TWI377065B

TWI377065B -

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Publication number: TWI377065B
Application number: TW98111670A
Authority: TW
Inventors: Null Null
Priority date: 2009-04-08
Filing date: 2009-04-08
Publication date: 2012-11-21
Also published as: TW201036621A

Description

1377065 申請案號第98111670號替換本 101.6.19 六、發明說明：【發明所屬之技術領域】本發明是有關於一種靈芝液，特別是指-種經由機械式研磨製成之靈芝子實體懸浮液。 ' 機械【先前技術】奈米/次微米產品是近年來的熱門話題，近年來研究學者、業者與一般大眾的注意，右忍有鏗於奈米/次微米科技的重要性及潛力’很多國家已將奈米/次微米科技之相關研究列為優先考量，且-但將奈米/次微米科技應用於食品材料上，將對全球的食品系統造成革命性的改變，雖献目前奈米/次微米食品材料的研究尚屬於起步階段，_材粒子細小後，應可提高吸收率，而有助於嬰兒、老人或消化系統不良者攝取必須的營養物質，且可應用於中草藥，使藥效更容易發揮，為達到這些長遠的目標，食品材料的奈米/次微米化技術與產品性質的瞭解是一項重要的課題。 7中草藥中’靈芝子實體被公認為具有多項保健功能，如抗腫瘤、調節免疫機能、降血脂、降血壓、降血糖、抗病毒、抗過敏、抗氧化、抗輻射、抑制血小板凝集與止痛等，無論是以完整子實體或萃取物食用，靈芝確實是相田女全的中草藥，甚至給予高劑量，例如：每日30 g之乾燥子實體萃取物，經證實亦無顯著的毒性反應，且利用動物實驗評估靈芝的營養價值和毒性，結果顯示靈芝並無基因毒性（genotoxicity)。據估計，2001年全世界靈芝的產量為4800噸，中國大 3 1377065 申請案號第98111670號替換本 101.6.19 陸是最大生產國（3500噸），約有200萬人食用靈芝，即每人每年約消耗2.5公斤，消費者健康意識的增加，使靈芝產品市場將逐年成長。但市面上的靈芝產品很多種，常見的為：整株靈芝、靈芝切片、膠囊、顆粒、錠丸與液狀飲品 (靈芝茶、靈芝酒、靈芝糖漿及靈芝飲料）等，依其有效成份含量而言，品質優劣參差不齊。雖然靈芝保健食品是目前食品市場的熱門商品’然而，其有效成分（例如幾丁質、春醣、β-葡聚糖）萃取不易、溶解度低，於人體内的吸收亦不甚理想，且萃取後之靈芝渣（多為膳食纖維之成分 )又常被視為廢棄物，而造成資源的浪費。經申請人研究，若能將完整之靈芝子實體粒徑奈米/次微米化處理，製成奈米/次微米級靈芝子實體懸浮液，不但有助於靈芝多醣等活性成分的釋出，而獲得完整的活性成分，及增加活性成分的吸收與利用外，還可從中獲得奈米/ 次微米化之膳食纖維，而同時減少廢棄物的產生。【發明内容】因此’本發明之目❾，即在提供一種經由機械式研磨製成，且具有細小靈芝微粒的靈芝子實體懸浮液。於是，本發明靈芝子實體懸浮液，為靈芝子實體盥可食性乳化劑-起經由機械式研磨後所得到含有靈芝微粒之靈芝子實體懸浮液，並含有纖維質、幾丁質，& 葡聚糖，且靈芝微粒之體積平均粒經小於1〇 μιη，該可食性乳化劑是啊80,且該可食性乳化劑含量是$芝子實體重量的 4 申請索號第98111670號替換本 101.6.19 一本發月之功效·透過機械式研磨來製造靈芝子實體懸汗液的方式，除了無須萃取即可獲得完整的活性成分外，還可大幅提高纖維質比表面積，且纖維質之後續利用無須再粉碎’可作為-良好的膳食纖維添加物。【實施方式】有關本發明之前述及其他技術内容、特點與功效，在以下配合參考圖式之一個較佳實施例的詳細說明中將可清楚的呈現》 # j發明靈芝子實體懸浮液的較佳實施例，係由整株靈芝子實體經過機械式研磨處理製成，該靈芝子實體懸浮液包：經：機械式研磨靈芝子實體所得到之靈芝微粒，及與靈芝子實體一起進行研磨之可食性乳化劑。機械式奈米研磨乃是利用機械能量、研磨介質（研磨珠）間的磁撞作用’以及漿料·流體與流體·研磨珠間的剪切力，將大粒子破碎與分散，常用於塗料或奈米粉體製備。機械式研磨機配有冷卻系統，可避免高溫的產生，有利於對熱敏感之有機材料的處理，例如食品㈣與中草藥等。以下即以傳統熱水萃取法獲得之萃取物，與本發明直接將靈芝子實心機械切磨進行奈米/讀米化處理後之致芝子貫體懸浮液進行比較說明。照組：傳統埶皮萃南將靈芝子實體清洗後置於70°C供箱中，以熱風乾燥至水份殘餘約10%左右。利用粉碎機1377065 Application No. 98111670 Replacement 101.6.19 VI. Description of the Invention: [Technical Field] The present invention relates to a Ganoderma lucidum liquid, in particular to a suspension of Ganoderma lucidum fruit body prepared by mechanical grinding . 'Mechanical [Prior Art] Nano/submicron products are a hot topic in recent years. In recent years, researchers, practitioners and the general public have paid attention to the importance and potential of nano/submicron technology. Many countries Research on nano/submicron technology has been prioritized, and – the application of nano/submicron technology to food materials will revolutionize the global food system, although currently available in nano/ The research on sub-micron food materials is still in its infancy. After the particles are small, they should increase the absorption rate, and help the infants, the elderly or the digestive system to get the necessary nutrients, and can be applied to Chinese herbal medicines. It is easier to play, and in order to achieve these long-term goals, the understanding of nano/submicron technology and product properties of food materials is an important issue. 7 Chinese herbal medicine 'Ganoderma lucidum fruit body is recognized as having many health functions, such as anti-tumor, regulating immune function, lowering blood fat, lowering blood pressure, lowering blood sugar, anti-virus, anti-allergic, anti-oxidation, anti-radiation, inhibiting platelet aggregation and pain relief, etc. Whether it is eaten as a whole fruiting body or extract, Ganoderma lucidum is indeed a Chinese herbal medicine of the same phase, even given a high dose, for example: 30 g of dried fruiting body extract per day, confirmed to have no significant toxicity, and utilized Animal experiments evaluated the nutritional value and toxicity of Ganoderma lucidum. The results showed that Ganoderma lucidum had no genotoxicity. It is estimated that the production of Ganoderma lucidum in the world was 4,800 tons in 2001, and China's Big 3 1377065 application number No. 98111670 replaced this 101.6.19. Land is the largest producer (3,500 tons), and about 2 million people consume Ganoderma lucidum, that is, each person. About 2.5 kilograms per year, the increase in consumer health awareness will make the market of Ganoderma lucidum products grow year by year. However, there are many kinds of Ganoderma lucidum products on the market. The common ones are: whole ganoderma lucidum, ganoderma lucidum slices, capsules, granules, ingots and liquid drinks (Ganoderma lucidum tea, Ganoderma lucidum wine, Ganoderma lucidum syrup and Ganoderma lucidum beverage), etc. In terms of quality, the quality is not good. Although Ganoderma lucidum health food is a popular commodity in the current food market, however, its active ingredients (such as chitin, spring sugar, β-glucan) are difficult to extract, have low solubility, and are not well absorbed in the human body, and are extracted. The latter ganoderma slag (mostly the ingredient of dietary fiber) is often regarded as waste, which causes waste of resources. According to the applicant's research, if the complete Ganoderma lucidum fruit body particle size nano/submicron treatment can be made into nano/sub-micron Ganoderma lucidum fruit body suspension, it not only contributes to the release of active ingredients such as Ganoderma lucidum polysaccharides. In addition to obtaining the complete active ingredient and increasing the absorption and utilization of the active ingredient, it is also possible to obtain nano/micronized dietary fiber therefrom while reducing waste generation. SUMMARY OF THE INVENTION Therefore, the object of the present invention is to provide a suspension of Ganoderma lucidum fruit body which is produced by mechanical grinding and has fine ganoderma lucidum particles. Therefore, the suspension of the Ganoderma lucidum fruit body of the present invention is an edible emulsifier of the Ganoderma lucidum fruit body - a suspension of the Ganoderma lucidum fruit body containing the Ganoderma lucidum microparticles obtained by mechanical grinding, and contains fibrous, chitin, & The polysaccharide, and the volume average particle size of the ganoderma lucidum particles is less than 1 〇μιη, the edible emulsifier is 80, and the edible emulsifier content is 4 of the weight of the shiji entity. The application number is 9811670, which replaces the 101.6. 19 The effect of a month of the moon · The method of making the suspension of the ganoderma lucidum fruit body by mechanical grinding, in addition to obtaining the complete active ingredient without extracting, can also greatly increase the specific surface area of the fiber, and the subsequent utilization of the fiber is not necessary Re-crushing 'can be used as a good dietary fiber supplement. [Embodiment] The foregoing and other technical contents, features and effects of the present invention will be apparent from the following detailed description of a preferred embodiment of the accompanying drawings. The preferred embodiment is prepared by mechanically grinding the whole Ganoderma lucidum fruit body, and the Ganoderma lucidum fruit body suspension package is: mechanically grinding the Ganoderma lucidum microparticles obtained from the Ganoderma lucidum fruit body, and grinding together with the Ganoderma lucidum fruit body. Edible emulsifier. Mechanical nano-grinding is the use of mechanical energy, the magnetic collision between the grinding medium (abrasive beads) and the shear force between the slurry, fluid and fluid and the grinding beads to break and disperse large particles, often used in coatings or Preparation of nano powder. The mechanical grinder is equipped with a cooling system to avoid the generation of high temperature and is suitable for the treatment of heat sensitive organic materials such as food (4) and Chinese herbal medicine. The following is an extract obtained by a conventional hot water extraction method, which is described in comparison with the present invention, in which a solid mechanical cutting of a ganoderma lucidum is carried out to carry out a nano/reading treatment. Photo group: traditional suede extract South Ganoderma lucidum fruit body is cleaned and placed in a box at 70 ° C, dried by hot air to about 10% residual moisture. Using a shredder

Co.，Taiwan)細碎成粉末備用。取3〇〇 g靈芝子實體粉末加 1377065 申請案號第98111670號替換本 101.6.19 入4500 mL蒸餾水，於1〇〇艺加熱2小時，趁熱以60 mesh 筛網先行粗濾’所得瀘、液再以Whatman No.4濾、紙（Co., Taiwan) finely pulverized into powder for use. Take 3〇〇g Ganoderma lucidum fruit body powder plus 1370065 Application No. 98111670 Replace this 101.6.19 into 4500 mL of distilled water, heat for 2 hours in 1〇〇 art, and heat filter with 60 mesh sieve. Liquid and then Whatman No.4 filter, paper (

