201212825 六、發明說明： 【發明所屬之技術領域】 本發明係指利用冬蟲夏草(C〇rdyCepS sinensis)固態發酵發酵物添加於 家禽飼料中以促進家禽性成熟之新應用。一種冬蟲夏草固態發酵物促 進提尚體重生長、雞冠色澤、睪固酮生成與StAR基因表現量分別為 7%、I25%、I33%、(對照組與蟲草線普j”呵_2 85mg知比較) 及曲精細管發育之飼料添加劑，添加劑為包括發酵物所提供腺苷 (Adenosine)為〇.95mg-2.85mg/kg本發明係有關於用以提高家禽生精作 用和睪固酮生成的飼料添加劑，主要含冬蟲夏草固態發酵物飼料添加 〇 劑中可應用之粉劑及水溶性散劑及預混劑 【先前技術】 已知的冬蟲夏草麵有四百多種，其中最具代表性的有兩種，一種是 中華蟲草咖⑼治），另一種是蛹蟲草、cordyceps ，《本草綱目》記載冬蟲夏草有保肺益腎、止血化瘦、治癆嗽 功效；《本草綱目拾遺》也指出它能益肺腎、補精髓、止血化痰；《中 藥大辭典》則說明，冬蟲夏草味甘酸、性平、氣香、入肺腎二經，可 強壯、益肺腎、補虛損、益精氣、解毒、止血化痰。 Q 蟲草萃取物具有降血糖的功效，L〇ea/. (2004)指出冬蟲夏草子實體萃 取物及人工培養之冬蟲夏草菌絲萃取物，可以降低鏈脲菌素 (streptozotocin, STZ)誘發之糖尿病大鼠高血糖症，且人工培養之功效 與天然子實體相同’亦可減緩糖尿病造成之雄性大鼠體重的減輕，經 動物實驗證明，可增加經四氧嘴咬誘導所產生之糖尿病小鼠誘發糖展 病之小鼠胰臟之胰島素分泌及減少胰島素之代謝。人工培養之冬蟲夏 草菌絲抗氧化力比野生冬蟲夏草高約他如倍，蟲草多醣體發現具有清 除自由基的能力。蟲草素為蜗蟲草(C〇rdyCepsmilitaris)主要的二級代謝 物’可誘導犬及人類乳癌細胞產生〉周亡小體。蟲草素也可有效降低一 氧化氣、前列腺素、介白素IL-1和腫瘤壞死因子的生成量，同時抑制 發炎因子iNOS、COX-2之mRNA與蛋白質的表現。 3 201212825 蟲草50%甲醇萃取物可刺激人類單核球細胞，1〇〇%甲醇萃取物可抑 制人類週邊血液單核細胞分泌IL_2及IFN_y。以罹患淋巴瘤的小鼠口 服冬蟲夏草萃取物，可增加巨噬細胞的吞噬能力，並且提高小氡免 力對抗沙門氏菌感染。 蟲草(Cordyceps sinensis)所含之腺苷、脲苷及鳥苷為冬蟲夏草有效成分 之一。腺苷較具有明顯藥理作用，能降低冠狀動脈的阻力，在心肌缺 氧時能發揮對心肌的保護作用。冬蟲夏草菌絲體萃取物處理小鼠初代 培養之萊狄吉氏細胞，具刺激細胞合成類固醇之效果。冬蟲夏草給予 尚未性成熟及性成熟的小鼠，三天及七天的試驗皆顯著升高血漿睪固 酮之水平。 〇 本發明為以家禽--雞為試驗動物，研究冬蟲夏草固態發酵物促進家禽促 進家禽性成熟的應用，試驗結果證實可以促進家禽生精作用和睪固酮 生成’方完成本發明。 【發明内容】 本發明重點為(一)飼料中添加冬蟲夏草固態發酵物作為公雞飼料添加 劑。（二）利用體重生長、雞冠色澤、睪丸切片觀察生精細管發育、睪 固酮生成與基因表現為指標，達到促進生長與性成熟，縮短飼養過程 與改善雞隻增重的應用。 ❹ 本發明另外提供一種冬蟲夏草固態發酵物促進提高體重生長、雞冠色 澤、睪固_生成與StAR基因表現量分別為7%、125%、133%、155%(對 照組與蟲草、線苦L9mg-2.85/kg t匕較)及曲精細管發育之飼料添加劑添 加劑為包括發酵物所提供腺苷(Adenosine)為〇 95mg_2 85 mg/kg。 本發明中最佳實施例中，腺苷為1.9 mg/kg。 【實施方式】 本實驗使用固態發酵物技術，經培養後腺苷含量平均可達丨988mg/g， 與天然蟲草子實體之腺苷含量2.47mg/g約略相等。 4 201212825 一、 冬蟲夏草（Cbr办活化與增菌 本發明使用冬蟲夏草（Cor办ce/w咖⑼也Sacc.菌株，利用固雄發 酵進行培養，以期產生高量之腺苷（adenosine)。將蟲草菌株以pDA (potato dextrose agar)平板繼代後於22°C下培養14天。而後將pDB 培養液放入500 mL之三角錐瓶内，於殺菌後加入無菌水於平板声養基 上並將菌絲刮取下來，將菌液接種於三角錐瓶内，並於22〇c恒溫下， 轉速控制在150 rpm，培養7天活化增菌。 二、 固態培養基 本發明使用以200g小麥為發酵基質，加入10%酵母抽出物、〇1〇/〇 O CaC〇3'0-05%' MgS〇4 · 7H20'0.1% NaH2P04'50% H2〇 > 〇.〇5〇/0 KH2P04 及1%glucose。將預活化菌液接種lOmL於固態培養基中，培養於23 °C，培養7天。 三、 腺苦（adenosine)分析 將 adenosine 標準品以 15% methanol 分別稀釋成 0、20、40、60、80 及100 pg/ml，再以高效液相層析儀分析並繪製標準曲線。發酵產物樣 品收集後以45°C熱風乾燥12小時，以均質機將樣品均質成為粉末 後，將各樣品稱取lg裝入15mL離心管中，再加入i〇mL 15% methanol，置入100°C烘箱中萃取1小時。再將萃取液以轉速 14000xg離心10分鐘，再將上清液收集後以0.22pm filter過濾後裝 入1.5mL微量離心管中以HPLC分析。 四、 HPLC分析條件 移動相為0.02MKH2P〇4 : methanol(85 : 15)，樣品注入HPLC體積為 20μ卜流速lml/min，UV偵測器偵測波長254nm，分析管柱為高效卡 氏逆相層析柱 RP-18 endcapped (5μιη)。 五、 雞隻及飼料： 由4週齡黑羽土雞，共72隻，逢機分至四組，每組3重複，每重複 5 201212825 6隻雞，以籠為重複單位。將Cor办ce声你msb取rAJ Sacc.固態發 酵產物添加於飼料中’以發酵物内所含腺苷為添加量為指標，分別為 對照組 0 mg/kg、0.95mg/kg、1.9 mg/kg、2.85mg/kg。 六、雞冠色差計量 雞隻犧牲後以游標卡尺進行量測最大長度及最大高度，計算其面積。 雞冠以手術刀沿雞隻頭骨切下，置於玻璃平板之上，隨後即以色差儀 讀取。 七、 睪丸組織切片檢查(評估生精作用） 福馬林固定之睪丸樣品進行修整至2-4 mm3，以95%及100%酒精 脫水後’將組織浸泡於二甲苯中加以透明，石蠟包埋及切片脫蠟後以 蘇木精與伊紅染色法（hematoxyline and eosin stain, HE)染色，再以 顯微鏡觀察切片。 八、 血液中睪固酮分析 以RIA放射線免疫分析法測定睪固酮。樣品中加入抗睪固酮的血清與 3H-testosterone，共培養在室溫下兩小時後，以冰停止反應。加入活性 碳’活性碳則是可吸附3H-testosterone/testosterone與抗血清結合後的 複合物，因此利用活性碳去除結合的部分，在4°C下反應15分鐘 後’將活性碳離心沉殿，測剩下游離的3H-testosterone，估算出含有睪 固酮量。 