TWI830382B

TWI830382B - Prunus salicina ferment, manufacturing method thereof, and uses thereof

Info

Publication number: TWI830382B
Application number: TW111135002A
Authority: TW
Inventors: 林詠翔; 賴柏穎; 林煥祐
Original assignee: 大江生醫股份有限公司
Priority date: 2021-09-15
Filing date: 2022-09-15
Publication date: 2024-01-21
Also published as: CN115804801A; CN115804739A; TW202313087A; TW202313082A

Abstract

A plum（ Prunus salicina） ferment is obtained by anaerobic fermenting what, being extracted with solvent from a plum. Furthermore, the plum ferment is further used for preparing a composition for whitening, enhancing mitochondrial activity, reducing skin spots.

Description

秋姬李發酵物、其製作方法和其用途Akihime plum fermented product, its preparation method and its uses

本發明關於一種秋姬李發酵物，特別是涉及一種秋姬李發酵物、其製作方法和其用於製備美白、提升粒線體活性及減少肌膚斑的用途。The present invention relates to a fermented product of Prunus prune, in particular to a fermented product of Prunus prune, its preparation method and its use for whitening, increasing mitochondrial activity and reducing skin spots.

自有機及/或天然的飲食概念興起後，生技公司及食品業者積極投入關於天然植物的相關產品的研發。為使植物相關產品對身體健康助益有科學驗證的基礎，植物的活性成分分析及功效評估成為產品開發的重點項目。Since the rise of the concept of organic and/or natural diet, biotechnology companies and food industry have actively invested in the research and development of natural plant-related products. In order to provide a basis for scientific verification of the benefits of plant-related products to human health, the analysis of active ingredients and efficacy evaluation of plants have become key items in product development.

被譽為日本李王品種的秋姬李（ Prunus salicina），是由日本山梨縣所孕育出的新品種。秋姬李果實碩大，果皮鮮濃紅潤，果肉肥厚細密，味道濃甜且香氣濃郁。並且，因其透紅的外型，更被封為「日本李姬」。因此，生技公司及食品業者積極研究秋姬李的活性成分分析及功效評估並據以開發成為相關產品。 Prunus salicina , known as the king plum variety in Japan, is a new variety bred in Yamanashi Prefecture, Japan. The fruit of Qiuji plum is huge, with fresh, thick and red skin, thick and fine flesh, sweet taste and rich aroma. Moreover, because of her rosy appearance, she was even dubbed the "Japanese Princess". Therefore, biotechnology companies and food industry companies are actively studying the analysis and efficacy evaluation of Qiu Ji Li's active ingredients and developing related products accordingly.

在一些實施例中，一種秋姬李（ Prunus salicina）發酵物是由一秋姬李經一溶劑萃取後再經由一厭氧發酵而得。 In some embodiments, a Prunus salicina fermentation product is obtained by extracting Prunus salicina with a solvent and then undergoing anaerobic fermentation.

在一些實施例中，此厭氧發酵係以酵母菌及/或乳酸菌進行厭氧發酵。In some embodiments, the anaerobic fermentation is performed using yeast and/or lactic acid bacteria.

在一些實施例中，一種秋姬李發酵物用於製備美白的組合物的用途。In some embodiments, a fermented product of Prunus prunus is used to prepare a whitening composition.

在一些實施例中，此秋姬李發酵物具有減少黑色素及/或抑制黑色素生成的作用。In some embodiments, the Qiuji Plum fermentation product has the effect of reducing melanin and/or inhibiting melanin production.

在一些實施例中，此秋姬李發酵物具有抑制酪胺酸酶活性的作用。In some embodiments, the fermented product of Prunus prunus L. has the effect of inhibiting tyrosinase activity.

在一些實施例中，一種秋姬李發酵物用於製備提升粒線體活性的組合物的用途。In some embodiments, a fermentation product of Prunus cinnabar is used to prepare a composition for increasing mitochondrial activity.

在一些實施例中，一種秋姬李發酵物用於製備減少肌膚色素斑的組合物的用途。In some embodiments, a fermented product of Prunus prunus is used to prepare a composition for reducing skin pigment spots.

在一些實施例中，此色素斑為深層棕色斑、可見斑點或其組合。In some embodiments, the pigmented spots are deep brown spots, visible spots, or a combination thereof.

在一些實施例中，前述的秋姬李發酵物包含2,5-呋喃二甲醇（2,5-bis-(hydroxymethyl)furane）、5-羥基糠酸（5-Hydroxymethyl-2-furonic acid）、反式-4-羥基環己烷羧酸（trans-4-Hydroxycyclohaxane carboxlic acid）、酪醇（Tyrosol）、6-羥基-6-(羥甲基)-2H-吡喃-3(6H)-酮（6-Hydroxy-6- (hydroxymethyl)-2H-pyran-3(6H)-one）、2-羥基-1-(5-(羥甲基)呋喃-2-基)丙烷-1-酮（2-hydroxy-1-(5-(hydroxymethyl)furan-2- yl)propan-1-one）或其組合。In some embodiments, the aforementioned Akihime plum fermentation product contains 2,5-bis-(hydroxymethyl)furane, 5-Hydroxymethyl-2-furonic acid, trans-4-Hydroxycyclohaxane carboxlic acid, tyrosol, 6-hydroxy-6-(hydroxymethyl)-2H-pyran-3(6H)-one (6-Hydroxy-6- (hydroxymethyl)-2H-pyran-3(6H)-one), 2-Hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one (2 -hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one) or combinations thereof.

在一些實施例中，一種秋姬李發酵物的製作方法，其包括以一溶劑萃取一秋姬李以得到一秋姬李萃取物、以及厭氧發酵秋姬李萃取物以得到秋姬李發酵物。In some embodiments, a method for making a plum fermented product includes extracting a plum extract with a solvent, and anaerobically fermenting the plum extract to obtain a fermented plum extract. things.

綜上，任一實施例的秋姬李發酵物，其能提升粒線體活性或提升膚況。換言之，任一實施例的秋姬李發酵物適用於製備提升粒線體活性或提升膚況的組合物。在一些實施例中，秋姬李發酵物或其所製得的組合物還具有下列一種或多種功能：美白、減少黑色素、抑制黑色素生成、抑制酪胺酸酶活性、減少肌膚棕色斑、及減少肌膚斑點。In summary, the fermented product of Prunus prunus in any embodiment can increase mitochondrial activity or improve skin condition. In other words, the fermented product of Prunus prunus in any embodiment is suitable for preparing a composition for improving mitochondrial activity or improving skin condition. In some embodiments, Qiuji Plum fermentation or the composition prepared therefrom also has one or more of the following functions: whitening, reducing melanin, inhibiting melanin production, inhibiting tyrosinase activity, reducing brown skin spots, and reducing Skin spots.

以下將描述本案的部分具體實施態樣。在不背離本案精神下，本案尚可以多種不同形式之態樣來實踐，不應將保護範圍限於說明書所具體陳述的條件。Some specific implementation aspects of this case will be described below. Without departing from the spirit of this case, this case can still be practiced in many different forms, and the scope of protection should not be limited to the conditions specifically stated in the description.

於本文中，在提及含量時所使用的含量單位「%」通常是指重量百分比。In this article, the content unit "%" used when referring to content usually refers to weight percentage.

請參閱圖1。在一些實施例中，秋姬李（ Prunus salicina）發酵物是由是由一秋姬李經一溶劑萃取（步驟S10）後再經由一厭氧發酵（步驟S11）而得。 See Figure 1. In some embodiments, Prunus salicina fermentation product is obtained from Prunus salicina through a solvent extraction (step S10) and then anaerobic fermentation (step S11).

在一些實施例中，在萃取步驟（即步驟S10）中，進行萃取的秋姬李可為秋姬李的植株、根、莖、葉、或果實。在一些實施例中，進行萃取的秋姬李為秋姬李的去除種子的帶皮果實。In some embodiments, in the extraction step (ie, step S10), the Prunus pumila that is extracted can be the plants, roots, stems, leaves, or fruits of Prunus truncatula. In some embodiments, the extracted Prunus prunus is the skin-bearing fruit of Prunus truncatula from which seeds have been removed.

在一些實施例中，在萃取步驟（即步驟S10）中，進行萃取的秋姬李可為原材料（如，完整的去除種子的帶皮果實），或為經物理前處理而分解成碎片、顆粒或粉末等細小尺寸的型態。所採用的物理前處理可包括下列至少一種：粗碎、切碎、打碎、剪碎、搗碎、及研磨。In some embodiments, in the extraction step (i.e., step S10), the extracted Qiuji Plum can be raw materials (such as intact fruits with skins from which seeds have been removed), or can be decomposed into fragments or particles after physical pretreatment. or powder and other fine-sized forms. The physical pretreatment used may include at least one of the following: coarse crushing, chopping, smashing, shearing, pounding, and grinding.

在一些實施例中，在萃取步驟（即步驟S10）中，進行萃取的秋姬李可為剛採收並收集的秋姬李、乾燥後的秋姬李或冷凍過的秋姬李。舉例來說，在萃取步驟（即步驟S10）中，利用溶劑對乾燥後的秋姬李進行萃取。In some embodiments, in the extraction step (ie, step S10), the Qiuji plums to be extracted may be freshly harvested and collected Qiuji plums, dried Qiuji plums or frozen Qiuji plums. For example, in the extraction step (ie step S10), the dried Akihime plum is extracted with a solvent.

在一些實施例中，在萃取步驟（即步驟S10）中，進行萃取的溶劑可為水或酒精。在一些實施例中，在萃取步驟（即步驟S10）中，進行萃取的溶劑為水。In some embodiments, in the extraction step (ie step S10), the solvent used for extraction may be water or alcohol. In some embodiments, in the extraction step (ie step S10), the solvent used for extraction is water.

在一些實施例中，可先將秋姬李打碎成秋姬李顆粒，然後再以溶劑進行萃取。舉例來說，將秋姬李去子帶皮果實打碎成粒徑為12 mm以下的秋姬李顆粒，再將秋姬李顆粒以水進行萃取。In some embodiments, Prunus prunus can be broken into Prunus prunus granules first, and then extracted with a solvent. For example, the peeled and peeled fruits of Prunus caerulea are broken into prune granules with a particle size of less than 12 mm, and then the prune granules are extracted with water.

在一些實施例中，以溶劑於80-100℃下萃取秋姬李0.5-1小時以得到秋姬李萃取液，然後再加入菌種進行厭氧發酵來得到秋姬李發酵物。舉例來說，可將秋姬李（如粒徑12 mm以下的秋姬李顆粒）浸泡在95℃水中0.5小時，使秋姬李中的效性成分溶解至水中而得到秋姬李萃取液。In some embodiments, plum blossoms are extracted with a solvent at 80-100°C for 0.5-1 hour to obtain plum plum extract, and then strains are added to perform anaerobic fermentation to obtain plum fermentation product. For example, plum blossoms (such as plum blossoms with a particle size of less than 12 mm) can be soaked in water at 95°C for 0.5 hours to dissolve the active ingredients in plums into the water to obtain plum blossom extract.

在一些實施例中，在萃取步驟（即步驟S10）中，溶劑與秋姬李的重量比為10-30：1-3。在一些實施例中，在萃取步驟（即步驟S10）中，溶劑與秋姬李的重量比為10：1。In some embodiments, in the extraction step (ie, step S10), the weight ratio of solvent to Qiuji Plum is 10-30:1-3. In some embodiments, in the extraction step (ie, step S10), the weight ratio of the solvent to Qiuji Plum is 10:1.

在一些實施例中，在萃取完成後添加菌種之前，可先將秋姬李萃取液冷卻至室溫以得到冷卻的秋姬李萃取液，然後再殖入菌種。In some embodiments, before adding the bacterial strain after the extraction is completed, the Prunus prunus extract may be cooled to room temperature to obtain a cooled Prunus prunus extract, and then the bacterial strain may be added.

在一些實施例中，秋姬李萃取液是在未移除萃取材料（即包含秋姬李）的情況下，進行厭氧發酵步驟（即步驟S11）。In some embodiments, the extract of Prunus prune is subjected to an anaerobic fermentation step (ie, step S11) without removing the extraction material (ie, containing Prunus prune).

在一些實施例中，厭氧發酵步驟（即步驟S11）係為在沒有持續供應氧氣的環境中進行發酵。在一些實施例中，在厭氧發酵步驟（即步驟S11）中，係以保鮮膜包覆發酵桶槽外身與蓋孔，阻隔外部空氣進入發酵桶槽內。In some embodiments, the anaerobic fermentation step (ie, step S11) is performed in an environment without a continuous supply of oxygen. In some embodiments, in the anaerobic fermentation step (ie step S11), the outer body and cover hole of the fermentation barrel are covered with plastic wrap to block external air from entering the fermentation barrel.

在一些實施例中，厭氧發酵步驟（即步驟S11）係加入複數菌種進行厭氧發酵5-10日以得到秋姬李發酵物。在一些實施例中，厭氧發酵步驟（即步驟S11）係加入複數菌種進行厭氧發酵2-6日以得到秋姬李發酵物。In some embodiments, the anaerobic fermentation step (i.e., step S11) is to add multiple strains of bacteria and perform anaerobic fermentation for 5-10 days to obtain the fermented product of Prunus prunus. In some embodiments, the anaerobic fermentation step (i.e. step S11) is to add multiple strains of bacteria and perform anaerobic fermentation for 2-6 days to obtain the fermented product of Prunus prunus.

在一些實施例中，此菌種為一酵母菌及/或一乳酸菌。在一些實施例中，此菌種為一酵母菌及一乳酸菌。In some embodiments, the bacterial species is a yeast and/or a lactic acid bacteria. In some embodiments, the bacterial species is a yeast and a lactic acid bacteria.

在一些實施例中，厭氧發酵步驟（即步驟S11）係為兩階段發酵。在一些實施例中，厭氧發酵步驟（即步驟S11）係於秋姬李萃取液中依序添加一酵母菌及一乳酸菌進行發酵。In some embodiments, the anaerobic fermentation step (ie step S11) is a two-stage fermentation. In some embodiments, the anaerobic fermentation step (ie, step S11) is to sequentially add a yeast and a lactobacillus to the Prunus prune extract for fermentation.

請參閱圖2。在一些實施例中，厭氧發酵步驟（即步驟S11）係於秋姬李萃取液中添加酵母菌進行第一次發酵以得到第一初發酵液（步驟S111）後，再於第一初發酵液中添加乳酸菌進行第二次發酵以得到第二初發酵液（步驟S112）。See Figure 2. In some embodiments, the anaerobic fermentation step (i.e., step S11) is performed by adding yeast to the Prunus pumila extract for the first fermentation to obtain the first primary fermentation liquid (step S111), and then in the first primary fermentation. Lactic acid bacteria are added to the liquid for a second fermentation to obtain a second primary fermentation liquid (step S112).

在一些實施例中，步驟S111係於秋姬李萃取液中殖入0.2%啤酒酵母（如，寄存編號 BCRC20271的啤酒酵母）並在28℃-37℃下發酵1-3天，以得到第一初發酵液。在一些實施例中，殖入啤酒酵母後的秋姬李萃取液係在30℃下發酵3天。In some embodiments, step S111 is to inoculate 0.2% brewer's yeast (such as brewer's yeast with registration number BCRC20271) into the plum extract and ferment it at 28°C-37°C for 1-3 days to obtain the first Initial fermentation liquid. In some embodiments, the plum extract after colonization with brewer's yeast is fermented at 30°C for 3 days.

在一些實施例中，在步驟S111中，藉由添加酵母菌於秋姬李萃取液中，得以使秋姬李萃取液發酵而生成酒精，藉以有利於提取出秋姬李內的有效成份。In some embodiments, in step S111, yeast is added to the Plum extract, so that the Plum extract can be fermented to generate alcohol, thereby facilitating extraction of the active ingredients in Plum extract.

在一些實施例中，第一初發酵液的pH值≦4.0，且其糖度約為3.6°Bx（白利糖度，Degrees Brix）。在一些實施例中，第一初發酵液的pH值為3.95。In some embodiments, the pH value of the first primary fermentation broth is ≦4.0, and its sugar content is approximately 3.6°Bx (Degrees Brix). In some embodiments, the pH value of the first primary fermentation broth is 3.95.

在一些實施例中，步驟S112係於第一初發酵液中殖入0.06%胚芽乳酸桿菌（如，寄存編號 BCRC910760的胚芽乳酸桿菌）並在28℃-37℃下發酵1-3天，以得到第二初發酵液。在一些實施例中，殖入胚芽乳酸桿菌後的第一初發酵液係在30℃下發酵3天。In some embodiments, step S112 is to colonize the first primary fermentation broth with 0.06% Lactobacillus plantarum (for example, Lactobacillus plantarum with registration number BCRC910760) and ferment it at 28°C-37°C for 1-3 days to obtain Second primary fermentation broth. In some embodiments, the first primary fermentation broth after colonization with Lactobacillus plantarum is fermented at 30°C for 3 days.

