ganoderma lucidum

Chief Technical Officer：Jingyi Guan

Marketing Director：Xin Wang

Planting Happiness

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TABLE OF CONTENTS

BACKGROUND INTRODUCTIO N

TEAM INFORMATION 4PRODUCT SUMMARY 4GANODERMA LUCIDUM 4TRITERPENES 5MEVALONATE PYROPHOSPHATE DECARBOXYLASE (MVD) 6GROBACTERIUM TUMEFACIENS-MEDIATED TRANSFORMATION 8GPD PROMOTER 9

SECTION A: RESEARCH , DEVELOPMENT AND PRODUCTION 6

CLONING FLOW CHART 11HYPHAE COLLECTION 11OBTAINING TARGET GENE MVD 11CONSTRUCTION OF OVER-EXPRESSION VECTOR GL-GPE 12OBTAINING AGROBACTERIUM STRAINS CONTAINING TI PLASMID GL-MVD 16AGROBACTERIUM-MEDIATED TRANSFORMATION & SELECTION AND SCREENING 19TRITERPENES OVER-EXPRESSION STRAINS 20PASSAGE STABILITY 21OPTIMAL TIME LENGTH OF PLANTING 21PLANTING 22SUPERCRITICAL CO2 FLUID EXTRACTION 23ELECTUARY PRODUCT 23

SECTION B: EFFICACY AND SAFETY TESTING

EFFICACY TESTING 26SAFETY TESTING 30MAMMALIAN TOXICITY TESTING 31ENVIRONMENTAL IMPACT 32

SECTION C: MARKETING

TARGET MARKET 34MARKETING PLAN 34YERHERB® IN THE MEDIA 35COST ANALYSIS AND PRICING 36

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CONSUMER PERCEPTION OF THE PRODUCT 37REFERENCES 39

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Background Introduction

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Team Information

Mr. Ye, founder of Yerherb Inc., was born the rural areas of Guangdong, China.

At the age of 18, he started his career working at various traditional herb shops in

different large cities. Intelligent, hard working, honest, Mr. Ye was promoted to main

buyer position over the years. Due to the wars in Mainland China, Mr. Ye and his wife

fled to Hong Kong, subsequently raising a family of eight children there. By the time

Mr. Ye decided to immigrate to the United States, he had become a shareholder of the

herb chain he worked at.

When Mr. Ye moved to Los Angeles in 1979, he immediately saw the vast

business opportunity due to the lack of availability of traditional Chinese herb in

United State. Yerherb officially opened its first storefront as a small family operation.

Its reputation grew rapidly in the US, the herb products sells many places in US.

Jingyi Guan and Xin Wang was entered in this company in 2005, they were in a

project group developing the Ganederma lucidum herbal tea product that will be talk

in details below.

Product Summary

The product produced is a Ganoderma lucidum extract herbal tea, with multi-

functions on people health. It has higher concentration of active ingredient and the

cost of the product is lower than the Ganoderma lucidum products present in the

market now.

Ganoderma lucidum

Ganoderma lucidum or Lingzhi mushroom, which has a shape of semi-circle or

kidney-like, is a sort of medical mushroom and has been used as popular remedy in

traditional Chinese medicine for more than 2,000 years. In most of Chinese ancient

medical works, we can see the similar description about G.lucidum-------a super

panacea. It is effective for treatment or prevention of neurasthenia, high blood

pressure, arrhythmia and enhancement of resistance of various diseases. What is

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more, from recent study, G.lucidum still has efficacy of curing cancer, diabetes and

even inhibiting senility. Nowadays the mushroom has been listed in the American

Herbal Pharmacopoeia and Therapeutic Compendium.

Triterpenes

Based on the previous study about the active ingredients in G.lucidum, the

efficacy mentioned to a great extent relates to chemical “triterpenes”. Triterpenes is a

series of secondary metabolites which belong to lanostane when the mushroom

forming fruiting body. At least 170 different triterpenes have been exacted from

G.lucidum so far. Here is a image of the structures of three typical triterpenes (or

ganoderic acid).

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Triterpenes can inhibit secretion of histamine from mast cells which can cause

allergy and inflammation. What is more, ganoderol A, ganoderol B, ganoderic acid Y

which belongs to triterpenes can robustly inhibit cholesterol synthesis by interrupting

the synthetic pathway from acetate to cholesterol. Fe2+—ascorbic acid which can

cause peroxide of lipid and 1,2,3-Benzenetriol which cause oxidation of erythrocyte

membrane can also be inhibited by triterpenes. In recent study, triterpenes has been

known that has effect in suppressing the growth and proliferation of cancer cells and

inducing apoptosis in a variety of leukemia, lymphoma, and myeloma cells by

increasing the amount of T lymphocyte and enhancing the activity of NK cells, IL-2,

tumor necrosis factors and macrophages.

However, the content of triterpenes is very low in wild-type G.lucidum, which

results in high cost in pharmaceutical industry.

Mevalonate Pyrophosphate Decarboxylase (MVD)

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Triterpenes is synthesized through the MVA (mevalonate) pathway. (Figure) And

during the pathway, a lot of enzymes,including HMGR,FPS, SQS and MVD, show

essential functions. Among these enzymes, MVD (mevalonate pyrophosphate

decarboxylase) is a key enzyme in catalyzing mevalonate-5PP into isopentenyl-PP

within the process of synthesis of Triterpenes.

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MVA pathway1. Acetyl-CoA acetyltransferase, AACT; 2. 3-hydroxy-3-methylglutary-CoA synthase, HMGS; 3. 3-hydroxy-3-methylglutary-CoA reductase, HMGR; 4. mevalonate kinase, MK; 5.phosphomevalonate kinase, MPK; 6. pyrophosphomevalonate decarboxylase, MVD; 7. isopenteny-diphophate isomerase, IDI; 8. famesyl diphosphate synthase, FPPs; 9. squalene synthase, SQS; 10. 2, 3-oxidosqualene-lanosterol cyclase, OSC;11. geranlgeranyl-PP synthase, FPS

Gene mvd which encoding protein MVD（Genbank: HQ5964）, and has a not

very long length of 1203bp. The complete sequence which is crucial for obtaining or

amplifying the gene has been known nowadays.(figure)

Agrobacterium-Mediated Transformation System for Fungi

Tumor inducing plasmid (Ti) is a kind of vector that originally used in

Agrobacterium-mediated transformation to plants. Target gene could be inserted into a

region called ”T-DNA” on the Ti plasmid which would be integrated into genome of

the Agrobacterium-infected plant cells by recombination. Now of Agrobacterium-

mediated transformation system for fungi has been well-established and shows a very

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Sequence of gene mvd from Genbank

good transforming efficiency by modifying the T-DNA sequence to make it include

homologous region with fungi. Additionally, specific selection markers and promoters

to fungi are utilized in such a system.

Gpd Promoter

Strong promoters are the sequences that have high affinity with RNA polymesare

and can promote synthesis of mRNA highly efficiently. gpd (glyceraldehyde 3-

phosphate dehydrogenase,GAPDH) promoter is a strong promoter in large

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Agrobacterium-mediated transformationA: Agrobacterium tumefaciens B: Agrobacterium genome C: Ti Plasmid : a: T-DNA , b: Vir genes , c: Replication origin , d: Opines catabolism genes D: Plant cell E: Mitochondria F: Chloroplast G: Nucleus

filamentous fungi which can be used for constructing the over-expression vectors. The

sequence of the promoter has been known by cloning the 5’ flanking region of the gpd

gene.

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Section A: Research, Development

and Production

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Research and Development

Cloning flow chart：

Hyphae Collection

Before all the experiments we would conduct, tissues of the fungi in specific

stage which would produce mRNA we wanted should be obtained. The wild type

strain G20 will be cultured in the PDA liquid medium, which with 150rmp shaking

bed incubation for 7 days under 28 . Then the hyohae could be collected by gauze. ℃

Obtaining Target Gene mvd

To obtain the target gene mvd, isolation of total RNA for synthesis of cDNA

should be accomplished first. Our team used liquid nitrogen to grind hyohae. After

homogenization, chloroform and isopropanol were used for isolation of RNA. The

isolated RNA was purified by Dnase and phenol. The quality of the total RNA was

proved good based on the result of gel electrophoresis.

The total RNA we acquired were then utilized in synthesis of cDNA by reverse-

transcription. For the reason that fungi is eukaryote which has poly(A) tail at 3’end of

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mRNA, Oligo(dT) and reverse-transtripatase (M-MLVRTase) were used respectively

as primer and DNA polymerase during the reaction.

