CN117165459B

CN117165459B - Hansenula polymorpha of grape juice and application thereof

Info

Publication number: CN117165459B
Application number: CN202311421591.5A
Authority: CN
Inventors: 徐焕焕; 李文浩; 阚洪玲; 王冠凤; 董艳美; 钱晓路; 李晓天; 郭学平
Original assignee: Bloomage Biotech Co Ltd
Current assignee: Bloomage Biotech Co Ltd
Priority date: 2023-10-31
Filing date: 2023-10-31
Publication date: 2024-02-23
Anticipated expiration: 2043-10-31
Also published as: CN117165459A

Abstract

The invention provides Hansenula polymorpha serving as grape juice and application thereof, and relates to the technical field of microorganisms. The grape juice has Hansenula polymorphaHanseniaspora uvarum) ZS02 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of 26056. In the invention, the Hansenula polymorpha is applied to the process of fermenting the juice by the Piteguo for the first time, thereby providing precious strain resources for the technical field of juice processing. In addition, the glucose content in the juice of the Piteguo can be reduced by fermenting the Piteguo with Hansenula polymorpha. In addition, the Piteguo fermentation liquor obtained by the invention has better free radical scavenging capability, can play a role in protecting skin barrier and repairing damaged skin barrier, and can also reduce inflammatory reaction.

Description

Hansenula polymorpha of grape juice and application thereof

Technical Field

The application relates to the technical field of microorganisms, in particular to Hansenula polymorpha containing grape juice and application thereof.

Background

Pitot fruit is a unique ancient tree species growing on foot of Taizi, has a planting history of more than 1000 years, and is a local cultivar of Xinjiang pear line in apple subfamily of Rosaceae. The Piteguo has the characteristics of strong tree vigor, long tree age, low soil requirement, cold resistance due to damp and damp preference, disease and insect resistance, strong adaptability and the like. The Piteguo fruit has sour and sweet taste, warm nature, contains various amino acids, vitamins, potassium, calcium, iron and other mineral elements necessary for human body, and has various health care functions of moistening lung and nourishing stomach, relieving diarrhea and quenching thirst, softening blood vessels, dispelling alcohol effect and protecting liver and the like.

At present, the main way of processing the Piteguo is to prepare Piteguo juice, and researches are mainly focused on aspects of Piteguo juice beverage formulas, nutritional ingredients and the like. For example, patent CN114223818A discloses a beer fruit medlar beverage formula with effects of clearing heat, moistening lung and nourishing liver and a preparation method thereof, patent CN113974029a discloses a beer fruit tea compound beverage and a preparation method thereof, patent CN114097958A discloses a beer fruit juice beverage with an anti-alcohol effect and a preparation method thereof, and patent CN112586699a discloses a beer fruit paste and a preparation method thereof. The Chinese patent publication No. CN112450346A discloses a preparation method of sugar-free fermented juice of Piteguo, which selects two yeasts (Angel yeast, hansen yeast, and Lactobacillus casei) to ferment and prepare the fermented juice of Piteguo. The production of a Piteger's cured juice by fermentation of Piteger's cured fruit is a relatively viable way of processing Piteger's fruit. At present, common strains for preparing the Piteguo juice comprise lactic acid bacteria and Saccharomyces cerevisiae, but the composite strain has complex fermentation process and higher production cost. And no report is available at present on preparation of a fermented filtrate of the Piteguo by using non-Saccharomyces cerevisiae or separating strains from the Piteguo and fermenting the Piteguo by using endophyte.

In addition, the current Piteguo processing mainly relates to Piteguo juice, the research direction is mainly focused on aspects of Piteguo juice beverage formula, nutrition ingredients and the like, and no application report of Piteguo fermentation filtrate in cosmetics is seen.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a Hansenula polymorpha which is obtained by screening endophyte grape juice of Piteguo from cured PiteguoHanseniaspora uvarum) ZS02 is preserved in China general microbiological culture Collection center (CGMCC) for 11 and 7 days in 2022, the preservation address is North Chen Silu No. 1 and 3 in the Korean area of Beijing, the preservation number is CGMCC No.26056, and the strain is non-Saccharomyces cerevisiae.

On the one hand, the application provides a Hansenula polymorpha of grape juiceHanseniaspora uvarum) The Hansenula polymorpha is Hansenula polymorpha ZS02, and is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 26056.

