This patent application describes a method for extracting chitin from animal biomass in a single step using enzymatic hydrolysis in an acid medium. The method involves homogenizing raw material containing chitin and adding enzymes active in acid conditions. This allows for proteolysis and solubilization of insoluble materials in a single step to extract chitin. The extracted chitin can then be further processed into products like chitosan, oligo-chitin, and glucosamines. The single-step enzymatic extraction method reduces the need for multiple extraction and washing steps compared to conventional chemical extraction methods.
8- IJRANSS-ANTIMICROBIAL POTENTIAL OF MARINE ACTINOMYCETESRavindragouda Patil
- 47 actinomycetes were isolated from mangrove swamp samples in Tamil Nadu, India, with most isolated from sediment.
- 34 isolates exhibited antagonism against shrimp pathogens Vibrio alginolyticus, V. harveyi, and V. parahaemolyticus.
- Isolate A10 showed the strongest inhibitory activity against all three pathogens, inhibiting growth by ≥20mm.
- Isolate A10 was identified as Streptomyces spp. based on chemotaxonomic and microscopic analysis.
Production of Spirulina in semicontinuous cultivation using medium recycleIJERA Editor
The biomass of cyanobacteria Spirulina is rich in bioactive compounds, with main applications in food and pharmaceutical industry. The objective of this study was to evaluate the effect of nutrient consumption on the growth kinetics of Spirulina sp. LEB 18 grown in semicontinuous cultivation in raceway bioreactor. Zarrouk medium was used, maintaining the original concentrations of carbon, nitrogen and phosphorus, while the other nutrients varied by 25%, 50%, 75%, and 100%. Chemical determinations were performed in the biomass for each growth cycle. Spirulina sp. LEB 18 exhibited cell growth until approximately 130 days of experiment. The maximum specific growth rate (0.184 d-1) and productivity (48.03 mg L-1 d-1) were observed in the trial with 50% Zarrouk medium, except for nitrogen, phosphorus and carbon. Nitrogen and phosphorus concentrations reduced up to 100% and 64.8%, respectively. No significant difference (p <0.0003) was observed in the protein concentrations for all tests. The results showed that Spirulina sp. LEB 18 can provide high growth rate and productivity, as well as production of proteic biomass when cultivated with lower nutrients concentration in Zarrouk medium, thus reducing production costs.
Development of a Bioactive Food Additive for Controlling of Fungal GrowthIJEAB
Fresh foods have a great importance in human nutrition. However, they are marketed with greatly reduced shelf life mainly due to fungal spoilage. In this work, cell wall degrading enzymes produced by Trichoderma asperellum T00 (TCWDE) were immobilized onto cashew gum polysaccharide (CGP) in order to evaluate the potential use of this material as food additive aiming to increase the shelf life by inhibiting fungal growth. Results from factorial design (32) evidenced that the best conditions for TCWDE immobilization was achieved with 20 min of reaction using 1 mmol L-1 of NaIO4. On these conditions it was observed 91% of retention yield for NAGase (30.1 ± 0.38 U mL-1), 41% for chitinase (0.67 ± 0.05 U mL-1), and 24% for β-1,3-glucanase (0.017± 0.001 U mL-1). CGP/TCWDE was effective for growth inhibition of Aspergillus fumigatus and Penicillium sp. and the inhibition mechanism seems to involve changes in the cell wall of those microorganisms. Finally, the CGP/TCWDE presented high stability after drying, maintaining enzymatic and biological activity after 200 days of storage at room temperature (25 ºC).
This document discusses the isolation and characterization of microorganisms from dairy effluent for use in activated sludge treatment. Fifteen bacterial isolates were obtained from dairy plant effluent samples. The isolates were characterized based on their cultural, morphological, biochemical and enzymatic properties. Several isolates showed amylase, urease and gelatinase activity. Most isolates were able to utilize glucose and other carbohydrates. Through characterization, microorganisms were identified that could potentially break down dairy effluent organic pollutants in activated sludge treatment.
This document describes the development of a sensitive in vitro bioassay to quantify the biological activity of phorbol esters in Jatropha oil. The researchers:
1) Confirmed that exposure to Jatropha oil triggered the same cellular response in MDCK cells as the model phorbol ester TPA.
2) Showed that Jatropha oil's phorbol esters activate protein kinase C, similarly to TPA.
3) Measured the expression of COX-2, an inflammation-related gene, in MDCK cells exposed to Jatropha oil and used this to quantify phorbol ester activity in terms of TPA toxic equivalents (TEQ).
The document discusses the antimicrobial properties of chitosan and its applications in plant disease control. Chitosan exhibits antimicrobial activity against viruses, bacteria, fungi and oomycetes. The level of suppression varies based on factors like molecular weight and chemical composition. It has been used as a seed coating agent to improve germination and as a foliar treatment to increase photosynthetic rates in plants. Studies have found chitosan and its derivatives can restrict the growth of pathogens and be effective for controlling plant diseases when applied as a soil amendment or foliar treatment.
The study investigated using essential oils to reduce bacteria in Artemia fransicana, a live food for aquaculture. Origanum vulgare completely inhibited hatching. All treatments significantly reduced presumed Vibrio counts. None significantly reduced general bacteria. Rosmarinus offcinalis reduced presumed Vibrio without inhibiting hatching. It showed potential for further study to identify maximum safe concentrations and effects on nutrition and bioaccumulation.
This document describes the development of an active and intelligent starch-based biodegradable food packaging system. Polyvinyl alcohol (PVA) and starch are used as the base polymers and cross-linked with glutaraldehyde to form films. Propolis extract is added as an active agent for its antimicrobial properties. Anthocyanin extracted from red cabbage is used as an intelligent agent due to its pH-responsive color change. Different film formulations are prepared by varying the concentration of propolis extract. The films are characterized through various tests to analyze their mechanical properties, water vapor transmission rate, moisture retention capability, swelling degree, biological leaching ability, colorimetric response, and antimicrobial activity. Films containing 20
8- IJRANSS-ANTIMICROBIAL POTENTIAL OF MARINE ACTINOMYCETESRavindragouda Patil
- 47 actinomycetes were isolated from mangrove swamp samples in Tamil Nadu, India, with most isolated from sediment.
- 34 isolates exhibited antagonism against shrimp pathogens Vibrio alginolyticus, V. harveyi, and V. parahaemolyticus.
- Isolate A10 showed the strongest inhibitory activity against all three pathogens, inhibiting growth by ≥20mm.
- Isolate A10 was identified as Streptomyces spp. based on chemotaxonomic and microscopic analysis.
Production of Spirulina in semicontinuous cultivation using medium recycleIJERA Editor
The biomass of cyanobacteria Spirulina is rich in bioactive compounds, with main applications in food and pharmaceutical industry. The objective of this study was to evaluate the effect of nutrient consumption on the growth kinetics of Spirulina sp. LEB 18 grown in semicontinuous cultivation in raceway bioreactor. Zarrouk medium was used, maintaining the original concentrations of carbon, nitrogen and phosphorus, while the other nutrients varied by 25%, 50%, 75%, and 100%. Chemical determinations were performed in the biomass for each growth cycle. Spirulina sp. LEB 18 exhibited cell growth until approximately 130 days of experiment. The maximum specific growth rate (0.184 d-1) and productivity (48.03 mg L-1 d-1) were observed in the trial with 50% Zarrouk medium, except for nitrogen, phosphorus and carbon. Nitrogen and phosphorus concentrations reduced up to 100% and 64.8%, respectively. No significant difference (p <0.0003) was observed in the protein concentrations for all tests. The results showed that Spirulina sp. LEB 18 can provide high growth rate and productivity, as well as production of proteic biomass when cultivated with lower nutrients concentration in Zarrouk medium, thus reducing production costs.
Development of a Bioactive Food Additive for Controlling of Fungal GrowthIJEAB
Fresh foods have a great importance in human nutrition. However, they are marketed with greatly reduced shelf life mainly due to fungal spoilage. In this work, cell wall degrading enzymes produced by Trichoderma asperellum T00 (TCWDE) were immobilized onto cashew gum polysaccharide (CGP) in order to evaluate the potential use of this material as food additive aiming to increase the shelf life by inhibiting fungal growth. Results from factorial design (32) evidenced that the best conditions for TCWDE immobilization was achieved with 20 min of reaction using 1 mmol L-1 of NaIO4. On these conditions it was observed 91% of retention yield for NAGase (30.1 ± 0.38 U mL-1), 41% for chitinase (0.67 ± 0.05 U mL-1), and 24% for β-1,3-glucanase (0.017± 0.001 U mL-1). CGP/TCWDE was effective for growth inhibition of Aspergillus fumigatus and Penicillium sp. and the inhibition mechanism seems to involve changes in the cell wall of those microorganisms. Finally, the CGP/TCWDE presented high stability after drying, maintaining enzymatic and biological activity after 200 days of storage at room temperature (25 ºC).
This document discusses the isolation and characterization of microorganisms from dairy effluent for use in activated sludge treatment. Fifteen bacterial isolates were obtained from dairy plant effluent samples. The isolates were characterized based on their cultural, morphological, biochemical and enzymatic properties. Several isolates showed amylase, urease and gelatinase activity. Most isolates were able to utilize glucose and other carbohydrates. Through characterization, microorganisms were identified that could potentially break down dairy effluent organic pollutants in activated sludge treatment.
This document describes the development of a sensitive in vitro bioassay to quantify the biological activity of phorbol esters in Jatropha oil. The researchers:
1) Confirmed that exposure to Jatropha oil triggered the same cellular response in MDCK cells as the model phorbol ester TPA.
2) Showed that Jatropha oil's phorbol esters activate protein kinase C, similarly to TPA.
3) Measured the expression of COX-2, an inflammation-related gene, in MDCK cells exposed to Jatropha oil and used this to quantify phorbol ester activity in terms of TPA toxic equivalents (TEQ).
The document discusses the antimicrobial properties of chitosan and its applications in plant disease control. Chitosan exhibits antimicrobial activity against viruses, bacteria, fungi and oomycetes. The level of suppression varies based on factors like molecular weight and chemical composition. It has been used as a seed coating agent to improve germination and as a foliar treatment to increase photosynthetic rates in plants. Studies have found chitosan and its derivatives can restrict the growth of pathogens and be effective for controlling plant diseases when applied as a soil amendment or foliar treatment.
The study investigated using essential oils to reduce bacteria in Artemia fransicana, a live food for aquaculture. Origanum vulgare completely inhibited hatching. All treatments significantly reduced presumed Vibrio counts. None significantly reduced general bacteria. Rosmarinus offcinalis reduced presumed Vibrio without inhibiting hatching. It showed potential for further study to identify maximum safe concentrations and effects on nutrition and bioaccumulation.
This document describes the development of an active and intelligent starch-based biodegradable food packaging system. Polyvinyl alcohol (PVA) and starch are used as the base polymers and cross-linked with glutaraldehyde to form films. Propolis extract is added as an active agent for its antimicrobial properties. Anthocyanin extracted from red cabbage is used as an intelligent agent due to its pH-responsive color change. Different film formulations are prepared by varying the concentration of propolis extract. The films are characterized through various tests to analyze their mechanical properties, water vapor transmission rate, moisture retention capability, swelling degree, biological leaching ability, colorimetric response, and antimicrobial activity. Films containing 20
CULTIVATION OF OSCILLATORIA SP IN DAIRY WASTE WATER IN TWO STAGE PHOTO BIOREA...civej
This paper presents an integrated approach to cultivate microalgae in dairy wastewater and to
investigate the capability of the organism for biodiesel production. The present study was carried out
using tolerant strains of microalgae collected from dairy effluent treatment plant, Kochi. Selected blue
green algal strains were mass cultured in the laboratory and acclimatized using different concentrations
of synthetic effluent. Blue green algal filaments were immobilized inside the primary and secondary
photobioreactors. The experiment was conducted in two stages including batch and continuous
treatment. The stage 1 of the experiment was designed for the reduction of physical impurities and the
nutrients. Stage 2 was designed mainly for the cultivation of blue green algae in dairy waste water by
utilizing the extra nutrients . Reduction of 94 -99.5% in phosphate was observed after 48 h of treatment
in the primary and secondary photobioreactors. The level of phosphate, total hardness, ammoniacal
nitrogen in the MSE was reduced by 97%,93 %, 81% respectively. BOD was reduced to 370mg L-1 from
1500 mg L-1 after 48 hrs of treatment in the primary reactor. COD was reduced to 85 mg L -1 from an
initial value of 1500 mg L -1 from medium strength effluent (MSE) and 90-95 % removal of COD was
also obtained from high strength effluent(HSE) during the study period. Biomass developed within the
reactor was harvested at every 15 days intervals from the secondary reactor and analyzed for lipids and
fattyacids. Presence of C14:0, C16:0,C18:0, C18:1 and C18:2 fatty acids strongly supports its abilility for
biodiesel production.
Hepatoprotective and antioxidant effects of Azolla microphylla based gold nan...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
Our present study sought to evaluate hepatoprotective and antioxidant effects of methanol extract of Azolla microphylla phytochemically synthesized gold nanoparticles (GNaP) in acetaminophen (APAP) - induced hepatotoxicity of fresh water common carp fish.
