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:
OVERVIEW
Gardenia 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 fragrant white 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 anti-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 the body It is mainly used to treat dysphoria, agrypnia, jaundice, gonorrhea, thirst, conjunctival congestion, angina, hematemesis, non-traumatic bleeding, hematodiarrhoea, hemuresis, pathopyretic ulcer, sprain, and swelling pain [4] According to recent studies, the oil extract of
G jaminoides has been traditionally utilized as a natural yellow dye and has applications in food additives, dyestuffs, ornamental plant cultivation, antiseptics, and new medicines The plant's oval-shaped fruits, which ripen to a reddish-golden color in late fall, are employed in traditional Chinese herbal medicine to address various health issues.
Gardenia jasminoidesfruit 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 activityin vitroandin vivo.
G jasminoides essential oil is rich in volatile components such as aliphatic acids, ketones, aldehydes, esters, alcohols, and aromatic derivatives The composition of this essential oil varies due to differences in processing temperature and duration, with high temperatures potentially transforming unstable components like iridoids into volatile compounds Given the pharmacological properties of G jasminoides oil and advancements in extraction techniques, significant efforts have been made to optimize extraction methods Gas chromatography-mass spectrometry (GC/MS) is the primary technique used to analyze the volatile components of G jasminoides The essential oil yield from Gardenia jasminoides flowers is approximately 0.02% v/w based on fresh weight.
G jasminoidesis 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 fromG 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 (1/6)-α-D- 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, Shanzhiside methyl ester (I), phloyoside (II), chlorotuberside (III), penstemonoside (IV) [3] By some common isolation method such as solvent partition separation, classic column chromatography, preparative high-performance liquid chromatography (prep-HPLC), high-speed countercurrent chromatography (HSCCC), and other isolation methods, at least 15 iridoids, including iridoids, iridiod glucosides, secoiridoids, and secoiridoid glucosides, have been isolated and identified [3] Many studies have indicated that geniposide has various beneficial health effects, including anti-inflammatory, antidepressive, anti-diabetic, and antithrombotic qualities, as well as protection against lipopolysaccharide (LPS)-induced apoptotic liver damage
A study analyzed the concentrations of geniposide, gardenoside, geniposidic acid, and chlorogenic acid in 68 samples collected from various locations in China and Korea The findings revealed average concentrations of 56.37 ± 26.24 µg/mg for geniposide, 49.57 ± 18.78 µg/mg for gardenoside, 3.15 ± 3.27 µg/mg for geniposidic acid, and 0.69 ± 0.39 µg/mg for chlorogenic acid.
The optimal solvent extraction conditions for Gardenia jaminoides were found to be a 51.3% ethanol/water mixture at an extraction temperature of 70.4°C for 28.6 minutes, yielding 10.9% geniposide and 2.497% total phenolic compounds Additionally, ultrasound-assisted extraction achieved maximum geniposide yields of 4.1% using water with a solid/liquid ratio of 1:30 at 70°C for 30 minutes.
Crocin is a naturally occurring carotenoid chemical compound found in the flowers of turmeric and gardenia This is a diester formed from gentiobiose disaccharide and crocetin dicarboxylic acid.
The 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
Crocin and its derivatives from Gardenia jaminoides are recognized for being less toxic, less allergenic, and more environmentally friendly compared to saffron Additionally, these compounds are utilized in the treatment of various disorders, including weight loss, sexual dysfunction, and premenstrual syndrome.
G jaminoideswere 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].
Gardenia jasminoides contains several phenolic acids, including 3,5-di-O-caffeoyl-4-O-(3-hydroxy-3-methyl)glutaroylquinic acid, 4-O-sinapoyl-5-O-caffeoyl-quinic acid, 3,5-di-O-caffeoylquinic acid, and chlorogenic acid Additionally, a new lignin glucoside, (+)-(7S,8R,8’R)-lyoniresinol 9-O-β-D-(6″-O-trans-sinapoyl) glucopyranoside, has been identified in Gardenia jasminoides.
Gardenia jaminoides contains various terpenoids, including secoridoids and monoterpenoids Notable compounds identified in this plant are 6’-O-trans-Sinapoyljasminoside C, 6’-O-trans-Sinapoyljasminoside A, and several jasminosides such as jasminoside C, B, G, K, I, H, S, J, M, and N Additionally, it features 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, and jasminodiol, along with other sinapoyl derivatives.
Terpenoids, particularly those with a small number of carbon atoms, can be found in the volatile oil Terpenoids are extracted and isolated in similar methods that iridoids are [3].
Extracts from the fruit of G jasminoides are promising dietary supplements rich in antioxidants Both water and ethanol extracts exhibit significant antioxidant activity, with the water extract showing superior performance The IC50 values for various radical-scavenging activities indicate strong efficacy, with values of 0.14 mg/ml for DPPH and 0.21 mg/ml for ABTS Additionally, these extracts demonstrate notable reducing power and inhibition of linoleic acid oxidation, alongside enhanced superoxide dismutase and catalase activities The antioxidant properties are closely associated with the levels of phenolic and flavonoid compounds Furthermore, purified crocin displays considerable antioxidative activity, comparable to butylated hydroxyanisole at specific concentrations.
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 signal impairment [20] Geniposide improved impaired glucose tolerance and hyperinsulinemia in individuals with hereditary type 2 diabetes caused by visceral fat accumulation [21] In diabetic mice, geniposide (200 mg/kg and 400 mg/kg) was demonstrated to be an effective hypoglycemic drug, considerably lowering blood glucose, insulin, and triglyceride levels in a dose-dependent way [22] Geniposide also reduced diabetic vascular damage by reducing monocytic adherence to human umbilical vein endothelial cells and the production of cell adhesion molecules produced by high glucose [23].
