阅读说明：本技术 二裂酵母发酵产物及其制备方法和用途 (Yeast cracking fermentation product and preparation method and application thereof ) 是由 李海珍 李海燕 于 2021-08-26 设计创作，主要内容包括：本发明公开了二裂酵母发酵产物及其制备方法和用途,该制备方法包括以下步骤：将长双歧杆菌接种于种子培养基中,厌氧培养活化,得到种子液；将种子液接种至发酵培养基中,调节发酵液初始pH值为6.5～7.0,厌氧发酵培养；所得发酵液离心,取上清液,为二裂酵母发酵产物；所述的种子培养基和发酵培养基,是取豆渣5～10g、甘蔗渣1～5g,补水至1kg。本发明采用豆渣和甘蔗渣作为双歧杆菌培养基,利用豆渣和甘蔗渣丰富多样的营养成分进一步强化提升双歧杆菌的有益代谢产物。本发明的二裂酵母发酵产物滤液在化妆品中具有较好的应用效果：具有优异的促进屏障修复相关基因表达、促进胶原蛋白合成的功效性能。(The invention discloses a yeast schizosaccharomyces cerevisiae fermentation product, a preparation method and application thereof, wherein the preparation method comprises the following steps: inoculating bifidobacterium longum into a seed culture medium, and performing anaerobic culture and activation to obtain a seed solution; inoculating the seed liquid into a fermentation culture medium, adjusting the initial pH value of the fermentation liquid to 6.5-7.0, and performing anaerobic fermentation culture; centrifuging the obtained fermentation liquor, and taking supernatant as a secondary fission yeast fermentation product; the seed culture medium and the fermentation culture medium are prepared by taking 5-10 g of bean dregs and 1-5 g of bagasse and adding water to 1 kg. The invention adopts the bean dregs and the bagasse as the culture medium of the bifidobacterium and utilizes rich and diversified nutrient components of the bean dregs and the bagasse to further strengthen and promote beneficial metabolites of the bifidobacterium. The secondary fission yeast fermentation product filtrate has better application effect in cosmetics: has excellent efficacy performance of promoting the expression of barrier repair related genes and promoting the synthesis of collagen.)

1. The preparation method of the yeast for secondary fission is characterized by comprising the following steps:

(1) inoculating bifidobacterium longum into a seed culture medium, and performing anaerobic culture and activation to obtain a seed solution;

(2) inoculating the seed liquid into a fermentation culture medium, adjusting the initial pH value of the fermentation liquid to 6.5-7.0, and performing anaerobic fermentation culture;

(3) centrifuging the fermentation liquor obtained in the step (2), and taking supernatant as a secondary fission yeast fermentation product;

the seed culture medium and the fermentation culture medium are prepared by taking 5-10 g of bean dregs and 1-5 g of bagasse and adding water to 1 kg.

2. The method of claim 1, wherein: and (3) carrying out low-temperature pasteurization on the obtained secondary fission yeast fermentation product, filtering, and adding a preservative to obtain the industrially usable secondary fission yeast fermentation product.

3. The method of claim 1, wherein: the Bifidobacterium longum in the step (1) is Bifidobacterium longum ATCC 15697.

4. The method of claim 1, wherein: and crushing the bean dregs and the bagasse, and then screening the crushed bean dregs and bagasse with a screen of 40-80 meshes.

5. The method of claim 1, wherein: in the step (1), the inoculation amount of the bifidobacterium longum accounts for 1-3% of the mass of the seed culture medium; the temperature of the anaerobic culture is 35-40 ℃, and the culture time is 20-30 h; the obtained seed liquid contains Bifidobacterium longum at a concentration of 1 × 10⁶～1×10⁸CFU/mL。

6. The method of claim 1, wherein: in the step (2), the seed liquid accounts for 3-5% of the volume of the fermentation medium; the culture temperature of the anaerobic fermentation culture is 36-38 ℃, and the culture time is 40-60 h.

