(Đồ án HCMUTE) enzymatic hydrolysis of geniposide from gardenia jaminoides to produce genipin as a pigment precursor and crosslinking agent

MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING Ho Chi Minh City, August, 2022 SKL 0 0 9 1 5 2 SUPERVISOR:

MINISTRY OF EDUCATION AND TRAINING

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION

FACULTY FOR HIGH QUALITY TRAINING

Ho Chi Minh City, August, 2022

SKL 0 0 9 1 5 2

SUPERVISOR: VO THI NHA NGUYEN VINH TIEN STUDENT: DANG HOANG DUC

HO DAC LOC

GRADUATION THESIS FOOD TECHNOLOGY

ENZYMATIC HYDROLYSIS OF GENIPOSIDE FROM GARDENIA JAMINOIDE

TO PRODUCE GENIPIN AS A PIGMENT PRECURSOR

AND CROSSLINKING AGENT

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION

FACULTY FOR HIGH QUALITY TRAINING

GRADUATION PROJECT

Thesis code 2022-18116009

ENZYMATIC HYDROLYSIS OF GENIPOSIDE

FROM GARDENIA JAMINOIDE TO PRODUCE

GENIPIN AS A PIGMENT PRECURSOR AND

NGUYEN VINH TIEN, ASSOC PROF.

Ho Chi Minh City, August 2022

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION

FACULTY FOR HIGH QUALITY TRAINING

GRADUATION PROJECT

Thesis code 2022-18116009

ENZYMATIC HYDROLYSIS OF GENIPOSIDE

FROM GARDENIA JAMINOIDES TO PRODUCE

GENIPIN AS A PIGMENT PRECURSOR AND

NGUYEN VINH TIEN, ASSOC PROF.

Ho Chi Minh City, August 2022

DECLARATION

Except where there is clear acknowledgment and reference to the work of others, we thus declarethat all content and materials included in and presented in this thesis are our original creations.Additionally, we guarantee that the materials acknowledged in the thesis have been citedappropriately and properly in line with requirements

, August 2022Signature

ACKNOWLEDGEMENT

"No research without action, no action without research" is a well-known quote attributed toKurt Lewin (2012) Indeed, in order to finish and attain the current outcome, we have addressedand conquered all the difficulties and obstacles inherent in and throughout the project Inaddition, our beloved professors, classmates, and families have made a substantial contributionthat stimulates and supports us

Therefore, we would like to sincerely thank all the lecturers in charge of the Department of FoodTechnology, Faculty of Chemical and Food Technology, and Ho Chi Minh City University ofTechnology and Education for providing us with valuable knowledge and the best equipment andfacilities to complete our thesis

We would also want to express our gratitude to our cherished supervisors, PhD Vo Thi Nga andAssoc Prof Nguyen Vinh Tien, who have passionately guided and shared their teachingexpertise and experience in order for us to complete this thesis

Sincerely, we would like to thank Ms Ho Thi Thu Trang of the Department of Food Technologyfor allowing and assisting us in using the available measuring instruments and equipment at theFaculty of Chemical and Food Technology laboratory

Nonetheless, we would want to express our gratitude to our classmates for supporting andorganizing the thesis's complex experiment

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TABLE OF CONTENTS

CHAPTER 1: OVERVIEW 1

1.1 Gardenia jasminoides 1

1.1.1 Overview of G.jasminoides 1

1.1.2 Chemical constituents 1

1.1.2.1 Volatile components in G jasminoides 1

1.1.2.2 Iridoids and iridoid glycoside 1

1.1.2.3 Crocins and their derivatives 2

1.1.2.4 Phenolic compounds 4

1.1.2.5 Terpenoids 4

1.1.3 Biological activities 4

1.1.3.1 Antioxidant activity 4

1.1.3.2 Antidiabetes 5

1.1.3.3 Antidepressant activity 5

1.1.3.4 Effects of blood circulation 6

1.2 Geniposide 6

1.3 Genipin 7

1.4 Gardenia Blue 10

1.5 Reasearch about gardenia blue pigment production 11

1.6 Cross-linking of genipin in chitosan film 12

1.7 Reasearch about forming crosslinking with genipin 13

CHAPTER 2: MATERIAL AND METHOD 15

2.1 Materials 15

2.2 Research process diagram 15

2.3 Method 16

2.3.1 Pigments from genipin processing 16

2.3.1.1 Extracting geniposide from seed of G Jasminoides in ethanol 16

2.3.1.2 Treating geniposide with cellulase to obtain a hydrolysate 16

2.3.1.3 Extracting genipin from the hydrolysate by ethyl acetate 17

2.3.1.4 Reacting the product comprising genipin with amine 17

2.3.2 Chitosan-genipin film processing 17

2.3.3 Procedure to investigate the optimal pH of the enzymatic hydrolysis of geniposide 18

2.3.4 Procedure to investigate the optimal duration of the enzymatic hydrolysis of geniposide 19

2.3.5 Procedure to investigate the optimal enzyme concentration of the enzymatic hydrolysis of geniposide 19

2.3.6 Procedure to investigate the optimal pH of the reaction between genipin and pigment precursors 20

2.3.7 Procedure to investigate the optimal duration of the reaction between genipin and pigment precursors 21

2.3.8 Procedure to investigate the different amine of the reaction between genipin and amine 21

2.3.9 Procedure to investigate the optimal pH of the reaction between genipin and protein extracted from Phaseolus lunatus 22

