Deproteinization Rate Research Articles

Valorizing Shrimp Shell as a Source of Chitin with Crude Enzymes from Germinated Winter Wheat and Buckwheat

Chitin is a valuable resource found in shrimp shells. Typically, the industrial process for isolating chitin from crustacean shells involves using chemical methods that require large amounts of acid and alkaline. While commercial protease application is a more environmentally friendly method, it can be costly. Germinated grains can be a source of low-cost protease for chitin isolation. This study aimed to assess the effectiveness of crude enzymes extracted from germinated winter wheat (Triticum aestivum) and buckwheat (Fagopyrum esculentum) to isolate crude chitin from white shrimp shells. The proteolytic and chitinolytic activities of the crude enzyme extracts were determined and applied in the deproteinization step during chitin isolation from shrimp shells. The deproteinization rate was assessed, the crude chitin yield was compared, and the isolated chitins were characterized through Fourier Transform Infrared (FTIR) spectroscopy analysis and x-ray diffraction (XRD) analysis. Crude enzymes extracted from germinated winter wheat and buckwheat exhibited proteolytic activity of approximately 0.49 U/mL and 0.46 U/mL, respectively. Using winter wheat extract and buckwheat extract acted on demineralized shrimp shells exerted a deproteinization rate of 53.6 ± 1.0% and 58.0 ± 2.3% and yielded 30.3 ± 5.31% and 29.2 ± 3.99% crude chitin, respectively. FTIR spectra of the isolated chitins showed amide I, amide II, amide III, asymmetric stretching of the C-O-C bridge, O-H stretching, N-H stretching and asymmetric C-H stretching, typical functional groups for chitin. The crystallinity index for commercial chitin, chemical-isolated chitin, winter wheat extract-isolated chitin and buckwheat extract-isolated chitin was 86.49%, 88.74%, 88.82% and 75.87%, respectively. Buckwheat extract-isolated chitin, deacetylated with lower crystallinity, warrants further investigation. Crude enzymes from winter wheat and buckwheat to remove protein from demineralized shrimp shells can be a more environmentally friendly method to valorize shrimp shells as a chitin source.

Extraction of Chitin from Black Soldier Fly (Hermetia illucens) and Its Puparium by Using Biological Treatment.

Chitin is the second-largest natural polymer polysaccharide in nature. Due to its important physical and chemical properties and excellent biocompatibility, safety, and biodegradability, it is widely used in agriculture, medicine, food, environmental protection, and other fields. However, traditional extraction methods cause environmental pollution and damage the structure of chitin. Bioprocessing is an emerging technology that shows great potential. In this research, the puparia and adults of black soldier fly (BSF) (Hermetia illucens L.) were used as raw materials. A continuous fermentation method was designed to extract chitin, by using Bacillus subtilis S4 and Acetobacter pasteurianus AS1.41. The Fourier transform infrared spectroscopy identification results showed that the extracted sample was α-chitin. Under continuous fermentation conditions, the deproteinization (DP) rate, demineralization (DM) rate, chitin yield (CY), and deacetylation degree (DD) of puparium chitin were 33.33%, 94.92%, 59.90%, and 18.52%, respectively. Meanwhile, the DP rate, DM rate, CY, and DD of adult chitin were 46.63%, 90.93%, 47.31%, and 37.38%, respectively. For BSF, B. subtilis S4 had a certain DP ability, and A. pasteurianus AS1.41 had a good DM effect. Moreover, BSF at different developmental stages could affect CY, and a higher concentration of NaOH was more favorable for deacetylation. Overall, simultaneous continuous fermentation could be a new biological approach to extract chitin from BSF.

Simultaneous Preparation of Chitin and Flavor Protein Hydrolysates from the By-Products of Shrimp Processing by One-Step Fermentation with Lactobacillus fermuntum.

One-step fermentation, inoculated with Lactobacillus fermentum (L. fermentum) in shrimp by-products, was carried out to obtain chitin and flavor protein hydrolysates at the same time. The fermentation conditions were optimized using response surface methodology, resulting in chitin with a demineralization rate of 89.48%, a deproteinization rate of 85.11%, and a chitin yield of 16.3%. The surface of chitin after fermentation was shown to be not dense, and there were a lot of pores. According to Fourier transform infrared spectroscopy and X-ray diffraction patterns, the fermented chitin belonged to α-chitin. More than 60 volatiles were identified from the fermentation broth after chitin extraction using gas chromatography-ion transfer spectrometry analysis. L. fermentum fermentation decreased the intensities of volatile compounds related to unsaturated fatty acid oxidation or amino acid deamination. By contrast, much more pleasant flavors related to fruity and roasted aroma were all enhanced in the fermentation broth. Our results suggest an efficient one-step fermentation technique to recover chitin and to increase aroma and flavor constituents from shrimp by-products.

