MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING Ho Chi Minh City, January 2024 EFFECT OF HIGH PRESSURE AND H
INTRODUCTION
Problem
The silkworm is the larva (the active immature form of an insect) or caterpillar of the
Bombyx mori moth Silk has been made for at least 5000 years in China (Dhanushree et al, 2022) The main components of silk are fibroin and sericin, which play an important role in increasing strength and hardness and maintaining the structural integrity of the cocoon The amount of protein in both main components accounts for about 75% and 25% of the total silk weight, respectively (Biswal et al, 2022) Sericin silk is a natural polymer produced by silkworms that is frequently removed by the textile industry but may be recovered and reused Nearly 50,000 tons of sericin are generated globally as a by-product of the degumming process, causing economic as well as environmental concerns (Vaishnav & Singh, 2023) Wastewater from industry is discharged, leading to an increase in the level of chemical oxygen demand (COD) and biological oxygen demand (BOD) Consequently, wastewater from the silk industry leads to water and environmental pollution (Fabiani et al, 1996) There have been ongoing efforts to recover and reuse it as a natural biopolymer in a variety of applications Sericin has recently been researched for its biological, physicochemical, and structural properties, indicating that it has considerable potential in the cosmetic, food, and textile industries In biomedicine and pharmaceuticals, sericin is considered a functional food due to its antioxidant properties, and various studies have reported that dietary intake of sericin significantly reduces cholesterol levels, serum, and free fatty acids (Barajas-Gamboa et al, 2016) In foods, sericin has the potential to improve color and texture while inhibiting polyphenol oxidase enzyme activity (Thongsook et al, 2011)
The presence of highly hydrophobic amino acids and its antioxidant capacity make sericin applicable in the food and cosmetic industries In order to completely exploit the supply of sericin found in silkworm cocoons for biomedical and food applications, environmental pollution is to be minimized (Kunz et al, 2016) In this report, the objective is to consider the physicochemical, biological, and structural properties of sericin by the method of protein extraction from silkworm cocoons At the same time, determine the influence of the properties and dynamics of sericin by the methods of high temperature and
3 high pressure, as well as the antimicrobial capacity of sericin The fractionation of sericin into various components is carried out by dissolving it in hot water for different time periods, during which the sericin undergoes hydrolytic cleavage (Gulrajani et al, 1992) Sericin is likewise degraded by this approach, however the decomposition preserves sericin's key characteristics (Rangi et al, 2015).
Research objective
The study was conducted to investigate the effects of high pressure and high temperature on the extraction of sericin from silkworm cocoons
-Determine the optimal temperature of sericin extract by investigating different temperatures
-Determine the optimal time for sericin extraction by investigating different times
-Simultaneously, evaluate the physicochemical properties and biological properties (antioxidant, antibacterial) of sericin with the aim of evaluating sericin as a potential raw material for the food and pharmaceutical industries.
Object and scope of the research
-Research Object: Sericin extract from silkworm cocoons, Bombyx mori
-Scope of the study: This study was carried out on a laboratory scale
-Research topic on the influence of high-temperature and high-pressure extraction methods on the sericin content extracted from silkworm cocoon shells At the same time, evaluate some properties of the sericin solution.
Research content
Investigate and evaluate the effects of temperature and time on sericin content obtained from the high temperature and high pressure (HTHP) extraction methods:
- Evaluate chemical composition of silk cocoon: total protein, lipid, carbohydrate, ash and moisture content
- Effect of temperature on the yield of sericin extraction from silk cocoon
- Effect of extraction time on the yield of sericin extraction from silk cocoon
- Study the structure of silk sericin through SDS-Page, FTIR spectroscopy
- Study the characteristics of silk sericin through antioxidant, antibacterial
- Study the amino acid compound in silk sericin.
Scientific and practical significance
Preliminary research on the content and bioactivity of sericin obtained from silkworm cocoon shells From there, assess the potential application of sericin in food A number of factors affecting the variability of the basic characteristics of sericin extract have been identified The application of sericin not only helps create innovation in the food industry but also contributes to environmental protection by limiting industrial waste from the garment industry
LITERATURE REVIEW
Overview of Silkworm Cocoon
Silk is a natural fiber produced by silk worms, including those belonging to the
Bombycidae, Saturnidae, and Lasiocampidae families, as well as spiders (Humenik et al.,
2011) [9] Mulberry silk, derived from Bombyx mori, a member of the Bombycidae family, is specifically obtained from worms fed with mulberry leaves The cultivation of these silk worms, which requires human care due to historical domestication, is known as sericulture
Sericulture is a demanding sector where the cultivation of mulberry trees (Moraceae family, Morus genus) and the reproduction of silk worms are key components The primary objective is to acquire both yarn and textile goods This process involves preserving eggs, breeding silk worms, disease prevention, feeding with mulberry leaves, collecting mature larvae, and transferring them to the area for cocoon formation (Takeda, 2009) Globally, approximately 100 thousand tons of silk are produced each year Among these, China contributes 70 %, followed by Brazil, Japan, India, Thailand, and Vietnam (Pescio.,et al 2008)
Silkworms feed on leaves, with these leaves containing 81.72 % water, 0.57 % fat, 1.55 % protein, 1.47 % fiber, and 14.21 % carbohydrates Leaf proteins are synthesized by the silk gland cells of the silkworm, and immediately after, they are stored in the lumen, where they transform into silk fibers During the spinning process, these silk fibers pass through the anterior gland and are then ejected through the die opening The result is a delicate double fibroin filament, coated by a gum called sericin Sericin aids in the formation of the silk cocoon by acting as a binder, maintaining its structural integrity The obtained oval-shaped structure serves as a safe haven during the larval metamorphosis process, transitioning from a larva to a pupa (Patel & Modasiya, 2011; Takeda, 2009)
