Microencapsulation of licorice essential oil by chitosan gelatin complex

Trang 1 MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING GRADUATION PROJECT FOOD TECHNOLOGY MICROENCAPSULATI

OVERVIEW

Gelatin

Gelatin has a rich history spanning 4000 years, originating with the Egyptians who used collagen-based glue for wooden objects This gel-like substance, derived from animal connective tissues such as skin, bones, and tendons, has been widely extracted and utilized in homes and industries for over 2000 years The late nineteenth century saw significant advancements in industrial production, leading to microbiologically safe gelatin that meets standard specifications Gelatin, a high molecular weight polypeptide, is specifically derived from collagen, which is the primary protein in animal connective tissues The term "gelatin" became common around 1700, originating from the Latin word 'gelatus', meaning film or frozen, and is strictly used to refer to proteinaceous materials derived from collagen, despite sometimes being confused with other gelling agents.

Gelatin is primarily composed of 85-90% protein, 0.5-2% mineral salts, and 8-13% water It includes all essential amino acids except tryptophan and typically contains only trace amounts of cystine Notably, gelatin is free from cholesterol and purines, and it boasts significantly higher levels of glycine and proline—10 to 20 times more than found in other proteins.

Gelatin is considered an incomplete protein because it contains only 9 of the 10 essential amino acids, resulting in a lower nutritional value compared to proteins found in milk and eggs Additionally, the amino acid composition of gelatin can vary based on the source of the raw materials and the production methods used.

Gelatin's molecular structure is composed of 18 distinct amino acids arranged in a specific cyclic sequence, resulting in a polypeptide chain containing approximately 1000 amino acids This primary structure features an amino acid group at one end and a carboxyl group at the other.

Gelatin is characterized by a Gly-X-Y structure, where X predominantly consists of proline and Y is primarily hydroxyproline This structure leads to the formation of a helical shape from three intertwined polypeptide chains, creating a secondary structure In its tertiary form, the helix spirals around itself, resulting in a rope-like molecular configuration known as a protofibril (Djagny, Wang, & Xu, 2001).

There are many ways to classify gelatin such as: by production materials and production methods

Classification according to production materials

Gelatin is primarily sourced from mammals, particularly swine and cows, which has traditionally been the most common raw material for its production However, concerns over mad cow disease have significantly impacted the availability of these sources In contrast, gelatin derived from fish skin and bones is regarded as superior in quality compared to other origins.

Classification according to production method

Gelatin grade A is produced through the acid treatment of raw materials, resulting in lower viscosity and gel strength compared to grade B, which is derived from base treatment Additionally, there are various other types of gelatin available, including cold water-soluble gelatin, hydrolyzed gelatin, and esterified gelatin.

Commercial gelatin is a pure, dry, and odorless substance that ranges from very pale yellow to amber in color, with a moisture content of 9 to 12% Its unique structure, characterized by ionic charges along the polypeptide chain, allows for excellent solubility and a remarkable capacity to absorb water, swelling up to 10 times its weight in both cold and warm water Gelatin fully dissolves at temperatures around 50-60°C, with a faster dissolution rate occurring at 90-95°C The properties of gelatin are influenced by various factors, including pH, raw materials, temperature, concentration, time, and processing methods.

Gelatin is an amphoteric substance due to its amine and carboxyl groups, which allows it to carry a positive charge in acidic conditions (H+) and a negative charge in basic conditions (OH−) The isoelectric points for type A gelatin range from 6 to 9.5, while type B gelatin falls between 4.5 and 5.6.

Gel-forming properties – gel strength

Gelatinization is a crucial factor in assessing the quality of gelatin when frozen, primarily determined by its gel strength, measured in bloom Bloom refers to the weight in grams needed to depress the surface of a gel, created by a tube with a diameter of 14.7 mm, to a depth of 4 mm For optimal results, a gel mass containing 6.67% gelatin should be maintained at 10°C for a duration of 16 to 18 hours.

Gelatin plays a crucial role in marshmallow production due to its excellent foam stability It's essential to select the appropriate type of gelatin, as different varieties exhibit varying levels of foam stability Additionally, the foaming properties can be enhanced by incorporating sodium lauryl sulfate within permissible limits.

The viscosity of a gelatin solution is influenced by several factors, including temperature, concentration, ionic content, pH, and molecular weight Specifically, viscosity increases with higher concentration and decreases with rising temperature At temperatures below 20°C, the solution forms a gel, while between 20°C and 30°C, it can exist as either a gel or a viscous solution with unstable viscosity Above 35°C, gelatin molecules fragment, resulting in unbound molecules regardless of concentration.

Gelatin forms a gel-reversible solution The freezing point depends on the proline and hydro proline content

Gelatin effectively binds with water, stabilizes foam, adjusts viscosity, withstands heat treatments, and maintains a soft structure without deformation With a low energy content of approximately 14.7 KJ/g, it is ideal for use in confectionery products containing 6-9% gelatin, offering a low-calorie option that is free from sugar and fat.

Brighten Skin: Gelatin provides essential proteins to improve skin moisture and elasticity, promote healing, and prevent wrinkles, sagging and sun damage

Gelatin enhances joint health by deeply penetrating the skin, promoting healing in connective tissues, alleviating joint pain, and improving cartilage elasticity, ultimately making your joints more durable and resilient.