在一些實施例中，在步驟S112中，藉由添加乳酸菌於第一初發酵液中，得以使第一初發酵液內的葡萄糖被消耗而降低糖度，並且產生乳酸而降低第一初發酵液的pH值。於此，降低第一初發酵液的pH值有利於進一步提取出秋姬李內的其他不同有效成分。In some embodiments, in step S112, by adding lactic acid bacteria to the first primary fermentation liquid, the glucose in the first primary fermentation liquid is consumed to reduce the sugar content, and lactic acid is produced to reduce the sugar content of the first primary fermentation liquid. pH value. Here, lowering the pH value of the first fermentation liquid is conducive to further extracting other different active ingredients in Qiuji Plum.

在一些實施例中，第二初發酵液的pH值≦3.5，且其糖度約為1.5°Bx。在一些實施例中，第二初發酵液的pH值為3.32。In some embodiments, the pH value of the second primary fermentation broth is ≦3.5, and its sugar content is approximately 1.5°Bx. In some embodiments, the pH value of the second primary fermentation broth is 3.32.

在一些實施例中，於步驟S112後，可進一步將第二初發酵液過濾，以得到初發酵過濾液。在一些實施例中，以200目數的濾網過濾第二初發酵液，以得到初發酵過濾液。In some embodiments, after step S112, the second primary fermentation liquid can be further filtered to obtain a primary fermentation filtrate. In some embodiments, the second primary fermentation liquid is filtered through a 200 mesh filter to obtain the primary fermentation filtrate.

在一些實施例中，還可將第二初發酵液或初發酵過濾液在55℃-65℃下進行減壓濃縮，以得到發酵濃縮液。在一些實施例中，減壓濃縮可在60℃下進行。In some embodiments, the second primary fermentation liquid or primary fermentation filtrate can also be concentrated under reduced pressure at 55°C-65°C to obtain a fermentation concentrate. In some embodiments, concentration under reduced pressure can be performed at 60°C.

在一些實施例中，可將前述的初發酵過濾液或發酵濃縮液進行過濾而得到發酵原液。在一些實施例中，此過濾步驟可以200目數的濾網進行。In some embodiments, the aforementioned primary fermentation filtrate or fermentation concentrate can be filtered to obtain fermentation stock solution. In some embodiments, this filtration step can be performed with a 200 mesh filter.

在一些實施例中，發酵原液的pH值≦3.5，且其糖度約為1.5°Bx。在一些實施例中，發酵原液的pH值為3.2。In some embodiments, the pH value of the fermentation stock solution is ≦3.5, and its sugar content is approximately 1.5°Bx. In some embodiments, the pH value of the fermentation stock solution is 3.2.

在一些實施例中，可添加寡糖至發酵原液中來將發酵原液的糖度調整至24°Bx，進而得到糖度調整後發酵原液。在一些實施例中，寡糖係由3-10個單醣分子聚合而成的低聚糖。在一些實施例中，寡糖係為，但不限於，果寡糖、半乳寡糖、木寡糖或異麥芽寡糖。在一些實施例中，寡糖為含20%-50%異麥芽寡糖的寡糖溶液。In some embodiments, oligosaccharides can be added to the fermentation stock solution to adjust the sugar content of the fermentation stock solution to 24°Bx, thereby obtaining a fermentation stock solution with adjusted sugar content. In some embodiments, the oligosaccharide is an oligosaccharide polymerized from 3 to 10 monosaccharide molecules. In some embodiments, the oligosaccharide is, but is not limited to, fructooligosaccharide, galactooligosaccharide, xylo-oligosaccharide, or isomaltooligosaccharide. In some embodiments, the oligosaccharide is an oligosaccharide solution containing 20%-50% isomaltooligosaccharide.

在一些實施例中，發酵原液還可進一步以醇類與水進行液相-液相分配萃取而得到醇類可溶部。在一些實施例中，醇類可為正丁醇，且此醇類可溶部為正丁醇可溶部。In some embodiments, the fermentation stock solution can be further subjected to liquid-liquid phase distribution extraction with alcohol and water to obtain the alcohol-soluble part. In some embodiments, the alcohol can be n-butanol, and the alcohol-soluble portion is n-butanol-soluble portion.

在一些實施例中，正丁醇可溶部可進一步以醇類為沖提液進行分離而得到多種化合物。In some embodiments, the n-butanol soluble part can be further separated using alcohols as eluent to obtain a variety of compounds.

應能理解的是，可依實際需求以厭氧發酵所得的第二初發酵液、初發酵過濾液、發酵濃縮液、發酵原液、糖度調整後發酵原液、醇類可溶部、分離得的化合物或其任意組合作為秋姬李發酵物。It should be understood that the second primary fermentation liquid, primary fermentation filtrate, fermentation concentrate, fermentation stock solution, sugar content-adjusted fermentation stock liquid, alcohol soluble fraction, and isolated compounds obtained by anaerobic fermentation can be used according to actual needs. or any combination thereof as Akihime plum ferment.

在一些實施例中，前述的秋姬李發酵物包含2,5-呋喃二甲醇（2,5-bis-(hydroxymethyl)furane）、5-羥基糠酸（5-Hydroxymethyl-2-furonic acid）、反式-4-羥基環己烷羧酸（trans-4-Hydroxycyclohaxane carboxlic acid）、酪醇（Tyrosol）、6-羥基-6-(羥甲基)-2H-吡喃-3(6H)-酮（6-Hydroxy-6-(hydroxymethyl)-2H-pyran-3(6H)-one）、2-羥基-1-(5-(羥甲基)呋喃-2-基)丙烷-1-酮（2-hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one）或其組合。在一些實施例中，秋姬李發酵物可由2,5-呋喃二甲醇、5-羥基糠酸、反式-4-羥基環己烷羧酸、酪醇、6-羥基-6-(羥甲基)-2H-吡喃-3(6H)-酮及2-羥基-1-(5-(羥甲基)呋喃-2-基)丙烷-1-酮所構成。In some embodiments, the aforementioned Akihime plum fermentation product contains 2,5-bis-(hydroxymethyl)furane, 5-Hydroxymethyl-2-furonic acid, trans-4-Hydroxycyclohaxane carboxlic acid, tyrosol, 6-hydroxy-6-(hydroxymethyl)-2H-pyran-3(6H)-one (6-Hydroxy-6-(hydroxymethyl)-2H-pyran-3(6H)-one), 2-Hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one (2 -hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one) or combinations thereof. In some embodiments, the plum fermentation product may be composed of 2,5-furandimethanol, 5-hydroxyfuroic acid, trans-4-hydroxycyclohexanecarboxylic acid, tyrosol, 6-hydroxy-6-(hydroxymethyl It is composed of 2H-pyran-3(6H)-one and 2-hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one.

在一些實施例中，前述的秋姬李發酵物具有美白的能力。換言之，秋姬李發酵物施予一個體時能美白此個體的肌膚。因此，秋姬李發酵物適用於製備美白的組合物。In some embodiments, the aforementioned Qiuji Plum fermentation product has whitening ability. In other words, when applied to an individual, the Qiuji plum ferment can whiten the individual's skin. Therefore, Qiuji plum ferment is suitable for preparing whitening compositions.

在一些實施例中，秋姬李發酵物具有減少黑色素、抑制黑色素生成、抑制酪胺酸酶活性、或其任意組合等能力。換言之，秋姬李發酵物施予一個體時能減少黑色素、抑制黑色素生成、抑制酪胺酸酶活性、或其任意組合。因此，秋姬李發酵物適用於製備減少黑色素、抑制黑色素生成、抑制酪胺酸酶活性、或其任意組合的組合物。In some embodiments, the fermented product of Prunus pumila has the ability to reduce melanin, inhibit melanin production, inhibit tyrosinase activity, or any combination thereof. In other words, when administered to an individual, the fermented product of Prunus cinnabar can reduce melanin, inhibit melanin production, inhibit tyrosinase activity, or any combination thereof. Therefore, the fermented product of Prunus prunus is suitable for preparing a composition that reduces melanin, inhibits melanin production, inhibits tyrosinase activity, or any combination thereof.

在一些實施例中，前述的秋姬李發酵物具有提升粒線體活性的能力。換言之，秋姬李發酵物施予一個體時能提升此個體的粒線體活性。因此，秋姬李發酵物適用於製備提升粒線體活性的組合物。In some embodiments, the aforementioned fermented product of Prunus pumila has the ability to increase mitochondrial activity. In other words, when administered to an individual, Akihime plum ferment can increase the individual's mitochondrial activity. Therefore, the fermented product of Prunus cinnabar is suitable for preparing a composition for improving mitochondrial activity.

在一些實施例中，前述的秋姬李發酵物具有減少肌膚棕色斑及/或減少肌膚斑點的能力。換言之，秋姬李發酵物施予一個體時能減少此個體肌膚的棕色斑及/或斑點。因此，秋姬李發酵物適用於製備減少肌膚棕色斑及/或減少肌膚斑點的組合物。In some embodiments, the aforementioned Akihime plum ferment has the ability to reduce brown spots on skin and/or reduce skin spots. In other words, when administered to an individual, the fermented product of Prunus cinnabar can reduce brown spots and/or spots on the individual's skin. Therefore, the fermented product of Prunus prunus is suitable for preparing a composition for reducing brown spots on skin and/or reducing skin spots.

在一些實施例中，前述的個體可為人。In some embodiments, the aforementioned individual may be a human.

在一些實施例中，製備所得的組合物可為醫藥組合物、食品組合物、化妝品組合物或保養品組合物。In some embodiments, the prepared composition can be a pharmaceutical composition, a food composition, a cosmetic composition or a skin care composition.

在一些實施例中，當前述的組合物為醫藥組合物時，此醫藥組合物包含有效含量的秋姬李發酵物。其中，此醫藥組合物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（orally）、或局部地（topically）投藥劑型。In some embodiments, when the aforementioned composition is a pharmaceutical composition, the pharmaceutical composition includes an effective content of the fermented product of Prunus prunus. Wherein, the pharmaceutical composition can be made suitable for administration enterally, parenterally, orally, or topically using techniques well known to those skilled in the art. Dosage form.

在一些實施例中，經腸道或口服地投藥劑型可為，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）或類似的物。In some embodiments, the dosage form for enteral or oral administration may be, but is not limited to, a tablet, a troche, a lozenge, a pill, or a capsule. , dispersible powder or granule, solution, suspension, emulsion, syrup, elixir, slurry or similar.

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）[例如，無菌的水性溶液（sterile aqueous solution）或分散液（dispersion）]、無菌的粉末（sterile powder）、外部製劑（external preparation）或類似的物。In some embodiments, the dosage form for parenteral or topical administration may be, but is not limited to, injection [eg, sterile aqueous solution or dispersion], sterile sterile powder, external preparation or similar.

在一些實施例中，注射品的投藥方式可為，但不限於，腹膜內注射（intraperitoneal injection）、皮下注射（subcutaneous injection）、表皮內注射（intraepidermal injection）、皮內注射（intradermal injection）、肌肉內注射（intramuscular injection）、靜脈內注射（intravenous injection）或病灶內注射（intralesional injection）。In some embodiments, the administration method of the injection may be, but is not limited to, intraperitoneal injection, subcutaneous injection, intraepidermal injection, intradermal injection, intramuscular injection Intramuscular injection, intravenous injection or intralesional injection.

在一些實施例中，含有有效含量的秋姬李發酵物的醫藥組合物可進一步包含被廣泛地使用於藥物製造技術的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（excipient）、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）以及類似的物。關於選用的載劑的種類與數量是落在熟習此項技術的人士的專業素養與例行技術範疇內。其中，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 pharmaceutical composition containing an effective content of the fermentation product of Prunus quinquefolia may further comprise a pharmaceutically acceptable carrier that is widely used in pharmaceutical manufacturing technology. In some embodiments, the pharmaceutically acceptable carrier may be one or more of the following carriers: solvent, buffer, emulsifier, suspending agent, decomposing agent ( decomposer), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent ), gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes and the like. The types and amounts of carriers selected are within the professionalism and routine skills of those familiar with this technology. Among them, the solvent as a pharmaceutically acceptable carrier can be water, normal saline, phosphate buffered saline (PBS) or alcohol containing aqueous solution (alcohol containing aqueous solution).

在一些實施例中，含有有效含量的秋姬李發酵物的醫藥組合物可利用熟習此技藝者所詳知的技術而被製造成一適合於局部地施用於皮膚上的外部製劑（external preparation）。在一些實施例中，外部製劑包括，但不限於：乳劑（emulsion）、凝膠（gel）、軟膏（ointment）、乳霜（cream）、貼片（patch）、擦劑（liniment）、粉末（powder）、氣溶膠（aerosol）、噴霧（spray）、乳液（lotion）、乳漿（serum）、糊劑（paste）、泡沫（foam）、滴劑（drop）、懸浮液（suspension）、油膏（salve）以及繃帶（bandage）。In some embodiments, a pharmaceutical composition containing an effective amount of Prunus quince ferment can be manufactured into an external preparation suitable for topical application to the skin using techniques well known to those skilled in the art. In some embodiments, external preparations include, but are not limited to: emulsions, gels, ointments, creams, patches, liniments, powders ( powder), aerosol, spray, lotion, serum, paste, foam, drop, suspension, ointment (salve) and bandage (bandage).

在一些實施例中，當前述的醫藥組合物為外部製劑時，此醫藥組合物可由有效含量的秋姬李發酵物與為熟習此項技藝者所詳知的一基底（base）相混合而製成。In some embodiments, when the aforementioned pharmaceutical composition is an external preparation, the pharmaceutical composition can be prepared by mixing an effective content of Prunus quince ferment and a base well known to those skilled in the art. become.

在一些實施例中，此基底可包含有一或多種選自於下列的添加劑（additives）：水、醇（alcohols）、甘醇（glycol）、碳氫化合物（hydrocarbons）[諸如石油膠（petroleum, jelly）以及白凡士林（white petrolatum）]、蠟（wax）[諸如石蠟（paraffin）以及黃蠟（yellow wax）]、保存劑（preserving agents）、抗氧化劑（antioxidants）、界面活性劑（surfactants）、吸收增強劑（absorption enhancers）、安定劑（stabilizing agents）、膠凝劑（gelling agents）[諸如卡波普®974P （carbopol®974P）、微結晶纖維素（microcrystalline cellulose）以及羧基甲基纖維素（carboxymethylcellulose）]、活性劑（active agents）、保濕劑（humectants）、氣味吸收劑（odor absorbers）、香料（fragrances）、pH調整劑（pH adjusting agents）、螯合劑（chelating agents）、乳化劑（emulsifiers）、閉塞劑（occlusive agents）、軟化劑（emollients）、增稠劑（thickeners）、助溶劑（solubilizing agents）、滲透增強劑（penetration enhancers）、抗刺激劑（anti-irritants）、著色劑（colorants）以及推進劑（propellants）等。有關這些添加劑的選用與數量是落在熟習此項技術的人士的專業素養與例行技術範疇內。In some embodiments, the base may include one or more additives selected from the group consisting of: water, alcohols, glycols, hydrocarbons (such as petroleum, jelly) ) and white petrolatum], waxes [such as paraffin and yellow wax], preserving agents, antioxidants, surfactants, absorption enhancers absorption enhancers, stabilizing agents, gelling agents (such as carbopol® 974P, microcrystalline cellulose and carboxymethylcellulose) ], active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants, and Propellants, etc. The selection and amounts of these additives are within the expertise and routine skills of those skilled in the art.

在一些實施例中，當前述的組合物為食品組合物時，此食品組合物包含特定含量的秋姬李發酵物。其中，此食品組合物的型態可為粉末、顆粒、溶液、膠體或膏體。In some embodiments, when the aforementioned composition is a food composition, the food composition includes a specific content of Akihime plum fermentation product. The food composition may be in the form of powder, granule, solution, colloid or paste.

在一些實施例中，含有秋姬李發酵物的食品組合物可為食品產品或食品添加物（food additive）。In some embodiments, the food composition containing the fermented product of Prunus quince L. can be a food product or a food additive.

在一些實施例中，含有秋姬李發酵物的食品產品可為飲料（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）等。在一些實施例中，含有秋姬李發酵物的食品產品可更包括一佐劑。舉例來說，佐劑可為麥芽糖糊精（Maltodextrin）、蘋果酸、蔗糖素、檸檬酸、水果香料、蜂蜜香料、甜菊糖苷或其組合等。關於選用的載劑的種類與數量是落在熟習此項技術的人士的專業素養與例行技術範疇內。In some embodiments, the food product containing the fermented product of Prunus quince can be beverages, fermented foods, bakery products, health foods, or dietary supplements. )wait. In some embodiments, the food product containing the fermented product of Prunus prunus truncatula may further include an adjuvant. For example, the adjuvant may be maltodextrin, malic acid, sucralose, citric acid, fruit flavors, honey flavors, steviol glycosides or combinations thereof. The types and amounts of carriers selected are within the professionalism and routine skills of those familiar with this technology.

