schlievert, 2015 ). This is attributed to the need for monolaurin to overcome the protective outer membrane barrier of Gram-negative bacteria. In addition to direct membrane disruption, monolaurin may also affect the metabolic processes of Gram-negative bacteria. It has been suggested that monolaurin can inhibit the synthesis of essential cellular components, thereby impairing bacterial growth and replication (Ghany et al., 2024 ). Furthermore, monolaurin has been shown to have a synergistic effect when combined with other antimicrobial agents, enhancing the overall antibacterial efficacy against Gram-positive and -negative bacteria. This synergy is particularly important in the context of rising antibiotic resistance, as it suggests that monolaurin could be used to enhance the effectiveness of existing antibiotics against resistant strains (Krislee et al., 2019 ). 4. Conclusion This study investigated the antibacterial activity of a novel compound, Hydrolyzed PKO-RPSO Blend (HPRB), against Gram-negative and Gram-positive bacteria. The research focused on evaluating the composition of HPRB and its effectiveness in inhibiting the growth of common human pathogens, including Staphylococcus aureus , Escherichia coli , and Salmonella typhi . The production of HPRB involved the hydrolysis of PKO and RPSO using lipase, resulting in four different formulations (HPRB-A, HPRB-B, HPRB-C, and HPRB-D) with varying proportions of PKO and RPSO. HPRB-C and HPRB-D exhibited the highest levels of 1-monolaurin. HPRB-C and HPRB-D demonstrated significant antibacterial activity against all three bacterial strains. The study also found that the RPSO component of HPRB contributed to its antibacterial activity, possibly due to the presence of phytonutrients such as total carotene, vitamin E, and squalene. Microscopy images further confirmed the antibacterial efficacy of HPRB-C and HPRB-D. The study's findings highlight the potential of HPRB, particularly HPRB-C and HPRB-D, as natural antibacterial agents. The presence of 1-monolaurin and phytonutrients in these formulations contributes to their effectiveness in inhibiting the growth of both Gram-positive and Gram-negative bacteria. Further research is needed to investigate the in vivo safety and efficacy of HPRB and explore its potential applications in the food and pharmaceutical industries. Declarations Conflict of Interest The authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this paper. Author Contribution Ilmi Fadhilah Rizki wrote the main manuscript text and prepared all the dataFrisda Rimbun Panjaitan, Manda Edy Mulyono, and Brahmani Dewa Bajra prepared the data and reviewed the manuscripts Acknowledgement This research was funded by Badan Pengelola Dana Perkebunan Kelapa Sawit (BPDPKS) through the Grant Riset Sawit (GRS) PRJ-363/DPKS/2022. 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Antibiotic Residues, Antimicrobial Resistance and Intervention Strategies of Foodborne Pathogens. Antibiotics 13 (4), 1–7. https://doi.org/10.3390/antibiotics13040321 (2024). Zeng, F. K. et al. Enzymatic production of monoacylglycerols with camellia oil by the glycerolysis reaction. JAOCS J. Am. Oil Chemists’ Soc. 87 (5), 531–537. https://doi.org/10.1007/s11746-009-1533-x (2010). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6181954","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":432901595,"identity":"a130d1ad-6ec6-4206-a3bd-2d08c36764d8","order_by":0,"name":"Ilmi Fadhilah Rizki","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIie3RMUvDQBTA8ScH7wbPdL0joF/hSiAK7YdJpi4iEUE6SEgRkkU7u/UrCIKTwwsFu2TwIyiBzp06SfFO6XYpdhO5/xQ4frx3FwCf7w+GHIDGwBLzgQAaNPJJYQ5kN2GGNJawLRH1bgKGgCXwTSySye7FAnaYEt0MLgLGlu04y6NAtZP28uUMeuHizb3Y0SPR6+gKGZ72G40xhult9LCUoKbnWReZr3CelgxiVWgxNKQMBUnQjXCu+DNlYwlfGyKHqOpfkLq0RNgpOkZ5sCWcusl0ZMm1IUmEwt6FpFB3wvliJ9X904rWg3RWVc+q+Mz7s2rx0WaUH/cEf3caZ/ZnmRFC70dM+0zx+Xy+f9wXOiNW8oXIVm4AAAAASUVORK5CYII=","orcid":"","institution":"Indonesia Oil Palm Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Ilmi","middleName":"Fadhilah","lastName":"Rizki","suffix":""},{"id":432901596,"identity":"3156df4a-4322-4b96-8ae9-0ea365c7606d","order_by":1,"name":"Frisda Rimbun Panjaitan","email":"","orcid":"","institution":"Indonesia Oil Palm Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Frisda","middleName":"Rimbun","lastName":"Panjaitan","suffix":""},{"id":432901597,"identity":"b3446e8c-cdda-42dc-8361-26a9efe04233","order_by":2,"name":"Manda Edy Mulyono","email":"","orcid":"","institution":"Indonesia Oil Palm Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Manda","middleName":"Edy","lastName":"Mulyono","suffix":""},{"id":432901598,"identity":"c2128c97-1a6e-437f-aec6-17a1c26c0253","order_by":3,"name":"Brahmani Dewa Bajra","email":"","orcid":"","institution":"Indonesia Oil Palm Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Brahmani","middleName":"Dewa","lastName":"Bajra","suffix":""}],"badges":[],"createdAt":"2025-03-08 04:53:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6181954/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6181954/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79572159,"identity":"146b1494-da80-4487-be57-1a1d293558e2","added_by":"auto","created_at":"2025-03-31 10:51:07","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":63710,"visible":true,"origin":"","legend":"\u003cp\u003eA schematic diagram of HPRB production via enzymatically hydrolysis\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6181954/v1/c5022c12fe704fff631201fa.jpeg"},{"id":79571794,"identity":"398d4360-1124-4aff-a329-a85bfc53ac64","added_by":"auto","created_at":"2025-03-31 10:43:07","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":107309,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of inhibition diameter in HPRB - C and HPRB - D treated with \u003cem\u003eS. aureus, E. coli\u003c/em\u003e, and \u003cem\u003eS. typhi\u003c/em\u003e. In each petridish, the HPRB used was diluted to 10% (250 mg/mL), 30% (500 mg/mL), 50% (1000 mg/mL), 100% (HPRB oil without dilution), negative control (Tween80), and positive control (penicillin and vancomycin)\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6181954/v1/54dcd2fcbd606502f1424773.jpeg"},{"id":79571797,"identity":"cbe8cd8d-e062-43cc-a3d3-9401a05f0eb3","added_by":"auto","created_at":"2025-03-31 10:43:07","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":131041,"visible":true,"origin":"","legend":"\u003cp\u003eDetermination of minimum inhibitory concentration of HPRB. The bacterial growth on 96-well plate with gradient of decreasing HPRB concentration and addition of 0.125% TTC\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6181954/v1/cc41380ea21fd44cba0ce2cd.jpeg"},{"id":79572862,"identity":"49f7c611-e172-45dc-a9fe-717402f4db8e","added_by":"auto","created_at":"2025-03-31 10:59:07","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":119222,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopy image of (A) untreated \u003cem\u003eS. \u003c/em\u003eaureus (positive control); (B) \u003cem\u003eS. \u003c/em\u003eaureus treated with HPRB-C 500 mg/mL; (C) \u003cem\u003eS. \u003c/em\u003eaureus treated with HPRB-D 500 mg/mL; (D) untreated \u003cem\u003eE. coli \u003c/em\u003e(positive control); (E) \u003cem\u003eE. coli \u003c/em\u003etreated with HPRB-C 250 mg/mL; (F) \u003cem\u003eE. coli \u003c/em\u003etreated with HPRB-D 250 mg/mL; (G) untreated \u003cem\u003eS. typhi \u003c/em\u003e(positive control); (H) \u003cem\u003eS. typhi \u003c/em\u003etreated with HPRB-C 500 mg/mL; and (I) S. typhi treated with HPRB-D 500 mg/mL\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6181954/v1/b110a132bfa06b6832006d16.jpeg"},{"id":80545431,"identity":"cf262d7f-f5f3-43c0-82b8-df1da0a41620","added_by":"auto","created_at":"2025-04-14 13:48:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1737460,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6181954/v1/4764b707-f8ea-40d2-beae-4ae1fc1ecf66.