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

INTRODUCTION

Research rationale

Modern civilization leads to the increasing demand of its civilians toward superiority in life So countless products are created to meet the needs of purchasers Tea is the most influential drink during the development of human history until the present According to some articles and studies, Asia – especially Southwest China, is considered the cradle of tea culture globally At first, tea was used as a herbal remedy until it was recognized as a daily drink Finally, tea is seen as an artistic activity that should be enhanced in creativity and study as well as enjoyment Currently, in the world and Vietnam particularly, tea not only plays a trending role as a quenching beverage, but it is also equally important as herbal medicine Drinking tea appropriately brings many benefits to human health since tea is an anti-cancer agent in various models such as liver, breast, lung or ancreas, and esophagus Also, tea is genuinely beneficial in some certain medic conditions such as high blood pressure and cholesterol, etc (Hara, 2011)

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 promotehealth; 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.

Research objective

The overall goal of the research is to develop a process for producing instant tea from Ganoderma subresinosum, supply to the market in order to improve the quality tea products, bring economic efficiency, contribute to the diversification of tea products in the market, and expand the application of the Ganoderma subresinosum fungus Futhermore, the study is to creating an innovative product to the present market

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.

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.

Limitations

The process of studying on the production of instant tea from Ganoderma subresinosum may have some certain limitations:

Distrbutive growing, difficulty in transporting and preserving are some factors cause high-cost manufacturing

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.

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

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.

LITERATURE REVIEW

Overview 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)

Classification of the genus Ganoderma (D Nguyen, 1985)

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

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

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

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

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 –

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

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:

- No adverse reaction between solvents and solvents

- Easily separates the substances by the solvent

- Safe, non-toxic, do not catch fire and explosion

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

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

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

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.

Overview about 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 18 th 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 20 th 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

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

- Lactose has a sweet taste, no aftertaste The sweetness of lactose is 30% of that of sacharose

- Lactose can cause symptoms of 'lactose intolerance' syndrome at doses of 3g or more in people with lactose-hydrolyzed lactose deficiency

- In the DEF pharma report, lactose is safe for diabetics, because of the amount of carbohydrates consumed in the diet of the diabetic, as the amount of 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

- 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)

Figure 5: Stevia rebaudiana Bertoni Botanical characteristics:

- Genus Stevia has more than 100 varieties, but only Stevia rebaudiana

Bertoni has sweet taste and can be grown Stevia is a perennial herb, grown more 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,

- 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,

- 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

- 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

- 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

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

- 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

Figure 7: the diagram of process of making instant tea powder

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

MATERIALS, RESEARCH CONTENTS, AND METHODS

Materials, chemicals, and equipments

- Main material: Ganoderma subresinosum was collected from Ha Giang

Province The harvested Ganoderma subresinosum was put into preliminary treatment Then, it was dried at 60 o C until the moisture content reachs 10 – 13 % The treated Ganoderma subresinosum was stored under cool temperature

- Additional ingredients: Lactose and Stevia and Licorice extracts

Table 3.1 Experiment chemicals, experimental equipment and instruments

No Chemicals Origin of production

29 Test tube cleaning brush Vietnam

3.1.3 Reseach scope and time table

+ 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 contents

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

- Determination of total moisture content of raw material;

- Determination of total ash content of raw material;

- Determination of total crude protein content of raw material

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

Research methods

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

Research content 2 To study factors that affect the extraction of

Ganoderma subresinosum following the single-factor model

Experiment 4: Research on the effect of the ultrasonic treatment time on the efficiency of extracting polysaccharide in Ganoderma subresinosum

Samples are treated with ultrasonic waves at different intervals of 0, 2, 4, 6 minutes This experiment follows the single-factor model Other extraction parameters are fixed, on the basis of which the experiment is arranged as Table 3.2

Table 3.1 Experimental design to study the effect of ultrasonic treatment time on the extraction efficiency of polysaccharide in Ganoderma subresinosum

Processing time by ultrasonic waves (minutes), (Sound intensity 100 Db)

•Ratio of material/solventi: 1/15 (g/ml)

Amount of total polysaccharide obtained

The extract is filtered through the filter paper 3 times By determining the content of total polysaccharide, the most suitable formula can be selected Each formula is repeated 3 times

Experiment 5: Research on the size of material on the efficiency of extracting polysaccharide in Ganoderma subresinosum

Samples in different size: 5, 3, 1, 0.2 cm were treated with ultrasonic waves with sound intensity of 100 Db This experiment follows the single-factor model Other extraction parameters are fixed, on the basis of which the experiment is arranged as Table 3.3

Table 2.3 Experimental design to study the size of material on the extraction efficiency of polysaccharide in Ganoderma subresinosum

(cm) Fixed Factor Monitor factor

• Time of ultrasonic wave treating will be selected from experiment 4

Amount of total polysaccharide obtained

The extract is filtered through the filter paper 3 times By determining the content of total polysaccharide, the most suitable formula can be selected Each formula has 3 times of repeatation

Experiment 6: Research on selecting solvent concentration to extract polysaccharide from Ganoderma subresinosum

