發(fā)布時間：2019-06-01 22:48

【摘要】： 隨著人們生活水平的提高,高膽固醇含量膳食的攝入也逐漸增多,過高的膽固醇攝入量容易引發(fā)高膽固醇血癥。通常,血液中膽固醇濃度每上升1 %,心腦血管疾病的死亡率就會上升2 %。因此,保持人體內(nèi)膽固醇含量在正常范圍內(nèi)對于預防心腦血管疾病是至關(guān)重要的。國外研究表明許多乳酸菌在體內(nèi)和體外都有降低膽固醇的功效。本文通過對傳統(tǒng)發(fā)酵食品中降膽固醇乳酸菌的篩選,鑒定,生物學活性,安全性,粘附和定殖及其降膽固醇的作用的研究,得出如下結(jié)論: 1.本研究以高膽固醇源培養(yǎng)基MRSO-CHOL為模型,從傳統(tǒng)發(fā)酵食品中分離純化得到66株具有降低膽固醇能力的乳酸菌菌株,從中篩選出5株具有較好降低膽固醇能力的菌株;在模擬消化環(huán)境下比較5株菌株的耐酸性、膽汁耐受能力和膽鹽降解(�；悄懰徕c)能力,最終確定GUO菌株為篩選目標菌株。GUO菌株經(jīng)傳統(tǒng)微生物鑒定方法和API 50CH細菌鑒定系統(tǒng)鑒定,確定GUO菌株為植物乳桿菌(Lactobacilluss plantarum)。 2. Lactobacillus plantarum GUO的生物學特性結(jié)果顯示:該菌株的最高,最低和最適生長溫度分別為43℃,12℃和30℃;最高,最低和最適起始生長pH分別為9.4,3.6和5.7。Lactobacillus plantarum GUO采用MRS培養(yǎng),2 h進入對數(shù)生長期,11 h進入穩(wěn)定期,生長比較旺盛;而采用SKM培養(yǎng)則生長比較緩慢,產(chǎn)酸能力也很弱。進一步研究發(fā)現(xiàn),Lactobacillus plantarum GUO的MRS培養(yǎng)液4℃保藏條件下,S值為15.2 d,而在脫脂奶中的S值為116.3 d。 3.益生菌的粘附和定殖是發(fā)揮其益生作用的第一步,也是最重要的一步。Lactobacillus plantarum GUO對Caco-2細胞的粘附結(jié)果表明:該菌株對Caco-2細胞的粘附在一定濃度和時間范圍內(nèi)存在量效關(guān)系,當加入細菌濃度達到108 cfu/mL、孵育2 h時,粘附趨于飽和;穩(wěn)定期的Lactobacillus plantarum GUO對Caco-2細胞的粘附效果最好。pH對于粘附作用具有顯著影響,在酸性范圍粘附性最強;Ca~(2+)、Mg~(2+)并沒有參與到Lactobacillus plantarum GUO對Caco-2細胞的粘附。因此,Lactobacillus plantarum GUO對Caco-2細胞的粘附是多種因素共同作用的結(jié)果,其中粘附素與受體的結(jié)合占主導地位。 4.采用PCR-DGGE研究了Lactobacillus plantarum GUO在大鼠腸道內(nèi)的定殖特性:結(jié)果表明:大鼠持續(xù)攝入Lactobacillus plantarum GUO發(fā)酵乳制品10 d后,Lactobacillus plantarum GUO在大鼠腸道的結(jié)腸、回腸、盲腸部位定殖,但在十二指腸和直腸部位未定殖。Lactobacillus plantarum GUO在腸道定殖結(jié)腸部位定殖時間最長,停止灌胃后11 d仍有少量定殖;在結(jié)腸部位定殖量最高,灌胃停止3 d后檢測回腸定殖量達108 cfu/g;回腸其次,最高約為107 cfu/g;盲腸僅第3組以及回腸第2組有定殖,約105 cfu/g。 5.進一步對Lactobacillus plantarum GUO的體外降膽固醇作用研究發(fā)現(xiàn),Lactobacillus plantarum GUO主要是通過同化與共沉淀兩種方式共同降低培養(yǎng)基中的膽固醇,其中同化作用占63.5 %;共沉淀占36.5 %。在此基礎(chǔ)上,對Lactobacillusplantarum GUO可能存在的降膽固醇相關(guān)酶進行了研究。采用硫酸銨沉淀、Sephacryl S-100層析、SDS-PAGE電泳等分離純化得到了降膽固醇相關(guān)的酶,該酶的分子量為38,000 Da,最適底物濃度為150μg/mL,最適ATP濃度為2.5 mg/mL,最適pH為7.0,最適溫度為55℃;在60℃以下,pH 6.0～9.0,酶活在30 min內(nèi)基本保持穩(wěn)定。 6.本研究采用耐藥性試驗、Ames試驗,急性毒性(經(jīng)口LD50)試驗,90 d喂養(yǎng)試驗和致畸試驗評價了Lactobacillus plantarum GUO的安全性。試驗結(jié)果表明,在實驗條件下,Lactobacillus plantarum GUO對氟哌酸(NOR)和慶大霉素(GEN)兩種抗生素不敏感;對頭孢拉定(CEF)、四環(huán)素(TET)中度敏感;對氯霉素(CMP)和紅霉素(ERY)高度敏感。Lactobacillus plantarum GUO的Ames試驗結(jié)果為陰性,無致突變作用;無急性毒性,最大耐受劑量MTD均大于20000 mg/kg。90 d喂養(yǎng)試驗結(jié)果表明:在實驗條件下,試驗動物生長期間體征指數(shù)正常;臟體比,血液學指標無顯著差異;各劑量組動物臟器未發(fā)現(xiàn)明顯組織學病變。致畸試驗結(jié)果表明:在實驗條件下,各劑量組試驗動物未見有母體毒性,胚胎毒性和致畸性。