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antibacterial Activity of Hydrolized Palm Kernel Oil and Red Palm Super Olein Blend against Gram negative and Gram-positive Bacteria","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe escalating prevalence of foodborne illnesses and the alarming rise of antibiotic-resistant bacteria pose a significant threat to global health (M et al., 2020). The World Health Organization (WHO) estimates that millions fall ill each year from foodborne pathogens, with the Southeast Asia region alone experiencing a staggering 150\u0026nbsp;million cases and over 175,000 deaths annually (World Health Organization, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The impact of these illnesses is particularly devastating for vulnerable populations such as young children, the elderly, and the immunocompromised, often leading to severe complications and exacerbating malnutrition (Tao et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The situation is further compounded by the limitations of current treatments, including antibiotics, due to the emergence of multidrug-resistant pathogens (Wu \u0026amp; Zeng, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe urgent need for novel and sustainable antibacterial agents has spurred extensive research into natural products, particularly those derived from plants. These plant-based antimicrobials offer several advantages over conventional antibiotics, including a broader spectrum of activity, reduced susceptibility to resistance development, and fewer side effects (Arip et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The ability of plant-derived compounds to target multiple bacterial components simultaneously, such as cell membranes, protein synthesis, and DNA replication, contributes to their efficacy and reduces the likelihood of resistance emergence (Li et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Furthermore, Kouitcheu et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) observed the synergistic effects when combining plants with antibiotics enhanced antibacterial activity, indicating the potential for plant-derived compounds to complement existing treatment strategies. Cheong et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) also emphasized the importance of exploring natural products, including plants, as sources of novel antibacterial agents to address the growing challenge of bacterial resistance.\u003c/p\u003e \u003cp\u003ePalm oil and its derivatives have garnered significant attention among the various natural products explored for their antimicrobial potential (Habibiasr et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Hovorkov\u0026aacute; et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Palm kernel oil (PKO), a widely available and sustainable resource, is rich in medium-chain fatty acids (MCFAs) and is known for its broad-spectrum antibacterial activity. In their research, Laloučkov\u0026aacute; et al. (2019) found that PKO has potential antibacterial effects against Gram-positive bacteria, including those causing mastitis. Ustadi et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) research study showed that waste palm cooking has an in vitro antibacterial effect against Staphylococcus aureus. Furthermore, the latest palm oil derivative, red palm super olein (RPSO), contains concentrated phytonutrients, including carotene and vitamin E, which may contribute to their antimicrobial properties (Mulyono et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBuilding upon this foundation, the present study focuses on a novel compound, Hydrolyzed PKO-RPSO Blend (HPRB), created by combining PKO and RPSO through hydrolysis. The unique composition of HPRB, encompassing MCFAs, acylglycerols, and phytonutrients, presents a promising avenue for developing a potent and broad-spectrum antibacterial agent. This research aims to comprehensively analyze the composition of HPRB and evaluate its antibacterial activity against common human pathogens, including \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, and \u003cem\u003eSalmonella typhi\u003c/em\u003e. Furthermore, the study seeks to identify the specific component within HPRB that is responsible for its antibacterial effects, providing valuable insights for future development and applications.\u003c/p\u003e \u003cp\u003eBy elucidating HPRB's antibacterial potential and key components, this study contributes to the growing body of knowledge on natural antimicrobials. The findings may pave the way for developing novel, effective, and sustainable strategies to combat foodborne illnesses and address the pressing challenge of antibiotic resistance.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals\u003c/h2\u003e \u003cp\u003eThe RPSO was manufactured in the Oleofood Laboratory of the Indonesian Oil Palm Research Institute (IOPRI), Medan (Mulyono et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). First, the PKO was obtained from a local palm oil mill in North Sumatra, Indonesia. The chemicals used in this process include the immobilized lipase (Novozymes (N435), Denmark), Mueller Hinton Agar (MHA), Mueller Hinton Broth (MHB), and 2,3,5-triphenyl tetrazolium chloride (TTC) (Himedia Laboratories PVT, Mumbai, India). The TTC (1.25%) was prepared with sterile distilled water and stored in sterile flasks covered with aluminum paper at 2 to 8\u003csup\u003eo\u003c/sup\u003eC. Tween-80 (Sigma-Aldrich, St. Louis, MO, USA) was used as a solvent.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Microorganisms\u003c/h2\u003e \u003cp\u003eThe antibacterial activity of samples alone and in combination with antibiotics such as vancomycin and penicillin was tested against three bacterial strains, \u003cem\u003eE. coli (\u003c/em\u003eATCC 25922), \u003cem\u003eS. aureus (\u003c/em\u003eATCC 25923), and \u003cem\u003eS. typhi (\u003c/em\u003eATCC 1408), purchased from the Faculty of Medicine Sumatera Utara University (North Sumatra, Indonesia). The inoculum suspension was obtained from the 24 h culture colonies and suspended in 0.9% sodium chloride (NaCl) sterile aqueous solution. The density was adjusted to 0.5 McFarland standard (10\u003csup\u003e8\u003c/sup\u003e colony-forming unit [CFU]/mL) turbidity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Production of hydrolyzed PKO and RPSO blend\u003c/h2\u003e \u003cp\u003eLipase-catalyzed hydrolysis produced Hydrolized PKO and RPSO Blend (HPRB) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The HPRB formulations were prepared by mixing PKO and RPSO in four different combinations: HPRB - A (20% PKO, 80% RPSO), HPRB - B (40% PKO, 60% RPSO), HPRB - C (60% PKO, 40% RPSO), and HPRB - D (80% PKO, 20% RPSO). The hydrolysis involved blending PKO and RPSO according to the respective ratio with 8% Novozymes 435 and water at 10% (w/w) relative to the oil. The reactants were mixed via magnetic stirring, and the reaction was maintained at 50\u003csup\u003eo\u003c/sup\u003eC. After 48 h, the result was filtered to remove the catalysts, and monolaurin-rich HPRPB was obtained. The monolaurin-rich HPRB was then characterized, including fatty acid, acylglycerol, phytonutrient composition, and lipase residue.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Characterization of HPRB\u003c/h2\u003e \u003cp\u003e \u003cem\u003eFatty acids composition\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe fatty acid composition was determined as fatty acid methyl esters via Gas Chromatography-2010 (Shimadzu, Japan) and a flame ionization detector (FID) equipped with a Shimadzu AOC-20i autosampler (Shimadzu, Japan). The fatty acid composition used DB-23 gas chromatography column (Agilent, USA) with increasing oven temperature from 90 to 208 ◦C (7 ◦C/min) and maintained at 208 ◦C for 5 min. The injector was set to 260 ◦C, and the nitrogen gas column flow was set at 1 mL/min.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAcylglycerol composition\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe acylglycerol composition was determined using the Shimadzu GCMS-QP2010 Plus (Shimadzu, Japan) equipped with a Shimadzu AOC-20i auto-injector (Shimadzu, Japan), an Agilent DB-5HT column (Agilent, USA), and a helium mobile phase at 100 psi. The injector was set at 325\u0026deg;C, while the interface and ion source were at 280\u0026deg;C. The oven temperature was initially set at 100\u0026deg;C for 1 min, increased to 223\u0026deg;C at 30\u0026deg;C/min, and later 1\u0026deg;C/min to 227\u0026deg;C. The final increase in temperature was at a rate of 5\u0026deg;C/min to 360\u0026deg;C for 10 min. Samples were prepared by silyl derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA).\u003c/p\u003e \u003cp\u003e \u003cem\u003eSqualene\u003c/em\u003e \u003c/p\u003e \u003cp\u003eSqualene was analyzed using Shimadzu GCMS-QP2010 Plus (Shimadzu, Japan) equipped with an Agilent HP-5MS column (Agilent, USA). Samples were prepared by saponification in the presence of ascorbic acid. The unsaponifiable fraction was injected into the GC\u0026ndash;MS at 1:50 ratio split injection mode. The helium gas was used at a pressure of 100 psi, while the injector, interface, and ion source temperatures were set at 325\u0026deg;C, 280\u0026deg;C, and 250\u0026deg;C, respectively. The oven temperature was initially set at 50\u0026deg;C for 2 minutes and increased at a rate of 10\u0026deg;C/minute to 300\u0026deg;C for 5 minutes.