7. The method of claim 1, wherein: in the step (3), the centrifugation is carried out at 3000-6000 r/min for 15-30 min.

8. The method of claim 2, wherein:

performing low-temperature pasteurization, wherein the temperature of inactivation treatment is 60-80 ℃, and the time of inactivation treatment is 20-50 min;

the filtration is carried out by utilizing a ceramic membrane filter, and the filtrate sequentially passes through ceramic membrane filter columns with the particle size of 1.5 mu m and 0.6 mu m;

the preservative is more than one of polyalcohol, phenoxyethanol, ethylhexyl glycerol, p-hydroxyacetophenone, ammonium benzoate or potassium sorbate;

the polyalcohol is more than one of butanediol, pentanediol or hexanediol.

9. A split yeast fermentation product, characterised in being produced by the method of any one of claims 1 to 8.

10. Use of the yeast for secondary splitting of claim 9 in cosmetics.

Technical Field

The invention relates to the field of microbial fermentation, in particular to a secondary fission yeast fermentation product and a preparation method and application thereof.

Background

The yeast (BIFIDA FERMENT FILTRATE) is metabolite obtained by fermenting Bacillus bifidus, and mainly contains organic acids (lactic acid, citric acid and succinic acid), vitamins (B1, B2, B6, B12, pantothenic acid and folic acid), bacteriocin (mostly water-soluble polypeptide), polysaccharide, protein and amino acid.

Lactic acid in the yeast bifida fermentation product is a natural moisturizing factor, exists in human skin, and has the effects of effectively removing fine lines and wrinkles, accelerating cutin renewal and the like; folic acid can activate epidermal cells of skin, promote water maintenance and nutrient absorption, and reduce keratinization rate of skin, thereby achieving the purposes of beautifying and nourishing skin and tenderizing skin; in addition, active ingredients such as amino acids, lipids, polysaccharides, adenosine, vitamins, trace minerals, vitamin B groups and the like can moisten and nourish the skin, help the skin to resist the damage of the external environment and pressure to the skin, simultaneously can promote various protein gene expressions related to the epidermal differentiation and the skin barrier function, and play the effects of wrinkle removal, repair, moisture preservation and skin elasticity enhancement. Therefore, the secondary fission yeast fermentation product has great market application prospect. However, the preparation process of the secondary fission yeast fermentation product filtrate used for cosmetic raw materials is complex at present, and exogenous nutrient components are required to be continuously supplemented to promote the fermentation growth of bifidobacteria, so that some exogenous additive components are remained in metabolites, and the production cost of fermentation is increased.

Disclosure of Invention

The invention aims to provide a preparation method of a yeast for bifidus fermentation, which effectively improves the anti-aging capability of the obtained fermentation product by adopting bean dregs and bagasse as culture media, and specifically realizes the promotion of the expression of barrier repair related genes and the improvement of fibroblast activity and COL-I protein expression level. These effects are not achieved by the prior art methods.

The purpose of the invention is realized by the following technical scheme:

the preparation method of the yeast for secondary fission comprises the following steps:

(1) inoculating bifidobacterium longum into a seed culture medium, and performing anaerobic culture and activation to obtain a seed solution;

(2) inoculating the seed liquid into a fermentation culture medium, adjusting the initial pH value of the fermentation liquid to 6.5-7.0, and performing anaerobic fermentation culture;

(3) centrifuging the fermentation liquor obtained in the step (2), and taking supernatant as a secondary fission yeast fermentation product;

preferably, the secondary fission yeast fermentation product is subjected to low-temperature pasteurization, filtration and preservative addition to obtain the industrially usable secondary fission yeast fermentation product.

Bifidobacterium longum, preferably Bifidobacterium longum ATCC 15697, as described in step (1);

the seed culture medium and the fermentation culture medium are prepared by taking 5-10 g of bean dregs and 1-5 g of bagasse and adding water to 1 kg; wherein the bean dregs and the bagasse are crushed and then pass through a screen with 40-80 meshes; the bean dregs contain a large amount of various nutrient elements such as protein, dietary fiber, amino acid, vitamin, mineral substance and the like, and provide a natural N source for the growth of bifidobacterium; in addition, the abundant polysaccharide components in the bagasse are utilized to provide a natural C source for the growth of the bifidobacteria.