Selecting the suitable solvent concentration will improve compound extraction efficiency This experiment follows the single-factor model Other extraction parameters are fixed To investigate extraction efficiency with ethanol solvents, these ranges of ethanol concentration are implemented They are 60, 70, 80, 90, 96%

Table 3.3 Experimental design to select solvents concentration for extracting polysaccharide from Ganoderma subresinosum

• Ratio of material/solvent: 1/15 (g/ml)

• Time of ultrasonic wave treating will be selected from experiment 4

• Size of material will be selected from experiment 5

Amount of total polysaccharide obtained

The extract is filtered through the filter paper 3 times By determining the content of total polysaccharide, the most suitable solvent concentration can be selected Each formula is repeated 3 times

Experiment 7: Research on selecting the ratio of Ganoderma subresinosum material with solvent

The research on selecting the ratio of Ganoderma subresinosum material with solvent to get the highest amount of total polysaccharide is designed following the Table 3.5 This experiment follows the single-factor model

Table 3.4 Research on selecting the ratio of Ganoderma subresinosum material with solvent for extracting total polysaccharide

Formula Material/solvent ratio (w/v) Fixed Factor Monitor factor

• Time of ultrasonic wave treating will be selected from experiment 4

• Size of material will be selected in experiment 5

• Ethanol solvent concentration will be selected from experiment 6

Amount of total polysaccharide obtained

The extract is filtered through the filter paper 3 times By determining the content of total polysaccharide, the most suitable material/solvent ratio (w/v) can be selected Each formula is repeated 3 times

Experiment 8: Research on selecting the time of extraction to achieve total polysaccharides from Ganoderma subresinosum

The time affects the efficiency of the extraction process Through the amount of total polysaccharide obtained, the best extraction time will be determined To conduct survey at the following time intervals: 30, 60, 90, 120 minutes The other conditions of extraction will be fixed This experiment follows the single-factor model

Table 3.5 Experimental study on selecting the time of extraction

• Time of ultrasonic wave treating will be selected from experiment

• Size of material was selected from experiment 5

• Ethanol solvent concentration will be selected from experiment 6

• The ratio of material/solvents will be selected from experiment 7

Amount of total polysaccharide obtained

The extract is filtered through the filter paper 3 times By determining the content of total polysaccharides, the most suitable the time of extraction can be selected Each experiment has 3 times of repeatation

Experiment 9: Studying on selecting temperature for extraction to achieve total polysaccharide from Ganoderma subresinosum

Extraction temperature affects the content of total polysaccharide significantly

To find the optimum extraction temperature, the survey at the following temperature ranges 60, 70, 80, 90 o C are conducted The experiment follows the single-model factor

Table 3.6 The effect of temperature on efficiency of polysaccharide extracted from Ganoderma subresinosum

• Time of ultrasonic wave treating will be selected from experiment

• Size of material will be selected from experiment 5

• Ethanol solvent concentration will be selected from experiment 6

• The ratio of material/solvents will be selected from experiment 7

• Extraction time will be selected from experiment 8

Amount of total polysaccharide obtained

The extract is filtered through the filter paper 3 times By determining the content of total polysaccharide, the most suitable temperature of extraction can be selected Each formula is repeated 3 times

Research content 2: Research on choosing the suitable ratios of

Ganoderma subresinosum extract and additive ingredients to produce instant tea

Experiment 10: Research on choosing the suitable ratios of Ganoderma Subresinosum extract and additive ingredients to achieve the expected instant tea from Ganoderma subresinosum

With the extract achieved from content 2, the research on the mixing ratio between Ganoderma subresinosum extract and auxiliary materials will be studied The Licorice and Stevia were extracted by ethanol 70% at the temperature of 40 –

45 o C The liquid extracts were evaporated until released almost solvent to get solid extracts

These solid extracts were used to mix with Lactose and Ganoderma subresinosum extract to create instant tea from Ganoderma subresinosum

The experimental design for determining the suitable ratio of Ganoderma Subresinosum extract and herbal additive extracts is shown in the Table 3.8

The procedure of making instant tea product from Ganoderma subresinosum and additive ingredients

Source: Food industries research institude

Figure 9: The expected procedure of making instant tea product from

Ganoderma subresinosum and additive ingredients Process description

Ganoderma subresinosum: raw materials have to meet the requirements of quality, fresh, not crushed damage;

Slicing: This process makes raw material become smaller in order to facilitate the soaking process, shorten the injection process

Extraction: To soak and extract the active ingredients in Ganoderma subresinosum as much as possible Ganoderma subresinosum is soaked in solvent, then find out the suitable material size, the solvent concentration, the time, the material/solvent ratio, the suitable temperature when extracting to get the highest amount of total polysaccharide

Concentrated process: To concentrate to remove solvent from the extract to facilitate mixing and shorten drying time The rotavap evaporator system is used to concentrate the extract in a certain ratio

Mixing: This process is to find out the most suitable ratio between extract of Ganoderma subresinosum and additive materials by Ganoderma subresinosum extract and the auxiliary materials A homogeneous mixture with a certain ratio will be created

Drying: The process of drying is to reduce the moisture content of the product, facilitate product quality and storage time

Packaging: Pakage is the final stage of the production process For powder form, tea powder must be packed in a tetra pack packaging

