Final thesis - Lecithin

LIST OF TABLESTable 2.1 Composition of crude lecithin obtained from various sources...4Table 4.1 Composition of rice bran oil gum sludge...18Table 4.2 Effect of shaking time and acetone

Foremost, according to the co-operation between Naresuan University and Nong LamUniversity, Ho Chi Minh City about internship, I would like to thank Faculty of Agriculture,Natural and Resources and Environment, Naresuan University and Faculty of Food Scienceand Technology, Nong Lam University, Ho Chi Minh City for giving me a chance to aninternship in Thailand for 3 months

In addition to that, I would especially like to thank all lecturers in the Faculty ofAgriculture, Natural and Resources and Environment, especially, my advisors Assit Prof Dr.Riantong Singanusong and Assit Prof Dr Umargar Haruna who guided, advised, encouragedand helped me in my thesis during three months I also would like to thank Asst Prof Dr.Orawan Kritsunankul for their invaluable help and suggestion on my experiment and project

I could not complete this research without the lots of help I received cheerfully fromeverybody We experienced great things together and they have shown me their enthusiastichelp

I also would like to express my gratitude to Dr Nguyen Minh Xuan Hong forsuggestion, support, helpful and guidance Furthermore, I would like to thank all my Thai andVietnamese friends who help and support not only in my thesis, but also in my life duringthree months become successful, happy, comfortable and cheerful

Finally, I am greatly thankful to my family for their inspiration and endless love

Nguyen Tan Phat

Nowadays, lecithin is used very widely in food industry because of high benefit that itprovides It can be used as emulsifiers, dispersants, wetting agents, viscosity modifiers,surfactants or nutritional supplements In the past, lecithin was extracted from many varioussources such as egg yolks, soybean, sunflower or rapeseeds In this study, the extraction oflecithin from rice bran gum sludge was carried out by following the methods of Ceci,Constenla, and Crapiste (2008) with totally 6 treatments which composes 3 ratios ofgum/acetone (1:1; 1:1.5; 1:2) and 2 shaking time (30 and 60 minutes) After extraction, theresult has shown that at 1:1.5 wet gums to acetone ratio were achieved the highest yield oflecithin (9.99 ± 1.00 %) Then, the obtained lecithin was analyzed acid value and moisturecontent The acid value was decreased when increasing the shaking time and the acid valuehas reached the highest value at the sample which has 1:1 gum to acetone ratio and 30minutes shaking (25.75 ± 0.11) The moisture content was determined by Moisture analyzerand there is no significant difference between the means of the result Rice bran gum sludgecontains much of phospholipid; therefore, the obtained lecithin has highest antioxidantactivity (25.57 - 39.52%) Normally, lecithin has high solubility in oil However, in this study,the solubility in oil of obtained lecithin is very low The reason maybe is the temperature ofoil is 240C