The proteins in gelatin help repair leaky gut wall damage, rebuilding the protective lining of the intestines

Gelatin supports detoxification due to its high glycine content, which helps counteract the inflammatory effects of methionine often associated with excessive meat consumption Additionally, the glycine and glutamic acid found in gelatin are essential for the production of glutathione, a vital detoxifier that protects the liver, manages toxins, and aids in the elimination of heavy metals.

The glycine in gelatin helps your body fight stress hormones and reduce anxiety, which in turn helps you get a better night's sleep

Gelatin is a component of lozenges, suppositories, isotonic solutions containing 0.5 - 0.7% gelatin

As an ingredient in an antiseptic used as an artificial tear

Used to produce hard and soft capsules, which prevent pharmaceuticals from being exposed to light and oxygen

Gelatin is used to manufacture hard and soft capsules

As a base of ointments and pastes such as toothpaste

Applications in the field of science and technology

Gelatin glue is used in electro-molding, waterproofing, dyeing and microscopy coating

A useful emulsifying agent when combined with liquids and other aerosols to create culture media.

Chitosan

Chitosan is a linear polysaccharide derived from chitin, a macromolecule found in the shells of shrimp, crabs, and other crustaceans As a renewable and inexhaustible resource primarily located in coastal areas, chitin holds significant research potential as a biomaterial across various fields Structurally, chitin consists of N–acetyl–D–glucosamine subunits linked by β(1–4) glycosidic bonds, forming the poly [β–(1–4)–2–acetamido–2–deoxy–D–glucopyranose] structure.

Chitin deacetylation is the process of transforming chitin into chitosan by removing the acetyl group (-NHCOCH3) and converting it to an amino group (-NH2) This transformation typically involves alkaline treatments, such as concentrated sodium hydroxide or high-temperature potassium hydroxide The deacetylation reaction is illustrated in Figure 1.2.

Figure 1.3 Chitin deacetylation reaction to chitosan

Chitosan, a polymer derived from chitin, has gained significant attention in food applications due to its unique properties The primary distinction between chitin and chitosan lies in the functional group at the C-2 position; chitin contains a hydroxyl and N-acetylamine group, while chitosan features an amino group Composed of D-glucosamine chains connected by β-(1,4)-glycoside bonds, chitosan offers promising benefits for food preservation and safety.

6 shown in Figure 1.5 On the market, chitosan can be obtained in two forms: yellowish flakes or powder, as shown in Figure 1.6

The viscosity of chitosan solution is affected by many factors such as temperature, pH, concentration, molecular weight, degree of reduction and extraction method

The molecular weight of chitosan significantly impacts its physicochemical properties, with low and high molecular weight variations affecting viscosity, hydrophilicity, moisture content, and thermal stability Low molecular weight chitosan exhibits reduced interaction with hot sulfuric acid, resulting in a lower degree of hydrolysis compared to its high molecular weight counterpart Furthermore, low molecular weight chitosan can penetrate bacterial cells, effectively inhibiting RNA transcription.

7 causing bacterial cell death In addition, chitosan with high molecular weight is considered to be more stable [9]

The ability to absorb moisture and expand

The water absorption capacity of chitosan is influenced by its inherent moisture and storage conditions Research indicates that as the degree of reduction decreases, the water content in dried chitosan increases Furthermore, higher concentrations of crosslinking agents lead to a reduction in chitosan's expansion properties Additionally, water absorption impacts viscosity, resulting in decreased tensile strength Prolonged storage of chitosan can also elevate its moisture content.

Chitosan is soluble in various organic and inorganic acids, with common solvents including methanoic acid, acetic acid, hydrochloric acid (1% HCl), diluted nitric acid, and lactic acid, with 1% acetic acid being the most frequently used However, concentrated acetic acid at high temperatures can lead to chitosan depolymerization Notably, chitosan remains insoluble in phosphoric and sulfuric acids at elevated temperatures (95 - 121°C) and is also insoluble in water The solubility of chitosan is influenced by factors such as the degree of deacetylation, extraction method, time, temperature, concentration, and molecular weight.

Chitosan is a unique molecule characterized by the presence of functional groups -OH and -NH2, which classify it as both an alcohol and an amine This structure allows for various chemical reactions at the functional group positions, enabling the formation of O– and N– substituents.

In addition to some chemical properties, chitosan possesses properties such as antibacterial, biodegradability, non-toxicity, etc Here are some common biological properties of chitosan:

Chitosan is bioavailable as it is a natural polymer with no toxic effects and readily biodegradable

Chitosan accelerates bone formation with the formation of cells

Chitosan exhibits a hemostatic effect that enhances wound healing by promoting the formation of granulation tissue and epithelial cells This process minimizes wound surface contraction, ultimately reducing scar formation.

It has the ability to inhibit the growth of some fungi and is widely used as a fungicide

It shows anti-cancer ability (inhibits the growth of tumor cells)

It acts as a cholesterol-lowering agent

Chitosan exhibits a hemostatic effect that enhances wound healing by promoting granule and epithelial cell formation, minimizing wound surface contraction, and reducing scar formation When combined with hemostatic drugs, chitosan solution not only accelerates hemostasis but also decreases swelling and facilitates rapid healing Additionally, chitosan serves as an effective cholesterol-lowering agent.

