阅读说明：本技术 一种莲子活性多糖、莲子活性物质及其提取方法和应用 (Lotus seed active polysaccharide, lotus seed active substance, extraction method and application thereof ) 是由 卢旭 雷乙 涂金金 许艺娴 郑宝东 马小清 于 2020-06-05 设计创作，主要内容包括：本发明涉及莲子活性物质提取技术领域,尤其涉及一种莲子活性多糖、莲子活性物质及其提取方法和应用。将莲子依次经过内肽酶和端肽酶复合酶的第一次酶解和胰蛋白酶的第二次酶解,酶解后过滤得到酶解液；将酶解液经过一次以上离心醇析得到莲子活性物质。其可以通过简单的方法和较低的成本将糖蛋白分离出莲子多糖和莲子蛋白,并保持其活性,更为重要的是可以实现产业化发展；莲子活性物质,其包括高活性和高纯度的莲子多糖和莲子蛋白,其具有较强的抗氧化、抗菌以及益生元功能；可以在益生元及抗氧化制品中应用。(The invention relates to the technical field of extraction of lotus seed active substances, in particular to lotus seed active polysaccharide, a lotus seed active substance, an extraction method and application thereof. Sequentially carrying out first enzymolysis on lotus seeds by using an endopeptidase and terminal peptidase compound enzyme and second enzymolysis by using trypsin, and filtering after enzymolysis to obtain an enzymolysis liquid; and centrifuging and alcohol-separating the enzymolysis liquid for more than one time to obtain the lotus seed active substance. The lotus seed polysaccharide and the lotus seed protein can be separated from the glycoprotein by a simple method and at a lower cost, the activity of the lotus seed polysaccharide and the lotus seed protein is kept, and more importantly, the industrial development can be realized; the lotus seed active substance comprises lotus seed polysaccharide and lotus seed protein with high activity and high purity, and has stronger antioxidant, antibacterial and prebiotic functions; can be applied to prebiotics and antioxidant products.)

1. The extraction method of the lotus seed active substance is characterized by comprising the following steps of:

s1 includes: sequentially carrying out first enzymolysis on lotus seeds by using an endopeptidase and terminal peptidase compound enzyme and second enzymolysis by using trypsin, and filtering after enzymolysis to obtain an enzymolysis liquid;

s2 includes: and centrifuging and alcohol-separating the enzymolysis liquid for more than one time to obtain the lotus seed active substance.

2. The method for extracting lotus seed active substances as claimed in claim 1, wherein step S1 further comprises the steps of treating lotus seeds before enzymolysis: adding water into lotus seeds, pulping, standing and precipitating for 6-10 hours at the temperature of below 4 ℃ to obtain lotus seed liquid.

3. The method for extracting lotus seed active substance as claimed in claim 1, wherein: the pH value of the first enzymolysis is 5.8-6.2, the enzyme adding amount of the compound enzyme is 280-320U/g, the enzymolysis temperature is 55-60 ℃, and the enzymolysis time is 360-390 min;

or/and

the enzyme adding amount of the second enzymolysis is 800-900U/g, and the enzymolysis time is 30-40 min.

4. The method for extracting lotus seed active substance as claimed in claim 1, wherein step S2 includes a first centrifugation alcohol precipitation: and (3) enabling the concentration of ethanol in the enzymolysis liquid to be 35-38%, and centrifuging to obtain a first supernatant and a first precipitate.

5. The method for extracting lotus seed active substance as claimed in claim 4, wherein the step S2 further comprises a second centrifugation alcohol precipitation: and (3) enabling the ethanol concentration of the first supernatant to be 52-55%, and centrifuging to obtain a second supernatant and a second precipitate.

6. The method for extracting lotus seed active substance as claimed in claim 5, wherein the step S2 further comprises a third centrifugation alcohol precipitation: and (4) enabling the ethanol concentration of the second supernatant to be 78-80%, and centrifuging to obtain a third supernatant and a third precipitate.

7. The method for extracting lotus seed active substance as claimed in any one of claims 1 to 6, further comprising step S3, wherein the precipitates obtained by more than one centrifugal alcohol precipitation are respectively subjected to tangential flow ultrafiltration, and the molecular weight cut-off of the ultrafiltration membrane is 1 KD.