Supercritical fluid extraction of G jasminoides oil and geniposide demonstrates antidepressant efficacy Genipin functions as an antidepressant by influencing glycolysis, gluconeogenesis, the TCA cycle, and lipid metabolism in the liver The antidepressant mechanism of geniposide remains to be fully elucidated.
6 connected to an increase in serotonin levels in the striatum and hippocampus of mice, as well as monoamine oxidase B [25][26].
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 to avoid arteriosclerosis and thrombosis [27].
Geniposide
The structure of geniposide was discovered in the 1960s Geniposide is one of the major iridoid glycosides in the fruit ofG jasminoides.
Fig 1 2 The structural formula of geniposide
Geniposide, a colorless compound, can be hydrolyzed by beta-glucosidase to produce genipin, which reacts with amino acids such as glycine, lysine, and phenylalanine to form a stable blue pigment suitable for food coloring Notably, the oral bacteria Actinomyces naeslundii and Actinomyces viscosus, known for their role in initiating tooth decay, contain beta-glucosidase that may react with compounds in gardenia fruit, potentially leading to a blue coloration in saliva This reaction is currently under investigation for its potential use in developing a reagent to target bacteria responsible for tooth decay.
Geniposide, a natural product with a biomolecular structure and low toxicity, has gained attention for its potential applications as a binding material Recent studies highlight its use in gelatin-bound formulations as a bioadhesive for wound dressings and bone grafts, indicating that geniposide could serve as a novel and safe cross-linking agent.
Glutaraldehyde is the most frequently used binding agent, but it raises similar toxicity concerns as geniposide In forensic science, geniposide is emerging as a promising method for tracing fingerprints on paper-based materials As a natural and environmentally friendly product, geniposide also holds potential as a raw material for drug manufacturing.
Geniposide exhibits a diverse range of pharmacological effects, including neuroprotective, antidiabetic, hepatoprotective, anti-inflammatory, analgesic, antidepressant-like, cardioprotective, antioxidant, immune-regulatory, antithrombotic, and antitumoral properties These effects position geniposide as a promising therapeutic agent for various diseases, such as Alzheimer's disease, Parkinson’s disease, diabetes and its complications, ischemia and reperfusion injury, and hepatic disorders.
. Fig 1 3.Hydrolysis of geniposide to genipin [29]
Cellulase is a more economical option compared to β-glucosidase, effectively catalyzing the removal of over 90 percent of the sugar moiety from geniposide Furthermore, it has the capability to degrade plant cell walls.
Genipin
Genipin is an aglycone derived from geniposide, an iridoid glycoside present inG.jasminoides.
Fig 1 4 Chemical structure of genipin
Genipin, an aglycone derivative derived from the hydrolysis of geniposide found in G jasminoides fruits, has been traditionally utilized in Chinese medicine to alleviate symptoms of type 2 diabetes, headaches, inflammation, and liver disorders Although colorless in its pure form, genipin can produce blue particles when it reacts with amino acids and proteins, making it a valuable natural dye in textiles and food production Additionally, genipin serves as an effective natural crosslinking agent for collagen, gelatin, proteins, and chitosan, employing two methods to cross-link materials with primary amine groups The mechanism involves a nucleophilic attack by an amino group on genipin's olefinic carbon, leading to a two-step reaction that covalently bonds genipin to the polymer This process generates a tautomeric aldehyde from an unstable intermediate, which then undergoes further reaction with an amine group from another polymer, resulting in additional covalent bond formation and cross-linking.
Genipin, as a crosslinking agent, exhibits cytotoxicity that is significantly lower—between 5000 and 10,000 times—than that of glutaraldehyde and other chemical agents Its optimal crosslinking conditions are achieved at temperatures ranging from 25 to 45 degrees Celsius and a pH level between 7.4 and 8.5.
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
Genipin has demonstrated protective effects against hepatic ischemia/reperfusion injury in rats Pharmacological studies indicate that it prevents lipid peroxidation and nitric oxide formation in rat paw edema, showcasing its potential anti-inflammatory, antithrombotic, and antiangiogenesis properties Additionally, research highlights genipin's ability to cross-link proteins, positioning it as a safer alternative to more toxic cross-linkers like glutaraldehyde for the mechanical reinforcement of various tissues and implants, including heart, nerve guide, cartilage, and trachea tissues.
Fig 1 5.Garnedia blue reaction between genipin and primary amines
Genipin reacts spontaneously with principal amines in amino acids and proteins in the presence of oxygen, resulting in the formation of water-soluble blue pigments However, the exact mechanism behind the production of these blue pigments from genipa fruits remains uncertain, along with the composition of the pigments themselves.
10 the consequence of the polymerization and dehydrogenation of multiple intermediate pigments by oxygen radicals, producing high molecular weight, water-soluble polymers [42].
Gardenia Blue
Gardenia blue is a widely used natural colorant in Asia, known for its stable dark blue hue that remains unchanged when mixed with other colorings It is soluble in water, propylene glycol, and ethanol, but not in organic solvents, and exhibits better heat resistance than most colorants This practically odorless substance has low hygroscopicity and maintains its stability and tonality across a pH range of 4.0 to 5.0 While calcium and aluminum ions have minimal impact on its color, tin and iron ions can enhance its depth Gardenia blue is commonly used in a variety of products, including frozen desserts, confections, baked goods, jams, noodles, beverages, wine, liqueurs, processed seafood, and agricultural items Additionally, genipin, derived from the fruits of Gardenia jasminoides Ellis, is produced by adding -glucosidase to iridoid glycosides Although genipin is colorless, it interacts with primary amino acids and protein hydrolysates to create a blue tint, making it a valuable natural dye for food, cosmetics, and textiles.
