research of the instant tea production process from ganoderma subresinosum

Keywords : Ganoderma subresinosum, Polysaccharide, Instant tea product, extraction process, antioxidant activity... The results of research on the effect of ultrasonic treatment time on

THAI NGUYEN UNIVERSITY

UNIVERSITY OF AGRICULTURE AND FORESTRY

HOANG MAI ANH

RESEARCH OF THE INSTANT TEA PRODUCTION PROCESS FROM GANODERMA SUBRESINOSUM

BACHELOR THESIS

Study Mode : Full time

Major : Food Technology

Faculty : Advanced Education Programs Batch : 2017 - 2022

Thai Nguyen, 2022

Thai Nguyen University of Agriculture and Forestry

Degree Program Bachelor of Food Technology

Student name HOANG MAI ANH

Ganoderma subresinosum, which grows on decayed trunks, belongs to the genus

of Ganoderma This species of fungus is claimed to have diverse pharmaceutical effects owing to the plentiful contained bioactive substances Nonetheless, most of current products are raw-sliced caps and fruiting bodies of Ganoderma subresinosum Lack of ready-to-use products from Ganoderma subresinosum on

present market results in scanty consumption of this fungus itself Therefore, this study focused on the process of producing instant tea from Ganoderma subresinosum on a laboratory scale To produce this kind of instant tea, the total

polysaccharide content will be extracted In which, the highest total polysaccharides were extracted in solvent ethanol 80% by using ultrasonic treatment time at 4 minutes with sound intensity 100 Db The corresponding chosen ratio for solvent per material is 1/20 In addition to this, the extracted time and temperature are 90 minutes and 80oC respectively Besides, the ability of scavenging free radicals of instant tea from Ganoderma subresinosum was conducted The instant tea product was made from Ganoderma subresinosum

extract and other herbal extracts with the sensory quality were evaluated by the sensory panel Other characteristics of instant tea from Ganoderma subresinosum had also been analized

Keywords : Ganoderma subresinosum, Polysaccharide, Instant

tea product, extraction process, antioxidant activity Number of pages: 70

Date of submission: June 1st, 2022

ACKNOWLEDGEMENT

First and foremost, I would like to earnestly express my sincere thank to the Lecturers of Thai Nguyen University of Agriculture and Forestry, especially the teachers in the Advance Education Programme, the Faculty of Biotechnology and Food Technology, the Institution of Life-Science, and last but not least Chemical and Supplies Offices of Institution of Life-Science for giving me the golden opportunity to learn and be learned during four years

I am dearly obligated to MSc Dinh Thi Kim Hoa, a Lecturer in the Department of Biotechnology and Food Technology, who always gave me wholehearted guidance in the process of studying and conducting my graduate research

I would also like to express my special thanks of gratitude to MSc Luu Hong Son and MSc Nguyen Thuong Tuan, teachers, and friends in the Department of Biotechnology and Food Technology as well as the Institution of Life-science for guiding and supplementing my laboratory skill and knowledge, and for always creating favorable conditions for me in the research process

I am overwhelmed in all gratefulness to acknowledge my depth to my family and friends for cheering me up during my graduation thesis

Thai Nguyen, June 02, 2022

Sincerely

Hoang Mai Anh

1.5 Scientific and practical significance 8

1.5.1 The scientific meanings of the topic 8

1.5.2 The practical meanings of the topic 9

CHAPTER II LITERATURE REVIEW 10

2.1 Overview of Ganoderma subresinosum 10

2.1.1 Characteristics of Ganoderma subresinosum 10

2.1.2 Distribution of Ganoderma subresinoisum 12

2.1.3 Overview of the chemical compositions of Ganoderma subresinosum 13

2.1.4 Overview of biological effects (pharmacology) of Ganoderma subresinosum 16

2.1.5 Product from Ganoderma subresinosum 18

2.2 Overview of herbal extraction process 19

2.2.1 Overview of extraction solvents and extraction methods 19

2.2.2 Overview of advantages and disadvantages of herbal extraction 21

2.3 Overview about instant tea 21

2.3.1 Origin of instant tea 21

2.3.2 Overview of additional material for the production of instant tea 22

2.3.3 Process of making instant tea powder 27

2.3.4 Production and consumption of instant tea in the country and in the world 28

CHAPTER III MATERIALS, RESEARCH CONTENTS, AND METHODS 31

3.1 Materials, chemicals, and equipments 31

3.1.1 Materials 31

3.1.2 Chemicals and equipments 31

3.1.3 Reseach scope and time table 32

3.2 Research contents 32

3.3 Research methods 33

3.3.1 Experimental design method 33

3.3.2 Analytical method 42

3.3.3 Sensory assessment method according to TCVN 3215-79 48

3.3.4 Data statistical analysis methods 49

CHAPTER IV RESULTS AND DISCUSSION 50

4.1 Analytical results of the chemical composition of Ganoderma subresinosum 50

4.2 The results of factors that affect the extraction of total polysaccharide from Ganoderma Subresinosum 50

4.2.1 The results of research on the effect of ultrasonic treatment time on the efficiency of total polysaccharide extracted from Ganoderma subresinosum 50

4.2.2 The results of selecting the size of material on the efficiency of extracting polysaccharides in Ganoderma subresinosum 51

4.2.3 The results of the study on selecting solvent concentration to extract polysaccharides from Ganoderma subresinosum 53

4.2.4 The results of selecting the ratio of raw materials Ganoderma subresinosum and solvent 53

4.2.5 Research results of selecting the extraction time total polysaccharide from Ganoderma subresinosum 54

4.2.6 Research results of selecting temperature for extraction of total polysaccharide from Ganoderma subresinosum 55

4.3 Research on choosing the suitable ratios of Ganoderma subresinosum extract and additive ingredients 56

4.4 The results of the research on the drying temperature of mixture of Ganoderma subresinosum extract and additive ingridients to produce instant tea 58

4.5 The complete process for producing instant tea Ganoderma subresinosum 59

4.6 Quality evaluation of instant tea products from extracted polysaccharide of Ganoderma subresinosum 62

4.6.1 Evaluation of the antioxidant capacity of instant tea from Ganoderma subresinosum 62

4.6.2 Evaluation on the sensory assessment of instant tea product from Ganoderma subresinosum based on TCVN 3215-79 63

4.6.3 The result of microbiological analysis in instant tea from Ganoderma subresinosum according to 46/2007/QD-BYT and QCVN 8-1:2001/BYT 64