\u003c/p\u003e \u003cp\u003e \u003cem\u003eTotal carotene\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe HPRB sample was dissolved in hexane and analyzed using a Shimadzu UV-1700 UV\u0026ndash;Visible spectrophotometer (Shimadzu, Japan) at 446 nm. The total carotene was calculated as follows:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:Total\\:carotene\\:\\left(ppm\\right)=\\frac{383\\times\\:volume\\times\\:absorbance\\:at\\:446\\:nm}{mass\\:of\\:sample\\times\\:100}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eVitamin E composition\u003c/em\u003e \u003c/p\u003e \u003cp\u003eVitamin E was analyzed with ultra-performance liquid chromatography (UPLC) (Waters 600 system, USA), equipped with an autosampler and ultraviolet (UV) detector. The analysis was performed in the Inertsil ODS-3 column (Inertsil, USA) with methanol as the mobile phase with a 1 mL/min flow rate. The injection volume was 20 \u0026micro;L, and the UV absorbance was measured at 292 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Disc Diffusion Assay of Antibacterial Activity\u003c/h2\u003e \u003cp\u003eThe anti-bacterial activity for all HPRB combinations was determined using the disc diffusion method, and the best formula against \u003cem\u003eE. coli (\u003c/em\u003eATCC 25922), \u003cem\u003eS. aureus (\u003c/em\u003eATCC 25923), and \u003cem\u003eS. typhi (\u003c/em\u003eATCC 1408) were identified. The HPRB combinations were adjusted to several concentrations, 10% (250 mg/ml), 30% (500 mg/ml), and 50% (1000 mg/ml), using Tween 80. The bacterial suspension (0.1 ml) was placed in a 10 mL agar medium test tube and homogenized to achieve 0.5 McFarland unit turbidity (1.5 x 10\u003csup\u003e8\u003c/sup\u003e CFU/mL). The suspension was then spread over a petri dish containing solidified MHA using a hockey stick and allowed to dry. Sterilized discs (diameter: 6 mm) were dipped into each HPRB formulation and placed on the agar surface inoculated with the respective bacteria. Subsequently, the petri dishes were incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 48 h. Tween 80 was used as a negative control, and two standard antibiotics, penicillin (10 mcg/disc) and vancomycin (30 mcg/disc) acted as the positive controls. The HPRB anti-bacterial activity was determined based on the diameter of the inhibition zone around the disc. Each experiment was carried out in triplicates, and the results obtained were subjected to an analysis of variance (ANOVA) test in the Statistical Package for Social Sciences (SPSS) version 25 (IBM, USA). The confidence level was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Determination of Minimum Inhibitory Concentration (MIC)\u003c/h2\u003e \u003cp\u003eMinimum Inhibitory Concentration (MIC) was determined using a microdilution test in 96-well plates according to the National Committee for Clinical Laboratory Standards with modification. Successive dilutions half ranging of HPRB and antibiotics were prepared by adding 50 \u0026micro;L of Mueller-Hinton broth (MHB) to achieve concentrations between 2 and 1000 mg/ml for HPRB and 0.5 to 250 mg/ml for the antibiotics in the wells. Each well's bacterial suspension density (50 \u0026micro;L) was 10\u003csup\u003e6\u003c/sup\u003e CFU/mL, prepared by diluting 0.5 McFarland suspension 100 times. The 96-well plates were prepared in triplicates, covered, and sealed tightly to prevent evaporation, and incubated at 37\u0026deg;C for 22 h. The bacterial growth was visualized by adding 20 \u0026micro;l of 2,3,5-triphenyl tetrazolium chloride (0.125%) solution to each well and incubated for two hours to observe the color change.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Bacterial Staining Assay\u003c/h2\u003e \u003cp\u003eAfter different treatment from the Minimum Inhibitory Concentration (MIC) procedure, the bacteria suspensions were transferred gently to the microscope slide. The slide is then heated over a gentle flame to help the cell adhesion to the glass slide and prevent the loss of bacteria during rinsing. The gram staining started with an added crystal violet stain over the slide. After 5 minutes, the stain is poured off, and the excess stain is rinsed with tap water. The iodine solution was poured off for 1 minute over the slide and rinsed with tap water. A few drops of decolorizer (mixed solvent of ethanol and acetone) were added to the slide for 10 seconds and rinsed with tap water. For the last step, the slide is counterstained with fuchsin solution for 40\u0026ndash;60 seconds and washed off with tap water. The slide was then air-dried and examined under a microscope under oil immersion.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Result and Discussion","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Characterization of HPRB\u003c/h2\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e3.1.1 Fatty Acid Composition of HPRB\u003c/h2\u003e \u003cp\u003eThe fatty acid compositions of HPRB formulations, PKO, and RPSO were analyzed using gas chromatography with flame ionization detection (GC/FID) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). PKO, derived from the palm kernel, is rich in medium-chain fatty acids (MCFAs), notably lauric acid (C12:0). In contrast, RPSO is characterized by a high content of long-chain fatty acids (LCFAs), particularly oleic acid (C18:1) and palmitic acid (C16:0). RPSO is derived from CPO, initially made into red palm olein (RPO), and fractionated through dry fractional crystallization to obtain a red palm super olein (RPSO) consisting of high oleic acid and low palmitic acid. Dry fractional crystallization was carried out by temperature adjustment without additives, concentrating the natural phytonutrients contained in CPO, such as carotene, vitamin E, and squalene (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e) (Mulyono et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFatty acid composition (wt.%) of HPRB, PKO, and RPSO\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eComposition\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHPRB - A\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHPRB - B\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHPRB - C\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHPRB - D\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePKO (E)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRPSO (F)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC6:0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e0.10 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15 ± 0,00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.19 ± 0,00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC8:0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.60 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.17 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e1.82 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.42 ± 0.16\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.16 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC10:0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.57 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.14 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e1.77 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.38 ± 0.13\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.06 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC12:0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.95 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.52 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e28.61 ± 0.25\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e39.06 ± 0.84\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e49.78 ± 0.07\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.17 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC14:0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.61 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.52 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e9.55 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.91 ± 0.06\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.02 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.89 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC16:0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e30.98 ± 0.05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.78 ± 0.05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e20.32 ± 0.15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.27 ± 0.32\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.21 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e35.91 ± 0.15\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC16:1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.14 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.12 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e0.08 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.17 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC18:0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.58 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.27 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e2.93 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.58 ± 0.07\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.15 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.87 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC18:1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39.64 ± 0.06\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.98 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e27.93 ± 0.