TWI774168B - Morus alba ferment, manufacturing method thereof and use thereof - Google Patents
Morus alba ferment, manufacturing method thereof and use thereof Download PDFInfo
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- TWI774168B TWI774168B TW109146336A TW109146336A TWI774168B TW I774168 B TWI774168 B TW I774168B TW 109146336 A TW109146336 A TW 109146336A TW 109146336 A TW109146336 A TW 109146336A TW I774168 B TWI774168 B TW I774168B
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- white mulberry
- skin
- fermented product
- fermentation
- mulberry fermented
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Abstract
Description
本發明係關於一種白桑葚發酵物及其製備方法與用途,特別係關於白桑葚發酵物用於製備改善肌膚狀況及/或抑制脂肪堆積之組合物的用途。The present invention relates to a white mulberry fermented product and its preparation method and use, in particular to the use of the white mulberry fermented product for preparing a composition for improving skin condition and/or inhibiting fat accumulation.
愛美是人的天性,肌膚美容及瘦身減脂已成為現代人重視的一大課題,不僅女性費盡心力追求肌膚美白及苗條體態,近年來男生注重於皮膚美白、保健保養的指數節節攀升,而皮膚之所以會變黑,其作用機制在於皮膚每天都直接或間接的暴露於日光照射下,為了保護皮膚不受紫外線的傷害,人體會合成黑色素(melanin)來防禦陽光中的紫外線,該黑色素會導致皮膚出現暗沉蠟黃、斑點或痘斑。It is human nature to love beauty. Skin beauty and slimming and fat reduction have become a major topic that modern people pay attention to. Not only women strive for skin whitening and slimming, but in recent years, boys focus on skin whitening and health care. The reason why the skin turns dark is that the skin is directly or indirectly exposed to sunlight every day. In order to protect the skin from ultraviolet rays, the human body will synthesize melanin to defend against ultraviolet rays in the sun. Can cause dull, sallow, blotchy, or acne-prone skin.
另外,隨著年齡增長肌膚會產生皺紋或鬆弛等問題,該皮膚皺紋或鬆弛已知係由於皮膚纖維母細胞之膠原蛋白產生能力降低而引起;而老化會導致肌膚的含水量降低,使皮膚失去緊緻和柔嫩,其與玻尿酸的流失有關,玻尿酸可以用於提升肌膚的保濕程度,同時加強肌膚表層的保護力。In addition, with age, the skin will develop wrinkles or sagging, which is known to be caused by the reduction of the collagen production capacity of dermal fibroblasts; and aging will reduce the skin's moisture content, causing the skin to lose Firming and softening, it is related to the loss of hyaluronic acid, which can be used to increase the moisture level of the skin and strengthen the protection of the skin's surface layer.
現今社會,自然有機及天然成分保養品概念逐漸興起,生技公司及保養品相關業者積極投入天然植物的相關產品研發,為使天然植物類產品對於人體健康保健有實質科學驗證的基礎,植物的成分分析及功效評估成為產品開發的重點項目。In today's society, the concept of natural organic and natural ingredients skin care products is gradually emerging, and biotechnology companies and skin care products related industries are actively investing in the research and development of natural plant-related products. Component analysis and efficacy evaluation have become key items in product development.
白桑葚(Morus alba ,又名white mulberry)係一種生長迅速的中小型桑樹,其可生長到10-20公尺高,且其通常被認定為壽命相較短的樹種,原產於中國北部和印度,目前廣泛種植於世界各地,包括:美國、土耳其、墨西哥、澳大利亞、阿根廷及伊朗等地區,其主要由蠶絲業者所種植目的是用以餵養生產中所使用的蠶,白桑葚葉子可長達30厘米呈鋸齒圓形狀,而其果實約長1-1.5厘米,並在成熟時可供食用。White mulberry ( Morus alba , also known as white mulberry) is a fast-growing small and medium-sized mulberry tree, which can grow to 10-20 meters high, and it is usually regarded as a short-lived tree species, native to northern China and India is currently widely planted around the world, including: the United States, Turkey, Mexico, Australia, Argentina, and Iran. It is mainly grown by silk producers to feed the silkworms used in production. The leaves of white mulberries can grow as long as 30 cm in the shape of a serrated circle, while the fruit is about 1-1.5 cm long and is edible when ripe.
一般熟知的桑葚果實是呈紅色或者黑紫色,然白桑葚果實係為白色,因此亦被稱為「白玉王」,其口感比普通黑桑葚更甜一些,而成熟的白桑葚帶有一點紫色,待其全熟透後,甜度可達到19度;白桑葚含有的熱量不高,適合身體肥胖者食用,且其含有蛋白質、脂肪、維生素、碳水化合物及膳食纖維等微量元素,營養價值豐富。然而,白桑葚是否具有其他功效及用途還未被廣泛了解與應用。The well-known mulberry fruit is red or black-purple, but the white mulberry fruit is white, so it is also called "white jade king", its taste is sweeter than the ordinary black mulberry, and the mature white mulberry has a little purple, After it is fully cooked, the sweetness can reach 19 degrees; the white mulberry contains low calories and is suitable for obese people, and it contains trace elements such as protein, fat, vitamins, carbohydrates and dietary fiber, and is rich in nutritional value. However, whether white mulberry has other effects and uses has not been widely understood and applied.
有鑑於此,本發明之目的在於提供一種白桑葚發酵物,其中該白桑葚發酵物係由一白桑葚果實經由水萃取所得之一白桑葚水萃取物,再經由一酵母菌以及一乳酸菌進行一前發酵,再以一醋酸菌進行一後發酵而獲得。In view of this, the object of the present invention is to provide a kind of white mulberry fermented product, wherein this white mulberry fermented product is a white mulberry water extract obtained by a white mulberry fruit through water extraction, and then through a yeast and a lactic acid bacteria to carry out a It is obtained by pre-fermentation and post-fermentation with an acetic acid bacteria.
在本發明的一些實施例中,該酵母菌之添加量為0.01-0.5%(v/v)、該乳酸菌之添加量為0.01-0.25%(v/v)、及該醋酸菌之添加量為1-20%(v/v)。In some embodiments of the present invention, the addition amount of the yeast is 0.01-0.5% (v/v), the addition amount of the lactic acid bacteria is 0.01-0.25% (v/v), and the addition amount of the acetic acid bacteria is 1-20%(v/v).
在本發明的一些實施例中,該前發酵之發酵時間為1-3天,且該後發酵之發酵時間為3-10天。In some embodiments of the present invention, the fermentation time of the pre-fermentation is 1-3 days, and the fermentation time of the post-fermentation is 3-10 days.
在本發明的一些實施例中,將該白桑葚發酵物進一步以200-400網目之網篩進行網篩過濾,並於55-65o C進行減壓濃縮,再添加40-70%異麥芽寡糖進行規格調整,再於95-105o C下加熱100-120分鐘進行滅菌。In some embodiments of the present invention, the white mulberry fermented product is further filtered with a mesh screen of 200-400 mesh, and concentrated under reduced pressure at 55-65 ℃ , and then 40-70% isomalt is added. The oligosaccharides are sized and sterilized by heating at 95-105 o C for 100-120 minutes.
在本發明的一些實施例中,該白桑葚發酵物之總多酚含量為該白桑葚水萃取物之總多酚含量的2-5倍。In some embodiments of the present invention, the total polyphenol content of the white mulberry fermentation product is 2-5 times the total polyphenol content of the white mulberry water extract.
本發明之另一目的在於提供一種前述白桑葚發酵物用於製備改善肌膚狀況及/或抑制脂肪堆積之組合物的用途。Another object of the present invention is to provide a use of the aforementioned white mulberry fermented product for preparing a composition for improving skin condition and/or inhibiting fat accumulation.
在本發明的一些實施例中,該改善肌膚狀況係為降低黑色素生成、提升皮膚纖維母細胞粒線體活性、提升玻尿酸分泌、減少紫外線色斑、提升肌膚含水量、提升肌膚彈力、及/或促進膠原蛋白增生。In some embodiments of the present invention, the improving skin condition is reducing melanin production, increasing skin fibroblast mitochondrial activity, increasing hyaluronic acid secretion, reducing UV pigmentation, increasing skin moisture content, increasing skin elasticity, and/or Promote collagen growth.
在本發明的一些實施例中,該組合物係透過降低發炎反應因子生成及抑制酪胺酸酶活性以達到該降低黑色素生成的功效,其中該發炎反應因子係為一氧化氮。In some embodiments of the present invention, the composition achieves the effect of reducing melanin production by reducing the production of an inflammatory response factor and inhibiting the activity of tyrosinase, wherein the inflammatory response factor is nitric oxide.
在本發明的一些實施例中,白桑葚發酵物的濃度為至少0.5%(v/v)。In some embodiments of the present invention, the concentration of the white mulberry fermentation product is at least 0.5% (v/v).
在本發明的一些實施例中,該組合物是一醫藥品、一食品或一保養品。In some embodiments of the present invention, the composition is a pharmaceutical product, a food product or a skin care product.
本發明之另一目的在於提供一種白桑葚發酵物的製備方法,係包括將一白桑葚經由水萃取所得之一白桑葚水萃取物,經由一酵母菌以及一乳酸菌進行一前發酵,再以一醋酸菌進行一後發酵,並以200-400網目之網篩過濾,再於55-65o C進行減壓濃縮,再添加40-70%異麥芽寡糖調整後,於95-105o C下加熱100-120分鐘進行滅菌;其中該酵母菌之添加量為0.01-0.5%(v/v)、該乳酸菌之添加量為0.01-0.25%(v/v)、及該醋酸菌之添加量為1-20%(v/v);其中該前發酵之發酵時間為1-3天,且該後發酵之發酵時間為3-10天。Another object of the present invention is to provide a method for preparing a white mulberry fermented product. Acetobacter is subjected to a post-fermentation, filtered with a 200-400 mesh sieve, concentrated under reduced pressure at 55-65 o C, and adjusted by adding 40-70% isomalt oligosaccharide, then adjusted at 95-105 o C Heating for 100-120 minutes for sterilization; wherein the addition amount of the yeast is 0.01-0.5% (v/v), the addition amount of the lactic acid bacteria is 0.01-0.25% (v/v), and the addition amount of the acetic acid bacteria 1-20% (v/v); wherein the fermentation time of the pre-fermentation is 1-3 days, and the fermentation time of the post-fermentation is 3-10 days.
以下將配合圖式進一步描述本案的部分具體實施態樣,下述所列舉的實施例係用以闡明本發明,並非用以限定本發明之範圍,任何熟習此技藝者,在不脫離本發明之精神和範圍內,當可做些許更動與潤飾,不應將保護範圍限於說明書所具體陳述的條件。Some specific implementations of this case will be further described below in conjunction with the drawings. The following examples are used to illustrate the present invention, but not to limit the scope of the present invention. The scope of protection should not be limited to the conditions specifically stated in the description, while some changes and modifications may be made within the spirit and scope.
本案使用Excel軟體進行統計分析。數據以平均值±標準差(SD)表示,各組之間的差異以學生t檢驗(student’s t-test)進行分析。圖式中「*」代表p值小於0.05,「**」代表p值小於0.01,以及「***」代表p值小於0.001。當「*」越多時,代表統計上的差異越顯著。而「###」符號代表與控制組相比p值小於0.001。The case used Excel software for statistical analysis. Data are presented as mean ± standard deviation (SD), and differences between groups were analyzed by Student's t-test. In the graph, "*" represents a p-value less than 0.05, "**" represents a p-value less than 0.01, and "***" represents a p-value less than 0.001. The more "*", the more significant the statistical difference. The "###" symbol represents a p-value less than 0.001 compared to the control group.
本文中所使用數值為近似值,所有實驗數據皆表示在正負20%的範圍內,較佳為在正負10%的範圍內,最佳為在正負5%的範圍內。The numerical values used herein are approximate, and all experimental data are expressed within the range of plus or minus 20%, preferably within the range of plus or
本文中之「wt%」係指重量百分比,而「vol%」係指體積百分比。關於本文中所使用之「白桑葚」通常是指白桑葚果實。Herein, "wt%" refers to weight percent, and "vol%" refers to volume percent. As used herein, "white mulberry" generally refers to the white mulberry fruit.