九、反轉錄聚合酶鏈鎖反應分析StAR基因表現 RNA 加入 4μ1 RT buffer (5x Frist-Strand Buffer)、Ιμΐ 0.1M DTT、Ιμΐ RNA 反轉錄酶和Ιμΐ RNAase inhibitor，於50°C下反應一小時後於701： 下15分鐘中止反應，產物即為20plcDNA，取2plcDNA加入〇.4μΐ U/μΙ Taq DNA polymerase、Ιμΐ 1〇 mM dNTP、5 μΐ 1 OX PCR buffer 後加 入2μ120μΜ引子，如下表： 4 201212825201212825 VI. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a new application of using a solid fermentation fermented product of Cordyceps sinensis (C〇rdyCepS sinensis) to be added to poultry feed to promote sexual maturity of poultry. A solid fermentation of Cordyceps sinensis promotes weight gain, cockscomb color, cremasterone production and StAR gene expression levels of 7%, I25%, and I33%, respectively (compared with the control group and Cordyceps sinensis) A feed additive for fine tube development, the additive is adenosine provided by the fermented product (Adenosine) is 〇.95mg-2.85mg/kg. The present invention relates to a feed additive for improving spermatogenesis and production of testosterone, mainly containing Cordyceps sinensis. Solid-state fermented feed additive-added powders and water-soluble powders and premixes [Prior Art] There are more than 400 kinds of known Cordyceps sinensis, the most representative of which are two kinds, one is Chinese Cordyceps (9) Governance), the other is Cordyceps militaris, cordyceps, "Compendium of Materia Medica" records that Cordyceps sinensis has the effect of protecting the lungs and kidneys, stopping bleeding and thinning, and treating phlegm; "Compendium of Compendium of Materia Medica" also points out that it can benefit lung and kidney, replenish the essence, and stop bleeding. Huayu; "Chinese Medicine Dictionary" shows that Cordyceps sinensis is sweet and sour, sexually flat, fragrant, into the lung and kidney, can be strong, benefit lung and kidney, tonic loss, benefit essence, detoxification, stop Blood phlegm. Q Cordyceps extract has the effect of lowering blood sugar, L〇ea/. (2004) pointed out that Cordyceps sinensis fruit extract and artificially cultured Cordyceps sinensis mycelium extract can reduce streptozotocin (STZ) induction Hyperglycemia in diabetic rats, and the effect of artificial culture is the same as that of natural fruiting bodies' can also alleviate the weight loss of male rats caused by diabetes. It has been proved by animal experiments that it can increase the diabetes caused by the induction of tetraoxide mouth bite. The insulin secretion of the pancreas of mice induced by glucocorticoid disease and the metabolism of insulin were reduced. The anti-oxidation power of the artificially cultivated Cordyceps sinensis was higher than that of the wild Cordyceps sinensis, and the Cordyceps polysaccharide was found to have the ability to scavenge free radicals. It is the main secondary metabolite of C〇rdyCepsmilitaris, which can induce the development of canine and human breast cancer cells. Cordycepin can also effectively reduce monoxide, prostaglandins, interleukin IL-1 and tumors. The amount of necrosis factor produced, while inhibiting the expression of inflammatory factors iNOS, COX-2 mRNA and protein. 