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A holistic approach to managing palm oil mill effluent (POME): biotechnological advances in the sustainable reuse of POME

Wu TY, Mohammad AW, Jahim JM, Anuar N

Biotechnol Adv, 2009 Jan-Feb;27(1):40-52.
PMID: 18804158 DOI: 10.1016/j.biotechadv.2008.08.005

During the last century, a great deal of research and development as well as applications has been devoted to waste. These include waste minimization and treatment, the environmental assessment of waste, minimization of environmental impact, life cycle assessment and others. The major reason for such huge efforts is that waste generation constitutes one of the major environmental problems where production industries are concerned. Until now, an increasing pressure has been put on finding methods of reusing waste, for instance through cleaner production, thus mirroring rapid changes in environmental policies. The palm oil industry is one of the leading industries in Malaysia with a yearly production of more than 13 million tons of crude palm oil and plantations covering 11% of the Malaysian land area. However, the production of such amounts of crude palm oil result in even larger amounts of palm oil mill effluent (POME), estimated at nearly three times the quantity of crude palm oil. Normally, POME is treated using end-of-pipe processes, but it is worth considering the potential value of POME prior to its treatment through introduction of a cleaner production. It is envisaged that POME can be sustainably reused as a fermentation substrate in the production of various metabolites, fertilizers and animal feeds through biotechnological advances. The present paper thus discusses various technically feasible and economically beneficial means of transforming the POME into low or preferably high value added products.
A phytotoxicity test of bulrush (Scirpus grossus) grown with diesel contamination in a free-flow reed bed system

Al-Baldawi IA, Abdullah SR, Anuar N, Suja F, Idris M

J Hazard Mater, 2013 May 15;252-253:64-9.
PMID: 23500791 DOI: 10.1016/j.jhazmat.2013.01.067

In this study, bulrush (Scirpus grossus) was subjected to a 72 day phytotoxicity test to assess its ability to phytoremediate diesel contamination in simulated wastewater at different concentrations (0, 8700, 17,400 and 26,100mg/L). Diesel degradation by S. grossus was measured in terms of total petroleum hydrocarbon (TPH-D). The TPH-D concentration in the synthetic wastewater was determined with the liquid-liquid extraction method and gas chromatography. S. grossus was found to reduce TPH-D by 70.0 and 80.2% for concentrations of 8700 mg/L and 17,400mg/L, respectively. At a diesel concentration of 26,100mg/L, S. grossus died after 14 days. Additionally, the biomass of S. grossus plants was found to increase throughout the phytotoxicity test, confirming the ability of the plant to survive in water contaminated with diesel at rates of less than 17,400mg/L.
A point-of-care cassette test for detection of Strongyloides stercoralis

Noordin R, Osman E, Kalantari N, Anuar NS, Gorgani-Firouzjaee T, Sithithaworn P, et al.

Acta Trop, 2022 Feb;226:106251.
PMID: 34808116 DOI: 10.1016/j.actatropica.2021.106251

Strongyloides stercoralis is a parasite that causes strongyloidiasis worldwide. It may lead to a life-long infection in immunocompetent people and hyperinfection in immunosuppressed patients. A point-of-care (POC) rapid test is helpful for patient diagnosis in resource-limited settings and as a detection tool in elimination/control programs. Previously, we reported a rapid IgG4 dipstick test (Ss Rapid®) for Strongyloides suitable for a laboratory setting. A POC cassette format of the test, which is field-applicable, has since been developed. Here, we report on a laboratory-based evaluation of the Ss Rapid® cas sette test on 285 sera. We assessed the diagnostic sensitivity of the Ss Rapid® cas sette with 32 sera, comprising samples from larval and/or DNA positive individuals from three countries. Additionally, we also tested samples from 33 seropositive endemic areas residents. We evaluated the diagnostic specificity of the test using 220 samples, comprising sera from other infections (n = 101), allergy cases with high IgE antibodies (n = 4), and blood donors (n = 115). The test showed high diagnostic sensitivity (97%, 31/32), and all sera of the seropositive endemic residents were reactive. It also showed high diagnostic specificity (94.5%, 208/220), and all false-positive samples tested negative after sera adsorption using recombinant NIE-coated microsphere beads. Additionally, we showed that the test worked with spiked whole blood samples. The study results showed that the SsRapid® cas sette test merits further laboratory and field evaluations.
Fulltext Arsenic Resistance and Biosorption by Isolated Rhizobacteria from the Roots of Ludwigia octovalvis

Titah HS, Abdullah SRS, Idris M, Anuar N, Basri H, Mukhlisin M, et al.

