微生態(tài)制劑的研制與應(yīng)用課件

1、微生態(tài)制劑的研制與應(yīng)用 1微生態(tài)制劑的定義和類型微生態(tài)制劑是在微生態(tài)學(xué)理論的指導(dǎo)下，調(diào)整生態(tài)失調(diào)（microdysbiosis）保持微生態(tài)平衡（microeubiosis），提高宿主（人、動植物）健康水平或增進(jìn)健康佳態(tài)（wellbeing）的生理性活菌制品（微生物）及其代謝產(chǎn)物以及促進(jìn)這些生理菌群生長繁殖的物質(zhì)制品。 目前國際上已將其分成3個類型:益生菌（Probiotics）益生元（Prebiotics）合生素（Synbiotics）益生菌，又稱益生素，是指投入后通過改善宿主腸道菌群生態(tài)平衡而發(fā)揮有益作用，達(dá)到提高宿主（人和動物）健康水平和健康佳態(tài)的活菌制劑及其代謝產(chǎn)物。其基本指導(dǎo)思想是用人

2、或動物正常生理菌群（normal microbiota）的成員，經(jīng)過選種和人工繁殖，通過各種途徑和劑型制成活菌制劑，然后再以投入方式使其回到原來環(huán)境，發(fā)揮自然的生理作用。目前應(yīng)用于人體的益生菌有雙歧桿菌、乳桿菌、腸球菌、大腸桿菌、枯草桿菌、蠟樣芽腸桿菌、地衣芽胞桿菌、丁酸梭菌和酵母菌等。 Probiotics are defined viable microorganisms, sufficient amounts of which reach the intestine in an active state and thus exert positive health effectsProb

3、iotic foods contain living probiotic microorganisms in an adequate matrix and in sufficient concentration, so that after their ingestion, the postulated effect is obtained, and is beyond that of usual nutrient suppliers.The term “probiotics” was created in the 1950s used in 1965 for live bacteria an

4、d spores as animal feed supplements that should help limiting the use of antibiotics in animal husbandry. The first generally accepted definition was given in 1989: a probiotic is “a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balan

5、ce”.Pharmaceutical products with live bacteria have also been on the market for a long time, although not labeled as “probiotic”, and for years without a sufficient proof of efficiency.Health Claims for ProbioticsComposition and effects of probiotic foods and their detection methods need to be clear

6、ly defined.1. A primary prerequisite is that such foods be healthy and safe, and free of pathogenic and toxic effects.2. Postulated health effects have to be proven by clinical studies in humans. In vitro studies and animal experimental analyses only give indications to possible health relevant effe

7、cts. They may be useful for identifying mechanisms of action, or for the search for new probiotics.3. Clinical studies should follow clearly defined study goals and a randomized, double-blind and placebo controlled design. Their results should be confirmed by independent research teams, and document

8、ed in peerreviewed scientific journals and be documented according to the rules of “good clinical practice” (GCP).6. The effectiveness of a probiotic and therefore the lowest concentration of probiotic microorganisms in the product from which a health effect may still be expected, depends on the kin

9、d of probiotic microorganism, the claimed effect, the duration of application, the food matrix, and, last but not least, the target group. Often 108109 probiotic bacteria per day are mentioned as the minimum amount for probiotic effects. A probiotic product should guarantee the ingestion of that num

10、ber of probiotic microorganisms at the end of its shelf life, which was used in the studies substantiating its health effect7. Probiotic effects are target specific. The effect of probiotic microorganisms on study participants may vary with age, health and gender, diet, residence and environment, e.

11、g. rural or urban etc. There are differences with respect to maturity or efficiency of the immune system to the predominant microflora and/or to hygiene standards. This has the consequence that results from studies in children/aged subjects, in diseased people or from the Third World cannot be trans

12、ferred without further examination to adults, healthy people or people from industrialized countries, respectively. On the other hand this meansparticularly in the case of small experimental groups and/or a small number of studiesthat inconsistent results do not necessarily cast doubt on the investi

13、gated probiotic effect, but more likely on its transferability from the participants of a successful study to the general population. “more studies are necessary to find out which section of the population may profit from a probiotic and under which conditions”.Selection criteria. Safe for humans, i

14、.e. free of pathogenic and toxic effects. Origin from the intestinal tract of healthy persons, as such microorganisms are regarded safe for humans and best adapted to the ecosystem of the gut. Tolerance to gastric and bile acid as well as sufficient resistance against digestive enzymes enable the su

15、rvival during the passage through stomach and upper intestinal tract, and have health-promoting effects in the gut. As the decrease in pH of the ingested food in the stomach is low due to the buffer capacity of the gastric acid, resistance against gastric acid is less critical than tolerance of the

