Isolation of heat resistant Bacillus strains as potential probiotics for Paralichthys olivaceus

Kai-Min Niu,Woo-Do Lee,Jeong-Min Lim,Kang-Woong Kim,Soo-Ki Kim 한국수산해양기술학회(구 한국어업기술학회) 2017 한국수산해양기술학회 학술발표대회 Vol.2017 No.11

Effects of dietary multi-strain probiotics supplementation in a low fishmeal diet on growth performance, nutrient utilization, proximate composition, immune parameters, and gut microbiota of juvenile olive flounder (Paralichthys olivaceus)

Niu, Kai-Min,Khosravi, Sanaz,Kothari, Damini,Lee, Woo-Do,Lim, Jeong-Min,Lee, Bong-Joo,Kim, Kang-Woong,Lim, Sang-Gu,Lee, Sang-Min,Kim, Soo-Ki Elsevier 2019 FISH AND SHELLFISH IMMUNOLOGY Vol.93 No.-

<P><B>Abstract</B></P> <P>A 12-week feeding trial was conducted to evaluate the effects of multi-strain probiotics (MSP) in a low fish meal (FM) diet on overall performance, gut microbiota, selected non-specific immune responses and antioxidant enzyme activities of olive flounder (<I>Paralichthys olivaceus</I>) juveniles. A total of 225 healthy olive flounders (initial mean body weight, 13.5 ± 0.01 g) were randomly separated into 3 groups of 75 fish, each group having three replicates of 25 fish; first group was fed with a FM-based control diet (Con), 2nd group was fed with a low-FM diet containing a blend of plant and animal protein meals replacing 30% of the FM protein (FM30), and 3rd group was fed with the FM30 diet supplemented with 10<SUP>8</SUP>–10<SUP>9</SUP> CFU kg<SUP>−1</SUP> of the MSP (Pro). With the exception of lipid retention, which was significantly lower in fish fed the FM30 diet compared to the other two treatments, no other statistically significant differences were recorded with respect to any of the other growth and nutrient utilization parameters. Myeloperoxidase and lysozyme activities of fish fed the Pro diet were much higher and significantly different than those of fish fed the FM30 diet. Glutathione peroxidase activity was significantly higher in Pro- than in Con-fed fish, which, in turn, was significantly higher than FM30-fed fish. Expression of immune-related genes including IL-1β, IL-6, and TNF-α was markedly upregulated in livers of the fish fed Pro diet compared to those fed the Con and FM30 diets. Furthermore, supplementation of MSP in FM30 diet enriched the <I>Lactobacillus</I> abundance in the fish gut as well as predictive gene functions in relation to lipid and carbohydrate metabolisms. These data suggested that the MSP could reduce the potential adverse effects of the low-FM diet and might be used as a healthy immunostimulant for olive flounder.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Low fishmeal diet (FM30) significantly affected the lipid retention of olive flounder. </LI> <LI> The multi-strain probiotics (MSP) improved the activities of myeloperoxidase, lysozyme, and glutathione peroxidase. </LI> <LI> The MSP supplement markedly upregulated the immune-related gene expression of IL-1β, IL-6, and TNF-α in the liver. </LI> <LI> The MSP supplement enriched the predictive gene functions in relation to lipid and carbohydrate metabolisms. </LI> <LI> The MSP could be used as a healthy immunostimulant for olive flounder. </LI> </UL> </P>

Characteristics of Chinese chives (Allium tuberosum) fermented by Leuconostoc mesenteroides

홍희옥,Kai-Min Niu,이준형,조상범,한성구,김수기 한국응용생명화학회 2016 Applied Biological Chemistry (Appl Biol Chem) Vol.59 No.3