8. A lotus seed active substance extracted by the extraction method of lotus seed active substance as defined in any one of claims 1 to 6.

9. Use of a lotus seed active substance according to claim 8 in prebiotics and anti-oxidant preparations.

10. The lotus seed active polysaccharide is characterized in that: it is mainly composed of polymeric sugar consisting of galactose, glucose, glucuronic acid and galacturonic acid in a molar ratio of 18.25:74.46:2.38: 4.91.

Technical Field

The invention relates to the technical field of extraction of lotus seed active substances, in particular to lotus seed active polysaccharide, a lotus seed active substance, an extraction method and application thereof.

Background

The agricultural products contain rich functional components, and the extraction and utilization of natural effective components have developed into an important direction for the deep processing of agricultural products in the world, and are a main way for improving the added value of the agricultural products and expanding the application range of the products. The lotus seed is a high-grade nourishing food and is popular with consumers at home and abroad. Jianning lotus seeds have become a post industry for Jianning in Fujian province and other characteristics and farmers to become rich. However, the problems of low utilization rate of lotus seed raw materials, single product structure, old processing technology, unreasonable industrial structure, lack of processing technology and the like are still outstanding at present, and the sustainable development of the special agricultural product processing industry is severely restricted. The natural polysaccharide from plant sources is used as an important natural active substance, and has great development potential and attractive industrial prospect in the aspects of resisting tumors, viruses, aging, oxidation, ulcer, blood sugar and the like in recent years.

The polysaccharide can be separated by various methods (including ethanol precipitation, membrane separation, column chromatography, etc.).

The invention patent (201810016008.5) provides a method for obtaining proteoglycan containing galacturonic acid glycan from mulberry, which adopts water extraction, enzymolysis (cellulase, papain and amylase), dialysis, centrifugation, alcohol precipitation, anion purification and gel column separation. Although column chromatography has high separation efficiency, a gel column is expensive, is usually applied to a laboratory to prepare a small amount of pure products, is not suitable for large-scale industrial use, and has low pertinence of adopted cellulase, papain and amylase, so that raw materials are wasted and the extraction rate is low.

At present, the ultrasonic coupling membrane separation and classification method (201610476325.6) for preparing the lycium barbarum polysaccharides with different molecular weights is mostly adopted in membrane separation operation, membrane separation and classification purification with different molecular weights are adopted, and multi-stage membrane separation operation is relatively complex. For example, the polysaccharide is separated by an ethanol fractional precipitation mode, for example, the glabrous sarcandra herb extractum residue polysaccharide, the preparation method and the application (201710135706.2) of the polysaccharide and the preparation method (201910646558.X) of the lentinan with different biological activities are disclosed, 30%, 60% and 90%, 20%, 50% and 70% of ethanol is used for carrying out fractional treatment on the raw material polysaccharide, but the pertinence is not strong.

The lotus seed polysaccharide is an important functional component in lotus seeds, and particularly, the extraction of the polysaccharide which is in a tight combination state with protein is still a difficult problem. The content of protein in the lotus seed is higher, about 17%, so that the purity and the biological activity of the lotus seed polysaccharide are improved by adopting a moderate low-temperature hydrolysis method, and the deep development and utilization of nutrient products such as lotus seed protein and the like are facilitated. The lotus seed polysaccharide which is found to be in a state of tightly combining polysaccharide and protein is glycoprotein, wherein the content of protein is 39.29 percent, and the content of polysaccharide is 37.78 percent.

Disclosure of Invention

Technical problem to be solved

In view of the above disadvantages and shortcomings of the prior art, the present invention provides a method for extracting lotus seed active substance, which can separate the lotus seed polysaccharide and lotus seed protein from glycoprotein by a simple method and at a low cost, and keep the activity thereof, and more importantly, can realize the industrial development;

correspondingly, the invention also provides the lotus seed active substance extracted by the extraction method, which comprises high-activity and high-purity lotus seed polysaccharide and lotus seed protein, and has stronger antioxidant, antibacterial and prebiotic functions;

correspondingly, the invention also provides lotus seed active polysaccharide which has stronger antioxidant, antibacterial and prebiotic functions.

Correspondingly, the invention also provides application of the lotus seed active substance in prebiotics and antioxidant products.