Figure 2.1 Bombyx mori L’s cocoon 2.1.2 The chemical composition silkworm cocoon
The structural composition of the silk cocoon layer involves two main proteins, fibroin and sericin Fibroin, serving as the central component, is a fibrous protein, while sericin, acting as the adhesive element, is a globular protein that wraps around the fibers and binds them together Additionally, certain impurities such as carbohydrates, salts, and waxes, referred to as "non-sericin" constituents, contribute to the water repellent properties of the silk cocoon
Concerning various silkworm types and methods for extracting components based on nutrition sources, the cocoon primarily consists of fibroin, sericin, and additional impurities such as pigments, waxes, carbohydrates, and phytochemicals These components make up approximately 75–83 %, 17–25 %, and around 1–4 % of the cocoon respectively (Hossein Biganeh., et al 2022) The amino acid residues present in silk proteins can be categorized into three classes, encompassing charged residues like aspartic acid, polar residues like serine, and hydrophobic residues like glycine
2.1.3 Biological characteristics of silk cocoon
In recent years, there has been a growing interest in the biological properties of silkworm, especially its main proteins such as fibroin and sericin Scientific studies have focused on exploring the pharmacological effects of silkworm and its specific components These applications include cardiovascular protection, antioxidant capabilities, cancer cell inhibition, diabetes treatment, lipid reduction, gastric mucosa protection, and improvement of skin health (Biganeh.,et al 2022) Silkworm has also been extensively researched and
7 applied in drug distribution and tissue engineering Importantly, its toxicity falls within an acceptable range
Although there is still a significant barrier to the widespread use of sericin in biomedical applications, particularly concerning reports of sensitivity in the medical field, thanks to the unique properties of this type of protein, representing a protein family, sericin has great potential for application in the food, cosmetics, and biopharmaceutical sectors An important condition is to ensure invisibility to the immune system, which can be achieved through packaging sericin in protective nano devices and maintaining low usage.
Overview of Sericin
Sericin can be extracted from silk fibers by removing it from the fibroin component Since only the fibroin part is essential in the silk industry, the removal of sericin is necessary and is typically carried out through the degumming process, after which sericin is discarded Recycling sericin from degumming water not only helps reduce the load in wastewater but also produces a biopolymer with various important properties There are several methods for extracting sericin, including boiling [69], alkaline treatment [70], organic solvents lactic acid and citric acid [35] Another method involves using high temperature and high pressure to eliminate sericin from silk fibers
Silk secirin an innate luminous globular protein, is obtained from the silk cocoon of
Bombyx mori (Ki CS, 2007; Poza P, 2002; Wu JH, 2007) Its chemical structure is depicted in Figure 2.2
Where R = -CH3 or -CH2C6H4OH
Figure 2.2 Chemical structure of silk sericin ( Saha., et al 2019)
Fibroin serves as the core structure of the silk fiber, while sericin acts as the adhesive substance enveloping it Additionally, the composite structure of fibroin consists of the amino acids Gly-Ser-Gly-Ala-Gly-Ala, forming beta-pleated sheets referred to as "β- keratin." Simultaneously, hydrogen bonds are established between chains and side chains, both above and below the plane of the hydrogen bond network In this context, R represents
H (glycine), R represents CH3 (alanine), and R represents CH2OH (serine).( Saha., et al 2019)
Sericin is a natural polymer found in the cocoon of the B mori silkworm, connecting with fibroin molecules through hydro bonds, constituting 25 – 30 % of the total cocoon weight Sericin is a hydrophilic protein with the ability to dissolve in hot water To separate sericin from fibroin or the silkworm cocoon, degumming techniques can be employed and implemented through various methods (Saha., et al 2019) Sericin typically exists in an amorphous state, exhibiting adhesive properties, enveloping fibroin protein fibers, forming the cocoon's structure, and aiding in maintaining the integrity of the cocoon's structure
Sericin is a macromolecule of hydrophilic amino containing hydrophilic amino acids, comprising a total of 18 amino acids It possesses potent polar groups, such as amino, carboxyl, and hydroxyl groups, which can engage in copolymerization, crosslink formation, and amalgamation with other polymers (Lamboni.,et al 2010) The elemental composition analysis of sericin reveals it consists of 6 % hydrogen, 46.5 % carbon, 16.5 % nitrogen, 31
% oxygen, and 0.9 % sulfur (Kunz., et al, 2016; Jena,K., et al, 2018) The CN ratio shows a correlation with hot water solubility; thus, a lower C/N ratio results in higher solubility in hot water (Jena,K., et al, 2018)
Sericin exhibits a molecular weight ranging from 10 to 300 kDa, depending on the solvent used (acidic or alkaline solvents) for sericin dissolution The content and molecular size of sericin are influenced by factors such as temperature, pH, processing time, and solvent concentration (Zhang YQ, 2002) ).The silk cocoon is divided into three layers of sericin-outer, middle, and inner-each containing 15 %, 10.5 %, and 4.5 % sericin respectively (Cao, T.-T., & Zhang, Y.-Q 2016) , as illustrated in Figure 2.3 Sericin remains insoluble in cold water but becomes soluble in hot water, as the extended protein molecules break down into smaller fractions under high-temperature conditions, making them easily hydrolyzed and dispersed (Cao, T.-T., & Zhang, Y.-Q., 2016; Zhang YQ, 2002)
Figure 2.3 : Three layers of sericin in a silk cocoon (Saha., et al 2019)
The amino acid content in sericin extracted using various methods is presented in table 2.1 Although there are slight variations in the percentage of amino acids in sericin extracted by different methods, the main amino acid components in sericin remain consistent Serine is the predominant amino acid in sericin (around 30 %), while aspartic acid and glycine make up about 10-20 % The amount of methionine in sericin decreases with increasing temperature, with significantly higher levels compared to sericin extracted by other methods Additionally, the amount of tyrosine in urea-extracted sericin is significantly lower than in sericin extracted by other methods Furthermore, sericin extracted by heat contains the highest levels of methionine and cysteine, sulfur-containing amino acids that can form double-helical structures (Aramwit, Pornanong; et al)