Preferably, in the step (1), the inoculation amount of the bifidobacterium longum accounts for 1-3% of the mass of the seed culture medium; the temperature of the anaerobic culture is 35-40 ℃ (particularly preferably 36-38 ℃), and the culture time is 20-30 h; the obtained seed liquid contains Bifidobacterium longum at a concentration of 1 × 10⁶～1×10⁸CFU/mL。

Preferably, in the step (2), the seed liquid accounts for 3-5% of the volume of the fermentation medium; the culture temperature of the anaerobic fermentation culture is 36-38 ℃, and the culture time is 40-60 h; adjusting the pH value of the fermentation liquor refers to adjusting by using citric acid-sodium citrate buffer solution;

in the step (3), centrifuging at 3000-6000 r/min for 15-30 min;

performing low-temperature pasteurization, wherein the temperature of inactivation treatment is 60-80 ℃, and the time of inactivation treatment is 20-50 min, so as to finish the inactivation treatment of microorganisms;

and filtering by using a ceramic membrane filter, and removing fine impurities from the filtrate by sequentially passing the filtrate through ceramic membrane filter columns of 1.5 mu m and 0.6 mu m.

The preservative is more than one of polyalcohol, phenoxyethanol, ethylhexyl glycerol, p-hydroxyacetophenone, ammonium benzoate or potassium sorbate;

the polyalcohol is more than one of butanediol, pentanediol or hexanediol.

The yeast for secondary fission prepared by the method can be applied to cosmetics.

Compared with the prior art, the invention has the following advantages and effects:

(1) the invention adopts the bean dregs and the bagasse as the culture medium of the bifidobacterium and utilizes rich and diversified nutrient components of the bean dregs and the bagasse to further strengthen and promote beneficial metabolites of the bifidobacterium.

(2) After the fermentation product is centrifugally treated, respectively collecting the lower bifidobacterium longum and the residual culture medium material and the upper bifidobacterium longum metabolite; the upper-layer bifidobacterium longum metabolite is processed to obtain a bifidus yeast fermentation product filtrate, and the lower-layer bifidobacterium longum and the culture medium excess can be recycled, so that the product cost is further reduced.

(3) The secondary fission yeast fermentation product filtrate has better application effect in cosmetics: has excellent efficacy performance of promoting the expression of barrier repair related genes and promoting the synthesis of collagen.

Drawings

FIG. 1 is a bar graph of the fold amplification of the genes involved in barrier repair.

FIG. 2 is a bar graph of fibroblast activity.

FIG. 3 is a histogram of COL-I protein expression levels.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1

A preparation method of a yeast fermentation product of fission yeast comprises the following steps:

(1) inoculating Bifidobacterium longum (ATCC 15697) into seed culture mediumPerforming medium anaerobic fermentation culture to obtain a seed solution; wherein the inoculation amount of Bacillus bifidus is 3% of the volume of the seed culture medium, the culture temperature is 38 deg.C, the culture time is 30h, and the concentration of the seed solution is 1 × 10⁸CFU/mL; the seed culture solution is prepared by taking 10g of bean dregs and 3g of bagasse and adding water to 1 kg; wherein the bean dregs and the bagasse are crushed and then screened by a 60-mesh screen.

(2) Inoculating the cultured seed solution into a sterilized fermentation culture medium according to 5% of the volume of the culture medium, adjusting the pH of the feed liquid in the fermentation tank to 7.0 by using a citric acid-sodium citrate buffer solution with the concentration of 0.01mol/L, and carrying out anaerobic fermentation culture at the culture temperature of 38 ℃ for 60 hours; the fermentation medium is prepared by taking 10g of bean dregs and 3g of bagasse and adding water to 1 kg.

(3) Respectively collecting the lower Bifidobacterium longum and the residual culture medium material and the upper Bifidobacterium longum metabolite by using a horizontal centrifuge with the rotating speed of 6000r/min and the centrifugation time of 15 min; pasteurizing the upper layer product, inactivating at 68 deg.C for 30 min; filtering with ceramic membrane filter, and sequentially passing the upper filtrate through ceramic membrane filter columns of 1.5 μm and 0.6 μm to remove fine impurities; the added preservative is 0.96 percent (calculated by the volume of the fermentation liquor obtained in the step (2); the same below) of butanediol, 0.5 percent of pentanediol, 0.5 percent of hexanediol and 0.3 percent of p-hydroxyacetophenone for compounding to obtain the yeast for fermenting the secondary fission.