In another aspect, the present application also provides a formulation comprising any one of A1) to A5):

a1 Hansenula polymorpha of grape juiceHanseniaspora uvarum)；

A2 Hansenula polymorpha of grape juiceHanseniaspora uvarum) A bacterial agent;

a3 Hansenula polymorpha of grape juiceHanseniaspora uvarum) Dead bacterial suspension;

a4 Hansenula polymorpha of grape juiceHanseniaspora uvarum) A metabolite;

a5 Hansenula polymorpha of grape juiceHanseniaspora uvarum) An extract;

wherein the Hansenula polymorpha is Hansenula polymorpha ZS02, and is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of 26056.

In another aspect, the present application also provides a composition comprising Hansenula polymorpha or a formulation as described above.

In a preferred embodiment, the method for culturing Hansenula polymorpha in grape juice comprises: inoculating Hansenula polymorpha with grape juice into a culture medium to be cultured at the temperature of 25-40 ℃ for 40-60 h; preferably, 48 h is incubated at 30 ℃.

Specifically, the culture temperature may be any one of the temperature values of 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃,30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃, and the like, and the culture time may be any one of the temperature values of 40 h, 41 h, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h, 49 h, 50 h, 51 h, 52 h, 53 h, 54 h, 55 h, 56 h, 57 h, 58 h, 59 h, 60 h, and the time value thereof.

The culture medium of Hansenula polymorpha in grape juice comprises the following components: a carbon source and a nitrogen source.

Wherein the carbon source can be selected from one or more of glucose, sucrose and fructose; the nitrogen source is selected from one or more of beef extract, peptone, and yeast extract.

Preferably, the culture medium is YPD medium, which may include: yeast extract 10.0 g/L, peptone 20.0 g/L, glucose 20.0 g/L.

In another aspect, the application also provides application of the Hansenula polymorpha or the preparation or the composition in preparation of the Piteguo fermentation broth.

In another aspect, the present application also provides a method for preparing a beer fermentation broth, the method comprising: the Piteguo is fermented by using the Hansenula polymorpha or the preparation or the composition.

Preferably, the fermentation comprises fermentation at a fermentation temperature of 25-40 ℃ and a fermentation speed of 100-300 rpm of 40-60 h.

Specifically, the fermentation temperature may be any one of 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃,30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃ and any one of the temperature values, the fermentation speed may be any one of 100 rpm, 110 rpm, 120 rpm, 130 rpm, 140 rpm, 150 rpm, 160 rpm, 170 rpm, 180 rpm, 190 rpm, 200 rpm, 210 rpm, 220 rpm, 230 rpm, 240 rpm, 250 rpm, 260 rpm, 270 rpm, 280 rpm, 290 rpm, 300 rpm and any one of the temperature values, and the fermentation time may be any one of the time values 40 h, 41 3834, 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h, 49 h, 50 h, 51 h, 52 h, 53 h, 54 h, 55 h, 56 h, 57 h, 58 h, 59 h, 60 h and any one of the time values.

More preferably, the fermentation comprises 48 to h at a fermentation temperature of 30℃and a fermentation speed of 200 rpm.

In a preferred embodiment, the preparation method of the Piteguo fermentation broth comprises the following steps:

crushing and filtering the Piteguo ripe fruits to obtain Piteguo raw juice;

sterilizing the Piteguo juice to obtain a fermentation medium;

step three, inoculating Hansenula polymorpha in grape juice into the fermentation medium, and fermenting 40-60 h at the temperature of 25-40 ℃ and the speed of 100-300 rpm to obtain a Piteger fruit fermentation liquid.

Preferably, the method further comprises the step of filtering the Piteguo fermentation broth for the purpose of removing the bacteria.

Preferably, the method further comprises the step of removing impurities from the fermentation broth of Piteguo so as to remove proteins produced during fermentation of Hansenula polymorpha.

On the other hand, the application also provides the Piteguo fermentation broth prepared by the method.

Preferably, the beer fruit fermentation broth is a low-sugar or sugar-free beer fruit fermentation broth.

More preferably, the total sugar content in the Piteguo fermentation broth is less than or equal to 0.5g/100mL.

In a preferred embodiment, the total sugar content in the fermented juice of the Pitot-cone obtained by fermentation of Hansenula polymorpha in grape juice is as low as 0.0857g/100mL.

In another aspect, the present application also provides a product comprising a beer fermentation broth as described above.

In another aspect, the application also provides the application of the Hansenula polymorpha or the preparation or the composition or the Piteguo fermentation broth or the product in reducing the sugar content of substances.

Preferably, the sugar is selected from one or more of glucose, sucrose, fructose, lactose, and soluble starch; more preferably, the sugar is selected from one or more of glucose, sucrose, fructose.