在一些實施例中，含有秋姬李發酵物的食品添加物可為調味料、甜味料、香料、pH值調整劑、乳化劑、著色料或穩定劑等。In some embodiments, food additives containing fermented products of Prunus prunus can be seasonings, sweeteners, spices, pH adjusters, emulsifiers, colorants or stabilizers, etc.

在一些實施例中，當前述的組合物為化妝品組合物或保養品組合物。換言之，化妝品組合物或保養品組合物包含有效含量的秋姬李發酵物。In some embodiments, the aforementioned composition is a cosmetic composition or skin care composition. In other words, the cosmetic composition or skin care composition contains an effective content of the fermented product of Akihime plum.

在一些實施例中，含有秋姬李發酵物的化妝品組合物或保養品組合物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。In some embodiments, the cosmetic composition or skin care composition containing the fermented substance of Prunus prunus can be in any of the following forms: lotion, gel, gel, mud mask, lotion, cream, lipstick, foundation, Pressed powder, powder, cleansing oil, cleansing milk, facial cleanser, shower gel, shampoo, hair conditioner, sunscreen lotion, hand cream, nail polish, perfume, essence and facial mask.

在一些實施例中，含有秋姬李發酵物的化妝品組合物或保養品組合物可視需要更包含外用品可接受成分。在一些實施例中，外用品可接受成分例如可為乳化劑、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 cosmetic composition or skin care product composition containing the fermented substance of Prunus quince L. may further contain ingredients acceptable for external use if necessary. In some embodiments, acceptable ingredients for topical products may be, for example, emulsifiers, penetration enhancers, softeners, solvents, excipients, antioxidants, or combinations thereof.

下列範例中若無特別敘明，所進行的實驗步驟是在室溫（約25℃）且常壓（1 atm）下進行。Unless otherwise stated in the following examples, the experimental steps were performed at room temperature (approximately 25°C) and normal pressure (1 atm).

例一Example 1

A. 原料：A. Raw materials:

1. 乾燥後的秋姬李（ Prunus salicina）的帶皮果實（去除種子），產地：中國，以下簡稱「秋姬李果實」。 1. Dried fruits with skin of Prunus salicina (seeds removed), origin: China, hereafter referred to as "Prunus salicina fruits".

2. 二次水，又稱RO水（逆滲透；Reverse Osmosis）或二次蒸餾水，以下簡稱「水」。2. Secondary water, also known as RO water (Reverse Osmosis) or double distilled water, hereinafter referred to as "water".

3. 啤酒酵母，其購自食品工業發展研究所生物資源保存及研究中心（BCRC）且具有寄存編號 BCRC20271。3. Brewer's yeast, purchased from the Bioresources Conservation and Research Center (BCRC) of the Institute for Development of the Food Industry with accession number BCRC20271.

4. 胚芽乳酸桿菌，其購自BCRC且具有寄存編號 BCRC910760。4. Lactobacillus plantarum, purchased from BCRC and having accession number BCRC910760.

5. 寡糖溶液，其為以水與異麥芽寡糖所配製成的20%-50%異麥芽寡糖溶液。5. Oligosaccharide solution, which is a 20%-50% isomaltooligosaccharide solution prepared with water and isomaltooligosaccharide.

B. 製備流程：B. Preparation process:

1. 將秋姬李果實以粉碎機（型號：12 Speed Blender；廠牌：Osterizer）進行打碎，以形成粒徑12 mm以下的秋姬李顆粒。1. Crush the plum fruit with a crusher (Model: 12 Speed Blender; Brand: Osterizer) to form plum particles with a particle size of less than 12 mm.

2. 將水加熱至95℃後，投入秋姬李顆粒，並且使秋姬李顆粒於95℃的水中浸泡0.5小時，以形成秋姬李萃取液。於此，投入的秋姬李顆粒與水的重量比為1：10。2. After heating the water to 95°C, add the Prunus prunus granules and soak them in the water at 95°C for 0.5 hours to form the Prunus truncatula extract. Here, the weight ratio of the Qiuji plum particles and water added is 1:10.

3. 於冷卻後的秋姬李萃取液（含有秋姬李）中殖入0.2%啤酒酵母，並在30℃下厭氧發酵3天，以得到第一初發酵液。第一初發酵液的pH值為3.95，且其糖度約為3.6°Bx。於此，厭氧發酵全程是在發酵桶槽內進行，並且厭氧發酵全程皆以保鮮膜包覆發酵桶槽外身與蓋孔，以阻隔外部空氣進入發酵桶槽內。3. Inoculate 0.2% beer yeast into the cooled Akihime plum extract (containing Akihime plum), and ferment anaerobically at 30°C for 3 days to obtain the first primary fermentation liquid. The pH value of the first fermentation broth is 3.95, and its sugar content is approximately 3.6°Bx. Here, the entire process of anaerobic fermentation is carried out in the fermentation barrel, and the outer body and cover hole of the fermentation barrel are covered with plastic wrap throughout the anaerobic fermentation process to block external air from entering the fermentation barrel.

4. 於第一初發酵液中殖入0.06%胚芽乳酸桿菌，並在30℃下厭氧發酵3天，以得到第二初發酵液。第二初發酵液的pH值為3.32，且其糖度約為1.5°Bx。於此，厭氧發酵全程是在發酵桶槽內進行，並且厭氧發酵全程皆以保鮮膜包覆發酵桶槽外身與蓋孔，以阻隔外部空氣進入發酵桶槽內。4. Inoculate the first primary fermentation broth with 0.06% Lactobacillus plantarum and ferment anaerobically at 30°C for 3 days to obtain the second primary fermentation broth. The pH value of the second primary fermentation broth is 3.32, and its sugar content is approximately 1.5°Bx. Here, the entire process of anaerobic fermentation is carried out in the fermentation barrel, and the outer body and cover hole of the fermentation barrel are covered with plastic wrap throughout the anaerobic fermentation process to block external air from entering the fermentation barrel.

5. 將第二初發酵液以200目數的濾網進行過濾以得到初發酵過濾液。5. Filter the second primary fermentation liquid through a 200-mesh filter to obtain the primary fermentation filtrate.

6. 將濃縮機（型號：Rotavapor R-100；廠牌：BUCHI）溫度設定為60℃後，利用濃縮機對初發酵過濾液進行減壓濃縮而得到發酵濃縮液。6. After setting the temperature of the concentrator (model: Rotavapor R-100; brand: BUCHI) to 60°C, use the concentrator to concentrate the primary fermentation filtrate under reduced pressure to obtain the fermentation concentrate.

7. 將發酵濃縮液以200目數的濾網進行過濾以得到發酵原液。發酵原液的pH值為3.2，且其糖度約為1.5°Bx。7. Filter the fermentation concentrate through a 200-mesh filter to obtain the fermentation stock solution. The pH value of the fermentation stock solution is 3.2, and its sugar content is approximately 1.5°Bx.

8. 於發酵原液中添加20%-50%異麥芽寡糖溶液以將發酵原液的糖度調整為24°Bx，以得到秋姬李發酵物A。8. Add 20%-50% isomaltooligosaccharide solution to the fermentation stock solution to adjust the sugar content of the fermentation stock solution to 24°Bx to obtain Akihime plum fermentation product A.

例二Example 2

A. 原料：A. Raw materials:

與例一所使用的原料皆相同。The raw materials used in Example 1 are the same.

B. 製備流程：B. Preparation process:

1. 將秋姬李果實以粉碎機進行打碎，以形成粒徑12 mm以下的秋姬李顆粒。1. Crush the plum fruit with a grinder to form plum particles with a particle size of less than 12 mm.

3. 於冷卻後的秋姬李萃取液（含有秋姬李）中殖入0.2%啤酒酵母，並在30℃下發酵3天，以得到第一初發酵液。於此，發酵全程是在發酵桶槽內進行，但發酵全程皆未以保鮮膜包覆發酵桶槽外身與蓋孔。3. Inoculate 0.2% beer yeast into the cooled Akihime plum extract (containing Akihime plum) and ferment it at 30°C for 3 days to obtain the first primary fermentation liquid. Here, the entire fermentation process is carried out in the fermentation barrel, but the outer body and cover hole of the fermentation barrel are not covered with plastic wrap throughout the fermentation process.

4. 於第一初發酵液中殖入0.06%胚芽乳酸桿菌，並在30℃下發酵3天，以得到第二初發酵液。於此，發酵全程是在發酵桶槽內進行，但發酵全程皆未以保鮮膜包覆發酵桶槽外身與蓋孔。4. Inoculate 0.06% Lactobacillus plantarum into the first primary fermentation broth, and ferment at 30°C for 3 days to obtain the second primary fermentation broth. Here, the entire fermentation process is carried out in the fermentation barrel, but the outer body and cover hole of the fermentation barrel are not covered with plastic wrap throughout the fermentation process.

7. 將發酵濃縮液以200目數的濾網進行過濾以得到發酵原液。7. Filter the fermentation concentrate through a 200-mesh filter to obtain the fermentation stock solution.

8. 於發酵原液中添加20%-50%異麥芽寡糖溶液以將發酵原液的糖度調整為24°Bx，以得到秋姬李發酵物B。8. Add 20%-50% isomaltooligosaccharide solution to the fermentation stock solution to adjust the sugar content of the fermentation stock solution to 24°Bx to obtain Akihime plum fermentation product B.

例三Example 3

秤取10.0 mg的沒食子酸（Gallic acid）置於10 mL容量瓶中，然後以水（H ₂O）定量至10 mL，以得到沒食子酸的儲備溶液（stock solution）（即含1000ppm的沒食子酸）。將沒食子酸的儲備溶液稀釋10倍，即100 μL沒食子酸的儲備溶液加900 μL的水，以得到100 μg/mL沒食子酸的初始溶液（即含100ppm的沒食子酸）。然後，依據下表一配製0 μg/mL、20 μg/mL、40 μg/mL、60 μg/mL、80 μg/mL、及100 μg/mL的沒食子酸的標準溶液，並分別取100 μL的各濃度的標準溶液至玻璃試管中。加入500 μL的福林酚試劑（Folin-Ciocalteu's phenol reagent，購自Merck）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400 μL的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200 μL的標準反應溶液至96孔板中，並測量其在750 nm下的吸光值，以獲得標準曲線。 Weigh 10.0 mg of gallic acid (Gallic acid) and place it in a 10 mL volumetric flask, and then quantify it with water (H ₂ O) to 10 mL to obtain a stock solution of gallic acid (that is, containing 1000ppm gallic acid). Dilute the stock solution of gallic acid 10 times, that is, add 900 μL of water to 100 μL of the stock solution of gallic acid, to obtain an initial solution of 100 μg/mL gallic acid (i.e., containing 100 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 below, and take 100 μg/mL of gallic acid solution respectively. Add μL of the standard solution of each concentration into a glass test tube. Add 500 μL of Folin-Ciocalteu's phenol reagent (purchased from Merck) into each glass test tube, mix evenly with the standard solution and let stand for 3 minutes, then add 400 μL of 7.5% sodium carbonate, mix evenly, and react for 30 minutes to obtain the standard reaction solution. Take 200 μL of the standard reaction solution into a 96-well plate, and measure its absorbance value at 750 nm to obtain a standard curve.

表一標準溶液（μg/mL） 0 20 40 60 80 100 初始溶液（μL） 0 20 40 60 80 100 水（μL） 100 80 60 40 20 0 Table I Standard solution (μg/mL) 0 20 40 60 80 100 Initial solution (μL) 0 20 40 60 80 100 Water (μL) 100 80 60 40 20 0

分別取例一所製得的秋姬李萃取液和秋姬李發酵物A，以及例二所製得的秋姬李發酵物B作為樣本。將各樣本以水稀釋10倍後取100 μL到離心管中。接著，加入500 μL的福林酚試劑至各玻璃試管中與樣本混合均勻並靜置3分鐘後，再加入400 μL的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200 μL的待測反應溶液至96孔板中，並測量待測反應溶液於750 nm下的吸光值。Take the plum extract and plum fermentation product A prepared in Example 1, and the fermentation material B prepared in example 2 as samples respectively. Dilute each sample 10 times with water and put 100 μL into a centrifuge tube. Next, add 500 μL of folinol reagent into each glass test tube, mix evenly with the sample, and let stand for 3 minutes. Then add 400 μL of 7.5% sodium carbonate, mix evenly, 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 that there are no 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.

接著，利用標準曲線與內插法將待測反應溶液的吸光值換算成總多酚（Polyphenol）含量，並回乘稀釋倍數以得到實際總多酚含量。Then, use the standard curve and interpolation method to convert the absorbance value of the reaction solution to be tested into the total polyphenol content, and multiply the dilution factor back to obtain the actual total polyphenol content.

於此，可得到秋姬李萃取液的總多酚含量為95.4ppm（即為95.4 μg/mL），秋姬李發酵物B的總多酚含量為213.913ppm（即為213.913 μg/mL），以及秋姬李發酵物A的總多酚含量為283.47ppm（即為283.47 μg/mL），如圖3所示。由此可知，相較於發酵前（即秋姬李萃取液），經發酵後的秋姬李發酵物的總多酚含量明顯增加。其中，經微生物厭氧發酵後，可增加總多酚含量3倍（297%）。並且，經厭氧發酵所得的秋姬李發酵物A的總多酚含量明顯多於經有氧發酵所得的秋姬李發酵物B。多酚類物質為富含於植物中的抗氧化物質，經文獻證實其可有效減少體內自由基的形成。由此可知，經厭氧發酵所得的秋姬李發酵物能具有較高的總多酚含量，進而能提升個體的抗氧化活性。Here, it can be obtained that the total polyphenol content of the plum extract is 95.4ppm (95.4 μg/mL), and the total polyphenol content of the fermented product B is 213.913ppm (213.913 μg/mL). And the total polyphenol content of Qiuji Plum fermentation product A is 283.47ppm (that is, 283.47 μg/mL), as shown in Figure 3. It can be seen that compared with that before fermentation (i.e., the extract of Prunus prunus), the total polyphenol content of the fermented Prunus chinensis fermentation product after fermentation increased significantly. Among them, after microbial anaerobic fermentation, the total polyphenol content can be increased by 3 times (297%). Moreover, the total polyphenol content of the fermented product A obtained by anaerobic fermentation was significantly higher than that of the fermented product B obtained by aerobic fermentation. Polyphenols are antioxidant substances rich in plants. Literature has proven that they can effectively reduce the formation of free radicals in the body. It can be seen from this that the fermented product of Prunus pumila obtained through anaerobic fermentation can have a higher total polyphenol content, which in turn can improve the antioxidant activity of individuals.

例四Example 4

A. 材料與儀器：A. Materials and instruments:

1. 細胞株：小鼠黑色素瘤細胞，購自ATCC（American type culture collection），細胞編號6475，以下簡稱B16F10細胞。1. Cell line: Mouse melanoma cells, purchased from ATCC (American type culture collection), cell number 6475, hereinafter referred to as B16F10 cells.

2. 細胞培養基：DMEM（Dulbecco’s modified Eagle’s medium，購自Gibco，產品編號12800017），添加10% FBS（Fetal Bovine Serum，購自Gibco，產品編號10437-028）以及1%抗生素（購自Gibco，產品編號15240-062）。2. Cell culture medium: DMEM (Dulbecco's modified Eagle's medium, purchased from Gibco, product number 12800017), added with 10% FBS (Fetal Bovine Serum, purchased from Gibco, product number 10437-028) and 1% antibiotics (purchased from Gibco, product number 10437-028) No. 15240-062).

3. 麴酸（Kojic acid），購自Sigma，產品編號K3125。3. Kojic acid, purchased from Sigma, product number K3125.

4. BSA的儲備溶液：以水及BSA（購自Sigma，產品編號A9647-100G）配製而成。4. BSA stock solution: Prepared with water and BSA (purchased from Sigma, product number A9647-100G).

5. 細胞裂解試劑：以RIPA Lysis and Extraction Buffer（購自Thermo，產品編號89900）及PMSF（phenylmethylsulfonyl fluorid，購自Thermo，產品編號36978）配製而成。5. Cell lysis reagent: prepared from RIPA Lysis and Extraction Buffer (purchased from Thermo, product number 89900) and PMSF (phenylmethylsulfonyl fluorid, purchased from Thermo, product number 36978).