Example 2

A preparation method of a yeast fermentation product of fission yeast comprises the following steps:

(1) inoculating Bifidobacterium longum (ATCC 15697) in seed culture medium, and performing anaerobic fermentation culture to obtain seed solution; wherein the inoculation amount of Bifidobacterium is 2% of the volume of the seed culture medium, the culture temperature is 37 deg.C, the culture time is 24 hr, and the strain concentration is 3.0 × 10⁷CFU/mL; the seed culture solution is prepared by taking 5g of bean dregs and 1g of bagasse and adding water to 1 kg; wherein the bean dregs and the bagasse are crushed and then screened by a 60-mesh screen.

(2) Inoculating the cultured seed solution into a sterilized fermentation culture medium according to 4% of the volume of the culture medium, adjusting the pH of the feed liquid in the fermentation tank to 6.6 by using a citric acid-sodium citrate buffer solution with the concentration of 0.01mol/L, and carrying out anaerobic fermentation culture at the culture temperature of 37 ℃ for 48 hours; the fermentation medium is prepared by taking 7g of bean dregs and 2g of bagasse and supplementing water to 1 kg.

(3) Respectively collecting the lower Bifidobacterium longum and the residual culture medium material and the upper Bifidobacterium longum metabolite by using a horizontal centrifuge with the rotation speed of 5000r/min and the centrifugation time of 20 min; pasteurizing the upper layer product, and inactivating at 65 deg.C for 30 min; filtering with ceramic membrane filter, and sequentially passing the upper filtrate through ceramic membrane filter columns of 1.5 μm and 0.6 μm to remove fine impurities; the added preservative is 0.96 percent of butanediol, 0.5 percent of pentanediol, 0.5 percent of hexanediol and 0.3 percent of p-hydroxyacetophenone for compounding to obtain the yeast fermentation product.

Example 3

A preparation method of a yeast fermentation product of fission yeast comprises the following steps:

(1) inoculating Bifidobacterium longum (ATCC 15697) in seed culture medium, and performing anaerobic fermentation culture to obtain seed solution; wherein the inoculation amount of Bacillus bifidus is 1% of the volume of the seed culture medium, the culture temperature is 36 deg.C, the culture time is 20 hr, and the strain concentration is 1.0 × 10⁶CFU/mL; the seed culture solution is prepared by taking 5g of bean dregs and 1g of bagasse and adding water to 1 kg; wherein the bean dregs and the bagasse are crushed and then screened by a 60-mesh screen.

(2) Inoculating the cultured seed solution into a sterilized fermentation culture medium according to 2% of the mass of the culture medium, adjusting the pH of the feed liquid in the fermentation tank to 6.5 by using a citric acid-sodium citrate buffer solution with the concentration of 0.01mol/L, and carrying out anaerobic fermentation culture at the culture temperature of 36 ℃ for 40 hours; the fermentation medium is prepared by taking 5g of bean dregs and 1g of bagasse and adding water to 1 kg.

(3) Respectively collecting the lower Bifidobacterium longum layer, the rest culture medium and the upper Bifidobacterium longum metabolite by using a horizontal centrifuge with the rotation speed of 4000r/min and the centrifugation time of 25 min; pasteurizing the upper layer product, and inactivating at 65 deg.C for 30 min; filtering with ceramic membrane filter, and sequentially passing the upper filtrate through ceramic membrane filter columns of 1.5 μm and 0.6 μm to remove fine impurities; the added preservative is 0.96 percent of butanediol, 0.5 percent of pentanediol, 0.5 percent of hexanediol and 0.3 percent of p-hydroxyacetophenone for compounding to obtain the yeast fermentation product.

Comparative example 1

A preparation method of a yeast fermentation product of fission yeast comprises the following steps:

(1) inoculating Bifidobacterium longum (ATCC 15697) in seed culture medium, and performing anaerobic fermentation culture to obtain seed solution; wherein the inoculation amount of the bifidobacterium is 3 percent of the volume of a seed culture medium, the culture temperature is 38 ℃, the culture time is 24 hours, and the seed culture solution is prepared by adding water to 1kg of skimmed milk powder 5g, glucose 1.5g and lactose 1.5 g;

(2) inoculating the cultured seed solution into a sterilized fermentation culture medium for anaerobic fermentation culture, adjusting the pH of the feed liquid in a fermentation tank to 6.5 by using a 0.01mol/L citric acid-sodium citrate buffer solution, and carrying out anaerobic fermentation culture at 38 ℃ for 48 h; the fermentation medium is composed of 3g of glucose, 1.5g of casein peptone, 3.0g of beef peptone, 2.0g of hydrolyzed milk protein and 1kg of water;