CHAPTER V CONCLUSIONS AND RECOMMENDATION 66

5.1 Conclusion 66

5.2 Recommendation 66

CHAPTER VI REFERENCES 67

CHAPTER VII APPENDIX 70

7.1 Ballot for product sensory assessment score 70

7.3.7 Effect that the mixing ratio affects product quality 77

7.3.8 Sensory temperature affects product quality 78

7.4 The results of radical scarvenging ability of instant tea powder from

Ganoderma subreinosum and Acid ascorbic 79

7.5 Line charts 80 7.5.1 Line chart of D-glucose standard curve in the method of determination of total polysaccharides by Phenol-sulfuric acid analysis method for Total Polysaccharide 80 7.5.2 Line chart of radical scarvenging ability of instant tea powder from

Ganoderma subresinosum and Acid ascorbic 81

LIST OF TABLE

Table 3.1 Experiment chemicals, experimental equipment and instruments 27

Table 3.2 Experimental design to study the effect of ultrasonic treatment time on the extraction efficiency of polysaccharide in Ganoderma subresinosum 34

Table 3.3 Experimental design to study the size of material on the extraction efficiency of polysaccharide in Ganoderma subresinosum 35

Table 3.4 Experimental design to select solvents concentration for extracting polysaccharide from Ganoderma subresinosum 36

Table 3.5 Research on selecting the ratio of Ganoderma subresinosum material with solvent for extracting total polysaccharide 37

Table 3.6 Experimental study on selecting the time of extraction 38

Table 3.7 The effect of temperature on efficiency of polysaccharide extracted from Ganoderma subresinosum 39

Table 3.8: The mixing ratio of Ganoderma subresinosum extract and herbal additive extracts 41

Table 3.9 Experimental design to investigate the effect of drying temperature on instant tea sensory quality 42

Table 3.10 Instant tea weight coefficient for sensory evaluation 48

Table 3.11: Scores of quality levels 49

Table 4.1 Ash, moisture and protein contents of Ganoderma subresinosum 50

Table 4.2 Results of the effect of the ultrasonic treatment time on the efficiency of extraction of total polysaccharide from Ganoderma subresinosum 51

Table 4.3 Results of the effect of material size on the efficiency of extraction of total polysaccharide from Ganoderma subresinosum 52

Table 4.4 The effect of the solvent concentration on the efficiency of extracting total polysaccharide from Ganoderma subresinosum 53

Table 4.5 Effects of material/solvent ratio on the efficiency of extracting total polysaccharides in Ganoderma subresinosum 54

Table 4.6 Effect of extraction time on the efficiency of extracting total polysaccharide in Ganoderma subresinosum 54

Table 4.7 Effect of temperature on extraction efficiency of polysaccharide in

Ganoderma subresinosum 55

Table 4.8 The effects of ratios of Ganoderma Subresinosum extract and additive

ingredients on the sensory quality of the product 57 Table 4.9 The effects of drying temperature 58 Table 4.10 The antioxidant activity of instant tea powder from Ganoderma subresinosum and Acid ascorbic 62

Table 4.11 The sensory assessment of instant tea product from Ganoderma subresinosum 63

Table 4.12 The results of microbiological analysis of in instant tea product from

LIST OF FIGURE

Figure 1: Ganoderma subresinosum 11

Figure 2: Some Products developed from Ganoderma subresinosum 18

Figure 3: Structural formula of lastose 23

Figure 4: Lactose powder 23

Figure 5: Stevia rebaudiana Bertoni 24

Figure 6: Glycyrrhixa glabra L 26

Figure 7: Diagram of process of making instant tea powder 27

Figure 8: Instant tea products in the world and Vietnam market 30

Figure 9: The expected procedure of making instant tea product from Ganoderma subresinosum and additive ingredients 40

Figure 10: Diagram of instant tea production process 60

The diversification of tea products has been clearly demonstrated in recent years Besides dried tea products, ready-to-drink tea products, tea bags, instant tea products are also available In the age of technology development, producing time is highly considered to be the priority Therefore, the beverage industry is required to launch products that accommodate both quality and time-saving Accordingly, instant tea is the product that meets this conditional requirement Not only quenching thirst at once, but instant tea also provides a great deal of energy and water, and eventually, it is a source of vitamin C to help strengthen the body’s resistance, protect cells from the harmful effect of free radicals For the economic aspect, instant tea is absolutely a potential good for development

Throughout the development of human history, plants that belong to Fungi

have been considered as an essential element that used as a main component for brewing beer process or bread making or even tea producing Some of them are said to be poisonous despite their very attractive and eye-catching appearance In contrast, mushrooms with an intimidating appearance are used as daily nourishment Among which, for certain species of mushrooms, is studied as a

medicinal or health-promoting herb (Boston & Ma 02115 +1495‑1000, 2020)

Ganoderma lucidum Kast has primevally been known as a mystical medicine that

can be used only by the emperors Through time, Garnodema species became

known as a health-boosting medication that even a commoner could afford Based on the facts that had been signified from ancient people who used this tonic derived from Ganoderma lucidum Kast A number of related reports have been made and

are also in the making, which has shown that Ganoderma lucidum Kast has great

pharmacological value According to the International Journal of Medicinal Mushroom, different Ganoderma funguses with different colors are the same

species It is clear that 6 different types of colored – funguses can be found due to their change of external circumstances such as moisturization, light, nutrition, and the total carbon dioxide in the atmosphere As a result, based on controlled ecological factors and cultivating process, getting different Garnodema fungi is at

hand (Solomon, n.d.) Based on the DNA features as well as biological characteristics, there are two groups – Ganoderma applanatum and Ganoderma lucidum Kast – branched from Ganoderma Despite having been in the same genus

of Ganoderma, Ganoderma applatum is recognized as a wood- demolishing

species whereas G lucidum Kast is stated to be a pharmaceutical species

(Petruzzello, 2022) According to two main documents of ancient China named Shen Nong manuscript and Summary typescript, Ganoderma lucidum Kast can

also be classified by their color as white lingzhi ( 白芝), yellow lingzhi ( 黃 芝), red lingzhi ( 赤芝), violet lingzhi ( 紫芝), black lingzhi ( 黑 芝), green lingzhi (青芝) Generally, this mushroom species is believed to have many therapeutic effects namely cancer, cardiovascular disease, chronic bronchitis, asthma or neurastheria, (Sanodiya et al., 2009) Particularly, Black- colored Ganoderma lucidum Kast which has another scientific name as Ganoderma Subresinosum (D

