TW201036621A - Suspension liquid of Ganoderma lucidum fruiting bodies and nano/submicron scale suspension liquid of Ganoderma lucidum fruiting bodies - Google Patents

Abstract

Disclosed are suspension liquid of Ganoderma lucidum fruiting bodies and nano/submicron scale suspension liquid of Ganoderma lucidum fruiting bodies, wherein the suspension liquid of Ganoderma lucidum fruiting bodies is the liquid containing Ganoderma lucidum particles obtained by mechanical grinding of Ganoderma lucidum fruiting bodies, which also contains fiber, chitin, and β -glucan. The average particle diameter of Ganoderma lucidum particles is less than 10 μ m. Such suspension liquid of Ganoderma lucidum fruiting bodies made by mechanical grinding is characterized in that the complete active ingredients can be obtained without any extraction operation. In addition, fiber surface area can be substantially increased, no further grinding operations are needed in the subsequent use of fiber, and can be used as a good meal fiber additives.

Description

201036621 VI. Description of the Invention: [Technical Field] The present invention relates to a ganoderma lucidum liquid, which is a suspension of a body of Ganoderma lucidum produced by mechanical grinding. ^ [Prior Art] Ο Nano/submicron products are a hot topic in recent years. In recent years, the attention of research scholars, operators and the general public has the importance and potential of nano/sub-microtechnology. Many countries have Prior research on nano/submicron technology is a priority, and – the application of nano/submicron technology to food materials will revolutionize the global food system, although currently nano/submicron The research on food materials is still in its infancy, but after the particles of the food are small, it should increase the absorption rate, and help the infants, the elderly or the digestive system to get the necessary nutrients, and it can be applied to the Chinese herbal medicine to make it easier to play. In order to achieve this long-term goal of the county's recognition of ημ Α 'jw two, the understanding of nano/sub-micron technology and product properties of food materials is an important issue. In Chinese herbal medicine, Ganoderma lucidum fruit body shells have been publicly advertised as having many health functions such as anti-tumor, regulating immune function, limiting nt cabin, lowering blood fat, lowering blood pressure, lowering blood sugar, anti-virus, anti-allergy, anti-oxidation, Anti-light shot, inhibition of platelet clotting and analgesia, whether it is eaten with intact fruiting bodies or extracts, it is safe to use Chinese herbal medicines, even high doses, such as: dried fruit extracts per 曰%^, It was confirmed that there was no significant toxicity, and the nutritional value and toxicity of Ganoderma lucidum were evaluated by animal experiments. The results showed that Ganoderma lucidum had no genotoxicity. It is estimated that the production of Ganoderma lucidum in the world was ridiculed in 2001. China's Big 3 201036621 is the largest producer (3,500 tons), and about 2 million people consume Ganoderma lucidum, which consumes about 2.5 kilograms per person per year. The increase in consciousness 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, its active ingredients (such as chitin, sugar-supplying, 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, not only help the release of active ingredients such as Ganoderma lucidum polysaccharide, 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 Accordingly, it is an object of the present invention to provide a suspension of Ganoderma lucidum fruit body which is produced by mechanical grinding and which has fine ganoderma lucidum particles. Another object of the present invention is to provide a suspension of Ganoderma lucidum fruit body which is produced by mechanical grinding and has nano/submicron, graded ganoderma lucidum particles. Thus, the suspension of the Ganoderma lucidum fruit body of the present invention is obtained by mechanically grinding the Ganoderma lucidum fruit body to obtain a suspension of Ganoderma lucidum fruit bodies containing the ganoderma lucidum particles, and contains cellulosic, chitin, and β-glucan, and the ganoderma lucidum particles The volume average particle size is less than 10 μηη. 201036621 Thus, the nano/sub-micron-sized suspension of Ganoderma lucidum fruit body of the present invention is a centrifugal supernatant obtained by mechanically grinding a ganoderma lucidum fruit body suspension containing Ganoderma lucidum, and then centrifuging. The nano/sub-micron Ganoderma lucidum fruit body suspension contains cellulosic, chitin, and glucan, and the volume average particle size of the ganoderma lucidum particles is less than 2 μm. The effect of the invention: the method for manufacturing the suspension liquid of the ganoderma lucidum fruit body by mechanical grinding, in addition to obtaining the complete active ingredient without extracting, the fiber specific surface area can be greatly improved, and the subsequent utilization of the fiber material does not need to be pulverized again. Can be used as a good dietary fiber supplement. The above and other technical contents, features, and advantages of the present invention will be apparent from the following detailed description of the preferred embodiments. The preferred embodiment of the suspension of the Ganoderma lucidum fruit body of the present invention is prepared by mechanically grinding the entire Ganoderma lucidum fruit body suspension, and the Ganoderma lucidum fruit body suspension comprises the Ganoderma lucidum microparticles obtained by mechanically grinding the Ganoderma lucidum fruit body, and An edible emulsifier that is ground together with the fruiting body of Ling. Mechanical nano-grinding is the use of mechanical energy, the collision between the grinding medium (abrasive beads) and the cutting force between the slurry_fluid and the fluid grinding beads to break and disperse large particles, often used in coatings or nanopowders. Preparation. The mechanical grinder is equipped with a cooling system to avoid high temperatures and facilitate the treatment of heat sensitive organic materials such as food materials and Chinese herbal medicines. The following is an extract