本文所述之「有效濃度」或「有效量」係表示能有效降低黑色素生成、提升皮膚纖維母細胞粒線體活性、提升玻尿酸分泌、抑制脂肪堆積、降低發炎反應因子生成、抑制酪胺酸脢活性、減少紫外線色斑、提升肌膚含水量、提升肌膚彈力、及/或促進膠原蛋白增生所需本發明之白桑葚發酵物的濃度。有效濃度依所作用的對象而可能不同,但可藉由例如劑量遞增試驗(Dose escalation)以實驗決定其有效濃度。The "effective concentration" or "effective amount" described herein means that it can effectively reduce the production of melanin, increase the mitochondrial activity of skin fibroblasts, increase the secretion of hyaluronic acid, inhibit the accumulation of fat, reduce the production of inflammatory response factors, and inhibit the production of tyrosinase The concentration of the white mulberry fermented product of the present invention required for activity, reducing UV spots, increasing skin moisture content, improving skin elasticity, and/or promoting collagen proliferation. The effective concentration may vary depending on the target, but the effective concentration can be determined experimentally by, for example, dose escalation.
在一些實施例中,白桑葚發酵物係由白桑葚水萃取物與酵母菌及乳酸菌進行一前發酵程序,再以一醋酸菌進行一後發酵程序所得。In some embodiments, the white mulberry fermented product is obtained by performing a pre-fermentation process with the water extract of white mulberry, yeast and lactic acid bacteria, and then performing a post-fermentation process with an acetic acid bacteria.
在一些實施例中,該白桑葚水萃取物包括白桑葚及水,該白桑葚與水的比例為1:5-20,並根據總重加入5%葡萄糖,使其糖度(Degrees Brix)>8.5,並於95o C加熱60分鐘。In some embodiments, the white mulberry water extract comprises white mulberry and water, the ratio of the white mulberry and water is 1:5-20, and 5% glucose is added according to the total weight, so that its Brix (Degrees Brix)>8.5 , and heated at 95 o C for 60 minutes.
於前發酵程序中,先添加0.01%-0.5%的酵母菌至白桑葚水萃取物內,並將白桑葚水萃取物與酵母菌發酵1日至2日後,再添加0.01%-0.25%乳酸菌進行發酵1日至2日以完成前發酵之程序。換言之,該酵母菌之添加量為0.01-0.5%(v/v),且該乳酸菌之添加量為0.01-0.25%(v/v),其中該前發酵之發酵時間為1-3天,並於28℃-37℃下進行。In the pre-fermentation process, first add 0.01%-0.5% yeast to the white mulberry water extract, and ferment the white mulberry water extract and yeast for 1 to 2 days, and then add 0.01%-0.25% lactic acid bacteria. Fermentation 1 to 2 days to complete the previous fermentation process. In other words, the addition amount of the yeast is 0.01-0.5% (v/v), and the addition amount of the lactic acid bacteria is 0.01-0.25% (v/v), wherein the fermentation time of the pre-fermentation is 1-3 days, and It is carried out at 28°C-37°C.
於後發酵程序中,添加1-20%的醋酸菌靜置發酵3-10天,待Brix值小於3.9且pH值等於3.7±1時即終止發酵。In the post-fermentation procedure, add 1-20% acetic acid bacteria for 3-10 days, and stop the fermentation when the Brix value is less than 3.9 and the pH value is equal to 3.7±1.
在一些實施例中,酵母菌可以是啤酒酵母(Saccharomyces cerevisiae ),在一些實施例中,乳酸菌可以是嗜熱鏈球菌(Streptococcus thermophilus )、瑞士乳酸菌(Lactobacillus helveticus )、或植物乳桿菌(Lactobacillus plantarum ),在一些實施例中,醋酸菌可以是乙酸醋酸菌(Acetobacter aceti )。In some embodiments, the yeast may be Saccharomyces cerevisiae , and in some embodiments, the lactic acid bacteria may be Streptococcus thermophilus , Lactobacillus helveticus , or Lactobacillus plantarum , in some embodiments, the acetic acid bacteria can be acetic acid bacteria ( Acetobacter aceti ).
在一些實施例中,白桑葚發酵物可進一步進行網篩過濾、減壓濃縮、規格調整及加熱滅菌。In some embodiments, the white mulberry fermented product can be further subjected to mesh filtration, reduced pressure concentration, specification adjustment and heat sterilization.
在一些實施例中,網篩過濾以200目數至400目數的網孔之濾網進行過濾,並於55-65o C進行減壓濃縮。In some embodiments, the mesh filtration is performed with a mesh filter with a mesh size of 200 to 400 mesh, and concentrated under reduced pressure at 55-65 ° C.
在一些實施例中,調整規格可透過添加40-70%的賦形劑(excipient)來調整發酵原液的糖度。在一些實施例中,用以調整糖度的賦形劑可為異麥芽寡糖。In some embodiments, the specification can be adjusted by adding 40-70% excipients to adjust the Brix of the fermentation stock. In some embodiments, the excipient used to adjust the sugar content may be isomalt-oligosaccharide.
在一些實施例中,加熱滅菌程序可為將原料混合液在95℃至105℃下滅菌100-120分鐘,並將滅菌後的混合液冷卻至室溫(即25℃至30℃)以形成白桑葚發酵物。在一些實施例中,滅菌後的原料混合液可採取自然降溫的方式冷卻至室溫。In some embodiments, the heat sterilization procedure may be to sterilize the raw material mixture at 95°C to 105°C for 100-120 minutes, and cool the sterilized mixture to room temperature (ie, 25°C to 30°C) to form white Mulberry fermented product. In some embodiments, the sterilized raw material mixture can be cooled to room temperature by natural cooling.
在一些實施例中,白桑葚發酵物的pH值為4.0±1.0,且白桑葚發酵物的糖度為39±2。In some embodiments, the pH of the white mulberry fermentation is 4.0±1.0, and the Brix of the white mulberry fermentation is 39±2.
在一些實施例中,該白桑葚發酵物的製備方法,係包括將一白桑葚經由水萃取所得之一白桑葚水萃取物,經由一酵母菌以及一乳酸菌進行一前發酵,再以一醋酸菌進行一後發酵,並以200-400網目之網篩過濾,再於55-65o C進行減壓濃縮,再添加40-70%異麥芽寡糖調整後,於95-105o C下加熱100-120分鐘進行滅菌;其中該酵母菌之添加量為0.01-0.5%(v/v)、該乳酸菌之添加量為0.01-0.25%(v/v)、及該醋酸菌之添加量為1-20%(v/v);其中該前發酵之發酵時間為1-3天,且該後發酵之發酵時間為3-10天。In some embodiments, the preparation method of the white mulberry fermented product includes extracting a white mulberry water extract obtained from a white mulberry through water, performing a pre-fermentation through a yeast and a lactic acid bacteria, and then using an acetic acid bacteria Carry out a post-fermentation, and filter with a 200-400 mesh sieve, then concentrate under reduced pressure at 55-65 o C, add 40-70% isomalt oligosaccharide for adjustment, and heat at 95-105 o C Sterilize for 100-120 minutes; wherein the addition amount of the yeast is 0.01-0.5% (v/v), the addition amount of the lactic acid bacteria is 0.01-0.25% (v/v), and the addition amount of the acetic acid bacteria is 1 -20% (v/v); wherein the fermentation time of the pre-fermentation is 1-3 days, and the fermentation time of the post-fermentation is 3-10 days.
在一些實施例中,前述白桑葚發酵物用於製備改善肌膚狀況及/或抑制脂肪堆積之組合物的用途。在一些實施例中,該改善肌膚狀況係為降低黑色素生成、提升皮膚纖維母細胞粒線體活性、提升玻尿酸分泌、減少紫外線色斑、提升肌膚含水量、提升肌膚彈力、及/或促進膠原蛋白增生。在一些實施例中,該組合物係透過降低發炎反應因子生成及抑制酪胺酸酶活性以達到該降低黑色素生成。在一些實施例中,該發炎反應因子係為一氧化氮。其中,受體可為人。In some embodiments, the aforementioned white mulberry fermented product is used to prepare a composition for improving skin condition and/or inhibiting fat accumulation. In some embodiments, the improving skin condition is reducing melanin production, increasing skin fibroblast mitochondrial activity, increasing hyaluronic acid secretion, reducing UV pigmentation, increasing skin moisture content, increasing skin elasticity, and/or promoting collagen hyperplasia. In some embodiments, the composition achieves the reduction in melanin production by reducing inflammatory response factor production and inhibiting tyrosinase activity. In some embodiments, the inflammatory response factor is nitric oxide. Wherein, the receptor can be human.
在一些實施例中,該白桑葚發酵物的濃度為至少0.5%(v/v)。In some embodiments, the concentration of the white mulberry fermentation is at least 0.5% (v/v).
在一些實施例中,該組合物是一醫藥品、一食品或一保養品。換言之,此醫藥品、食品或保養品包含有效含量的白桑葚發酵物。In some embodiments, the composition is a pharmaceutical product, a food product, or a skin care product. In other words, the medicine, food or skin care product contains an effective content of white mulberry fermented product.
在一些實施例中,前述之醫藥品可利用熟習此技藝者所詳知的技術而被製造成適合於經腸道地、非經腸道地(parenterally)、口服地、或局部地(topically)投藥劑型。In some embodiments, the aforementioned pharmaceutical products can be manufactured for parenterally, parenterally, orally, or topically using techniques well known to those skilled in the art Dosage form.
在一些實施例中,經腸道或口服的投藥劑型可為,但不限於,錠劑(tablet)、片劑(troche)、***錠(lozenge)、丸劑(pill)、膠囊(capsule)、分散性粉末(dispersible powder)或細顆粒(granule)、溶液、懸浮液(suspension)、乳劑(emulsion)、糖漿(syrup)、酏劑(elixir)、濃漿(slurry)或類似之物。在一些實施例中,非經腸道地或局部地投藥劑型可為,但不限於,注射品(injection)、無菌的粉末(sterile powder)、外部製劑(external preparation)或類似之物。在一些實施例中,注射品的投藥方式可為皮下注射(subcutaneous injection)、表皮內注射(intraepidermal injection)、皮內注射(intradermal injection)或病灶內注射(intralesional injection)。In some embodiments, the dosage form for parenteral or oral administration can be, but is not limited to, a tablet, troche, lozenge, pill, capsule , dispersible powder or granule, solution, suspension, emulsion, syrup, elixir, slurry or the like. In some embodiments, the dosage form for parenteral or topical administration can be, but is not limited to, an injection, sterile powder, external preparation, or the like. In some embodiments, the injection can be administered by subcutaneous injection, intraepidermal injection, intradermal injection or intralesional injection.
在一些實施例中,前述之醫藥品可包含被廣泛地使用於藥物製造技術之醫藥上可接受的載劑(pharmaceutically acceptable carrier)。在一些實施例中,醫藥上可接受的載劑可為下列載劑中一種或多種:溶劑(solvent)、緩衝液(buffer)、乳化劑(emulsifier)、懸浮劑(suspending agent)、分解劑(decomposer)、崩解劑(disintegrating agent)、分散劑(dispersing agent)、黏結劑(binding agent)、賦形劑、安定劑(stabilizing agent)、螯合劑(chelating agent)、稀釋劑(diluent)、膠凝劑(gelling agent)、防腐劑(preservative)、潤濕劑(wetting agent)、潤滑劑(lubricant)、吸收延遲劑(absorption delaying agent)、脂質體(liposome)以及類似之物。關於選用之載劑的種類與數量是落在熟習此項技術之人士的專業素養與例行技術範疇內。在一些實施例中,作為醫藥上可接受的載劑的溶劑可為水、生理鹽水(normal saline)、磷酸鹽緩衝液(phosphate buffered saline,PBS)、或含有醇的水性溶液(alcohol containing aqueous solution )。In some embodiments, the aforementioned pharmaceutical products may include pharmaceutically acceptable carriers that are widely used in pharmaceutical manufacturing techniques. In some embodiments, the pharmaceutically acceptable carrier can be one or more of the following carriers: solvent, buffer, emulsifier, suspending agent, disintegrant ( decomposer), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, glue gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes, and the like. Regarding the type and quantity of the carrier selected, it falls within the scope of the professional quality and routine skills of those who are familiar with the technology. In some embodiments, the solvent as a pharmaceutically acceptable carrier can be water, normal saline, phosphate buffered saline (PBS), or an alcohol containing aqueous solution ).
