• Journal of Microbiology and Biotechnology
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• 제33권9호
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• pp.1228-1237
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• 2023

The CRISPR-Cas system has emerged as the most efficient genome editing technique for a wide range of cells. Delivery of the Cas9-sgRNA ribonucleoprotein complex (Cas9 RNP) has gained popularity. The objective of this study was to develop a quantitative polymerase chain reaction (qPCR)-based assay to quantify the double-strand break reaction mediated by Cas9 RNP. To accomplish this, the dextransucrase gene (dsr) from Leuconostoc citreum was selected as the target DNA. The Cas9 protein was produced using recombinant Escherichia coli BL21, and two sgRNAs were synthesized through in vitro transcription to facilitate binding with the dsr gene. Under optimized in vitro conditions, the 2.6 kb dsr DNA was specifically cleaved into 1.1 and 1.5 kb fragments by both Cas9-sgRNA365 and Cas9-sgRNA433. By monitoring changes in dsr concentration using qPCR, the endonuclease activities of the two Cas9 RNPs were measured, and their efficiencies were compared. Specifically, the specific activities of dsr365RNP and dsr433RNP were 28.74 and 34.48 (unit/㎍ RNP), respectively. The versatility of this method was also verified using different target genes, uracil phosphoribosyl transferase (upp) gene, of Bifidobacterium bifidum and specific sgRNAs. The assay method was also utilized to determine the impact of high electrical field on Cas9 RNP activity during an efficient electroporation process. Overall, the results demonstrated that the qPCR-based method is an effective tool for measuring the endonuclease activity of Cas9 RNP.

• Asian-Australasian Journal of Animal Sciences
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• 제20권11호
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• pp.1699-1704
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• 2007

This experiment was carried out to evaluate the effects of adding encapsulated-glucose, glucose, sorbic acid or prefermented juice of epiphytic lactic acid bacteria (FJLB) on the fermentation quality and residual mono- and disaccharide composition of Italian ryegrass (Lolium multiflorum Lam) silages. The additive treatments were as follows: (1) control (no addition), (2) encapsulated-glucose addition at 0.5% for glucose, (3) glucose addition at 1%, (4) sorbic acid addition at 0.1%, (5) FJLB addition at a theoretical application rate of $2.67{\times}10^5$ CFU (colony forming unit) $g^{-1}$, on a fresh weight basis of Italian ryegrass. Although control and encapsulated-glucose treatments had higher contents of butyric acid (33.45, 21.50 g $kg^{-1}$ DM) and ammonia-N/Total nitrogen (114.91, 87.01 g $kg^{-1}$) as compared with the other treated silages, the fermentation in all silages was clearly dominated by lactic acid. This was well indicated by the low pH (4.38-3.59), and high lactic acid/acetic acid (4.39-22.97) and lactic acid content (46.85-121.76 g $kg^{-1}$ DM). Encapsulated-0.5% glucose and glucose addition increased lactic acid/acetic acid, and significantly (p<0.05) decreased ammonia-N/total nitrogen, and the contents of butyric acid and total volatile fatty acids (VFAs) as compared with the control. However, there were higher butyric acid and lower residual mono-and di-saccharides on the two treatments as compared with sorbic acid and FJLB addition, and their utilization efficiency of water soluble carbohydrates (WSC) was lower than that of both sorbic acid and FJLB additions. Sorbic acid addition showed the lowest content of ethanol and ammonia-N/total nitrogen, and the highest content of residual fructose and total mono-and disaccharides as well as the higher lactic acid/acetic acid value. Sorbic acid addition decreased the loss of mono-and disaccharides, and inhibited the activity of clostridial and other undesirable bacteria, and greatly increased the utilization efficiency of fermentable substrates by epiphytic LAB. FJLB addition had the lowest pH value and the highest lactic acid content among all additive treatments, with the most intensive lactic acid fermentation occurring in FJLB treated silage. This resulted in the faster accumulation of lactic acid and faster pH reduction. Sorbic acid and FJLB addition depressed clostridia or other undesirable bacterial fermentation which decreased the WSC loss and saved the fermentable substrate for lactic acid fermentation.