obtained by a conventional hot water extraction method, and the present invention directly compares the Ganoderma lucidum fruit body suspension after mechanically grinding the nano/micron treatment of the Ganoderma lucidum fruit body. Control group: traditional hot water extraction, the Ganoderma lucidum fruit body was washed and placed in 7 (TC oven, dried by hot air to about 10% residual moisture. Using a crusher (RT-04, Rong Tsong Iron Co., Taiwan) Powdered for use. Take 300 g of Ganoderma lucidum fruit body powder, add 4500 mL of steamed water, heat at 100 °C for 2 hours, heat it with 60 mesh sieve, and then use the sputum to filter with Whatman No.4 (paper) Whatman, Springfield Mill, UK) Filtration to remove the fine residue, the yellow filtrate is obtained as the extract, and the residue is further added with 4500 mL of distilled water and extracted as above. Three hot water extractions are performed for each sample, and the filtrate is mixed. Concentrate to a suitable volume under vacuum, calculate the concentration rate and record the experimental group: mechanically grind the entire Ganoderma lucidum fruiting body, including the following steps (1) pre-grinding. Scale the ganoderma lucidum fruiting body, wash the diced and add 250 mL 4 ° C deionized water, whipped with a shredder (Blender 7012S, Waring Commercial, USA) for 5 minutes, then poured into a 600 mL high-profile beaker, with deionized water (150 mL,) remaining in the stir Fruiting body in the shredder The coarse material was washed out, allowed to stand overnight at 4 ° C, the fibrous structure was relaxed, and then whipped at 20,000 rpm for 10 minutes with a high speed homogenizer (Polytron PT 3000, Kinematica AG, Switzerland) (full temperature ice bath to avoid temperature) Rise), the sub-solid particles are less than 300 μηι, and then mechanically ground. Step (2) Mechanical grinding. 201036621 Grinding the sub-step (1) using a nano-fine grinding machine (MiniPur, NETZCH-Feinmahltechnik GmbH, German) For solid particles, the grinding is divided into two stages. In the first stage, cerium-zirconium beads with a particle size of 0.8 mm are used as grinding media, and in the second stage, cerium-zirconium beads with a particle diameter of 0.3 mm are used as grinding media, and the feed rate is 360 mL. /min. In order to explore the influence of operating conditions on the product, 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 fruiting body. After the preparation of the suspension, the chemical and physical properties of the control group and the experimental group were compared and analyzed as follows: (1) Α·β-glucan (β-D-glucans) content determination β-glucan was quantified by fluorescent dye Aniline blue. After centrifugation of the ground Ganoderma lucidum fruit body suspension (2000×g, 10 min), Filter No.4 filter paper, take appropriate amount of sample solution, add 0.3 N NaOH to make the total 〇W product 3 mL, stir for 30 minutes, adjust the pH to 11.5 with 1 N HC1, add 50 mM Na2HP04-Na0H buffer with pH 11.5 (including 0.5 Μ NaCl) and bring up to 10 mL. 2 mL of the above solution was added, 0.2 mL of aniline blue (1 mg/mL) was added, mixed with a Vortex shake, and allowed to stand for 2 hours, and detected by a fluorescent detector (excitation wavelength 395 nm, emission wavelength * 495 nm). The β-glucan content of the sample was calculated by preparing a standard curve with different concentrations of Lentinan. Β. Determination of total dietary fiber 7 201036621 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 Chinese herbal medicines meet this specification). C. Chitin content determination Take 400 mg of Ganoderma lucidum fruit body suspension with 6 N HC1 at 1 〇〇. The mixture was refluxed and hydrolyzed for 5 hours, cooled to room temperature, and then transferred to Whatman No. 4 filter paper. 1 mL of the filtrate was dried at 45 to 50 ° C under reduced pressure and used. The dried hydrolyzed product is dissolved back into the steaming 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 The particle size distribution of the obtained ganoderma lucidum microparticles was measured using a dynamic light scattering particle size analyzer (Nanotrac 150, Microtrac Inc., USA). The measurement range is from 0.8 nm to 6500 nm. If it is outside this range, Bayi 1J is measured by LS 230 particle size analyzer manufactured by Beckman Coulter (CA, USA). Its particle size is 201036621. The measurement range is 0.4 μιη to 2000 μιη. After the light source is filtered, it is detected. The lower limit is up to 40 nm. Deionized water was used as a blank test and measured at 25 °C. After the ground suspension of the Ganoderma lucidum fruit body is properly diluted, it is directly measured by an instrument. The analysis software (FLEX Software, Microtrac Inc., USA) was used to analyze the scattering signals, and the Doppler shifts of the particles were calculated to obtain the particle size distribution percentage, the mean particle size, the median particle size, and the like. Distribution parameters. B. Microscopic observation 〇 The optical structure (Optiphot-Pol, Nikon, Japan) was used to observe the structure of the crude ganoderma lucidum and the change of the ganoderma lucidum particles in the initial stage of grinding. The microstructure of the obtained Ganoderma lucidum microparticles was observed by a scanning electron microscope (SEM). After the sample is diluted, it is applied to a glass slide, dried at room temperature, and the sample is adhered to the aluminum table with silver glue. The surface of the sample is plated with a gold film by an ion laminator under vacuum, and observed by SEM. The microstructure of the sample. The microstructure of the obtained Ganoderma lucidum microparticles was also observed using a transmission electron microscope (TEM) (JEM-1230, JEOL Co. Ltd, Japan), and the measurement results of the particle size distribution Ο were confirmed. After the sample is properly diluted, the sample suspension is taken up by a TEM-specific 200 mesh mineral carbon copper mesh (01800-F, Ted Pella, Inc., USA), dried in a dry box, and the dried sample is directly observed by TEM. The desired digital image was obtained by a CCD camera system (DualVision CCD, Gatan Inc., USA). The electron source used in TEM is a hot cathode electron gun (

