PRACTICAL TRAINING REPORT This practical training report is submitted for the partial requirement for Bachelor Degree By: JESSICA ASTELIA 14.I1.0130

  

DETERMINATION OF CHOLESTEROL OXIDATION

PRODUCTS IN PORK FLOSS BY GAS CHROMATOGRAPHY

• – FLAME IONIZATION DETECTOR

PRACTICAL TRAINING REPORT

  This practical training report is submitted for the partial requirement for Bachelor Degree

  

By:

JESSICA ASTELIA

14.I1.0130

  

DEPARTMENT OF FOOD TECHNOLOGY

FACULTY OF AGRICULTURAL TECHNOLOGY

SOEGIJAPRANATA CATHOLIC UNIVERSITY

  

DETERMINATION OF CHOLESTEROL OXIDATION

PRODUCTS IN PORK FLOSS BY GAS CHROMATOGRAPHY

• – FLAME IONIZATION DETECTOR

  

Practical Training at Fu Jen Catholic University, New Taipei City, Taiwan

By:

Jessica Astelia

Student ID : 14.I1.0130

  

Faculty : Agricultural Technology

This Practical training report has been approved and supported by examiner in

th

  

Practical Training Exam on 7 of March 2017:

th

  Semarang, 7 of March 2017 Department of Food Technology Faculty of Agricultural Technology Soegijapranata Catholic University

  Practical Training Advisor I Practical Training Advisor II Prof. Bin-Huei Chen Dr. Ir. B. Soedarini, MP.

  

PREFACE

  Praise the Lord because of His grace and blessing, author would have the opportunity to undergo the practical training and finish the report. This report is complete accountability from the practical training which was done in Fu Jen Catholic University,

  th th New Taipei City, Taiwan that take place from January 4 until February 28 2017.

  During the training, the author did the research entitled: “Determination of Cholesterol Oxidation Products in Pork Floss by Gas Chromatography – Flame Ionization Detector”. This report was written as a requirement to acquire Bachelor Degree of Food Technology in Soegijapranata Catholic University, Semarang, Indonesia.

  The author would not be able to finish this task alone, and only by support and guidance given by people around the author, this report could be finished. Special thanks for:

  1. Dr. V. Kristina Ananingsih, ST, MSc. as a Dean of Faculty of Agricultural Technology, Soegijapranata Catholic University for giving the opportunity to join this practical training.

  2. Prof. Bin-Huei Chen as the advisor from Fu Jen Catholic University for giving guidance and supporting all the time during the practical training.

  3. Dr. Ir. B. Soedarini, MP. as the advisor from Soegijapranata Catholic University for always taking care and giving advices during the practical training.

  4. Che-Wei, Lauren, Jerry, Kyle, Yi-Fen, Jun-Yu, and Hua as the students in Fu Jen Catholic University (EP310) and all of the friends from Taiwan that author can’t mentioned one by one who always support and accompany author.

  5. My family who always support and cheer everyday

  6. Denny and Fia who has been the practical training mates during the practical training program.

  Semarang, March 7

  th

  2017 Author

  

TABLE OF CONTENTS

APPROVAL PAGE ....................................................................................................................... i

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

  

LIST OF TABLES

  

LIST OF FIGURES

  

1. INTRODUCTION

  1.1. Background of Practical Training

  Nowadays, people not only looking for the delicious food, but also pay attention to the nutritional value contained. From here, we know how important food technologist is to fulfill the demands of consumers. We should be aware for the changing of food products demand. These facts become the main reason to conduct the practical training for students from Food Technology Department, Soegijapranata Catholic University, Semarang, Indonesia. Besides, we also can exchange some knowledge about the development of food technology in each country. This program is one of the requirement to gain Bachelor Degree in Faculty of Agricultural Technology, Department of Food Technology, Soegijapranata Catholic University, Semarang, Indonesia.

  Food Science Department, Fu Jen Catholic University, New Taipei City, Taiwan, is chosen as the practical training work because this faculty is advanced in the field of food processing and development, which is become important recently. During this practical training, one of the graduate student will help us to doing the research and become the assistant. This practical training program give opportunity to do research abroad and experiencing another culture from global citizen.

  1.2. Purpose

  The practical training in Fu Jen Catholic University, New Taipei City, Taiwan has purpose:

  1. To give an experience about doing food research abroad with the new environment

  2. To give an opportunity to adapt with new circumtances and society in another country.

1.3. Time and Place

  The practical training was conducted in Food Science Department, Fu Jen Catholic

  th

  University, New Taipei City, Taiwan. This activity took place between January 4 until

  th February 28 2017.