(II) technical scheme

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

a method for extracting lotus seed active substances comprises the following steps of: s1 includes: sequentially carrying out first enzymolysis on lotus seeds by using an endopeptidase and terminal peptidase compound enzyme and second enzymolysis by using trypsin, and filtering after enzymolysis to obtain an enzymolysis liquid; s2 includes: and centrifuging and alcohol-separating the enzymolysis liquid for more than one time to obtain the lotus seed active substance.

Further, step S1 includes the treatment of lotus seeds before enzymolysis: adding water into lotus seeds, pulping, standing and precipitating for 6-10 hours at the temperature of below 4 ℃ to obtain lotus seed liquid.

Further, the pH value of the first enzymolysis is 5.8-6.2, the enzyme adding amount of the compound enzyme is 280-320U/g, the enzymolysis temperature is 55-60 ℃, and the enzymolysis time is 360-390 min;

or/and

the enzyme adding amount of the second enzymolysis is 800-900U/g, and the enzymolysis time is 30-40 min.

Further, step S2 includes a first centrifugation alcohol-precipitation: and (3) enabling the concentration of ethanol in the enzymolysis liquid to be 35-38%, and centrifuging to obtain a first supernatant and a first precipitate.

Further, step S2 further includes a second centrifugation alcohol precipitation: and (3) enabling the ethanol concentration of the first supernatant to be 52-55%, and centrifuging to obtain a second supernatant and a second precipitate.

Further, step S2 further includes a third centrifugation alcohol analysis: and (4) enabling the ethanol concentration of the second supernatant to be 78-80%, and centrifuging to obtain a third supernatant and a third precipitate.

The invention also provides an extraction method of any lotus seed active substance, which further comprises the step S3 of respectively carrying out tangential flow ultrafiltration on precipitates obtained by more than one centrifugal alcohol precipitation, wherein the molecular weight cut-off of an ultrafiltration membrane is 1 KD.

The invention also provides the lotus seed active substance extracted by the extraction method of any lotus seed active substance.

The invention also provides application of the lotus seed active substance in prebiotics and antioxidant products.

The invention also provides lotus seed active polysaccharide which is mainly composed of polymeric sugar consisting of galactose, glucose, glucuronic acid and galacturonic acid according to the molar ratio of 18.25:74.46:2.38: 4.91.

The lotus seed active polysaccharide has the structure of → 6-Glc-1 → is a main chain, and has a linkage mode of → 3-Glc-1 → branched chain substitution at the C3 position, and the main signal value of the 13CNMR spectrum is basically consistent with the 13CNMR spectrum shown in FIG. 7.

(III) advantageous effects

Compared with the prior art, the method has the advantages that,

1. the invention uses specific endopeptidase and terminal peptidase to carry out directional hydrolysis, so that polysaccharide and protein in the polysaccharide protein in lotus seeds are separated; the trypsin is hydrolyzed for the second time, so that the protein is further hydrolyzed, and the protein and the polysaccharide with high activity can be conveniently separated by an alcohol precipitation method. The preparation method provided by the invention has the advantages that the used equipment is simple, the method steps are easy to implement, the high-purity and high-activity lotus seed protein and polysaccharide are obtained, the utilization rate of raw materials is improved, the cost is indirectly reduced, and the industrialized large-scale production can be realized. The preparation of the lotus seed polysaccharide is not decolorized and deproteinized, and column separation and purification of the polysaccharide are not carried out, so that the obtained high-purity lotus seed polysaccharide component avoids polysaccharide loss caused by the column separation and purification process of the polysaccharide, and in addition, a large amount of cost and expense of column chromatography, complicated operation steps and lower yield are saved.

2. The lotus seed polysaccharide powder obtained by the invention has high component purity, clear structure and controllable quality, has antioxidant activity and proliferation activity in an in vitro antioxidant test and a bifidobacterium growth test, has a glycosidic bond with the structure of general polysaccharide prebiotics, has potential prebiotic potential, and can be applied to the fields of food, medicine, health care products and the like.