Table 2.1 The amino acid composition of sericin extracted using various methods (in mole%) (Aramwit et al 2009)
Amino acid Extraction method of sericin
The working conditions of the enzyme restrict the use of this method for extraction purposes (Sothornvit, R., et al, 2010) The hot water extraction method is the most common due to the mentioned constraints for sericin extraction In this method, silk is heated in hot distilled water without adding any chemicals Both time and temperature play important roles in the amount of extracted sericin Although this method causes a reduction in the quality of sericin, the extent of the reduction is not to the point where sericin loses its essential properties It has been reported that temperature can be applied to the system through boiling at atmospheric pressure (Aramwit, Pornanon; et al 2009), HTHP (high temperature, high pressure) (Gupta, D., et al, 2014; Haggag, K.,2007) However, the HTHP method for sericin extraction does not use chemicals but produces smoke, is messy, time- consuming, and may damage fibroin (Aramwit, Pornanong; 2009) In this report, sericin was extracted from silk cocoon using the high-temperature and high-pressure method Below is the basic procedure for this method
To extract sericin using the high temperature and high-pressure method with distilled water, you can follow these general steps:
- Autoclave machine or pressure cooker
+ Obtain silk cocoons from silkworms
+ Sort and clean the cocoons to remove any impurities
+ Place the cleaned silk cocoons inside the autoclave machine Add the calculated amount of distilled water
-Adjust Parameters: Set the autoclave machine or pressure cooker to high temperature and high pressure conditions The specific temperature and pressure values may vary, but commonly, temperatures around 121 °C in 15 and 30 mininutes
-Cooling: After the extraction process is complete, allow the autoclave to cool down before opening it
-Separate Sericin: Extract the silk cocoons from the autoclave and separate the sericin from the fibroin This can be done by washing the cocoons and gently separating the sericin layer
-Characterization: Optionally, you can characterize the extracted sericin for its properties, such as molecular weight, composition, and functional groups
+ The specific conditions and duration may need to be optimized based on the equipment used and the desired properties of the extracted sericin
+ Always consider safety precautions when working with high temperatures and pressures
+ This general procedure provides an overview, and the actual process may be adapted based on specific research or production requirements.
Applications of Sericin
A reported study has demonstrated the antibacterial activity of (Bungthong, C., et al, 2021) Extracted through a degumming process involving sodium carbonate, sericin has shown inhibitory effects against E coli bacteria, a common foodborne pathogen (Senakoon, Waraluk, et al (2009) Sericin obtained through water degumming has exhibited the ability to inhibit S aureus, another foodborne pathogenic bacterium
Due to the presence of polyphenols and flavonoids, sericin exhibits antioxidant properties (Kunz, Regina Inês, et al, 2016) As a result, it has been identified that the ability of amino acids, hydroxyl groups, high-molecular-weight sericin, polyphenols, and flavonoids to donate hydrogen contributes to the reduction and discoloration of DPPH (FAN, JIN‐BO, et al 2009) Therefore, sericin is a valuable multifunctional substance that can be explored for applications in the cosmetics industry and food preservation, as a natural and safe component against oxidative processes affecting food quality and shelf life (Miguel, Gabriela Andrea, et al)
The powder made from sericin (5–30 %) and silk fibers (70–95 %) exhibits anti- static and moisture-absorbing properties (Kundu, Subhas C., et al 2008) Silk sericin, with its high water solubility, becomes a crucial element in the cosmetics industry (Yazicioglu, Alkin, et al, 2017) Sericin not only acts as an adhesive but is also widely recognized in skincare, haircare, and nail products, contributing to improved skin elasticity and anti- wrinkle effects (Gillis G., Bojanowski, et al, 2009) Particularly noteworthy is sericin's ability to inhibit apoptosis and stimulate the synthesis of collagen type I Additionally, it surpasses vitamin C in its anti-aging capabilities by effectively limiting oxidative stress (Kitisin, T., et al 2013).
Applications of Sericin in the Food Industry
2.4.1 In the food packaging and food coating
The current primary materials for food packaging are synthetic polymers Synthetic polymers are non-biodegradable and non-renewable, contributing to environmental pollution This issue can be mitigated by utilizing biodegradable polymers as alternatives to synthetic polymers (Takechi, T., & Takamura, H 2014) Sericin has been discovered to exhibit excellent reactivity and possesses numerous high biological functions, such as biodegradability, biocompatibility, antibacterial properties, and antioxidant capabilities (Zhang YQ, 2002)
Another article suggests that the use of a coating material containing sericin, chitosan, aloe vera, and glycerol has the potential to extend the shelf life of tomatoes when stored at 25°C and 70% relative moisture This material helps maintain the quantity of fruits and prevents the aging process, creating conditions similar to those after harvest ATR–FTIR analysis indicates that the coating material does not affect the structure of the fruits (Takechi, T., & Takamura, H, 2014)
Sericin is used in the production of bread When combined with 2–4 g sericin/1 kg of flour to make bread, there is a tendency to reduce the height and bulk density of the bread, as well as alter its color However, sericin maintains the uniform internal surface structure and preserves the original flavor (Takechi, T., & Takamura, H, 2014)
Another author reports the use of sericin in salad dressing (Takechi, T., & Takamura,
H, 2014) The author asserts that sericin, lacking immune-stimulating properties (Takechi, T., & Takamura, H, 2014), can be employed as an emulsifying agent instead of natural food emulsifiers like egg yolk and casein, which occasionally pose the risk of potential allergic reactions Furthermore, the emulsifying activity of sericin can be enhanced through acylation with oleic acid (Ogino, M., et al 2006).
