Development of drinking product from the quinoa seed (chenopodium quinoa)

Our study focused on the following content The optimization of enzymatic hydrolysis using response surface methodology to study the influence of the variables enzyme concentration, hydro

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE

Supervisor : Dr Tran Thi Lan Huon

AGRICULTURAL UNIVERSITY PRESS - 2017

DECLARATION

I hereby declare that this thesis is my own work and effort and that it has not been submitted anywhere for any degree Where other sources of information have been used, they have been acknowledged

Hanoi, May 10th, 2017 Master candidate

Le My Hanh

ACKNOWLEDGEMENTS

I would like to express the deepest thanks to my supervisor Dr Tran Thi Lan Huong for her guidance during the time I conduct the research She provided me the material to perform all experiments and taught me how stuff works She opened my mind for any kind of research conversation, and always being a good listener I am also grateful to Msc Nguyen Huy Bao who taught me how to process data on design expert software and some skills in Microsoft He helped me better understand the theories related to optimization and sensory evaluation Moreover, he used to encourage me whenever I was depressed during this study

Sincerest thanks to my colleagues in the Department of Quality Management and Food Safety who helped me to do some works in the office Therefore, I could spend more time to study and complete my research thesis

The students in the Faculty of Food science and Technology are Chuc and Linh who helped me a lot in the experiments of this study I would like to express my thanks

to them

Last but most definitely not least, I would like to express my deepest gratitude to

my parents and the rest of the family who always emphasized the importance of education It is not the tiniest bit exaggerated to say this thesis wouldn’t exist without some “parental guidance” throughout my (numerous) years in education

Hanoi, May 10th, 2017 Master candidate

Le My Hanh

TABLE OF CONTENTS

Declaration i

Acknowledgements ii

Table of contents iii

List of abbreviations iv

List of tables v

List of figures vi

Thesis abstract vii

Chapter 1 Introduction 1

1.1 Introduction 1

1.2 Objective 2

Chapter 2 Literature Review 3

2.1 Overview of material 3

2.2 Cereal and grain for non-alcohol beverage 14

Chapter 3 Materials and methods 18

3.1 Materials 18

3.2 Research content 18

3.3 Methods 18

Chapter 4 Results and discussion 26

4.1 Analysing the main chemical content 26

4.2 Enzymatic hydrolysis optimization of quinoa starch 27

4.3 Effect of sterilization regime (temperature and time) on product quality 33

Chapter 5 Conclusion and recommendations 40

5.1 Conclusion 40

5.2 Recommendations 40

Reference 41

Appendix 50

LIST OF ABBREVIATIONS

Acronym Abbreviations

ANOVA Analyze of variance

CFU Colony forming unit

FAO Food and Agriculture Organization

ULS Unstructured line scale

RSM Response surface methodology

SICA Agricultural Census and Information System WHO World Health Organization

LIST OF TABLES

Table 2.1 Amino acids composition of quinoa seed, barley, soybean, and wheata 6

Table 2.2 Comparison of essential amino acids content of barley, corn and wheat to FAO/WHO suggested requirement 7

Table 2.3 Mineral composition whole quinoa seed, dehulled quinoa seed, quinoa flour, oat, barley (mg/100 g) 9

Table 3.1 Factors and their levels for the Box-Behnken design 21

Table 3.2 Experimental design and responses of the dependent variables to the hydrolysis parameters 22

Table 4.1 The main chemical content of quinoa seed (% dry weight) 26

Table 4.2 Amino acid profiles (g/100g protein) 27

Table 4.3 Responses of the dependent variables to the hydrolysis parameters 28

Table 4.4 Regression coefficients of the predicted second-order polynomial models for the total sugar content, score of color and score of appearance 29

Table 4.5 Experimental data of verification of predicted technical parameters of enzyme 33

Table 4.6 The number of microbe of sample sterilized at 1100C (CFU/ml) 35

Table 4.7 The numerous of microbe of sample sterilized at 1100C in 10min (CFU/ml) 36

LIST OF FIGURES

Fig 2.1 Structure α-amylase (Payan, 2004) 13

Fig 2.2 Commercial rice milk 14

Fig 2.3 Commercial soy milk 16

Fig 2.4 Commercial oat milk 16

Fig 3.1 Flow chart of producing quinoa milk 19

Fig 3.2 Unstructured line scale for training and sensorial evaluation 25

Fig 4.1 Surface plot of the total sugar content (Y1) as a function of concentration of enzyme and temperature at time of 80 min and a function of temperature and time at concentration of 0,15% enzyme 30

Fig 4.2 Surface plot of the score of color (Y2) as a function of concentration of enzyme and temperature at time of 80 min and as a function of temperature and time at concentration of 0.15% enzyme 31

Fig 4.3 Surface plot of the score of appearance (Y3) as a function of time and temperature at concentration of 0.15% enzyme and as a function of temperature and enzyme concentration at time of 63.1 min 32

Fig 4.4 Contour plot showing optimal values of responses 32

Fig 4.5 Effect of sterilization on color of product 33

Fig 4.6 Distribution of response level on hedonic scale 37

Fig 4.7 Average scores of quinoa milk and rice milk 38

Fig 4.8 Flow chart of producing process of quinoa milk with the attached technological parameters 39

THESIS ABSTRACT

The quinoa milk was processed from quinoa seeed (Chenopodium quinoa) which has high nutrition value aiming to diversification of beverage products in the market Our study focused on the following content