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.72 ± 0.57\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.05 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e44.97 ± 0.13\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC18:2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.23 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.91 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e6.41 ± 0.03\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.11 ± 0.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.16 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13.23 ± 0.03\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC18:3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0,23 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.17 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e0.11 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.06 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.28 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC20:0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.28 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.24 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e0.19 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.08 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.33 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC20:1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.14 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.12 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e0.10 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.09 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.08 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.14 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΣSFA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e48.59 ± 0.08\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e56.67 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e65.34 ± 0.15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e73.95 ± 0.68\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e82.69 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e41.19 ± 0.16\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΣUSFA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.40 ± 0.08\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43.32 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e34.65 ± 0.15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.99 ± 0.68\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17.30 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e58.80 ± 0.16\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΣMUFA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39.93 ± 0.06\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.22 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e28.12 ± 0.11\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e21.82 ± 0.57\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.14 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e45.29 ± 0.13\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΣPUFA\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e11.46 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.09 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e6.53 ± 0.04\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.17 ± 0.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.16 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13.51 ± 0.03\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eResults are reported as mean ± SD, n = 3\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, lauric acid (49.78%) was the predominant fatty acid in PKO, followed by myristic acid (16.02%). In contrast, RPSO contained the highest amount of oleic acid (44.97%), followed by palmitic acid (35.91%). The highest SFA content was observed in PKO (82.69%), followed by HPRB-D (73.95%), HPRB-C (65.34%), HPRB-B (56.67%), HPRB-A (48.59%), and RPSO (41.19%). Conversely, unsaturated fatty acid (USFA) and polyunsaturated fatty acid (PUFA) compositions were most abundant in RPSO and least abundant in PKO. The highest monounsaturated fatty acid (MUFA) content was found in RPSO (45.39%), followed by HPRB-A (39.93%), HPRB-B (34.22%), HPRB-C (28.12%), HPRB-D (21.82%), and PKO (15.14%). The fatty acid composition of the HPRB formulations was influenced by the relative proportions of PKO and RPSO used in their production. As the proportion of PKO increased in HPRB, the levels of lauric (C12:0) and myristic (C14:0) acids also increased, ranging from 8.95–39.06% and 3.61–12.91%, respectively. Conversely, an increase in the RPSO level in HPRB resulted in a corresponding increase in oleic (C18:1) and palmitic (C16:0) acids, ranging from 21.72–39.64% and 14.27–30.98%, respectively.\u003c/p\u003e \u003cp\u003eThe varying fatty acid profiles of these oils are expected to influence the antimicrobial properties of the resulting HPRB formulations. Medium-chain fatty acids (MCFAs), particularly lauric and myristic acids have been shown to possess broad-spectrum antibacterial activity against various bacteria, including human pathogens bacteria (Casillas-Vargas et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Desbois \u0026amp; Smith, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Matsue et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Okukawa et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, in this study, HPRB was hydrolyzed enzymatically, incorporating both PKO and RPSO, to enrich the composition of fatty acids and their corresponding acyglycerols composition, to enhance the potential antimicrobial activity of these formulations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e3.1.2 Acyglycerol Composition of HPRB\u003c/h2\u003e \u003cp\u003eThe HPRB production process resulted in a unique acylglycerol profile that sets it apart from the original raw materials, PKO and RPSO. This distinction in composition significantly impacts the anti-bacterial properties of the various HPRB formulations. The analysis of the acylglycerol composition, as presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e, highlights the diversity in the distribution of different acylglycerol classes across the HPRB formulations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAcylgycerol composition (wt.%) of HPRB, PKO, and RPSO\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eAcylglycerol species\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHPRB A\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHPRB B\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHPRB C\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eHPRB D\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ePKO (E)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRPSO (F)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e\u003cb\u003eFFA\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.42 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.04 ± 0.19\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.75 ± 0.27\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.11 ± 0.30\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eO\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.82 ± 0.44\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.59 ± 0.13\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.52 ± 0.14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.11 ± 0.06\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.53 ± 0.12\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.70 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.44 ± 0.14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.36 ± 0.09\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e14.94\u003c/b\u003e ± \u003cb\u003e0.47\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e9.07\u003c/b\u003e ± \u003cb\u003e0.20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e9.63\u003c/b\u003e ± \u003cb\u003e0.20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e9.21\u003c/b\u003e ± \u003cb\u003e0.36\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.53\u003c/b\u003e ± \u003cb\u003e0.12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"9\" rowspan=\"10\"\u003e \u003cp\u003e\u003cb\u003eMAG\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-Ca-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.53 ± 0.06\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCa--\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.20 ± 0.59\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-La-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.79 ± 0.06\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.76 ± 0.39\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.30 ± 0.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLa--\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.95 ± 1.15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e17.54 ± 0.72\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24.69 ± 1.