Int J Microbiol, 2018;2018:3101498.
PMID: 30723505 DOI: 10.1155/2018/3101498

Certain rhizobacteria can be applied to remove arsenic in the environment through bioremediation or phytoremediation. This study determines the minimum inhibitory concentration (MIC) of arsenic on identified rhizobacteria that were isolated from the roots of Ludwigia octovalvis (Jacq.) Raven. The arsenic biosorption capability of the was also analyzed. Among the 10 isolated rhizobacteria, five were Gram-positive (Arthrobacter globiformis, Bacillus megaterium, Bacillus cereus, Bacillus pumilus, and Staphylococcus lentus), and five were Gram-negative (Enterobacter asburiae, Sphingomonas paucimobilis, Pantoea spp., Rhizobium rhizogenes, and Rhizobium radiobacter). R. radiobacter showed the highest MIC of >1,500 mg/L of arsenic. All the rhizobacteria were capable of absorbing arsenic, and S. paucimobilis showed the highest arsenic biosorption capability (146.4 ± 23.4 mg/g dry cell weight). Kinetic rate analysis showed that B. cereus followed the pore diffusion model (R2 = 0.86), E. asburiae followed the pseudo-first-order kinetic model (R2 = 0.99), and R. rhizogenes followed the pseudo-second-order kinetic model (R2 = 0.93). The identified rhizobacteria differ in their mechanism of arsenic biosorption, arsenic biosorption capability, and kinetic models in arsenic biosorption.
Arsenic toxicity on Ludwigia octovalvis in spiked sand

Titah HS, Abdullah SR, Mushrifah I, Anuar N, Basri H, Mukhlisin M

Bull Environ Contam Toxicol, 2013 Jun;90(6):714-9.
PMID: 23595348 DOI: 10.1007/s00128-013-0996-5

Wilting, especially of the leaves, was observed as an initial symptom of arsenate [As(V)] to Ludwigia octovalvis (Jacq.) P. H. Raven. The plants tolerated As(V) levels of 39 mg kg⁻¹ for as long as 35 days of exposure. After 91 days, the maximum concentration of As uptake in the plant occurred at As(V) concentration of 65 mg kg⁻¹ while As concentration in the stems, roots and leaves were 6139.9 ± 829.5, 1284.5 ± 242.9 and 1126.1 ± 117.2 mg kg⁻¹, respectively. In conclusion, As(V) could cause toxic effects in L. octovalvis and the plants could uptake and accumulate As in plant tissues.
Fulltext Cell-based influenza vaccine: current production, halal status assessment, and recommendations towards Islamic-compliant manufacturing

Zulkarnain NN, Anuar N, Abd Rahman N, Sheikh Abdullah SR, Alias MN, Yaacob M, et al.

Hum Vaccin Immunother, 2021 07 03;17(7):2158-2168.
PMID: 33539195 DOI: 10.1080/21645515.2020.1865044

Influenza virus is a life-threatening pathogen that infects millions of people every year, with annual mortality in the hundreds of thousands. The scenario for controlling infection has worsened with increasing numbers of vaccine hesitancy cases reported worldwide due to objections on safety, religious and other grounds. Uses of haram (impermissible) and mashbooh (doubtful) ingredients in vaccine production has raised doubts among Muslim consumers and consequently stimulated serious vaccine hesitancy. To address this major problem, we have reviewed and recommended some alternatives appropriate for manufacturing cell-based influenza vaccine which comply with Islamic laws and consumers' needs. Intensive assessments of current influenza vaccine production in both scientific and Islamic views have led to the identification of four main ingredients deemed impermissible in novel sharia-compliant (approved by Islamic laws) vaccine manufacturing. Only some of these impermissible components could be replaced with halal (permissible) alternatives, while others remain impermissible due to unavailability and unsuitability.
Fulltext Chemicals Constituents Isolated from Cultivate Alpinia conchigera Griff. and Antimicrobial Activity

Taib MNAM, Anuar N, Hanafiah KM, Al-Shammary AAK, Saaid M, Awang K

Trop Life Sci Res, 2020 Apr;31(1):159-178.
PMID: 32963717 DOI: 10.21315/tlsr2020.31.1.10