The optimization of enzymatic hydrolysis using response surface methodology to study the influence of the variables (enzyme concentration, hydrolysis temperature and hydrolysis time) on the variability of total sugar content and sensory properties (color and appearance) of quinoa milk Validation of models showed a good agreement between experimental results and the predicted responses The results indicated that all three variables had a significant impact on responses The hydrolysis temperature had the most significant effect on the total sugar content However, the variable which had the most significant effect on the score of color and appearance of quinoa milk as well was hydrolysis time The optimal hydrolysis parameters were the enzyme concentration

of 0.12%, hydrolysis temperature of 91.9°C and time of 69.6 min according to the response surface analysis Under this condition, the total sugar content, score of color and score of appearance was 83.95mg/ml, 8.47 and 8.5 respectively

The quality and shelf-life of product is greatly determined by sterilization regime The impact of twelve different regimes of heat sterilization, defined by their combinations of temperature and time (temperature: 1100C, 1150C and 1210C, time: 5min, 7min, 10min and 15min) on the color and microbiological properties of products was evaluated In term of color, the three samples which sterilized at 1100C in 5, 7 and

10 min achieved the highest score of color The sample sterilized at 1100C in 10 min was the best which successfully met requirements about microbiological criteria according National technical regulation for soft drinks, 2010 (QCVN 6-2:2010/BYT) The result of sensory evaluation by panel indicated that the average scores of both quinoa milk and Korean rice milk that were in the range from “like extremely” to

“dislike extremely” were 6.25 and 6.22 respectively Although quinoa milk is a new product in Viet Nam, it was slightly liked by the panel

Finally, the process of producing quinoa milk was built with the attached technological parameters

CHAPTER 1 INTRODUCTION 1.1 INTRODUCTION

Drinks play an important role in human’s life It provides body not only a big amount of water involved in the metabolism of the cell but also a huge amount of nutrients, vitamins and minerals to compensate for the loss of energy and nutrients have been consumed in the activity of human (Hien, 2006)

Nowadays, because of busy lifestyle, the time spending for consuming food

in daily life is very short Furthermore, people pay more attention about their health Therefore, the ready to drink providing full of nutrition for body is quite necessary In Vietnamese beverage industry, consumers tend to use natural products such as green tea or fruit juice although the price is higher than that of carbonated beverage Besides, many kinds of seed is also nutritious source of raw material used in manufacturing of drinking product for the consumer such as soy beans, rice, corn

Quinoa grains have presented in the world 7000 years ago at South America (Bazile et al., 2016) but it is quite new in Viet Nam Despite of early appearance, the potential and benefits of quinoa have recently been known by researchers in other countries Studies have been performed in an increasing number of countries The number of countries growing quinoa has risen rapidly from 8 in 1980 to 75 in 2014, with a further 20 countries which sowed quinoa for the first time in 2015 (Bazile and Baudron, 2015) In Vietnam, quinoa plants were grown and developed in the period

of time between 1986 and 2000 with HV1 specie in many provinces, the yield from 14.0 to 20.6 kg / ha (Trinh Ngoc Duc, 2001) Nowadays, they are grown in Quang Tri, An Giang province and Viet Nam University of Agriculture This kind of seed attracts the attention of many researches in different countries because of its nutritional value (James, 2009) especially protein Its protein levels are similar to those found in milk and higher than those present in cereals such as wheat, rice and maize (James, 2009) Furthermore, quinoa seed has highest content of bioactive compounds compared to other cereals and pseudo-cereals (Hirose et al., 2010) Therefore, we conduct the research on the topic: “Development of drinking product from the quinoa seed (Chenopodium quinoa)” to diversify of beverage products in the market

- Enzymatic hydrolysis optimization of quinoa starch

- Determining the appropriate condition of sterilization

- Determining degree of preference of consumer toward product

- Building the process of producing quinoa milk

CHAPTER 2 – LITERATURE REVIEW 2.1 OVERVIEW OF MATERIAL

2.1.1 Quinoa seed

2.1.1.1 History and distribution

Quinoa has been recognized for centuries as an important food crop in the high Andes of South America (Tapia, 1982) The name quinoa in the Quechua and Aymara languages means ‘Mother Grain’ and this crop occupied a place of prominence in the Inca Empire next only to maize (Bhargava and Ohri, 2016) After that, quinoa seed did not receive much attention from people until the 1980s, the market for quinoa began expanding in Europe and North America mainly in the health-food sector and the demand was met partly by imports from South America and by the development of quinoa in new regions outside its center of origin (Bhargava and Ohri, 2016)

According to Rojas (1998) the geographical distribution of quinoa in the region extends from 5°N in Southern Colombia, to 43°S in the Xth Region of Chile During the last decade, quinoa has been started extensive cultivation in Chile, Ecuador, Argentina, and Colombia (Rojas, 1998) There were research projects on quinoa, such as SICA (Agricultural Census and Information System)

of the Agricultural Ministry of Ecuador; Quinuacoche CANOE program promoted by the Latin American Foundation in Colombia; Provincial Congress for Quinoa promoted by the Deputies Chamber of Salta, Argentina; Program of Encouragement for Business Design; and Innovation promoted by the Euro Chile Foundation (Taboada et al., 2011)