6. 蛋白質測定試劑：以Bio-Rad Protein Assay Dye Reagent Concentrate（購自Bio-Rad，產品編號5000006）和其4倍體積的水稀釋配製而成。6. Protein determination reagent: Prepared by diluting Bio-Rad Protein Assay Dye Reagent Concentrate (purchased from Bio-Rad, product number 5000006) and 4 times its volume of water.

7. 10 mM L-多巴（L-DOPA，3,4-二羥苯丙氨酸）：以9.8 mg L-多巴（購自Sigma，產品編號D9628-5G）和5 mL 0.1mM 磷酸鹽緩衝液（phosphate buffer，pH7.0，購自Gibco，產品編號14200-075）配製而成。7. 10 mM L-DOPA (3,4-dihydroxyphenylalanine): 9.8 mg L-DOPA (purchased from Sigma, Product No. D9628-5G) and 5 mL 0.1mM phosphate It was prepared from phosphate buffer (phosphate buffer, pH7.0, purchased from Gibco, product number 14200-075).

8. 酵素免疫分析儀（ELISA reader），購自BioTek公司（美國）。8. Enzyme immunoassay analyzer (ELISA reader), purchased from BioTek Company (USA).

B. 試驗流程：B. Test process:

1. 將B16F10細胞以每孔1.5×10 ⁵個的密度，接種於含細胞培養基的6孔培養盤中，並在37 ℃下培養24小時。於此，B16F10細胞分為五個試驗組別，其分別為：空白組、麴酸組、實驗組A、實驗組B與實驗組C。各組進行三重複（意即各組各有三孔）。 1. Inoculate B16F10 cells into a 6-well culture plate containing cell culture medium at a density of 1.5×10 ⁵ cells per well, and culture at 37°C for 24 hours. Here, B16F10 cells are divided into five experimental groups, which are: blank group, kojic acid group, experimental group A, experimental group B and experimental group C. Each group was performed in triplicate (meaning each group had three holes).

2. 培養24小時後，將各組更換為實驗培養基，並於37℃下繼續培養48小時。其中，空白組的實驗培養基為不含樣品或麴酸的細胞培養基；麴酸組的實驗培養基為含有200 μg/mL的麴酸的細胞培養基，其中麴酸已被廣泛認知為具有降低黑色素生成效果的物質；實驗組A的實驗培養基為含有0.0625% (v/v) 的例一製得的秋姬李萃取液的細胞培養基；實驗組B的實驗培養基為含有0.5% (v/v) 的例二製得的秋姬李發酵物B的細胞培養基；以及實驗組C的實驗培養基為含有0.5% (v/v) 的例一製得的秋姬李發酵物A的細胞培養基。2. After 24 hours of culture, replace each group with the experimental medium and continue to culture at 37°C for 48 hours. Among them, the experimental culture medium of the blank group is a cell culture medium that does not contain samples or kojic acid; the experimental culture medium of the kojic acid group is a cell culture medium containing 200 μg/mL kojic acid, of which kojic acid has been widely recognized to have the effect of reducing melanin production. substance; the experimental medium of experimental group A is a cell culture medium containing 0.0625% (v/v) of the plum extract prepared in Example 1; the experimental medium of experimental group B is a cell culture medium containing 0.5% (v/v) The cell culture medium of the fermented substance B of Prunus prunus L. prepared in Example 2; and the experimental medium of experimental group C is the cell culture medium of the fermented substance A of Prunus chinensis prepared in Example 1 containing 0.5% (v/v).

3. 移除培養後的各組的實驗培養基，並以DPBS進行潤洗2次。3. Remove the experimental culture medium of each group after culture and rinse twice with DPBS.

4. 於潤洗後，添加200ul 細胞裂解試劑至各孔中以裂解細胞，形成細胞裂解液。4. After rinsing, add 200ul of cell lysis reagent to each well to lyse the cells to form a cell lysis solution.

5. 將各組離心試管離心使細胞沉澱。5. Centrifuge each group of centrifuge tubes to pellet the cells.

6. 收集各組離心試管內的上清液以進行蛋白定量。6. Collect the supernatant in each group of centrifuge tubes for protein quantification.

7. 標準曲線建立：依據下表二於96孔盤中配製0 mg/mL、0.15625 mg/mL、0.3125 mg/mL、0.625 mg/mL、1.25 mg/mL、及2.5 mg/mL的BSA的標準溶液。接著，加入200 μL的蛋白質測定試劑至各孔內與標準溶液混合均勻並靜置5分鐘以得到標準反應溶液，測量其在595 nm下的吸光值，以獲得標準曲線。7. Standard curve establishment: Prepare the standards of 0 mg/mL, 0.15625 mg/mL, 0.3125 mg/mL, 0.625 mg/mL, 1.25 mg/mL, and 2.5 mg/mL BSA in a 96-well plate according to Table 2 below. solution. Then, add 200 μL of protein assay reagent into each well, mix with the standard solution, and let stand for 5 minutes to obtain a standard reaction solution. Measure its absorbance value at 595 nm to obtain a standard curve.

表二標準溶液（mg/mL） 0 0.15625 0.3125 0.625 1.25 2.5 BSA的儲備溶液（mL） 0 0.625 1.25 2.5 5 10 水（mL） 10 9.375 8.75 7.5 5 0 Table II Standard solution (mg/mL) 0 0.15625 0.3125 0.625 1.25 2.5 BSA stock solution (mL) 0 0.625 1.25 2.5 5 10 Water (mL) 10 9.375 8.75 7.5 5 0

8. 各組上清液蛋白質定量：於96孔盤每孔中分別加入1 μL的步驟6所收集的各組離心試管內的上清液，並分別加入9 μL的水，混合均勻以得到各組的上清液蛋白質定量溶液。接著，加入200 μL的蛋白質測定試劑至各孔內與各組的樣品蛋白質定量溶液混合均勻並靜置5分鐘以得到待測反應溶液，測量其在595 nm下的吸光值。利用標準曲線與內插法將待測反應溶液的吸光值換算成蛋白質定量值。8. Quantification of protein in the supernatant of each group: Add 1 μL of the supernatant of each group of centrifuge tubes collected in step 6 to each well of the 96-well plate, and add 9 μL of water, and mix evenly to obtain each Set the supernatant protein quantification solution. Then, add 200 μL of protein determination reagent into each well, mix it evenly with the sample protein quantitative solution of each group, and let it stand for 5 minutes to obtain the reaction solution to be tested, and measure its absorbance value at 595 nm. Use the standard curve and interpolation method to convert the absorbance value of the reaction solution to be measured into a protein quantitative value.

9. 將含有10mg蛋白質定量值的各組離心試管內的上清液加到新的96孔盤的對應孔中，並於每孔加入細胞裂解試劑使每孔最終體積為100 μL（即，使各組的蛋白質濃度相同，皆為100 mg/mL）。9. Add the supernatant from each set of centrifuge tubes containing 10mg protein quantitative value to the corresponding well of a new 96-well plate, and add cell lysis reagent to each well to make the final volume of each well 100 μL (i.e., make The protein concentration of each group is the same, 100 mg/mL).

10. 於每孔加入10 μL 10 mM L-多巴，並於37℃下避光反應20分鐘以進行氧化反應。10. Add 10 μL of 10 mM L-dopa to each well and incubate at 37°C in the dark for 20 minutes to perform oxidation reaction.

11. 反應20分鐘後，利用酵素免疫分析儀測量每孔405 nm的吸光值（OD ₄₀₅值）。 11. After 20 minutes of reaction, use an enzyme immunoassay analyzer to measure the absorbance value at 405 nm (OD ₄₀₅ value) of each well.

C. 試驗結果：C. Test results:

所有組別的相對酪胺酸酶活性係依下列公式計算：相對酪胺酸酶活性（%）=（各組OD ₄₀₅值/空白組OD ₄₀₅值）×100%。 The relative tyrosinase activity of all groups was calculated according to the following formula: relative tyrosinase activity (%) = (OD ₄₀₅ value of each group/OD ₄₀₅ value of the blank group) × 100%.

空白組與其他各組，以及實驗組B與實驗組C的試驗結果之間的統計學顯著差異是以學生t檢驗（student t-test）統計分析得到。於圖式中，「*」代表在與空白組比較下其p值小於0.05、「**」代表在與空白組比較下其p值小於0.01，以及「***」代表在與空白組比較下其p值小於0.001；而於圖式中，「#」代表在與實驗組B比較下其p值小於0.05、「##」代表在與實驗組B比較下其p值小於0.01，以及「###」代表在與實驗組B比較下其p值小於0.001。The statistically significant differences between the test results of the blank group and other groups, as well as experimental group B and experimental group C, were obtained by statistical analysis using student t-test. In the figure, "*" means that the p value is less than 0.05 when compared with the blank group, "**" means that the p value is less than 0.01 when compared with the blank group, and "***" means that the p value is less than 0.01 when compared with the blank group. Its p value is less than 0.001; and in the diagram, "#" means that its p value is less than 0.05 when compared with experimental group B, "##" means that its p value is less than 0.01 when compared with experimental group B, and " ###" means that its p value is less than 0.001 when compared with experimental group B.

請參閱圖4。空白組未使用樣品或麴酸進行處理，因此空白組的試驗結果代表B16F10細胞在正常的生理代謝情況下的表現。於此，在設定空白組的相對酪胺酸酶活性為100%的情況下，麴酸組的相對酪胺酸酶活性為85.2%，實驗組A的相對酪胺酸酶活性為97.7%，實驗組B的相對酪胺酸酶活性為71.7%，而實驗組C的相對酪胺酸酶活性為60%。也就是說，相對於空白組，麴酸組的相對酪胺酸酶活性顯著降低約14.8%，實驗組A的相對酪胺酸酶活性降低約2.3%，實驗組B的相對酪胺酸酶活性顯著降低約28.3%，而實驗組C的相對酪胺酸酶活性顯著降低約40%。並且，相對於實驗組B，實驗組C亦顯著降低約11.7%的相對酪胺酸酶活性。See Figure 4. The blank group was not treated with samples or kojic acid, so the test results of the blank group represent the performance of B16F10 cells under normal physiological metabolism. Here, when the relative tyrosinase activity of the blank group is set to 100%, the relative tyrosinase activity of the koji acid group is 85.2%, and the relative tyrosinase activity of the experimental group A is 97.7%. The relative tyrosinase activity of group B was 71.7%, while the relative tyrosinase activity of experimental group C was 60%. That is to say, compared with the blank group, the relative tyrosinase activity of the koji acid group was significantly reduced by about 14.8%, the relative tyrosinase activity of experimental group A was reduced by about 2.3%, and the relative tyrosinase activity of experimental group B was significantly reduced by about 14.8%. The relative tyrosinase activity of experimental group C was significantly reduced by approximately 28.3%, while the relative tyrosinase activity of experimental group C was significantly reduced by approximately 40%. Moreover, compared to experimental group B, experimental group C also significantly reduced the relative tyrosinase activity by about 11.7%.

酪胺酸酶是生物體內黑色素合成的關鍵酵素， L-多巴在酪胺酸酶的作用下，會產生多巴醌（Dopaquinone）並進而形成黑色素。因此透過藉由黑色素的檢測，可評估酪胺酸酶活性，並評估黑色素細胞產生黑色素的能力。Tyrosinase is the key enzyme for the synthesis of melanin in the body. Under the action of tyrosinase, L-dopa will produce dopaquinone and then form melanin. Therefore, by detecting melanin, tyrosinase activity can be evaluated and the ability of melanocytes to produce melanin can be evaluated.

由實驗結果可知，秋姬李萃取液、經厭氧發酵所得的秋姬李發酵物A、以及經有氧發酵所得的秋姬李發酵物B皆能降低酪胺酸酶活性。而經厭氧發酵所得的秋姬李發酵物A具有顯著降低酪胺酸酶活性的能力，且相較麴酸、秋姬李萃取液及經有氧發酵所得的秋姬李發酵物B優異。換言之，經厭氧發酵所得的秋姬李發酵物能明顯降低酪胺酸酶活性，藉以抑制及/或減少黑色素的合成，進而有益於減少個體的黑色素以及提升個體皮膚的通透與亮白程度。It can be seen from the experimental results that the plum extract, the fermented product A obtained by anaerobic fermentation, and the fermented product B obtained by aerobic fermentation can all reduce tyrosinase activity. The fermented product A of Prunus pumila obtained through anaerobic fermentation has the ability to significantly reduce the tyrosinase activity, and is superior to koji acid, Prunus prunus extract and the fermented product B obtained from aerobic fermentation. In other words, the Qiuji Plum ferment obtained through anaerobic fermentation can significantly reduce the tyrosinase activity, thereby inhibiting and/or reducing the synthesis of melanin, which is beneficial to reducing individual melanin and improving the transparency and whitening of individual skin. .

例五Example 5

A. 材料與儀器：A. Materials and instruments:

1. 細胞株：小鼠黑色素瘤細胞，購自ATCC，細胞編號6475，以下簡稱B16F10細胞。1. Cell line: Mouse melanoma cells, purchased from ATCC, cell number 6475, hereinafter referred to as B16F10 cells.

4. 1N NaOH：以水及NaOH（購自Sigma，產品編號221465）配製而成。4. 1N NaOH: Prepared with water and NaOH (purchased from Sigma, product number 221465).

5. 胰蛋白酶：以10X胰蛋白酶（購自Thermo，產品編號15400-054）和其9倍體積的DPBS稀釋配製而成。即，將10X胰蛋白酶進行十倍稀釋。5. Trypsin: Prepared by diluting 10X trypsin (purchased from Thermo, product number 15400-054) and 9 times its volume of DPBS. That is, dilute 10X trypsin tenfold.

6. 酵素免疫分析儀（ELISA reader），購自BioTek公司（美國）。6. Enzyme immunoassay analyzer (ELISA reader), purchased from BioTek Company (USA).

B. 試驗流程：B. Test process:

1. 將B16F10細胞以每孔1.5×10 ⁵個的密度，接種於每孔含2 mL的細胞培養基的6孔培養盤中，並在37 ℃下培養24小時。於此，B16F10細胞分為五個試驗組別，其分別為：空白組、麴酸組、實驗組A、實驗組B與實驗組C。各組進行三重複（意即各組各有三孔）。 1. Inoculate B16F10 cells into a 6-well culture plate containing 2 mL of cell culture medium in each well at a density of 1.5×10 ⁵ cells per well, and culture at 37°C for 24 hours. Here, B16F10 cells are divided into five experimental groups, which are: blank group, kojic acid group, experimental group A, experimental group B and experimental group C. Each group was performed in triplicate (meaning each group had three holes).

2. 培養24小時後，將各組更換為2 mL實驗培養基，並於37℃下繼續培養48小時。其中，空白組的實驗培養基為不含樣品或麴酸的細胞培養基；麴酸組的實驗培養基為含有200 μg/mL的麴酸的細胞培養基，其中麴酸已被廣泛認知為具有降低黑色素生成效果的物質；實驗組A的實驗培養基為含有0.0625%(v/v) 的例一製得的秋姬李萃取液的細胞培養基；實驗組B的實驗培養基為含有0.5%(v/v) 的例二製得的秋姬李發酵物B的細胞培養基；以及實驗組C的實驗培養基為含有0.5%(v/v) 的例一製得的秋姬李發酵物A的細胞培養基。2. After 24 hours of culture, replace each group with 2 mL of experimental medium and continue to culture at 37°C for 48 hours. Among them, the experimental culture medium of the blank group is a cell culture medium that does not contain samples or kojic acid; the experimental culture medium of the kojic acid group is a cell culture medium containing 200 μg/mL kojic acid, of which kojic acid has been widely recognized to have the effect of reducing melanin production. substance; the experimental medium of experimental group A is a cell culture medium containing 0.0625% (v/v) of the plum extract prepared in Example 1; the experimental medium of experimental group B is a cell culture medium containing 0.5% (v/v) The cell culture medium of the fermented product B of Prunus prunus L. prepared in Example 2; and the experimental culture medium of experimental group C is the cell culture medium of the fermented product A of Prunus azuris prepared in Example 1 containing 0.5% (v/v).

4. 於潤洗後，添加200 μL胰蛋白酶至各孔中反應3分鐘。反應後，添加細胞培養基以終止反應。而後收集各孔中的懸浮細胞與細胞培養基至對應的離心試管內。4. After rinsing, add 200 μL trypsin to each well and react for 3 minutes. After the reaction, cell culture medium was added to terminate the reaction. Then collect the suspended cells and cell culture medium in each well into the corresponding centrifuge tube.

5. 將各離心試管離心使細胞沉澱、移除各組離心試管內的上清液，再以DPBS清洗並懸浮沉澱細胞，然後反覆進行2次後，得到以200μL DPBS回溶的細胞懸浮液。5. Centrifuge each centrifuge tube to pellet the cells, remove the supernatant in each group of centrifuge tubes, and then wash and suspend the precipitated cells with DPBS. Repeat this twice to obtain a cell suspension that is redissolved in 200 μL DPBS.