Chitosan, a natural and non-toxic substance, is extensively utilized in food technology as a food additive to preserve flavor, color, and emulsion in fruit juices Its antibacterial and antifungal properties, along with its film-forming capabilities, make chitosan an effective agent for food preservation.

Licorice essential oil

Licorice essential oil, known for its distinctive scent and subtle woody aroma, contains volatile compounds and solutes It is rich in Glycyrrhizin, a substance renowned for its potent antibacterial and anti-inflammatory properties Additionally, this essential oil is frequently used in oral medications, aiding in liver detoxification and protection against Carbon tetrachloride damage, as well as in the treatment of hepatitis B.

Licorice is a flowering plant native to Asia, characterized by its perennial underground stem that can extend up to 2 meters This stem produces trunks that reach heights of 0.5 to 1.5 meters, featuring compound, feathery leaves with 9-17 ovate leaflets The plant showcases light purple, butterfly-shaped flowers, and its fruit is a legume, with the glabra species being smooth and straight, while the uralensis species is curved and hairy In addition to Northern licorice, commonly known as licorice, there are other variants such as Southern licorice, which differ in morphological characteristics and uses It's essential to distinguish between these types to avoid confusion.

Wild Licorice, also known as Tho Licorice or Gia Licorice, is a member of the Scrophulariaceae family This herb is commonly utilized as an alternative to Northern Licorice for treating conditions such as fever and cassava poisoning.

1.3.2 Biological effects of licorice essential oil

Historically, Western medicine viewed licorice primarily as a supplementary remedy, appreciated for its sweetness that made prescriptions more palatable In contrast, Eastern medicine recognized licorice as a potent therapeutic agent, integrating it into the majority of medicinal prescriptions for its ability to treat various ailments.

Today, there are many clinical studies that have proven the effects of licorice

Detoxification effect: Very strong detoxification with toxins of diphtheria, poison of porpoises, snakes, shock phenomenon, tetanus toxin This effect is believed to be of glycyrrhizin

Effects on respiration: Helps relieve cough, loosen phlegm, or is used in combination in cough remedies

The antispasmodic effects on gastrointestinal smooth muscle play a crucial role in treating irritable bowel syndrome, alleviating symptoms and promoting digestive health Additionally, these effects aid in the healing of gastric ulcers and inhibit the histamine-induced increase in gastric secretion This treatment approach is also effective in relieving constipation and addressing ulcers in the digestive system, providing benefits similar to those of cortisol.

Effects on the liver: Hepatoprotective in chronic hepatitis and increased biliary excretion Anti-hepatitis and anti-allergy

Effects on the kidneys: Helps diuretic

Licorice can be utilized as a natural alternative for cortisol in the treatment of Addison's disease, also known as primary adrenal insufficiency This condition results from inadequate production of the steroid hormone cortisol due to issues with the adrenal glands By mimicking the effects of cortisol, licorice serves as an effective replacement therapy for those suffering from adrenal insufficiency.

Other effects: anti-oxidant, prevent atherosclerosis, reduce belly fat accumulation, antibacterial, and lower blood sugar, improve the body's immunity

Licorice soothe some diseases, limit the growth of bacteria, help the wound not to become infected

D-limonene, found in orange peels, is recognized for its numerous health benefits, including its roles as an anti-depressant, digestive aid, anti-inflammatory, diuretic, antispasmodic, sedative, and anti-bacterial agent It effectively treats digestive disorders, mouth ulcers, and neutralizes stomach acid, while also promoting healthy intestinal function and liver support Moreover, scientific studies have demonstrated D-limonene's potential in preventing certain types of cancer, highlighting its significance in overall health maintenance.

Microencapsulation technique

Microencapsulation is a technique that involves enclosing substrates—whether solid, liquid, or gas—within an ultra-thin shell to preserve their integrity and prevent quality degradation or loss This method allows for the controlled release of the encapsulated materials under specific conditions The size of microencapsulated particles can vary from a few nanometers to several micrometers, and they may consist of one or multiple layers with varying thickness surrounding the core.

Since the 1950s, spray drying microencapsulation has gained popularity in production due to its flexibility, temperature control, and cost-effectiveness This technique yields high-quality microencapsulated particles with sizes under 40 micrometers However, a notable drawback is the reliance on water-soluble coatings, as the process necessitates input in the form of a solution or suspension.

Coatings used in microencapsulation include gum acacia, maltodextrin, various polysaccharides like alginate and carboxymethylcellulose, and proteins such as milk serum and pea protein Carrageenan is a popular choice for spray drying due to its pseudoplasticity, which aids in creating a smooth, spherical microencapsulation and enhances adhesion between the coating and core Its properties of emulsification, palatability, and biodegradability make it widely utilized Additionally, maltodextrin, a hydrolyzed form of starch, is favored for its film-forming capabilities, flavor and fat binding, low cost, and high water solubility, while also minimizing stickiness and agglomeration in powder formation.

Freeze drying is a cost-effective microencapsulation technique that enhances the durability, temperature stability, and shelf life of various ingredients, including inorganic and organic salts, enzymes, and odor compounds This highly productive method can be operated continuously or in batches, making it versatile for different applications While extensively studied in the pharmaceutical industry, freeze drying is gaining traction in the food sector as well The technology utilizes fats and lipid carriers, such as palm oil, beeswax, cocoa butter, and kernel oil, to optimize the encapsulation process.

