The outer membrane of Gram-negative bacteria is the outermost layer of cellular environment in which numerous biophysical and biochemical processes are in action sustaining viability. Advances in cell engineering enable modification of bacterial genetic information that subsequently alters membrane physiology to adapt bacteria to specific purposes. Surface display of a functional molecule on the outer membranes is one of strategies that directs host cells to respond to a specific extracellular matter or stimulus. While intracellular expression of a functional peptide or protein fused to a membrane-anchoring motif is commonly practiced for surface display, the method is not readily applicable to exogenous or large proteins inexpressible in bacteria. Chemical conjugation at reactive groups naturally occurring on the membrane might be an alternative, but often compromises fitness due to non-specific modification of essential components. Herein, we demonstrated two distinct approaches that enable site-specific decoration of the outer membrane with a fluorescent agent in Escherichia coli. An unnatural amino acid genetically incorporated in a surface-exposed peptide could act as a chemoselective handle for bioorthogonal dye labeling. A surface-displayed α-helical domain originating from a part of a selected heterodimeric coiled-coil complex could recruit and anchor a green fluorescent protein tagged with a complementary α-helical domain to the membrane surface in a site- and hetero-specific manner. These methods hold a promise as on-demand tools to confer new functionalities on the bacterial membranes.
Objective: This study was conducted to determine the optimal dose of novel iron amino acid complexes (Fe-Lys-Glu) by measuring laying performance, egg quality, egg iron (Fe) concentrations, and blood biochemical parameters in laying hens. Methods: A total of 1,260 18-week-old healthy Beijing White laying hens were randomly divided into 7 groups with 12 replicates of 15 birds each. After a 2-wk acclimation to the basal diet, hens were fed diets supplemented with 0 (negative control, the analyzed innate iron content was 75.06 mg/kg), 15, 30, 45, 60, and 75 mg Fe/kg as Fe-Lys-Glu or 45 mg Fe/kg from FeSO4 (positive control) for 24 wk. Results: Results showed that compared with the negative and positive control groups, dietary supplementation with 30 to 75 mg Fe/kg from Fe-Lys-Glu significantly (linear and quadratic, p<0.05) increased the laying rate (LR) and average daily egg weight (ADEW); hens administered 45 to 75 mg Fe/kg as Fe-Lys-Glu showed a remarkable (linear, p<0.05) decrease in feed conversion ratio. There were no significant differences among all groups in egg quality. The iron concentrations in egg yolk and serum were elevated by increasing Fe-Lys-Glu levels, and the highest iron content was found in 75 mg Fe/kg group. In addition, hens fed 45 mg Fe/kg from Fe-Lys-Glu had (linear and quadratic, p<0.05) higher yolk Fe contents than that with the same dosage of FeSO4 supplementation. The red blood cell (RBC) count and hemoglobin content (linear and quadratic, p<0.05) increased obviously in the groups fed with 30 to 75 mg Fe/kg as Fe-Lys-Glu in comparison with the control group. Fe-Lys-Glu supplementation also (linear and quadratic, p<0.05) enhanced the activity of copper/zinc-superoxide dismutase (Cu/Zn-SOD) in serum, as a result, the serum malonaldehyde content (linear and quadratic, p<0.05) decreased in hens received 60 to 75 mg Fe/kg as Fe-Lys-Glu. Conclusion: Supplementation Fe-Lys-Glu in laying hens could substitute for FeSO4 and the optimal additive levels of Fe-Lys-Glu are 45 mg Fe/kg in layers diets based on the quadratic regression analysis of LR, ADEW, RBC, and Cu/Zn-SOD.