Gardenia fruit, traditionally used in Chinese medicine, exhibits pharmacological properties such as blocking liver apoptosis, providing neuroprotective and anti-depressive effects, and possessing anti-inflammatory actions Genipin, a compound derived from gardenia, is known to spontaneously crosslink with proteins, collagen, gelatin, and chitosan, although the specifics of its interaction with primary amines remain unclear It has been investigated as a safer alternative to glutaraldehyde for biochemical crosslinking in tissues, particularly as a fixative for heterograft tissues and as a biodegradable covering for sutures in connective tissue repair While limited in vitro studies suggest that genipin may have genotoxic potential, its ability to form crosslinks with DNA is still uncertain, and gardenia blue has been the focus of various genotoxicity studies in Japan.
Current studies fail to satisfy regulatory standards for marketing products with gardenia blue in the U.S and European markets due to undefined purity levels and the inaccessibility of study data.
Recent studies published in peer-reviewed literature indicate that the primary objective of assessing gardenia blue and genipin was not met These substances were evaluated in a Good Laboratory Practices (GLP)-compliant test battery, adhering to EFSA, OECD, and FDA guidelines for genotoxicity and toxicity testing This evaluation aims to support the global marketing of gardenia blue as a natural food colorant, pending approval from the US FDA, the European Union, and a favorable safety opinion from JECFA, an FAO/WHO agency Genipin was specifically assessed as a potential contaminant that may arise from intestinal bacteria interacting with gardenia blue in food products The comprehensive genotoxicity assessment included bacterial reverse mutation assays, in vitro mammalian micronucleus and chromosome aberration assays, as well as combined micronucleus/comet assays conducted in male and female mice.
The formulation comprised 24.8% Gardenia blue, 69.5% dextrin, 4.6% water, and 1.2% other components, with residual genipin levels below 10 ppm To verify concentrations, samples from the top, middle, and bottom of each formulation were analyzed by OpAns, LLC, and Alera Laboratories, both located in Durham, NC The results indicated that the chemical formulations maintained stability throughout the experiment.
To comply with the OECD test guideline, the bacterial mutagenicity test for genipin utilized a top concentration exceeding the 5000 g/plate nominal dose limit However, subsequent tests adhered to the guideline by maintaining concentrations within 10% of the maximum specified limit.
Reasearch about gardenia blue pigment production
Gardenia blue is a water-soluble natural color widely used in the food, pharmaceutical, and cosmetics industries It is derived from geniposide, a compound found in Gardenia Jasminoides Ellis of the Rubiaceae family, which is processed with α-glucosidase to create genipin This genipin then reacts with an amino acid to produce the gardenia blue pigment However, the pigment produced through this method tends to be dark, low in color value, and of poor quality, making it unsuitable for applications like beverages To address this issue, a new production method involving ultra-filtration has been developed to enhance the color value of gardenia blue pigment.
To obtain high color value gardenia blue pigment, genipin is combined with an amino acid to eliminate residual geniposide, followed by filtrate extraction An alternative method involves using large mesh non-polar resin to remove a-crocin before β-glucosidase treatment However, these methods are costly and complex, making them unsuitable for large-scale industrial use Therefore, there is a pressing need for a novel, simple method that is ideal for commercial applications in producing brilliant gardenia blue pigment.
Lili Li et al (2015) developed an effective method for recovering genipin from Eucommia ulmoides bark using a continuous approach that combines ultrasonic and microwave pretreatments with enzymatic hydrolysis and simultaneous extraction (EHSE) The pretreatment involved microwaving 1.0 g of dry bark powder with 10 mL of deionized water for 10 minutes at 500 W Optimal hydrolysis conditions included a cellulase concentration of 0.5 mg/mL, a pH of 4.0, a 24-hour incubation period, and a temperature of 40 °C After incubation, 10 mL of ethanol was added for ultrasonic extraction of genipin for 30 minutes, achieving a yield of 1.71 mol/g Scanning electron micrographs indicated significant structural changes in the plant due to EHSE, confirming its suitability for genipin extraction from Eucommia ulmoides bark and potentially other plants.
Weerapath Winotapun et al (2013) conducted research on genipin, an iridoid aglycone, produced directly from crude gardenia fruit using a one-pot method This innovative approach utilized a single cellulase to disrupt plant cells and cleave sugar molecules, enhancing the release of intracellular iridoids and converting geniposide to genipin The biocatalysis process involved eco-friendly ethyl acetate for product extraction, which aided in partial purification and minimized genipin degradation By employing 10 mg/mL cellulase and incubating for 24 hours at 50°C and pH 4, along with in situ extraction, they achieved a high purity genipin yield of 58.83 mg/g, representing increases of 12.38 and 1.72 times compared to processes lacking enzyme or in situ extraction.
Cross-linking of genipin in chitosan film
Genipin is a promising crosslinking agent for chitosan films due to its reactivity with nucleophilic groups, such as amino groups This unique property enhances the structural integrity and functionality of chitosan-based materials.
Genipin undergoes a ring-opening reaction to form an intermediate aldehyde group through the nucleophilic attack by chitosan amino groups If allowed to react with a nucleophilic reagent, further polymerization of genipin molecules can occur Crosslinked materials exposed to air develop a dark blue color due to oxygen radical-induced polymerization and interactions with amino groups The color of genipin crosslinked chitosan films transitions from clear to blue or brownish, influenced by the pH during cross-linking Researchers suggest that these color changes result from the formation of distinct crosslinked chitosan structures interacting with genipin The degree of crosslinking varies significantly with pH, peaking around pH 7 Additionally, crosslinking with genipin enhances the mechanical properties and water resistance of chitosan films.
Fig 1 6 Crosslinking reaction between chitosan and genipin
Reasearch about forming crosslinking with genipin
Adriana Bigi et al (2002) investigated the feasibility of crosslinking gelatin films with genipin to enhance their stability The study involved a comprehensive evaluation of the mechanical, chemical, and thermal properties of samples treated with varying doses of genipin solutions, focusing on the extent of crosslinking achieved.
