Hanseniaspora uvarum and use thereof

Abstract

The present invention relates to the technical field of microorganisms. Provided are Hanseniaspora uvarum and the use thereof. The Hanseniaspora uvarum ZS02 is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC NO. 26056. The Hanseniaspora uvarum is used in the fermentation process of Pyrus ussuriensis Maxim fruit juice for the first time, which provides a valuable strain resource for the technical field of fruit juice processing. Moreover, the use of the Hanseniaspora uvarum in the fermentation of Pyrus ussuriensis Maxim can reduce the sugar content in the Pyrus ussuriensis Maxim fruit juice. In addition, the obtained fermentation liquid of Pyrus ussuriensis Maxim has good free-radical scavenging capacity, has the effects of protecting the skin barrier, repairing damage to the skin barrier, resisting aging and tightening the skin, and can reduce inflammatory responses.

Description

A kind of grape juice spore-bearing Hansenula and its application

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on October 31, 2023, with application number 202311421591.5 and invention name “A Grape Juice Spore-bearing Hansen Yeast and Its Applications”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present application relates to the field of microbial technology, and in particular to a Hansenula sporangiophora vitis vinifera and its application. Background Art

[0003] The Pit Fruit, originally known as the Pi Nang Fruit, also known as the Sour Pear and Pi Tai Fruit, is a unique ancient tree species that grows at the foot of Taizi Mountain. With a cultivated history of over 1,000 years, it is a local cultivar of the Xinjiang Pyrus family, part of the Rosaceae subfamily. The Pit Fruit is characterized by strong vigor, long lifespan, relaxed soil requirements, a preference for shade and humidity, cold tolerance, resistance to pests and diseases, and strong adaptability. The Pit Fruit has a sweet and sour taste and a warm nature. It contains a variety of amino acids, vitamins, and essential minerals such as potassium, calcium, and iron. It has numerous health benefits, including moistening the lungs and stomach, relieving diarrhea and thirst, softening blood vessels, and protecting the liver from alcohol intoxication.

[0004] Currently, the primary method for processing pitaya is pitaya juice, with research primarily focusing on the formulation and nutritional composition of pitaya juice beverages. For example, patent CN114223818A discloses a pitaya-wolfberry beverage formula and preparation method for its heat-clearing, lung-moistening, and liver-nourishing effects; patent CN113974029A discloses a pitaya-tea compound beverage and its preparation method; patent CN114097958A discloses a pitaya-juice beverage with hangover-relieving properties and its preparation method; and patent CN112586699A discloses a pitaya paste and its preparation method. Chinese invention patent publication number CN112450346A, "A Method for Producing Sugar-Free Pitaya Fermented Juice," utilizes two yeast strains (Angel Yeast and Chr. Hansen) and Lactobacillus casei to ferment pitaya juice. Using ripe pitaya fruit to ferment and produce ripe pitaya juice is a relatively viable method for processing pitaya. Currently, the most commonly used strains for producing beer juice are lactic acid bacteria and Saccharomyces cerevisiae. However, the fermentation process for mixed strains is complex and the production cost is high. Furthermore, there are no reports of using non-Saccharomyces cerevisiae yeasts or strains isolated from ripe beer fruit and fermented with these endophytes to produce beer fruit fermentation filtrate.

[0005] In addition, currently pitaya processing mainly involves pitaya juice, and its research direction is mainly focused on the formulation and nutritional components of pitaya juice beverages. There are no reports on the application of pitaya fermentation filtrate in cosmetics.

[0006] Summary of the Invention

[0007] In order to solve the problems in the prior art, the present invention provides a pitaya endophyte Hanseniaspora uvarum ZS02 screened from the ripened pitaya fruit. It was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on November 7, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCCNO.26056, and the fungus is a non-brewer's yeast.

[0008] On the one hand, the present application provides a Hanseniaspora uvarum, wherein the Hanseniaspora uvarum is Hanseniaspora uvarum ZS02, which is deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms with a deposit number of CGMCC NO.26056.

[0009] On the other hand, the present application also provides a preparation containing any one of A1) to A5):

[0010] A1) Hanseniaspora uvarum;

[0011] A2) Hanseniaspora uvarum (Hanseniaspora uvarum)

[0012] A3) a suspension of dead Hanseniaspora uvarum in grape juice;

[0013] A4) Metabolites of Hanseniaspora uvarum;

[0014] A5) Hanseniaspora uvarum grape juice extract;

[0015] The Hansenula sporogenes is Hansenula sporogenes ZS02, which is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms with a deposit number of CGMCCNO.26056.

[0016] On the other hand, the present application also provides a composition, comprising the aforementioned Hansenula sporangiophora vitis vinifera or the aforementioned preparation.

[0017] In an optional embodiment, the method for culturing Hansenula sporangioides comprises: inoculating Hansenula sporangioides into a culture medium and culturing at 25° C.-40° C. for 40-60 hours; optionally, culturing at 30° C. for 48 hours.

[0018] Furthermore, the culture temperature may be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, or any temperature value therein, and the culture time may be 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h, 49h, 50h, 51h, 52h, 53h, 54h, 55h, 56h, 57h, 58h, 59h, 60h, or any time value therein.

[0019] The culture medium of Hansenula sporangiophora vitis vinifera comprises a carbon source and a nitrogen source.

[0020] The carbon source may be selected from one or more of glucose, sucrose, and fructose; the nitrogen source may be selected from one or more of beef extract, peptone, and yeast extract.

[0021] Optionally, the culture medium is YPD medium, which may include: 10.0 g / L yeast extract, 20.0 g / L peptone, and 20.0 g / L glucose.

[0022] On the other hand, the present application also provides the use of the aforementioned Hansenula sporangiophora vitis vinifera or the aforementioned preparation or the aforementioned composition in preparing a pitaya fermentation broth.