The current research status of sericin
Currently, research on sericin in the field of food technology is attracting attention from the scientific community and the food industry worldwide Sericin, a protein derived from silk, is being explored and applied in various applications within the food industry Some studies focus on using sericin as a thickening agent, preservative, or antioxidant in food Its biological properties, including its ability to form gels and adhesion, offer potential for enhancing the quality and stability of food products Moreover, sericin is being investigated to create protective coatings to shield food from oxidation, contamination, and to maintain freshness and longevity
While still in the research and development phase, the application of sericin in food technology holds significant potential for improving the quality and attractiveness of food products, thereby creating opportunities for innovation in the food industry
Sericin is still being researched in various fields:
- Medical applications: There's particular interest in the medical potential of sericin Research focuses on medical material applications, including tissue regeneration, self- dissolving properties, and even the production of smart medical products
- Textiles and Fashion: Sericin is considered a potential raw material in the textile and fashion industries Its flexibility can lend unique characteristics to textile products
- Processing and Extraction: Research into sericin processing and extraction methods revolves around optimizing processes to ensure high quality and efficiency
- Pharmaceutical and Healthcare applications: Several studies emphasize sericin's potential in antioxidant, antibacterial properties, and other features in pharmaceuticals and healthcare products
Table 2.2 Pharmaceutical and Healthcare Applications
Anticancer activity Pandiarajan J., et al(2011)
Anti-tyrosinase activity Kumar J.P., et al ( 2017)
Kumar J.P., et al(1981) Anti-inflammatory activity Kaewkorn W., et al (2012) Anti-aging activity Wu J.-H., et al ( 2007)
Anticancer activity Khampieng T., et al ( 2015)
Deenonpoe R.,et al (2019) Anti-tyrosinase activity Kundu S.C.,et al (2008)
- Medical Applications: There's particular interest in the medical potential of sericin Research focuses on medical material applications, including tissue regeneration, self- dissolving properties, and even the production of smart medical products ( Fakharany, et al 2020)
- Textiles and Fashion: Sericin is considered a potential raw material in the textile and fashion industries Its flexibility can lend unique characteristics to textile products (Kumar, et al 2022)
- Processing and Extraction: Research into sericin processing and extraction methods revolves around optimizing processes to ensure high quality and efficiency (Lamboni et al 2015)
The research on sericin in Vietnam is drawing attention from both the research community and related industries Sericin, a protein derived from silk, is being explored and applied across various fields in Vietnam, including healthcare, food industry, pharmaceuticals, and textile industry
Studies on sericin in Vietnam are focused on optimizing the extraction process from silk and analyzing its properties and applications in different sectors (Le; et al., 2022)
In the healthcare sector, sericin is being researched for applications in tissue regeneration, the production of smart medical materials, and other advanced medical products
Within the food industry, sericin is being considered to enhance the quality and preservation characteristics of food products
In pharmaceuticals and healthcare, research is concentrating on sericin's antioxidant, antibacterial properties, and other beneficial characteristics for health-oriented product development
While still in progress, research on sericin in Vietnam holds significant potential for extensive development and application across various domains, especially when combining fundamental research with practical applications
The research findings on the extraction of silk proteins from silkworm cocoons, conducted by Le Hong Van and colleagues, were investigated using three methods: Na2CO3 salt utilization, neutral soap application, and the high-temperature, high-pressure method It was found that the extraction of sericin and fibroin by boiling silkworm cocoons in pure water at a temperature of 126°C and a pressure of 0.14 Mpa for 5 hours was determined to be the optimal method The sericin and fibroin powders were assessed to have high purity and were easily soluble in water Consequently, the powders of sericin and fibroin are regarded as potential raw materials for application in the cosmetic or food industry (Le; et al., 2022).
MATERIALS AND METHODS
Materials
The B mori silkworm cocoons used in the study are from silkworm farms in the Nam Dinh area After a small portion of the shell is sliced apart to reveal the silkworm pupae, the cocoons will be collected and transported to facilities that need silkworm cocoon shell materials The cocoon shell of the silkworm must be structurally intact, unstained, and purified
The silkworm harvest is in the fall from September 5 to 10 and ends in November every year, and the group purchases cocoon shells near the end of September.
Equipment for study
Table 3.1 Chemicals used in the study
2 Sodium carbonate (Na2CO3) China
7 Disodium phosphate (Na2HPO4) China
14 Folin Ciocalteu phenol reagent Germany
Table 3.2 Equipment used in the study
1 2 and 4-digit analytical balance Sartorius, Germany
7 Ultraviolet–Visible (UV-VIS) spectrophotometry Japan
An investigation of the raw material composition in the cocoon shells of silkworms (moisture, ash content, protein content, lipid, carbohydrates)
Investigate the effects of sericin extraction using high- temperature and high-pressure methods at different temperatures and times