A metabolomic study was conducted to identify key metabolic components, which are correlated with the growth of 4-year-old Pinus koraiensis seedlings harvested at actively height growing season (May 18th). Among 105 individual metabolites identified by GC/MS analysis, alanine, threonine, oleic acid, and butanoic acids were negatively correlated with both height and weight of 4-year-old seedlings, while malic acid, xylose, glucose, d-turanose and inositol had positive correlation with various growth parameters. During the actively growing season, the concentrations of both amino acids and organic acids in the main stem of Superior seedling group were lower but the photosynthates such as mono-saccharide and sucrose were higher than in other seedling groups such as Intermediate and Inferior. Interestingly, d-turanose, an analogue of sucrose that is not metabolized in higher plants but used as carbon source by many organisms including numerous species of bacteria and fungi, showed the highest correlation (r=0.896, p<0.001) with height of 4-year-old seedlings, indicating that possible interaction with mycorrhizal organisms. Therefore we suggest that several metabolites selected in this study may be used as metabolic markers for complex traits in P. koraiensis.
Journal of the Korean Society of Food Science and Nutrition
/
v.40
no.1
/
pp.94-101
/
2011
Squid (Todarodes pacificus) is processed as dried or seasoned-dried products and its catch gradually increased from 270,298 M/T in 2005 to 367,940 M/T in 2008 in Korea. Squid processing by-product (viscera) was usually discarded as a waste resulting in environmental problem. In order to utilize squid viscera for more value-added products, a natural squid seasoning was developed by fermenting with Aspergillus oryzae koji. Squid viscera at 5, 10 and 15% salt concentrations with fixed levels of 5% koji and 30% water was fermented at room temperature. The quality properties of squid fermented products such as amino-N, TMA, VBN, total viable cell count, pH and total acidity were determined at different fermentation periods. The contents of amino-N, TMA, and VBN of squid seasoning at 5% salt concentration fermented for 14 days were the highest. Based on amino-N content, squid viscera at 5% koji fermented for 14 days was selected for further assays: the content of moisture, crude protein, crude lipid, crude ash, and carbohydrate were 5.98, 35.19, 33.08, 11.30, and 14.45%, respectively. The content of glutamate, alanine, leusine and lysine were 7.06, 12.34, 9.90 and 10.22%, respectively. The $IC_{50}$ values of DPPH scavenging and $\beta$-glucuronidase inhibitory activity were 12.89 and 12.58 mg/mL, respectively. A natural squid seasoning was manufactured by mixing fermented squid viscera and an ingredient. Based on the results of sensory evaluation, the fermented squid viscera seasoning was almost equal to other natural complex seasonings such as anchovy, cow meat, and fisheries seasoning.
This study aimed to examine the possibility of upcycling extracts of Angelica keiskei and Oenanthe javanica juice by-products through comparing enzyme extraction (EE) and complex extraction (CE) methods to increase the extraction yield and flavor of materials. A higher extraction yield was obtained for free amino acid content with EE and CE for A. keiskei and O. javanica juice by-products, respectively, and a higher extraction efficiency was achieved with juice by-products than with extracts prepared from raw materials before juice production. The content of major amino acids varied depending on the extraction method used. When used according to the characteristics of the extract, their use as a functional material was confirmed along with improvement in the flavor of the food. Consistently high extraction yields for organic acid and sugar levels were obtained with CE in A. keiskei and O. javanica juice by-products. The DPPH radical scavenging ability and TPC were consistently high with CE in A. keiskei and O. javanica juice by-products; the increase in extracted content was likely because of the reaction between the ethanol used for CE and the phenolic compounds. However, because the antioxidant capacity of the juice by-product extracts was somewhat lower than that of the extracts from raw materials before juice production, the amount used should be reviewed. The TFC was found to be higher in extracts obtained with EE than with CE for A. keiskei juice by-products; however, no significant difference was observed between EE and CE in the O. javanica juice by-products. Through this study, the taste compounds and antioxidant properties of extracts obtained from juice by-products produced after the production of A. keiskei and O. javanica green juice were analyzed, and the availability of high value-added materials was confirmed. Based on these research results, expanding specific R&D for practical use should be explored.
