TWI726303B - Lactococcus lactis gen3013 strain and a composition for preventing or treating cancer comprising the same - Google Patents
Lactococcus lactis gen3013 strain and a composition for preventing or treating cancer comprising the same Download PDFInfo
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- TWI726303B TWI726303B TW108112856A TW108112856A TWI726303B TW I726303 B TWI726303 B TW I726303B TW 108112856 A TW108112856 A TW 108112856A TW 108112856 A TW108112856 A TW 108112856A TW I726303 B TWI726303 B TW I726303B
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Abstract
Description
本發明係關於新穎乳酸乳球菌(Lactococcus lactis)GEN3013菌株及包含其之用於預防或治療癌症之組合物。具體地，本發明之乳酸乳球菌GEN3013菌株不僅發揮抑制癌細胞本身增殖之作用，而且還發揮降低癌細胞移動性及抑制血管生成之作用，該作用顯著優於已知之乳酸乳球菌株。 The present invention relates to a novel strain of Lactococcus lactis (Lactococcus lactis) GEN3013 and a composition containing it for preventing or treating cancer. Specifically, the Lactococcus lactis GEN3013 strain of the present invention not only inhibits the proliferation of cancer cells, but also reduces the mobility of cancer cells and inhibits angiogenesis, which is significantly better than the known Lactococcus lactis strains.
此外，本發明之乳酸乳球菌GEN3013菌株顯示出抗炎、抗氧化及免疫增強之活性。 In addition, the Lactococcus lactis GEN3013 strain of the present invention exhibits anti-inflammatory, anti-oxidant and immune-enhancing activities.
由於不同之社會及環境因素，癌症發病率每年都在增加。癌症係受到嚴重關注之代表性疾病，同時抗癌治療在全球範圍內得到了廣泛研究，包括細胞位準之基礎研究。然而，導致癌症之機制尚未完全清楚地確立。此外，為了預防復發並考慮到實現完全康復之困難，仍然存在對新抗癌劑之不斷增長之需求。 Due to different social and environmental factors, the incidence of cancer is increasing every year. Cancer is a representative disease that has received serious attention. At the same time, anti-cancer treatments have been extensively studied worldwide, including basic research on cell level. However, the mechanism that causes cancer has not yet been fully and clearly established. In addition, in order to prevent recurrence and take into account the difficulty of achieving complete recovery, there is still a growing demand for new anticancer agents.
同時，治療癌症之高成本包括直接醫療費用，以及諸如經濟活動減少、康復成本及患者護理成本之間接費用，此可能給癌症患者家庭及所有社會組織帶來經濟負擔。因此，現在需要特別低成本且安全之新型抗癌劑。At the same time, the high cost of cancer treatment includes direct medical expenses, as well as indirect expenses such as reduced economic activities, rehabilitation costs, and patient care costs, which may bring economic burdens to cancer patients’ families and all social organizations. Therefore, there is a need for a new type of anticancer agent that is particularly low-cost and safe.
在癌症中，腫瘤微環境藉由不受限之細胞增殖、來自缺氧之血管生成、控制浸潤癌組織之免疫細胞之功能而得以保存，其中細胞經由血液或淋巴管來移動或轉移到其他組織，最終導致死亡。In cancer, the tumor microenvironment is preserved by unrestricted cell proliferation, angiogenesis from hypoxia, and the function of controlling immune cells that infiltrate cancer tissues, in which cells move or metastasize to other tissues through the blood or lymph vessels , Which eventually leads to death.
經開發用於治療癌症之合成抗癌劑抑制癌細胞之代謝以抑制細胞增殖或誘導細胞凋亡。另外，雙鏈螺旋之DNA在細胞分裂期間展開以抑制DNA複製程序，基於該抑制程序來阻斷細胞分裂並誘導細胞凋亡，最終抑制癌細胞之增殖或消除癌細胞。然而，合成之抗癌劑甚至可以攻擊正常細胞。因此，引起藥物誘導之副作用，例如脫髮、腹瀉或發燒。已經開發了用於降低副作用及增強抗癌活性之靶向抗癌劑，該抗癌劑靶向涉及癌症產生之突變基因而非正常細胞，以增強合成抗癌劑。然而，針對大多數靶向抗癌劑之抗性機制在2至3年內表現。在此情況下，靶向抗癌劑不再對治療有效。Synthetic anticancer agents developed for the treatment of cancer inhibit the metabolism of cancer cells to inhibit cell proliferation or induce cell apoptosis. In addition, the DNA of the double-stranded helix unfolds during cell division to inhibit the DNA replication program, based on the inhibition program to block cell division and induce cell apoptosis, and ultimately inhibit the proliferation of cancer cells or eliminate cancer cells. However, synthetic anticancer agents can even attack normal cells. Therefore, it causes drug-induced side effects, such as hair loss, diarrhea, or fever. Targeted anticancer agents for reducing side effects and enhancing anticancer activity have been developed. The anticancer agents target mutated genes involved in cancer production rather than normal cells to enhance the synthesis of anticancer agents. However, resistance mechanisms against most targeted anticancer agents manifest within 2 to 3 years. In this case, targeted anticancer agents are no longer effective for treatment.
癌症特徵之一係由於不受限增殖而在組織中產生缺氧，隨後產生異常微血管以在低氧環境下存活、接收營養物並保持增殖。因此，已開發出靶向血管生成相關因子之抗癌劑並將其用作患者之治療劑。然而，雖然血管生成相關之抗癌劑拮抗血管生成，但它不能充分抑制癌細胞之快速增殖。因此，將它與其他抗癌劑聯合給予臨床患者。One of the characteristics of cancer is the generation of hypoxia in the tissue due to unrestricted proliferation, followed by abnormal capillaries to survive in a hypoxic environment, receive nutrients, and maintain proliferation. Therefore, anticancer agents targeting angiogenesis-related factors have been developed and used as therapeutic agents for patients. However, although angiogenesis-related anticancer agents antagonize angiogenesis, they cannot sufficiently inhibit the rapid proliferation of cancer cells. Therefore, it is administered to clinical patients in combination with other anticancer agents.
最近，對抗癌劑及腫瘤微環境之積極及充滿活力之研究已經導致開發免疫腫瘤劑，例如用於控制患者免疫力之免疫檢查點抑制劑，該免疫檢查點抑制劑現在用作治療劑。特別地，已知用PD-1/PD-L1治療對皮膚或肺癌患者具有高度響應性，並且改善生活品質。免疫腫瘤劑控制腫瘤微環境中免疫細胞之功能，以抑制癌細胞之增殖並增加免疫細胞之活性。然而，免疫腫瘤劑並非對所有患者都表現出相同之抗癌作用。此外，尚未明確地確立特定於免疫腫瘤劑之任何生物標誌物。存在幾個問題，例如，在JAK-STAT基因發生變化的情況下沒有藥物作用，由於所產生之抗體之特徵而導致自體免疫疾病之風險，治療之高成本及其類似問題。Recently, active and energetic research on anticancer agents and tumor microenvironment has led to the development of immuno-oncology agents, such as immune checkpoint inhibitors for controlling the immunity of patients, which are now used as therapeutic agents. In particular, it is known that treatment with PD-1/PD-L1 is highly responsive to skin or lung cancer patients and improves the quality of life. The immuno-oncology agent controls the function of immune cells in the tumor microenvironment to inhibit the proliferation of cancer cells and increase the activity of immune cells. However, immuno-oncology agents do not show the same anti-cancer effect in all patients. In addition, any biomarkers specific to immuno-oncology agents have not been clearly established. There are several problems. For example, there is no drug effect when the JAK-STAT gene changes, the risk of autoimmune diseases due to the characteristics of the antibodies produced, the high cost of treatment and similar problems.
癌症係由於暴露於毒素誘導之化學物質、由於壓力引起之DNA損傷、病毒或有害細菌感染、慢性炎症等而引起的。此誘導癌症之機制不限於特定之癌症，並且幾乎在所有癌症中都觀察到。因此，已知減輕由感染引起之炎症及減少異常高濃度之活性氧物質(reactive oxygen specy; ROS)之抗氧化活性不僅降低了對癌細胞之刺激，而且還減輕了癌細胞之增殖。Cancer is caused by exposure to toxin-induced chemicals, DNA damage due to stress, viral or harmful bacterial infection, chronic inflammation, etc. The mechanism for inducing cancer is not limited to a specific cancer, and is observed in almost all cancers. Therefore, it is known to reduce inflammation caused by infection and reduce the antioxidant activity of abnormally high concentrations of reactive oxygen specy (ROS) not only to reduce the stimulation of cancer cells, but also to reduce the proliferation of cancer cells.
免疫力及體內平衡之保持在醫療照護中係最重要的。當免疫力降低時，暴露於危險環境或感染容易引起疾病。始終用抗癌劑及抗生素對疾病進行治療削弱了體內免疫力並減緩了用於緩解或治療疾病之藥物作用，從而可能導致繼發性疾病。The maintenance of immunity and homeostasis is the most important thing in medical care. When immunity is reduced, exposure to dangerous environments or infections can easily cause disease. Always use anti-cancer agents and antibiotics to treat diseases, weaken the body's immunity and slow down the effects of drugs used to alleviate or treat diseases, which may lead to secondary diseases.
駐留在人體消化道中之乳酸菌藉由降解纖維狀材料及複合蛋白質來發揮提供有用營養物之作用。因此，藉由改善包括人在內之動物胃腸道中之腸道微生物環境而使宿主在健康方面受益之活微生物統稱為益生菌。The lactic acid bacteria residing in the human digestive tract degrade fibrous materials and complex proteins to provide useful nutrients. Therefore, living microorganisms that benefit the host's health by improving the intestinal microbial environment in the gastrointestinal tract of animals including humans are collectively referred to as probiotics.