Whatman，Springfield Mill，UK)過濾以除去細微殘渣，可得黃色滤液，即為萃取液。殘渣再加入4500 mL蒸餾水，且如上進行萃取。每一樣品進行三次熱水萃取，將所得濾液混合，於真空下濃縮至適量體積，計算濃縮率並紀錄之〇實驗組：機械式研磨整铁雷芏子實體，包含以下步麻丄步驟（一）研磨前處理。秤取靈芝子實體，洗淨切丁後加入250 mL 4乞去離子水，以攪碎器（Blender 7012S，Waring Commercial, USA) 攪打5分鐘後倒入600 mL的高型燒杯中，以去離子水（ 150 mL、4°C )將殘留在攪碎器中的子實體粗碎物洗出，於 4°C下靜置隔夜，使纖維組織鬆弛，再以高速均質機（ Polytron PT 3000，Kinematica AG，Switzerland)於 20,000 rpm下攪打10分鐘（全程冰浴以避免溫度上升），使子實體顆粒小於300 μιη，再進行機械式研磨。步驟（二）機械式研磨。使用奈米細磨機（MiniPur，NETZCH- Feinmahltechnik GmbH，German)研磨上述步驟（一）所得之子實體顆粒，研磨需分為二個階段。第一階段使用粒徑0.8 mm之釔錯珠為研磨介質，第二階段則改用粒徑0.3 mm之釔锆珠為研磨介質，進料速度360 mL/min ^為探討操作條件對產品之影 6 1377065 申請案號第98111670號替換本 101.6.19 響，選擇不同的固形物濃度、研磨介質填充量及攪拌軸轉速進行實驗，於研磨過程中，每30分鐘取樣進行粒徑分析〇完成上述靈芝子實體懸浮液之製備後，將針對對照組與實驗組所得之靈芝液進行化性與物性分析比較，分別如下所述： (一）化性分析 Α. β-葡聚糖（β-D-glucans)含量測定利用螢光染劑Aniline blue定量β-葡聚糖。取研磨後之靈芝子實體懸浮液離心（ 2000xg，10 min)後，以Whatman Νο·4濾紙過濾，取適量樣品溶液，加入0.3 N NaOH使總體積為3 mL，攪拌30分鐘，以1 N HC1調整pH至11.5，加入 50 mM 之 pH 11.5 的 Na2HP04-Na0H buffer (含 0.5 Μ NaCl)，並定容至10 mL。取上述溶液2 mL，力口入0.2 mL aniline blue ( 1 mg/mL)，以Vortex震盪混合均勻後，靜置 2小時，以螢光檢測器檢測（激發波長395 nm，放射波長 495 nm )。以不同濃度之Lentinan製備標準曲線，計算樣品中之β-葡聚糖含量。 Β.總膳食纖維測定採用 AOAC 991.43 (enzymatic-gravimetric method)和 AOAC 993.21 (nonenzymatic-gravimetric method)兩種方法。AOAC 991.43是目前廣為學者所接受之測定方法，AOAC 993.21則是一種不使用酵素的簡便方法（此法只適用於總膳食纖維> 10%，澱粉< 2%之樣品，包括靈芝之部分中草 7 1377065 申請案號第98111670號替換本 101.6.19 藥均符合此規範）。 C.幾丁質（Chitin)含量測定取400 mg靈芝子實體懸浮液，以6 N HC1於100°C下迴流水解5小時，冷卻至室溫後以Whatman Νο·4濾紙過濾，取1 mL濾液於45〜50°C下減壓乾燥後備用。將乾燥之水解產物回溶於蒸餾水中。取1 mL上述水解產物之稀釋溶液加入 0.25 mL 4% acetylacetone，於 90°C 下加熱 1 小時，冷卻後加入2 mL ethanol並震盪使沉澱物溶解，隨後加入0.25 mL Ehrlich reagent，呈色後，於波長530 nm下測定其吸光值。利用不同濃度之 glucosamine hydrochloride ( 5〜50 pg/mL)製作標準曲線，並以 l，4-anhydro-N-acetyl-2-deoxy-D-glucopyranose equivalent 計算幾丁質含量。 (二）物性分析 A.粒徑分佈測定使用動態光散射粒徑分析儀（dynamic light scattering particle size analyzer) (Nanotrac 150，Microtrac Inc” USA) ，測定所得之靈芝微粒的粒徑分佈，該儀器的測定範圍0.8 nm至6500 nm。如超出該範圍，貝ij改用 Beckman Coulter (CA, USA)生產之LS 230粒徑分析儀進行量測，其粒徑量測範圍0.4 μηι至2000 μηι，光源經濾光處理後，偵測下限可達40 nm »以去離子水作為空白測試，並於25°C下進行測定。將研磨後之靈芝子實體懸浮液經適當稀釋後，直接以儀器測定之。利用分析軟體（FLEX Software，Microtrac Inc.，USA)分析散射訊號，計算粒子之Doppler shifts以求 8 1377065 申請案號第98111670號替換本 101.6.19 得粒徑分佈百分比、平均粒徑（mean particle size )、中間粒徑（median particle size)等粒徑分佈參數。 B.顯微觀察使用光學顯微鏡（Optiphot-Pol，Nikon，Japan)觀察靈芝粗碎後之組織結構，以及研磨初期靈芝顆粒之變化情形。以掃描式電子顯微鏡（SEM)觀察所得靈芝微粒的顯微結構。樣品經稀釋後，塗抹於載玻片上，置於室溫下乾燥，用銀膠將樣品黏著於鋁台上，於真空狀態下以離子覆膜器將樣品表面鍍上金膜後，以SEM觀察樣品之顯微結構。另亦使用穿透式電子顯微鏡（TEM ) ( JEM-1230，JEOL Co. Ltd, Japan)觀察所得靈芝微粒之顯微結構，確認粒徑分佈之測量結果。樣品經適當稀釋後，以TEM專用之200 mesh 鍍碳銅網（ 01800-F，Ted Pella，Inc.，U.S.A.)沾取樣品懸浮液，置於乾燥箱中乾燥，乾燥後之樣品直接以TEM觀察，並以 CCD 照相系統（DualVision CCD，Gatan Inc.，US A )结取所需之數位影像。TEM所用之電子源為熱陰極電子搶（ LaB6燈絲），最大加速電壓為120KV。 (三）靈芝子實體懸浮液之穩定性處理與分析 A.界面活性劑之添加選擇適當的研磨操作條件（轉速：3600 rpm，介質直徑：0.2 mm ;時間：90分鐘），於研磨前加入不同種類、濃度和HLB值之可食性乳化劑。選用之乳化劑有：1.離子型界面活性劑（ionic surfactant )，例如脂肪酸鹽類（屬陰離子型界面活性劑）。2.非離子型界面活性劑（nonionic 9 1377065 申請案號第98111670號替換本 101.6.19 surfactant)，例如（1)蔗糖龍（sugar ester)(選擇 hlb 值分別為3、7、11和15之蔗糖酯）;⑴聚山梨醇酐脂肪酸醋（_ 85、80、60、20 和 Tween 65、20，HLB 值分別為 1·8 4·3 6·7、8.6、10.6、16.7 )。上述所選用之乳化劑濃度，是以研磨之固形物含量為基礎，加入5%、1〇%、2〇% 和 50%。 Β·儲存性試驗將研磨後及經穩定處理之靈芝子實體懸浮液分別放置在4°C和室溫下，於儲存期間（〇、〇 5、丨、2、6、12 ' 24 48 72 96小時）’取樣進行濁度分析和粒徑分析。於此試驗中，將添加少許防腐劑，以避免微生物的生長。 C.濁度測定濁度的高低與溶液中粒子之數量有關，若樣品中之奈米/次微米粒子聚集成大粒子，甚至沉澱，則懸浮粒子總數下降致使上層溶液濁度下降。可由濁度初步判斷靈芝子實體懸洋液之穩定性，進而淘汰不適用的乳化劑。使用攜Whatman, Springfield Mill, UK) is filtered to remove the fine residue to give a yellow filtrate which is the extract. The residue was further added with 4500 mL of distilled water and extracted as above. Three hot water extractions were performed for each sample, and the obtained filtrate was mixed, concentrated under vacuum to an appropriate volume, and the concentration ratio was calculated and recorded. Experimental group: mechanically ground iron-streaked fruit body, including the following step of paralysis (1) ) Pre-grinding treatment. Grab the Ganoderma lucidum fruiting body, wash the diced, add 250 mL of 4 乞 deionized water, whipped with a masher (Blender 7012S, Waring Commercial, USA) for 5 minutes, then pour into a 600 mL high-shaped beaker to go The ionized water (150 mL, 4 ° C) washed out the fruit body coarse residue remaining in the masher, and left at 4 ° C overnight to relax the fiber structure, and then a high-speed homogenizer (Polytron PT 3000, Kinematica AG, Switzerland) was whipped at 20,000 rpm for 10 minutes (full ice bath to avoid temperature rise), the fruiting body particles were less than 300 μηη, and then mechanically ground. Step (2) Mechanical grinding. The fruiting body particles obtained in the above step (1) were ground using a nano fine grinding machine (MiniPur, NETZCH-Feinmahltechnik GmbH, German), and the grinding was carried out in two stages. In the first stage, the erbium beads with a particle size of 0.8 mm were used as the grinding medium. In the second stage, the cerium-zirconium beads with a diameter of 0.3 mm were used as the grinding medium. The feed rate was 360 mL/min ^ to explore the operating conditions. 6 1377065 Application No. 98111670 Replace this 101.6.19 ring, select different solid concentration, grinding medium filling amount and stirring shaft speed for experiment. During the grinding process, sample the particle size every 30 minutes to complete the above-mentioned Ganoderma lucidum. After the preparation of the fruiting body suspension, the chemical and physical properties of the control group and the experimental group were compared and analyzed as follows: (1) Chemical analysis Α. β-glucan (β-D- Glucans) Determination of β-glucan by fluorescent dye Aniline blue. After centrifugation of the ground Ganoderma lucidum fruit body suspension (2000xg, 10 min), filter with Whatman Νο·4 filter paper, take appropriate amount of sample solution, add 0.3 N NaOH to make the total volume 3 mL, stir for 30 minutes, to 1 N HC1 Adjust the pH to 11.5 and add 50 mM Na2HP04-Na0H buffer (containing 0.5 Μ NaCl) at pH 11.5 and bring up to 10 mL. Take 2 mL of the above solution, dip into 0.2 mL aniline blue (1 mg/mL), mix well with Vortex, and let stand for 2 hours. Detect with a fluorescent detector (excitation wavelength 395 nm, emission wavelength 495 nm). The β-glucan content in the sample was calculated by preparing a standard curve with different concentrations of Lentinan. Β. Total dietary fiber was measured using AOAC 991.43 (enzymatic-gravimetric method) and AOAC 993.21 (nonenzymatic-gravimetric method). AOAC 991.43 is currently accepted by a wide range of scholars. AOAC 993.21 is a simple method of not using enzymes (this method is only applicable to total dietary fiber > 10%, starch < 2% of samples, including parts of Ganoderma lucidum) Zhongcao 7 1377065 Application No. 98111670 replaces this 101.6.19 drug in accordance with this specification). C. Chitin content determination 400 mg Ganoderma lucidum fruit body suspension, 6 N HC1 reflux hydrolysis at 100 ° C for 5 hours, cooled to room temperature, filtered with Whatman Ν ο 4 filter paper, 1 mL filtrate Dry at 45~50 ° C under reduced pressure and set aside. The dried hydrolyzed product is dissolved back in distilled water. Add 1 mL of the diluted solution of the above hydrolyzate to 0.25 mL of 4% acetylacetone, heat at 90 ° C for 1 hour, add 2 mL of ethanol after cooling and shake to dissolve the precipitate, then add 0.25 mL of Ehrlich reagent, after coloring, The absorbance was measured at a wavelength of 530 nm. Standard curves were prepared using different concentrations of glucosamine hydrochloride (5 to 50 pg/mL) and chitin content was calculated as l,4-anhydro-N-acetyl-2-deoxy-D-glucopyranose equivalent. (2) Physical property analysis A. Particle size distribution measurement Using a dynamic light scattering particle size analyzer (Nanotrac 150, Microtrac Inc. USA), the particle size distribution of the obtained ganoderma lucidum microparticles was measured. The measurement range is from 0.8 nm to 6500 nm. If it is outside this range, Beij is converted to a LS 230 particle size analyzer manufactured by Beckman Coulter (CA, USA). The particle size measurement range is 0.4 μηι to 2000 μηι. After filtering, the detection limit is up to 40 nm » Deionized water is used as a blank test and measured at 25 ° C. The ground suspension of the Ganoderma lucidum fruit body is appropriately diluted and directly measured by an instrument. Analytical scattering software (FLEX Software, Microtrac Inc., USA) was used to analyze the scattering signal, and the Doppler shifts of the particles were calculated to obtain the particle size distribution percentage and the average particle size (mean particle size) in order to replace the 101.6.19 application No. 9811670. ), particle size distribution parameters such as median particle size B. Microscopic observation Observed Ganoderma lucidum using an optical microscope (Optiphot-Pol, Nikon, Japan) The microstructure and the change of Ganoderma lucidum particles in the initial stage of grinding. The microstructure of the obtained Ganoderma lucidum particles was observed by scanning electron microscopy (SEM). The sample was diluted, applied to a glass slide, and dried at room temperature. The silver paste adhered the sample to the aluminum table, and after depositing the gold film on the surface of the sample with an ion laminator under vacuum, the microstructure of the sample was observed by SEM. A transmission electron microscope (TEM) (JEM) was also used. -1230, JEOL Co. Ltd, Japan) Observed the microstructure of the obtained Ganoderma lucidum microparticles and confirmed the measurement results of the particle size distribution. After appropriate dilution, the sample was TEM-specific 200 mesh carbon-coated copper mesh (01800-F, Ted Pella). , Inc., USA) Dip the sample suspension, dry it in a dry box, dry the sample directly by TEM, and take the desired number with a CCD camera system (DualVision CCD, Gatan Inc., US A) Image. The electron source used in TEM is hot cathode electron grab (LaB6 filament), and the maximum accelerating voltage is 120KV. (III) Stability treatment and analysis of Ganoderma lucidum fruit body suspension A. Addition of surfactant is appropriate The grinding conditions (rotation speed: 3600 rpm, media diameter: 0.2 mm; time: 90 minutes) were added with different types, concentrations and HLB values of the edible emulsifier before grinding. The emulsifiers used are: 1. Ionic ionic surfactants, such as fatty acid salts (anionic surfactants). 