CN111363687B

CN111363687B - Lu's combined yeast strain, rose fermentation liquor containing same and application

Info

Publication number: CN111363687B
Application number: CN202010247861.5A
Authority: CN
Inventors: 杨立新; 郎八一; 晏秀祥
Original assignee: Kunming Institute of Botany of CAS
Current assignee: Kunming Institute of Botany of CAS
Priority date: 2020-04-01
Filing date: 2020-04-01
Publication date: 2022-05-24
Anticipated expiration: 2040-04-01
Also published as: CN111363687A

Abstract

The invention provides a Saccharomyces cerevisiae strain and a rose fermentation liquid containing the same, and a preparation method and application thereof. The preservation number of the strain is CGMCC No. 19335. The rose sugar is derived from the traditional rose sugar in the Dali Baijiu area, and is obtained by purifying the traditional rose sugar diluent in the Dali Baijiu area, which is cultured for different fermentation years by one or more of a PDA culture medium, a wort culture medium, a Chaudhur culture medium, a YPD culture medium and a Sabouraud culture medium. The roux combined yeast strain and the rose fermentation liquor containing the roux combined yeast strain can obviously increase the content of total polyphenol and total flavone of the rose liquid, thereby improving the antioxidant activity and tyrosinase inhibitory activity of the rose liquid. It can be directly eaten as functional food or used as main raw material or auxiliary material of functional cosmetics for resisting senility, whitening skin and the like in the field of cosmetic application.

Description

Lu's combined yeast strain, rose fermentation liquor containing same and application

The technical field is as follows:

the invention belongs to the field of functional food fermentation and functional cosmetic application, and particularly relates to a zygosaccharomyces rouxii strain capable of enhancing functional food or cosmetics, a rose fermentation liquid containing the zygosaccharomyces rouxii strain and application of the rose fermentation liquid.

The background art comprises the following steps:

the rose sugar is a characteristic medicinal and edible food in Yunnan region, mainly focuses on Kunming, Wenshan, Dali and Lijiang, etc., wherein the rose sugar is naturally fermented under the long-standing name of Dali Bai nationality, and is mainly influenced by fermentation environment and plant resource microorganisms in the fermentation process. Therefore, microorganisms play an extremely important role in the flavor, texture and activity of fermented foods. Moreover, fermentation promotes complex flavor components, and the compounds jointly influence the physicochemical properties and the physiological activity of the fermented product.

The Zygosaccharomyces rouxii (Zygosaccharomyces rouxii) belongs to the genus Zygosaccharomyces, Saccharomyces, Zygosaccharomyces, phylogenetically, in the kingdom of eukaryotes. The colony is round, glossy, flat and neat in edge. The zygosaccharomyces rouxii is a common high-permeability-resistant yeast, and is mainly used for producing products such as soy sauce, sweet sauce, flavor enhancer and the like by fermentation traditionally. In the industrial production, the arabitol sugar can be produced by fermentation. In the field of food technology, Saccharomyces rouxii (Zygosaccharomyces rouxii) can also be used for culturing and adsorbing heavy metal cadmium. However, the application in functional foods and functional cosmetics is hardly reported.

Polyphenols and flavonoids are widely present in plants and are natural antioxidants that are capable of scavenging active oxygen and free radicals in the body and preventing oxidative stress effects. Oxidative stress is an important factor causing aging and diseases, while polyphenol and flavonoid compounds contain polyhydroxy structures, can be chelated with metal ions to form complexes, can inhibit aging and related diseases caused by lipid oxidation and the like caused by metals, can also be chelated with copper ions at the active part of tyrosinase to form tyrosinase activity inhibition, and in recent years, polyphenol and flavonoid compounds are paid attention by the cosmetic industry and have important prospects in the development of functional foods and cosmetics.

According to the invention, by carrying out microbial separation on the traditional rose sugar in the Dali white region, a Lu's combined yeast strain (Zygosaccharomyces rouxii) is separated and identified and added into the rose liquid for fermentation, the total polyphenol and total flavone contents of the rose liquid after fermentation are obviously improved, and the antioxidant activity and tyrosinase inhibitory activity of the rose liquid are increased. Therefore, the Zygosaccharomyces rouxii strain capable of enhancing the activity of functional foods or cosmetics is provided, and has important application value for the development of functional foods and cosmetics.