在一些實施例中,前述之任一組合物可為食用組合物。換言之,食用組合物包含特定含量的白桑葚發酵物。在一些實施例中,前述之食用組合物可為食品產品或食品添加物(food additive)。在一些實施例中,食品產品可為但不限於:飲料(beverages)、發酵食品(fermented foods)、烘培產品(bakery products)、健康食品(health foods)或膳食補充品(dietary supplements)。In some embodiments, any of the foregoing compositions may be edible compositions. In other words, the edible composition contains a specific amount of white mulberry fermented product. In some embodiments, the aforementioned edible composition may be a food product or a food additive. In some embodiments, the food products can be, but are not limited to, beverages, fermented foods, bakery products, health foods, or dietary supplements.
在一些實施例中,前述之食用組合物可以口服方式施予受體。其中,食用組合物的型態可為粉末、顆粒、溶液、膠體或膏體。In some embodiments, the aforementioned edible compositions can be administered to a recipient orally. Wherein, the form of the edible composition can be powder, granule, solution, colloid or paste.
在一些實施例中,前述之任一組合物可為化妝品或保養品。換言之,化妝品或保養品包含特定含量的白桑葚發酵物。In some embodiments, any of the foregoing compositions can be a cosmetic or skin care product. In other words, cosmetics or skin care products contain a specific content of white mulberry fermented product.
在一些實施例中,前述之化妝品或保養品可為下列任一種型態:化妝水、凝膠、凍膜、泥膜、乳液、乳霜、唇膏、粉底、粉餅、蜜粉、卸妝油、卸妝乳、洗面乳、沐浴乳、洗髮精、護髮乳、防曬乳、護手霜、指甲油、香水、精華液及面膜。在一些實施例中,前述之化妝品或保養品可視需要更包含外用品可接受成分。在一些實施例中,外用品可接受成分可例如為乳化劑、滲透促進劑、軟化劑、溶劑、賦型劑、抗氧化劑、或其組合。In some embodiments, the aforementioned cosmetics or skin care products can be in any of the following forms: lotion, gel, jelly film, mud film, lotion, cream, lipstick, foundation, pressed powder, powder, makeup remover, makeup remover Milk, facial cleanser, body wash, shampoo, conditioner, sunscreen, hand cream, nail polish, perfume, serum and mask. In some embodiments, the aforementioned cosmetics or skin care products may further include externally acceptable ingredients as needed. In some embodiments, topical acceptable ingredients may be, for example, emulsifiers, penetration enhancers, emollients, solvents, excipients, antioxidants, or combinations thereof.
實施例一、Embodiment 1. 白桑葚發酵物Fermented white mulberry 的製備preparation
首先,將白桑葚(Morus alba ,白桑葚原產地為土耳其)與水以1:10(即,白桑葚為一倍重量,水為十倍重量)的比例混合均勻,再添加5%葡萄糖溶液將糖度調整為8.5,並於95℃下加熱60分鐘,以獲得白桑葚水萃取物。First, mix the white mulberry ( Morus alba , the origin of white mulberry is Turkey) and water at a ratio of 1:10 (that is, the white mulberry is one time by weight, and the water is ten times the weight), and then add 5% glucose solution. The sugar content was adjusted to 8.5, and heated at 95° C. for 60 minutes to obtain a white mulberry water extract.
接著,於白桑葚水萃取物中殖入0.1%啤酒酵母(Saccharomyces cerevisiae )(購自食品工業發展研究所生物資源保存及研究中心(BCRC),寄存編號BCRC20271)並在約30℃下發酵1天,以得到第一初發酵汁液。接著,於第一初發酵汁液中殖入0.05%嗜熱鏈球菌(Streptococcus thermophilus )(購自BCRC,寄存編號BCRC910636)並在約30℃下發酵1天,以得到第二初發酵汁液。最後,於第二初發酵汁液中殖入5%乙酸醋酸菌(Acetobacter aceti )(購自BCRC,寄存編號BCRC11688)並在約30℃下發酵5天,以得到第三初發酵汁液。Then, 0.1% brewer's yeast ( Saccharomyces cerevisiae ) (purchased from the Biological Resource Conservation and Research Center (BCRC), Institute of Food Industry Development, accession number BCRC20271) was propagated into the white mulberry water extract and fermented at about 30°C for 1 day , to obtain the first primary fermentation juice. Next, 0.05% Streptococcus thermophilus (purchased from BCRC, accession number BCRC910636) was propagated into the first primary fermentation juice and fermented at about 30° C. for 1 day to obtain a second primary fermentation juice. Finally, 5% acetic acid bacteria ( Acetobacter aceti ) (purchased from BCRC, accession number BCRC11688) was propagated in the second primary fermentation juice and fermented at about 30°C for 5 days to obtain the third primary fermentation juice.
再將第三初發酵汁液以目數200的網孔之濾網進行網篩過濾,再於約60℃下進行減壓濃縮,以得到發酵原液。最後,添加60%異麥芽寡糖至發酵原液後在約100℃下滅菌2小時,以得到白桑葚發酵物。Then, the third primary fermentation juice is filtered through a mesh screen with a mesh size of 200, and then concentrated under reduced pressure at about 60° C. to obtain a fermentation stock solution. Finally, after adding 60% isomalt oligosaccharide to the fermentation stock solution, it was sterilized at about 100° C. for 2 hours to obtain a white mulberry fermented product.
於此,分別對白桑葚水萃取物以及白桑葚發酵物進行糖度檢測。其中,白桑葚水萃取物的糖度約為8.5,而白桑葚發酵物的糖度約為40。Here, the sugar content of the white mulberry water extract and the white mulberry fermented product were detected respectively. Among them, the sugar content of white mulberry water extract is about 8.5, and the sugar content of white mulberry fermented product is about 40.
實施例Example 二、總多酚含量測試2. Total polyphenol content test
秤取10.0mg的沒食子酸(Gallic acid,購自Sigma,料號:G7384)置於10mL容量瓶中,然後以水(H2 O)定量至10mL,以得到沒食子酸的儲備溶液(stock solution)。將沒食子酸的儲備溶液稀釋10倍,即100μL沒食子酸的儲備溶液加900μL的水,以得到100μg/mL沒食子酸的初始溶液(即含1000ppm的沒食子酸)。然後,依據下表一配置0μg/mL、20μg/mL、40μg/mL、60μg/mL、80μg/mL、及100μg/mL之沒食子酸的標準溶液,並分別取100μL之各濃度的標準溶液至玻璃試管中。加入500μL之福林酚試劑(Folin-Ciocalteu's phenol reagent,購自Merck,料號:1.09001.0100)至各玻璃試管內與標準溶液混合均勻並靜置3分鐘後,再加入400μL之7.5%碳酸鈉混合均勻後反應30分鐘以得到標準反應溶液。取200μL之標準反應溶液至96孔板中,並測量其在750nm下之吸光值,以獲得標準曲線。10.0 mg of gallic acid (Gallic acid, purchased from Sigma, material number: G7384) was weighed into a 10 mL volumetric flask, and then quantified to 10 mL with water (H 2 O) to obtain a stock solution of gallic acid (stock solution). The stock solution of gallic acid was diluted 10-fold, ie, 100 μL of the stock solution of gallic acid plus 900 μL of water, to obtain an initial solution of 100 μg/mL gallic acid (ie, containing 1000 ppm of gallic acid). Then, prepare standard solutions of gallic acid of 0 μg/mL, 20 μg/mL, 40 μg/mL, 60 μg/mL, 80 μg/mL, and 100 μg/mL according to Table 1, and take 100 μL of the standard solutions of each concentration. into a glass test tube. Add 500 μL of Folin-Ciocalteu's phenol reagent (purchased from Merck, part number: 1.09001.0100) to each glass test tube and mix it with the standard solution and let stand for 3 minutes, then add 400 μL of 7.5% sodium carbonate After mixing uniformly, the reaction was carried out for 30 minutes to obtain a standard reaction solution. Take 200 μL of the standard reaction solution into a 96-well plate, and measure its absorbance at 750 nm to obtain a standard curve.
表一
將實驗組的測試樣品(即實施例一的白桑葚發酵物)及對照組的測試樣品(即實施例一的白桑葚水萃液)分別以水稀釋20倍。將各樣本取100μL到玻璃試管中。接著,加入500μL之福林酚試劑至玻璃試管中與樣本混合均勻並靜置3分鐘後,再加入400μL之7.5%碳酸鈉混合均勻後反應30分鐘以得到待測反應溶液。將裝有待測反應溶液的玻璃試管進行震盪以確保無氣泡後,取200μL之待測反應溶液至96孔板中,並測量待測反應溶液於750nm下之吸光值,接著,各樣本對應的待測反應溶液的吸光值先除以樣本的糖度,再利用標準曲線以內插法換算成總多酚含量。The test sample of the experimental group (ie the white mulberry fermented product of Example 1) and the test sample of the control group (ie the white mulberry water extract of Example 1) were diluted 20 times with water respectively. Take 100 μL of each sample into a glass test tube. Next, add 500 μL of Folin phenol reagent to the glass test tube and mix with the sample evenly and let it stand for 3 minutes, then add 400 μL of 7.5% sodium carbonate, mix well, and react for 30 minutes to obtain the reaction solution to be tested. After shaking the glass test tube containing the reaction solution to be tested to ensure no air bubbles, take 200 μL of the reaction solution to be tested into a 96-well plate, and measure the absorbance value of the reaction solution to be tested at 750 nm. The absorbance value of the reaction solution to be tested was first divided by the sugar content of the sample, and then converted into the total polyphenol content by interpolation of the standard curve.
總多酚含量結果如圖1所示,對照組的總多酚含量為90ppm及實驗組的總多酚含量為250ppm,與對照組相較之下,樣品組的總多酚含量有顯著的提升。於此,可推斷出稀釋前的白桑葚水萃物及白桑葚發酵物(即經本案實施例一製備方式所製得的白桑葚水萃物及白桑葚發酵物)的實際總多酚含量分別為1800ppm及5000ppm。由此可知,白桑葚透過微生物發酵後,可提升總多酚含量約2.77倍。即,相對於白桑葚水萃物,白桑葚發酵物會大量釋出總多酚,並能提升抗氧化活性,可有效減少自由基累積。The results of total polyphenol content are shown in Figure 1. The total polyphenol content of the control group is 90 ppm and the total polyphenol content of the experimental group is 250 ppm. Compared with the control group, the total polyphenol content of the sample group has been significantly improved. . Here, it can be inferred that the actual total polyphenol content of the white mulberry water extract and the white mulberry fermented product (that is, the white mulberry water extract and the white mulberry fermented product obtained by the first preparation method of this case) before the dilution are respectively 1800ppm and 5000ppm. It can be seen that the total polyphenol content of white mulberry can be increased by about 2.77 times after microbial fermentation. That is, compared with the white mulberry water extract, the white mulberry fermented product will release a large amount of total polyphenols, and can enhance the antioxidant activity, which can effectively reduce the accumulation of free radicals.
實施例 三、抗發炎能力之一氧化氮檢測 Example 3. Detection of Nitric Oxide, One of Anti-inflammatory Ability
為探討白桑葚發酵物是否具有降低發炎反應之效果,本實施例將巨噬細胞RAW264.7作為細胞平台培養,再透過脂多醣(lipopolysaccharide,LPS)處理誘導發炎反應產生,並偵測發炎反應因子一氧化氮(NO)的生成量,以評估樣品是否達到降低發炎反應之效果。In order to investigate whether the fermentation product of white mulberry has the effect of reducing inflammatory response, in this example, macrophage RAW264.7 was cultured as a cell platform, and then lipopolysaccharide (LPS) treatment was used to induce inflammatory response, and inflammatory response factors were detected. The amount of nitric oxide (NO) generated was used to evaluate whether the sample achieved the effect of reducing the inflammatory response.