TABLE OF CONTENT

ACKNOWLEDGEMENT i

ABSTRACT ii

TABLE OF CONTENT iii

LIST OF TABLES iv

LIST OF FIGURES vi

LIST OF ABBREVIATIONS vii

CHAPTER 1: INTRODUCTION 1

1.1 Introduction 1

1.2 Research Aim 2

1.3 Research Scope 2

CHAPTER 2: LITERATURE REVIEW 3

2.1 Introduction about rice bran 3

2.2 Rice bran oil (RBO) 5

2.3 Lecithin 7

2.4 Extraction of lecithin 8

2.5 Application of lecithin 11

CHAPTER 3 MATERIALS AND METHODS 13

3.1 Materials 13

3.2 Chemicals 13

3.3 Apparatus 13

3.4 Methodology 13

3.4.1 Method for lecithin extraction from degumming by-product 13

3.4.2 Analysis 15

3.4.2.1 Yield 15

3.4.2.2 Acid value 15

3.4.2.3 Moisture content 15

3.4.2.4 Antioxidant activity (DPPH) 15

3.4.2.5 Solubility in oil 17

3.4.3 Statistical analysis 17

CHAPTER 4 RESULTS AND DISCUSSIONS 18

4.1 Composition of weight gum sample 18

4.2 Effect of shaking time and acetone to gum ratio and on the extraction of lecithin 18

4.3.1 Acid value 20

4.3.2 Moisture content 21

4.3.3 Antioxidant activity (DPPH) 22

4.3.4 Solubility in oil 23

CHAPTER 5 CONCLUSIONS AND RECOMMENDATIONS 24

5.1 Conclusions 24

5.2 Recommendations 24

REFERENCES 25

APPENDIX 28

LIST OF TABLES

Table 2.1 Composition of crude lecithin obtained from various sources 4Table 4.1 Composition of rice bran oil gum sludge 18Table 4.2 Effect of shaking time and acetone to gum ratio on the extraction of dried gum 19Table 4.3 Effect of the shaking time and acetone to gum ratio on the Acid value of the lecithin 20Table 4.4 Effect of shaking time and acetone concentration on the acid moisture content of lecithin 21Table 4.5 Effect of shaking time and acetone concentration on the antioxidant activity of

lecithin 22Table 4.6 Effect of shaking time and acetone concentration on the solubility in oil of lecithin 23

LIST OF FIGURES

Figure 2.1 Structure of rice kernel 3

Figure 2.2 Process of chemical refining RBO 6

Figure 2.3 Structure of soybean lecithin 7

Figure 2.4 Some phosphatides in soybean lecithin 7

Figure 2.5 Flowchart for the lecithin processing 10

Figure 2.6 Some products containing lecithin 11

Figure 3.1 Flowchart of lecithin production 14

Figure 4.1 Appearance of lecithin after extraction with acetone 19

Rice bran lecithinSoybean lecithin

CHAPTER 1: INTRODUCTION

1.1 Introduction

Rice is the top of food crop in the world, and rice bran is a by-product which isobtained after rice processing Rice bran is the cuticle between the paddy husk and the ricegrain (Amarasinghe, Kumarasiri, & Gangodavilage, 2009) Long time ago, rice bran wasmainly used for animal feed without any extraction methods Thus, in recent year foodindustry has been developing in rice bran extraction to obtain wishful oil, protein, lecithin orother substances (Orthoefer, 1996)

Nowadays, lecithin is very commonly used in food, pharmaceutical and cosmeticindustries About 25-30% used in margarine, 25-30% used in baking chocolate and ice cream,10-20% for technical products, 3-5% in cosmetic and approximately 3% for pharmaceutical(Wu & Wang, 2004) In food, lecithin provides about a dozen functions, including as anemulsifier, as a wetting agent, for viscosity reduction, as a release agent and for crystallizationcontrol Lecithin also provides functions in numerous industrial applications as well (List,2015) In addition, native and modified lecithin is widely employed as multifunctionalingredients in food industries (Cabezas, et al , 2009) Besides, soybean phospholipids are alsowidely used as emulsifiers, wetting agents and baking improvers (Patil, Galge, & Thorat,2010)

Lecithin contains mainly of phosphatidylcholine (PC), phosphatidylethanolamine(PE), phosphatidylinositol (PI) and other minor compounds such as phosphatidic acid (PA),triglycerides, carbohydrates, etc (Cabezas et al., 2009) Lecithin usually comes from sourcessuch as soybean, egg, milk, rapeseed, cottonseed, and sunflower Crude soybean lecithintypically contains 18% PC, 14% PE, 9% PI, 2 % minor phospholipids (PL), 11% glycolipids,5% complex sugars and 37% neutral oil (Wu and Wang, 2004)

Alternatively, according to Van Nieuwenhuizen (2014) & Prasad (2015), rice bran has

a highest content of lecithin (PL 53.4%) Therefore, extraction of lecithin from rice bran oilwould be necessary due to its high potential in food industry for use as emulsifier Moreover,lecithin is mostly nontoxic in acute oral studies, short-term oral studies and sub-chronic

dermal studies in animal It is also neither a reproductive toxicant, nor is it mutagenic(Lecithin, 2001).