In 1993, a project titled ‘Quinoa-a multipurpose crop’ for EC’s agricultural diversification was initiated in the European Union (Jacobsen et al., 2003a) This led to setting up of laboratories in Scotland and France, and field trials in England, Denmark, the Netherlands and Italy Quinoa has been evaluated as a potential crop in Denmark, Poland, Sweden, Italy, and Greece (Bhargava and Srivastava, 2013) It has also been successfully tested in North America and Africa and has been cultivated in the US since the early 1980s and commercially produced since the mid-1980s in the Colorado Rockies, especially in the San Luis Valley (Bhargava and Srivastava, 2013) The North American Quinoa

Producers Association was formed in 1988 and a small processing plant was started for the crop produced in the area Production has also been attempted in California, New Mexico, Oregon, and Washington In Canada, cultivation is done in Saskatchewan and Manitoba most of which is organic (Bhargava and Srivastava, 2013)

The Asian experiment on quinoa introduction has been quite impressive with the crop showing good adaptation and abundant yield in the Indian subcontinent Quinoa was successfully introduced in India in the early 1990s and exhaustive field trials have proved its cultivation as an alternative winter crop for the North Indian Plains (Bhargava et al., 2007) It was introduced in Pakistan in

2007 in the central Punjab to lessen the dependence of the common people on conventional crops (Bhargava and Ohri, 2016) Field tests have been done in Japan in the climatic conditions of Southern Kanto District of Japan (Ujiie et al., 2007) Field tests in Kenya have shown seed yield up to 9 t/ha and biomass yield

up to 15 t/ha indicating high seed yield comparable to that in the Andean region (Mujica et al., 2001) A partnership between the Danish Company Eghojgaard and the Egyptian Natural Oil Company (NATOIL) has been constituted since the year 2007 for promoting quinoa in Egypt (Bhargava and Srivastava, 2013) Quinoa was formally put in field trials in the Sinai Peninsula with 13 varieties and strains being tested in deserts of South Sinai governorate (near Nuwaiba city) which proved to be a success (Shams, 2011) Recent introduction in Morocco has shown a high potetial of adaptation in the country (Hirich et al., 2014)

Today quinoa is presently cultivated or tested in 95 countries of the world This global expansion of quinoa looks set to continue as increasing numbers of countries are testing quinoa (Bazile et al., 2016)

2.1.1.2 Chemical composition of quinoa seed

Quinoa is a very interesting food due to its complete nutritional characteristics Some main chemicals of quinoa seed are discussed following

a Carbohydrate

Starch is the main carbohydrate in quinoa, located in primarily the perisperm (Prego and Maldonado, 1998) It makes up approximately 58.1-64.2% of the dry matter, of which 11% is amylose (Repo-Carrasco et al., 2003)

Quinoa starch granules are smaller in size than that of common cereals, having

a polygonal form with a diameter of 2 µm Being rich in amylopectin, it has excellent freeze-thaw stability and thus is an ideal thickener in frozen foods and other applications where resistance to retrogradation is desired (Ahamed et al., 1998)

According to Repo-Carrasco, these starches could provide an interesting alternative to replace chemically modified starches (Repo-Carrasco and Molina, 1991) Genetic variation of starch granule size in the Bolivian quinoa collection ranged from 1 to 28 µm, this variable can be used to make different mixtures with cereals and legumes, and establish the functional nature of quinoa (Rojas et al., 2010)

b Protein

The mean protein content reported in the literature for quinoa seed (QS) is 12–23% (Abugoch et al., 2008) Compared to cereal grains, the total protein content of QS (16.3% dry basis (db)) is higher than that of barley (11% db), rice (7.5% db), or corn (13.4% db), and is comparable to that of wheat (15.4% db) (Abugoch et al., 2008) Quinoa contains all ten essential amino acids (Vega‐Gálvez et al., 2010; Meneguetti et al., 2011) Relative to cereal grains, quinoa proteins (QPs) are particularly high in lysine, the limiting amino acid in most cereal grains (Table 2.1) Their essential amino acid balance is excellent because

of a wider amino acid range than in cereals and legumes (Ruales and Nair, 1993), with higher lysine (5.1–6.4%) and methionine (0.4–1%) contents (Bhargava et al., 2003; Prakash and Pal, 1998) QPs have higher histidine content than barley, soy, or wheat proteins, while the methionine and cystine content of quinoa is adequate for children (2–12 years old) and adults (Table 2.2), it is similar to that

of barley and soy, and lower than the amounts in wheat According to the FAO/WHO suggested requirements (Table 2.2) for a 10-year-old children, QPs have adequate levels of aromatic amino acids (phenylalanine and tyrosine) and similarly in histidine, isoleucine, threonine, phenylalanine, tyrosine, and valine contents By comparison (Table 2.2), lysine and leucine in QPs are limiting amino acids for 2–5-year-old infants or children, while all the essential amino acids of this protein are sufficient according to FAO/WHO suggested requirements for 10–12- year-old children Therefore, quinoa could be a good source of proteins for feeding infants and children