材料與儀器 1.細胞株:小鼠巨噬細胞RAW264.7,取自美國典型培養物保存中心(American Type Culture Collection,ATCC® ,Cat.TIB-71)。 2.培養基:將DMEM改良培養基(Dulbecco’s modified Eagle’s medium,DMEM,購自Gibco,Cat.11965-092)添加額外成分使其含有10 vol%胎牛血清(Fetal Bovine Serum,FBS,購自Gibco,Cat.10437-028)及1% 青黴素-鏈黴素(購自Gibco,Cat. 15140122)。 3.磷酸緩衝鹽溶液(PBS溶液):購自Gibco,產品編號為Cat.14200-075。 4.脂多醣(lipopolysaccharide,LPS):購自Sigma,產品編號為SI-L2880-25MG。 5.亞硝酸鹽檢測試劑盒(Griess reagent kit):購自Life technologies,產品編號為1445263。 6.白桑葚水萃取物及白桑葚發酵物:由本案實施例一之製備方式所製得。Materials and Instruments 1. Cell line: mouse macrophage RAW264.7, obtained from American Type Culture Collection (American Type Culture Collection, ATCC® , Cat. TIB-71). 2. Medium: DMEM modified medium (Dulbecco's modified Eagle's medium, DMEM, purchased from Gibco, Cat. 11965-092) was added with additional components to contain 10 vol% fetal bovine serum (Fetal Bovine Serum, FBS, purchased from Gibco, Cat. .10437-028) and 1% penicillin-streptomycin (purchased from Gibco, Cat. 15140122). 3. Phosphate buffered saline solution (PBS solution): purchased from Gibco, product number Cat. 14200-075. 4. Lipopolysaccharide (LPS): purchased from Sigma, product code is SI-L2880-25MG. 5. Griess reagent kit: purchased from Life technologies, product number 1445263. 6. White mulberry water extract and white mulberry fermented product: prepared by the preparation method of Example 1 of this case.
實驗步驟: 1. 實驗將會分為實驗組(實驗組A及實驗組B)、控制組、以及對照組進行;其中該實驗組A為白桑葚水萃取物組,實驗組B為白桑葚發酵物組;各組分別進行三重複試驗: 2. 將RAW264.7細胞以 1 x 104 cells/well的密度種入96孔細胞培養盤,培養液體積為200 μl/well,於37℃、5%CO2 恆溫培養箱中培養24小時。 3. 將待測孔盤中的培養基移除後,於實驗組及對照組中加入LPS(濃度為200 ng/ml);同時,實驗組A之培養液額外加入白桑葚水萃取物,使其濃度為0.5 mg/mL;實驗組B之培養液額外加入白桑葚發酵物,使其濃度為0.5 mg/mL,作用24小時。(使用不含FBS的培養基配置) 4. 取一96孔盤,每孔加入130 μl的二次水,並將反應完之步驟3每孔中取出150 μl培養液加入上述已經含有130 μl二次水的96孔盤中。 5. 配置Griess試劑(reagent A: B=1:1),取20 μl加入96孔盤各孔中之培養液避光反應30分鐘。 6. 以ELISA讀盤機(購自BioTek),測定O.D.548 nm波長之吸光值(OD值讀值越大,表示NO的含量濃度越高) 。 7. 使用 student t-test 進行統計分析(*表p <0.05,**表p <0.01,***表p <0.001)。Experimental steps: 1. The experiment will be divided into experimental group (experimental group A and experimental group B), control group, and control group; wherein the experimental group A is the white mulberry water extract group, and the experimental group B is the white mulberry fermentation RAW264.7 cells were seeded into 96-well cell culture plates at a density of 1 x 10 4 cells/well, the volume of culture medium was 200 μl/well, and the cells were incubated at 37°C, 5 24 hours in a %CO 2 constant temperature incubator. 3. After removing the medium in the well plate to be tested, LPS (concentration of 200 ng/ml) was added to the experimental group and the control group; at the same time, the culture medium of experimental group A was additionally added with white mulberry water extract to make it. The concentration was 0.5 mg/mL; the culture medium of experimental group B was additionally added with white mulberry fermented product to make the concentration of 0.5 mg/mL, and it was treated for 24 hours. (Use a medium without FBS) 4. Take a 96-well plate, add 130 μl of secondary water to each well, and add 150 μl of the culture medium from each well of step 3 after the reaction to the above-mentioned 130 μl secondary water. water in a 96-well dish. 5. Prepare Griess reagent (reagent A: B=1:1), add 20 μl of the culture medium to each well of a 96-well plate, and react in the dark for 30 minutes. 6. Using an ELISA reader (purchased from BioTek), measure the absorbance at OD548 nm (the larger the OD reading, the higher the NO concentration). 7. Statistical analysis was performed using student t-test (* table p < 0.05, ** table p < 0.01, *** table p < 0.001).
實驗結果Experimental results
參閱圖2,其為經本發明白桑葚水萃取物及白桑葚發酵物處理後之一氧化氮相對生成量改變圖,一氧化氮係為重要之發炎反應因子,故本實施例以一氧化氮之生成量作為發炎反應能力之指標。如圖所示,對照組相對控制組之一氧化氮生成量為122.86%,實驗組A(白桑葚水萃取物)相對控制組之一氧化氮生成量為116.33%,而實驗組B(白桑葚發酵物)相對控制組之一氧化氮生成量為106%。由該實驗結果可知,與對照組相比白桑葚水萃取物及白桑葚發酵物分別降低一氧化氮生成量約6.53%及16.86%,且經白桑葚發酵物處理之RAW264.7巨噬細胞之一氧化氮生成量顯著低於對照組,表示RAW264.7巨噬細胞經白桑葚發酵物處理後可降低發炎反應因子之生成,顯示白桑葚發酵物具有抵抗發炎反應及分泌抗發炎因子之功效,且其效果優於白桑葚水萃取物,而分泌抗發炎因子可以達到降低黑色素沉澱的效果。Referring to FIG. 2 , it is a graph showing the change in the relative generation amount of nitric oxide after the white mulberry water extract and the white mulberry fermented product of the present invention are treated. Nitric oxide is an important inflammatory response factor. The amount of production was used as an indicator of inflammatory response capacity. As shown in the figure, the production of nitric oxide in the control group was 122.86% relative to the control group, the production of nitric oxide in the experimental group A (white mulberry water extract) relative to the control group was 116.33%, while the experimental group B (white mulberry water extract) produced 116.33% of nitric oxide. Fermentation) was 106% nitric oxide production relative to the control group. It can be seen from the experimental results that compared with the control group, the white mulberry water extract and the white mulberry fermented product reduced the production of nitric oxide by about 6.53% and 16.86%, respectively, and the RAW264.7 macrophages treated with the white mulberry fermented product were significantly reduced. The production of nitric oxide was significantly lower than that of the control group, indicating that RAW264.7 macrophages could reduce the production of inflammatory response factors after treatment with white mulberry fermentation, indicating that white mulberry fermentation had the effect of resisting inflammatory response and secreting anti-inflammatory factors. And its effect is better than that of white mulberry water extract, and the secretion of anti-inflammatory factors can reduce the effect of melanin precipitation.
實施例Example 四、酪胺酸酶活性檢測4. Detection of tyrosinase activity
酪胺酸酶是生物體內黑色素合成的關鍵,酪胺酸酶會催化酪胺酸轉變為L-多巴(L-Dopa),接著產生多巴醌(Dopaquinone)進而形成黑色素。於此,藉由檢測酪胺酸酶活性來了解白桑葚發酵物對於黑色素細胞產生黑色素的能力影響,當酪胺酸酶活性低時,黑色素細胞產生黑色素的能力就會相對降低。Tyrosinase is the key to melanin synthesis in living organisms. Tyrosinase catalyzes the conversion of tyrosine into L-Dopa, and then produces Dopaquinone to form melanin. Here, the tyrosinase activity was detected to understand the effect of white mulberry fermentation on the ability of melanocytes to produce melanin. When the tyrosinase activity was low, the ability of melanocytes to produce melanin was relatively reduced.
材料與儀器 1. 細胞株:小鼠黑色素瘤細胞B16F10,購自美國典型培養物保存中心(American Type Culture Collection,ATCC® ,No.6475),以下簡稱B16F10細胞。 2. 培養基:將DMEM改良培養基(Dulbecco’s modified Eagle’s medium,DMEM,購自Gibco,Cat. 12100-038)添加額外成分使其含有10 vol% FBS(fetal bovine Serum,購自Gibco,產品編號10438-026)及1% 青黴素-鏈黴素(購自Gibco,Cat. 15140122)。 3. 磷酸緩衝鹽溶液(PBS溶液):購自Gibco,產品編號10437-028。 4. 麴酸(Kojic acid):購自Sigma-Aldrich,產品編號K3125-5G。 5. 全稱放射免疫沉澱法緩衝液蛋白質裂解液(RIPA Lysis Buffer (PMSF)):購自Sigma-Aldrich,產品編號10837091001。 6. 蛋白質定量套組(Bio-rad Protein Assay,購自Biotek,產品編號Epoch)。 7. 10 mM L-多巴(L-Dopa):將9.8 mg的L-多巴(購自Sigma-Aldrich,產品編號D9628-5G)溶於5mL的0.1mM,pH值7.0的PBS溶液中。 8. 胰蛋白酶:10X Trypsin-EDTA(購自Gibco)以1X PBS溶液稀釋10倍。 9. 白桑葚水萃取物及白桑葚發酵物:由本案實施例一之製備方式所製得。Materials and instruments 1. Cell line: mouse melanoma cell B16F10, purchased from American Type Culture Collection (American Type Culture Collection, ATCC® , No. 6475), hereinafter referred to as B16F10 cell. 2. Medium: DMEM modified medium (Dulbecco's modified Eagle's medium, DMEM, purchased from Gibco, Cat. 12100-038) was added with additional components to contain 10 vol% FBS (fetal bovine Serum, purchased from Gibco, product number 10438-026) ) and 1% penicillin-streptomycin (purchased from Gibco, Cat. 15140122). 3. Phosphate buffered saline solution (PBS solution): purchased from Gibco, product number 10437-028. 4. Kojic acid: purchased from Sigma-Aldrich, product number K3125-5G. 5. Full name Radioimmunoprecipitation Buffer Protein Lysis Buffer (RIPA Lysis Buffer (PMSF)): purchased from Sigma-Aldrich, product number 10837091001. 6. Protein quantification kit (Bio-rad Protein Assay, purchased from Biotek, product number Epoch). 7. 10 mM L-Dopa (L-Dopa): 9.8 mg of L-Dopa (purchased from Sigma-Aldrich, product number D9628-5G) was dissolved in 5 mL of 0.1 mM PBS, pH 7.0. 8. Trypsin: 10X Trypsin-EDTA (purchased from Gibco) diluted 10 times in 1X PBS solution. 9. White mulberry water extract and white mulberry fermented product: obtained by the preparation method of Example 1 of this case.
實驗步驟:Experimental steps:
於每一孔中加入2mL上述培養基,並將B16F10細胞以1.5 x 105 的每孔密度種入一6孔盤中,於37°C下,培養24小時。2 mL of the above medium was added to each well, and B16F10 cells were seeded in a 6-well dish at a density of 1.5 x 10 5 per well and incubated at 37°C for 24 hours.
本實驗分為控制組、對照組、實驗組A及實驗組B。其中,各組加入2mL的新鮮DMEM培養液。而對照組之培養液額外加入麴酸(Kojic acid),使其濃度為0.5 mg/mL,其中麴酸已被廣泛認知為具有抑制細胞酪胺酸酶活性之物質;實驗組A之培養液額外加入白桑葚水萃取物,使其濃度為0.5 mg/mL;實驗組B之培養液額外加入白桑葚發酵物,使其濃度為0.5 mg/mL。各組進行三重複,並反應培養48小時。The experiment was divided into control group, control group, experimental group A and experimental group B. Among them, 2 mL of fresh DMEM medium was added to each group. The culture medium of the control group was additionally added with Kojic acid at a concentration of 0.5 mg/mL, of which kojic acid has been widely recognized as a substance that inhibits cellular tyrosinase activity; the culture medium of experimental group A was additionally White mulberry water extract was added to make its concentration 0.5 mg/mL; the culture medium of experimental group B was additionally added white mulberry fermented product to make its concentration 0.5 mg/mL. Three replicates were performed for each group, and the reaction was incubated for 48 hours.
培養完畢後,將培養液移除,以1x PBS()清洗兩次。再加入胰蛋白酶-EDTA對細胞作用3分鐘,回收懸浮的細胞於15 mL離心管,以400 xg離心5分鐘使細胞沉澱。After incubation, the medium was removed and washed twice with 1x PBS(). Then trypsin-EDTA was added to act on the cells for 3 minutes, the suspended cells were recovered in a 15 mL centrifuge tube, and the cells were pelleted by centrifugation at 400 xg for 5 minutes.
再將沉澱細胞以1x PBS清洗兩次後,以200 μL細胞裂解液讓細胞懸浮,震盪混合後以12,000 xg離心20分鐘。After the pelleted cells were washed twice with 1x PBS, the cells were suspended with 200 μL of cell lysate, mixed by shaking, and centrifuged at 12,000 x g for 20 minutes.