LaB6 filament) with a maximum accelerating voltage of 120 KV. (III) Stability treatment and analysis of suspension of Ganoderma lucidum fruiting body A. Addition of surfactant agent 9 201036621 Select appropriate grinding operation conditions (rotation speed: 36 rpm, medium diameter: 0.2 mm; time: 9 〇 minutes), Edible emulsifiers of different types, concentrations and HLB values were added prior to milling. The emulsifiers used are: ionic surfactants, such as fatty acid salts (anionic surfactants). 2. Non-ionic surfactant (n〇ni〇nie SUrfaCtant), for example (1) sugar ester (selecting sucrose esters with HLB values of 3, 7, 11 and 15); (2) The sorbitan fatty acid S曰 (Span 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 72 96 hours) , sampling for turbidity analysis and particle size analysis. In this type 5 test, a small amount of preservative will be added to avoid the growth of microorganisms. c. Turbidity measurement 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 sink; the total number of suspended particles decreases, causing the turbidity of the upper solution to decrease. The stability of the solid suspension of Ganoderma lucidum can be judged by turbidity, and the emulsifier which is not suitable can be eliminated. Use

f Good turbidity °T (P〇rtable turbidimeter, model 2100P, HACH p y, u’s·a.) Measure the change of turbidity during storage, before the detection

Correction of Gelex Secondary Standards (turbidity 〇 1 〇 NTU 10 201036621, NTU of 0- 1 and 0_1000 NTU), taking the ground and stabilized suspension of Ganoderma lucidum fruit body, diluted appropriately Take 15 mL into the water sample test bottle and measure it directly by turbidity meter, the unit is NTU. D. Zeta potential Determination of interface potential is an indicator of the attraction or repulsive force between particles. The enhancement of this value indicates that the more stable the particle is, it is an effective tool for measuring the surface properties of the particle. On the instrument, the voltage of the fixed value of the particle is given, 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. The interface potential analyzer (Zeta (10) printing (10) can analyze the interface potential in real time. Corresponding to the pH value, emulsifier concentration and time curve and results, since the measurement of the interface potential is an important indicator of the stability of the nano/submicron particles, the analysis of the data can infer the nano/submicron. The possible reasons for the re-aggregation of 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, and understand nano/ The surface characteristics of submicron-sized Ganoderma lucidum particles are an important basis for further modification of the surface properties of particles in the future. E·Microscopic observation using optical microscopy 〇ptiph〇t_p〇1, Nik〇n, Japan) Observed changes in the re-aggregation of nano/sub-micron Ganoderma lucidum particles. In addition, observation by a transmission electron microscope (TEM) (jEM_1230, JEOL c〇Ltd, Japan) The microstructure of the nano/sub-micron-sized Ganoderma lucidum particles during storage was confirmed by the measurement and re-aggregation of the particle size cloth, and the obtained sample was observed by a scanning electron microscope (Scannmg electron micr〇sc〇pe, SEM). Microscopy 11 201036621 Structure 0 (4) 28-day sub-acute toxicity test according to the "Good Practices for Non-Clinical Testing of Drugs" announced by the Department of Health, and the toxicity test method in the "Method for Assessment of Health Food Safety" for 28 days. Toxicity test. Changes in animal body weight and food consumption were measured during the administration of the test substance. 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 last dose group and the control group. A. Animal treatment Animals were used for 5-6 weeks ICR mice from the Animal Center of National Taiwan University Medical College. Each dose group (four) male and female 12 animals. Before the toxicity test, the animals were first acclimatized for one week in an animal room with temperature, humidity and light, and freely ingested normal feed and water. The feeding experiment was started one week later, and the mice were sacrificed after a continuous day. Β·Ganoderma Lucidum In this trial, the low-dose and medium-dose groups were 〇2 and 〇 2 g/kg/day, respectively, and the high-dose group was 2 g/kg/day. The low dose (〇g/kg/day), medium dose (0_2 g/kg/day), and high dose (2 g/kg/day) are the same daily doses, 1〇 times, and 1 for the recommended daily intake of adults. 〇〇 times. C. Determination of serum biochemical value The blood sample of the animal was taken from the carotid artery blood into a serum separation tube, and the serum sample taken after centrifugation at 3000 xg for 15 minutes. The analysis items are as follows: high density lipoprotein (HDL), low density lipoprotein 12, 201036621 (low density lipoprotein, LDL), glutamic oxaloacetic transaminase (GOT), valine pyruvate Glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatinine (CRE), total cholesterol (cholesterol, CHO), triglyceride (TG), sodium Ions (Na+), potassium ions (K+), gas ions (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), hemoglobin (Hb) ), hematocrit (Hct), mean corpuscular (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (platelets) , PLT), lymphocytes (LYMPH), red cell distribution width-coefficient of variation (RDW-CV), jk platelet distribution width (PDW), mean platelet volume (mean platelet volume) , MPV), platelet-large cell range (P-LCR), and the like. E. Histopathological section The organs of all animals, liver, kidney, heart, spleen, lung, testicular, parasitoid and uterus and ovary were removed and placed in 10% 13 201036621 formalin for one week. After sectioning, hemat xylin and staining were added as 'microscopically observed'. Histopathological section interpretation was commissioned by the Taiwan Institute of Animal Science and Technology. The following is a description of the process and branch of the suspension of Ganoderma lucidum fruit body of the present invention: (1) The relationship between the grinding process and the particle size is as shown in Table 1. Under the same grinding time (90 min), the rotation speed is discussed (2310~) The effect of 3570 rpm), feed concentration (〇.5~2 〇g/mL) and grinding bead loading (80~14〇mL) on the Ganoderma lucidum nano/micronization process. Table 1 No. Centrifugal speed (rom) Ganoderma lucidum feed concentration (g/mL) Grinding beads filling amount (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 7 2940 1 η 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 diameter of the product. . 14 201036621 The above-mentioned pretreatment of the finely ground suspension of Ganoderma lucidum at a fixed rotation speed of 36 rpm, a fixed feed rate of 360 mL/min and a fixed filling amount of 14 ,, and different solid concentration and two kinds of grinding beads ( 0.8 mm, 0.3 mm yttrium zirconium beads were ground 3 〇, 6 〇, 9 〇, 12 〇, 15 〇, 18 〇 and minutes. It was found that the pre-treated Ganoderma lucidum finely divided suspension had a particle size of less than 300 μm, but most of it was still larger than 15 μm. Therefore, it is necessary to use staged grinding for the nano/submicron of the Ganoderma lucidum fruiting body. That is, the fine grinding is first performed with the granules, and the grinding is continued until the particle size falls below about 5 〇μ〇1.