The invention content is as follows:

the first purpose of the invention is to provide a Ludwith combined yeast strain with the preservation number of CGMCC No. 19335. The Zygosaccharomyces rouxii strain is identified by Beijing Optimalaceae Biotechnology Co., Ltd, is deposited in Kunming plant research institute of Chinese academy of sciences, and is numbered as TFR-1. And the strain is preserved in China general microbiological culture Collection center on 13.01.2020, wherein the preservation address is as follows: no. 3 of West Lu No.1 of Beijing, Chaoyang, and the collection number of the culture collection center of the institute of microbiology of Chinese academy of sciences is CGMCC No. 19335.

The second purpose of the invention is to provide a source of the above zygosaccharomyces rouxii strain, which is obtained by purifying one or more of PDA culture medium, wort culture medium, Chaudhuri culture medium, YPD culture medium and Sabouraud culture medium by culturing traditional rose sugar diluent in the white kingdom region with different fermentation periods.

The third purpose of the invention is to solve the key technical problem of providing a preparation method capable of enhancing the activity of functional foods or cosmetics.

In order to solve the technical problems, the invention provides the following technical scheme:

A Saccharomyces cerevisiae Zygosaccharomyces rouxii with preservation number of CGMCC No.19335 is provided.

The Saccharomyces cerevisiae strain is a Saccharomyces rouxii strain derived from traditional rose sugar in the Dalbergia, and is obtained by purifying traditional rose sugar dilution in the Dalbergia with different fermentation periods by one or more of PDA culture medium, wort culture medium, Chao's culture medium, YPD culture medium and Sa's culture medium.

A strain of zygosaccharomyces rouxii as described, the age of rose-sugar fermentation is a time point or time points in 15 days-20 years of traditional rose-sugar fermentation; the Dali Bai region refers to one or more regions of Dali ancient city, Dali Happy continent, Dali He Qing, Dali Wei mountain, Dali Jianchuan, Happy ancient town and Dali Er Source; the traditional rose sugar diluent is prepared by diluting traditional rose sugar with sterile water by 10-10%⁶Doubled rose sugar solution.

The application of the zygosaccharomyces rouxii strain in functional food fermentation.

The application of the zygosaccharomyces rouxii strain in producing functional cosmetic raw materials.

The Zygosaccharomyces rouxii is applied to the preparation of antioxidant activity and tyrosinase inhibition active agents.

The invention also provides a rose fermentation liquor containing the Zygosaccharomyces rouxii, which is prepared from the following components in parts by weight: 20-30% of rose petal, 10-30% of white sugar, 10-30% of brown sugar, 1-10% of honey, 1-5% of edible white spirit, 40-60% of sterile water and 4.15 x 10%⁶1-5% of CFU/ml Lu's combined yeast strain liquid.

A rose fermentation liquor, which is obtained by adding the zygosaccharomyces rouxii strain into rose liquor for fermentation, and is prepared by the following steps:

(1) raw materials: the materials are prepared according to the following weight ratio: 20-30% of rose petals, 10-30% of white sugar, 10-30% of brown sugar, 1-10% of honey, 1-5% of edible white spirit, 40-60% of sterile water and 4.15 x 10⁶1-5% of CFU/ml Lu's combined yeast strain liquid;

(2) and (3) sterilization: sterilizing the fermentation tank under high pressure, sterilizing the raw materials with ethanol before filling and before inoculating, sterilizing with ultraviolet for 45-90min after filling and before inoculating;

(3) Inoculation: adding 4.15 x 10 weight parts of the sterilized fermentation tank and the raw materials in the step (2)⁶One of 1-5% of CFU/ml Zygosaccharomyces rouxii bacterial liquid, and mixing uniformly;

(4) and (3) fermentation: sealing the fermentation tank obtained in the step (3), controlling the fermentation temperature to be 20-37 ℃ and the fermentation time to be 24-720 h;

(5) and (4) sample preservation, namely centrifuging the sample obtained by fermentation in the step (4) at 4 ℃ for 15min at 4000rmp/min, collecting supernatant, and preserving at-20 ℃.