2.3.10 Procedure to investigate the differences of chitosan-genipin films in different genipin concentration 23

2.3.10.1 Color measurement 24

2.3.10.2 Moisture content 25

2.3.10.3 FTIR – Fourier Transform Infrared Spectroscopy 25

2.3.10.4 Thickness 25

2.3.10.5 Tensile strength and elongation 25

2.3.11 Statistical analysis 25

CHAPTER 3 : RESULTS AND DISCUSSION 26

3.1 Factors affecting the hydrolysis reaction of geniposide 26

3.1.1 pH 26

3.1.2 Time of enzymatic reaction 30

3.1.3 Enzyme concentration 33

3.2 Factors affecting the reaction to produce blue pigments from genipin 36

3.2.1 pH 36

3.2.2 Time of pigment forming reaction 39

3.2.3 Types of amine-containing compounds 40

3.2.4 Effect of pH on protein of Lima bean when reacting with genipin 43

3.2.4.1 Uv-vis of pigment solution when genipin act with protein from Phaseolus lunatus in different pH 44

3.2.4.2 FTIR of residue of proteinwhen acting with protein from Phaseolus lunatus in different pH 45

3.3 Properties of chitosan-genipin films 46

3.3.1 Uv-vis of films when change genipin content 46

3.3.2 FTIR of films when change genipin content 48

3.3.3 Moisture content of genipin – chitosan films (%) 49

3.3.4 Mechanical properties of genipin – chitosan films (Thickness, TS and EL) 50 3.3.5 Swelling content of genipin – chitosan films 51

CHAPTER 4: CONCLUSION 52

REFERENCES 53

LIST OF FIGURES

Fig 1 1 Structure of crocin 3

Fig 1 2 The structural formula of geniposide 6

Fig 1 3.Hydrolysis of geniposide to genipin [29] 7

Fig 1 4 Chemical structure of genipin 8

Fig 1 5 Garnedia blue reaction between genipin and primary amines 9

Fig 1 6 Crosslinking reaction between chitosan and genipin 13

Fig 2 1 Pigments from genipin producing diagram 15

Fig 2 2 Chitosan-genipin film producing diagram 16

Fig 3 1 Gardenia blue solution in different pH of enzymatic reaction 26

Fig 3 2 (a) UV spectra of genipin solution after enzymatic reaction in different pH of enzymatic reaction (b) Absorbance of 310 nm of genipin solution after enzymatic reaction in different pH of enzymatic reaction 27

Fig 3 3 (a) UV-vis spectra of genipin solution after ethyl acetate extracted in different pH of enzymatic reaction (b) Absorbance of 310 nm of genipin solution after ethyl acetate extracted in different pH of enzymatic reaction 28

Fig 3 4 (a) UV-vis spectra of gardenia blue pigment in different pH of enzymatic reaction (b) Absorbance of 590 nm of gardenia blue pigment in different pH of enzymatic reaction 28

Fig 3 5 Gardenia blue solution in different time of enzymatic reaction 30

Fig 3 6 (a) UV-vis spectra of genipin solution after enzymatic reaction in different time of enzymatic reaction (b) Absorbance of 310 mn of genipin solution after enzymatic reaction in different time of enzymatic reaction 31

Fig 3 7 (a) UV-Vis spectra of genipin solution after ethyl acetate extraction in different time of enzymatic reaction (b) Absorbance of 310 nm of genipin solution after ethyl acetate extraction in different time of enzymatic reaction 31

reaction in different enzyme concentration 34

Fig 3 12 (a) UV-Vis spectra of genipin solution after ethyl acetate extracted in different enzyme concentration (b) Absorbance of 310 nm of genipin solution after ethyl

acetate extracted in different enzyme concentration 35

Fig 3 13 (a) UV-vis spectra of gardenia blue solution in different enzyme concentration.

(b) Absorbance of 310 nm of gardenia blue solution in different enzyme concentration.35

Fig 3 14 Gardenia blue solution in different pH of the formation of garnedia bluereaction 37

Fig 3 15 (a) UV-vis spectra of gardenia blue solution in different pH of the formation

of garnedia blue reaction (b) Absorbance of 590 nm of gardenia blue solution in

different pH of the formation of garnedia blue reaction 37

Fig 3 16 (a) UV-vis spectra of gardenia blue solution in different time of the formation

of garnedia blue reaction (b) Absorbance of 590 nm of gardenia blue solution in

different time of the formation of garnedia blue reaction .39Fig 3 17 Color difference of solution after reacting with different amines 41