6. 將細胞懸浮液以液態氮冷凍約10秒後，置於室溫約30分鐘至完全解凍。6. Freeze the cell suspension in liquid nitrogen for about 10 seconds, then place it at room temperature for about 30 minutes until it is completely thawed.

7. 完全解凍後，將離心試管離心。而後移除離心試管內的上清液後，加入120 μL 1N NaOH至各離心試管後，置於60℃的乾浴槽加熱1小時使黑色素溶出，以獲得各組的待檢測樣本。7. Once completely thawed, centrifuge the tube. After removing the supernatant from the centrifuge tubes, add 120 μL 1N NaOH to each centrifuge tube, and place it in a dry bath at 60°C for 1 hour to dissolve the melanin to obtain the samples to be tested in each group.

8. 各組取100 μL各組的待檢測樣本至96孔培養盤，並利用酵素免疫分析儀測量每孔405 nm的吸光值（OD ₄₀₅值）。 8. Take 100 μL of the sample to be tested from each group into a 96-well culture plate, and use an enzyme immunoassay analyzer to measure the absorbance value at 405 nm (OD ₄₀₅ value) of each well.

C. 試驗結果：C. Test results:

所有組別的相對黑色素含量係依下列公式計算：相對黑色素含量（%）=（各組OD ₄₀₅值/空白組OD ₄₀₅值）×100%。 The relative melanin content of all groups was calculated according to the following formula: relative melanin content (%) = (OD ₄₀₅ value of each group/OD ₄₀₅ value of the blank group) × 100%.

請參閱圖5。空白組未使用樣品或麴酸進行處理，因此空白組的試驗結果代表B16F10細胞在正常的生理代謝情況下的表現。於此，在設定空白組的相對黑色素含量為100%的情況下，實驗組A的相對黑色素含量為107.3%，實驗組B的相對黑色素含量為104.7%，而實驗組C的相對黑色素含量為87.9%。也就是說，相對於空白組，實驗組A的相對黑色素含量提升約7.3%，實驗組B的相對黑色素含量提升約4.7%，而實驗組C的相對黑色素含量顯著降低約12.1%。並且，相對於實驗組B，實驗組C的相對黑色素含量亦顯著降低約16.8%。See Figure 5. The blank group was not treated with samples or kojic acid, so the test results of the blank group represent the performance of B16F10 cells under normal physiological metabolism. Here, when the relative melanin content of the blank group is set to 100%, the relative melanin content of experimental group A is 107.3%, the relative melanin content of experimental group B is 104.7%, and the relative melanin content of experimental group C is 87.9 %. In other words, compared with the blank group, the relative melanin content of experimental group A increased by approximately 7.3%, the relative melanin content of experimental group B increased by approximately 4.7%, and the relative melanin content of experimental group C significantly decreased by approximately 12.1%. Moreover, compared with experimental group B, the relative melanin content of experimental group C also significantly decreased by about 16.8%.

由此可知，秋姬李萃取液及經有氧發酵所得的秋姬李發酵物B皆無法降低B16F10細胞的黑色素含量，反而提升B16F10細胞的黑色素含量。而經厭氧發酵所得的秋姬李發酵物A具有顯著降低黑色素含量的能力，且相較秋姬李萃取液及經有氧發酵所得的秋姬李發酵物B優異。換言之，經厭氧發酵所得的秋姬李發酵物能減少黑色素及/或抑制黑色素生成，藉以減少個體的黑色素，進而提升個體皮膚的通透與亮白程度。It can be seen from this that neither the plum extract nor the fermented substance B obtained by aerobic fermentation can reduce the melanin content of B16F10 cells, but instead increase the melanin content of B16F10 cells. The fermented product A of Prunus pumila obtained through anaerobic fermentation has the ability to significantly reduce the melanin content, and is superior to the Prunus prunus extract and the fermented product B obtained through aerobic fermentation. In other words, the Qiuji plum ferment obtained through anaerobic fermentation can reduce melanin and/or inhibit melanin production, thereby reducing an individual's melanin, thereby improving the transparency and whitening of the individual's skin.

例六Example 6

A. 材料與儀器：A. Materials and instruments:

1. 細胞株：人類皮膚纖維母細胞，購自BCRC（Bioresource Collection and Research Center，生物資源保存及研究中心），細胞編號60153，以下簡稱CCD-966SK細胞。1. Cell line: Human dermal fibroblasts, purchased from BCRC (Bioresource Collection and Research Center), cell number 60153, hereinafter referred to as CCD-966SK cells.

2. 細胞培養基：MEM（Minimum essential medium，購自Gibco，產品編號11095080），添加10% FBS（Fetal Bovine Serum，購自Gibco，產品編號10437-028）、1% 青黴素-鏈黴素（購自Gibco，產品編號15140122）、1 mM 丙酮酸鈉（sodium pyruvate，購自Gibco，產品編號11360-070）、1.5 g/L 碳酸氫鈉（sodium bicarbonate，購自Sigma，產品編號S5761-500G）以及0.1 mM非必需氨基酸（non-essential amino acids，購自Gibco，產品編號11140050）。2. Cell culture medium: MEM (Minimum essential medium, purchased from Gibco, product number 11095080), added with 10% FBS (Fetal Bovine Serum, purchased from Gibco, product number 10437-028), 1% penicillin-streptomycin (purchased from Gibco, product number 15140122), 1 mM sodium pyruvate (purchased from Gibco, product number 11360-070), 1.5 g/L sodium bicarbonate (purchased from Sigma, product number S5761-500G), and 0.1 mM non-essential amino acids (purchased from Gibco, product number 11140050).

3. 胰蛋白酶：以10X胰蛋白酶（購自Gibco，產品編號15400-054）和其9倍體積的DPBS稀釋配製而成。3. Trypsin: Prepared by diluting 10X trypsin (purchased from Gibco, product number 15400-054) and 9 times its volume of DPBS.

4. Assay buffer：以10X Assay buffer（MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit中的試劑，購自BD，產品編號BDB551302）和其9倍體積的DPBS稀釋配製而成，並置於恆溫水浴槽使其溫度維持於 37℃。4. Assay buffer: Prepared by diluting 10X Assay buffer (reagent in MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit, purchased from BD, product number BDB551302) and 9 times its volume of DPBS, and place it in a constant temperature water bath to bring it to temperature Maintain at 37°C.

5. JC-1 作用試劑：以130 μL DMSO（購自ECHO，產品編號DA1101-000000-72EC）溶解JC-1 dye（MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit中的試劑，購自BD，產品編號BDB551302）以形成JC-1的儲存溶液。接著，再以JC-1的儲存溶液和其99倍體積的Assay buffer稀釋配製而成。5. JC-1 action reagent: Dissolve JC-1 dye (reagent in MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit, purchased from BD, product number BDB551302) with 130 μL DMSO (purchased from ECHO, product number DA1101-000000-72EC) ) to form a storage solution of JC-1. Then, the stock solution of JC-1 and its 99-fold volume of Assay buffer were diluted to prepare the solution.

6. 流式細胞儀，購自BD。6. Flow cytometer, purchased from BD.

B. 試驗流程：B. Test process:

1. 將CCD-966SK細胞以每孔1×10 ⁵個的密度，接種於每孔含2 mL的細胞培養基的6孔培養盤中，並在37 ℃下培養24小時。於此，CCD-966SK細胞分為四個試驗組別，其分別為：空白組、實驗組A、實驗組B與實驗組C。各組進行三重複（意即各組各有三孔）。 1. Inoculate CCD-966SK cells into a 6-well culture plate containing 2 mL of cell culture medium in each well at a density of 1×10 ⁵ cells per well, and culture at 37°C for 24 hours. Here, CCD-966SK cells are divided into four experimental groups, which are: blank group, experimental group A, experimental group B and experimental group C. Each group was performed in triplicate (meaning each group had three holes).

2. 培養24小時後，將各組更換為實驗培養基，並於37℃下繼續培養24小時。其中，空白組的實驗培養基為不含樣品的細胞培養基；實驗組A的實驗培養基為含有0.0625%(v/v) 的例一製得的秋姬李萃取液的細胞培養基；實驗組B的實驗培養基為含有0.5%(v/v) 的例二製得的秋姬李發酵物B的細胞培養基；以及實驗組C的實驗培養基為含有0.5%(v/v) 的例一製得的秋姬李發酵物A的細胞培養基。2. After 24 hours of culture, replace each group with the experimental medium and continue to culture at 37°C for 24 hours. Among them, the experimental culture medium of the blank group is a cell culture medium without samples; the experimental culture medium of the experimental group A is a cell culture medium containing 0.0625% (v/v) of the plum extract prepared in Example 1; the experimental culture medium of the experimental group B The culture medium is a cell culture medium containing 0.5% (v/v) of the Akihime fermentation product B prepared in Example 2; and the experimental culture medium of the experimental group C is a cell culture medium containing 0.5% (v/v) of the Akihime fermented in Example 1. Cell culture medium of plum ferment A.

4. 於潤洗後，添加200 μL胰蛋白酶至各孔中反應5分鐘。反應後，添加細胞培養基以終止反應。而後收集各孔中的懸浮細胞與細胞培養基至對應的離心試管內，將各離心試管離心使細胞沉澱。4. After rinsing, add 200 μL trypsin to each well and react for 5 minutes. After the reaction, cell culture medium was added to terminate the reaction. Then collect the suspended cells and cell culture medium in each well into the corresponding centrifuge tube, and centrifuge each centrifuge tube to precipitate the cells.

5. 移除各組離心試管內的上清液，並以DPBS清洗沉澱細胞2次後，再以100μL JC-1 作用試劑重新懸浮細胞，混合均勻並避光作用15分鐘。5. Remove the supernatant from the centrifuge tubes in each group, wash the precipitated cells twice with DPBS, then resuspend the cells with 100 μL JC-1 action reagent, mix evenly, and incubate in the dark for 15 minutes.

6. 作用後，將各組離心試管離心並移除上清液，再以Assay buffer清洗沉澱細胞2次。6. After the action, centrifuge the centrifuge tubes of each group and remove the supernatant, and then wash the precipitated cells twice with Assay buffer.

7. 於清洗後，於各組離心試管中添加500μL DPBS重新懸浮細胞以形成細胞懸浮液。7. After washing, add 500 μL DPBS to each set of centrifuge tubes to resuspend the cells to form a cell suspension.

8. 將流式細胞儀的激發光設定為488 nm，散射光設定為527 nm（FITC）及590 nm（PE）後，利用流式細胞儀檢測各組的綠色（527 nm（FITC））螢光訊號以及紅色（590 nm（PE））螢光訊號。8. After setting the excitation light of the flow cytometer to 488 nm and the scattered light to 527 nm (FITC) and 590 nm (PE), use the flow cytometer to detect the green (527 nm (FITC)) fluorescent light in each group. optical signal and red (590 nm (PE)) fluorescent signal.

C. 試驗結果：C. Test results:

所有組別的相對粒線體活性係依下列公式計算：相對粒線體活性（%）=（各組紅色螢光訊號值/空白組紅色螢光訊號值）×100%。The relative mitochondrial activity of all groups was calculated according to the following formula: relative mitochondrial activity (%) = (red fluorescence signal value of each group/red fluorescence signal value of the blank group) × 100%.

空白組與其他各組的試驗結果之間的統計學顯著差異是以學生t檢驗（student t-test）統計分析得到。於圖式中，「*」代表在與空白組比較下其p值小於0.05、「**」代表在與空白組比較下其p值小於0.01，以及「***」代表在與空白組比較下其p值小於0.001。Statistically significant differences between the test results of the blank group and other groups were obtained using student t-test statistical analysis. In the figure, "*" means that the p value is less than 0.05 when compared with the blank group, "**" means that the p value is less than 0.01 when compared with the blank group, and "***" means that the p value is less than 0.01 when compared with the blank group. Its p-value is less than 0.001.

請參閱圖6。空白組未使用樣品進行處理，因此空白組的試驗結果代表CCD-966SK細胞在正常的生理代謝情況下的表現。於此，在設定空白組的相對粒線體活性為100%的情況下，實驗組A的相對粒線體活性為126.9%，實驗組B的相對粒線體活性為120.95%，而實驗組C的相對粒線體活性為145.76%。也就是說，相對於空白組，實驗組A的相對粒線體活性顯著提升約26.9%，實驗組B的相對粒線體活性顯著提升約20.95%，而實驗組C的相對粒線體活性顯著提升約45.76%。See Figure 6. The blank group was not processed with samples, so the test results of the blank group represent the performance of CCD-966SK cells under normal physiological metabolism. Here, when the relative mitochondrial activity of the blank group is set to 100%, the relative mitochondrial activity of experimental group A is 126.9%, the relative mitochondrial activity of experimental group B is 120.95%, and the relative mitochondrial activity of experimental group C is 120.95%. The relative mitochondrial activity was 145.76%. That is to say, compared with the blank group, the relative mitochondrial activity of experimental group A was significantly increased by about 26.9%, the relative mitochondrial activity of experimental group B was significantly increased by about 20.95%, and the relative mitochondrial activity of experimental group C was significantly increased. An increase of approximately 45.76%.

由此可知，秋姬李萃取液、經厭氧發酵所得的秋姬李發酵物A、以及經有氧發酵所得的秋姬李發酵物B皆能明顯提升粒線體活性。而經厭氧發酵所得的秋姬李發酵物A具有顯著提升粒線體活性的能力，且相較秋姬李萃取液及經有氧發酵所得的秋姬李發酵物B優異。粒線體是細胞的發電機，扮演能量傳遞的重要角色，當其活性越高，代表細胞生理活動越旺盛。換言之，經厭氧發酵所得的秋姬李發酵物能提升粒線體活性，藉以提升細胞生理活動、並且調控細胞凋亡與老化。It can be seen from this that the Prunus quinquefolia extract, the Prunus quinquefolia fermented product A obtained through anaerobic fermentation, and the Prunus quinquefolia fermented product B obtained through aerobic fermentation can significantly increase mitochondrial activity. The fermented product A of Prunus prunus, obtained through anaerobic fermentation, has the ability to significantly increase mitochondrial activity, and is superior to the Prunus azure extract and the fermented product B obtained through aerobic fermentation. Mitochondria are the generators of cells and play an important role in energy transmission. The higher their activity, the more vigorous the physiological activities of the cells. In other words, the Qiuji Plum fermentation product obtained through anaerobic fermentation can increase mitochondrial activity, thereby increasing cell physiological activities and regulating cell apoptosis and aging.

例七Example 7

A. 材料與儀器：A. Materials and instruments:

2. 細胞培養基：MEM（Minimum essential medium，購自Thermo，產品編號61100061），添加10% FBS（Fetal Bovine Serum，購自Gibco，產品編號10437-028）、1% 青黴素-鏈黴素（購自Gibco，產品編號15140122）、1 mM 丙酮酸鈉（sodium pyruvate，購自Gibco，產品編號11360-070）、1.5 g/L 碳酸氫鈉（sodium bicarbonate，購自Sigma，產品編號S5761-500G）以及0.1 mM非必需氨基酸（non-essential amino acids，購自Gibco，產品編號11140050）。2. Cell culture medium: MEM (Minimum essential medium, purchased from Thermo, product number 61100061), added with 10% FBS (Fetal Bovine Serum, purchased from Gibco, product number 10437-028), 1% penicillin-streptomycin (purchased from Gibco, product number 15140122), 1 mM sodium pyruvate (purchased from Gibco, product number 11360-070), 1.5 g/L sodium bicarbonate (purchased from Sigma, product number S5761-500G), and 0.1 mM non-essential amino acids (purchased from Gibco, product number 11140050).

3. Assay buffer：以10X Assay buffer（MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit中的試劑，購自BD，產品編號BDB551302）和其9倍體積的DPBS稀釋配製而成，並置於恆溫水浴槽使其溫度維持於 37℃。3. Assay buffer: Prepared by diluting 10X Assay buffer (reagent in MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit, purchased from BD, product number BDB551302) and 9 times its volume of DPBS, and place it in a constant temperature water bath to bring it to temperature Maintain at 37°C.

4. JC-1 作用試劑：以130 μL DMSO（購自ECHO，產品編號DA1101-000000-72EC）溶解JC-1 dye（MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit中的試劑，購自BD，產品編號BDB551302）以形成JC-1的儲存溶液。接著，再以JC-1的儲存溶液和其99倍體積的Assay buffer稀釋配製而成。4. JC-1 action reagent: Dissolve JC-1 dye (reagent in MitoScreen Flow Cytometry Mitochondrial Membrane Potential Detection Kit, purchased from BD, product number BDB551302) with 130 μL DMSO (purchased from ECHO, product number DA1101-000000-72EC) ) to form a storage solution of JC-1. Then, the stock solution of JC-1 and its 99-fold volume of Assay buffer were diluted to prepare the solution.