According to the rose fermentation liquor, the DPPH free radical clearance rate is 78-100% when the rose fermentation liquor is at the concentration of 0.1-10%; the tyrosinase inhibition rate is 2-87%.

The invention also provides a preparation method of the rose fermentation liquor, which comprises the following steps:

(1) raw materials: the materials are prepared according to the following weight ratio: 20-30% of rose petals, 10-30% of white sugar, 10-30% of brown sugar, 1-10% of honey, 1-5% of edible white spirit, 40-60% of sterile water and 4.15 x 10⁶1-5% of CFU/ml Lu's combined yeast liquid;

(5) and (5) sample preservation, namely centrifuging the sample obtained by fermentation in the step (4) at 4 ℃ for 15min at 4000rmp/min, collecting supernatant, and preserving at-20 ℃.

In addition, the application of the rose fermentation liquor in preparing functional foods or anti-aging and whitening functional cosmetics is provided.

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

the invention provides a zygosaccharomyces rouxii strain capable of enhancing the activity of functional food or cosmetics and a rose fermentation liquid containing the zygosaccharomyces rouxii strain. The strain and the rose fermentation liquor containing the strain can be applied to functional food and cosmetics as a strategy for enriching total polyphenol and total flavone. The rose liquid fermentation product of the zygosaccharomyces rouxii can be directly eaten as functional food or used as a main raw material or an auxiliary material of functional cosmetics such as anti-aging and whitening cosmetics in the field of cosmetic application.

The Zygosaccharomyces rouxii strain is identified by Beijing Optimala Biotechnology Limited, is preserved in Kunming plant research institute of Chinese academy of sciences, and is numbered as TFR-1. And the culture is preserved in China general microbiological culture Collection center on 13.01.2020, with the preservation address: the preservation number of No. 3 of Xilu No.1 of Beijing, Chaoyang, and the strain preservation center of the institute of microbiology of Chinese academy of sciences is CGMCC No. 19335.

Description of the drawings:

FIG. 1 shows the culture pattern of a Zygosaccharomyces rouxii strain of the present invention.

The specific implementation mode is as follows:

the following description will further explain the substance of the present invention by using the embodiments of the present invention with reference to the accompanying drawings, but the present invention is not limited thereto.

Example 1

Isolation and characterization of a strain of Saccharomyces rouxii.

1. Isolation and purification of the strains

Diluting traditional rose sugar purchased from different fermentation years in the region of the great care white with sterile water for 10 percent²Spreading on PDA culture medium plate with spreader, and culturing at 28 deg.CCulturing in a box, and after the colonies grow out, performing colony purification by adopting a Z-shaped plate marking method. The different fermentation periods of the rose sugar refer to one time point or a plurality of time points in 15 days to 20 years of the traditional rose sugar fermentation; the Dali Bai region refers to one or more regions of Dali ancient city, Dali Happy continent, Dali He Qing, Dali Wei mountain, Dali Jianchuan and Dali Er Source; the traditional rose sugar dilution means that the traditional rose sugar is diluted by 10-10 percent of sterile water ⁶The rose liquid after doubling.

2. Bacterial colony characteristics and cell morphology

The bacteria on the purified PDA culture medium plate are all milky white, the surface is smooth and moist, and the size of the bacterial colony is moderate. The colony morphology in PDA medium plates is shown in FIG. 1.

3. Molecular biological characterization of strains

The purified colonies were picked, genomic DNA was extracted using TSINGKE plant DNA extraction kit (universal), and fungal universal primers were used for the forward primer ITS1 and reverse primer ITS4 according to the following specifications: template: 1-3ul, forward primer: 1ul (10P), reverse primer: 1ul (10P), enzyme mix: 15ul (2X), ddH₂Genomic DNA was amplified after O supplementation to 30 ul. The PCR amplification conditions were: pre-denaturation at 94 ℃ for 5min was followed by the following cycles: denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 30s, 35 cycles, and extension at 72 ℃ for 5 min. The PCR product was detected by 1.0% agarose gel electrophoresis and recovered by cutting the gel to obtain a product, Saccharomyces Rouxii, which was deposited at Kunming plant institute of Chinese academy of sciences under the accession number TFR-1 and was classified and named as Saccharomyces Rouxii. Meanwhile, the culture is preserved in China general microbiological culture Collection center (CGMCC) on 13.01.2020, with the preservation number of CGMCC 19335.

