The understanding of the structure morphology of oil-rich emulsion from enzyme-assisted extraction processing(EAEP)was a critical step to break the oil-rich emulsion structure in order to recover oil.Albeit EAEP metho...The understanding of the structure morphology of oil-rich emulsion from enzyme-assisted extraction processing(EAEP)was a critical step to break the oil-rich emulsion structure in order to recover oil.Albeit EAEP method has been applied as an alternative way to conventional solvent extraction method,the structure morphology of oil-rich emulsion was still unclear.The current study aimed to investigate the structure morphology of oil-rich emulsion from EAEP using 3 D confocal Raman imaging technique.With increasing the enzymatic hydrolysis duration from 1 to 3 h,the stability of oil-rich emulsion was decreased as visualized in the 3 D confocal Raman images that the protein and oil were mixed together.The subsequent Raman spectrum analysis further revealed that the decreased stability of oil-rich emulsion was due to the protein aggregations via SS bonds or protein-lipid interactions.The conformational transfer in protein indicated the formation of a compact structure.展开更多
Objective To explore the optimum condition for complex enzyme-assisted extraction of galanthamine from Lycoris aurea by L_9（3~4） orthogonal array design and separation effect of cation exchange resin on galanthamine...Objective To explore the optimum condition for complex enzyme-assisted extraction of galanthamine from Lycoris aurea by L_9（3~4） orthogonal array design and separation effect of cation exchange resin on galanthamine. Methods Cellulase and pectinase solution was used as the extraction solvent. The effects of p H value of enzyme, amount of complex enzyme, dissociation time, and enzymatic hydrolysis temperature on the extraction results were investigated. Results The optimal conditions were obtained as follows： ratio of solid to liquid（g：mL） 1：10, pH value 4.5, amount of complex enzymes 4%, enzymatic hydrolysis temperature 50oC, and reaction time 2.0 h. Under these conditions, the extraction yield of galanthamine was 0.0294%. In addition, D-001 cation exchange resin was selected for separation of galanthamine. The separation conditions were that adsorption flow rate was 3 BV/h with reagent of pH2 and the desorption flow rate was 3 BV/h with 70% ethanol solution containing 1.5 mol/L ammonia. After separation, the content of galanthamine was increased to 12.31%. Conclusion The results provide a reference for industrial production of galanthamine.展开更多
The effect of pre-extraction treatment of Centella asiatica(L.)using different pre-treatment parameters,namely,pH(4.0-6.0)and treatment time(15-105 min),in combination with enzymatic pretreatment,on centelloid extract...The effect of pre-extraction treatment of Centella asiatica(L.)using different pre-treatment parameters,namely,pH(4.0-6.0)and treatment time(15-105 min),in combination with enzymatic pretreatment,on centelloid extracts obtained by ultrasound-assisted extraction was investigated.It was observed that the pre-extraction treatment of plant samples at all tested pH values in combination with enzymatic deconstruction led to distinct synergistic effects.Liberation of centelloids was found to occur mainly at two different pretreatment regimes,namely,Phase I(Proton-induced pectin relaxation and rapid glycoside liberation),which led to maximum glycoside yield at pH 4.0 within 15 min(18.20±0.15 mg/g madecassoside and 8.51±0.15 mg/g asiaticoside),and Phase II(Enzyme-mediated collapse and aglycone re-adsorption),in which,at pH 5.0-6.0,the role of enzymatic contribution in enhancing the extraction of aglycones was found to be negligible.Optimum yields and quality of centelloid aglycones were achieved at pH 6.0,where maximal yields were obtained at 60 min and declined thereafter.The observed decline in centelloid aglycone yields at prolonged treatment time could be attributed to the extensive enzymatic fragmentation and subsequent collapse of the disrupted plant matrix,leading to non-specific re-adsorption of the bioactive triterpenes.This study provides a sustainable and cost-effective framework for optimizing extract composition through precise pH and temporal control,offering an efficient methodology for the green separation of bioactive centelloids.展开更多