2. Nonionic surfactant (nonionic 9 1377065 application No. 98111670 replaces this 101.6.19 surfactant), for example (1) sugar ester (selecting hlb values of 3, 7, 11 and 15 respectively) Sucrose ester); (1) Polysorbate fatty acid vinegar (_ 85, 80, 60, 20 and Tween 65, 20, HLB values are 1·8 4·3 6·7, 8.6, 10.6, 16.7). The emulsifier concentration selected above is based on the solid content of the mill and is added at 5%, 1%, 2% and 50%. Β·Storage test The ground and stabilized Ganoderma lucidum fruit body suspensions were placed at 4 ° C and room temperature during storage (〇, 〇5, 丨, 2, 6, 12 ' 24 48 72 96 hours) ) 'Sampling for turbidity analysis and particle size analysis. In this test, a small amount of preservative will be added to avoid the growth of microorganisms. C. Determination of turbidity The level of turbidity is related to the amount of particles in the solution. If the nano/submicron particles in the sample aggregate into large particles or even precipitate, the total number of suspended particles decreases, causing the turbidity of the upper solution to decrease. The stability of the suspension liquid of Ganoderma lucidum can be preliminarily judged by turbidity, and the emulsifier which is not suitable can be eliminated. Use

帶 ^濁度 at ( portable turbidimeter, model 21 OOP，HACH company，U，S·Α·)測定儲存期間濁度的變化，檢測前先以標準品（Gelex Secondary Standards)(濁度分別為〇_10 Ντυ 、〇·1〇0 NTU和〇_1〇00 NTU)校正，取研磨及穩定處理後之靈芝子實體懸浮液，經適當稀釋，取15 mL·置入水樣檢測瓶中，直接以濁度計測定之，單位為NTU。 D·界面電位（Zetapotentia丨）測定界面電位是敕子間吸引力或排斥力的指標，該值的增 10 1377065 申請案號第98111670號替換本丨〇丨.<U9 加，顯示粒子越穩定，是測量粒子表面性質的有效工具。將稀溶液置於谓測儀上，賦予粒子固定值的電I，觀察粒子的移動速率，由移動速率與時間的相關性可導出粒子表面的相關性質。界面電位分析儀（加potent )可即時分析界面電位對應#值、乳化劑添加濃度及時間的變化曲線圖及結果，由於界面電位粒子穩定性的重要指標，藉由該數據的分析/;=奈卡求/次微核靈芝微粒再聚集的可能原因，進而探討其基理論。配合現有之濁度計及雷射粒徑分析儀等，將可使產品穩定性之解析更加完備且具時效性，同時瞭解奈米米級靈芝微粒的表面特性，是將來進一步修飾粒子表面性質的重要依據。 E.顯微觀察使用光學顯微鏡（〇ptiph()t_PQl，Nik(m，hpan)觀察奈米/次微米級靈芝微粒再聚集之變化情形。此外，以穿透式電子顯微鏡（讓）（脑_123G，卿L c。ud，—η)觀察儲存期間奈米/次微米級靈芝微粒的顯微結構，以確認挪分佈的測量及再聚集之結構，並以掃描式電子顯微鏡；The turbidity change during storage was measured with a turbidity at (portable turbidimeter, model 21 OOP, HACH company, U, S·Α·), and the standard (Gelex Secondary Standards) was used before the test (the turbidity was 〇10, respectively). Ντυ, 〇·1〇0 NTU and 〇_1〇00 NTU) Correction, take the grinding and stable treatment of the suspension of Ganoderma lucidum fruit body, after appropriate dilution, take 15 mL · placed in the water sample test bottle, directly turbid Determined by the meter, the unit is NTU. D. Interface potential (Zetapotentia丨) The interface potential is an indicator of the attraction or repulsive force between the scorpions. This value is increased by 10 1377065. Application No. 98111670 is replaced by the 丨〇丨. U9 plus, showing that the particles are more stable. Is an effective tool for measuring the surface properties of particles. The dilute solution is placed on the pre-measuring instrument, and the fixed value of the electric I is given to the particle, and the moving rate of the particle is observed. The correlation between the moving rate and the time can be used to derive the relevant properties of the particle surface. Interface potential analyzer (plus potent) can instantly analyze the interface potential corresponding to # value, emulsifier concentration and time curve and results, due to the important indicators of interface potential particle stability, by the analysis of the data /; The possible reasons for the re-aggregation of the cardo/micronuclear Ganoderma lucidum particles, and then explore its basic theory. With the existing turbidity meter and laser particle size analyzer, the analysis of product stability can be more complete and time-sensitive. At the same time, understanding the surface characteristics of nanometer-grade Ganoderma lucidum particles will further modify the surface properties of the particles in the future. Important reference. E. Microscopic observation Using an optical microscope (〇ptiph()t_PQl, Nik(m, hpan) to observe changes in the re-aggregation of nano/sub-micron-sized Ganoderma lucidum microparticles. In addition, with a transmission electron microscope (Let) (brain _ 123G, Qing L c.ud, η) Observing the microstructure of nano/sub-micron Ganoderma lucidum particles during storage to confirm the measurement of the distribution and re-aggregation of the distribution, and to scan electron microscopy;

Sc_ing e丨ectron microsc〇pe，随）觀察所得樣品的顯微結構0 (四）28天亞急毒性試驗「依衛生署公告的「藥品非臨床試驗優良操作規範」，和健康食品安全性評估方法」中的毒性試驗方法進行28天亞急毒性試驗。在試驗物質給予期_量㈣H重及食物 11 1377065 申請案號第98111670號替換本 101.6.19 消耗量之變化。剖檢前收集血液樣品，進行血液及血清生化分析。試驗物質給予期間結束，進行剖檢，以肉眼觀察及記錄動物的器官與組織之變化，並測量主要臟器重量。最高劑量組與對照組進行組織病理檢驗。 A. 動物處理動物則採用自台大醫學院動物中心的5_6週ICR小鼠，每劑量組使用雄、雌各12隻動物。在進行毒性測試前，動物首先在具溫度、溼度及光照之動物房適應一週，並自由攝取正常飼料及水。一週後開始餵食實驗，連續28天後，犧牲小鼠。 B. 靈芝劑量在本試驗中，低劑量及中劑量組則分別採0.02及0.2 g/kg/day，高劑量組採用 2 g/kg/day。此低劑量（0.02 g/kg/day)、中劑量（0.2 g/kg/day)、高劑量（2 g/kg/day) 分別為成人每曰建議攝取量之同劑量、10倍及100倍。 C. 血清生化值之測定動物的血液樣本取自頸動脈血放入血清分離管，經 3000 xg 15分鐘離心後取的血清樣本。分析項目如下：高密度脂蛋白（high density lipoprotein, HDL)、低密度脂蛋白 (low density lipoprotein, LDL )、麵氨酸氨基轉化酶（ glutamic oxaloacetic transaminase，GOT)、麵氨酸丙酮酸氨基轉化酶（glutamic pyruvic transaminase, GPT)、血液尿素氮（blood urea nitrogen, BUN)、肌氨酸針（creatinine, CRE )、總膽固醇（cholesterol，CHO )、三酸甘油脂（ 12 1377065 申請案號第98111670號替換本 10丨.6.19 triglyceride, TG )、鈉離子（Na+ )、鉀離子（K+ )、氣離子 (C1-)、葡萄糖（Glucose)等。 D.血液分析動物的血液樣本取自頸動脈血放入含EDTA抗凝劑試管採集1 mL全血，分析項目如下：白血球（white blood cell，WBC)、紅血球（red blood cell，RBC )、血紅素（ hemoglobin, Hb)、血球容積比（hematocrit，Hct)、平均紅血球容積（mean corpuscular, MCV)、平均紅血球血紅素（ mean corpuscular hemoglobin, MCH)、平均红血球血紅球濃度(mean corpuscular hemoglobin concentration, MCHC)、血小板（platelets, PLT )、淋巴球數目（lymphocytes, LYMPH )、紅血球平均寬度（red cell distribution width-coefficient of variation, RDW-CV)、血小板紅 jk 球（platelet distribution width，PDW)、平均血小板容積（mean platelet volume, MPV )、血小板大細胞範圍（platelet-large cell range，P-LCR)等。 Ε·組織病理切片 ' 將所有動物之組織，肝臟、腎臟、心臟、脾臟、肺臟、睪丸、副睪及子宮卵巢等標的器官取下後，放入10% formal’in固定一星期。切片後以hematoxylin and eosin染色後，以顯微鏡觀察。組織病理切片判讀委託台灣動物科技研究所進行。以下即針對本發明靈芝子實體懸浮液之製程與分析進行說明： 13 1377065 申請案號第98111670號替換本 HH.6.19 (一）、研磨製程與粒徑之關係如表1所示研磨條件，於相同研磨時間（90 min)下，探討轉速（2310〜3570 rpm)、進料濃度（0.5〜2.0 g/mL) 和研磨珠填充量（80〜140 mL)對靈芝奈米/次微米化製程之影響。表1 編號離心轉速 (rpm ) 靈芝進料濃度 (g/mL ) 研磨珠填充量 (mL) 1 3570 0.5 140 2 3570 0.5 110 3 3570 0.5 80 4 3570 1.0 140 5 3570 2.0 140 6 2940 0.5 140 7 2310 0.5 140 如圖1所示，轉速提高會增加研磨珠間的碰撞次數和強度，致使溫度快速上升並促使粒子再聚集，造成平均粒徑的增加。提高進料濃度可降低粒子間的平均距離並使研磨珠碰撞範圍内存在較多的粒子而增加研磨效率。填充量過高則可能會限制研磨珠於研磨過程中的運動，降低碰撞速度，致使研磨效率降低。由上述實驗結果可知，在奈米/ 次微米級靈芝子實體懸浮液的研磨製程中，使用較高的固形物濃度，並降低研磨轉速及研磨珠的填充量，應可降低產品的體積平均粒徑。將刖述刖處理所得之靈芝細碎懸浮液於36〇〇卬爪之固定轉速、360 mL/min之固定進料速率和14〇之固定填充量，以及不同固形物濃度與兩種研磨珠大+ (0.8 mm、0.3 mm 釔锆珠）下研磨 30、6〇、9〇、12〇、15〇、18〇和 27〇分鐘發見’’二剷處理後之靈芝細碎懸浮液，其粒徑雖可小於 14 1377065 101.6.19 申請案號第98111670號替換本 300 μπι，但絕大部分仍大於15〇 μιη，因此，欲將靈芝子實體奈米/次微米化需採用階段性研磨，亦即先以0.8 mm纪錯珠細磨，待粒徑降至約5〇 μιη以下，再改以〇 3 mm釔锆珠繼續研磨。如圖2所示，研磨時間亦影響粒徑，隨著研磨時間的增加，體積平均粒徑減少，於研磨6〇分鐘，體積平均粒徑之減少速率較高，隨後趨缓，研磨9〇分鐘後，靈芝微粒之體積平均粒徑約為1.2 μπι。繼之改以〇3 mm锆珠研磨9〇分鐘後’則體積平均粒徑小力i 〇㈣。因所得懸浮液之粒徑分佈範圍較寬，故粒子間易再聚集而沉澱，雖然數目平均粒徑比體積平均粒徑小很多，但為求更佳的品質，本發明是以體積平均粒徑為指標。由圖3、4所示之粒徑分析結果可知，將上述以〇3爪爪釔鍅珠研磨180分鐘所得靈芝子實體懸浮液以i〇〇〇〇g離心 1〇分鐘，除去較大之顆粒後，離心上清液之粒徑分佈範圍為28〜578 nm (圖3)，體積平均粒徑約1〇5nm，其中67% 的粒子小於100 nm，就食品而言，小於i _應足可增加其於禮内的吸收’離心上清液之粒子皆小於i μπι，其固形物含量雖較低，但因研磨而被萃提出之活性成分則皆仍存留下來。而離心沉降物之顆粒尺寸小於1〇 μιη (圖4)，質地細緻，可作為保養品（如面膜）或敷料、人工皮膚之基材 ’且因富含幾Τ質及纖維素’所以可作為_良好的腾食纖維添加物。如圖5所示’顯微觀察研磨後之靈芝微粒，新鮮靈芝 15 1377065 申請案號第98111670號替換本丨01619 子實體經blender授打後，以掃描式電子顯微鏡（se⑷觀察’由圖5B中清楚可見靈芝子實體之樹狀分枝骨骼菌絲。經適當均質處理後’該骨絡菌絲變的更為細碎、鬆散（如圖5C所示），且其大小約在數十至數百微米，而此一狀態將有利於後續之研磨處理。圖5D為經兩段式研磨後，利用穿透式電子顯微鏡（TEM)㈣之結果，圖中圓形顆粒為靈芝子實體懸浮液中的奈米粒子，由seale ^可明顯的看到’單-顆粒確可達（M _以下之奈米級尺度，但因顆粒間的再聚集使整體之粒徑範圍變大，可達數百奈米，即為次微米級尺度。此-結果亦驗證了上餘好析儀所測得之結果’並再次確認本發明使用之介質研磨製程，確實可使靈芝子實體研磨後之靈芝微粒降至奈米/次微米尺产。