5. Hoechst染劑，購自Thermo，產品編號D 33342。5. Hoechst dye, purchased from Thermo, product number D 33342.

6. 顯微鏡，購自ZEISS，產品編號Axio Vert A1。6. Microscope, purchased from ZEISS, product number Axio Vert A1.

B. 試驗流程：B. Test process:

1. 將CCD-966SK細胞以每孔1×10 ⁴個的密度，接種於每孔含0.5 mL的細胞培養基的24孔培養盤中，並在37 ℃下培養24小時。於此，CCD-966SK細胞分為四個試驗組別，其分別為：空白組、實驗組A、實驗組B與實驗組C。各組進行三重複（意即各組各有三孔）。 1. Inoculate CCD-966SK cells into a 24-well culture plate containing 0.5 mL of cell culture medium in each well at a density of 1×10 ⁴ cells per well, and culture at 37°C for 24 hours. Here, CCD-966SK cells are divided into four experimental groups, which are: blank group, experimental group A, experimental group B and experimental group C. Each group was performed in triplicate (meaning each group had three holes).

3. 移除培養後的各組的實驗培養基，並以DPBS進行潤洗1次。3. Remove the experimental culture medium of each group after culture and rinse once with DPBS.

4. 於潤洗後，添加300μL JC-1 作用試劑並輕微搖晃以確保孔盤底部皆有JC-1 作用試劑覆蓋後，避光靜置 15 分鐘。4. After rinsing, add 300μL JC-1 reagent and shake slightly to ensure that the bottom of the well plate is covered with JC-1 reagent, then let it stand in the dark for 15 minutes.

5. 避光靜置後，移除各組的JC-1 作用試劑，並以DPBS進行潤洗1次。5. After standing in the dark, remove the JC-1 reagent from each group and rinse once with DPBS.

6. 於潤洗後，添加Hoechst染劑（1:20000 稀釋），並避光染色5分鐘。6. After rinsing, add Hoechst dye (1:20000 dilution) and stain for 5 minutes in the dark.

7. 於避光染色後，移除各組的Hoechst染劑，並添加500μL細胞培養基，以維持細胞在拍攝過程中的健康狀態。7. After staining in the dark, remove the Hoechst dye from each group and add 500 μL of cell culture medium to maintain the health of the cells during the shooting process.

8. 利用顯微鏡拍攝各組的染色結果。其中，於染色結果中，紅色螢光係代表正常的粒線體的訊號；綠色螢光係代表細胞凋亡的粒線體的訊號；而藍色螢光係代表細胞核的訊號。8. Use a microscope to photograph the staining results of each group. Among them, in the staining results, the red fluorescence represents the signal of normal mitochondria; the green fluorescence represents the signal of apoptotic mitochondria; and the blue fluorescence represents the signal of the nucleus.

C. 試驗結果：C. Test results:

請參閱圖7。空白組的綠色螢光比例明顯高於紅色螢光。相對於空白組，實驗組A的綠色螢光比例明顯下降，紅色螢光比例明顯上升，其表示秋姬李萃取液能明顯提升粒線體活性，即明顯減少細胞凋亡。相對於空白組，實驗組B的綠色螢光與紅色螢光比例近似於空白組，然其綠色螢光強度明顯下降，其表示經有氧發酵所得的秋姬李發酵物B能些微提升粒線體活性，即些微減少細胞凋亡。相對於空白組，實驗組C的綠色螢光比例明顯下降，紅色螢光比例明顯上升，其表示經厭氧發酵所得的秋姬李發酵物A能明顯提升粒線體活性，即明顯減少細胞凋亡。See Figure 7. The proportion of green fluorescence in the blank group was significantly higher than that of red fluorescence. Compared with the blank group, the proportion of green fluorescence in experimental group A significantly decreased, and the proportion of red fluorescence increased significantly, which indicated that the plum extract can significantly increase mitochondrial activity, that is, significantly reduce cell apoptosis. Compared with the blank group, the ratio of green fluorescence to red fluorescence in the experimental group B is similar to that of the blank group, but its green fluorescence intensity decreases significantly, which means that the Qiuji plum fermented product B obtained through aerobic fermentation can slightly improve the grain line. body activity, that is, slightly reducing cell apoptosis. Compared with the blank group, the proportion of green fluorescence in experimental group C decreased significantly, and the proportion of red fluorescence increased significantly, which indicates that the fermented product A of Prunus pumila obtained through anaerobic fermentation can significantly increase mitochondrial activity, that is, significantly reduce cell apoptosis. Death.

由此可知，秋姬李萃取液、經厭氧發酵所得的秋姬李發酵物A、以及經有氧發酵所得的秋姬李發酵物B皆能提升粒線體活性。而經厭氧發酵所得的秋姬李發酵物A具有顯著提升粒線體活性的能力，且相較秋姬李萃取液及經有氧發酵所得的秋姬李發酵物B優異。粒線體是細胞的發電機，扮演能量傳遞的重要角色，當其活性越高，代表細胞生理活動越旺盛。換言之，經厭氧發酵所得的秋姬李發酵物能提升粒線體活性，藉以提升細胞生理活動、並且調控細胞凋亡與老化。It can be seen from this that the plum extract, the fermented product A obtained by anaerobic fermentation, and the fermented product B obtained by aerobic fermentation can all increase mitochondrial activity. The fermented product A of Prunus prunus, obtained through anaerobic fermentation, has the ability to significantly increase mitochondrial activity, and is superior to the Prunus azure extract and the fermented product B obtained through aerobic fermentation. Mitochondria are the generators of cells and play an important role in energy transmission. The higher their activity, the more vigorous the physiological activities of the cells. In other words, the Qiuji Plum fermentation product obtained through anaerobic fermentation can increase mitochondrial activity, thereby increasing cell physiological activities and regulating cell apoptosis and aging.

例八Example 8

A. 試驗流程：A. Test process:

令12位欲美白或提升膚況的20至55歲受試者每日飲用一瓶50 mL試驗飲品（其含有6 mL的使用例一所製得的秋姬李發酵物A，其餘為水和甜味劑），連續飲用8週（即56日）。12 subjects aged 20 to 55 who wanted to whiten or improve their skin condition were asked to drink a bottle of 50 mL test drink every day (which contained 6 mL of Qiuji plum fermented product A prepared in usage example 1, and the rest was water and Sweetener), drink continuously for 8 weeks (i.e. 56 days).

受試者於開始飲用前（臉部已清潔，第0週）及飲用56日（臉部已清潔，第8週）後，進行肌膚檢測。肌膚檢測為依據不同檢測項目，使用對應的儀器及測量方式，紀錄臉部肌膚的數值、並拍攝使用前及使用後的照片。肌膚檢測項目有肌膚斑點（skin spot）及肌膚棕色斑（skin brown spot）。並且，於使用前及使用後進行檢測時，受試者所在的測試區域的溫度與濕度為一致，以減少外界的溫濕度等因素會對肌膚所造成的影響。The subjects conducted skin tests before starting to drink (face was cleansed, week 0) and after drinking for 56 days (face was cleansed, week 8). Skin testing is based on different testing items, using corresponding instruments and measurement methods to record facial skin values and take photos before and after use. Skin testing items include skin spots and skin brown spots. Moreover, when testing before and after use, the temperature and humidity of the test area where the subject is located are consistent to reduce the impact of external factors such as temperature and humidity on the skin.

肌膚斑點係使用購自美國Canfield scientific公司的VISIA高階數位膚質檢測儀（VISIA Complexion Analysis System）對受試者在飲用試驗飲品前，以及飲用56日後的面部肌膚進行檢測。此檢測儀係透過可見光（白光）拍攝高解析度的肌膚圖像，並以內建軟體根據肉眼可見的色素斑點數量與面積進行分析以得到可代表皮膚的斑點狀況的一數值（以下稱肌膚斑點程度值）。並且，得到的肌膚斑點程度值越高，說明肌膚斑點程度越高。然後，再以下列公式計算出相對肌膚斑點程度：相對肌膚斑點程度（%）=（各組肌膚斑點程度值 /使用前肌膚斑點程度值）×100%。Skin spots were measured using the VISIA Complexion Analysis System purchased from Canfield Scientific in the United States to test the subjects' facial skin before drinking the test drink and 56 days after drinking it. This detector takes high-resolution skin images through visible light (white light), and uses built-in software to analyze the number and area of pigment spots visible to the naked eye to obtain a value that represents the spot status of the skin (hereinafter referred to as skin spots). degree value). Moreover, the higher the obtained skin spot level value is, the higher the skin spot level is. Then, the relative skin spot degree is calculated using the following formula: relative skin spot degree (%) = (skin spot degree value of each group / skin spot degree value before use) × 100%.

肌膚棕色斑係使用購自美國Canfield scientific公司的VISIA高階數位膚質檢測儀（VISIA Complexion Analysis System）對受試者在飲用試驗飲品前，以及飲用56日後的面部肌膚進行檢測。此檢測儀係透過RBX偏振光技術進行臉部肌膚拍攝，偵測肉眼不可見的真皮層黑色素斑以得到可代表皮膚的棕色斑狀況的一數值（以下稱肌膚棕色斑程度值）。並且，得到的肌膚棕色斑程度值越高，說明肌膚棕色斑程度越高。然後，再以下列公式計算出相對肌膚棕色斑程度：相對肌膚棕色斑程度（%）=（各組肌膚棕色斑程度值 /使用前肌膚棕色斑程度值）×100%。The brown spots on the skin were measured using the VISIA Complexion Analysis System purchased from Canfield Scientific in the United States to test the subjects' facial skin before drinking the test drink and 56 days after drinking it. This detector uses RBX polarized light technology to take pictures of facial skin, detect melanin spots in the dermis that are invisible to the naked eye, and obtain a value that can represent the brown spot status of the skin (hereinafter referred to as the skin brown spot level value). Moreover, the higher the obtained skin brown spot degree value is, the higher the skin brown spot degree is. Then, the relative skin brown spot degree is calculated using the following formula: relative skin brown spot degree (%) = (skin brown spot degree value of each group / skin brown spot degree value before use) × 100%.

B. 試驗結果：B. Test results:

1. 關於「肌膚斑點」的檢測結果1. About the test results of "skin spots"

參照圖8。將12位受試者第0週的肌膚斑點程度值視為100%的相對肌膚斑點程度。此時，在第8週（即持續飲用試驗飲品8週後），其平均相對肌膚斑點程度為96.4%。換言之，相較於飲用試驗飲品前（第0週），持續飲用8週含有秋姬李發酵物A的試驗飲品後可使此些受試者的相對肌膚斑點程度減少3.6%，並且改善人數比率達66.7%（8位）。由此可知，經厭氧發酵所得的秋姬李發酵物確實可減少肌膚斑點或淡化可見斑點，並改善受試者肌膚狀況，進而提升肌膚質感。Refer to Figure 8. The skin spot level values of the 12 subjects at week 0 were regarded as 100% relative skin spot level. At this time, at week 8 (that is, after 8 weeks of continuous drinking of the test drink), the average relative skin spot degree was 96.4%. In other words, compared with before drinking the test drink (week 0), continuous drinking of the test drink containing Akihime plum ferment A for 8 weeks can reduce the relative skin spots of these subjects by 3.6%, and improve the number ratio Reaching 66.7% (8th place). It can be seen from this that the Qiuji plum ferment obtained through anaerobic fermentation can indeed reduce skin spots or lighten visible spots, and improve the skin condition of the subjects, thereby improving the skin texture.

2. 關於「肌膚棕色斑」的檢測結果2. About the test results of "brown spots on skin"

參照圖9。將12位受試者第0週的肌膚棕色斑程度值視為100%的相對肌膚棕色斑程度。此時，在第8週（即持續飲用試驗飲品8週後），其平均相對肌膚棕色斑程度為93.3%。換言之，相較於飲用試驗飲品前（第0週），持續飲用8週含秋姬李發酵物A的試驗飲品後可使此些受試者的相對肌膚棕色斑程度減少6.7%，並且改善人數比率達66.7%（8位）。由此可知，經厭氧發酵所得的秋姬李發酵物確實可減少肌膚棕色斑或深層班，並改善受試者肌膚狀況，進而提升肌膚質感。Refer to Figure 9. The skin brown spot degree value of the 12 subjects at week 0 was regarded as 100% of the relative skin brown spot degree. At this time, at week 8 (that is, after 8 weeks of continuous drinking of the test drink), the average relative skin brown spot degree was 93.3%. In other words, compared with before drinking the test drink (week 0), continuous drinking of the test drink containing Akihime plum ferment A for 8 weeks can reduce the relative skin brown spots of these subjects by 6.7%, and improve the number of The ratio reaches 66.7% (8th place). It can be seen from this that the fermented Akihime plum ferment obtained through anaerobic fermentation can indeed reduce brown spots or deep spots on the skin, improve the skin condition of the subjects, and thereby enhance the skin texture.

例九Example 9

A. 材料與儀器：A. Materials and instruments:

1. 高解析液相層析質譜儀：串連超高效液相層析儀（Ultimate 3000 HPLC, Thermo Fisher Scientific）與高解析度軌道式離子阱質譜儀（Q-EXACTIVE System with Ion Max Source，Thermo Fisher Scientific）測定，單位為 m/z。1. High-resolution liquid chromatography mass spectrometer: tandem ultra-high performance liquid chromatography (Ultimate 3000 HPLC, Thermo Fisher Scientific) and high-resolution orbital ion trap mass spectrometer (Q-EXACTIVE System with Ion Max Source, Thermo Fisher Scientific), the unit is m/z.

2. 高效能液相層析儀（High Performance Liquid Chromatography，HPLC）： Hitachi chromaster 5260系列；沖提溶劑輸送係Hitachi chromaster 5110；管柱恆溫裝置Hitachi chromaster 5310；光二極體陣列偵測器(Diode Array Detector, DAD)係Hitachi chromaster 5430，偵測波長為210 nm。2. High Performance Liquid Chromatography (HPLC): Hitachi chromaster 5260 series; extraction solvent delivery system Hitachi chromaster 5110; column thermostat Hitachi chromaster 5310; photodiode array detector (Diode Array Detector, DAD) is Hitachi chromaster 5430, with a detection wavelength of 210 nm.

3. HPLC分析管柱：Mightysil RP-18 GP 250（Kanto，250 x 4.6 mm，5μm）。3. HPLC analysis column: Mightysil RP-18 GP 250 (Kanto, 250 x 4.6 mm, 5μm).

4. 管柱層析（Column Chromatography）填充材料：4. Column Chromatography packing materials:

(1) 大孔樹脂：Diaion HP-20，購自三菱化學公司，日本。(1) Macroporous resin: Diaion HP-20, purchased from Mitsubishi Chemical Company, Japan.

(2) 正相矽膠：Merck Kieselgel 60，40-63 um，購自默克，德國，產品編號Art. 9385。(2) Normal phase silica gel: Merck Kieselgel 60, 40-63 um, purchased from Merck, Germany, product number Art. 9385.

(3) 逆相矽膠：Merck LiChroprep® RP-18，40-63 um，購自默克，德國，產品編號Art. 0250。(3) Reverse phase silica: Merck LiChroprep® RP-18, 40-63 um, purchased from Merck, Germany, product number Art. 0250.

5. 溶劑（solvent）：甲醇（methanol），購自默克台灣。5. Solvent: methanol, purchased from Merck Taiwan.

6. 聚偏氟乙烯薄膜過濾（Polyvinylidene fluoride membrane filters，PVDF），孔徑0.22微米，購自Millipore，美國。6. Polyvinylidene fluoride membrane filters (PVDF), pore size 0.22 micron, purchased from Millipore, USA.

7. 樣品：(1)例一所製得的秋姬李萃取液，以及(2)例一所製得的秋姬李發酵物A。7. Samples: (1) Prunus prunus extract prepared in Example 1, and (2) Fermented product A of Prunus truncatula prepared in Example 1.

B. HPLC分析流程：B. HPLC analysis process:

1. 秋姬李萃取液及秋姬李發酵物A，係以聚偏氟乙烯薄膜過濾進行過濾。為了將秋姬李萃取液及秋姬李發酵物A的脂溶性成分與水溶性成分分離，以正丁醇為溶劑，將例一所製得的秋姬李萃取液及秋姬李發酵物A進行液相-液相分配萃取，分別得到秋姬李萃取液的正丁醇可溶部（脂溶性成分）和秋姬李萃取液的水可溶部（水溶性成分），以及秋姬李發酵物A的正丁醇可溶部和秋姬李發酵物A的水可溶部。1. Plum extract and Plum ferment A are filtered through polyvinylidene fluoride membrane filtration. In order to separate the fat-soluble components and water-soluble components of the plum extract and plum ferment A, n-butanol was used as the solvent, and the plum extract and ferment A prepared in Example 1 were separated. Liquid-liquid phase partition extraction was performed to obtain the n-butanol soluble part (fat-soluble component) of the plum extract and the water-soluble part (water-soluble component) of the plum extract, as well as the fermentation of the plum. The n-butanol soluble part of substance A and the water-soluble part of Akihime plum fermentation substance A.

