Lactic acid bacteria and yeast co-fermented chrysanthemum product as well as preparation method and application thereof

Abstract

The invention discloses a lactic acid bacteria and yeast co-fermented chrysanthemum product and a preparation method and application thereof, the product comprises a chrysanthemum fermented product and a metagen generated by co-fermenting chrysanthemum with lactic acid bacteria and yeast, and the ratio of the chrysanthemum fermented product to the metagen is 1: 10-1: 20; the lactobacillus is lactobacillus fermentum ZJU2303-RH15, the preservation number of the lactobacillus fermentum ZJU2303-RH15 is CGMCC (China General Microbiological Culture Collection Center) 1.60150, the saccharomycetes is Saccharomyces cerevisiae ZJU2014-RH05, and the preservation number of the saccharomycetes is CGMCC 2.6673. The whitening and anti-wrinkle composition has the advantages of good whitening and anti-wrinkle functions and low production cost, and has a wide market prospect in preparation of cosmetics with whitening and anti-wrinkle functions.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and in particular relates to a chrysanthemum product co-fermented by lactic acid bacteria and yeast, a preparation method and application thereof. Background Art

[0002] Functional microorganisms and their fermented products have a wide range of uses in the food and cosmetics fields. In recent years, postbiotics, which are fermented and inactivated by probiotics and composed of probiotic cell components and their fermentation products, have become a research and development hotspot and have demonstrated a variety of skin care and beauty effects.

[0003] For example, patent document CN202311565607.X discloses a strain of Lactobacillus plantarum CCFM1354 and its postbiotics that target anti-sugar, anti-aging and skin health improvement, which can be used to prepare topical or oral products that target anti-sugar, anti-aging and skin health improvement.

[0004] Patent document CN202410186954.X discloses a fermented mucus lactobacillus JYLF-315 for improving skin aging and its postbiotic preparation and application, which can inhibit skin pathogens, reduce skin inflammation, and slow down skin aging.

[0005] Patent document CN202311458890.6 discloses a probiotic postbiotic composition for improving the scalp environment, as well as its preparation method and application. The composition includes postbiotics obtained by fermenting two strains of bacteria, Lactobacillus paracasei ProSci-101 and Lactobacillus fermentum ProSci-602. The postbiotics have the functions of cell repair, promoting elastin production and dermis metabolism, thereby achieving the purpose of improving the scalp environment and preventing hair loss.

[0006] Chrysanthemum has become an important food and cosmetic raw material because it is rich in natural products.

[0007] For example, patent document CN202311537592.6 discloses the application of a compound containing celery seed volatile oil and calendula extract, and a polysaccharide composition. Celery seed volatile oil and calendula extract are compounded and used. The synergistic effect between the two within a certain proportion range can achieve 1+1>2 antioxidant, whitening, anti-ultraviolet, antibacterial and other effects. On this basis, after adding any one of kelp polysaccharide, sargassum polysaccharide, ginkgo polysaccharide or platycodon polysaccharide, the resulting polysaccharide gel mask and cream have better moisturizing and antibacterial functions, and at the same time extend the shelf life of the product.

[0008] Patent document CN202111200150.3 discloses a device and method for preparing golden chrysanthemum volatile oil with whitening and freckle-removing effects. The skin care products prepared using golden chrysanthemum liposomes have the effects of moisturizing, nourishing, whitening, anti-aging, anti-acne and relieving skin inflammation.

[0009] Patent document CN201810908915.0 discloses a total flavonoid extract of snow chrysanthemum, a preparation method thereof, and an application in cosmetics. The total flavonoid extract of snow chrysanthemum has strong ultraviolet absorption in the UVB and UVA bands, has a good inhibitory effect on tyrosinase, and can be used as a plant additive in the development of sunscreen and whitening cosmetics.