[0023] On the other hand, the present application also provides a method for preparing pitaya fermentation broth, the method comprising: fermenting pitaya using the above-mentioned Hansenula sporangiophora vitis vinifera or the above-mentioned preparation or the above-mentioned composition.

[0024] Optionally, the fermentation includes fermenting at a fermentation temperature of 25° C.-40° C. and a fermentation rotation speed of 100-300 rpm for 40-60 hours.

[0025] Furthermore, the fermentation temperature may be 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or any temperature value therein, and the fermentation speed may be 100rpm, 110rpm, 120rpm, 130rpm, 140rpm, 150rpm, 160rpm, 170rpm, 180rpm, 190rpm, 200rpm, 210rpm m, 220rpm, 230rpm, 240rpm, 250rpm, 260rpm, 270rpm, 280rpm, 290rpm, 300rpm and any speed value therein, and the fermentation time can be 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h, 49h, 50h, 51h, 52h, 53h, 54h, 55h, 56h, 57h, 58h, 59h, 60h and any time value therein.

[0026] More optionally, the fermentation comprises fermenting for 48 hours at a fermentation temperature of 30° C. and a fermentation speed of 200 rpm.

[0027] In an optional embodiment, the method for preparing the pitaya fermentation broth comprises:

[0028] Step 1: crush the ripened pitaya fruit and filter to obtain pitaya fruit juice;

[0029] Step 2: sterilizing the pitaya juice to obtain a fermentation medium;

[0030] Step 3: inoculate Hansenula sporogenes into the fermentation medium, and ferment at 25° C.-40° C. and 100-300 rpm for 40-60 hours to obtain a pitaya fermentation liquid.

[0031] Optionally, the method further comprises filtering the pitaya fermentation liquor in order to remove the bacteria.

[0032] Optionally, the method further comprises the step of removing impurities from the pitaya fermentation liquor to remove proteins produced during the fermentation of Hansenula sporogenes in grape juice.

[0033] On the other hand, the present application also provides the pitaya fermentation broth prepared by the above method.

[0034] Optionally, the pitaya fermentation liquid is a low-sugar or sugar-free pitaya fermentation liquid.

[0035] More optionally, the total sugar content in the pitaya fermentation broth is ≤0.5g / 100mL.

[0036] In an optional embodiment, the total sugar content in the pitaya fermented juice obtained by fermentation with Hansenula sporogenes can be as low as 0.0857 g / 100 mL.

[0037] On the other hand, the present application also provides a product, which comprises the above-mentioned pitaya fermentation liquid.

[0038] On the other hand, the present application also provides the use of the aforementioned Hansenula sporangiophora vitis vinifera, the aforementioned preparation, the aforementioned composition, the aforementioned pitaya fermentation broth, or the aforementioned product in reducing the sugar content of a substance.

[0039] Optionally, the sugar is selected from one or more of glucose, sucrose, fructose, lactose, and soluble starch; more optionally, the sugar is selected from one or more of glucose, sucrose, and fructose.

[0040] On the other hand, the present application also provides the use of the aforementioned Hansenula sporangiophora vitis vinifera, the aforementioned preparation, the aforementioned composition, the aforementioned pitaya fermentation broth, or the aforementioned product in degrading sugar.

[0041] Optionally, the sugar is selected from one or more of glucose, sucrose, fructose, lactose, and soluble starch; more optionally, the sugar is selected from one or more of glucose, sucrose, and fructose.

[0042] The Hansenula sporogenes described in the present application can effectively utilize glucose, sucrose, and fructose in the culture medium as carbon sources. Therefore, it can play a role in reducing the content of glucose, sucrose, and fructose in the material during the fermentation process, and can be used to prepare sugar-free or low-sugar products.

[0043] On the other hand, the present application also provides the use of the aforementioned Hansenula sporangiophora vitis vinifera, the aforementioned preparation, the aforementioned composition, the aforementioned pitaya fermentation broth, or the aforementioned product in anti-inflammatory and antioxidant activities for purposes other than disease diagnosis and treatment.

[0044] The concentration of the pitaya fermentation broth can be selected from any value among 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%.

[0045] Optionally, the concentration of the pitaya fermentation liquid is 2%-10% by mass, more preferably 10%. Within this concentration range, the pitaya fermentation liquid has a certain degree of inhibitory effect on reactive oxygen species (ROS) generated before and after UV damage.

[0046] Optionally, the concentration of the pitaya fermentation broth is 2%-10% by mass, more preferably 5%-10%. The pitaya fermentation broth at a concentration of 2% significantly inhibits the secretion of IL-1β and TNF-α by macrophages, while concentrations of 5% and 10% significantly inhibit the secretion of IL-1β, IL-6, and TNF-α by macrophages.

[0047] On the other hand, the present application also provides the use of the aforementioned Hansenula vitis spores, or the aforementioned preparation, or the aforementioned composition, or the aforementioned pitaya fermentation broth, or the aforementioned product in preventing and / or repairing skin barrier damage for non-disease diagnosis and treatment purposes.

[0048] On the other hand, the present application also provides the use of the aforementioned Hansenula sporangiophora vitis vinifera, or the aforementioned preparation, or the aforementioned composition, or the aforementioned pitaya fermentation broth, or the aforementioned product in anti-aging for non-disease diagnosis and treatment purposes.

[0049] On the other hand, the present application also provides the use of the aforementioned Hansenula vitis spores, or the aforementioned preparation, or the aforementioned composition, or the aforementioned pitaya fermentation broth, or the aforementioned product in skin tightening for non-disease diagnosis and treatment purposes.

[0050] Wherein, the skin tightening is achieved by increasing collagen content and / or inhibiting glycation reaction.

[0051] The method for increasing collagen content is to promote the expression of type I collagen.

[0052] On the other hand, the present application also provides the use of the aforementioned Hansenula sporangiophora vitis vinifera, or the aforementioned preparation, or the aforementioned composition, or the aforementioned pitaya fermentation broth, or the aforementioned product in promoting the expression of type I collagen (COLI protein) for non-disease diagnosis and treatment purposes.