The extrusion microencapsulation technique is mainly applied to volatile and unstable odorous materials with the use of carbohydrate lattice as encapsulation This method is often used

Microencapsulation offers notable advantages, including excellent efficiency that extends product shelf life due to limited air diffusion through the carbohydrate coating However, this technique has limitations, such as its applicability only to solutions with low concentrations (around 8%); at higher concentrations, stability decreases, and the substrate may easily diffuse out and oxidize Additionally, the production process is costly, and the resulting microencapsulated particles tend to be relatively large, ranging from 50 to 100 microns, with packaging materials primarily restricted to maltodextrin and starch.

Microencapsulation technique using rotating disk

The microencapsulation process involves pouring a substrate and encapsulation mixture into a rotating disc, where centrifugal force breaks the encapsulation into small particles These microencapsulated particles are then rapidly cooled to enhance the hardness of their outer shell Particles that do not combine with the substrate are filtered through a sieve and returned to the feed For optimal results, substrate particles should be between 100 – 150 μm in diameter, and quick cooling of the microencapsulation is essential This rotary disc technique offers a promising alternative to traditional methods like spray drying and freeze drying, delivering higher yields at comparable operating costs.

Microencapsulation technique using fluidized bed technology

Microencapsulation using fluidized bed technology offers significant flexibility by utilizing various encapsulation materials, including polysaccharides, proteins, emulsifiers, fats, cellulose derivatives, and gums This method involves suspending solid particles in a controlled chamber, maintaining specific temperature and humidity conditions The resulting microencapsulated particles are uniformly sized, typically ranging from 50 to 500 micrometers.

This method utilizes concentrated encapsulation in a molten state, resulting in a significantly reduced microencapsulation time and lower energy consumption due to minimal water evaporation Consequently, this leads to a substantial decrease in production costs These remarkable benefits make this technique highly popular in the industry for encapsulating various food ingredients and additives, including ascorbic acid, baking powder, and flavoring agents.

Currently, two primary methods for generating condenser droplets exist: simple and complex Both methods share a similar microencapsulation mechanism, differing mainly in their phase separation techniques In the simple method, solvates facilitate phase separation, while the complex method relies on the interaction between two oppositely charged polymers To create complex capacitor droplets, a positively charged polymer solution is mixed with a negatively charged polymer solution in a solvent, typically water The substrate is first dispersed in the positively charged polymer solution, followed by the addition of the negatively charged solution The coating deposition occurs as the two polymers attract each other, a process that can be expedited by incorporating salts or adjusting pH and temperature Furthermore, the coating thickness can be tailored to specific requirements.

12 by controlling the addition of a second macromolecular compound When using this technique, care must be taken to create the right environmental conditions to avoid clustering of particles

The microencapsulation process using the coagulation droplet method involves several key steps: (a) dispersing the substrate within a macromolecular solution, (b) separating the shell materials from this solution, (c) ensuring the shell adheres to the material needing protection, and (d) allowing the particles to continue adhering until a complete protective film is established.

Microencapsulation technique using fluidized bed technology

Microencapsulation using fluidized bed technology offers versatility with various encapsulation materials, including polysaccharides, proteins, and emulsifiers This method maintains uniform particle sizes between 50 to 500 µm, providing greater flexibility compared to other techniques Additionally, it reduces encapsulation time and energy consumption by minimizing water evaporation, leading to significant cost savings Due to these advantages, fluidized bed technology is widely adopted in the food industry for encapsulating ingredients and additives like ascorbic acid, baking powder, and flavoring agents.

Microencapsulation using the condenser droplet technique is prevalent in the food industry, where droplet coagulation separates an aqueous polymeric colloid solution into two distinct phases One phase comprises numerous colloidal-rich droplets that are interconnected, while the other phase, known as the equilibrium liquid, consists of a copolymer solution (Soper & Thomas, 2000) Currently, there are two approaches to generating condenser droplets: simple and complex methods.

The microencapsulation mechanisms of these two methods are fundamentally similar, differing primarily in their phase separation techniques In the simpler method, solutions facilitate phase separation, whereas the complex method relies on the interaction between two oppositely charged polymers for this process.

To create complex droplets, a common method involves mixing two oppositely charged macromolecular compounds in a solvent, typically water, where the substrate is dispersed in a positively charged polymer solution A negatively charged polymer solution is then added, and the deposition of the coating onto the substrate occurs through electrostatic attraction between the two polymers The process can be accelerated by adjusting pH and temperature values or adding salts, while the coating thickness can be controlled by regulating the addition of the second macromolecular compound However, careful control of environmental conditions is necessary to prevent particle clustering.

Positively charged colloids, such as gelatin and agar, contrast with negatively charged colloids like carboxymethylcellulose and gum Arabic To achieve a negatively charged state, it may be necessary to dilute these colloids with water or adjust the pH, depending on their isoelectric point It's essential that these reactions take place at temperatures above the gelation point; otherwise, the colloids will not remain in the liquid phase, leading to the formation of droplets.

Studies related to microencapsulation and chitosan

Chen Tan et al developed polysaccharide-based nanoparticles through a multi-polyelectrolyte complex of chitosan (CS) and Gum Arabic (GA) to serve as an effective delivery system for curcumin The study's findings demonstrate the potential of this innovative approach in enhancing curcumin delivery.