Then change to 0.3 mm error beads, as shown in Figure 2, the grinding time also affects the particle size. As the grinding time increases, the volume average particle size decreases. At 6G minutes of grinding, the volume average particle size decreases at a higher rate. After slowing down, after grinding for 9 minutes, the volume average particle size of the ganoderma lucidum particles is about 丨.2陴. After grinding for 9 minutes with G 3 _ zirconium beads, the volume average particle size is less than 1 G μηι. Because the particle size distribution range of the (IV) floating liquid is wide, the particles are easily aggregated 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. The diameter is an indicator. From the results of the granule pinch analysis shown in Figs. 3 and 4, it can be seen that the above-mentioned suspension of Ganoderma lucidum fruit body obtained by grinding the 〇3 face 钇 lead bead for just a minute is centrifuged for 1 〇 minutes to remove larger particles, and the supernatant is centrifuged. The particle size distribution of k is 28~578 nm (Fig. 3). The bulk of the body is about 105 nm, and 67% of the particles are less than 100 nm. It can increase its absorption in the body, centrifuge the supernatant, and save the private labor. The particles of the moon liquid are less than 1 μη!, although the solid content is low, but the active ingredient is extracted by grinding. All still retain 15 201036621 down ° and the particle size of the centrifugal drop is less than 1G_ (Figure 4), the texture is fine, can be used as a skin care product (such as mask) or dressing, artificial skin substrate and is rich in chitin and Cellulose' is therefore a good dietary fiber supplement. As shown in Fig. 5, the ground Ganoderma lucidum microparticles were observed microscopically, and the fresh Ganoderma lucidum fruiting bodies were carried by the blender and observed by scanning electron microscopy (SEM). The dendritic branches of the Ganoderma lucidum fruiting body were clearly visible from Fig. 5B. Road hyphae. After proper homogenization, the osteochords become more finely divided and loose (e.g., =5C), and their size is about tens to hundreds of micrometers, and this state will facilitate subsequent grinding treatment. Figure 5D shows the results of observation by a transmission electron microscope (TEM) after two-stage grinding. The circular particles in the figure are the nanoparticles in the suspension of the fruiting body of the spirit, which can be clearly seen from the pure The particles can reach the nanometer scale below μ1μιη, but the particle size range of the whole becomes larger due to the re-aggregation between the particles, which is up to several hundred nanometers, which is the sub-micron scale. This result also verified the results of the above particle size analyzer and reconfirmed that the medium grinding process used in the present invention can indeed reduce the Ganoderma lucidum particles after grinding of the Ganoderma lucidum fruit body to the nano/submicron scale. Since the Ganoderma lucidum fruiting body is rich in cellulose and the chitin texture is rough and firm, the particle size of these fibrous substances is lowered, and in addition to increasing the specific surface area, the taste of the product can be improved. The physiological effect of the fiber is related to the adsorption of the surface. Therefore, by increasing the specific surface area of the fiber, the physiological activity can be increased. The particle size of the dietary fiber is reduced from 〇1 to i (4). The specific surface area can be increased by 1 〇〇. The recommended intake of succumb fiber can be reduced to 1% of the original recommended amount. Below nm, it is recommended to take 16 201036621 to reduce it to one thousandth of the original recommended amount. In other words, by adding 50 to 500 ppm of nano/submicron dietary fiber to 500 mL of drinking water, the recommended daily intake of dietary fiber (25 g) is achieved. Based on this point of view, the centrifugation supernatant of the suspension of Ganoderma lucidum fruit body not only retains the physiological active ingredients unique to Ganoderma lucidum, but also provides a high surface area dietary fiber. (2) Relationship between grinding process and active ingredients

As shown in Table 2, after the medium was ground, the content of β-glucan was significantly increased, and the grinding was carried out with a larger grinding medium (particle size 0.8 mm), and the content of β-glucan in the product was extracted with hot water ( About 0.01 mg/mL) is similar. As the particle size decreases and the grinding time increases, the β-glucan content increases four times that of hot water extraction products, and is higher than the products collected on the market (0.0023 ~ 0.0202 mg/mL). Table 2