The study reveals that the difference in the number of free e-amino groups before and after crosslinking increases with genipin concentration, reaching up to approximately 85% Concurrently, the film's deformability decreases as Young's modulus (E) increases Differential scanning calorimetry (d.s.c.) findings indicate that crosslinking significantly reduces swelling in physiological solutions and enhances the thermal stability of the samples Notably, films treated with genipin concentrations above 0.67% exhibit similar properties, highlighting genipin's effective stabilizing effect Although a minor gelatin release of 2% was noted after one month in buffer solution, the mechanical, thermal, and swelling characteristics of the films closely resemble those of glutaraldehyde crosslinked gelatin, suggesting that genipin, being less cytotoxic, is a promising alternative for crosslinking gelatin biomaterials.
Biopolymeric films crosslinked with genipin, a natural reagent, show significant promise for food packaging, as highlighted by Nataliya Kildeeva et al (2020) Their research examines how the functional group ratio in the chitosan-genipin system affects film absorption in both visible and ultraviolet spectra, along with its sorption, physical, and mechanical properties The degree of chitosan crosslinking in these films was determined through experimental data on film swelling and water vapor sorption isotherms Notably, crosslinking with genipin enhances the swelling, water resistance, and mechanical properties of the films.
MATERIAL AND METHOD
Materials
G.jasminoides powder were obtained from the local market (Thu Duc District, Vietnam). Cellulase derived fromTrichoderma 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) All chemicals were analytical grade.
Research process diagram
Fig 2 1 Pigments from genipin producing diagram
Fig 2 2 Chitosan-genipin film producing diagram
Method
2.3.1.1 Extracting geniposide from seed of G Jasminoides in ethanol
Geniposide was extracted from G jasminoides seed powder using a 50% ethanol solution A total of 10 g of the material was dissolved in 100 mL of the solvent, which was stirred at 50 °C for one hour in a covered beaker to prevent evaporation After soaking, the solution was allowed to settle, filtered, and the filtrate was collected in a separate beaker for storage, with the process repeated three times The initial extract was dark red-brown, gradually fading with each additional solvent, and all extracts were combined.
2.3.1.2 Treating geniposide with cellulase to obtain a hydrolysate
The extraction in2.3.1.1.(100 mL) was evaporated to obtain geniposide.
In a study on the extraction of Gardenia jasminoides bark, it was found that 1 g of the bark yielded approximately 0.08 g of geniposide when extracted with 50% ethanol (mL/mL) Additionally, the extraction process produced about 0.267 g of geniposide from 100 mL of the solution.
Cellulase was dissolved in 50 mM citric acid-NaOH buffers at various pH levels (4.0 to 7.0) to optimize enzyme-assisted extraction conditions The amount of cellulase used in the enzymatic reaction ranged from 0.1 to 0.5 g per 1 g of geniposide.
A solution was prepared by dissolving a mixture of cellulase and 0.267 g of geniposide in 100 mL of citric acid-NaOH buffer This solution was then stirred on a hot plate magnetic stirrer at 50 °C for a duration of 2 to 10 hours to facilitate the enzymatic reaction that produces genipin.
2.3.1.3 Extracting genipin from the hydrolysate by ethyl acetate
Ethyl acetate (50 mL) was added to the hydrolysate, and the mixture was shaken multiple times to ensure complete dissolution of genipin After allowing the mixture to settle, the organic phase was collected and this process was repeated three times The combined extracts were then evaporated to isolate genipin for subsequent procedures.
2.3.1.4 Reacting the product comprising genipin with amine
The genipin in2.3.1.3was dissolved with 50 mL 50% (mL/mL) EtOH.
The optimal amount of amine is typically between 1 to 3 moles for every mole of genipin In section 2.3.1.3, the quantity of genipin used was 0.008 g, which corresponds to approximately 3 x 10^{-5} moles, given that the molar mass of genipin is 266 g/mole.
In this thesis, for the reaction to take place completely, we added the large amount of amine 100 mL of 0.1 M MSG was prepared by dissolving 1.69 g MSG in 100 mL distilled water.
MSG solution was added to the genipin in ethanol The reaction was taken place in 75 o C in the range from 2 to 12 hours.
After the reaction, the mixture was dryed by vacuum dryer to obtain the pigment powder.
The genipin in2.3.1.3was dissolved with 50 mL of distilled water.
The chitosan (CH) film was prepared by dissolving 2% (m/v) chitosan powder in 1% (v/v) acetic acid on a magnetic stirrer, followed by the addition of 30% (v/m) glycerol The film solution, consisting of 20 mL and cast in 90 mm diameter Petri plates, incorporated genipin at concentrations ranging from 0.0025 to 0.01 moles per mole of NH2, with the optimal amount detailed in section 3.8.1 The films were dried at 40°C for 72 hours, then manually peeled off for analysis of various properties, including absorption spectrum via UV-vis spectra, FTIR, color measurement, moisture content, mechanical properties (thickness, tensile strength, elongation), and swelling content.
2.3.3 Procedure to investigate the optimal pH of the enzymatic hydrolysis of geniposide
The hydrolysate (10 mL) contained geniposide in 2.3.1.1 was poured into 7 vials, then it were evaporated to obtain geniposide.
To prepare a 200 mL solution of a 50 mM citric acid-NaOH buffer, first, prepare seven beakers of 100 mM citric acid solution by dissolving 1.92 g of citric acid in 100 mL of distilled water for each beaker Next, create a 0.1 M NaOH solution by dissolving 0.4 g of NaOH in 100 mL of distilled water Use this NaOH solution to adjust the pH of the citric acid solutions in the seven beakers to the desired values of 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0 Finally, add distilled water to each beaker until the total volume reaches 200 mL.