The positive amine groups of CS have a strong interaction with the negatively charged GA at pH 4.0 and the mixing ratio is 1:1, forming stable and homogeneous nanoparticles

These hydrophilic nanoparticles are thought to make curcumin water soluble and can be dispersed in water

The FTIR spectrum of CS-GA nanoparticles exhibited significant alterations in the Carbonyl-Amide region, with the -NH oscillation shifting from 1596 cm-1 to 1560 cm-1 Additionally, the absorption peak at 1423 cm-1 in the nanoparticles intensified compared to that of CS-GA particles These modifications indicate the presence of electrostatic interactions between NH3+ CS and COO- groups.

In a study by Franco Furlani et al., complex synthesis involving polysaccharides was conducted using two water-soluble biopolymers: chitosan hydrochloride and sodium hyaluronan By mixing chitosan with low molecular weight hyaluronan, electrostatic interactions were enhanced Key findings indicate that the polymer weight ratio and the physicochemical properties of chitosan significantly influence the size and homogeneity of the colloid This research enhances the understanding of chitosan and hyaluronan complexes, highlighting their potential applications in the food and biomedical industries.

A study by Hamid Rajabi et al evaluated the formation of chitosan-gum Arabic nanoparticles through ionic gelation, confirming the interaction between the functional groups (-COO- and -NH3+) of chitosan and gum Arabic via Fourier infrared (FTIR) spectroscopy and X-Ray diffraction (XRD) Transmission electron microscopy (TEM) revealed smooth, spherical shapes and uniform particle size distribution The microencapsulation yield varied from 29.12% to 52.34%, attributed to the enhanced attractive forces between the positive and negative groups of these biological compounds and an increase in Zeta potential.

Daniele Baiocco et al successfully fabricated oil-encapsulated microparticles using hexylsalicylate (HS), gum Arabic, and chitosan, with glutaraldehyde serving as a cross-linker Their study reveals that the stirring speed significantly influences the morphology of the microencapsulated particles Additionally, experimental microscopic analysis indicates that while the breaking force of HS microparticles increases, there is no significant change in particle diameter.

Some applications of microencapsulation in food

Microencapsulation technology is extensively utilized across various sectors, including agriculture, pharmaceuticals, cosmetics, textiles, and printing, with a significant impact in the food industry Its growing popularity stems from its effectiveness in enhancing product stability throughout storage and processing.

In today's health-conscious society, foods enriched with probiotics and antioxidants are increasingly popular However, these beneficial compounds can be sensitive to environmental factors like temperature, pH levels, transport conditions, and digestive enzymes To maintain their quality and effectiveness, these products are frequently microencapsulated.

Fat microencapsulation technology to produce soluble fat milk powder

Fat is prone to oxidation, which can lead to an unpleasant rancid smell, particularly at high temperatures In the past, producing high-fat powdered milk often resulted in difficulties during product recall, as the milk powder would stick to the walls of processing equipment due to a fat layer forming on the granules This melting at elevated temperatures caused particles to clump together, compromising product quality To address these issues, researchers have developed microencapsulation techniques that protect fats by limiting their exposure to oxygen and high temperatures, thereby reducing oxidation and enhancing the overall quality and recovery efficiency of the product.

Polyphenols have garnered significant interest in recent research due to their potential health benefits, leading to their incorporation into functional food, nutrition, and pharmaceutical products However, a major challenge in utilizing polyphenols lies in their astringent taste, which can render products unpalatable to consumers, thereby hindering their widespread application in the food industry.

The product faces several limitations, prompting the microencapsulation of polyphenols prior to incorporation This process effectively reduces and masks unpleasant flavors, enhancing consumer acceptance while ensuring the stability of these compounds throughout storage.

Microencapsulation technology for probiotics is widely utilized in the industry to guarantee the safe delivery of beneficial probiotics to the small intestine This method protects probiotics from being compromised by microorganisms, enzymes, and varying pH levels, ensuring their efficacy in products like yogurt and cheese.

Table 1.1 Some examples of probiotic microencapsulation in cheese products

K- Carrageenan Skim milk Alginate/starch

Table 1.2 Some examples of probiotic microencapsulation in yogurt products

B.bifidum Extrusion Gellan/xanthan gum

L.acidophilus Spray drying Maltodextrin/gum arabic

MATERIALS AND METHODS

Materials and equipment

Table 2.1 Materials and chemicals used

UV-Vis Spectrophotometer UH - 3500 Hitachi, Switzerland

Ret Basic Heated Magnetic Stirrer IKA, Germany

Lab 885 pH meter SI Analytics, Germany

Analytical balance 4 digits Sartorius, Germany

Method

The zeta test was conducted to assess the charge variation of chitosan, gelatin, and complex colloidal particles at a 0.1% (w/v) concentration in response to changes in solution pH Measurements were carried out at 25 ºC using a Zetasizer Pro instrument, following the methodology established by Duhoranimana et al (2017), with pH adjustments made using 0.1N HCl and NaOH.