The Hansenula polymorpha in the grape juice can effectively utilize glucose, sucrose and fructose in a culture medium as carbon sources, so that the glucose, sucrose and fructose content in substances can be reduced in the fermentation process, and the Hansenula polymorpha can be further used for preparing sugar-free or low-sugar products.

In another aspect, the application also provides the application of the Hansenula polymorpha or the preparation or the composition or the Piteguo fermentation broth or the product in anti-inflammatory and anti-oxidation for the purpose of non-disease diagnosis and treatment.

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

Preferably, the concentration of the Piteguo fermentation liquor is 2% -10% in percentage by mass; more preferably 10%. Within this concentration range, the beer fruit broth has some degree of inhibition of Reactive Oxygen Species (ROS) generated before and after ultraviolet injury.

Preferably, the concentration of the Piteguo fermentation liquor is 2% -10% in percentage by mass; more preferably, 5% -10%. Wherein, the Piteguo fermentation liquor has remarkable inhibition effect on the secretion of IL-1 beta and TNF-alpha by macrophages at the concentration of 2 percent; the concentrations of 5% and 10% have remarkable inhibiting effect on the secretion of IL-1 beta, IL-6 and TNF-alpha by macrophages.

In another aspect, the present application also provides the use of Hansenula polymorpha or the formulation or the composition or the Piteguo fermentation broth or the product in the prevention and/or repair of damaged skin barrier for non-disease diagnostic therapeutic purposes.

The invention has the following beneficial effects:

1. the invention provides a strain of endophytic non-saccharomyces cerevisiae-hansenula polymorpha of grape juice screened from PiteguoHanseniaspora uvarum) ZS02 is applied to the preparation process of the beer-fruit fermented juice for the first time, provides precious strain resources for the technical field of juice processing, and has great application value. In addition, because the strain is selected from endophytes of the Piteguo, and exogenous strains are not selected, the strain has higher safety compared with other exogenous strains.

2. The glucose-containing Hansenula polymorpha is adopted to ferment the Piteguo, so that the sugar content in the Piteguo juice can be reduced, and the sugar content of the Piteguo juice is reduced from 10.82g/100mL to 0.0857g/100mL after fermentation.

3. The Piteguo fermentation liquor obtained by the invention has better free radical scavenging capability, can play a role in protecting skin barrier and repairing damaged skin barrier, can also inhibit macrophage induced by LPS from secreting IL-1 beta, IL-6 and TNF-alpha, reduce inflammatory reaction, and can be used in antioxidant or anti-inflammatory cosmetics.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute an undue limitation to the application. In the drawings:

FIG. 1 is a schematic diagram of the filtrates obtained with different single colony fermentations;

FIG. 2 is a diagram of observation of the bacterial cell shape (. Times.100).

Biological preservation information:

hansenula polymorpha strain for grape juiceHanseniaspora uvarum) ZS02, 11 months of 2022The strain is preserved in China general microbiological culture Collection center (CGMCC) for 7 days, the preservation number is CGMCC NO.26056, and the preservation address is North Silu No. 1 and No. 3 in the Korean region of Beijing city.

Detailed Description

In order to more clearly illustrate the general concepts of the present application, the following detailed description is made by way of example with reference to the accompanying drawings. In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art that the invention may be practiced without one or more of these details. In other instances, well-known features have not been described in detail in order to avoid obscuring the invention.

The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer.

Wherein, grape juice has Hansenula polymorphaHanseniaspora uvarum) ZS02 is preserved in China general microbiological culture Collection center (CGMCC) for 11 and 7 days in 2022, and the preservation address is 1 # 3 of North Silu of the Chaoyang area of Beijing city, and the preservation number is 26056 of CGMCC.

Pichia kluyveri (CGMCC No. 2.4488), mechnia mermairei (CGMCC No. 2.3314) and hansenula grape juice (CGMCC No. 2.3213) are purchased from the China general microbiological culture Collection center; saccharomyces cerevisiae (ATCC 9080) was purchased from the American type culture Collection.

In the following embodiments, unless specified otherwise, the reagents or apparatus used are conventional products available commercially without reference to the manufacturer.

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

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.

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

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-80.0 g/L, pH are 6.5+ -0.2.