In this experiment, 0.0053 g of cellulase was added to each vial, and the mixture of cellulase and geniposide was dissolved in 10 mL of citric acid-NaOH buffer The vials were then covered and incubated at 50 °C for 6 hours The genipin content was subsequently analyzed using UV-VIS spectroscopy.
Ethyl acetate (5 mL) was added to each hydrolysate and the mixture was shaken multiple times to ensure complete dissolution of genipin After allowing the mixture to settle, the organic phase was collected, and this process was repeated three times The genipin content was then analyzed using UV-vis spectroscopy.
The ethyl acetate containing genipin was evaporated, and the genipin was subsequently dissolved in 5 mL of 50% ethanol A 0.1 M MSG solution was prepared by dissolving 1.69 g of MSG in 100 mL of distilled water Finally, 10 mL of the MSG solution was added to each vial containing genipin in ethanol.
19 reaction was taken place in 75 o C for 10 hours The genipin content in this step was compared by UV-vis and color measurement.
2.3.4 Procedure to investigate the optimal duration of the enzymatic hydrolysis of geniposide
10 mL of the hydrolysate contained geniposide in2.3.1.1was poured into each 5 vial, then were evaporated to obtain geniposide.
To prepare a 200 mL solution of 50 mM citric acid-NaOH buffer at pH 4.5, first, dissolve 1.92 g of citric acid in 100 mL of distilled water to create a 100 mM citric acid solution Next, prepare a 0.1 M NaOH solution by dissolving 0.4 g of NaOH in 100 mL of distilled water Use the 0.1 M NaOH solution to adjust the pH of the 100 mM citric acid solution to 4.5 Finally, add distilled water to the adjusted solution until the total volume reaches 200 mL.
Each vial received 0.0053 g of cellulase, which was then combined with geniposide and dissolved in 10 mL of citric acid-NaOH buffer The vials were sealed and maintained at a temperature of 50 °C.
2 to 10 hours (2, 4, 6, 8, and 10 hours) The genipin content in this step was compared by UV- vis.
Ethyl acetate (5 mL) was added to each hydrolysate and shaken multiple times to ensure complete dissolution of genipin After allowing the mixture to settle, the organic phase was collected, and this process was repeated three times The genipin content was then analyzed using UV-vis spectroscopy.
The ethyl acetate contained genipin before it was evaporated The genipin was dissolved with 5 mL50% (mL/mL) EtOH 100 mL of 0.1 M MSG was prepared by dissolving 1.69 g MSG in
In this experiment, 100 mL of distilled water was used, and 10 mL of MSG solution was added to each vial containing genipin in ethanol The reaction occurred at a temperature of 75 °C for a duration of 10 hours The genipin content was subsequently analyzed through UV-vis spectroscopy and color measurement techniques.
2.3.5 Procedure to investigate the optimal enzyme concentration of the enzymatic hydrolysis of geniposide
The hydrolysate contained geniposide in 2.3.1.1 (10 mL) was added to each 5 vial, then evaporated to obtain geniposide.
Preparation of pH 4.5 buffer solution (similar to 2.3.4)
RESULTS AND DISCUSSION
Factors affecting the hydrolysis reaction of geniposide
Cellulase activity is influenced by pH, with optimal performance for cellulase from Trichoderma reesei occurring between 4.5 and 7.5 However, determining the ideal pH for maximizing genipin extraction is crucial, as specific pH levels can lead to side reactions that produce undesirable blue products, ultimately reducing yield Additionally, genipin may undergo hydrolysis in acidic conditions Therefore, selecting the appropriate operating pH is essential to achieve high enzyme activity while minimizing product degradation.
Figure 3.1 illustrates the variation in blue pigment intensity across seven samples with pH levels ranging from 4 to 7 during the enzymatic reaction The sample at pH 4.5 exhibited the darkest blue hue, suggesting that pH 4.5 may be the most optimal for the enzymatic reaction To confirm this finding, additional experiments, including UV-vis spectra and color measurements, were conducted.
Fig 3 1 Gardenia blue solution in different pH of enzymatic reaction
The UV λmax of genipin is observed at 310 nm As illustrated in Figure 3.2, the signal corresponding to genipin at this wavelength varies across different pH levels from 4 to 7 Notably, in Figure 3.2a, the concentration of genipin in the sample at pH 4.5 is higher compared to the other samples.
We drew figure 3.2b by using the absorbance in 310 mn wavelength for easily observing. a) b)
The UV spectra of genipin solution were analyzed following enzymatic reactions at various pH levels Additionally, the absorbance at 310 nm of the genipin solution was measured after these enzymatic reactions across different pH conditions.
Figure 3.3 confirms the findings of Figure 3.2, indicating that the highest genipin content is found in the pH 4.5 sample after extraction with ethyl acetate In Figure 3.3a, the genipin concentration at 310 nm is notably greater in the pH 4.5 sample compared to the others Figure 3.3b presents the absorbance data at 310 nm for clearer observation.
The UV-vis spectra of genipin solution, following ethyl acetate extraction at various pH levels during the enzymatic reaction, are illustrated in Fig 3.3(a) Additionally, Fig 3.3(b) presents the absorbance at 310 nm of the genipin solution extracted under the same conditions.
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.
The pigment produced from the reaction between genipin and amino groups serves as an indicator of genipin levels in enzymatic reactions As shown in Figure 3.4, the optimal pH for this enzymatic reaction is 4.5, with a peak absorption at 590 nm, corresponding to the UV λ max of gardenia blue pigment.