Lecithin is a generic term to designate any group of yellow-brownish fatty substancesoccurring in animal and plant tissues, which are amphiphilic - they attract both water and fattysubstances (and so are both hydrophilic and lipophilic) and used for smoothing food textures,dissolving powders (emulsifying), homogenizing liquid mixtures, and repelling stickingmaterials It is also a mixture of glycerophospholipids which includes phosphatidylcholine,phosphatidylinositol and phosphatidic acid

1.2 Research Aim

The aims of this research were as follows:

1 To extract lecithin from the degumming by-product of rice bran oil refining

2 To determine the yield and analyze moisture content, acid value, antioxidant activityand solubility in oil of extracted lecithin

3 To compare the antioxidant activity between extracted lecithin and commerciallecithin

1.3 Research Scope

This research was limited to the laboratory extraction of lecithin from the degummingsludge obtained during the refining process of RBO It was also concern with thedetermination of acid value, antioxidant activity, solubility in oil and moisture content ofextracted lecithin

Gum sludge was kindly supplied at Surin Bran Oil Company Limited, Thailand

CHAPTER 2: LITERATURE REVIEW

2.1 Introduction about rice bran

Rice is one of the most importance food grains in the world It is a main staple food inThailand, Viet Nam and almost Asian countries Rice is also the main export produce ofThailand and Vietnam Following by USDA and Foreign Agricultural Services (2010) thereare approximately 20 million tons of rice are produced in Thailand every year, whichproduces about 1.3 million tons of rice bran (Thanonkaew et al., 2012) Rice bran is a by-product (8%) of the rice milling industry which consists of pericarp, the seed coat, the nucleusand the aleuronic layer Rice bran was chosen due to its granular structure, insolubility inwater, chemical stability and local availability (Montanher, Oliveira, & Rollemberg, 2005)

Figure 2.1 Structure of rice kernel

(Sources: Orthoefer ,1996)

The composition of rice bran depends on the rice type, climatic conditions, and riceprocessing methods The oil content in rice bran varies from 12-25 wt % and approximately95-98% of the oil is extractable (Amarasinghe and Gangodavilage, 2004)

Rice bran is rich in protein, lipids, dietary fiber, vitamins and minerals (Luh, Barber,and de Barber, 1991) However, in the past those nutrients have been mainly used for animalfeed, not for human consumption Therefore, many recent researches which give the way to

develop rice processing so as to produce more valuable products suitable for humanconsumption

Rice bran has a characteristic bland flavor, slightly bitter and sweet Rice bran has beenused in foods as full-fat rice bran, defatted rice bran, and in the form of rice bran oil and proteinconcentrates Full-fat rice bran and defatted rice bran have been incorporated into many bakeryproducts such as multigrain breads, doughnuts, pancakes, waffle mixes, muffins, specialtybreads and cookies, in breakfast cereals, and in deep-fried preparations Defatted rice bran hasbeen used in a wide variety of other products such as protein supplements, binder ingredientfor meat and sausage products, and raw material for the production of hydrolyzed vegetableproteins The commercial application of rice bran includes the introduction of rice bran cereal,granola bar, and fruit and fiber bars (Prakash and Ramaswamy, 1996)

The amount of lipids after extraction is about 21.9-23% of the bran dry weight andcontained 88.1-89.2% neutral lipids, 6.3-7% glycolipids, and 4.5-4.9% phospholipids Neutrallipids included 83-85.5% triacylglycerols, 5.9-6.8% monoacylglycerols and small amounts ofdiacylglycerols, sterols and free fatty acids The main phospholipids were phosphatidylcholine, phosphatidyl ethanolamine, phosphatidyl inositol and phosphatic acid but they werepresent in small amounts (Hemavathy and Prabhakar, 1987)

The major compositions which were found in lecithin from other kinds of vegetable

and seed oil sources are given in Table 2.1.