Materials and Methods:
GNaP were prepared by green synthesis method using methanol extract of Azolla microphylla. Twenty four fishes weighing 146 ± 2.5 g were used in this experiment and these were divided into four experimental groups, each comprising 6 fishes. Group 1 served as control. Group 2 fishes were exposed to APAP (500 mg/kg) for 24 h. Groups 3 and 4 fishes were exposed to APAP (500 mg/kg) + GNaP (2.5 mg/kg) and GNaP (2.5 mg/kg) for 24 h, respectively. The hepatoprotective and antioxidant potentials were assessed by measuring liver damage, biochemical parameters, ions status, and histological alterations.
Results:
APAP exposed fish showed significant elevated levels of metabolic enzymes (LDH, G6PDH and MDH), hepatotoxic markers (GPT, GOT and ALP), reduced hepatic glycogen, lipids, protein, albumin, globulin, increased levels of bilirubin, creatinine, and oxidative stress markers (TBRAS, LHP and protein carbonyl), altered the tissue enzymes (SOD, CAT, GSH-Px and GST) non-enzyme (GSH), cellular sulfhydryl (T-SH, P-SH and NP-SH) levels, reduced hepatic ions (Ca2+, Na+ and K+), and abnormal liver histology. It was observe that GNaP has reversal effects on the levels of above mentioned parameters in APAP hepatotoxicity.
Conclusion:
Azolla microphylla phytochemically synthesized GNaP protects liver against oxidative damage and tissue damaging enzyme activities and could be used as an effective protector against acetaminophen-induced hepatic damage in fresh water common carp fish.
This document describes a study that developed a method to determine off-flavor compounds geosmin and 2-MIB in live fish using in vivo solid-phase microextraction (SPME). Two kinetic calibration methods were investigated: on-fiber standardization and measurement using predetermined sampling rates. Both methods were validated using traditional analysis of samples collected through lethal sampling. The developed in vivo SPME method was then applied to determine geosmin and 2-MIB levels in live fish in a recirculating aquaculture system, with detection limits below human sensory thresholds.
Phytase from Bacillus cereus MTCC 10072 was purified about 10.75 fold to apparent homogeneity with a recovery of 34% referred to the phytase activity in the crude extract. The monomeric enzyme displayed molecular weight of 45 KDa and showed maximum activity at temperature 60 ºC and pH 6.5. Iso electric point of the purified enzyme was found to be 5.6. Substrate specificity studies showed it is highly specific to its substrate and maximum relative activity of 128% was obtained with calcium phytate. Activity was unaffected or moderately stimulated by a range of metal ions with only Ca2+ exerting (118%) stimulatory effect. The enzyme is significantly thermo stable at 60 ºC and retains a significantly greater proportion of maximal activity at physiological temperatures. This may render it of industrial interest. Further to check the applicability of the enzyme effect of different doses of crude enzyme (10, 25, 50 and 100 units) in dephosphorylation of animal feed was evaluated. Up to 66 h of incubation, the animal feed was monitored for the released inorganic phosphate content present in the feed. An enzyme dose of 100U and 50U of crude phytase enzyme per flask were found suitable to liberate enough amount of inorganic phosphorus in case of poultry and pig feed respectively.
This document summarizes a study that investigated how incorporating rosemary essential oil (REO) at concentrations of 0.5%, 1.0%, and 1.5% affects the properties of chitosan-based films. The main findings were:
1) Incorporating REO up to 1.5% decreased the film's solubility in water by about 25% and water absorption by 85% due to interactions between chitosan and REO.
2) REO improved film transparency from 4.97 for neat chitosan films to 7.61 and reduced light transmission, especially in the UV range, by over 25%.
3) Films containing REO showed greater antibacterial activity against foodborne
This document discusses chitin and chitosan. It begins with a brief history of the discovery of chitin. It then discusses the sources of chitin in nature, including the exoskeletons of arthropods, cell walls of fungi, and shells of mollusks. The document outlines the isolation and production processes of chitin and chitosan from sources like shellfish and fungi. It also summarizes some of the key applications and properties of chitin and chitosan.
Comparative study between film and coating packaging based onJesus Almaraz Muñoz
This study compared the effectiveness of a shrimp concentrate film and coating at preserving fish sausages during chilled storage. The coating increased the lag phase of total microorganisms and enterobacteria to 15 and 10 days, respectively, while the film drastically inhibited the growth of these microbes. Both helped maintain quality by decreasing pH and volatile bases during storage, with greater effects seen from the film. The coating kept sausage properties similar to controls for 25 days while the film resulted in a lower moisture and pH product resembling pickled sausage that remained stable for at least 42 days.
HHP processing of avocado paste at 600 MPa for 3 minutes significantly increased total extractable carotenoid levels by about 56%. During storage of the processed paste for 40 days at 4°C, carotenoid levels declined but remained higher than in unprocessed paste. ORAC values, a measure of antioxidant capacity, decreased after processing but increased during storage, indicating carotenoids contribute minimally to total antioxidant capacity. The study showed HHP processing and refrigerated storage retained avocado carotenoids and bioactive compounds within the product's sensory shelf life.
Biotechnological applications in agriculture a new source of edibleSiamak Alizade
This document summarizes research on using the Terminalia belerica plant for various biotechnological applications. Key points:
- The plant's seeds can be used to produce a new edible oil, comprising 37% of the dry kernel weight. Analysis found the oil contains fatty acids similar to olive oil.
- Byproducts of oil extraction include an oilcake containing high levels of nitrogen (8.34%) and proteins (60% digestible) suitable for use as biofertilizer.
- Other byproducts include tannins extracted from the fruit pulp, suitable for use in leather/medicine, and antioxidants like gallic acid in the seed coat with potential to preserve vegetable oils.
Gelatin-based nanocomposite films were prepared with zinc oxide nanoparticles (ZnO-NPs) and glycerol to develop sensitive layers for monitoring relative humidity in food packaging. The incorporation of ZnO-NPs and glycerol induced changes in the films' morphology, structure, water contact angle and vapor permeability. Electrical characterization showed the nanocomposite films were highly sensitive to changes in relative humidity, responding positively with a sensitivity of 99.47%. The results suggest gelatin-ZnO nanocomposite films have potential for use in sensors to monitor relative humidity conditions important for food quality and safety.
HPP processing of avocados and guacamole provides several benefits:
1) It inactivates spoilage microorganisms like bacteria and molds, extending shelf life to 40 days while maintaining sensory quality.
2) Pathogens like L. monocytogenes and Salmonella are reduced by over 5 log cycles.
3) Color and nutrients are largely preserved since HPP does not use heat, inactivating enzymes like PPO by only 50% and maintaining carotenoid and antioxidant levels.
4) Refrigerated storage after HPP further controls enzyme activity and microbes, protecting quality for the extended shelf life.
INVESTIGATION ON ISOLATED AND PURIFIED LIPOXYGENASE FROM AVOCADO IN THE PRESE...ljmonking
This document describes research on isolating and characterizing lipoxygenase (LOX) enzyme from avocado. Key points:
- LOX was isolated from avocado through a multi-step purification process involving acetone extraction, pH adjustment, salt precipitation, and ultracentrifugation.
- The purified LOX's activity was tested using linolenic acid as a substrate. Its optimum pH was found to be 6.5 and optimum temperature was 40°C.
- Kinetic parameters Km and Vmax for LOX were determined using the Lineweaver-Burk method with varying linolenic acid concentrations. Characterization of avocado LOX could help minimize its detrimental effects on
The document summarizes a study that investigated the chemical composition and antioxidant activity of a glycoprotein purified from the seaweed Undaria pinnatifida. The key findings are:
1) The purified glycoprotein (UPGP) consisted of 42.53% carbohydrate and 57.47% protein.
2) UPGP showed dose-dependent antioxidant activities in various assays both before and after simulated digestion, though activities decreased in the gastric phase and increased in the duodenal phase.
3) UPGP also showed protective effects against oxidative DNA damage both before and after certain phases of simulated digestion.
This document characterizes various properties of an exopolysaccharide (EPS) produced by Lactobacillus kefiranofaciens ZW3 isolated from Tibetan kefir, including its molecular weight, solubility, water and oil binding capacities, structure as viewed by scanning electron microscopy and atomic force microscopy, rheological properties under different conditions, melting point, and degradation temperature. The molecular weight was determined to be 5.5 x 10^4 Da by gel permeation chromatography. Solubility in water was 14.2% and water and oil binding capacities were 496.0% and 884.74% respectively. Scanning electron microscopy showed a smooth surface structure and atomic force microscopy revealed an almost
IRJET- Scavenging Efficiency of Azolla Pinnata in Effluent as Remediation AgentIRJET Journal
This document summarizes a study on using the aquatic fern Azolla pinnata to remediate effluent from a sugar cane industry. Key findings include:
1) Azolla pinnata showed significant growth in diluted effluent (25% concentration) and helped reduce levels of pollutants like BOD, COD, and suspended solids.
2) Parameters like chlorophyll content, relative growth rate, and seed germination increased for Azolla grown in diluted effluent, indicating uptake of nutrients promoted growth.
3) Treated effluent supported Azolla biomass production and was suitable for irrigation, while the biomass could be used as fertilizer due to accumulated
Characterization of fresh moringa oleifera beverageAlexander Decker
This document characterizes a fresh Moringa oleifera beverage made from 50% moringa leaf extract, 38% pineapple juice, and 12% carrot extract. Proximate and chemical analyses were conducted on the beverage. The beverage contained 2.9g/100ml of protein, 1.02mg of iron, and 159.14mg/100ml of vitamin C. After 8 weeks of storage under different conditions, 78% of the initial vitamin C content was retained, even under sunlight. No microbial growth was observed under any storage conditions, and sensory evaluation found the product remained acceptable. The beverage is concluded to be an excellent way to provide nutrients to malnourished individuals and consumers.
Characterization, treatment and recovery of fish byproduct as a stable bio-fe...Skyfox Publishing Group
National fish production reached in 2013 an amount of 1,245,912 tons. Now the pelagic fish industry generates a
significant amount of waste up to 60%. The physicochemical analyses of these fish by-products showed a wealth of organic material
including proteins (87.4% dry weight) and minerals (9% dry weight). These components and others are capitalizing to be used for
agricultural or agri-food. Biotransformation of biotechnologically in fish products was performed in the laboratory. Fresh fish byproducts
were ground and mixed with a carbon-rich source of carbohydrates. The mixture was inoculated with selected fermentation
yeast. The fermentation of evolution was controlled by monitoring chemical parameters (pH, dry matter, ash, total nitrogen) and
microbiological (FMAT, Enterobacteriaceae: fecal and total Coliforms, yeasts). Then the product thus stabilized at a pH of 3.8 has
undergone a second treatment. A hydrolyzate rich in amino acids and trace elements was obtained with excellent hygienic quality after a
total elimination of pathogens. Its use will be as factors for fertilizer and soil amendment. We produced three biostimulants necessary for
plant growth: Rooting fertilizer, Plant elongation fertilizer and production fertilizer. The application tests on these biofertilizers in the
laboratory were performed and have allowed obtaining promising results.
This document describes a study that developed and characterized intelligent food packaging films containing a biohybrid material based on anthocyanins from eggplant peel and laponite. The biohybrid was produced by adsorbing anthocyanins onto the surface of laponite. Chitosan films were produced containing this biohybrid. The films with biohybrid were thicker, less soluble in water, and changed color when exposed to different pH solutions, indicating they could be used to monitor food freshness based on pH changes. The films were able to change color from gray to red or yellow depending on acidic or basic conditions, similarly to how anthocyanins change color with pH. The films were tested for monitoring meat freshness at different temperatures by correlating film
ABSTRACT In the present study, Chitin has been extracted from Agaricus bisporus (Button mushroom). The obtained chitin was converted into the more useful chitosan and the crude chitosan extract was measured for its absorption maxima by UV Spectrophotometer and the maximum peak at 265nm was observed. FT-IR spectroscopy was done to identify the functional groups present in the chitosan which was analyzed between the ranges of 4000–400 cm-1. Chitosan was characterized by significant amide bands at 3265.49 cm−1. The absorbance bands of 1402.25, 1153.43, 900.76 and 445.56 cm-1 indicates CH2 stretching, CH stretching, C=O stretching in secondary amide respectively which confirms the structure of chitosan. The antioxidant activity of chitosan was determined by DPPH free radical scavenging assay and the value gained is 65.90% at 250 mg/ml which is due to the presence of rutin, gallic acid, caffeic acid and catechin in the phenolic composition of Agaricus bisporus. Finally, in vitro antibacterial screening of chitosan from Agaricus bisporus was performed against selected clinical isolates and the zone of inhibition shows highest activity in Bacillus subtilis, P. aeruginosa followed by K. pneumonia, and Acinetobacter baumannii. These findings suggest that the Agaricus bisporus act as the potential source to produce eco-friendly chitin and chitosan in the development of drugs, artificial bone and raw material for food industries in near future.
Key-words: Agaricus bisporus, Chitosan, Antioxidants, Antibacterial activity, UV spectroscopy, FTIR spectroscopy
Protein Extraction and Purification of Soybean Flakes and Meals Using a Lime ...IJMER
This document describes a study that investigated extracting and purifying protein from soybean flakes and meals using lime treatment followed by ultrafiltration. Key findings include:
- Soybean flakes and meals were produced from whole soybeans using conventional methods. Flakes had higher protein and carbohydrate extraction than meals after treatments.
- Lime treatment at 0.5 moles resulted in the highest extraction of total dissolved solids (TDS) from both flakes and meals, compared to water, NaOH, or other lime concentrations.