The detection method involved in the following examples is as follows:

total sugar was measured by phenol sulfuric acid method:

and (3) making a standard curve: accurately weighing standard glucose 20 mg in a 500 mL volumetric flask, adding water to the scale, respectively sucking 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8 mL, respectively adding purified water to 2.0 mL, and then adding 6% phenol 1.0 mL and concentrated sulfuric acid 5.0 mL. Shaking, cooling, standing at room temperature for 20 min, measuring absorbance at 490, nm, taking 2.0 mL water as blank, taking glucose concentration as abscissa, and absorbance Y as ordinate to draw standard curve, wherein the formula of the drawn standard curve is Y=17.6x-0.0003, R ² =0.999。

Glucose was measured by a biosensing analyzer: centrifuging the cultured bacterial liquid at 6000 rpm for 10 min, and taking supernatant obtained after centrifugation, and detecting the glucose concentration by a biosensing analyzer.

Example 1 isolated culture and identification of Strain

Materials: commercially available beer ripens.

The experimental method comprises the following steps:

sampling from pulp and juice of Piteger ripened fruit, adding into MRS liquid culture medium, culturing at 25deg.C at rotation speed of 200 rpm for 2 d to obtain culture solution; the culture broth was plated onto MRS solid medium and incubated at 25 ℃. According to the characteristics of colony morphology, color, microscopic thallus morphology and the like, 10 different single colonies (with the number of 1-10) in MRS solid culture medium are selected and respectively inoculated into the Piteger cured fruit juice for fermentation, and the colors of the different colony fermented fruit juice are slightly different, as shown in figure 1. According to the smell and color of the fermentation liquor, strains (namely bacterial colonies 1-4) with unique aromatic smell (ester smell) and lighter color of the fermentation liquor after fermentation are selected from the strains so as to facilitate the subsequent purification process and ensure that the products can be kept stable. After 2 d culture by MRS liquid culture medium, the thalli are collected and sent to China megaorganism for 18S rDNA sequencing, and the bacterial colony 1-4 is found to be the same strain by sequencing, and the specific identification result is as follows.

Identification of strains:

the determination result of the 18S rDNA gene sequence of the strain is shown as SEQ ID NO: 1. The sequence comparison of the detected gene sequence is carried out by utilizing the National Center for Biotechnology Information (NCBI) website, and the result shows that the homology of the 18S rDNA sequence of the strain and Hansenula polymorpha in grape juice is highest, and the similarity is more than 99%. Thus, it is considered that the strain selected from Piteguo's cured fruit pulp or its juice is Hansenula polymorpha strainHanseniaspora uvarum) Is a non-saccharomyces cerevisiae.

The grape juice Hansenula polymorpha is named ZS02 and is preserved in China general microbiological culture Collection center (CGMCC) for 11 and 7 days in 2022, wherein the preservation address is North Chen Xilelu No. 1 and 3 in the Korean region of Beijing, and the preservation number is CGMCC No.26056.

Example 2 morphological and biological characterization of Hansenula polymorpha CGMCC No.26056

1. Colony and fungus shape observation

Culturing activated Hansenula polymorpha CGMCC NO.26056 thallus in YPD solid culture medium at 30deg.C, and observing colony morphology; a few bacteria were picked up with an inoculating loop to sterile purified water, and the morphology of the bacteria was observed under a microscope.

The observation results are shown in fig. 2: the thalli are oval, spherical or lemon; the colony is round, milky white, opaque, neat in edge, smooth in surface and convex in middle.

2. Carbon source utilization experiment

Strain activation: inoculating Hansenula polymorpha CGMCC No.26056 to YPD solid culture medium, and culturing at 30deg.C for 48 h.

Fermentation: inoculating activated Hansenula polymorpha CGMCC No.26056 strain into a triangular flask containing YPD liquid culture medium, and shaking culturing at 30deg.C and 200 rpm for 48 h with a inoculation amount of Hansenula polymorpha monoclonal strain of 100mL in 250 mL triangular flask. Wherein glucose, sucrose, lactose, fructose and soluble starch are used as carbon sources of the culture medium respectively (the content of each carbon source is 20 g/L). After completion, biomass (OD) ₆₀₀ ) The glucose and total sugar content in the fermentation supernatant were also measured by a biosensing analyzer and a phenol sulfuric acid method, and the results are shown in Table 1.

TABLE 1 carbon utilization of Hansenula polymorpha in grape juice

As shown in Table 1, the Hansenula polymorpha CGMCC No.26056 can use glucose, sucrose and fructose as carbon sources, and has lower utilization rate of lactose and soluble starch, and correspondingly, the total sugar content is relatively higher after fermentation is finished.