3 201212825 Cordyceps 50% methanol extract Exciting human monocyte cells, 1% methanol extract can inhibit the secretion of IL_2 and IFN_y by human peripheral blood mononuclear cells. Oral administration of Cordyceps extract in mice with lymphoma can increase the phagocytic capacity of macrophages and improve Adenine is resistant to Salmonella infection. Adenosine, uridine and guanosine contained in Cordyceps sinensis are one of the active constituents of Cordyceps sinensis. Adenosine has obvious pharmacological effects, can reduce the resistance of coronary artery, and is hypoxic in myocardial It can exert the protective effect on the myocardium. The mycelium extract of Cordyceps sinensis can treat the Leydig cells in the primary culture of mice, and stimulate the cells to synthesize steroids. Cordyceps sinensis administered mice that were not sexually mature and sexually mature, and the three-day and seven-day tests significantly increased the level of plasma steroids. 〇 The present invention is to use poultry-chicken as a test animal to study the application of solid fermentation of Cordyceps sinensis to promote the sexual maturity of poultry in poultry, and the test results confirmed that the spermatogenesis and the production of testosterone can be promoted to complete the present invention. SUMMARY OF THE INVENTION The present invention focuses on (1) adding a solid fermented product of Cordyceps sinensis to a feed as a cock feed additive. (2) Using weight growth, cockscomb color, and sputum pill to observe the development of fine tube, steroid formation and gene expression as indicators, to promote growth and sexual maturity, shorten the feeding process and improve the weight gain of chickens. ❹ The present invention further provides a solid fermentation product of Cordyceps sinensis to promote weight growth, cockroach color, tamping _ generation and StAR gene expression amounts of 7%, 125%, 133%, and 155%, respectively (control group and cordyceps, line bitter L9mg- 2.85/kg t匕) and the feed additive for the development of the fine tube are adenosine (Adenosine) including 发酵95mg_2 85 mg/kg. In a preferred embodiment of the invention, the adenosine is 1.9 mg/kg. [Embodiment] In this experiment, the solid fermentation technology was used, and the adenosine content after culture was 丨 988 mg/g on average, which was approximately equal to the adenosine content of the natural Cordyceps fruit body 2.47 mg/g. 4 201212825 I. Cordyceps sinensis (Cbr office activation and enrichment) The present invention uses Cordyceps sinensis (Cor ce/w coffee (9) also Sacc. strain, cultured with Guxiong fermentation, in order to produce high amount of adenosine (adenosine). After subculture with pDA (potato dextrose agar) plate, culture at 14 ° C for 14 days. Then pDB culture medium was placed in a 500 mL flask, after sterilization, sterile water was added to the plate sonicator and the bacteria were added. The silk was scraped off, and the bacterial liquid was inoculated into a triangular flask, and the temperature was controlled at 150 rpm and the growth rate was controlled at 150 rpm for 7 days to activate the bacteria. 