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-M-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.52 ± 0.03\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM--\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.15 ± 0.09\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.22 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.61 ± 0.14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.91 ± 0.12\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP--\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.80 ± 0.08\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.36 ± 0.05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.10 ± 0.07\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.67 ± 0.24\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eO--\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.63 ± 0.14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.17 ± 0.06\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.70 ± 0.23\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.43 ± 1.18\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.40 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS--\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.29 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.58 ± 0.03\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.68 ± 0.16\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e3.58\u003c/b\u003e ± \u003cb\u003e0.15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e24.79\u003c/b\u003e ± \u003cb\u003e1.36\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e30.18\u003c/b\u003e ± \u003cb\u003e1.57\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e44.93\u003c/b\u003e ± \u003cb\u003e3.09\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e0.40\u003c/b\u003e ± \u003cb\u003e0.02\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"11\" rowspan=\"12\"\u003e \u003cp\u003e\u003cb\u003eDAG\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLa-Cp\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.27 ± 1.27\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLa-Ca\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.61 ± 0.05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.99 ± 0.05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.75 ± 0.13\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLaLa-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.62 ± 0.04\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.52 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.75 ± 0.13\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.93 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.15 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLa-La\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.22 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.52 ± 0.17\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.81 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8.90 ± 0.41\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.44 ± 0.03\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLaM-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.49 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.05 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.78 ± 0.20\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLa-M\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.24 ± 0.03\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.33 ± 0.16\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.20 ± 0.61\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.14 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLa-P\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.67 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.77 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.46 ± 0.32\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.05 ± 0.05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLaO-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.33 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.71 ± 0.07\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.12 ± 0.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP-P\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.12 ± 0.12\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eP-O\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.18 ± 0.11\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eO-O\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.85 ± 0.19\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e1.83\u003c/b\u003e ± \u003cb\u003e0.06\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e8.37\u003c/b\u003e ± \u003cb\u003e0.20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e16.39\u003c/b\u003e ± \u003cb\u003e0.43\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e24.40\u003c/b\u003e ± \u003cb\u003e1.94\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.78 ± 0.00\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e2.14\u003c/b\u003e ± \u003cb\u003e0.18\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"15\" rowspan=\"16\"\u003e \u003cp\u003e\u003cb\u003eTAG\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLaLaCo\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.71 ± 0.06\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLaLaCp\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.11 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.18 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.78 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.61 ± 0.34\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLaLaCa\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.90 ± 0.11\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLaLaLa\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.28 ± 0.03\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.53 ± 0.15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.10 ± 0.30\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.57 ± 0.75\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e27.72 ± 0.35\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLaLaM\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.02 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.34 ± 0.04\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.78 ± 0.35\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.83 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20.58 ± 0.15\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLaLaP\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.17 ± 0.17\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.65 ± 0.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.07 ± 0.18\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.37 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLaLaO\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.31 ± 0.05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.64 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.47 ± 0.47\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.71 ± 0.04\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLaMP\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.71 ± 0.04\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLaMO\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.30 ± 0.30\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.97 ± 0.04\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLaMS\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.04 ± 0.00\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLaOP\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.52 ± 0.14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePPP\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.50 ± 0.04\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.52 ± 0.06\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.57 ± 0.20\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.26 ± 0.2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.07 ± 0.25\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.10 ± 0.25\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePOP\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20.19 ± 1.11\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.83 ± 0.33\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.53 ± 0.06\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.68 ± 0.40\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.27 ± 0.04\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e24.96 ± 1.54\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePOO\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e45.11 ± 1.21\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.02 ± 0.56\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e20.57 ± 1.77\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.90 ± 1.