  

Figure 1. Map of Fu Jen Catholic University, New Taipei City, Taiwan

  The red indicates the location of Fu Jen Catholic University which is located in No. 510 Zhongzheng Rd, Xinzhuang District, New Taipei City, 24205, Taiwan (R.O.C) (Telephone : (02) 29052000).

2. INSTITUTION PROFILE

2.1. Fu Jen Catholic University

  Fu Jen Catholic University (FJU) is the first university in China that founded in Beijing in 1925 by the Bendedictines of Saint Vincent Archabbey. Fu Jen Catholic University is also called Fu Jen or Fu Da. Moved by Christian understanding of love and inspired by the high ideals of Confucian education; it adopted the name “Fu Jen” to give expression to its universal vision and mission realized through holistic education. Fu Jen also hopes to serve society through various additional academic programs and community services. Fu Jen University has history of 80 years, and provides the country with good educated students characterized by integrated physical, social, intellectual, aesthetic, moral, and spiritual development which have contributed greatly in all fields in society. Fu Jen is noted for attracting foreign students from another country include Indonesia. Fu Jen provides 11 colleges such as Liberal Arts, Arts, Communication, Education, Medicine, Science and Engineering, Foreign Languages, Human Ecology, Law, Social Sciences, Management; 48 departments, 47 master’s programs, 23 in-service master’s programs, 11 Ph.D. programs and School of Continuing Education. The land capacity of the university is about 35 hectars and current student enrollment is 26,000. The University has about 120 relations with the other universities in the world.

  2.2. Department of Food Science

  In 1963, Department of Family Studies and Nutrition Sciences was established and grouped into the Family Studies and the Nutrition Sciences section. Nutrition Sciences section was combined with the Food Sciences section as the Department of Nutrition and Food Sciences in 1971. The Graduate Institute of Nutrition and Food Sciences was established in 1983 and started to offer a master’s degree program. The doctoral program was joined to the Institute in 1995. Food Sciences section became an individual department in 2006. The Department of Food Science offers Bachelor’s degree program and Master’s degree program.

  

Figure 3. Logo of Department of Food Science FJU

  2.3. Mission of Faculty

  Uphold the spirit of pursuing truth, goodness, beauty and holiness, the Department of Food Science at Fu Jen Catholic University integrates basic sciences with latest technology for excellence education, research, and service. We are committed to promote the healthier, tastier and safer for improving eating quality, human health and wellness.

3. RESEARCH PROJECT 3.1. Research Overview

  The topic of the research is “Determination of Cholesterol Oxidation Products in Pork Floss by Gas Chromatography – Flame Ionization Detector”. There are seven kinds of standard solutions. Then there are five pork floss samples extracted with QuEChERS method at day one, five, and ten. The objective of this research is to determine the different formation of COPs in pork floss during storage at room temperature. The advisor of this research is Prof. Bin-Huei Chen.

3.2. Background of Research

  Nowadays, people more consider about the relationship between food products and health. Many studied been widely encourage to improve people dietary habits. Processed meat products are highly consumed by people, however they are not considered healthy because a high consumption of meat products may be increase the risk of several cancer (WHO, 2015). Cholesterol contents of meat and meat products varied considerably, but in general it is less than 70 mg/100 g except for edible offal and it is assumed that one-third of daily intake come from meat and meat products (Chizzolini et al., 1999). Cholesterol is an important biological compound, but its oxidation products can be harmful for human health.

  Cooking is essential as it improves taste, digestibility, and extends shelf life. However, this method is related to the formation of Cholesterol Oxidation Products (COPs). Cooking, dehydration during storage, and radiation are some of the main causes of cholesterol oxidation in food products of animal origin. COPs have been proven to be cytotoxic, mutagenic, and carcinogenic, and also considered to be a primary factor responsible for atherosclerosis (Khan et al., 2015). In general, healthy human plasma contains 12.6 mg/L of COPs and consumption of foods containing COPs will increases

  3.3. Pork Floss Pork floss is a popular traditional Chinese dried meat with a light and crispy texture.

  People usually used it as a topping for many foods or eat it as a snack. It is prepared by boiling fresh raw pork until the muscle fibers could easily separate. Followed by cooling, pressing, and then adding the various additives, such as sugar, salt, monosodium glutamate, edible oil, sauce, and spices. Then the cooked pork stir fried for about 1 hour until turned out to brown-colored in a shredded form (Liao et al., 2009). However, no information is available for the variety and amount of COPs in pork floss.