2. Solid medium The present invention used 200 g of wheat as a fermentation substrate. Add 10% yeast extract, 〇1〇/〇O CaC〇3'0-05%' MgS〇4 · 7H20'0.1% NaH2P04'50% H2〇> 〇.〇5〇/0 KH2P04 and 1%glucose The pre-activated bacterial solution was inoculated with 10 mL in solid medium and cultured at 23 ° C for 7 days. 3. Adenosine analysis The adenosine standard was diluted to 0, 20, 40, 60 with 15% methanol, respectively. 80 and 100 pg/ml, and then analyze and draw a standard curve with high performance liquid chromatography. The product samples were collected and dried by hot air at 45 ° C for 12 hours. After homogenizing the samples into powders, each sample was weighed into lg into a 15 mL centrifuge tube, and then i〇mL 15% methanol was added and placed at 100 ° C. The mixture was extracted in an oven for 1 hour. The extract was centrifuged at 14,000 x g for 10 minutes, and the supernatant was collected, filtered through a 0.22 pm filter, and loaded into a 1.5 mL microcentrifuge tube for HPLC analysis. 0.02MKH2P〇4 : methanol(85 : 15), sample injection HPLC volume 20μb flow rate lml/min, UV detector detection wavelength 254nm, analytical column for efficient Cartesian reverse phase chromatography column RP-18 endcapped ( 5μιη). 5. Chickens and feed: From 4 weeks old black-feathered chicken, a total of 72, divided into four groups, each group of 3 repetitions, each repeat 5 201212825 6 chickens, with cages as repeating units. Cor ce sound your msb take rAJ Sacc. Solid-state fermentation products are added to the feed'. The amount of adenosine contained in the fermented product is used as the index, which is 0 mg/kg, 0.95 mg/kg, 1.9 mg/kg, respectively. 2.85mg/kg. 6. The color difference of the cockscomb is measured by the vernier caliper after sacrifice. The maximum length and height, the area is calculated. Scalpel along the crest skull birds cut, placed on a glass plate, followed by a color difference meter that is read in. 7. Tissue biopsy (evaluation of spermatogenesis) Samples of formalin-fixed testicles were trimmed to 2-4 mm3, dehydrated with 95% and 100% alcohol, and the tissue was immersed in xylene for transparency, paraffin embedding and The sections were deparaffinized and stained with hematoxyline and eosin stain (HE), and sections were observed under a microscope. 