CN101897430A - Ganoderma lucidum fruit body suspension and nano/sub-micron grade ganoderma lucidum fruit body suspension - Google Patents
Ganoderma lucidum fruit body suspension and nano/sub-micron grade ganoderma lucidum fruit body suspension Download PDFInfo
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Abstract
The invention provides ganoderma lucidum fruit body suspension and nano/sub-micron grade ganoderma lucidum fruit body suspension which is liquid containing ganoderma particles obtained by mechanically grinding ganoderma lucidum fruit body and contains cellulose, chitin and beta-glucans, and average grain diameter of the ganoderma particles is less than 10mum. The mechanically grinded ganoderma lucidum fruit body suspension can acquire complete active ingredients without extraction and greatly improve specific surface area of the cellulose; and the cellulose needs not to be crushed any more in subsequent utilization and can be taken as a good dietary fiber additive.
Description
Technical field
The present invention relates to a kind of lucidum liquid, particularly relate to a kind of ganoderma lucidum fruitbody suspension of making via mechanical polishing.
Background technology
Nanometer/time micron product is a hot issue in recent years, caused the researcher in recent years, dealer and general popular attention, because the importance and the potentiality of nanometer/time micron science and technology, a lot of countries classify the nanometer/correlative study of time micron science and technology to pay the utmost attention to as, and with nanometer/time micron technological applications on food material, and the food system in the whole world is caused revolutionary change, though the research of nanometer/time micron food material still belongs to the starting stage at present, after but the food materials particle is tiny, should improve absorptivity, and help the baby, the bad person of old man or digestive system absorbs necessary nutriment, and can be applicable to Chinese herbal medicine, make drug effect be easier to performance, for reaching these long-range targets, the understanding of the nanometer of food material/time micron technology and product property is an important problem.
In Chinese herbal medicine, ganoderma lucidum fruitbody is acknowledged as has multinomial health care, as antitumor, regulate immunity function, reducing blood lipid, hypotensive, hypoglycemic, antiviral, antiallergy, anti-oxidant, radioresistance, suppress platelet aggregation and pain relieving etc., no matter be edible with complete fructification or extract, glossy ganoderma is quite safe Chinese herbal medicine really, even give high dose, for example: every day 30g dry fructification extract, verified do not have a significant toxic reaction yet, and utilize nutritive value and the toxicity of zoopery assessment glossy ganoderma, the result shows that glossy ganoderma there is no genotoxicity (genotoxicity).
According to estimates, calendar year 2001 the output of whole world glossy ganoderma be 4800 tons, China is largest production state (3500 tons), and 2,000,000 people's edible glossy ganodermas are arranged approximately, consumes 2.5 kilograms for each person every year approximately, the increase of consumer health consciousness will be grown up ganoderma lucidum product market year by year.But ganoderma lucidum product on the market is a variety of, common is: whole strain glossy ganoderma, glossy ganoderma section, capsule, particle, ingot ball and aqueous drink (ganoderma tea, Lucid Ganoderma wine, Ganoderma Syrupus and Ganoderma lucidum drink) etc., according to its effective component content, quality good or not is uneven.Though ganoderma health-care food is the much-sought-after item of present food products market, yet, its active ingredient (for example chitin, few candy, beta glucan) extraction is difficult for, solubility is low, absorption in human body is also not satisfactory, and the glossy ganoderma slag (mostly being the composition of dietary fiber) after the extraction often is regarded as discarded object again, and causes the waste of resource.
Study through the applicant, if can be with complete ganoderma lucidum fruitbody grain diameter nano/time micron processing, make nanometer/sub-micron grade ganoderma lucidum fruitbody suspension, except helping disengaging of glossy ganoderma polysaccharide isoreactivity composition, and the acquisition complete active ingredients, and increase outside the absorption and utilization of active component, also can therefrom obtain the dietary fiber of nanometer/time micronization, and reduce generation of waste simultaneously.
Summary of the invention
The objective of the invention is to make via mechanical polishing providing a kind of, and have the ganoderma lucidum fruitbody suspension of tiny glossy ganoderma particulate.
Another object of the present invention is to make via mechanical polishing providing a kind of, and has the ganoderma lucidum fruitbody suspension of nanometer/sub-micron grade glossy ganoderma particulate.
Ganoderma lucidum fruitbody suspension of the present invention, for ganoderma lucidum fruitbody via mechanical polishing after the resultant ganoderma lucidum fruitbody suspension that contains the glossy ganoderma particulate, and contain cellulosic, chitin and beta glucan, and the volume average particle size of glossy ganoderma particulate is less than 10 μ m.
Nanometer of the present invention/sub-micron grade ganoderma lucidum fruitbody suspension, be with ganoderma lucidum fruitbody via mechanical polishing after the resultant ganoderma lucidum fruitbody suspension that contains the glossy ganoderma particulate, again through centrifugal resulting centrifuged supernatant, this nanometer/sub-micron grade ganoderma lucidum fruitbody suspension contains glossy ganoderma particulate, cellulosic, chitin and beta glucan, and the volume average particle size of glossy ganoderma particulate is less than 2 μ m.
Beneficial effect of the present invention is: see through the ganoderma lucidum fruitbody suspension made from mechanical polishing, can obtain the complete active ingredients except extracting just, also can significantly improve the cellulosic specific area, and cellulosic later use need not be pulverized again, can be used as a kind of good dietary fiber additive.