Test sample β-glucan content (mg/mL) (β-l,3-D-glucan) Hot water extraction 0.010±0.0 0.8 mm bead grinding 0.0096+0.0004 0.3 mm Jiuzhu beads grinding 0.0398±0·0003 Green (Amazon Biotechnology 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 )·········· The hot water or organic solvent is extracted, although the active ingredient can be obtained, but the extraction amount is limited, and there are still problems in the treatment of wastewater, organic solvent and residue after processing. In the mechanical grinding process, not only the dietary fiber such as cellulose and chitin can be obtained in the suspension of Ganoderma lucidum fruit body, but also the active ingredient can be broken by the crushing of the cell wall of the Ganoderma lucidum fruit body during the fine grinding process. Released. As shown in Table 3, there is a similar phenomenon in the chitin content. The product obtained by grinding the medium obtained by the medium grinding is obtained from the grinding of the Taihaomen, υ·δ mm medium. . However, if the obtained product is removed by centrifugation, the chitin of the supernatant contains the mesh, and the width of the gentleman's ruler is reduced, but it is still about 5 times that of the general ... 7 product. The removed particles contain a cryptic chitin. Since the hot water extraction can only extract water solubility (4), the content of chitin in the 3 extracts is very low, and most of them are left in the medicine. Containing 35~40% chitin, 4〇~50% glucan, the rest is ',,, pigment (mainly nitrogenous, good source of dietary fiber, such as crushing and thinning fruiting bodies) Grinding to nano/sub-micron size can not only replace the extraction 'products will also preserve all the components of the fruiting body (rich in chitin, cellulose and other wax fiber), and because of the small particles, it helps the body to absorb, Therefore, this nano/second "Shenzhizhizi (4) suspension or its concentrated product will be suitable as an additive for health foods.

-- (2) 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 and rapid integration of Ganoderma lucidum particles. Larger particle clusters, the increase in particle size causes the gravity effect to be much greater than the Brownian motion effect of 18 201036621, which in turn produces significant sedimentation. After 4 hours of standing under it for 24 hours, the particle size was analyzed, and the particles were aggregated and formed into tens of micrometers. The volume average particle size increased to 9.35 μιη (as shown in Fig. 6), and the abrasive product was considered for subsequent processing. For example, the stability of cold storage, cold, east drying or high temperature sterilization, etc., for the particle size analysis of the fruiting body suspension after cold (Russian, 24 +) and high pressure steam elimination g (12H 15 minutes) The difference in particle size distribution before and after processing. The Ganoderma lucidum particles in the S-substantial suspension are cold; in the east process, they will accumulate 'solution; the east will appear to be separated from the water. The clusters formed by the aggregation are cotton-like and difficult to physically or mechanically (such as shock, Homogenization, ultrasonic vibration 4)) Disperse, cold; the particle size distribution of the East Sub-submerged suspension is shown in Figure 7, the volume average particle size is 109 μηι. High steam sterilization also causes severe re-aggregation between the particles, forming a mass of clusters of particles, and it is difficult to disperse it like physical or mechanical force as cold beam treatment, and its particle size distribution range is 5.9~824.5 _ The volume average particle size is 132 _ (as shown in Figure 8). The suspension of the Ganoderma lucidum fruit body obtained by the machine grinding method has a wide distribution range and is easy to accumulate and precipitate, but the solid content is high, and it is rich in chitin and cellulose. The suspension of the Ganoderma lucidum fruit body or its concentrated product will be Suitable as an additive to health foods. (4) The stability of the centrifugation supernatant of the suspension of Ganoderma lucidum fruit body is shown in Table 4. 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 product The higher the stability. Grinding the centrifugation supernatant product of the 3 G min of the Shiba solid suspension (1 〇g for 10 minutes), after 21 days of storage volume 19 201036621, the flat granules were increased from 0 195 to 199 μπι, but grinded I8 min of Ganoderma lucidum, the supernatant product of the solid w float (centrifugation for 1 〇 minutes), the volume average particle size increased from G.126 to 〇.137 μηι, the increase rate is less than 1 ( %), showing increased grinding time helps particle stability