8. Analysis of testosterone in blood Determination of testosterone by RIA radioimmunoassay. The anti-tanosterone-containing serum was added to the sample and 3H-testosterone, co-cultured for two hours at room temperature, and the reaction was stopped with ice. Adding activated carbon 'activated carbon is a complex that can adsorb 3H-testosterone/testosterone and antiserum, so the activated carbon is used to remove the bound part, and after reacting at 4 ° C for 15 minutes, the activated carbon is centrifuged. The free 3H-testosterone was left to measure and the amount of testosterone was estimated. Nine, reverse transcription polymerase chain reaction analysis StAR gene expression RNA Add 4μ1 RT buffer (5x Frist-Strand Buffer), Ιμΐ 0.1M DTT, Ιμΐ RNA reverse transcriptase and Ιμΐ RNAase inhibitor, react at 50 ° C for one hour At 701: the reaction was stopped in the next 15 minutes. The product was 20 pl cDNA. 2 pl cDNA was added to 〇.4μΐ U/μΙ Taq DNA polymerase, Ιμΐ 1〇mM dNTP, 5 μΐ 1 OX PCR buffer and 2μ120μΜ primer was added, as shown in the following table: 4 201212825
Primer Forward Reverse β-actin TGATATTGCTGCGCTCGTTG CATGGCTGGGGTGTTGAAG StAR GCTGCCATCTCCTACCAACA AGCACCGAACACTCACAAA 產物進行電泳後分析基因表現量。 實施例1·冬蟲夏草固態發酵發酵物發酵期間對腺苷含量之影響 利用固態發酵基質接種冬蟲夏草後利用HPLC分析發酵〇,7，14,21天腺 苷含量之變化。結果如圖丨及圖2所示，腺苷滯留時間約為第757分 〇 鐘，發酵過程以第7天達到最高為1.9mg/g，之後逐漸下降。 實施例2·難隻餵飼冬蟲夏草固態發酵物對難隻髏重、難冠紅綠指數和 血液中睪固酮濃度之影響 將冬蟲夏草嶋發酵物與雞隻飼料混合’添加劑量以每公斤飼料添加 含發酵物所含腺苷mg量為依據，分為對照組（無添加)、〇 %、丨9、2沾 mg/kg 〇 ' 飼料配方組成如下表 飼料配方 使用百分比 玉米 62 豆粉 25 魚粉 10 磷酸鈣 0.8 石灰石粉 0.7 鹽 0.4 胺基酸 0.5 膽鹼 0.1 維生素及礦物質 0.5 總重 100 7 201212825 結果如圖3所示’雞隻傲飼6週後秤重，以腺苷LQmg/kg添加量體重 最高為2.55kg比照組2.38 kg增加7%。雞冠紅潤程度以雞冠紅綠指數 表示，結果如圖4所示。隨冬蟲夏草添加比對照組顯著提高增加紅綠 指數，且以1.9 mg組1.8為最高比對照組0.8增加I25%。測量雞隻血 中睪固酮濃度結果如圖5所示，蟲草添加組均可提高睪固酮濃度，並 且以1.9 mg組效果最佳2.8 ng/mL比對照組1.2 ng/mL增加1330/〇。 實施例3·難隻飯飼冬蟲夏草固態發酵物對難隻生精作用及睪固酮合成 之StAR基因表現之影響 雞隻餵飼冬蟲夏草發酵物對睪丸組織切片結果，如圖6結果所示。對 照組：曲細精管中僅有精原細胞(Spermatogonia)存在，在蟲草腺苷0.95 mg組可觀察少數曲細精管有多量精子(spermatozoa)，而1.9mg組所有 曲細精管管腔精子量甚多，但2.85 mg組並無精子及可見少數精細胞 (spermatid)。以RT-PCR分析StAR基因表現，結果如圖7所見，冬蟲 夏草發酵物對StAR基因表現量，以添加量2.85mg組有最高表現量 1.15比對照組0.45增加155%表現。 結論： 雞隻飼料中添加冬蟲夏草固態發酵物，可以促進體重生長、雞冠色澤、 睪丸生精細管中精子生精作用、睪固酮生成&StAR基因表現量’因此 達到促進生長與性成熟，縮短飼養過程與改善雞隻增重的應用，緣此 本發明揭示冬蟲夏草固態發酵物可用於製造促進家禽性成熟之飼料添 加劑的新應用。 【圖式簡單說明】 【主要元件符號說明】 ePrimer Forward Reverse β-actin TGATATTGCTGCGCTCGTTG CATGGCTGGGGTGTTGAAG StAR GCTGCCATCTCCTACCAACA AGCACCGAACACTCACAAA The product was analyzed for gene expression after electrophoresis. Example 1 The effect of the solid fermentation fermentation of the Cordyceps sinensis on the adenosine content during fermentation The inoculum of the Cordyceps sinensis was inoculated with the solid fermentation substrate, and the adenosine content of the fermented mash was analyzed by HPLC for 7, 14, 21 days. As a result, as shown in Fig. 2 and Fig. 2, the retention time of adenosine was about 757 minutes, and the fermentation process reached a maximum of 1.9 mg/g on the 7th day, and then gradually decreased. Example 2: Difficult to feed only the solid fermented product of Cordyceps sinensis on the hard-to-weight, difficult-to-crude red-green index and the concentration