Table 3.7: The mixing ratio of Ganoderma subresinosum extract and herbal additive extracts

After obtaining the instant tea product, based on sensory assessment, the most appropriate ratio will be selected

The sensory assessment of products is conducted followed TCVN 3215 - 79

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

Experiment 11: Research on the drying temperature of mixture to produce instant tea from Ganoderma subresinosum

Table 3.8 Experimental design to investigate the effect of drying temperature on instant tea sensory quality

Based on the sensory quality of instant products, the most suitable temperature of drying process will be determined

3.3.2.1 Methods of determination of moisture content followed the TCVN 9741:2013

• Principle: Use the heat to separate the moisture in the material, while retaining all the substances in the material Therefore the drying temperature should not be too high or too low If the temperature is too high, the moisture will evaporate quickly, but it is easy to change the chemical properties of the substances, especially volatile substances, which will affect the analysis results If the temperature is too low, the steam is released slowly, causing time and substances to change Moisture was determined by drying to constant mass

Determinations of moisture follow TCVN 9741:2013 standard

Principle of the method: The method is based on drying the tea samples to constant mass under defined conditions

To conduct moisture determination must use:

Analytical balance with permissible deviation not exceeding 0.001g The oven can be controlled to adjust the temperature (105˚C) or (120˚C)

The weighing bowl is made of glass, porcelain or aluminum with a diameter of 50mm and a lid

• Procedure: Take samples and crush them, use a analytical balance to weigh 2g of Ganoderma subresinosum, with an error not exceeding 0.001g, put them in a bowl of known weight Place the bowl containing the sample in the oven and raise the temperature to 105ºC Dry for about 6 hours, take the bowl containing the sample and place it in the desiccator, make it cool Then weigh and record the results After the first weighing, re-dry the sample at the above temperature for 1 hour until the mass is constant When necessary, repeat these operations until the difference in results between two successive weighing does not exceed 0.005g

Moisture is determined by drying to constant mass

Moisture (W) is calculated as a percentage of mass by the formula

W: Moisture of food (%) m: Weight of sample before drying (g) m1: Weight of sample after drying (g)

The test result is the mean of two parallel determinations, the difference between them not exceeding 0,2%

3.3.2.2 Determination method of ash content according to the TCVN 8124:2019

•Principle: Fire ingredients or food products at a temperature of 500 - 600 °

C of the burning and volatile organic substances, the remaining inorganic matter is the ash component

Instruments and equipment used to conduct the test should be used:

Analytical balance with error not exceeding ± 0.01g

The crucible is made by porcelain or metal

The furnace can be adjusted to 500 - 600ºC

Desiccator, below to desiccant matter

Electric stove or alcohol lamp

• Procedure: Fire a porcelain crucible or metal bowl washed, in the oven with temperature from 500 to 600ºC to constant weight Cool in a desiccator and weigh on the analytical balance to the nearest 0.005g

RESULTS AND DISCUSSION

Analytical results of the chemical composition of Ganoderma subresinosum

The result of moisture, ash and protein content of Ganoderma subresinosum are all shown in Table 4.1

Table 4.11 Ash, moisture and protein contents of Ganoderma subresinosum

Table 4.1 shows the ash and moisture as well as crude protein components of the initial material Ganoderma subresinosum which had been put into a preliminary drying preservation with initial moisture content of 10 ~ 13% On the basis of dry material, the moisture content accounts for about 4.35% in Ganoderma subresinosum Thus, there will be no difficulty in preserving this kind of fungus

The ash content of Ganoderma subresinosum is quite low, it accounts for 1.47%

As for the content of crude protein, it is 6.87% - a quite high number in protein content of Ganoderma subresinosum Compared to a study of non-volatile components of Ganoderma lucidum, this fungus contains 1.8% ash and 7-8% crude protein (Mau et al., 2001) The results of non-volatile components of

Ganoderma subesinosum appears tobe smaller than those of Ganoderma lucidum.

The results of factors that affect the extraction of total polysaccharide from

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

Ultrasonic waves have the effect of breaking chemical bonds, breaking cells, and assisting the ability to extract substances contained in materials, so ultrasonic treatment will improve the extraction efficiency of compounds, substances presented Ganoderma subresinosum

Table 4.12 Results of the effect of the ultrasonic treatment time on the efficiency of extraction of total polysaccharide from Ganoderma subresinosum The ultrasonic treatment time

(Values in the same row with different exponents have significant differences at the level α = 0.05)

Table 4.2 shows the amount of total polysaccharide extracted with the different ultrasonic treatment time The total polysaccharide content increases steadily when the samples treated with increasing duration of 0, 2, 4 minutes but decreases when treated in 6 minutes by ultrasonic waves Sample that undergos no ultrasonic treatment time has the lowest total polysaccharide content at 4.96 (mg/g) Samples that are treated under ultrasonic waves had much higher total polysaccharide contents On the treatment of ultrasonic waves of 2 minutes, the content of polysaccharide reaches 9.06 (mg/g) The highest amount of total polysaccharide at 12.25 (mg/g) with the treatment duration of 4 minutes is highest value because the longer time of treatment impacting on the material causes the material structure to be broken completetly However, when the treating time increases to 6 minutes, the content of polysaccharide tends to decrease This is due to the prolonged time, large energy, high temperature, the destruction of active polysaccharide and the undesirable compounds will also be extracted Therefore, the process of treatment with ultrasonic waves for 4 minutes is selected for the next experiments In compared with the reseach work “ of Son L et al “the research on the extraction of polysaccharides from fungus Ganoderma lucidum” The overall results appears to be the same, but the total polysaccharide of Ganoderma subresinosum underwent ultrasonic treatment with different parameters of time is much higher than that of Ganoderma lucidum (Luu S et al, 2020)