由於靈芝子實體富含纖維素及幾丁質，質地粗糙且堅勒’將這些纖維物質的粒徑下降，除提高比表面積外，亦可改善產品進食之口感。齡纖維之生理功效肖其吸附作用有關，因此，提高膳食纖維之比表面積，;增進其生理活性。將膳食纖維之粒徑由〇1 mm下降至丨pm，曰比表面積可提高⑽#，膳食纖維之建議攝取量則可減為原，議量的百分之一，若粒徑下降至1〇〇⑽以下，建議攝取量則可減至原建議量的千分之_。換言之，於5⑼扯之飲用水中加入50〜500 ppm之奈米/次微米級腾食纖維，即可達到腊食纖维之每日建議攝取量（25g)。基於這個觀點，靈芝子實體懸浮液之離心上清液不但保有靈芝特有的生理 /舌f生成为，更額外提供高表面積之膳食纖维。 16 1377065 * 申請案號第981Π670號替換本 101.6.19 * (二）研磨製程與有效成分之關係如表2所示，經介質研磨後，β-葡聚糖的含量明顯増加，以較大研磨介質（粒徑0.8 mm)進行研磨，產品中的β-. 葡聚糖含量與熱水萃取產品（約0.01 mg/mL)相近，隨著介質粒徑的降低及研磨時間的增加，β-葡聚糖含量增加為熱水萃取產品的4倍’且高於市面上所收集到的商品（〇,〇〇23 ~ 0.0202 mg/mL )。表2 測試樣品 β-葡聚糖含量（mg/mL) (β-l ,3-D-gIucan ) 熱水萃取 0.010 + 0.0 ' 0.8 mm記錯味研磨 0.0096±0.0004 ~ 0.3 mm纪錯珠研磨 0.0398+0.0003 Green (Amazon Biotechnolosy Co.，Ltd) 0.0023 土 0.0001 Ex (Syngen Biotech Co·，Ltd) 0.01+0.0001 Gene (Genefrem Biotechnology Co., Ltd) 0.02±0.0001 Natsuki (Hill-Top Food Co., Ltd) 0.0079+0.0001 Join-Yes (Join-Yes International Co., Ltd) 0.0025+0.0 ~~~~ 各樣品皆進行三次取樣測試’：=次平均值+SD " ~~ 一般的市售商品’其聲稱之有效成分多以熱水或有機溶劑來提取’雖可取得該有效成分，但提取量有限，且尚有加工後之廢水、有機溶劑及殘渣處理問題。以機械式研磨的製程方式，不但可使靈芝子實體懸浮液中具有纖維素、幾丁質等腾食纖維，亦可藉由細磨過程中靈芝子實體細胞壁的破碎，促使有效成份的釋出。如表3 .所示，幾丁質含量亦有類似的現象，以ο」爪以介質研磨所得之產品中幾丁質含量高☆ 〇8 mm介質研磨所獲得的產品，但若以離心方式將所得產品中之較大的顆粒移除後，上清液之幾丁質含量則大幅下降，但仍約為一般 17 1377065 101.6.19 申請案號苐98111670號替換本熱水卒取所得產品的5肖，可見該被移除之顆粒含有豐富的幾丁質。由於熱水提取幾乎只可抽得水溶性物質，故其提取液中之幾丁質含量甚低，絕大部分都殘存在藥潰中。樂凌約含有35〜4G%幾丁質’ 4G〜5G%p_葡聚糖，其餘為以黑色素（_anines)為主之含氮物’為良好的膳食纖維來源’如將子實體粉碎並細磨至奈米/次微米尺寸，不僅可取代萃取，產品令亦將保存子實體的所有成分（富含幾丁質有助於人體的吸收，、纖維素等膳食纖維），且因粒子小所以此奈求/次微求級靈芝子實體懸浮液或其濃縮產品將適於作為保健食品之添加物。表3 測試樣品熱水萃取機械式研磨 4 丁質濃度（mg/mL) 0 05+0.0018 〇 · 8紀錯珠研磨 1.14+0.0116 一 0.3記錯珠研磨 1 -98+0.01 43 _〇.3釔锆珠研磨德之離心上清& 0-25 + 0.001 8 離心上清液··丨〇〇〇〇g，1 〇分鐘各樣*皆進行=次取樣測試，=次平珀佶+SD 1 (三）靈芝子實體懸浮液之穩定性如圖6〜8所示，靈芝子實體經研磨後，高濃度的靈芝微粒及寬廣的粒徑分佈，促使靈芝微粒間相互碰撞並迅速聚集成較大之微粒團簇，粒徑的增加致使重力效應遠大於布朗運動效應，進而產生明顯的沉降現象。於下靜置 24小時後分析其粒徑，微粒已聚集並形成數十微米之團簇，體積平均粒徑增至9·35 μηι (如圖6所示），考量研磨產品於後續加工，如冷凍貯藏、冷凍乾燥或高溫滅菌加工等之安定性，針對冷凍（-2(TC，24小時）及高壓蒸汽滅菌（ 18 1377065 ' 申請案號第98111670號替換本 101.6.19 . 121°C，15分鐘）後之子實體懸浮液進行粒徑分析，以探討加工前後之粒徑分佈差異》靈芝子實體懸浮液之靈芝微粒於冷凍過程中會再聚集，解凍後似有離水之相分離現象，聚集形成的微粒團簇呈棉絮狀並難以物理或機械力（如震盪、均質、超音波震盪處理）將之分散，冷凍後子實體懸浮液之粒徑分佈如圖7 ，體積平均粒徑為1〇9 pm。高壓蒸汽滅菌處理亦會造成微粒間嚴重的再聚集作用，形成呈團塊狀之微粒團簇，並如同冷凍處理般難以物理或機械力將之分散，其粒徑分佈範圍5.9〜824.5 μιη，體積平均粒徑為132 μηι (如圖8所示機 . 械式研磨所得之靈芝子實體懸浮液，粒徑分佈範圍寬廣，易再聚集沉澱，但固形物含量高，富含幾丁質、纖維素，此靈芝子實體懸浮液或其濃縮產品將適於作為保健食品之添加物。 (四）靈芝子實髅懸浮液之離心上清液的穩定性如表4所示，研磨時間的改變，除了會影響靈芝微粒平均粒徑外，也間接影響儲存安定性，研磨時間越長者，產。。的穩定性越高。研磨3〇分鐘之靈芝子實體懸浮液的離心上清液產品（1000〇g離心10分鐘），經21天的儲存體積平均粒徑由〇. 195增加為丨丨99 μηι，但研磨180分鐘之靈芝子實體懸浮液的上清液產品（1〇〇〇〇g離心1〇分鐘），體積平均粒杈由〇. 126增為〇〖37 μηΊ，增加率低於丨〇%，顯示增加研磨時間有助於粒子的穩定性。 19 1377065 申請案號第98111670珑替換本 101.6.19 表4___ 餘存時間（天） —趙積平均粒徑（“m) It量平均粒5徑（/^)2丨· Λ ^ ^ )__( volume mean diameter) ( nnmK〇. a：____ ， 30 0.195 0.610 1.199 v uum〇( 0.103 ϊγ mean aia 0.127 meter ) 0 131 60 0.158 0.236 0.341 0.079 0.107 0 090 90 0.151 0.173 0.202 0.078 0.070 0 092 120 0.149 0.149 0.153 0.090 0.073 0 078 150 0.139 0.143 0.138 0.071 0.056 0 070 180 CXL 0 Λ η/ 0.126 0.133 0.137 0.066 0.068 0.064 研磨30〜90分鐘：以0.8 mm記錯珠進行之第一階段研磨結果。，研磨120~18〇分鐘：以0.3 mm釔锆珠進行之笛二階段研磨結罢。接著，就凍乾、高壓蒸汽滅菌及濃縮處理對靈芝子實體懸浮液之離心上清液的影響做一探討。如圖9所示’先將靈芝上清液經凍乾後，再分散於水溶液中’並以Nanotrac 150粒徑分析儀測定粒徑分佈（粒禮1測上限為6.54 μιη )，由結果可知，康乾處理會促使靈芝微粒間再聚集。凍乾後，粒徑分佈範圍寬廣，有相當比例微粒之粒徑大於6.54 μηι (礙於儀器量測上限而無法測得 )，其體積平均粒徑為1.177 μιη。如圖10a所示，離心上清液經高壓蒸汽滅菌處理後仍保有相當好的安定性，滅菌處理後，待溫度下降至室溫即量測其粒徑’所有粒子皆小於1 μηι，粒徑分佈範圍36〜8〇〇 nm’體積平均粒徑為140 nm’且仍有56%的微粒維持在 100 nrn以下。觀察其於室溫下貯存28天（如圖i〇b所示）及1年（如圖10c所示）之粒徑變化，粒徑分佈雖有往大粒徑偏移，但偏移幅度甚小，而其體積平均粒徑亦只分別辦至165 nm和373 nm，顯示離心上清液經高溫作用後仍十分 20 1377065 101.6.19 申請案號第98111670號替換本穩定’粒子於貯存期間之再聚集現象並不顯著。藉由離心雖可將離心上清液之粒徑分佈降至1 μηι以下 ’並使其具有較佳之穩定性，但相對的，離心上清液之固形物含量及靈芝微粒數則大幅減少。為提高固形物濃度，以減壓濃缩方式降低離心上清液之水分含量，並於不同濃縮倍率（concentration ratio)下取樣，量測其粒徑變化。濃縮倍率之計算是以原液體積除上最終濃縮液體積。圖u與表5為離心上清液經2、4 ' 8、12及2〇倍濃縮後之粒徑分析結果，低於4倍濃縮之離心上清液，其粒徑變化不大，體積平均粒徑只增加33%，且微粒皆小於i μηι。濃縮8倍以上時，粒子間開始有聚集現象產生，並使粒徑分佈往大粒徑偏移’粒徑分佈大於i μιηβ就本實施例而言，為提高產品固形物濃度並兼顧安定性，將離心上清液濃縮“倍應可符合所需。上清液濃縮倍率 (濃度） 0 [3.4 mg/mL) 2 (6,8 mg/mL) 4 (13.6 mg/mL) 體積平均粒徑 (U m)Sc_ing e丨ectron microsc〇pe, with) observation of the microstructure of the sample 0 (4) 28-day sub-acute toxicity test "according to the "Good Practices for Non-Clinical Testing of Drugs" published by the Department of Health, and health food safety assessment methods The toxicity test method was carried out for a 28-day sub-acute toxicity test. During the test substance administration period _ quantity (four) H weight and food 11 1377065 application number No. 98111670 replace the change of 101.6.19 consumption. Blood samples were collected prior to necropsy for blood and serum biochemical analysis. At the end of the test substance administration period, a necropsy was performed to visually observe and record changes in the organs and tissues of the animals, and measure the weight of the main organs. Histopathological examination was performed in the highest dose group and the control group. A. Animal Treatment Animals were treated with 5-6 weeks ICR mice from the Animal Center of National Taiwan University Medical College. Each dose group used 12 male and female animals. Prior to the toxicity test, the animals were first acclimatized for one week in an animal room with temperature, humidity and light, and were free to ingest normal feed and water. The feeding experiment was started one week later and the mice were sacrificed after 28 consecutive days. B. Ganoderma Lucidum In this test, 0.02 and 0.2 g/kg/day were taken in the low- and medium-dose groups, respectively, and 2 g/kg/day in the high-dose group. This low dose (0.02 g/kg/day), medium dose (0.2 g/kg/day), and high dose (2 g/kg/day) are the same dose, 10 times and 100 times the recommended intake for each adult. . C. Determination of serum biochemical values Animal blood samples were taken from carotid blood into a serum separation tube and centrifuged at 3000 xg for 15 minutes. The analysis items are as follows: high density lipoprotein (HDL), low density lipoprotein (LDL), glutamic oxaloacetic transaminase (GOT), and amino acid pyruvate aminotransferase (glutamic pyruvic transaminase, GPT), blood urea nitrogen (BUN), creatinine (CRE), total cholesterol (cholesterol, CHO), triglyceride (12 1377065 Application No. 98111670) Replace 10丨.6.19 triglyceride, TG ), sodium ion (Na+ ), potassium ion (K+ ), gas ion (C1-), glucose (Glucose), and the like. D. Blood analysis Animal blood samples were taken from carotid blood and 1 mL of whole blood was collected from EDTA anticoagulant tubes. The analysis items were as follows: white blood cell (WBC), red blood cell (RBC), heme. (hemoglobin, Hb), hematocrit (Hct), mean corpuscular (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) Platelets (PLT), lymphocytes (LYMPH), red cell distribution width-coefficient of variation (RDW-CV), platelet distribution width (PDW), mean platelet volume (mean platelet volume, MPV), platelet-large cell range (P-LCR), and the like. Ε · Histopathological section ' Remove all the tissues of the animal, liver, kidney, heart, spleen, lungs, testicles, sputum, uterus and ovary, and put them in 10% formal'in for one week. After sectioning, staining with hematoxylin and eosin was observed under a microscope. Histopathological section interpretation was commissioned by the Taiwan Institute of Animal Science and Technology. The following is a description of the process and analysis of the suspension of the Ganoderma lucidum fruit body of the present invention: 13 1377065 Application No. 98111670 Replace the HH.6.19 (I), the relationship between the grinding process and the particle size is as shown in Table 1, Under the same grinding time (90 min), the rotational speed (2310~3570 rpm), the feed concentration (0.5~2.0 g/mL) and the grinding bead filling amount (80~140 mL) were applied to the Ganoderma lucidum nano/submicron process. influences. Table 1 No. Centrifugal Speed (rpm) Ganoderma Lucidum Feed Concentration (g/mL) Abrasive Bead Filling Volume (mL) 1 3570 0.5 140 2 3570 0.5 110 3 3570 0.5 80 4 3570 1.0 140 5 3570 2.0 140 6 2940 0.5 140 7 2310 0.5 140 As shown in Figure 1, an increase in the number of revolutions increases the number and intensity of collisions between the beads, causing the temperature to rise rapidly and causing the particles to reaggregate, resulting in an increase in the average particle size. Increasing the feed concentration reduces the average distance between the particles and results in more particles in the impact zone of the grinding beads, increasing the grinding efficiency. Excessive filling may limit the movement of the beads during the grinding process and reduce the collision speed, resulting in reduced grinding efficiency. It can be seen from the above experimental results that in the grinding process of the nano/sub-micron-sized Ganoderma lucidum fruit body suspension, the use of a higher solid concentration, and the reduction of the grinding speed and the filling amount of the grinding beads should reduce the volume average particle size of the product. path. The fixed speed of the Ganoderma lucidum finely divided