自20世紀90年代Metchnikoff公佈關於乳酸菌延長壽命之研究結果以來，對乳酸菌之抗癌活性之研究已經開始。具體來說，Bogdanov等人公佈保加利亞乳桿菌顯著抑制作為腹水腫瘤細胞系之肉瘤180之生長，該細胞系被移植到試驗動物之腹水中。此外，Matsuzaki等人公佈當將乾酪乳桿菌給予作為與人類癌症轉移類似之癌症模型之3LL及Line-10肝細胞瘤時，向肺及淋巴結之轉移被顯著抑制。他們還揭示了當經由小鼠之靜脈內注射(100 mg/kg×4)，將乾酪乳桿菌連續給予經常發生轉移之B16-F10黑素瘤時，實現了優異之癌症治療效果。雖然許多研究人員試圖開發具有抗癌活性之菌株，但還沒有開發出作為藥物之具有顯著抗癌活性之菌株之任何案例。Since Metchnikoff published the results of research on the extension of life of lactic acid bacteria in the 1990s, research on the anti-cancer activity of lactic acid bacteria has begun. Specifically, Bogdanov et al. announced that Lactobacillus bulgaricus significantly inhibited the growth of sarcoma 180 as an ascites tumor cell line, which was transplanted into the ascites of experimental animals. In addition, Matsuzaki et al. announced that when Lactobacillus casei was administered to 3LL and Line-10 hepatocellular tumors, which are cancer models similar to human cancer metastasis, metastasis to the lungs and lymph nodes was significantly inhibited. They also revealed that when intravenous injection (100 mg/kg×4) in mice, L. casei was continuously administered to B16-F10 melanoma that frequently metastasized, an excellent cancer treatment effect was achieved. Although many researchers have tried to develop strains with anti-cancer activity, they have not yet developed any cases of strains with significant anti-cancer activity as drugs.
基於上述，在本發明人開發並研究了具有顯著抗癌活性之益生菌的同時，證實乳酸乳球菌GEN3013菌株不僅具有抑制癌細胞增殖、降低癌細胞移動性、抑制血管生成之作用，還有抗炎及抗氧化活性，此等活性都在本發明中達到。Based on the above, while the present inventors have developed and studied probiotics with significant anti-cancer activity, it has been confirmed that Lactococcus lactis GEN3013 strain not only has the effects of inhibiting cancer cell proliferation, reducing cancer cell mobility, and inhibiting angiogenesis, but also Inflammation and antioxidant activity, these activities are all achieved in the present invention.
技術問題technical problem
本發明係鑑定具有抗癌及抗炎作用之菌株，研究該菌株之種系發生特徵，並利用發現結果來開發新穎抗癌劑或用於炎性疾病或免疫疾病之治療劑。The present invention is to identify strains with anti-cancer and anti-inflammatory effects, study the phylogenetic characteristics of the strains, and use the findings to develop novel anti-cancer agents or therapeutic agents for inflammatory diseases or immune diseases.
因此，本發明之目的係提供一種具有優異之預防或治療癌症效果之新穎乳酸乳球菌菌株。Therefore, the object of the present invention is to provide a novel strain of Lactococcus lactis with excellent cancer prevention or treatment effects.
另外，本發明之目的在於提供一種用於預防或治療癌症之醫藥組合物，該醫藥組合物包含乳酸乳球菌菌株。In addition, the object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising a strain of Lactococcus lactis.
此外，本發明之目的係提供包含乳酸乳球菌菌株之用於預防或改善癌症之食品組合物或動物飼料組合物。問題之解決方案 In addition, the object of the present invention is to provide a food composition or an animal feed composition containing a strain of Lactococcus lactis for preventing or improving cancer. The solution to the problem
為了實現上述目的，本發明提供了乳酸乳球菌GEN3013菌株。該菌株於2017年12月13日保藏在韓國生物科學及生物技術研究所附屬之生物資源中心，保藏號為KCTC13426BP，命名為乳酸乳球菌GNC3013，於2018年4月3日更名為乳酸乳球菌GEN3013。In order to achieve the above objective, the present invention provides the Lactococcus lactis GEN3013 strain. The strain was deposited in the Biological Resource Center affiliated to the Korea Institute of Bioscience and Biotechnology on December 13, 2017, with the deposit number KCTC13426BP, named Lactococcus lactis GNC3013, and changed its name to Lactococcus lactis GEN3013 on April 3, 2018 .
此外，本發明提供用於預防或治療癌症之醫藥組合物，該醫藥組合物包含乳酸乳球菌GEN3013菌株。具體地，乳酸乳球菌GEN3013菌株可以指菌株本身或包括藉由裂解菌株獲得之細胞質級分。In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, the pharmaceutical composition comprising Lactococcus lactis GEN3013 strain. Specifically, the Lactococcus lactis GEN3013 strain may refer to the strain itself or include the cytoplasmic fraction obtained by lysing the strain.
或者，本發明提供預防或治療癌症之方法，該方法包括以治療有效量給予有需要之受試者乳酸乳球菌GEN3013菌株。如本文所用之術語「受試者」可以係哺乳動物，較佳人。Alternatively, the present invention provides a method for preventing or treating cancer, the method comprising administering a GEN3013 strain of Lactococcus lactis to a subject in need thereof in a therapeutically effective amount. The term "subject" as used herein may be a mammal, preferably a human.
特別地，本發明之乳酸乳球菌GEN3013菌株之特徵在於表現出抗癌、抗炎、抗氧化及增強免疫力之所有作用。In particular, the Lactococcus lactis GEN3013 strain of the present invention is characterized by exhibiting all the effects of anti-cancer, anti-inflammatory, anti-oxidation and immunity enhancement.
在本發明中，癌症可以係但不限於黑素瘤、鱗狀細胞癌、乳腺癌、頭頸癌、甲狀腺癌、軟組織肉瘤、骨肉瘤、睾丸癌、前列腺癌、卵巢癌、膀胱癌、皮膚癌、腦癌、血管肉瘤、肥大細胞瘤、白血病、淋巴瘤、肝癌、肺癌、胰腺癌、胃癌、腎癌、結腸癌、造血腫瘤或其轉移性癌症。較佳地，在本發明中，癌症可以係肺癌、結腸癌、胃癌、乳腺癌或肝癌。In the present invention, cancer can be, but not limited to, melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, skin cancer, Brain cancer, angiosarcoma, mast cell tumor, leukemia, lymphoma, liver cancer, lung cancer, pancreatic cancer, stomach cancer, kidney cancer, colon cancer, hematopoietic tumor or its metastatic cancer. Preferably, in the present invention, the cancer may be lung cancer, colon cancer, stomach cancer, breast cancer or liver cancer.
在本發明中，炎性疾病可以係但不限於骨關節炎、風濕性關節炎、痛風、強直性脊柱炎、腱炎、腱膜炎、風濕熱、狼瘡、纖維肌痛、牛皮癬性關節炎、哮喘、異位性皮膚炎、克羅恩病或潰瘍性結腸炎。In the present invention, the inflammatory disease can be, but not limited to, osteoarthritis, rheumatoid arthritis, gout, ankylosing spondylitis, tendinitis, aponeuritis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, Asthma, atopic dermatitis, Crohn’s disease, or ulcerative colitis.
本發明之乳酸乳球菌GEN3013菌株可以藉由抑制癌細胞之增殖及降低癌細胞之移動性而表現出抗癌作用。此外，本發明之乳酸乳球菌GEN3013菌株可藉由抑制血管內皮生長因子(vascular endothelial growth factor; VEGF)、血管生成素1(Ang1)及血管生成素2(Ang2)之表現而表現出抗癌作用。The Lactococcus lactis GEN3013 strain of the present invention can exhibit anti-cancer effects by inhibiting the proliferation of cancer cells and reducing the mobility of cancer cells. In addition, the Lactococcus lactis GEN3013 strain of the present invention can exhibit anti-cancer effects by inhibiting the expression of vascular endothelial growth factor (VEGF), Angiopoietin 1 (Ang1) and Angiopoietin 2 (Ang2) .
而且，本發明之乳酸乳球菌GEN3013菌株可藉由抑制TNF-α之表現而表現出抗炎症及抗癌作用。具體地，TNF-α係在慢性或急性炎症反應，例如人體感染、外傷、敗血症或風濕性關節炎期間由免疫細胞分泌之細胞因子。如果TNF-α之濃度增加，則會引起細胞內脂質及葡萄糖代謝之損害。雖然TNF-α被稱為誘導細胞壞死之細胞因子，但據報道，當TNF-α之持續刺激傳遞給細胞時，它會影響細胞內代謝以產生致癌基因。因此，細胞之異常增殖發生，最終促進癌症之發展。Moreover, the Lactococcus lactis GEN3013 strain of the present invention can exhibit anti-inflammatory and anti-cancer effects by inhibiting the expression of TNF-α. Specifically, TNF-α is a cytokine secreted by immune cells during chronic or acute inflammation, such as human infection, trauma, sepsis or rheumatoid arthritis. If the concentration of TNF-α increases, it will cause damage to intracellular lipid and glucose metabolism. Although TNF-α is known as a cytokine that induces cell necrosis, it has been reported that when the continuous stimulation of TNF-α is delivered to cells, it affects intracellular metabolism to produce oncogenes. Therefore, abnormal cell proliferation occurs, which ultimately promotes the development of cancer.