Distinguishing functional exosomes and other extracellular vesicles as a nucleic acid cargo by the anion-exchange method.

The development of a new large‐scale purification protocol is required for research on the reliable bioactivity and drug discovery of extracellular vesicles (EVs). To address this issue, herein, we propose an effective method for preparing high‐performance exosomes (EXOs) by using an anion‐exchange method. Cytotoxic T‐lymphocyte (CTL) EVs from 4 L of culture supernatant through a 220 nm cut‐off filter are divided into two populations at a deproteinization rate of over 99.97%, which are eluted at low (0.15 M–0.3 M) and high (0.3 M–0.5 M) NaCl concentrations (approximately 2 × 1012 and 1.5 × 1012 particles, respectively) through the anion‐exchange column chromatography. The former are abundant in EXO proteins, including late endosome‐associated proteins and rab‐family and integrin‐family proteins, and functional micro (mi) RNAs, and have bioactivity for preventing tumour metastasis by depleting mesenchymal cell populations in the primary tumour lesions. By contrast, the latter is microvesicle (MV)‐like particles including DNA, core histone and ribosomal proteins, and GC‐rich miRNAs with unknown function, and are easily phagocytosed by mannose receptor+ Kupffer cells. Thus, the anion‐exchange method is suitable for the large‐scale separation of bioactive EXOs and MV‐like EVs as a cargo for dangerous nucleic acids at high‐purity.

Microbial Conversion of Shrimp Heads to Proteases and Chitin as an Effective Dye Adsorbent.

As a green and effective technique in the production of a large number of valuable products, the microbial conversion of chitinous fishery wastes is receiving much attention. In this study, protease production using the Paenibacillus mucilaginosus TKU032 strain was conducted on culture media containing several common types of chitinous fishery by-products serving as the carbon and nitrogen (C/N) nutrition source. Among the chitinous wastes, 1.5% (w/v) shrimp head powder (SHP) was found to be the most appropriate nutritional source for protease production when a maximal enzyme activity of 3.14 ± 0.1 U/mL was observed on the 3rd day of the culture period. The molecular mass of P. mucilaginosus TKU032 protease was estimated to be nearly 32 kDa by the polyacrylamide gel electrophoresis method. The residual SHP obtained from the culture medium was also considered to be utilized for chitin extraction. The deproteinization rate of the fermentation was estimated to be 45%, and the chitin obtained from fermented SHP (fSHP) displayed a similar characteristic Fourier-transform infrared spectroscopy (FTIR) profile as that from SHP. In addition, SHP, fSHP, and chitins obtained from SHP and fSHP were investigated for their adsorptive capacity of nine types of dyes, and chitin obtained from fSHP displayed a good adsorption rate on Congo Red and Red No. 7, at 99% and 97%, respectively. In short, the results provide potential support for the utilization of SHP in the production of P. mucilaginosus TKU032 protease via the fermentation as well as the preparation of chitin from fSHP as an effective dye adsorbent.

Green, Simple, and Effective Process for the Comprehensive Utilization of Shrimp Shell Waste.

An environmentally friendly approach for the comprehensive utilization of shrimp shell waste was reported. Instant catapult steam explosion (ICSE) was employed for shrimp shell waste pretreatment. After ICSE, lower crystallinity and greater surface areas of shrimp shells were achieved, which significantly enhanced the extraction of chitin. Compared to the traditional method, weaker organic acid (HCOOH) and much lower dosages of KOH (90% molar less) were used, and chitin with a high demineralization rate (98.2%) and deproteinization rate (97.7%) was obtained. The wastewater was neutralized by simply intermixing, and it was recycled as a potential plant fertilizer because it contained more oligopeptides, calcium, and potassium, but it was less salty and therefore non-toxic to plants. The whole process produced less solid waste and no waste water. The obtained chitin also showed a low degree of acetylation (50.5%), which demonstrates the potential for environmentally friendly preparation of chitosan in dilute alkali through ICSE.