• Asian-Australasian Journal of Animal Sciences
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• 제15권11호
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• pp.1653-1658
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• 2002

The objectives of this study were to examine calpain activity and meat tenderness by three different feeding patterns in Korean native cattle (KNC). Total forty-five animals were assigned each fifteen in long term restriction feeding (LTFR), long-term restriction feeding and hormone treatment (LTFR-tH), and short term non-restriction feeding (STFNR), respectively. Concentrate was restricted based on body weight in exp 1 and 2. However, it was fed ad libitum in exp. 3. Hormonal implantation was made with $M-PO^{TM}$ for bulls and with $F-TO^{TM}$ for heifers at 18, 20, 22 months of age in exp. 2. Animals were purchased (3-5 month old) from local cattle market and managed in two local farms and university research unit at three different years. Animals were slaughtered at 24 months for long-term trial and at 18 month for short term-trial. Loin and tender loin muscle was used for calpain activity and meat quality. Calpain proteolytic system was not changed by treatment. However, calpastatin activity was low in short-term trial. The calpain and calpastatin activity is reciprocal relationship, therefore, the high calpain activity may effect on quality grade. The shear force value was decreased as the processing of aging after postmortem. On the other hand, the cooking loss was significantly higher in short-term than in long-term trial, and then gradually decreased by the aging. Hormone implants to increase meat yield influenced to calpastatin activity more powerfully than calpain activity to meat tenderness. In meat color-a*, there was not significant difference in loin. Meat color-b* was decreased as postmortem aging time increased in tenderloin. Western blots were done to learn whether these proteins are degraded during postmortem storage and whether this degradation temporally parallels the decrease of shear force value. Vinculin was detected at 0 day and 1 day and degraded after 3 day. In conclusion, Calpain activity was affected slightly on meat tenderness. But meat tenderness was influenced by calpastatin, more effectively.

• Asian-Australasian Journal of Animal Sciences
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• 제15권6호
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• pp.838-843
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• 2002

The objectives of this study were to examine growth performance and meat quality by three different feeding patterns in Korean native cattle (KNC). In each of 3 years, fifteen KNC were randomly assigned in a (3 feeding management)${\times}$(3 sex) factorial design experiment; thus, in total, there were 5 animals in each of the 9 treatments. The three feeding management treatments were longterm (24 month) restriction feeding (LTFR), long-term restriction feeding-hormone implant (LTFR-tH), and short-term (18 month) nonrestriction feeding (STFNR). Three sexes were bull, steer, and heifer. Concentrate diet was fed restriction-feeding method based on body weight in LTFR and LTFR-tH. However, the diet was fed ad libitum in STFNR. Hormonal implantation was made three times with M-$PO^{TM}$ for bulls and with F-$TO^{TM}$ for heifers at 18, 20, 22 month of age in LTFR-tH. Animal were purchased from the local cattle market and managed in two local farms and at the university research unit. Animals were slaughtered at 24 months for long-term trial and at 18 month for short-term trial. The growth rate was the highest in bulls and the lowest in heifers. However, the differences were diminished in F-$TO^{TM}$ implanted heifers. The average daily gain was high in STFNR due to ad libitum feeding. The carcass grade was similar among the treatments on percentage bases. Hormonal implants improved significantly the meat quality grade in all sexes. Castration increased body fat content and improved meat quality grade by intramuscular fat deposition. In conclusion, long-term feeding and hormone treatment increased meat quality grade more than short-term feeding. However, ADG was higher in the short-term trial although feedefficiency was lower.

• Animal Bioscience
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• 제34권3_spc호
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• pp.393-404
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• 2021

Objective: This study compared the catechin composition of different tea byproducts and investigated the effects of dietary supplementation with green tea byproducts on the accumulation of abdominal fat, the modulation of lipid metabolism, and the inflammatory response in red feather native chickens. Methods: Bioactive compounds were detected, and in vitro anti-obesity capacity analyzed via 3T3-L1 preadipocytes. In animal experiments, 320 one-day-old red feather native chickens were divided into 4 treatment groups: control, basal diet supplemented with 0.5% Jinxuan byproduct (JBP), basal diet supplemented with 1% JBP, or basal diet supplemented with 5×106 colony-forming unit (CFU)/kg Bacillus amyloliquefaciens+5×106 CFU/kg Saccharomyces cerevisiae (BA+SC). Growth performance, serum characteristics, carcass characteristics, and the mRNA expression of selected genes were measured. Results: This study compared several cultivars of tea, but Jinxuan showed the highest levels of the anti-obesity compound epigallocatechin gallate. 3T3-L1 preadipocytes treated with Jinxuan extract significantly reduced lipid accumulation. There were no significant differences in growth performance, serum characteristics, or carcass characteristics among the groups. However, in the 0.5% JBP group, mRNA expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) were significantly decreased. In the 1% JBP group, FAS, ACC and peroxisome proliferator-activated receptor γ levels were significantly decreased. Moreover, inflammation-related mRNA expression levels were decreased by the addition of JBP. Conclusion: JBP contained abundant catechins and related bioactive compounds, which reduced lipid accumulation in 3T3-L1 preadipocytes, however there was no significant reduction in abdominal fat. This may be due to a lack of active anti-obesity compounds or because the major changes in fat metabolism were not in the abdomen. Nonetheless, lipogenesis-related and inflammation-related mRNA expression were reduced in the 1% JBP group. In addition, dietary supplementation with tea byproducts could reduce the massive amount of byproducts created during tea production and modulate lipid metabolism and the inflammatory response in chickens.

• Animal Bioscience
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• 제37권4호
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• pp.689-696
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• 2024

Objective: Our study examined the impact of propriety blends of Bacillus strain probiotics on the performance, egg quality, and faecal microflora of laying hens. Methods: A total of 183 Institut de selection Animale (ISA) brown laying hens aged 23 weeks with an average body weight of 1,894±72 g were randomly allocated into 3 groups as control (corn-soybean meal based diet, CON), 0.5 g/kg Enterosure probiotics (ET1, 3×10⁸ colony-forming unit [CFU]/kg feed), and 5 g/kg Enterosure probiotics (ET2, 3×10⁹ CFU/kg feed) administered in mashed form. At the completion of each phase hen day egg production (HDEP), average egg weight (AEW), feed intake, and faecal microbiota were evaluated. Results: HDEP and AEW were higher (p<0.05) in the ET2-supplemented diet in phase 3 (week 9 to 12) compared with CON. Egg mass (EM) was higher (p<0.05) in phase 2 at ET2, and also higher (p<0.05) in phase 3 at the ET1 and ET2-supplemented diets compared with CON. Feed conversion ratio was lower (p<0.05) in phase 3 at the ET1 and ET2-supplemented diets, with ET2 being the lowest compared with ET1 and CON. Yolk colour was higher (p<0.05) in the ET-supplemented diets at phase 3 compared with CON. Bifidobacterium spp. was higher (p<0.05) in the ET2- supplemented diet compared with CON in phase 2, while in In phase 3, Lactobacillus spp. and Bifidobacterium spp. were higher (p<0.05) in the ET-supplemented diets compared with CON. Coliforms were lower (p<0.05) in the ET-supplemented diets compared with CON in phase 3. Conclusion: The propriety blends of Bacillus strain probiotics supplements at 0.5 g/kg and 5 g/kg could improve the production and quality of eggs with more significance at 5 g/kg for HDEP, AEW and EM, which was achieved via the increase of beneficial microbiomes such as Lactobacillus spp., Bifidobacterium spp., and the decrease of pathogenic microbiomes like Escherichia coli and Coliforms which was speculated to improve gut barrier function and the reproductive hormone.

• 한국축산식품학회지
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• 제40권6호
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• pp.1014-1032
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• 2020

This work aimed to investigate the psychobiotic effects of six bacterial strains on the mind and behavior of male Wistar rats. The probiotic (PRO) group (n=7) were rats pre-treated with antibiotics for 7 days followed by 14-day probiotic administration, antibiotics (ANT) group (n=7) were rats treated with antibiotics for 21 days without probiotics. The control (CON) group (n=7) were rats that received sham treatment for 21 days. The six bacterial strains with probiotic properties were mostly isolated from Thai fermented foods; Pedicoccus pentosaceus WS11, Lactobacillus plantarum SK321, L. fermentum SK324, L. brevis TRBC 3003, Bifidobacterium adolescentis TBRC 7154 and Lactococcus lactis subsp. lactis TBRC 375. The probiotics were freeze-dried into powder (6×10⁹ CFU/5 g) and administered to the PRO group via oral gavage. Behavioral tests were performed. The PRO group displayed significantly reduced anxiety level and increased locomotor function using a marble burying test and open field test, respectively and significantly improved short-term memory performance using a novel object recognition test. Antibiotics significantly reduced microbial counts in rat feces in the ANT group by 100 fold compared to the PRO group. Probiotics significantly enhanced antioxidant enzymatic and non-enzymatic defenses in rat brains as assessed using catalase activity and ferric reducing antioxidant power assay, respectively. Probiotics also showed neuroprotective effects with less pyknotic cells and lower frequency of vacuolization in cerebral cortex. This multi-strain probiotic formulation from Thai fermented foods may offer a potential to develop psychobiotic-rich functional foods to modulate human mind and behaviors.

• 한국가금학회지
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• 제33권4호
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• pp.303-308
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• 2006

본 연구는 산란계 사료내 갯벌 미생물 유래 단백질 분해 효소제를 첨가하였을 때 계란 품질, 영양소 소화율 및 혈청 내 총 단백질 함량에 미치는 영향을 알아보기 위해 실시하였다. 사양 시험은 29주령 ISA brown 갈색계 252수를 공시하며 8주간 실시하였다. 처리구는 옥수수-대두박 위주의 사료인 기초 사료구(CON; basal diet), 기초 사료내 단백질 분해 효소제 0.05% 첨가구(PROI; basal diet+0.05% pretense), 0.1% 첨가구 (PRO2; basal diet+0.1% protease)로서 처리구당 7반복, 반복당 12수씩 완전임의 배치하였다. 전체 사양시험 기간 동안, 산란율은 처리구간에 유의적인 차이를 보이지 않았다(P>0.05). 난황고의 변화량은 PRO1 처리구가 CON 처리구와 비교하여 유의적으로 높게 나타났다(P<0.05). 난중의 변화량은 PRO2처리구가 CON과 PRO1 처리구와 비교하여 유의적으로 높게 나타났다(P<0.05). 난각 품질, 난황색, haugh unit 및 난황 계수에서는 처리구간에 유의적인 차이를 보이지 않았다(P>0.05). 건물 소화율에서는 CON과 PRO2처리구가 PRO1 처리구와 비교하여 유의적으로 높게 나타났고(P<0.05), 질소 소화율에서는 PRO2 처리구가 CON 처리구와 비교하여 유의적으로 높게 나타났다(P<0.05). 혈청내 총 단백질 함량에서는 처리구간에 유의적인 차이를 보이지 않았다(P>0.05). 결론적으로, 산란계 사료내 갯벌 미생물 유래 단백질 분해 효소제의 첨가는 난황고, 난중 및 영양소 소화율을 개선시켰다.

• 한국가금학회지
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• 제34권1호
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• pp.9-14
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• 2007

본 연구는 산란계 사료내 갯벌 미생물 유래 단백질 분해 효소제를 첨가하였을 때 계란 품질, 영양소 소화율 및 혈청 내 총 단백질 함량에 미치는 영향을 알아보기 위해 실시하였다. 사양 시험은 29주령 ISA brown 갈색계 252수를 공시하였고, 사양 시험은 8주간 실시하였다. 옥수수-대두박 위주의 기초 사료구(CON; basal diet), 단백질 분해 효소제 0.05, 0.10% 첨가구(PRO1; PRO2)로 처리구당 7반복, 반복당 12수씩 완전임의 배치하였다. 전체 사양 시험 기간 동안, 산란율은 처리구간에 유의적인 차이를 보이지 않았다(P>0.05). 난황고의 변화량은 PRO1 처리구가 CON 처리구와 비교하여 유의적으로 높게 나타났다(P<0.05). 난중의 변화량은 PRO2 처리구가 CON과 PRO1 처리구와 비교하여 유의적으로 높게 나타났다(P<0.05). 난각 품질, 난황색, haugh unit 및 난황 계수에서는 처리구간에 유의적인 차이를 보이지 않았다(P>0.05). 건물 소화율에서는 CON과 PRO2 처리구가 PRO1 처리구와 비교하여 유의적으로 높게 나타났고(P<0.05), 질소 소화율에서는 PRO2 처리구가 CON 처리구와 비교하여 유의적으로 높게 나타났다(P<0.05). 혈청내 총 단백질 함량에서는 처리구간에 유의적인 차이를 보이지 않았다(P>0.05). 결론적으로, 산란계 사료내 갯벌 미생물 유래 단백질 분해 효소제의 첨가는 난황고, 난중 및 영양소 소화율을 개선시켰다.

• 생명과학회지
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• 제30권10호
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• pp.860-866
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• 2020

본 연구는 음식폐기물을 활용하여 갈색거저리를 사육한 후에 산란계에 급이시 나타나는 계란의 특성을 분석하기 위해 수행되었다. 계란의 두께는 처리군에서 약간 얇아지는 경향성을 보였지만, 신선도를 나타내는 Haugh unit는 처리군이 대조군에 비교하여 높아지는 것으로 나타났고, 특히 5%가 가장 높았다. 난황의 색상은 5% 급이 군에서 명도와 황색도가 낮아지는 반면에 적색도가 증가되는 경향성을 보였다. 5% 급이군은 조지방과 조단백이 대조군과 유사하게 형성되는 것으로 나타났지만, 10%와 15% 급이군들은 대조군보다 조지방 및 조단백 함량이 낮은 것으로 나타났다. 그러나 탄수화물 함량은 10%와 15% 급이군들에서 높게 형성되는 것으로 관찰되었다. 처리군들에서 구성아미노산은 대조군에 비교하여 필수아미노산보다 비필수아미노산의 비율의 증가가 높은 것으로 나타났다. 항산화물질의 지표가 되는 폴리페놀의 함량은 5% 급이군에서 가장 높은 것으로 나타났기 때문에, 계란의 저장성 및 항산화에 영향을 줄 수 있는 것으로 추정된다. 따라서 고단백 및 고지방 사료인 갈색거저리를 보충 사료원으로 활용할 때 계란의 영양성분 함량에 중용한 변화를 유발할 수 있고 계란의 신선도를 개선할 수 있는 것으로 제의된다.