The cDNA products we obtained is a mixture of cDNA from different mRNA. To

get specific cloning product of mvd gene, we designed a pair of primer(named MVD-

O1 and MVD-O2) based on the cDNA sequence from GenBank (No.HQ596495.1).

Furthermore, we introduce two restriction sites respectively into these two primers

(sequences with underlines):

MVD-O1: 5’- ACTGggatccATGAGCGTATACCAAG-3’ (Bam HI)

MVD-O2: 5’-ACTGtctagaTCACTTCGGAAGGCC-3’ (Xba I)

Then we conducted PCR for specific amplification of mvd gene using the MVD-

O1 and MVD-O2 primers and total cDNA and successfully obtained target gene mvd.

Construction of Over-Expression Vector GL-GPE

Before construction of the over expression vector GL-GPE, strong promoter gpd

is needed to be purified from total DNA and specifically amplification. Based on the

sequence of gpd we got from other lab, we designed the specific primers GL-GPD-F1

and GL-GPD-R1 and introduce BstX1 site and Aat II site respectively into the

flanking region of the primers.The regions with underlines are BstX1 site and Aat II

site.

GL-GPD-F1:5’-GATCgacgtcTCCAAAGCCGCTCTCATGG-3’ (Aat II)

GL-GPD-R1:5’-GATCccaacatggtggAGGGGGATGAAGAGTGAG-3’ (BstX I)

After we obtained the specific amplified products, we created recombinant

plasmid PGL-GPD with plasmid pCAMBIA 1300.

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Diagram of pCAMBIA 1300 plasmid

pCAMBIA 1300 plasmid is a Agrobacterium binary vector for plant or fungi

transformation. This plasmid is a shuttle vector which can be replicated and expressed

in both E.coli and Agrobacterium. It has two selective markers, one is hgyR

(hygromycin resistance) which is used for plant or fungi cells selection and the other

marker is KanR (kanamycin resistance) which is used for bacteria cells selection. We

digested the pCAMBIA 1300 plasmids and gdp promoter fragments with BstX1 and

Aat II. Then we used T4 ligase to ligate the pCAMBIA 1300 plasmid vector and the

gpd fragments. And we got ligation product pGL-GPD.

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Construction of binary vector pGL-GPD

Aat II

Competent E.coli Cells were created by heat shock with Ca2+ and mixed with

ligation product we got from last step. Then we incubated the solution and plated the

solution onto kanamycin LB medium for selection of transformant colonies. After

incubation, we took individual colonies on the plates, purified plasmid from cells

using kit and did RFLP (restriction fragment length polymorphism) for screening.

Within the screening, we extracted the plasimds from transformant cells. After

digestion by BstX I and Aat II, we did agrose gel electrophoresis with EtBr and

identified the recombinant colonies which can show a band of inserted fragment. Then

we cultured cells from those colonies and isolated vectors pGL-GPD. Here is the gel

electrophoresis result.

The inserted gpd promoter replaced the CAmv35S promoter which was used for

expression of higher plant genes. But the plasmid still needed another gpd promoter

with different direction for following inserted target gene expression. For that, we

designed two primers for specifically amplification gpd promoter fragments with two

different restriction sites (which were located in MCS):

GL-GPD-F2: 5’-GATCggtaccTCCAAAGCCGCTCTCATGG-3’ (KpnI)

GL-GPD-R2: 5’-GATCgaattcAGGGGGATGAAGAGTGAG-3’ (EcoR I)

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The digestion of plasmid pGL-GPD by BstX I and Aat II1,2: pgl-GPD; 3,4: The digestion by BstX I and Aat II; M: DL2000 DNA Marker

Same method of digestion and ligation of inserted fragments and plasmids were

used here as above with enzymes KpnI and EcoR I.

After transformation and selection for transfromants, we did PCR screening and

agrose gel electrophoresis using GL-GPD-F2 and GL-GPD-R2 primers for obtaining

the recombinant starins. Then we cultured the strains and isolated over-expression

vector named GL-GPE.