Alpinia conchigera Griff. is a plant species from the family Zingiberaceae. Coloquially known as wild ginger, Alpinia conchigera Griff. is used as food condiment and for traditional treatment of skin diseases. Isolation studies to identify bioactive compounds of rhizomes of Alpinia conchigera yielded seven compounds; 1'S-1'-acetoxychavicol acetate (1), trans-p-coumaryl diacetate (2), p-hydroxycinnamyl acetate (3), 1'S-1'-hydroxychavicol acetate (4) p-hydroxybenzaldehyde (5), stigmasterol (6) and β-sitosterol (7). Compounds 1, 2 and 5 were evaluated for antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA). Among the compounds tested, Compound 1 showed good antimicrobial activity against the strain of MRSA with minimum inhibition concentration (MIC) value of 0.5 mg/mL. Meanwhile, Compounds 2 and 5 exhibited moderate activity with MIC value between 1.0 and 2.0 mg/mL. These findings indicate antimicrobial potential of 1'S-1'-acetoxychavicol acetate (1), compound derived from rhizome of Alpinia conchigera Griff. against MRSA, which warrant further investigation.
Fulltext Combining ability for germination traits in Jatropha curcas L

Islam AK, Anuar N, Yaakob Z, Ghani JA, Osman M

ScientificWorldJournal, 2013;2013:935981.
PMID: 24222756 DOI: 10.1155/2013/935981

Six parents of Jatropha curcas were crossed in half diallel fashion, and the F 1s were evaluated to determine the combining ability for nine germination parameters. The ratio between general combining ability (GCA) and specific combining ability (SCA) variances indicated preponderance of additive gene action for all the characters except germination percentage, time of 50% germination, seedling length, and seedling vigor index. The parents P 1 and P 2 were the best general combiner for most of the characters studied. The cross P 1 × P 5 was the best specific combiner for speed of emergence, germination percentage, germination energy, germination index, and seedling vigor index, the cross P 2 × P 5 for mean germination time, time of 50% germination, and seedling length, and the cross P 4 × P 5 for number of days to first germination. The germination percentage varied from 58.06 to 92.76% among the parents and 53.43 to 98.96% among the hybrids. The highest germination (98.96%) was observed in hybrid P 2 × P 4, and none of the hybrids or parents showed 100% germination. The highest germination index (GI) and seedling vigor index (SVI) were found in hybrid P 1 × P 5 and P 2 × P 5, respectively. The results of this study provide clue for the improvement of Jatropha variety through breeding program.
Comparative performance of free surface and sub-surface flow systems in the phytoremediation of hydrocarbons using Scirpus grossus

Al-Baldawi IA, Abdullah SR, Suja F, Anuar N, Mushrifah I

J Environ Manage, 2013 Nov 30;130:324-30.
PMID: 24113536 DOI: 10.1016/j.jenvman.2013.09.010

Two types of flow system, free surface flow (FSF) and sub-surface flow (SSF), were examined to select a better way to remove total petroleum hydrocarbons (TPH) using diesel as a hydrocarbon model in a phytotoxicity test to Scirpus grossus. The removal efficiencies of TPH for the two flow systems were compared. Several wastewater parameters, including temperature (T, °C), dissolved oxygen (DO, mgL(-1)), oxidation-reduction potential (ORP, mV), and pH were recorded during the experimental runs. In addition, overall plant lengths, wet weights, and dry weights were also monitored. The phytotoxicity test using the bulrush plant S. grossus was run for 72 days with different diesel concentrations (1%, 2%, and 3%) (Vdiesel/Vwater). A comparison between the two flow systems showed that the SSF system was more efficient than the FSF system in removing TPH from the synthetic wastewater, with average removal efficiencies of 91.5% and 80.2%, respectively. The SSF system was able to tolerate higher diesel concentrations than was the FSF system.
Cytotoxicity evaluation of ketoprofen found in pharmaceutical wastewater on HEK 293 cell growth and metabolism

Zulkarnain NN, Anuar N, Johari NA, Sheikh Abdullah SR, Othman AR

Environ Toxicol Pharmacol, 2020 Nov;80:103498.
PMID: 32950717 DOI: 10.1016/j.etap.2020.103498