[0053] On the other hand, the present application also provides the use of the aforementioned Hansenula sporangiophora vitis vinifera, or the aforementioned preparation, or the aforementioned composition, or the aforementioned pitaya fermentation broth, or the aforementioned product in inhibiting glycosylation reactions for non-disease diagnosis and treatment purposes.

[0054] The inhibition of the glycation reaction is achieved by inhibiting the generation of AGEs induced by the glycation intermediate product D-glyceraldehyde.

[0055] The present invention has the following beneficial effects:

[0056] 1. This invention provides a strain of non-Saccharomyces yeast, Hanseniaspora uvarum ZS02, isolated from the pitaya fruit. It is used for the first time in the production of pitaya fruit juice, providing a valuable bacterial resource for juice processing technology and possessing significant application value. Furthermore, because this strain is isolated from pitaya fruit endophytes, rather than using exogenous strains, it is safer than other exogenous strains.

[0057] 2. The use of the grape juice Hansenula sporogenous yeast to ferment pitaya can reduce the sugar content in the pitaya juice. After fermentation, the sugar content of the pitaya juice is reduced from 10.82g / 100mL to 0.0857g / 100mL.

[0058] 3. The pitaya fermentation liquid obtained by the present invention has good free radical scavenging ability, can protect the skin barrier, repair damaged skin barrier, anti-aging, inhibit skin glycation, and tighten the skin. It can also inhibit LPS-induced macrophage secretion of IL-1β, IL-6 and TNF-α, reduce inflammatory response, and can be used in antioxidant, anti-sugar and / or anti-inflammatory cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0060] FIG1 is a schematic diagram of filtrates obtained by fermentation using different single bacterial colonies;

[0061] FIG2 is a diagram showing bacterial morphology (magnification: ×100).

[0062] Biodeposit Information:

[0063] A strain of grape juice spore-bearing Hanseniaspora uvarum ZS02 was deposited in the General Microbiology Center of the China Culture Collection Administration (CGMCC) on November 7, 2022, with the deposit number CGMCC NO.26056, and the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0064] In order to more clearly illustrate the overall concept of the present application, the following is a detailed description of the embodiments in conjunction with the accompanying drawings. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0065] If no specific conditions are specified in the examples, the experiments were carried out according to conventional conditions or conditions recommended by the manufacturer.

[0066] Among them, Hanseniaspora uvarum ZS02 was deposited in the General Microbiology Center of China Culture Collection Administration (CGMCC) on November 7, 2022. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.26056.

[0067] Pichia kluyveri (CGMCC NO.2.4488), Maggimycosis (CGMCC NO.2.3314), and Hansenula vitis spores (CGMCC NO.2.3213) were purchased from the General Microbiology Center of China Culture Collection Administration; Saccharomyces cerevisiae (ATCC 9080) was purchased from the American Type Culture Collection.

[0068] Unless otherwise specified, in the following embodiments, the reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market.

[0069] YPD liquid medium: yeast extract 10.0 g / L, peptone 20.0 g / L, glucose 20.0 g / L.

[0070] YPD solid medium: yeast extract 10.0 g / L, peptone 20.0 g / L, glucose 20.0 g / L, agar powder 20.0 g / L.

[0071] MRS solid medium: peptone 10.0 g / L, beef extract 8.0 g, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween-80 1.0 g / L, pH 6.5±0.2, agar 14.0 g / L.

[0072] MRS liquid medium: peptone 10.0 g / L, beef extract 8.0 g, yeast extract 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween-80 1.0 g / L, pH 6.5±0.2.

[0073] The detection methods involved in the following embodiments are as follows:

[0074] Phenol sulfuric acid method for total sugar determination:

[0075] Prepare a standard curve: Accurately weigh 20 mg of standard glucose into a 500 mL volumetric flask, add water to the mark, and pipette 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 mL of each aliquot. Make up to 2.0 mL with purified water, then add 1.0 mL of 6% phenol and 5.0 mL of concentrated sulfuric acid. Shake well and cool. After standing at room temperature for 20 minutes, measure the absorbance at 490 nm. Use 2.0 mL of water as a blank and follow the same color development procedure. Draw a standard curve with glucose concentration as the horizontal axis and absorbance Y as the vertical axis. The equation for the standard curve is Y = 17.6x - 0.0003, R 2 =0.999.

[0076] Glucose measurement using a biosensor analyzer: The cultured bacterial solution was centrifuged at 6000 rpm for 10 min, and the supernatant obtained after centrifugation was taken to detect the glucose concentration using a biosensor analyzer.

[0077] Example 1 Isolation, culture and identification of bacterial strains

[0078] Ingredients: Commercially available ripened bitter oranges.

[0079] Experimental methods:

[0080] Samples were collected from the pulp and juice of ripened Betula fruit and added to MRS liquid medium. After incubation at 200 rpm and 25°C for 2 days, a culture medium was obtained. The culture medium was then spread onto MRS solid medium and incubated at 25°C. Based on colony morphology, color, and microscopic bacterial morphology, ten individual colonies (numbered colonies 1-10) were selected from the MRS solid medium and inoculated into ripened Betula fruit juice for fermentation. The fermented juices showed slight color differences between the different colonies, as shown in Figure 1. Based on the odor and color of the fermentation broth, strains (i.e., colonies 1-4) exhibiting a distinct aromatic odor (ester aroma) and lighter color after fermentation were selected for subsequent purification and product stability. After 2 days of incubation in MRS liquid medium, the bacteria were collected and sent to BGI for 18S rDNA sequencing. Sequencing confirmed that colonies 1-4 were the same strain, with detailed identification results as follows.