A new method for obtaining the photoluminescence of footwear impression by using 1,2-indandione (1,2-IND) solution, which is a latent fingerprint-developing reagent, was studied. A binary complex of DL-alanine and 1,2-IND was prepared by spraying a DL-alanine solution and the 1,2-IND solution (an amino acid sensitive reagent) onto dry or wet origin footwear impression deposited on the surface of printed A4 paper. This binary complex reacts with the trace metal component in the footwear impression to form a ternary complex that exhibits photoluminescence. However, when 5-methylthioninhydrin (5-MTN) solution was used instead of 1,2-IND, no consistent photoluminescence was observed even under identical treatment conditions. In addition, when footwear impressions treated with DL-alanine and 1,2-IND solutions were stored under various temperature conditions (30, 40 and $50^{\circ}C$) and various humidity conditions (30 %, 40 %, 50 % and 60 % RH), the contrast between the footwear impression and the background decreased. Optimal footwear impression photoluminescence was obtained when the footwear impressions treated with DL-alanine and 1,2-IND solutions were stored at $30^{\circ}C$ and 30 % RH for 1 h. The sensitivity of the developed method was ccompared with the sensitivities of three known methods - black gelatin lifting, 2,2'-dipyridyl treatment, and 8-hydroxyquinoline treatment. The results showed that the sensitivity of the developed method was worse than that of the black gelatin lifting method but better than that of 2,2'-dipyridyl or 8-hydroxyquinoline treatment method.
Fish protein hydrolysates(FPH) prepared from defatted mackerel meal by proteases such as complex enzymes, bromelain, alcalase, $\alpha-chymotrypsin,$ trypsin, papain and pepsin were tested for inhibitory activity against angiotensin-I converting enzyme(ACE). Among proteases tested, the hydrolysates obtained from the treatment of complex enzymes or bromelain showed relatively higher activity. ACE inhibitory activity of the hydrolysates increased until hydrolysis of 8 hrs, and was stable by heat treatment for 20min at $100^{\circ}C.$ From the profiles of fractionation of the hydrolysates with Bio-gel P-2, the most active fraction had about MW 1,450 and it's amino acid was abundant in Asp, Glu, Lys, Leu, Val and Ala. $IC_{50}\;(amounts\;of\;inhibitors\;needed\;for\;50\%\;inhibition)$ of the active fraction of the hydrolysates obtained from the treatment of complex enzyme and bromelain was 90 and $130 {\mu}g,$ respectively.
Truong, Anh Duc;Hong, Yeojin;Lee, Janggeun;Lee, Kyungbaek;Tran, Ha Thi Thanh;Dang, Hoang Vu;Nguyen, Viet Khong;Lillehoj, Hyun S.;Hong, Yeong Ho
Asian-Australasian Journal of Animal Sciences
/
v.32
no.5
/
pp.614-628
/
2019
Objective: The inhibitory leukocyte immunoglobulin-like receptors (LILRBs) play an important role in innate immunity. The present study represents the first description of the cloning and structural and functional analysis of LILRB1 and LILRB3 isolated from two genetically disparate chicken lines. Methods: Chicken LILRB1-3 genes were identified by bioinformatics approach. Expression studies were performed by transfection, quantitative polymerase chain reaction. Signal transduction was analyzed by western blots, immunoprecipitation and flow cytometric. Cytokine levels were determined by enzyme-linked immunosorbent assay. Results: Amino acid homology and phylogenetic analyses showed that the homologies of LILRB1 and LILRB3 in the chicken line 6.3 to those proteins in the chicken line 7.2 ranged between 97%-99%, while homologies between chicken and mammal proteins ranged between 13%-19%, and 13%-69%, respectively. Our findings indicate that LILRB1 and LILRB3 subdivided into two groups based on the immunoreceptor tyrosine-based inhibitory motifs (ITIM) present in the transmembrane domain. Chicken line 6.3 has two ITIM motifs of the sequence LxYxxL and SxYxxV while line 7.2 has two ITIM motifs of the sequences LxYxxL and LxYxxV. These motifs bind to SHP-2 (protein tyrosine phosphatase, non-receptor type 11) that plays a regulatory role in immune functions. Moreover, our data indicate that LILRB1 and LILRB3 associated with and activated major histocompatibility complex (MHC) class I and ${\beta}2-microglobulin$ and induced the expression of transporters associated with antigen processing, which are essential for MHC class I antigen presentation. This suggests that LILRB1 and LILRB3 are transcriptional regulators, modulating the expression of components in the MHC class I pathway and thereby regulating immune responses. Furthermore, LILRB1 and LILRB3 activated Janus kinase2/tyrosine kinase 2 (JAK2/TYK2); signal transducer and activator of transcription1/3 (STAT1/3), and suppressor of cytokine signaling 1 genes expressed in Macrophage (HD11) cells, which induced Th1, Th2, and Th17 cytokines. Conclusion: These data indicate that LILRB1 and LILRB3 are innate immune receptors associated with SHP-2, MHC class I, ${\beta}2-microglobulin$, and they activate the Janus kinase/signal transducer and activator of transcription signaling pathway. Thus, our study provides novel insights into the regulation of immunity and immunopathology.
This work was carried out to investigate effects of the freezing/thawing method on duck meat kept in a freezer for a month. The meats used were breast muscle collected from Korean native ducks (KND) that were fed for 8 weeks (2.8 kg of live weight). Forty-five samples were used after being frozen in storage for one month and were then divided into 5 treatments (3 replications/treatment, 3 samples/replication). Five treatments (CON, FFFT, FFST, SFFT and SFST) were control groups (CON) and four were experimental groups, using $2{\times}2$ complex factors with two freezing methods (fast freezing, FF, $-50^{\circ}C$ in a deep freezer; slow freezing, SF, $-20^{\circ}C$ in a common freezer) and two thawing methods (fast thawing, FT, 5 h $12^{\circ}C$ with flow water; slow thawing, ST, 24 h $5^{\circ}C$ in a refrigerator). Lightness of KND meat in FF and FT groups was lower than that of control (P<0.05). Yellowness of KND meat of the ST group was higher than that of control (P<0.05). Cooking loss (CL) and water holding capacity (WHC) of KND meat in the control were lower than those of the freezing and thawing groups (P<0.01, P<0.05), but shear force (SF) of the control was higher than that of other groups (P<0.01). Moisture content of the ST group was higher than that of the FT group (P<0.05), and protein content of the FF group was higher than that of control (P<0.05). Stearic acid (C18:0) of the SF group was higher than that of the FF group (P<0.05). Arachidonic acid (C20:4n6) of control was higher than that of the SF and ST groups (P<0.01, P<0.05). Alanine, aspartic acid, glutamic acid, serine, and tyrosine content of the control were lower than that of the freezing and thawing groups (P<0.05). These results show that freezing and thawing methods affect meat color, shear force, cooking loss, and WHC-related water content.
Journal of the Korean Society of Food Science and Nutrition
/
v.37
no.8
/
pp.1025-1029
/
2008
Fresh green tea leaf extracts were prepared by different enzyme treatment conditions, such as concentration, treating time and treating temperature using complex enzyme, Rapidase TF, and then extracted for 30 min at $80^{\circ}C$ to investigate their physicochemical properties. The results showed that free sugar content in every sample tended to increase, especially glucose content was increased up to 7.25 times compared to the control. Total amino acid was barely affected by the enzyme treatment and caffeine content was increased with reaction temperature. Total polyphenol and total catechin content was increased according to the amount of enzyme added and reaction temperature. Regardless of enzyme treatment conditions, composition of catechins were epigallocatechin, epicatechin, epicatechin gallate and epigallocatechin gallate by descending order of the content. Gallic acid content increased up to 0.04% and $45^{\circ}C$ with no further significant changes thereafter. From the results above, we could conclude that a simple and new method to extract green tea materials directly from fresh green tea leaves with improved extract ratio may be introduced by adding $0.08{\sim}0.1%$ of Rapidase TF to heat treated fresh green tea leaves and keeping temperature at $37{\sim}45^{\circ}C$ for $180{\sim}240\;min$ in order to skip existing complicated procedures.
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