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

The bioactive chemicals concentrate mostly in the cell wall of Ganoderma subresinosum Therefore, slicing and cutting into small pieces in order to disrupt the outer shell structure and fungal tissue structure As a results, it creates favorable conditions for the active ingredients to escape, the smaller the size of the material, the higher the extraction efficiency Thickness of materials consequently inprove the extraction efficiency of the compound, substances presented Ganoderma subresinosum

Table 4.13 Results of the effect of material size on the efficiency of extraction of total polysaccharide from Ganoderma subresinosum

The size of material (cm) 5 3 1 0.2

(Values in the same row with different exponents have significant differences at the level α = 0.05)

Table 4.3 shows the content of polysaccharide affected by the size of materials The total amount of extracted polysaccharide increases gradually as the size gets smaller At the thickness of 5 cm, the total polysaccharide obtained from

Ganoderma subresinosum is 6.67 (mg/g) Likewise, at the thickness of 3 cm, the total polysaccharides was 8.80 (mg/g) It can be seen that these values are statistical difference at the level α = 0.05 In addition to this, the highest recorded amount of total polysacchride obtained from Ganoderma subresinosum is 11.44 (mg/g) at 0.2 cm thick Followed by the thickness of 1 cm in size, the achieved polysaccharide is 11.30 mg/g However, there is no significant different at α = 0.05 Conclusively, based on Table 4.3, the total content of polysaccharide with 1cm and 0.2 cm in thicknesss has a slightly different in number Therefore, to optimize the extraction process, save time and energy in crushing raw materials, as well as making it easier for the filtration process after extraction, the thickness of 1 cm in size of a slice of materials is chosen for the following experiments A research of extracting polysaccharide from Ganoderma lucidum to produce instant tea by Loi N T shows the effect of material size on the extracting efficiency of polysaccharide with different thickness parameters In which, among the size range, the author also refers that the smaller the size of the material gets, the better the extraction efficiency becomes (Nguyen L., 2017)

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

Ethanol is a solvent that is quite good at dissolving polar compounds Using ethanol at different concentrations results in different extraction effects The experiment to determine the effect of solvent concentration of ethanol on the efficiency of extracting polysaccharides is conducted and the results are obtained in Table 4.4

Table 4.14 The effect of the solvent concentration on the efficiency of extracting total polysaccharide from Ganoderma subresinosum

(Values in the same row with different exponents have significant differences at the level α = 0.05)

Through table 4.4, it is clear that extraction process using different solvent concentrations leads to the dissimilar obtained contents of total polysaccharide The content of polysaccharide increases gradually from 4.52 (mg/g) to 11,44 (mg/g) when the ethanol solvent concentration increases from 60% to 90% At ethanol 96%, the total polysaccharide decreases into 9.30 (mg/g) and there is a statistical difference with the α = 0.05 Similarly, the content of total polysaccharides obtained when increasing the concentration of ethanol from 80% to 90% also increases but there is no difference at the significant level α = 0.05 Therefore, to have the highest economic efficiency, the suitable concentration of ethanol selected for extracting was 80% and is used for the followed experiments This results is similar to a publication from a study of Son L H et al that indicates the selected concentration also 80 % ethanol for extracting Ganoderma lucidum In addition, some recent researchers also report that ethanol acts as an extracting solvent, will bring better extraction efficiency than other kind of solvent namely n-hexane, H2O

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

The amount of solvent whether more or less affects the extraction of substances in the raw materials If the amount of solvent is too small, it is just enough to make the material wet, so the extraction efficiency will be low

Conversely, if the amount of solvent used is too much, it will waste solvent So to find out the ratio of raw materials/solvents is necessary for the extraction and collection of products

Table 4.15 Effects of material/solvent ratio on the efficiency of extracting total polysaccharides in Ganoderma subresinosum Ratio of material/solvent (g/ml) 1/10 1/15 1/20 1/25

(Values in the same row with different exponents have significant differences at the level α = 0.05)

Table 4.5 reveals the content of total polysaccharide increases when the ratio of raw materials/solvents increases With a material/solvent ratio of 1/10, the lowest total polysaccharide achieved is just 8.73 (mg/g) When the amount of solvent used are higher with the ratio from 1/15 to 1/20, the amount of total polysaccharide also increases The highest amount of total polysaccharide reaches 11.66 (mg/g) at the ratio of 1/20 However, at the ratio of 1/25, the total polysaccharide slightly decreases to 11.41 (mg/g) but there is no difference among two fomulas at the significant level α

= 0.05 Therefore, to ensure extraction efficiency as well as to minimize production costs, the ratio of material/solvent of 1/20 is used for the extraction and applied for the next experiments