[0010] Patent documents CN201711055448.3, CN201711055449.8, CN201711062573.7 and CN201711054986.0 disclose antioxidant whitening compositions, moisturizing and soothing skin care compositions, anti-wrinkle skin care compositions, anti-aging skin care products and preparation methods thereof containing chrysanthemum cell extracts, showing that chrysanthemum cell extracts, as strong antioxidants, can promptly scavenge peroxyl free radicals, prevent inactivation of effective ingredients, and enhance whitening effects. When used together with loofah extract, sanguisorba officinalis root extract and perilla leaf extract, they have good synergistic anti-allergic effects, can promote skin metabolism, and reduce fine lines on the skin. When used together with walnut leaf extract and Acanthopanax senticosus extract, they have anti-aging activity.

[0011] Although many technical solutions for utilizing chrysanthemums have been disclosed in the prior art, there is still great potential for exploring the utilization value of chrysanthemums. How to better utilize chrysanthemums is also a hot topic of current research. Summary of the invention

[0012] The object of the present invention is to provide a lactic acid bacteria and yeast co-fermented chrysanthemum product, a preparation method and application thereof. The present invention has the advantages of good whitening and anti-wrinkle function and low production cost, and has broad market prospects in the preparation of cosmetics with whitening and anti-wrinkle functions.

[0013] The technical solution of the present invention is as follows: the lactic acid bacteria and yeast co-fermentation chrysanthemum product comprises chrysanthemum fermentation product and postbiotics generated by the lactic acid bacteria and yeast co-fermentation chrysanthemum, and the ratio of the chrysanthemum fermentation product to the postbiotics is 1:10 to 1:20.

[0014] In the aforementioned lactic acid bacteria and yeast co-fermented chrysanthemum product, the ratio of chrysanthemum fermentation product to postbiotics is 1:15.

[0015] In the aforementioned lactic acid bacteria and yeast co-fermented chrysanthemum product, the lactic acid bacteria is Lactobacillus fermentum ZJU2303-RH15, with a preservation number of CGMCC 1.60150, and the yeast is Saccharomyces cerevisiae ZJU2014-RH05, with a preservation number of CGMCC 2.6673.

[0016] The method for preparing the chrysanthemum product by co-fermentation of lactic acid bacteria and yeast comprises the following steps:

[0017] a. First, crush the chrysanthemum, add water and mix well, and then add brown sugar to obtain a fermentation medium. Each liter of the fermentation medium contains 4-6 parts of chrysanthemum and 1-3 parts of brown sugar, each part of which is 10g;

[0018] b. Activate lactic acid bacteria and yeast separately and inoculate them into the fermentation medium at a density of (0.5-1.5)×10 7 cfu / mL, ferment at 30±2℃ for 48-72h to obtain fermentation broth;

[0019] c. The fermentation liquid is centrifuged to obtain a supernatant and a residual portion, the supernatant is concentrated under reduced pressure to 15%-25% of the original volume, and then freeze-dried to obtain a chrysanthemum fermentation product;

[0020] d. Ultrasonic treatment and spray drying of the remaining portion of step c are performed to obtain postbiotics;

[0021] e. Compounding the chrysanthemum fermentation product and postbiotics in proportion to obtain a finished product.

[0022] In the aforementioned method for preparing a chrysanthemum product by co-fermentation of lactic acid bacteria and yeast, in step a, each liter of fermentation medium contains 5 parts of chrysanthemum and 2 parts of brown sugar.

[0023] In the above-mentioned method for preparing chrysanthemum products by co-fermentation of lactic acid bacteria and yeast, in step b, the inoculation density is 1×10 7 cfu / mL, fermentation for 60h.

[0024] In the aforementioned method for preparing a chrysanthemum product by co-fermentation of lactic acid bacteria and yeast, in the step c, the supernatant is concentrated and freeze-dried under the conditions of a vacuum degree of 65-75 KPa and a temperature of 55-60°C.

[0025] In the aforementioned method for preparing a chrysanthemum product by co-fermentation of lactic acid bacteria and yeast, in the step d, the remaining portion of the step c is subjected to ultrasonic treatment to break the cells in the remaining portion and then spray-dried.