將上清液取出至1.5 mL離心管中,進行蛋白質濃度的檢測:Remove the supernatant into a 1.5 mL centrifuge tube for protein concentration detection:
先將Bio-rad染劑與去離子水混合(體積比為1:4),分裝500 μL至微量離心管中。而後,分別於前述微量離心管中,加入10、8、6、4、2、1與0 μL 2mg/mL的BSA以製備標準濃度蛋白質。再加入2 μL測試樣本進行檢測。取出前述反應後之測試樣本200 μL於96孔盤中,以ELISA讀取儀(購自BioTek)測定595 nm的吸光值。First, mix Bio-rad stain with deionized water (1:4 by volume), and dispense 500 μL into microcentrifuge tubes. Then, 10, 8, 6, 4, 2, 1, and 0 μL of 2 mg/mL BSA were added to the aforementioned microcentrifuge tubes to prepare standard protein concentration. Add another 2 μL of test sample for detection. 200 μL of the test sample after the aforementioned reaction was taken out in a 96-well plate, and the absorbance at 595 nm was measured with an ELISA reader (purchased from BioTek).
接著,於每孔中加入400 μg蛋白質,再加入90 μL細胞裂解液,包括控制組及對照組。在避光環境下,在37°C下加入10 μL10M的L-Dopa,每10分鐘觀察一次,直到懸浮液變黑。之後再測定405 nm的吸光值。Next, 400 μg of protein was added to each well, followed by 90 μL of cell lysate, including control group and control group. In the dark, add 10 μL of 10 M L-Dopa at 37 °C and observe every 10 min until the suspension turns black. The absorbance at 405 nm was then measured.
酪胺酸酶含量的分析公式為:酪胺酸酶抑制性(%)=(樣本吸光值/控制組吸光值)×100%。所得數值再以微軟EXCEL軟體,利用Student t檢定進行統計分析,其結果如圖3所示。The analysis formula of tyrosinase content is: tyrosinase inhibition (%)=(sample absorbance/control group absorbance)×100%. The obtained values were then statistically analyzed with Microsoft EXCEL software and Student's t test, and the results are shown in Figure 3.
由圖3之結果可知,在本發明之白桑葚樣品的處理下,可達到抑制酪胺酸酶活性的功效。具體而言,對照組相對控制組之酪胺酸酶活性為69.87%,實驗組A(白桑葚水萃取物)相對控制組之酪胺酸酶活性為86.44%,而實驗組B(白桑葚發酵物)相對控制組之酪胺酸酶活性為74.13%。實驗組A與控制組相比酪胺酸酶活性降低約13.56%,實驗組B與控制組相比酪胺酸酶活性降低約25.87%,由此可知,實驗組B(白桑葚發酵物)之酪胺酸酶抑制能力又高於實驗組A(白桑葚水萃物),且本案白桑葚發酵物之功效近似於麴酸之抑制酪胺酸酶活性之效果。因此,藉由本發明實施例所製備之白桑葚發酵物,確實可降低酪胺酸酶活性,進而可減少黑色素的生成,故可應用於減少皮膚黑斑、提升肌膚光澤、白皙度肌膚美白的相關組合物的成分中。It can be seen from the results in FIG. 3 that the effect of inhibiting the activity of tyrosinase can be achieved under the treatment of the white mulberry sample of the present invention. Specifically, the tyrosinase activity of the control group relative to the control group was 69.87%, the tyrosinase activity of the experimental group A (white mulberry water extract) relative to the control group was 86.44%, and the experimental group B (white mulberry fermentation The tyrosinase activity relative to the control group was 74.13%. Compared with the control group, the tyrosinase activity of the experimental group A decreased by about 13.56%, and the tyrosinase activity of the experimental group B was decreased by about 25.87% compared with the control group. The tyrosinase inhibitory ability was higher than that of experimental group A (white mulberry water extract), and the efficacy of the white mulberry fermented product in this case was similar to that of kojic acid in inhibiting tyrosinase activity. Therefore, the white mulberry fermented product prepared by the embodiment of the present invention can indeed reduce the activity of tyrosinase, thereby reducing the generation of melanin, so it can be applied to reduce skin dark spots, improve skin luster, whiteness and skin whitening. components of the composition.
實施例五、黑色素生成檢測Embodiment 5. Detection of melanin production
於此,以ELISA讀盤機(enzyme-linked immunosorbent assay reader)測定黑色素瘤細胞株B16F10經白桑葚發酵物處理後,其黑色素含量的變化。Here, an enzyme-linked immunosorbent assay reader was used to measure the change of the melanin content of the melanoma cell line B16F10 after being treated with the white mulberry fermentation product.
材料與儀器 1. 細胞株:小鼠黑色素瘤細胞B16F10,購自美國典型培養物保存中心(American Type CμLtureCollection,ATCC®,No.6475),以下簡稱B16F10細胞。 2. 培養基:將DMEM (Dulbecco's modified minimal essential medium,購自Gibco,Cat. 12100-038)添加額外成分使其含有1 vol%的抗生素溶液(Antibiotic Antimycotic Solution,購自Gibco,15240-062)及10 vol%的FBS(fetal bovine Serum,購自Gibco,10437-028)。 3. 磷酸緩衝鹽溶液(PBS溶液):購自Gibco,產品編號10437-028。 4. 以二次蒸餾水配製1N NaOH(購自Sigma,產品編號221465)溶液。 5. 酵素免疫分析儀(ELISA reader,購自BioTek,產品編號FLx 800)。 6. 白桑葚水萃取物及白桑葚發酵物:由本案實施例一之製備方式所製得。Materials and Instruments 1. Cell line: mouse melanoma cell B16F10, purchased from American Type Culture Collection (American Type CμLture Collection, ATCC®, No. 6475), hereinafter referred to as B16F10 cell. 2. Medium: DMEM (Dulbecco's modified minimal essential medium, available from Gibco, Cat. 12100-038) was added with additional components to contain 1 vol% of an antibiotic solution (Antibiotic Antimycotic Solution, available from Gibco, 15240-062) and 10 vol% of FBS (fetal bovine Serum, available from Gibco, 10437-028). 3. Phosphate buffered saline solution (PBS solution): purchased from Gibco, product number 10437-028. 4. A solution of 1N NaOH (purchased from Sigma, product number 221465) was prepared in double distilled water. 5. Enzyme immunoassay analyzer (ELISA reader, purchased from BioTek, product number FLx 800). 6. White mulberry water extract and white mulberry fermented product: prepared by the preparation method of Example 1 of this case.
實驗步驟
實驗將會分為實驗組A、實驗組B、控制組及對照組四組進行,各組分別進行三重複試驗:
1.將B16F10細胞以每孔1.5×105
個的方式,接種於每孔含3ml培養基之6孔培養盤中。
2.將培養盤置於5%CO2
、37℃環境下,培養24小時。
3.而後,在不干擾附著細胞的情況下,移除每孔之培養基。
4.本實驗分為控制組、對照組、實驗組A及實驗組B。其中,各組加入2mL的新鮮DMEM培養液。而對照組之培養液額外加入麴酸(Kojic acid),使其濃度為0.5 mg/mL,其中麴酸已被廣泛認知為具有降低黑色素生成效果之物質;實驗組A之培養液額外加入白桑葚水萃取物,使其濃度為0.5 mg/mL;實驗組B之培養液額外加入白桑葚發酵物,使其濃度為0.5 mg/mL。各組進行三重複,並使其於37℃下培養24小時。
5.將反應完畢之實驗組A、實驗組B及對照組移至藍光箱中,使其在室溫(25±5℃)下接受藍光照射3小時。另外,控制組移至暗室中,使其在室溫下靜置3小時。
6.反應後,將細胞於37℃下培養48小時。
7.而後,移除培養基,並以PBS溶液清洗細胞2次。
8.將200μl胰蛋白酶(Trypsin-EDTA (10X),購自Gibco;產品編號15400-054)加至每孔中反應3分鐘。反應後,添加6 mL培養基溶液終止反應。而後收集各孔中之懸浮細胞與培養基至對應的15ml離心試管內,將各離心試管以400 xg離心5分鐘使細胞沉澱。
9.以PBS溶液清洗沉澱細胞二次後,再以200μL PBS溶液重新懸浮細胞。
10.將細胞懸浮液以液態氮冷凍10分鐘,再置於室溫約30分鐘至完全解凍。
11.完全解凍後,將試管以12,000g離心3分鐘。
12.移除上清液,再以120μL 1N氫氧化鈉溶液重新懸浮細胞沉澱後,使試管於60℃乾浴1小時,以獲得待檢測樣本。
13.將100μL待檢測樣本移入一96孔盤,使用ELISA讀盤機測量細胞溶液在450nm的吸光值。Experimental Procedures The experiment will be divided into four groups: experimental group A, experimental group B, control group and control group. Each group will carry out three repeated experiments: 1. B16F10 cells are inoculated in 1.5×10 5 cells per well. in 6-well plates containing 3 ml of medium per well. 2. Place the culture plate in an environment of 5% CO 2 and 37° C. for 24 hours. 3. Then, without disturbing the attached cells, remove the medium from each well. 4. This experiment is divided into control group, control group, experimental group A and experimental group B. Among them, 2 mL of fresh DMEM medium was added to each group. The culture medium of the control group was additionally added with kojic acid to a concentration of 0.5 mg/mL, of which kojic acid has been widely recognized as a substance that has the effect of reducing the production of melanin; the culture medium of the experimental group A was additionally added with white mulberry The water extract was added to make the concentration of 0.5 mg/mL; the culture medium of experimental group B was additionally added with white mulberry fermentation product to make the concentration of 0.5 mg/mL. Groups were replicated in triplicate and incubated at 37°C for 24 hours. 5. The experimental group A, the experimental group B and the control group after the reaction were moved to a blue light box, and subjected to blue light irradiation at room temperature (25±5°C) for 3 hours. Additionally, the control group was moved to a dark room and allowed to stand at room temperature for 3 hours. 6. After the reaction, the cells were incubated at 37°C for 48 hours. 7. Afterwards, the medium was removed and the cells were washed twice with PBS solution. 8. Add 200 μl of trypsin (Trypsin-EDTA (10X), purchased from Gibco; product number 15400-054) to each well for 3 minutes of reaction. After the reaction, 6 mL of medium solution was added to stop the reaction. Then, the suspended cells and medium in each well were collected into corresponding 15ml centrifuge tubes, and each centrifuge tube was centrifuged at 400 xg for 5 minutes to pellet the cells. 9. After washing the pelleted cells twice with PBS solution, resuspend the cells with 200 μL PBS solution. 10. Freeze the cell suspension in liquid nitrogen for 10 minutes and allow to thaw completely at room temperature for about 30 minutes. 11. After complete thawing, centrifuge the tube at 12,000 g for 3 minutes. 12. Remove the supernatant, resuspend the cell pellet with 120 μL of 1N sodium hydroxide solution, and dry the test tube at 60° C. for 1 hour to obtain the sample to be tested. 13.
實驗結果Experimental results
實驗組以及控制組的黑色素相對表現量係依下列公式計算:黑色素相對表現量(%)=(各組OD450 值/控制組OD450 值)×100%。因實驗係進行三重複,故將三重複實驗之結果平均後,顯示於圖4。The relative expression of melanin in the experimental group and the control group was calculated according to the following formula: relative expression of melanin (%)=(OD 450 value of each group/OD 450 value of control group)×100%. Since the experiment was performed in triplicate, the results of the triplicate experiments were averaged and shown in Figure 4 .
如圖4所示,在本發明之白桑葚樣品的處理下,可達到降低黑色素生成的功效。具體而言,對照組相對控制組之黑色素生成量為83.9%,實驗組A(白桑葚水萃取物)相對控制組之黑色素生成量為93.54%,而實驗組B(白桑葚發酵物)相對控制組之黑色素生成量為86.05%,實驗組A與控制組相比黑色素生成降低約6.46%,實驗組B與控制組相比黑色素生成降低約13.95%,且本案白桑葚發酵物之功效近似於麴酸之降低黑色素生成之效果。因此,本發明實施例所製備之白桑葚發酵物,確實可有效減少黑色素細胞所生成之黑色素,故可應用於減少皮膚黑斑、提升肌膚光澤、白皙度肌膚美白的相關組合物的成分中。As shown in FIG. 4 , under the treatment of the white mulberry sample of the present invention, the effect of reducing the production of melanin can be achieved. Specifically, the melanin production of the control group relative to the control group was 83.9%, the melanin production of experimental group A (white mulberry water extract) relative to the control group was 93.54%, and the experimental group B (white mulberry fermented product) was relatively control The melanin production of the group was 86.05%. Compared with the control group, the production of melanin in the experimental group A was reduced by about 6.46%, and the production of melanin in the experimental group B was reduced by about 13.95% compared with the control group. The effect of acid to reduce melanin production. Therefore, the white mulberry fermented product prepared in the embodiment of the present invention can indeed effectively reduce the melanin produced by melanocytes, so it can be used as an ingredient in a related composition for reducing skin dark spots, enhancing skin luster, and whitening skin.