suspension obtained by the treatment of the 〇〇卬于于 is fixed at a fixed speed of 36 〇〇卬, a fixed feed rate of 360 mL/min, and a fixed filling amount of 14 ,, and different solid concentrations and two kinds of grinding beads + (0.8 mm, 0.3 mm 钇 zirconium beads) under grinding 30, 6 〇, 9 〇, 12 〇, 15 〇, 18 〇 and 27 〇 minutes, see the ''two shovel treatment of Ganoderma lucidum finely divided suspension, although the particle size Can be less than 14 1377065 101.6.19 Application No. 98111670 replaces this 300 μπι, but most of it is still greater than 15〇μιη, therefore, it is necessary to use staged grinding for the nano/submicron of the Ganoderma lucidum fruit body, that is, first Finely grind with a 0.8 mm eclipse, until the particle size falls below about 5 〇μηη, and then continue grinding with 〇3 mm 钇 zirconium beads. As shown in Fig. 2, the grinding time also affects the particle size. As the grinding time increases, the volume average particle size decreases. After grinding for 6 minutes, the volume average particle diameter decreases at a higher rate, and then slows down, grinding for 9 minutes. Thereafter, the volume average particle diameter of the ganoderma lucidum particles is about 1.2 μm. Then, after grinding for 9 minutes with 〇3 mm zirconium beads, the volume average particle size is small, i 〇 (4). Since the obtained suspension has a wide particle size distribution range, the particles are easily reaggregated and precipitated. Although the number average particle diameter is much smaller than the volume average particle diameter, in order to obtain better quality, the present invention is a volume average particle diameter. For the indicator. From the results of particle size analysis shown in Figs. 3 and 4, it was found that the above-mentioned suspension of Ganoderma lucidum fruit body obtained by grinding the 〇3 claw claw beads for 180 minutes was centrifuged at i〇〇〇〇g for 1 minute to remove larger particles. After that, the centrifugation supernatant has a particle size distribution ranging from 28 to 578 nm (Fig. 3), and the volume average particle diameter is about 1 〇 5 nm, wherein 67% of the particles are smaller than 100 nm, and in the case of food, less than i _ is sufficient. Increasing the absorption in the ritual 'The supernatant of the centrifugation supernatant is less than i μπι, although the solid content is low, but the active ingredients extracted by grinding still exist. The centrifugal sediment has a particle size of less than 1〇μηη (Fig. 4). It has a fine texture and can be used as a skin care product (such as a mask) or a dressing, artificial skin substrate, and because it is rich in several tannins and cellulose. _Good dietary fiber supplements. As shown in Fig. 5, 'microscopic observation of the grounded Ganoderma lucidum particles, fresh Ganoderma lucidum 15 1377065 application No. 98111670, replacing the 丨01619 fruiting body after being manipulated by Blender, and scanning electron microscope (se(4) observation' from Figure 5B The dendritic mycelium of the Ganoderma lucidum fruiting body is clearly visible. After proper homogenization treatment, the mycelium of the bone is more finely divided and loose (as shown in Fig. 5C), and its size is about tens to hundreds. Micron, and this state will facilitate the subsequent grinding process. Figure 5D is the result of a two-stage grinding using a transmission electron microscope (TEM) (4), the circular particles in the suspension of Ganoderma lucidum fruit body Nanoparticles, by seale ^ can clearly see that 'single-particles are indeed reachable (M _ below the nanometer scale, but due to the re-aggregation between particles, the overall particle size range becomes larger, up to hundreds of Meter, which is the sub-micron scale. This results also verified the results measured by the analytical analyzer and reconfirmed that the medium grinding process used in the present invention can actually reduce the Ganoderma lucidum particles after grinding the Ganoderma lucidum fruit body. Nano/sub-micron size. The Ganoderma lucidum fruiting body is rich in cellulose and chitin, and the texture is rough and the Gule's particle size is reduced. In addition to increasing the specific surface area, it can also improve the taste of the product. The physiological effect of the aged fiber is absorbed. Related to the effect, therefore, increase the specific surface area of dietary fiber; increase its physiological activity. The particle size of dietary fiber decreased from 〇1 mm to 丨pm, the specific surface area of 曰 can be increased (10)#, and the recommended intake of dietary fiber can be reduced. For the original, one percent of the discussion, if the particle size drops below 1〇〇(10), it is recommended that the intake can be reduced to the original recommended amount of 千. In other words, add 50~ to the drinking water of 5(9) 500 ppm of nano/sub-micron-sized dietary fiber can achieve the daily recommended intake of wax fiber (25g). Based on this point of view, the centrifugation supernatant of the suspension of Ganoderma lucidum fruit body not only retains the unique physiology of Ganoderma lucidum / Tongue f is generated to provide extra high surface area dietary fiber. 16 1377065 * Application No. 981Π670 Replacement of this 101.6.19 * (2) The relationship between the grinding process and the active ingredients is shown in Table 2, and the media is ground. The content of β-glucan is obviously increased, and it is ground with a larger grinding medium (particle size 0.8 mm). The content of β-. glucan in the product is similar to that of hot water extraction product (about 0.01 mg/mL). With the decrease of the particle size of the medium and the increase of the grinding time, the β-glucan content is increased by 4 times that of the hot water extraction product and is higher than the products collected on the market (〇, 〇〇23 ~ 0.0202 mg/mL) Table 2 Test sample β-glucan content (mg/mL) (β-l,3-D-gIucan) Hot water extraction 0.010 + 0.0 ' 0.8 mm wrong taste grinding 0.0096±0.0004 ~ 0.3 mm Jiuzhu beads grinding 0.0398+0.0003 Green (Amazon Biotechnolosy Co., Ltd) 0.0023 0.0001 Ex (Syngen Biotech Co., Ltd) 0.01+0.0001 Gene (Genefrem Biotechnology Co., Ltd) 0.02±0.0001 Natsuki (Hill-Top Food Co., Ltd) 0.0079+0.0001 Join-Yes (Join-Yes International Co., Ltd) 0.0025+0.0 ~~~~ Each sample was tested three times ':==average value + SD " ~~ general commercial goods' claim The active ingredients are mostly extracted with hot water or organic solvents. 'Although the active ingredients can be obtained, but the extraction amount is limited. And there is still processing the waste water, an organic solvent and residue disposal problems. In the mechanical grinding process, not only can the somatic fiber in the suspension of Ganoderma lucidum fruit have cellulose, chitin, etc., but also the cell wall of the Ganoderma lucidum fruit body can be broken during the fine grinding process to promote the release of the active ingredient. . As shown in Table 3, the chitin content also has a similar phenomenon, in which the product obtained by grinding the medium with a high degree of chitin is ☆ 〇 8 mm medium grinding, but if it is centrifuged After the larger particles in the obtained product are removed, the chitin content of the supernatant is greatly reduced, but it is still about the general 17 1377065 101.6.19 application number 苐98111670 to replace the product obtained from the hot water pumping Xiao, it can be seen that the removed particles are rich in chitin. Since the hot water extraction can only extract water-soluble substances, the chitin content in the extract is very low, and most of them are left in the medicine. Leling contains about 35~4G% chitin '4G~5G%p_glucan, and the rest is nitrogen-containing (_anines)-based nitrogen-good as a good source of dietary fiber, such as crushing and thinning fruiting bodies Grinding to nano/sub-micron size, not only can replace the extraction, the product will also preserve all the components of the fruiting body (rich in chitin helps the body to absorb, dietary fiber such as cellulose), and because the particles are small This nevi/sub-leveling Ganoderma lucidum fruit body suspension or its concentrated product will be suitable as an additive for health foods. Table 3 Test sample Hot water extraction Mechanical grinding 4 Butyl concentration (mg/mL) 0 05+0.0018 〇· 8 Ji wrong beads grinding 1.14+0.0116 A 0.3 mark wrong bead grinding 1 -98+0.01 43 _〇.3钇Zirconium Beads Grinding Centrifugal Supernatant & 0-25 + 0.001 8 Centrifugal supernatant ··丨〇〇〇〇g, 1 〇 min *all = test sampling, = times flat 佶佶 + SD 1 (3) The stability of the suspension of Ganoderma lucidum fruit body is shown in Figure 6~8. After the grinding of the Ganoderma lucidum fruiting body, the high concentration of Ganoderma lucidum particles and the broad particle size distribution promote the collision between the Ganoderma lucidum particles and the rapid integration. The particle clusters, the increase in particle size, cause the gravity effect to be much larger than the Brownian motion effect, which in turn produces significant sedimentation. After standing for 24 hours, the particle size was analyzed, the particles were aggregated and formed into clusters of several tens of micrometers, and the volume average particle size was increased to 9·35 μηι (as shown in Fig. 6), and the abrasive product was considered for subsequent processing, such as The stability of frozen storage, freeze-drying or high-temperature sterilization, etc., for freezing (-2 (TC, 24 hours) and autoclaving (18 1377065 ' Application No. 98111670, replacing this 101.6.19. 