Table 2.1 Composition of crude lecithin obtained from various sources

2.2 Rice bran oil (RBO)

Rice bran oil is used very popular in several countries such as Japan, India, China andIndonesia as a cooking oil (Ghosh, 2007) and it is the oil which extracted from the germ andinner hull of rice (Katsri et al, 2008)

RBO has a very good shelf-life compared to other cooking oils because of antioxidantspresent in it Its low viscosity allows less oil to be absorbed during cooking, reducing overallcalories The typical composition of crude RBO is 81–84% triacylglycerols (TAG), 2–3%diacylglycerols (DAG), 1–2% monoacylglycerols (MAG), 2–6% free fatty acids (FFA), 3–4%wax, 0.8% glycolipids, 1–2% phospholipids (PL) and 4% unsaponifiable In comparison toother vegetable oils, crude RBO tends to contain higher levels of non-TAGs, most of whichare to be removed during refining processes The phospholipids are predominantly hydratablephosphatidylcholine (PC), phosphatidyl-inositol (PI) and non-hydratable phospholipids thatare calcium and magnesium salts of phosphatidic acid (PA) and phosphatidyl ethanolamine(PE) (Ghosh, 2007)

RBO has a very good balance in its fatty acid composition Moreover, it is rich inessential fatty acids; linoleic acid (32-38%) and linolenic acid (1-2%) The fatty acidcomposition of RBO is mainly monounsaturated fatty acid (MUFA), accounting for 40% oftotal fat This MUFA has been reported to reduce LDL cholesterol and increase HDLcholesterol (Katsri and Singanusong) The unsaturated fatty acids in RBO which consist of38.4% oleic acid, 34.4% linoleic acid and 2.2% linolenic acid while the saturated fatty acidscontain 21.5% palmitic acid and 2.9% stearic acid (Rukmini & Raghuram, 1991) The

refining process of the RBO is shown in Figure 2.2.

Rice bran oil also contains Y- oryzanol which helps to protect UV light induced lipidperoxidation and hence serves as a potential sunscreen agent The folic acid and its esters inthe Y- oryzanol prevent skin aging and promote hair growth (Prasad et al., 2011) Similar tovitamin E, the Y-oryzanol has more than four times as effective for preventing tissueoxidation (Patel and Naik, 2004)

Rice bran

Stabilization

Solvent treatment

Crude rice bran oil

Dewaxing → Wax sludge

Degumming → Gum sludge

Alkali treatment→ Soap stock

Bleaching

Chemically refined rice bran oil

Figure 2.2 Process of chemical refining RBO

(Source: Patel & Naik, 2004)

In Thailand, there has been growing interest in the use of colored rice extracts as anew source of anti-oxidative and anti-inflammatory effects This study investigates the effects

of different colored rice extracts in terms of their biological content, anti-oxidative activity,and their ability to reduce pro-inflammatory cytokines and matrix metalloproteinase (MMP)expression (Kitisin, Saewan, & Luplertlop, 2015) The Y-oryzanol in the RBO was alsorevealed to have the inhibitory effect against inflammatory disease is an in vitro assay (Islam

et al., 2008)

2.3 Lecithin

Lecithin is a mixture of acetone insoluble phospholipids, containing mainlyphosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), minorcompounds such as phosphatidic acid (PA) and other substances (triglycerides, carbohydrates,etc.) (Cabezas et al., 2009)

Figure 2.3 Structure of soybean lecithin (Source: Scholfield, 1981)

Figure 2.4 Some of phosphatides in soybean lecithin

(Source: Scholfield, 1981)

The lecithin fraction which is soluble in ethanol is rich in PC and very useful as aemulsifier for O/W emulsion The other fraction which is insoluble in ethanol is rich in PEand PI and is a good water in oil (W/O) emulsifying agent (Cabezas et al., 2009) Thesaturated fatty acids (SFA) consist of more in the PC-enriched fraction while the PI-enriched

fraction has more unsaturated fatty acids (UFA) (Wu & Wang, 2004) Egg yolks are a goodsource of phospholipids (PL) PL represents approximately 10% of the wet weight of the eggyolk, equivalent to about 22% of the total egg yolk solids In egg yolks, egg lecithin contains80.5% PC and 11.7% PE In addition, egg-yolk lecithin contains relatively more saturated FAthan does soybean lecithin, and it may have better oxidative stability than soybean lecithin.Thus, yolk lecithin may have certain unique applications in many food products (Palacios &Wang, 2005a).