A green and efficient deproteination method for polysaccharide from Meretrix meretrix Linnaeus by copper ion chelating aerogel adsorption

Abstract Polysaccharides (PMM) from Meretrix meretrix Linnaeus exhibit a variety of biological activities; however, conventional purification methods are costly, time consuming and introducing toxic agents. This paper aimed to develop a green and efficient method for the deproteinization of PMM by copper ion chelating aerogel (CCA) adsorption. First, CCA was successfully prepared by copper ion chelating composite aerogel (CA). Then, CCA adsorption was applied to remove protein mixed in PMM. The optimal parameter conditions of this deproteinization method were screened, using the deproteinization rate (Dr%), polysaccharide recovery rate (Rr%) and selectivity coefficient (Kc) as evaluation indicators, by single factor experiment. The optimal conditions were obtained as follows: CCA dose of 1.4%, pH value of 5.0 and adsorption temperature of 30 °C. Under these conditions, equivalent Dr% (93.31 ± 1.33%), higher Rr% (93.34 ± 1.08%) and Kc (14.01 ± 0.46) were achieved compared with the latest reported deproteinization report. Finally, the effect of deproteinization between CCA adsorption and Sevag method, which is the most classical deproteinization method, were compared to verify the practicability of CCA adsorption. As a result, CCA adsorption not only showed much higher Rr% as well as Kc, but also would not cause any reagent contamination and environment pressure. In conclusion, CCA adsorption would be a green and highly efficient method with great potential in large-scale environmentally sustainable deproteinization of polysaccharides in the future.

Recovery of Chitin and Protein from Shrimp Head Waste by Endogenous Enzyme Autolysis and Fermentation

The industrial processing of shrimp produces massive quantities of solid waste that is a notable source of animal protein, chitin, carotenoids, and other bioactive compounds that are not appropriately utilized. In the present study, chitin and protein extraction from shrimp head with autolysis and fermentation using Bacillus licheniformis were investigated. The results showed that when shrimp heads were autolyzed with a natural pH at 50°C for 4 h, the total amino acid nitrogen in the supernatant was 5.01 mg mL−1. Then, when a 50% (v/m) inoculum of the hydrolysate was incubated at 60°C for 10 h, a deproteinization rate of 88.3% could be obtained. The fermented supernatant was processed into a dry protein powder, while the residues were demineralized by 10% citric acid for chitin. The recovered protein powder contained 5.5% moisture, 11.5% ash, and 66.7% protein, while the chitin contained 3.5% moisture, 2.1% ash, and 3.1% protein. In addition, amino acids, minerals, heavy metals, the degree of acetylation, microstructure, and Fourier-transform infrared (FT-IR) spectroscopy results were analyzed. Furthermore, the statistics of the large scale trial after treatment with 20 kg of shrimp heads were analyzed. Thus, this work made the shrimp waste utilization environmentally sound and valuable.

The Deproteinization, Antioxidant Acticities and Inhibitory Effect on α-Amylase of Polysaccharides from Corn Silk

An approach of deproteinization of polysaccharides from corn silk (CSPs) was established to improve the purity and activities of CSPs by investigating Sevag method, trichloroacetic acid (TCA) method and HCl method. Deproteinization rate, polysaccharide retention rate, antioxidant activities and inhibition α-amylase effects were used as indicative mark to select suitable deproteinization method of CSPs. The crude CSPs was extracted by the hot water method. Results showed that TCA method had higher deproteinization rate and polysaccharide retention rate than that of Sevag method and HCl method, and the deproteinization optimum concentration of TCA was 4%. Under the optimized conditions, the deproteinization rate and polysaccharide retention rate were 53.68% and 64.13% respectively. In terms of antioxidant and α-amylase inhibitory effects, TCA-deproteinized CSPs (T-CSPs) had stronger activities, followed by Sevag-deproteinized CSPs (S-CSPs) and HCl-deproteinized CSPs (H-CSPs). Form a conclusion, TCA method was a good way to remove protein.

Effect of deproteinization methods on the antioxidant activity of polysaccharides extracted from Lentinus edodes stipe

Removal of protein from plant polysaccharide extract is often regarded as essential step for antioxidant activity evaluation. The aim of this study was to compare two different methods (neutral protease and Sevag) for efficient removal of protein from Lentinus edodes stipe polysaccharide extract. Orthogonal test was used to optimize the parameters of neutral protease deproteinization effects on polysaccharide extracted from Lentinus edodes stipe. Furthermore, antioxidant activity was also investigated by the two deproteinization methods. Results showed that the optimum parameters of deproteinization by neutral protease were pH of 6, temperature of 60 °C, time of 1 h, and enzyme amount 0.2%. Under these conditions, the polysaccharide retention rate was 92.29%, and the deproteinization rate was 84.30%. This result was higher than that of Sevag, which deproteinization rate and polysaccharide retention rate were 55.43% and 82.29%, respectively. The antioxidant activity of polysaccharide declined after the polysaccharide deproteinized for both techniques, however, the polysaccharide deproteinized by Sevag showed the lowest antioxidant activity. Therefore, neutral protease could be used for deproteinization of polysaccharide extracted from Lentinus edodes stipe.