  3.4. Cholesterol Oxidation Products

  Cholesterol is a steroid which is biosynthesized by all animal cells that has many metabolic functions. Animal products are the major source of cholesterol in the diet. Although cholesterol is a relatively stable compound, it can be oxidized under mild and harsh conditions, processing step, and storage time (Garcia-Marquez et al., 2014). Cholesterol oxidation reactions resulting a wide range of secondary products which called COPs. COPs are group of sterols that are similar in structure to cholesterol but contains an additional hydroxy, ketone or epoxide group on the sterol nucleus and/or a hydroxyl group on the side chain of their molecules (Hur et al., 2007).

  There are several factors that influence the formation of COPs: presence of oxygen, light (photo-oxidation), high temperatures, and other factors (Ahn et al., 2001). Thereby, we can delay their formation by using the packaging materials that can help avoid the entrance of air and light, stored it with the right temperature, and adding the antioxidant (Garcia-Marquez et al., 2014). COPs are considered more harmful than cholesterol and they have been shown to be cytotoxic, atherogenic, mutagenic and carcinogenic. So, a high consumption of COPs has an adverse effect on human health. The most common products of cholesterol oxidation found in foods are 7

  α- hydroxycholesterol (7

  3.5. Sample Extraction

  Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) is a method that we used for extracting sample in this research. However, the QuEChERS method is popular in the analysis of pestcides and other compounds in huge variety of food products. This method has important advantages over another extraction methods which are enable yielding high recovery rates for wide range of analytes, very accurate results, high sample throughput, low solvent consumption and very small waste generation (Lehotay

  et al.

  , 2005). This method involves two simple steps. First, the homogenized samples are extracted and partitioned using an organic solvent and salt solution. Then, the supernatant is purifying using dispersive solid-phase extraction (d-SPE) (Schenck and Hobbs, 2004). The main factors considered in initial extraction and extraction part are the type and amount of extraction solvent, sample amount, and sample/solvent ratio. For clean up stage, the major problem are the type and amount of sorbent and their selectivity (Rejczak and Tuzimski, 2015). Organic solvent extraction is then followed by derivatization and analyzed by gas chromatography.

  3.6. Gas Chromatography – Flame Ionization Detector Analysis (GC-FID)

  GC is a dynamic method for separation and detection volatile organic compounds in a gas mixture, which commonly used to estimate the concentration of a substance in the gas phase. There are several types of detector and we used FID for this research. FID will ionization the organic compound by burning the compounds in the hydrogen and air. This detector is the most common used for gas chromatography because it has high sensitivity for various components and worked at various concentrations (Grob & Barry, 2004). The detector is placed in the end of the column and will present the chromatogram results.

4. RESEARCH METHODOLOGY

  4.1. Materials

  Materials that used for this research are 5 different brand (©) of pork floss. A 味小寶 wèi xi ǎo bǎo (味全-wèi quán), B 義美 yìměi (義美-yìměi), C 得意的一天 déyì de yītiān (佳格-jiā gé), D 新東陽 xīn dōngyáng (新東陽-xīn dōngyáng ), E is lab-made, deionized water, acetone, extraction powder, d-SPE sorbent, pyridine, nitrogen, derivation agent (Sylon BTZ), and standard (7

  α-OH, 7β-OH, 5,6β-EP, 5,6α-EP, triol, 25-OH, 7-keto) purchased from Sigma (St. Louis, MO, USA), fresh pork, water, salt, sugar, monosodium glutamate, soy sauce, lard, spices, gravy.

  4.2. Statistics Tool

  Statistic tools that used for this research are An Agilent Technologies series HP6890 Gas Chromatographic system equipped with a flame ionization detector (GC-FID), centrifuge Sorvall RC5C High Speed Centrifuge, Du Pont, USA (Wilmington, Delaware, USA), centrifuge tube, eppendorf tubes, beaker glass, micropipette, tips, scale, filter, ceramic stone (10 x 25 mm), vortex mixer, syringe, pressure cookers, wooden mallet.