[0081] Strain identification:

[0082] The 18S rDNA gene sequence of this strain is shown in SEQ ID NO: 1. Sequence alignment using the National Center for Biotechnology Information (NCBI) website revealed that the strain's 18S rDNA sequence shared the highest homology with Hanseniaspora uvarum, with a similarity exceeding 99%. Therefore, the strain isolated from the pulp or juice of ripened Beer fruit is considered to be Hanseniaspora uvarum, a non-Saccharomyces yeast.

[0083] The above-mentioned Hansenula sporangiophora vitis vinifera, named ZS02, was deposited on November 7, 2022, at the General Microbiology Center of the China Culture Collection Administration (CGMCC), with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 26056. Example 2 Identification of the morphological characteristics and biological properties of Hansenula sporangiophora vitis vinifera CGMCC NO. 26056

[0084] 1. Observation of colony and bacterial morphology

[0085] Activated Hansenula vitis spores CGMCC NO.26056 cells were streaked and inoculated onto YPD solid medium at 30°C. The colony morphology was observed. A small amount of cells was picked up with an inoculating loop and transferred to sterile purified water. The cell morphology was observed under a microscope.

[0086] The observation results are shown in Figure 2: the bacteria are oval, spherical or lemon-shaped; the colonies are round, milky white, opaque, with neat edges, smooth surface and a convex center.

[0087] 2. Carbon source utilization experiment

[0088] Strain activation: Hansenula vitis spores CGMCC No. 26056 was inoculated into YPD solid medium and cultured at 30°C for 48 h.

[0089] Fermentation: The activated Hansenula sporogenes CGMCC NO.26056 strain was inoculated into a triangular flask containing YPD liquid medium. The inoculation amount was 100 mL per 250 mL triangular flask, and the inoculation amount was a single clone of Hansenula sporogenes. The culture was shaken at 30°C and 200 rpm for 48 hours. Glucose, sucrose, lactose, fructose, and soluble starch were used as carbon sources for the culture medium (each carbon source content was 20 g / L). After the completion of the culture, the biomass (OD 600 ), and the glucose and total sugar contents in the fermentation supernatant were determined by biosensor analyzer and phenol-sulfuric acid method. The results are shown in Table 1.

[0090] Table 1 Carbon source utilization of Hansenula sporeifera in grape juice

[0091] As shown in Table 1, the Hansenula sporogenes CGMCC No. 26056 in the present application can utilize glucose, sucrose, and fructose as carbon sources, but has a low utilization rate of lactose and soluble starch. Accordingly, the total sugar content after fermentation is relatively high.

[0092] 3. Nitrogen source utilization experiment

[0093] Strain activation: Hansenula vitis spores CGMCC No. 26056 was inoculated into YPD solid medium and cultured at 30°C for 48 h.

[0094] Fermentation: A single clone of the activated Hansenula sporogenes CGMCC NO.26056 strain was inoculated into a 250 mL Erlenmeyer flask containing YPD liquid medium. 100 mL of the liquid was added to a 250 mL Erlenmeyer flask and cultured at 30°C and 200 rpm for 48 hours. Ammonium sulfate, beef extract, peptone, and yeast extract were used as the sole nitrogen source for the culture medium (each nitrogen source at 10 g / L). After completion, the biomass (OD 600 ), and the glucose and total sugar contents in the fermentation supernatant were determined by biosensor analyzer and phenol-sulfuric acid method. The results are shown in Table 2.

[0095] Table 2 Nitrogen source utilization of Hansenula sporeifera in grape juice

[0096] As shown in Table 2, the Hansenula sporogenes CGMCC No. 26056 in the present application can utilize a single nitrogen source: beef extract, peptone, and yeast extract, but basically does not grow in a culture medium with ammonium sulfate as the single nitrogen source.

[0097] Example 3 Preparation of Pittosporum Fermented Juice

[0098] Test Example 1

[0099] 1) Crush the ripened Pitto fruit, filter through gauze to obtain the filtered juice, and filter through a 2.0 μm clarification plate, a 0.8 μm clarification plate, and a 0.22 μm filter membrane in sequence to obtain Pitto fruit juice.

[0100] 2) 100 mL of pitaya juice was measured and sterilized at 100° C. for 10 min to obtain a fermentation medium.

[0101] 3) A single colony of Hanseniaspora uvarum CGMCC No. 26056 was inoculated into the fermentation medium, and cultured in a shake flask at 200 rpm and 28° C. for 46 h to obtain a pitaya fermentation broth.

[0102] 4) The pitaya fermentation liquid was centrifuged at 6000 rpm for 10 min, and after the bacteria were removed by filtration, the supernatant was filtered through a 0.22 μm filter membrane under sterile conditions to obtain pitaya fermentation juice.

[0103] The phenol-sulfuric acid method and biosensor analyzer were used to determine the total sugar and glucose contents in Pittosporum fructus fermented juice.

[0104] The final test results showed that the total sugar content in the fermented pitaya juice of this test example was 0.117 g / 100 mL, and the glucose content was 0.011 g / 100 mL.

[0105] Test Example 2

[0106] 1) Crush the ripened Pitto fruit, filter through gauze to obtain the filtered juice, and filter through a 2.0 μm clarification plate, a 0.8 μm clarification plate, and a 0.22 μm filter membrane in sequence to obtain Pitto fruit juice.

[0107] 2) 100 mL of pitaya juice was measured and sterilized at 100° C. for 10 min to obtain a fermentation medium.

[0108] 3) A single colony of Hanseniaspora uvarum CGMCC No. 26056 was inoculated into the fermentation medium, and cultured in a shake flask at 200 rpm and 30° C. for 48 h to obtain a pitaya fermentation broth.

[0109] 4) The pitaya fermentation liquid was centrifuged at 6000 rpm for 10 min, and after the bacterial cells were removed by filtration, the supernatant was filtered through a 0.22 μm filter membrane under sterile conditions to obtain pitaya fermentation juice.