- Ultrafiltration purified the protein extracts from flakes and meals with 94-97% protein yield and removal of carbohydrates through diafiltration.
- Lime treatment is a potential
Bioproduction of bioactive compounds screening of bioproduction conditions of...ainia centro tecnológico
1. The document describes a study screening bioproduction conditions for microalgae and lichen symbionts to produce bioactive compounds. It examines the adaptation of various microalgae like Chlorella and Asterochloris erici to different culture media and processing methods.
2. The results show that Chlorella vulgaris was able to grow under mixotrophic conditions using various carbon sources and treatments like ultrasounds increased bioactivity. Asterochloris erici was adapted from solid to liquid culture and able to be cultured at large scale under autotrophic conditions.
3. The study demonstrates that bioproduction technologies can be used to obtain high-value compounds from microalgae and lichen
CULTIVATION OF OSCILLATORIA SP IN DAIRY WASTE WATER IN TWO STAGE PHOTO BIOREA...civej
This paper presents an integrated approach to cultivate microalgae in dairy wastewater and to
investigate the capability of the organism for biodiesel production. The present study was carried out
using tolerant strains of microalgae collected from dairy effluent treatment plant, Kochi. Selected blue
green algal strains were mass cultured in the laboratory and acclimatized using different concentrations
of synthetic effluent. Blue green algal filaments were immobilized inside the primary and secondary
photobioreactors. The experiment was conducted in two stages including batch and continuous
treatment. The stage 1 of the experiment was designed for the reduction of physical impurities and the
nutrients. Stage 2 was designed mainly for the cultivation of blue green algae in dairy waste water by
utilizing the extra nutrients . Reduction of 94 -99.5% in phosphate was observed after 48 h of treatment
in the primary and secondary photobioreactors. The level of phosphate, total hardness, ammoniacal
nitrogen in the MSE was reduced by 97%,93 %, 81% respectively. BOD was reduced to 370mg L-1 from
1500 mg L-1 after 48 hrs of treatment in the primary reactor. COD was reduced to 85 mg L -1 from an
initial value of 1500 mg L -1 from medium strength effluent (MSE) and 90-95 % removal of COD was
also obtained from high strength effluent(HSE) during the study period. Biomass developed within the
reactor was harvested at every 15 days intervals from the secondary reactor and analyzed for lipids and
fattyacids. Presence of C14:0, C16:0,C18:0, C18:1 and C18:2 fatty acids strongly supports its abilility for
biodiesel production.
Hepatoprotective and antioxidant effects of Azolla microphylla based gold nan...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
Our present study sought to evaluate hepatoprotective and antioxidant effects of methanol extract of Azolla microphylla phytochemically synthesized gold nanoparticles (GNaP) in acetaminophen (APAP) - induced hepatotoxicity of fresh water common carp fish.
Materials and Methods:
GNaP were prepared by green synthesis method using methanol extract of Azolla microphylla. Twenty four fishes weighing 146 ± 2.5 g were used in this experiment and these were divided into four experimental groups, each comprising 6 fishes. Group 1 served as control. Group 2 fishes were exposed to APAP (500 mg/kg) for 24 h. Groups 3 and 4 fishes were exposed to APAP (500 mg/kg) + GNaP (2.5 mg/kg) and GNaP (2.5 mg/kg) for 24 h, respectively. The hepatoprotective and antioxidant potentials were assessed by measuring liver damage, biochemical parameters, ions status, and histological alterations.
Results:
APAP exposed fish showed significant elevated levels of metabolic enzymes (LDH, G6PDH and MDH), hepatotoxic markers (GPT, GOT and ALP), reduced hepatic glycogen, lipids, protein, albumin, globulin, increased levels of bilirubin, creatinine, and oxidative stress markers (TBRAS, LHP and protein carbonyl), altered the tissue enzymes (SOD, CAT, GSH-Px and GST) non-enzyme (GSH), cellular sulfhydryl (T-SH, P-SH and NP-SH) levels, reduced hepatic ions (Ca2+, Na+ and K+), and abnormal liver histology. It was observe that GNaP has reversal effects on the levels of above mentioned parameters in APAP hepatotoxicity.
Conclusion:
Azolla microphylla phytochemically synthesized GNaP protects liver against oxidative damage and tissue damaging enzyme activities and could be used as an effective protector against acetaminophen-induced hepatic damage in fresh water common carp fish.
This document describes a study that developed a method to determine off-flavor compounds geosmin and 2-MIB in live fish using in vivo solid-phase microextraction (SPME). Two kinetic calibration methods were investigated: on-fiber standardization and measurement using predetermined sampling rates. Both methods were validated using traditional analysis of samples collected through lethal sampling. The developed in vivo SPME method was then applied to determine geosmin and 2-MIB levels in live fish in a recirculating aquaculture system, with detection limits below human sensory thresholds.
Phytase from Bacillus cereus MTCC 10072 was purified about 10.75 fold to apparent homogeneity with a recovery of 34% referred to the phytase activity in the crude extract. The monomeric enzyme displayed molecular weight of 45 KDa and showed maximum activity at temperature 60 ºC and pH 6.5. Iso electric point of the purified enzyme was found to be 5.6. Substrate specificity studies showed it is highly specific to its substrate and maximum relative activity of 128% was obtained with calcium phytate. Activity was unaffected or moderately stimulated by a range of metal ions with only Ca2+ exerting (118%) stimulatory effect. The enzyme is significantly thermo stable at 60 ºC and retains a significantly greater proportion of maximal activity at physiological temperatures. This may render it of industrial interest. Further to check the applicability of the enzyme effect of different doses of crude enzyme (10, 25, 50 and 100 units) in dephosphorylation of animal feed was evaluated. Up to 66 h of incubation, the animal feed was monitored for the released inorganic phosphate content present in the feed. An enzyme dose of 100U and 50U of crude phytase enzyme per flask were found suitable to liberate enough amount of inorganic phosphorus in case of poultry and pig feed respectively.
This document summarizes a study that investigated how incorporating rosemary essential oil (REO) at concentrations of 0.5%, 1.0%, and 1.5% affects the properties of chitosan-based films. The main findings were:
1) Incorporating REO up to 1.5% decreased the film's solubility in water by about 25% and water absorption by 85% due to interactions between chitosan and REO.
2) REO improved film transparency from 4.97 for neat chitosan films to 7.61 and reduced light transmission, especially in the UV range, by over 25%.
3) Films containing REO showed greater antibacterial activity against foodborne
This document discusses chitin and chitosan. It begins with a brief history of the discovery of chitin. It then discusses the sources of chitin in nature, including the exoskeletons of arthropods, cell walls of fungi, and shells of mollusks. The document outlines the isolation and production processes of chitin and chitosan from sources like shellfish and fungi. It also summarizes some of the key applications and properties of chitin and chitosan.
Comparative study between film and coating packaging based onJesus Almaraz Muñoz
This study compared the effectiveness of a shrimp concentrate film and coating at preserving fish sausages during chilled storage. The coating increased the lag phase of total microorganisms and enterobacteria to 15 and 10 days, respectively, while the film drastically inhibited the growth of these microbes. Both helped maintain quality by decreasing pH and volatile bases during storage, with greater effects seen from the film. The coating kept sausage properties similar to controls for 25 days while the film resulted in a lower moisture and pH product resembling pickled sausage that remained stable for at least 42 days.
HHP processing of avocado paste at 600 MPa for 3 minutes significantly increased total extractable carotenoid levels by about 56%. During storage of the processed paste for 40 days at 4°C, carotenoid levels declined but remained higher than in unprocessed paste. ORAC values, a measure of antioxidant capacity, decreased after processing but increased during storage, indicating carotenoids contribute minimally to total antioxidant capacity. The study showed HHP processing and refrigerated storage retained avocado carotenoids and bioactive compounds within the product's sensory shelf life.
Biotechnological applications in agriculture a new source of edibleSiamak Alizade
This document summarizes research on using the Terminalia belerica plant for various biotechnological applications. Key points:
- The plant's seeds can be used to produce a new edible oil, comprising 37% of the dry kernel weight. Analysis found the oil contains fatty acids similar to olive oil.
- Byproducts of oil extraction include an oilcake containing high levels of nitrogen (8.34%) and proteins (60% digestible) suitable for use as biofertilizer.
- Other byproducts include tannins extracted from the fruit pulp, suitable for use in leather/medicine, and antioxidants like gallic acid in the seed coat with potential to preserve vegetable oils.
Gelatin-based nanocomposite films were prepared with zinc oxide nanoparticles (ZnO-NPs) and glycerol to develop sensitive layers for monitoring relative humidity in food packaging. The incorporation of ZnO-NPs and glycerol induced changes in the films' morphology, structure, water contact angle and vapor permeability. Electrical characterization showed the nanocomposite films were highly sensitive to changes in relative humidity, responding positively with a sensitivity of 99.47%. The results suggest gelatin-ZnO nanocomposite films have potential for use in sensors to monitor relative humidity conditions important for food quality and safety.
HPP processing of avocados and guacamole provides several benefits:
1) It inactivates spoilage microorganisms like bacteria and molds, extending shelf life to 40 days while maintaining sensory quality.
2) Pathogens like L. monocytogenes and Salmonella are reduced by over 5 log cycles.
3) Color and nutrients are largely preserved since HPP does not use heat, inactivating enzymes like PPO by only 50% and maintaining carotenoid and antioxidant levels.
4) Refrigerated storage after HPP further controls enzyme activity and microbes, protecting quality for the extended shelf life.
INVESTIGATION ON ISOLATED AND PURIFIED LIPOXYGENASE FROM AVOCADO IN THE PRESE...ljmonking
This document describes research on isolating and characterizing lipoxygenase (LOX) enzyme from avocado. Key points:
- LOX was isolated from avocado through a multi-step purification process involving acetone extraction, pH adjustment, salt precipitation, and ultracentrifugation.
- The purified LOX's activity was tested using linolenic acid as a substrate. Its optimum pH was found to be 6.5 and optimum temperature was 40°C.
- Kinetic parameters Km and Vmax for LOX were determined using the Lineweaver-Burk method with varying linolenic acid concentrations. Characterization of avocado LOX could help minimize its detrimental effects on
The document summarizes a study that investigated the chemical composition and antioxidant activity of a glycoprotein purified from the seaweed Undaria pinnatifida. The key findings are:
1) The purified glycoprotein (UPGP) consisted of 42.53% carbohydrate and 57.47% protein.
2) UPGP showed dose-dependent antioxidant activities in various assays both before and after simulated digestion, though activities decreased in the gastric phase and increased in the duodenal phase.
3) UPGP also showed protective effects against oxidative DNA damage both before and after certain phases of simulated digestion.
This document characterizes various properties of an exopolysaccharide (EPS) produced by Lactobacillus kefiranofaciens ZW3 isolated from Tibetan kefir, including its molecular weight, solubility, water and oil binding capacities, structure as viewed by scanning electron microscopy and atomic force microscopy, rheological properties under different conditions, melting point, and degradation temperature. The molecular weight was determined to be 5.5 x 10^4 Da by gel permeation chromatography. Solubility in water was 14.2% and water and oil binding capacities were 496.0% and 884.74% respectively. Scanning electron microscopy showed a smooth surface structure and atomic force microscopy revealed an almost
IRJET- Scavenging Efficiency of Azolla Pinnata in Effluent as Remediation AgentIRJET Journal
This document summarizes a study on using the aquatic fern Azolla pinnata to remediate effluent from a sugar cane industry. Key findings include:
1) Azolla pinnata showed significant growth in diluted effluent (25% concentration) and helped reduce levels of pollutants like BOD, COD, and suspended solids.
2) Parameters like chlorophyll content, relative growth rate, and seed germination increased for Azolla grown in diluted effluent, indicating uptake of nutrients promoted growth.
3) Treated effluent supported Azolla biomass production and was suitable for irrigation, while the biomass could be used as fertilizer due to accumulated
Characterization of fresh moringa oleifera beverageAlexander Decker
This document characterizes a fresh Moringa oleifera beverage made from 50% moringa leaf extract, 38% pineapple juice, and 12% carrot extract. Proximate and chemical analyses were conducted on the beverage. The beverage contained 2.9g/100ml of protein, 1.02mg of iron, and 159.14mg/100ml of vitamin C. After 8 weeks of storage under different conditions, 78% of the initial vitamin C content was retained, even under sunlight. No microbial growth was observed under any storage conditions, and sensory evaluation found the product remained acceptable. The beverage is concluded to be an excellent way to provide nutrients to malnourished individuals and consumers.
Characterization, treatment and recovery of fish byproduct as a stable bio-fe...Skyfox Publishing Group
National fish production reached in 2013 an amount of 1,245,912 tons. Now the pelagic fish industry generates a
significant amount of waste up to 60%. The physicochemical analyses of these fish by-products showed a wealth of organic material
including proteins (87.4% dry weight) and minerals (9% dry weight). These components and others are capitalizing to be used for
agricultural or agri-food. Biotransformation of biotechnologically in fish products was performed in the laboratory. Fresh fish byproducts
were ground and mixed with a carbon-rich source of carbohydrates. The mixture was inoculated with selected fermentation
yeast. The fermentation of evolution was controlled by monitoring chemical parameters (pH, dry matter, ash, total nitrogen) and
microbiological (FMAT, Enterobacteriaceae: fecal and total Coliforms, yeasts). Then the product thus stabilized at a pH of 3.8 has
undergone a second treatment. A hydrolyzate rich in amino acids and trace elements was obtained with excellent hygienic quality after a
total elimination of pathogens. Its use will be as factors for fertilizer and soil amendment. We produced three biostimulants necessary for
plant growth: Rooting fertilizer, Plant elongation fertilizer and production fertilizer. The application tests on these biofertilizers in the
laboratory were performed and have allowed obtaining promising results.