Grinding time (minutes) 30 volume average particle size (β m ) volume mean diameter) 0.195 0.158 number average particle size (in m) X number mean diameter ) 0.127 90 0.341 , 0.092 0.078 research 30~----~~~ ~~~~° 0 068 ^ 8_(10) The first stage grinding results of the beads. -~钇Zirconium beads for the second stage grinding result 0.064 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, 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 measurement was 6.54 μη〇). Donggan treatment will promote the re-polymerization of Ganoderma lucidum particles. After the bed is dry, the particle size distribution is wider than $, and a certain proportion of the particles have a particle size larger than 6.54 μιη (which cannot be measured due to the upper limit of the instrument measurement). The particle size is 1.177 μηη. As shown in Figure 10a, the centrifuged supernatant retains a fairly good stability after autoclaving. After sterilization, the particle size is measured after the temperature drops to room temperature. Less than i μηι, the particle size distribution range is 36~8〇ζ (10), the volume average particle size is 140nm, and the ruthenium particles remain below 100 nm. It is observed to be stored at room temperature for 28 days (as shown in Figure i〇b) Show) 20 201036621 (as shown in Figure 10e) The particle size change 'particle size distribution has a large particle size' but the offset is very small, and its volume average particle size is only increased by -65 nm and 373 nm, respectively. Centrifugal supernatant is still very stable after high temperature action The re-aggregation phenomenon during the storage period is not significant. By centrifugation, the centrifugation distribution of the centrifugation supernatant can be reduced to below and the stability is stabilized, but the relative centrifugation supernatant solids 3 The amount of Ganoderma lucidum particles was greatly reduced. In order to increase the concentration of solids, the water content of the supernatant was reduced by concentration under reduced pressure, and the particle size was measured under different concentration ratios (C〇ncentration rati〇). The concentration ratio is divided into the volume of the final concentrate by the volume of the original solution. 胄u and Table 5 are the particle size analysis results after centrifugation and concentration of the supernatant by 2, 4, 8, 12 and 2G times. In the concentrated supernatant of 4 times concentrated, the particle size did not change much, the volume average particle size only increased by 33%, and the particles were all smaller than i μιη. When the concentration was more than 8 times, the aggregation between the particles began to occur and the particle size was generated. The distribution is shifted to a large particle size, and the particle size distribution is greater than 1 μηι. In the present embodiment, in order to improve the σ ΰ solid content and balance stability, the centrifugation supernatant is concentrated 4 to 6 times to meet the requirements. 21 201036621 Table 5 Concentration ratio of supernatant (concentrated Volume average particle size (U m) Particle size distribution range (um) Particles with a particle size of less than 1 U m contain Jin (Ό/λ 0 (3.4 mp/mi ^ 0.105 0.03-0.578 100 2 (6.8 mp/mi > 0.141 0.036 ～0.750 100 4 —mp/mL) 0.140 0.030-0.972 100 8 0.351 0.033 ～2.120 95 12 -X40.8 mp/mi ) 1.922 0 · 111 ～6.54 61 20 -J68.0 rnff/ηιΐΛ 1.880 0.859 ～6.54 61 (5) Effect of emulsifier on the stability of suspension of Ganoderma lucidum fruit body The turbidity is a simple physical parameter. For a stable dispersion of suspended solution, the turbidity should not be significantly different with time. Accordingly, the present invention performs turbidity analysis for various variables to be explored to screen for preferred emulsifiers. As shown in Table 6, the turbidity of the Ganoderma lucidum fruit body suspension after grinding without adding an emulsifier is higher than 1000 Ντυ, and the turbidity of the samples after dilution of 2, 5, 1 〇 and 50 times is over time. Gradually, the higher the dilution ratio, the smaller the rate of change. Low concentration (high dilution ratio) suspension turbidity ^ high concentration (low dilution ratio), showing that the particle concentration is an important parameter affecting the stability of the suspension. The higher the concentration, the higher the collision probability between particles. The phenomenon is also relatively obvious. On the whole, the aggregation between the particles is quite obvious, and the aggregation and sedimentation behavior of the suspension of the Ganoderma lucidum fruit body is obvious. 22 201036621

Resting time ------ turbidity (NTU) (hours) DF = 〇2 5 10 50 0 ΝΑ 575 230 107 20.1 1/4 ΝΑ 582 215 105 20 1 1/2 ΝΑ 595 209 103 19 7 1 ΝΑ 617 204 102 19 7 2 ΝΑ 718 25.6 101 19 5 4 ΝΑ ΝΑ 22.0 40.8 19 5 8 ΝΑ 27.8 20.3 21.4 1 0 1 24 ΝΑ 28.7 ~~ 21.7 18.3 i 7 J 20 〇48 ΝΑ 30.3 17.6 13.7 17 8 72 ΝΑ 38.3 16.7 13.1 1 Λ Λ 96 831 36.3 14.2 11.2 1 Ί A : dilution ratio -- 丄 4 · VJ

ΝΑ : >1000NTU In the present invention, sucrose vinegar (sugar "〖π, hlb respectively 3, 7, 11 and 1S), poly sorbitol, which is different in HLB (hydrophile phile_lip phile balance) Alcoholic fatty acid esters (85, 8〇, 6〇, 2〇 and Ding gift 11 65, 20, HLB 1 > 8, 4_3, 6 7 , 8 6 , 1〇_6 and 16.7, respectively) and fatty acids Glycerolipid (HLB is 38) as the initial screening of the emulsification J, the amount of addition is first fixed to 5% of the weight of the Ganoderma lucidum fruiting body

As shown in Fig. 12, respectively, the turbidity change rate is related to time under the diluting magnifications of Ganoderma lucidum fruit body suspensions of $, H) and 50 times, respectively. A turbidity change rate of negative values indicates a decrease in turbidity, and a positive value indicates an increase. With the exception of a few positive values, the turbidity trend of almost all tests was reduced. On the whole, 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 decreased with time. Ppm In the case of the highest dilution (four) 50 (Ganoderma lucidum particle concentration about 23 201036621 (01 mg / mL)), the rate of change of the most is +, and the stability of the polysorbate fatty acid S 曰 ritual agent is better, The absolute value of the change rate of turbidity after % hour is less than 30%. Under the low dilution ratio (5 times and 10 times) of the low-silk shaker, the turbidity change rate is not related to the emulsifier type and HLB value, and the absolute value of the turbidity change rate after % hour is greater than 8〇%. No significant stability effect. Therefore, if the turbidity is used as a criterion, the Jingyou Ganoderma lucidum fruit body suspension is 5/(> added amount of polysorbate fatty acid ester as a dispersing agent, and the concentration is 100 ppm (the most suitable dilution ratio is 5 times). The suspension of Ganoderma lucidum fruit body has better stability. From the analysis of the particle size of Table 7 (the concentration of Ganoderma lucidum microparticles is 1〇〇ppm), it can be seen that the suspension of Ganoderma lucidum fruit body without adding ritual agent is stored for 96 hours. The volume average particle size (MV) of the post-aggregation particles is 3 24, wherein the particles having a particle size of less than 1 μη (nano/submicron) account for 24.9% of the total mass of the Ganoderma lucidum fruit body. On the nanometer scale (less than _ nm), the total mass of Ganoderma lucidum fruit body is ❸2 〇 4%. When 5% emulsifier is added during grinding, the volume average particle size of fatty acid glyceride, Span 85 and Span 8 添加 is changed. The smaller amplitudes are 3.86, 2.90, 3.68 μΐη respectively. In addition, the interface potential value will contribute to the stability of the particles, and the interface potential value of the ginseng fruit body suspension without adding emulsifier is about _丨丨7. Examples of emulsifiers used, with Span 85 and Span 80 And Span 20 has a significant increase in the interfacial potential of the particles, and the interface potential values are -16.7, -13.8, and -13·7 mV, respectively. The above turbidity analysis, particle size analysis, and interface potential analysis results, the amount of emulsifier added. In 5% of cases, an emulsifier with a small HLB value helps to stabilize the suspension system of Ganoderma lucidum fruit body. Therefore, it is recommended to recommend sweet and sour vinegar (hlb 3), fatty acid glyceride (HLB 3.8), and Span 85 (HLB丨). 8) and 邡 8〇 (HLB 4.3), etc., the test of the effect of the amount of emulsifier added on the stability of the liquid storage system of the sage.