Evaluation of the chemical properties of sericin
Evaluation of the biochemical properties of sericin
Evaluation of the physicochemical properties of sericin
10 Necessary equipment such as petri dishes, beakers, pipettes, etc.
Methods
- Determination of amino acid composition
- Determine of antibacterial activity of sericin
- Determine the molecular weight of protein (SDS electrophoresis - page)
- Evaluation of Ultraviolet-visible spectroscopy of sericin extract
Figure 3.1: Diagram of experimental research 3.3.2 Extraction process of sericin from silkworm cocoon
The study was carried out with the following contents: "Effect of high pressure and temperature on sericin protein properties of silkworm Bombyx mori" The study was carried out using this method with some modifications (Belhaj Khalifa, I.,et al, 2012)
Figure 3.2: Flow chart of sericin extraction process from silkworm cocoons 3.3.3 Experimental procedure
A purified cocoon is a cocoon that has been cleaned of silk and pupa inside the cocoon a) Cutting
Chop into strands with diameter 1– 2 mm to increase extraction efficiency
Figure 3.3 Chopped silk cocoon shells b) Degumming method
There are many different degumming methods, typically using distilled water, liquid neutral detergent, and sodium carbonate solution evaluated by Raman spectroscopy, or the method high temperature and high pressure using distilled water to extract sericin High- temperature and high-pressure degumming (HTHP) method is used to extract sericin from silkworm cocoons Currently, some degumming methods commonly used in the industry include degumming with alkaline solutions (Cao & Zhang, 2016) This method involves heating silk cocoons in hot distilled water without the use of any additional chemicals Investigations into the extraction were conducted at various temperatures and times Temperature and extraction time have a significant impact on the amount of sericin that is extracted Prepare glass test tubes or an Erlenmeyer flask containing cocoon shells and distilled water in a ratio of 1:30
- At temperatures from 70, 80, 90 degrees Celsius, heated in a thermostatic bath
- At 100 degrees Celsius, the extraction is done using an electric stove, boiling water and using a thermometer to measure the required temperature
- At temperatures of 110, 120, and 130 degrees Celsius, using an autoclave machine.
Table 3.3 Arrange experiments to investigate sericin extraction at different temperatures and times
After gumming, the sericin and fibroin-mixed solution was filtrated through Qualitative Filter Paper Fibroin is insoluble in water, while sericin is hydrophilic and can dissolve in water This filtering procedure separates silk fibroin
The sericin is separated from the sericin solution using a centrifuge machine following filtration.
Research Methods
3.4.1.1 Method for moiture content determination
Purpose (Oven-Drying Method): Silkworm cocoon is heated in a drying cabinet to remove moisture The initial and final weights of the sample are measured Moisture content is calculated based on the weight loss
Procedure: Drying the petri dish and lid drying cabinet at 105 °C for 30 minutes (label the order number on both the dish and lid) Allow the dish to cool in a desiccator to room temperature, then accurately weigh the combined mass of both the dish and lid 3 g of silkworm cocoons was dried at 105 °C for 6 hours until a constant mass is achieved Calculate moisture content as a percentage of the total weight (Anuduang A.,et al, 2020) The moisture content is calculated using the formula:
G1: mass of plate and sample before drying
G2: mass of plate and sample after drying
3.4.1.2 Method for determining total ash content of cocoon
Purpose: The determination of total ash refers to the analytical process of quantifying the total inorganic residue left behind after the combustion or incineration of a sample This residue, commonly known as ash, includes minerals, salts, and other inorganic components present in the original material
Procedure: Based on the method (Anuduang A et al.): empty, clean, and dry crucible and record the weight 2 g of sample into the crucible and record the weight Heat gradually to prevent sample decomposition Transfer the dried sample to a muffle furnace at 600 o C for 7 hours until complete combustion occurs In case there is still black residue, take it out to cool, add a few drops of concentrated H2O2 or HNO3, and heat again until the residue turns white The crucible with the ashed sample were recorded total their weight
The ash content is calculated using the formula:
G1: mass of crucible with silk cocoon
G2: mass of crucible with ash
3.4.1.3 Determination of total nitrogen content by Kjeldahl method of cocoon
Purpose: Determine the nitrogen content of sericin from silkworm cocoon samples (Gupta, D., et al, 2014)
Procedure: The experiment was performed based on the method of Gupta et al (2013) and with some adjustments, specifically:
- Digestion: Silkworm cocoon samples were weighed to the nearest 0.5 g Then, the Kjeldahl tubes containing the sample were inserted into the machine, and 15 mL of concentrated
H2SO4 was slowly added to the tubes Acids decompose organic matter through oxidation and denitrification in the form of liberated ammonium sulfate (Martín, J., 2017) To speed up decomposition, add a catalyst This experiment used 2.5 g of K2SO4 to increase the boiling temperature, 0.075 g of CuSO4, and 0.075 g of TiO2 to catalyze the reaction This process taked place within 3 hours at 75% of the capacity of the Kjeldahl digestion machine
- Distillation: The distillation of the solution now takes place, and a small quantity of sodium hydroxide is added to convert the ammonium salt to ammonia The sample is distilled using a nitrogen distillation machine The released NH3 is recovered in H3BO3 with two indicators: bromocresol green (0.1%) and methyl red (0.1%)
- Titration: The amount of NH3 in the H3BO3 solution was determined with standard 0.1 N HCl From the volume of 0.1 N HCl used for determination, % total nitrogen or % protein could be calculated as following:
Content (%) of total nitrogen in the sample:
With: % Protein in the sample = % 𝟏
N: Nitrogen content in weight percent
VHCl: number of mL of 0.1 N HCl standard solution used for titration, mL m: mass of sample; g
K: correction factor for HCl concentration 0.1N (the titration factor is considered equal to 1 if a HCl 0.1 N standard tube was used)
1/17= conversion factor to protein of silk sericin
The blank test with distilled water (1 mL) was carried out simultaneously with the silk cocoon samples
3.4.1.3 Determine the lipid content by Soxhlet method of cocoon
The procedural steps presented in the following section are based on Nielsen's method (2009) with some modifications 2 g of chopped silkworm cocoons, wrap in filter paper, and place in a separatory funnel Add the solvent mixture of diethyl ether and petrodium ether into the separatory funnel and let it overflow into the solvent container so that the amount of solvent takes up at least 1/3 of the container and not more than 2/3 of the solvent container Install the condenser tube Heat the solvent and proceed with extraction for 6–8 hours until the lipid is completely extracted At the end of the extraction process, the cocoon sample in the filter paper is removed from the excess solvent (placed in a fume hood and then dried to a constant weight) The amount of lipid in the sample is calculated by the mass loss compared to the original cocoon sample (unextracted cocoon sample)
In which: m1: Initial sample mass m2: Sample mass after extraction
3.4.1.4 Determine the carbohydrate content of cocoon
The carbohydrate content in the water sample is determined from the components of protein, lipid, moisture, and ash, and is calculated using the following formula:
% Carbohydrate= 100% - % Protein -% Lipid - Moisture 3.4.2 Determination of the optimal temperature during the sericin extraction process