Description of drawings
Fig. 1 is the curve map that volume average particle size is ground rotating speed and input concentration relatively;
Fig. 2 is the curve map of milling time relative grain size;
Fig. 3 is the particle diameter distribution map of the centrifuged supernatant of ganoderma lucidum fruitbody suspension;
Fig. 4 is the particle diameter distribution map of the centrifugal sediment of ganoderma lucidum fruitbody suspension;
Fig. 5 is the striograph of fresh ganoderma lucidum fruitbody;
Fig. 6 is the striograph of ganoderma lucidum fruitbody after blender beats;
Fig. 7 is the striograph of ganoderma lucidum fruitbody after homogeneous is handled;
Fig. 8 is the striograph of ganoderma lucidum fruitbody after two-part mechanical lapping;
Fig. 9 is the particle diameter distribution map of ganoderma lucidum fruitbody suspension after 4 ℃ of storages;
Figure 10 is that ganoderma lucidum fruitbody suspension is through the particle diameter distribution map after the freeze drying;
Figure 11 is that ganoderma lucidum fruitbody suspension is through the particle diameter distribution map behind the autoclaving;
Figure 12 is the particle diameter distribution map of centrifuged supernatant after freezing processing of ganoderma lucidum fruitbody suspension;
Figure 13 is the centrifuged supernatant of ganoderma lucidum fruitbody suspension measures gained at once behind autoclaving a particle diameter distribution map;
Figure 14 is that the centrifuged supernatant of ganoderma lucidum fruitbody suspension is through autoclaving and the particle diameter distribution map after storing in 28 days;
Figure 15 is that the centrifuged supernatant of ganoderma lucidum fruitbody suspension is through autoclaving and the particle diameter distribution map after storing in 1 year;
Figure 16 is the centrifuged supernatant particle diameter distribution map of concentration not as yet of ganoderma lucidum fruitbody suspension;
Figure 17 is the particle diameter distribution map of centrifuged supernatant after 2 times of concentration of ganoderma lucidum fruitbody suspension;
Figure 18 is the particle diameter distribution map of centrifuged supernatant after 4 times of concentration of ganoderma lucidum fruitbody suspension;
Figure 19 is the particle diameter distribution map of centrifuged supernatant after 8 times of concentration of ganoderma lucidum fruitbody suspension;
Figure 20 is the particle diameter distribution map of centrifuged supernatant after 12 times of concentration of ganoderma lucidum fruitbody suspension;
Figure 21 is the particle diameter distribution map of centrifuged supernatant after 20 times of concentration of ganoderma lucidum fruitbody suspension;
Figure 22 be ganoderma lucidum fruitbody suspension through dilute 5 times and with the even mixed turbidity of emulsifying agent change curve in time;
Figure 23 be ganoderma lucidum fruitbody suspension through dilute 5 times and with the even mixed turbidity of emulsifying agent change curve in time;
Figure 24 be ganoderma lucidum fruitbody suspension through dilute 10 times and with the even mixed turbidity of emulsifying agent change curve in time;
Figure 25 be ganoderma lucidum fruitbody suspension through dilute 10 times and with the even mixed turbidity of emulsifying agent change curve in time;
Figure 26 be ganoderma lucidum fruitbody suspension through dilute 50 times and with the even mixed turbidity of emulsifying agent change curve in time;
Figure 27 be ganoderma lucidum fruitbody suspension through dilute 50 times and with the even mixed turbidity of emulsifying agent change curve in time.
The specific embodiment
The present invention is described in detail below in conjunction with drawings and Examples:
The embodiment of ganoderma lucidum fruitbody suspension of the present invention, be to handle through mechanical polishing by whole strain ganoderma lucidum fruitbody to make, this ganoderma lucidum fruitbody suspension comprises via the resulting glossy ganoderma particulate of mechanical polishing ganoderma lucidum fruitbody, reaches the edibility emulsifying agent that grinds with ganoderma lucidum fruitbody.
Mechanical polishing is the collision effect that utilizes between mechanical energy, abrasive media (grinding bead), and the shearing force between slurry-fluid and fluid-grinding bead, and broken and dispersion is usually used in coating or nano powder preparation with macroparticle.The mechanical polishing machine is furnished with cooling system, can avoid the generation of high temperature, helps the processing to heat sensitive organic material, for example food material and Chinese herbal medicine etc.
The following glossy ganoderma extract that just obtains with general water hot extraction's method directly compares explanation with ganoderma lucidum fruitbody with the ganoderma lucidum fruitbody suspension that mechanical polishing carries out after nanometer/time micron processing with the present invention.
Control group: general water hot extraction
Ganoderma lucidum fruitbody cleaned be placed in 70 ℃ of baking ovens, with heated-air drying to moisture content remaining about about 10%.(Taiwan) powdered in small, broken bits is standby for RT-04, Rong Tsong Iron Co. to utilize pulverizer.Get 300g ganoderma lucidum fruitbody powder and add 4500mL distilled water, in 100 ℃ of heating 2 hours, while hot with the 60mesh screen cloth coarse filtration of going ahead of the rest, gained filtrate is again with Whatman No.4 filter paper (Whatman, Springfield Mill, UK) filtering to remove trickle residue, can get yellow filtrate, just is extract.Residue adds 4500mL distilled water again, and as above extracts.Each sample carries out the water hot extraction three times, and gained filtrate is mixed, and is concentrated into an amount of volume under vacuum, calculating enrichment factor and record.
Experimental group: mechanical polishing is put in order the strain ganoderma lucidum fruitbody, comprises following steps:
Step (one) is ground pre-treatment.
Weigh ganoderma lucidum fruitbody, clean the back of dicing and add 4 ℃ of deionized waters of 250mL, with pulper (Blender7012S, Waring Commercial, USA) whipping was poured in the beaker in tall form of 600mL after 5 minutes, with deionized water (150mL, 4 ℃) the fructification coarse crushing thing that will remain in the pulper washes out, under 4 ℃, leave standstill overnight, make fibr tissue lax, again with high-shear homogenizer (Polytron PT 3000, Kinematica AG, Switzerland) in 20,000rpm beats 10 minutes down (omnidistance ice bath rises to avoid temperature), makes the fructification particle less than 300 μ m, carries out mechanical polishing again.
Step (two) mechanical polishing.
Use nanometer atomizer mill (MiniPur, NETZCH-Feinmahltechnik GmbH, German) the fructification particle of grinding above-mentioned steps () gained, grinding need be divided into two stages.Phase I uses the yttrium zirconium pearl of particle diameter 0.8mm to be abrasive media, and second stage is that the yttrium zirconium pearl of using particle diameter 0.3mm instead is an abrasive media, charging rate 360mL/min.For inquiring into the influence of operating condition to product, select different glossy ganoderma solid concentrations, abrasive media loading and agitator shaft speed to experimentize, in process of lapping, granularmetric analysis is carried out in sampling in per 30 minutes.
After finishing the preparation of above-mentioned ganoderma lucidum fruitbody suspension, will carry out voltinism and Physical Property Analysis at the lucidum liquid of control group and experimental group gained relatively, as described below respectively:
(1) voltinism analysis
A. beta glucan (the assay of β-D-glucans)
Utilize the quantitative beta glucan of fluorescein stain Aniline blue.Get the centrifugal (2000 * g of ganoderma lucidum fruitbody suspension after the grinding, 10min), filter with Whatman No.4 filter paper, get an amount of sample solution, it is 3mL that adding 0.3NNaOH makes cumulative volume, stirs 30 minutes, adjusts pH to 11.5 with 1N HCl, add the Na2HPO4-NaOH buffer (containing 0.5M NaCl) of the pH 11.5 of 50mM, and be settled to 10mL.Get above-mentioned solution 2mL, add 0.2mL aniline blue (1mg/mL), after mixing with the Vortex concussion, left standstill 2 hours, detect (excitation wavelength 395nm, radiation wavelength 495nm) with fluorescence detector.With the Lentinan preparation standard curve of variable concentrations, the beta glucan content in the calculation sample.
B. total dietary fiber is measured
Adopt AOAC 991.43 (enzymatic-gravimetric method) and two kinds of methods of AOAC 993.21 (nonenzymatic-gravimetric method).AOAC 991.43 is assay methods that widely scholar accepted at present, AOAC 993.21 is that (this method only is applicable to total dietary fiber＞10% to a kind of short-cut method that does not use ferment, the sample of starch＜2% comprises that the part Chinese herbal medicine of glossy ganoderma all meets this standard).
C. chitin (Chitin) assay
Get 400mg ganoderma lucidum fruitbody suspension,, filter with Whatman No.4 filter paper after being cooled to room temperature 100 ℃ of following back hydrolysis 5 hours with 6N HCl, it is standby behind 45～50 ℃ of following drying under reduced pressure to get 1mL filtrate.The hydrolysate of drying returned be dissolved in the distilled water.The dilute solution of getting 1mL said hydrolyzed product adds 0.25mL 4%acetylacetone, heated 1 hour down at 90 ℃, the cooling back adds 2mL ethanol and shakes makes the sediment dissolving, adds 0.25mL Ehrlich reagent subsequently, behind the colour generation, under wavelength 530nm, measure its light absorption value.Utilize glucosamine hydrochloride (5～50 μ g/mL) the production standard curve of variable concentrations, and with 1,4-anhydro-N-acetyl-2-deoxy-D-glucopyranose equivalent calculates chitin content.
(2) Physical Property Analysis
A. particle size distribution measuring
(Nanotrac 150 to use dynamic light scattering particle size analyzer (dynamic light scattering particle size analyzer), Microtrac Inc., USA), measure the particle diameter distribution of the glossy ganoderma particulate of gained, the measurement range 0.8nm to 6500nm of this instrument.As exceed this scope, just use instead Beckman Coulter (CA, USA) LS 230 particle size analyzers of Sheng Chaning measure, its particle size measuring scope 0.4 μ m to 2000 μ m, light source are after filter handling, the detecting lower limit can reach 40nm., and under 25 ℃, measure as skip test with deionized water.Ganoderma lucidum fruitbody suspension after grinding is after suitable dilution, direct with Instrument measuring.Utilize analysis software (FLEX Software, Microtrac Inc., USA) analyze the scattering signal, the Doppler shifts that calculates particle is in the hope of particle diameter distribute percentage, average grain diameter (mean particle size), median diameter particle diameter distributed constants such as (median particle size).
B. microexamination
Use light microscope (Optiphot-Pol, Nikon, the Japan) institutional framework after the coarse crushing of observation glossy ganoderma, and the variation situation of grinding initial stage ganoderma particles.Get the microstructure of glossy ganoderma particulate with sweep electron microscope (SEM) observation post.Sample is applied on the slide after dilution, places under the room temperature dryly, with elargol sample is attached on the aluminium platform, under the vacuum state with ion overlay film device with sample surfaces plated with gold film after, with the microstructure of SEM observation sample.(Japan) observation post gets the microstructure of glossy ganoderma particulate for JEM-1230, JEOL Co.Ltd, confirms the measurement result that particle diameter distributes also to use transmission electron microscope (TEM) in addition.Sample is after suitably diluting, 200mesh plating carbon copper mesh (01800-F with the TEM special use, Ted Pella, Inc., U.S.A.) pick sample suspension, place drying box dry, dried sample is directly with tem observation, and with the CCD photographic system (DualVision CCD, Gatan Inc. USA) pluck required digitized video.The used electron source of TEM is hot-cathode electric rifle (a LaB6 filament), and maximum accelerating potential is 120KV.
(3) stability of ganoderma lucidum fruitbody suspension is handled and is analyzed
A. the interpolation of interfacial agent
Select suitable grinding operation condition (rotating speed: 3600rpm, medium diameter: 0.2mm; Time: 90 minutes), the edibility emulsifying agent that before grinding, adds variety classes, concentration and HLB (hydrophile-lipophile balance, hydrophilic lipophilic equilibrium) value.The emulsifying agent of selecting for use has: 1. ionic interfacial agent (ionic surfactant), for example fatty acid salt (belonging to the anionic interfacial agent).2. non-ionic interfacial agent (nonionicsurfactant), for example (1) sucrose ester (sugar ester) (selecting the HLB value to be respectively 3,7,11 and 15 sucrose ester); (2) polysorbate acid anhydride fatty acid ester ( Span 85,80,60,20 and Tween 65,20, the HLB value is respectively 1.8,4.3,6.7,8.6,10.6,16.7).Above-mentioned selected emulsifier concentration is the solid content to grind, and adds 5%, 10%, 20% and 50%.
B. storage characteristics test
With grind the back and through the stable ganoderma lucidum fruitbody suspension of handling be placed on respectively 4 ℃ with room temperature environment under, between the storage life (0,0.5,1,2,6,12,24,48,72,96 hour), nephelometric analysis and granularmetric analysis are carried out in sampling.In this test, with a little anticorrisive agent of interpolation, to avoid microbial growth.
C. turbidimetric analysis turbidimetry
The height of turbidity is relevant with the quantity of particle in the solution, if the nanometer/time micro particles in the sample is gathered into macroparticle, even precipitation, the suspended particles sum descends, and causes the upper solution turbidity to descend.Can tentatively judge the stability of ganoderma lucidum fruitbody suspension by turbidity, and then eliminate inapplicable emulsifying agent.Use portable nephelometer (portableturbidimeter, model 2100P, HACH company, U.S.A.) variation of turbidity between the mensuration storage life detects preceding elder generation and proofreaies and correct with standard items (Gelex Secondary Standards) (turbidity is respectively 0-10NTU, 0-100NTU and 0-1000NTU), the ganoderma lucidum fruitbody suspension after getting grinding and stablizing processing, through suitably dilution, get 15mL and insert in the water sample detection bottle, directly measure with nephelometer, unit is NTU.
D. phase boundary potential (Zeta potential) is measured
Phase boundary potential is the index of attraction or repulsive force between particle, and the increase of this value shows that particle is stable more, is the effective tool of measuring particle surface character.Weak solution is placed on the detecting instrument, give the voltage of particle fixed value, observe the rate travel of particle,, can derive the relevant nature of particle surface by the correlation of rate travel and time.Phase boundary potential analyzer (Zeta potential analyzer) but the corresponding pH value of assay surface current potential, emulsifying agent interpolation concentration and the change curve of time and result, because the important pointer that is determined as nanometer/time micro particles stability of phase boundary potential, analysis by these data, can reason out the possible cause that nanometer/sub-micron grade glossy ganoderma particulate reassociates, and then inquire into its basic theory.Cooperating existing turbidity to take into account laser particle size analyzer etc., can make the parsing of product stability add complete and tool is ageing, understand the surface characteristic of nanometer/sub-micron grade glossy ganoderma particulate simultaneously, is the important evidence of further modifying particle surface character in the future.
E. microexamination
(Optiphot-Pol, Nikon Japan) observe the variation situation that nanometer/sub-micron grade glossy ganoderma particulate reassociates to use light microscope.In addition, with transmission electron microscope (TEM) (JEM-1230, JEOL Co.Ltd, Japan) microstructure of nanometer/sub-micron grade glossy ganoderma particulate between the observation storage life, to confirm measurement that particle diameter distributes and the structure that reassociates, and with sweep electron microscope (Scanning electron microscope, SEM) observation post gets the microstructure of sample.
(4) 28 days inferior acute toxicity tests
During giving, substances measures the variation of the weight of animals and food consumption quantity.Collect blood sample before cuing open inspection, carry out blood and serum biochemistry analysis.Substances finishes during giving, and cuts open inspection, with perusal and the organ of record animal and the variation of tissue, and measures main organs weight.Maximum dose level group and control group carry out the histopathology check.
A. animal is handled
Animal is to adopt the week ICR mouse from the 5-6 of the big medical college of platform animal center, and every dosage group is used male, female each 12 animal.Before carrying out toxotest, animal at first adapts to a week at the Animal House of tool temperature, humidity and illumination, and freely absorbs normal feed and water.Begin the feeding experiment after one week, after continuous 28 days, sacrifice mouse.
B. glossy ganoderma dosage
In this test, low dosage and in the dosage group be to adopt 0.02 and 0.2g/kg/day respectively, high dose group adopts 2g/kg/day.This low dosage (0.02g/kg/day), middle dosage (0.2g/kg/day), high dose (2g/kg/day) are respectively the same dosage, 10 times and 100 times that the adult advises intake every day.
C. the mensuration of serum biochemistry value
The blood sample of animal is taken from arteria carotis blood and is put into serum separator tube, the serum sample of getting after 3000 * g 15 minutes is centrifugal.Analysis project is as follows: HDL (high density lipoprotein, HDL), low-density lipoprotein (low density lipoprotein, LDL), amino invertase (the glutamic oxaloacetictransaminase of bran propylhomoserin, GOT), amino invertase (the glutamic pyruvic transaminase of bran propylhomoserin pyruvic acid, GPT), blood urea nitrogen (BUN) (blood urea nitrogen, BUN), creatinine (creatinine, CRE), T-CHOL (cholesterol, CHO), triglyceride (triglyceride, TG), sodium ion (Na
+), potassium ion (K
+), chlorion (Cl
-), glucose (Glucose) etc.
D. blood analysis
The blood sample of animal is taken from arteria carotis blood and is put into and contain EDTA anti-coagulants test tube and gather the 1mL whole blood, analysis project is as follows: white blood cell (white blood cell, WBC), red blood cell (red blood cell, RBC), ferroheme (hemoglobin, Hb), blood cell volumetric ratio (hematocrit, Hct), mean corpuscular volume (mean corpuscular, MCV), average red blood cell ferroheme (mean corpuscular hemoglobin, MCH), the average blood red ball concentration of red blood cell (mean corpuscular hemoglobin concentration, MCHC), blood platelet (platelets, PLT), lymph corpuscle number (lymphocytes, LYMPH), red blood cell mean breadth (red cell distributionwidth-coefficient of variation, RDW-CV), blood platelet red blood cell (platelet distribution width, PDW), average platelet volume (mean platelet volume, MPV), blood platelet maxicell scope (platelet-large cell range, P-LCR) etc.
E. tissue pathological slice
With the tissue of all animals, after target organs such as liver, kidney, heart, spleen, lungs, cover ball, secondary cover and uterus ovary take off, put into 10%formalin and fix a week.After dyeing with hematoxylin and eosin in the section back, with microscopic examination.The tissue pathological slice interpretation entrusts Taiwan animal science and technology research institute to carry out.
Below just describe at the processing procedure and the analysis of ganoderma lucidum fruitbody suspension of the present invention:
(1), grinds the relation of processing procedure and particle diameter
Grinding condition as shown in table 1, under identical milling time (90min), inquire into rotating speed (2310～3570rpm), input concentration (0.5～2.0g/mL) and (80～140mL) the influences of grinding bead loading to ganoderma nano/time micron processing procedure.
Table 1
As shown in Figure 1, rotating speed improves the collision frequency and the intensity that can increase between grinding bead, causes the temperature fast rise and impels particle to reassociate, and causes the increase of average grain diameter.Improve input concentration and can reduce interparticle average distance, and make and have more particle in the grinding bead collision scope and increase grinding efficiency.Loading is too high, may limit the motion of grinding bead in process of lapping, reduces impact velocity, causes grinding efficiency to reduce.By above-mentioned experimental result as can be known, in the grinding processing procedure of nanometer/sub-micron grade ganoderma lucidum fruitbody suspension, use higher solid concentration, and reduce the loading that grinds rotating speed and grinding bead, should reduce the volume average particle size of product.
The glossy ganoderma of aforementioned pre-treatment gained suspension in small, broken bits in the fixed rotating speed of 3600rpm, the fixedly feed rate of 360mL/min and the fixedly loading of 140mL, is reached different solid concentrations and two kinds of grinding bead sizes (0.8mm, 0.3mm yttrium zirconium pearl) and ground 30,60,90,120,150,180 and 270 minutes down.The glossy ganoderma of discovery after pre-treatment suspension in small, broken bits, though its particle diameter can be less than 300 μ m, but the overwhelming majority is still greater than 150 μ m, therefore, desire need adopt interim the grinding with ganoderma lucidum fruitbody nanometer/time micronization, just earlier with the fine grinding of 0.8mm yttrium zirconium pearl, treat that particle diameter reduces to below about 50 μ m, change again with 0.3mm yttrium zirconium pearl and continue to grind.
As shown in Figure 2, milling time also influences particle diameter, along with the increase of milling time, volume average particle size reduces, and in grinding 60 minutes, the minimizing speed of volume average particle size is higher, ease up subsequently, grind after 90 minutes, the volume average particle size of glossy ganoderma particulate is about 1.2 μ m.Continue change grind 90 minutes with 0.3mm zirconium pearl after, volume average particle size is less than 1.0 μ m.Particle size distribution range broad because of gained suspension precipitates so easily reassociate between particle, though number average grain diameter specific volume average grain diameter is little a lot, is the quality of having relatively high expectations, and the present invention is to be pointer with the volume average particle size.
By the granularmetric analysis result shown in Fig. 3,4 as can be known, grind 180 minutes gained ganoderma lucidum fruitbody suspension centrifugal 10 minutes with above-mentioned with 0.3mm yttrium zirconium pearl with 10000g, after removing bigger particle, the particle size distribution range of centrifuged supernatant is 28～578nm (Fig. 3), the about 105nm of volume average particle size, wherein 67% particle is less than 100nm, with regard to food, should increase its absorption in vivo less than 1 μ m, the particle of centrifuged supernatant is all less than 1 μ m, though its solid content is lower, but the active component that proposes because of grinding quilt collection all still remains.And the particle size of centrifugal deposition is less than 10 μ m (Fig. 4), and quality is careful, can be used as the base material of skin care products (as facial mask) or dressing, artificial skin, and because of being rich in chitin and cellulose, so can be used as a kind of good dietary fiber additive.
Shown in Fig. 5～8, the glossy ganoderma particulate after microexamination is ground, fresh ganoderma lucidum fruitbody (as shown in Figure 5) are observed with sweep electron microscope (SEM), by the arborization bone mycelia of clearly visible ganoderma lucidum fruitbody among Fig. 6 after blender beats.After suitably homogeneous is handled, (as shown in Figure 7) comparatively in small, broken bits, loose that this bone mycelia becomes, and its size about tens of to hundreds of microns, and this a kind of state will help follow-up milled processed.Fig. 8 is after two-part grinds, the result who utilizes transmission electron microscope (TEM) to observe, circular granular is the nano particle in the ganoderma lucidum fruitbody suspension among the figure, can significantly see by scale, single particle really can reach the following nanoscale yardstick of 0.1 μ m, but, can reach hundreds of nanometers, just the sub-micron grade yardstick because of intergranular reassociating makes whole particle size range become big.This a kind of result has also verified the result that above-mentioned particle size analyzer is measured, and reaffirms the medium milling processing procedure that the present invention uses, and can make the glossy ganoderma particulate after ganoderma lucidum fruitbody grinds reduce to nanometer/time micro-meter scale really.
Because ganoderma lucidum fruitbody is rich in cellulose and chitin, quality is coarse and tough and tensile, and the particle diameter decline with these fibrous matters except that increasing specific surface area, also can improve the mouthfeel of product feed.The physiological function of dietary fiber is relevant with its surperficial suction-operated, therefore, improves the specific area of dietary fiber, can promote its physiologically active.The particle diameter of dietary fiber is dropped to 1 μ m by 0.1mm, and specific area can improve 100 times, the suggestion intake of dietary fiber can be kept to former suggestion amount percentage one, if particle diameter drops to below the 100nm, the suggestion intake can reduce to thousand minutes of former suggestion amount one.Change speech, in the drinking water of 500mL, add nanometer/sub-micron grade dietary fiber of 50～500ppm, advise intake (25g) every day that just can reach dietary fiber.Based on this viewpoint, the centrifuged supernatant of ganoderma lucidum fruitbody suspension is possessed the distinctive physiologically active ingredient of glossy ganoderma, and the dietary fiber of high surface also additionally is provided.
(2) relation of grinding processing procedure and active ingredient
As shown in table 2, behind medium milling, the content of beta glucan obviously increases, grind with big abrasive media (particle diameter 0.8mm), (about 0.01mg/mL) is close for beta glucan content in the product and water hot extraction's product, along with the reduction of media particle size and the increase of milling time, beta glucan content increases to 4 times of water hot extraction's product, and is higher than collected on the market commodity (0.0023～0.0202mg/mL).
Table 2
General commercial goods, its active ingredient of claiming is many to be extracted with hot water or organic solvent, though can obtain this active ingredient, extracted amount is limited, and waste water, organic solvent and residue treatment problem after the processing are still arranged.In the processing procedure mode of mechanical polishing, have dietary fibers such as cellulose, chitin in the ganoderma lucidum fruitbody suspension except making, also can impel disengaging of effective ingredient by the fragmentation of ganoderma lucidum fruitbody cell membrane in the fine grinding process.
As shown in table 3, chitin content also has similar phenomenon, be higher than the product that the 0.8mm medium milling is obtained with chitin content in the product of 0.3mm medium milling gained, but after the bigger particle in the products obtained therefrom being removed with centrifugation, the chitin content of supernatant can decline to a great extent, but still be about 5 times of general water hot extraction's products obtained therefrom, as seen this particle that is removed contains rich chitin.Since hot water extracting almost take out only water-soluble substances, so the chitin content in its extract is very low, the overwhelming majority all remains in the dregs of a decoction.The dregs of a decoction contain 35～40% chitins approximately, 40～50% beta glucans, all the other are the nitrogenous thing based on melanin (melanines), be good dietary fiber source, as nanometer/time micron-scale is pulverized and be finely ground to fructification, not only can replace extraction, also will preserve all the components (being rich in dietary fibers such as chitin, cellulose) of fructification in the product, and because of particle little, this nanometer/sub-micron grade ganoderma lucidum fruitbody suspension or its concentrated product help absorption by human body, so will be suitable for the additive as health food.
Table 3
(3) stability of ganoderma lucidum fruitbody suspension
Shown in Fig. 9～11, ganoderma lucidum fruitbody is after grinding, the glossy ganoderma particulate of high concentration and broad particle diameter distribute, impel and collide and be gathered into rapidly bigger particulate cluster between the glossy ganoderma particulate mutually, the increase of particle diameter causes gravitational effect much larger than the Brownian movement effect, and then produces tangible sedimentation phenomenon.Under 4 ℃, leave standstill 24 hours its particle diameters of post analysis, particulate has been assembled and has been constituted tens of microns cluster, volume average particle size increases to 9.35 μ m (as shown in Figure 9), consider that abrasive product is in following process, as the stability of chilled storage, freeze drying or high-temperature sterilization processing etc., at (121 ℃ of freezing (20 ℃, 24 hours) and high pressure steam sterilizations, 15 minutes) after fructification suspension carry out granularmetric analysis, to inquire into the particle diameter distributional difference before and after the processing.
The glossy ganoderma particulate of ganoderma lucidum fruitbody suspension can reassociate in refrigerating process, dried up phenomenon of phase separation is seemingly arranged after thawing, the particulate cluster that assemble to constitute be flocculence and be difficult to physics or mechanical force (handling) as concussion, homogeneous, ultrasonic concussion will dispersion, the particle diameter of freezing back fructification suspension distributes as Figure 10, and volume average particle size is 109 μ m.High pressure steam sterilization is handled the effect of reassociating serious between particulate that also can cause, constitute and be crumby particulate cluster, and as be difficult to as the freezing processing physics or mechanical force will dispersion, its particle size distribution range 5.9～824.5 μ m, volume average particle size is 132 μ m (as shown in figure 11).The ganoderma lucidum fruitbody suspension of mechanical polishing gained, the particle size distribution range broadness, the precipitation that easily reassociates, but solid content height are rich in chitin, cellulose, and this ganoderma lucidum fruitbody suspension or its concentrated product will be suitable for the additive as health food.
(4) stability of the centrifuged supernatant of ganoderma lucidum fruitbody suspension
As shown in table 4, the change of milling time, except meeting influences glossy ganoderma particulate average grain diameter, also remote-effects stability in storage, milling time is got over the elder, and the stability of product is high more.Grind the centrifuged supernatant product (centrifugal 10 minutes of 10000g) of 30 minutes ganoderma lucidum fruitbody suspension, increase to 1.199 μ m through the long-pending average grain diameter of 21 days memories by 0.195, but grind the supernatant product (centrifugal 10 minutes of 10000g) of 180 minutes ganoderma lucidum fruitbody suspension, volume average particle size increases to 0.137 μ m by 0.126, increment rate is lower than 10%, and showing increases the stability that milling time helps particle.
Then, with regard to freeze-drying, high pressure steam sterilization and concentration discussion is done in the influence of the centrifuged supernatant of ganoderma lucidum fruitbody suspension.
Table 4
As shown in figure 12, earlier with the glossy ganoderma supernatant after freeze-drying, be scattered in the aqueous solution again, and measure particle diameter with Nanotrac 150 particle size analyzers and distribute (being limited to 6.54 μ m on the particle size measuring), by the result as can be known, the freeze-drying processing can impel between the glossy ganoderma particulate and reassociates.After the freeze-drying, the particle size distribution range broadness, the particle diameter that the significant proportion particulate is arranged is greater than 6.54 μ m (can't record for fear of instrument measures the upper limit), and its volume average particle size is 1.177 μ m.
Shown in Figure 13～15, centrifuged supernatant is still possessed goodish stability after high pressure steam sterilization is handled, after the sterilization treatment, treat that temperature drops to room temperature and just measures its particle diameter, all particles are all less than 1 μ m, particle size distribution range 36～800nm, volume average particle size is 140nm, and still has 56% particulate to maintain below the 100nm.Observe it stores 28 days (as shown in figure 14) and 1 year (as shown in figure 15) under room temperature change of size, though distributing, particle diameter has toward big particle diameter skew, but offset amplitude is very little, and its volume average particle size also only increases to 165nm and 373nm respectively, show that centrifuged supernatant is still very stable behind high temperature action, particle is not remarkable in the phenomenon that reassociates of lay up period.
Though reduce to below the 1 μ m by the centrifugal particle diameter of centrifuged supernatant can the distribution, and make its stability that has, but relative, the solid content of centrifuged supernatant and glossy ganoderma particle number can significantly reduce.For improving solid concentration, reduce the moisture of centrifuged supernatant in the mode of concentrating that reduces pressure, and, measure its change of size in different concentration rates (concentrationratio) sampling down.The calculating of concentration rate is to remove last final concentrate volume with the stoste volume.Figure 16～21 and table 5 are respectively centrifuged supernatant through 2,4,8,12 and 20 times of granularmetric analysis results after concentrating, and are lower than 4 times of concentrated centrifuged supernatant, and its change of size is little, and volume average particle size only increases by 33%, and particulate is all less than 1 μ m.When concentrating more than 8 times, begin to have clustering phenomena to produce between particle, and particle diameter is distributed toward big particle diameter skew, particle diameter distributes greater than 1 μ m.With regard to present embodiment,, centrifuged supernatant is concentrated 4～6 times should meet required for improving the product solid concentration and taking into account stability.