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

Extraction time affects extraction efficiency, energy and solvent cost If the extraction time is short, the active ingredients released are little, but when the extraction time is increased, energy is lost, the production process will last longer Therefore, it is very important to survey the time of extraction The results of this experiment are recorded in Table 4.6

Table 4.16 Effect of eaxtraction time on the efficiency of extracting total polysaccharide in Ganoderma subresinosum

(Values in the same row with different exponents have significant differences at the level α = 0.05)

The results in Table 4.6 show that the longer the extraction duration, the greater the amount of total polysaccharide obtained However, up to a certain time of extraction, the amount of polysaccharide increases very slowly When extracting for 30 minutes, the obtained polysaccharide content is low at exactly 7.40 (mg/g) The content of this active ingredient increases rapidly when extracting for 60 minutes, with polysaccharide content of 9.07 (mg/g), after 90 minutes of extraction, the highest amount of polysaccharide obtained is 11.57 (mg/g) When the raw materials are extracted for a longer time (120 minutes) the total polysaccharide tends to decrease Moreover, there is no difference in the content of polysaccharide obtained when extracted at 90 minutes and 120 minutes Therefore, to save time and energy for the duration of extraction as well as minimize the product cost, the extraction time of 90 minutes is choosen for releasing total polysaccharides from

Ganoderma Subresinosum As a result, this factor is applied for the next experiments

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

Temperature is one of the factors that greatly affects the extraction process When the extraction temperature is high, the porosity of the material and the viscosity decreases and the active ingredient dissolves more easily into the solvent However, temperature is also a limiting factor because the temperature too high can cause unnecessary reactions such as increasing the solubility of some impurities, difficulty in filtration, promoting chemical changes Therefore, they can cause change the quality of the extract with disadvantages and increase production costs The experiments at 60 o C, 70 o C,

80 o C and 90 o C are conducted and the results are presented in Table 4.7

Table 4.17 Effect of temperature on extraction efficiency of polysaccharide in Ganoderma subresinosum

(Values in the same row with different exponents have significant differences at the level α = 0.05)

According to Table 4.7, when the temperature increases, the content of polysaccharide tends to increase At 60 o C, the polysaccharide content is 8.47 (mg/g), then increases to 10.4 (mg/g) when the temperature reaches 70 o C With higher temperature of extraction process, from 80 o C to 90 o C, the amount of total polysaccharide achieved is higher also, they are 11.69 (mg/g) and 11.51 (mg/g) respectively However, there is no difference in the contents of polysaccharide obtained when extracted at the temperature of 80 o C and 90 o C at the significant level α = 0.05 Therefore, the temperature of 80 o C is selected for the extraction of active ingredients in Ganoderma subresinosum Extracting temperature is considered to be an crucial – affecting factor during the extraction of Ganoderma lucidum by many researchers Especially in a publication of Pham et al studing on the “preliminary prcessing, extraction total polysaccharide and triterpenoids from

Ganoderma lucidum fruiting body” refers the range of temperature from 60 to 90 minutes (Pham H et al, 2021).

Research on choosing the suitable ratios of Ganoderma subresinosum

Lactose acts as a adjuvant to help the active ingredient be distributed effectively and also to contribute to improving the sensory quality of the product Licorice and Stevia have a sweet taste so they can help to enhance the flavour and the taste the product, but Licorice and Stevia also have their own color and flavour, thus affecting the sensory properties of the product Thus, recipe selection must be based on flavour and taste harmony By using the 0-5 scale sensory score method with different mixing ratios, the sensory panel of 8 members evaluate the quality of products to choose the most suitable and favorite mixed formula

Table 4.18 The effects of ratios of Ganoderma Subresinosum extract and additive ingredients on the sensory quality of the product

Formula Flavor Taste State Color Total score Classification

F26 1.83 e 1.88 d 2.50 c 2.40 d 8.61 Low quality F27 2.35 d 2.00 d 1.96 d 2.65 d 8.96 Low quality F28 2.90 c 3.06 c 3.45 b 3.21 c 12.62 Medium F29 3.60 b 3.80 b 4.00 a 3.80 b 15.20 Quite good

(Values in the same column with different exponents differ at a significance level of ≤ 0.05)

From Table 4.8, it can be seen that formulas F28, F29 and F30 have quite good scores based on the rating scale of instant tea products from TCVN 3215-79 For the two formulas, F26 and F27, the average score achieved is very low The taste and flavor of the formulas F26 and F27 are rated the worst with a very low score because of the high presence of Licorice and Stevia In addition, the amount of added lactose is quite low, so the score of the flavor is quite low owing to its blandness F28 has adjusted for Licorice and Stevia ingredients, along with an increase amount of lactose, the score for the taste of tea has reached a pretty average level Beyond the formula group, F29 and F30 are two formulas with good scores because of the harmony of Lactose, Licorice and Stevia with the main ingredient Ganoderma subresinosum

Similarly, in terms of the state of tea, there is a clear difference between the score of each fomula With 2 formulas, F26 and F27, the score is low and the feedbacks are not positive F26 has the lowest points and followed by F27 have low points for the state of the tea because it contains many of the main ingredients,