Nguyen, 1985) is ranked among the six types As contained in many other kinds of Ganoderma Lucidum Kast, Ganoderma Subresinosum includes various types of

valuable pharmaceutical components specifically Lactone A, Adenosine, Acid oleic, Gemanium, Selenium, Polysaccharides, All of which help strengthen the

body's immune system, antioxidants, blood circulation, maintain and stabilize

blood pressure, enhance liver function, sedation assistance and deep sleep–boosting (N Nguyen, 2018)

By blending the extracts of certain herbs together with that of Ganoderma subresinosum, creating instant tea products includes numerous of uses and

benefits This tea acts as a functional beverage to promote health; moreover, it also meets the increasing demand of consumers for natural products In economic terms, instant tea products promote economic development, contributing to

diversifying product designs on the tea market Based on the discussion above, the project "Research on the process of producing instant tea from Ganoderma subresinosum " is deliberately conducted

to the present market

1.2.2 Detail objectives

Determining the effect of the following factors on an amount of total polysaccharides extracted: time, temperature, solvent, material/solvent ratio, and solvent concentration

Determining the Ganoderma subresinosum extract and additive ingredients

ratio for making instant tea;

Completing the process of producing instant tea from Ganoderma subresinosum extract

Evaluating the quality of the instant tea from Ganoderma subresinosum

1.3 Research questions

How to make products from Ganoderma subresinosum familiar to the consumers?

Ganoderma subresinosum is claimed to have extreme biological effects on

human health However, the process of collecting this fungus is challenging because of its scarce growing Subsequently, manufacturing product prize is a

considerable problem for manufacturers Difficulty in inputing step leads to limitation of different products Nevertheless, thank to the developing of agricultural technique, cultivating Ganoderma subresinosum is at hand Products

from Ganoderma subresinosum become diverse from raw materials to capsules or

pure powder Despite the number of products from Ganoderma subresinosum on

market, they are not yet developed as beverage products The research of making tea is a potential chance By developing a functional beverage from Ganoderma subresinosum, the functional food market will be diversified Consumers will have

more choices of products from Ganoderma species

How to develop instant tea product on the basis of extracting valuable biological active ingredients from Ganoderma subresinosum?

Polysaccharide contained in Ganoderma subresinosum is proved to have

many good influences on human health Hence, optimizing the withdrawal of total polysaccharide during the extraction of this fungus is neccessary This study is to research on the ability of extracting total polysaccharide present in Ganoderma subresinosum for the making of its instant tea Therefore, this research will also

improve the biological values of Ganoderma subresinosum

The process of extraction polysaccharide is not optimized

Limitation of time cause the imcomplement surey of the process namely aroma’s treatment, extraction of separately chemical components of material, entire evaporation of solvent presenting in the extract of material

1.5 Scientific and practical significance

1.5.1 The scientific meanings of the topic

To provide more scientific information about the Ganoderma subresinosum

To provide the parameters for the extraction of total polysaccharide from

Ganoderma subresinosum

To create suitable parameters for the production process of instant tea from

Ganoderma subresinosum at laboratory scale

1.5.2 The practical meanings of the topic

To create new products with high quality for comsumers’ health at a reasonable price;

To diversify the instant tea products on the market from Ganoderma subresinosum;

To create a new research direction on Ganoderma subresinosum, and

improve the output for this medicinal fungus

CHAPTER II LITERATURE REVIEW

2.1 Overview of Ganoderma subresinosum

2.1.1 Characteristics of Ganoderma subresinosum

Ganoderma subrenosisum, one type of fungus among countless of fungus

that belongs to the genus Ganoderma P.Kast in the Ganodermataceaes family

It is an oriental fungus with a woody texture and ivory black colour on its exterior It grows mainly on the trunks of decayed trees and takes nutrients from there for its growing progression (Ho, 2012) This plant mainly grows on many dead and decayed trunks which will be an ideal environment for the growth of this fungus Many decayed trunks from Cocos nucifera, Melaleuca, Prunus presica, Grinding tree, Senna Siamea, Peltophorum pterocarpum, Depending on the nutrient

source from the host trunk , Ganoderma subresinosum can grow with a single

fruiting body or a cluster (Mộc, 2021) Ganoderma subresinosum consists of two

main parts, the mushroom stalk and the mushroom cap The peduncle has an avarage length, unbranched, sometimes bent with slighly curvation Mushroom caps are fan-shaped or semicircular when mature, its size will change markedly over time Ganoderma subresinosum’s caps can appear many concentric, wavy

and scattered fringes

The genus of Ganoderma contains almost 300 species of wood-decaying

fungi in the family of Garnodermataceae Before cultivating, this species is used

only by the nobility They act as parasites since their nutrient comes mostly from saprobes on dead wood (Benzie & Wachtel-Galor, 2011) Since ancient times,

Ganoderma appeared to have been extremely rare They could only be seen upon

the decayed tree trunks in some abysmal forests For the consumption of

Ganoderma species, North America, Japan, Korea, Taiwan and China are some

countries that made up a large portion of the whole global Additionally, taxa of

Ganodermataceaes have also served as different traditional medicinal products

among Laos, Myanmar, Thailand and Vietnam - which is a group of nations that locates among Mekong Subregion (Hapuarachchi, 2019) However, with the modern technique, almost every species of Ganoderma are multiplicated and

cultivated under well control Each of every parts of the mushroom is used for

diseases treatment as Ganoderma genera consist of bioactive compounds

Bioactive substances are external- nutritional compositions, which appear in daily food with a very small amount Some medical dictionaries indicate that bioactive substances can actively react in living cells or making direct influences on living organisms Generally, several diseases namely anti-cancer, immunomodulation, antioxidation, and bacterial infections are some highlighting feature treatment of

Ganoderma species (Chen et al., 2019)

Source: Namlinhchiongtien.com

Figure 1: Ganoderma subresinosum

Classification of the genus Ganoderma (D Nguyen, 1985)