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Amplification pattern obtained with primers for fragment gpd from transformant

1-3: randomly chosen transfromants; P: GL-GPE as postive control; N: untransfromed E coli as negative control; M: DL 2000 DNA Marker

Obtaining Agrobacterium Strains Containing Ti Plasmid GL-MVD

In construction of mvd over-expression plsamid GL-MVD, GL-GPE vector and

mvd cDNA fragment was digested with Bam HI and Xba I and ligated with each other

by T4 ligase.

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Construction of over-expression vector GL-GPE

After using heat-shock with CaCl2 to make competent agrobacterium, GL-GPE

solution were mixed with the cells. Because the Plasmid has a kanamycin resistance

selection marker, we used LB medium with kanamycin to select transformants.

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Bam HI and Xba I digest

Full-length mvd gene

Construction of over-expression vector GL-MVD

Then we took transformant colonies and isolated plasmid which was used for

RFLP (restriction fragment length polymorphism) screening. During the process of

RFLP, we digested the plasmids with BamH I and Xba I and conducted the agrose gel

electrophoresis. The recombinant colonies were identified when the band of inserted

mvd gene (1203bp) shown on the gel.

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The digestion of plasmid GL-MVD byBamH I and Xba I1, 2: double restriction enzyme digestion of plasmid GL-MVD; M: DL 2000 DNA Marker

Selection for transformants on kanamycin medium

Agrobacterium Tumefaciens-Mediated Transformation

Before transformation of the GL-MVD plasmid into G20 Ganoderma lucidum

strain, protoplasts of the fungi were prepared. The grinded hyohae was treated with

lywallzyme to remove cell wall. Then the solution of Agrobacterium was mixed with

the protoplasts and incubated for infection. The mixture then was plated on

hygromycin PDA medium to select transformants.

And then these tranformants were subjected to chromosome DNA extraction for

PCR screening with gpd promoter primers GL-GPD-R2 and mvd primer MVD-O2 to

test if the fragment has been inserted into chromosome genome and if the gpd

promoter has fused with gene mvd. After obtaining amplification products, we did

agrose gel electrophoresis to identify recombinants which shown the band of inserted

mvd gene. And we named those recombinant strains “GMOEi”(i=1, 2. 3.......15)

(Figure)

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Selection of hygromycin-resitant transformants of G.Lucidum1,5: G20 as the negative control; 2, 3, 4, 6, 7, 8: The selected hygromycin-resistant transformants

Triterpenes Over-Expression Strains

After culturing such recombinant strain for 20 days, we tested and compared the

triterpenes contents of the over-expression strains with wile-type. In this experiment,

we used ultrasonic method to bread the cells and extract triterpenes by chloroform.

Then we used the vanillin perchloric acid colorimetric method to measure oleanic acid

content for establishing standard curve in standard solution and triterpenes content in

the strains by testing OD values under A245nm. At last, we found that triterpenes

content of one of the over-expression strains GMOE10 which was 3.5mg/100mg dry

weight was 101% more than the content of wild-type (G20).

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Amplification pattern obtained with primers for inserted gene mvd in genomic DNA isolated from GMOEs.

Lane 1, negative control with no template DNA; Lane 2, negative control with untransformed G.Lucidum; Lane 3, GL-MVD as positive control; Lane 4 to 18, GMOE1 to GMOE15; Lane M: DL 2000 DNA Marker

Detection of the triterpenes of GMOEs

Passage Stability

To ensure the passage stability of the insertion, we did subculture of the

GMOE10 strain respectively for 3/5/10 generations and test the triterpenes content.

The results of subculture show that triterpenes over-expression phenotype of the

GMOE10 was stable from generation to generation.

Number of generations Content of triterpenes(mg/mg dry weight)

3 3.6

5 3.4

10 3.5

Optimal Time Length of Planting

Though we obtained the over-expression strain, it was still not enough for

optimize triterpenes producing for the reason that the secretion of triterpenes are

different at different stages of life cycle of Ganoderma lucidum. So we designed an

experiment to find out the optimal time length of planting. Here we adopted the well-

developed planting method as in agricultural industry to culture the GMOE10 strain.

sawdust, bran, sucrose, gypsum, water will be mixed following a ratio equal to

73:25:1:1:65 and filled into LDPE bag as compost. The compost were then sterile and

adjusted to pH 8.0. After planting the 20-day GMOE10 mycelium in different bags,

we marked those bags with “15 days”, “35 days”, “55 days”, “75 days”, “90 days”

which meant different time length of growing for different bags. These mycelium

contained bags were incubated under 27 and 90% air humidity. (image)After℃

finishing incubation, we extracted triterpenes using ultrasonic and chloroform and

measured the contents of triterpenes of different samples. The result indicated that

optimal time length of planting was 75 days.