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.07 ± 0.05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e56.51 ± 1.85\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOOO\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.55 ± 0.49\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.95 ± 0.73\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.89 ± 0.09\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.59 ± 0.59\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.97 ± 0.09\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14.47 ± 0.03\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e79.64\u003c/b\u003e ± \u003cb\u003e0.67\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e57.77\u003c/b\u003e ± \u003cb\u003e1.37\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e43.80\u003c/b\u003e ± \u003cb\u003e2.21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e21.46\u003c/b\u003e ± \u003cb\u003e0.78\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e99.23\u003c/b\u003e ± \u003cb\u003e0.00\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e96.93\u003c/b\u003e ± \u003cb\u003e0.04\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003eCo = caproate (C6:0), Cp = caprylate (C8:0), Ca = caprate (C10:0), La = laurate (C12:0), M = myristate (C14:0), P = palmitate (C16:0), O = oleate (C18:1), S = stearate (C18:0), ND not detected.\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003csup\u003ea\u003c/sup\u003e Mean ± standard deviation (\u003cem\u003en\u003c/em\u003e = 3)\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd colspan=\"8\"\u003e\u003csup\u003eb\u003c/sup\u003e Some acylglycerols might be undetected due to limit of detection.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhytonutrient composition (mg/kg) of HPRB, PKO, and RPSO\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eComposition\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHPRB – A\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHPRB - B\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHPRB - C\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHPRB - D\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePKO (E)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRPSO (F)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal carotene\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e604.08 ± 0.74\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e452.69 ± 1.42\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e303.29 ± 1.24\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e165.05 ± 2.33\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.18 ± 1.07\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c7\"\u003e \u003cp\u003e761.23 ± 1.64\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal vitamin E\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e867.54 ± 3.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e608.19 ± 10.29\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e251.32 ± 2.51\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e78.61 ± 1.63\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.60 ± 1.65\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c7\"\u003e \u003cp\u003e1118.17 ± 7.24\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eδ-Tocotrienol\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e142.64 ± 0.88\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e131.74 ± 1.70\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e102.46 ± 2.38\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e49.36 ± 0.43\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.60 ± 1.65\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c7\"\u003e \u003cp\u003e167.11 ± 2.60\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eγ-Tocotrienol\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e438.12 ± 0.86\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e336.39 ± 4.95\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e148.85 ± 2.85\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29.24 ± 2.06\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c7\"\u003e \u003cp\u003e549.16 ± 2.43\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα-Tocotrienol\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e230.57 ± 1.70\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e115.16 ± 6.78\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c7\"\u003e \u003cp\u003e313.02 ± 1.43\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eα-Tocopherol\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e56.21 ± 0.62\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e24.88 ± 0.40\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c7\"\u003e \u003cp\u003e88.87 ± 2.74\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSqualene\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e178.5 ± 3.53\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e131 ± 2.82\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e87.5 ± 2.12\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e51 ± 1.41\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e23.5 ± 2.12\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c7\"\u003e \u003cp\u003e257.5 ± 17.67\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eResults are reported as mean ± SD, n = 3\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e \u003cp\u003eHPRB-A stands out with the highest concentration of free fatty acids (14.94%) and TAGs (79.64%). In contrast, HPRB-D displays a markedly different profile, rich in MAGs (44.93%) and DAGs (24.40%). The abundance of these monoacylglycerols might contribute to enhanced anti-bacterial activity, as certain MAGs are known to disrupt bacterial cell membranes (Jackman et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). HPRB-C also shows a notable presence of MAGs (30.18%) and DAGs (16.39%), suggesting it may share some of the anti-bacterial properties observed in HPRB-D.\u003c/p\u003e \u003cp\u003eThe free fatty acid composition of HPRB is primarily characterized by the presence of palmitic, oleic, and stearic acids. The MAG fraction in HPRB is mainly composed of 1-monolaurin and 1-monomyristin. Notably, HPRB-D boasts the highest levels of both 1-monolaurin (24.69%) and 1-monomyristin (5.91%), while HPRB-C contains the second-highest amount of 1-monolaurin (17.54%). The prevalence of 1-monolaurin is particularly noteworthy due to its potent anti-bacterial (Ngatirah et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The formation of 1-monolaurin and 1-monomyristin in HPRB is attributed to the partial hydrolysis of PKO and RPSO during the lipase-catalyzed reaction. This process selectively cleaves ester bonds in triglycerides, leading to the generation of MAGs. The specific composition of MAGs is influenced by the fatty acid profile of the starting materials and the selectivity of the lipase enzyme (Monteiro et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Subroto, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zeng et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e3.1.3 Phytonutrient Composition of HPRB\u003c/h2\u003e \u003cp\u003eThe raw materials used to synthesize HPRB have limited nutrients. The PKO lacks phytonutrients, while RPSO has low MCFA. Thus, combining PKO and RPSO potentially increases the composition of MCFA and phytonutrients in the HPRB. The resulting HPRB in this study exhibited changes in the MCFA and phytonutrient contents, and MAG was present as the fatty acid. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e3\u003c/span\u003e details the phytonutrient composition of each HPRB formulation, PKO, and RPSO, consisting of total carotene, vitamin E and its isomers, and squalene. The RPSO has the highest phytonutrient among the oils: total carotene = 761.23 ppm, total vitamin E = 1118.17 ppm, and squalene = 257.5 ppm. Moreover, RPSO has low palmitic acid (saturated fatty acid) and high oleic acid (unsaturated fatty acid), as shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The double bond presence is attributed to the high solubility of unsaturated fats. RPSO is the primary source of phytonutrients in HPRB synthesis; thus, phytonutrients composition increases with higher RPSO inclusion in the formulation.