Species: Ganoderma subresinosum

In A field guide to mushrooms: North America, most of the Ganoderma in

general usually grow in the tropical forests – which have moisture levels and coniferous forests (McKnight & McKnight, 1998) In particular, Ganoderma subresinosum has the same type of living environment as that of Ganoderma lucidum These days, due to the devastations of many forests around the globe, it

is crucial to cultivate the species artificially Thus, the morphology of Ganoderma subresinosum may have some slight changes Ganoderma subresinosum is a

woody mushroom with ivory black color and a glossy exterior It contains two

main parts – stem and cap, in which the stem of the mushroom is a type of bending pillar The stem’s length of Ganoderma subresinosum depends on the cultivating

condition, it commonly stretches from 2 to 10 centimeters Likewise, the better the living condition is; the wider the Ganoderma subresinosum cap Besides, not

only having round-shaped, the mushroom also has an oval-shaped cap with a glossy upper surface The average diameter size would be in the range of 3 to 15 centimeters

Ganoderma genus prefers growing in some tropical areas which converges

suitable provisions namely: Moisture from 80-95 in percentage, ventilation, cellulose directly provision, 5.5 – 7 in pH range, enough light supply, (McKnight & McKnight, 1998)

This fungus is also called by several scientific names such as: Aumaroderma Subresinosum (Murrill) Corner and (Murrill) Teixeira 1992, or Fomes subresinosum Murrill, Polyporus mamelliporus Beeli, Trachyderma subresinosum

(Murrill) Imazeki (Hedwigia, 1983)

For some nations, there will be different names for Ganoderma subresinosum

due to the diversification of languages English name: Lingzhi, Ling Chih Chinese name: Black lingzhi Japanese name: Black Reshi Korean name: Black Yeongji Vietnamese name: Hac Chi, Linh Chi Den, Hac Linh Chi

2.1.2 Distribution of Ganoderma subresinoisum

In the world: A report conducted in 1938 by C.J.Humphrey indicated that there were several occurrences of Ganoderma subresinosum under various local

names shown sight in the South of Africa Those sights of Ganoderma subresinosum were considered to have been the fungus species that grow naturally

in the world Currently, the appearance of Ganoderma captured the attention of

people around the world Owing to its amazing functions the search for

Ganoderma species became larger Therefore, the occurrence frequency of Ganoderma subresinosum has been increasingly improved (Humphrey, 1938) In

addition to that, the Democratic Republic of Congo, Malaysia, Thailand, China, Kenya, Singapore, Zimbabwe, and Taiwan are some of the world’s nations that caught the occurrences of different kinds of Ganoderma subresinosum (Kadhila,

2010)

In Vietnam, Ganoderma subresinosum can be found in some mountainous

areas whose weather is tropical such as Thanh Hoa, Vinh Phuc, Furthermore, to preserve the genus of Ganoderma, there are some National Parks that grow Ganoderma species on a large scale for instance: Phuoc Binh National park (T

Nguyen, 2019)

2.1.3 Overview of the chemical compositions of Ganoderma subresinosum

Over the last few years, there has been increasingly umpteen studies related to the chemical compositions of Ganoderma genera in that this species carries

various bioactive substances Having originated partly in China, Ganoderma is

also put understudied by myriad of Chinese Professors As stated in China Times in 1996, Ganoderma genus contains some chemical compositions that create

superior influences Accordingly, some preliminary studies and related reports mentioned the disease treatments brought back by the bioactive substances that present in Ganoderma

Having been rooted in the same genus, despite the unidentical color Ganoderma subresinosum contains some resembling chemical substances and yet specific ones

Nevertheless, Ganoderma subresinosum appears to have limited production Hence,

there would be fewer studies or surveys conducted on this type of fungi around the globe and in Viet Nam particularly Fortunately, a small number of surveys were handled, and some of them concentrated on the influences that the present bioactive substances in Ganoderma subresinosum deliver

Most mushrooms including Ganoderma is composed of approximately 90% of water by weight Accordingly, the remaining 10% concluded of about 10-40% protein, 3-28% carbohydrate, 2-8% fat, 3 – 32% fiber, 8 – 10% ash Besides, there are also some found vitamins and minerals, potassium, calcium, phosphorus, magnesium, selenium, iron, zinc, and copper accounting for most of the mineral content A research was conducted to find that one type of fungus of Ganoderma genus consists of 1,8% ash, 26–28% carbohydrate, 3–5% crude fat, 57% crude fiber, and 7–8% crude protein (Mau et al., 2001) A study shows that Ganoderma subresinosum contains countless chemical substances accounting for mineral

content including potassium, calcium, phosphorus, magnesium, selenium, iron, zinc, copper, and some vitamins (Borchers et al., 1999)

A wide variety of bioactive molecules, such as terpenoids, steroids, phenols, nucleotides, and their derivatives, glycoproteins, and polysaccharides is consisted by this mushroom (Chang & Buswell, 1996) In Chapter 9 of Herbal Medicine: Biomolecular and Clinical Aspects; Ganodema was being focused on all aspects,

especially its components It was claimed that polysaccharides, triterpenes, and peptidoglycans are the three foremost physiologically active components in

Ganoderma subresinosum (Boh et al., 2007)

Polysaccharides: Ganoderma species are believed to contain many

high-molecular-weight polysaccharides These compounds carry many biological activities and are found in all parts of the fungus There are more than 200 types of polysaccharides isolated from fruiting bodies and spores of Ganoderma species These polysaccharides have a helical shape which immobilized by hydrogen bonds Having been found in the fruiting body of fungi, the water-soluble heteropolysaccharide is composed of simple sugars D-glucose, D-galactose, D-mannose, and D-xylose, consisting of β -glucan and glucurono- β -glucan This bioactive component is considered to be the major active chemical component in

Ganoderma subresinosum Linked together by glycosidic bonds of long

monosaccharides chains Insoluble fibers are considered indigestible fibers, typical dietary fibers of polysaccharides include cellulose, hemicellulose, β-Glucan, pectin, mucilage, lignin, (Bao et al., 2002) The strong biological activity of polysaccharides extracted from Ganoderma species spores and mycelium has a

tremendously good effect on certain ailments In addition to this, a study by a group of authors shown that polysaccharide‘s naturally occurring - β-glucans which are

present in fungi have enhancing effect on the immune system (Dalia et al., n.d.) A recent analysis pointed out the effect of β-glucans in the treatment of Covid – 19 (Mirończuk-Chodakowska et al., 2021)

Triterpenes: This group of natural substances has chain lengths that are multiples of five, namely methyl (monoterpenes) and β -carotene (tetraterpenes) The subclass of terpenoids is triterpenoid and its general formula is C13H48 In