For conventional methods, lecithin has been extracted from soy bean as it has a highamount and low cost However, many new commercial sources have continue to emergedwith the increased production of sunflower, canola (Lambourne, Covey, Chai, & Dunstan,1999) and rice bran oil (Adhikari & Adhikari, 1986)

According to Hussain (2015) rice bran lecithin (RBL) has more oxidative comparedwith soy lecithin due to its less content of linoleic and linolenic acids On the other hand, RBLalso consists of oryzanol and tocotrienols which gives it more value for being efficientemulsifier and growth promoter The main differences between the SL and RBL are thecomposition and characteristics, but the RBL has more phospholipids than SL Therefore,RBL can be more effectively replaced the SL (Machado, de Assis, Machado, & de Souza-Soares, 2014) RBL is also hypoallergenic; it can provide alternative for the food that needs toeliminate the use of SL (Asia pacific food industry, 2012) In addition, as a mention before,rice bran has a highest content of lecithin (53.4% PL) when compares with soy bean,sunflower and canola

2.4 Extraction of lecithin

Lecithin has been extracted from many sources such as eggs (Maximiano et al., 2008;Palacios & Wang, 2005b; Sreedevi, Joseph, Devi, & Hari, 2012; Wu & Wang, 2004), soybean(Wu & Wang, 2003), sunflower (Cabezas et al., 2009), canola (Xie & Dunford, 2016),rapeseeds (Sosada, 1992), etc However, soybean predominates than other vegetables andoilseed sources of lecithin because of its readily availability and its outstanding functionality(Wu & Wang, 2003)

Lecithin is obtained from a by-product of crude oil by hydration and separation(Machado et al., 2014) It is obtained from the gum generated during the degumming step ofvegetable oil refining Water and acids degumming are the conventional methods employed todegum vegetable oil But, in the past two decades, enzymatic degumming is used to hydrolyzephospholipids to increase oil yield (Xie & Dunford, 2016) During degumming, phosphatidesare converted to hydrated gum due to their affinity for water.

The purpose of degumming is to prevent the settling down during storage or transport,prevent acidulation, reduce the losses in neutralization and produce lecithin in commercialscale (Sengar,et al…, 2014) During soy lecithin production, the sludge of lecithin and wateremulsion obtained from the degumming centrifugation contains about 25-50% water, issometimes bleached using hydrogen peroxide and then dried to about 1% moisture Theobtained product is called unrefined or natural lecithin with 65-70% phospholipids and 30-35% crude soy oil The oil can later be removed by extraction with acetone to obtain therefined lecithin (Shurtleff and Aoyagi, 2016) Lecithin is obtained by drying the gum from thedegumming process (Adhikari and Adhikari, 1986) The crude lecithin gum contain alecithin/water/oil in a ratio 35:5:15, depending on the degumming processing condition(Lambourne et al., 1999)

The recovery of lecithin from the gum obtained after enzymatic degumming of crudecanola oil with phospholipase A2 (PLA2) and its ethanol fraction (Xie and Dunford, 2016).The ethanol soluble and insoluble fractions of the crude lecithin (CL) containlysophosphatidylcholine (45.25 g/100g) and phosphatidylinositol, phosphoric acid andlysophatidylinositol (31.41 g/100g) respectively The CLP and the ethanol fractions werefound to be of superior quality than commercial SL for O/W emulsion according to theirinvestigation

Another research which carried out by (Ceci, Constenla, and Crapiste, 2008), oil andlecithin were recovered from water degumming sludge of crude soybean oil by directextraction with cold acetone, extraction after removing the water under vacuum and solventpartition with hexane or ethanol There was no significant difference observed in the lecithinyield and about 720-807 g/kg of dried gum was obtained However, higher oil yield (up to588g/kg) was obtained when the water was removed before extraction

Egg-yolk lecithin was reported to be extracted from both deoiled yolk and undeoiledyolks using ethanol The yield and PL purity of the PC-enriched fractions of undeoiled yolkwere found to be 23.9 and 35.7 %, while that of deoiled yolk were 13.5 and 53.5% Therecovery of the total PL was 70% from deoiled which is higher than 60% from deoiled yolk Itwas also revealed that heating during the process had little effect on the PL extraction(Palacios and Wang, 2005b).