3. Nitrogen source utilization experiment

Fermentation: the activated Hansenula polymorpha CGMCC No.26056 strain is inoculated in a triangular flask containing YPD liquid culture medium in a 250 mL triangular flask liquid 100mL, and shake-cultured at 30 ℃ and 200 rpm to obtain 48 h. Wherein ammonium sulfate, beef extract, peptone, yeast extract are used as the only nitrogen sources (each nitrogen source content is 10 g/L) of the culture medium, respectively. After completion, biomass (OD) ₆₀₀ ) The glucose and total sugar content in the fermentation supernatant were also measured by a biosensing analyzer and a phenol sulfuric acid method, and the results are shown in Table 2.

TABLE 2 Nitrogen Source utilization of Hansenula polymorpha in grape juice

As shown in Table 2, the Hansenula polymorpha CGMCC No.26056 can use a single nitrogen source: beef extract, peptone, yeast extract, but essentially no growth in media with ammonium sulfate as the sole nitrogen source.

Example 3 preparation of Piteguo fermentation juice

Test example 1

1) Crushing the Piteguo, filtering with gauze to obtain filtrate, and sequentially filtering with 2.0 μm clarifying plate, 0.8 μm clarifying plate, and 0.22 μm filter membrane to obtain Piteguo juice.

2) Taking 100mL Piteguo juice, sterilizing at 100deg.C for 10 min to obtain fermentation medium.

3) Hansenula polymorpha of grape juiceHanseniaspora uvarum) Single colony of CGMCC No.26056 is inoculated into the fermentation culture medium, and is shake-flask cultured at 200 rpm and 28 ℃ for 46 h to obtain the Piteguo fermentation broth.

4) Centrifuging the Piteguo fermentation liquor for 10 min at the rotation speed of 6000 rpm, filtering to remove thalli, taking supernatant, and filtering with a 0.22 mu m filter membrane under the aseptic condition to finally obtain the Piteguo fermentation liquor.

And (3) measuring the total sugar and the glucose content in the fermented juice of the Piteguo by using a phenol sulfuric acid method and a biosensing analyzer.

Finally, the total sugar content in the beer fermentation juice of the test example is 0.117g/100mL, and the glucose content is 0.011g/100mL.

Test example 2

3) Hansenula polymorpha of grape juiceHanseniaspora uvarum) Inoculating single colony of CGMCC No.26056 into the fermentation medium, shake-culturing at 200 rpm and 30deg.C for 48 h to obtain Piteguo fermentation liquid。

The total sugar content in the beer fermentation juice of the test example is 0.0857g/100mL, and the glucose content is 0.007g/100mL. After the fermentation of the Hansenula polymorpha in the grape juice, the total sugar content in the fermented juice of the Piteguo is reduced from 10.82g/100mL to 0.0857g/100mL.

Test example 3

3) Hansenula polymorpha of grape juiceHanseniaspora uvarum) Single colony of CGMCC No.26056 is inoculated into the fermentation culture medium, and is shake-flask cultured at 200 rpm and 32 ℃ for 50 h to obtain the Piteguo fermentation broth.

The total sugar content in the beer fermentation juice of the test example is finally detected to be 0.0872g/100mL, and the glucose content is detected to be 0.008g/100mL.

Comparative example 1

The difference between comparative example 1 and test example 2 is only that the species used for fermenting the Pitot ripe fruit is different, and the species used in comparative example 1 is commercial Kluyveromyces Pichia pastoris CGMCC No.2.4488, and the Pitot fruit fermentation broth fermented by Kluyveromyces non-endophyte Kluyveromyces Pichia is finally obtained.

The total sugar content in the beer fermentation juice of the comparative example was 7.19g/100mL, and the glucose content was 1g/100mL.

Comparative example 2

Comparative example 2 and test example 2 differ only in the species used to ferment the brewery ripe fruit, and the species used in comparative example 2 was commercially available Saccharomyces cerevisiae ATCC 9080, eventually resulting in a brewery fermented juice fermented by Saccharomyces cerevisiae.

The total sugar content in the beer fermentation juice of the comparative example was 5.82g/100mL, and the glucose content was 1.5g/100mL.

Comparative example 3

Comparative example 3 and test example 2 differ only in the species used for fermenting the brewer's ripe fruit, and the species used in comparative example 3 was commercially available Mei Ji Yeast CGMCC No.2.3314, and finally a Piteger fruit fermented juice fermented by Mei Ji Yeast was obtained.

The total sugar content in the beer fermentation juice of the comparative example was 3.41g/100mL, and the glucose content was 0.6g/100mL.