121 °C, 15 After the minute, the fruiting body suspension is subjected to particle size analysis to investigate the difference in particle size distribution before and after processing. The ganoderma lucidum particles of the suspension of Ganoderma lucidum fruit body re-aggregate during the freezing process. After thawing, there seems to be separation from the water phase. The particle clusters are cotton-like and difficult to disperse by physical or mechanical forces (such as shock, homogenization, and ultrasonic vibration). The particle size distribution of the frozen fruit body suspension is shown in Figure 7. The volume average particle size is 1〇9. Pm. Autoclaving also causes severe re-aggregation between the particles, forming clusters of clusters of particles, and it is difficult to physically or mechanically disperse it as it is frozen. The cloth range is 5.9~824.5 μιη, and the volume average particle size is 132 μηι (as shown in Figure 8. The mechanical suspension of the Ganoderma lucidum fruit body suspension has a wide particle size distribution, easy to accumulate and precipitate, but the solid content is high, Rich in chitin and cellulose, this suspension of Ganoderma lucidum fruit body or its concentrated product will be suitable as an additive for health foods. (4) The stability of the centrifugation supernatant of the suspension of Ganoderma lucidum is shown in Table 4. It shows that the change of grinding time, in addition to affecting the average particle size of Ganoderma lucidum particles, also indirectly affects the storage stability. The longer the grinding time, the higher the stability. The centrifugation of the suspension of Ganoderma lucidum fruit body after 3 minutes of grinding The supernatant product (centrifuged at 1000 〇g for 10 minutes), the 21-day storage volume average particle size increased from 〇.195 to 丨丨99 μηι, but the supernatant product of the suspension of the body extract of Ganoderma lucidum for 180 minutes (1 〇〇〇〇g centrifugation for 1 ))), the volume average granules increased from 〇. 126 to 〇37 μηΊ, the increase rate is lower than 丨〇%, indicating that increasing the grinding time contributes to the stability of the particles. 19 1377065 No. 9 8111670珑Replace this 101.6.19 Table 4___ Remaining time (days)—Ze product average particle size (“m) It quantity average grain 5 diameter (/^) 2丨· Λ ^ ^ )__( volume mean diameter) ( nnmK :. a:____, 30 0.195 0.610 1.199 v uum〇( 0.103 ϊγ mean aia 0.127 meter ) 0 131 60 0.158 0.236 0.341 0.079 0.107 0 090 90 0.151 0.173 0.202 0.078 0.070 0 092 120 0.149 0.149 0.153 0.090 0.073 0 078 150 0.139 0.143 0.138 0.071 0.056 0 070 180 CXL 0 Λ η/ 0.126 0.133 0.137 0.066 0.068 0.064 Grinding for 30 to 90 minutes: The first stage grinding result with a 0.8 mm mark bead. Grinding for 120~18〇 minutes: two-stage grinding of the flute with 0.3 mm yttrium zirconium beads. Next, the effects of lyophilization, autoclaving and concentration treatment on the centrifugation supernatant of the body suspension of Ganoderma lucidum were discussed. As shown in Fig. 9, 'the supernatant of Ganoderma lucidum was lyophilized and then dispersed in an aqueous solution' and the particle size distribution was measured by a Nanotrac 150 particle size analyzer (the upper limit of the particle size measurement was 6.54 μιη). Kanggan treatment will promote the re-aggregation between Ganoderma lucidum particles. After lyophilization, the particle size distribution range is wide, and the particle size of the particles is relatively larger than 6.54 μηι (which cannot be measured due to the upper limit of the instrument measurement), and the volume average particle diameter is 1.177 μιη. As shown in Fig. 10a, the centrifuged supernatant retains a fairly good stability after autoclaving. After sterilization, the particle size is measured until the temperature drops to room temperature. All particles are less than 1 μηι. The distribution range is 36~8〇〇nm' volume average particle size is 140 nm' and 56% of the particles remain below 100 nrn. Observe the particle size change at room temperature for 28 days (as shown in Figure i b) and 1 year (as shown in Figure 10c). Although the particle size distribution has a large particle size shift, the offset is very high. It is small, and its volume average particle size is only 165 nm and 373 nm, respectively. It shows that after centrifugation of the supernatant, it is still very high. 20 1377065 101.6.19 Application No. 98111670 Replaces the stable 'particles during storage. The phenomenon of re-aggregation is not significant. Although the particle size distribution of the centrifugation supernatant can be reduced to less than 1 μηη by centrifugation and the stability is preferably maintained, the solid content of the centrifugation supernatant and the number of ganoderma lucidum particles are relatively reduced. In order to increase the solid content concentration, the moisture content of the centrifugal supernatant was reduced by a reduced pressure concentration method, and samples were taken under different concentration ratios to measure the particle size change. The concentration reduction is calculated by dividing the final concentrate volume by the volume of the stock solution. Figure u and Table 5 show the results of particle size analysis after centrifugation of the supernatant by 2, 4 ' 8, 12 and 2 times. The concentration of the supernatant is less than 4 times, and the particle size does not change much. The particle size is only increased by 33%, and the particles are all smaller than i μηι. When the concentration is more than 8 times, aggregation occurs between the particles, and the particle size distribution is shifted to the large particle size. The particle size distribution is larger than i μιηβ. In the present embodiment, in order to improve the solid content concentration of the product and to achieve stability, Concentrate the centrifugation supernatant to "should be as desired. Supernatant concentration ratio (concentration) 0 [3.4 mg/mL) 2 (6,8 mg/mL) 4 (13.6 mg/mL) Volume average particle size ( U m)

0.140 表5 粒徑分布範圍 (// m) 0.03-0.578 0.036 〜0.750 0.030 〜0.972 粒徑小於 m之微粒含晉（％、 1000.140 Table 5 Particle size distribution range (// m) 0.03-0.578 0.036 ~0.750 0.030 ~0.972 Particles smaller than m Particles containing gold (%, 100

100 100 (27.2 mg/mL) 12 (40.8 mg/mL) 20 (68.0 mg/mL) 0.351 0-033-2.120 95 1.922 1.880 〇· 111 〜6.54 〇_ 8 5 9〜6 · 5 4 61 ~ 1~ -----__ 0 1(五）乳化劑對靈芝子實體懸浮液穩定性之影響濁度是-量測簡易之物理參數，對穩定分散之懸浮溶液而言，其濁度不應隨時間而呈現顯著差異。因此 21 1377065 101.6.19 申請案號第98111670號替換本 Μ篩選較佳明針對所欲探討的各種變因，進行濁度分析，的乳化劑。如表6所示，未添加乳化劑之靈芝子實體經研磨後，靈芝子實體懸浮液濁度高於職麵，稀釋Η磨麦 5〇倍後之試樣的濁度均隨時間而逐漸下降，稀釋，濁度之變化率愈小。低濃度（高稀釋倍率）懸浮液之濁= 定性較U度（⑽釋倍率）佳，⑽餘濃度是料縣^液穩定性的重要參數，濃度愈高微粒間碰撞機率愈高現^也相5明顯。整體而言，微粒間的聚集相當明顯，致使靈之子實體懸浮液的聚集沉降行為明顯。100 100 (27.2 mg/mL) 12 (40.8 mg/mL) 20 (68.0 mg/mL) 0.351 0-033-2.120 95 1.922 1.880 〇· 111 ～6.54 〇_ 8 5 9~6 · 5 4 61 ~ 1~ -----__ 0 1 (5) Effect of Emulsifier on Stability of Ganoderma lucidum Fruit Body Suspension Turbidity is a simple physical parameter for measurement. For a stably dispersed suspension solution, the turbidity should not be with time. And there is a significant difference. Therefore, 21 1377065 101.6.19 Application No. 98111670 Replacement This is an emulsifier for turbidity analysis for various variables to be explored. As shown in Table 6, after the emulsifier-free Ganoderma lucidum fruit body was ground, the turbidity of the Ganoderma lucidum fruit body suspension was higher than that of the face, and the turbidity of the sample after the mashing of the mashed rice was gradually decreased with time. , dilution, the rate of change in turbidity is smaller. Low concentration (high dilution ratio) suspension turbidity = qualitatively better than U degree ((10) release rate), (10) residual concentration is an important parameter for the stability of the material reservoir. The higher the concentration, the higher the collision probability between particles. 5 obvious. On the whole, the aggregation between the particles is quite obvious, resulting in obvious aggregation and sedimentation behavior of the suspension of the fruit body.