Example 2

An experiment of increasing total polyphenols and total flavonoids in rose liquid fermentation with a strain of Saccharomyces rouxii.

Preparation of rose fermentation broth containing a strain of zygosaccharomyces rouxii:

(1) preparing materials: the ingredients are prepared according to the following components (weight ratio): 25% of rose petal and white0% of sugar, 25% of brown sugar, 0% of honey, 0% of edible white wine, 45% of sterile water and 4.15 x 10⁶5% of a strain of a mutant strain of CFU/ml of Saccharomyces rouxii (Zygosaccharomyces rouxii).

(2) And (3) sterilization: high-pressure sterilization, alcohol sterilization and ultraviolet sterilization.

(3) Inoculation: adding 4.15 x 10 of the fermentation tank and the raw materials sterilized in the step (2) according to the weight ratio⁶5 percent of CFU/ml strain of Zygosaccharomyces rouxii (Zygosaccharomyces rouxii) bacteria liquid and evenly mixed.

(4) Fermentation: and (4) sealing the fermentation tank obtained in the step (3), controlling the fermentation temperature to be 28 ℃, and fermenting for 24-720 h.

Table 1 raw material components weight specific gravity

TABLE 2 Total Polyphenol and Total Flavonoids content variation during fermentation

Example 3

Evaluation of rose liquid fermentation broth in functional food and cosmetics.

1. Rose fermentation broth was prepared according to example 2.

2. Evaluation of antioxidant activity:

mixing the rose liquid fermentation liquor of a sample to be detected with DPPH (final concentration of 100 mu M) for reaction, setting 3 repeated holes, simultaneously setting blank control holes and Trolox positive control holes of different samples, measuring OD values by an enzyme-labeling instrument at 30 ℃ for 1h, and calculating to obtain the oxidation resistance, wherein the detection wavelength is 515 nm.

Oxidation resistance (%) (1-well experiment OD)_515nmBlank well OD_515nm)×100％

3. Tyrosinase inhibitory activity evaluation:

mixing the rose liquid fermentation liquor of a sample to be detected with L-Dopa, adding tyrosinase (the final concentration is 25U/mL) to start reaction, setting 3 repeated holes, setting a blank control without the sample and a Kojic Acid positive control, measuring an OD value by an enzyme-labeling instrument at room temperature for 5min, and measuring the detection wavelength at 490 nm. And calculating to obtain the tyrosinase activity inhibition rate.

Tyrosinase activity inhibition (%) (1-sample OD)_490nmExperimental control well OD_490nm)×100

TABLE 3 in vitro antioxidant Effect of Rose fermentation broth samples

TABLE 4 inhibition of tyrosinase by rose fermentation broth samples

Example 4:

the zygosaccharomyces rouxii strain and the rose liquid fermentation liquid containing the zygosaccharomyces rouxii can be used for preparing food and sweetening agents, and can be directly eaten when being used as food or prepared by being matched with conventional food auxiliary materials.

The preparation process of the granules comprises the following steps: the strain of Zygosaccharomyces rouxii obtained in examples 1 and 2 and rose liquor fermentation broth 3 parts, dextrin 2 parts, and appropriate amount of 50% ethanol. The raw and auxiliary materials are prepared according to the conventional process for preparing granules, namely, the raw and auxiliary materials are firstly tested and weighed, soft material preparation, granulation, drying, granule finishing and finished product subpackaging. The specification of the granules is as follows: 20g per bag. Is a portable preparation, has simple ingredients, maintains the nutrient components to the maximum extent, can enhance the immunity, resist oxidation, inhibit tyramine enzyme and the like, is suitable for people of all ages, and is suitable for all kinds of people.

Example 5:

application of nasal spray

Nasal spray: 0.2 percent of Lu's combined yeast strain or rose liquid fermentation liquor

Sodium chloride 8mg

EDTA 1mg

Sodium phosphate buffer (pH6.5) 10mg

Polyethoxyether 10mg

Double distilled water 2ml

The preparation method comprises the following steps: adding one component into double distilled water with stirring until completely deeply decomposing, and adding the other component. After adding distilled water to 2ml, the solution was filtered on a sterile filter, bottled and partitioned according to the appropriate dose.