(3) performing pasteurization on the upper filtrate at the inactivation temperature of 65 ℃ for 30min by using a horizontal centrifuge with the rotation speed of 4000r/min and the centrifugation time of 25 min; filtering with ceramic membrane filter, and sequentially passing the upper filtrate through ceramic membrane filter columns of 1.5 μm and 0.6 μm to remove fine impurities; the added preservative is 0.96 percent of butanediol, 0.5 percent of pentanediol, 0.5 percent of hexanediol and 0.3 percent of p-hydroxyacetophenone for compounding to obtain the yeast fermentation product.

Efficacy testing

The effects of the fermentation products obtained in examples 1 to 3 and comparative example 1 on the aspects of promoting the expression of the genes related to barrier repair, promoting the synthesis of collagen and the like are examined and compared:

promotion of the expression of barrier repair-related genes

Using human keratinocytes (HaCat cells) as a model, the expression of the barrier repair-associated genes (TGM1, FLG, LOR, IVL and ABCA12) was determined.

Transglutaminase 1 gene TGM 1: the coding membrane-associated calcium-dependent thiolase, which is resistant to hydrolysis by proteases, is a key step in the terminal differentiation of keratinocytes into the cornified envelope, and is the material basis for the barrier function of the skin.

Silk fibroin FLG: is key to skin barrier, can reduce invasion of antigen and TEWL, and can release acidic substances to lower skin pH value and inhibit bacterial growth.

Loricrin LOR: is a main component of epidermal granulosa cell keratohyalin granules, and the skin barrier function is damaged by the fact that FLG gene mutation influences terminal differentiation of keratinocytes and expression abnormality thereof.

Inner coat protein IVL: cross-linked and deposited on the inner side of cell membrane under the catalysis of transglutaminase TGK to form water-insoluble cornified envelope.

ABCA12 gene: as an ABC family liposome carrier, the lamellar body limiting membrane is positioned and participates in lamellar body formation, and plays an important role in maintaining normal skin barrier function.

The test steps are as follows:

(1) cell inoculation: by 2.5X 10⁵Cells were seeded at cell/well density into 6-well plates in incubators (37 ℃, 5% CO)₂95% RH) overnight.

(2) Preparing liquid: the fermentation products of the examples and the comparative examples were prepared into a 5% volume fraction solution using complete medium (MEM + 10% FBS) as a solvent, and used as a working solution of a test substance for a fluorescence quantitative PCR test.

(3) Administration: each group is provided with 3 repeated holes, when the plating rate of the cells in the 6-hole plate reaches 40-50%, the medicine is administered in groups, and the medicine is cultured in an incubator (37 ℃ and 5% CO)₂95% RH) for 24 h.

(4) Collecting cells: after incubation and incubation of the samples for 24h, PBS washing was performed twice at 1 mL/well. After washing, 1mL of RNAioso Plus was added to each well, lysed cells were aspirated, collected into 1.5mL enzyme-free EP tubes, and stored in a freezer at-80 ℃.

(5) Fluorescent quantitative PCR test: RNA extraction was performed according to the RNAioso Plus instructions, reverse transcription was performed according to the reverse transcription kit, and fluorescent quantitative PCR reaction system preparation and on-machine detection were performed according to the SYBR Premix Ex TaqTM II fluorescent dye (TaKaRa DRR081A) product instructions.

(6) And (4) calculating a result: by using 2^-△△CTThe method performs a result calculation.

The results of fluorescent quantitative PCR detection of the barrier-associated genes by the fermentation products of examples 1 to 3 and comparative example 1 are shown in FIG. 1, and according to the results shown in FIG. 1, the fermentation products of examples 1 to 3 have significant promotion effects on the expression of the barrier repair-associated genes TGM1, FLG, LOR, IVL and ABCA12 at a concentration of 5%, which are higher than the effect of comparative example 1.