Purpose: Determine the optimal temperature in the process of extracting silkworm cocoons
In this experiment, different temperatures from 70 to 130 °C was tested for a fixed period of
Procedure: 0.5 g of chopped silkworm cocoons was soak into 30 mL of distilled water, then extracted proceduce was carried out in different temperatures After extraction, the solution will be filtered through filter paper to remove fibroin After filtration, the solution will be centrifuged to remove residue Sericin content (mg/mL) will be determined using the Lowry method The experiment was repeated at least three times
Table 3.4 Arrange the experiment to investigate the sericin extraction process at different temperatures
130 3.4.3 Determine the optimal time during the sericin extraction process
Purpose: Determine the optimal time in the process of separating silkworm cocoons In this experiment, different times in the table 3.5 was studied within the optimal temperature range
Procedure: 0.5 g of chopped silkworm cocoons was soak into 30 mL of distilled water, then extracted proceduce was carried out in different time After extraction, the solution will be filtered through filter paper to remove fibroin After filtration, the solution will be centrifuged to remove residue Sericin content (mg/mL) will be determined using the Lowry method The experiment was repeated at least three times
Table 3.5 Arrange the experiment to investigate the sericin extraction process at different times
Analysis method
3.5.1 Estimation of sericin quality and quantity the protein content by Lowry's method
Based on the research method of (Lowry, Rosebrough, Farr, & Randall, 1951), most proteins contain tyrosine and tryptophan [13] The amount of these amino acids depends on the type of protein Therefore, proteins of the same type have the same content of these amino acids When the protein reacts with the Folin reagent, a colored complex will form The color intensity of this complex is proportional to the tyrosine content, and tryptophan is also a protein Measure color intensity using a spectrophotometer at 660 - 750nm wavelength (Shen, C H 2023) Therefore, we can use the colorimetric method to determine protein content
3.5.1.2 The Lowry method to determine Protein content
Reagent A: 20 g Na2CO3 and 4g NaOH, 1.51 g K2C4H4O6 mixed in 1000 mL distilled water
Reagent B: weigh 0.5 g CuSO4ã5H2O and dissolve in 1% sodium citrate solution to make 100ml
Solution C: (only used during the day) is a mixture of 2 solutions A and B mixed in a ratio of 50:1
Folin–Ciocalteu reagent: Dilute 5 times with distilled water before use
Based on the standard protein chart (BSA), the protein content to be analyzed can be determined
Determine protein content using a spectrophotometer at 660 nm wavelength of the research solution (Lowry, O., et al, 1951)
Measure on the machine 3 times and take the average value
1 gram of standard BSA was dissolved it in 100 mL of distilled water (10 mg/mL) Then diluting the stock solution with distilled water to concentrations of 2, 4, 6, 8, and 10 to construct a standard graph
Take exactly 0.5 mL of solution containing BSA with different concentrations into a test tube, add 2.5 mL of mixed drug solution, shake well with a shaker, and leave for 20 minutes Then, add 0.1 mL of Folin reagent diluted five times to the test tube mixture, shake well, and leave at room temperature for 20 minutes Compare the color of the mixture at the 660 nm wavelength
Standard sample: replace the solution containing BSA with distilled water; the remaining steps are similar
Take the average value and build a regression line
Take 0.5 mL of protein solution to be tested and put it in a test tube, add 2.5 mL of mixed drug solution, shake well with a shaker and leave for 20 minutes Then, add 0.1 mL of Folin reagent diluted 5 times to the test tube mixture, shake well and leave at room temperature for 20 minutes Compare the color of the mixture at 660 nm wavelength
Standard sample: replace the solution containing protein with distilled water, the remaining steps are similar.
Characterization of sericin solutions from HTHP extraction method
Purpose: As proteins absorb the near-ultraviolet area due to the electron transfer of aromatic amino acids, tryptophan, tyrosine, and phenylalanine, determine the quality of the proteins
Procedure: The experiment was performed based on the method (Rangi et al., 2015) Sericin solution will be measured using a UV-Vis spectrophotometer at a wavelength of (190–300 nm) in the ultraviolet light range
Purpose: Polyacrylamide gel electrophoresis (PAGE) is perhaps the most popular analytical technique that aims to separate and characterize the molecular weight of proteins
Procedure : The implementation method is based on Trudel et al (1989): a) Prepare gel separation (Resolving gel) at the anode (12.5%)
Prepare the mixture according to the following Table 3.6
- Shake gently to form a homogeneous solution
- Use a micropipette to pump the solution into the mold without creating air bubbles until it is about 5-8cm high
- Gently add a layer of distilled water to the gel and wait for it to harden (15–20 minutes) b) Prepare stacking gel at the cathode (4%)
Prepare the mixture according to the following Table 3.7:
- Shake gently to dissolve completely into a homogeneous solution
- After the separation gel has completely solidified, suck out the water layer above
- Use a micropipette to pump the solution onto the separating gel layer so that no air bubbles are created c) Run electrophoresis
- Gently insert the gel mold into the electrophoresis unit
- Add electrophoresis buffer to the electrophoresis chamber
- Add 10 μL of mix sample solution into the wells in order
- Close the electrophoresis box and close the circuit at a voltage of 100 Volts
- Runs for about 2 hours at room temperature with a 30 mA constant current
- Electrophoresis buffer composition: (g/L) Glycine 14.4 g; SDS 1 g; Tris-base 3.02 g use double-distilled water to make 1 liter e) Staining after SDS-PAGE electrophoresis
- After completing the electrophoresis, remove the gel from the plate
- Soak in the protein dye solution and shake gently for 30 minutes to 1 hour
Transfer the gel to soak in the sample washing solution; change the solution several times until the gel is transparent; at this time, blue bands will appear, which are protein bands
Dyeing solution: acetic acid 10 % (v/v), Coomassie Blue (Coomassie Brillant Blue-R250) 0.25 %, and methanol 50 % (v/v)
De-staining solution: acetic acid 10 % (v/v), methanol 25 % (v/v), and 65 % water
Purpose: The purpose is to analyze the molecular structure of sericin and allow the research sample to be distinguished from other types of protein materials
Procedure: The infrared spectroscopy method was based on the method of (Gupta et al.) To identify the functional groups found in sericin, the FTIR spectra of samples of the compound were recorded using an FTIR spectrometer (Jasco, Japan) Spectra in the range of 4000–400 cm-1 were acquired using the KBr pelleting process (Gupta, D., et al, 2014)
Principle: The isoelectric point of a protein is the pH point at which the protein's charge is zero, meaning it has an equal number of protons and electrons This point is also known as the pI point (isoelectric point)
Purpose: Defined as the pH level at which an atom transmits no net electrical charge Procedure: The experiment is performed according to the following Table 3.8:
Table 3.8 Experimental arrangement for the isoelectric method of sericin extract
After adding the chemicals according to the above table to 5 test tubes, shake well and compare the turbidity of the test tubes Measure the pH of test tubes using a handheld pH meter
3.6.5 Evaluation of amino acid composition
Purpose: Determination of amino acid composition of sericin extract
Procedure: Experiments were performed using AOAC Official Method 994.12 Amino acids in feeds ((AOAC (2010)).