Enzymatic methods have been demonstrated to effectively enhance the gel properties of soybean protein.Current research primarily focuses on modifying commercially available soybean protein isolate(SPI)products,which i...Enzymatic methods have been demonstrated to effectively enhance the gel properties of soybean protein.Current research primarily focuses on modifying commercially available soybean protein isolate(SPI)products,which involves complex modification processes and poses challenges for industrialization.This study aimed to investigate the impact of enzyme-assisted extraction on structural,physicochemical and gelation properties of the derived SPI.Limited hydrolysis was achieved by subjecting defatted soy flour to hydrolysis using Flavorzyme,Neutrase and Protamex for varying durations while controlling the degree of hydrolysis(DH)below 10%.The enzyme-assisted extracted SPI(ESPI)prepared using Flavorzyme exhibited smaller particle sizes compared to those treated with Protamex and Neutrase,facilitating aggregation through hydrophobic interactions,hydrogen bonds,and disulfide bonds to form a denser and more uniform gel network structure.Minimal hydrolysis with Protamex exposed more active groups on protein molecules leading to improved formation of a gel network structure.On the other hand,Nuetrase leads to a reduced enhancement in protein surface’s hydrophobicity,indicating limited impact on the gelation properties of the protein.This research work provides novel insights into effectively improving the gelation properties of derived enzyme-assisted extracted SPI.展开更多
A novel method using ethanol and ultrasound to extract oil from cream obtained from enzyme-assisted aqueous extraction of soybean oil was developed.To evaluate the relationships between operating variables and free oi...A novel method using ethanol and ultrasound to extract oil from cream obtained from enzyme-assisted aqueous extraction of soybean oil was developed.To evaluate the relationships between operating variables and free oil yield and to maximize the free oil yield,response surface methodology was introduced in this work.The developed regression model was fitted with R2=0.9591.Optimized variables were:ethanol concentration of73%,ethanol addition volume of 0.55 L/kg,ultrasound power of 427 W,ultrasound time of 47 s,and ultrasound temperature of 53℃.The free oil yield from the cream under the above conditions was 92.6±3.4%.Scanning electron microscopy(SEM)was used to evaluate the effect of ultrasonic treatment on ethanoltreated cream,and the SEM images clearly showed that the ultrasound treatment affected dispersing and fracturing of the microstructure of ethanol-treated cream.展开更多
Human T-cell lymphophilic virus type 1(HTLV-1),the known retrovirus causing cancer in humans,is closely associated with adult T-cell leukemia/lymphoma and HTLV-1 associated myelopathy/tropical spastic paraparesis.Due ...Human T-cell lymphophilic virus type 1(HTLV-1),the known retrovirus causing cancer in humans,is closely associated with adult T-cell leukemia/lymphoma and HTLV-1 associated myelopathy/tropical spastic paraparesis.Due to its ability to evade the host's defense mechanisms,early tracking of HTLV-1 becomes crucial.In this study,we integrateλ-Exonuclease(λ-Exo)-assisted target recycling with a terminal deoxynucleotidyl transferase(TdT)-mediated template-free DNA extension process to develop an electrochemical analysis platform for the specific and sensitive detection of HTLV-1 DNA.During theλ-Exo-assisted target recycling,HTLV-1 DNA is recognized by hairpin DNA(Hp-DNA),forming double-stranded DNA(dsDNA)through DNA hybridization.The dsDNA,featuring blunt 5'terminal phosphorylation,is cleaved byλ-Exo,generating abundant short output sequence(sDNA).HTLV-1 DNA is released,initiating a cyclic hybridization-cleavage process.Subsequently,thiol-labelled capture DNA(CP-DNA)assembled on gold electrode surface captures a substantial amount of the generated sDNA,forming CP-DNA-sDNA nanostructures.When TdT and dNTPs are present on the electrode surface,the 3'-OH terminal of sDNA extends to generate long single-stranded DNA(ssDNA)structure.Methylene blue(MB)is selected as the electrochemical signal molecule.MB not only binds with ssDNA but also interacts specifically with dsDNA,resulting in a significantly enhanced electrochemical signal on modified electrode surface.The detection limit of HTLV-1 DNA is as low as 19 amol/L(S/N=3)when the two signal amplification strategies are combined.The analysis platform exhibits excellent analytical performance and holds promise as a novel tool for the early tracing and diagnosis of HTLV-1 DNA.展开更多
This study explores the potential of Cynara scoly mus L.(artichoke)waste as bioactive compounds resource with antimicrobial and antiviral properties.Three extracts were prepared using different methods:an ultrasound-a...This study explores the potential of Cynara scoly mus L.(artichoke)waste as bioactive compounds resource with antimicrobial and antiviral properties.Three extracts were prepared using different methods:an ultrasound-assisted extraction with EtOH:H_(2)O(1:1),an enzymatic pretreatment with Rapidase®Revelation Aroma,and a lactic fermentation for 10 days.The metabolomic profiles were compared using a UHPLC-HR-MS/Orbitrap spectrometer.While all extracts contained as main metabolites apigenin,luteolin,cynarine,chlorogenic acid,and caffeic acid,enzymatic pretreatment and fermentation resulted in significantly higher levels of secondary metabolites than the untreated waste extract.The results of antimicrobial and antiviral activities indicated that only pretreated and fermented extracts exhibited activity against Staphylococcus aureus,Bacillus cereus,and HSV-2 virus.These findings suggest that enzymatic pretreatment and lactic fermentation here used for the first time on artichoke waste could improve the bioactivity of its extracts,highlighting the use of C.scolymus by-products as a source of potential antimicrobial and antiviral constituents.展开更多