Ganoderma species, the triterpene stays under the form of lanostane, which is

involved in the synthesis of lanosterol Kubota et al succeeded in isolating the first

triterpenoids from Ganoderma species, which are ganoderals, ganoderiols, and

ganodermic acids (Kubota et al., 1982) This active constituent shows a significantly good impact on some types of diseases including anti-tumor, anti–complement activities Indeed, a survey by Bingji M et al emphasizes the effect

of Triterpenoids – which originates in the spores of G Lucidum (another species

roots in the same genus of Ganoderma subresinosum) (Bijing et al., 2011) A

recent discovery finds that triterpenoids from Ganoderma species as potential

inhibitors against the Dengue virus NS2B-NS3 protease through vỉtual screening (Shiv et al., 2019) The bitter taste of the mushroom itself is because of the amount triterpenens present in Ganoderma subresinosum

Peptidoglycans: These bioactive components are one of the largest macromolecules in bacterial cells, which normally form a mesh-like structure called a peptidoglycan sacculus enclosing the cytoplasmic membrane This polymer consists of units of N-acetylglucosamine and N-acetylmuramic acid The content of peptidoglycan contained in Ganoderma subresinosum will have a few

different uses However, antiviral and boosting the host immune system are some typical impacts of this bioactive compound (Zubing et al., 2005)

Polyphenols: In addition to the three main active ingredients in Ganoderma subresinosum, Polyphenol is also a useful bioactive molecule Polyphenols are

phytochemicals found in various plant species in nature, the group of this bioactive substance is best known as flavonoids Flavonoids are a group of phenolic compounds with a C6-C3-C6 framework, or in other words, a basic framework consisting of 2 benzene rings A and B connected by a 3-carbon chain Flavonoids are known to be a subclass with the ability to scavenge free radicals, inhibit enzymatic hydrolysis and oxidation, and have anti-inflammatory properties (Mohammed et al., 2011)

Other components: Besides the above boiative constituents, there are also lots of components that are discovered because of their existing effects Lactone A, a cyclic ester, consists of a 1-oxacycloalkan-2-one structure (−C(=O)−O−), or analogues having unsaturation or heteroatoms replacing one or more carbon atoms of the ring The total content of Lactone A takes responsibility for reducing the cholesterol level in blood Cellulose contains a chain of glucose units linked by β-1,4-glycosidic bonds, helping in Diabetes mellitus treatment, and constipation

Germanium focuses on stimulating the oxygen exchange, which results in well blood circulating (Gong et al., 2019)

Currently, there has beeen a study published in 2011 from a group of Vietnamese researchers that study on chemical comositions of a kind of Black lingzhi named Aumaroderma subresinosum In which, aside from other already-

discovered components, it is concluded that there are mainly unsaturated fatty covering about 80% of the total acids exist in the crude extract of the experiment’s sample (Doan et al., 2011) However, up to now, there has not been any other in-depth researchs on the species Ganoderma subresinosum

2.1.4 Overview of biological effects (pharmacology) of Ganoderma subresinosum

Ganoderma genus is considered to have the most remarkable

pharmacological effects among the existing mushrooms on the globe Specifically, most of the species roots from Ganoderma genus have similar influences on

human’s diseases Therefore, Ganoderma subresinosum has been in the same

branch as Ganoderma Lucidum also has these listed effects such as anti-tumor

activity immunomodulation, anti-oxidant, diabetes mellitus, liver and gastric injury and antiviral activity

Anti-tumor activity: The tumor is formed by the excessive growth of the

abnormal cell group in the body during cell division This type of solid mass tissue may affect bones, skins, and tissues Despite the fact that many tumors are not cancer, they still need prevention and treatment Several treatments though there have been, fore-prevention is still in need Accordingly, it is crucial to have a suitable diet and enough supplements of nutrilite contained in food A study in 2009 indicates that the total polysaccharides content found in Ganoderma extracts

plays an essential role in the anti-tumor activity (J Wang et al., 2009)

Immunomodulation: The immune system is able to detect a wide variety of

pathogens including viruses, parasitic worms, and cancer cells This network of biological processes is to protect an organism from any diseases caused by pathogens or any kinds of objects namely wood splinters Therefore, to boost this system’s ability, enough supplement of bioactive substances is needed In addition to this, Ganoderma

species is put ino studies by many researchers owing to its obvious bioactive

constituents In 1997, a study posted an evidence of immunostimulating by cytokines

immunological influencers (S Wang et al., 1997)

Antioxidant activity: Lipid peroxidation can be caused by free radicals, which

result in the damaging of cell membranes, cell tissue or even cell deaths Consequently, the host body that suffers all of those damages, is likely to be in great harm Ganoderma genus has been proved to be a promising fungus that

contains flavonoid - a type of bioactive component that has a great contribution to the antioxidant activity Accordingly, a study of Mau, Lin and Chen (2001) compared the antioxidant properties of several Ganoderma species In which, at

0.6 mg/mL, G lucidum, G lucidum antler, and G Tsugae show a percentage of

lipid oxidation of 2.30 -6.4% (Mau et al., 2001)

Diabetes mellitus: This type of illness is caused by the decline of insulin

response and production of the body Subsequently, it results in an abnormal increase of blood sugar levels In 1985, Hinoko et al conducted an experiment on

a mouse model, it is reported that ganoderan B from Ganoderma species are able

to increase the plasma insulin (Hinoko et al., 1985) Another experiment conduct edon mouse model by Tomoda et al in 1986 showed the significant hypoglycemic

effects in mice and the increase of plasma insulin levels in normal and loaded mice by the total present ganoderan B (Tomoda et al., 1986)

glucose-Liver and gastric injury: The polysaccharides in Ganoderma species are

effective in increasing plasma albumin, which is effective in protecting the liver and restoring liver function, especially in the treatment of hepatitis B Ganoderic acid R and S, also ganoderic acid A present in spores of Ganoderma fungus have

hepatoprotective effects induced by galactosamine In 2013, Wu et al performed

a study on methanol extracts and n-hexane fractions, dichloromethane, methanol extracts and compounds isolated from Ganoderma lucidum such asergosterol, and ganodermanotriol These substances have hepatoprotective effects in vitro and in vitro, increasing the degree of protection of hepatocytes against t-BHP (Wu et al.,

2013)