Fig 3 18 UV-vis spectra of gardenia blue solution in types of amine-containing

Phaseolus lunatus in different pH 44

Fig 3 21 FTIR of protein residue after being treated with genipin 45 Fig 3 22 UV-vis spectra of genipin-chitosan films 47

Fig 3 23 FTIR of genipin-chitosan films 48

LIST OF TABLES

Table 1 1 Composition of Crocin colorant in gardenia fruit 3

Table 2 1 Preparation of different amine solutions 22Table 2 2 Buffer solution preparation 23

Table 2 3 Chitosan film 24 Table 2 4 Chitosan film 24

Table 3 1 Color measurement of gardenia blue solution in different pH of enzymatic

Table 3 7 Mechanical properties of genipin-chitosan films 50

Table 3 8 Swelling content of genipin – chitosan films 51

ABSTRACT

Gardenia blue pigment is typically made from the raw material geniposide found in Gardenia

Jasminoides Ellis of Rubiaceae by processing geniposide with α-glucosidase to create genipin,

which then combines with an amino acid to form the color However, the resulting gardenia bluepigment is dark, has a low color value, and is of poor quality Therefore, it is unsuitable for someuses, including drinks

A second method for producing the gardenia blue pigment with a high color value involves filtering the gardenia blue pigment obtained from the reaction of genipin with an amino acid toremove the residual geniposide and then extracting the filtrate to obtain the high-color-valuegardenia blue pigment Another method involves putting the raw material geniposide through a

ultra-non-polar resin with a wide mesh to remove α-crocin prior to treating it with β-glucosidase.

However, due to the high cost and complexity of these procedures, they cannot be carried out on

a wide scale in the industry This study, focuses on the pH sensitive, enzyme concentration andtime of the enzymatic reaction between geniposide and cellulase, pH sensitive, type of pigmentprecursors, time of the pigment forming reaction And pH sensitive of reaction between genipin

and protein from Lima bean The result for producing pigment from Gardenia jasminoides are

pH 4.5, 0.2 g cellulase per 1 g geniposide, 6 hours are the optimal conditions for the enzymaticreaction between geniposide and cellulase The evaluated method for this research are UV-visand color measurement Moreover, pH 8, MSG, 10 hours are the optimal conditions for thepigment forming reaction The evaluated method for this research are UV-vis and colormeasurement Additionally, pH 10 is the optimal condition for the reaction between genipin andprotein from Lima bean Regarding the genipin-chitosan film, when changing the genipin/NH2

concentration, the crosslinking between chitosan and genipin also changed In addition, theconcentration of 0.0075 genipin/NH2 had the highest tensile strength Moreover, addingconcentration also causes moisture and swelling to decrease gradually

Keywords: Gardenia jasminoides, genipin, geniposide, gardenia blue, crossliking,

genipin-chitosan films

CHAPTER 1: OVERVIEW 1.1. Gardenia jasminoides

1.1.1 Overview of G.jasminoides

G jaminoides, an evergreen tree of the Rubiaceae family, is planted in many parts of China

under the Chinese name Zhi Zi It thrives in a variety of temperate climates and has fragrantwhite blooms [1] When the plant's oval-shaped fruits ripen in late fall, they become a reddish-golden hue [2]

G jaminoides possess a variety of biological functions, including diabetic,

anti-inflammatory, antidepressant, and antioxidant qualities, as well as the ability to improve sleep

quality [3] G.jasminoides herb has the ability to access the meridians of the heart, lungs, and

triple burner It has the ability to extinguish an evil fire, ease internal heat, and cool blood in thebody It is mainly used to treat dysphoria, agrypnia, jaundice, gonorrhea, thirst, conjunctivalcongestion, angina, hematemesis, non-traumatic bleeding, hematodiarrhoea, hemuresis,pathopyretic ulcer, sprain, and swelling pain [4] According to recent studies, the oil extract of

Gardenia jasminoides has antidepressant properties [5].

It has long been used as a natural yellow dye The exploitations of G jaminoides plants had been

involved in food additives, dyestuffs, cultivation of the ornamental plant, antiseptics, and newmedicines When the plant's oval-shaped fruits ripen in late fall, they become a reddish-goldenhue and are used in traditional Chinese herbal medicine to treat a variety of maladies [2]

Gardenia jasminoides fruit extract, which can be yellow, red, or blue, is commonly utilized as a

natural colorant in the food business [6]

In recent years, G jasminoides has been primarily focused on extraction methods Extracts obtained have demonstrated biological activity in vitro and in vivo.