The paradigm shifts from discarding horticulture byproducts to exploiting them for their bioactive potential has encouraged research on non-conventional extraction techniques such as extraction using microwave,ultraso...The paradigm shifts from discarding horticulture byproducts to exploiting them for their bioactive potential has encouraged research on non-conventional extraction techniques such as extraction using microwave,ultrasound,high-pressure,etc.Kinnow peels,a primary residue of kinnow processing industry,are profuse with polysaccharides,such as dietary fiber which can be isolated and used as a functional ingredient in fortified/enriched foods.In the present study,ultrasound-assisted extraction(UAE)and ultrasound-assisted-enzymatic extraction(UAEE)have been optimized to maximize dietary fiber yield from kinnow peel waste.Total dietary fiber(TDF)of 52.042±0.862%was observed using UAE at 38%amplitude,liquid to solid ratio(LSR)of 40 mL/g,at 44℃,after sonication time of 13 min,which increased to 60.974±0.827%upon sequential enzymatic treatment.The WHC,OHC,and GAC of extracted dietary fiber ranged between 8.17 and 9.24±0.11 g/g,2.96-4.68±0.09 g/g,and 1.56-2.28±0.04 mmol/g.The physicochemical characterization of dietary fiber using analytical instruments and its functional properties such as oil/water holding,and glucose adsorption capacities proved that kinnow peels dietary fiber can be used to develop fiber-enriched products.The extracted fiber has been used to develop cookies which showed good nutritional,sensory,and textural properties with an overall acceptability of 7.15-7.84.This research will be helpful in scale-up studies on the valorization of different citrus peels through an extraction approach.展开更多
Peanut oil is widely favored for its pleasant flavor and nutritional value.However,conventional processing techniques often lead to the loss of micronutrients in peanut oil and yield a product with a monotonous flavor...Peanut oil is widely favored for its pleasant flavor and nutritional value.However,conventional processing techniques often lead to the loss of micronutrients in peanut oil and yield a product with a monotonous flavor profile.This study examined the impact of various combined biological pretreatments of peanut germination and enzymatic hydrolysis on peanut oil,with an emphasis on the improvement of its physicochemical properties,nutritional quality,and flavor profile.It was demonstrated that the nutrient composition and flavor profile of peanut oil could be remarkably enhanced by moderate germination combined with enzymatic hydrolysis.The total phenolic content in peanut oil was increased by approximately 5-fold(from 27.53 to 153.54 mg/kg),and the resveratrol content was enhanced from 0.25 mg/kg to 0.57 mg/kg.Furthermore,the combined treatment resulted in approximately a 10-fold increase in total volatile content(from 3343.87 to 54,761.83μg/kg)compared with the untreated.Pyrazines,furans,and aldehydes were the predominant volatile compounds contributing to the desirable flavor of the treated peanut oil.Notably,2,3-dihydro-3,5-dihydroxy-6-methyl-4Hpyran-4-one and phenylacetaldehyde were identified as key contributors,primarily imparting caramel-like notes to the treated peanut oil.This study offers a viable strategy for producing premium peanut oil with optimized nutritional and sensory profiles.展开更多
基金the financial support received from National Natural Science Foundation of China(No.31430067 and 31601475)China Postdoctoral Science Foundation funded project(No.2017M610200)Heilongjiang Postdoctoral Foundation(No.LBH-Z17011)
文摘The understanding of the structure morphology of oil-rich emulsion from enzyme-assisted extraction processing(EAEP)was a critical step to break the oil-rich emulsion structure in order to recover oil.Albeit EAEP method has been applied as an alternative way to conventional solvent extraction method,the structure morphology of oil-rich emulsion was still unclear.The current study aimed to investigate the structure morphology of oil-rich emulsion from EAEP using 3 D confocal Raman imaging technique.With increasing the enzymatic hydrolysis duration from 1 to 3 h,the stability of oil-rich emulsion was decreased as visualized in the 3 D confocal Raman images that the protein and oil were mixed together.The subsequent Raman spectrum analysis further revealed that the decreased stability of oil-rich emulsion was due to the protein aggregations via SS bonds or protein-lipid interactions.The conformational transfer in protein indicated the formation of a compact structure.