此外，本發明之乳酸乳球菌GEN3013菌株增加IFN-γ之產生並減少IL-10之產生，從而可能表現出抗癌或免疫增強作用。IFN-γ係當發生免疫刺激時，由NK細胞及T淋巴細胞分泌之蛋白質。已知與質膜受體之反應藉由JAK-STAT信號來活化免疫細胞以抑制癌細胞之增殖或殺死癌細胞。In addition, the Lactococcus lactis GEN3013 strain of the present invention increases the production of IFN-γ and reduces the production of IL-10, thereby possibly exhibiting anti-cancer or immune-enhancing effects. IFN-γ is a protein secreted by NK cells and T lymphocytes when immune stimulation occurs. It is known that the reaction with plasma membrane receptors activates immune cells by JAK-STAT signal to inhibit the proliferation of cancer cells or kill cancer cells.
IL-10係巨噬細胞、T細胞、NK細胞及樹突細胞中炎症反應期間產生之免疫因子之一。IL-10增加PD1之表現，從而誘導腫瘤PD-L1之表現。因此，進行免疫細胞與腫瘤之間之信號傳遞以促進降低抗癌劑效果之機制。IL-10不斷誘導調節性T細胞之活性，其中調節性T細胞抑制殺死腫瘤細胞之正常T細胞活性。因此，已知如果IL-10在腫瘤微環境中之表現較高，則惡性腫瘤患者之預後不好。IL-10 is one of the immune factors produced during inflammation in macrophages, T cells, NK cells and dendritic cells. IL-10 increases the expression of PD1, thereby inducing the expression of tumor PD-L1. Therefore, signal transmission between immune cells and tumors is carried out to promote the mechanism of reducing the effect of anticancer agents. IL-10 continuously induces the activity of regulatory T cells, which inhibit normal T cell activity that kills tumor cells. Therefore, it is known that if IL-10 has a higher performance in the tumor microenvironment, the prognosis of patients with malignant tumors is not good.
本發明係關於用於預防或治療癌症之醫藥組合物，其特徵在於包含乳酸乳球菌GEN3013菌株；及抗癌劑或免疫腫瘤劑。或者，本發明提供預防或治療癌症之方法，該方法包括給予需要其之受試者乳酸乳球菌GEN3013菌株；及抗癌劑或免疫腫瘤劑。The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer, which is characterized by comprising the Lactococcus lactis GEN3013 strain; and an anticancer agent or an immuno-tumor agent. Alternatively, the present invention provides a method for preventing or treating cancer, the method comprising administering to a subject in need thereof the GEN3013 strain of Lactococcus lactis; and an anticancer agent or an immunoneoplastic agent.
抗癌劑可以係但不限於奧沙利鉑、培美曲塞、順鉑、吉西他濱、卡鉑、氟尿嘧啶(5-FU)、環磷醯胺、紫杉醇、長春新鹼、依託泊苷、多柔比星及其類似藥劑。Anticancer agents can be, but not limited to, oxaliplatin, pemetrexed, cisplatin, gemcitabine, carboplatin, fluorouracil (5-FU), cyclophosphamide, paclitaxel, vincristine, etoposide, doxorubicin Bixing and its similar agents.
免疫腫瘤劑可以係但不限於抗PD1、抗PDL1、抗PDL2、抗A2AR、抗B7-H3、抗B7-H4、抗BTLA、抗KIR、抗CTLA、抗Tim3或抗LAG3。The immuno-oncology agent may be, but is not limited to, anti-PD1, anti-PDL1, anti-PDL2, anti-A2AR, anti-B7-H3, anti-B7-H4, anti-BTLA, anti-KIR, anti-CTLA, anti-Tim3, or anti-LAG3.
在本發明中，乳酸乳球菌GEN3013菌株；及抗癌劑或免疫腫瘤劑可以依次或同時給予有需要之患者。In the present invention, Lactococcus lactis GEN3013 strain; and anticancer agent or immuno-tumor agent can be administered to patients in need sequentially or simultaneously.
本發明係關於用於預防或改善癌症之食品組合物或動物飼料組合物，該食品組合物或動物飼料組合物包含乳酸乳球菌GEN3013菌株。The present invention relates to a food composition or animal feed composition for preventing or ameliorating cancer. The food composition or animal feed composition comprises the Lactococcus lactis GEN3013 strain.
食品組合物可以係但不限於保健功能食品、乳製品、發酵產品或食品添加劑。本發明之作用 The food composition can be, but is not limited to, health functional foods, dairy products, fermented products or food additives. Effect of the invention
本發明之新穎乳酸乳球菌GEN3013菌株具有抑制不同癌細胞系增殖之作用。此外，本發明之乳酸乳球菌GEN3013菌株發揮降低癌細胞之移動性及抑制血管生成之作用，該作用明顯優於已知之乳酸乳球菌菌株。The novel Lactococcus lactis GEN3013 strain of the present invention has the effect of inhibiting the proliferation of different cancer cell lines. In addition, the Lactococcus lactis GEN3013 strain of the present invention exerts the effects of reducing the mobility of cancer cells and inhibiting angiogenesis, which is significantly better than the known Lactococcus lactis strains.
此外，本發明之乳酸乳球菌GEN3013菌株可用於炎性疾病或免疫疾病，因為它具有抗炎、抗氧化或免疫增強作用。In addition, the Lactococcus lactis GEN3013 strain of the present invention can be used for inflammatory diseases or immune diseases because it has anti-inflammatory, anti-oxidant or immune-enhancing effects.
特別地，本發明之乳酸乳球菌GEN3013菌株不僅在單獨給藥時表現出優異之抗癌作用，而且與各自給藥相比，當與其它抗癌劑或免疫腫瘤劑聯合給藥時具有更顯著之抗癌作用。In particular, the Lactococcus lactis GEN3013 strain of the present invention not only exhibits an excellent anticancer effect when administered alone, but also has a more significant effect when administered in combination with other anticancer agents or immuno-neoplastic agents compared to the respective administrations. Its anti-cancer effect.
本發明人不斷研究，發現具有顯著抗癌作用之益生菌，並最終發現新穎乳酸乳球菌GEN3013菌株顯示出優異之抗癌作用，從而實現了本發明。The inventor of the present invention has continued to study and found probiotics with significant anti-cancer effects, and finally found that the novel Lactococcus lactis GEN3013 strain shows excellent anti-cancer effects, thus realizing the present invention.
令人驚訝地，GEN3013菌株發揮抑制來自例如肺癌、結腸癌、胃癌、乳腺癌及肝癌之各種細胞系之增殖之顯著作用。Surprisingly, the GEN3013 strain exerts a significant effect of inhibiting the proliferation of various cell lines such as lung cancer, colon cancer, gastric cancer, breast cancer and liver cancer.
此外，當用GEN3013菌株處理癌細胞系時，癌細胞之移動性顯著降低。與正常細胞不同，即使在細胞發生損傷之情況下，癌細胞也能夠隨著增殖而移動。癌細胞移動性降低意味著癌症轉移之可能性降低。因此，GEN3013菌株具有抑制癌症轉移之作用。In addition, when the cancer cell line was treated with the GEN3013 strain, the mobility of the cancer cells was significantly reduced. Unlike normal cells, even when cells are damaged, cancer cells can move along with proliferation. The reduced mobility of cancer cells means that the possibility of cancer metastasis is reduced. Therefore, the GEN3013 strain has the effect of inhibiting cancer metastasis.
此外，GEN3013菌株可以抑制作為涉及血管生成之主要因子之VEGF(血管內皮生長因子)、Ang1(血管生成素1)及Ang2(血管生成素2)中之所有者之表現。In addition, the GEN3013 strain can inhibit the performance of the owners of VEGF (vascular endothelial growth factor), Ang1 (angiogenin 1) and Ang2 (angiogenin 2), which are the main factors involved in angiogenesis.
血管生成係癌細胞之特徵之一。一旦血管生成受到抑制，就可以阻止經由血管向癌細胞輸送營養物質以抑制癌細胞之增殖。Angiogenesis is one of the characteristics of cancer cells. Once angiogenesis is inhibited, it can prevent the delivery of nutrients to cancer cells through blood vessels to inhibit the proliferation of cancer cells.
免疫係人類健康之基本因素。免疫力下降導致有害細菌之繁殖、疾病之發生及慢性病之發展。此外，當免疫力由於抗生素或疾病之治療劑之副作用被抑制時，可能發展為繼發疾病。因此，本發明之GEN3013菌株在抗癌治療下增強免疫力以增強患者之抗癌活性。Immunity is the basic factor of human health. The decline in immunity leads to the reproduction of harmful bacteria, the occurrence of diseases and the development of chronic diseases. In addition, when immunity is suppressed due to the side effects of antibiotics or therapeutic agents for diseases, secondary diseases may develop. Therefore, the GEN3013 strain of the present invention enhances immunity under anti-cancer treatment to enhance the anti-cancer activity of patients.