This document describes a study that developed and characterized intelligent food packaging films containing a biohybrid material based on anthocyanins from eggplant peel and laponite. The biohybrid was produced by adsorbing anthocyanins onto the surface of laponite. Chitosan films were produced containing this biohybrid. The films with biohybrid were thicker, less soluble in water, and changed color when exposed to different pH solutions, indicating they could be used to monitor food freshness based on pH changes. The films were able to change color from gray to red or yellow depending on acidic or basic conditions, similarly to how anthocyanins change color with pH. The films were tested for monitoring meat freshness at different temperatures by correlating film
ABSTRACT In the present study, Chitin has been extracted from Agaricus bisporus (Button mushroom). The obtained chitin was converted into the more useful chitosan and the crude chitosan extract was measured for its absorption maxima by UV Spectrophotometer and the maximum peak at 265nm was observed. FT-IR spectroscopy was done to identify the functional groups present in the chitosan which was analyzed between the ranges of 4000–400 cm-1. Chitosan was characterized by significant amide bands at 3265.49 cm−1. The absorbance bands of 1402.25, 1153.43, 900.76 and 445.56 cm-1 indicates CH2 stretching, CH stretching, C=O stretching in secondary amide respectively which confirms the structure of chitosan. The antioxidant activity of chitosan was determined by DPPH free radical scavenging assay and the value gained is 65.90% at 250 mg/ml which is due to the presence of rutin, gallic acid, caffeic acid and catechin in the phenolic composition of Agaricus bisporus. Finally, in vitro antibacterial screening of chitosan from Agaricus bisporus was performed against selected clinical isolates and the zone of inhibition shows highest activity in Bacillus subtilis, P. aeruginosa followed by K. pneumonia, and Acinetobacter baumannii. These findings suggest that the Agaricus bisporus act as the potential source to produce eco-friendly chitin and chitosan in the development of drugs, artificial bone and raw material for food industries in near future.
Key-words: Agaricus bisporus, Chitosan, Antioxidants, Antibacterial activity, UV spectroscopy, FTIR spectroscopy
Protein Extraction and Purification of Soybean Flakes and Meals Using a Lime ...IJMER
This document describes a study that investigated extracting and purifying protein from soybean flakes and meals using lime treatment followed by ultrafiltration. Key findings include:
- Soybean flakes and meals were produced from whole soybeans using conventional methods. Flakes had higher protein and carbohydrate extraction than meals after treatments.
- Lime treatment at 0.5 moles resulted in the highest extraction of total dissolved solids (TDS) from both flakes and meals, compared to water, NaOH, or other lime concentrations.
- Ultrafiltration purified the protein extracts from flakes and meals with 94-97% protein yield and removal of carbohydrates through diafiltration.
- Lime treatment is a potential
Bioproduction of bioactive compounds screening of bioproduction conditions of...ainia centro tecnológico
1. The document describes a study screening bioproduction conditions for microalgae and lichen symbionts to produce bioactive compounds. It examines the adaptation of various microalgae like Chlorella and Asterochloris erici to different culture media and processing methods.
2. The results show that Chlorella vulgaris was able to grow under mixotrophic conditions using various carbon sources and treatments like ultrasounds increased bioactivity. Asterochloris erici was adapted from solid to liquid culture and able to be cultured at large scale under autotrophic conditions.
3. The study demonstrates that bioproduction technologies can be used to obtain high-value compounds from microalgae and lichen
Marine algae are a rich source of natural bioactive compounds with pharmaceutical, nutraceutical and cosmetic applications. Multi-omics approaches can be used to identify compounds of interest from algae. Notable compounds isolated from algae include carotenoids, fatty acids, antibiotics, and biosurfactants. Algae also have applications as human and animal food, in space research producing oxygen, and in energy production through biofuel and methane. Microalgae can be used to remediate wastewater by removing nutrients and excess carbon dioxide.
The global carotenoid market was valued at $1.5 billion in 2014 and astaxanthin accounted for 25% of that market. Haematococcus pluvialis is currently the main commercial source of natural astaxanthin but production is constrained by contamination, low growth rates, die off during culture changes, and the need for extraction from thick-walled cysts. This study developed an alternative production method where H. pluvialis was cultured in an improved medium in the green stage, achieving 34% higher cell yields, and then transferred to unfavorable conditions to produce red motile macrozoids containing 1.5% astaxanthin, avoiding current issues.
Production and isolation of chitosan by submerged and solid state fermentationNgcon35
This document summarizes a study that developed a method for producing and isolating chitosan through liquid and solid-state fermentation of Lentinus edodes. Under liquid fermentation, chitosan yields of 120 mg/L were achieved. Solid-state fermentation produced significantly higher yields of 6.18 g/kg, up to 50 times greater than other fungal methods. Solid-state fermentation provides an economical process for large-scale chitosan production with characteristics suitable for industrial applications.
Sponsor Day on animal feeding: Studies of feed additives in experimental cond...Irta
This document summarizes research on studying feed additives in experimental conditions. It describes various experimental infection models used to study Salmonella, E. coli, and Clostridium perfringens. It also discusses analyzing the gut microbiota using cloning, sequencing, and ion torrent analysis. Key findings include that the gut microbiota plays an essential role in digestive physiology and animal health, and can be modified by feed composition and additives, which can help reduce variance in productive parameters and improve farm economics.
Biodiesel from microalgae production methods - a reviewPriyakapriya
Microalgae are simple photosynthetic organisms that can be used to produce biodiesel. They grow rapidly using sunlight and carbon dioxide and can be cultivated in open ponds or enclosed photobioreactors. The microalgae are harvested and the oil is extracted, which can then be converted to biodiesel via transesterification. Additional co-products from biodiesel production include glycerol and omega-3 fatty acids. Microalgae represent a promising source of sustainable biofuel and other useful products.
An Investigation Into The Mechanisms Underlying Enhanced Biosulphidogenesis I...iosrjce
Anthropogenic activities like mining, processes of metallurgy and other chemical industries lead to
the discharge of a high amount of sulphate into the environment that causes serious problems to human health.
This paper illustrates the employment of thermophilic sulphate reducing bacteria for biosulphidogenesis. Two
different species have been isolated from hot water spring of Vajreshwari and Ganeshpuri,Thane, Maharashtra,
INDIA.The mechanism involved in biosulphidogenesis includes production of specific protein as well as
liberation of some extracellular polymeric compound (EPS) e.g. proteins, carbohydrate, acids etc. that are
produced during the microbial cell metabolism. These compounds plays an important role in the faster
reduction of sulphate and decrease in production rate of sulphide.The isolate was found to be of genus
Bacillusand type strain was found to be subtilis Zankar and licheniformis Sonali. The strain sequence were
deposited in NCBI database with accession number KJ939324 and KJ939325 respectively. The result highlights
the potential use of these organism in biosulphidogenesis.
This document discusses collaborations between universities and companies to bridge gaps. It provides examples of two collaborations:
1. A collaboration between a university and CWBI to develop a biofungicide from Pseudozyma flocculosa for controlling powdery mildew. Fundamental research in the 1980s led to field trials and a patented formulation in the 2000s.
2. A collaboration between CWBI and universities in Senegal, Burkina Faso, and other countries to identify microorganisms involved in traditional fermented foods and develop controlled starter cultures and processing techniques. Examples discussed include m'bannick milk in Senegal and soumbala flour in Burkina Faso. The goal
Screening of Biosurfactant Bioemulsifier Producing Bacteria from Petroleum Co...ijtsrd
The release of impurities in the environment, containing petroleum and petroleum cogitated products, is engenders of global being taint. It is also a hazardous for human and animal health, since many of these impurities have evidenced to be toxic and oncogenic. Hydrocarbon particles that are secreted into the environment are hard to get rid of, since they change state to surfaces and are captured by surface tension in a water immiscible stage. Bioremediation has tested to be an alternate to lessen the effects caused to impureness of soil and water, applying the metabolic abilities of microorganisms that can apply hydrocarbons as source of carbon and energy, or that can alter them by co metabolism. The proficiency of removal is directly related to the compound’s chemical structure, to its bioavailability deliberation, harmfulness, flexibility and approach and to the physicochemical situation present in the atmosphere. Perwez Qureshi | Dr. Reshma Jaweria "Screening of Biosurfactant/Bioemulsifier Producing Bacteria from Petroleum Contaminated Soil" Published in International Journal of Trend in Scientific Research and Development (ijtsrd), ISSN: 2456-6470, Volume-5 | Issue-5 , August 2021, URL: https://www.ijtsrd.com/papers/ijtsrd46276.pdf Paper URL: https://www.ijtsrd.com/biological-science/microbiology/46276/screening-of-biosurfactantbioemulsifier-producing-bacteria-from-petroleum-contaminated-soil/perwez-qureshi
The document discusses food waste management from food processing. It covers waste reduction strategies like waste minimization and water recycling. Common waste streams from food processing include solid waste and wastewater. Wastewater treatment typically involves pretreatment, primary treatment to remove solids, secondary biological treatment, and sludge treatment. The byproducts from treatment can be utilized when possible to recover resources.
Optimization of key process variables for enhanced refamycin b production in ...ijabjournal
In the present study of solid media conditions for the refamycin B yield by solid state fermentation was studied and optimized using both classical method and statistical design of experiments). Statistical analysis of the results of Plackett–Burman showed that the lower level of initial moisture , initial pH, barbital, glucose and to solid media, or increase in the concentration of xylose in the range tested, results in significant effect in refamycin B yield of AmycolatopsisrifamycinicaMTCC 14 by solid state
fermentation. The effect of change in the levels of initial moisture, initial pH, barbital, glucose and xylose
on the rfefamycin B yield was studied using central composite design methodology. Statistical analysis of
the data showed that all the independent process had significant effect on refamycin B yield. The interaction between initial moisture and initial pH, between initial moisture and barbital, between initial moisture and glucose, between initial moisture and xylose, between initial pH and xylose, between barbital and glucose, between barbital and xylose, and between glucose and xylose were significant when the response was refamycin B.
This document discusses the importance and potential of marine algal bioprospecting. It notes that marine algae are a unique and rich source of biodiversity that can help improve human life. Marine organisms have highly developed defense systems that allow them to survive in harsh conditions, and this has led to the evolution of many valuable natural compounds. The document outlines various compounds and products that have been obtained from micro and macro algae, including pigments, fatty acids, polysaccharides, and other chemicals with applications in pharmaceuticals, cosmetics, nutraceuticals, foods, and biofuels. It also discusses methods for cultivating and analyzing algae to obtain these compounds, including 'omics technologies and genetic engineering approaches.
Researchers at the University of North Carolina Wilmington collaborated with IKA Works, Inc. to create a 10 liter closed, stirred-tank photobioreactor to produce high densities of dinoflagellate microalgae. Using cultures of Amphidinium gibbosum and Alexandrium ostenfeldii/peruvianum, the photobioreactor generated equal biomass to eighty liters of static culture and cell counts of A. gibbosum reached up to 300 million cells per liter. The photobioreactor provides an effective system for growing dinoflagellates at scale while using fewer resources than traditional methods.
1. The document describes the extraction of glucosinolates (GLs), sulfur-containing secondary metabolites found in Brassicaceae plants, from seeds and oil cakes of Camelina sativa. Three rare GLs were obtained in yields up to 3.7% from extractions.
2. Efficient synthetic routes were developed for the gram-scale preparation of natural GLs. Methods were optimized for both extraction and synthesis to access GLs from 100mg to 1g.
3. Artificial GLs were designed and synthesized as new bioconjugational tools. GL-mannoside glycoconjugates were shown to act as myrosinase substrates while retaining affinity for lectins, demonstrating
Introducing the Laboratory of Aquaculture & Artemia Reference Center, Ghent U...Jean Dhont
The Laboratory of Aquaculture & Artemia Reference Center of Ghent University is a leading research and education center specialised in larval nutrition of aquatic organisms, microbial management, disease control, Artemia fundamental and applied research
The document outlines the student's industrial work experience at the Nigerian Institute of Oceanography and Marine Research (NIOMR). It describes the various departments at NIOMR, including Fish Technology, Biological Oceanography, Marine Biology, and Biotechnology. It then details several projects the student worked on, including microbial analysis of smoked fish, value addition processes like making fish crackers, and algae growth inhibition tests.
Microbial fermentation By Aneela SaleemAneelaSaleem
This document discusses different types of fermentation processes used in industry. It begins with an introduction and overview of fermentation media and microorganisms. It then describes the main types of fermentation processes - batch, fed-batch, and continuous fermentation - and factors that influence each type such as growth rate and flow rate. The document also covers solid state and submerged liquid fermentations. Important considerations for continuous fermentation are highlighted. Recent advances in fermentation technology are briefly mentioned at the end.