表6 於本發明令，選擇以不同HLB(hydr〇phileiip〇phiie bal e親水親脂均衡）之嚴糖醋（叫肛ester，HLB分別為3、7、il和15)、聚山梨醇酐脂肪酸酯（Span 85、8〇、 6〇、2〇和 Tween 65、20，HLB 分別為 1>8、4.3、6 7、8 6 寿16·7 )及脂肪酸甘油脂（HLB為3.8)作為初步篩 22 1377065 ' 申請索號苐兕〗丨1670號替換本 1〇ι.6.19 選的乳化劍，添加量先固定為相對於靈芝子實體重量之5 % ^ 2 M所7^ ’分别為$芝子實體懸浮液稀釋倍率5 210倍和5G倍下，濁度變化率隨時間之_。濁度變化李為負值表示濁度下降’正值則表示上升。除了少數的正值外，幾乎所有試驗的濁度變化趨勢均為下降。整體而S，稀釋倍率愈高，微粒間碰撞機率降低，聚集作用減緩，濁度隨時間的變化率趨緩，各稀釋倍率的濁度均隨時間的增加而下降。在最高稀釋倍率50的情況下(靈芝微粒濃度約議卿 (0.001 mg/mL))，濁度變化率最小，且以聚山梨醇針脂肪酸醋類乳化劑的穩定性較佳，於96小時後濁度變化率絕對值均小於3G %。於低稀釋倍率（5倍及1()倍）下，濁度變化率與乳化劑種類、HLB值似無關聯性，％小時後濁度：化率絕對值均大於80 % ’即無顯著穩定效果。 >因此，如以濁度作為判斷基準’就靈芝子實體懸浮液而呂，以5 %添加量之聚山梨醇酐脂肪酸酯作為分散劑，對濃度100 PPm (最高稀釋倍率50倍）之靈芝子實體懸浮液有較佳的穩定性。由表7之粒徑（靈芝微粒濃度為1〇〇 ppm)分析結果可知’未添加乳化劑之靈芝子實體懸浮液經靜置存放96小時後，靈芝微粒再聚集後的體積平均粒徑（MV)為3 24 pm ’其中’粒徑小於1 的微粒（奈米/次微米級）佔靈芝子實體總質量的24.9%，粒徑屬奈米尺度（小於10〇 nm)者 23 13/7065 101.6.19 申請索號第98111670號替換本二佔靈芝子貫體總質量的2〇4%。於研磨時時’添加脂肪竣甘油脂—及…者之趙積千: 粒，幅度較小，分別變為一.9。、— = 提同界面電位值將有助於微粒之穩定性，靈芝子實體懸浮液未添加乳化劑之界面電位值約_117 _，本實施例所^ 用乳化劑中，^Span 85、Span 8〇及—2〇對於微粒界面_ 電位的增加較為顯著，界面電位值分別為-16_7、_13.8及 13.7 mV。综合上述濁度分析 '粒徑分析和介面電位分析結果，於乳化劑添加量5%的情況下，HLB值小的乳化劑有助於穩定靈芝子實體懸浮液系統，因此，選擇蔗糖酯（HLb 3 )、脂肪酸甘油脂（HLB 3.8)、Span 85 (HLB ι8)及 span 80 (HLB 4 _ 3 )等，進行乳化劑添加量對靈芝子實體懸浮液系統穩定性影響之測試。 24 1377065 申請索號第98111670號替換本 101.6.19 ---1 測試樣品 ~ 7 pH 介面電位 (mV、體積平均粒徑 (Um) 數量平均粒徑無添加乳化剤靈芝子實體懸浮液 5.69 (JW m) -11.7 3.24 0 124 靈芝子實體上清液 5.47 -20.0 0.100 〇有添加乳化剤蔗糖酯（HLB 3) 5.64 -U.5 10.1 0 107 蔗糖酯（HLB 7) 5.66 -12.1 20.7 Λ 1 rn 蔗糖酯（HLB 11) 5.65 -10.4 21.9 0.101 蔗糖酯（HLB 15) 5.63 --二_ •10.3 14.8 Π 1 ΓΠ 脂肪酸甘油脂（HLB 3.8) 5.70 -10.7 3.86 ν . 1 V ^ 0.107 Span 85 (HLB 1.8) 5.67 -1 6.7 2.90 0 119 Span 80 (HLB 4.3) 5.63 -13.8 3.68 Π 1 ΠΟ Span 60 (HLB 6.7) 5.68 -10.9 6.52 0 114 Span 20 (HLB 8.6) 5.65 Γ -13.7 8.41 Π 1 1 1 Tween 65(HLB 10.6) 5.62 -8.8 23.3 〇 ι Λ? Tween 20(HLB 16.7) 5.64 -11.3 55.4 Λ 1 Λ 1 ν · 1 V/ 1 如表8、9所示，靈芝子實體懸浮液於不同乳化劑添加量（相對於靈芝子實體重量之5%、1〇% 、2〇%和5〇%)時之粒徑分析、界面電位量測結杲。由粒徑分佈結果可知，四種乳化劑中以Span 80之穩定效果較佳，其添加量1〇%時，可將微粒之體積平均粒徑降至2·29 μηι，並提高奈米和次微米粒子所佔的比例。觀察界面電位之變化情形可發現，提高四種乳化劑的添加量皆可增加靈芝子實體懸浮液之界面電位值，但高於20%則穩定效果有限β 25 1377065 申請案號第981U670號替換本 101.6.19 表8 乳化劑種類乳化劑添加量（％) 體積平均粒徑（μπι) 數量平均粒徑（μπι) 奈米粒子含量（wt%) 奈米/次微米粒子含量 (wt%) 無乳化劑 0 3.43±0·23 0.121 2.04 24.9 蔗糖酯（HLB 3) 5 10·5±0·16 0.106 1.99 12.6 脂肪酸甘油脂 (HLB 3.8) 5 3·91±0·05 0.107 2.73 17.2 10 5.94±0.36 0.116 2.85 26.3 20 9.06±0.56 0.102 2.91 15.6 Span 85 (HLB 1.8) _5 2.95±0·06 0.111 2.61 20.1 !〇_ 2·55±0·07 0.139 1.66 33.7 20 2.48±0·07 0.172 0.67 29.4 Span 80 (HLB 4.3) _5 3.78 + 0.14 0.108 3.11 21.8 10 2.29±(Μ4 0.126 3.03 40.6 20 3.51±0·11 0.138 1.93 34.3 表9 乳化劑種類界面電位（zeta potential) 5% 10% 20% 50% 蔗糖酯（HLB 3) 11.5±1·0 -12.9±0.2 -14.2±0.8 脂肪酸甘油脂 (HLB 3.8) -10·7±0·4 -13·8±0.3 16·6±1.1 • Span 85(HLB 1,8、 -15·7±0.9 -1 6.3±0·6 -21.2+0.8 -2 5.3 + 1 7 Span 80(HLB 4.3> -13·8±0·5 -14.1+0.4 -24.2±1·0 -24.7±Γ(Γ 在28天亞急毒性試驗期間，雄、雌性小鼠對照組及處理組之平均每日體重變化、平均每曰食物消耗量與平均每 ’雄、雌性小鼠處理組和對照組其檢測值皆在正常值範圍内，主曰水消耗量皆無顯著差異血液分析亦無明顯差異，要器官之組織切片檢查亦正常，所以在試驗期間並未造成死亡現象以及引發不良臨床徵兆。 ’不觀上述’靈之子實體含有大量粗纖维及木質素成分，且-般真菌細胞壁的成分除了纖維素外尚有幾丁二 =多黯等活性成分亦多存在於細胞壁中，本發明透過機械式研磨來製造靈芝子實體懸浮液的方式，除了無須萃取 26 1377065 申請索號第98111670號替換本丨01.6.19 即可獲得完整的活性成分外，還可大幅提高纖維質比表面積，且纖維質之後續利用無須再粉碎，可作為一良好的腾食纖維添加物。其中，研磨所得之靈芝子實體懸浮液雜後續離心處理後，離心沉降物中富含幾丁質及纖維素，其顆粒尺寸小於Η) μιη，質地細緻，可作為保養品（如面膜）或敷料、人工皮膚之基材，而離心上清液中，靈芝微粒粒徑皆屬奈米/次微米級，並保有完整之活性成份其幾丁質等有效成分為傳統熱水萃取之5倍以上，且含有部分粒徑較小之纖維素，同樣可用以作為保健食品之添加物，故媒實能達成本發明之目的。惟以上所述者，僅為本發明之較佳實施例而已，當不能以此限定本發明實施之範圍，即大凡依本發明申請專利範圍及發明說明内容所作之簡單的等效變化與修飾，皆仍屬本發明專利涵蓋之範圍内。【圖式簡單說明】圖1是體積平均粒徑相對研磨轉速與進料濃度之曲線圖；圖2是研磨時間相對粒徑之曲線圖；圖3是靈芝子實體懸浮液之離心上清液的粒徑分布圖圖4是靈芝子實體懸浮液之離心沉澱物的粒徑分布圖圖5是靈芝子實體之影像圖；說明靈芝子實體自尚未研磨至經過機械式研磨後之粒徑變化； 27 1377065 t 申請案號第98111670號替換本 101.6.19 圖6是靈芝子實體懸浮液經4。(：儲存後之粒徑分布圖；圖7是靈芝子實體懸浮液經過冷凍乾燥後之粒徑分布圖，圖8是靈芝子實體懸浮液經過高壓蒸氣滅菌後之粒經分布圖；圖9疋靈芝子實體懸浮液之離心上清液經冷陳處理後的粒徑分布圖；圖10是靈芝子實體懸浮液之離心上清液經高壓蒸氣滅鹵後之不同儲存時間的粒徑分布圖；圖11疋靈芝子實體懸浮液之離心上清液經不同倍數之濃縮後的粒徑分布圖；圖12是靈芝子實體與乳化劑一起機械式研磨製成之靈芝子實體懸浮液的濁度變化曲線圖；圖13是靈芝子實體與乳化劑一起機械式研磨製成之靈芝子實體懸浮液的濁度變化曲線圖；及圖14是靈芝子實體與乳化劑一起機械式研磨製成之靈芝子實體懸浮液的濁度變化曲線圖。 28 1377065 • 申請案號第98111670號替換本 101.6.19 • 【主要元件符號說明】無 29Table 6 In the present invention, the choice of different HLB (hydr〇phileiip〇phiie bal e hydrophilic lipophilic balance) of sweet and sour vinegar (called analester, HLB 3, 7, il and 15 respectively), polysorbate fat Acidate (Span 85, 8〇, 6〇, 2〇 and Tween 65, 20, HLB 1 > 8, 4.3, 6 7 , 8 6 life 16.7) and fatty acid glyceride (HLB 3.8) as preliminary Sieve 22 1377065 'Application No. 苐兕〗丨 1670 Replace the emulsified sword of 1〇ι.6.19, the amount of addition is fixed to 5% of the weight of the Ganoderma lucidum fruit body ^ 2 M 7 ^ ' respectively The dilution ratio of the fruit body suspension was 5 210 times and 5G times, and the turbidity change rate was over time. Change in turbidity A negative value for Lee indicates a decrease in turbidity. A positive value indicates an increase. With the exception of a few positive values, the turbidity trend of almost all tests was reduced. As a whole, S, the higher the dilution ratio, the lower the collision probability between particles, the slower the aggregation effect, the slower the change rate of turbidity with time, and the turbidity of each dilution ratio decreases with time. At the highest dilution ratio of 50 (Ganoderma lucidum microparticle concentration (about 0.001 mg / mL)), the turbidity change rate is the smallest, and the stability of the polysorbate needle fatty acid vinegar emulsifier is better, after 96 hours The absolute value of the turbidity change rate is less than 3G%. At low dilution ratios (5 times and 1 () times), the turbidity change rate is not related to the emulsifier type and HLB value. After % hour, the turbidity: the absolute value of the conversion rate is greater than 80% 'that is, there is no significant stability. effect. > Therefore, if the turbidity is used as the criterion for the determination of the suspension of Ganoderma lucidum fruit body, the sorbitol fatty acid ester of 5% is added as a dispersing agent, and the concentration is 100 PPm (the highest dilution ratio is 50 times). Ganoderma lucidum fruit body suspension has better stability. From the analysis results of the particle size of Table 7 (the concentration of Ganoderma lucidum particles is 1〇〇ppm), it is known that the volume average particle size of the Ganoderma lucidum microparticles after re-aggregation after 96 hours of standing storage without the addition of an emulsifier ) is 3 24 pm 'where particles with a particle size of less than 1 (nano/sub-micron) account for 24.9% of the total mass of the Ganoderma lucidum fruit body, and the particle size is of the nanometer scale (less than 10 〇 nm) 23 13/7065 101.6 .19 Application No. 98111670 replaces the second part of the total mass of Ganoderma lucidum. At the time of grinding, 'addition of fat glycerol fat-- and those of Zhao Jiqian: granules, which are smaller, become one-.9. , — = The same interface potential value will contribute to the stability of the particles. The interface potential value of the ginseng fruit body suspension without adding emulsifier is about _117 _, in this embodiment, the emulsifier is used, ^Span 85, Span 8〇 and -2〇 for the particle interface _ potential increase is more significant, the interface potential values are -16_7, _13.8 and 13.7 mV. Combining the above turbidity analysis 'particle size analysis and interface potential analysis results, the emulsifier with small HLB value helps to stabilize the suspension system of Ganoderma lucidum fruit body in the case of 5% emulsifier addition. Therefore, sucrose ester (HLb) is selected. 3), fatty acid glycerides (HLB 3.8), Span 85 (HLB ι8) and span 80 (HLB 4 _ 3), etc., the effect of the amount of emulsifier added on the stability of the suspension system of Ganoderma lucidum fruit body. 24 1377065 Application No. 98111670 Replacement 101.6.19 ---1 Test sample ~ 7 pH interface potential (mV, volume average particle size (Um) number average particle size without added emulsified 剤 Ganoderma lucidum fruit body suspension 5.69 (JW m ) -11.7 3.24 0 124 Ganoderma lucidum fruiting body supernatant 5.47 -20.0 0.100 添加 added emulsified sucrose ester (HLB 3) 5.64 -U.5 10.1 0 107 sucrose ester (HLB 7) 5.66 -12.1 20.7 Λ 1 rn sucrose ester ( HLB 11) 5.65 -10.4 21.9 0.101 Sucrose ester (HLB 15) 5.63 --II_10.3 14.8 Π 1 ΓΠ Fatty acid glyceride (HLB 3.8) 5.70 -10.7 3.86 ν . 