[0110] The phenol-sulfuric acid method and biosensor analyzer were used to determine the total sugar and glucose contents in Pittosporum fructus fermented juice.

[0111] The total sugar content of the pitaya fermented juice in this experimental example was 0.0857 g / 100 mL, and the glucose content was 0.007 g / 100 mL. After fermentation with Hansenula sporogenes, the total sugar content of the pitaya fermented juice was reduced from 10.82 g / 100 mL to 0.0857 g / 100 mL.

[0112] Test Example 3

[0113] 1) Crush the ripened Pitto fruit, filter through gauze to obtain the filtered juice, and filter through a 2.0 μm clarification plate, a 0.8 μm clarification plate, and a 0.22 μm filter membrane in sequence to obtain Pitto fruit juice.

[0114] 2) 100 mL of pitaya juice was measured and sterilized at 100° C. for 10 min to obtain a fermentation medium.

[0115] 3) A single colony of Hanseniaspora uvarum CGMCC No. 26056 was inoculated into the fermentation medium, and cultured in a shake flask at 200 rpm and 32° C. for 50 h to obtain a pitaya fermentation broth.

[0116] 4) The pitaya fermentation liquid was centrifuged at 6000 rpm for 10 min, and after the bacteria were removed by filtration, the supernatant was filtered through a 0.22 μm filter membrane under sterile conditions to obtain pitaya fermentation juice.

[0117] The phenol-sulfuric acid method and biosensor analyzer were used to determine the total sugar and glucose contents in Pittosporum fructus fermented juice.

[0118] The final test results showed that the total sugar content in the fermented pitaya juice of this test example was 0.0872 g / 100 mL, and the glucose content was 0.008 g / 100 mL.

[0119] Comparative Example 1

[0120] The only difference between Comparative Example 1 and Experimental Example 2 is the different strains used to ferment the ripened pitaya fruit. The strain used in Comparative Example 1 is the commercially available Pichia kluyveri CGMCC NO.2.4488, and the pitaya fermented juice fermented by the pitaya non-endophytic yeast Pichia kluyveri is finally obtained.

[0121] The total sugar content in the fermented pitaya juice of this comparative example was 7.19 g / 100 mL, and the glucose content was 1 g / 100 mL.

[0122] Comparative Example 2

[0123] The only difference between Comparative Example 2 and Experimental Example 2 is the different strains used for fermenting the ripened pitaya fruit. The strain used in Comparative Example 2 is the commercially available Saccharomyces cerevisiae ATCC 9080, and pitaya fruit fermented juice fermented by Saccharomyces cerevisiae is finally obtained.

[0124] The total sugar content of the fermented pitaya juice in this comparative example was 5.82 g / 100 mL, and the glucose content was 1.5 g / 100 mL.

[0125] Comparative Example 3

[0126] The only difference between Comparative Example 3 and Experimental Example 2 is the different strains used to ferment the ripened pitaya fruit. The strain used in Comparative Example 3 is the commercially available Maggi yeast CGMCC NO.2.3314, and finally the pitaya fruit fermented juice fermented by Maggi yeast is obtained.

[0127] The total sugar content in the fermented pitaya juice of this comparative example was 3.41 g / 100 mL, and the glucose content was 0.6 g / 100 mL.

[0128] Comparative Example 4

[0129] The only difference between Comparative Example 4 and Experimental Example 2 is the strain used to ferment the ripened pitaya fruit. The strain used in Comparative Example 4 is the commercially available Hansenula sporogenes CGMCC No. 2.3213, and the pitaya fruit fermented juice fermented by the commercially available Hansenula sporogenes CGMCC No. 2.3213 is finally obtained.

[0130] The total sugar content in the fermented pitaya juice of this comparative example was 2.75 g / 100 mL, and the glucose content was 1.0 g / 100 mL.

[0131] in conclusion

[0132] The results of Experimental Examples 1-3 and Comparative Examples 1-4 demonstrate that the total sugar content in the pitaya fermented juice obtained by fermenting pitaya with the presently described Hanseniaspora uvarum CGMCC No. 26056 yeast (endophytic in pitaya ripening fruit) significantly decreased. This indicates that, compared to other yeasts, Hanseniaspora uvarum has a superior ability to consume sugars. In an alternative experimental example, the total sugar content in the pitaya fermented juice was reduced from 10.82 g / 100 mL to 0.0857 g / 100 mL.

[0133] Example 4 Evaluation of the Antioxidant Efficacy of Pittosporum Fermentation Filtrate

[0134] This embodiment provides a method for preparing a fermentation filtrate of a maltose fruit, the method comprising the following steps:

[0135] 1) Crush the ripened Pitto fruit, filter through gauze to obtain the filtered juice, and filter through a 2.0 μm clarification plate, a 0.8 μm clarification plate, and a 0.22 μm filter membrane in sequence to obtain Pitto fruit juice.

[0136] 2) Measure 100 mL of pitaya fruit juice and sterilize it at 100°C for 10 min to obtain pitaya fruit juice filtrate.

[0137] 3) A single colony of Hanseniaspora uvarum CGMCC No. 26056 was inoculated into the sterile pitaya juice filtrate, and cultured in a shake flask at 200 rpm and 30° C. for 48 h to obtain pitaya fermentation broth.

[0138] 4) The pitaya fermentation broth was centrifuged at 6000 rpm for 10 min, and after filtering to remove bacterial cells, the supernatant was sterilized at 80° C. for 1 h to remove proteins produced during the fermentation of non-Saccharomyces cerevisiae yeast, and then filtered through a 0.22 μm filter membrane to obtain a pitaya fermentation filtrate.

[0139] The antioxidant effect was tested by irradiating HaCaT cells with UVA+UVB. The cells were exposed to samples before and after irradiation to detect the content of intracellular reactive oxygen free radicals (ROS) and to investigate the ability of pitaya fermentation filtrate to inhibit ROS production or scavenge ROS.