2.2.2 Suitable conditions to form gelatin-chitosan complexes

Recovery efficiency and turbidity measurements were used to determine the optimal pH and chitosan-gelatin weight ratio for complex formation

2.2.2.1 Investigate the optimal pH between gelatin and chitosan to complex formation

The investigation into the ideal pH conditions for forming chitosan-gelatin complexes was conducted following the methodology of Eratte et al, with slight modifications to enhance the process.

Dissolve 0.1 g of chitosan in 100 mL of 1% acetic acid solution, 0.1 g of gelatin in 100 mL of distilled water at room temperature to form 0.1% (w/v) solutions Then, the two solutions were mixed at ratios of chitosan:gelatin 1:1, 1:2, 1:4, 2:1, 4:1 and were magnetically stirred for 5 minutes The mixture was measured for absorbance at 600 nm using a UV-Vis spectrophotometer at various pH in therange from 2 to 9 pH of the mixture was adjusted by adding 0.1N HCl or NaOH and stirred continuously The pH with the highest absorbance was considered optimal for complex formation

2.2.2.2 Investigate the optimal ratio between chitosan and gelatin to complex formation

The chitosan and gelatin were mixed in various ratios of 1:1, 1:2, 1:4, 2:1, and 4:1, with pH adjusted to achieve the highest absorbance as determined in section 2.2.2.1 Following this, the mixture underwent centrifugation and filtration to isolate the solid component, which was then dried at 105ºC on a Petri dish until a constant weight was achieved The yield of the chitosan-gelatin complex was calculated using a specified formula.

In there: m1: petri dish volume (g); m2: product weight and petri dish after drying (g); mCS: original chitosan mass (g); mG: original gelatin mass (g)

2.2.3.1 Constructing a standard curve for licorice essential oil in hexane

The creation of a standard curve for licorice essential oil in hexane is essential for quantifying the oil in subsequent processes, enabling the calculation of microencapsulation efficiency.

The standard curve for licorice essential oil in hexane was established following the method outlined by Caciano Pelayo (2019) A stock solution was created at a concentration of 100 μg/ml by dissolving 0.01 g of essential oil in 100 mL of hexane The UV-Vis absorption spectrum of this solution was scanned across the 200-800 nm range to identify the maximum absorption wavelength, which was determined to be 248 nm Calibration curves were then generated using the absorbances from ten diluted solutions derived from the stock solution, with the results detailed in Table 2.3.

Table 2.3 Concentration of essential oil in hexane and the absorbance of the solution

No Volume of stock solution (mL)

Essential oil concentration (àg/mL)

Figure 2.1 Standard curve of essential oil in hexane at 248 nm

Table 2.4 : Mass ratio of components in microencapsulation system to total volume of 600 mL of water

Ratio of shell:core Mass of chitosan (g) Mass of gelatin (g) Mass of licorice essential oil (g)

Figure 2.2 Process diagram for microencapsulation of licorrice essential oil by chitosan-gelatin complex

The microencapsulation system, developed by Fernandes et al (2016) with modifications, utilizes a chitosan and gelatin ratio of 1:4 and a polymer shell to essential oil core ratio of 1:1, 2:1, and 4:1 Chitosan is dissolved in a 1% acetic acid solution, while gelatin is prepared in distilled water, both achieving a polymer concentration of 1% (w/v) Licorice essential oil and the emulsifier Tween80 (5% of the core) are gradually incorporated into the chitosan solution, followed by homogenization at 9000 rpm for 5 minutes The 1% gelatin solution is then added slowly, with further homogenization for another 5 minutes The pH is adjusted to 7.5 using 1N NaOH under continuous magnetic stirring for 30 minutes at room temperature, after which the mixture is refrigerated for at least 5 hours The final step involves centrifugation, collection of the supernatant, and drying of the microencapsulated powder either by convection at 55ºC or freeze-drying for 24 hours.

The recovery efficiency was calculated based on the mass ratio of the microencapsulated powder obtained after drying and the total dry matter present in the initial system

In there: CY (coacervate yield): recovery efficiency; mFS: Mass of the final solid obtained after microencapsulation and drying (g);

22 mIS: mass of initial dry matter used in the microencapsulation system (g)

2.2.5 Microencapsulation of licorice essential oil

The efficiency of licorice oil microencapsulation was determined by assessing the quantity of licorice essential oil present in the filtrate post-encapsulation, along with the amount of oil found on the surface of the microencapsulated powder.

• Determination of the amount of licorice essential oil in the filtrate

After centrifugation of the filtrate, 25 mL of hexane was added and stirred for 10 minutes to ensure the unencapsulated licorice essential oil was fully dissolved The mixture was then centrifuged at 6000 rpm for 10 minutes, allowing the lighter phase containing the essential oil to be recovered This phase was adjusted to a final volume of 100 mL with hexane, and the absorbance was measured at 248 nm to quantify the essential oil content using a standard curve.

• Determination of surface licorice essential oil

The surface essential oil content was measured using the method outlined by Caciano Pelayo (2019), with some modifications Initially, 0.5 g of microcapsule was dispersed in 15 mL of hexane and stirred continuously for 10 minutes The resulting filtrate was passed through filter paper, and the solids retained were washed three times with 15 mL of hexane each time The filtrate and washings were combined and adjusted to a final volume of 100 mL with hexane The absorbance of this solution was then measured at 248 nm, allowing for the determination of the surface essential oil amount (msurface) based on a standard curve.