1.1.2 Chemical constituents

1.1.2.1 Volatile components in G jasminoides

Aliphatic acids, ketones, aldehydes, esters, alcohols, and aromatic derivatives are the most

abundant volatile components in G jasminoides essential oil [5][7] Because of the differences in processing temperature and duration, the essential oil from G jasminoides includes varying

amounts and quantities of volatile components Furthermore, during high-temperature processing,unstable components such as iridoids may be partly transformed to volatile components [5]

Because of the pharmacological activity of G jasminoides oil and the availability of current extraction techniques, numerous efforts were put into the extraction of G jasminoides in order to

identify the best extraction method [3] The primary approach employed to detect volatile

components in G jasminoides was gas chromatography-mass spectrometry (GC/MS) [5] The

percentage yield of the essential oil of Gardenia jasminoides flowers was 0.02 % v/w (freshweight) [8]

1.1.2.2 Iridoids and iridoid glycoside

G jasminoides is high in iridoids and iridoid glycosides The content of iridoid glycosides may

vary from different regions at about 5-6% [9] There are 35 iridoids isolated from G jasminoides includes geniposide, 6β-hydroxy geniposide, geniposidic acid, gardenoside, 6α-hydroxy geniposide, 6-O-methylscandoside methyl ester, 6-O-methyldeacetylasperulosidic acid methyl

ester, 8-O-methylmonotropein methyl ester, Shanzhiside, Gardoside,

10-O-trans-sinapoylgeniposide, 6’’-O-trans-sinapoylgenipin gentiobioside, 6"-O-trans-p-coumaroylgenipin gentiobioside, 6’-O-sinapoylgeniposide, 6"-O-caffeoylgenipin gentiobioside, genipin 1-O-β-D-

apiofuranosyl (1/6)-β-D-glucopyranoside, genipin 1-O-α-D-apiofuranosyl glucopyranoside, 6β-hydroxy genipin, genipin, gardenoside, deacetylasperulosidic acid methyl ester, scandoside methyl ester,4’’-O-[(E)-p-coumaroyl] gentiobiosylgenipin, 6’-O-[(E)-sinapoyl] gardoside, Bartsioside, Gardenal-I, Gardenal-II, Gardenal-III, ixoroside, (+)-(7S,8R,8’R)- lyoniresinol 9-O-β-D-(6’’-O-trans-sinapoyl) glucopyranoside, 10-O-trans-sinpoylgeniposide,

(1/6)-α-D-Shanzhiside methyl ester (I), phloyoside (II), chlorotuberside (III), penstemonoside (IV) [3] Bysome common isolation method such as solvent partition separation, classic columnchromatography, preparative high-performance liquid chromatography (prep-HPLC), high-speedcountercurrent chromatography (HSCCC), and other isolation methods, at least 15 iridoids,including iridoids, iridiod glucosides, secoiridoids, and secoiridoid glucosides, have beenisolated and identified [3] Many studies have indicated that geniposide has various beneficialhealth effects, including anti-inflammatory, antidepressive, anti-diabetic, and antithromboticqualities, as well as protection against lipopolysaccharide (LPS)-induced apoptotic liver damage[3] A research determined the concentrations of geniposide, gardenoside, geniposidic acid, andchlorogenic acid in 68 samples from different locations of China and Korea to be 56.37 + 26.24µg/mg, 49.57 + 18.78 µg/mg, 3.15 + 3.27 µg/mg, and 0.69 + 0.39 µg/mg, respectively [10]

Gardenia jaminoides' optimum solvent extraction conditions were 51.3% ethanol/water

combined with an extraction temperature of 70.4oC for 28.6 minutes The yields of geniposideand total phenolic compounds were 10.9% and 2.497%, respectively, under these conditions [11].The other extraction method is ultrasound- and microwave-assisted extraction The followingwere discovered to be the optimal conditions for obtaining the maximum yields of geniposide

from G jaminoides utilizing ultrasound-assisted extraction: water with a solid/liquid ratio of

1:30 at 70°C for 30 minutes, giving 4.1% geniposide [12]

1.1.2.3 Crocins and their derivatives

Crocin is a naturally occurring carotenoid chemical compound found in the flowers of turmericand gardenia This is a diester formed from gentiobiose disaccharide and crocetin dicarboxylicacid

Molecular formula: C44H64O24

Molecular weight: 976.96 g/mol

Fig 1 1 Structure of crocinThe composition of the yellow pigments from the gardenia fruit is presented in the table

Table 1 1 Composition of Crocin colorant in gardenia fruit

1 Crocetin-digentiobiosyl ester (trans): Crocin 68.3

2 Crocetin-monogentiobiosyl-monoglucosyl ester 4.5

3 Crocetin-monogentiobiosyl ester (trans) 2.5

4 Crocetin-monogentiobiosyl ester (cis) 5.3

5 Crocetin-diglucosyl ester (trans) 15.3

6 Crocetin-monoglucosyl ester (trans) 2.5

7 Crocetin-monoglucosyl ester (cis) 0.9

Crocin and its derivatives derived from Gardenia jaminoides have been shown to be less toxic,

less allergenic, and more environmentally friendly than saffron [13] Crocin and its derivatives

derived from Gardenia jaminoides are also used to treat disorders such as weight loss, sexual

dysfunction, and premenstrual syndrome [3]

G jaminoides were extracted utilizing a homogenate extraction method in a 50/50 ethanol/water

solution, with a liquid/material ratio of 15:1 (v/w) and a particle size of 1.7 mm and an extraction

period of 41 seconds [14] The extraction yield of the edible yellow pigment from Gardenia

jaminoides was 50% greater when using the microwave-assisted extraction system than when

using the standard extraction method [15]

1.1.2.4 Phenolic compounds

Some phenolic acids have been found in Gardenia jaminoides such as

caffeoyl-4-O-(3-hydroxy-3-methyl)gluta-roylquinic acid, 4-O-sinapoyl-5-O-cafffeoyl-quinic acid, caffeoylquinic acid [16] and chlorogenic acid [2] One new lignin glucoside, (+)-(7S,8R,8’R)-lyoniresinol 9-O-β-D-(6″-O-trans-sinapoyl) glucopyranoside, has been found in G jasminoides.