炎性疾病之一個顯著特徵係活性氧物質(ROS)增加。人們早就知道ROS會破壞細菌並殺死人體細胞。此外，中等濃度之ROS作用於細胞之信號轉導途徑，而高濃度之ROS長時間暴露導致蛋白質、脂質及核酸之非特異性損傷。ROS在正常生理過程中起重要作用，如蛋白質磷酸化、轉錄因子之氧化/還原調節及離子通道。作為另一個主要功能，ROS涉及生物合成程序，包括甲狀腺激素之產生及細胞外基質之交聯鍵。過量ROS導致由線粒體功能障礙引起之神經變性疾病，例如神經源性肌肉無力、運動失調、視網膜炎、MELAS症候群、MERRF症候群、Kearns-Sayre症候群、眼瞼痙攣、色素性視網膜病、心臟傳導紊亂、腦脊液增加或心血管疾病。此外，眾所周知，大多數癌細胞具有高活性之ROS，導致細胞之異常增殖。因此，本發明之GEN3013菌株減少ROS並抑制癌細胞之增殖，從而防止了不同疾病之發展。A significant feature of inflammatory diseases is the increase in reactive oxygen species (ROS). It has long been known that ROS can destroy bacteria and kill human cells. In addition, moderate concentrations of ROS act on the signal transduction pathway of cells, while prolonged exposure to high concentrations of ROS causes non-specific damage to proteins, lipids, and nucleic acids. ROS plays an important role in normal physiological processes, such as protein phosphorylation, oxidation/reduction regulation of transcription factors, and ion channels. As another main function, ROS is involved in biosynthetic processes, including the production of thyroid hormones and the cross-linking of extracellular matrix. Excessive ROS leads to neurodegenerative diseases caused by mitochondrial dysfunction, such as neurogenic muscle weakness, ataxia, retinitis, MELAS syndrome, MERRF syndrome, Kearns-Sayre syndrome, blepharospasm, retinopathy pigmentosa, cardiac conduction disorders, cerebrospinal fluid Increased or cardiovascular disease. In addition, it is well known that most cancer cells have highly active ROS, leading to abnormal cell proliferation. Therefore, the GEN3013 strain of the present invention reduces ROS and inhibits the proliferation of cancer cells, thereby preventing the development of different diseases.
此外，本發明之GEN3013菌株增加IFN-γ之產生，IFN-γ係具有抑制腫瘤細胞增殖活性之由細胞毒性T細胞分泌之免疫因子，並且減少IL-10之產生，IL-10係由T調節細胞分泌之免疫因子，該免疫因子抑制細胞毒性T細胞之活性，從而產生有效免疫腫瘤。In addition, the GEN3013 strain of the present invention increases the production of IFN-γ, which is an immune factor secreted by cytotoxic T cells that inhibits the proliferation of tumor cells, and reduces the production of IL-10, which is regulated by T The immune factor secreted by the cell, the immune factor inhibits the activity of cytotoxic T cells, thereby producing an effective immune tumor.
當用GEN3013菌株；及已知之合成抗癌劑或免疫腫瘤劑或其組合中之任一者處理不同癌細胞系時，用GEN3013處理之癌細胞系比用抗癌劑處理之癌細胞系具有更顯著之抑制細胞增殖之效果。用GEN3013及抗癌劑之組合處理之癌細胞系比用GEN3013或抗癌劑中之任一者處理之癌細胞系具有更優異之抑制細胞增殖之效果。因此，GEN3013與已知之抗癌劑組合顯示出增加之抗癌活性。When any one of the GEN3013 strain; and known synthetic anticancer agents or immuno-neoplastic agents or combinations thereof is used to treat different cancer cell lines, the cancer cell lines treated with GEN3013 have better results than the cancer cell lines treated with anticancer agents. Significantly inhibit cell proliferation. The cancer cell line treated with the combination of GEN3013 and the anticancer agent has a better effect of inhibiting cell proliferation than the cancer cell line treated with either GEN3013 or the anticancer agent. Therefore, GEN3013 in combination with known anticancer agents showed increased anticancer activity.
因此，本發明之乳酸乳球菌GEN3013菌株發揮抑制癌細胞增殖、降低癌細胞移動性及抑制血管生成之作用。因此，本發明之乳酸乳球菌GEN3013菌株可以用作顯著之抗癌劑，也可以與常規之合成抗癌劑或免疫腫瘤劑聯合給藥。Therefore, the Lactococcus lactis GEN3013 strain of the present invention exerts the effects of inhibiting the proliferation of cancer cells, reducing the mobility of cancer cells, and inhibiting angiogenesis. Therefore, the Lactococcus lactis GEN3013 strain of the present invention can be used as a significant anticancer agent, and can also be administered in combination with conventional synthetic anticancer agents or immuno-tumor agents.
此外，GEN3013菌株可顯著降低由作為炎症誘導劑之LPS衍生之TNF-α之表現，以預防或治療炎性疾病及癌症。In addition, the GEN3013 strain can significantly reduce the expression of TNF-α derived from LPS as an inflammation inducer to prevent or treat inflammatory diseases and cancer.
本發明提供了藉由向體內給予乳酸乳球菌GEN3013菌株來預防或治療癌症、炎性疾病、免疫疾病等之方法。The present invention provides a method for preventing or treating cancer, inflammatory diseases, immune diseases, etc. by administering the GEN3013 strain of Lactococcus lactis into the body.
本發明之組合物可以用作藥物、保健功能食品、乳製品、發酵產品、食品添加劑或動物飼料。The composition of the present invention can be used as medicine, health functional food, dairy products, fermented products, food additives or animal feed.
在下文中，可以藉由以下實例獲得對本發明之更好理解，該等實例用於說明，但不應解釋為對本發明之限制。僅提供以下實例以向熟習此項技術者詳細解釋。[ 實例 1] 乳酸乳球菌 GEN3013 菌株之分離 In the following, a better understanding of the present invention can be obtained through the following examples, which are used for illustration, but should not be construed as limiting the present invention. Only the following examples are provided to explain in detail to those familiar with this technology. [ Example 1] Isolation of Lactococcus lactis GEN3013 strain
為了分離人腸中之乳酸菌，獲得了20個月齡嬰兒之糞便。藉由利用乳桿菌屬及雙歧桿菌屬之選擇性培養基從嬰兒糞便中分離菌株。In order to isolate the lactic acid bacteria from the human intestine, the stool of a 20-month-old baby was obtained. Strains are isolated from infant feces by using selective media of Lactobacillus and Bifidobacterium.
將收集之糞便樣品在含有0.85%NaCl之磷酸鹽緩衝鹽水(PBS)溶液中稀釋至10-5 、10-6 倍稀釋，然後將其鋪展在De Man Rogosa，Sharpe瓊脂(MRS肉湯； Difco, USA)之培養基中，加入0.5%半胱胺酸，並在37℃下進行厭氧培養48小時，以獲得菌落。然後使用菌落PCR及測序技術鑑定每個菌株之16S rRNA之基因序列。將與乳酸乳球菌高度相似(100%)之菌株命名為GEN3013(SEQ ID No.1)。Dilute the collected stool sample in phosphate buffered saline (PBS) solution containing 0.85% NaCl to 10 -5 and 10 -6 times dilution, and then spread it on De Man Rogosa, Sharpe agar (MRS broth; Difco, In the medium of USA), 0.5% cysteine was added, and anaerobic culture was carried out at 37°C for 48 hours to obtain colonies. Then use colony PCR and sequencing technology to identify the 16S rRNA gene sequence of each strain. The strain highly similar (100%) to Lactococcus lactis was named GEN3013 (SEQ ID No. 1).
將所選擇之菌株在MRS肉湯及BL肉湯中傳代培養，然後在80℃之冰箱中儲存在含有20%甘油之MRS肉湯及BL肉湯中。[ 實例 2] 乳酸乳球菌 GEN3013 菌株之鑑定 The selected strains were subcultured in MRS broth and BL broth, and then stored in MRS broth and BL broth containing 20% glycerol in a refrigerator at 80°C. [ Example 2] Identification of Lactococcus lactis GEN3013 strain
當分析該菌株之rRNA序列時，將其鑑定為乳酸乳球菌(Lactococcus lactis)。該菌株於2017年12月13日保藏在韓國生物科學及生物技術研究所附屬之生物資源中心，保藏號為KCTC13426BP，命名為乳酸乳球菌GNC3013，於2018年4月3日更名為乳酸乳球菌GEN3013。 1. 評估糖發酵性質When the rRNA sequence of this strain was analyzed, it was identified as Lactococcus lactis. The strain was deposited in the Biological Resource Center affiliated to the Korea Institute of Bioscience and Biotechnology on December 13, 2017, with the deposit number KCTC13426BP, named Lactococcus lactis GNC3013, and changed its name to Lactococcus lactis GEN3013 on April 3, 2018 . 1. Assess the properties of sugar fermentation
藉由使用API CHL套組(BioMetrieux Co. France)評估GEN3013菌株之碳水化合物發酵性質。結果示於下文表1中。
[表1]
為了評估GEN3013菌株之生物化學性質，使用API ZYM套組(BioMetrieux Co. France)分析酶活性。結果示於下文表2中。
[表2]
為了評估GEN3013菌株抑制有害細菌生長之效果，進行了圓盤擴散試驗。指示菌株係金黃色葡萄球菌KCTC3881、銅綠假單胞菌KCTC 2004、大腸桿菌KCTC 1682及無害利斯特菌KCTC 3586，將它們培養、鋪展在瓊脂平板上，並置於滅菌盤上。In order to evaluate the effect of GEN3013 strain on inhibiting the growth of harmful bacteria, a disc diffusion test was carried out. The indicator strains are Staphylococcus aureus KCTC3881, Pseudomonas aeruginosa KCTC 2004, Escherichia coli KCTC 1682 and innocuous Listeria KCTC 3586. They are cultivated, spread on agar plates, and placed on sterile plates.