Table 3 1.Color measurement of gardenia blue solution in different pH of enzymatic reaction
Note: different lowercase letters in a column illustrate significant differences (p < 0.05)
Color is a key characteristic of gardenia blue pigment, defined by three parameters: L, a, and b The L parameter indicates lightness, ranging from 0 to 100, while the a parameter measures the transition from greenness to redness, with values from -128 to +127 The b parameter represents the shift from blueness to yellowness, also spanning -128 to +127 A positive a value indicates a red hue, whereas a negative value suggests green Similarly, a b value greater than zero signifies yellow, while a value less than zero indicates blue.
Table 3.1 presents the color measurement results of gardenia blue across various pH levels during enzymatic reactions The blueness of gardenia blue serves as an indicator of genipin content at different pH values Notably, the sample at pH 4.5 exhibited the highest blueness (b value = -6.12±0.36), showing a significant difference compared to the other seven samples (p < 0.05) Conversely, the samples at pH 6.5 and 7.0 displayed the lowest blueness, indicating a reduced genipin content These findings from the color measurement experiment align with the results obtained from UV-VIS analysis.
The study investigated how varying pH levels influence genipin production to identify the optimal pH for cellulase activity It was found that the pH of the enzyme solution significantly affects enzyme performance Research by Lili Li et al (2015) indicates that the ideal pH range for cellulase is between 4.0 and 5.5.
A 2018 patent on gardenia blue production indicates that the optimal pH range for the enzymatic reaction is between 4.0 and 4.6 This finding aligns with the results of a study conducted by Lili Li et al in 2015, which also examined the impact of pH on enzyme activity.
30 and the patent which is mentioned before Therefore, pH 4.5 was chosen for the next experiments.
Time plays a crucial role in enzymatic reactions, as determining the optimal reaction time can enhance efficiency and reduce wasted time As illustrated in Figure 3.5, the intensity of blue pigment varies among five samples with reaction times ranging from 2 to 10 hours, with the 6-hour sample exhibiting the darkest blue hue This suggests that 6 hours may be the most effective duration for the enzymatic reaction To further validate this finding, additional experiments, including UV-vis spectra and color measurements, were conducted.
Fig 3 5 Gardenia blue solution in different time of enzymatic reaction
Figures 3.6 and 3.7 illustrate the impact of varying incubation times on genipin production Notably, figure 3.6 reveals a significant increase in genipin yield during the initial 6 hours, followed by a decline from the 6th to the 10th hour Additionally, figure 3.6a indicates that at 310 nm, the genipin concentration in the pH 4.5 sample surpasses that of the other samples Figure 3.6b was created using the absorbance data.
310 mn wavelength for easily observing.
The UV-vis spectra of genipin solution were analyzed following enzymatic reactions conducted over varying time periods The absorbance at 310 nm was measured to assess the changes in genipin solution as the enzymatic reaction progressed.
The UV-vis spectra of genipin solution, following ethyl acetate extraction, were analyzed at various enzymatic reaction times Notably, the absorbance at 310 nm of the genipin solution also varied with the duration of the enzymatic reaction.
Factors affecting the reaction to produce blue pigments from genipin
The amino groups in the pigment precursor may be effected by pH conditions Therefore, in this part, we did an optimal pH survey for the pigment forming reaction.
Figure 3.14 illustrates the variation in blue pigment intensity across seven samples with pH levels ranging from 2 to 10 hours during the reaction between genipin and MSG The samples at pH 7 and 8 exhibited the darkest blue hues, suggesting that these pH levels may be optimal for the enzymatic reaction Additional experiments, including UV-VIS spectra and color measurements, were conducted to further clarify these findings.
Fig 3 14 Gardenia blue solution in different pH of the formation of garnedia blue reaction a) b)
The UV-vis spectra of gardenia blue solution reveal variations in different pH levels during the formation of gardenia blue Additionally, the absorbance at 590 nm demonstrates how the pH influences the reaction process of gardenia blue formation.
The Figure 3.15 shown the information that the crosslinking process between the amino group (-
The reaction between gardenia blue and primary amine (NH2) is significantly influenced by pH levels, with optimal conditions occurring at neutral pH values of 7 and 8 Productivity of gardenia blue increases from acidic to neutral conditions, peaking at pH 8 before declining in basic environments Notably, the b value for the pH 8 sample is approximately -9.39, indicating a higher concentration of blue pigment compared to other samples.
Dan Yang et al (2011) found that in acidic media, amino groups did not interact with genipin In contrast, under basic conditions, the products exhibited a λmax value of 584 nm, resulting in mauve or dark red colors Notably, only the reaction in distilled water yielded blue pigments with the same λmax of 584 nm J Yamashita et al (2022) identified that the optimal pH for the formation of gardenia blue is approximately 7 to 8.
The kinetics of the reaction, as indicated by absorbance, increase with rising pH levels from 4 to 8 This phenomenon is attributed to the sensitivity of un-protonated amino groups to pH changes At lower pH levels, protonated amine groups are less reactive with genipin, demonstrating that higher pH enhances the reaction's effectiveness.
In alkaline conditions ranging from pH 8 to 10, genipin undergoes saponification to form genipinic acid, which then reacts with amino groups to create colors other than blue.
In conclusion, pH 8 was used as an optimal conditions for the reaction between genipin and primary amine to produce gardenia blue in this thesis.
Table 3.3.Color measurement of gardenia blue solution in different pH of the formation of garnedia blue
Note: different lowercase letters in a column illustrate significant differences (p < 0.05)
3.2.2.Time of pigment forming reaction
Following the identification of the optimal pH for producing the highest quality blue pigments, we further explored the impact of varying reaction times on genipin concentration in the samples.
Fig 3 16 (a)UV-vis spectra of gardenia blue solution in different time of the formation of pH L values a values b values
40 garnedia blue reaction.(b)Absorbance of 590 nm of gardenia blue solution in different time of the formation of garnedia blue reaction.