16、bacteria to bile acid and digestive enzymes in the small bowel. Detection of parameters enabling a (positive) influence on the intestinal flora like adhesion to intestinal epithelial cells, survival and reproducing capacity in the human large intestine, or production of antimicrobial substances.The

17、effects of probiotics may be classified in three modes of action (i) Probiotics might be able to modulate the hosts defences including the innate as well as the acquired immune system.This mode of action is most likely important for the prevention and therapy of infectious diseases but also for the

18、treatment of (chronic) inflammation of the digestive tract or parts thereof.In addition, this probiotic action could be important for the eradication of neoplastichostcells. (ii) Probiotics can also have a direct effect on other microorganisms,commensaland/or pathogenic ones.This principle is in man

19、y cases of importance for the prevention and therapy of infections and restorationof the microbial equilibrium in the gut. (iii) Finally,probiotic effects may be based on actions affecting microbial products like toxins, host products e.g.bile salts and food ingredients. Such actions may result in i

20、nactivation of toxins and detoxification of host and food components in the gut. Immune modulationProbiotics can influence the immune system by products like metabolites, cell wall components and DNA.Obviously,immune modulatory effects might be even achieved with dead probiotic bacteria or just prob

21、iotics-derived components like peptidoglycan fragments orDNA.Probiotic products are recognized by host cells The main target cells in that context are therefore gut epithelial and gut-associated immune cells. The interaction of probiotics with host (epithelial) cells by adhesion itself might already

22、 trigger a signalling cascade leading to immunemodulation. Release of soluble factors can trigger signalling cascades in immune cells or in epithelial cells which subsequently affect immune cells. However, probiotic and commensal bacterial adherence to gut epithelial cells in vivo has not been demon

23、strated. Rather, such bacteria adhere just to and invade the outer mucus layer but do not reach the epithelial cells themselvesThe uptake and transcytosis of bacteria is done by M- cells, and pass them down to dendritic cells (DCs) in the subepithelial dome region. Direct contact between probiotics

24、and host cells in the gut occurs by internalization of bacteria by DCs which are localized below the epithelial cells (non M-cells) However,probiotics are also able to protect the integrity of the mucosal gut barrier against the destructive action of enteropatho- genic Escherichia coli in a TLR-inde

25、pendent way.A direct anti-nflammatory effect of EcN on human gut epithelial cells (HCT15) could only be demonstrated for live bacteria but without direct contact. Rather a secreted factor mediated this effect by suppressing the TNFa-induced IL-8 transactivation by a mechanism in dependent of NF-kB i

26、nhibition Some probiotics are able to alter cytokine produc- tion by modulating cellular signal transduction.Theycaneither block degradation of the inhibitor IkB by inhibiting the ubiquitination of this inhibitor, by interfering with proteasome function or influencing RelA localisation via the recep

27、tor g-dependent signal cascade which in turn is activated through a peroxisome proliferatorProbiotic FoodFermented Milk Products with Probiotic Properties-Yogurt-like, solid or liquid milk products containing living probiotic bacteria are the most popular probiotic foodstuffsProbiotic CheeseOther Pr

28、obiotic Food and Food Ingredients All these products have in common that their production has been described in the scientific or patent literature, but that they have not been tested in clinical trials and that they did not stay on the market for long. Ice Cream, Sweets, Vegetable Food, Meat Produc

29、ts, Dried Probiotic Products, Microencapsulated Probiotics A prebiotic was first defined in 1995 as “a non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host h

30、ealth.”“A prebiotic is a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microflora that confers benefits upon host well being and health.”益生元（Prebiotics）是指能夠選擇性地促進(jìn)宿主腸道內(nèi)原有的一種或幾種有益細(xì)菌（益生菌）生長繁殖的物質(zhì)，通過有益菌的繁殖增多，抑制有害細(xì)菌生長，從而達(dá)到調(diào)整腸

31、道菌群，促進(jìn)機(jī)體健康的目的。這類物質(zhì)最早發(fā)現(xiàn)的是雙歧因子（bifidus factor）。如各種寡糖類物質(zhì)（oligosaccharides）或稱低聚糖。常見的有乳果糖（lactulose）、蔗糖低聚糖（oligosucrose）棉子低聚糖（oligofaffinose）、異麥芽低聚糖（oligomaltose）、玉米低聚糖（cornoligossacharides）和大豆低聚糖（soybean oligosaccha-rides）等。 這些糖類既不被人體消化系統(tǒng)消化和吸收，亦不被腸道菌群分解和利用，只能為腸道有益菌群如雙歧桿菌和乳桿菌利用，促進(jìn)有益菌的生長繁殖，抑制有害菌的生長，從而達(dá)到調(diào)整