將作為試驗菌株之乳酸乳球菌GEN3013置於滅菌盤上，在37℃下培養24小時，然後測量抑制區之大小。結果示於下文表3中。
[表3]
將GEN3013菌株在含有0.5%葡萄糖及0.05%半胱胺酸之MRS肉湯中傳代培養。為了根據培養基之pH變化來評估菌株是否存活，我們製備了含有0.5%葡萄糖及0.05%半胱胺酸之MRS肉湯，將其調節至pH 2、2.5、3及3.5。接種洗滌過之細菌，使得濃度為2×106
CFU/ml，在37℃下以非生長狀態培養2小時，計數活細菌。如下藉由比較接種前之活細菌數與接種後2小時之活細菌數來計算存活率。結果示於下文表4中。
存活率(%) = [(CFU/ml 120min)/(CFU/ml 0min)] x 100
[表4]
如上表4所示，在pH 2.0時，作為對照之鼠李糖乳桿菌LGG之存活率為0.26%，而乳酸乳球菌GEN3013菌株之存活率為16.90%，表明GEN3013菌株在高酸性環境中具有顯著之存活力。As shown in Table 4 above, at pH 2.0, the survival rate of Lactobacillus rhamnosus LGG as a control was 0.26%, while the survival rate of Lactococcus lactis GEN3013 strain was 16.90%, indicating that the GEN3013 strain is significant in a highly acidic environment的viability.
因此，證實由於乳酸乳球菌GEN 3013菌株具有良好之耐酸性，因此在酸性條件下之存活係有利的。 3. 評估腸黏附Therefore, it was confirmed that because the Lactococcus lactis GEN 3013 strain has good acid resistance, it is advantageous to survive under acidic conditions. 3. Assess intestinal adhesion
為了評估GEN3013菌株之腸黏附性，對從韓國基因庫購買之人上皮結直腸腺癌細胞之Caco-2細胞進行細胞黏附試驗。對於細胞培養，藉由添加20%胎牛血清(FBS，Gibco)、100 U/mL青黴素及100 μg/mL鏈黴素(Gibco)來使用最低必需培養基(MEM； Gibco，Carlsbad，CA，USA)。In order to evaluate the intestinal adhesion of the GEN3013 strain, a cell adhesion test was performed on Caco-2 cells of human epithelial colorectal adenocarcinoma cells purchased from the Korean Gene Bank. For cell culture, use the minimum essential medium (MEM; Gibco, Carlsbad, CA, USA) by adding 20% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco) .
用於實驗之細胞在37℃，5%CO2 及90%濕度條件下培養，其中培養基每兩或三天更換一次。當細胞分化達到峰值時，用磷酸鹽緩衝鹽水(PBS)洗滌細胞，用胰蛋白酶-EDTA溶液分離黏附之細胞，並置於12孔板中，使其具有1×105 細胞/孔。The cells used for the experiment were cultured at 37°C, 5% CO 2 and 90% humidity, where the medium was changed every two or three days. When the cell differentiation reached the peak, the cells were washed with phosphate buffered saline (PBS), and the adherent cells were separated with trypsin-EDTA solution, and placed in a 12-well plate to have 1×10 5 cells/well.
將經傳代培養之分離菌株接種於培養20天之細胞中，使其量為1×107 CFU/ml，並在37℃，5%CO2 中培養2小時。用PBS洗滌兩次後，在含有0.05%半胱胺酸之MRS肉湯中計數活細菌。藉由比較初始數與最終數來計算黏附率，其中基於作為對照之鼠李糖乳桿菌LGG之值，資料以倍數單位來表示。The subcultured isolated strains were inoculated into cells cultured for 20 days to make the amount 1×10 7 CFU/ml, and cultured at 37°C and 5% CO 2 for 2 hours. After washing twice with PBS, live bacteria were counted in MRS broth containing 0.05% cysteine. The adhesion rate was calculated by comparing the initial number with the final number, and the data was expressed in multiple units based on the value of Lactobacillus rhamnosus LGG as a control.
計算結果如第1圖所示，從中證實GEN3013菌株具有與作為對照之鼠李糖乳桿菌LGG相似之腸黏附。因此，發現GEN3013菌株具有優異之腸黏附性，因此具有較高腸適應性。 4. 抗生素抗性試驗The calculation results are shown in Figure 1, which confirms that the GEN3013 strain has intestinal adhesion similar to that of the control Lactobacillus rhamnosus LGG. Therefore, it was found that the GEN3013 strain has excellent intestinal adhesion and therefore higher intestinal adaptability. 4. Antibiotic resistance test
為了評估GEN3013菌株之抗生素抗性，測量最小抑制濃度值。使藉由傳代培養具有最大活性之乳酸菌在培養基中鋪展，置於Etest(BioMetrieux Co. France)中培養24小時，並測量產生透明區之MIC值。結果示於表5中。
[表5]
如上表5所示，發現該菌株對氨苄青黴素、四環素、慶大黴素、卡那黴素及鏈黴素之抗生素具有敏感性。當參考EFSA(efsa雜誌2012;10(6):2740)中指出之截止值時，證實該菌株可安全用作乳酸菌。[ 實例 4] 乳酸乳球菌 GEN3013 菌株抑制腫瘤增殖之效果 As shown in Table 5 above, the strain was found to be sensitive to antibiotics such as ampicillin, tetracycline, gentamicin, kanamycin and streptomycin. When referring to the cut-off value indicated in EFSA (efsa magazine 2012;10(6):2740), it is confirmed that this strain can be safely used as a lactic acid bacteria. [ Example 4] Lactococcus lactis GEN3013 strain inhibiting tumor growth
為了評估乳酸乳球菌GEN3013是否在不同癌細胞系中顯示出抗癌活性，進行了MTT測定。In order to evaluate whether Lactococcus lactis GEN3013 shows anti-cancer activity in different cancer cell lines, MTT assay was performed.
肺癌(A549、H1975、HCC827、H1299、SW900)，結腸癌(HCT116、LoVo、SNU-C2A)，胃癌(SNU216、AGS、MKN-28、MKN-1、SNU-601、SNU-1)，乳腺癌(Hs578T、BT20、MDA-MB-231)及肝癌(HepG2、Hep3B)之細胞系用作人類癌細胞系用於檢測，而結腸癌(CT26，MC38)、肺癌(LLC1)及乳腺癌(4T1)之細胞系用作小鼠癌細胞系。Lung cancer (A549, H1975, HCC827, H1299, SW900), colon cancer (HCT116, LoVo, SNU-C2A), stomach cancer (SNU216, AGS, MKN-28, MKN-1, SNU-601, SNU-1), breast cancer (Hs578T, BT20, MDA-MB-231) and liver cancer (HepG2, Hep3B) cell lines are used as human cancer cell lines for detection, while colon cancer (CT26, MC38), lung cancer (LLC1) and breast cancer (4T1) The cell line is used as a mouse cancer cell line.
將癌細胞系以1～5×103
個細胞/孔之量置於96孔板中。24小時後，加入0.5%乳酸菌樣品並培養72小時，然後處理試劑MTT(溴化3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑噻唑藍)在每個孔中反應2小時，使活細胞與線粒體反應，將黃色MTT變成紫色。然後全部移除含有MTT之培養基，在每個孔中加入100 μl DMSO，藉由使用Multiplate Reader、以540 nm處之吸光度來測量紫色之濃度。源自人之癌細胞系及源自小鼠之癌細胞系之測試結果顯示在下表6及7中。
[表6]
如上表6及7所示，GEN3013菌株在抑制人及小鼠來源之癌細胞系上之細胞增殖方面發揮了顯著作用。As shown in Tables 6 and 7, the GEN3013 strain has played a significant role in inhibiting cell proliferation on human and mouse-derived cancer cell lines.
此外，將GEN3013抑制人源癌細胞系生長之效果作為IC50值來評估，如下表8所示。
[表8]
韓國專利申請第10-2001-0012973號揭示了乳酸屬乳酪對於SNU-1之IC50 值為54 μg/ml，對於SNU-C2A之IC50 值為75 μg/ml，而乳酸乳球菌(Lactococcus lactis)屬乳酸對於SNU-1之值為11 μg/ml，對於SNU-C2A之值為23 μg/ml。Korean Patent Application No. 10-2001-0012973 discloses that the IC 50 value of lactic acid cheese for SNU-1 is 54 μg/ml, and the IC 50 value for SNU-C2A is 75 μg/ml. However, Lactococcus lactis ) The value of lactic acid is 11 μg/ml for SNU-1 and 23 μg/ml for SNU-C2A.
如上表8所示，GEN3013對於SNU-1之IC50
值為6.43 μg/ml，對於SNU-C2A之IC50
值為8.82 μg/ml，該等值遠低於先前研究中已知之其他菌株之IC50
值。因此，證實GEN3013顯示出抑制不同癌細胞生長之顯著效果。[ 實例 5] 乳酸乳球菌 GEN3013 菌株降低癌細胞移動性之作用 As shown in Table 8 above, the IC 50 value of GEN3013 for SNU-1 is 6.43 μg/ml, and the IC 50 value for SNU-C2A is 8.82 μg/ml, which is much lower than the
與正常細胞不同，癌細胞即使在細胞發生損傷時也會隨著增殖而移動。為了評估GEN3013菌株是否降低癌細胞之移動性，進行了傷口癒合測定。將A549(5×105 個細胞)及HCT116(6×105 個細胞)菌株固定在6孔板上。當90%至95%之細胞增殖時，我們在一定間隔內以1000p尖端損傷細胞。用PBS或GEN3013菌株處理細胞24小時，藉由顯微鏡觀察細胞之移動性。結果如第2圖所示。Unlike normal cells, cancer cells move with proliferation even when the cells are damaged. To evaluate whether the GEN3013 strain reduces the mobility of cancer cells, a wound healing assay was performed. The A549 (5×10 5 cells) and HCT116 (6×10 5 cells) strains were fixed on a 6-well plate. When 90% to 95% of the cells proliferate, we damage the cells with a 1000p tip within a certain interval. The cells were treated with PBS or GEN3013 strain for 24 hours, and the mobility of the cells was observed under a microscope. The result is shown in Figure 2.