Secondly, synthesis of collagen

When the skin is irradiated by ultraviolet, excessive active oxygen is generated in cells, so that aging-related genes are expressed, inflammation cascade reaction is induced, the expression quantity of elastin and collagen is reduced, and the skin has aging phenomena such as relaxation, wrinkles and the like. UVA stimulates human skin fibroblasts, resulting in degradation of collagen, particularly type I collagen (COL-I).

This test is intended to evaluate whether or not the fermentation products of examples 1 to 3 and comparative example 1 have a promoting effect on the secretion of COL-I by detecting the COL-I content in fibroblasts after UVA stimulation and comparing the change in the content with that of the solvent control group.

And (3) testing the cytotoxicity of the fibroblasts, wherein the specific testing steps are as follows:

(1) cell culture: preparing cell suspension 24h before test, inoculating the cell suspension into 96-well cell culture plate, 100 μ L per well, 8000 cells per well, 37 deg.C, 5% CO₂And culturing for 24 h.

(2) Exposure: the stock solution was discarded from the wells, and 100. mu.L of each well was added to a blank sample group of 100. mu.L of a test sample (test sample is a test sample solution prepared by adding a proper amount of complete medium (DMEM + 10% FBS) to the fermentation product of example 1 to prepare a volume percentage of 0.5%, 1.0%, 5.0%, 10%, 15%, 20%, 30%, 40%) of complete medium (DMEM + 10% FBS). 37 ℃ and 5% CO₂Incubate for 24 h. Cell morphology and characteristics were observed under phase contrast inverted microscope.

(3) MTT test: mu.L of 5mg/mL MTT solution was added to each well and the incubator was incubated for 3. + -. 0.5 h. And removing liquid in the holes, adding 100 mu L of DMSO into each hole, placing the hole in an oscillator for oscillation for 10-15 min, and then measuring the absorption value at the wavelength of 570nm of the microplate reader.

(4) Data processing: the data of each group are expressed as mean ± standard deviation, and the relative cell activity (viatility) of each group is calculated based on the cell activity of the control group as 100%.

The cells used in this test were human dermal fibroblasts (HFF-1) from the Shanghai cell Bank of Shanghai Life sciences institute of Chinese academy of sciences. The relative cell activity of human dermal fibroblasts at various concentrations of the sample of example 1 is shown in FIG. 2. in comparison to the blank sample set, the relative cell activity of human dermal fibroblasts was 73.98% at a 20% sample of example 1 (relative cell activity higher than 70% was considered by the industry as a safe concentration). Therefore, the addition amount of the composition is 1-20% of the safe administration concentration of the cells.

Third, detection of COL-I content

(1) Inoculation: human skin fibroblasts were cultured at 5.0X 10⁴Was inoculated into 6-well plates at 37 ℃ with 5% CO₂Incubating in an incubator overnight;

(2) administration: when the cell plating rate in the 6-well plate reaches 50% -60%, the drug is administered in four groups (negative control group, blank control group, test sample group 1, test sample group 2), and each group is provided with 3 multiple wells. Complete medium (DMEM + 10% FBS) was added to the negative control group and the blank control group, and complete medium (DMEM + 10% FBS) containing the test substance at a concentration of 5% (v/v) and 10% (v/v) was added to the test sample groups 1 and 2, respectively. 37 ℃ and 5% CO₂The incubator continues to culture for 24 h.

(3) Irradiation: the test sample group 1, the test sample group 2 and the negative control group were subjected to a treatment of 40 mJ. cm^-2UVA irradiation of (1), ultraviolet irradiation dose (mJ. cm)^-2) Radiation intensity (mW/cm)²) X irradiation time (min), blank group did not need irradiation. After irradiation, the medium was changed from each group to complete medium (DMEM + 10% FBS), 37 ℃ with 5% CO₂The incubator is used for 24 h.

(4) And (3) detecting the content of COL-I: the supernatant was collected and stored at-80 ℃ and the cytokines were measured using an ELISA kit.

(5) And (3) data analysis: data are presented as mean ± standard deviation, and data are analyzed using SPSS, considering that differences are statistically significant if p < 0.05.

As shown in FIG. 3, the products of examples 1-3 and comparative example 1 of the present invention showed a significant increase in the expression level of COL-I protein (p <0.05) compared to the negative control (UVA +) at a dose of 10%.

According to the test of promoting the expression of the barrier repair gene and promoting the formation of collagen, the split yeast fermentation product filtrate has the anti-aging effect.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.