Ganoderma subresinosum Since the smell and taste of this mushroom is quite typical, it will dis-flavored the pleasant flavors of added Lcorice and Stevia

As for the color of the tea, F26, F27 and F28 also receives not high marks In contrast, the F30 formula is highly appreciated thanks to the harmony of both minor ingredients and main ingredients

Finally, F30 with the highest score of 16.04 with a fairly good rating is selected to complete the process of making instant tea from the extract of

The results of the research on the drying temperature of mixture of Ganoderma

Ganoderma subresinosum extract and additive ingridients to produce instant tea

Drying is a very important step in the production of instant tea, the purpose of drying is to reduce moisture in the product, for easy storage and to facilitate the next process Temperature is one of the factors that can cause changes of the chemical compositions in the products If the temperature is too low, it will take long time, easily spoil and waste materials, which also greatly affects the taste of the product In contrast, if the temperature is too high, it will burn the sugar as well as the other chemical compositions of the dissolved tea compounds

To find the most suitable temperature for drying, the 3 formulas with different drying temperatures of 35 o C, 45 o C and 55 o C are implemented respectively The results of this study are shown in Table 4.9

Table 4.19 The effects of drying temperature on sensory quality of the product

Formula Flavor Taste State Color Total score Classification

(Values in the same column with different exponents differ at a significance level of ≤ 0.05)

Overall, the formula F33 has a higher average score than the formula 32 and the formula F31

The F31 formulation has a rather low score on flavor and taste, as the low drying temperature and insufficient drying time cause the dough to become gelatinous, which does not dry quickly and has a rather sticky texture This directly affects the quality of the finished tea For F32 formula, the score for flavor and taste is much higher because there is a suitable drying temperature in a certain time period So the finished tea product has a better color However, F33 achieves the highest score of the 3 recipes for flavor and taste at 55 o C drying process The color and status of F32 and F33 also have a much higher score than F30, F33 gives a slightly yellow color - the typical color of tea products The F32 has a lighter color and the F31 has a pale yellowish white The color and state of the F31 seems unclear due to the lack of temperature and drying time

In summary, with a drying temperature of 55 o C, formula F33 has an average score better than the other two formulas, F32 and F31 The factor scores of F33 are also generally higher than that of the other formulations especially in terms of color and state of the tea Therefore, based on the scoring table, the formula F33 with a drying temperature of 55 o C is chosen for this experiment.

The complete process for producing instant tea Ganoderma subresinosum

From technological parameters obtained from the above experiments, the process of producing instant tea from Ganoderma subresinosum is described below

Figure 10: Diagram of instant tea production process

Condensing Cutting to 1cm slice

10g Lactose : 68.5g Licorice extract : 0.5g Stevia extract : 1g

Solvent: Alcohol 80 0 Material/solvent ratio: 1/20 Time : 90 min Temperature : 80 o C

Extraction process of Licorice and Stevia

Vacuum condensing by rotovap Time : 3h Temperature : 60 o C Pressure : 175 bar (Ethanol)

Complete instant tea product Preliminary treatment

- Raw materials: Ganoderma subresinosum is not moldy, crushed or spoiled These raw materials are put into a preliminary treatment to eliminate the dirt, impurities

- Drying: Ganoderma subresinosum is dried at 45 o C until reaching the moisture of 10 - 13% in order not to be molddy for the use of preservation

- Cutting: The whole Ganoderma subresinosum cap and fruiting body are cut to slice of 1cm in thickness The extraction process will be faster owing to the material undersize Therefore, the bioactive components quickly dissolve in the solvent

- Soaking and extracting: Materials are soaked in ethanol solvent 80%, with extraction temperature is 80 o C, raw material/solvent ratio is 1/20 (g/ml) and extraction for 90 minutes The obtained extracts are collected and prepared for the condensing step

Soaking the of Stevia and Licorice materials in a temperature of 40 - 45 o C, for 3 hours, in 70 o alcohol to get Stevia and Licorice extract

- Condensing the extracts: The purpose of condensing is to remove solvent to increase the dry matter content of the product When it becomes a thick, viscous form, a liquid extract is obtained and ready for the drying process

- Drying liquid extracts: The liquid extracts will be dried to reach the moisture of 8 - 10%

- Grinding: After drying process, the solid extracts are firm and difficult to mix with addtive components Hence, it is necessary to grind them to powder form

- Mixing: Three extract powders from Ganoderma subresinosum, Stevia and

Licorice and lactose will be mixed following the ratios of 10/68.5/0.5/1 respectively The mixture is mixed well to ensure the harmony of taste and flavor

- Drying: The purpose of this drying step is to reduce the moisture of the final products to 3 - 5% The temperature of 45 0 C will be applied to get safe humidity and for better preservation

- Packing: The mixed and dried extracts will be weighed and packed in a certain unit.