3. The extraction method is simple, easy to operate and suitable for large-scale production, the separation of specific lotus seed polysaccharide components can be realized, and the obtained oligosaccharide monomer has prebiotics and antioxidant effects. The invention can fill the blank of the domestic production mode of single component of high-purity lotus seed polysaccharide, provides a basis for the full development and utilization of the lotus seed polysaccharide and other nutrient components, is beneficial to improving the industrial value of lotus seeds, expands the effective path for utilizing byproducts in the deep processing of lotus seeds, and simultaneously provides a new direction for the high-purity production of other natural polysaccharides.

Drawings

FIG. 1 is DEAE-FAST-FLOW elution curve of lotus seed polysaccharide;

FIG. 2 is a sephadex elution profile of LSPS 2;

FIG. 3 shows the results of HPLC testing of the components of LSPS 2;

FIG. 4 shows the antioxidant activity and bifidobacteria enrichment of the alcohol extracts obtained from solutions of different ethanol concentrations;

FIG. 5 is a confocal microscope photograph of Bifidobacterium growth under different alcohol analyte treatments;

FIG. 6 is an IR spectrum of lotus seed active polysaccharide;

FIG. 7 is a characteristic 13CNMR map of lotus seed active polysaccharide;

FIG. 8 is a characteristic 1HNMR map of lotus seed active polysaccharide product.

Detailed Description

For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.

[ first embodiment ] to provide a toner

The invention provides a method for extracting lotus seed active substances, which comprises the following steps of: s1 includes: sequentially carrying out first enzymolysis on lotus seeds by using an endopeptidase and terminal peptidase compound enzyme and second enzymolysis by using trypsin, and filtering after enzymolysis to obtain an enzymolysis liquid; s2 includes: and centrifuging and alcohol-separating the enzymolysis liquid for more than one time to obtain the lotus seed active substance.

The invention uses specific endopeptidase and terminal peptidase to carry out directional hydrolysis, hydrolyzes the core connecting part of protein particles and polysaccharide which are closely connected, separates polysaccharide and protein in the polysaccharide protein in lotus seeds, and carries out slow reaction for the first enzymolysis; under the condition that the tightly connected parts of the protein particles are in a loose state through first enzymolysis, trypsin is used for second enzymolysis to be rapidly combined with the action sites of the specific peptide fragments at the loose peripheral parts of the lotus seed protein particles, so that the rapid reaction is realized, the protein is further hydrolyzed into polypeptide, and the protein, the polypeptide and the polysaccharide with high activity can be conveniently separated and obtained at the centrifugal alcohol precipitation stage.

The embodiment realizes the simultaneous separation of the protein, the polypeptide and the polysaccharide in the lotus seeds, improves the utilization rate of raw materials, and the obtained protein, the polypeptide and the polysaccharide have strong antioxidant activity and prebiotics function.

The method of the embodiment is simple, easy to operate and suitable for large-scale production, the separation of the specific lotus seed polysaccharide component can be realized, and the obtained oligosaccharide monomer has prebiotics and antioxidant effects. The invention can fill the blank of a domestic single-component production mode of high-purity lotus seed polysaccharide, provides a basis for the full development and utilization of the lotus seed polysaccharide and other nutritional ingredients, is beneficial to improving the industrial value of lotus seeds, expands the effective path for utilizing byproducts in lotus seed deep processing, and simultaneously provides a new direction for the high-purity production of other natural polysaccharides.

In order to improve the efficiency of the first enzymolysis and enable the separation of the protein to be more thorough, further, the pH value of the first enzymolysis is 5.8-6.2, the enzyme adding amount of the compound enzyme is 280-320U/g, the enzymolysis temperature is 55-60 ℃, and the enzymolysis time is 360-390 min;

or/and

the enzyme adding amount of the second enzymolysis is 800-900U/g, and the enzymolysis time is 30-40 min.

In the embodiment, the enzyme adding amount and the enzymolysis time of the second enzymolysis are limited, and the purpose is to stop the reaction before the small molecular peptide segment is formed, so that the waste and the formation of a large amount of free amino acids caused by the excessive hydrolysis of the protein are avoided, and the production of the lotus seed protein powder is facilitated. Wherein, the enzyme adding amount U/g represents the enzyme activity unit added per g of lotus seed dry powder.

Further, step S1 includes the treatment of lotus seeds before enzymolysis: adding water into lotus seeds, pulping, standing and precipitating for 6-10 hours at the temperature of below 4 ℃ to obtain lotus seed liquid.