Comparative example 4

The only difference between comparative example 4 and test example 2 is the strain used for fermenting the Piteguo's cured fruit, the strain used in comparative example 4 is commercially available Hansenula polymorpha CGMCC No.2.3213, and the beer fermentation broth fermented by commercially available Hansenula polymorpha CGMCC No.2.3213 is finally obtained.

The total sugar content in the beer fermentation juice of the comparative example was 2.75g/100mL, and the glucose content was 1.0 g/100mL.

Conclusion(s)

As can be seen from the results of test examples 1 to 3 and comparative examples 1 to 4, hansenula polymorpha in grape juice (endophytic in Piteger's ripe fruit) as described in the present applicationHanseniaspora uvarum) The total sugar content in the fermented juice of the Piteguo obtained by fermenting the Piteguo with CGMCC NO.26056 is obviously reduced, namely, compared with other yeasts, the Hansenula polymorpha has better sugar consumption capability. In the preferred test example, the total sugar content in the fermented juice of the Piteguo was reduced from 10.82g/100mL to 0.0857g/100mL.

Example 4 evaluation of antioxidant efficacy of Piteguo fermentation filtrate

The embodiment provides a preparation method of a Piteguo fermentation filtrate, which comprises the following steps:

2) Taking 100mL juice, sterilizing at 100deg.C for 10 min to obtain juice.

3) Hansenula polymorpha of grape juiceHanseniaspora uvarum) Inoculating single colony of CGMCC No.26056 into the sterile Piteguo juice filtrate, and shake-culturing at 200 rpm and 30 ℃ for 48 h to obtain Piteguo fermentation liquor.

4) Centrifuging the Piteguo fermentation liquor at 6000 rpm for 10 min, filtering to remove thalli, taking supernatant, sterilizing at 80 ℃ for 1 h to remove proteins generated in the non-Saccharomyces cerevisiae fermentation process, and filtering with a 0.22 mu m filter membrane to finally obtain Piteguo fermentation filtrate.

The antioxidant effect adopts UVA+UVB to irradiate HaCaT cells, and the cells are contacted with a sample before and after irradiation to detect the content of Reactive Oxygen Species (ROS) in the cells, so that the capacity of the Piteguo fermentation filtrate to inhibit the generation of ROS or remove ROS is examined.

1. Experimental materials

The preparation method comprises the steps of fermenting the filtrate of the Piteguo, filtering the juice of the Piteguo, carrying out HaCaT on human skin keratinocytes, carrying out fetal bovine serum (Gibco), carrying out ROS detection kit (Kelvin), carrying out DMEM (Kelvin), and carrying out trypsin (Kelvin).

2. Instrument and equipment

Carbon dioxide incubator (SANYO), inverted microscope (OLYMPUS, CKX 41), digital display thermostatic water bath (large instrument factory in gold altar city), ultra clean bench (SCB-1520, manufactured by Haer instruments Co., ltd., beijing east Asian), multifunctional microplate reader (TECAN, spark), thermostatic microplate fast vibrator (kylin medical instrument factory in Hai Meng city), UV-8 type ultraviolet lamp box (Beijing electric light source institute), flow cytometer (BD).

3. Experimental method

3.1 Preparation of sample solutions

The solution is prepared by serum-free culture solution DMEM, and filtered and sterilized by a 0.22 mu m filter membrane, and the experimental concentrations of the fermented filtrate of the Piteguo and the filtrate of the Piteguo juice are 2%, 5% and 10%.

3.2 Ultraviolet injury repair efficacy

And (3) paving: taking logarithmic growth phase HaCaT cells at 1×10 ⁵ Inoculating 1 mL cell suspension in 12-well plate at a density of 1/mL, and placing in a carbon dioxide incubator at 37deg.C and 5% CO ₂ Culture 24 h.

And (3) irradiation: irradiation group, cover preservative film, UVA 2000. Mu.W/cm ² Is irradiated with UVB at 700. Mu.W/cm for 1 h ² The negative control group was covered with aluminum foil paper without irradiation for 3 min.