of testosterone in the blood. Mix the Cordyceps sinensis fermented material with the chicken feed. The amount of additive is added to the fermentation per kg of feed. The amount of adenosine contained in the substance is based on the control group (no added), 〇%, 丨9, 2 沾mg/kg 〇' feed formula consists of the following table feed formula use percentage corn 62 soy flour 25 fish meal 10 calcium phosphate 0.8 limestone powder 0.7 salt 0.4 amino acid 0.5 choline 0.1 vitamins and minerals 0.5 total weight 100 7 201212825 The results shown in Figure 3 'chicken proud after 6 weeks of weighing, with adenosine LQmg / kg added the highest weight It increased by 7% for 2.35 kg of the 2.35 kg comparison group. The degree of rosy rosin is expressed in the cockscomb red-green index, and the results are shown in Figure 4. The addition of Cordyceps sinensis significantly increased the red-green index compared with the control group, and the highest in the 1.9 mg group was 1.8% compared with the control group 0.8. The results of measuring the concentration of testosterone in chicken blood were as shown in Fig. 5. The concentration of testosterone in the Cordyceps group increased the concentration of guanosterone in the 1.9 mg group and increased by 1300 ng/mL in the control group by 1.2 ng/mL. Example 3 The effect of the solid fermented material of Cordyceps sinensis on the performance of the StAR gene and the performance of the StAR gene of the testosterone synthesis The results of the tissue sectioning of the testosterone of the fermented chicken Cordyceps sinensis were shown in Fig. 6. In the control group, only spermatogonial cells (Spermatogonia) existed in the seminiferous tubules. In the 0.95 mg group of Cordyceps adenosine, a small number of spermatozoa (spermatozoa) were observed, while in the 1.9 mg group, all the seminiferous tubules were in the 1.9 mg group. There is a large amount of sperm, but there is no sperm and a few spermatozoa (spermatid) in the 2.85 mg group. The performance of StAR gene was analyzed by RT-PCR. The results are shown in Fig. 7. The performance of the StAR gene in the fermentation of the Cordyceps sinensis was up to 155% in the group with the added amount of 2.85 mg, which was 155% higher than that of the control group. Conclusion: The solid feed of Cordyceps sinensis in chicken feed can promote the growth of body weight, the color of the cockscomb, the spermatogenesis of spermatozoa in the fine tube, the production of steroids & the expression of StAR gene, thus promoting growth and sexual maturity and shortening the feeding process. In order to improve the weight gain of chickens, the present invention discloses that a solid fermented product of Cordyceps sinensis can be used for the manufacture of a new application of a feed additive for promoting sexual maturity in poultry. [Simple description of the diagram] [Description of main component symbols] e