\u003c/p\u003e \u003cp\u003eThe antibacterial activity of carotenoids is often linked to their ability to modulate oxidative stress. The bioactive properties of carotenoids are not limited to their antioxidant effects; they also exhibit direct antibacterial activity. For example, studies have shown that carotenoids can inhibit the growth of both Gram-positive and Gram-negative bacteria, suggesting a broad spectrum of antimicrobial action (Karpiński \u0026amp; Adamczak, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In addition to their antioxidant and antibacterial properties, carotenoids have been implicated in various therapeutic applications. The extraction and application of carotenoids in food and pharmaceutical industries are also gaining traction. In this research, the synergy between carotenoids and other bioactive compounds, such as lauric acid in monoacylglycerol composition, can further amplify their health benefits, including enhanced antibacterial effects\u003c/p\u003e \u003cp\u003eAnother research also indicates that tocotrienols can modulate immune responses, which may enhance the ability to combat bacterial infections. For instance, tocotrienol treatment has been shown to influence the expression of pro-inflammatory cytokines in macrophages, suggesting a role in the regulation of inflammatory responses that are critical during bacterial infections (Qureshi et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This immunomodulatory effect may be particularly beneficial in managing infections caused by multi-drug resistant pathogens, as tocotrienols have demonstrated the ability to inhibit the growth of both Gram-positive and Gram-negative bacteria, including Staphylococcus aureus (Hartmann et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This suggests that tocotrienols may not only support the immune system but also exert direct antimicrobial effects, making them a promising candidate for the development of natural antibacterial agents. Tocotrienols have been suggested to work synergistically with carotenoids enhancing their antibacterial effects and providing a multi-faceted approach to infection management. Furthermore, their antioxidant properties may help to mitigate oxidative stress associated with bacterial infections, thereby protecting host tissues and improving overall health outcomes (Shrum et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.1.4 Anti-Bacterial Activity of HPRB\u003c/h2\u003e \u003cp\u003eThe antibacterial activities of the HPRB formulations were evaluated by measuring the inhibition zone diameters (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The most effective formulations were HPRB-C and HPRB-D, which exhibited the largest inhibition zones against all three bacterial strains. Specifically, HPRB-C (500 mg/ml) displayed inhibition zones of 20.4 mm, 10.85 mm, and 14.52 mm against \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003eS. typhi\u003c/em\u003e. Similarly, HPRB-D (250 mg/ml) showed inhibition zones of 19.47 mm, 14.72 mm, and 13.87 mm against the same strains. The minimum inhibitory concentrations (MICs) for HPRB-C and HPRB-D were also lower than those for HPRB-A (1000 mg/ml; 14.15 mm, 13.57 mm, and 10.77 mm) and HPRB-B (1000 mg/ml; 16.4 mm, 9.12 mm, and 13.7 mm), further supporting their superior antibacterial activity. The raw materials, PKO (palm kernel oil) and RPSO (refined, bleached, and deodorized palm stearin oil), showed either no inhibition (NI) or significantly smaller inhibition zones (PKO: 8.65 mm, 8.22 mm, 12.1 mm; RPSO: 8 mm, 8.55 mm, 9.85 mm) compared to the HPRB formulations, highlighting the enhanced antibacterial properties achieved through the formulation process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eInhibition zone diameter and minimum inhibitory concentration of HPRB\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u003cem\u003eSalmonella typhi\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInhibition diameter (mm)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMIC (mg/ml)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInhibition diameter (mm)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMIC (mg/ml)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInhibition diameter (mm)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eMIC (mg/ml)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPRB – A\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.15 ± 0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e13.57 ± 0.14\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10.77 ± 0.05\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPRB – B\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e16.4 ± 0.015\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.12 ± 0.03\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.7 ± 0.00\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPRB – C\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.4 ± 0.065\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10.85 ± 0.03\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e14.52 ± 0.21\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHPRB – D\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.47 ± 0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.72 ± 0.47\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.87 ± 0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePKO (E)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.65 ± 0.05\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNI*\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.22 ± 0.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNI*\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12.1 ± 0.26\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNI*\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRPSO (F)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8 ± 0.1\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.55 ± 0.02\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1000\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e9.85 ± 0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eThe inhibition zone includes the diameter of the disk (6 mm). Values of inhibition diameter are given as mean ± SD, n = 3. Different letters are significantly different in each column by Tukey’s test (P \u0026lt; 0.05). (*NI: No inhibition)\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, visually demonstrates the antibacterial activity of HPRB-C and HPRB-D against \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003eS. typhi\u003c/em\u003e. The clear zones of inhibition surrounding the HPRB-treated discs indicate the effective suppression of bacterial growth. The varying diameters of these zones reflect differences in susceptibility among the bacterial strains and potentially concentration-dependent effects of the HPRB formulations. The inclusion of positive (penicillin and vancomycin) and negative (Tween 80) controls further validates the observed antibacterial activity and ensures the reliability of the assay.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e illustrates the determination of the minimum inhibitory concentration (MIC) of HPRB against three bacterial strains: \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, and \u003cem\u003eSalmonella typhi\u003c/em\u003e, using a 96-well plate setup with decreasing HPRB concentrations and the colorimetric indicator TTC. The red color, indicative of bacterial growth, diminishes with increasing HPRB concentration, allowing visual identification of the MIC as the lowest concentration without red color. The figure enables comparison of MICs across strains, revealing varying susceptibilities and the potential of HPRB as an antibacterial agent.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe microscopy images presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e provide a visual representation of the antibacterial efficacy of HPRB-C and HPRB-D against \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003eS. typhi\u003c/em\u003e. The untreated bacteria in the positive control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG) exhibit robust growth, forming dense and confluent bacterial colonies that stain darkly with crystal violet, indicating intact cell walls. In contrast, the treated samples reveal varying degrees of bacterial inhibition that correspond to the minimum inhibitory concentrations (MICs) determined through quantitative assays.