To determine the optimal reaction time for genipin with NH2, we analyzed six samples over a period of 2 to 12 hours, measuring their UV-vis absorbance The results indicated that the absorbance at the UV λ max of 590 nm, associated with the blue pigment of gardenia blue, was lowest in the 2-hour sample, with subsequent measurements at 4 and 8 hours showing increased absorbance Notably, the 10-hour sample exhibited the highest absorbance, while the 12-hour sample showed a decrease Therefore, a reaction time of 10 hours is optimal, resulting in a dark blue color with L = 43.32 ± 0.06 and b = -6.26 ± 0.89, as detailed in Table 3.4.
Table 3 4 Color measurement of gardenia blue solution in different time of the formation of garnedia blue reaction Time L values a values b values
Note: different lowercase letters in a column illustrate significant differences (p < 0.05)
3.2.3.Types of amine-containing compounds
In our experiments, we utilized genipin in conjunction with monosodium glutamate (MSG) as the amine However, the effectiveness of other amines compared to MSG remains uncertain Therefore, we conducted an investigation into various amines to determine the most suitable option for the genipin-amine reaction.
Figure 3.17 shows the color change of the solution when different amines react with genipin Six cuvettes with 6 different colors can be seen.
Fig 3 17 Color difference of solution after reacting with different amines
(1) ethanolamine (2) glycine (3) urea (4) diethanolamine (5) n-pentylamine (6) MSG
Fig 3 18.UV-vis spectra of gardenia blue solution in types of amine-containing compounds
In our study to determine the optimal type of amine for use with genipin, we examined six different amines under optimal reaction conditions and measured their UV-Vis spectra from 380 to 760 nm The results, illustrated in Figure 3.18, indicate that amines exhibit peaks at wavelengths between 580 and 600 nm, while amino acids peak around 590 to 600 nm Notably, the amino acids MSG and Gly displayed a blue color and the highest absorbance within the 580 nm wavelength range.
The analysis of Table 3.5 reveals that the samples of MSG and Gly exhibit a dark blue color, with measurements of L = 44.19±1.92 and L = 41.07±1.31, and b values of -8.87±0.29 and -10.24±0.09, respectively In contrast, primary amines such as ethanolamine and n-pentylamine show relatively high absorbance in the 580-600 nm range, but their color reaction is less intense than anticipated, as indicated by b values of 7.26±0.49 for ethanolamine and 3.72±0.39 for n-pentylamine For a more precise representation of the colors, refer to Figure 3.17.
The results for 43 diethanolamine exceeded expectations, demonstrating a higher absorbance compared to n-pentylamine; however, the solution did not achieve a blue color Additionally, the Urea sample exhibited minimal absorption peaks in the wavelength range of 580-600 nm.
Table 3 5 Color measurement of gardenia blue solution in different amines
MSG 44.19±1.92 b -4.46±1.68 a -8.87±0.29 b Ure 62.81±0.42 d 0.78±0.68 b 2.40±0.10 c Ethanolamine 55.3±1.07 cd -2.87±0.45 a 7.26±0.49 f Glycine 41.07±1.31 a -0.16±0.43 b -10.24±0.09 a Diethanolamine 54.18±0.80 c -0.43±0.23 b 6.03±0.57 e n-pentylamine 56.54±0.46 d -4.11±1.14 a 3.72±0.39 d
Note: different lowercase letters in a column illustrate significant differences (p < 0.05)
3.2.4 Effect of pH on protein of Lima bean when reacting with genipin
After finding the best amine, when working with genipin, the aim is to find the best blue pigment.
We explored the potential reaction between genipin and protein, questioning if it would behave similarly to its reaction with amines Our investigation focused specifically on the interaction between genipin and Lima bean protein.
Figure 3.19 shows the color change of the solution when the mung bean protein is reacted with genipin at different pH As the pH increases, the color is also darker.
Fig 3 19.Color difference of reaction between genipin and Phaseolus lunatus extraction in different pH
3.2.4.1 Uv-vis of pigment solution when genipin act with protein from Phaseolus lunatus in different pH a) b)
Fig 3 20 (a)UV-vis spectra of solution when genipin act with protein from Phaseolus lunatus in different pH.(b)Absorbance of 590 nm of when genipin act with protein from
Phaseolus lunatus in different pH
After reacting Lima bean protein with genipin in a reaction buffer, we measured the solution using UV-Vis spectroscopy to identify the optimal genipin content Four samples were tested at varying pH levels from 9 to 12 As shown in Figure 3.20, the UV λmax of the blue pigment from gardenia blue is observed at 590 nm The sample at pH 10 exhibited the highest absorbance, indicating it is the optimal condition for the reaction This suggests that Lima bean protein is more soluble in a buffer at pH 10 compared to other pH levels.
3.2.4.2 FTIR of residue of proteinwhen acting with protein from Phaseolus lunatus in different pH
Fig 3 21.FTIR of protein residue after being treated with genipin
The reaction of bean extract with genipin results in a color change, and the detailed structure of the bean residue is analyzed using FTIR spectroscopy The FT-IR spectra of the bean residues, measured at pH levels ranging from 9 to 12, reveal a sharp peak at 1040 cm\(^{-1}\), attributed to hydroxyl groups, likely from genipin Additionally, two characteristic peaks at 1619 cm\(^{-1}\) and 1528 cm\(^{-1}\) correspond to amide I (C=O stretching vibrations) and amide II (C-N stretching and N-H bending vibrations), respectively Notably, a change in the amide I peak is observed when the bean protein interacts with genipin.
Properties of chitosan-genipin films
Following our exploration of the factors influencing blue color production from genipin, we are now examining the properties of the genipin-chitosan film to assess its quality.