Compared with the traditional high-temperature water bath polysaccharide extraction mode, the method can reduce the functional value and purity of the polysaccharide, and the preparation process of the embodiment adopts low temperature, which is beneficial to avoiding the oxidation reaction of the polysaccharide and the polymerization with other substances in the extraction process, so that the higher purity and better antioxidant activity of the lotus seed polysaccharide, protein and polypeptide are maintained.

Furthermore, the treatment of the lotus seeds also comprises,

1. selecting and drying: selecting fresh lotus seeds with full and undamaged particles, screening and decoring the lotus seeds, and drying the lotus seeds at the temperature of 40-45 ℃ until the water content is less than 11-13%;

2. crushing and sieving: weighing the dried lotus seeds, crushing the lotus seeds in a crusher, and sieving the crushed lotus seeds with a 30-40-mesh sieve to obtain lotus seed powder;

3. adding water for compounding: putting the obtained lotus seed powder into a conical flask, adding deionized water according to the material-liquid ratio of 1: 9-1: 10, and uniformly mixing and stirring to obtain a compound material;

4. washing starch with water: pulping the obtained compound feed liquid for 4-5 min by using a pulping machine, washing filter residues by using deionized water with the weight 4-6 times that of the lotus seed residues, combining filtrates, standing and precipitating for 8h at 4 ℃, centrifuging to remove the precipitate to obtain lotus seed starch, combining the obtained supernatant and the washed lotus seed filter residues, stirring, and adding 0.2mol/L acetic acid-sodium acetate buffer solution;

the invention constructs the high-efficiency extraction integration technology of the lotus seed polysaccharide and the byproducts by combining the biotechnology and the modern food engineering technology, and simultaneously extracts the lotus seed polysaccharide, the lotus seed starch, the lotus seed protein and the lotus seed oligopeptide in the production process, so that each step is fully applied, and the production process is greatly simplified. In addition, before each step of alcohol precipitation and sedimentation, the solution is centrifuged to remove micromolecular alcohol-soluble substances such as lotus seed polyphenol, flavone and the like, so that impurities are prevented from being brought into lotus seed polysaccharide and protein, the purity of the product is ensured, and simultaneously, enzyme in the solution can be inactivated by ethanol.

Further, step S2 includes a first centrifugation alcohol-precipitation: and (3) enabling the concentration of ethanol in the enzymolysis liquid to be 35-38%, and centrifuging to obtain a first supernatant and a first precipitate.

Specifically, the obtained enzymolysis liquid is cooled to room temperature, is centrifuged for 20min at the rotating speed of 4000r/min to obtain filtrate, is added with absolute ethyl alcohol to ensure that the concentration of the ethyl alcohol in the enzymolysis liquid reaches 35-38%, and is centrifuged for 20min at the rotating speed of 4000r/min to obtain a first supernatant and a first precipitate; separating the obtained first precipitate to obtain the lotus seed protein.

Further, step S2 further includes a second centrifugation alcohol precipitation: and (3) enabling the ethanol concentration of the first supernatant to be 52-55%, and centrifuging to obtain a second supernatant and a second precipitate. Specifically, absolute ethyl alcohol is added into the first supernatant to adjust the concentration of the ethyl alcohol, and the mixture is centrifuged for 20min at the rotating speed of 4000 r/min. Separating the second precipitate to obtain the lotus seed polysaccharide.

Further, step S2 further includes a third centrifugation alcohol analysis: and (4) enabling the ethanol concentration of the second supernatant to be 78-80%, and centrifuging to obtain a third supernatant and a third precipitate. Specifically, absolute ethyl alcohol is added into the second supernatant to adjust the concentration of the ethyl alcohol, and the mixture is centrifuged for 20min at the rotating speed of 4000 r/min. Separating the third precipitate to obtain lotus seed protein; the third supernatant is the lotus seed oligopeptide.

In the embodiment, the alcohol precipitation is performed through fractional centrifugation and the first centrifugal alcohol precipitation is performed, so that the enzyme activity is enabled on one hand, and the lotus seed protein is precipitated on the other hand; the centrifugal alcohol precipitation of the second time and the third time is to obtain lotus seed polysaccharide and lotus seed oligopeptide.