2. 使用HPLC對各樣品的組分進行分析，以得到各樣品的指紋圖譜。於此，以甲醇（A）與水（B）為溶劑，額外添加0.1%甲酸，並設定流速為1 mL/min。沖提條件：0分鐘時，甲醇（A）: 水（B）＝2:98；10分鐘時，A:B＝2:98；40分鐘時，A:B＝70:30；50分鐘時，A:B＝100:0；60分鐘時，A:B＝100:0。其中，樣品為秋姬李萃取液樣品（包含秋姬李萃取液的正丁醇可溶部和秋姬李萃取液的水可溶部）以及秋姬李發酵物A樣品（包含秋姬李發酵物A的正丁醇可溶部和秋姬李發酵物A的水可溶部），並且樣品濃度皆為50 mg/mL，注射量為10 μL。其中，於HPLC分析時，管柱溫度為40 ℃。2. Use HPLC to analyze the components of each sample to obtain the fingerprint of each sample. Here, methanol (A) and water (B) were used as solvents, additional 0.1% formic acid was added, and the flow rate was set to 1 mL/min. Elution conditions: at 0 minutes, methanol (A): water (B) = 2:98; at 10 minutes, A:B = 2:98; at 40 minutes, A:B = 70:30; at 50 minutes, A:B=100:0; at 60 minutes, A:B=100:0. Among them, the samples are the plum extract sample (including the n-butanol soluble part of the plum extract and the water-soluble part of the plum extract) and the fermented product A sample of the plum fermented product (including the fermentation product of the plum The n-butanol soluble part of substance A and the water-soluble part of plum fermentation substance A), and the sample concentration is 50 mg/mL, and the injection volume is 10 μL. Among them, during HPLC analysis, the column temperature was 40°C.

C. HPLC分析結果：C. HPLC analysis results:

請參閱圖10。秋姬李萃取液的HPLC指紋圖譜較無明顯波鋒，且峰度較低。而相對於秋姬李萃取液，秋姬李發酵物A的HPLC指紋圖譜有較多明顯波鋒（波鋒02-S、01、03、05、08、04、06、07以及09），且峰度較高。也就是說，相對於秋姬李萃取液，經由微生物厭氧發酵所得的秋姬李發酵物得以大幅增加其所含有的化合物，甚至是具有秋姬李萃取液所未含有的化合物。See Figure 10. The HPLC fingerprint of Plum extract has no obvious wave front and low kurtosis. Compared with the Qiuji Plum extract, the HPLC fingerprint of Qiuji Plum fermentation product A has more obvious wave fronts (wave fronts 02-S, 01, 03, 05, 08, 04, 06, 07 and 09), and The kurtosis is higher. That is to say, compared to the Prunus akiu extract, the fermentation product of Prunus akiu obtained through microbial anaerobic fermentation can significantly increase the number of compounds it contains, and even contains compounds that are not contained in the Plum extract.

由此可知，經厭氧發酵所得的秋姬李發酵物相對於秋姬李萃取液具有更加豐富且多元化的化合物組成。It can be seen from this that the fermented product of Prunus quinquefolius obtained through anaerobic fermentation has a richer and more diversified compound composition than the Prunus quinquefolius extract.

例十Example 10

A. 材料與儀器：A. Materials and instruments:

1. 核磁共振光譜儀（Nuclear Magnetic Resonance Spectrometer，NMR）：1D與2D光譜使用Ascend 400 MHz，Bruker Co.，Germany，以δ表示化學位移（chemical shift），單位為 ppm。1. Nuclear Magnetic Resonance Spectrometer (NMR): Ascend 400 MHz, Bruker Co., Germany was used for 1D and 2D spectra. Chemical shift is represented by δ, and the unit is ppm.

2. 高解析液相層析質譜儀：串連超高效液相層析儀（Ultimate 3000 HPLC, Thermo Fisher Scientific）與高解析度軌道式離子阱質譜儀（Q-EXACTIVE System with Ion Max Source，Thermo Fisher Scientific）測定，單位為 m/z。2. High-resolution liquid chromatography mass spectrometer: tandem ultra-high performance liquid chromatography (Ultimate 3000 HPLC, Thermo Fisher Scientific) and high-resolution orbital ion trap mass spectrometer (Q-EXACTIVE System with Ion Max Source, Thermo Fisher Scientific), the unit is m/z.

3. 中壓液相層析儀（Medium pressure liquid chromatography，MPLC）：CombiFlash® Rf+，Teledyne ISCO，Lincoln，NE。3. Medium pressure liquid chromatography (MPLC): CombiFlash® Rf+, Teledyne ISCO, Lincoln, NE.

4. 高效能液相層析儀（High Performance Liquid Chromatography，HPLC）： Hitachi chromaster 5260系列；沖提溶劑輸送係Hitachi chromaster 5110；管柱恆溫裝置Hitachi chromaster 5310；光二極體陣列偵測器(Diode Array Detector, DAD)係Hitachi chromaster 5430，偵測波長為210 nm。4. High Performance Liquid Chromatography (HPLC): Hitachi chromaster 5260 series; elution solvent delivery system Hitachi chromaster 5110; column thermostat Hitachi chromaster 5310; photodiode array detector (Diode Array Detector, DAD) is Hitachi chromaster 5430, with a detection wavelength of 210 nm.

5. HPLC分析管柱：Mightysil RP-18 GP 250（Kanto，250 x 4.6 mm，5μm）。5. HPLC analysis column: Mightysil RP-18 GP 250 (Kanto, 250 x 4.6 mm, 5μm).

6. HPLC分離管柱：Luna® 5μm C18(2) 100 Å（250 x 10 mm），購自Phenomenex，美國。6. HPLC separation column: Luna® 5μm C18(2) 100 Å (250 x 10 mm), purchased from Phenomenex, USA.

7. 管柱層析（Column Chromatography）填充材料：7. Column Chromatography packing materials:

8. 薄層色層分析（Thin-Layer Chromatography）：8. Thin-Layer Chromatography:

(1) TLC鋁片：薄層層析片，塗覆矽膠60 F ₂₅₄（0.25 mm），購自默克，德國。 (1) TLC aluminum sheet: Thin layer chromatography sheet, coated with silica gel 60 F ₂₅₄ (0.25 mm), purchased from Merck, Germany.

(2) TLC鋁片：薄層層析片，塗覆RP-18 F _254-S（0.25 mm），購自默克，德國。 (2) TLC aluminum sheet: Thin layer chromatography sheet, coated with RP-18 F _254-S (0.25 mm), purchased from Merck, Germany.

9. 紫外光燈（UV Lamp）：UVP UVGL-25，波長為 254 nm及365 nm。9. Ultraviolet light lamp (UV Lamp): UVP UVGL-25, wavelength is 254 nm and 365 nm.

10. 溶劑（solvent）：正己烷（n-hexane）、乙酸乙酯（ethyl acetate）、丙酮（acetone）、甲醇（methanol）、乙醇（ethanol）、乙腈（acetonitrile）、氯仿-d1（氘化程度99.5%）、甲醇-d4（氘化程度99.5%）、重水（deuterium oxide，氘化程度＞99.8％）、以及二甲基亞碸-d6（氘化程度＞99.9%），皆購自默克台灣。10. Solvent: n-hexane, ethyl acetate, acetone, methanol, ethanol, acetonitrile, chloroform-d1 (deuteration degree 99.5%), methanol-d4 (deuterated degree 99.5%), heavy water (deuterium oxide, deuterated degree >99.8%), and dimethyl sulfoxide-d6 (deuterated degree >99.9%), all purchased from Merck Taiwan.

11. 樣品：例一所製得的秋姬李發酵物A。11. Sample: Fermented product A of Qiuji plum prepared in Example 1.

B. 化合物分離與結構鑑定流程：B. Compound isolation and structure identification process:

1. 為了將秋姬李發酵物的脂溶性成分與水溶性成分分離，以正丁醇為溶劑，將5 L例一所製得的秋姬李發酵物A進行液相-液相分配萃取，得到12.6 g正丁醇可溶部（脂溶性成分）以及101.9 g水可溶部（水溶性成分）。1. In order to separate the fat-soluble components and water-soluble components of the fermented product of Prunus prune, use n-butanol as the solvent to perform liquid-liquid phase partition extraction on 5 L of the fermented product A of Prunus prune prepared in Example 1. 12.6 g of the n-butanol soluble part (fat-soluble component) and 101.9 g of the water-soluble part (water-soluble component) were obtained.

2. 功效化合物分離純化過程中，係依據生物活性導引分離方法（Bioassay guided fractionation）；並且，以降低黑色素細胞的黑色素的功效，做為分層及其後續細分離部的選擇依據。2. During the isolation and purification process of functional compounds, the bioassay guided fractionation method is used; and the effect of reducing melanin in melanocytes is used as the basis for the selection of stratification and subsequent subdivision fractions.

3. 12.6 g正丁醇可溶部繼續以Diaion HP-20大孔樹脂管柱層析（column chromatography）分離法進行初步分離，依序以純水、純水-甲醇體積比1:1、甲醇為沖提液，得到3個分離部（以下稱BUF1分離部、BUF2分離部以及BUF3分離部）。3. 12.6 g of n-butanol soluble part will continue to be preliminarily separated by Diaion HP-20 macroporous resin column chromatography separation method, followed by pure water, pure water-methanol volume ratio 1:1, and methanol. As the eluate, three separation parts (hereinafter referred to as BUF1 separation part, BUF2 separation part and BUF3 separation part) were obtained.

4. 取BUF1分離部並以逆向-中壓液相層析儀（RP-MPLC）對BUF1分離部進行再分離，以得到多種沖提物。於此，沖提液由水至甲醇線性沖提，沖提時間60分鐘，流速每分鐘10毫升。後續利用薄層色層分析（TLC鋁片：薄層層析片，塗覆矽膠60 F254（0.25 mm），購自默克，德國）合併相似結果的沖提物，而得到3個次分離部（以下稱BUF1-1分離部、BUF1-2分離部以及BUF1-3分離部）。4. Take the BUF1 separation part and re-separate the BUF1 separation part with reverse phase-medium pressure liquid chromatography (RP-MPLC) to obtain a variety of eluates. Here, the elution liquid is linearly eluted from water to methanol, the elution time is 60 minutes, and the flow rate is 10 ml per minute. Subsequent thin-layer chromatography analysis (TLC aluminum flakes: thin-layer chromatography flakes, coated with silica gel 60 F254 (0.25 mm), purchased from Merck, Germany) was used to combine the extracts with similar results to obtain three sub-separations. (Hereinafter referred to as BUF1-1 separation part, BUF1-2 separation part and BUF1-3 separation part).

5. 將BUF1-2分離部經逆相-高效率液相層析純化（甲醇/水=1/9），而得到二化合物TCI-GSF-01與TCI-GSF-03。經氫-核磁共振光譜（1H-NMR）分析其化學結構後，經文獻比對確認化合物TCI-GSF-01為2,5-呋喃二甲醇（2,5-bis-(hydroxymethyl)furane），如圖11所示；以及，化合物TCI-GSF-03為5-羥基糠酸（5-Hydroxymethyl-2-furonic acid），如圖12所示。5. Purify the separation part of BUF1-2 through reverse-phase high-efficiency liquid chromatography (methanol/water=1/9) to obtain two compounds TCI-GSF-01 and TCI-GSF-03. After analyzing its chemical structure by hydrogen nuclear magnetic resonance spectroscopy (1H-NMR), it was confirmed through literature comparison that compound TCI-GSF-01 is 2,5-bis-(hydroxymethyl)furane, such as As shown in Figure 11; and, compound TCI-GSF-03 is 5-Hydroxymethyl-2-furonic acid, as shown in Figure 12.

6. 另取BUF-2分離部並以逆向-中壓液相層析儀（RP-MPLC）對BUF-2分離部進行再分離，以得到多種沖提物。於此，沖提液由水至甲醇線性沖提，沖提時間100分鐘，流速每分鐘10毫升。後續利用薄層色層分析（TLC鋁片：薄層層析片，塗覆RP-18 F254-S（0.25 mm），購自默克，德國）合併相似結果的沖提物，而得到6個次分離部（以下稱BUF2-1分離部、BUF2-2分離部、BUF2-3分離部、BUF2-4分離部、BUF2-5分離部以及BUF2-6分離部）。6. Take another BUF-2 separation part and re-separate the BUF-2 separation part with reverse phase-medium pressure liquid chromatography (RP-MPLC) to obtain a variety of eluates. Here, the elution liquid is linearly eluted from water to methanol, the elution time is 100 minutes, and the flow rate is 10 ml per minute. Subsequent thin-layer chromatography analysis (TLC aluminum flakes: thin-layer chromatography flakes, coated with RP-18 F254-S (0.25 mm), purchased from Merck, Germany) was used to combine the extracts with similar results, and 6 Secondary separation section (hereinafter referred to as BUF2-1 separation section, BUF2-2 separation section, BUF2-3 separation section, BUF2-4 separation section, BUF2-5 separation section and BUF2-6 separation section).

7. 將BU2-2分離部經逆向-HPLC純化（甲醇/水=3/17），而得到化合物TCI-GSF-05。經氫-核磁共振光譜（1H-NMR）與碳-核磁共振光譜（13C-NMR）分析其化學結構後，經文獻比對確認化合物TCI-GSF-05為6-羥基-6-(羥甲基)-2H-吡喃-3(6H)-酮（6-hydroxy-6- (hydroxymethyl)-2H-pyran-3(6H)-one），如圖14至圖15所示。7. Purify the BU2-2 separation part through reverse-HPLC (methanol/water=3/17) to obtain compound TCI-GSF-05. After analyzing its chemical structure by hydrogen-nuclear magnetic resonance spectroscopy (1H-NMR) and carbon-nuclear magnetic resonance spectroscopy (13C-NMR), it was confirmed through literature comparison that compound TCI-GSF-05 is 6-hydroxy-6-(hydroxymethyl )-2H-pyran-3(6H)-one (6-hydroxy-6- (hydroxymethyl)-2H-pyran-3(6H)-one), as shown in Figures 14 to 15.

8. 將BU2-3分離部經逆向-HPLC純化（甲醇/水=1/4），而得到化合物TCI-GSF-04。經氫-核磁共振光譜（1H-NMR）分析其化學結構後，經文獻比對確認化合物TCI-GSF-04為反式-4-羥基環己烷羧酸（trans-4-hydroxycyclohaxane carboxlic acid），如圖13所示。8. Purify the BU2-3 separation part through reverse-HPLC (methanol/water=1/4) to obtain compound TCI-GSF-04. After analyzing its chemical structure by hydrogen nuclear magnetic resonance spectroscopy (1H-NMR), it was confirmed through literature comparison that compound TCI-GSF-04 is trans-4-hydroxycyclohaxane carboxlic acid. As shown in Figure 13.

9. 將BUF2-4分離部經逆向-HPLC純化（甲醇/水=1/3），而得到化合物TCI-GSF-07。經氫-核磁共振光譜（1H-NMR）分析其化學結構後，經文獻比對確認化合物TCI-GSF-07為酪醇（tyrosol），如圖16所示。9. Purify the BUF2-4 separation part through reverse-HPLC (methanol/water=1/3) to obtain compound TCI-GSF-07. After analyzing its chemical structure by hydrogen nuclear magnetic resonance spectroscopy (1H-NMR), it was confirmed that compound TCI-GSF-07 was tyrosol through literature comparison, as shown in Figure 16.

10. 將BUF2-5分離部經逆向-HPLC純化（甲醇/水=3/7），而得到化合物TCI-GSF-09。經氫-核磁共振光譜（1H-NMR）與碳-核磁共振光譜（13C-NMR）分析其化學結構後，經文獻比對確認化合物TCI-GSF-09為2-羥基-1-(5-(羥甲基)呋喃-2-基)丙烷-1-酮（2-hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one），如圖17至圖18所示。10. Purify the BUF2-5 separation part through reverse-HPLC (methanol/water=3/7) to obtain compound TCI-GSF-09. After analyzing its chemical structure by hydrogen-nuclear magnetic resonance spectroscopy (1H-NMR) and carbon-nuclear magnetic resonance spectroscopy (13C-NMR), it was confirmed through literature comparison that compound TCI-GSF-09 is 2-hydroxy-1-(5-( Hydroxymethyl)furan-2-yl)propan-1-one (2-hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one), as shown in Figures 17 to 18.