This study was conducted to investigate the characteristics of bioactive compounds in Chinese chives juice (FC) fermented with Leuconostoc mesenteroides SK1962, a lactic acid bacteria isolated from Kim-chi. L. mesenteroides SK1962 only (LO) was used as comparison. The pH of FC gradually decreased from 6.21 to 4.23 during the 48-h incubation period, which was similar to that of LO. The growth of L. mesenteroides SK1962 in FC was higher compared with that in LO for various incubation times, with the exception of the 8-h incubation. Total polyphenol and flavonoid contents in FC were higher as compared with those in LO during incubation, leading to increased antioxidant activity in FC at different incubation times. Moreover, FC was more effective than LO in reduction of superoxide free radical production in primary bovine mammary alveolar cells. In addition, FC demonstrated antibacterial properties against pathogenic bacteria such as Listeria monocytogenes, Pantoea agglomerans, Haemophilus parasuis, Salmonella gallinarum, Escherichia coli O157, and Burkholderia. sp. Although LO also showed antibacterial effects against the above-mentioned pathogenic bacteria, its antibacterial activities were generally lower compared with those of FC. The results show that the antioxidant and antibacterial activities in Chinese chives was induced by fermentation with L. mesenteroides SK 1962. In conclusion, fermentation may lead to an increase in bioactive compounds including total polyphenol and flavonoid.

Characteristics of Eggshell Powder as Carriers of Probiotics

Woo-Do Lee(이우도),Kai-Min Niu(우개민),Jeong-Min Lim(임정민),Kwon-Jung Yi(이권정),Bong-Joo Lee(이봉주),Kang-Woong Kim(김강웅),Kyoung-Duck Kim(김경덕),Sang-Woo Hur(허상우),Hyon-Sob Han(한현섭),Soo-Ki Kim(김수기) 한국생명과학회 2018 생명과학회지 Vol.28 No.1

계란 가공 부산물인 난각(ES: Eggshell)은 탄산칼슘 함량이 높아 사료에 첨가하여 칼슘원으로 이용되고 있다. 본 연구에서는 ES를 생균제의 부형제인 운반체로서 활용 가능성을 처음으로 시도하였다. L. plantarum을 대두박(SBM: Soybean meal), 난각조각(ESL: Eggshell powder with large particles), 난각미세분말(ESF: Eggshell powder with fine particles), 그리고 이들의 복합운반체인 SBM+ESL과 SBM+ESF에 생균제를 흡착시켜 그 부착상태를 주사전자현미경으로 확인하였다. 이 중 복합운반체인 SBM+ESF는 상온에서 4주 동안 pH 7~8을 유지하면서 L. plantarum의 가장 높은 생존율을 보였다. 본 연구에 사용한 모든 생균제들은 보존기간 동안 4℃에서는 높은 생존율을 보였다. 30℃에서는 유산균수는 크게 감소하였으나, B. licheniformis는 높은 생존율을 유지하였고 B. subtilis, B. amyloliquefaciens와 S. cerevisiae는 2 log10 (CFU/g)정도 감소하였다. 상기 연구결과는 사료의 칼슘원으로 이용되는 난각미세분말(ESF)을 대두박과 혼합하여 사용하면 B. licheniformis를 비롯한 일부 생균제의 생존성을 향상시켜 부형제(운반체)로도 사용할 수 있음을 밝혔다. Eggshell (ES) is a by-product of table eggs with high content of calcium carbonate which can be used as a calcium source in feed. In this study, we have first illuminated the potential application of ES as a novel carrier for probiotics. The carriers used in the study include a SBM (Soybean meal), ESL (Eggshell powder with large particles), ESF (Eggshell powder with fine particles), and the complex carriers (SBM+ESL, SBM+ESF). The structure of carriers absorbed by L. plantarum was confirmed by SEM image. Among these carriers, the complex carrier SBM+ESF showed the highest viability of L. plantarum with pH 7~8 during four weeks storage at room temperature. The SBM+ESF was further tested as a carrier for various probiotic strains at 4℃ or 30℃. All the probiotic strains showed high viability at 4℃ storage. However, a significant reduction of Lactobacillus cells was observed at 30℃ storage. B. lichenifomis maintained high viability whereas B. subtilis, B. amyloliquefaciens, and S. cerevisiae showed the reduction of 2 log10 (CFU/g). These results suggest that if the ESF as a calcium source in feed was mixed with SBM, it can be used as an effective complex carrier for improving the viability of some probiotics including B. licheniformis.