如第2圖所示，基於所損傷之A549及HCT116細胞在0 h之細胞條件，在24小時，A549及HCT116分別具有65.31±1.69%及33.82±5.86%之癌細胞活性移動性。當用GEN3013處理時，A549及HCT116中細胞之移動性分別顯著降低至17.60±5.19%及13.50±8.55%。As shown in Figure 2, based on the cell conditions of the injured A549 and HCT116 cells at 0 h, at 24 hours, A549 and HCT116 have 65.31±1.69% and 33.82±5.86% of cancer cell activity mobility, respectively. When treated with GEN3013, the mobility of cells in A549 and HCT116 were significantly reduced to 17.60±5.19% and 13.50±8.55%, respectively.
因此，證實GEN3013菌株抑制癌症轉移，因此表現出優異之抗癌活性。[ 實例 6] 乳酸乳球菌 GEN3013 菌株抑制血管生成之作用 Therefore, it was confirmed that the GEN3013 strain inhibited cancer metastasis and therefore exhibited excellent anti-cancer activity. [ Example 6] Lactococcus lactis GEN3013 strain inhibits angiogenesis
為了評估GEN3013菌株是否抑制血管生成(血管生成係癌細胞之主要特徵之一)，進行了血管生成相關因子之表現試驗。In order to evaluate whether the GEN3013 strain inhibits angiogenesis (one of the main characteristics of angiogenic cancer cells), the performance test of angiogenesis-related factors was performed.
用GEN3013處理HCT116細胞系24小時，從中獲得RNA以合成cDNA。同樣，在正常PCR中分析血管內皮生長因子(vascular endothelial growth factor; VEGF)之表現，同時在即時PCR中分析Ang1及Ang2之表現。結果如第3及4圖所示。The HCT116 cell line was treated with GEN3013 for 24 hours, from which RNA was obtained to synthesize cDNA. Similarly, analyze the performance of vascular endothelial growth factor (VEGF) in normal PCR, and analyze the performance of Ang1 and Ang2 in real-time PCR. The results are shown in Figures 3 and 4.
如第3圖所示，與對照相比，用GEN3013處理之組具有顯著降低之VEGF之121同種型及165同種型之表現。此外，189同種型之表現在對照中係不顯著的，而在用GEN3013處理之組中未發現189同種型之表現。As shown in Figure 3, compared with the control, the group treated with GEN3013 had significantly reduced expression of the 121 isotype and the 165 isotype of VEGF. In addition, the performance of the 189 isotype was not significant in the control, and no performance of the 189 isotype was found in the group treated with GEN3013.
此外，如第4圖所示，在Ang1及Ang2(該等物質係除VEGF以外之涉及血管生成之因子)之表現方面，在用GEN3013處理之組中，很少觀察到Ang1之表現，同時未觀察到Ang2之表現。In addition, as shown in Figure 4, in terms of the performance of Ang1 and Ang2 (these substances are factors involved in angiogenesis other than VEGF), in the group treated with GEN3013, the performance of Ang1 was rarely observed, and there was no The performance of Ang2 was observed.
因此，證實GEN3013抑制血管生成並因此阻止營養物藉由血管遞送到癌細胞中，導致抑制癌細胞增殖之顯著效果。[ 實例 7] 乳酸乳球菌 GEN3013 菌株之抗炎作用 Therefore, it was confirmed that GEN3013 inhibits angiogenesis and thus prevents the delivery of nutrients to cancer cells through blood vessels, resulting in a significant effect of inhibiting the proliferation of cancer cells. [ Example 7] Anti-inflammatory effect of Lactococcus lactis GEN3013 strain
為了評估GEN3013菌株之抗炎作用，將作為小鼠巨噬細胞之RAW264.7細胞用GEN3013菌株處理24小時，然後用炎症誘導劑LPS 100 ng/ml處理6小時以獲得RNA。In order to evaluate the anti-inflammatory effect of the GEN3013 strain, RAW264.7 cells, which are mouse macrophages, were treated with the GEN3013 strain for 24 hours, and then treated with the
從1 μg RNA合成cDNA，並用於在即時PCR中評估TNF-α之表現，TNF-α已知為炎症因子。結果如第5圖所示。CDNA was synthesized from 1 μg RNA and used to evaluate the performance of TNF-α in real-time PCR. TNF-α is known as an inflammatory factor. The result is shown in Figure 5.
如第5圖所示，當藉由LPS之TNF-α表現增加之程度定義為1相對指數時，用GEN3013處理之組具有不超過0.5之TNF-α表現增加程度。由於TNF-α也已知為癌症誘導劑，TNF-α表現顯著降低之結果意味著GEN3013具有抗炎及抗癌活性。[ 實例 8] 乳酸乳球菌 GEN3013 菌株之 T 細胞活性，以及控制 IFN- γ 及 IL-10 產生之效果 As shown in Figure 5, when the degree of increase in TNF-α expression by LPS is defined as a relative index of 1, the group treated with GEN3013 has an increase in TNF-α expression of no more than 0.5. Since TNF-α is also known as a cancer inducer, the result of a significant decrease in TNF-α performance means that GEN3013 has anti-inflammatory and anti-cancer activities. [ Example 8] T cell activity of Lactococcus lactis GEN3013 strain , and the effect of controlling the production of IFN- γ and IL-10
PBMC藉由使用Ficoll從人血液中分離，用RBC裂解緩衝液移除紅血球，並在LS柱及MACS緩衝液中分離單核細胞。PBMC is separated from human blood by using Ficoll, red blood cells are removed with RBC lysis buffer, and monocytes are separated in LS column and MACS buffer.
將GEN3013置於96孔板中，每孔加入5×103 個分離之單核細胞，稍微混合，以便與菌株發生反應，並培養2小時。藉由反應2小時，GEN3013將單核細胞分化成巨噬細胞。Place GEN3013 in a 96-well plate, add 5×10 3 separated monocytes to each well, mix slightly to react with the strain, and incubate for 2 hours. After 2 hours of reaction, GEN3013 differentiated monocytes into macrophages.
在使單核細胞與GEN3013反應之同時，將表現CD4及CD8之T細胞與MACS緩衝液及LS柱中剩餘之PBMC細胞分離。將分離之T細胞在100 μl RPMI培養基中稀釋，使得其量為5×104 個細胞，置於含有單核細胞及GEN3013之孔中，並培養48小時以活化免疫。While making monocytes react with GEN3013, the T cells expressing CD4 and CD8 are separated from the MACS buffer and the remaining PBMC cells in the LS column. The separated T cells were diluted in 100 μl RPMI medium to make the amount of 5×10 4 cells, placed in the wells containing monocytes and GEN3013, and cultured for 48 hours to activate immunity.
一段時間後，將每孔中之細胞培養物收集在1.5 ml管中，單獨分離上清液，並在ELISA套組中測量IFN-γ及IL-10之產生。結果顯示在第6及7圖中。After a period of time, the cell culture in each well was collected in a 1.5 ml tube, the supernatant was separated separately, and the production of IFN-γ and IL-10 was measured in the ELISA kit. The results are shown in Figures 6 and 7.
如第6圖所示，與用大腸桿菌處理之對照相比，用GEN3013處理之組增加了IFN-γ之產生超過兩倍，IFN-γ係具有腫瘤細胞增殖抑制活性之由細胞毒性T細胞分泌之免疫因子。As shown in Figure 6, compared with the control treated with Escherichia coli, the group treated with GEN3013 increased the production of IFN-γ more than twice, which is secreted by cytotoxic T cells with tumor cell proliferation inhibitory activity. The immune factor.
此外，如第7圖所示，與用大腸桿菌處理之對照相比，用GEN3013處理之組較少產生IL-10，IL-10係由T調節細胞分泌之抑制細胞毒性T細胞活性之免疫因子。In addition, as shown in Figure 7, compared with the control treated with E. coli, the group treated with GEN3013 produced less IL-10, which is an immune factor secreted by T regulatory cells that inhibits the activity of cytotoxic T cells .
因此，證實GEN3013控制涉及抗癌及免疫活性之T細胞，以及T細胞分泌之免疫因子之產生。[ 實例 9] 乳酸乳球菌 GEN3013 菌株之抗氧化活性 Therefore, it was confirmed that GEN3013 controls the production of T cells involved in anti-cancer and immune activity, and the production of immune factors secreted by T cells. [ Example 9] Antioxidant activity of Lactococcus lactis GEN3013 strain
為了評估GEN3013之抗氧化活性，用GEN3013處理A549癌細胞系24小時，然後用0.5 uM H2O2處理5小時，並用FACs設備中之DCFDA螢光染料測量細胞中ROS之量。結果如下表9及第8圖所示。
[表9]
如上表9所示，單獨用PBS處理之組具有4.66%之ROS，而僅用H2O2處理之組具有64.8%之ROS增加量。用0.1%或0.5% GEN3013處理之組分別具有54.6%及35.6%之ROS。因此，發現GEN3013表現出降低ROS量之作用，並且GEN3013濃度之增加導致ROS之抑制增加。As shown in Table 9 above, the group treated with PBS alone had ROS of 4.66%, while the group treated with H2O2 only had a 64.8% increase in ROS. The groups treated with 0.1% or 0.5% GEN3013 had 54.6% and 35.6% ROS, respectively. Therefore, it was found that GEN3013 exhibited the effect of reducing the amount of ROS, and an increase in the concentration of GEN3013 resulted in an increase in the inhibition of ROS.