3,5-di-O-1.1.2.5 Terpenoids

Secoiridoids and monoterpenoids are among the terpenoids found in Gardenia jaminoides

including 6’-O-trans-Sinapoyljasminoside C, 6’-O-trans-Sinapoyljasminoside A,rehmapicrogenin, jasminoside C, jasminoside B, jasminoside G, jasminoside K, jasminoside I,jasminoside H, epi-jasminoside H, 6’-O-trans-sinapoyljasminoside L, jasminoside S,Jasminoside J, 6’-O-trans-Sinapoyljasminoside B, 6’-O-trans-Sinapoyljasminoside L,jasminoside M, jasminoside N, jasminoside C, 6-O-b-D-xylopyranosyl-b-D-glucopyranosyl(2E)-3,7-dimethylocta-2,6-dienoate, 6-O-b-D-glucopyranosyl-b-D-glucopyranosyl (2E)-3,7-dimethylocta-2,6-dienoate, jasminoside E, sacranoside B, jasminodiol, jasminoside H,jasminoside I, 6’-O-sinapoyljasminoside A, 6’-O-sinapoyljasminoside C [3]

Terpenoids, particularly those with a small number of carbon atoms, can be found in the volatileoil Terpenoids are extracted and isolated in similar methods that iridoids are [3]

1.1.3 Biological activities

1.1.3.1 Antioxidant activity

1.1.3.2 Antidiabetes

Type 2 diabetes is caused by insulin resistance G Jasminoides water extracts increase insulin sensitivity in steroid-induced insulin-resistant rats, with an optimum dosage of 200 mg/kg of G.

jasminoides water extract [19] Genipin improved age-related insulin resistance, which was

linked to improvements in hepatic oxidative stress, mitochondrial dysfunction, and insulin signalimpairment [20] Geniposide improved impaired glucose tolerance and hyperinsulinemia inindividuals with hereditary type 2 diabetes caused by visceral fat accumulation [21] In diabeticmice, geniposide (200 mg/kg and 400 mg/kg) was demonstrated to be an effective hypoglycemicdrug, considerably lowering blood glucose, insulin, and triglyceride levels in a dose-dependentway [22] Geniposide also reduced diabetic vascular damage by reducing monocytic adherence

to human umbilical vein endothelial cells and the production of cell adhesion moleculesproduced by high glucose [23]

1.1.3.3 Antidepressant activity

Supercritical fluid extraction of G jasminoides oil and geniposide shown antidepressant efficacy.

Genipin acts as an antidepressant by modulating glycolysis/gluconeogenesis, the TCA cycle, andlipid metabolism in the liver [24] The antidepressant mechanism of geniposide may be

connected to an increase in serotonin levels in the striatum and hippocampus of mice, as well asmonoamine oxidase B [25][26]

1.1.3.4 Effects of blood circulation

G jasminoides hot water extracts did not increase the proliferation of cultured vascular smooth

muscle cells, but did preferentially stimulate endothelial cell proliferation, which may help toavoid arteriosclerosis and thrombosis [27]

The structure of geniposide was discovered in the 1960s Geniposide is one of the major iridoid

glycosides in the fruit of G jasminoides.

Molecular formula: C17H24O10

Molecular weight: 388.36 g/mol

Fig 1 2 The structural formula of geniposideGeniposide, which is colorless, can be hydrolyzed with beta-glucosidase to give genipin, whichreacts with amino acids (glycine, lysine, phenylalanine) to provide a blue pigment, stable underheat, light, and pH, can be used as a food coloring The oral bacteria Actinomyces naeslundii andActinomyces viscosus, which contribute to the initiation and induction of tooth decay in humans,contain beta-glucosidase that should induce a reaction with compounds in the fruit of thegardenia fruit for blue color in saliva, this reaction is being studied for applications to create areagent for bacteria that can cause tooth decay

Since it is a natural product, and biomolecular structure with low toxicity, geniposide hasrecently been investigated as a binding material in various applications One of the more recentdiscoveries is the use of gelatin-bound geniposides as a bioadhesive, in wound dressings, andbone grafts This has shown geniposide to have potential as a new and safe cross-linking agent

The most common binding agents found was glutaraldehyde However, glutaraldehyde hadsimilar toxicity concerns in the same tests as geniposide In the field of forensic science,geniposide is being considered as a new method to develop the ability to trace fingerprints onpaper-based materials Since geniposide is a natural, environmentally friendly product, it is apotential raw material for drug manufacturing