  4.3.Methods 4.3.1. Pork Floss Preparation

  Sample E that we used for this research was made in Fu Jen Laboratory. First step, 1200 gram of fresh pork was boiled with the pressure cookers for 1 hour until the muscle fibers could easily separate. After that, cooling it for a while and using a wooden mallet to smash the muscle fiber and shredded it. Followed by mixing it with the various additives, such as 20% sugar, 1.6% salt, 1% monosodium glutamate, 8% soysauce, 30% gravy, and 0.1% spices, and stir fried the cooked pork. After 25 minutes, adding 14%

  4.3.2. Sample Extraction

  Standard solutions were prepared by dissolved the standards with pyridine. For sample preparation, two grams of sample was dissolved in 10 ml of deionized water in the centrifuge tubes. A ceramic stone homogenizers was put into the solution and then vortex it for one minute. Then, the sample was added by 10 ml acetone and vortex it for one minute. After that, added the extraction powder which consist of MgSO

  4 4 g and Na

  Acetate 1 g. The tube was vortexed for one minute and centrifuged with 3000 rcf

  o

  (relative centrifugal force) for 10 minutes at 4

  C. The supernatant (4 mL) from the centrifugation process was collected and added to the d-SPE sorbent which consists of 900 mg MgSO

  4 , 300 mg PSA, and 300 mg C18EC. The tube was vortexed for one o

  minute and centrifuged with 3000 rcf for 10 minutes at 4

  C. Take 1 mL of the supernatant into 1.5 mL eppendorf tube and dried it with nitrogen. After that, dissolving it with 100

  μl pyridine and vortexing it. The solution was filtered through a 0.22 μm syringe filter (Nylon) . Take 40 uL of sample solution, then add 20 μL of internal standard and 40

  μL of derivatization agent (sylon BTZ). Let the samples derivatized for 1 hour in the dark at room temperature . Next, 1 μL of the resulting solution injected into GC-FID. Each sample was done in duplicate so there will be ten times extraction.

  4.3.3. Gas Chromatography – Flame Ionization Detector Condition

  The derivatized extracts were analysed by Gas Chromatography composed of Agilent technologies series HP6890 and a flame ionization detector (FID). A DB-5MS capillary column (30 m × 0.25 mm I.D., 0.25

  μm ) was purchased from Agilent Technologies (Palo Alto, CA, USA). A split injection ratio that we used was 1:1. Injection volume was 1 μL and the flow rate was 1 mL/min. Helium was used as the carrier gas. The

  o o o

  initial oven temperature set as 230 C and raised to 290 C for 10 C/min and maintain for 9 minutes with the total time being 15 minutes. The condition of injection was set at

  o o

  280

  C, the detector temperature was 310

  C, the flow rate of H

  2 was at 45 mL/min, and

4.3.4. Statistical Analysis

  Statistical analysis was performed by one-way analysis of variation (ANOVA) using SAS 9.4 and Duncan’s multiple range test was employed to differentiate the significance among mean values (p < 0.05).

5. RESULT AND DISCUSSION

5.1.QuEChERS Method Development

  Sample we used for this research is pork floss. There are five different samples of pork floss extracted in three different days. The different days is aimed to know the changes of different COPs during storage at room temperature. First, two grams of sample was dissolved in 10 mL of deionized water in the centrifuge tubes. A ceramic stone homogenizers was put into the solution and then vortex it for one minute. Then, the sample was added by 10 mL acetone and vortex it for one minute. The addition of an inorganic salt into a mixture of solution causes salting out which will separate the solution into two-layer. The extraction powder consists of MgSO

  4 4 g which contributes

  to eliminate water and Na Acetate 1 g as the buffer to control the polarity of the extraction solvents. The tube was vortexed for one minute and centrifuge with 3000 rcf

  o

  for 10 minutes at 4

  C. The supernatant (4 mL) from the centrifugation process collected and added to the d-SPE sorbent which consists of 900 mg MgSO

  4 , 300 mg PSA, and

  300 mg C18EC. PSA and C18 is commonly applied to remove impurities such as sugar, fatty acid, organic acid, and lipid (Rejczak and Tuzimski, 2015).

  Then the tube was vortex for one minute and centrifuge with 3000 rcf for 10 minutes at

  o 4 C to increase distribution of the SPE material and contribute the clean-up process.