因此，證實GEN3013菌株發揮抑制細胞中ROS引起之細胞損傷及減少癌細胞增殖之作用。[ 實例 10] 乳酸乳球菌 GEN3013 菌株與抗癌劑聯合用於抑制癌細胞增殖之作用 ( 活體內試驗 ) Therefore, it was confirmed that the GEN3013 strain can inhibit cell damage caused by ROS in cells and reduce the proliferation of cancer cells. [ Example 10] Combination of Lactococcus lactis GEN3013 strain and anticancer agent to inhibit the proliferation of cancer cells ( in vivo test )
將乳酸乳球菌GEN3013菌株在含有0.5%葡萄糖及0.05%半胱胺酸之MRS液體培養基(Difco)中於37℃培養24小時，使待用於試驗之細菌裂解。The Lactococcus lactis GEN3013 strain was cultured in MRS liquid medium (Difco) containing 0.5% glucose and 0.05% cysteine at 37°C for 24 hours to lyse the bacteria to be used in the test.
在試驗中使用A549(人肺癌細胞)、LLC1(小鼠肺癌細胞)、HCT116、LoVo(人結腸癌細胞)及MC38(小鼠結腸癌細胞)。人癌細胞系在RPMI培養基中培養，而小鼠癌細胞系在DMEM培養基中培養。所有癌細胞系之培養基含有10%熱滅活之胎牛血清，100 U/ml青黴素及100 μg/ml鏈黴素(Gibco Life Technologies，Grand Island，NY，U.S.A.)。使用細胞系進行測試，同時在37℃及5%CO2 下培養。A549 (human lung cancer cells), LLC1 (mouse lung cancer cells), HCT116, LoVo (human colon cancer cells) and MC38 (mouse colon cancer cells) were used in the experiment. Human cancer cell lines are cultured in RPMI medium, while mouse cancer cell lines are cultured in DMEM medium. The culture medium of all cancer cell lines contains 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco Life Technologies, Grand Island, NY, USA). The cell line was used for testing, and cultured at 37°C and 5% CO 2 at the same time.
奧沙利鉑或培美曲塞用作抗癌劑。為了評估藉由抗癌劑及乳酸菌之組合處理來抑制癌細胞之增殖，基於以下方法進行實驗。將不同之癌細胞系稀釋並以1至2×103 之量置於6孔板之每個孔中，固定24小時，然後用乳酸菌及/或抗癌劑處理每個孔，每兩到三天更換一次培養基。將用乳酸菌及/或抗癌劑處理144小時之板用4%福爾馬林處理30分鐘以固定細胞，用PBS洗滌兩次，用結晶紫溶液染色5分鐘，並再次用蒸餾水洗滌，觀察細胞增殖情況。結果如第9圖所示。Oxaliplatin or pemetrexed are used as anticancer agents. In order to evaluate the inhibition of the proliferation of cancer cells by the combined treatment of anticancer agents and lactic acid bacteria, experiments were conducted based on the following methods. Dilute different cancer cell lines and place 1 to 2×10 3 in each well of a 6-well plate, fix for 24 hours, and then treat each well with lactic acid bacteria and/or anticancer agents, every two to three Change the medium once a day. The plate treated with lactic acid bacteria and/or anticancer agent for 144 hours was treated with 4% formalin for 30 minutes to fix the cells, washed twice with PBS, stained with crystal violet solution for 5 minutes, and washed again with distilled water to observe the cells Proliferation. The result is shown in Figure 9.
如第9圖所示，與用抗癌劑處理之癌細胞系相比，用GEN3013處理之癌細胞系表現出對癌細胞增殖之優異抑制。特別地，僅用GEN3013處理之MC38小鼠結腸癌細胞及LLC1小鼠肺癌細胞顯示出由GEN3013引起之對於癌細胞增殖之幾乎完全抑制。As shown in Figure 9, cancer cell lines treated with GEN3013 exhibited superior inhibition of cancer cell proliferation compared to cancer cell lines treated with anticancer agents. In particular, MC38 mouse colon cancer cells and LLC1 mouse lung cancer cells treated with GEN3013 only showed almost complete inhibition of cancer cell proliferation caused by GEN3013.
此外，用GEN3013菌株與抗癌劑組合處理之癌細胞系比用GEN3013或抗癌劑中之任一者處理之癌細胞系發揮更顯著之抑制細胞增殖之作用。[ 實例 11] 乳酸乳球菌 GEN3013 菌株與抗癌劑聯合用於抑制腫瘤增殖之作用 ( 活體內試驗 ) In addition, the cancer cell line treated with the combination of the GEN3013 strain and the anticancer agent exerts a more significant effect of inhibiting cell proliferation than the cancer cell line treated with either GEN3013 or the anticancer agent. [ Example 11] Combination of Lactococcus lactis GEN3013 strain and anticancer agent to inhibit tumor proliferation ( in vivo test )
在製備腫瘤模型之前，將乳酸菌樣品給予小鼠2週以增加腸黏附及免疫力。然後將2×105 個MC38癌細胞皮下注射到每組8只C57BL/6小鼠之右髖周圍以製備腫瘤模型。在注射腫瘤細胞之同時，將乳酸菌樣品口服給予動物模型三週(從週一到週六)。將口服給藥之乳酸菌樣品以每只小鼠CFU 1*109 之量在200 μl PBS中稀釋。在誘導癌症後之每週一及週四腹膜內注射奧沙利鉑(3 mg/kg，Sellekchem)或抗PD1(2 mg/kg，BioXCell)作為抗癌劑。在用乳酸菌；及奧沙利鉑或抗PD1，及組合中之任一者來處理之組中觀察抑制腫瘤之作用。結果如第10及11圖所示。Before preparing the tumor model, lactic acid bacteria samples were given to mice for 2 weeks to increase intestinal adhesion and immunity. Then, 2×10 5 MC38 cancer cells were subcutaneously injected around the right hip of each group of 8 C57BL/6 mice to prepare tumor models. Simultaneously with the injection of tumor cells, samples of lactic acid bacteria were orally administered to the animal model for three weeks (from Monday to Saturday). The orally administered lactic acid bacteria samples were diluted in 200 μl PBS with an amount of 1*109 CFU per mouse. Oxaliplatin (3 mg/kg, Sellekchem) or anti-PD1 (2 mg/kg, BioXCell) was injected intraperitoneally every Monday and Thursday after cancer induction as an anticancer agent. The anti-tumor effect was observed in the group treated with lactic acid bacteria; and oxaliplatin or anti-PD1, and any one of the combination. The results are shown in Figures 10 and 11.
如第10及11圖所示，與單獨給予GEN3013或抗癌劑相比，聯合給藥顯示出更顯著之抑制腫瘤之效果。[ 實例 12] 乳酸乳球菌 GEN3013 菌株與抗癌劑聯合用於抑制腫瘤增殖之作用 ( 活體內試驗 ) As shown in Figures 10 and 11, compared with GEN3013 or an anticancer agent administered alone, the combined administration showed a more significant tumor suppressing effect. [ Example 12] Combination of Lactococcus lactis GEN3013 strain and anticancer agent to inhibit tumor proliferation ( in vivo test )
基於實例11之結果，進行動物試驗以分析藉由GEN3013使免疫細胞侵入腫瘤。完成測試後，將腫瘤及脾臟從小鼠體內分離、裂解，並分離免疫細胞。分離之免疫細胞與螢光抗體反應，螢光抗體根據功能對應於免疫細胞之標記，然後藉由FACs分析。結果如第12至15圖所示。Based on the results of Example 11, animal experiments were performed to analyze the invasion of immune cells into tumors by GEN3013. After the test is completed, the tumor and spleen are separated and lysed from the mouse, and immune cells are separated. The separated immune cells react with fluorescent antibodies. The fluorescent antibodies correspond to the markers of immune cells according to their functions, and then analyzed by FACs. The results are shown in Figures 12 to 15.
如第12至15圖所示，與對照相比，用GEN3013處理之組具有增加之CD4 T細胞、CD8 T細胞、CD8效應T細胞及NK細胞之分佈，該等細胞對於抗癌免疫應答起重要作用。控制T細胞活性之調節性T細胞之數量顯著減少。 As shown in Figures 12 to 15, compared with the control, the group treated with GEN3013 has an increased distribution of CD4 T cells, CD8 T cells, CD8 effector T cells and NK cells, which are important for the anti-cancer immune response effect. The number of regulatory T cells that control T cell activity is significantly reduced.
在用GEN3013及抗癌劑之組合治療之組中，增加之免疫應答更顯著。 In the group treated with the combination of GEN3013 and anticancer agents, the increased immune response was more significant.
從結果證實，由GEN3013引起之對於免疫細胞之活性之控制涉及抑制腫瘤增殖。 It was confirmed from the results that the control of immune cell activity caused by GEN3013 involves the inhibition of tumor proliferation.
˙保藏機構：韓國生物科學及生物技術研究所 ˙Preservation institution: Korea Institute of Bioscience and Biotechnology
˙保藏號：KCTC13426BP ˙Preservation number: KCTC13426BP
˙保藏日期：2017年12月13日 ˙Preservation date: December 13, 2017
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寄存機構：食品工業發展研究所生物資源保存及研究中心 Depository institution: Biological Resources Conservation and Research Center of Food Industry Development Institute
寄存日期：2019/06/13 Deposit Date: 2019/06/13
寄存號碼：BCRC910903 Deposit number: BCRC910903
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寄存國家：韓國 Host country: South Korea
寄存機構：韓國菌種中心(KCTC) Depository organization: Korea Culture Center (KCTC)
寄存日期：2017/12/13 Deposit Date: 2017/12/13
寄存號碼：KCTC13426BP Deposit number: KCTC13426BP
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第1圖係顯示鼠李糖乳桿菌LGG及GEN3013菌株對Caco-2細胞之細胞黏附之結果。Figure 1 shows the results of cell adhesion of Lactobacillus rhamnosus LGG and GEN3013 strains to Caco-2 cells.