Geniposide displays a wide spectrum of in vitro and in vivo pharmacological effects, including

neuroprotective, antidiabetic, hepatoprotective, anti-inflammatory, analgesic, antidepressant-like,cardioprotective, antioxidant, immune-regulatory, antithrombotic, and antitumoral effects, andthese pharmacological effects lay the foundation for geniposide of being a potential therapeuticagent for the treatment of several diseases, such as Alzheimer's disease (AD), Parkinson’sdisease (PD), diabetes and diabetic complications, ischemia and reperfusion injury, and hepaticdisorders [28]

.Fig 1 3.Hydrolysis of geniposide to genipin [29]

Cellulase is less costly than β-glucosidase and catalyzes the removal of the sugar moiety fromgeniposide to a desirable degree of more than 90 percent Additionally, it may degrade the plantcell wall [29]

1.3 Genipin

Genipin is an aglycone derived from geniposide, an iridoid glycoside present in G.jasminoides.

Molecular formula: C11H14O5

Molecular weight: 226.23 g/mol

Fig 1 4 Chemical structure of genipinGenipin is an aglycone derivative that comes from the hydrolysis of geniposide It is taken from

the fruits of the plant G jasminoides Genipin has been traditionally used in habitual Chinese

medicine to mitigate the symptoms of type 2 diabetes, headache, inflammation, and hepaticdisorders, among other conditions Genipin is colorless on its own, but when it reacts with aminoacids and protein, it may spontaneously produce blue particles Because of these properties,genipin has been successfully used as a natural dye in the production of textiles and food [30].Genipin is a fantastic crosslinking agent that may be acquired naturally and is used forcrosslinking collagen, gelatin, proteins, and chitosan It is known to use two different methods tocross-link materials containing primary amine groups [31] The mechanism postulated by Zhuand Park is based on the ring-opening reaction of genipin An amino group may trigger thisreaction through a nucleophilic assault on the olefinic carbon atom of genipin After this stage, atwo-step reaction is carried out to complete the grafting of the genipin onto the polymer through

a covalent bond using the amino group A tautomeric aldehyde is produced when an unstableintermediate created in the process eventually collapses The newly planted aldehyde group isthen subjected to an additional assault from an amine group originating from a different polymer,which results in the development of another covalent bond resulting in the formation of thecross-link [32]

Compared to any other crosslinking agent, including glutaraldehyde, the cytotoxicity of genipin

as a crosslinking agent is between 5000 and 10,000 times lower[33] In contrast to otherchemical crosslinking agents, the circumstances most conducive to establishing its crosslinkingcapabilities include temperatures between 25 and 45 degrees Celsius and a pH range from 7.4 to8.5 [34]

Traditional Chinese medicine has long utilized genipin as a treatment for inflammatory,jaundice-related, and hepatic disorders [35] Its use as a herbal remedy for liver problems is

supported by its reported protective effects against hepatic ischemia/reperfusion injury in rats.Additional pharmacological studies revealed genipin's prevention of lipid peroxidation and theformation of nitric oxide in rat paw edema, in addition to its potential anti-inflammatory,antithrombotic, and antiangiogenesis activities [36] [37] In addition, some research examinedthe protein cross-linking abilities of genipin, employing it as an alternative to cross-linkers withhigher toxic (such as glutaraldehyde) for the mechanical defense of tissues and implants (such asheart, nerve guide, cartilage, and trachea tissues) [38] [39] [40]

Fig 1 5.Garnedia blue reaction between genipin and primary aminesGenipin easily undergoes a spontaneous reaction with the principal amines found in amino acidsand proteins in the presence of oxygen to create water-soluble blue pigments [41] Uncertaintyexists over the precise mechanism of genipin-based blue pigment production obtained fromgenipa fruits, as well as the makeup of the pigment It is hypothesized that the blue pigments are

the consequence of the polymerization and dehydrogenation of multiple intermediate pigments

by oxygen radicals, producing high molecular weight, water-soluble polymers [42]

1.4 Gardenia blue

Gardenia blue is a natural colorant widely used in Asia It has a characteristic dark blue colorthat is unalterable with other colorings It is soluble in aqueous solutions of water, propyleneglycol, and ethanol but not in organic solvents The colorant resists heat better than most Thesubstance is practically odorless and has a weak hygroscopicity The stability and tonality of thecolorant are unaffected by PH variations at a PH value of 1 percent aqueous solution of 4.5+0.5.Calcium and aluminum ions have little effect on the colorant, whereas tin and iron ions can cause

a deepening of the color Frozen desserts, confections and baked goods, jams, noodles, beverages,wine and liqueurs, processed seafood, and agricultural products are all commonly colored withgardenia blue Genipin, the aglycone of geniposide, is produced commercially by adding -

glucosidase to a combination of iridoid glycosides isolated from the fruits of Gardenia

jasminoides Ellis Despite genipin's lack of color on its own, the interaction with primary amino

acids and hydrolysates of proteins provide a blue tint serving as a natural dye that is useful forfood, cosmetics, and textiles