Evaluation of the antioxidant activity of sericin
Principle: The test evaluates antioxidants ability to neutralize the (ABTS + ) stable radical cation, a blue-green chromophore with maximum absorption at 734 nm, whose intensity decreases in antioxidant presence (Ratnavathi, C V., & Komala, V V., 2016) The degree of blue-green discoloration is determined by the sample concentration, reaction duration, and intrinsic antioxidant activity It is measured as an abrupt drop in absorbance to 734 nm
Purpose: The ABTS assay measures the relative ability of antioxidants to scavenge the ABTS generated in aqueous phase (Ratnavathi, C V., & Komala, V V 2016)
Procedure: Based on the method (Pellegrini, N et al.): ABTS is a stable free radical cation ABTS is soluble in water at a concentration of 7mM Prepare a potassium persulfate solution with a concentration of 2.45 mM Prepare a cationic solution ABTS + , including 250 L of ABTS (7 mM) and 250 L of K2S2O8 (2.45 mM) Allow the mixture to stand in the dark at room temperature for 12–16 hours before use Stoichiometrically, ABTS and potassium on sulfate react in a 1:0.5 ratio, resulting in incomplete oxidation of ABTS, with absorbance peaking over 6 hours [18] The radical was stable in this form for more than two days when stored in the dark at room temperature [18] Dilute the ABTS solution with distilled water until the absorbance is 0.7 ± 0.02 at 734 nm
Then, the mixture consisted of 900 L of adjusted ABTS + solution and 100 L of sericin solution at different concentrations After giving the mixture a gentle shake, it was allowed to sit at room temperature for ten minutes in the dark Next, take a mixture measurement at
734 nm Distilled water was used as a blank sample
The free radical scavenging activity % ABTS was calculated according to the following equation ( Wangsawat et al., 2021):
ABTS radical scavenging (%) = [(Abs control – Abs sample )/Abs control ] × 100
Abscontrol: is the absorbance of the control reaction
Abssample: is the absorbance of the extract
The antiradical activity was expressed as IC50 (mg/mL), which represented the extract concentrations scavenging 50% of ABTS radicals.
Evaluation of antibacterial activity of sericin
Principle: Inhibition against foodborne pathogenic bacteria Potential inhibitory effect against both Gram-negative and Gram-positive bacteria based on optical density measurement (OD600nm) (Seo, S J., et al 2023)
Purpose: Investigating the antibacterial ability of the sericin that extracted by the water degumming method as well as antibacterial activity based on different concentrations of sericin
Escherichia coli (VTCC-B-482) and Staphylococcus aureus (VTCC-B-480) bacterial strains used in this study were purchased from the Institute of Microbiology and Biotechnology (Vietnam National University, Hanoi) Prepare bacterial culture materials: bacterial strains used to evaluate the antibacterial activity of sericin samples are
Staphylococcus aureus and Escherichia coli S Aureus and E Coli was cultivated in Peptone solution at 37 o C for 24 hours After 24 hours, the S Aureus and E Coli strains were diluted by taking 1 mL of the strain into a test tube containing 9 mL of peptone and NaCl solution Shake the test tube well; this solution has a dilution of 10 -1 Continue to take 1 mL of the same from the test tube with a dilution of 10 -1 into the test tube containing 9 mL of the second dilution solution Mix the solution well to obtain a solution with a dilution of 10 -2 Continue to proceed as above until reaching a dilution of 10 -5 OD660 nm=0.01
Sterilize Peptone medium (Glucose 1 %, Peptone 5 %, Yeast 2.5 %, Agar 20 %) and the necessary tools with an autoclave set at 121°C for 15 minutes The autoclave helps remove any potential contaminants and ensures a sterile environment
Stock solution: Sericin agar dilution was prepared by adding 6, 7, 8, 9, 10, and 11 mL of HTHP extracted sericin in 100 mL of sterilize Peptone medium, with the corresponding sericin concentrations of 0.56, 0.66, 0.75, 0.84, 0.94, and 1.03 mg/mL
Experiment: 100 L of bacteria solution (OD660 nm=0.01) was applied into petri plate Then, 20mL Sericin agar dilution with various sericin concentration was applied Mix the medium with the bacteria thoroughly by rotating the petri dish clockwise and counterclockwise Let it stand for 15 minutes Finally, the sample was incubated in an incubator at 37 o C for 24 hours [22] Read the results
The experiments were repeated at least three times The above operations are all performed under sterile conditions ((Biological Safety Cabinet (BSC))
How to calculate the number of colonies per 1 mL sample:
N = Number of colonies per ml or g of product Σ c = Sum of all colonies on all plates counted n1 = Number of plates in first dilution counted n2 = Number of plates in second dilution counted d = Dilution from which the first counts were obtain
RESULTS AND DISCUSSIONS
Raw silk cocoon composition
Conducting measurements of the parameters of Bombyx mori silk cocoon shells, including determining moisture content, ash, and total protein content as presented in section 3.4 The results obtained are shown in Table 4.1 below
Table 4.1 Some Parameters of Bombyx Mori Silk Cocoon Shell Raw Material