The preparation process of the lotus seed polysaccharide does not carry out decoloration and deproteinization treatment, and does not carry out column separation and purification of the polysaccharide, thereby obtaining the lotus seed polysaccharide with high purity, avoiding the polysaccharide loss caused by the column separation and purification process of the polysaccharide, and saving a large amount of expense and expense of column chromatography, complicated operation steps and lower yield. The embodiment forms a new continuous, efficient and directional preparation process of polysaccharide and hydrolyzed protein with different molecular weights by an integrated technology of separating the lotus seed polysaccharide and byproducts by high-throughput fractional alcohol precipitation, realizes a technical means of parallel separation and purification of lotus seed polysaccharide components and hydrolyzed protein by multiple channels, and realizes high-purity separation of a certain lotus seed polysaccharide and protein components from raw materials by specific and accurate ethanol concentration. The lotus seed polysaccharide and the lotus seed protein are subjected to alcohol precipitation into a plurality of groups through different alcohol concentrations of graded alcohol precipitation, so that components with effects of oxidation resistance and intestinal flora can be selected more favorably, and the problem that the traditional alcohol precipitation step has no pertinence is avoided.

Further, the method also comprises a step S3 of respectively carrying out tangential flow ultrafiltration on precipitates obtained by more than one centrifugal alcohol precipitation, wherein the molecular weight cut-off of the ultrafiltration membrane is 1 KD.

Specifically, the first precipitate, the second precipitate and the third precipitate are respectively added into deionized water to prepare solutions with the concentration of 35 percent, and tangential flow ultrafiltration is respectively carried out; the ultrafiltration mode is equal-volume ultrafiltration and room-temperature injection water is added, and the multiple of ultrafiltration and water addition is 4-5 times; the material of the flow ultrafiltration membrane is a cellulose triacetate membrane, and the molecular weight cut-off is 1 KD.

And respectively obtaining a first solution, a second solution and a third solution by the first precipitation, the second precipitation and the third precipitation through tangential flow ultrafiltration. The invention further purifies the polysaccharide and other fractions with different molecular weights precipitated by alcohol by adopting a tangential flow ultrafiltration technology, and can avoid the phenomena of molecular weight reduction and agglomeration of the polysaccharide or protein after the separation by the traditional ultrafiltration membrane technology.

Further, the method also comprises the step of freeze drying: and diluting the obtained first solution, second solution and third solution with deionized water respectively, and freeze-drying to obtain lotus seed protein powder, lotus seed polysaccharide powder and lotus seed protein powder respectively. And freeze-drying the third supernatant to obtain the lotus seed oligopeptide powder.

[ second embodiment ] to provide a medicine for treating diabetes

The present embodiment also provides the lotus seed active substance extracted by the method for extracting a lotus seed active substance according to the first embodiment. Respectively lotus seed protein powder, lotus seed polysaccharide powder and lotus seed oligopeptide powder.

The lotus seed polysaccharide powder has a total solid weight of more than 85%, and weight average molecular weight of 3.058 × 10⁴Da; lotus seedThe polysaccharide is monosaccharide composition comprising galactose, glucose, glucuronic acid and galacturonic acid, and the molar ratio is 18.25:74.46:2.38: 4.91; the main stretching vibration absorption peak of the infrared characteristic spectrum of the lotus seed polysaccharide is basically consistent with that in the infrared characteristic spectrum shown in figure 6.

The lotus seed polysaccharide has the structure → 6-Glc-1 → is the main chain, and the C3 position has the linkage of → 3-Glc-1 → branched chain substitution, and the main signal value of the 13C NMR spectrum is basically consistent with the 13C NMR spectrum shown in FIG. 7.

The lotus seed polysaccharide powder has high component purity, clear structure and controllable quality, has antioxidant activity and proliferation activity in an in vitro antioxidant test and a bifidobacterium growth test, has a glycosidic bond with the structure of general polysaccharide prebiotics, has potential prebiotic potential, and can be applied to the fields of food, medicine, health care products and the like.

[ third embodiment ]

The embodiment also provides application of the lotus seed active substance in prebiotics and antioxidant products.

In order to better understand the above technical solutions, exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention can be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.