Example 6:

application of whitening cosmetics

The formula (W%) of the whitening cream is as follows:

0.5 percent of Ludwith combined yeast strain or rose liquid fermentation liquor

Stearic acid 8.0

C16 alcohol 2.0

Self-emulsifying monoglyceride 2.0

Hydrogenated lanolin 2.0

Liquid paraffin 12.0

Glycerol 7.0

Emulsifier 1.5

0.2% of preservative

Essence 0.2

Deionized water to 100

The cosmetic with the formula is prepared by a conventional cosmetic preparation method.

Example 7:

application of anti-aging cosmetics

The formula of the emulsion (W%):

deionized water to 100ml

Example 8:

toning lotion application

Toning lotion formula

Water phase: 0.35 g of sodium polyacrylate, 4 g of glycerol, 2.5 g of 1, 3-butanediol, 2.5 g of witch hazel extract, 50.5 g of vitamin B, 0.5 g of arbutin and EDTA-Na 250 mg; deionized water, appropriate amount, add to 100 ml. The preparation method comprises the following steps: 0.3 g of sodium polyacrylate is dissolved in 70ml of water, stirred and fully swelled. Slowly adding the rest components in the prescription, and stirring continuously; then 0.2% of the yeast strain or rose liquid fermentation broth was added and stirred to homogenize the mixture. Adding deionized water to 100ml, and packaging.

Example 9:

application of repairing frost

The prescription of the repair cream comprises: 6 g of glycerol, 1.5 g of carbomer, 1.5 g of triethanolamine, 6 g of propylene glycol, 0.2 g of ethyl hydroxybenzoate, 1.2 g of tanshinone extract, a proper amount of torreya grandis essential oil and deionized water, and the total weight is 100 g. The preparation method comprises the following steps: adding carbomer into deionized water, stirring, standing overnight to swell completely, adding glycerol, and adjusting pH with triethylamine to increase gel matrix viscosity. And uniformly mixing the tanshinone extract, the propylene glycol and the deionized water according to the prescription amount. Adding carbomer gel, adding 0.5% zygosaccharomyces rouxii or rose liquid fermentation broth, and ethyl hydroxybenzoate, mixing, stirring, adding distilled water to desired volume, and grinding.

The zygosaccharomyces rouxii strain capable of enhancing the activity of functional food or cosmetics is derived from the traditional rose sugar in the white region of the Dali, and is prepared from the following components in parts by weight: 20-30% of rose petals, 10-30% of white sugar, 10-30% of brown sugar, 1-10% of honey, 1-5% of edible white spirit, 40-60% of sterile water and 4.15 x 10⁶1-5% of CFU/ml Lu's combined yeast strain (Zygosaccharomyces rouxii) bacterial liquid, and can remarkably increase the content of total polyphenol and total flavone of the rose liquid under the conditions of fermentation temperature of 20-37 ℃ and fermentation time of 24-720 h, thereby improving the antioxidation of the rose liquidActivity and tyrosinase inhibitory activity. It can be directly eaten as functional food or used as main raw material or auxiliary material of functional cosmetics for resisting senility, whitening skin and the like in the field of cosmetic application.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made by those skilled in the art without departing from the spirit and scope of the invention.

Claims

1. A yeast strain of Saccharomyces rouxii (Zygosaccharomyces rouxii) Zygosaccharomyces rouxii) The preservation number of the strain is CGMCC No. 19335.

2. Use of the strain of Saccharomyces rouxii according to claim 1 for functional food fermentation.

3. Use of the strain of Saccharomyces rouxii according to claim 1 for the production of functional cosmetics.

4. Use of the strain of Saccharomyces rouxii as claimed in claim 1 for the preparation of antioxidant and tyrosinase inhibiting agents.

5. The rose fermentation liquor is characterized by being prepared from the following components in parts by weight: 20-30% of rose petals, 10-30% of white granulated sugar, 10-30% of brown sugar, 1-10% of honey, 1-5% of edible white spirit, 40-60% of sterile water and 4.15 × 10⁶CFU/ml in claim 1 of the Roots joined yeast strain bacterial liquid 1-5%.

6. Use of the rose fermentation broth of claim 5 for the preparation of functional foods or anti-aging and whitening functional cosmetics.