實施例六、皮膚細胞粒線體活性實驗Example 6. Skin cell mitochondrial activity experiment
為探討白桑葚發酵物對皮膚細胞粒線體功能的影響,本實施例以流式細胞儀評估人類皮膚纖維母細胞CCD-966sk經白桑葚發酵物處理後,其粒線體活性的變化。本實驗以JC-1聚集能力作為粒線體活性之指標,在膜電位正常的健康粒線體中,JC-1以多聚體(polymer)形式存在,而當粒線體膜電位降低時,JC-1會變成單體(monomer),其可當作粒線體活性評估的指標。In order to explore the effect of white mulberry fermentation product on the mitochondrial function of skin cells, flow cytometry was used to evaluate the changes in mitochondrial activity of human skin fibroblasts CCD-966sk treated with white mulberry fermentation product. In this experiment, the aggregation ability of JC-1 was used as an indicator of mitochondrial activity. In healthy mitochondria with normal membrane potential, JC-1 exists in the form of a polymer, and when the mitochondrial membrane potential decreases, JC-1 exists in the form of a polymer. JC-1 becomes a monomer, which can be used as an indicator for the assessment of mitochondrial activity.
材料與儀器 1. 細胞株:人類皮膚纖維母細胞CCD-966sk(BCRC No. 60153)。 2. 細胞培養基:含有10 vol% FBS(購自Gibco)之基礎培養基。其中,基礎培養基是由Eagle’s最低限度基本培養基(Eagle’s minimum essential medium(MEM),購自Gibco,產品編號15188-319)額外添加成分使其含有1 mM 丙酮酸鈉(sodium pyruvate,購自Gibco)、1.5 g/L 碳酸氫鈉(sodium bicarbonate,購自Sigma)及0.1 mM 非必需胺基酸(non-essential amino acid solution,購自Gibco)所配製而成。 3. 磷酸緩衝鹽溶液(PBS溶液):購自Gibco。 4. 粒線體膜電位偵測套組(BDTM MitoScreen(JC-1) kit,型號551302)。其中,粒線體膜電位偵測套組具有JC-1染料(凍乾)及10X分析緩衝液。於使用前,以1X PBS將10X分析緩衝液稀釋10倍,以形成1X分析緩衝液。加入130 μL DMSO至JC-1染劑(凍乾)中,以形成JC-1儲備溶液。然後,再以1X分析緩衝液稀釋JC-1儲備溶液,以形成JC-1工作試劑(JC-1 working solution;即JC-1粒線體特異性染劑)。稀釋倍率為JC-1儲備溶液比1X分析緩衝液為1:100。 5. 胰蛋白酶:10X Trypsin-EDTA(購自Gibco)以1X PBS溶液稀釋10倍。 6. 流式細胞儀:購自BD Pharmingen公司,型號BDTM Accuri C6 Plus。 7. 白桑葚水萃取物及白桑葚發酵物:由本案實施例一之製備方式所製得。Materials and Instruments 1. Cell Line: Human skin fibroblasts CCD-966sk (BCRC No. 60153). 2. Cell culture medium: basal medium containing 10 vol% FBS (purchased from Gibco). Among them, the basal medium is made of Eagle's minimum essential medium (MEM), purchased from Gibco, product number 15188-319) with additional components to make it contain 1 mM sodium pyruvate (sodium pyruvate, purchased from Gibco), 1.5 g/L sodium bicarbonate (purchased from Sigma) and 0.1 mM non-essential amino acid solution (purchased from Gibco) were prepared. 3. Phosphate buffered saline solution (PBS solution): purchased from Gibco. 4. Mitochondrial membrane potential detection kit (BD TM MitoScreen(JC-1) kit, model 551302). Among them, the mitochondrial membrane potential detection kit has JC-1 dye (lyophilized) and 10X analysis buffer. 10X Assay Buffer was diluted 10-fold in IX PBS prior to use to form IX Assay Buffer. 130 μL of DMSO was added to JC-1 stain (lyophilized) to form a JC-1 stock solution. The JC-1 stock solution was then diluted with 1X assay buffer to form the JC-1 working solution (ie, JC-1 mitochondrial specific stain). The dilution ratio was 1:100 for JC-1 stock solution to 1X assay buffer. 5. Trypsin: 10X Trypsin-EDTA (purchased from Gibco) diluted 10 times in 1X PBS solution. 6. Flow cytometer: purchased from BD Pharmingen Company, model BDTM Accuri C6 Plus. 7. White mulberry water extract and white mulberry fermented product: prepared by the preparation method of Example 1 of this case.
實驗步驟 1. 將CCD-966sk細胞以每孔1×105 個的方式,接種於每孔含2 mL細胞培養基之6孔培養盤中。 2. 將培養盤的各孔中的培養基更換成2 mL實驗培養基。其中,實驗組A的實驗培養基為含有0.5 mg/mL的實施例一得到的白桑葚水萃取物。實驗組B的實驗培養基為含有0.5 mg/mL的實施例一得到的白桑葚發酵物之細胞培養基。控制組的實驗培養基為單純的細胞培養基(即不含白桑葚樣品)。 3. 將培養盤置於5%CO2 、37 ℃下,培養24小時。 4. 移除培養盤中的實驗培養基並以1 mL的1X PBS溶液潤洗2次。 5. 將200 μL胰蛋白酶加至每孔中並在暗處反應5分鐘。反應後,添加細胞培養基終止反應。將各孔中之細胞與細胞培養基收集至個別對應的1.5 mL離心管內,並將含有細胞與細胞培養基之離心管以400 xg離心10分鐘。 6. 於離心後移除上清液,再以1 mL的1X PBS溶液重新回溶或轉移細胞沉澱物至15 mL離心管以形成細胞懸浮液。 7. 將含有細胞懸浮液的離心管以400 xg離心5分鐘。 8. 於離心後移除各離心管中的上清液並加入100 μL的JC-1工作試劑至各離心管。 9. 將各離心管中的細胞沉澱物與JC-1工作試劑渦旋均勻並在避光處理下培養15分鐘。 10. 15分鐘後,將各離心管以400 xg離心5分鐘。 11. 於離心後,移除各離心管中的上清液、以1 mL的1X PBS溶液回溶各試管中的細胞沉澱物並以400 xg離心5分鐘,此步驟重複兩遍。 12. 於離心後,移除各離心管中的上清液、以500 μL的1X PBS重新懸浮細胞,以得到待測細胞液。 13. 以流式細胞儀量測各孔的待檢測細胞中細胞粒線體的膜電位,以進行粒線體活性分析。因實驗係進行三重複,故將各組的三重複實驗之結果平均以得平均值,然後以控制組的平均值為100%之相對JC-1聚集量,將實驗組的平均值換算為相對JC-1聚集量,如圖5所示。Experimental steps 1. CCD-966sk cells were seeded in a 6-well culture dish containing 2 mL of cell culture medium per well at 1×10 5 cells per well. 2. Replace the medium in each well of the plate with 2 mL of experimental medium. Wherein, the experimental medium of experimental group A was the white mulberry water extract obtained in Example 1 containing 0.5 mg/mL. The experimental medium of experimental group B was a cell culture medium containing 0.5 mg/mL of the white mulberry fermented product obtained in Example 1. The experimental medium of the control group was pure cell culture medium (ie, no white mulberry sample). 3. Place the culture plate in 5% CO 2 at 37°C for 24 hours. 4. Remove the experimental medium from the plate and rinse twice with 1 mL of 1X PBS solution. 5. Add 200 μL of trypsin to each well and react in the dark for 5 minutes. After the reaction, cell culture medium was added to terminate the reaction. Collect the cells and cell culture medium in each well into a corresponding 1.5 mL centrifuge tube, and centrifuge the centrifuge tube containing the cells and cell culture medium at 400 x g for 10 minutes. 6. After centrifugation, remove the supernatant and re-dissolve the cell pellet with 1 mL of 1X PBS or transfer the cell pellet to a 15 mL centrifuge tube to form a cell suspension. 7. Centrifuge the tube containing the cell suspension at 400 x g for 5 minutes. 8. After centrifugation, remove the supernatant from each centrifuge tube and add 100 μL of JC-1 working reagent to each centrifuge tube. 9. Vortex the cell pellet in each centrifuge tube with JC-1 working reagent and incubate in the dark for 15 minutes. 10. After 15 minutes, centrifuge each tube at 400 x g for 5 minutes. 11. After centrifugation, remove the supernatant from each centrifuge tube, re-dissolve the cell pellet in each tube with 1 mL of 1X PBS solution and centrifuge at 400 x g for 5 min. Repeat this step twice. 12. After centrifugation, remove the supernatant from each centrifuge tube and resuspend the cells with 500 μL of 1X PBS to obtain the cell solution to be tested. 13. Measure the mitochondrial membrane potential of the cells to be tested in each well by flow cytometry to analyze mitochondrial activity. Since the experiment was carried out in three repetitions, the results of the three repeated experiments in each group were averaged to obtain the average value, and then the average value of the control group was taken as 100% of the relative accumulation of JC-1, and the average value of the experimental group was converted into a relative value. The aggregated amount of JC-1 is shown in Figure 5.
實驗結果Experimental results
參閱圖5,其為經本發明白桑葚水萃取物及白桑葚發酵物處理之實驗組與未經處理之控制組之JC-1聚集相對量比較長條圖。由該圖可知,實驗組A之相對JC-1聚集量約為97.5%,然實驗組B之相對JC-1聚集量約為117.19%。換言之,與控制組相較之下,實驗組B的人類皮膚纖維母細胞的粒線體活性提升約17.19%。顯示白桑葚發酵物可提升皮膚細胞之粒線體活性,而達到提升皮膚細胞活性之效果。Referring to FIG. 5 , it is a bar graph comparing the relative amount of JC-1 aggregation in the experimental group treated with the white mulberry water extract and the white mulberry fermented product of the present invention and the untreated control group. It can be seen from the figure that the relative aggregate amount of JC-1 in experimental group A is about 97.5%, while the relative amount of JC-1 aggregate in experimental group B is about 117.19%. In other words, compared with the control group, the mitochondrial activity of human skin fibroblasts in experimental group B increased by about 17.19%. It is shown that the white mulberry fermented product can enhance the mitochondrial activity of skin cells and achieve the effect of enhancing the activity of skin cells.
實施例七、分泌玻尿酸能力檢測Embodiment 7. Detection of the ability to secrete hyaluronic acid
於此,以人類玻尿酸檢測套組配合酵素免疫分析儀(ELISA reader),測定人類初代皮膚角質細胞HPEK-50經白桑葚發酵物處理後,其細胞分泌玻尿酸的變化。Here, the human hyaluronic acid detection kit was used in conjunction with an ELISA reader to measure the changes in the secretion of hyaluronic acid from human primary skin keratinocytes HPEK-50 treated with white mulberry fermentation.
材料與儀器 1.細胞株:人類初代皮膚角質細胞(Human primary epidermal keratinocytes)HPEKp(CELLnTEC公司(瑞士),HPEK-50)。 2.培養基:無血清之角質細胞培養液(keratinocyte-SFM)(Gibco公司,美國,編號為#10724-011)。 3.人類玻尿酸檢測套組(Human Hyaluronic acid, HA ELISA Kit):購自Cusabio Biotech。 4.酵素免疫分析儀(ELISA reader),購自BioTek公司。 5.白桑葚水萃取物及白桑葚發酵物:由本案實施例一之製備方式所製得。Materials and Instruments 1. Cell line: Human primary epidermal keratinocytes (Human primary epidermal keratinocytes) HPEKp (CELLnTEC Company (Switzerland), HPEK-50). 2. Culture medium: serum-free keratinocyte-SFM (Gibco Company, USA, No. #10724-011). 3. Human Hyaluronic acid (HA ELISA Kit): purchased from Cusabio Biotech. 4. Enzyme immunoassay analyzer (ELISA reader), purchased from BioTek Company. 5. White mulberry water extract and white mulberry fermented product: prepared by the preparation method of Example 1 of this case.