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Content of triterpenes at different generation

Contents of triterpenes of incubation of different time length

Number of days for planting Content of triterpenes(mg/mg dry weight)

15 2.8

35 4.1

55 6.5

75 9.7

90 8.1

Scale-up ProductionPlanting

To obtain enough Ganoderma lucidum as raw material, we built up green houses

for large-scale planting. Same method including 75-days culturing as what we

mentioned above was used for planting. Additionally, beside temperature and air

humidity, light was another factor needed attention. Too strong or too weak light

would inhibit the growth of the fruiting body of the fungi.

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Supercritical CO2 Fluid Extraction

To further enhance the efficiency and safety and lower the cost and

environmental contamination of triterpenes extration in Industrial production, we

decided to adopt the supercritical CO2 fluid extraction（ SFE） method. Such a

method is based on the principle that when the supercritical CO2 fluid gets in touch

with and dislsoves the mixture, compositions which have different polarities, boiling

points and molecular weights could be separated under different pressures and

temperatures. Then when the fluid returns to CO2 gas, the extract could be

precipitated. Because the method works under a temperature range from 35 to℃

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Planting G.lucidum

40 , the volatile compositions could effectively maintain after extraction. We℃

purchased a 40L SFE equipment which combined with ultrasonic breaking from

Taiwan Supercritical Technology CO., LTD, and such mixed extraction method

provided a dramatically enhancement in efficiency of extraction from 3.5% to 41%.

Electuary Product

To produce electuary for G.Lucidum tea, the extract was evenly mixed with

dextrin with a ratio of 1:20 to make extractum. After squeezing trough the screen

mesh (mesh number=20) to make powders, 70 heat was used to dry the powders℃

and finished products were packaged. And we also produced canned drinks from dry

powdered.

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40L SFE equipment

40L SFE equipment40L SFE equipment

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Image of our products

Section B: Efficacy and Safety

Testing

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Efficacy Testing

Ganoderma lucidum extract on mice test approved by FDA

The effects of Ganoderma lucidum extracts on the immune function and

growth of mice under the experimental incubation were studied. The results

showed that the weight of mice gained in the testing groups were higher than the

control groups after they fed on the extracts, and their abdomen phagocytic index

and phagocytosis rate of macrophages increased significantly after they fed on the

extracts for 30 days and did injections for 7 days. The rates between the mice 's

liver and spleen weights to their body weights increased compared with the

negative control. The results suggested that Ganoderma lucidum extracts

enhanced the mice 's immune function.

The material and protocol for the test approved by FDA:

Preparation of the chicken blood with red blood cells

Extract 1ml chicken blood from the live healthy chicken wing. Transfer the

chicken blood into 5ml Alsvers solution. Made a 5% of the cells suspension with

sterilized saline.

Preparation of Ganoderma lucidum extract water used in our product

Method used was same as our product extraction as previously discussed.

Testing on different groups of mice

The animals used in this experiment are pure white mice, weight 16g-18g.

These mice were divided into 6 groups at random, each group of 10, with free

feeding standard feed. Group 1 ~ 4 group drank Ganoderma lucidum extract

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water, the concentration respectively 3. 2 mg/mL, 11. 2 mg/mL, 32. 0 mg/mL and

140 mg/mL, in feeding after 30 days, testing groups of mice did celiac injection

with the different concentrations of Ganoderma lucidum extract of 1 mL/day for

7 days. Group 5 was negative control group, which drank distilled water. 30 days

after, they were injected of saline by 1 mL/day for 7 days. The group 6 as the

positive control group, which had the same breeding method as the negative

control group. Before two days of the death of the mice, the abdominal cavity

injection was performed on them with cyclophosphamide (final concentration of

0. 75 mg/m, L), 1 mL/day, continuous (2 days). In 24 hours after the last

injection, they were executed by cervical dislocation method. Recorded each feed

quantity and water quantity, and weighted each mouse in different groups,

calculated the mean of each group.