[0026] The aforementioned lactic acid bacteria and yeast co-fermented chrysanthemum product is used as a whitening and anti-wrinkle functional ingredient in cosmetics, and the addition ratio in the cosmetics is 0.5% to 1.5%.

[0027] In the aforementioned application, the addition ratio is 1%.

[0028] Compared with the prior art, the present invention uses brown sugar and chrysanthemum as fermentation substrates, and utilizes lactic acid bacteria and yeast to co-ferment and prepare chrysanthemum fermentation products and postbiotics. The fermentation raw materials are highly safe and contain rich active substances. By preferably using specific lactic acid bacteria and specific yeasts, the two are synergistic, the obtained chrysanthemum fermentation products have high total antioxidant activity, and the obtained chrysanthemum fermentation products and the obtained postbiotics also have a synergistic effect. After the two are compounded in an appropriate proportion, they have good whitening and anti-wrinkle functions. In addition, the combination of specific lactic acid bacteria and specific yeasts can also significantly increase the yield of postbiotics, thereby reducing product costs. In summary, the present invention has the advantages of good whitening and anti-wrinkle functions and low production costs, and has broad market prospects in the preparation of cosmetics with whitening and anti-wrinkle functions. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with the embodiments, but they are not intended to limit the present invention.

[0030] The test methods used in the following examples and comparative examples are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0031] The lactic acid bacteria refers to Lactobacillus fermentum ZJU2303-RH15 (hereinafter referred to as RH15), which was deposited in the China General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on October 26, 2023, with the deposit number CGMCC 1.60150.

[0032] The yeast refers to Saccharomyces cerevisiae ZJU2014-RH05 (hereinafter referred to as RH05), which was deposited in the China General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on December 23, 2021, with the deposit number CGMCC 2.6673.

[0033] Both RH15 and RH05 originated from food fermentation experiments conducted by the College of Biosystems Engineering and Food Science of Zhejiang University. They were obtained by streaking single colony culture, and rRNA sequencing results confirmed that they were classified as Lactobacillus rhamnosus and Saccharomyces cerevisiae.

[0034] Both RH15 and RH05 have been publicly deposited and are strains available to the public.

[0035] MRS liquid medium, used for RH15 activation, commercially available or self-prepared, 1L, including the following components: peptone 10g, beef extract 10g, yeast extract 5g, diammonium hydrogen citrate 2g, glucose 20g, sodium acetate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.5g, manganese sulfate 0.2g, Tween 80 1.5mL, dilute to 1L, sterilize at 121℃ for 15min;

[0036] YPD liquid culture medium, used for RH05 activation, commercially available or self-prepared, includes the following components per 1 L: 20 g peptone, 10 g yeast extract, 20 g glucose, fixed to 1 L, sterilized at 121°C for 15 min.

[0037] Brown sugar, in compliance with standard "T / CNFIA 110-2018 Brown sugar".

[0038] Chrysanthemum, in line with the standard "T / CNHFA111.170-2024 Health food raw material chrysanthemum".

[0039] The experimental methods involved in the following examples and comparative examples are as follows:

[0040] 1. Total antioxidant activity of chrysanthemum fermentation product (component I).

[0041] The total antioxidant activity (T-AOC) was determined using a kit (Nanjing Jiancheng Bioengineering Research Institute) according to the instructions. At 37°C, the absorbance value of the reaction system increased by 0.01 per milliliter of fermentation liquid per minute, which was 1 T-AOC unit, expressed as U / mL.

[0042] 2. Yield of postbiotics (component II).

[0043] After removing the fermentation broth, i.e. the chrysanthemum fermentation product (component I), the centrifuged bacterial cells and other precipitates are ultrasonically treated and spray-dried to obtain the postbiotics (component II), which are mainly lactic acid bacteria and yeast cells. Therefore, the bacterial density in the fermentation broth at the end of fermentation is used to characterize the yield of postbiotics (component II).