[0140] 1. Experimental Materials

[0141] Piper melongena fermentation filtrate, Piper melongena fruit juice filtrate, human skin keratinocytes HaCaT, fetal bovine serum (Gibco), ROS detection kit (KeyGen Biotechnology), DMEM culture medium (KeyGen Biotechnology), trypsin (KeyGen Biotechnology).

[0142] 2. Instruments and equipment

[0143] Carbon dioxide incubator (SANYO), inverted microscope (OLYMPUS, CKX41), digital constant temperature water bath (Jintan Zhongda Instrument Factory), clean bench (Beijing Donglian Har Instrument Manufacturing Co., Ltd., SCB-1520), multifunctional microplate reader (TECAN, Spark), constant temperature microplate fast oscillator (Haimen Qilin Medical Instrument Factory), UV-8 ultraviolet light box (Beijing Electric Light Source Research Institute), and flow cytometer (BD).

[0144] 3. Experimental methods

[0145] 3.1 Preparation of sample solution

[0146] The solution was prepared with serum-free culture medium DMEM and sterilized by filtration with a 0.22 μm filter membrane. The experimental concentrations of the pitaya fermentation filtrate and pitaya juice filtrate were 2%, 5%, and 10%.

[0147] 3.2 UV damage repair effect

[0148] Plating: HaCaT cells in logarithmic growth phase were taken and plated at 1×10 5 Cells were seeded at a density of 100 cells / mL in a 12-well plate, with 1 mL of cell suspension per well, and cultured in a carbon dioxide incubator at 37°C and 5% CO2 for 24 h.

[0149] Irradiation: Irradiation group, covered with plastic wrap, UVA 2000μW / cm 2 The intensity of irradiation was 1h, and UVB was 700μW / cm 2 The negative control group was covered with aluminum foil and not irradiated.

[0150] Sample Processing: After irradiation, the old culture medium was discarded. The experimental groups were treated with 2%, 5%, and 10% pitaya fermentation filtrate and pitaya juice filtrate, respectively. The model and negative control groups were treated with 1 mL of serum-free culture medium per well. Cultures were continued for 24 hours, after which the culture medium was discarded and the cells were washed twice with PBS. 1.5 mL of DCFH-DA was added to each well and incubated in a cell culture incubator for 30 minutes, mixing every 5 minutes to ensure sufficient probe binding. The probes were discarded, and the cells were washed twice with prewarmed serum-free culture medium. 1 mL of serum-free culture medium was added to each well and incubated at 37°C for 10 minutes. After washing once with PBS, the cells were trypsinized, washed twice with PBS, and resuspended in 300 μL of PBS. The cells were analyzed by flow cytometry, and the mean fluorescence intensity was recorded.

[0151] 3.3 UV damage protection effect

[0152] Plating: HaCaT cells in logarithmic growth phase were taken and plated at 1×10 5 Cells were seeded at a density of 100 cells / mL in a 12-well plate, with 1 mL of cell suspension per well, and cultured in a carbon dioxide incubator at 37°C and 5% CO2 for 24 h.

[0153] Sample processing: discard the old culture medium, add the fermentation filtrate of pitaya fruit and the filtrate of pitaya fruit juice at concentrations of 2%, 5%, and 10%, respectively; add serum-free culture medium to the model group and negative control group, 1 mL per well, and continue culturing for 24 hours.

[0154] Irradiation: Cover the irradiation group with plastic wrap, UVA at 2000μW / cm 2 The intensity of irradiation was 1h, and UVB was 700μW / cm 2 The cells were irradiated at an intensity of 100 nm for 3 minutes, while the negative control group was covered with aluminum foil and not irradiated. After irradiation, the culture medium was discarded, serum-free culture medium was added, and the cells were cultured for another 3 hours. After the culture was completed, the culture medium was discarded and the cells were washed twice with PBS. 1.5 mL of DCFH-DA was added to each well and placed in a cell culture incubator for 30 minutes. The cells were mixed every 5 minutes to allow the probe to fully bind. The probe was discarded and washed twice with preheated serum-free culture medium. 1 mL of serum-free culture medium was added to each well and incubated at 37°C for 10 minutes. After washing once with PBS, the cells were trypsinized, washed twice with PBS, resuspended in 300 μL of PBS, and detected by flow cytometry. The average fluorescence intensity was recorded and the ROS inhibition rate was calculated.

[0155] The calculation method of ROS inhibition rate is:

[0156] (1) Where:

[0157] T—the average of three fluorescence intensities of the tested sample;

[0158] C—the average of three times of fluorescence intensity of negative control;

[0159] C0—the average of three fluorescence intensities of the naked cell group.

[0160] 3. Experimental results

[0161] The above experimental results are shown in Table 3.

[0162] Table 3 Effect of Pittosporum fructification filtrate on the content of reactive oxygen species (ROS)

[0163] As shown in Table 3, the pitaya fermentation filtrate provided by the present invention exhibited a certain degree of inhibitory effect on reactive oxygen species (ROS) generated before and after UV damage at 2%, 5%, and 10% concentrations. The pitaya fermentation filtrate had the highest ROS inhibition rate at a concentration of 10%. This indicates that the pitaya fermentation filtrate provided by the present invention has excellent oxidative protection and repair effects, with the 10% pitaya fermentation filtrate exhibiting the best oxidative protection and repair effects, and can be added to cosmetics as an antioxidant ingredient.

[0164] Within the same concentration range, the protection against UV damage from pitaya fermentation filtrate was superior to that from pitaya juice filtrate without microbial fermentation. Furthermore, pitaya fermentation filtrate demonstrated superior UV damage repair, while unfermented pitaya juice filtrate did not.