Mice abdominal cavity macrophage specimen preparation

Mice treated by intraperitoneal injection of 5% chicken blood red blood cells,

each mouse injected by 0.5ml. The cervical dislocation method executed after 2

hours of injection. Instantly by intraperitoneal injection of 0. 5 mL saline, gently

rubbed the abdominal cavity for 1 min, cut open abdominal skin, and made a

small cut in muscle layer, transferred peritoneal fluid on the clean slide, incubated

for 30 min at 37℃. Using 10% Giemsa staining before observe under the

microscope. Direct observed under microscope, there were 2000 macrophage in

each mouse. The phagocytosis rate and phagocytic index were being calculated:

Phagocytosis Percent = (phagocytose chicken blood red blood cell number of

macrophages present 2000) x 100%

Phagocytic index = total number of chicken blood red blood cells being

phagocytized ÷ number of macrophages present phagocytose chicken blood red

blood cells

Determine the ratio of the organ weight to weight of the mice

After executed of the mice immediately, opened the abdomen and chest,

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quickly removed the liver, lung, spleen, thymus, they were weighing respectively.

Calculated organ to body weight ratio and the average value of 10 mice in each

group.

Result and Discussion:

Effect on mice abdominal cavity macrophage phagocytosis

Microscopy results show in table1, four different concentrations of Ganoderma

lucidum in experimental mice abdominal cavity macrophage phagocytosis rates

compared with negative control group have obvious improvement, increased in 20.

16% ~ 69. 79%, and got a significant or extremely significant difference. The

concentration with 3. 20 mg/mL Ganoderma lucidum group phagocytosis rate

increased the most, the rest of the groups phagocytosis rate increased slightly. Positive

control group was lower than negative group by 42. 99%. This shows that Ganoderma

lucidum extract water had enhanced the experimental mice abdominal cavity

macrophage for chicken blood red blood cells phagocytosis rate, improved the cellular

immune function of mice.

Table1: Effect on mice abdominal cavity macrophage phagocytosis

Test group Concentration

(mg/ml)

Phagocytosis

rate(%)

Phagocytosis

index

Negative group 0 25. 53± 0. 67 1. 50± 0. 21

Positive group 0 14. 55± 1. 84 1. 28± 0.13

Ganoderma lucidum groups 3.2 43. 34± 2. 11 1. 34± 0. 20




From the data shows in table 2, by feeding with Ganoderma lucidum extract water,

the mice growing average weight is greater than the control group, different

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concentration of Ganoderma lucidum group had different weight gain. The

concentration with 3.2 mg/ml increased the most. The highest concentration of

treatment group, weight gain was not the most. The reason might be that with the

increase of Ganoderma lucidum component content in extract solution create the

bitter taste of the drinking water, make high concentration group water consumption

lower than the control group and low concentration group, which affect weight gain.

Table 2 shows the effect on the body weight of the mice


(mg/ml)

Average of Body

weight in the

group (10 mice)

Percentage of

increase (%)

Negative group 0 0. 1479 ---

Ganoderma lucidum groups 3.2 0. 2382 61. 05




As shows in table 3, mice in different concentrations of Ganoderma lucidum groups

by drinking Ganoderma lucidum extract water after 30 days and 7 days of injection,

the liver, spleen and thymus to body weight ratios were significantly increased than

the control group, and had no obvious adverse effect on the lungs. Spleen and thymus

are immune organs, the liver has detoxification function. From this result, the long-

term use of a certain amount of Ganoderma lucidum extract solution can improve the

body's detoxification ability. In this experiment, the development of immune organs in

mice had promoted effectively, it had the effect that increased in the body's immune

function.

Table3: Effect on the ratios of organs to the body weights.


(mg/ml)

Liver/body

weight

Spleen to

body/

weight

Lungs/body

weight

Thymus/

body weight

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Negative group 0 4. 59± 0.

49

0. 60± 0. 02 0. 67± 0. 06 0. 24± 0. 02

Positive group 0 5. 54± 0.

10

0. 38± 0. 01 0. 60± 0. 07 0. 16± 0. 05

Ganoderma lucidum groups 3.2 5. 14± 0.

55

0. 72± 0. 02 0. 69± 0. 04 0. 19± 0. 04


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0. 89± 0. 08 0. 73± 0. 21 0. 23± 0. 12


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0. 80± 0. 12 0. 66± 0. 05 0. 33± 0. 12


80

0. 87± 0. 10 0. 70± 0. 01 0. 19± 0. 13

Safety Testing

There are two types of safety tests were performed. They are ELISA and PCR tests.