Quality evaluation of instant tea products from extracted polysaccharide of

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

DPPH free radical scavenging activity is one of the most effective, and simple antioxidant activity experiment The samples that has the ability to capture DPPH free radicals proves that they contain several compounds capable of donating hydrogen or transfering electrons to free radicals directly The results of the DPPH free radicals scavenging experiments were performed with instant tea powder samples containing Ganoderma subresinosum extract and a positive control Ascorbic acid with a concentration range of 31-500 (àg/ml) and 10-50 (àg/ml) respectively, shown in Table 4.10

Table 4.20 The antioxidant activity of instant tea powder from Ganoderma subresinosum and Acid ascorbic

Ganoderma subresinosum instant tea powder

Table 4.10 indicates the ability of scarvenging radicals of both samples and their IC50 values In which, the ability of free radicals scarvening of instant tea powder from Ganoderma subresinosum has been proved to be existed by the chaing of color and the values of scarvenging abilty In addition to this, at the concentration range of 31-500 àg/ml, the scarvenging radical ability in percentage of instant tea powder from Ganoderma subresinosum extract fluctuates in the range of 31.91 ± 0.29 to 73.14 ± 0.14 Wheares with the positive control sample – Acid ascorbic, in a range of 10-50 àg/ml, its scarvenging radicals activity in percentage vary from 39.69 ± 0.18 to 84.47 ± 0.22 Besides, the results shows that instant tea powder from

Ganoderma subresinosum surely have the antioxidant activity but not as strong as that of Acid ascorbic Since the IC50 of sample of instant tea powder from

Ganoderma subresinosum is 200.27 ± 1.51 àg/ml much higher than 19.36 ± 1.03 àg/ml of Acid Ascorbic A science project research from Institution of Biotechnology and Food technology, Industrial University of Ho Chi Minh City also study on the antioxidant activity of Ganoderma lucidum Accordingly, the value IC50 of Ganoderma lucidum is 306.939 àg/ml, much higher than IC50 of Ganoderma subresinosum with the same range of concentration (Duong, 2016) As for the positive control sample Ascorbic acid, there are also numerous of studies using it as control sample confronted with the herbal extract samples to evaluate its ability of free radicals scarvenging Specially in a report from a group of author, it can be seen that Acid ascobic has the similar number of IC50 at estimately 20.98 ± 0.3 àg/ml at the concentration range of 0 to 60 àg/ml (Nguyen Y et al, 2019)

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

Table 4.21 The sensory assessment of instant tea product from Ganoderma subresinosum

The sensory chracterisitics of the research product

1 Color Light yellow, pure Light yellow, a bit pure

The harmony of main ingredients and additional ingredients

Ganoderma subresinosum with additional ingredients namely Stevia and Licorice

4 Taste Soft sweet, non-odd taste Soft sweet from lactose and Stevia

Table 4.11 shows that all of sensory characteristic of researched instant tea product from Ganoderma subresinosum appears to meet the requirements about state, flavor, taste and color based on TCVN 3215-79

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

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

No Parameters Units Requirement Results

1 Bacillus cereus CFU/g - Not detected

5 Staphylococus aureus CFU/g - Not detected

6 Total of mold and yeast count CFU/g - Not detected

7 Total aerobic microbial count CFU/g - Not detected

Table 4.12 indicates the amount of microoganism count presented in the researched instant tea from Ganoderma subresinosum In addition to this, all of the listed microoganisms on the Table 4.12 are not detected in CPU or MNP per gram of sample Conclusively, this instant tea product from Ganoderma subresinosum has met the requirements

4.6.4 Evaluation of standards on heavy metal present in instant tea from Ganoderma subresinosum based on QCVN 8-2:2011/BYT

Table 4.13 The results of heavy metal count present in instant tea product from Ganoderma subresinosum

No Heavy metal Requirement Unit Results

1 Pb ML: 2.0 Ppm Not- detected

2 Hg ML: 0.05 Ppm Not-detected

3 Cd ML: 1.0 Ppm Not-detected

Table 4.13 shows that there is no indicator of heavy metal present in the researched instant tea product from Ganoderma subresinosum

4.6.5 Preliminary cost calculation for 5g piece of instant tea product from Ganoderma subresinosum

Table 4.14 The preliminary cost calculation for 5g piece of instant tea product from Ganoderma subresinosum

No Expense items Unit Cost (VND)

The prize of one piece 7 000

The prize of one pack (20 pieces) 140 000

Table 4.14 shows the preliminary cost for 1 piece (5g) of instant tea product from Ganoderma subresinosum For one pack of tea, the prize will be 140.000 VND, containing 20 pieces Compared with the present instant tea products on the market, the researched Ganoderma subresinosum instant tea is a bit higher in prize The high prize is due to the high expense of manufacturing cost Inspite of its high cost, this researched instant tea product from Ganoderma subresinosum has tons of valuable bioactive components which results in improving the comsumers’ health In addition, it is affordable for the comsumers to purchase this kind of beverage owing to its good influences Therefore, the prize 140 000 VND for a pack (20 pieces) is considered resonable.

CONCLUSIONS AND RECOMMENDATION

Conclusion

Ganoderma subresinosum has the chemical component content as follows:

+ Crude protein content accounted for 6.87%

From the results of the research, there are some conclusions as follows:

- During the extraction process of Ganoderma subresinosum, the most suitable factors to have the highest amount of total polysaccharides content are: + Solvent is alcohol with the concentration of 80%;

+ Time of extraction process is 90 minutes;

+ Temperature of extraction process is 80 o C;

+ Ratio of material/solvent is 1/20 (w/v)

- The mixing ratio of ingredients to create instant tea from Ganoderma subresinosum is determined with 10g Ganoderma subresinosum, 68.5g Lactose,

0.5g Licorice solid extract and 1g Stevia solid extract

- The drying temperature of instant tea was determined with the temperature of 55 o C.