The experimental results revealed that the moisture content of silkworm cocoon shells is 5.6% This finding was consistent with results of the research of Kweon et al Kwweon reported a moisture content of 5.9–6.4 % in the cocoon shells Hegazy et al indicated that fresh silkworm cocoon shells have a moisture content ranging from 10–12 % (Kweon H and Jo Y Y 2012) To preserve silkworm cocoon shells at room temperature, extend shelf life, and prevent microbial growth, various drying methods are commonly employed before introducing the cocoon shells to the market The final moisture content of the cocoon shells will depend on the specific drying methods applied Despite belonging to the same B mori species, environmental variations and cultivation conditions could also impact the characteristics of silkworm cocoon shells
The ash composition in silkworm cocoon shells typically includes inorganic minerals and elements that remain after the organic components have been burned or incinerated Ash content is a measure of the total mineral content in a material
Table 4.1 showed that the ash content in silkworm cocoon shells is 0.9 % This result differed from the findings of Kweon et al.which reported an ash content in cocoon shells ranging from 0.3 % to 0.4 % However, it was aligned with the results of Gulrajani et al where the ash content could vary between 0.8 % and 5.2 % This indicated that the specific minerals present in silkworm cocoon ash could vary depending on many factors such as the silkworm's diet, environmental conditions, and processing methods
The total protein content of silkworm cocoon using the Kjeldahl method is 85.2 % The typical composition of silk consists of approximately 75–83 % fibroin and 17–25 % sericin, which might include secondary metabolites, wax, pigments, carbohydrates, and various other impurities (Heliyon, et al 2022) The total protein content in silkworm cocoons comprises over 90 %, including fibroin and sericin proteins The experimental results from our group slightly differed from those reported by Heliyon et al in 2022 Despite both studies focusing on B.mori, variations in the living environment and diet also influenced the overall protein content in silkworm cocoons.
Determination of the optimal temperature during the sericin extraction process
To investigate the effect of temperature on the extraction process of sericin with distilled water as solvent under temperature conditions of 90, 100, 110, and 121, 130 o C for a fixed time of 30 minutes, as presented in Section 3.2 The obtained results are shown in
Temperature ( o C) Figure 4.1: Sericin content in various temperature extraction
Based on the results in Figure 4.1, it showed that sericin concentration progressively was raised from low to high at under 100 o C temperatures At temperatures of 70 o C, 80 o C,
90 o C, and 100 o C, there is a slight increase in sericin concentration, but unclearly Specifically, at these temperatures, the sericin concentrations were 2.74±0.183; 4.77±0.29; 5.67± 0.17; and 7.50±0.17 mg/mL The sericin concentration obtained at the above temperatures is relatively low, and the sericin yield is correspondingly lower (Wang et al, 2021) This also indicated that molecular decomposition of sericin hardly occurred until treatment at temperatures (70, 80, 90, and 100 o C) for 30 min As a reference to the decomposition of sericin at low temperatures (Jang, M J et al, 2017), when silk cocoons were treated with water at the above temperatures for 30 min, a very small amount of sericin was extracted, inferring a low degumming process rate in Figure 4.1 The results of the extraction experiment showed that the extraction and decomposition of sericin molecules hardly took place at the above temperatures within 30 minutes
When extraction temperature was over 100 o C, sericin content was extremely increased For example: 30.7±0.5, 37.17±0.6 and 30.5±0.4 mg/mL at 110 o C, 120 o C and 130 oC respectively are 3 times higher than under 100 o C temperature From 110 o C to 120 o C, higher temperature raised the sericin content However, after 120 o C, the higher sericin content was not happened At 130 o C, the sericin concentration decreased compared to the sericin concentration at 120 o C
Concentration of extraction (mg/mL)
In conclusion, it showed that the sericin concentration is highest at 120 o C comparing to the surveyed temperatures The conclusion is similar to previous studies of (Saha et al, 2019), the molecular decomposition of sericin depends on the processing temperature (Wang et al, 2021) It was suggested that the temperature affects the absorption of sericin concentration (Wang et al, 2021)
As the increasing temperature, the thermodynamics of the molecules increase, so the extraction process took easily place, and the obtained sericin content also gradually increased However, the nature of sericin is protein, which could be denatured by temperature or extraction time Therefore, once the optimal sericin content extraction conditions were achieved, the sericin would begin to denature and be removed during the filtration and centrifugation steps, causing the decreasing of sericin content
The results also showed that at 130 o C, the obtained sericin concentration was 30.25±0.4 mg/mL, significantly reduced that compared to 120 o C extraction When the temperature was increased, protein molecules were changed their structure and lose their activity Under the effect of temperature, the molecule was dilated because the secondary bonds were broken, leading to the release of some functional groups (SH, NH2, COOH) Then the protein molecules bond together without any rules, forming a precipitate
The results of the ANOVA analysis of the investigated temperature during the sericin extraction process are presented in Appendix 2 The coefficient showed a P value of less than 0.05 (p