The FDA required manufactures to to label their products with regards to eight

specific allergens: milk, eggs, fish, shellfish, peanuts, wheat, soybeans and tree nuts

since 2005. These allergens are responsible for over 90% of the documented food

allergen-related cases. The most common and preferred methods approved by FDA

for allergens testing in food industry are ELISA and PCR.

The testing methods have been developed that can now detect allergens in finished

products even at very low concentration as per million (ppm) range. By performing

this test, our product can be detected if it contains any popular allergen at the

molecular level. The allergens then can be avoiding during manufacturing or being

labelled on the product box for alert.

ELISA methods detect the actual allergen protein molecule by binding antibodies to

the allergen and then using an enzyme-linked conjugate to create a colorimetric

change that can be measured.

The PCR methods, which are more sensitive and detect the DNA molecules of

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these allergens, can be used in raw and finished products and are not affected by the

heating process, because DNA typically remains intact after being exposed to the high

temperatures most foods. As food allergens are becoming an increasingly important

issue in food safety. These two tests are crucial before the food products hit the

market. Form the results of ELISA and PCR test. There was not any popular allergen

ingredient being detected in our product.

Allergen tests

Popular allergens ELISA PCR

milk None None

eggs None None

fish None None

shellfish None None

peanuts None None

soybeans None None

tree nuts None None

Ganoderma lucidum has been used in China for long time, there are several

beneficial effects of Ganoderma lucidum have been claimed. The majority of these

claims have not been studied in controlled clinical trials, but there has been an

abundance of clinical use, as well as in vitro and animal testing data support its safety.

The safety of using it is further supported by common use of Ganoderma lucidum as

an edible mushroom and broad exposure to consumers with no adverse effects

reported.

Mammalian Toxicity Testing

In 2012, a double-blind, placebo-controlled, parallel group interventional trial was

performed by using our product. There were 50 generally healthy volunteers (age 18-

65 years, BMI 19-35) were administered beverages containing placebo (control) and

our herbal tea includes Ganoderma lucidum active ingredient (1.5g/day) for 12 weeks.

After drinking for 12 weeks, there was no adverse effect on both groups. The group

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consumed the active ingredient reported they got better sleep, and felt the herbal tea

reduce their feeling of stress. The BMIs for test group remained and for some obese

people their BMI reduced. Some people reported their skin looks healthier. The results

of this study indicate that Ganoderma lucidum at a dose of 15 g/person/day was

functioned in adults in the general population without any adverse effect.

Functional trial on 50 volunteers

Adverse

effects

Sleep better Feel less

stressful

Better looks

skin

Getting

infected by

disease

during 12

weeks

Blood

cholesterol

levels

reduced

Control

group

None 3 people

reported

2 people

reported

4 people

reported

8 people

reported

2 people

reported

Testing

group

None 42 people

reported

38 people

reported

39 people

reported

2 people

reported

35 people

reported

Environmental Impact

Our product is pure Ganoderma lucidum extract powder with dextrin. Both

ingredients were approved by FDA to use in the food industry. From the experiment

test on animals and allergen tests by ELISA and PCR, there was no undesirable side

effect for healthy adults. And as our planting environment is in the greenhouse under

very restrict controls, there is not any negative effect to the environment. The species

is also planted in America continent for a long time in some area. So there is not any

issue with biological invasion.

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Section C: Marketing

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Target Market

Our product is facing healthy or sub-healthy state adults and seniors. The product is

designed for people living in stressful environment. These people may associate with

sleeping disorder, anxiety and immune function reduce. The product is also aim to

people with chronic alcohol drinking habit. These people may have higher blood

pressure and unhealthy liver, which can be reduced by keep drinking our product. Our

product also designed to all kinds of people want to keep a good look skin, as it has

anti-aging function by keeping consuming it. It is also applicable to vegetarians.

Marketing Plan

Step1: set up the goal as achieve sales 50,000 boxes of product per month within six

months.

Step2: evaluation of the internal situation of the business marketing the product

Competition: our competitors could be supplements products, medicines, and

other functional herbal drinks.