The flowchart for obtaining lecithin is presented in Figure 2.5

Rice bran

↓Pre-cleaning

↓Cooking

↓Expansion

↓Screening

↓Drying

↓Oil extraction

↓Miscela

↓Dessolventizer

↓Degumming

↓Lecithin

Figure 2.5 Flowchart for the lecithin processing

(Source: Machado et al., 2014)

The crude lecithin obtained after degumming which usually undergoes purification toobtain the pure lecithin The CSL was found to contain approximately 18% PC beforepurification which is not enough for the pharmaceutical applications.

2.5 Application of lecithin

In food industry, lecithin used for a lot of product such as margarine or bakingchocolate as a emulsifier and also used for cosmetics and pharmaceuticals as a ingredients

Figure 2.6 Some of products containing lecithin

The emulsification properties of different lecithin products are a function of their

"hydrophilic-lipophilic balance" (HLB) The HLB describes the relative composition of thewater-loving (polar) and fat-loving (non-polar) elements of an emulsifier such as lecithin

Typically, lecithin products are added to the oil phase for oil-in- water emulsions Inwater-in-oil emulsions, they must be added to the oil phase Depending on the type of lecithin

being used, heating to 120°f will aid in dispersion and can improve handling and mixingcharacteristics

Lecithin products allow control of viscosity in liquid and semi-liquid products Forexample, chocolate and compound coatings utilize lecithin to control viscosity Lecithingreatly reduces the surface tension of fats, allowing the particles of chocolate, sugar and othermilk products to be coated This coating improves the foodstuff particle flow ability andsignificantly improves mix ability Typical usage levels for viscosity modification range from0.2%-0.6% of total product weight

Lecithin products promote easy and complete separation of food from food contactsurfaces

Lecithin helps form a stable film barrier that prevents adhesion of food products to oneanother Direct incorporation, as in baked goods, allows for better machinability andminimized sticking to the mixing vessel Best results are obtained when the lecithin is surfaceapplied versus direct incorporation, such as on processed cheese slices Regulatorycompliance should be reviewed when direct incorporation is practiced to comply with FCCstandards of identity

CHAPTER 3 MATERIALS AND METHODS

3.1 Materials

In order to achieve the objectives of this research, the extraction of lecithin from ricebran gum sludge would have followed the methods of Ceci, Constenla, and Crapiste (2008).Two main materials were used in this study are gum sludge was kindly supplied at Surin BranOil Company Limited, Thailand and cold acetone as a solvent

- BUCHI rotary vacuum evaporator R-124

- Orbital Shaker N-Biotek 101M

- Vacuum Filter EYELA A_1000S

- Moisture Analyzer SARTORMS MA 40

- Vortex mixer

- Spectrophotometer genesis 20

- Centrifuge Handi-spin

3.4 Methodology

3.4.1 Method for lecithin extraction from degumming by-product

The sample and solvent which used for this study are gum sludge and cold acetone The experimental design which used for this study is Complete Randomized Designwith 2 factors and 3 replications Totally 18 treatments were investigated

Factors used in this study are:

o Factor 1: ratio of gum/acetone, composed of 3 ratios: 1:1; 1:1.5 & 1:2

o Factor 2: shaking time, composed of 2 durations: 30 min & 60 min

In order to achieve the objectives of this research, the extraction of lecithin from ricebran gum sludge will follow the methods of Ceci, Constenla, and Crapiste (2008) (Figure3.1)

The gumming sludge obtained from the crude rice bran oil extraction will beevaporated under vacuum (70mmHg) in a rotary vacuum evaporator at 600C and thecentrifuged The residue then will be extracted with acetone The ratio of 1:1 1:1.5 1:2 w/v ofacetone to gum will be used with continuous shaking for 30 & 60 min After resting for 15min, the extract will be filtered and the residue extracted for another two times with acetone(1:1 w/v) under the same condition The lecithin will finally be obtained by drying the residue

in a rotary vacuum evaporator at 125 mmHg at 400C The lecithin yield will be determined

Evaporate under vacuum at 70 mmHg using rotary vacuum

evaporator at 60oC

Extract the gum with cold acetone using gum/solvent ratio (1:1; 1:1.5; 1:2 w/v), with

continuousshaking for 30 & 60 minutes