Table 2.1 Amino acids composition of quinoa seed, barley,

soybean, and wheata

Amino acid

Quinoa seed Barley

pearled Soybean raw Wheat durum mg/g protein

Table 2.2 Comparison of essential amino acids content of barley, corn and

wheat to FAO/WHO suggested requirement

Amino acids

Quinoa seeda

Barley pearled

a

Soybeans raw

Wheat durum

a

FAO/WHO suggested requirementsb

mg/g protein year old

2-5-year old

10-12-Adu-lt Histidine 28.8 22.5 27.6 23.5 19 19 16 Isoleucine 35.7 36.5 44.5 38.9 28 28 28 Leucine 59.5 98.2 72 68.1 66 44 19 Lysine 54.2 37.2 57.8 22.1 58 44 16 Methyonnine

and Cystine 36.2 41.3 28.9 22.7 25 22 17 Phenyalanine

and Tyrosine 60.9 84.7 84.8 85.9 63 22 19 Threonine 29.8 34 38.6 26.7 34 28 9 Tryptophan 11.4 16.6 12 12.8 11 9 5 Valine 42.1 49 57.1 41.6 35 25 13

Source: a USDA (2005); b Friedman and Brandon (2001))

c Lipid

QS have been considered an alternative oilseed crop due to their lipidic fraction (Koziol, 1993) Besides the high content and good biological quality of their proteins, QS have an interesting lipid composition of about 1.8–9.5% (Wood et al., 1993; Ranhotra et al., 1993) Quinoa has an oil content (7% dry basis) higher than corn (4.9% dry basis) and lower than soy (20.9% dry basis) (Koziol, 1993) According to Przybylski et al (1994), QS lipids contain high amounts of neutral lipids in all the seed fractions analyzed (Przybylski et al., 1994) Triglycerides are the major fraction present, accounting for over 50% of the neutral lipids Diglycerides are present in whole seeds and contribute 20% of the neutral lipid fraction Lysophosphatidyl ethanolamine and phosphatidyl

choline are the most abundant (57%) of the total polar lipids (Przybylski et al., 1994) Some researchers have characterized the fatty acid composition of quinoa lipids as follows: total saturated 19–12.3%, mainly palmitic acid; total monounsaturated 25–28.7%, mainly oleic acid, and total polyunsaturated 58.3%—chiefly linoleic acid (about 90%) ( Oshodi, 1999; Ranhotra et al., 1993; Ryan et al., 2007) Omega-6 and omega-3 fatty acids are essential fatty acids because they cannot be synthesized by humans, who must obtain them from foods The essential fatty acids are metabolized to longer chain fatty acids of 20 and 22 carbon atoms Linoleic acid is metabolized to arachidonic acid and linolenic acid to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) EPA and DHA play important roles in prostaglandin metabolism, thrombosis and atherosclerosis, immunology and inflammation, and membrane function ( Youdim et al., 2000) The fatty acid profile of QS is similar to corn and soybean oil (Youdim et al., 2000; Kozioł, 1992; Oshodi, 1999) Essential fatty acids are important acids, like linoleic and linolenic acids, that are necessary substrates in animal metabolism Linoleic acid (C18:2) is one of the most abundant polyunsaturated fatty acids (PUFA ) identified in QS; PUFAs have several positive effects on cardiovascular disease ( Keys and Parlin, 1966) and improved insulin sensitivity (Lovejoy, 1999)

The oil fraction of QS has high quality and is highly nutritious, based on the fact that it has a high degree of unsaturation, with a polyunsaturation index of 3.9–4.7 In this fraction, not only the fatty acid composition is important Another important feature is the natural presence of a high amount of vitamin E ( a-tocopherol), 0.59–2.6 mg/100 g in the seeds (Ryan et al., 2007), which acts as a natural defense against lipid oxidation (Ng et al., 2007) This fact could lead to a very stable oil from QS, with vitamin E acting as a natural antioxidant The tocopherol content in quinoa whole flour has been reported as 3.1–5.5 mg/100 g (Ruales and Nair, 1993; Ryan et al., 2007) The chemical stability of the lipids in quinoa flour was studied by Ng et al (2007), who found that the lipids were stable during 30 days, and this stability is due to vitamin E present naturally

d Vitamin and minerals

QS are also rich in micronutrients such as minerals and vitamins Table 2.3 shows the mineral content of QS and quinoa flour The main minerals are potassium, phosphorus, and magnesium (Table 2.3) According to the National

Academy of Sciences (2004) the magnesium, manganese, copper, and iron present in 100 g of QS cover the daily needs of infants and adults, while the phosphorus and zinc content in 100g is sufficient for children, but covers 40– 60% of the daily needs of adults The potassium content can contribute between 18% and 22% of infant and adult requirements, while the calcium content can contribute 10% of the requirements However, the mineral content of QS is higher than that of cereals like oat (except phosphorus) or barley, especially that

of potassium, magnesium, and calcium (Table 2.3)

In their research, Konishi et al (2004) found that abrasion of QS (for saponin elimination) caused specifically a decrease in calcium content (Konishi

et al., 2004) On the other hand, they found that the distribution of minerals in

QS revealed that phosphorus and magnesium were localized in embryonic tissue,while calcium and potassium were present in the pericarp (Table 2.3).