基金Hunan Provincial Science and Technology Department(2012TP4002-3)Zhangjiajie Science and Technology Bureau(2014YB17)
文摘Objective To explore the optimum condition for complex enzyme-assisted extraction of galanthamine from Lycoris aurea by L_9（3~4） orthogonal array design and separation effect of cation exchange resin on galanthamine. Methods Cellulase and pectinase solution was used as the extraction solvent. The effects of p H value of enzyme, amount of complex enzyme, dissociation time, and enzymatic hydrolysis temperature on the extraction results were investigated. Results The optimal conditions were obtained as follows： ratio of solid to liquid（g：mL） 1：10, pH value 4.5, amount of complex enzymes 4%, enzymatic hydrolysis temperature 50oC, and reaction time 2.0 h. Under these conditions, the extraction yield of galanthamine was 0.0294%. In addition, D-001 cation exchange resin was selected for separation of galanthamine. The separation conditions were that adsorption flow rate was 3 BV/h with reagent of pH2 and the desorption flow rate was 3 BV/h with 70% ethanol solution containing 1.5 mol/L ammonia. After separation, the content of galanthamine was increased to 12.31%. Conclusion The results provide a reference for industrial production of galanthamine.
基金the National Science,Research and Innovation Fund(NSRF)[RE-KRIS/FF67/024].
文摘The effect of pre-extraction treatment of Centella asiatica(L.)using different pre-treatment parameters,namely,pH(4.0-6.0)and treatment time(15-105 min),in combination with enzymatic pretreatment,on centelloid extracts obtained by ultrasound-assisted extraction was investigated.It was observed that the pre-extraction treatment of plant samples at all tested pH values in combination with enzymatic deconstruction led to distinct synergistic effects.Liberation of centelloids was found to occur mainly at two different pretreatment regimes,namely,Phase I(Proton-induced pectin relaxation and rapid glycoside liberation),which led to maximum glycoside yield at pH 4.0 within 15 min(18.20±0.15 mg/g madecassoside and 8.51±0.15 mg/g asiaticoside),and Phase II(Enzyme-mediated collapse and aglycone re-adsorption),in which,at pH 5.0-6.0,the role of enzymatic contribution in enhancing the extraction of aglycones was found to be negligible.Optimum yields and quality of centelloid aglycones were achieved at pH 6.0,where maximal yields were obtained at 60 min and declined thereafter.The observed decline in centelloid aglycone yields at prolonged treatment time could be attributed to the extensive enzymatic fragmentation and subsequent collapse of the disrupted plant matrix,leading to non-specific re-adsorption of the bioactive triterpenes.This study provides a sustainable and cost-effective framework for optimizing extract composition through precise pH and temporal control,offering an efficient methodology for the green separation of bioactive centelloids.
基金supported by the National Natural Science Foundation of China under Grant No.32472484Zhejiang Provincial Natural Science Foundation of China under Grant No.LQ24C200009Beijing Science and Technology Plan under Grant No.Z231100003723003 and Food Science and Technology Foundation of CIFST-Mengniu Nutrition&Health Youth Fund(2023M04).