[0044] Lactic acid bacteria count: in accordance with the method specified in "GB 4789.35-2023 National Food Safety Standard Food Microbiology Examination Lactic Acid Bacteria Examination".

[0045] Yeast count: in accordance with the method specified in "GB 4789.15-2016 National Food Safety Standard Food Microbiology Examination Mold and Yeast Count".

[0046] 3. Determination of tyrosinase inhibition rate (representing whitening function).

[0047] Reference (ZHAO Y et al. Identification and molecular mechanism of novel tyrosinase inhibitory peptides from the hydrolyzate of 'Fengdan' peony (Paeonia ostii) seed meal proteins: Pepti domics and in silico analysis. LWT-Food Science & Technology, 2023 (180): 114695.), add 1.0 mL of sample solution and 0.5 mL of tyrosinase solution to the sample tube (T), add 1.0 mL of sample solution and 0.5 mL of buffer to the sample background tube (T0), add 1.0 mL of buffer and 0.5 mL of tyrosinase solution to the enzyme reaction tube (C), and add 1.5 mL of buffer to the solvent background tube (C0). After incubation in a 37°C water bath for 10 minutes, 2.0 mL of L-DOPA solution was added. After reacting at room temperature for 5 minutes, the absorbance was measured at 475 nm. Kojic acid was used as a positive control. The tyrosinase inhibition rate (%) was calculated according to the following formula:

[0048] Tyrosinase inhibition rate (%) = [1-(T-T0) / (C-C0)] × 100%,

[0049] Wherein: T represents the absorbance of the sample group; T0 represents the absorbance of the sample background group; C represents the absorbance of the enzyme reaction group; C0 represents the absorbance of the solvent background group.

[0050] 4. Determination of hyaluronidase inhibition rate (representing anti-wrinkle function).

[0051] Reference (Liu Lijun et al. Determination of the content of sodium hyaluronate in raw materials by spectrophotometry. Beverage Industry, 2023, 26(1): 18-21.), add 50μL of the sample solution to be tested, 4.85mL of 0.2mol / L sodium hyaluronate buffer and 50μL of 1.0mg / mL hyaluronidase solution into a centrifuge tube, shake well and add 50μL of 3.44mg / mL sodium hyaluronate solution, react at 37℃ for 15min, measure the absorbance of the solution at 400nm wavelength, record it as A1, replace the enzyme solution with buffer solution and measure the absorbance as sample control Ac1, the blank control is to replace the enzyme solution and sample solution with buffer solution, and measure the absorbance as Ac2, and replace the sample with buffer solution and measure the absorbance as A0, with sodium hyaluronate as the positive control. Calculate the hyaluronidase activity inhibition rate (%) according to the following formula,

[0052] Hyaluronidase inhibition rate (%) = [1-(Ac1-A1) / (Ac2-A0)] × 100%,

[0053] Wherein: Ac1 represents the absorbance of the sample control group; A1 represents the absorbance of the sample group; Ac2 represents the absorbance of the blank control group; A0 represents the absorbance of the blank group.

[0054] 5. Synergistic effect evaluation: The synergistic effect evaluation adopts Jin (Zhengjun)'s formula combined with the index Q value. First, the efficacy value E is calculated.

[0055] E=(E 试验 -E 对照 ) / E 试验 ×100%,

[0056] Then the Jin (Zhengjun) formula was used to calculate the combination index Q value.

[0057] Q=E Ⅰ+Ⅱ / (E Ⅰ +E Ⅱ -E Ⅰ ·E Ⅱ ),

[0058] E Ⅰ+Ⅱ E is the effect of two factors with expected synergistic effects when used together. Ⅰ 、E Ⅱ The Q value is the effect of two factors that are expected to have synergistic effects when applied separately. The meaning of Q value: 0.85≤Q≤1.15 is simple addition (+); Q>1.15 is enhancement (++); Q<0.85 is antagonism (-). The larger the Q value, the stronger the synergistic effect.