  Take 1 ml of the supernatant into 1.5 mL eppendorf tube and dried it with nitrogen to remove solvents (Derewiaka and Molinska, 2015). After that, dissolve it with 100 μl pyridine and vortex it. The solution was filtered through a 0.22

  μm Nylon filter with a syringe . Take 40 uL of sample solution, then add 20 μL of internal standard and 40 μL of derivatization agent (sylon BTZ). Let the samples derivatized for 1 hour in the dark at room temperature . Derivatization is a chemical process to increase the compound volatility so it will be more suitable for analysis (Kangani et al., 2008). Chemical derivatization often require to improve the peak symmetry. Next, 1

  μL of the resulting

5.2.Gas Chromatography Method Development

  Gas Chromatography principles is vaporized a liquid sample to a gas then carries it through a column with an inert gas carrier. The column has a stationary phase that interacts differently with each compound, which will involve a different retention time. The detector will response and convert it to an electrical signals resulting a gas chromatogram we can analyze. (Kangani et al., 2008). The advantage of using gas chromatography are the analysis time relatively short and have high sensitivity.

  

Figure 4. Principle of Gas Chromatography - Flame Ionization Detector

  (Source :

  

)

  The test of two initial temperatures (230 and 250°C), two flow rate (0.5 and 1.0 mL/min), and two split ratios (10:1 and 1:1) was conducted to optimized the gas chromatography method for this research. The result indicated that lower initial temperature (230°C) will improve the peak symmetry and no thermal degradation was observed (Chen et al., 1994). Furthermore, at a higher flow rate (1.0 mL/min), all of the COPs showed better separation and shorter retention time (< 15 minutes). Moreover, the result also showed that a lower split ratio (1:1) will improve the peak symmetry. Hence, the initial temperature of 230°C, the flow rate of 1.0 mL/min, and the split ratio 1:1 was chosen. In general, method optimization requires a proper choice of initial temperature,

5.3. Identification Method

  We used two methods for identification of the COPs in pork floss sample. (1) Compare the retention time of COPs standard with pork floss sample which shows in Table 1. (2) Add COPs standard into sample solution for co-chromatography which shows in Figure 5.

  a b

  Table 1. Retention ti of COPs me (Rt), retention factor (κ) and separation factor (α) and cholesterol by using GC-FID

  Peak Compound Rt (min)

  κ α Number

  1.5 - - - Solvent

  1 6.91 3.607 1.377 5α-cholestane

  2 8.95 4.967 1.074 7α-OH cholesterol

  3 9.5 5.333 1.129

  4 10.53 6.020 1.056 7β-OH

  5 11.04 6.360 1.022 5,6β-EP

  6 11.25 6.500 1.122 5,6α-EP triol

  7 12.44 7.293 1.090 25-OH

  8 13.43 7.953 1.023 _a 7-keto

  9 13.7 8.133 1.023

  κ=(t R R = retention factors of peak 1, k = retention factor of peak 2 _c α= κ ^{2 / κ 1 , κ 1 2} Numbers in parentheses represent values between two neighboring peaks

• – t )/ t , t = retention time of solvent peak, t = retention time of COPs _b

  Table 1 shows retention time, retention factor ( κ), and separation factor (α). To have a good separation,

  κ should be in the range of 1 ~ 10 (or 0.5 to 20 in extreme cases) and α value is more than 1 (α=1 means the peaks overlap each other). Our experiment shows good separation method as the retention factors were in the range of 3.607 ~ 8.133 and all of the α values were >1.

  1

  3

  5

  4

  2

  7

  6 8 9

Figure 5. Co-Chromatography

  

Peaks: (1) internal standard (5 α-cholestane), (2) 7α-OH, (3) cholesterol, (4) 7β-OH, (5) 5,6β-EP, (6) 5,6α-

EP, (7) triol, (8) 25-OH, (9) 7-keto. The blue line presents the sample solution The red line presents the sample solution added with standard solution.

  Figure 5. shows the chromatograms of the internal standard when added into sample. The blue line shows the sample solution while the red line shows the sample soulution added with standard solution. Then we can see that the COPs in pork floss were identified.

5.4. Quantification Method

  Internal standard calibration curve is a common quantification method in instrument analysis, for example in LC or GC. There are several factors to choose the internal standards: (1) the structure of the internal standard should be similar with the analyte (Figure 6.), (2) the internal standard peak should be separate individually (Figure 7.), and (3) the compound should not be present in the sample (EPA Method 8000C, 2003).