Antiviral activity: The Ganodermic acid present in Ganoderma subresinosum

has anti-inflammatory, antibacterial, antiseptic, and anti-allergic effects Clinical studies have shown that the polysaccharides present in Ganoderma subresinosum

help to nourish the body, and make the body healthy, thereby enhancing the body's immunity Ganoderma subresinosum’s bioactive compositions help the body to

have the ability to prevent and destroy harmful agents such as viruses, and bacteria Especially the ability to find and destroy harmful cancer cells and inhibit the growth of cancer cells growth of HIV cells According to Wang et al the

inhibition of virus replication from Ganoderic acid Y – an isolated bioactive substance in Ganoderma species The virus coating prevention is inhibited by the maximum rate of 83.9% at about 20 μg/mL of Ganoderic acid Y (S Wang et al., 1997)

2.1.5 Products from Ganoderma subresinosum

In the world: Ganoderma subresinosum originated in the same Ganoderma

genus as well as many other species of Lingzhi Various kinds of Ganoderma

fungus are used to make herbal medicine in countless nations namely USA, Brazil, China, and Japan, Currently, there are studies on the effects of Ganoderma subresinosum on human body with each specific condition and group of

pathologies In addition to specific diseases such as enhancing immunity, oxidant or anti-viral, effects on diabetics Other unspecified effects are also clearly indicated such as increasing nutrients and energy for brain cells, reducing fatigue, improving sleep, being a tonic for ill people, people eating poorly, stabilizing blood pressure are also the influences of Ganoderma Subresinosum on users There

anti-are many products from Ganoderma subresinosum in capsules typed

Source: Wikipedia.com Source: Jkendai.com Source: Japan.com

Figure 2: Some products developed from Ganoderma subresinosum

In Vietnam, there are no official documents also commercial product on the use of this species However, there are some products made from raw-mushroom cap and

fruiting body that are develop to meet the need of comsumers These products of

Ganoderma subresinosum are reported to have the similar beneficial influences as

those – that are studied by many authors in the world

2.2 Overview of herbal extraction process

2.2.1 Overview of extraction solvents and extraction methods

Extraction generally is the physico-chemical process that represents the withdrawal of a solute in a liquid or a solid by another solute – which is called “solvent” by a diffusion of substances of different concentrations (Abubakar & Haque, 2020) As in term of pharmacy, to extract is to seperate the medicinal active portions of plant tissues from the inactive or inert components by untilizing some of the seletive solvents present in standard procedures The extraction of a liquid sample is called liquid- extraction whereas the extraction of solutes dissolved in solids is solid-extraction During the extraction process, the amount of active substance obtained and the extraction efficiency will likely be affected by various factors Those factors may be counted as size, type of materials, kind of solvents, extraction time, required temperature, etc Extraction in chemical industry and production line is an important process which contributes to the production of valuable products directly Futhermore, extraction these days is used widely since it brings about many economical benefits and high automatically capability

2.2.1.1 Overview of extraction solvents

The extraction solvent is one of the most affecting factor that has influece on extraction process Therefore, the appropriate chosen solvent gives the effective result of extracted sample Despite the large number of different solvents used in the extraction process, they can be categorized by some typical characteristics such as polarity, viscosity, and surface tension Generally, non-polar chemicals dissolve easily in less polar solvents, however substances having many polar groups are difficult to dissolve in less polar solvents In contrast, strong polar solvents dissolve molecules with numerous polar groups easily but are difficult to dissolve less polar ones Solvents can be classified based on their polarity ability (Muhamad et al., 2017)

- Non-polar solvents: petroleum ether, gasoline, hexane, heptane, benzene, toluene,

- Less polar solvents: chloroform, dichloromethane, acetone, - Strong-polar solvents: Water, glycerin, ethanol,

Besides, the viscosity and surface tension of the solvent are also considered as important as polarity ability The lower viscosity and the smaller surface tension the solvents have, the higher the absorption of the solvent into the medicinal sample creating a favourable condition for extraction process

There are some certain conditions for solvent selection in extraction process: - Do not corrode equipment

- Easy to find, cheap

- No adverse reaction between solvents and solvents - Easily separates the substances by the solvent - Safe, non-toxic, do not catch fire and explosion

2.2.1.2 Extraction method

Each medicinal material has its own unique properties Therefore, finding an appropiate solvent for the exact meterial to give an effective results is very crucial Based on the nature of the medicinal plants and solvents, as well as the conditions of the infrastructure and experimental equipment, and finally the scale of the experiment There are several methods that can be put into application: soak, immersion, upstream extraction, or other methods Moreover, supercritical liquid extraction and ultrasonic extraction are also required in other modern equiment The ganeral techniques of extracting a medicinal plant often includes various technologies such as maceration, infusion, precolation, digestion, decoction, etc

Maceration method

In this process, materials or processed plant samples are put into a glass jar or a stainless steel container with a lid It is unsuitable to use a plastic jar or bottle since the bottle itself can be reactive or reflective to the solvents, which can directly affect the result of extraction process Subsequently, pure solvents are slowly poured into the sample flask Soaked samples are preserved under room temperature in the duration of 24 to 48 hours or even 72 hours This length of time imprenates the plant cell of materials with the fitting solvents leading to the disolving of sample’s natural compound Afterward, it is vital to filter strictly by filter paper to retrieve the extract mixture Consequently, extraction solution and

material residues are likely to be seperated thoroughly by either filteration or decantation

2.2.2 Overview of advantages and disadvantages of herbal extraction

Advantages of herbal extract

For medicinal plants that are subjected to the extraction method, the resulting product is likely have a lower impurity content, along with the percentage of active ingredients that will likely be higher than the percentage of active ingredients in the original herbal medicine In case of liquid extracts, they will have the same ratio of active ingredients as in the original herbal medicine In addition, herbs or medicinal herbs are very compact, light, and easy to transport It can be easily used as an intermediate or applied medicinal preparation technologies in the form of tablets, capsules or syrups, etc

Disadvantages of herbal extract

Medicinal herbs are the finished product of the extraction process, so medicinal herbs must go through numourous steps Since going through low-temperature processing, the finished product is susceptible to microorganisms such as bacteria, mold or yeast In addition, the medicinal plant itself is the medium that provides nutrients for microorganisms to thrive