[0165] Example 5 Evaluation of the anti-inflammatory efficacy of pitaya fermentation filtrate

[0166] Anti-inflammatory Effect Lipopolysaccharide (LPS) was used to stimulate mouse macrophages to secrete inflammatory factors. The anti-inflammatory effect of the pitaya fermentation filtrate was investigated by quantitatively detecting the expression of inflammatory factors.

[0167] 1. Experimental Materials

[0168] The pitaya fermentation filtrate and pitaya juice filtrate prepared by the method of Example 4, mouse macrophage Raw264.7, fetal bovine serum (Gibco), Mouse TNF-α ELISA Kit (Proteintech), Mouse IL-6 ELISA Kit (Proteintech), Mouse IL-1β ELISA Kit (Proteintech), DMEM culture medium (Keygen Biotechnology), and human Pro-Collagen I alpha 1 ELISA kit (abcam).

[0169] 2. Instruments and equipment

[0170] Carbon dioxide incubator (SANYO), inverted microscope (OLYMPUS, CKX41), digital constant temperature water bath (Jintan Zhongda Instrument Factory), clean bench (Beijing Donglian Har Instrument Manufacturing Co., Ltd., SCB-1520), multifunctional microplate reader (TECAN, Spark), constant temperature microplate fast oscillator (Haimen Qilin Medical Instrument Factory), UV-8 ultraviolet light box (Beijing Electric Light Source Research Institute), and flow cytometer (BD).

[0171] 3. Experimental methods

[0172] 3.1 Preparation of sample solution

[0173] LPS: Prepare a stock solution with a concentration of 500,000 units / mL using serum-free 1640 culture medium, sterilize by filtration through a 0.22 μm filter membrane, store in a refrigerator at -20°C, and dilute to a working solution of 40,000 units / mL before use.

[0174] Sample solution: Prepare the sample solution with LPS solution and filter it through a 0.22 μm filter membrane for sterilization.

[0175] 3.2 Sample processing

[0176] Raw264.7 cells were cultured at a rate of 1 × 10 5 The cells were inoculated with 50 μg / mL of culture medium in a 24-well plate and cultured at 37°C, 5% CO2 for 24 h. Samples were added, and LPS was used as a model control. The cells were cultured for another 24 h. The cell supernatant was collected and centrifuged. Inflammatory factors were detected according to the ELISA kit instructions.

[0177] 3.3 Detection of inflammatory factor expression

[0178] Collect the cell supernatant and centrifuge at 1000 rpm for 10 minutes, and take the supernatant for detection. Set up blank wells (no sample and enzyme-labeled reagent), standard wells, and test sample wells respectively. Accurately add 100 μL of the standard sample to the enzyme-labeled coated plate, and add 100 μL of the test sample to the test sample well. Add the sample to the bottom of the enzyme-labeled plate well and shake gently to mix. Seal the plate with a sealing film and incubate at 37°C for 120 minutes. Dilute the 20-fold concentrated washing solution 20 times with distilled water and set aside. Discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds, then discard it. Repeat this 4 times and pat dry. Add 100 μL of antibody working solution to each well and incubate for 60 minutes. Repeat the wash once. Add 100 μL of HRP working solution to each well and incubate for 40 minutes. Repeat the wash once. Add 100 μL of TMB working solution to each well and incubate for 15-20 minutes. Add 100 μL of stop solution to each well to terminate the reaction. The blank well was used to adjust the plate to zero and the absorbance (OD value) was measured at a wavelength of 450 nm.

[0179] 4. Experimental results

[0180] The above experimental results are shown in Table 4.

[0181] Table 4 Effect of Pittosporum fructus fermentation filtrate on TNF-α secretion

[0182] Table 5 Effect of Pittosporum fructus fermentation filtrate on IL-6 secretion

[0183] Table 6 Effect of Pittosporum fructus fermentation filtrate on IL-1β secretion

[0184] As can be seen from Tables 4, 5, and 6, the pitaya fermentation filtrate provided by the present invention has a significant inhibitory effect on the secretion of IL-1β and TNF-α by macrophages within a concentration range of 2%; and has a significant inhibitory effect on the secretion of IL-1β, IL-6, and TNF-α by macrophages within a concentration range of 5% and 10%, indicating that the pitaya fermentation filtrate provided by the present invention has a good anti-inflammatory effect and can be added to cosmetics as an anti-inflammatory raw material. The inhibitory effect of the pitaya fermentation filtrate is better than that of the pitaya juice filtrate that has not been fermented by microorganisms.

[0185] Example 6 Evaluation of the Skin Tightening Effect of Piper methysticum Fermentation Filtrate

[0186] 1. Effects of samples on type I collagen in FB cells (human dermal fibroblasts)

[0187] 1. Detection method: In this example, an immunofluorescence assay was used to determine the relative mean fluorescence intensity of COLI in FB cells using a semi-quantitative technique, which can be used to determine the effect of the test sample on COLI protein expression.

[0188] 2. Detection index: Calculation of the relative mean fluorescence intensity of COLI (%): By comparing with the control group, the relative mean fluorescence intensity of COLI before and after the sample treatment was detected.

[0189] 3. Group settings are shown in Table 7.

[0190] Table 7 Group settings

[0191] 4. Detection steps

[0192] Cell inoculation: Take cells in the logarithmic growth phase, use DMEM medium containing 10% FBS, and when the cell density reaches about 80%, digest with 0.05% trypsin, centrifuge at 1000 rpm for 5 minutes, resuspend and count, inoculate into 24-well plates, and culture at 37°C, 5% CO2.

[0193] Sample Treatment: 24 hours after seeding, the culture medium was aspirated and replaced with fresh DMEM medium containing 10% FBS for the control group. The sample groups were co-treated with fresh medium containing the corresponding sample concentrations. Incubate in an incubator for 72 hours. After exposure, discard the samples, wash with PBS, and fix the cells with cold methanol.