這表明Lactobacillus plantarum GUO是安全的,可以作為益生菌進行深度的開發(fā)利用。 7.由于Lactobacillus plantarum GUO具有良好的功能特性和生物學活性,為了更好地發(fā)揮其益生作用,將其應(yīng)用于產(chǎn)品中具有更重要的現(xiàn)實意義。在單因素實驗基礎(chǔ)上設(shè)計并優(yōu)化了Lactobacillus plantarum GUO的發(fā)酵乳配方Lactobacillus plantarum GUO發(fā)酵乳的基本配方為:15 %SKM,5 %葡萄糖,3 %發(fā)酵劑。Lactobacillus plantarum GUO發(fā)酵乳飲料的基本配方為:發(fā)酵基料30 %,蔗糖13 %,果膠0.3 %,檸檬酸鈉0.1 %。 8.為了了解Lactobacillus plantarum GUO在產(chǎn)品中的降膽固醇功效,參照保健食品功能性評價方法和程序,將Lactobacillus plantarum GUO發(fā)酵乳灌喂高血脂模型大鼠。結(jié)果表明:與對照組相比,含有活菌的Lactobacillus plantarum GUO發(fā)酵乳能夠顯著降低大鼠體內(nèi)的TC和LDL-C;而滅活的Lactobacillus plantarum GUO發(fā)酵乳則沒有此功能。這表明Lactobacillus plantarum GUO在大鼠體內(nèi)也具有良好的降低膽固醇功效。
[Abstract]:With the improvement of people's living standard, the intake of high-cholesterol diet is also increasing, and the high cholesterol intake can easily lead to hypercholesteremia. In general, the cholesterol concentration in the blood is increased by 1%, and the mortality of cardiovascular and cerebrovascular diseases will increase by 2%. Therefore, the content of cholesterol in the human body is essential to the prevention of cardiovascular and cerebrovascular diseases within the normal range. The foreign research shows that many lactic acid bacteria have the effect of reducing cholesterol in vivo and in vitro. This paper studies the screening, identification, biological activity, safety, adhesion and colonization of cholesterol-lowering lactic acid bacteria in traditional fermented food and the effect of reducing cholesterol. 1. The study was conducted with a high cholesterol source medium, MSRO-CHOL, In the model,66 strains of lactic acid bacteria with reduced cholesterol ability were isolated and purified from the traditional fermented food, and 5 strains with better cholesterol-lowering ability were selected from the strain; and 5 strains were compared under the simulated digestion environment. The ability of acid resistance, bile tolerance and bile salt degradation (sodium taurocholate) was determined, and the GUO strain was determined to be a screen. Standard strain. The GUO strain was identified by the traditional microbial identification method and the API 50CH bacterial identification system, and the GUO strain was determined to be Lactobacillus plantarum. (um).2. The biological characteristics of Lactobacillus planarum GUO showed that the highest, minimum and optimum growth temperature of the strain were 43 鈩，

本文編號：2490639