Table 2.3 Mineral composition whole quinoa seed, dehulled quinoa seed,

quinoa flour, oat, barley (mg/100 g)

Whole QSa Dehulled QSa Quinoa flour b,c Oatd Barleyd

Phosphorous 411 414.9 22-462 734 221 Potassium 732 656 714-855 566 280 Magnesium 502 467.9 161-232 235 79

Note: n.r.: not reported

Source:a Konishi et al (2004); b Ranhotra et al (1993);

c Oshodi et al (1999); d USDA (2005)

The vitamin content (Table 2.4) is also interesting, because QS have high levels of vitamin B6 and total folate, whose amounts in 100g can cover the requirements of children and adults The riboflavin content in 100g contributes

80% of the daily needs of children and 40% of those of adults (National Academy of Sciences, 2004) The niacin content does not cover the daily needs, but is beneficial in the diet Thiamin values in quinoa are lower than those in oat

or barley, but those of niacin, riboflavin, vitamin B6, and total folate are higher (Ranhotra et al., 1993)

Table 2.4 Vitamin composition quinoa flour, oat, barley (mg/100g)

Quinoa flour a,b Oatb Barleyb

Saponins are compounds that contain sugar chains and a triterpenoid aglycone (sapogenin) in their structure (Sparg et al., 2004) They are categorized according to the number of sugar chains in their structure as mono-, di-, or tridesmosidic Four main structures of sapogenins have been identified in quinoa: doleanolic acid, hederagenin, phyolaccagenic acid, and 30-o-methylspergulagenat (Zhu et al., 2002) The major carbohydrates are glucose, arabinose and galactose Besides, 20 triterpene saponins have been isolated from different parts of Chenopodium quinoa (flowers, fruits, seed coats, and seeds) (Zhu et al., 2002; Kuljanabhagavad et al., 2008)

The sapogenin content in seeds of sweet genotypes varied from 0.02%

to 0.04% and in seeds of bitter genotypes from 0.14% to 2.3% (Güçlü-Üstündağ and Mazza, 2007) These values are higher than those in soybean and oat, but lower than in green pea and yucca (Güçlü-Üstündağ and Mazza, 2007)

2.1.1.3 Present and future uses of quinoa seed

Quinoa is well adapted to extreme weather conditions, and it is currently produced by Bolivia, Peru, Ecuador, Chile, Argentina, and Colombia It is basically exported as dry and saponin-free quinoa, with Europe and the USA as the main consumers Future uses can be wide-ranging, like textured and fermented products There are many ways in which it can be consumed: cooked,

AS flour, extruded Quinoa meat substitute has been introduced in Europe (Tellers, 2008) There are several developments with quinoa flour at a smaller scale, like bread, cookies, muffins, pasta, snacks, drinks, flakes, breakfast cereals, baby foods, beer, diet supplements, and extrudates (Ahamed et al., 1997; Bhargava et al., 2006; Caperuto et al., 2001; Chauhan et al., 1992; Doğan and Karwe, 2003) Coulter and Lorenz (1991) obtained extruded corn grits–quinoa blends that had high protein quality and solubility and an acceptable sensory evaluation (Coulter and Lorenz, 1991) Caperuto et al (2000) developed gluten-free quinoa spaghetti and obtained a product without loss of solids and acceptable weight and volume increase upon cooking, while the adhesiveness of the cooked product was not very high (Caperuto et al., 2001) The product was sensorially accepted by the panelists

Quinoa flour does not have good baking properties like wheat gluten proteins The wheat proteins are able to form a viscoelastic network when flour is

mixed with water to form dough, and these viscoelastic properties allow the use

of wheat to produce bread and other processed foods (Shewry et al., 1995) Quinoa bread has been made by including 10% of wheat flour (Chauhan et al., 1992) There are some gluten-free products without good baking properties for celiac groups, and quinoa provides an opportunity to develop gluten-free cereal-based products (Gallagher et al., 2004) Dogan and Karwe (2003) showed that quinoa can be used to make novel, healthy, extruded, snack-type food products (Doğan and Karwe, 2003) They got a good product with maximum expansion, minimum density, high degree of gelatinization, and low water solubility index (16% feed moisture content, 1300C die temperature, and 375 rpm screw speed) Quinoa has shown a high nutritional value and only recently is being used as a novel functional food However, it is very important to increase and promote QS production, diversify production, and enhance its consumption An important aspect to consider for promoting quinoa consumption is to inform consumers of the good properties of quinoa and let them incorporate it in their daily diet as a healthy, nutritious, good tasting, and versatile food Alternatively, it is necessary

to develop new functional products that can be available on the market for the ordinary user, and scale them up to industrial level

2.1.2 TermamylR 120L (Alpha amylase)

TermamylR 120L is a kind of Bacterial thermophilic alpha-amylase which can act at pH=6.0-6.4 in pre-liquefaction at 80-850C and continuous liquefaction

at 105-1100C (Chandrasekaran, 2015) It belongs to a family of endo-amylases that catalysis the initial hydrolysis of starch into shorter oligosaccharides through the cleavage of α-D-(1-4) glycosidic bonds ( Iulek et al., 2000) Neither terminal glucose residues nor α-1,6-linkages can be cleaved by α-amylase (Whitcomb and Lowe, 2007) The end products of α-amylase action are oligosaccharides with varying length with an α-configuration and α-limit dextrins (Van Der Maarel et al., 2002), which constitute a mixture of maltose, maltotriose, and branched oligosaccharides of 6-8 glucose units that contain both α-1,4 and α-1,6 linkages (Whitcomb and Lowe, 2007) Others amylolytic enzymes participate in the process of starch breakdown, but the contribution of α-amylase is the most important for the initiation of this process (Tangphatsornruang et al., 2005) The amylase has a three-dimensional structure capable of binding to substrate and, by the action of highly specific catalytic groups, promote the

breakage of the glycoside links (Iulek et al., 2000) The protein contains 3 domains: A, B, and C (Fig 2.1) The A domain is the largest, presenting a typical barrel shaped (β/α)8 super structure The B domain is inserted between the A and