3.3.1 Uv-vis of films when change genipin content
Fig 3 22.UV-vis spectra of genipin-chitosan films
This experiment investigates the effects of varying genipin concentrations on crosslinking in chitosan samples, evaluated through absorbance measurements Five samples were prepared, with one being pure chitosan and the others containing increasing genipin concentrations from 0.0025 to 0.01 The absorption spectra, illustrated in Figure 3.22, reveal that pure chitosan exhibits weak bands below 400 nm, while genipin-containing films show strong absorption bands in the 260-700 nm range Notably, the absorbance peaks at approximately 600–610 nm, with the 0.01 genipin/NH2 sample demonstrating the highest absorbance, which decreases with lower genipin concentrations The absorption band at 610 nm is linked to the radical polymerization of genipin, influenced by atmospheric oxygen during chitosan crosslinking Additionally, a peak in absorbance is observed in the 280-290 nm range.
The absorption trend observed at approximately 600-610 nm closely resembles that of the 48 trend The incorporation of the chitosan amino group into the genipin heterocycle leads to the formation of a heterocyclic amine, which is associated with the absorption band in the range of 280-290 nm Additionally, there is an increase in peak intensity within the 280-290 nm range.
290 nm shows a greater degree of genipin binding by chitosan at a high genipin/NH 2 ratio [57].
3.3.2 FTIR of films when change genipin content
Fig 3 23 FTIR of genipin-chitosan films
After allowing the film to dry, we performed FTIR measurements to analyze the structural changes in chitosan when interacting with genipin Figure 3.23 displays four samples with genipin concentrations ranging from 0.0025 to 0.01, alongside a control chitosan sample The FTIR spectrum of chitosan reveals several absorption bands, with peaks at 1653 cm\(^{-1}\) and 1549 cm\(^{-1}\) corresponding to N-H bending in amide II and C=O stretching in amide I, respectively Additionally, the band associated with the unique asymmetric stretching of the C-O-C polysaccharide structure is also noted.
FTIR studies on genipin-treated biopolymers revealed additional peaks at 1281, 1423, and 1639 cm\$^{-1}\$ after cross-linking with 0.0025 genipin/NH2, indicating C-O-C asymmetric stretching, CH3 bending of the methyl ester, and C=C ring stretching These findings suggest that the carboxymethyl group of genipin reacted with the amino group of chitosan to form a secondary amide Furthermore, as the concentration of genipin in the chitosan solution increased, the FTIR peak shifted from 1638 to 1653 cm\$^{-1}\$, indicating a broader FTIR spectrum.
The analysis reveals an increasing trend at the peak of 1281 cm\(^{-1}\) and a decreasing trend at 1423 cm\(^{-1}\) The band at 1112 cm\(^{-1}\) is attributed to the C-N stretch of the tertiary aromatic amine from the cross-linked genipin nitrogen iridoid, which is covalently bonded to chitosan Furthermore, the band at 1070 cm\(^{-1}\) in the cross-linked spectrum is significantly stronger than that of pure genipin, indicating its association with the cross-linking process Notably, the peak at 1070 cm\(^{-1}\) decreases with increasing genipin concentration, suggesting that higher genipin concentrations enhance the cross-linking ability.
3.3.3 Moisture content of genipin – chitosan films (%)
Table 3 6 Moisture content of genipin – chitosan films genipin/ NH2content Moisture content (%)
Note: different lowercase letters in a column illustrate significant differences (p < 0.05)
Measuring the moisture content of the film is essential to observe the changes that occur when genipin cross-links with chitosan As indicated in Table 3.6, the moisture content of the samples showed a gradual decrease compared to the control samples.
The study presents four samples with increasing concentrations of genipin in a chitosan solution, ranging from 0.0025 to 0.01 genipin/NH2 The interaction between genipin and chitosan appears to alter the structure of genipin, resulting in the formation of membranes that effectively block the penetration of water molecules.
3.3.4 Mechanical properties of genipin – chitosan films (Thickness, TS and EL)
Table 3 7.Mechanical properties of genipin-chitosan films
Thicknes(mm) Tensile strength(N/mm 2 ) Elongation (%)
Note: different lowercase letters in a column illustrate significant differences (p < 0.05)
This experiment aims to evaluate the tensile strength, elongation, flexibility, and mechanical resistance of films, which are crucial for assessing their integrity and sustainability when varying genipin concentrations We investigated four genipin concentrations, including a control sample without genipin, with the highest concentration being 0.01 genipin/NH2 and the lowest at 0.0025 genipin/NH2 The required amount of crosslinking reagent for the reaction is less than that needed for gel formation in a 2% chitosan solution, leading to an increase in solution concentration during film formation as the solvent evaporates Notably, the film produced at this genipin/NH2 ratio remained colorless for 24 hours post-evaporation before turning blue and becoming water-repellent.
Reducing the genipin content in the chitosan solution surprisingly increased the strength of the film According to Table 3.7, films with a concentration of 0.0075 genipin/NH2 exhibited greater durability compared to those with 0.01 genipin/NH2, indicating that the crosslinking in the chitosan solution was not fully achieved at the higher concentration.
51 completed before the solvent evaporated the macromolecular chains of chitosan and may also result from the chains' immobilization in the equilibrium state, where more intermolecular interactions take place [57]
3.3.5 Swelling content of genipin – chitosan films
Table 3 8 Swelling content of genipin – chitosan films
Genipin/ NH2content Swelling content (%)
Note: different lowercase letters in a column illustrate significant differences (p < 0.05)
This experiment demonstrates that the water repellency of the film varies with different concentrations of genipin As shown in Table 3.8, the swelling ratio of the 0.0025 genipin/chitosan film is the highest, while lower genipin concentrations lead to a decrease in swelling ratios Additionally, the swelling ratios of the crosslinked chitosan films decrease as the concentration of genipin increases.
Increasing the genipin content in the crosslinking process with chitosan leads to denser crosslinking, which hinders water penetration and limits the internal structure's moisture absorption.