Inefficient ketoprofen removal from pharmaceutical wastewater may negatively impact the ecosystem and cause detrimental risks to human health. This study was conducted to determine the cytotoxicity effects of ketoprofen on HEK 293 cell growth and metabolism, including cyclooxygenase-1 (COX-1) expression, at environmentally relevant concentrations. The cytotoxic effects were evaluated through the trypan blue test, DNS assay, MTT assay, and the expression ratio of the COX-1 gene. The results of this study show insignificant (p > 0.05) cytotoxic effects of ketoprofen on cell viability and cell metabolism. However, high glucose consumption rates among the treated cells cause an imitation of the Warburg effect, which is likely linked to the development of cancer cells. Apart from that, the upregulation of COX-1 expression among the treated cells indicates remote possibility of inflammation. Although no significant cytotoxic effects of ketoprofen were detected throughout this study, the effects of prolonged exposure of residual ketoprofen need to be evaluated in the future.
Fulltext Detection of secreted antimicrobial peptides isolated from cell-free culture supernatant of Paenibacillus alvei AN5

Alkotaini B, Anuar N, Kadhum AA, Sani AA

J Ind Microbiol Biotechnol, 2013 Jun;40(6):571-9.
PMID: 23508455 DOI: 10.1007/s10295-013-1259-5

An antimicrobial substance produced by the Paenibacillus alvei strain AN5 was detected in fermentation broth. Subsequently, cell-free culture supernatant (CFCS) was obtained by medium centrifugation and filtration, and its antimicrobial activity was tested. This showed a broad inhibitory spectrum against both Gram-positive and -negative bacterial strains. The CFCS was then purified and subjected to SDS-PAGE and infrared spectroscopy, which indicated the proteinaceous nature of the antimicrobial compound. Some de novo sequencing using an automatic Q-TOF premier system determined the amino acid sequence of the purified antimicrobial peptide as Y-S-K-S-L-P-L-S-V-L-N-P (1,316 Da). The novel peptide was designated as peptide AN5-1. Its mode of action was bactericidal, inducing cell lysis in E. coli ATCC 29522 and S. aureus, and non-cell lysis in both S. marcescens and B. cereus ATCC 14579. Peptide AN5-1 displayed stability at a wide range of pH values (2-12) and remained active after exposure to high temperatures (100 °C). It also maintained its antimicrobial activity after incubation with chemicals such as SDS, urea and EDTA.
Fulltext Development and characterisation of ibuprofen-loaded nanoemulsion with enhanced oral bioavailability

Anuar N, Sabri AH, Bustami Effendi TJ, Abdul Hamid K

Heliyon, 2020 Jul;6(7):e04570.
PMID: 32775730 DOI: 10.1016/j.heliyon.2020.e04570

Lipophilic compounds constitute a majority of therapeutics in the pipeline of drug discovery. Despite possessing enhanced efficacy and permeability, some of these drugs suffer poor solubility necessitating the need of a suitable drug delivery system. Nanoemulsion is a drug delivery system that provides enhanced solubility for poorly soluble drugs in an attempt to improve the oral bioavailability. The purpose of this study is to develop a nanoemulsion system using ibuprofen as a model drug in order to investigate the potential of this colloidal system to enhance the absorption of poorly water-soluble drugs. Ibuprofen loaded-nanoemulsion with different drug concentrations (1.5, 3 and 6% w/w) were formulated from olive oil, sucrose ester L-1695 and glycerol using D-phase emulsification technique. A pseudoternary phase diagram was utilised to identify the optimal excipient composition to formulate the nanoemulsion system. In vitro diffusion chamber studies using rodent intestinal linings highlighted improved absorption profile when ibuprofen was delivered as nanoemulsion in comparison to microemulsions and drug-in-oil systems. This was further corroborated by in vivo studies using rat model that highlighted a two-fold increase in ibuprofen absorption when the drug was administered as a nanoemulsion relative to drug-in-oil system. On the other hand, when ibuprofen was administered as microemulsions, only a 1.5-fold increase in absorption was observed relative to drug-in-oil system. Thus, this study highlights the potential of using nanoemulsion as a drug delivery system to enhance the oral bioavailability of hydrophobic drugs.
Do the physical and environment PETTLEP elements predict sport imagery ability?

Anuar N, Williams SE, Cumming J

Eur J Sport Sci, 2017 Nov;17(10):1319-1327.
PMID: 28950801 DOI: 10.1080/17461391.2017.1377290

The present study aimed to examine whether physical and environment elements of PETTLEP imagery relate to the ability to image five types of sport imagery (i.e. skill, strategy, goal, affect and mastery). Two hundred and ninety participants (152 males, 148 females; Mage = 20.24 years, SD = 4.36) from various sports completed the Sport Imagery Ability Questionnaire (SIAQ), and a set of items designed specifically for the study to assess how frequently participants incorporate physical (e.g. 'I make small movements or gestures during the imagery') and environment (e.g. 'I image in the real training/competition environment') elements of PETTLEP imagery. Structural equation modelling tested a hypothesised model in which imagery priming (i.e. the best fitting physical and environment elements) significantly and positively predicted imagery ability of the different imagery types (skill, β = 0.38; strategy, β = 0.23; goal, β = 0.21; affect, β = 0.25; mastery, β = 0.22). The model was a good fit to the data: χ2(174) = 263.87, p
Fulltext Effects of different drying methods on essential oil yield and component profile of Polygonum minus root extract

Azhari, N. A. M., Markom, M., Ismail, I., Anuar, N.