2.3 Overview about instant tea

2.3.1 Origin of instant tea

Tea is considered the most dominant beverage of all time Throughout human history, tea-based beverages have been modified in various ways Accordingly, there are many types of products developed towards commercialization Among tea products present currently on the market, instant tea products are quite potential Back to it’s spendor history In terms of history and biology, all experts have come to a conclusion of a term “Asia – mostly western of northern of China, is the cradle of tea culture” At first, tea was only considered a medicinal herb for skin diseases It was not until 2737 BC, Shen Nong discovered that tea leaves could be mixed with boiling water and used as an antidote By 618-907 BC, tea was

spread and flourished in Japan The 18th has marked a turning point on the way to reach the world, countries such as the US, UK, and India also welcome it as a drink

to enjoy Not until 1885 did John William Brown of Huddersfield United Kingdom

patent a paste In which, the mixing of the concentrated tea extracts, evaporated milk and sugar, finally together in the dilution with boiling water results in a cup of tea As the habitat of enjoying tea in tea rooms in United State became obsolete and largely disappeared in the early 20th century A type of tea packaged quickly added into the Bristish and Canadian diets during the World War II However, not until 1946 did instant tea become commercialized in the United States Up to now, the United States become the third largest tea importer in the world just after United Kingdom and Russia (Saltmarsh, 1992)

2.3.2 Overview of additional material for the production of instant tea

2.3.2.1 Lactose

Definition: Lactose has scientific name as O - β - D - galactopyranosyl- (1 →

4) -D-glucopyranose A disaccharide, made up of one β D - galactose molecule and one β D-glucose molecule; linked together by β - 1-4 glicozid bonds, abundant in mammalian milk

Properties: Lactose is easy to dissolve in water, mild sweet taste, pleasant,

neutral and less hygroscopic, easy to granulate, easy to dry

Role: During digestion, lactose is hydrolyzed to galactose and glucose In

which galactose is an important nutrient in creating myelin membranes, it encapsulates nerves and speeds up the transmission of nerve signals, so it plays an important role in brain development Lactose is also considered a smart sugar Lactose is also used as substrate for beneficial dairy bacteria, facilitating them to multiply and eliminate potentially pathogenic bacteria, helping to maintain the stomach environment, and make the stomach and intestines healthy and strong The final product of lactose metabolism is lactic acid, which is significant in helping to acidify the gut and aid in protein digestion

carbohydrates in the diet of the diabetic can be up to 255g while the amount of lactose added to the drug is very small In addition, lactose is absorbed and digested more slowly than saccharose and glucose Furthermore, lactose does not cause tooth decay by enzymes The end product of lactose metabolism is lactic acid, which is significant in helping to acidify the gut and aid in protein digestion (Dekker et al., 2019)

In general, lactose is used as a popular pharmaceutical adjuvant because of its low cost, availability, low hygroscopicity, chemical stability, mild sweetness, complete water solubility and minimal incompatibility with pharmaceutical substances and other excipients α-lactose is an adjuvant used in the manufacture of solid drugs such as tablets which are prepared by wet granulation or straight pressing (pelleting without moisture or heat treatment), capsules or powders inhaler (α-lactose acts as a carrier) β-lactose is used as a filler and stick in the straight pelletizing process

Source: wikipedia.com Source: wikipedia.com

Figure 3: Structural formula of lastose Figure 4: Lactose powder

2.3.2.2 Stevia rebaudiana Bertoni Origin:

- Scientific name: Stevia rebaudiana Bertoni

- Stevia is native to South America, commonly known as molasses, sugar

grass It was brought to Europe in 1887 Naturally native to the Amambay and Iquacu regions of the Brazilian and Paraguay border, today is grown in abundance around the world and is used as a medicinal herb its The Asians

took the herb to dry and used it in everyday life as a herbal remedy, the part used mostly on the ground (Anh, 2018)

than 6 months and start to turn to wood after 6 months

- Roots: The roots of perennial Stevia are very strong, with feathers from

0-30cm at a depth of 20-30 cm from the ground, spreading cluster roots with a diameter of 40cm

- Stem: dusty stem, round branches, hairy young stems, green old body purple with average height of either 50-60 cm or 80-90 cm when well developed with a diameter of 5-8mm

- Leaves have three prominent veins, the annex is feather-shaped, the leaf edge is serrated Both stems and leaves taste very sweet The first flowers grow at the leaf edge and cluster on the tops

Stevia rebaudiana Bertoniflowers are clustered in a sphere containing many small ivory-white tubular flowers Each flower cluster is usually about 2cm long and has a gentle scent Flowers bloom in October and last until February next year

Distribution: Nowadays, Stevia is grown in many countries such as Brazil,

China, Japan, Mexico, Taiwan, Thailand, Argentina, Paraguay, Israel, USA,

Vietnam…

Nutritional and chemical composition

- The nutritional composition of Stevia leaves contains protein content from

9.8% to 20.4%, fat from 1.9% to 5.9%, carbohydrates from 35.5 to 61.9% and

reducing sugar from 3.3% to 6.9%

- The main ingredients in Stevia are diterpenoid glycosides, including main

types: Stevioside (5-10%), rebaudioside A (2-4%), rebaudisoside C (1-2%) and dulcoside A (0.5- 1%) and two subtypes rebaudisoside D and E, Some macronutrients and trace minerals are also found in Stevia : Ca, Mg, Fe, Mn, Sr,

Cu, Cr, Cd

- Stevia is 300 times sweeter than cane sugar because the main active

ingredient is steviol (it's a glycoside) Therefore, clover is used in foods to treat diseases such as diabetes, high blood pressure Steviozit in Stevia after

hydrolysis will give 3 molecules steviol and isosteviol Steviol is 300 times sweeter than sucrose (Do Huy Bich et al 2003) [42], low in energy, not fermented, not degraded but has a delicious taste, can be used to replace sugar in the diet The important property of these glucosides is that they can sweeten foods and beverages without being harmful to humans, making it easy and economical to harvest

Uses of Stevia:

- Stevia is used to help people with diabetes significantly improve their health,

stabilize sugar gliders blood glucose content In addition, Stevia also works to reduce

blood pressure, reduce inflammation (gingivitis, stomach pain, )

- On the other hand, Stevia is also used for food production such as

confectionery, soft drinks It is also used in food production for diabetics and for weight loss is used to replace the sweetness of sugar

- In the field of Stevia cosmetics with anti-inflammatory and antifungal

effects is used for shampoos, anti-inflammatory, acne reduction, sebum reduction to improve skin look brighter

2.3.2.3 Glycyrrhixa glabra L Origin:

- Scientific name: Glycyrrhiza uralensis fish and Glycyrrhixa glabra L,

belonging to the butterfly family (Fabaceae)