前述化合物的名稱及化學結構式如下表三所示。The names and chemical structural formulas of the aforementioned compounds are shown in Table 3 below.

表三化合物名稱化學結構式 TCI-GSF-01 2,5-呋喃二甲醇式(I) TCI-GSF-03 5-羥基糠酸式(II) TCI-GSF-04 反式-4-羥基環己烷羧酸式(III) TCI-GSF-05 6-羥基-6-(羥甲基)-2H-吡喃-3(6H)-酮式(IV) TCI-GSF-07 酪醇式(V) TCI-GSF-09 2-羥基-1-(5-(羥甲基)呋喃-2-基)丙烷-1-酮式(VI) Table 3 compound Name Chemical structural formula TCI-GSF-01 2,5-furandimethanol Formula (I) TCI-GSF-03 5-Hydroxyfuroic acid Formula (II) TCI-GSF-04 trans-4-hydroxycyclohexanecarboxylic acid Formula (III) TCI-GSF-05 6-hydroxy-6-(hydroxymethyl)-2H-pyran-3(6H)-one Formula (IV) TCI-GSF-07 Tyrosol Formula(V) TCI-GSF-09 2-Hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one Formula (VI)

例十一Example 11

A. 材料與儀器：A. Materials and instruments:

B. 試驗流程：B. Test process:

1. 將B16F10細胞以每孔1.5×10 ⁵個的密度，接種於含細胞培養基的6孔培養盤中，並在37 ℃下培養24小時。於此，B16F10細胞分為八個試驗組別，其分別為：空白組、麴酸組、化合物01組、化合物03組、化合物04組、化合物05組、化合物07組與化合物09組。各組進行三重複（意即各組各有三孔）。 1. Inoculate B16F10 cells into a 6-well culture plate containing cell culture medium at a density of 1.5×10 ⁵ cells per well, and culture at 37°C for 24 hours. Here, B16F10 cells were divided into eight test groups, which were: blank group, kojic acid group, compound 01 group, compound 03 group, compound 04 group, compound 05 group, compound 07 group and compound 09 group. Each group was performed in triplicate (meaning each group had three holes).

2. 培養24小時後，將各組更換為實驗培養基，並於37℃下繼續培養48小時。其中，空白組的實驗培養基為不含樣品或麴酸的細胞培養基；麴酸組的實驗培養基為含有200 μg/mL的麴酸的細胞培養基，其中麴酸已被廣泛認知為具有降低黑色素生成效果的物質；化合物01組的實驗培養基為含有100 μM 的例九分離所得的化合物TCI-GSF-01的細胞培養基；化合物03組的實驗培養基為含有100 μM 的例九分離所得的化合物TCI-GSF-03的細胞培養基；化合物04組的實驗培養基為含有100 μM 的例九分離所得的化合物TCI-GSF-04的細胞培養基；化合物05組的實驗培養基為含有100 μM 的例九分離所得的化合物TCI-GSF-05的細胞培養基；化合物07組的實驗培養基為含有100 μM 的例九分離所得的化合物TCI-GSF-07的細胞培養基；以及化合物09組的實驗培養基為含有100 μM 的例九分離所得的化合物TCI-GSF-09的細胞培養基。2. After 24 hours of culture, replace each group with the experimental medium and continue to culture at 37°C for 48 hours. Among them, the experimental culture medium of the blank group is a cell culture medium that does not contain samples or kojic acid; the experimental culture medium of the kojic acid group is a cell culture medium containing 200 μg/mL kojic acid, of which kojic acid has been widely recognized to have the effect of reducing melanin production. The experimental medium of the compound 01 group is a cell culture medium containing 100 μM of the compound TCI-GSF-01 isolated in Example 9; the experimental medium of the compound 03 group is a cell culture medium containing 100 μM of the compound TCI-GSF-01 isolated in Example 9. The cell culture medium of 03; the experimental culture medium of the compound 04 group is the cell culture medium containing 100 μM of the compound TCI-GSF-04 isolated in Example 9; the experimental culture medium of the compound 05 group is the cell culture medium containing 100 μM of the compound TCI-GSF-04 isolated in Example 9 The cell culture medium of GSF-05; the experimental culture medium of the compound 07 group is a cell culture medium containing 100 μM of the compound TCI-GSF-07 isolated in Example 9; and the experimental culture medium of the compound 09 group is a cell culture medium containing 100 μM of the compound TCI-GSF-07 isolated in Example 9. Cell culture medium of compound TCI-GSF-09.

C. 試驗結果：C. Test results:

空白組與其他各組的試驗結果之間的統計學顯著差異是以學生t檢驗統計分析得到。於圖式中，「*」代表在與空白組比較下其p值小於0.05、「**」代表在與空白組比較下其p值小於0.01，以及「***」代表在與空白組比較下其p值小於0.001。Statistically significant differences between the test results of the blank group and other groups were obtained by statistical analysis using Student's t test. In the figure, "*" means that the p value is less than 0.05 when compared with the blank group, "**" means that the p value is less than 0.01 when compared with the blank group, and "***" means that the p value is less than 0.01 when compared with the blank group. Its p-value is less than 0.001.

請參閱圖19。空白組未使用樣品或麴酸進行處理，因此空白組的試驗結果代表B16F10細胞在正常的生理代謝情況下的表現。於此，在設定空白組的相對黑色素含量為100%的情況下，麴酸組的相對黑色素含量為82.5%，化合物01組的相對黑色素含量為53.5%，化合物03組的相對黑色素含量為85.7%，化合物04組的相對黑色素含量為80.6%，化合物05組的相對黑色素含量為61.6%，化合物07組的相對黑色素含量為83.3%，而化合物09組的相對黑色素含量為86.1%。也就是說，相對於空白組，麴酸組的相對黑色素含量顯著降低約17.5%，化合物01組的相對黑色素含量顯著降低約46.5%，化合物03組的相對黑色素含量顯著降低約14.3%，化合物04組的相對黑色素含量顯著降低約19.4%，化合物05組的相對黑色素含量顯著降低約38.4%，化合物07組的相對黑色素含量顯著降低約16.7%，而化合物09組的相對黑色素含量顯著降低約13.9%。See Figure 19. The blank group was not treated with samples or kojic acid, so the test results of the blank group represent the performance of B16F10 cells under normal physiological metabolism. Here, when the relative melanin content of the blank group is set to 100%, the relative melanin content of the koji acid group is 82.5%, the relative melanin content of the compound 01 group is 53.5%, and the relative melanin content of the compound 03 group is 85.7% , the relative melanin content of compound 04 group is 80.6%, the relative melanin content of compound 05 group is 61.6%, the relative melanin content of compound 07 group is 83.3%, and the relative melanin content of compound 09 group is 86.1%. That is to say, compared with the blank group, the relative melanin content of the koji acid group was significantly reduced by about 17.5%, the relative melanin content of the compound 01 group was significantly reduced by about 46.5%, the relative melanin content of the compound 03 group was significantly reduced by about 14.3%, and the relative melanin content of the compound 04 group was significantly reduced by about 14.3%. The relative melanin content of the compound 05 group was significantly reduced by about 19.4%, the relative melanin content of the compound 05 group was significantly reduced by about 38.4%, the relative melanin content of the compound 07 group was significantly reduced by about 16.7%, and the relative melanin content of the compound 09 group was significantly reduced by about 13.9% .

由此可知，化合物TCI-GSF-01、TCI-GSF-03、TCI-GSF-04、TCI-GSF-05、TCI-GSF-07及TCI-GSF-09皆能顯著降低黑色素。其中，化合物TCI-GSF-01相較其他化合物，具有較為優異的降低黑色素含量的能力。換言之，秋姬李發酵物所包含的2,5-呋喃二甲醇（2,5-bis-(hydroxymethyl)furane）、5-羥基糠酸（5-Hydroxymethyl-2-furonic acid）、反式-4-羥基環己烷羧酸（trans-4-Hydroxycyclohaxane carboxlic acid）、酪醇（Tyrosol）、6-羥基-6-(羥甲基)-2H-吡喃-3(6H)-酮（6-Hydroxy-6-(hydroxymethyl)-2H-pyran-3(6H)-one）和2-羥基-1-(5-(羥甲基)呋喃-2-基)丙烷-1-酮（2-hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one）皆具有減少黑色素及/或抑制黑色素生成的能力，藉以有益於減少個體的黑色素，進而提升個體皮膚的通透與亮白程度。It can be seen that compounds TCI-GSF-01, TCI-GSF-03, TCI-GSF-04, TCI-GSF-05, TCI-GSF-07 and TCI-GSF-09 can significantly reduce melanin. Among them, compound TCI-GSF-01 has an excellent ability to reduce melanin content compared with other compounds. In other words, 2,5-bis-(hydroxymethyl)furane, 5-Hydroxymethyl-2-furonic acid, trans-4 contained in the fermentation product of Akihime plum -Trans-4-Hydroxycyclohaxane carboxlic acid, Tyrosol, 6-Hydroxy-6-(hydroxymethyl)-2H-pyran-3(6H)-one (6-Hydroxy -6-(hydroxymethyl)-2H-pyran-3(6H)-one) and 2-hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one (2-hydroxy-1 -(5-(hydroxymethyl)furan-2-yl)propan-1-one) all have the ability to reduce melanin and/or inhibit melanin production, thereby benefiting from reducing individual melanin and thereby improving individual skin transparency and whitening. degree.

S10:步驟 S11:步驟 S111:步驟 S112:步驟 S10: Steps S11: Steps S111: Steps S112: Step

圖1是一實施例的秋姬李發酵物的製作方法的流程圖。圖2是圖1中步驟S11的一實施例的流程圖。圖3是秋姬李萃取液、秋姬李好氧發酵物及秋姬李發酵物的總多酚含量檢測結果的柱狀圖。圖4是相對酪胺酸酶活性的細胞實驗結果的柱狀圖。圖5是相對黑色素含量的細胞實驗結果的柱狀圖A。圖6是相對粒線體活性的細胞實驗結果的柱狀圖。圖7是粒線體活性的細胞實驗結果的螢光染色圖。圖8是第0週及第8週的相對肌膚斑點程度的人體實驗結果的柱狀圖。圖9是第0週及第8週的相對肌膚棕色斑程度的人體實驗結果的柱狀圖。圖10是秋姬李萃取液及秋姬李發酵物的HPLC指紋圖譜。圖11是TCI-GSF-01的氫-核磁共振光譜圖譜。圖12是TCI-GSF-03的氫-核磁共振光譜圖譜。圖13是TCI-GSF-04的氫-核磁共振光譜圖譜。圖14是TCI-GSF-05的氫-核磁共振光譜圖譜。圖15是TCI-GSF-05的碳-核磁共振光譜圖譜。圖16是TCI-GSF-07的氫-核磁共振光譜圖譜。圖17是TCI-GSF-09的氫-核磁共振光譜圖譜。圖18是TCI-GSF-09的碳-核磁共振光譜圖譜。圖19是相對黑色素含量的細胞實驗結果的柱狀圖B。 Figure 1 is a flow chart of a method for producing a fermented product of Akihime plum according to one embodiment. FIG. 2 is a flow chart of an embodiment of step S11 in FIG. 1 . Figure 3 is a bar graph showing the detection results of the total polyphenol content of Plum extract, Plum aerobic fermentation and Plum fermentation. Figure 4 is a bar graph of cell experiment results relative to tyrosinase activity. Figure 5 is histogram A of cell experimental results relative to melanin content. Figure 6 is a bar graph of cellular experimental results relative to mitochondrial activity. Figure 7 is a fluorescent staining picture of the results of cellular experiments on mitochondrial activity. Figure 8 is a bar graph of human experiment results of relative skin spot levels at weeks 0 and 8. Figure 9 is a histogram of human experiment results of relative skin brown spot levels at weeks 0 and 8. Figure 10 is the HPLC fingerprint of Plum extract and Plum fermentation material. Figure 11 is the hydrogen-nuclear magnetic resonance spectrum of TCI-GSF-01. Figure 12 is the hydrogen-nuclear magnetic resonance spectrum of TCI-GSF-03. Figure 13 is the hydrogen-nuclear magnetic resonance spectrum of TCI-GSF-04. Figure 14 is the hydrogen-nuclear magnetic resonance spectrum of TCI-GSF-05. Figure 15 is the carbon-nuclear magnetic resonance spectrum of TCI-GSF-05. Figure 16 is the hydrogen-nuclear magnetic resonance spectrum of TCI-GSF-07. Figure 17 is the hydrogen-nuclear magnetic resonance spectrum of TCI-GSF-09. Figure 18 is the carbon-nuclear magnetic resonance spectrum of TCI-GSF-09. Figure 19 is a histogram B of cell experimental results relative to melanin content.

Claims

一種秋姬李(Prunus salicina)發酵物，其中該秋姬李發酵物是由一秋姬李經一溶劑萃取後再經由一厭氧發酵而得，該溶劑為水或酒精，該厭氧發酵係以酵母菌及乳酸菌進行厭氧發酵。 A Prunus salicina fermentation product, wherein the Prunus salicina fermentation product is obtained by extracting a Prunus salicina with a solvent and then subjecting it to an anaerobic fermentation. The solvent is water or alcohol, and the anaerobic fermentation system Anaerobic fermentation with yeast and lactic acid bacteria.

如請求項1所述的秋姬李發酵物，其中該秋姬李發酵物包含2,5-呋喃二甲醇、5-羥基糠酸、反式-4-羥基環己烷羧酸、酪醇、6-羥基-6-(羥甲基)-2H-吡喃-3(6H)-酮、2-羥基-1-(5-(羥甲基)呋喃-2-基)丙烷-1-酮或其組合。 The Akihime plum fermented product as claimed in claim 1, wherein the Akihime plum fermented product contains 2,5-furandimethanol, 5-hydroxyfuroic acid, trans-4-hydroxycyclohexanecarboxylic acid, tyrosol, 6-hydroxy-6-(hydroxymethyl)-2H-pyran-3(6H)-one, 2-hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one or its combination.

一種如請求項1所述的秋姬李發酵物用於製備美白的組合物的用途。 The use of the fermented Akihime plum fermented product as described in claim 1 for preparing a whitening composition.

如請求項3所述的用途，其中該秋姬李發酵物具有減少黑色素及/或抑制黑色素生成的作用。 The use as described in claim 3, wherein the Akihime plum fermentation product has the effect of reducing melanin and/or inhibiting melanin production.

如請求項3所述的用途，其中該秋姬李發酵物具有抑制酪胺酸酶活性的作用。 The use as described in claim 3, wherein the fermented Akihime plum ferment has the effect of inhibiting tyrosinase activity.

一種如請求項1所述的秋姬李發酵物用於製備提升粒線體活性的組合物的用途。 The use of the fermented product of Prunus prunus as described in claim 1 for preparing a composition for improving mitochondrial activity.

一種如請求項1所述的秋姬李發酵物用於製備減少肌膚色素斑的組合物的用途。 The use of the fermented Akihime plum as described in claim 1 for preparing a composition for reducing skin pigment spots.

如請求項7所述的用途，其中該色素斑為深層棕色斑、可見斑點或其組合。 The use as described in claim 7, wherein the pigmented spots are deep brown spots, visible spots or a combination thereof.

如請求項3至8中任一項所述的用途，其中該秋姬李發酵物包含2,5-呋喃二甲醇、5-羥基糠酸、反式-4-羥基環己烷羧酸、酪醇、6- 羥基-6-(羥甲基)-2H-吡喃-3(6H)-酮、2-羥基-1-(5-(羥甲基)呋喃-2-基)丙烷-1-酮或其組合。 The use as described in any one of claims 3 to 8, wherein the Akihime plum fermentation product contains 2,5-furandimethanol, 5-hydroxyfuroic acid, trans-4-hydroxycyclohexanecarboxylic acid, tyrosine Alcohol, 6- Hydroxy-6-(hydroxymethyl)-2H-pyran-3(6H)-one, 2-hydroxy-1-(5-(hydroxymethyl)furan-2-yl)propan-1-one or combinations thereof .

一種秋姬李發酵物的製作方法，包括：以一溶劑萃取一秋姬李以得到一秋姬李萃取物；以及厭氧發酵該秋姬李萃取物以得到該秋姬李發酵物，該溶劑為水或酒精，該厭氧發酵係以酵母菌及乳酸菌進行厭氧發酵。 A method for making a plum fermented product, including: extracting a plum extract with a solvent to obtain a plum extract; and anaerobically fermenting the plum extract to obtain the fermented product, the solvent It is water or alcohol, and the anaerobic fermentation is carried out by yeast and lactic acid bacteria.