International Food Research Journal, 2020;27(1):96-102.
MyJurnal

Polygonum minus is a plant rich with bioactive components that contribute to food, pharmaceutical, and perfume industries. However, high moisture content in fresh plants will allow
microbial activity that leads to the degradation of plant quality. This can be prevented by
drying the fresh plants to preserve the characteristics of their bioactive components. The
present work was conducted to determine the effect of different drying methods such as
air-drying, oven-drying (40 and 60°C), and freeze-drying on essential oil (EO) yield and
chemical compounds of P. minus roots. For comparison purposes, all samples were extracted
by maceration with n-hexane at room temperature. Then, the samples were analysed and
identified by using gas chromatography-mass spectrometry (GC-MS). The highest EO yield
extract was obtained from freeze-drying (4.15 ± 0.5), followed by air-drying (3.79 ± 0.19). EO
yield from oven-drying at 40 and 60°C was 3.4 ± 0.14 and 0.86 ± 0.04, respectively. Results
showed that by increasing the drying temperature, the EO yield would decrease and cause a
loss of major chemical compounds in the P. minus root. Air-drying was found to be the best
method in preserving the presence of important chemical compound in P. minus roots such as
β-caryophyllene (1.43%), pentadecane (4.34%), hexadecanoic acid (3.91%) and oleic acid
(3.97%).
Evaluation of morphological changes of Staphylococcus aureus and Escherichia coli induced with the antimicrobial peptide AN5-1

Alkotaini B, Anuar N, Kadhum AA

Appl Biochem Biotechnol, 2015 Feb;175(4):1868-78.
PMID: 25427593 DOI: 10.1007/s12010-014-1410-4

The mechanisms of action of AN5-1 against Gram-negative and Gram-positive bacteria were investigated by evaluations of the intracellular content leakage and by microscopic observations of the treated cells. Escherichia coli and Staphylococcus aureus were used for this investigation. Measurements of DNA, RNA, proteins, and β-galactosidase were taken, and the results showed a significant increase in the cultivation media after treatment with AN5-1 compared with the untreated cells. The morphological changes of treated cells were shown using transmission electron microscopy (TEM) and atomic force microscopy (AFM). The observations showed that AN5-1 acts against E. coli and against S. aureus in similar ways, by targeting the cell wall, causing disruptions; at a high concentration (80 AU/ml), these disruptions led to cell lysis. The 3D AFM imaging system showed that at a low concentration of 20 AU/ml, the effect of AN5-1 is restricted to pore formation only. Moreover, a separation between the cell wall and the cytoplasm was observed when Gram-negative bacteria were treated with a low concentration (20 AU/ml) of AN5-1.
Exploring measurement tools to optimise hospital physician distribution

Suan NAM, Soelar SA, Rani RA, Anuar NA, Aziz KAA, Chan HK, et al.

Med J Malaysia, 2024 Mar;79(2):222-233.
PMID: 38553930

INTRODUCTION: Equitable healthcare delivery is essential and requires resources to be distributed, which include assets and healthcare workers. To date, there is no gold standard for measuring the correct number of physicians to meet healthcare needs. This rapid review aims to explore measurement tools employed to optimise the distribution of hospital physicians, with a focus on ensuring fair resource allocation for equitable healthcare delivery.
MATERIALS AND METHODS: A literature search was performed across PubMed, EMBASE, Emerald Insight and grey literature sources. The key terms used in the search include 'distribution', 'method', and 'physician', focusing on research articles published in English from 2002 to 2022 that described methods or tools to measure hospital-based physicians' distribution. Relevant articles were selected through a two-level screening process and critically appraised. The primary outcome is the measurement tools used to assess the distribution of hospital-based physicians. Study characteristics, tool advantages and limitations were also extracted. The extracted data were synthesised narratively.
RESULTS: Out of 7,199 identified articles, 13 met the inclusion criteria. Among the selected articles, 12 were from Asia and one from Africa. The review identified eight measurement tools: Gini coefficients and Lorenz curve, Robin Hood index, Theil index, concentration index, Workload Indicator of Staffing Need method, spatial autocorrelation analysis, mixed integer linear programming model and cohortcomponent model. These tools rely on fundamental data concerning population and physician numbers to generate outputs. Additionally, five studies employed a combination of these tools to gain a comprehensive understanding of physician distribution dynamics.
CONCLUSION: Measurement tools can be used to assess physician distribution according to population needs. Nevertheless, each tool has its own merits and limitations, underscoring the importance of employing a combination of tools. The choice of measuring tool should be tailored to the specific context and research objectives.
Fulltext Gonococcal conjunctivitis: A case report