1 V ^ 0.107 Span 85 (HLB 1.8) 5.67 - 1 6.7 2.90 0 119 Span 80 (HLB 4.3) 5.63 -13.8 3.68 Π 1 ΠΟ Span 60 (HLB 6.7) 5.68 -10.9 6.52 0 114 Span 20 (HLB 8.6) 5.65 Γ -13.7 8.41 Π 1 1 1 Tween 65 (HLB 10.6 ) 5.62 -8.8 23.3 〇ι Λ? Tween 20(HLB 16.7) 5.64 -11.3 55.4 Λ 1 Λ 1 ν · 1 V/ 1 As shown in Tables 8 and 9, the amount of suspension of Ganoderma lucidum fruit body in different emulsifiers (relative) Particle size analysis, interface potential at 5%, 1%, 2%, and 5% of the weight of the body weight of Ganoderma lucidum The results of particle size distribution show that the stability of Span 80 is better among the four emulsifiers, and the volume average particle size of the particles can be reduced to 2·29 μηι when the addition amount is 1〇%. The ratio of nano- and micro-micron particles. Observing the change of interface potential, it can be found that increasing the addition amount of four emulsifiers can increase the interface potential of the suspension of Ganoderma lucidum fruit body, but the stability effect is limited above 20%. β 25 1377065 Application No. 981U670 Replacement 101.6.19 Table 8 Emulsifier Type Emulsifier Addition Amount (%) Volume Average Particle Size (μπι) Number Average Particle Size (μπι) Nanoparticle Content (wt%) Nano /Submicron particle content (wt%) No emulsifier 0 3.43±0·23 0.121 2.04 24.9 Sucrose ester (HLB 3) 5 10·5±0·16 0.106 1.99 12.6 Fatty acid glyceride (HLB 3.8) 5 3·91± 0·05 0.107 2.73 17.2 10 5.94±0.36 0.116 2.85 26.3 20 9.06±0.56 0.102 2.91 15.6 Span 85 (HLB 1.8) _5 2.95±0·06 0.111 2.61 20.1 !〇_ 2·55±0·07 0.139 1.66 33.7 20 2.48 ±0·07 0.172 0.67 29.4 Span 80 (HLB 4.3) _5 3.78 + 0.14 0.10 8 3.11 21.8 10 2.29±(Μ4 0.126 3.03 40.6 20 3.51±0·11 0.138 1.93 34.3 Table 9 Emulsifier type interface potential (zeta potential) 5% 10% 20% 50% sucrose ester (HLB 3) 11.5±1·0 -12.9±0.2 -14.2±0.8 fatty acid glycerides (HLB 3.8) -10·7±0·4 -13·8±0.3 16·6±1.1 • Span 85 (HLB 1,8, -15·7±0.9 - 1 6.3±0·6 -21.2+0.8 -2 5.3 + 1 7 Span 80(HLB 4.3> -13·8±0·5 -14.1+0.4 -24.2±1·0 -24.7±Γ(Γ in 28 days During the subacute toxicity test, the average daily body weight change and the average food consumption per ounce of male and female mice in the control group and the treatment group were the normal values of the average male and female mice treated and control groups. Within the range, there was no significant difference in the main sputum water consumption. There was no significant difference in blood analysis. The tissue biopsy of the organs was also normal, so no death was caused during the trial and bad clinical signs were caused. 'The above-mentioned 'Ling's fruiting body contains a large amount of crude fiber and lignin components, and the components of the fungal cell wall, in addition to cellulose, are also present in the cell wall. Mechanical grinding to produce a suspension of Ganoderma lucidum fruiting body, in addition to the extraction of 26 1377065 application No. 98111670 to replace the original 丨 01.6.19 to obtain the complete active ingredients, can also significantly increase the specific surface area of the fiber, and fiber Subsequent use of the quality does not require further pulverization, and can be used as a good dietary fiber additive. Among them, after the centrifugal treatment of the suspension of the Ganoderma lucidum fruit body suspension, the centrifugal sedimentation is rich in chitin and cellulose, and the particle size is less than Η) μιη, the texture is fine, and can be used as a skin care product (such as a mask) or a dressing. The substrate of artificial skin, and the centrifugation supernatant, the particle size of the ganoderma lucidum particles are nanometer / sub-micron, and retain the complete active ingredients, such as chitin and other active ingredients more than 5 times the traditional hot water extraction, Moreover, cellulose containing a small part of the particle size can also be used as an additive for health foods, so that the medium can achieve the object of the present invention. The above is only the preferred embodiment of the present invention, and the scope of the invention is not limited thereto, that is, the simple equivalent changes and modifications made by the scope of the invention and the description of the invention are All remain within the scope of the invention patent. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph of volume average particle diameter versus grinding speed and feed concentration; Fig. 2 is a graph of grinding time versus particle diameter; Fig. 3 is a centrifugation supernatant of a suspension of Ganoderma lucidum fruit body Figure 4 is the particle size distribution of the centrifuged sediment of the Ganoderma lucidum fruiting body suspension. Figure 5 is an image of the Ganoderma lucidum fruiting body; it shows the particle size change of the Ganoderma lucidum fruiting body from not grinding to mechanical grinding; 1377065 t Application No. 98111670 Replacement 101.6.19 Figure 6 is a suspension of Ganoderma lucidum fruit body. (: particle size distribution after storage; Figure 7 is the particle size distribution of the suspension of Ganoderma lucidum fruit body after lyophilization, Figure 8 is the particle distribution of the suspension of Ganoderma lucidum fruit body after autoclaving; Figure 9疋The particle size distribution of the centrifugation supernatant of the suspension of Ganoderma lucidum fruit body after cold treatment; Figure 10 is the particle size distribution of the centrifugation supernatant of the suspension of Ganoderma lucidum fruit body suspension after high pressure steam elimination; Fig. 11 is a graph showing the particle size distribution of the centrifugation supernatant of the suspension of Ganoderma lucidum fruit body after different times; Figure 12 is the turbidity change of the suspension of Ganoderma lucidum fruit body by mechanical grinding of Ganoderma lucidum fruit body together with emulsifier Figure 13 is a graph showing the turbidity of a suspension of Ganoderma lucidum fruit body mechanically ground by a Ganoderma lucidum fruit body together with an emulsifier; and Figure 14 is a ganoderma lucidum produced by mechanically grinding a Ganoderma lucidum fruit body together with an emulsifier. Turbidity curve of solid suspension 28 1377065 • Application No. 98111670 Replacement 101.6.19 • [Main component symbol description] No 29

Claims

1377065 Application No. 98111670 Replacement 101.6.19 VII. Patent Application Range: pv month 1% amendment replacement page announcement 1. A suspension of Ganoderma lucidum fruit body, mechanically ground together with Ganoderma lucidum fruiting body and edible emulsifier After that, a suspension of Ganoderma lucidum fruit body containing Ganoderma lucidum particles is obtained, and contains cellulose, chitin, and β-glucan, and the volume average particle diameter of the ganoderma lucidum particles is less than 1 〇μπι, and the edible emulsifier is span 80. And the edible emulsifier content is 10% by weight of the ganoderma lucidum fruiting body. 30 1377065 Application No. 98111670 Replacement 101.6.19 IV. Designation of Representative Representatives: (1) The representative representative of the case is: Figure (3). (2) A brief description of the symbol of the representative figure: None 5. If there is a chemical formula in this case, please disclose the chemical formula that best shows the characteristics of the invention: