阅读说明：本技术 一种黄精植物甾醇多糖蛋白的制备方法及其活性制品 (Preparation method of polygonatum sibiricum phytosterol polysaccharide protein and active product thereof ) 是由 陈蕙如 陈茂达 于 2021-08-18 设计创作，主要内容包括：本发明公开一种黄精植物甾醇多糖蛋白的制备方法,以黄精植物发酵物进入肠胃道后的肝肠循环吸收后,由下腔动脉路径进入泌尿道系统血管循环引发的局部作用。为此,本发明所要解决的技术问题在于现有发酵技术中从发酵物分离多糖蛋白,并解决提取率较低,且生物活性不高的问题,同时,本发明还公开一种黄精植物甾醇多糖蛋白的活性制品,其具有促骨盆底骨骼肌强度,及抗血管收缩功效。(The invention discloses a preparation method of polygonatum sibiricum phytosterol polysaccharide protein, which is a local effect caused by the circulation of blood vessels of a urinary tract system from a inferior vena cava path after a polygonatum sibiricum plant fermentation product enters the gastrointestinal tract and is absorbed by the liver and intestine circulation. Therefore, the invention aims to solve the technical problems of separating polysaccharide protein from a fermentation product in the prior fermentation technology, solving the problems of low extraction rate and low biological activity, and simultaneously discloses an active product of polygonatum plant sterol polysaccharide protein, which has the effects of promoting the strength of skeletal muscles of the pelvic floor and resisting vasoconstriction.)

1. A preparation method of polygonatum sibiricum phytosterol polysaccharide protein is characterized by comprising the following steps:

(1) pretreatment: taking the root of fresh rhizoma Polygonati, removing attachments on the surface of rhizoma Polygonati, weighing (wet weight) and taking as the measuring basis, cutting rhizoma Polygonati into pieces, adding 4-8 times of water into the crushed rhizoma Polygonati, and making the crushed rhizoma Polygonati into pulp by using a pulp homogenizer or a colloid mill;

(2) primary extraction: filtering the rhizoma polygonati homogenate obtained in the step (1) by adopting a filtering method, and respectively collecting a filter cake and filtrate, wherein the filtrate is a first extracting solution;

(3) secondary extraction: re-suspending the sealwort filter cake obtained in the step (2) in water with the weight of 3-5 times, adjusting the pH of the solution to 5.0-7.0, filtering and collecting filtrate to obtain a second extracting solution;

(4) enzymolysis: mixing the filtrates, adding 0.1-5.0 wt% of compound zymolyte of rhizoma Polygonati, and stirring at 30-50 deg.C for 6-12 hr; the composite zymolyte is prepared by mixing the following components in a mass ratio of 1-3: 0.5-2 of cellulase and papain.

(5) And (3) precipitation treatment: adjusting the pH value of the enzymatic hydrolysate obtained in the step (4) to 3.0-4.5, adding a sulfate solution, uniformly stirring, standing, treating the filtrate by using a centrifugal method, and collecting the filtrate;

(6) and (3) drying: and (4) performing ultrafiltration treatment on the filtrate obtained in the step (5), collecting trapped fluid, and performing spray drying to obtain powder, namely the crude polygonatum polysaccharide protein composition.

2. The method for preparing polygonatum kingianum phytosterol polysaccharide protein according to claim 1, wherein in the step (4), the method for preparing the composite zymolyte fermentation product comprises the following steps: taking cleaned pineapple raw materials, setting enzymolysis conditions as a material-liquid ratio of 1: 10(m/V), adding 2% of cellulase CellulaseACCF-4740, at a temperature of 55 ℃, pH4.5 and a reaction time of 2.5h, carrying out enzymolysis, after the hydrolysis of the cellulase is finished, inactivating the enzyme in a boiling water bath for 15min, centrifuging at 9000r/min, and collecting supernatant to obtain pineapple hydrolysate; after the enzyme activity of the pineapple enzymatic hydrolysate is inactivated, adjusting the pH value to 6.2-6.6, sterilizing at 121 ℃ by high-pressure steam for 20min, inoculating 1 volume percent of pentose slice bacteria liquid activated by MRS culture medium into the pineapple enzymatic hydrolysate, standing in a constant-temperature incubator at 37 ℃ and fermenting; after the fermentation is finished, centrifuging at 9000r/min, and collecting supernatant to obtain the compound zymolyte.

3. The method for preparing polygonatum kingianum phytosterol proteoglycan according to claim 1, wherein in the step (4), the raw material for enzymolysis is a mixture of filtrates obtained by primary and secondary extraction.

4. The method for preparing polygonatum kingianum phytosterol polysaccharide protein according to claim 1, wherein in the step (4), the method for preparing polygonatum kingianum polysaccharide protein comprises the following steps: extracting the extract at 4 deg.C for 44-50h, filtering, slowly adding ammonium sulfate into the filtrate at an addition amount of 0.5-1.5g/mL, stirring continuously during adding ammonium sulfate to prevent partial supersaturation of ammonium sulfate, standing for 45-50h, centrifuging at 4 deg.C for 5-10min at 25000g, redissolving the precipitate with acidic buffer solution, dialyzing at 4 deg.C with dialysis bag with molecular weight of 5000Da, centrifuging the dialysate at 20000g for 10min, and collecting the supernatant as the prepared expansin solution;

wherein, the components of the homogenization buffer solution are as follows: 25 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 1.5mmol/L sodium metabisulfite, 2mmol/L EDTA, 0.1 wt% Triton X-100, pH 7.0; the components of the extracting solution are as follows: 15 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 1.0mmol/L ethylene diamine tetraacetic acid, 1.5mmol/L sodium metabisulfite, 0.5mol/L NaCl, pH6.0; the preparation of the acidic buffer solution comprises the following steps: 2.5g of sodium acetate is dissolved in water, the pH is adjusted to 4.0 with glacial acetic acid, and the volume of the water is adjusted to 1L.

5. The method for preparing polygonatum plant sterol polysaccharide protein according to claim 1, wherein in the step (5), the sulfate added to the solution can be ferric sulfate, zinc sulfate or sodium sulfate or a mixture thereof.

6. An active product of polygonatum kingianum phytosterol polysaccharide protein is characterized by comprising the following components in parts by weight: 10-20 parts of maca enzyme extract, 20-48 parts of rhizoma polygonati enzyme extract, 15-25 parts of medlar enzyme extract, 15-20 parts of Chinese yam enzyme extract, 3-8 parts of beer yeast extract, 3-8 parts of trehalose extract, 15-30 parts of pumpkin seed extract, 4-8 parts of taurine extract, 5-10 parts of L-arginine extract, 13-18 parts of maltodextrin and 0.2-1 part of magnesium stearate.

7. The active product of polygonatum kingianum phytosterol proteoglycan according to claim 6, wherein the concentration of said active product is 65% -88%.

Technical Field

The invention relates to the technical field of natural plant extracts, in particular to a preparation method of polygonatum sibiricum phytosterol polysaccharide protein and an active product thereof.

Background

Rhizoma Polygonati (Polygonatum sibiricum Red.) is dried rhizome of plant of Liliaceae. Rhizoma Polygonati is used as the medicine by root and stem. Has effects in invigorating qi , invigorating spleen, moistening lung, and invigorating kidney. Can be used for treating weakness of spleen and stomach, asthenia, xerostomia, dry cough due to lung deficiency, insufficiency of essence and blood, and diabetes due to internal heat. Sweet in nature and flavor, neutral in nature and mild in nature, and entering lung, spleen and kidney meridians. Can be used for treating yin deficiency, lung dryness, dry cough with little phlegm, and chronic cough due to lung and kidney yin deficiency, for treating spleen and stomach weakness, invigorating spleen yin, invigorating spleen qi, and treating dizziness, soreness of waist and knees, premature gray hair, and diabetes due to kidney essence deficiency.

Disclosure of Invention

One object of the present invention is: provides a preparation method of polygonatum kingianum phytosterol polysaccharide protein and an active product thereof, which are used for solving the problems in the prior art.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of polygonatum sibiricum phytosterol polysaccharide protein comprises the following steps:

(1) pretreatment: taking the root of fresh rhizoma Polygonati, removing attachments on the surface of rhizoma Polygonati, weighing (wet weight) and taking as the measuring basis, cutting rhizoma Polygonati into pieces, adding 4-8 times of water into the crushed rhizoma Polygonati, and making the crushed rhizoma Polygonati into pulp by using a pulp homogenizer or a colloid mill;

(2) primary extraction: filtering the rhizoma polygonati homogenate obtained in the step (1) by adopting a filtering method, and respectively collecting a filter cake and filtrate, wherein the filtrate is a first extracting solution;

(3) secondary extraction: re-suspending the sealwort filter cake obtained in the step (2) in water with the weight of 3-5 times, adjusting the pH of the solution to 5.0-7.0, filtering and collecting filtrate to obtain a second extracting solution;

(4) enzymolysis: mixing the filtrates, adding 0.1-5.0 wt% of compound zymolyte of rhizoma Polygonati, and stirring at 30-50 deg.C for 6-12 hr; the composite zymolyte is prepared by mixing the following components in a mass ratio of 1-3: 0.5-2 of cellulase and papain.

(5) And (3) precipitation treatment: adjusting the pH value of the enzymatic hydrolysate obtained in the step (4) to 3.0-4.5, adding a sulfate solution, uniformly stirring, standing, treating the filtrate by using a centrifugal method, and collecting the filtrate;

(6) and (3) drying: and (4) performing ultrafiltration treatment on the filtrate obtained in the step (5), collecting trapped fluid, and performing spray drying to obtain powder, namely the crude polygonatum polysaccharide protein composition.

As a preferred technical solution, in the step (4), the preparation method of the composite zymolyte fermentation product comprises: taking cleaned pineapple raw materials, setting enzymolysis conditions as a material-liquid ratio of 1: 10(m/V), adding 2% of cellulase CellulaseACCF-4740, at a temperature of 55 ℃, pH4.5 and a reaction time of 2.5h, carrying out enzymolysis, after the hydrolysis of the cellulase is finished, inactivating the enzyme in a boiling water bath for 15min, centrifuging at 9000r/min, and collecting supernatant to obtain pineapple hydrolysate; after the enzyme activity of the pineapple enzymatic hydrolysate is inactivated, adjusting the pH value to 6.2-6.6, sterilizing at 121 ℃ by high-pressure steam for 20min, inoculating 1 volume percent of pentose slice bacteria liquid activated by MRS culture medium into the pineapple enzymatic hydrolysate, standing in a constant-temperature incubator at 37 ℃ and fermenting; after the fermentation is finished, centrifuging at 9000r/min, and collecting supernatant to obtain the compound zymolyte.

As a preferable technical solution, in the step (4), the enzymolysis raw material is a mixture of filtrates obtained by primary and secondary extraction.

As a preferred technical solution, in the step (4), the preparation method of the polygonatum polysaccharide protein comprises the following steps: extracting the extract at 4 deg.C for 44-50h, filtering, slowly adding ammonium sulfate into the filtrate at an addition amount of 0.5-1.5g/mL, stirring continuously during adding ammonium sulfate to prevent partial supersaturation of ammonium sulfate, standing for 45-50h, centrifuging at 4 deg.C for 5-10min at 25000g, redissolving the precipitate with acidic buffer solution, dialyzing at 4 deg.C with dialysis bag with molecular weight of 5000Da, centrifuging the dialysate at 20000g for 10min, and collecting the supernatant as the prepared expansin solution;

wherein, the components of the homogenization buffer solution are as follows: 25 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 1.5mmol/L sodium metabisulfite, 2mmol/L EDTA, 0.1 wt% Triton X-100, pH 7.0; the components of the extracting solution are as follows: 15 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 1.0mmol/L ethylene diamine tetraacetic acid, 1.5mmol/L sodium metabisulfite, 0.5mol/L NaCl, pH6.0; the preparation of the acidic buffer solution comprises the following steps: 2.5g of sodium acetate is dissolved in water, the pH is adjusted to 4.0 with glacial acetic acid, and the volume of the water is adjusted to 1L.

In a preferable embodiment, in the step (5), the sulfate added to the solution may be ferric sulfate, zinc sulfate, or sodium sulfate, or a mixture thereof.

An active product of polygonatum kingianum phytosterol polysaccharide protein comprises the following components in parts by weight: 10-20 parts of maca enzyme extract, 20-48 parts of rhizoma polygonati enzyme extract, 15-25 parts of medlar enzyme extract, 15-20 parts of Chinese yam enzyme extract, 3-8 parts of beer yeast extract, 3-8 parts of trehalose extract, 15-30 parts of pumpkin seed extract, 4-8 parts of taurine extract, 5-10 parts of L-arginine extract, 13-18 parts of maltodextrin and 0.2-1 part of magnesium stearate.

As a preferred technical scheme, the concentration of the active product is 65-88%.

The invention has the beneficial effects that: disclosed is a process for preparing polygonatum rhizome phytosterol polysaccharide protein, which comprises the steps of carrying out liver-intestine circulation absorption after polygonatum rhizome plant fermentation products enter gastrointestinal tract, entering the vascular circulation of urinary tract system from inferior cavity artery path for local action. Therefore, the invention aims to solve the technical problems of separating polysaccharide protein from a fermentation product in the prior fermentation technology, solving the problems of low extraction rate and low biological activity, and simultaneously discloses an active product of polygonatum plant sterol polysaccharide protein, which has the effects of promoting the strength of skeletal muscles of the pelvic floor and resisting vasoconstriction.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments.

A preparation method of polygonatum sibiricum phytosterol polysaccharide protein comprises the following steps:

(1) pretreatment: taking the root of fresh rhizoma Polygonati, removing attachments on the surface of rhizoma Polygonati, weighing (wet weight) and taking as the measuring basis, cutting rhizoma Polygonati into pieces, adding 4-8 times of water into the crushed rhizoma Polygonati, and making the crushed rhizoma Polygonati into pulp by using a pulp homogenizer or a colloid mill;

(2) primary extraction: filtering the rhizoma polygonati homogenate obtained in the step (1) by adopting a filtering method, and respectively collecting a filter cake and filtrate, wherein the filtrate is a first extracting solution;

(3) secondary extraction: re-suspending the sealwort filter cake obtained in the step (2) in water with the weight of 3-5 times, adjusting the pH of the solution to 5.0-7.0, filtering and collecting filtrate to obtain a second extracting solution;

(4) enzymolysis: mixing the filtrates, adding 0.1-5.0 wt% of compound zymolyte of rhizoma Polygonati, and stirring at 30-50 deg.C for 6-12 hr; the composite zymolyte is prepared by mixing the following components in a mass ratio of 1-3: 0.5-2 of cellulase and papain.

(5) And (3) precipitation treatment: adjusting the pH value of the enzymatic hydrolysate obtained in the step (4) to 3.0-4.5, adding a sulfate solution, uniformly stirring, standing, treating the filtrate by using a centrifugal method, and collecting the filtrate;

(6) and (3) drying: and (4) performing ultrafiltration treatment on the filtrate obtained in the step (5), collecting trapped fluid, and performing spray drying to obtain powder, namely the crude polygonatum polysaccharide protein composition.

In the step (4), the preparation method of the composite zymolyte fermentation product comprises the following steps: taking cleaned pineapple raw materials, setting enzymolysis conditions as a material-liquid ratio of 1: 10(m/V), adding 2% of cellulase CellulaseACCF-4740, at a temperature of 55 ℃, pH4.5 and a reaction time of 2.5h, carrying out enzymolysis, after the hydrolysis of the cellulase is finished, inactivating the enzyme in a boiling water bath for 15min, centrifuging at 9000r/min, and collecting supernatant to obtain pineapple hydrolysate; after the enzyme activity of the pineapple enzymatic hydrolysate is inactivated, adjusting the pH value to 6.2-6.6, sterilizing at 121 ℃ by high-pressure steam for 20min, inoculating 1 volume percent of pentose slice bacteria liquid activated by MRS culture medium into the pineapple enzymatic hydrolysate, standing in a constant-temperature incubator at 37 ℃ and fermenting; after the fermentation is finished, centrifuging at 9000r/min, and collecting supernatant to obtain the compound zymolyte.

In the step (4), the enzymolysis raw material is a mixture of the filtrates after primary and secondary extraction.

In the step (4), the preparation method of the polygonatum polysaccharide protein comprises the following steps: extracting the extract at 4 deg.C for 44-50h, filtering, slowly adding ammonium sulfate into the filtrate at an addition amount of 0.5-1.5g/mL, stirring continuously during adding ammonium sulfate to prevent partial supersaturation of ammonium sulfate, standing for 45-50h, centrifuging at 4 deg.C for 5-10min at 25000g, redissolving the precipitate with acidic buffer solution, dialyzing at 4 deg.C with dialysis bag with molecular weight of 5000Da, centrifuging the dialysate at 20000g for 10min, and collecting the supernatant as the prepared expansin solution;

wherein, the components of the homogenization buffer solution are as follows: 25 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 1.5mmol/L sodium metabisulfite, 2mmol/L EDTA, 0.1 wt% Triton X-100, pH 7.0; the components of the extracting solution are as follows: 15 mmol/L4-hydroxyethyl piperazine ethanesulfonic acid, 1.0mmol/L ethylene diamine tetraacetic acid, 1.5mmol/L sodium metabisulfite, 0.5mol/L NaCl, pH6.0; the preparation of the acidic buffer solution comprises the following steps: 2.5g of sodium acetate is dissolved in water, the pH is adjusted to 4.0 with glacial acetic acid, and the volume of the water is adjusted to 1L.

In the step (5), the sulfate added to the solution can be ferric sulfate, zinc sulfate or sodium sulfate or a mixture thereof.

An active product of polygonatum kingianum phytosterol polysaccharide protein comprises the following components in parts by weight: 10-20 parts of maca enzyme extract, 20-48 parts of rhizoma polygonati enzyme extract, 15-25 parts of medlar enzyme extract, 15-20 parts of Chinese yam enzyme extract, 3-8 parts of beer yeast extract, 3-8 parts of trehalose extract, 15-30 parts of pumpkin seed extract, 4-8 parts of taurine extract, 5-10 parts of L-arginine extract, 13-18 parts of maltodextrin and 0.2-1 part of magnesium stearate.

The concentration of the active product is 65% -88%.

Rhizome of Polygonatum sibiricum contains steroid saponin, and 2 furostanol type saponins and 2 spirostanol type saponins are separated. In addition, the polygonatum polysaccharide A, B, C is contained, the relative molecular mass of the polygonatum polysaccharide A, B, C is more than 20 million Dalton, and the molecular mass is determined by the molar ratio of glucose (glucose), mannose (mannose) and galacturonic acid (galacturonic acid) as 6: 26: 1, condensation to obtain; and containing polygonatum oligosaccharide A, B, C, wherein the relative molecular masses are 1630, 862 and 472 respectively, and the molecular masses are determined by the molar ratio of fructose (fructose) to glucose (8: 1. 4: 1 and 2: 1 is condensed. The effective components act on physiological influences on the action of pathogenic microorganisms of human bodies, and in-vitro tests show that the water extract (1: 320) of the sealwort has inhibition effects on typhoid bacillus, staphylococcus aureus and acid-fast bacillus, and 2 percent of sealwort has inhibition effects on common pathogenic fungi in a Sasa medium to different degrees. Has remarkable inhibitory effect on hyperglycemia caused by epinephrine, and has effects of improving energy and relieving fatigue. The components have antioxidant effect, and can increase mouse liver superoxide dismutase (SOD) activity and reduce myocardial lipofuscin content in experimental animals. Has regulating effect on cardiovascular, and the water extract can significantly increase the flow of anaesthesia canine coronary artery, resist rabbit myocardial ischemia caused by hypophysin, resist T wave increase caused by hypophysin, promote early recovery of T wave abnormality, and enhance tolerance of mouse to anoxia. The activity of Na +, K + -ATP enzyme in the cell membrane of the mouse can be improved after the mouse enters the gastrointestinal tract by the oral liquid for 10 days. The 40% rhizoma polygonati alcohol extract is injected in an abdominal cavity (0.3 ml/mouse), so that the doping rate of cell DNA to 3H-TdR in the myocardial, liver and spleen tissues of a mouse can be improved. The rhizoma polygonati decoction is perfused (0.5 g/mouse) to reduce the cAMP and cGMP contents of the plasma of the mouse, and the cGMP contents are reduced more, so that the cAMP/cGMP ratio is larger than that of a control group, the cAMP and cGMP contents of spleen tissues are increased, but the cAMP/cGMP ratio is not changed obviously.

According to verification, the invention can achieve the effects of resisting local microvascular contraction and enhancing endogenous hormones and enhancing the strength of pelvic floor muscles after being eaten for 4-12 hours, and based on the fact that the main active component of the invention is polygonatum vegetable polysaccharide protein, active ingredients extracted from vegetable sterol polysaccharide protein and other phytochemicals are matched, and trace elements such as sodium, potassium, calcium, magnesium, phosphorus, zinc, copper and iron in plants can act on a cell enzyme system after entering circulation, so that the balance of intracellular and extracellular information and nucleic acid biosynthesis is maintained; on the other hand, the yam polysaccharide, the medlar polysaccharide and the pumpkin seed extract jointly act on gland cells of a host to induce endocrine regulation, taurine and L-arginine in a supplementary formula can be matched to stimulate gland organs such as prostate cells, kidney accessory gland tissue cells and the like to promote the activity of the gland cells, and the reactivity of action potential of pelvic floor muscles is enhanced, so that the brain and intestine axis endocrine regulation function of an organism is enhanced, the brain and intestine axis endocrine regulation function is easy to be absorbed by a human body, and no uncomfortable symptom is caused when 98% of low-age people take the brain and intestine axis endocrine regulation function for a long time.

The food therapy value is as follows:

strengthening bones and muscles, benefiting essence and marrow, and whitening hair: rhizoma polygonati, rhizoma corydalis, root of Chinese wolfberry, Chinese arborvitae twig and leaf of Chinese arborvitae, and radix astragali. One stone is boiled, one stone is steamed with the starter, and one stone is glutinous rice, like the common wine. (" mu")

② replenishing essence: wolfberry fruit (good for winter harvest) and Siberian solomonseal rhizome. Grinding into fine powder, mixing the two medicines, smashing into blocks, kneading into cakes, drying, smashing into powder, and refining with honey into pills, such as the size of phoenix tree seed. Fifty pills are taken every time, and the pills are taken with warm water. ("Qixiao Liang Fang" medlar pill)

③ treating weakness of the spleen and the stomach, and tiredness and weakness: rhizoma polygonati, radix codonopsitis and rhizoma dioscoreae are steamed to chicken. ("Manual of Chinese herbal medicine for rural areas in Hunan)

Fourthly, treating cough and bloody sputum due to lung diseases: fresh rhizoma polygonati with two roots and two heads and crystal sugar with one sugar. It is administered after being stewed with boiling water. (& Min east materia Medica)

Treating lung with tuberculosis, weakness after illness: rhizoma polygonati is five money or two money. Decocting with water or stewing pork. ("Manual of Chinese herbal medicine for rural areas in Hunan)

Sixthly, treating flaccidity of lower limbs of the children: one for rhizoma polygonati and two for winter honey. It is administered after being stewed with boiling water. (& Min east materia Medica)

Seventhly, treating stomach heat and thirst: six-money of rhizoma polygonati, five-money of rehmannia glutinosa and Chinese yam, four-money of trichosanthes root and ophiopogon root. Decocting with water. ("Shandong Chinese herbal handbook")

Eighthly, treating eyes, tonifying liver qi and improving eyesight: once jin of turnip seed (washed with water), and twice jin of Huang Jing seed (steamed with turnip seed for nine times and dried by aeration). The raw materials are smashed into powder. The porridge is taken with porridge for two changes every day, and is taken with warm water after being taken every noon and evening. (& lt & ltShenghui Fang & gt & ltBrassica napus & gt powder)

Ninthly, treating the symptoms of qi obstruction, chronic wind entering pulse, scabbing of nose, color failure and itchy skin: rhizoma Polygonati root (peeled and cleaned) for two jin. In the middle of the day, the rice is soft, the rice is steamed in the rice steamer, and the rice in two buckets is cooked to the degree, and the people do not need to take the rice at any time. ("holy economical book")

R for enterobiasis: eight-money sealwort is stewed for oral administration with one or two of crystal sugar. [ (Fujian Chinese medicine (6)): 44, 1965).

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention and the technical principles used, and any changes or substitutions which can be easily conceived by those skilled in the art within the technical scope of the present invention disclosed herein should be covered within the protective scope of the present invention.