第2圖係顯示癌細胞系未處理或用GEN3013菌株處理時癌細胞之移動性之結果。Figure 2 shows the results of cancer cell mobility when the cancer cell line is untreated or treated with the GEN3013 strain.
第3圖顯示了藉由PCR確認之未經處理或用GEN3013菌株處理之HCT116細胞系中VEGF之表現。Figure 3 shows the expression of VEGF in the HCT116 cell line without treatment or treated with GEN3013 strain confirmed by PCR.
第4圖顯示了藉由即時PCR確認之未經處理或用GEN3013菌株處理之HCT116細胞系中Ang1及Ang2之表現。Figure 4 shows the performance of Ang1 and Ang2 in the HCT116 cell line untreated or treated with GEN3013 strain confirmed by real-time PCR.
第5圖顯示未經處理或用GEN3013菌株處理之RAW264.7細胞中，藉由LPS之TNF-α表現之增加。Figure 5 shows the increase in TNF-α expression by LPS in RAW264.7 cells untreated or treated with GEN3013 strain.
第6圖係顯示藉由ELISA套組證實之GEN3013菌株增加IFN-γ產生之作用之結果。Figure 6 shows the results of the effect of GEN3013 strain on increasing IFN-γ production confirmed by the ELISA kit.
第7圖係顯示藉由ELISA套組證實之GEN3013菌株抑制IL-10產生之作用之結果。Figure 7 shows the results of the inhibition of IL-10 production by the GEN3013 strain confirmed by the ELISA kit.
第8圖係顯示GEN3013菌株抑制活性氧物質之作用之結果。Figure 8 shows the results of the inhibition of reactive oxygen species by strain GEN3013.
第9圖係顯示當用GEN3013菌株或抗癌劑(奧沙利鉑或培美曲塞)或其組合中之任一者處理癌細胞系時細胞增殖之結果。Figure 9 shows the results of cell proliferation when any one of the GEN3013 strain or an anticancer agent (oxaliplatin or pemetrexed) or a combination thereof is used to treat a cancer cell line.
第10圖係顯示當GEN3013菌株或抗癌劑(奧沙利鉑)分別或組合給予腫瘤誘導之C57BL/6小鼠時抑制腫瘤之作用之圖。Figure 10 is a graph showing the tumor-inhibiting effect of GEN3013 strain or anticancer agent (oxaliplatin) separately or in combination to tumor-induced C57BL/6 mice.
第11圖係顯示當GEN3013菌株或用於治療癌症之抗體(抗PD1)分別或組合給予腫瘤誘導之C57BL/6小鼠時抑制腫瘤之作用之圖。Figure 11 is a graph showing the tumor-inhibiting effect of GEN3013 strain or an antibody for cancer treatment (anti-PD1) administered separately or in combination to tumor-induced C57BL/6 mice.
第12圖係當GEN3013菌株或抗癌劑(奧沙利鉑)分別或組合給予腫瘤誘導之C57BL/6小鼠時，從小鼠脾臟中分離之免疫細胞中獲得之結果。Figure 12 shows the results obtained from immune cells isolated from mouse spleen when GEN3013 strain or anticancer agent (oxaliplatin) was administered separately or in combination to tumor-induced C57BL/6 mice.
第13圖係當GEN3013菌株或抗癌劑(奧沙利鉑)分別或組合給予腫瘤誘導之C57BL/6小鼠時，從小鼠腫瘤中分離之免疫細胞中獲得之結果。Figure 13 shows the results obtained from immune cells isolated from mouse tumors when GEN3013 strain or anticancer agent (oxaliplatin) was administered separately or in combination to tumor-induced C57BL/6 mice.
第14圖係當將GEN3013菌株或用於治療癌症之抗體(抗-PD1)分別或組合給予腫瘤誘導之C57BL/6小鼠時，從小鼠脾臟中分離之免疫細胞中獲得之結果。Figure 14 shows the results obtained from immune cells isolated from mouse spleen when GEN3013 strain or an antibody for cancer treatment (anti-PD1) was administered separately or in combination to tumor-induced C57BL/6 mice.
第15圖係當將GEN3013菌株或用於治療癌症之抗體(抗-PD1)分別或組合給予腫瘤誘導之C57BL/6小鼠時，從小鼠腫瘤中分離之免疫細胞中獲得之結果。Figure 15 shows the results obtained from immune cells isolated from mouse tumors when GEN3013 strain or an antibody for cancer treatment (anti-PD1) was administered to tumor-induced C57BL/6 mice separately or in combination.
<110> 南韓商韓國億諾生物有限公司(Genome and Company) <110> Genome and Company, South Korea
<120> 乳酸乳球菌GEN3013菌株及包含其之用於預防或治療癌症之組合物 <120> Lactococcus lactis GEN3013 strain and composition containing it for preventing or treating cancer
<160> 1 <160> 1
<170> KoPatentIn 3.0 <170> KoPatentIn 3.0
<210> 1 <210> 1
<211> 1496 <211> 1496
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 乳酸乳球菌(Lactococcus lactis)GEN3013 <223> Lactococcus lactis GEN3013
<400> 1
Claims (14)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020180042912A KR101980525B1 (en) | 2018-04-12 | 2018-04-12 | Lactococcus lactis GEN3013 and composition for preventing or treating cancers comprising the same |
| KR10-2018-0042912 | 2018-04-12 |
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| Publication Number | Publication Date |
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| TW201946637A TW201946637A (en) | 2019-12-16 |
| TWI726303B true TWI726303B (en) | 2021-05-01 |
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| Application Number | Title | Priority Date | Filing Date |
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| TW108112856A TWI726303B (en) | 2018-04-12 | 2019-04-12 | Lactococcus lactis gen3013 strain and a composition for preventing or treating cancer comprising the same |
Country Status (3)
| Country | Link |
|---|---|
| KR (1) | KR101980525B1 (en) |
| TW (1) | TWI726303B (en) |
| WO (1) | WO2019199048A1 (en) |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101864347B1 (en) * | 2017-09-06 | 2018-06-05 | 주식회사 지놈앤컴퍼니 | Lactobacillus rhamnosus LM1019 and composition for preventing and treating obesity or diabetes mellitus comprising the same |
| KR102298533B1 (en) | 2020-10-30 | 2021-09-07 | 주식회사 아미코스메틱 | Antianging cosmetic composition comprising green tea fermented extract fermented by inoculating lactococcus lactis strain |
| KR102391017B1 (en) * | 2021-07-13 | 2022-04-29 | 한국식품연구원 | Composition for preventing, improving or treating cancer comprising the Lactococcus lactis WiKim0133 |
| CN113462738A (en) * | 2021-08-24 | 2021-10-01 | 蓝科医美科学技术(吉林)有限公司 | Fermentation extraction process of ginseng extract |
| CN114381411B (en) * | 2022-03-23 | 2022-05-27 | 山东中科嘉亿生物工程有限公司 | Lactococcus lactis JYLL-60 and application thereof in preparation of product for improving immunity |
| CN114686402B (en) * | 2022-04-25 | 2023-06-20 | 善恩康生物科技（苏州）有限公司 | Lactococcus lactis subspecies lactis HFY14 and application thereof |
| CN116121110A (en) * | 2022-10-28 | 2023-05-16 | 益加生物科技成都有限公司 | Lactococcus lactis YJ0801 with strong antibacterial and anti-inflammatory effects and application thereof |
| KR102645456B1 (en) * | 2023-04-24 | 2024-03-13 | 주식회사 메디오젠 | Lactococcus lactis MG5474 strain having immune enhancing and intestinal health promoting activity and composition composing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| KR100411198B1 (en) * | 2001-03-13 | 2003-12-18 | 매일유업주식회사 | Formulation for inhibiting proliferation of tumor cell line |
| KR100654383B1 (en) * | 2006-02-08 | 2006-12-06 | 재단법인서울대학교산학협력재단 | Food composition comprising ADI fraction separated from Lactococcus lactis for inhibiting proliferation of tumor cell |
| KR100979448B1 (en) * | 2008-03-06 | 2010-09-02 | 건국대학교 산학협력단 | Functional beverage for health adding whey protein hydrolysate and probiotic bacteria and process for preparing the same |
| US8338162B2 (en) * | 2009-04-17 | 2012-12-25 | Anaeropharma Science, Inc. | Obligately anaerobic mutant lactic acid bacterium and preparation method therefor, and expression vector functioning in obligately anaerobic lactic acid bacterium |
| WO2016196605A1 (en) * | 2015-06-01 | 2016-12-08 | The University Of Chicago | Treatment of cancer by manipulation of commensal microflora |
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2018
- 2018-04-12 KR KR1020180042912A patent/KR101980525B1/en active IP Right Grant
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- 2019-04-11 WO PCT/KR2019/004262 patent/WO2019199048A1/en active Application Filing
- 2019-04-12 TW TW108112856A patent/TWI726303B/en active
Non-Patent Citations (1)
| Title |
|---|
| Kyoung Jun Han, "Anticancer and Anti-Inflammatory Activity of Probiotic Lactococcus lactis NK34", Journal of Microbiology and Biotechnology, 2015, pages 1697-1701. * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2019199048A1 (en) | 2019-10-17 |
| KR101980525B1 (en) | 2019-05-21 |
| TW201946637A (en) | 2019-12-16 |
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