Recommendation

- There should be some in-depth researches on process of producing instant tea from Ganoderma subresinosum to improve the typical smell of the fungus

- It is very necessary to evaluate the bioactiveness of total polysaccharide obtained from the extraction process

- It is crucial to investigate the time of preservation of instant tea product during the storage.

APPENDIX

Ballot for product sensory assessment score

1 Test for quality score (TCVN 3215-79)

2 Full name of the tester:……… … Age……… Sex…………

4 Experiment Sensory evaluation by scoring method for instant tea from

Ganoderma subresinosum – Nam Lim Den

Evaluate the scores for the indicators by a 5-point scale as in the table (below table)

How to score: score on a 5-point scale

Tea powder product is brown color, instant tea water is light brown, characteristic of the product

Tea powder product is light brown, soluble tea water is light brown, not specific to the product

The product has a light brown powder that, when dissolved in water, gives the product a bright yellow or non- concentrated reddish brown color

The product has a light brown powder that, when dissolved in water, produces a very light yellow color or a dark reddish- brown color that is not concentrated for the product

The soluble tea powder is not yellow- brown, or white, and the water is colorless or almost white

A natural, pleasant aroma characteristic of the smell of

No strange smell or smell, strong and durable

It has a pleasant natural aroma, characteristic of the smell of

Ganoderma subresinosum root and stem

No strange flavour or burning smell, but not strong or durable

The scent is very mild, pleasant but not specific to the smell

Ganoderma subresinosum tubers and stems There is no strange flavour or smell, the fragrance is not strong and not durable

There is almost no characteristic aroma for the scent of

There is no strange flavour or smell, the fragrance is not strong and not durable

There is no smell, there is a strong, rancid smell

The very sweet taste of

5 4 3 2 1 characteristic of the taste of

The aftertaste has a cool sweet taste, not bitter cool sweetness of

, the taste is very bad taste of

, or just the sweet taste of

Glycyrrhixa glabra L and Stevia rebaudiana Bertoni

Tea is a fine powder, homogeneous, porous, lint- free, no clumping, no residue or layering when dissolved in water

Tea is a fine powder, less homogeneous, lint-free, lump-free, water-soluble without sediment or layered

The powder is less fine, heterogeneous, lint-free, slightly lumpy, when dissolved in water, there is an uneven dispersion, but at a very low level

Large, heterogeneous clump, when dissolved in water, there is uneven dispersion

Large lump, or marshmallow, sticky, when dissolved in water layered and uneven

(TCVN 3218-1993, Ministry of Science - Technology and Environment,

Data processing

7.3.1 Results about the effect of the ultrasonic treatment time on the efficiency of extraction total polysaccharide from Ganoderma Subresinosum

Sum of Squares df Mean Square F Sig

Duncan time N Subset for alpha = 0.05

Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3,000

7.3.2 The results of study on selecting material size to extract polysaccharides from Ganoderma subresinosum

Sum of Squares df Mean Square F Sig

Duncan formula N Subset for alpha = 0.05

Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3,000

7.3.3 The results of study on selecting solvent concentration to extract polysaccharides from Ganoderma subresinosum

Sum of Squares df Mean Square F Sig

Duncan concentration N Subset for alpha = 0.05

Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3,000

7.3.4 The results of selecting the ratio of raw materials Ganoderma Subresinosum

Sum of Squares df Mean Square F Sig

Duncan ratio N Subset for alpha = 0.05

Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3,000

7.3.5 Research results of selecting the time to extract total polysaccharides from Ganoderma subresinosum

Sum of Squares df Mean Square F Sig

Duncan a time N Subset for alpha = 0.05

Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3,000

7.3.6 Research results of selecting temperature for extraction of total polysaccharides from Ganoderma subresinosum

Sum of Squares df Mean Square F Sig

Duncan a temperature N Subset for alpha = 0.05

Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3,000

7.3.7 Effect that the mixing ratio affects product quality color

Duncan a formula N Subset for alpha = 0.05

Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3,000 state

Duncan a formula N Subset for alpha = 0.05

Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3,000 taste

Duncan a formula N Subset for alpha = 0.05

Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3,000 flavor

Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3,000

7.3.8 Sensory temperature affects product quality color

Duncan a formula N Subset for alpha = 0.05

Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3,000 flavor

Duncan a formula N Subset for alpha = 0.05

Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3,000 taste

Duncan a formula N Subset for alpha = 0.05

Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3,000 state

Duncan a formula N Subset for alpha = 0.05

Means for groups in homogeneous subsets are displayed a Uses Harmonic Mean Sample Size = 3,000

7.4 The results of radical scarvenging ability of instant tea powder from Ganoderma subreinosum and Acid ascorbic

Con Mean Std Deviation Minimum Maximum

IC50 Mean Std Deviation Minimum Maximum

Line charts

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