[0194] Staining: Remove the cell slides, block with BSA for 30 minutes, incubate with COLI antibody overnight, discard the primary antibody, wash with PBS, add corresponding secondary antibodies and DAPI and incubate in the dark, finally add mounting medium, use fluorescence microscope to take pictures under the same exposure conditions, and use software to analyze the relative average fluorescence intensity of COLI protein in the cells.

[0195] 5. Data analysis: Calculate the relative mean fluorescence intensity (%) using the following formula:

[0196] After background removal, the readings were normalized using the control group as the baseline and expressed as Mean ± SD. Analyses were performed using Graphpad Prism statistical software. Intergroup comparisons were performed using the t-test. *P < 0.05 indicates a statistically significant difference, **P < 0.01 indicates a statistically significant difference, and ***P < 0.001 indicates an extremely significant difference.

[0197] 6. Judgment criteria: If the relative mean fluorescence intensity (%) is greater than 100% and is statistically different from the control group, the sample at this concentration is determined to be able to increase the relative mean fluorescence intensity of the COLI protein.

[0198] 7. The results are shown in Table 8.

[0199] Table 8 Detection results of relative fluorescence intensity of COLI protein by samples

[0200] 2. Effects of samples on AGEs content in FB cells

[0201] 1. Detection method: Immunofluorescence assay was used to determine the relative mean fluorescence intensity of AGEs in FB cells using a semi-quantitative technique, which can be used to determine the effect of the test sample on the generation of AGEs.

[0202] 2. Detection index: Calculation of the relative mean fluorescence intensity (%) of AGEs: By comparing with the control group, the relative mean fluorescence intensity of AGEs before and after the sample treatment was detected.

[0203] 3. Group settings are shown in Table 9.

[0204] Table 9 Group settings

[0205] 4. Detection steps

[0206] Cell inoculation: Take cells in the logarithmic growth phase, use DMEM medium containing 10% FBS, and when the cell density reaches about 80%, digest with 0.05% trypsin, centrifuge at 1000 rpm for 5 minutes, resuspend and count, inoculate into 24-well plates, and culture at 37°C, 5% CO2.

[0207] Sample Treatment: 24 hours after seeding, the culture medium was aspirated and replaced with fresh culture medium supplemented with 45 mg / L D-glyceraldehyde alone for the control group. The sample groups were treated with fresh culture medium supplemented with 45 mg / L D-glyceraldehyde and the corresponding sample concentrations. The cells were incubated in an incubator for 72 hours. After exposure, the samples were discarded, washed with PBS, and the cells were fixed with cold methanol.

[0208] Staining: The cell slides were removed, blocked with BSA for 30 minutes, incubated with AGEs antibody overnight, the primary antibody was discarded, and the slides were washed with PBS. The corresponding secondary antibodies and DAPI were added and incubated in the dark. Finally, the mounting medium was added. The slides were photographed under the same exposure conditions using a fluorescence microscope, and the relative average fluorescence intensity of AGEs in the cells was analyzed using software.

[0209] 5. The results are shown in Table 10.

[0210] Table 10 Detection results of relative fluorescence intensity of AGEs by samples

[0211] In summary, the results of the immunofluorescence test showed that treatment with 5% pitaya fermentation filtrate could enhance the relative fluorescence intensity of COLI protein in human dermal fibroblasts by 9%, with a statistically significant difference (P < 0.05); 5% pitaya fermentation filtrate could inhibit the formation of AGEs induced by the glycation intermediate D-glyceraldehyde, and the result was statistically significant (P < 0.01). The above results indicate that pitaya fermentation filtrate has the effect of tightening the skin.

[0212] The foregoing is merely an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should all be included within the scope of the claims of the present application.

Claims

1. A grape juice spore-bearing Hansen yeast (Hanseniaspora uvarum), characterized in that The Hansenula vitis spores is Hansenula vitis spores ZS02, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.26056.

2. A preparation, characterized in that The preparation contains any one of A1) to A5): A1) Hanseniaspora uvarum; A2) Grape juice spore-forming Hansen yeast (Hanseniaspora uvarum); A3) a suspension of dead Hanseniaspora uvarum in grape juice; A4) Metabolites of Hanseniaspora uvarum; A5) Hanseniaspora uvarum extract from grape juice; The Hansenula vitis spores is Hansenula vitis spores ZS02, which is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with a deposit number of CGMCC NO.26056.

3. A composition, characterized in that The composition comprises the Hansenula vitis spores according to claim 1 or the preparation according to claim 2.

4. Use of the Hansenula sporogenes of claim 1, the preparation of claim 2, or the composition of claim 3 in preparing pitaya fermentation broth.

5. A method for preparing a pitaya fermentation liquid, characterized in that: The method comprises: fermenting pitaya using the Hansenula sporogenes of claim 1 or the preparation of claim 2 or the composition of claim 3.

6. The pitaya fermentation liquid prepared by the method according to claim 5.

7. A product, characterized in that The product comprises the pitaya fermentation broth as claimed in claim 6.

8. Use of the Hansenula sporogenes of claim 1, the preparation of claim 2, the composition of claim 3, the pitaya fermentation liquid of claim 6, or the product of claim 7 in reducing the sugar content of a substance.

9. Use of the Hansenula sporogenes of claim 1, the preparation of claim 2, the composition of claim 3, the pitaya fermentation broth of claim 6, or the product of claim 7 in anti-inflammatory and / or anti-oxidative treatment for purposes other than disease diagnosis and treatment.

10. Use of the Hansenula sporogenes of claim 1, the preparation of claim 2, the composition of claim 3, the pitaya fermentation broth of claim 6, or the product of claim 7 in preventing and / or repairing skin barrier damage for non-disease diagnosis and treatment purposes.

11. Use of the Hansenula sporogenes of claim 1, or the preparation of claim 2, or the composition of claim 3, or the pitaya fermentation liquid of any one of claims 6, or the product of claim 7 in anti-aging, skin tightening and / or anti-glycation for non-disease diagnosis and treatment purposes.

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