1. (19) United States
(12) Patent Application Publication (10) Pub. No.: US2014/0100361 A1
US 201401.00361A1
Le Roux et al. 43) Pub. Date: Apr. 10, 20149
(54) EXTRACTION OF CHITINS IN A SINGLE (30) Foreign Application Priority Data
STEP BY ENZYMATIC HYDROLYSIS IN AN
ACID MEDIUM May 26, 2011 (FR) ....................................... 115458O
(75) Inventors: Karine Le Roux, Milly-la-Foret (FR): O O
Jean-Pascal Berge, Saint-Ave (FR): Publication Classification
RiseBy hapeleurline (51) Int.Cl./ . s COSB 37/08 (2006.01)
Saint-Etienne-de-Montluc (FR); (52) U.S. Cl
Abdellah Arhaliass, Saint-Nazaire (FR) CPC .................................... C08B 37/003 (2013.01)
(73) Assignee: IFREMER (INSTITUT FRANCAIS USPC ............................................. 536/20: 435/274
DE RECHERCHE POUR
LEXPLOITATION DE LA MER), (57) ABSTRACT
Issy-les-Moulineaux (FR)
A method ofenzymatic extraction ofchitin is realized in a
(21) Appl. No.: 14/122,427 single step wherein the chitin is obtained by enzymatic
1-1. hydrolysis of raw material constituted by animal biomass
(22) PCT Filed: May 25, 2012 including chitin, the enzymatic hydrolysis using an enzyme
act1Ve 1n ac1d medium. Also d1SCIOSed 1S arOceSS Of Ot1m1(86). PCT No.: PCT/FR2O12/05118O ive in acid medium. Also disclosed is a p foptimi
S371 (c)(1),
(2), (4) Date: Nov. 26, 2013
Raw rateria
Washing, dying, giding
zation ofthe methodofenzymaticextractionofchitin,as well
as the chitin susceptible to be obtained by the method of
enzymatic extraction.
Reaction edium
sowent + acid
control ofpH and temperature
homogenization
2 enzyme additio:
Soisbie phase
pigments, tigids, Sigars, i.e.ai
Saits, airino acids, peptides...}
Neutralizatio aid
separatios by categories
of compouds
Enzymatic hydroEysis inan acid
nedin =
proteoysis and Scitizatio:
of the litera sats
filtration f rising
insibie phase
= CH3S
bleaching aidfor
deacetylation
foration of chitosan,
oligo-chiti chitosai,
gucosamines
2. Patent Application Publication Apr. 10, 2014 US 2014/0100361 A1
Reaction medium
{so went + acid}
contro ofpH and temperatuie
Raw materia
Washing, dying, giding
homogenization
2} enzyme addition
Enzymatic hydrolysis in an acid
medium =
proteoiysis and soiuxilization
of the irrera sats
fitration f rising
Soluble phase
pigments, tipids, Sugars, minera:
saits, aimino acids, peptides...}
insolubie phase
= CHENS
bleachingandfor
deacetylation
Neutralization aid
Separation by categories
of compounds r
iformation of chitosan,
oligo-critifchitosa,
gucosamines
F.G. 1
3. US 2014/0100361 A1
EXTRACTION OF CHTINS IN A SINGLE
STEP BY ENZYMATIC HYDROLYSIS IN AN
ACID MEDUM
FIELD OF INVENTION
0001. The present invention relates to the field of the
recovery ofbiomass, preferably animal biomass, more pref
erably marine and/or entomological biomass. In particular,
the present invention relates to a method for the enzymatic
extraction of chitin in a single step, from animal biomass
elements comprising chitin, preferably from marine and/or
entomologicalby-products,usinganactiveenzymeinanacid
medium.
BACKGROUND OF INVENTION
0002 The production and consumption of marine prod
ucts, and particularly of crustaceans, notably prawns, is
increasing each year. By-products (heads and shells) gener
ally represent more than 50% ofthe fresh weight ofcrusta
ceans. Theusethereofisthus amajorissuegiventheVolumes
involved and also the slow natural biodegradability thereof.
Chitin is the main product derived from these by-products.
0003 Moreover, entomophagy is a common dietary prac
ticeinsomecountries whichistendingtodevelop worldwide.
Indeed, insects represent an advantageous dietary resource
due to the nutritional qualities thereof. Furthermore, insect
production offers a very advantageous environmentally
friendly alternative compared to the production ofotherani
mal proteins. The by-products obtained from insect protein
production include chitin-rich shells, as for crustaceans.
0004 Chitin is the second most plentiful polysaccharide
on the surface ofthe Earth after cellulose. It does not have a
single chemical structure, but several, since it includes
polysaccharides consisting of N-acetyl-B-D-glucosamine
units and D-glucosamine units.
0005 Chitinpartiallyformstheexoskeleton ofinsectsand
crustaceans and the wall of fungi and bacteria. Chitin thus
represents 20 to 30% of the shells of crustaceans. Besides
chitin, the exoskeleton ofcrustaceans contains 20 to 40% of
proteins, 30 to 60% ofmineralsand 0to 14% offat (Waldeck
J. Daum G., Bisping B. and Meinhardt F., Appl. Env, Micro
biol., 2006, 72 (12), 7879-7885). Chitin thus represents 3 to
60% oftheshellsofinsects. Besideschitin, theexoskeletonof
insects contains 20 to 80% ofproteins, 1 to 20% ofminerals
and 10 to 50% offat (“Forest insects as food: humans bite
back. Proceedings ofa workshop onAsia-Pacific resources
andtheirpotential fordevelopment, 19-21 Feb. 2008,Chiang
Mai, Thailand FAO). Whether for crustaceans or for
insects, the proportions of the various constituents vary
according to the species, age, genus and may fluctuate
according to the seasons and environmental conditions.
Chitin extraction conditions should thus be adapted accord
ing to the raw material used (Tolaimate A. Desbrieres J.
Rhazi M.andAlaguiA., Polymer, 2003, 44(26), 7939-7952).
0006 Chitin is found incrustaceanandinsectby-products
in the form ofchitin/proteinS/minerals complexes. It is usu
ally extracted in two “chemical extraction' steps:
0007 demineralization by means ofacid hydrolysis, to
remove minerals; and
0008 deproteinization by means ofbase hydrolysis, to
remove proteins.
0009 Chitin extraction from marine by-products is cur
rently carried out on an industrial scaleby means of"chemi
Apr. 10, 2014
cal extraction'. Chitin extraction from insects has not been
very developed to date but has already been the subject of
studies, essentially using a chemical process (cicada chitin:
Sajomsang W. and Gonil P. Mat. Science Engineering C,
2010, 30(3), 357-363; silkworm pupa chitin: Paulino A.,
Simionato J. Garcia J. and Nozaki J. Carbohydrate Poly
mers, 2006, 64, 98-103; bumblebee chitin: Majtan J., Bil
ikova K. Markovic O. Grog, J., Kogan G. andSimuth J., Int.
J. Biol. Macromol., 2007, 40,237-241).
0010. A third optional bleaching step, for example using
Sodium hypochlorite, is frequently used to remove residual
pigments. Washing operations, generally with water, are
required between these various steps.
0011 Chitincan thenbereadilydeacetylated, forexample
using sodium hydroxide, to produce chitosan.
0012 Chitin is conventionally extracted for a wide range
ofapplications: medical,pharmaceutical, dietary, food,tech
nical (water filtration and depollution), etc. Indeed, chitin,
chitosan and the derivatives thereof, particularly the oligo
mers thereof, are biocompatible, biodegradable and non
toxic. Thetype ofapplication depends on the physicochemi
cal characteristics of chitin and the derivatives thereof. In
particular, chitosan may be particularly used for producing
mulching film, Stomach protection gels, but also for active
ingredient encapsulation, waste water filtration, cartilage
replacement, tissue regeneration, etc.
0013 Industrial chitin extraction from marine by-prod
ucts is essentially implemented in emerging countries. Con
ventional chemical extraction uses large quantities of
reagents (essentially hydrochloric acid, Sodium hydroxide
andbleachingagents)whichareharmful foroperators,equip
mentandthe environment. Furthermore, thebasicdeprotein
ization step is generally performed hot and thus requires a
high energy input. Moreover, the washing steps give rise to
very large volumes ofpolluted effluents, which are techni
cally difficult and expensive to recycle.
0014. Oneoftheproblems associated with currentextrac
tion methods is thepossibility ofchitin being denatured dur
ing the process (Crini G., Badot P. and Guibal E., Chitine et
Chitosan. Du polymered l'application, 2009, Presses Uni
versitaires de Franche-Comté).
0015 Studies have shown that chitin could be extracted
using biological methods, notably by means of enzymatic
extraction or microbiological fermentation, particularly for
the deproteinization step.
0016. Of the research on the fermentation process, the
studies conducted by Beaney use the exoskeleton of the
Nephrops norvegicus prawn as the study Substance (Beaney
P. Lizardi-Mendoza.J. andHealy M.,J. Chem. Tech. Biotech.,
2005,80, 145-150). In this study, chitinisextractedby means
oflactic fermentation in thepresenceofbacterialstrains for5
days at 30°C. Theacidification ofthe medium, due to lactic
acid production by the bacteria, results in partial demineral
ization while the bacteria carry out deproteinization. In this
study, the pH decreases to a value of 3.5 after 7 days of
fermentation. However, under these conditions, the chitin
extracted still contains 13% ofproteins and 14% minerals. A
purer chitin may then be obtained by performing further
chemical treatments. This typeofmethodis thus not suitable
fordirectlyobtainingahigh-quality chitin,limitingtheappli
cations thereof.
0017. A further microbial fermentation study was con
ducted to extractchitin from redcrab shellsby co-fermenting
the shells in the presence oftwo bacteria: firstly, Lactobacil
4. US 2014/0100361 A1
lus paracasei tolerans KCTC-3074, which is a lactic acid
producing bacterium, and secondly Serratia marcescens
FS-3,which isanextracellularprotease-producingbacterium
(JungW.,JoG., Kuk.J., Kim K. andPark R. Appl. Microbiol.
Biotechnol., 2006, 71,234-237). Co-fermentation was main
tained for7 days at30° C. and resulted in a demineralization
rate of97.2% and a deproteinization rate of merely 52.6%.
Thechitin obtainedwas notcharacterisedin this studybutthe
low deproteinization rate is limiting in terms of the use
thereof.
0018. A further microbial fermentation study was con
ducted by the same team, with a bacterial strain producing
proteases for deproteinizing and demineralizing marine by
products (Jo G., JungW. KukJ. Oh K. KimY. and Park R.
Carbohydratepolymers, 2008, 74, 504-508). A fermentation
testwasconductedfor7daysat30°C.,in thepresenceof10%
of bacterial strain, and resulted in a deproteinization and
demineralization rate of 84% and 47%, respectively. As
above, the demineralization is due to the pH decrease over
time (pH 5.6 after 7 days of fermentation), associated with
bacterial acid production. The low degree of purity of the
chitins obtained is limiting in terms of the applications
thereofand this method, like the previous method, involves
the drawback ofrequiring a very long reaction time.
0019. The best demineralization and deproteinization
yields by means of a two-step fermentation process were
obtained by Waldeck (Waldeck J. Daum G., Bisping B. and
Meinhardt F., Appl. Env, Microbiol., 2006, 72 (12), 7879
7885).Afterfermentingfor6daysfrom42to55°C.,followed
by a 3hour lactic acid treatment, the residual protein content
is less than 10% and the demineralization rate is equal to
98.8%. As in the study by Jo et al., the reaction time is
relatively long.
0020 Chitin extraction by means ofa fermentation pro
cess thus results in a chitin with a higher residual protein
content than in the case of chemical extraction and further
treatments are frequently required to improvethe demineral
ization. Furthermore,thereactiontimesare muchlongerthan
with the chemical process.
0021 Chitin extraction by means ofa biological process
may also be performed using enzymatic extraction.
0022. A method for extracting chitin comprising the
removalofproteinsbymeansoftheenzymaticactivity offish
viscera was proposed in the international patent application
WO 86/06082. In particular, the method described in this
patent application comprises the extraction of chitin from
prawn shells by means ofdemineralization with an acid fol
lowed by deproteinization using fish viscera, optionally pre
ensiledat pH 1.2-2.5. The raw material, i.e. the prawn shells,
is first ensiled in a Sulphuric acid solution. Ensiling makes it
possible to store the raw material before use and enables the
demineralization thereof. Secondly,thepre-ensiled shellsare
placed in contact with fish viscera for deproteinization. The
characteristics of the chitin obtained using this two-step
method are not specified.
0023. Enzymatic extraction may also be performed using
apurifiedenzyme,generallyaproteolyticenzyme.This is,for
example, the case in the study conducted by N. Gagné, with
the use ofchymotrypsin orpapain for extracting chitin from
prawn shells (Gagné N. “Production ofchitin and chitosan
from crustacean wasteandtheiruseasfoodprocessing aid',
1993 McGill University—Montreal, doctoral thesis). Aftera
conventional chemical demineralization step, the proteins
present are hydrolysed by the enzymes. The optimal depro
Apr. 10, 2014
teinization conditionsparticularly involveapH of8.0-8.7 for
chymotrypsin and papain. Under the conditions used, the
residual protein content is very low (1.3% and 2.8% forchy
motrypsin and papain, respectively).
0024. The same type ofmethod was used by Synowiecki
and Al Khateeb for conducting the enzymatic digestion of
prawn shells, previously demineralized with hydrochloric
acid, usingalcalase, at 55° C. and pH8.5 (Synowiecki J. and
Al Khateeb, Food Chemistry, 2000, 68, 147-152).