• Determination of licorice essential oil encapsulation efficiency

Due to the challenges in directly measuring encapsulated essential oil, we calculated the amount by subtracting the percentages of essential oil found in the filtrate and on the surface of the microcapsules from 100% The percentages of surface licorice oil and the microencapsulation yield were determined using specific formulas.

Where: mfiltrate: volume of licorice essential oil in the filtrate (g); msurface: volume of licorice oil on the surface (g); men: initial mass of licorice essential oil used for encapsulation (g);

EY: Encapsulation efficiency for licorice essential oil (%)

S%: percentage of surface licorice essential oil

F%: percentage of licorice essential oil in the filtrate

Solubility

The solubility of the microcapsule powder was determined by adding 0.5 g of the microcapsule to 50 mL of water at different pH (3, 7, and 9) with continuous magnetic stirring for

After 30 minutes, the mixture was filtered, and 20 mL of the supernatant was transferred to a petri dish, which was then dried at 105°C until a constant weight was achieved, with this process repeated three times The solubility was calculated using a specified formula.

In there: m1: mass of petri dish and solids after drying (g); m2: initial petri dish mass (g);

%msol: the solubility (%); a = 0.2 g is the amount of microencapsulated powder dispersed in 20 mL of solution.

Swelling ability

Place 1 g of microencapsulated powder in a 10 mL measuring cylinder, record the volume of the initial powder (V1) Then, 7 mL of solvent (water) was added and the cylinder was covered to prevent solvent evaporation Allow the mixture to stand in the dark for 2 h and then record the volume of solids after swelling (V2) The volume difference (V2 – V1)*100/V1 is the swelling ability of the microcapsule powder

RESULTS AND DISCUSSION

The zeta potential of the chitosan-gelatin complex

The formation of the chitosan:gelatin complex is attributed to the electrostatic attraction between positively charged chitosan and negatively charged gelatin To assess the charge characteristics of these polymers, the zeta potentials of gelatin, chitosan, and the chitosan:gelatin complex (1:4 w/w) were measured Figure 3.1 illustrates the variation in zeta potential for 0.1% (w/w) gelatin and chitosan across a pH range of 2.0 to 9.0.

Figure 3.1 Zeta potential of 0.1% (w/v) chitosan, gelatin, and chitosan:gelatin complex 1% solutions at different pH

In the pH range of 2-4, the zeta potential of gelatin decreases from 14.3 to -3.3 mV as pH increases At acidic pH levels, both gelatin and chitosan carry a positive charge due to the amino groups in their structure existing in the -NH3+ form As the pH rises, the positive charge diminishes, resulting in a decrease in zeta potential.

Gelatin exhibits a negative charge and zeta potential at pH levels above 4.5 due to the presence of carboxyl groups that can convert to carboxylate in basic conditions In contrast, chitosan maintains a solely positive zeta potential, as it lacks carboxyl groups.

Complex formation arises from electrostatic attraction and charge neutralization when substances acquire opposite charges As illustrated in Figure 5.1, at pH levels above 4.5, chitosan carries a positive charge while gelatin holds a negative charge, enabling them to form a stable complex.

Effect of pH on the formation of chitosan-gelatin complex

Turbidity measurement was conducted to quantitatively assess the complexation between chitosan and gelatin The optical absorbance at 600 nm was used to evaluate turbidity, which correlates with the density of solid particles in the solution.

Ze ta Pot e n tial ( m V) pH

25 solution Therefore, we investigated the change in turbidity of the mixture when changing the pH in the range of 2.0 - 9.0 and the ratio between chitosan:gelatin from 1:4 to 4:1

Figure 3.2 Absorbance at 600 nm of the mixture of two chitosan:gelatin solutions at different pH and polymer ratios

At pH levels below 5.5, there was minimal complex precipitation observed in the solution, aligning with predictions based on zeta potentials As the pH increased from 5.5 to 7.5, turbidity rose, indicating the formation of solid chitosan-gelatin complexes due to gelatin acquiring a more negative charge However, when the pH surpassed 7.5, the absorbance of the samples decreased, suggesting that the chitosan-gelatin complexes disintegrated into smaller, soluble fragments, which in turn reduced the turbidity of the solution.

Figure 3.3 Maximum absorbance of chitosan - gelatin mixture at different ratios

The ratio between the polysaccharides is a factor affecting the charge balance between the biopolymers in the mixture This is one of the important factors affecting complex formation as

0.8 pH=7.5 (1:4) pH=7.0 (1:2) pH=8.0 (1:1) pH=9 (2:1) pH=8.5 (4:1)

Ma x im u m ab so rb an ce

26 well as product recovery The experiment was conducted with samples with chitosan: gelatin ratio of 1:4, 1:2, 1:1, 2:1, 4:1

The absorbance of various polymer ratios was analyzed, with the results indicating that each ratio exhibited distinct maximum absorption levels, as shown in Figure 3.3 Notably, samples with a chitosan-to-gelatin ratio of 1:4 demonstrated higher absorbance values compared to the other samples, suggesting that this specific ratio facilitates the formation of a greater amount of chitosan-gelatin complex.