C domains and is attached to the A domain by disulphide bond The C domain has a β sheet structure linked to the A domain by a simple polypeptide chain and seems to be an independent domain with unknown function The active site (substrate-binding) of the α-amylase is situated in a long cleft located between the carboxyl end of the A and B domains The calcium (Ca2+) is situated between the A and B domains and may act in the stabilization of the three-dimensinoal structure and as allosteric activator Binding of substrate analogs suggest that Asp206, Glu230 and Asp297 participate in catalysis (Muralikrishna and Nirmala, 2005) The substrate-binding site contains 5 subsites with the catalytic site positioned at subsite 3 Substrate can bind to the first glucose residue in subsite 1

or 2, allowing cleavage to occur between the first and second or second and third glucose residues (Whitcomb and Lowe, 2007)

Fig 2.1 Structure α-amylase (Payan, 2004)

The most widespread applications of α-amylases are in the starch industry, wich are used for starch hydrolysis in the starch liquefaction process that converts starch into fructose and glucose syrups (Nielsen and Borchert, 2000) The enzymatic conversion of all starch includes: gelatinization, which involves

the dissolution of starch granules, thereby forming a viscous suspension; liquefaction, which involves partial hydrolysis and loss in viscosity; and saccharification, involving the production of glucose and maltose via further hydrolysis (Gupta et al., 2003)

Enzymatic hydrolysis by using alpha amylase is an important step in the processing of beverage products from starchy grains Deswal and his coworkers conducted the research on optimization of enzymatic production process of oat milk (Deswal et al., 2013) In Viet Nam, Nguyen Minh Thuy and her coworkers has researched enzymatic hydrolysis optimization of rice starch for rice milk processing aiming to obtain the optimal technical parameters of enzyme alpha-amylase (Thuy and Tuyen, 2015)

2.2 CEREAL AND GRAIN FOR NON-DAIRY MILK

During recent years, non-dairy milk types (vegetable milk), such as soymilk, coconut milk, almonds milk, rice milk, peanut milk and oat milk, have been an increased demand from consumers due to their high functional properties The cereal and grain milks also do not contain cholesterol or lactose; hence, these milk types are preferred by someone who are vegetarians, have special diet or who are lactose intolerant (Ismail, 2015) Some of them are discussed following

Rice milk is made from boiled rice (both brown rice and white rice versions are available) It has a watery consistency and to help it taste more like cow’s milk, flavor enhancers like brown rice syrup and vanilla are often added Most brands have about the same number of calories as 2% cow’s milk and about half the fat Unlike other plant-based dairy substitutes, rice milk has little or no fiber

By comparing the nutritive values of rice milk to cow milk, it can be seen rice milk provides higher carbohydrate and calcium contents than fresh cow milk, while the latter contains higher total fat, cholesterol and protein contents Hence, the rice milk can be used as a good nutrient source when supplemented with appropriate amount of protein

2.2.2 Soymilk

Soymilk is essentially the water extract of soybeans The traditional method

of producing soymilk involves soaking the soybeans, followed by wet grinding, filtering and cooking (Kwok and Niranjan, 1995)

Soymilk contains high amounts of iron, unsaturated fatty acids (linoleic acid) and niacin, but low amounts of fat, carbohydrates and calcium content as compared to cow’s and human milks (Khodke et al., 2014)

Soymilk is cholesterol, gluten and lactose free, while containing phytochemicals Therefore, it is considered as important healthy drink for people who are allergic to cow’s milk proteins or have lactose intolerance and those who have special health or religious diet requirements (Obadina et al., 2013; Khodke

et al., 2014; Tripathi et al., 2015) Soymilk is also a rich source of soluble and insoluble dietary fibers, and isoflavones whose presence in everyday diet is very important (Obadina et al., 2013) The phytochemicals present in soymilk are health promoting (Tripathi et al., 2015) and as a result, the use of soymilk in daily diet is highly recommended

Soymilk is a popular beverage with abundant vegetable protein in Asian countries As a nutrient-rich beverage, soymilk consumption has sustained a growth rate of 21% per year in the US (Wrick, 2003)

Fig 2.3 Commercial soy milk 2.2.3 Oat milk

Oat milk is made from oat groats (the hulled kernel of the oat grain) and water; some brands are a mix of oats and other grains

Oat milk is excellent sources of soluble dietary fiber (β-glucan) (Tiwari and Cummins, 2012), significant amount of phytochemicals (Peterson, 2001), rich in protein, lipids, minerals, and vitamins (Singh et al., 2013) Various literatures have reported that consumption of oat milk has been shown to decrease plasma cholesterol and LDL cholesterol concentrations in healthy individuals (Bekers et al., 2001)

Fig 2.4 Commercial oat milk

2.2.4 Quinoa milk

Quinoa milk, which may have potential for consumption directly as milk or

in milky products, may in the near future be of significant consumption (Jacobsen et al., 2003b) It is a high-quality, nutritive, and healthy product, easily digested by the school children who suffer from malnutrition (Jacobsen et al., 2003b) Lívia de L de O Pineli et al (2015) conducted the research aiming to low glycemic index and increase protein content in novel quinoa milk The result shown that product were improved for lipids, sodium, proteins and glycemic index, in comparison with nutrition facts and literature data of some plant-based milks, including one commercial quinoa milk (Pineli et al., 2015) It will be necessary to extend the production areas of quinoa cultivars for milk production, and to build processing plants for milk in areas of quinoa production, for the benefit of farmers, consumers, and rural agroindustries (Mujica et al., 2000a) An aspect of great importance will be the dissemination of the use of this highly nutritive and tasty product among consumers, who may be people unable to ingest animal lactose or casein (Jacobsen et al., 2003b) Quinoa milk might be an alternative to the most common vegetable milk, from soybean