Extraction of polysaccharides under vacuum condition from Lentinus edodes stipe and their antioxidant activities in vitro.

To improve the efficiency of polysaccharide extraction, vacuum technology was used to breakdown the cell wall to extract polysaccharides from Lentinus edodes stipe, and the optimal parameters were optimized by response surface methodology. Then the antioxidant activities in vitro of the polysaccharides were evaluated. Results showed that the optimal conditions for polysaccharide extraction were temperature of 62°C, vacuum of 0.08MPa, stirring speed of 1200r/min, time of 25min and material/liquid ratio of 1:26 (g/mL). Under these conditions, the yield of polysaccharide was 4.28%. The decolorization rate and deproteinization rate of polysaccharides treated by kaolin and hydrogen peroxide were higher than that of activated carbon. However, there was no significant influence on the polysaccharide retention rate, that was about 80%. Results also indicated that polysaccharide treated with activated carbon could obtain higher antioxidant activity.

Study on the Deproteinization of Polysaccharides from Lethariella spp.

The objective of this study was to discuss the method for deproteinization of the polysaccharides from Lethariella spp. Based on the single factor experiment, the orthogonal design was applied to select the optimal condition for the deproteinization of polysaccharides. The deproteinization rate and polysaccharide retention rate was used as indexes which were determined by UV-Vis spectrometer. The optimal extraction conditions were as follows: (Ratio of n-butyl alcohol to chloroform was 0.20, dosage of mixture was 20%, deproteinization duration was 10 min and times of deproteinization were five. Under this optimal condition, deproteinization rate and polysaccharide retention rate of polysaccharides was 55.37% and 60.34%, respectively. The process was suitable and feasible for the deproteinization of polysaccharides in Lethariella spp. extreact.

Deproteinization of Distillery Yeast Biomass Waste by Protease-producing Bacillus megaterium PB 4

The distillery yeast biomass (DYB), a waste by-product from distilleries is one of the under-utilized protein sources to be exploited for the production of protein hydrolysates. In this study, a neutral protease produced from Bacillus megaterium PB4 isolated from agro wastes enriched soil exhibited significant deproteinization of distillery yeast biomass to yield protein hydrolysates. Among three protease producing strains isolated, the maximum protease production of 120.3 ± 1.4 U mL-1 was exhibited by PB4 grown in Luria-Bertani (LB) broth supplemented with casein at pH 7.0 and 30°C. The minimal media supplemented with various carbon and nitrogen sources, glucose and peptone significantly improved the protease production in PB4. The effect of metal ions such as Zn2+, Mg2+, Mn2+, Cu2+ at 0.5 and 1.0 mM final concentration in the media on protease production indicated that Zn2+ (1.0 mM) was favorable in enhancing the protease production. The Km and Vmax of PB4 protease for casein substrate was 0.6 U ml-1 and 217.3 μM min-1 mg-1 protein, respectively. The deproteinization rate for distillery yeast biomass was 84 and 76.4% using culture and crude enzyme extracted from PB4, respectively. To the best of our knowledge, deproteinization of distillery yeast biomass using proteolytic bacterial isolate has never been demonstrated before. The experimental results in the present study suggest that Bacillus megaterium PB4, can be utilized for the eco-friendly, economical production of protein hydrolysates and may be considered as a potential candidate in various biotechnological applications.

Antioxidant production and chitin recovery from shrimp head fermentation with Streptococcus thermophilus

Fermentation of shrimp head was conducted using Streptococcus thermophilus to produce antioxidant and recover chitin. Fermentation conditions were found to be 10% shrimp head concentration, 5% glucose concentration, 1.2%(v/v) inoculum size, and 64 h of incubation time at 42°C to attain an initial pH of 5.00 with response surface method optimization and the actual deproteinization rate obtained was 93.59%. Antioxidant activity in the supernatant fluid increased greatly during fermentation, the DPPH radical scavenging ability of the culture supernatant was about 98.70%. The concent-ration of astaxanthin, phenolic compounds, and free amino acid in the culture supernatant was 1.774 μg/mL, 589.69 μg gallic acid equivalents/mL, and 796.978mg/mL, respectively. Comparison of the FT-IR spectra and X-ray diffraction (XRD) analysis among commercial chitin (CTa), chitin prepared by the S. thermophilus fermentation (CTb), and chitin prepared by chemical treatments (CTc) demonstrated that CTb had the highest degree of deacetylation.