實驗步驟Experimental procedure
實驗將會分為實驗組A、實驗組B、控制組(未添加白桑葚樣本)三組進行: 1. 將人類初代皮膚角質細胞以每孔1×104 個的密度,接種於每孔培養於每孔含2ml培養基之96孔培養盤中,並於實驗組A之培養液額外加入白桑葚水萃取物,使其濃度為0.5 mg/mL;於實驗組B之培養液額外加入白桑葚發酵物,使其濃度為0.5 mg/mL。各組進行三重複,並於37℃下,培養24小時。 2. 每孔取100μL的培養基,然後加到預塗(pre-coated)的ELISA盤中,於37°C下培養2小時。 3. 移除每個孔中的培養基,勿進行清洗。 4. 於每個孔中添加100μL的生物素抗體(Biotin-antibody),並於37°C下培養1小時。 5. 反應完畢後,移除每孔之培養基,並以洗滌緩衝液(Wash Buffer)200μL進行清洗,每次清洗靜置2分鐘,重複此步驟三次。最後一次清洗完成後,透過抽吸除去所有剩餘的洗滌緩衝液。(確認完全清除液體) 6. 向每個孔中加入100μL HRP-抗生物素蛋白(HRP-avidin),並在37°C下培養1小時。 7. 反應完畢後,重複步驟6之清洗方式。 8. 於每孔中添加90μL TMB底物(TMB Substrate),並37°C下避光反應15-30分鐘。 9. 於每孔中添加50μL終止液(Stop Solution),輕輕敲打培養盤以確保其充分混合。 10. 利用酵素免疫分析儀(ELISA reader)於5分鐘內測量每孔450nm之吸光值。 11. 所得結果以Excel軟體進行統計分析。The experiment will be divided into three groups: experimental group A, experimental group B, and control group (without the addition of white mulberry samples): 1. Inoculate human primary skin keratinocytes at a density of 1×10 4 per well into each well of culture In a 96-well culture plate containing 2 ml of medium in each well, the water extract of white mulberry was additionally added to the culture medium of experimental group A to make the concentration of 0.5 mg/mL; the culture medium of experimental group B was additionally added white mulberry fermentation substance to a concentration of 0.5 mg/mL. Each group was replicated in triplicate and incubated at 37°C for 24 hours. 2. Take 100 μL of medium per well, add it to a pre-coated ELISA plate, and incubate at 37°C for 2 hours. 3. Remove medium from each well without washing. 4. Add 100 μL of Biotin-antibody to each well and incubate at 37°C for 1 hour. 5. After the reaction is completed, remove the medium from each well and wash with 200 μL of Wash Buffer. Each wash is allowed to stand for 2 minutes, and this step is repeated three times. After the last wash, remove all remaining wash buffer by aspiration. (Confirm complete removal of liquid) 6. Add 100 μL of HRP-avidin to each well and incubate at 37°C for 1 hour. 7. After the reaction is completed, repeat the cleaning method in step 6. 8. Add 90 μL of TMB Substrate to each well and incubate at 37°C for 15-30 minutes in the dark. 9. Add 50 μL of Stop Solution to each well and tap the plate lightly to ensure adequate mixing. 10. Use an ELISA reader to measure the absorbance at 450 nm in each well within 5 minutes. 11. Statistical analysis of the obtained results was performed with Excel software.
實驗結果:Experimental results:
圖6是經本發明白桑葚水萃取物或白桑葚發酵物處理後,角質細胞分泌玻尿酸的結果圖。具體而言,實驗組A之相對玻尿酸分泌約為130%,然實驗組B之相對玻尿酸分泌約為170%。換言之,與控制組相較之下,實驗組A及實驗組B的相對玻尿酸分泌率皆有提升,其中,實驗組A(白桑葚水萃取物)之相對玻尿酸分泌與控制組相比提升30%,而實驗組B(白桑葚發酵物)之相對玻尿酸分泌與控制組相比提升70%,由此可知,白桑葚發酵物之提升玻尿酸分泌的能力相較白桑葚水萃取物優異。本實施例的結果顯示,本發明白桑葚發酵物具有促進玻尿酸分泌的功效,藉此有益於保留皮膚膠原蛋白、增加皮膚含水量,並提供皮膚彈性和柔韌性。FIG. 6 is a graph showing the results of keratinocytes secreting hyaluronic acid after being treated with the white mulberry water extract or the white mulberry fermented product of the present invention. Specifically, the relative hyaluronic acid secretion of experimental group A was about 130%, while the relative hyaluronic acid secretion of experimental group B was about 170%. In other words, compared with the control group, the relative hyaluronic acid secretion rates of both the experimental group A and the experimental group B increased. Among them, the relative hyaluronic acid secretion of the experimental group A (white mulberry water extract) increased by 30% compared with the control group Compared with the control group, the relative hyaluronic acid secretion of experimental group B (white mulberry fermented product) increased by 70%. It can be seen that the ability of white mulberry fermented product to increase hyaluronic acid secretion is better than that of white mulberry water extract. The results of this example show that the white mulberry fermented product of the present invention has the effect of promoting the secretion of hyaluronic acid, thereby helping to retain skin collagen, increase skin water content, and provide skin elasticity and flexibility.
玻尿酸可以防止皮膚自然老化,並保護皮膚免於太陽紫外線、煙草煙霧和空氣中污染物質之危害。玻尿酸亦具有幫助增加皮膚水份,使肌膚結構緊密澎潤,以減少皮膚細紋和皺紋的出現。另外,玻尿酸在傷口癒合中也起著關鍵作用,當皮膚細胞需要修復或受損時其濃度亦會增加,且其應用於皮膚傷口已被證明可以減輕傷口的大小且減緩疼痛,亦可幫助降低傷口細胞感染風險。Hyaluronic acid prevents natural aging of the skin and protects the skin from the sun's UV rays, tobacco smoke and air pollutants. Hyaluronic acid also helps increase skin moisture and tightens the skin structure to reduce the appearance of fine lines and wrinkles. In addition, hyaluronic acid also plays a key role in wound healing. Its concentration increases when skin cells need to be repaired or damaged, and its application to skin wounds has been shown to reduce wound size and pain, and can also help reduce Risk of wound cell infection.
另外,玻尿酸對於骨關節炎亦具有幫助,實驗證實每天食用80-200毫克玻尿酸至少兩個月,即可顯著減輕骨關節炎患者的膝關節疼痛,亦有助於減輕胃酸反流症狀,而玻尿酸具有出色的保濕性,其亦常用於治療乾眼症、減緩骨質疏鬆及減輕膀胱疼痛症候群等諸多功效。In addition, hyaluronic acid is also helpful for osteoarthritis. Experiments have shown that consuming 80-200 mg of hyaluronic acid every day for at least two months can significantly reduce knee pain in patients with osteoarthritis, and also help reduce gastric acid reflux symptoms. With excellent moisturizing properties, it is also commonly used in the treatment of dry eyes, slowing osteoporosis and reducing bladder pain syndrome.
實施例八、抑制脂肪堆積功效試驗Embodiment 8. Test on the efficacy of inhibiting fat accumulation
脂肪細胞內以油滴(Lipid droplet)的形式貯存脂肪。基此,本次試驗分析染色後的油滴,以觀察細胞內油滴的數量,藉以確認脂肪堆積的狀態。後續,再將染劑溶出並分析以作為量化的數值指標。Fat is stored in fat cells in the form of lipid droplets. Based on this, the dyed oil droplets were analyzed in this experiment to observe the number of oil droplets in the cells, thereby confirming the state of fat accumulation. Subsequently, the dye was dissolved and analyzed as a quantitative numerical indicator.
本次試驗採用小鼠骨髓基質細胞(後續簡稱OP9細胞),OP9細胞購自美國典型培養物保存中心(American Type Culture Collection,ATCC® )之OP9細胞株(ATCC CRL-2749)。Mouse bone marrow stromal cells (hereinafter referred to as OP9 cells) were used in this experiment, and OP9 cells were purchased from the OP9 cell line (ATCC CRL-2749) of the American Type Culture Collection (ATCC ® ).
首先,取24孔培養盤將每孔接種8×104
個OP9細胞及500μL培養基(Medium),該培養基為MEMAM(Minimum Essential Medium Alpha Medium,購自Gibco,產品編號Cat. 12000-022)細胞培養液加入20%之胎牛血清(Fetal Bovine Serum,購自Gibco,產品編號Cat.10437-028)及0.1%之青黴素/鏈黴素(Penicillin-streptomycin,購自Gibco,產品編號Cat.15240-062),在37℃下培養7天。此7天的細胞培養期間每隔3天更換培養基。7天後,以顯微鏡(ZEISS;放大倍率400x)觀察細胞內油滴形成,藉以確認細胞已完全分化為脂肪細胞。First, take a 24-well culture plate and inoculate each well with 8×10 4 OP9 cells and 500 μL of medium (Medium), which is MEMAM (Minimum Essential Medium Alpha Medium, purchased from Gibco, product number Cat. 12000-022)
然後,將分化完成的脂肪細胞分為二組:實驗組A、實驗組B與控制組。Then, the differentiated adipocytes were divided into two groups: experimental group A, experimental group B and control group.
實驗組:依照每孔500μL培養基含有125μg的由本案實施例一之方式製備而成的白桑葚水萃取物(實驗組A)或白桑葚發酵物(實驗組B),使其濃度為0.25 mg/mL,並在37℃下培養7天。在7天的細胞處理期間每隔3天更換培養基。Experimental group: 500 μL medium per well contains 125 μg of white mulberry water extract (experimental group A) or white mulberry fermented product (experimental group B) prepared by the method of Example 1 of this case, and its concentration is 0.25 mg/ mL and incubated at 37°C for 7 days. The medium was changed every 3 days during the 7 days of cell treatment.
控制組:不作任何處理,即不額外添加其他化合物至含分化後的脂肪細胞的分化培養基中,在37℃下培養7天。此7天的細胞處理期間每隔3天更換培養基。Control group: without any treatment, that is, without additionally adding other compounds to the differentiation medium containing differentiated adipocytes, and cultured at 37° C. for 7 days. The medium was changed every 3 days during this 7-day cell treatment period.
接下來,依據下列步驟進行油紅O的染色。於7天細胞處理後,將培養基移除,以1mL之磷酸鹽緩衝溶液(Phosphate buffered saline, PBS)清洗脂肪細胞兩次,再加入1mL之10%甲醛並於室溫下反應30分鐘以固定脂肪細胞。接著移除甲醛後以1mL之PBS輕輕地清洗脂肪細胞兩次,接著於每孔細胞內加入1mL之60%異丙醇,反應1分鐘後,移除異丙醇並加入1mL之油紅O作用溶液與脂肪細胞反應,於室溫下反應1小時,接著移除與脂肪細胞作用的油紅O作用溶液並迅速地以1mL之60%異丙醇進將脂肪細胞行脫色5秒鐘,再使用顯微鏡觀察並拍攝細胞。Next, oil red O staining was performed according to the following procedure. After 7 days of cell treatment, the medium was removed, the adipocytes were washed twice with 1 mL of Phosphate buffered saline (PBS), and then 1 mL of 10% formaldehyde was added and reacted at room temperature for 30 minutes to fix the fat. cell. After removing the formaldehyde, the adipocytes were gently washed twice with 1 mL of PBS, and then 1 mL of 60% isopropanol was added to each well of cells. After 1 minute of reaction, the isopropanol was removed and 1 mL of Oil Red O was added. The action solution reacted with adipocytes, reacted at room temperature for 1 hour, then removed the oil red O action solution that acted on adipocytes and quickly decolorized the adipocytes with 1 mL of 60% isopropanol for 5 seconds. Use a microscope to observe and photograph the cells.