Effect of ratio of chitosan and gelatin on complex formation

We selected the pH level that produced the highest turbidity for each chitosan:gelatin ratio Following this, we centrifuged the mixtures at their optimal pH, obtained and dried the resulting solid complex, and measured the recovery efficiencies for each chitosan:gelatin ratio The recovery efficiency indicates the percentage of the initial chitosan and gelatin that was recovered as a complex.

Table 3.1: Recovery efficiency of chitosan – gelatin complex at different rates

Chitosan: Gelatin Complex recovery efficiency

Table 3.1 illustrates the efficiency of chitosan-gelatin complex recovery across various chitosan-gelatin ratios, revealing that a 1:4 ratio yields the highest recovery at 85.17%, followed by 74.76% for the 1:2 ratio The efficiency of complex recovery diminishes with a decrease in gelatin content, consistent with turbidity measurements presented in section 3.2 These findings suggest that exceeding the 1:4 chitosan-gelatin ratio results in excess chitosan, which cannot be effectively recovered through centrifugation.

The formation of the chitosan-gelatin complex is significantly influenced by pH levels and the chitosan to gelatin mass ratio Experimental findings indicate that optimal microencapsulation occurs at a pH of 7.5 and a chitosan to gelatin ratio of 1:4.

Microencapsulation efficiency

We optimized the pH and chitosan:gelatin ratio for the encapsulation of licorice essential oil, using the chitosan:gelatin mixture as the shell and the essential oil as the core Our study focused on how varying shell:core weight ratios affected the efficiency of essential oil encapsulation.

Encapsulation efficiency = 100% - %essential oil in filtrate - % surface essential oil

Table 3.2 Microencapsulation of licorice essential oil

Shell: core Essential oil in filtrate (%)

Research indicates that the shell-to-core ratio significantly influences microencapsulation efficiency As the shell-to-core ratio increases from 1:1 to 4:1, the encapsulation efficiency of essential oils rises from 55.95% to 84.08%, demonstrating an acceptable level of efficiency This improvement can be attributed to the increased amount of shell material available to envelop the essential oil droplets, thereby enhancing encapsulation efficiency.

Table 3.3 Licorice essential oil recovery efficiency

Sample Ratio Essential oil recovery efficiency (%) shell: core 1:1 23.94 ± 0.06 shell: core 2:1 45.91 ± 0.51 shell: core 4:1 72.02 ± 0.09

Higher shell:core ratios lead to increased microcapsule recovery efficiency, as indicated in Table 3.3 This phenomenon occurs because lower shell:core ratios result in a greater amount of unencapsulated oil, which precipitates with the complex and ultimately reduces recovery efficiency.

The results clearly show that the 4:1 shell:core ratio was the appropriate for the microencapsulation of licorice essential oil by the chitosan-gelatin complex

Swelling capacity of microcapsules

Figure 3.4 Swelling of two microecapsule samples in water

The swelling capacity of microencapsulated powder is crucial for its practical application, with research indicating that this capacity varies with different shell:core ratios Among three tested samples, the 4:1 shell:core ratio exhibited the highest swelling at 446.3% This increased swelling can be attributed to the hydrophilic nature of the biopolymer shells, composed of chitosan and gelatin, which readily absorb water In contrast, the hydrophobic licorice essential oil shows limited interaction with water, leading to a greater swelling capacity as the shell content increases.

Solubility

Figure 3.5 Solubility of microencapsulated samples at different pH

Sw el li ng c ap ac ity (% )

The solubility of microencapsulated powder is crucial for its practical application and industrial production This study examined the solubility of microencapsulated powder samples under various pH conditions, revealing optimal solubility at pH 3.5 In acidic environments, the microencapsulated powder demonstrates superior solubility compared to neutral or basic conditions, primarily due to the presence of chitosan Specifically, at pH 3.5, solubility levels ranged from 33.3% to 28.2%, while at pH 5.5, solubility decreased to 24.7% to 19.5% At pH 7.5, solubility further declined to a range of 21.2% to 17.8% The acidic pH facilitates the breakdown of bonds within the polymer chains, enhancing the solubility of the microencapsulated powder.

The study of the chitosan-gelatin microencapsulation system with licorice essential oil highlights the impact of various factors on the formation of the microencapsulation complex Licorice essential oil, rich in vitamins C, D, B, and E, is susceptible to oxidation and external influences such as light, temperature, and pH, making effective encapsulation crucial The research demonstrates that while the microencapsulation process does not negatively affect the licorice essential oil, it provides essential protection against harsh environments, including the digestive system This study paves the way for utilizing vitamin-rich essential oils in commercial applications, such as flavoring powders for cakes and candies, and in the development of functional foods like tablets, by employing a chitosan-gelatin coating to safeguard sensitive components during production and shield them from environmental degradation.

The limitations in research scope, survey results, experimental duration, data quality, and equipment challenges have impacted our findings With additional time for research and development, I aim to enhance the accuracy of microencapsulation techniques significantly.

❖ Diversify research data to demonstrate diversity in microencapsulation technology with different core types

❖ Using new methods and materials

❖ Bring practical applied research in food production

Apendix 1 The zeta potential of gelatin and chitosan in solution pH

Apendix 2 Absorbance of chitosan-gelatin complex at different pH pH Chitosan:gelatin

Apendix 3 The efficiency of chitosan-gelatin complex recovery at different ratios

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The zeta potential of gelatin and chitosan in solution

Absorbance of chitosan-gelatin complex at different pH