^ As shown in Tables 8 and 9, when the suspension of Ganoderma lucidum fruit body is added to different emulsifiers (relative to 5%, 10%, 20% and 50% of the weight of Ganoderma lucidum fruiting body)

The particle 彳 two knife analysis, interface potential measurement results. From the results of particle size distribution, it is known that the stability of Span 8G is better among the four emulsifiers. The addition amount of the emulsifier can reduce the volume average particle size of the particles to 2.29 μηη, and increase the content of nanoparticles and sub- and m-particles. proportion. Observing the change of the interface potential, it can be found that the addition of the four emulsifiers can increase the interface potential of the suspension of Ganoderma lucidum fruit body, but the stability effect is limited when it is higher than 20%. 25 201036621 Factory Table 8 Emulsifier Type Emulsifier Addition (%) Volume Average Particle Size (μηι) Quantity Average Particle Size (μηι) Nanoparticle Content (wt%) Nano/Submicron Particle Content Γλ»/会,无Emulsifier 0 3.43±〇.2S 0.121 2.04 24 Q Sucrose ester (HLB 3) 5 !〇·5±〇.16 0.106 1.99 1 ο A fatty acid glyceride (HLB 3.8) __5 10 3-91+0.05 5-94 + 0.36 0.107 2.73 1 2.0 17.2 0.116 2.85 26 3 20 9.06+0.56 0.102 2.91 1 ς £ Span 8 5 (HLB 1.8) —5 2.95±〇.ftfi 0.111 2.61 20.1 2.55 + 0.07 0.139 1.66 33.7 20 2.48±〇.〇 7 Γ 0.172 0.67 20 A Span 80 (HLB 4.3) 5 3.78±0.14 0.108 3.11 21 8 10 Γ2.29 + 0.14 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 -12.910.2 -14.2 + 0.8 fatty acid glycerides (HLB 3.8) ·10_7±0·4 -13.810.3 16.6+1.1 Span 85 (HLB 1.8) -15.7+0.9 -16.3 + 0.6 -21.2 + 0.8 -2 S 3 + 1 7 Span 80(HLB 4.3) -13.8±0·5 -14.1+0.4 -24.2±l_〇-24.7 + 1 .〇28 days Asian emergency During the toxicity test, there was no significant difference in mean daily body weight change, average food consumption per meal, and average water consumption per liter of water in male and female control groups, and blood analysis in male and female mice treated and control groups. There was no significant difference. The detection values were all within the normal range, and the biopsy of the main organs was normal. Therefore, no deaths were caused during the trial and adverse clinical signs were caused. The above-mentioned 'Ganoderma lucidum fruiting body contains a large amount of crude fiber and lignin component' and generally the components of the fungal cell wall are chitinous in addition to cellulose. Active ingredients such as Ganoderma lucidum polysaccharide are also present in the cell wall, and the present invention is mechanically Grinding to make the suspension of Ganoderma lucidum fruit body, in addition to the no-extraction extraction 26 201036621 can obtain the complete active ingredients, can also greatly increase the specific surface area of the fiber, and the subsequent use of the fiber does not need to be crushed, can be used as a good Take advantage of fiber additives. Wherein, the ground suspension of the Ganoderma lucidum fruit body obtained by grinding is subjected to subsequent centrifugation, and the centrifugation sediment is rich in chitin and cellulose, and the particle size is smaller than Η), the texture is fine, and can be used as a skin care product (such as a mask) or The dressing is applied to the substrate of the skin, and in the centrifugation supernatant, the particle size of the ganoderma lucidum particles is nanometer/submicron, and the intact active ingredient is retained, and the chitin 0 胄 active ingredient is the traditional hot water extraction 5 More than a multiple, and a cellulose having a partial particle size is also available as an additive to health foods, and the object of the present invention can be achieved. The above is only the preferred embodiment of the present invention, and is not limited to the scope of the present invention, that is, the simple equivalent of the patent application and the description of the invention according to the present invention. Variations and modifications are still within the scope of the invention. [Simple description of the diagram] 〇® 1 is a graph of volume average particle size versus grinding speed and feed concentration; 'Fig. 2 曲线 grinding time relative particle size curve; 疋 疋 疋 子 子 体 体Figure 2 shows the particle size distribution of the centrifuged sediments of the Ganoderma lucidum fruiting body suspension. Figure 5 is an image of the Ganoderma lucidum fruiting body; it shows that the Ganoderma lucidum fruiting body has not been ground until after mechanical grinding. Particle size change; 27 201036621 Figure 6 is the particle size distribution of the suspension of Ganoderma lucidum fruit body after storage; Figure 7 is the particle size distribution of the suspension of Ganoderma lucidum fruit body after cold beam drying; Figure 8 is the Ganoderma lucidum fruiting body The post-granulation distribution of the suspension after autoclaving; Figure 9 is the particle size distribution of the centrifuged supernatant of the suspension of Ganoderma lucidum fruit body after freezing; Figure 1 is the centrifugation supernatant of the suspension of Ganoderma lucidum fruiting body Figure 9 shows the particle size distribution of the centrifuged supernatant of the suspension of Ganoderma lucidum fruit body after concentration; Figure 12 shows the particle size distribution of the supernatant of the suspension of Ganoderma lucidum fruit body; Turbidity curve of the suspension of Ganoderma lucidum fruit body after mechanical grinding; Figure 13 is a graph showing the turbidity of the suspension of Ganoderma lucidum fruit body after mechanical grinding of Ganoderma lucidum fruit body together with emulsifier; The turbidity curve of the suspension of Ganoderma lucidum fruit body after mechanically grinding together with the emulsifier. 28 201036621 [Explanation of main component symbols]