Antioxidant and Antibacterial Activities of Lactobacillus-fermented Artemisia annua L. as a Potential Fish Feed Additive

Ah-Ran Lee(이아란),Kai-Min Niu(우개민),Su-Kyung Kang(강수경),Sung-Gu Han(한성구),Bong-Joo Lee(이봉주),Soo-Ki Kim(김수기) 한국생명과학회 2017 생명과학회지 Vol.27 No.6

최근 양어에서는 전염병, 환경오염, 어분 가격 상승 등으로 어려움을 가지고 있다. 본 연구는 쑥에서 분리한 L. plantarum SK3494를 이용하여 전통 약초인 개똥쑥을 발효시켜 in vitro 생리활성을 측정하고 어류용 사료첨가제로서의 가능성을 연구하였다. 개똥쑥의 발효시 유산균수는 9.38 log10 CFU/ml이며, pH는 4.1로 나타났다. 발효개똥쑥의 항산화활성은 DPPH 측정과 MAC-T 세포 내 과산화물 감소 실험에서 뛰어난 효과를 보였다. 발효개똥쑥은 어류 병원균인Photobacterium damselae subsp. damselae와 Vibrio ichthyoenteri에 대하여 강한 항균활성을 나타내었다. 따라서 유산균을 이용한 개똥쑥 발효물은 양식용 사료첨가제로써 가능성을 가지고 있는 것으로 판단되었다. Fermented medical herbs using Lactobacilli have attracted significant attention due to their enhanced biological activities. A traditional medicinal plant, Artemisia annua L., was fermented using a probiotic strain, L. plantarum SK3494. The strain was isolated from Artemisia princeps var. orientalis and molecularly identified through sequence similarities and phylogenetic tree analysis. The antioxidant activity of L. plantarum-fermented A. annua L. (LFA) was determined using the DPPH free radical scavenging assay. Cellular antioxidant activity of LFA was examined using the superoxide radical reduction assay in MAT-C cells. Total polyphenol contents (TPC) and flavonoid contents (TFC) of LFA were determined. The antibacterial activity of LFA against fish pathogens was also determined in this study. The viable cell number (9.38 log10 CFU/ml) and pH (4.1) results showed good adaptive ability of the selected strain during fermentation. LFA was found to have enhanced antioxidant activity compared to non-fermented A. annua L. (NFA) based on the DPPH assay. Cellular antioxidant activity was present in both LFA and NFA. After 24 hr and 48 hr of fermentation, the LFA also showed antibacterial activities against fish pathogens Photobacterium damselae subsp. damselae and Vibrio ichthyoenteri. These results suggest that L. plantarum-fermented A. annua L. may have potential as a feed additive in aquaculture.

Complete genome sequence of Paenibacillus konkukensis sp. nov. SK3146 as a potential probiotic strain

( Hae-in Jung ),( Sungkwon Park ),( Kai-min Niu ),( Sang-won Lee ),( Damini Kothari ),( Kwon Jung Yi ),( Soo-ki Kim ) 한국축산학회 2021 한국축산학회지 Vol.63 No.3

Paenibacillus konkukensis sp. nov., SK3146 is a novel strain isolated from a pig feed. Here, we present complete genome sequence of SK3146. The genome consists of a single circular genome measuring 7,968,964 bp in size with an average guanine + cytosine (G+C) content of 53.4%. Genomic annotation revealed that the strain encodes 151 proteins related to hydrolases (EC3), which was higher than those in Bacillus subtilis and Escherichia coli. Diverse kinds of hydrolases including galactosidase, glucosidase, cellulase, lipase, xylanase, and protease were found in the genome of SK3146, coupled with one bacteriocin encoding gene. The complete genome sequence of P. konkukensis SK3146 indicates the immense probiotic potential of the strain with nutrient digestibility and antimicrobial activity functions.

Effect of dietary supplementation of a phytogenic blend containing Schisandra chinensis, Pinus densiflora, and Allium tuberosum on productivity, egg quality, and health parameters in laying hens

Moon, Seung-Gyu,Lee, Sung-Kwang,Lee, Woo-Do,Niu, Kai-Min,Hwang, Won-Uk,Oh, Jong-Seok,Kothari, Damini,Kim, Soo-Ki Asian Australasian Association of Animal Productio 2021 Animal Bioscience Vol.34 No.2

Objective: This study was conducted to investigate the supplementary effect of a phytogenic blend (SPA: a mixture containing fermented Schisandra chinensis pomace, fermented Pinus densiflora needle extract, and Allium tuberosum powder in the ratio of 2:2:1) on egg production, egg quality, blood constituents, and visceral organs in laying hens. Methods: A total of 135 Hy-line brown laying hens (48-wk-old) were randomly allocated to three dietary treatments with 5 replicates of 9 hens. The control group (CON) was fed a basal diet (no exogenous SPA addition) and the experimental groups were fed the basal diet containing SPA at the level of 0.1% and 0.3% for 6 weeks. Results: The feed intake was significantly improved in SPA supplemented groups as compared with the control (p<0.05). However, egg production, daily egg mass, and feed conversion ratio were not different among the dietary treatments (p>0.05). For egg quality traits, only Haugh unit (HU) was significantly improved in SPA (0.3%) (p<0.05) as compared with other groups. However, HU was not affected during 4-wk of storage at 18℃ among the dietary treatments (p>0.05). Furthermore, SPA supplementation did not affect the blood biochemical constituents except for the phosphate content, which was significantly higher in SPA groups than the CON group (p<0.05). There were no significant differences in visceral organ characteristics and immune indicators (immunoglobulin A [IgA], IgG, and IgM) in SPA or CON groups. Conclusion: This study suggested that the supplementation of SPA may have beneficial effects on feed intake and egg quality in laying hens.

말분변 미생물을 이용한 주스박 사료의 발효 특성

황원욱,김겸헌,우개민,임정호,우제훈,채현석,박남건,김수기,Hwang, Won-Uk,Kim, Gyeom-Heon,Niu, Kai-Min,Lim, Joung-Ho,Woo, Jae-Hoon,Chae, Hyun-Seok,Park, Nam-Geon,Kim, Soo-Ki 한국초지조사료학회 2017 한국초지조사료학회지 Vol.37 No.4

본 연구에서는 주스박에 토착하고 있는 미생물을 분리하고 이를 발효에 이용하여 사료적 가치를 평가하고자 하였다. 주스박과 대두박을 원료로 하여 바실러스, 효모 그리고 유산균을 혼합 접종하여 수분 60%에서 고체발효 후의 pH변화, 총 균수의 변화와 같은 발효특성과 원료의 시간대별 일반성분의 변화를 확인하고자 하였다. 사과박, 당근박, 포도박, 감귤박에서 발효에 이용하기 위한 토착균주들을 분리, 동정하였으며 분리균 중 Lactobacillus plantarum, Weissella cibaria 및 Bacillus subtilis를 선택하여 농산부산물 고체발효에 이용하였다. 발효 주스박의 원료로는 사과박, 당근박, 포도박, 감귤박과 각 박을 동일비율로 혼합한 혼합박을 이용하여 5개의 시험구를 구성하였다. 위의 박들을 대두박과 2:3의 비율로 혼합하였으며 수분은 60%로 하여 발효 출발점에 바실러스, 12시간대에 효모 그리고 24시간에 유산균을 단계적으로 접종하여 $35^{\circ}C$에서 총 48시간 동안 발효하였다. 발효에 의한 pH변화는 발효 전후에서 사과박, 당근박, 포도박, 감귤박 그리고 혼합박에서 각각 $6.25{\rightarrow}4.47$, $6.23{\rightarrow}4.41$, $6.12{\rightarrow}4.66$, $5.49{\rightarrow}4.56$ 그리고 $5.45{\rightarrow}4.40$로 낮아지는 변화를 보였다. 발효 시간에 따른 생균수를 조사한 결과 발효 최초 접종시 초기 균수인 $10^7$에 비해 12, 24 그리고 48시간 발효 후에 모든 처리구에서 $10^8{\sim}10^9$로 증식되면서 발효가 지속적으로 진행되어 선별한 균주와 단계별 균주의 접종조건이 주스박을 발효함에 있어 적합한 것으로 판단되었다. 발효 초기인 12시간까지 젖산의 생성이 급격히 진행되었으며 사과박, 당근박, 포도박, 감귤박 그리고 혼합박에서 각각 632.40, 726.74, 674.29, 647.39 그리고 748.77 mMol/g으로 생성되었다. 발효 시간에 따른 건물함량은 초기 건물함량인 60%에서 12, 24시간 발효 후 당근박의 건물함량이 각각 54.84%, 56.66%로 다른 처리구들보다 유의적으로 높은 값을 보였으며(p<0.05), 포도박은 45.3%, 44.15%로 낮은 건물량을 보였다(p<0.05). 전반적으로 건물함량은 원물의 20%정도가 감소되었으며, 발효 시간에 따라서는 크게 변하지 않았다. 발효 시간대별 NDF는 발효 12시간째 모든 주스박에서 많은 감소를 보였으며 24시간부터 서서히 감소 또는 유지를 하였으며, 24시간대에는 포도박과 사과박이 가장 높은 경향을 보였다(p<0.10). ADF는 전반적으로 발효 시간에 따라 큰 변화를 보이지 않았으나, 사과박은 발효초기에 비해 12시간 때부터 급격히 감소하는 경향을 보였다 (p<0.10). 발효 시간에 따른 조회분은 쥬스박 별로 차이를 보이지 않았으나 (p<0.05), 전체적으로 감소되는 경향을 보였다. 주스박을 이용한 말용 사료를 개발하기 위해 사과박, 당근박, 포도박 그리고 말분변을 접종하였을 때 이들을 이용하는 우점 균주를 분리할 수 있었다. 이들 균주 중에서 발효에 유용하게 사용할 수 있는 바실러스와 유산균을 선별하였다. 바실러스, 효모 그리고 유산균을 3단계로 12시간 간격으로 각각 순서에 따라 접종하였다. 각 균주의 접종이후 12시간부터는 총 균수가 각각 $10^8$ 이상으로 유지하였다. 48시간 배양 후는 바실러스, 효모 그리고 유산균이 거의 균등하게 성장하였으며 이러한 주스박 발효물을 이용하여 말 사료로 이용하면 식품부산물을 사료자원으로 이용할 수 있을 뿐만 아니라 말을 위한 생균제의 급여로 In vitro fermentation was conducted to figure out alternative fiber sources for horse feed. For the development of value-added products as a horse feed resource, the pomaces from apple, carrot, grape, and citrus were fermented under solid-state conditions in the presence of 60% soybean meal with 40% of each fruit pomace at 60% of moisture content. Lactobacillus plantarum SK3873, Lactobacillus plantarum SK3893, Weissella cibaria SK3880, and Bacillus subtilis SK3889 were isolated from the fermented fruit pomace by inoculation of horse feces. For the growth of Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum, they were inoculated in 3-step order at 0, 12, and 24 h, respectively. The fruit pomace was fermented for 48 h at $35^{\circ}C$. The pH of the apple, carrot, grape, citrus and all mixed pomaces decreased from 5.45~6.25 to 4.40~4.77. Microbial growth was maintained at $10^8{\sim}10^9cfu/g$. After 12 and 24 h incubation, dry matter of carrot pomace were highest at 54.84 and 56.66%, respectively (P<0.05) and that of grape pomace was lower than others during fermentation (P<0.05). Dry matter was generally reduced by about 20%. NDF decreased gradually or maintained after 24 h, indicating the fiber degradation. Ash content tended to decrease during fermentation. After 48 hours fermentation, Bacillus, yeast and Lactobacillus showed an excellent growth by using juice by-products. These results suggest that fermented juice pomace has a potential as horse feedstuff with probiotics to maintain beneficial microflora in horse gut.