  We prepare the COPs standard solution in different concentration between 0.4 ~ 4 ppm, except for the 5,6 β-EP range is 0.4 ~ 28 ppm. Then we calculate the (COPs peak area/ISTS area, As/Ai) and (COPs standard concentration/ISTD concentration, C/Ci) in

  Excel (Figure 8.). Linearity was determined by the values of determination coefficient

  2

  obtained from calibration curves. The equations and determination coefficients (R ) for

  2

  each analyte are presented in Table 2. The R for seven COPs were > 0.9964 which means the standard calibration curve have a good linearity. We used the following formula to calculate the COPs amount in each pork floss: As : COPs sample area Ai : Internal standard area b : Intercept in calibration curve a : Calibration slope Ci : Concentration of internal standard V : Extraction volume

  Figure 6. The chemical structure of (a) 5- α-Cholestane (b) Cholesterol

(c) Seven COPs

  

Figure 7. GC Chromatogram of COPs standards

Peaks: (1) internal standard (5 α-cholestane), (2) 7α-OH, (3) cholesterol, (4) 7β-OH, (5) 5,6β-EP, (6) 5,6α-

EP, (7) triol, (8) 25-OH, (9) 7-keto.

  Reagent peaks shown without number labelling

  Table 2. Linearity and Regression Equations from Calibration Curves COPs standards Calibration Curve Equation R

  2

  7α-OH y = 0.5816x + 0.0033 0.9976 7β-OH y = 0.5949x + 0.0044 0.9980

  5,6β-EP y = 0.5993x + 0.0012 0.9989 5,6α-EP y = 0.5418x + 0.0016 0.9984 ^{min 2 4 6 8 10 12 14 pA 10 15 20 25 30 35 40 45 FID1 A, (QQQ\17021305.D) 1 2 3 4 5 6 7 8 9}

5.5.Analysis of Cholesterol Oxidation Products (COPs) in Pork Floss

  The different COPs were analyzed by Gas Chromatography according to the method previously described. Identification of COPs was based on the comparison of the retention times of the samples with the standards (Yurchenko et al., 2016). The concentrations of COPS were calculated using the peak area of COPS over the peak area of the internal standard. The area ratios of COPS to internal standard were determined at six different concentrations, which are 0.02, 0.04, 0.12, 0.2, 0.32, and 0.4 ppm. (a) _{A A A A A A A A A} A _{A A A A A A A A A A} ^A A A A _{A A}

  (b) _{A A A A A} A _{A A A A A A B B A}

  (c) _{A A A A A A B A A A A A A A A A B B A A} AB (d) _{A A A A A} A _{A A A A A A A A} A

  (e) _{C A A A A A A A B AB B} A _{B B A}

  b

  a

  2966.39±95.73

  a

  2866.79±112.48

  a

  7β-OH 2434.31±438.08

  a

  3346.89±353.90

  a

  3399.60±253.80

  a

  3012.47±119.32

  Sample A Day 1 Day 5 Day 10 7α-OH

  4316.59±106.32

  5,6β-EP 28181.69±724.27

  ab

  3622.38±338.48

  b

  7-keto 3087.62±144.29

  a

  1088.12±104.10

  a

  1033.62±110.57

  a

  25-OH 827.65±513.43

  a

  2307.49±268.51

  a

  a

  1443.32±165.92

  a

  a

  3152.88±794.02

  a

  3422.74±90.90

  a

  7-keto 2863.45±59.40

  a

  1756.10±219.46

  a

  2221.23±1.13

  a

  25-OH 1341.54±598.39

  1536.00±122.58

  30983.09±1823.22

  a

  1680.14±137.31

  a

  triol 1426.22±320.62

  a

  1923.25±69.58

  a

  1864.51±448.44

  a

  5,6α-EP 2077.52±161.73

  a

  27072.17±3809.51

  a

• triol 664.41±92.11

  b

  Figure 9. shows the changes of different COPs during storage at room temperature in sample A to sample E. The separation between different compounds has been successfully achieved. The result showed that the most predominant oxidized cholesterol from sample A to sample E were 7

  a

  a

  1053.94±112.52

  b

  512.66±25.74

  b

  5,6α-EP

  a

  971.66±146.70

  a

  874.13±70.36

  a

  5,6β-EP 663.62±57.08

  1098.37±114.37

  a

  a

  852.17±68.74

  a

  7β-OH 1319.83±217.90

  a

  1045.73±85.30

  a

  978.93±192.14

  a

  1402.94±149.62

  Sample B Day 1 Day 5 Day 10 7α-OH

  

Table 3. Changes of Different COPs (ng/g) during Storage at Temperature Room

(a) Sample A; (b) Sample B; (c) Sample C; (d) Sample D; and (e) Sample E

  α-OH, 7β-OH, 5,6β-EP, triol, and 7-keto.