後續,將染色後的各組再依下列步驟進行油紅O的定量。加入100%異丙醇於染色之細胞中,並置於振盪器上反應10分鐘以溶解油滴,接著取100μL至96孔培養盤中,以測量ELISA讀取儀(BioTek)讀取各組之OD510nm 讀值。其中,利用Excel軟體進行student t-test以決定兩個樣本群體之間是否在統計上具有顯著差異,如圖7所示(圖式中「*」代表p值小於0.05,「**」代表p值小於0.01,以及「***」代表p值小於0.001。當「*」越多時,代表統計上的差異越顯著)。Subsequently, the dyed groups were then quantified for Oil Red O according to the following steps. Add 100% isopropanol to the stained cells, and place on a shaker to react for 10 minutes to dissolve the oil droplets, then take 100 μL into a 96-well culture dish to measure the OD of each group with an ELISA reader (BioTek). 510nm reading. Among them, use Excel software to perform student t-test to determine whether there is a statistically significant difference between the two sample groups, as shown in Figure 7 ("*" in the diagram represents p value less than 0.05, "**" represents p Values less than 0.01, and "***" represent p-values less than 0.001. The more "*", the more statistically significant the difference).
參考圖7,將控制組的脂肪油滴含量視為1(即100%)時,實驗組B的脂肪油滴含量為82.6%,意即經由白桑葚發酵物處理之後能顯著地減少約17.4%的脂肪堆積量。此結果顯示,白桑葚發酵物能有效阻斷脂肪細胞的增大,以減少脂肪細胞中油滴的含量,而達到抑制脂肪堆積之功效。Referring to Figure 7, when the fatty oil droplet content of the control group is regarded as 1 (i.e. 100%), the fatty oil droplet content of the experimental group B is 82.6%, which means that it can be significantly reduced by about 17.4% after being treated with the white mulberry fermented product. of fat accumulation. The results show that the white mulberry fermented product can effectively block the enlargement of adipocytes, so as to reduce the content of oil droplets in adipocytes, and achieve the effect of inhibiting fat accumulation.
實施例九、白桑葚酵素飲之皮膚改善功效試驗Embodiment 9. Skin improvement efficacy test of white mulberry enzyme drink
令10位肌膚較暗沉、鬆垮不緊緻之受試者每日飲用一瓶50 mL白桑葚酵素飲(其含有12vol%實施例一中所得到的白桑葚發酵物與88vol%水),連續飲用4週。並且,於飲用前(即第0週)及飲用4週後(即第4週),以VISIA肌膚檢測儀(VISIA Complexion Analysis System,購自美國Canfield公司)及皮膚表面濕度測試儀(Corneometer CM825,購自德國C+K公司)檢測肌膚狀況。於此,以VISIA肌膚檢測儀量測肌膚紋理、肌膚紫外線色斑、肌膚皺紋、肌膚彈性及膠原蛋白之改變情況,並且以皮膚表面濕度測試儀量測肌膚含水量。Make 10 subjects with dull skin, loose and not firm, drink a bottle of 50 mL white mulberry enzyme drink (which contains 12 vol% of the white mulberry fermented product obtained in Example 1 and 88 vol% of water) every day, Drink continuously for 4 weeks. And, before drinking (that is, the 0th week) and after 4 weeks of drinking (that is, the 4th week), use a VISIA skin detector (VISIA Complexion Analysis System, purchased from Canfield, USA) and a skin surface humidity tester (Corneometer CM825, Purchased from C+K Company, Germany) to detect skin condition. Here, the VISIA skin tester was used to measure the changes of skin texture, skin UV spots, skin wrinkles, skin elasticity and collagen, and the skin moisture content was measured by the skin surface humidity tester.
請參閱圖8。該圖係受試者飲用含本發明之白桑葚發酵物之酵素飲後之紫外線色斑之平均改善百分比。其中,當受試者於飲用前之平均紫外線色斑含量為100%時,飲用4週後之平均紫外線色斑含量為93.3%。換言之,受試者飲用含本發明之白桑葚發酵物之酵素飲4週後,平均紫外線色斑含量減少6.7%。由此可見,本發明之白桑葚發酵物具有降低紫外線色斑生成之功效,以達到淡斑之效果。See Figure 8. The graph shows the average improvement percentage of ultraviolet pigmentation after subjects drank the enzyme drink containing the white mulberry fermented product of the present invention. Among them, when the average content of UV spots before drinking was 100%, the average content of UV spots after 4 weeks of drinking was 93.3%. In other words, after the subjects drank the enzyme drink containing the white mulberry fermented product of the present invention for 4 weeks, the average UV-stained content was reduced by 6.7%. Thus, it can be seen that the white mulberry fermented product of the present invention has the effect of reducing the generation of ultraviolet stains, so as to achieve the effect of lightening.
請參閱圖9。該圖係受試者飲用含本發明之白桑葚發酵物之酵素飲後之肌膚含水量之平均改善百分比。其中,當受試者於飲用前之平均肌膚含水量為100%時,飲用4週後之平均肌膚含水量為120.2%。換言之,受試者飲用含本發明之白桑葚發酵物之酵素飲4週後,平均肌膚含水量提升20.2%,達統計上之顯著(「***」符號代表與飲用前相比p值小於0.001)。由此可見,本發明之白桑葚發酵物具有提升肌膚含水量,鎖住肌膚水分,以及提升肌膚保濕效果之功能。See Figure 9. The graph is the average percentage improvement of skin moisture content after subjects drink the enzyme drink containing the white mulberry fermented product of the present invention. Among them, when the average skin moisture content of the subjects before drinking was 100%, the average skin moisture content after drinking for 4 weeks was 120.2%. In other words, after the subjects drank the enzyme drink containing the white mulberry fermented product of the present invention for 4 weeks, the average skin water content increased by 20.2%, which was statistically significant (the "***" symbol represents that the p value is less than that before drinking 0.001). It can be seen that the white mulberry fermented product of the present invention has the functions of increasing the moisture content of the skin, locking the moisture in the skin, and enhancing the moisturizing effect of the skin.
請參閱圖10。該圖係受試者飲用含本發明之白桑葚發酵物之酵素飲後之肌膚彈力之平均改善百分比。其中,當受試者於飲用前之平均肌膚彈力為100%時,飲用4週後之平均肌膚彈力為104%。換言之,受試者飲用含本發明之白桑葚發酵物之酵素飲4週後,平均肌膚彈力提升4%,達統計上之顯著。由此可見,本發明之白桑葚發酵物具有提升肌膚彈力之功效,使肌膚更加有彈性。See Figure 10. The graph is the average percentage improvement of skin elasticity after subjects drink the enzyme drink containing the white mulberry fermented product of the present invention. Among them, when the average skin elasticity of the subjects before drinking was 100%, the average skin elasticity after drinking for 4 weeks was 104%. In other words, after the subjects drank the enzyme drink containing the white mulberry fermented product of the present invention for 4 weeks, the average skin elasticity increased by 4%, which was statistically significant. It can be seen that the white mulberry fermented product of the present invention has the effect of enhancing skin elasticity, making the skin more elastic.
請參閱圖11。該圖係受試者飲用含本發明之白桑葚發酵物之酵素飲後之肌膚膠原蛋白之平均改變百分比。其中,當受試者於飲用前之平均肌膚膠原蛋白含量為100%時,飲用4週後之平均肌膚膠原蛋白含量為115.5%。換言之,受試者飲用含本發明之白桑葚發酵物之酵素飲4週後,平均肌膚膠原蛋白含量提升15.5%,達統計上之顯著。由此可見,本發明之白桑葚發酵物具有提升肌膚膠原蛋白含量之功效,膠原蛋白與皮膚的彈性有關,能延緩皮膚的老化並減少皺紋、改善橘皮組織,以使肌膚更加有彈性。See Figure 11. The graph is the average percentage change of skin collagen after subjects drink the enzyme drink containing the white mulberry fermented product of the present invention. Among them, when the average skin collagen content of the subjects before drinking was 100%, the average skin collagen content after drinking for 4 weeks was 115.5%. In other words, after the subjects drank the enzyme drink containing the white mulberry fermented product of the present invention for 4 weeks, the average skin collagen content increased by 15.5%, which was statistically significant. It can be seen that the white mulberry fermented product of the present invention has the effect of increasing the content of collagen in the skin. Collagen is related to the elasticity of the skin, and can delay the aging of the skin, reduce wrinkles, improve cellulite, and make the skin more elastic.
參照圖8至圖11,相比飲用前(第0週),持續4週飲用白桑葚發酵飲可使肌膚紫外線色斑含量減少約6.7%,使肌膚含水量顯著提升約20.2%,使肌膚彈力提升約4%,以及使肌膚膠原蛋白含量顯著提升15.5%。由此可知,長期使用白桑葚發酵飲可改善肌膚狀況,即白桑葚發酵物具改善肌膚狀況之功效。Referring to Figure 8 to Figure 11, compared to before drinking (week 0), drinking white mulberry fermented drink for 4 weeks can reduce the content of UV spots on the skin by about 6.7%, significantly increase the water content of the skin by about 20.2%, and make the skin elastic Increases by about 4%, and significantly increases skin collagen content by 15.5%. It can be seen that long-term use of white mulberry fermented drink can improve skin condition, that is, white mulberry fermented product has the effect of improving skin condition.
綜上所述,根據本發明任一實施例的白桑葚發酵物,其可於製備改善肌膚狀況及/或抑制脂肪堆積的組合物。換言之,前述之組合物具有下列一種或多種功能:降低黑色素生成、提升皮膚纖維母細胞粒線體活性、提升玻尿酸分泌、降低發炎反應因子生成、抑制酪胺酸酶活性、減少紫外線色斑、提升肌膚含水量、提升肌膚彈力、促進膠原蛋白增生以及抑制脂肪堆積。To sum up, according to any embodiment of the white mulberry fermented product of the present invention, it can be used to prepare a composition for improving skin condition and/or inhibiting fat accumulation. In other words, the aforementioned composition has one or more of the following functions: reducing melanin production, enhancing mitochondrial activity of dermal fibroblasts, enhancing hyaluronic acid secretion, reducing inflammatory response factor production, inhibiting tyrosinase activity, reducing ultraviolet pigmentation, enhancing Skin moisture content, improve skin elasticity, promote collagen proliferation and inhibit fat accumulation.
無none
圖1係為本發明之實施例二的白桑葚水萃取物及白桑葚發酵物總多酚含量之長條圖。 圖2係為本發明之實施例三的實驗組與控制組之相對一氧化氮生成量比例圖。 圖3係為本發明之實施例四的實驗組與控制組之相對酪胺酸酶活性圖。 圖4係為本發明之實施例五的實驗組與控制組之相對黑色素生成比例圖。 圖5係為本發明之實施例六的實驗組與控制組之相對皮膚細胞粒線體活性圖。 圖6係為本發明之實施例七的實驗組與控制組之相對玻尿酸生成比例圖。 圖7係為本發明之實施例八的實驗組與控制組之相對脂肪油滴含量比例圖。 圖8係為本發明之實施例九的試驗者使用白桑葚發酵物飲4週後之相對紫外線色斑變化圖。 圖9係為本發明之實施例九的試驗者使用白桑葚發酵物飲4週後之相對肌膚含水量變化圖。 圖10係為本發明之實施例九的試驗者使用白桑葚發酵物飲4週後之相對肌膚彈力變化圖。 圖11係為本發明之實施例九的試驗者使用白桑葚發酵物飲4週後之相對膠原蛋白變化圖。FIG. 1 is a bar graph showing the total polyphenol content of the white mulberry water extract and the white mulberry fermented product according to Example 2 of the present invention. FIG. 2 is a graph showing the relative nitric oxide generation ratio between the experimental group and the control group according to the third embodiment of the present invention. FIG. 3 is a graph showing the relative tyrosinase activity of the experimental group and the control group in Example 4 of the present invention. FIG. 4 is a graph showing the relative melanin production ratio between the experimental group and the control group in Example 5 of the present invention. FIG. 5 is a graph showing the relative mitochondrial activity of skin cells in the experimental group and the control group in Example 6 of the present invention. 6 is a graph showing the relative production ratio of hyaluronic acid between the experimental group and the control group in Example 7 of the present invention. FIG. 7 is a graph showing the relative proportion of fatty oil droplets in the experimental group and the control group in Example 8 of the present invention. FIG. 8 is a graph showing the change of relative ultraviolet light spots after the experimenter in Example 9 of the present invention used the white mulberry fermented drink for 4 weeks. FIG. 9 is a graph showing the change of relative skin water content after the experimenter in Example 9 of the present invention consumed the white mulberry fermented drink for 4 weeks. Fig. 10 is a graph showing the relative skin elasticity change of the experimenter in Example 9 of the present invention after using the white mulberry fermented drink for 4 weeks. Fig. 11 is a graph showing the relative changes in collagen after the experimenter in Example 9 of the present invention consumed the white mulberry fermented drink for 4 weeks.
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