Table 5
(5) emulsifying agent is to the influence of ganoderma lucidum fruitbody stability of suspension
Turbidity is the easy physical parameter of a kind of measurement, and for the aaerosol solution of stable dispersion, its turbidity should not present significant difference in time.Therefore, the present invention is directed to various changes that the institute desire inquires into because of, carry out nephelometric analysis, with the emulsifying agent of screening.
As shown in table 6, the ganoderma lucidum fruitbody that does not add emulsifying agent is after grinding, and ganoderma lucidum fruitbody suspension turbidity is higher than 1000NTU, and the turbidity of the sample after dilute 2,5,10 and 50 times all descends in time and gradually, and dilution ratio is height more, and the rate of change of turbidity is littler.The stable higher concentration of the turbidity of low concentration (highly diluted multiplying power) suspension (low dilution ratio) is good, shows that particle concentration is the important parameter that influences stability of suspension, and concentration heals and collides more height of probability between high particulate, and clustering phenomena is also obvious relatively.Generally speaking, the gathering between particulate is quite obvious, causes the gathering sedimentation behavior of ganoderma lucidum fruitbody suspension obvious.
Table 6
In the present invention, selection is with different HLB (hydrophile-lipophile balance, hydrophilic lipophilic equilibrium) Zhi sucrose ester (sugar ester, HLB is respectively 3,7,11 and 15), polysorbate acid anhydride fatty acid ester (Span 85 (sorbester p37), Span 80 (sorbester p37), Span 60 (sorbester p37), Span 20 (sorbester p37) and Tween 65 (polysorbate65), Tween 20 (polysorbas20), HLB is respectively 1.8,4.3,6.7,8.6,10.6 and 16.7) and fatty acid glycerine fat (HLB is 3.8) as the emulsifying agent of Preliminary screening, addition is fixed as earlier with respect to 5% of ganoderma lucidum fruitbody weight.
Figure 22～27 are respectively ganoderma lucidum fruitbody suspension through diluting 5 times (Figure 22,23), 10 times (Figure 24,25) and 50 times (Figure 26,27), and add after various emulsifying agents evenly mix turbidity rate of change relation in time respectively.The turbidity rate of change is that negative value represents that turbidity descends, and rises on the occasion of expression.Except minority on the occasion of, the turbidity variation tendency of nearly all test is decline.
Generally speaking, dilution ratio is higher, and the collision probability reduces between particulate, and aggtegation is slowed down, and turbidity rate over time eases up, the turbidity of each dilution ratio all in time increase and descend.
Under the situation of highly diluted multiplying power 50 (the about 100ppm of glossy ganoderma particle concentration (0.001mg/mL)), turbidity rate of change minimum, and with the stability of polysorbate acid anhydride fatty acid ester emulsifying agent, turbidity rate of change absolute value is all less than 30% after 96 hours.Under low dilution ratio (5 times and 10 times), turbidity rate of change and emulsifying agent kind, HLB value are like onrelevant, and turbidity rate of change absolute value does not just have remarkable stablizing effect all greater than 80% after 96 hours.
Therefore, as with turbidity as judgment standard, with regard to ganoderma lucidum fruitbody suspension, with the polysorbate acid anhydride fatty acid ester of 5% addition as dispersant, the stability that the ganoderma lucidum fruitbody suspension of concentration 100ppm (50 times of highly diluted multiplying powers) is had.
Analysis result as can be known by the particle diameter (the glossy ganoderma particle concentration is 100ppm) of table 7, the ganoderma lucidum fruitbody suspension that does not add emulsifying agent through leave standstill deposit 96 hours after, volume average particle size after the glossy ganoderma particulate reassociates (MV) is 3.24 μ m, wherein, particle diameter accounts for 24.9% of ganoderma lucidum fruitbody gross mass less than the particulate (nanometer/sub-micron grade) of 1 μ m, and particle diameter belongs to nanoscale (less than 100nm) person and accounts for 2.04% of ganoderma lucidum fruitbody gross mass.When adding 5% emulsifying agent when grinding, the volume average particle size amplitude of variation of adding fatty acid glycerine fat, Span 85 and Span is less, becomes 3.86,2.90,3.68 μ m respectively.In addition, improve the stability that the phase boundary potential value will help particulate, ganoderma lucidum fruitbody suspension does not add the phase boundary potential value pact-11.7mV of emulsifying agent, present embodiment uses in the emulsifying agent, comparatively remarkable for the increase of particulate phase boundary potential with Span 85, Span 80 and Span 20, the phase boundary potential value is respectively-16.7 ,-13.8 and reaches-13.7mV.
Comprehensive above-mentioned nephelometric analysis, granularmetric analysis and phase boundary potential analysis result, under the situation of emulsifying agent addition 5%, the emulsifying agent that the HLB value is little helps to stablize ganoderma lucidum fruitbody suspension system, therefore, below just select sucrose ester (HLB 3), fatty acid glycerine fat (HLB 3.8), Span 85 (HLB 1.8) and Span 80 (HLB 4.3) etc., carry out two-part mechanical lapping with ganoderma lucidum fruitbody is actual, carry out the emulsifying agent addition the sex test of ganoderma lucidum fruitbody suspension system stability.
Table 7
Shown in table 8,9, ganoderma lucidum fruitbody suspension in the different emulsifiers addition (with respect to ganoderma lucidum fruitbody weight 5%, 10%, 20% and 50%) time granularmetric analysis, phase boundary potential measurement.By the particle diameter distribution results as can be known, with the stablizing effect of Span 80, during its addition 10%, the volume average particle size of particulate can be reduced to 2.29 μ m in four kinds of emulsifying agents, and improve nanometer and time shared ratio of micro particles.Observing the variation situation of phase boundary potential can find, the addition that improves four kinds of emulsifying agents all can increase the phase boundary potential value of ganoderma lucidum fruitbody suspension, but it is limited to be higher than 20% stablizing effect.
Table 8
Table 9
During 28 days inferior acute toxicity tests, average every day of the changes of weight of male, female mice control group and processed group, average every day food consumption quantity with average every day water consumption all do not have significant difference, hero, female mice processed group and control group blood analysis be no significant difference also, its detected value is all in range of normal value, the histotomy inspection of major organs is also normal, so in do not cause death phenomenon and cause bad clinical s sign of duration of test.