  25-OH - - - 7-keto 1425.00±19.85

  1083.83±32.41

  triol 1458.69±237.83

  a

  a

  1329.12±97.01

  a

  1454.16±45.31

  a

  5,6α-EP 1734.00±210.37

  a

  27673.46±121.90

  a

  26899.24±1370.45

  a

  5,6β-EP 23909.37±4865.89

  2643.30±327.82

  a

  a

  2505.07±37.69

  a

  7β-OH 2336.69±63.57

  a

  3320.56±310.19

  a

  3242.09±406.80

  a

  7α-OH 2364.28±620.24

  Total Amount 5475.80±536.56 4301.72±389.39 5863.33±974.23 Sample C Day 1 Day 5 Day 10

  a

  1693.63±515.34

  Total Amount 41337.20±2421.81 46438.10±2867.28 41753.68±5468.75

  Sample D Day 1 Day 5 Day 10

  a a a

  900.25±163.69 1195.51±193.14 1386.72±183.40 7α-OH

  a a a

  803.24±158.91 1055.85±71.33 1161.05±120.86 7β-OH

  a a a

  3351.07±25.64 3595.74±690.14 3785.99±744.97 5,6β-EP

• 5,6α-EP

  a a a

  triol 3240.15±222.70 3282.74±474.05 4263.11±742.75

• 25-OH

  a a a

  7-keto 1229.94±73.91 1555.90±191.72 1232.17±2.96 Total Amount 9524.65±644.85 10685.74±1620.38 11829.04±1794.94

  Sample E Day 1 Day 5 Day 10

  a b ab

  906.48±118.54 582.83±23.55 825.74±104.80 7α-OH

  b a c

  952.91±178.95 1368.57±18.76 594.72±34.33 7β-OH

  a a a

  1298.88±42.05 1453.08±135.43 1349.93±57.72 5,6β-EP

• 5,6α-EP

  b b a

  triol 509.48±19.94 461.04±56.75 727.24±26.67

• 25-OH

  a a a

  7-keto 1039.06±191.26 1286.16±328.49 1431.49±61.07 Total Amount 4706.81±550.74 5151.68±562.98 4929.12±284.59 Values of content are MEAN±SD of two replicates.

  Different letters whitin a row indicate significant differences between different day. _* Data expressed as ng/g.

• means no detected

  The result is presented in Table 3. We can see that the COPs of pork floss varied significantly during storage. In general, the pork floss contains higer level of COPs during storage time. Among the tested samples, the highest total COPs content (4643.8±286.72 µg/g) was observed in Sample A at Day 5 and the lowest (430.17±38.93 µg/g) in Sample B at Day 5. As we can see there is a significant difference in 7α-OH, 7β-OH, 5,6β-EP, triol, and 7-keto in some samples. The formation of triol and 7-keto for some samples was higher compared to the other compounds. This result is similar to that of Monahan et al., (1992) who reported a significant increase of total amount of COPs in cooked pork products during storage. The COPs contents after four days storage were higher than that after two days storage. due to a high variation of ingredients in each sample. The oxidation of cholesterol in food is influenced by many factors, such as food composition, presence of anti-oxidants, storage condition, food processing, and other factors like moisture (Min et al., 2016). Badiani et al. (2002) also reported that different cooking method will produce different moisture content of meat products which lead to different levels of cholesterol products. Overall, from this research we can see that COPs contents in pork floss will be higher following a longer storage time.

6. CONCLUSIONS AND RECOMMENDATION

  6.1. Conclusions

• QuEChERS extraction method and Gas Chromatography with Flame Ionization Detector are suitable for the analysis of COPs in pork floss.
• We can use GC-FID combined with DB-5MS capillary column to separate the internal standard (5

  α-cholestane), 7α-OH, cholesterol, 7β-OH, 5,6β-EP, 5,6α-EP, triol, 25-OH, 7-keto whitin 19 minutes.

• The most predominant COPs in pork floss detected were 7

  α-OH, 7β-OH, 5,6β-EP, triol, and 7-keto.

• In some of pork floss, we found that COPs content will increase during storage.

  6.2. Recommendation

  However, further investigation is needed to elucidate the impact of autoxidation on pork floss.