文摘Enzymatic methods have been demonstrated to effectively enhance the gel properties of soybean protein.Current research primarily focuses on modifying commercially available soybean protein isolate(SPI)products,which involves complex modification processes and poses challenges for industrialization.This study aimed to investigate the impact of enzyme-assisted extraction on structural,physicochemical and gelation properties of the derived SPI.Limited hydrolysis was achieved by subjecting defatted soy flour to hydrolysis using Flavorzyme,Neutrase and Protamex for varying durations while controlling the degree of hydrolysis(DH)below 10%.The enzyme-assisted extracted SPI(ESPI)prepared using Flavorzyme exhibited smaller particle sizes compared to those treated with Protamex and Neutrase,facilitating aggregation through hydrophobic interactions,hydrogen bonds,and disulfide bonds to form a denser and more uniform gel network structure.Minimal hydrolysis with Protamex exposed more active groups on protein molecules leading to improved formation of a gel network structure.On the other hand,Nuetrase leads to a reduced enhancement in protein surface’s hydrophobicity,indicating limited impact on the gelation properties of the protein.This research work provides novel insights into effectively improving the gelation properties of derived enzyme-assisted extracted SPI.
基金the National High-tech R&D Program of China(863 Program)(grant number 2013AA102104)the open-end fund from the Key Laboratory of Soybean Biology of Chinese Education Ministry,Northeast Agricultural University(grant numberSB12C01)+1 种基金the Special Fund for the Establishment of Modern Agricultural R&D Systems(grant number nycytx-004)the National Research Center of Soybean Engineering and Technology,and the Northeast Agricultural University for the support of this project
文摘A novel method using ethanol and ultrasound to extract oil from cream obtained from enzyme-assisted aqueous extraction of soybean oil was developed.To evaluate the relationships between operating variables and free oil yield and to maximize the free oil yield,response surface methodology was introduced in this work.The developed regression model was fitted with R2=0.9591.Optimized variables were:ethanol concentration of73%,ethanol addition volume of 0.55 L/kg,ultrasound power of 427 W,ultrasound time of 47 s,and ultrasound temperature of 53℃.The free oil yield from the cream under the above conditions was 92.6±3.4%.Scanning electron microscopy(SEM)was used to evaluate the effect of ultrasonic treatment on ethanoltreated cream,and the SEM images clearly showed that the ultrasound treatment affected dispersing and fracturing of the microstructure of ethanol-treated cream.
基金financially supported by Central Leading Local Science and Technology Development Fund Project(guikeZY22096017)Natural Science Foundation of Guangxi Province(2024GXNSFDA010036)National Natural Science Foundation of China(22164014,U23A2089).
文摘Human T-cell lymphophilic virus type 1(HTLV-1),the known retrovirus causing cancer in humans,is closely associated with adult T-cell leukemia/lymphoma and HTLV-1 associated myelopathy/tropical spastic paraparesis.Due to its ability to evade the host's defense mechanisms,early tracking of HTLV-1 becomes crucial.In this study,we integrateλ-Exonuclease(λ-Exo)-assisted target recycling with a terminal deoxynucleotidyl transferase(TdT)-mediated template-free DNA extension process to develop an electrochemical analysis platform for the specific and sensitive detection of HTLV-1 DNA.During theλ-Exo-assisted target recycling,HTLV-1 DNA is recognized by hairpin DNA(Hp-DNA),forming double-stranded DNA(dsDNA)through DNA hybridization.The dsDNA,featuring blunt 5'terminal phosphorylation,is cleaved byλ-Exo,generating abundant short output sequence(sDNA).HTLV-1 DNA is released,initiating a cyclic hybridization-cleavage process.Subsequently,thiol-labelled capture DNA(CP-DNA)assembled on gold electrode surface captures a substantial amount of the generated sDNA,forming CP-DNA-sDNA nanostructures.When TdT and dNTPs are present on the electrode surface,the 3'-OH terminal of sDNA extends to generate long single-stranded DNA(ssDNA)structure.Methylene blue(MB)is selected as the electrochemical signal molecule.MB not only binds with ssDNA but also interacts specifically with dsDNA,resulting in a significantly enhanced electrochemical signal on modified electrode surface.The detection limit of HTLV-1 DNA is as low as 19 amol/L(S/N=3)when the two signal amplification strategies are combined.The analysis platform exhibits excellent analytical performance and holds promise as a novel tool for the early tracing and diagnosis of HTLV-1 DNA.