Sample treatment: after irradiation, the old culture solution was discarded, the experimental group was added with the fermented filtrate of Piteguo and the filtrate of Piteguo juice, the concentration of the fermented filtrate of Piteguo and the filtrate of Piteguo juice were 2%, 5% and 10%, respectively, the model group and the negative control group were added with serum-free culture solution, each well was 1. 1 mL, the culture was continued for 24. 24 h, the culture solution was discarded, and the culture solution was washed with PBS for 2 times. 1.5 mL of DCFH-DA is added into each hole, the mixture is put into a cell culture box for incubation for 30 min, and the mixture is uniformly mixed every 5 min to fully combine probes. The probes were discarded and washed twice with pre-warmed serum-free medium and incubated at 37℃for 10 min with 1 mL serum-free medium per well. After washing 1 time with PBS, cells were digested with pancreatin, washed 2 times with PBS, resuspended with 300. Mu.L of PBS, and the average fluorescence intensity was recorded.

3.3 Protective effect against ultraviolet injury

Sample treatment: the old culture solution was discarded, the fermented filtrate of Piteguo and the filtrate of Piteguo juice were added at concentrations of 2%, 5% and 10%, respectively, and the model group and the negative control group were added with serum-free culture solution at 1 mL per well, followed by further culturing at 24 h.

And (3) irradiation: irradiating the group cover preservative film, and using 2000 mu W/cm for UVA ² Is irradiated for 1 h, UVB 700. Mu.W/cm ² The negative control group was covered with aluminum foil paper without irradiation for 3 min. After the irradiation, the culture medium was discarded, and the serum-free culture medium was added to continue the culture of 3 h. After the completion of the incubation, the culture medium was discarded, and washed with PBS 2 times. 1.5 mL of DCFH-DA is added into each hole, the mixture is put into a cell culture box for incubation for 30 min, and the mixture is uniformly mixed every 5 min to fully combine probes. The probes were discarded and washed twice with pre-warmed serum-free medium and incubated at 37℃for 10 min with 1 mL serum-free medium per well. After washing 1 time with PBS, cells were digested with pancreatin, washed 2 times with PBS, resuspended with 300. Mu.L of PBS, detected with a flow cytometer, the average fluorescence intensity was recorded, and the ROS inhibition was calculated.

The ROS inhibition rate calculation method comprises the following steps:

（1）

(1) Wherein:

t-the average of 3 times of fluorescence intensity of the sample;

c—3 averages of fluorescence intensity of negative control;

c0-3 averages of the fluorescent intensity of the naked cell group.

3. Experimental results

The results of the above experiments are shown in Table 3.

TABLE 3 Effect of Piteguo fermentation filtrate on Reactive Oxygen Species (ROS) content

As shown in Table 3, the beer fermentation filtrate provided by the invention has a certain degree of inhibition effect on Reactive Oxygen Species (ROS) generated before and after ultraviolet injury at 2%, 5% and 10%, and the beer fermentation filtrate has the highest inhibition rate on ROS generation at 10% concentration. The invention shows that the Piteguo fermentation filtrate has better oxidation protection and repair effects, and the 10% Piteguo fermentation filtrate has optimal oxidation protection and repair effects and can be used as an antioxidant raw material to be added into cosmetics.

In the same concentration range, the ultraviolet injury protection effect of the beer fruit fermentation filtrate is better than that of the beer fruit juice filtrate which is not fermented by microorganisms. And, the fermented filtrate of the Piteguo shows a good effect of repairing ultraviolet damage, whereas the unfermented filtrate of the Piteguo juice does not have the effect.

Example 5 evaluation of anti-inflammatory efficacy of Piteguo fermentation filtrate

The anti-inflammatory efficacy adopts Lipopolysaccharide (LPS) to stimulate the macrophages of mice, so that the macrophages secrete inflammatory factors, and the anti-inflammatory efficacy of the fermented filtrate of the Piteguo is examined by quantitatively detecting the expression of the inflammatory factors.

1. Experimental materials

The beer fermentation filtrate and beer juice filtrate prepared as described in example 4, mouse macrophage Raw264.7, fetal bovine serum (Gibco), mouse TNF-. Alpha.ELISA Kit (proteontech), mouse IL-6 ELISA Kit (proteontech), mouse IL-1. Beta. ELISA Kit (proteontech), DMEM medium (Kelly organism), human Pro-Collagen I alpha 1 ELISA Kit (abcam).

2. Instrument and equipment

3. Experimental method

3.1 Preparation of sample solutions

LPS: a mother solution with the concentration of 50 ten thousand units/mL is prepared by using a serum-free 1640 culture solution, filtered and sterilized by a 0.22 mu m filter membrane, stored in a refrigerator at the temperature of minus 20 ℃, and diluted into an action solution with the concentration of 4 ten thousand units/mL before use.

Sample solution: the sample solution was prepared with LPS working solution and filtered through a 0.22 μm filter.