Our advantages: Our product is multi-functional herbal tea, which not includes

any harmful product. It is very easy to take and it taste better. During the

production it has the lower cost compared to the product exist in the market in

nowadays. Our product has much higher concentration of the active

ingredient, and our pricing is better for the consumers.

Step3: Description of the ideal customer for the product including age, household

income, geographic location, work situation.

Our ideal customer are healthy and sub-healthy adults or senior people with higher

incomes. The product is aim in large cities in US.

Step4: Create marketing strategies.

There will be several types of promotions discussed as following.

Step5: Create the marketing budget.

The cost will be talked in details later.

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Yerherb® in the Media

There will be several promotions of this product to be well known and reach

our target sales goal. At the beginning of the sales, the TV advertisement and

Radio advertisement of the brand and the product will be distributed for familiar

and well known. The mainly information of the advertisement is about or brand

name and the multi-functions of this new kind of herbal drink.

As our pricing is higher than regular herbal teas. Our herbal tea product with

several functions will aim to people have higher consumption ability. For this

reason, there will be brochures distributed in the airport and on the flights to

reach more high income costumers. There will also be online video

advertisements, as people using computers may have issues with sub-healthy are

our major target costumers.

The posters will be shown in poplar pharmacies and markets. And there will be

some free samples provided to the consumers interested in the product. There will

be some sales promoters in the market to introduce the background and function

of our product to consumers in details.

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Brochures

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Poster

Cost Analysis and Pricing

The equipment will produce 500 tons Ganoderma lucidum extract from 2,200

tons of raw materials in each year. There will be 40 greenhouses, 7 acres in total

for the planting of the Ganoderma lucidum raw material. For each greenhouse, it

will produce about 600 Ganoderma lucidum plants. The total price of the planting

is about $500,000 per year. The science and development cost will be $150,000

per year (Chemical reagent, Enzymes and vector, PCR & post-PCR analysis, G20

strain, lab equipment). The dextrin used costs about $200,000 per year. The cost

of the processing equipment will be $100,000 for once. The maintenance and

labor cost for each year is about $100,000. The logistics of our product is about

$500,000 per year. The promotion and advertising costs are $1,000,000 dollars

per year. Our product is 28 tea bags per box (approximately 30g/ bag). The price

of our product in the marketplace will be labelled as 16 dollars per box. We will

also have canned drinks，which includes 30g of active ingredient plus water per

can in 350ml. This is better for the people who do not like hot drinks. The canned

drinks are very convenient to carry and drink at any time for the consumers. The

price for the can drink will be 4 dollars per can.

Image of tea bag

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image of canned drink

Consumer Perception of the Product

The existing herbal teas in the market are approximately 6 to 7 dollars per box.

They could be our major competitors. As most of the herbal tea in the market now

only has one or few functions or just with some herbal or fruit flavors, they

cannot be considered as functional herbal tea. Our product includes very precious

ingredient. Which has the same value as ginseng, cubilose. By doing market

research, most of ginseng tea in market in US are imported. It is difficult to get

them in America’s popular supermarkets. Our product is planted in the US and

compared to other precious herbal product it has higher active ingredient. It is the

best choice for the people to consume with long time to maintain their health and

enjoy the herbal flavor drinks.

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Reference：1.Shi,L., Qin,L., Xu,Y., Ren,A., Fang,X., Mu,D., Tan,Q. and Zhao,M. (2012). Molecular cloning, characterization, and function analysis of a mevalonate pyrophosphate decarboxylase gene from Ganoderma lucidum. Mol. Biol. Rep. 39 (5), 6149-6159 2.Shi,L. (2012). The developmet of agrobacterium tumefaciens-mediated transformation and its application in the study of triterpenes biosynthesis of the meidical fungus Ganoderma lucidum [D]. Nanjing Agricultural University.3.Cheng, C., Yue, Q., Wu, X., Wu, Z., Song, X., Tao, S.. . Guo, D. (2010). Cytotoxic triterpenoids from Ganoderma lucidum.Phytochemistry, 71(13), 1579-1585. 4.Sliva, D. (2006). Ganoderma lucidum in cancer research.Leukemia Research, 30(7), 767-768. 5.LI Xiufen, QIAN G Yufeng , YI Huilan, LI Xianlei ( School of Lif e Science and Technology , Shanx i University , Taiyuan 030006,China

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ganoderma lucidum

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