32、腸道正常菌群的目的。其它尚有一些有機(jī)酸及其鹽類，如葡萄糖酸和葡萄糖酸鈣以及我國的某些中草藥類，如人參、黨參、黃芪等或茶葉提取物亦能起到益生元的作用。 合生素（Synbiotics）是指益生菌和益生元同時并存的制劑。此類制品是以益生菌和益生元同時并用，服用后到達(dá)腸腔可使進(jìn)入的益生菌在益生元的作用下，再行繁殖增多，使之更有利于發(fā)揮抗病、保健的有益作用。 2.微生態(tài)制劑的國內(nèi)外研究和生產(chǎn)概況日本是世界上研制開發(fā)和利用微生態(tài)制劑較早的國家之一，其產(chǎn)品主要是雙歧桿菌活菌制劑。據(jù)報道至今在日本生產(chǎn)這類制劑年產(chǎn)值達(dá)200億日元以上的企業(yè)已有10余家。品種分3大類：雙歧桿菌食品包括雙歧酸奶，雙歧桿菌乳制品、雙

33、歧桿菌面包及餅干類雙歧桿菌保健食品（含雙歧因子）/以雙歧桿菌促生因子為中心的特定保健食品（包括強(qiáng)化寡糖類食品及雙歧桿菌、乳桿菌培養(yǎng)物的提取物等）雙歧桿菌藥品包括單菌制劑和聯(lián)菌制劑，其劑型有粉劑、顆料劑、錠劑、膠囊劑和微膠囊劑等多種。 國際上對開發(fā)新微生態(tài)制品的主要方向從單純的“益生菌”或“益生元”轉(zhuǎn)向結(jié)構(gòu)合理、效果更加優(yōu)越的“合生素”這一方面。即“益生菌”和“益生原”同時并存或并用的制劑。據(jù)日本報道：實(shí)驗(yàn)研究已經(jīng)證明，在雙歧桿菌活菌制劑中加入雙歧因子（例如各種類型低聚糖）后，其效果比不加的制劑提高10100倍。 正在開發(fā)能使活菌制劑有更好穩(wěn)定性的新劑型，例如腸溶膠囊和微膠囊劑型。它們不僅能保持

34、活菌在產(chǎn)品中延長存活時間，而且人體服用時更能通過胃酸這個屏障。保證有更多益生菌進(jìn)入腸道而使其發(fā)揮有益的作用。 增加活菌制劑中的活菌數(shù)量（為108109/g）。 利用分子生物學(xué)和遺傳工程技術(shù)、改造生理性細(xì)菌的遺傳基因，將外源性有益基因轉(zhuǎn)入生理性細(xì)菌中，構(gòu)建成優(yōu)良的工程菌株，從而研制出更多更有效的新型微生態(tài)制劑，造福于人類。 3微生態(tài)制劑的主要作用機(jī)理微生態(tài)制劑與其它藥物不同，從理論上講，它優(yōu)于抗生素，克服了應(yīng)用抗生素所造成的菌群失調(diào)、耐藥菌株的增加以及藥物的毒副反應(yīng)。實(shí)踐證明，微生態(tài)制劑的優(yōu)越性即健康人群使用它來增進(jìn)健康素質(zhì)，提高健康水平，達(dá)到防病治病目的，其作用機(jī)理有下列幾個方面：3.1生態(tài)平

35、衡理論 微生態(tài)學(xué)認(rèn)為，人體、動植物體表及體內(nèi)寄居著大量的正常微生物群。宿主、正常微生物群和外環(huán)境構(gòu)成一個微生態(tài)系統(tǒng)。在正常條件下，這個系統(tǒng)處于動態(tài)平衡狀態(tài)。這一方面對宿主有利，能輔助宿主進(jìn)行某些生理過程；另一方面對寄居的微生物有利，使之保持一定的微生物群落組合，維持其生長與繁殖。 在微生態(tài)系統(tǒng)內(nèi)微群落水平中，少數(shù)優(yōu)勢群對整個群落起著決定作用，而在微種群內(nèi)部中優(yōu)勢個體對整個群落起著控制作用。一旦因種種原因而失去優(yōu)勢種群，則微群落就會解體。若失去優(yōu)勢個體，則優(yōu)勢種群更替，并改變了微生態(tài)平衡。 一旦因種種原因而失去優(yōu)勢種群，則微群落就會解體。若失去優(yōu)勢個體，則優(yōu)勢種群更替，并改變了微生態(tài)平衡。例如，