Take a broad view of above-mentioned, ganoderma lucidum fruitbody contains a large amount of crude fibres and lignin composition, and the composition of general fungal cell wall still has chitin except cellulose, be present in the cell membrane as glossy ganoderma polysaccharide isoreactivity composition also more, the present invention sees through the mode that mechanical polishing is made ganoderma lucidum fruitbody suspension, can obtain the complete active ingredients except extracting just, also can significantly improve the cellulosic specific area, and cellulosic later use need not be pulverized again, can be used as a kind of good dietary fiber additive.Wherein, the ganoderma lucidum fruitbody suspension that grinds gained is again after follow-up centrifugal treating, be rich in chitin and cellulose in the centrifugal deposition, its particle size is less than 10 μ m, quality is careful, can be used as the base material of skin care products (as facial mask) or dressing, artificial skin, and in the centrifuged supernatant, the glossy ganoderma diameter of particle all belongs to nanometer/sub-micron grade, and possess complete active ingredient, active ingredients such as its chitin are more than 5 times of general water hot extraction, and contain the less cellulose of part particle diameter, equally can be in order to the additive as health food.
Claims (15)
1. a ganoderma lucidum fruitbody suspension is characterized in that, is to be made via mechanical polishing by ganoderma lucidum fruitbody, and contains glossy ganoderma particulate, cellulosic, chitin and beta glucan, and the volume average particle size of glossy ganoderma particulate is less than 10 μ m.
2. ganoderma lucidum fruitbody suspension as claimed in claim 1 is characterized in that, particle diameter accounts for more than 10% of glossy ganoderma gross mass less than the glossy ganoderma particulate of 1 μ m.
3. ganoderma lucidum fruitbody suspension as claimed in claim 2 is characterized in that chitin content is higher than 0.1mg/mL, and cellulosic content is higher than 0.1mg/mL, and beta glucan content is higher than 0.01mg/mL.
4. as claim 1,2 or 3 described ganoderma lucidum fruitbody suspension, it is characterized in that, also comprise the edibility emulsifying agent that carries out mechanical polishing with ganoderma lucidum fruitbody.
5. ganoderma lucidum fruitbody suspension as claimed in claim 4 is characterized in that, this edibility emulsifying agent is to be selected from non-ionic interfacial agent, and this edibility emulsifier content is 0.1～25% of a ganoderma lucidum fruitbody weight.
6. ganoderma lucidum fruitbody suspension as claimed in claim 5 is characterized in that, the hydrophilic lipophilic equilibrium value of edibility emulsifying agent is between 1～5.
7. ganoderma lucidum fruitbody suspension as claimed in claim 6 is characterized in that, this edibility emulsifying agent is to be selected from by polysorbate acid anhydride fatty acid ester, fatty acid glycerine fat, and the colony that is formed of mixing of polysorbate acid anhydride fatty acid ester and fatty acid glycerine fat.
8. ganoderma lucidum fruitbody suspension as claimed in claim 7 is characterized in that, polysorbate acid anhydride fatty acid ester is the colony that is formed of mixing that is selected from sorbester p17, sorbester p37 and sorbester p17 and sorbester p37.
9. nanometer/sub-micron grade ganoderma lucidum fruitbody suspension, it is characterized in that, be with ganoderma lucidum fruitbody via mechanical polishing after, again through centrifugal resulting centrifuged supernatant, this nanometer/sub-micron grade ganoderma lucidum fruitbody suspension contains glossy ganoderma particulate, cellulosic, chitin and beta glucan, and the volume average particle size of glossy ganoderma particulate is less than 2 μ m.
10. nanometer as claimed in claim 9/sub-micron grade ganoderma lucidum fruitbody suspension is characterized in that particle diameter accounts for more than 60% of total glossy ganoderma fraction of particle less than the glossy ganoderma particulate of 1 μ m.
11., it is characterized in that the concentration range of glossy ganoderma particulate is between 3mg/mL～100mg/mL as claim 9 or 10 described nanometers/sub-micron grade ganoderma lucidum fruitbody suspension.
12. nanometer as claimed in claim 10/sub-micron grade ganoderma lucidum fruitbody suspension is characterized in that, the volume average particle size of glossy ganoderma particulate is less than 250nm, and the particle diameter of glossy ganoderma particulate is less than 1 μ m.
13. nanometer as claimed in claim 12/sub-micron grade ganoderma lucidum fruitbody suspension is characterized in that the particle diameter of the glossy ganoderma particulate more than 50% is less than 100nm.
14. nanometer as claimed in claim 13/sub-micron grade ganoderma lucidum fruitbody suspension is characterized in that the concentration range of glossy ganoderma particulate is between 3mg/mL～20mg/mL.
15., it is characterized in that chitin concentration is greater than 0.1mg/mL as claim 9,10,12 or 13 described nanometers/sub-micron grade ganoderma lucidum fruitbody suspension.
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| CN102240189A (en) * | 2011-05-20 | 2011-11-16 | 南京航空航天大学 | Standing wave type ultrasonic dust collector and dustcollection method thereof |
| CN115068424A (en) * | 2022-07-15 | 2022-09-20 | 中农弘仁纳米生物科技(北京)有限公司 | Preparation method and application of nanoscale wall-broken ganoderma lucidum spore powder |
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| CN102132864A (en) * | 2011-02-22 | 2011-07-27 | 江苏安惠生物科技有限公司 | Edible and medicinal nano fungus fiber for inducing proliferation of probiotics and production method thereof |
| CN102240189A (en) * | 2011-05-20 | 2011-11-16 | 南京航空航天大学 | Standing wave type ultrasonic dust collector and dustcollection method thereof |
| CN115068424A (en) * | 2022-07-15 | 2022-09-20 | 中农弘仁纳米生物科技(北京)有限公司 | Preparation method and application of nanoscale wall-broken ganoderma lucidum spore powder |
| CN115068424B (en) * | 2022-07-15 | 2024-05-24 | 中农弘仁纳米生物科技(北京)有限公司 | Preparation method and application of nanoscale wall-broken ganoderma lucidum spore powder |
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