CHAPTER 3 MATERIALS AND METHODS 3.1 MATERIALS

Quinoa seeds were provided by Faculty of Agronomy, Vietnam National University of Agriculture which harvested in autumn season It belongs to sweet cultivar named “Atlas” from Netherlands Sweet condensed milk from Vinamilk Company was purchased from local market Vanilla was purchased from local market as well Enzyme α–amylase (Termamyl) from Novozyme which is a kind

of Bacterial thermophilic alpha-amylase This enzyme was stored at 40C in fridge

3.2 RESEARCH CONTENT

- Optimization technical parameters (temperature, time and enzyme dose)

of enzyme Termamyl which hydrolyze quinoa starch

- Effect of sterilization regime (temperature and time) on product quality

- Evaluating degree of reference of consumer toward product

- Building the process of producing quinoa milk

3.3 METHODS

3.3.1 Technological method

Process of producing quinoa milk

Fig 3.1 Flow chart of producing quinoa milk Quinoa seed: As with numerous agricultural products, harvesting quinoa seeds will lead to co-mingling of the seeds with other components found in the field and transportation process, and these foreign materials need to be removed to make quinoa seeds suitable for human consumption Firstly, small and low density objects such as: straw, leaves, undeveloped quinoa seeds, dirt, weed seed and dust were removed by screening Secondly, quinoa seeds were dried in convection drying equipment at temperature of 50oC until they reach the

“equilibrium moisture” content (around 14%) The purpose of drying is to lower the moisture content in order to guarantee conditions favorable for further processing Drying is also advantageous to quinoa seeds quality in that it can

bottle, sealing Sterilisation

Product

inactivate bacteria, yeasts and moulds that can decrease shelf-life and pose food safety risks

Grinding: After the procedure of drying, quinoa seeds were subjected to a dry grinding to reduce the size of quinoa seeds Therefore, starch was more rapidly absorb water This was leading to provoke starch gelatinization and improve the subsequent enzymatic treatment

Starch gelatinizing: Water was added in the proportion 1:5 (quinoa:water) and the mixture was heated at 800C for 10 minutes When starch is heated in water, granules absorb water and swell The absorption of water by amorphous regions within the granules destabilizes their crystalline structure, resulting in the loss of birefringence (Parker and Ring, 2001; Donovan, 1979)

Enzymatic treatment: After the gelatinization step, it was the procedure of enzyme treatment The slurry was heated to 900C and then enzyme Termamyl was added with the concentration of 0.075% v/v This process took place over a period of 60 minutes This step was needed to attain the low viscosity and the certain sugar which the consumer demands of these products

Filtrating: After treating the enzyme Termamyl, the procedure of filtration was applied to obtain the liquid which did not contain any solid The beverage was filtered with a cloth

Mixing: After filtrating, sweetened condensed milk and vanilla were added into the liquid to improve not only sensory value but also nutritious value of product

Filling into the bottle and sealing: The liquid is filing into the glass bottle and sealing to prepare for the sterilization

Sterilizing: This is the important step contributing to ensuring microbiological safety The equipment used to conduct this procedure was autoclave The technical parameters employed were temperature of 1100C and time of 10 minutes

3.3.2 Experimental design

3.3.2.1 Optimization of enzymatic hydrolysis of quinoa starch

Response surface methodology (RSM) was applied to identify optimum levels of three variables of the enzyme concentration (%), hydrolysis temperature

(°C) and hydrolysis time (min) regarding of three responses — total sugar content, score of color and score of appearance of quinoa milk The coded and uncoded independent variables used in the RSM design are listed in Table 3.1 Ranges of enzyme concentration (X1), temperature (X2) and time (X3) were selected based on preliminary experimental results The experiments were designed according to the Box Behnken design using 15 experimental runs as shown in Table 3.2 Data were analyzed by multiple regressions through the least-square method A second-order polynomial equation was used to express the responses as a function of the independent variables as follows

Y = a0 + ∑ 𝑎iXi + ∑ 𝑎iiXi2 + ∑ 𝑎ijXiXj (1) where Yk represents the measured response variables, a0 is a constant, ai, aii and

aij are the linear, quadratic and interactive coefficients of the model, respectively

Xi and Xj are the levels of the independent variables

Software ‘‘Design-expert’’ (version 10.0.3, Stat-Ease Inc., Minneapolis, USA) was used to statically analyze and draw response surface plots and contour plots

Table 3.1 Factors and their levels for the Box-Behnken design

Variable Symbol Coded variable levels

Enzyme concentration (%) X1 0.05 0.10 0.15 Temperature (oC) X2 80 90 100

Table 3.2 Experimental design and responses of the dependent variables to

the hydrolysis parameters

Temperature (0C)

Time (min)

3.3.2.2 Effects of sterilization regime on quality of product

Aiming to ensure microbiological quality and prolong self-life of product, sterilization is an important step in processing of production of this product