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe images show that HPRB-D at 500 mg/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) completely inhibits \u003cem\u003eS. aureus\u003c/em\u003e growth, resulting in a clear background devoid of any visible bacterial colonies. In contrast, HPRB-C at the same concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) shows only partial inhibition, with a few scattered and faintly stained bacterial cells visible. This observation aligns with the MIC data, indicating that a higher concentration of HPRB-C is needed to achieve complete inhibition of \u003cem\u003eS. aureus\u003c/em\u003e. For \u003cem\u003eE. coli\u003c/em\u003e, both HPRB-C and HPRB-D at 250 mg/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF, respectively) appear to significantly reduce bacterial growth compared to the untreated control. However, HPRB-D treatment results in a clearer background with fewer and fainter bacterial cells, suggesting a stronger inhibitory effect compared to HPRB-C at the same concentration. This observation is consistent with the MIC data, which shows that HPRB-D has a lower MIC against \u003cem\u003eE. coli\u003c/em\u003e than HPRB-C. In the case of \u003cem\u003eS. typhi\u003c/em\u003e, both HPRB-C and HPRB-D at 500 mg/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI, respectively) completely inhibit bacterial growth, resulting in a clear background with no visible bacterial colonies. This observation is in agreement with the MIC data, which shows that both compounds have the same MIC against \u003cem\u003eS. typhi\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe primary action of HPRB against Gram-positive bacteria is from monolaurin ability to integrate into and disrupt the lipid bilayer of the bacterial cell membrane. This disruption is facilitated by the lipophilic nature of monolaurin, allowing it to interact with the lipid components of the membrane (Farhanghi et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). When monolaurin penetrates the membrane, it alters the membrane's integrity, leading to increased permeability and eventual cell lysis. This mechanism is particularly effective against Gram-positive bacteria, which possess a thick peptidoglycan layer that is more susceptible to such disruptions compared to the outer membrane of Gram-negative bacteria (Dai et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In addition to direct membrane disruption, monolaurin has been shown to inhibit the growth of bacteria by interfering with their metabolic processes. For instance, it can inhibit the synthesis of essential cellular components, which is vital for bacterial growth and replication. This dual action—disrupting the membrane while also affecting metabolic pathways—enhances the overall antibacterial efficacy of monolaurin.\u003c/p\u003e \u003cp\u003eGram-negative bacteria are composed of a lipid bilayer and a layer of lipopolysaccharides (LPS). This outer membrane serves as a protective barrier, making Gram-negatve bacteria inherently more rresistant to many antimicrobial agents, including monolaurin (Sedlaříková et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). While monolaurin is effective against Gram-positive bacteria, its activity against Gram-negative bacteria is typically lower, as the LPS layer can impede the penetration of monolaurin into the bacterial cell (Buňková et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eResearche indicates that monolaurin can interact with the lipid components of the Gram-negative bacterial membrane, leading to increased permeability and potential cell lysis. However, the effectiveness of monolaurin against Gram-negative bacteria often requires higher concentrations compared to Gram-positive bacteria (Ukachukwu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). For instance, studies have shown that monolaurin can exhibit antibacterial activity against E. coli, but the concentration required for significant effects is typically higher than that needed for Gram-positive organisms (Mueller \u0026amp; Schlievert, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This is attributed to the need for monolaurin to overcome the protective outer membrane barrier of Gram-negative bacteria. In addition to direct membrane disruption, monolaurin may also affect the metabolic processes of Gram-negative bacteria. It has been suggested that monolaurin can inhibit the synthesis of essential cellular components, thereby impairing bacterial growth and replication (Ghany et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, monolaurin has been shown to have a synergistic effect when combined with other antimicrobial agents, enhancing the overall antibacterial efficacy against Gram-positive and -negative bacteria. This synergy is particularly important in the context of rising antibiotic resistance, as it suggests that monolaurin could be used to enhance the effectiveness of existing antibiotics against resistant strains (Krislee et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study investigated the antibacterial activity of a novel compound, Hydrolyzed PKO-RPSO Blend (HPRB), against Gram-negative and Gram-positive bacteria. The research focused on evaluating the composition of HPRB and its effectiveness in inhibiting the growth of common human pathogens, including \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, \u003cem\u003eEscherichia coli\u003c/em\u003e, and \u003cem\u003eSalmonella typhi\u003c/em\u003e. The production of HPRB involved the hydrolysis of PKO and RPSO using lipase, resulting in four different formulations (HPRB-A, HPRB-B, HPRB-C, and HPRB-D) with varying proportions of PKO and RPSO. HPRB-C and HPRB-D exhibited the highest levels of 1-monolaurin. HPRB-C and HPRB-D demonstrated significant antibacterial activity against all three bacterial strains. The study also found that the RPSO component of HPRB contributed to its antibacterial activity, possibly due to the presence of phytonutrients such as total carotene, vitamin E, and squalene. Microscopy images further confirmed the antibacterial efficacy of HPRB-C and HPRB-D. The study's findings highlight the potential of HPRB, particularly HPRB-C and HPRB-D, as natural antibacterial agents. The presence of 1-monolaurin and phytonutrients in these formulations contributes to their effectiveness in inhibiting the growth of both Gram-positive and Gram-negative bacteria. Further research is needed to investigate the in vivo safety and efficacy of HPRB and explore its potential applications in the food and pharmaceutical industries.\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this paper.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eIlmi Fadhilah Rizki wrote the main manuscript text and prepared all the dataFrisda Rimbun Panjaitan, Manda Edy Mulyono, and Brahmani Dewa Bajra prepared the data and reviewed the manuscripts\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research was funded by Badan Pengelola Dana Perkebunan Kelapa Sawit (BPDPKS) through the Grant Riset Sawit (GRS) PRJ-363/DPKS/2022. The authors thank the Oil Palm Downstream Research Group, particularly Oleofood Laboratory team members, for their roles in this research.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and analyzed during the current study are stored in a controlled access repository at the Indonesian Oil Palm Research Institute. Due to the sensitive nature of the data, they are not publicly available. However, reasonable requests for access can be directed to the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArip, M. et al. Review on Plant-Based Management in Combating Antimicrobial Resistance - Mechanistic Perspective. \u003cem\u003eFront. 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Oil Chemists\u0026rsquo; Soc.\u003c/em\u003e \u003cb\u003e87\u003c/b\u003e (5), 531\u0026ndash;537. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11746-009-1533-x\u003c/span\u003e\u003cspan address=\"10.1007/s11746-009-1533-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2010).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antibacterial activity, Red palm super olein, palm kernel oil, monolaurin, phytonutrients","lastPublishedDoi":"10.21203/rs.3.rs-6181954/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6181954/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study investigated the antibacterial activity of a novel compound, Hydrolyzed Palm Kernel Oil and Red Palm Super Olein Blend (HPRB), against Gram-negative (\u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eSalmonella typhi\u003c/em\u003e) and Gram-positive (\u003cem\u003eStaphylococcus aureus\u003c/em\u003e) bacteria. HPRB was synthesized through enzymatic hydrolysis with four different ratios of palm kernel oil (PKO) and red palm super olein (RPSO). The antibacterial activity was evaluated using disc diffusion and microdilution methods. The results showed that HPRB-C (60% PKO, 40% RPSO) and HPRB-D (80% PKO, 20% RPSO) exhibited the highest antibacterial activity against all bacteria tested, attributed to the high concentration of 1-monolaurin (17.54% and 24.99%, respectively). The phytonutrient content also contributed to the antibacterial activity of HPRB. HPRB has the potential to be used as a natural antibacterial agent.\u003c/p\u003e","manuscriptTitle":"Antibacterial Activity of Hydrolized Palm Kernel Oil and Red Palm Super Olein Blend against Gram negative and Gram-positive Bacteria","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-31 10:43:02","doi":"10.21203/rs.3.rs-6181954/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"48f281e1-33c4-4cf8-ac72-6390682ac40e","owner":[],"postedDate":"March 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46096978,"name":"Biological sciences/Microbiology"},{"id":46096979,"name":"Biological sciences/Microbiology/Antimicrobials"}],"tags":[],"updatedAt":"2025-04-14T13:40:02+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-31 10:43:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6181954","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6181954","identity":"rs-6181954","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}