Long used in traditional Chinese medicine, gardenia fruit has been shown to havepharmacological qualities including the ability to block liver apoptosis, neuroprotective and anti-depressive effects, and anti-inflammatory action [43] [37] [44] Although the crosslinkingprocess between genipin and molecules containing a primary amine is still unclear, genipinspontaneously produces crosslinks with protein, collagen, gelatin, and chitosan [45] Genipin hasbeen researched as a glutaraldehyde-alternative for inducing biochemical crosslinking in tissuebecause of its inherent capacity to crosslink, especially as a fixative for heterograft tissues [45].Moreover, as a biodegradable covering for sutures used to mend rupturing connective tissue [46]

Limited in vitro work reveals that genipin has genotoxic potential, however it is unclear if it

forms crosslinks with DNA [47] Gardenia blue has already been the subject of severalgenotoxicity studies in Japan

These studies, however, do not meet the current regulatory requirements for marketing productscontaining gardenia blue in U.S or European markets because the purity of the gardenia blueused in them was not clearly defined, the study data are not easily accessible (they have not been

published in the peer-reviewed literature), and most importantly, the studies did not meet thestudy's primary objective Gardenia blue and genipin were tested in a Good Laboratory Practices(GLP)-compliant test battery in accordance with current EFSA, OECD, and FDA guidances ongenotoxicity and toxicity testing in order to facilitate the global marketing of gardenia blue as anatural food colorant following approval by the US Food and Drug Administration (FDA), theEuropean Union, and a positive safety opinion from JECFA, an FAO/ WHO agency [48] As a

potential contaminant in the gardenia blue hue that can be created in vivo as a result of intestinal

bacteria interactions with gardenia blue utilized in food products, genipin was assessed

Including bacterial reverse mutation assays, in vitro mammalian micronucleus and chromosome

aberration assays, and combined micronucleus/comet assays carried out in male and female mice,the results of the full assessment of genotoxicity are provided here

Gardenia blue made up 24.8% of the formulation, which also contained dextrin (69.5%), water(4.6%), and other components (1.2%); the residual genipin level was under 10 ppm In order tocheck concentrations, samples taken from the top, middle, and bottom portions of each chemicalformulation were sent to OpAns, LLC, Durham, NC, and Alera Laboratories, LLC, Durham, NC.These tests revealed that the chemical formulations remained stable throughout the experiment

In order to ensure that the OECD test guideline was followed regardless of any deviation fromthe nominal dose of 5000 g/plate as determined by chemical analysis of the dose formulation, thetop concentration tested for the bacterial mutagenicity test of genipin was higher than the upperlimit specified by the OECD test guideline; in subsequent tests, a concentration that was within10% of the maximum specified by the guidelines was considered acceptable [48]

1.5 Reasearch about gardenia blue pigment production

Gardenia blue is a water-soluble natural color that is frequently utilized in the food,pharmaceutical, and cosmetics sectors Nowadays, the gardenia blue pigment is often made from

the raw material geniposide found in Gardenia Jasminoides Ellis of the Rubiaceae by processing

geniposide with α-glucosidase to form genipin, which then interacts with an amino acid toproduce the gardenia blue pigment [49] The gardenia blue pigment created by this technique, onthe other hand, is dark, has a low color value, and is of poor quality As a result, it is unsuitablefor various uses, such as drinks [50] A method of producing gardenia blue pigment with a highcolor value that involves ultra-filtering the gardenia blue pigment obtained from the reaction of

genipin with an amino acid to remove residual geniposide and then extracting the filtrate toobtain the gardenia blue pigment with a high color value [51] The alternative method involvesrunning geniposide through a large mesh non-polar resin to remove a-crocin before treating itwith β-glucosidase [52] However, because these methods are expensive and complicated, theyare inappropriate for large-scale industrial use As a result, there is still a need for a novelmethod that is simple to run and ideal for commercial applications for creating gardenia bluepigment that is brilliant and suitable for industrial applications [49]

According to Lili Li et al (2015), to recover genipin from Eucommia ulmoides bark, acontinuous approach based on the combination of ultrasonic and microwave pretreatmentsfollowed by enzymatic hydrolysis and simultaneous extraction (EHSE) has been developed.During the pretreatment phase, a combination of 1.0 g dry bark powder and 10 mL deionizedwater was microwaved for 10 minutes at 500 W The best settings for the hydrolysis stage were

as follows: 0.5 mg/mL cellulase concentration, 4.0 pH of enzyme solution, 24 h incubationperiod, and 40 °C incubation temperature Following incubation, 10 mL ethanol was added toextract genipin by ultrasonic for 30 minutes The yield of genipin after EHSE treatment mightreach 1.71 mol/g Furthermore, scanning electron micrographs demonstrated that EHSE caused asubstantial structural disturbance in the plant The results showed that the EHSE technique was asuitable option for preparing genipin from Eucommia ulmoides bark and other plants [53]

Weerapath Winotapun et al (2013) had research about genipin, an iridoid aglycone, which wasproduced directly in one pot from crude gardenia fruit The approach depended on theemployment of a single cellulase to damage plant cells while simultaneously cleaving off sugarmolecules, increasing the release of intracellular iridoids and converting geniposide to genipin.During the biocatalysis, the product was extracted using eco-friendly ethyl acetate, whichprovided partial purification and reduced genipin degradation Using 10 mg/mL cellulase and a24-hour incubation at 50 C, pH 4, combined with in situ extraction, genipin with high purity wasproduced at 58.83 mg/g, which increased 12.38 and 1.72 times when compared to processes

without either enzyme or in situ extraction [29].