基金the Tuscany Region for co-funding the research project TUSCAVIA-“from TUScany and his Cultivations new Anti-VIral and Antibacterial substances”(POR FESR Toscana 2014-2020-Azione 1.1.5-Sub-azione A1)The authors acknowledge CISUP-Centre for Instrumentation Sharing-University of Pisa for the use of Operetta CLS imaging facility and Mass Spectrometry facility.
文摘This study explores the potential of Cynara scoly mus L.(artichoke)waste as bioactive compounds resource with antimicrobial and antiviral properties.Three extracts were prepared using different methods:an ultrasound-assisted extraction with EtOH:H_(2)O(1:1),an enzymatic pretreatment with Rapidase®Revelation Aroma,and a lactic fermentation for 10 days.The metabolomic profiles were compared using a UHPLC-HR-MS/Orbitrap spectrometer.While all extracts contained as main metabolites apigenin,luteolin,cynarine,chlorogenic acid,and caffeic acid,enzymatic pretreatment and fermentation resulted in significantly higher levels of secondary metabolites than the untreated waste extract.The results of antimicrobial and antiviral activities indicated that only pretreated and fermented extracts exhibited activity against Staphylococcus aureus,Bacillus cereus,and HSV-2 virus.These findings suggest that enzymatic pretreatment and lactic fermentation here used for the first time on artichoke waste could improve the bioactivity of its extracts,highlighting the use of C.scolymus by-products as a source of potential antimicrobial and antiviral constituents.
基金Name of the organization:Council of Scientific and Industrial Research(CSIR),India.
文摘The paradigm shifts from discarding horticulture byproducts to exploiting them for their bioactive potential has encouraged research on non-conventional extraction techniques such as extraction using microwave,ultrasound,high-pressure,etc.Kinnow peels,a primary residue of kinnow processing industry,are profuse with polysaccharides,such as dietary fiber which can be isolated and used as a functional ingredient in fortified/enriched foods.In the present study,ultrasound-assisted extraction(UAE)and ultrasound-assisted-enzymatic extraction(UAEE)have been optimized to maximize dietary fiber yield from kinnow peel waste.Total dietary fiber(TDF)of 52.042±0.862%was observed using UAE at 38%amplitude,liquid to solid ratio(LSR)of 40 mL/g,at 44℃,after sonication time of 13 min,which increased to 60.974±0.827%upon sequential enzymatic treatment.The WHC,OHC,and GAC of extracted dietary fiber ranged between 8.17 and 9.24±0.11 g/g,2.96-4.68±0.09 g/g,and 1.56-2.28±0.04 mmol/g.The physicochemical characterization of dietary fiber using analytical instruments and its functional properties such as oil/water holding,and glucose adsorption capacities proved that kinnow peels dietary fiber can be used to develop fiber-enriched products.The extracted fiber has been used to develop cookies which showed good nutritional,sensory,and textural properties with an overall acceptability of 7.15-7.84.This research will be helpful in scale-up studies on the valorization of different citrus peels through an extraction approach.
基金support from the Department of Science and Technology of Shandong Province under grant number 2023TZXD076.
文摘Peanut oil is widely favored for its pleasant flavor and nutritional value.However,conventional processing techniques often lead to the loss of micronutrients in peanut oil and yield a product with a monotonous flavor profile.This study examined the impact of various combined biological pretreatments of peanut germination and enzymatic hydrolysis on peanut oil,with an emphasis on the improvement of its physicochemical properties,nutritional quality,and flavor profile.It was demonstrated that the nutrient composition and flavor profile of peanut oil could be remarkably enhanced by moderate germination combined with enzymatic hydrolysis.The total phenolic content in peanut oil was increased by approximately 5-fold(from 27.53 to 153.54 mg/kg),and the resveratrol content was enhanced from 0.25 mg/kg to 0.57 mg/kg.Furthermore,the combined treatment resulted in approximately a 10-fold increase in total volatile content(from 3343.87 to 54,761.83μg/kg)compared with the untreated.Pyrazines,furans,and aldehydes were the predominant volatile compounds contributing to the desirable flavor of the treated peanut oil.Notably,2,3-dihydro-3,5-dihydroxy-6-methyl-4Hpyran-4-one and phenylacetaldehyde were identified as key contributors,primarily imparting caramel-like notes to the treated peanut oil.This study offers a viable strategy for producing premium peanut oil with optimized nutritional and sensory profiles.