Anuar N, Idris NS

Malays Fam Physician, 2018;13(3):27-28.
PMID: 30800230

We report a case of acute gonococcal conjunctivitis in a 36-year-old woman who presented with eye redness and a history of discharge for one month. Prior to presenting to us, she was treated for bacterial conjunctivitis with three courses of local antibiotics by three different clinics after brief assessments and without improvement. The final diagnosis of gonococcal conjunctivitis was made after a complete history was elicited and supported by the presence of Neisseria Gonorrhoeae in the eye swab culture test. She and her sexual partner were treated successfully with intramuscular Ceftriaxone and oral Azithromycin. This case highlights the importance of complete history taking, including sexual history, which translates into early recognition and treatment, thus preventing complications.
Fulltext Gonococcal conjunctivitis: a case report

Anuar, N., Idris, N.S.

Malaysian Family Physician, 2018;13(3):27-28.
MyJurnal

We report a case of acute gonococcal conjunctivitis in a 36-year-old woman who presented with
eye redness and a history of discharge for one month. Prior to presenting to us, she was treated for
bacterial conjunctivitis with three courses of local antibiotics by three different clinics after brief
assessments and without improvement. The final diagnosis of gonococcal conjunctivitis was made
after a complete history was elicited and supported by the presence of Neisseria Gonorrhoeae in
the eye swab culture test. She and her sexual partner were treated successfully with intramuscular
Ceftriaxone and oral Azithromycin. This case highlights the importance of complete history taking,
including sexual history, which translates into early recognition and treatment, thus preventing
complications. (Copied from article).
Imagery training for Malaysian paralympics athletes - an important step towards sports equity

Anuar N, Bahar N

Br J Sports Med, 2023 Mar 13.
PMID: 36914222 DOI: 10.1136/bjsports-2023-106809
Isolation and identification of a new intracellular antimicrobial peptide produced by Paenibacillus alvei AN5

Alkotaini B, Anuar N, Kadhum AA, Sani AA

World J Microbiol Biotechnol, 2014 Apr;30(4):1377-85.
PMID: 24272828 DOI: 10.1007/s11274-013-1558-z

A wild-type, Gram-positive, rod-shaped, endospore-forming and motile bacteria has been isolated from palm oil mill sludge in Malaysia. Molecular identification using 16S rRNA gene sequence analysis indicated that the bacteria belonged to genus Paenibacillus. With 97 % similarity to P. alvei (AUG6), the isolate was designated as P. alvei AN5. An antimicrobial compound was extracted from P. alvei AN5-pelleted cells using 95 % methanol and was then lyophilized. Precipitates were re-suspended in phosphate buffered saline (PBS), producing an antimicrobial crude extract (ACE). The ACE showed antimicrobial activity against Salmonella enteritidis ATCC 13076, Escherichia coli ATCC 29522, Bacillus cereus ATCC 14579 and Lactobacillus plantarum ATCC 8014. By using SP-Sepharose cation exchange chromatography, Sephadex G-25 gel filtration and Tricine SDS-PAGE, the ACE was purified, which produced a ~2-kDa active band. SDS-PAGE and infrared (IR) spectroscopy indicated the proteinaceous nature of the antimicrobial compound in the ACE, and liquid chromatography electrospray ionization mass spectroscopy and de novo sequencing using an automatic, Q-TOF premier system detected a peptide with the amino acid sequence F-C-K-S-L-P-L-P-L-S-V-K (1,330.7789 Da). This novel peptide was designated as AN5-2. The antimicrobial peptide exhibited stability from pH 3 to 12 and maintained its activity after being heated to 90 °C. It also remained active after incubation with denaturants (urea, SDS and EDTA).

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