- Morphological characteristics: Glycyrrhixa glabra L is a perennial plant,

the stem can be up to 1-1.5m high; The whole plant has very small hairs, double leaves, ovoid, pointed tip, intact edge is 2 to 2.5 cm long Licorice flowers when blooming are light purple, butterfly petals, flowers bloom in summer and autumn, long flowers 14-22mm The fruit is curved in sickle shape, 3-4cm long, 6-8cm wide, dark brown, the fruit surface has many long leaves, in the fruit there are 2-8 small flat seeds, grayish brown or light black green, shiny

Source: Vinmec.com

Figure 6: Glycyrrhixa glabra L

Chemical composition

- Glycyrrhizin belongs to the olean group which is a saponin, naturally found

in licorice root in the salt form of potassium and calcium It accounts for 10-14% in dry medicinal herbs, it has a sweet taste, 60 times sweeter than saccarozo sugar - The second most important group of active ingredients in licorice is flavonoids with a content of 3 - 4% The main flavonoids of Glycyrrhixa glabra L

are liquiritin, isoliquiritin and liquiritigenin

- In addition, in the roots of Glycyrrhixa glabra L, the main ingredients are

polysaccharice and some monosaccharides

Uses of Glycyrrhixa glabra L

- Glycyrrhixa glabra L has a very strong detoxifying effect on leukocytes

toxins, snake toxins, dizziness, tetanus poison

- Effects as octison: Increases the volume of water and salt in the body, causing water retention, and at the same time treating ulcers in the digestive system Glycyrrhixa glabra L is also combined with other herbs to treat coughs,

stomach pain, stomach ulcers, and boils

- Glycyrrhixa glabra L is also used in medicine as a flavour Glycyrrhixa glabra L root contains Glycyrrhizin which is a saponin belonging to olean group,

content from 10-14% in dry ingredients, only in the subsoil, has a very sweet taste (60 times saccharose) This is the most important saponin of the root Glycyrrhixa glabra L In traditional medicine: Glycyrrhixa glabra L has a sweet taste, is

calculated on 12 meridians, has the effect of clearing heat, detoxifying, laxative, spleen tonic, and regulating medicinal herbs

2.3.3 Process of making instant tea powder

Materials

Wash

Instant tea Additional

2.3.4 Production and consumption of instant tea in the country and in the world

Instant tea products these days are diverse owing to the various requirements of consumers Consequently, the ingredients or formation of instant tea products becomes multifarious The fundamental part of each instant tea product is tea powder mixed with the flavored powder from many other edible herbs There are various tea brands presented in Viet Nam market including Lipton, Nestea, Cozy tea, Matcha, Instant milk tea, In addtion to this, all of the listed brands develop their products by diversifying the flavor by making a combination of pure tea powder with many herbs powder as well as fruits or flower extracts For instance, instant tea with flavor from peach, lychee, apple, rose, chrysanthemum, red artichoke this kind of instant tea product is called additive tea The combination of tea powder with flavored powders from herbs together with boiling water, makes a very convenient way of enjoying tea Not only served as a quenching thurst, instant tea products also give myriad of health benefits Each instant tea product contains each advantages:

- Meditation Tea: is used to treat diseases such as colds, coughs, stomach aches and alcohol help For instance: Artichoke tea: Red artichoke is rich in antioxidants Therefore, this tea can help you prevent diseases caused by the accumulation of free radicals Drinking this tea can prevent cancer, avoid skin aging, prevent aging process, cardiovascular disease , Chamomile tea helps with sedation, pain relief, cancer prevention, and anti-aging support

In the world’s market

Tea is a typical beverage of Asia, but in recent years, tea has gained popularity all over the world Due to its popularity and convinience, instant tea products was developed as a quick quenching beverage

A survey shown a numourous of instant tea premix prducts from nations all over the globe In which, an Indonesian tea brand owned by Unilever, announced the lauch of new 3-in1 instant tea products in June 2019 This product consists of two new flovours : caramel and Teh Tarik

In the same year, a company located in Gujarat, India announced to introduce a tea premix belongs to the instant tea product chains

In Viet Nam market

Instant tea in Vietnam is extremely dynamic and develops with the needs of its users In addition to the function of heat, beverage is also a product to enhance health and reduce the risk of obesity, cancer,

Though there are myriad of instant tea products from Ganoderma lucidum

created and presented in the market Instant tea products from Ganoderma Subresinosum is so far have not put into reseach and developed

Source: Alamy.com Source: indiamart.com

Source: amazon.com Source: deciclik.com

Figure 8: Instant tea products in the world and Vietnam market

CHAPTER III MATERIALS, RESEARCH CONTENTS, AND METHODS

3.1 Materials, chemicals, and equipments

3.1.1 Materials

- Main material: Ganoderma subresinosum was collected from Ha Giang

Province The harvested Ganoderma subresinosum was put into preliminary

treatment Then, it was dried at 60oC until the moisture content reachs 10 – 13 % The treated Ganoderma subresinosum was stored under cool temperature

- Additional ingredients: Lactose and Stevia and Licorice extracts

3.1.2 Chemicals and equipments

Table 3.1 Experiment chemicals, experimental equipment and instruments Experiment chemicals

Experimental equipments

3.1.3 Reseach scope and time table

- Location:

+ Laboratory of Faculty of Biotechnology and Food Technology

+ Institution of Life-Science, Thai Nguyen University of Agriculture and Forestry - Implementation time: February 2022 to June 2022

- The research was carried out in the laboratory scale

Research content 2 To study factors that affect the extraction of total polysaccharide from Ganoderma subresinosum following the single-factor model

- Research the effect of the ultrasonic treatment time on the efficiency of

extracting polysaccharide in Ganoderma subresinosum;

- Reseach on the size of Ganoderma subresinosum;

- Research on the concentration of solvent; - Research on material/solvent ratio;

- Research on the time of extraction;

- Research on the temperature of extraction

Research content 3: Research on choosing the suitable ratios of Ganoderma subresinosum extract and additive ingredients to create instant tea

Research content 4: Research on the drying temperature of the mixture to produce instant tea from Ganoderma subresinosum

Research content 5: The quality evaluation of instant tea from Ganoderma subresinosum and preliminary calculation of product price from Ganoderma subresinosum

3.3 Research methods

3.3.1 Experimental design method

Research content 1 To study on the chemical compositions of Ganoderma subresinosum

The moisture content will be analyzed by method of determination of moisture content followed the TCVN 9741:2013

The ash content will be determined by method of determination of ash according to the TCVN 8124:2019

The crude protein content will be determined by the determination of total Nitrogen content – Kjeldahl principle and crude Protein calculation followed the TCVN 8099-1:2015