However, the sensory properties are degraded because of high temperature Therefore, the impact of twelve different regimes of heat sterilization, defined by their combinations of temperature and time (Table 3.3) on the color and microbiological properties of samples was evaluated

Table 3.3 Sterilization regimes

Run Temperature (0C) Time (min)

3.3.2.3 Consumer sensory evaluation

A sample achieving both the best sensory quality and the hygiene and safety quality measured consumer liking on a 9 point hedonic scale where 1 =

“extremely disliked” and 9 = “extremely liked” In parallel with the quinoa milk, commercially marketed product (Korean rice milk) was also appreciated by the panel to compare the differences in favorability of the two products The procedure was carried out for the purpose of exploration prior to bringing the product to market

3.3.3 Analytical methods

3.3.3.1 Analyzing the main chemical contents

a Determining amino acid component by high-performance liquid chromatography method (HPLC)

b Determination of moisture content by drying the sample to a constant weight

c Determining lipid content by Soxhlet system

d Determining protein by Kjeldahl method

e Determination the total sugar content

The techniques used for total sugar quantification are an adaptation of DNS method using the 3-5-dinitrosalicylic acid - DNS modified (Miller, 1959)

3.3.3.2 Analyzing microorganism criteria

Microbiological quality of sterilized quinoa milk was controlled by determining the following parameters: total aerobic microorganisms (CFU) according to TCVN 4884:2005, the number of coliforms (CFU) according to TCVN 6848:2007, the colony forming units of yeasts and moulds according to TCVN 8275-1:2009, the number of E coli according TCVN 7924-2 : 2008, the number of Staphylococcus aureus according TCVN 4830-1 : 2005 the number of Clostridium perfringen according TCVN 4991:2005, the Streptococci faecal according TCVN 6189-2:1996 and Pseudomonas aeruginosa according ISO 16266:2006

3.3.3.3 Sensorial analysis method

a Determining the score of color and score of appearance

The products were evaluated color and appearance by trained panelists Unstructured line scale (ULS) was used in training panelists and evaluating the products The use of ULS was reliable and sensitive to product differences (Galvez and Resurreccion, 1990) The line scale was anchored 100 mm from each end, with the end points of color (brown and milk white) and appearance

(homogeneous, inhomogeneous) After evaluating, panelists rate the intensity of color and structure characteristic by marking a vertical mark across the horizontal rating line These marks are converted to numerical data by measuring the distance from the origin (“weak”) of the line to the vertical mark

Brown Milk white

Inhomogeneous Homogenous

Fig 3.2 Unstructured line scale for training and sensorial evaluation

b Determining degree of reference of consumer toward product

The sensory evaluation for quinoa milk and Korean rice milk was conducted by hedonic scale rating test using 60 untrained panelists The panelists were asked to score for preference on a scale of 1 to 9, which including Like Extremely, Like Very Much, Like Moderately, Like Slightly, Neither Like or Dislike, Dislike Slightly, Dislike Moderately, Dislike Very Much, and Dislike Extremely (Ha Duyen Tu, 2006)

3.3.3.4 Data analyze

The data obtained from the experiment of consumer sensory evaluation which were subjected to analysis of variance (ANOVA) to determine significant difference The data of score of color were subjected on Megastat software The data obtained from the optimal experiment were subjected on Design Expert software (version 10.0.3, Stat-Ease Inc., Minneapolis, USA)

CHAPTER 4 RESULTS AND DISCUSSION 4.1 ANALYSING THE MAIN CHEMICAL CONTENT

In Viet Nam, quinoa plant has been quite new which have not planted widely Therefore some main chemicals of seeds examined were analyzed to compare with that of other cultivars which were planted in the world The results were presented in Table 4.1

Table 4.1 The main chemical content of quinoa seed

The main characteristic of the quinoa seeds is the special quality of its amino acid composition The result of amino acid analysis in the seeds examined

is presented in table 4.2

Table 4.2 Amino acid profiles (g/100g protein)

No Amino acid Experimental data

in lysine which is the limiting amino acid in most cereals According to the FAO/WHO suggested requirements (as mentioned in Table 2.2) for a 2-5-year-old children, 10-12-year-old children and adult, QPs in the seeds examined have adequate levels of histidine, isoleucine, threonine, lysine, leucine, tryptophan and valine contents It is clearly that the protein quality of quinoa planted in Viet Nam was quite good

4.2 ENZYMATIC HYDROLYSIS OPTIMIZATION OF QUINOA STARCH 4.2.1 Fitting the model

The combined effects of enzyme concentration (X1), hydrolysis temperature (X2) and hydrolysis time (X3) on total sugar content (Y1), score of color (Y2) and score of appearance (Y3) are presented in Table 4.3 Table 4.4 shows the coefficients of variables in the models calculated using the least square technique and their statistical significances were judged using an ANOVA test at a significant level of 0.05 For any of the terms in the model, a large regression coefficient and a small p-value would indicate a more significant effect on the respective response variables (Khuri and Cornell, 1987) ANOVA showed that the resultant second-order polynomial model adequately represented the experimental data with the coefficient of multiple determination (R2) of 0.9501; 0.9334 and 0.9401 for the total sugar content, score of color and score of appearance, respectively

Table 4.3 Responses of the dependent variables to the hydrolysis

parameters

Run

Technical parameters

Total sugar content (mg/ml)

Score of color

Score of appearance

Enzyme

concentration

(%)

Temperature (0C)

Time (min)