0025. A method for producing chitin involving an enzy
matic hydrolysis step has been patented (CN1715255). This
method offers a general approach for processing the raw
material sincecompounds otherthan chitinarealso extracted
from prawn shells. In particular, this method comprises an
enzymatic hydrolysis step followed by solvent extract. The
solidportionobtained is thenplacedinthepresenceofhydro
chloricacidtoperform demineralizationandfinishextracting
chitin.
0026. All the enzymatic chitin extraction methods cur
rently described involvean independentconventional chemi
cal demineralization step, before or after the enzymatic
hydrolysis step. In this way, even ifthe deproteinization step
is carriedoutusingabiological method,there is still achemi
calstep requiring washing operations andproducingpolluted
effluents and liable to affect the properties of the extracted
chitin.
0027. The current methods are thus not satisfactory and
thereisaneedforchitinextractionmethodswhicharesimple,
rapid, efficient, inexpensive and more environmentally
friendly. These methods should be suitable for producing
chitins wherein thepurity iscompatible with usein the food,
dietetic or cosmetic industries. Furthermore, the chitins pro
ducedshould meetthespecifications requiredtobeprocessed
into chitosan, oligo-chitosan orglucosamines,etc. Inparticu
lar, the degree of polymerization of the chitin should be
sufficiently high and it should not be denatured during the
process.
0028 Moreover, further compounds can potentially be
recovered using crustacean and insect by-products, particu
larly in nutraceuticals, dietetics or cosmetics. Indeed, these
marine and insect by-products contain soluble compounds
Suchas lipids,pigments, Sugars, mineral salts, aminoacids or
peptides. Targeted extractions of these soluble compounds
have been developed, such as for example the extraction of
pigmentsand inparticularofastaxanthin which is usedin the
food industry (U.S. Pat. No. 7,241,463). However, these
methods targeted on Soluble compound extraction require
further steps to conduct chitin extraction.
0029. In current chitin extraction methods, chitin is
obtained in Solid form and the liquid extraction phases, con
taining soluble compounds of potential interest, are not
recovered.Thislackofrecovery canparticularlybeexplained
by the poor quality ofthe Soluble compounds present in the
liquid phases. Indeed, in these chitin extraction methods,
soluble compounds are frequently degraded due to the rela
tively severe conditions used.
0030 Therefore, there is a need for chitin purification
usinga methodthatrespects theinitial structurethereofmore
and is also suitable forbeingassociated with soluble product
co-extraction.
0031. TheApplicantconducted research inordertoobtain
full recovery of the portions of animal biomass containing
chitin, notably full recovery of marine by-products, in par
ticular crustacean shells and entomological biomass, in par
5. US 2014/0100361 A1
ticularinsectshells. Inparticular,co-extraction methods were
studied with a view to the advantages thereof in relation to
targeted extractions.
SUMMARY
0032. The invention thus relates toa method fortheenzy
maticextraction ofchitin,characterisedin thatsaid methodis
carried out in a single step, hereinafter referred to as the
“single step', wherein chitin is obtained by the enzymatic
hydrolysis ofanimal biomass comprising chitin, said enzy
matic hydrolysis using an active enzyme in an acid medium.
0033 Accordingto oneembodiment, saidsinglestepisan
enzymatic hydrolysis intended for deproteinizing and dem
ineralizing marine by-products simultaneously.
0034. According to one embodiment, saidactive enzyme
in an acid medium is a protease having a broad spectrum of
activity inan acid medium, preferably pepsin ora stableacid
protease.
0035. Accordingto oneembodiment,theenzyme concen
trationused forhydrolysis is 0.1 to 75%,preferably5 to30%,
more preferably from approximately 23 to approximately
27% in weight relative to the weight of the protein mass
estimated in the raw material.
0036. According to one embodiment, the acid medium is
obtained by means ofthe presence of an acid, preferably a
dietary acid, morepreferably phosphoric acid orformic acid.
0037 Accordingtooneembodiment, saidanimalbiomass
comprising chitin comprises marine by-products, preferably
marine by-products obtained from crustaceans, preferably
prawns, crabs orkrill, orfrom cephalopods,preferably squid
or cuttlefish.
0038 Accordingtooneembodiment, saidanimalbiomass
comprising chitin comprises insect by-products, preferably
insect by-products obtained from beetles orhymenoptera.
0039. According to one embodiment, said methodfurther
comprises operationsforwashing,dryingand/orgrindingthe
raw material, preferably water washing, cold drying and/or
grinding operations resulting in fragments less than 1 mm in
S17C.
0040. According to one embodiment, said methodfurther
comprisesreactionmediumtreatmentoperationsattheendof
the enzymatic hydrolysis, said operations comprising opera
tionsforseparatingtheSolidandliquidphases, rinsingand/or
dryingtheinsolubleportion,preferably operations consisting
offiltration, rinsing with waterand/oroven-drying.
0041. The invention also relates to a method for optimis
ing said method forthe enzymatic extraction ofchitin, char
acterized in that said optimization method comprises at least
one ofthe following steps:
0042 a) selecting the pH oftheacid medium in the range
pH-0-2, preferably pH-0-1.5, preferably pH-0-1,82 82 82
wherepH isthepHatwhichtheenzymeexhibits maximum
activity,
0043 b) selecting the temperature ofthe acid medium in
therangeTit.0-20°C.,preferablyTit.0-15°C., preferably
T+0-10° C., where T is the temperature at which the
enzyme exhibits maximum activity,
0044 c) determining the mineral and protein content of
the raw material,
0045 d) calculating the acid concentration to be used in
the reaction medium, accordingto the mineral content ofthe
raw material, itbeing understood that, according to one pre
Apr. 10, 2014
ferred embodiment, the pH is selected such that the reaction
medium is maintained throughout the enzymatic hydrolysis
atthe pH selected in step a).
0046 e) calculating the proportion ofenzyme to be used
with respect to the protein content ofthe raw material,
0047 f)determiningthe reaction timeforobtaining chitin
orthe chitin derivatives sought.
0048. The invention further relates to chitin that can be
obtained by means ofthe method accordingto the invention.
0049. The invention also relates to chitosan that can be
obtained by deacetylating chitin according to the invention.
0050. Theinventionalso relatesto acompositioncompris
ing chitin accordingto the invention and/or chitosan accord
ing to the invention.
0051. Theinvention also relates to a pharmaceutical com
position comprising chitin according to the invention and/or
chitosan according to the invention, a cosmetic composition
comprising chitin according to the invention and/or chitosan
according to the invention, a medical device comprising
chitinaccordingtotheinventionand/orchitosanaccordingto
the invention.
0.052 Theinventionalso relatestoafoodproduct,a nutra
ceutical composition, a dietetic composition, a food Supple
ment or a functional food comprising chitin according to the
invention and/or chitosan according to the invention.
0053. Theinventionalso relatesto acompositioncompris
ing chitin accordingto the invention and/or chitosan accord
ing to the invention for the use thereofin water treatment,
filtration and/or water depollution.
0054 The invention also relates to a texturingagent com
prising chitin according to the invention and/or chitosan
according to the invention.
DEFINITIONS
0055. In the present invention, the following terms are
defined as follows:
0056 “Chitin’ refers to N-acetyl-glucosamineandglu
cosamine polysaccharides.
0057 "Chitosan” refers to chitin deacetylation prod
ucts. The borderline between chitosan and chitin con
sists ofa 50% degree ofacetylation: below, the com
pound is called chitosan, above, chitin.
0.058 Animal biomass” refers to all organic matter of
animal origin.
0059) “Marineby-products” refers to parts not used by
the food industry in marine products, particularly crus
tacean shells and heads.
0060 “Entomological by-products”or“insectby-prod
ucts” refers to parts not used by the food industry in
entomological products, particularly insect shells and
heads.
0061 “Degreeofpolymerization”referstothelengthof
apolymerchain,particularly chitin.Thedegree ofpoly
merization consists of the number of monomer units
forming the polymer chain.
0062 “Crystallinity index' refers to the proportion of
material found in the crystalline state.
0063 “Demineralization” referstoamethodforremov
ing minerals.
0.064 “Deproteinization” refers to a method for remov
ing proteins.
0065 “Depolymerization” referstothereductionofthe
length ofthe polymeric chain ofchitin.
6. US 2014/0100361 A1
0066 “Deacetylation” refers to the removal of acetyl
groups and corresponds to the transition from chitin to
chitosan.
0067 “Moisturecontent” refers to the mass percentage
ofwater contained in a sample.
0068 “Proteincontent” refers to the masspercentageof
protein contained in a sample.
0069. “Mineral content” refers to the mass percentage
ofminerals contained in a sample.
0070 "Chitin content” refers to the mass percentage of
chitin contained in a sample.
0071 "Approximately,placedbeforea number, means
more or less 10% ofthe nominal value ofthe number.
0072 Unless specified otherwise, the percentages are
mass percentages.
DETAILED DESCRIPTION
0073. The present invention relates to a method for the
enzymatic extraction of chitin in a single step, from a raw
material obtained from animal biomass and comprising
chitin,preferably a raw material made ofmarine by-products
and/orentomologicalby-products, usingan activeenzyme in
an acid medium, preferably a protease, the acid used being
preferably a dietary acid, this method also being suitable for
extracting soluble compounds such as lipids, pigments, Sug
ars, mineral salts, amino acids orpeptides.
0074. In the present invention, the two key steps of the
conventional method forextracting chitin, i.e. demineraliza
tion in an acid medium and deproteinization in an alkaline
medium, are merged into a single step. This merging into a
single step is enabled through the use ofan enzyme wherein
the optimal activity pH is acidic: the enzyme performs the
deproteinization, while the acidic pH makes it possible to
carry outthe demineralization simultaneously.
0075. The method according to the invention only com
prising a single key step, it offers the advantage ofreducing
rinsing-related material losses between the two steps ofthe
conventional method. This method also makes it possible to
decrease the reagentand solvent consumption and limit pol
luted effluent production. This method is thus both inexpen
sive and environmentally friendly.
0076. Theconditions used in the method according to the
present invention are such that the biological activity of
chitins and the native structure thereofare preserved better
than in the extraction methods existing to date.
0077. The method according to the present invention
offers the advantage ofenabling destructuration ofthe crus
tacean and/or insect by-product matrix by separating chitin,
proteins and minerals, these three major constituents being
initially strongly linked.
0078. The method according the invention comprises an
enzymatic hydrolysis step in an acid medium performing
demineralization and deproteinization simultaneously. The
minerals and proteins are detached from the solid phase and
carried in the liquid phase.
0079 According to one embodiment, the method accord
ing to the present invention may comprise, in addition to the
enzymatichydrolysisstepinanacid medium,preparationand
processing operations:
0080 preparing the raw material,
0081 preparing a reaction medium according to the
optimal conditions for enzymatic activity and compris
ing at least one acid,
Apr. 10, 2014
0082 mixing the raw material prepared in the reaction
medium, homogenizing and adding enzyme,
0.083 enzymatic hydrolysis step with controlled tem
perature, pH and stirring:
I0084 simultaneous deproteinization and demineraliza
tion reactions foran optimized time,
0085 separating the soluble and insoluble portions of
the “reaction liquor.
I0086) washing the “insoluble' portion, drying and
packaging,
0.087 optionally, characterising the extracted products.
0088 Raw Material
I0089. The term "raw material', according to the present
invention,denotestheanimalbiomasscomprisingchitinused
forextracting chitin, preferably marineby-products used for
extracting chitin and/or entomological by-products used for
extracting chitin.
0090 According to one embodiment, the raw material
used in the method according to the present invention com
prises marine by-products, preferably crustaceans, prawns,
crabs, krill, more preferably crustacean shells and heads:
accordingto oneparticularembodimentofthe invention, the
raw material is obtained from cephalopods, preferably squid
orcuttlefish.
0091. According to one embodiment, the raw material
used in the method according to the present invention com
prises entomological by-products, preferably from beetles
such as the Tenebrio molitorbeetle, hymenoptera suchasthe
Hermetia illucens fly, more preferably insect shells and
heads.
0092. The raw material preparation operation should be
suitable for retaining the qualities thereofwhile meeting the
requirements ofthe method.
0093. According to oneembodimentofthe presentinven
tion,theraw materialpreparation comprises cleaning, drying
and/orgrinding operations.
0094. According to one embodiment, the raw material is
cleaned with water.
0.095 According to one embodiment, the raw material is
driedfrom 1 hourto36hourspreferably forapproximately 18
hours,preferablyinventilatedair,preferablyata temperature
of 5 to 35° C., more preferably of approximately 12° C.
According to one embodiment, the raw material is ground to
obtain fragments having a maximum diameter equal to
approximately 10mm,preferably havingadiameterlessthan
approximately 1 mm.
0096. According to one embodiment, the raw material,
preferably prepared by cleaning, drying and grinding, is
stored prior to extraction at a temperature between -30 and
-10°C., preferably at -20°C., preferably limiting the pres
ence ofoxygen.
0097. Reaction Medium
0098. The term “reaction medium', according to the
present invention, denotes the medium wherein the enzy
matic hydrolysis reaction in an acid medium takes place.
0099. The reaction medium preparation should account
forthe enzyme activity conditions used Such as thetempera
ture, solventandpH. Theselectionoftheseconditions makes
it possible to optimize the reaction time and yields.