Study on Deproteinization of Polysaccharide from Corn Silk with Cetyl Trimethyl Ammonium Bromide

To study the optimum deproteination conditions of polysaccharide from corn silk with cetyl trimethyl ammonium bromide(CTAB). The removal rate of protein and residual rate of polysaccharide was chosen as the index. The optimal deproteination condition of CTAB was that NaCl was 0.010g, pH was 6.0, the volume ratio of CTAB was 10%, dissociated time was 24h. Deproteinization rate was 58.17%, the residual rate of polysaccharide was 25.29%.

Deproteinization of Polysaccharide from the Stigma Maydis by Chitosan Method

This work was deproteinization in crude polysaccharide from the Stigma maydis by Chitosan method. According to deproteinization rate and polysaccharide loss rate as indexs, the optimized condition as follows: 0.1mL of chitosan, 12°C of temperature, 40 min of still time, and pH 7. The result showed that the deproteinization rate was 73.6% and the polysaccharide loss rate was 71.5%.

Response Surface Optimization of Deproteinization from Shrimp Shell by Pepsin

For the purpose of establishing a method of deprotein from the shrimp shell powder that is ready to prepare chitin by pepsin, which is a environment more friendly method compare to the other traditional methods. One factor experiment was applied to study the optimal deproteinization conditions., The data of one factor experiments indicates that the proper catalytic condition might be as follows, pH1.5, temperature 35°C, enzyme dose 500U/g, and the process time 4 hours, take the cost of production into account. On the basis of these results, response surface methodology (RSM) was used to study the optimal process conditions, the data indicates that the optimal condition occurs at 700 U/g, 36.09°C, and 5 hours, and the maximum deproteinization rate approaches 8.47%.

Study on Deproteinization and Decoloration in Fucoidan

Sevag, trichloroacetic acid and combining Sevag and TCA were used to remove protein from Fucoidan. And the conditions of deproteinization and undeproteinization were discussed to find the influence on the decoloration in Fucoidan. The results showed that methods of combining Sevag and TCA were better than others. By handling Sevag twice, using 1.3 mL TCA every 10 mL polysaccharose solution, the deproteinization rate reached 78.68%, and the polysaccharose loss rate was 28.89%. The decolourization ratio of stock solution was 29.13%, meanwhile, that ratio of deproteinization solution highly was 85.17%.

Deproteinization of Polysaccharide from the Stigma Maydis by Sevag Method

The aim of this work is to deproteinize crude polysaccharide from the Stigma maydis by sevag method. According to deproteinization rate and polysaccharide residual rate, the optimized process was deproteinization number (4 times), time of oscillating (11min), the volume ratio of sample to sevag reagent (2:1), the volume ratio of chloroform to n-butyl alcohol (5:1), deproteinization rate was 64.2%, the polysaccharide loss rate was 34.3%, and the purity of polysaccharide was increased from 11.5% to 32.3%.

Rapid purification of polysaccharides using novel radial flow ion-exchange by response surface methodology from Ganoderma lucidum

Radial flow chromatography (RFC) packed with ion-exchange resin was used to purify the Ganoderma lucidum crude polysaccharides. The effects of sample concentration, processing capacity, sample flow-rate and elution flow-rate on purification efficiency were investigated using single and response surface experiments (RSM). The purification efficiency of RFC was compared with that of axial column and traditional chemical method. RSM analysis showed good correspondence between experimental and predicted values. The adjusted coefficient of determination ( R adj 2 ) for the model was 92.85%. Probability value ( p < 0.005) demonstrated high significance for the regression model. The optimal conditions for the purification of G. lucidum crude polysaccharides with RFC were determined: sample concentration of 10 mg/ml, sample volume of 100 ml, sample flow-rate of 2 ml/min and elution flow-rate of 40 ml/min. The deproteinization rate, decoloration rate and polysaccharide recovery rate under the optional chromatography conditions were 80.35%, 88.52% and 85.06%, respectively. The comparative test results indicated that the best purification efficiency was obtained by RFC within the shortest time.