3.2 Sample processing

Raw264.7 cells were grown at 1X 10 ⁵ inoculating/mL to24-well plate, at 37℃with 5% CO ₂ Culturing 24 h, adding sample, and culturing 24 h with LPS as model control. Cell supernatants were collected and centrifuged and inflammatory factors were detected according to ELISA kit instructions.

3.3 Detection of inflammatory factor expression

Cell supernatants were collected and centrifuged at 1000 rpm for 10 min, and the supernatants were assayed. Blank holes (without adding sample and enzyme-labeled reagent), standard holes and holes of sample to be tested are respectively arranged. And accurately adding 100 mu L of standard substances on the enzyme-labeled coated plate, and adding 100 mu L of samples to be detected into the holes of the samples to be detected. And (3) adding the sample to the bottom of the hole of the enzyme-labeled plate, and mixing the sample with light shaking. Incubation was carried out for 120 min at 37℃after membrane sealing with a sealing plate. The 20-fold concentrated washing solution is diluted with distilled water for later use. Discarding the liquid, spin-drying, filling each well with the washing liquid, standing for 30 s, discarding, repeating the above steps for 4 times, and drying. 100. Mu.L of antibody working solution was added to each well and incubated for 60 min. The washing was repeated 1 time. 100 mu L of HRP working solution was added to each well and incubated for 40 min. The washing was repeated 1 time. Each well was incubated with 100. Mu.L of TMB working solution for 15-20 min. 100. Mu.L of stop solution was added to each well to terminate the reaction. Absorbance (OD) was measured at the wavelength of blank Kong Diaoling, 450 nm.

4. Experimental results

The results of the above experiments are shown in Table 4.

TABLE 4 Effect of Piteguo fermentation filtrate on TNF-alpha secretion

TABLE 5 Effect of Piteguo fermentation filtrate on IL-6 secretion

TABLE 6 Effect of Piteguo fermentation filtrate on IL-1 beta secretion

From tables 4, 5 and 6, the beer fruit fermentation filtrate provided by the invention has remarkable inhibition effect on secretion of IL-1 beta and TNF-alpha by macrophages in the concentration range of 2%; the concentration ranges of 5% and 10% have remarkable inhibition effect on the secretion of IL-1 beta, IL-6 and TNF-alpha by macrophages, which indicates that the Piteguo fermentation filtrate provided by the invention has a better anti-inflammatory effect, can be used as an anti-inflammatory raw material to be added into cosmetics, and has better inhibition effect than Piteguo fermentation filtrate without microbial fermentation.

The foregoing is merely exemplary of the present application and is not intended to limit the present application. Various modifications and changes may be made to the present application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc. which are within the spirit and principles of the present application are intended to be included within the scope of the claims of the present application.

Claims

1. Hansenula polymorpha of grape juiceHanseniaspora uvarum) The method is characterized in that the Hansenula polymorpha is Hansenula polymorpha ZS02, and is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of No.26056.

2. A formulation comprising any one of A1) to A2):

a1 Hansenula polymorpha of grape juiceHanseniaspora uvarum)；

A2 Hansenula polymorpha of grape juiceHanseniaspora uvarum) A bacterial agent;

3. A composition comprising hansenula polymorpha according to claim 1 or the formulation according to claim 2.

4. Use of hansenula polymorpha in grape juice according to claim 1 or the formulation according to claim 2 or the composition according to claim 3 for the preparation of a fermentation broth of a beer.

5. A method of preparing a beer fermentation broth, the method comprising: fermenting Piteguo with Hansenula polymorpha according to claim 1 or a formulation according to claim 2 or a composition according to claim 3.

6. The beer fermentation broth made by the method of claim 5.

7. A product comprising the beer fermentation broth of claim 6.

8. Use of hansenula polymorpha in grape juice according to claim 1 or the formulation according to claim 2 or the composition according to claim 3 for reducing the sugar content of a culture medium; the sugar is selected from one or more of glucose, sucrose, fructose, lactose and soluble starch.

9. Use of hansenula polymorpha in grape juice according to claim 1 or the formulation according to claim 2 or the composition according to claim 3 or the beer fermentation broth according to claim 6 or the product according to claim 7 for anti-inflammatory, anti-oxidant purposes for non-diagnostic treatment of diseases.

10. Use of hansenula polymorpha in grape juice according to claim 1 or the formulation according to claim 2 or the composition according to claim 3 or the beer fermentation broth according to claim 6 or the product according to claim 7 for the prevention and/or repair of uv-induced damage of the skin barrier for non-disease diagnostic therapeutic purposes.