0100. Accordingtooneembodiment,thereactionmedium
is maintained during the enzymatic hydrolysis at a tempera
turebetween2 and80°C., preferablybetween35 and45° C.,
morepreferably fromapproximately37 toapproximately 40°
C.
7. US 2014/0100361 A1
0101. According to one embodiment, the temperature of
the reaction medium is adapted to the enzyme used so that
saidenzymehasaquasi-optimalactivitythroughouttheenzy
matic hydrolysis.
0102) Accordingtooneembodiment,thereaction medium
is maintained during the enzymatic hydrolysis at a tempera
tureintherangeTit.0to20°C., preferablyTit.0to 15°C.,
preferably T0 to 10°C., where T is the temperature at
which the enzyme exhibits maximum activity. The tempera
tureselectedshould notinducethedegradation oftheenzyme
orinhibittheactionthereof.Advantageously,thetemperature
ofthe reaction medium is less than T. So as to limitenergy
consumption.
0103) According to one embodiment, the pH ofthe reac
tion medium is 0.5 to 6.5, preferably from 1.8 to 3.8, more
preferably from approximately 1.9 to approximately 2.1. If
the enzyme is pepsin, the pH of the reaction medium is
preferably from approximately 1.9 to approximately 2.1.
0104. According to one embodiment, the pH ofthe reac
tion medium is acidic and the value thereofis adapted to the
enzyme used so that said enzyme has an optimal activity.
01.05
tion medium is in the range pH-t2, preferably pH-t1.5.
preferably pH-1, where pH is the pH at which the
enzyme exhibits maximum activity. The pH selected should
beacidic toensurethatthechitin extractionyieldis sufficient.
0106. Accordingtooneembodiment,thereaction medium
is ready for use when the temperature and pH conditions
selected for the enzymatic hydrolysis reaction are stabilized.
0107 According to a first embodiment, the reaction
medium comprises at least one acid. According to a second
embodiment, the reaction medium further comprises a sol
vent such as water or an aqueous solution.
0108. According to oneembodimentofthepresentinven
tion, the acid used is preferably a dietary acid, preferably
phosphoric acid or formic acid.
0109 Whentheacidused intheenzymatichydrolysisstep
is a dietary acid, the products extracted by means of the
methodaccordingtothepresentinventionoffertheadvantage
of being suitable for easier use in the food and cosmetic
SectOrS.
0110. According to one embodiment, the acid concentra
tioninthereaction medium is from 0.1 to 6 mol-L', prefer
ably from 0.8 to 2.8 mol-L, more preferably from 0.9 to 1
mol-L".
0111. According to one embodiment, the acid concentra
tion in the reaction medium is adaptedto the mineral content
oftheraw material usedsothatthepHofthereaction medium
is acidic and remains constant throughout the enzymatic
hydrolysis.
0112
0113. According to one embodiment, the enzyme used in
the presentinvention is an activeenzyme in an acid medium,
preferablyaproteasehavingabroadspectrum ofactivityinan
acid medium, preferably pepsin ora stable acid protease.
0114. Accordingto oneembodiment,theenzymeconcen
tration in the reaction medium is adapted to the protein con
tentofthe raw material used. Accordingto one embodiment,
theenzymeconcentration is0.1 to 75%, preferably 5 to30%,
more preferably from approximately 23 to approximately
27% by mass in relation to the protein mass estimated in the
raw material.
According to one embodiment, the pH ofthe reac
Enzyme
Apr. 10, 2014
0115 Reaction Conditions
0116. According to one embodiment, the raw material is
mixed with the reaction medium andthe resulting mixture is
optionallyhomogenized by stirring for 0to 30 minutes, pref
erably for3to 10 minutes, morepreferablyforapproximately
5 minutes.
0117. According to one embodiment, the ratio between
the weight ofraw material prepared and the volume ofreac
tion medium is 1:60 to 2:1, preferably 1:7 to 1:3, more pref
erably equal to 1:5.
0118. According to one embodiment, the ratio between
the weight ofraw material prepared and the volume ofreac
tion medium is adapted to the size ofthe fragments of raw
material prepared. In particular, account is taken ofthe fact
that,whenthesizeofthefragmentsofraw material decreases,
Solventabsorption increases andthat, consequently, it is nec
essary to increase the Volume ofreaction medium.
0119) Adding raw material into the acid reaction medium
may give rise to the formation offoam dueto theproduction
ofcarbon dioxideduetothepresenceofcalcium carbonatein
the exoskeleton ofcrustaceans and insects.Accordingto one
embodiment, the vessel used for performing enzymatic
hydrolysis has a suitable Volume for preventing the foam
liable to form from overflowing. The riskoffoam production
increases when the temperature of the acid before mixing
increases.
I0120 According to one embodiment, the temperature of
thereaction mediumbeforeaddingtheraw materialis5to 65°
C., preferably 20 to 30°C., more preferably approximately
25°C. In this embodiment, the temperature of the reaction
medium is selectedin orderto beless than the temperatureat
which the enzymatic hydrolysis is to be conducted, so as to
limit foam formation when adding raw material.
I0121 According to a first embodiment, the enzyme is
added directly into the homogenized reaction medium
optionally containing the raw material.
0122. According to a second embodiment, the enzyme is
solubilized in water, or in a solution, preferably an aqueous
Solution,andisaddedintothehomogenized reaction medium
containing the raw material.
I0123. According to one embodiment, the enzymatic
hydrolysis reaction is performed under stirring so as to opti
mize the contact between the raw material and the enzyme.
0.124. According to one embodiment, the initial pH and
temperature conditions ofthe reaction medium are kept con
stant throughout the enzymatic hydrolysis reaction. Accord
ingtoafurtherembodiment,theinitialpHand/ortemperature
conditions of the reaction medium are not kept constant
throughout the enzymatic hydrolysis reaction.
0.125. According to one embodiment, the enzymatic
hydrolysis reaction is performed in areactorequipped with a
device for regulating the temperature. According to a first
embodiment, said reactorisadouble-jacketreactorwherein a
heat transfer fluid circulates, the temperature of said fluid
beingpossibletocontrol.Accordingtoasecondembodiment,
said reactor is equipped with a heating resistor, thetempera
ture ofsaid resistor being suitable forbeing controlled.
I0126. According to a first embodiment, the pH is stable
throughoutthe enzymatic hydrolysis. According to a second
embodiment, the pH is adjusted, during the enzymatic
hydrolysis reaction, to the pKa value between the acid used
and calcium carbonate by adding a concentrated acid solu
tion, the acid being the same as that used in the reaction
medium.
8. US 2014/0100361 A1
0127. According to one embodiment, the duration ofthe
enzymatic hydrolysis is from 30 minutes to 24 hours,prefer
ably from 1 hour to 12 hours, preferably from 3 hours to 8
hours, more preferably approximately 6 hours.
0128. According to one embodiment, the duration ofthe
enzymatichydrolysis is adaptedto theactivity oftheenzyme
usedforconductingtheenzymatic hydrolysis reaction, to the
acid used and to the raw material.
0129. According to one embodiment, the duration ofthe
enzymatic hydrolysis is adapted according to the features
sought fortheendproducts, such as the degree ofpurity, the
degree ofpolymerization and the degree ofacetylation.
0130. According to one embodiment, theenzymatic reac
tion produces a reaction liquor comprising soluble and
insoluble portions.
0131 Products Separation
0.132. According to one embodiment, the soluble and
insolubleportions ofthe reaction liquorare separated by any
suitable means known to those skilled in the art.
0.133 According to a first embodiment, the soluble and
insoluble portions are separated by filtration. According to
one embodiment, the filtration is performed by a filtration
system preserving the integrity of the structures of the
extracted compounds. According to a further embodiment,
the filtration is performed by a membrane press filtration
system. According to a further embodiment, the filtration is
performed on a filter cloth, preferably on bolting cloth.
0134. Accordingtoa secondembodiment,the solubleand
insoluble portions are separated by centrifugation.
0135 According to one embodiment, the insoluble por
tion of the reaction liquor very predominantly contains
chitins and the soluble portion contains various compounds
Suchas lipids,pigments, Sugars, mineral salts, aminoacids or
peptides.
0136. According to one embodiment, the insoluble por
tion is rinsed using a solvent. According to a first embodi
ment, the solvent is water or an aqueous Solution. This
embodiment is preferred ifthechitins are subsequently used
fordietary applications. Accordingto a secondembodiment,
the insoluble portion is first rinsed with water or an aqueous
Solution and then with a bleaching agent such as hydrogen
peroxide, Sodium hypochlorite orpotassium perSulphateand
is rinsedagain with wateroranaqueous solution.Thissecond
embodiment is preferred ifbleachingofthechitins is sought.
In this embodiment, the bleaching agents used are in accor
dance with legislation.
0.137 Chitins are very hygroscopic substances, wherein
the biological activity may be degraded by an increase in
temperature.
0138 According to one embodiment, the filtered and
rinsed insoluble portion is then dried for 8 to 16 hours, pref
erably for approximately 12 hours, in an oven wherein the
temperature is preferably less than 100° C., preferably
between50and95°C.,morepreferablyapproximately90°C.
0.139. Accordingtooneembodiment,thefilteredinsoluble
portion is neutralized with sodium hydroxide. According to
oneembodiment, the insoluble portion is freeze-dried.
0140. According to one embodiment, the dried and/or
freeze-dried insoluble portion is packaged in vessels such as
glass orplastic bottles or vacuum pouches and storedprefer
ably atambienttemperature in a dry place. Accordingto one
particularembodiment, theinsolubleportion(chitin)isstored
ata temperature less than ambienttemperature, preferably at
Apr. 10, 2014
atemperaturefrom -30 to0°C., morepreferably from -20to
-10°C., morepreferably at approximately -20°C.
0.141. Accordingtoafirstembodiment, thesolubleportion
is centrifuged. According to a second embodiment, the
soluble portion is dialysed and ultrafiltered. According to a
third embodiment, the compounds from the neutralized
soluble portion are extracted using organic solvents. The
organic oraqueous solvents arethen evaporatedto beable to
obtain the compounds ofinterest.
0142. The technique for processing the soluble phase is
dependent on the nature ofthe compounds to be recovered.
0143. ExtractionYields
0144 Controlling the reaction medium (enzyme concen
tration, pH and temperature) according to the raw material
used makes itpossible to control the yieldand thebiochemi
cal and physicochemical characteristics of the chitins
obtained.Theoretically,byextendingthehydrolysistime,the
degree ofpolymerization tends to be reduced.
0145 The mass extraction yield ofthe insoluble portions
(Yd) is dependenton the nature ofthe raw material, acidand
enzyme used and is calculated using the following formula:
Yd%=100*(dried insoluble weight)/(dry raw material
weight)
0146 According to one embodiment, the insoluble por
tionpredominantly contains chitins andresidualproteinsand
minerals not removed during the enzymatic hydrolysis reac
tion.
0147 Applying conventional chemical extraction treat
ment to the insoluble portions obtained using the method
according to the present invention makes it possible to esti
mate the residual impurity content in the insoluble portion.
Indeed,this treatment is suitablefor removingthe majority of
residual proteins and minerals.
0.148. The degree ofchitin purity (D purity) is estimated
by means of gravimetry, by measuring the mass of the
insoluble sample before and after treating the insoluble por
tions with 1.25 mol-L' sodium hydroxide at 90° C. for 1
hour. As mentioned above, this treatment is suitable for
removing residual proteins and minerals. The estimated
degree ofpurity is calculated using the following formula:
D-purity=100*(mass ofinsoluble portion aftertreat
ment)/(mass ofinsoluble portion beforetreat
ment)
0149 Accordingtooneembodiment,theestimateddegree
of chitin purity (D'purity) is greater than 75%, preferably
greater than 80%, more preferably greater than 85%, more
preferably greater than 90%.
0150. According to one embodiment, the mass residual
proteincontentinthedriedinsolubleportionisless than20%,
preferably less than 15%, preferably less than 10%, more
preferably less than 5%.
0151. According to oneembodiment, the massproportion
ofproteins removed by the method according to the present
invention is greater than 80%, preferably greater than 85%,
more preferably greater than 90%, more preferably greater
than 95%.
0152. According to one embodiment, the weight amount
ofresidual mineral in the dried insoluble portion is less than
5%, preferably less than 3%, more preferably less than 1%.
0153. According to one embodiment, the weight amount
ofminerals removed by the method according to the present
invention is greater than 95%, preferably greater than 97%,
more preferably greater than 99%.
9. US 2014/0100361 A1
0154 According to one embodiment, an additional
bleachingoperation isperformedontheinsolubleportion, for
removing pigments along with a portion ofthe residual pro
teins and minerals.
0155 According to one embodiment, an additional
deacetylation operation is performed on the insoluble por
tion, for producing chitosan and removing a portion of the
residual proteins.
0156 Features ofthe Chitins Extracted
0157 Accordingtooneembodiment,thechitins extracted
using the method according to the present invention may be
usedas is or converted into chitosan, chitin oligomers, chito
san oligomers or optionally N-acetylated glucosamines.
0158. The method according to the present invention is
Suitableforobtainingawiderangeofchitin qualityinrespect
ofthedegreeofpurityandpolymerization.Theotherfeatures
(pattern distribution, C, B and Y form) are dependent on the
nature of the raw material and not on the features of the
method according to the invention.