1.6 Cross-linking of genipin in chitosan film

Because of its ability to react with nucleophilic groups like amino groups, genipin is an excellentcandidate for use as a crosslinking agent in chitosan films The reaction mechanism of chitosan

with genipin found that genipin undertook a ring-opening reaction to form an intermediatealdehyde group due to the nucleophilic attack by chitosan amino groups [54] Furtherpolymerization of the genipin molecules may take place if they are allowed to react with anucleophilic reagent Crosslinked materials exposed to air take on a dark blue coloring This isbecause the oxygen radical-induced polymerization of genipin and its interaction with aminogroups cause crosslinked materials to take on this color [55] The color of genipin crosslinkedchitosan films changed from their initial clear state to either a blue or brownish hue, and thischange was dependent on the pH value at the time of cross-linking [54] These scientistshypothesized that the color shifts were caused by the formation of distinct structures ofcrosslinked chitosan due to the interaction between primary amino groups on chitosan and eitherthe original genipin or the polymerized version of genipin The degree to which the genipin-crosslinked chitosan films were crosslinked significantly varied on the pH values at which theywere crosslinked, with the degree being highest around pH 7 It has been reported thatcrosslinking with genipin improves the mechanical properties and water resistance of chitosanfilms

Fig 1 6 Crosslinking reaction between chitosan and genipin

1.7 Reasearch about forming crosslinking with genipin

Adriana Bigi et al (2002) reported that the feasibility of crosslinking gelatin films with genipin

to stabilize them was studied using mechanical, chemical, and thermal evaluation of samplestreated with genipin solutions at various doses The amount of crosslinking, measured as the

difference in the number of free e-amino groups before and after crosslinking, increases withgenipin concentration up to roughly 85% Simultaneously, the film’s deformability diminishes asYoung's modulus E; rises Furthermore, as demonstrated by the findings of the d.s.c research,crosslinking causes a considerable reduction in swelling in physiological solution and improvesthe thermal stability of the samples The results obtained from films treated with genipin atconcentrations of more than 0.67 percent are remarkably similar, indicating that genipin has agood stabilizing effect Despite the small gelatin release (2%) observed after one month of buffersolution storage, the mechanical, thermal, and swelling properties of the films are very similar tothose previously obtained for glutaraldehyde crosslinked gelatin, suggesting that genipin, which

is far less cytotoxic, can be considered a viable alternative for crosslinking gelatin biomaterials[56]

According to Nataliya Kildeeva et al (2020), biopolymeric films crosslinked by genipin, anatural reagent, should offer great potential in food packaging The effect of the functional groupratio in the chitosan-genipin system on film absorption in the visible and ultraviolet parts of thespectrum, as well as sorption, physical, and mechanical characteristics, has been investigated.The degree of chitosan crosslinking in films formed from genipin-containing solutions wascalculated using experimental data on film swelling and water vapor sorption isotherms.Crosslinking with genipin increases the swelling, water resistance, and mechanicalcharacteristics of the films [57]

CHAPTER 2: MATERIAL AND METHOD 2.1 Materials

G.jasminoides powder were obtained from the local market (Thu Duc District, Vietnam).

Cellulase derived from Trichoderma reesei, protein from Lima bean, ethanol, sodium hydroxide,

citric acid, acetic acid, acid hydrochloric, potassium sorbate, ethyl acetate, monosodium

glutamate, ethanolamine, dithanolamine, n-pentylamine, urea, glycine, chitosan powder, glycerol

and buffer chemicals were purchased from a chemical local store (District 10, Vietnam) Allchemicals were analytical grade

2.2 Research process diagram

Fig 2 1 Pigments from genipin producing diagram

Fig 2 2 Chitosan-genipin film producing diagram

2.3.1 Pigments from genipin processing

2.3.1.1 Extracting geniposide from seed of G Jasminoides in ethanol

The geniposide in the G jasminoides seed powder was extracted using an ethanol extraction

solution The material (10 g) was dissolved in 100 mL of 50% (mL/mL) EtOH The beaker wascovered to prevent the solvent from being evaporated, and placed it on the hot plate magneticstirrer and stirred it at 50 oC for 1 hour The solution after soaking was allowed to settle, theextract will be filtered, then put the filtrate into another beaker and stored it Repeat it 3 times.The extract obtained was first dark red-brown, then faded in subsequent solvent additions Theextracts were pooled together

2.3.1.2 Treating geniposide with cellulase to obtain a hydrolysate