29

Claims (1)

201036621 VII. Patent application scope: L A suspension of Ganoderma lucidum fruit body, which starts to grasp π. The body of Maling is served by mechanical grinding and contains the quality of chitosan particles, chitin, and β_” sugar diameter is less than 丨. _. And the volume average particle of the ganoderma lucidum particle ^ according to the patent scope of the first item of the Ganoderma lucidum fruit body suspension, its wide, to, 1 μΐΏ of Ganoderma lucidum particles accounted for 1% of the total mass of Ganoderma lucidum 3. In the second patent range, the chitin content is higher than that of the suspension of Ganoderma lucidum fruit body described in 项. 4. mg/mL. 5. 6. According to the scope of patent application No. 3, the fiber content is higher than 〇. According to the scope of patent application No. 4, the glucan content is higher than the liquid in accordance with the scope of the patent application, and also includes a suspension of Ganoderma lucidum fruit body, which is 1 mg/mL. The edible liquid of the Ganoderma lucidum fruit body suspension, which is 机械.〇1 mg/mL, 2 or 5 of the above-mentioned suspension bodies of the Ganoderma lucidum fruit body, is mechanically ground. 7. According to the scope of the patent application, the edible property Emulsifier is the child of the spirit (4) a floating liquid, the edible emulsifier content being from a nonionic surfactant, and 8. 0.1 to 25% by weight of the sub-entity of the seventh embodiment of the patent application. wherein the edible emulsifier is ^The fruiting body suspension of the spirit of 9. According to f, please patent the range 帛8 ^ value" in 1~18. Among them, the edible emulsifier has a suspension value of 1 to 1 灵. 30 201036621 10. The suspension of Ganoderma lucidum fruit body according to claim 9 of the patent application, wherein the edible emulsifier has an HLB value of 1 to 5. 11. The suspension of Ganoderma lucidum fruit body according to claim 10, wherein the edible emulsifier is selected from the group consisting of vitamin fat, polysorbate fatty acid fat, fatty acid glycerolipid, and borrowing and glycolipid a group consisting of a mixture of polysorbate fatty acid fat and fatty acid glyceride. 12. The suspension of Ganoderma lucidum fruit body according to the scope of claim 5, wherein the polysorbate fatty acid ester is a group selected from the group consisting of span 8〇, span85, and O span 80, span85. 13. The suspension of Ganoderma lucidum fruit body according to claim 10, wherein the edible emulsifier is selected from the group consisting of polysorbate fatty acid fat, fatty acid glyceride, and polysorbate fatty acid fat, fatty acid a group of glycerides mixed. 14. The suspension of Ganoderma lucidum fruit body according to claim 13, wherein the polysorbate fatty acid ester is selected from the group consisting of span 8 〇, span 85, and span 80 and Span 85. 〇15·—A nano/sub-micron-sized suspension of Ganoderma lucidum fruit body, which is a centrifugal supernatant obtained by mechanically grinding the Ganoderma lucidum fruit body and then centrifuging, and the nano/sub-micron Ganoderma lucidum fruit body suspension is suspended. The liquid contains cellulosic, chitin, and β-glucan, and the volume average particle diameter of the ganoderma lucidum particles is less than 2 μ. 16. The nano/sub-micron ganoderma lucidum-solid suspension according to claim 15 wherein the ganoderma lucidum particles having a particle size smaller than i _ occupies more than 60% of the total lings-chicken granule content. 17. The nano/submicron spirit 31 201036621 medicinal suspension according to claim 15 or 16 of the patent application, wherein mg/mL 〜1〇〇 mg/mL. The concentration range of Ganoderma lucidum microparticles is in the range of 18. According to the scope of the patent application, the county is located in the county, "the item is the sub-micron-sized ganoderma lucidum fruit body suspension liquid", wherein the volume average particle size of the ganoderma lucidum particles is small. Nm 'and the size of the Ganoderma lucidum particles is less than 1. 19. According to the scope of the patent application, item 18, extracting, A, water/sub-micron ganoderma lucidum fruit body suspension, wherein more than 5% of the 100 nm 〇 之 particles have a particle size of less than 20. Apply for a patented range of physical suspensions, where, ~20 mg/mL. The concentration of nano-Ganoderma lucidum microparticles in the 19th sub-micron range of Ganoderma lucidum is between 3 mg/mL and the concentration of nano/chitin in 19 items is greater than (M 21_ according to the scope of the patent application, μ, 18, micron-sized suspension of Ganoderma lucidum fruit body, of which, mg/mL.