0159. Accordingtooneembodiment,thechitins extracted
usingthe methodaccordingtothepresent inventionaresimi
lar in form to the natural form ofchitin. In other words, the
chitins extracted using the method according to the present
invention are not denatured or only slightly denatured in
relation to natural chitin.
0160 Accordingtooneembodiment,theestimateddegree
ofpurity ofthe chitins extracted using the method according
to the present invention is greater than 85%, preferably
greater than 90%, more preferably greater than 95%.
0161 Thedegreeofpurity ofthechitinsobtainedusingthe
method according to the present invention is sufficient to be
able to convert said chitin in the form of chitosan, chitin
oligomers, chitosan oligomers and glucosamines.
0162 According to one embodiment, the degree ofpoly
merization ofthe chitins isestimatedby calculatingbasedon
the average molecular mass ofsaid chitins. Accordingto one
embodiment, the average molecular mass of the chitins is
estimated by calculatingbased on the intrinsic viscosity. The
intrinsic viscosity may be determined using the method
described by Poirier et al. (Poirier, M. and Charlet, G., Car
bohydrate Polymers, 2002, 50, 363-370).
0163 According to one embodiment, the degree ofpoly
merization ofthe chitins extracted using the method accord
ing to thepresentinventionis from 1.10 to 1.10, preferably
from 1.10 to 1.107, more preferablyfrom 1.10 to 1.10.
0164. According to oneembodiment, the degreeofacety
lation ofthe chitins extracted using the method according to
the present invention is from 80% to 100%, preferably from
90% to 98%, more preferably from 95% to 97%.
0.165 According to one embodiment, the crystallinity
index ofthe chitins extracted using the method according to
the present invention is from 10% to 70%, preferably from
20% to 50%, more preferably from 30% to 40%.
0166 Soluble Compounds
0167 According to one embodiment, the soluble sub
stances extracted using the method according to the present
invention may be peptides, pigments, Sugars and mineral
salts.Theuseofdietary acidinthis methodenables the useof
these compounds in the food, dietetic and nutraceutical sec
tOrS.
0168 The present invention thus offers the advantage of
limiting the quantity ofwaste since all Substances other that
chitin extracted using the method according to the invention
can also be used or recovered.
Apr. 10, 2014
0169. According to one embodiment, the pigments
extractedusingthemethodaccordingtothepresentinvention
are astaxanthin.
BRIEF DESCRIPTION OF THE DRAWINGS
(0170 FIG. 1 represents a scheme of the method for
extracting chitin according to the invention.
EXAMPLES
0171 The invention will be understood more clearly on
readingthefollowing example, illustratingthepresentinven
tion in a non-limiting fashion
Example 1
Enzymatic Hydrolysis Using Pepsin in the Presence
ofPhosphoricAcid
0172 Materials
(0173 The raw material usedis the raw Panaeus vannamei
prawn exoskeleton. The raw material is dried at 12° C. in
ventilated air and ground to produce fragments less than 1
mm in size.The raw materialprepared is stored at -20°C. in
a WaCl.
0.174. The reagent used to maintain theacidic pH is phos
phoricacid.Theacidconcentration is calculatedaccordingto
theinitial mineralcontentintheraw materialprepared. Foran
initial mineral contentof25% in weightrelative to theweight
ofdryraw material, a 0.94 mol-L' phosphoric acid solution
is used to keep the pH ofthe reaction medium around 2.
(0175. The acid protease used is pepsin (CAS 9001-75-6,
supplier: Sigma, activity: 8112 U/mg). It is stored in powder
form at +4°C. It is solubilised in distilled water for 15 min
beforebeing introducedintothereaction medium. The quan
tity ofenzymeadded in this example is equivalent to 25% of
the estimated protein mass in the initial raw material. In this
way, for a sample of 5 g ofraw material having a moisture
content ofapproximately 15% andaprotein content ofclose
to 40%, this isequivalent to8.5% ofenzyme in relation tothe
raw material, i.e. a quantity ofpepsin of0.43g.
(0176)
0177 5g ofraw material, prepared as describedabove, is
weighed. The composition ofthe dry extract is determined
according to the analytical methods described below.
(0178 A 0.94mol-L' phosphoricacidsolution(25mL)is
preheated to 30° C. and added to the raw material. The mix
ture is stirred for 5 min so that it homogenises. The pH
measured with a pH-meter should be stable and be between
1.9 and 2.1.
0179 Pepsin (0.43 g), previously solubilised in 1 mL of
water,isadded to thereaction medium.Themixture isheated
to 40°C. on a hotplate and incubated in an oven kept at 40°
C.1° C.
0180. After6hoursofincubation,themixtureisfilteredon
bolting cloth and rinsed with plenty of distilled water. The
retentate is resuspended in distilled water, the mixture is
stirred for 10 min beforebeing filtered and rinsedagain with
water.Thesolidfractionobtainedis transferredintoacupand
dried overnight at 90° C. in an oven. The mass ofdry extract
obtained (m=1.29g) is suitable forcalculating theextraction
yield, i.e. 30.26% w/w.
Protocol
10. US 2014/0100361 A1
0181 Analyses
0182. The moisture content ofthe sample is measured by
means ofgravimetry, by measuring the mass ofthe sample
before and after being placed overnight at 105°C.
0183 The mineral content is determined by means of
gravimetry, by measuring the mass ofthe sample before and
after incineration at 600° C. for 6 hours.
0184 The protein content is estimated by means ofgas
chromatographybyassayingthetotalaminoacids. Itcan also
be measured by means ofa colorimetric assay (Lowry, BSA,
Bradford orCoomassieblue)orby meansofa Kjeldahlassay.
0185. The chitin content can be measured by means of
gravimetry, by measuring the mass ofthe sample before and
after the following treatments:
0186 for the raw material: treatment for 60 minutes
with 1NHClatambienttemperature, followedby 1.25N
NaOH at 90° C. for 120 minutes and finally bleaching
with 33% hydrogen peroxide and acetone;
0187 for the hydrolysis products, the treatment is lim
ited to a treatment with 1.25 N NaOH at 90° C. for one
hour.
0188 The molecular mass ofthe chitins is estimated by
calculatingbased on the intrinsic viscosity. The intrinsic vis
cosity can be determined using the method described by
Poirier etal., which is based on the Mark-Houwink equation
(Poirier, M. and Charlet, G., Carbohydrate Polymers, 2002,
50, 363-370). In this way, the intrinsic viscosity was deter
minedby measuring the reduced viscosity using Solutions of
variouschitinconcentrations inN,N-dimethylacetamidecon
taining 5% LiCl. The apparatus used is an Ubbelohde capil
lary viscometer. The viscometerconstant K is 0.3 cSt/s. The
measurement Volume is 15 mL.
0189 Thedegreeofpolymerizationiscalculatedusingthe
molecular mass ofthe chitins.
0190. The degree ofacetylation is estimated by means of
protein liquid NMR, according to the method described by
Einbu A. Varum K., 2008. Chitin (20 mg) is solubilized in 1
mLofDC1 (7.6N in DO, Euriso-top) with magnetic stirring
atambient temperature for5hours.The "H NMR analysis is
performed at 300 K using a Bruker ALS300 spectrometer
(300 MHz, referenceTMSP 0.00ppm). The degreeofacety
lation is then calculated basedon the intensity ofthecharac
teristic proton NMR signals, according to the formula given
by Einbu et al.
0191 The crystallinity index is determined by means of
X-raydiffraction.Thediffractometerusedisa Bruker-axS D8
Discover (Karlsruhe, Germany). Radiation is produced in a
coppertube(CuKC-1.5405A)and thebeamsproducedare
recorded every 10 min. Using the spectra obtained, the
method forcalculating the crystallinity index is based on the
ratio between theareas ofthecrystalline Zones overthe total
area (Osario-Madrazo A. David L., Trombotto S. Lucas J.
M., Peniche-Covas C. and Domard A. Carbohydrate Poly
mers, 2011, 83, 1730-1739).
(0192
0193 After6hoursofenzymatichydrolysiswithpepsinin
the presence of phosphoric acid, the extraction yield is
30.26+0.32% w/w. The composition of the dry extract
obtainedcan becomparedto thatofthe fully dried raw mate
rial orthe prepared raw material used in this example (table
1):
Results and Discussion
Apr. 10, 2014
TABLE 1
mois
ture minerals proteins chitin lipids Sugars
fully dry
8W
material:
% by mass O% 25% 40% 30% ND ND
prepared
8W
material:
% by mass 14.55% 21.25% 34% 25.5% 3.5%. 1.2%
dry
extract:
% by mass O% O.99 10.98 88.42 ND ND
(+0.03%) (+1.01%) (+1.22%)
per 100 g Og 0.30 g 3.32 g 26.76 g ND ND
ofdry raw
material
ND: not determined
0194 In view ofthecomposition ofthe dry raw material,
the quantities of minerals and proteins removed using the
method are 98.5% and91.7%, respectively.
0.195 The residual mineral and protein contents (table 1)
are those found in the unprocessed end product, without a
bleaching step. Applying a bleaching agent or washing with
Sodium hydroxide enhances the degree ofpurity.
(0196. The degree ofacetylation measured by NMR is, in
this example, in the region of95%. The molecular weight of
this sample is in the region of 10 to 10 g/mol and the
crystallinity index35%. These featuresaresimilarto those of
native chitin.
0197) Theperformances ofthis examplecan be enhanced
by increasing the quantity of pepsin used. In this way, the
experiment was conducted with a pepsin concentration of
41% with respectto thequantity ofproteinspresentintheraw
material, instead of 25% previously. The degree of chitin
purityincreases (96.78%insteadof88.42%)as thedeprotein
ization isenhanced(92.00% ofproteins removed)along with
the demineralization (99.23% ofminerals removed).
1.A methodfortheenzymaticextractionofchitin,wherein
said method is carried out in a single step, wherein chitin is
obtainedby theenzymatichydrolyzis ofraw materialconsti
tuted by animal biomass comprising chitin, said enzymatic
hydrolyzis using an active enzyme in an acid medium.
2.A methodaccordingto claim 1, wherein said single step
is an enzymatic hydrolysis fordeproteinizinganddemineral
izing said raw material simultaneously.
3. A method according to claim 1, wherein said enzyme
active in an acid medium is a protease having a broad spec
trum of activity in an acid medium, preferably pepsin or a
stable acid protease.
4. A method according to claim 1, wherein the enzyme
concentration usedforhydrolysis is 0.1 to 75%,preferably 5
to 30%, more preferably from approximately 23 to approxi
mately 27% in weight relative to the estimated weightofthe
protein in the raw material.
5.A methodaccordingtoclaim 1,whereintheacidmedium
is obtainedby means ofthe presence ofan acid, preferably a
dietary acid, morepreferably phosphoric acid orformic acid.
6.Amethodaccordingto claim 1, whereinanimalbiomass
comprising chitin comprises marine by-products, preferably
11. US 2014/0100361 A1
marine by-products obtained from crustaceans, preferably
prawns, crabs orkrill, orfrom cephalopods,preferably squid
or cuttlefish.
7. A method according to claim 1, wherein said animal
biomass comprising chitin comprises insect by-products,
preferably insect by-products obtained from beetles or
hymenoptera.
8. A method according to claim 1, further comprising
operations forwashing, drying and/orgrindingtheraw mate
rial, preferably water washing, cold drying and/or grinding
operations.
9.Amethodaccordingtoclaim 1, furthercomprising reac
tion medium treatmentoperationsattheendoftheenzymatic
hydrolyzis, said operations comprising operations for sepa
rating the Solid and liquid phases, rinsing and/or drying the
insoluble portion.
10. A method foroptimizingthe method fortheenzymatic
extraction ofchitindescribedin claim 1, wherein said method
comprises at least one ofthe following steps:
a) selecting the pH of the acid medium in the range
pHenzi-2, preferably pHenzit1.5, preferably pHenz+1,
where pHenz is the pH at which the enzyme exhibits
maximum activity,
b)electingthetemperatureoftheacid medium intherange
Tenzi-20° C., preferably Tenzi.15° C., preferably
Tenzi-10°C.,whereTenzis thetemperatureatwhich the
enzyme exhibits maximum activity,
c) determining the mineral and protein content ofthe raw
material,
d) calculating the acid concentration to be used in the
reactionmedium,accordingtothemineral contentofthe
Apr. 10, 2014
raw material. Such thatthepH is maintainedthroughout
the enzymatic hydrolysis atthe pH selected in step a),
e) calculating the proportion of enzyme to be used with
respect to the protein content ofthe raw material,
f) determining the reaction time forobtaining chitin orthe
chitin derivatives sought.
11. Chitin that can be obtained by means ofthe method
according to claim 1.
12. Chitosan that can be obtained by deacetylating chitin
according to claim 11.
13. Composition comprising chitin according to claim 11.
14. A pharmaceutical composition comprising chitin
according to claim 11.
15.Acosmetic compositioncomprisingchitinaccordingto
claim 11.
16.A medical device comprising chitin accordingto claim
11.
17. A food product, nutraceutical composition, dietetic
composition,foodSupplementorfunctional food comprising
chitin according to claim 11.
18.Acomposition comprisingchitinaccordingto claim 11
fortheuse thereofin watertreatment, filtrationand/ordepol
lution.
19.Atexturingagentcomprising chitin accordingto claim
11.
20. A method according to claim 2, wherein said enzyme
active in an acid medium is a protease having a broad spec
trum of activity in an acid medium, preferably pepsin or a
stable acid protease.