36、由于抗生素、放射治療、手術(shù)和過敏性疾患等因素引起正常菌群變化，微生態(tài)平衡遭到破壞，即生態(tài)和菌群遭受失調(diào)，引起一系列臨床癥狀。如雙重感染和免疫力降低等。利用宿主體內(nèi)的正常微生物優(yōu)勢菌群成員的益生菌，制成的微生態(tài)制劑，可以調(diào)節(jié)失調(diào)的菌群，使宿主體內(nèi)恢復(fù)正常的微生態(tài)平衡，達(dá)到防病治病的目的。3.2生物屏障理論生物屏障理論又稱生物拮抗理論，腸道內(nèi)正常菌群直接參與機(jī)體生物防御的屏障結(jié)構(gòu)，包括化學(xué)屏障和生物屏障?；瘜W(xué)屏障是指腸內(nèi)主要菌群的代謝產(chǎn)物例如乙酸、乳酸、丙酸、過氧化氫及細(xì)菌素等活性物質(zhì)，可阻止或殺滅病原微生物在體內(nèi)的定植。生物屏障是指定植于粘膜或皮膚上皮細(xì)胞之間的正常菌群所形成的生物膜樣結(jié)構(gòu)，通過

37、定植保護(hù)作用影響過路菌或外來致病的定植、占位、生長和繁殖。微生態(tài)制劑中的益生菌就是這類正常菌群中的成員，可參與生物屏障結(jié)構(gòu)，發(fā)揮生物拮抗作用。3.3生物奪氧理論根據(jù)正常微生物群的自然定植規(guī)律，人或動物出生時是無菌的，出生后不久就被一系列微生物細(xì)菌定植了。定植的順序先是需氧菌，后是兼性厭氧菌，隨后的是厭氧菌。厭氧菌之所以不能先定植，是因?yàn)樽匀簧硟?nèi)有過多的氧。在需氧或兼性厭氧菌生長一段時期后，由于氧被大量消耗，從而提供了厭氧菌生長條件，厭氧菌才能生長。厭氧菌雖然不能先定植，但是整個微生態(tài)系統(tǒng)中其數(shù)量上占據(jù)首位，并保持著一定的生態(tài)平衡。利用無毒、無害、非致病性微生物（如蠟桿芽胞桿菌等）暫時在腸道內(nèi)

38、定植，使局部環(huán)境中氧分子濃度降低，氧化還原電位下降，造成適合正常腸道優(yōu)勢菌生長的微環(huán)境，促進(jìn)厭氧菌大量繁殖生長，最終達(dá)到微生態(tài)平衡。 3.4三流循環(huán)學(xué)說三流循環(huán)其主要內(nèi)容是能量流、物質(zhì)流及基因流的循環(huán)。能量流即能源運(yùn)轉(zhuǎn)，正常微生物群的內(nèi)部與其宿主保持著能源交換和運(yùn)轉(zhuǎn)的關(guān)系。正常微生物與正常微生物之間所存在著能源的交換關(guān)系。近年已從電子顯微鏡的觀察中發(fā)現(xiàn)，人和動物腸上皮細(xì)胞的微絨毛（microvilli）與正常細(xì)菌細(xì)胞壁上的菌毛（pili）極為貼近，并發(fā)現(xiàn)有物質(zhì)交換的現(xiàn)象發(fā)生。物質(zhì)流即物質(zhì)交換正常生理菌群的能源與物質(zhì)均依賴于宿主，不存在宏觀生態(tài)學(xué)中的生產(chǎn)者、消費(fèi)者和分解者的區(qū)別。但都存在著降解（

39、catabolison）與合成（anabolism)的代謝。降解與合成是微生物代謝中的必然途徑，這與宿主細(xì)胞的功能是一致的。正常生理微生物菌群與宿主細(xì)胞通過降解和合成代謝進(jìn)行物質(zhì)交換。裂解的細(xì)胞與細(xì)胞外酶可為微生物利用，而微生物產(chǎn)生的酶、維生素、刺激素以及微生物降解的細(xì)胞成分也可為宿主細(xì)胞利用，如此反復(fù)進(jìn)行著物質(zhì)交換。 基因流即基因交換在正常微生物之間有著廣泛的基因（即DNA）交換，例如耐藥因子（R因子）、產(chǎn)毒因子等都可在正常微生物之間通過物質(zhì)的傳遞進(jìn)行交換。微生態(tài)制劑可以作為非特異性免疫調(diào)節(jié)因子，促進(jìn)機(jī)體吞噬細(xì)胞的吞噬能力和促進(jìn)B淋巴細(xì)胞產(chǎn)生抗體的能力。這不僅可以抑制腐敗菌和致病菌的生長，還可降解腸道的有毒物質(zhì)（如氨、酚、內(nèi)毒素等），保證微生態(tài)系統(tǒng)中的能量流、物質(zhì)流和基因流的正常運(yùn)轉(zhuǎn)。 4微生態(tài)制劑的應(yīng)用 4.1微生態(tài)制劑的作