[0059] For specific steps, please refer to the literature (Liu Lijuan et al. Effects of Smilax glabra, Prunus mume and their combination on inflammatory factors and Notch signaling pathway in skin lesions of mice with psoriasis. Journal of Traditional Chinese Medicine, 2022, 63(19): 1880-1887.)

[0060] Example 1: A method for preparing a chrysanthemum product by co-fermentation of lactic acid bacteria and yeast, comprising the following steps:

[0061] a. Prepare fermentation medium (1L, w / v): grind 5 parts of chrysanthemum, add water and mix well, add 2 parts of brown sugar, each weighing 10g, and adjust the volume to 1L;

[0062] b. RH15 and RH05 were activated and inoculated into the fermentation medium at a density of 0.5×10 7 cfu / mL, ferment at 30±2℃ for 48h to obtain fermentation broth;

[0063] c. The fermentation liquid is centrifuged to obtain a supernatant and a remainder, and the supernatant is concentrated to 15% to 25% (preferably 20%) of the original volume under the conditions of a vacuum degree of 65 to 75 KPa and a temperature of 55 to 60° C., and then freeze-dried to obtain a chrysanthemum fermentation product (referred to as component I);

[0064] d. treating the remaining portion of step c with ultrasonic waves to break the cells inside the portion, and then spray drying the portion to obtain postbiotics (referred to as component II);

[0065] e. Compounding the chrysanthemum fermentation product and postbiotics in a certain proportion to obtain a finished product.

[0066] The chrysanthemum fermentation product obtained in step c was tested, and the yield of the postbiotics obtained in step d was tested. The test results are as follows:

[0067] The total antioxidant activity of chrysanthemum ferment (fraction I) = 64.6% ± 8.5%.

[0068] Postbiotic (component II) yield (bacterial density) = 6.55 × 10 8 ±1.07×10 8 cfu / mL.

[0069] e. Compounding the chrysanthemum fermentation product and postbiotics in proportion to obtain a finished product.

[0070] Example 2: Based on Example 1, in step b, the inoculation density of RH15 and RH05 was changed to 1.5×10 7 cfu / mL, ferment at 30±2℃ for 72h, obtain fermentation liquid, and keep the rest unchanged. The test results are as follows:

[0071] The total antioxidant activity of chrysanthemum ferment (fraction I) = 64.8% ± 4.2%.

[0072] Postbiotics (component II) yield (bacterial density) = 6.93 × 10 8 ±1.48×10 8 cfu / mL.

[0073] Example 3: Based on Example 1, in step b, the inoculation density of RH15 and RH05 was changed to 1×10 7 cfu / mL, ferment at 30±2℃ for 60h, obtain fermentation liquid, and keep the rest unchanged. The test results are as follows:

[0074] The total antioxidant activity of chrysanthemum ferment (fraction I) = 66.3% ± 7.1%.

[0075] Postbiotic (component II) yield (bacterial density) = 8.65 × 10 8 ±1.20×10 8cfu / mL.

[0076] Comparative Example 1: Based on Example 1, RH05 was omitted in step b, and RH15 was activated and the inoculation density was 1×10 7 cfu / mL was inoculated into the fermentation medium and fermented at 30±2℃ for 60 hours to obtain the fermentation liquid, and the rest remained unchanged. The test results are as follows:

[0077] The total antioxidant activity of chrysanthemum ferment (fraction I) = 22.5% ± 4.4%.

[0078] Postbiotics (component II) yield (bacterial density) = 4.08 × 10 8 ±2.35×10 7 cfu / mL.

[0079] Comparative Example 2: Based on Example 1, RH15 was omitted in step b, and RH05 was activated and inoculated at a density of 1×10 7 cfu / mL was inoculated into the fermentation medium and fermented at 30±2℃ for 60 hours to obtain the fermentation liquid, and the rest remained unchanged. The test results are as follows:

[0080] The total antioxidant activity of chrysanthemum ferment (fraction I) = 23.8% ± 6.8%.

[0081] Postbiotic (component II) yield (bacterial density) = 3.65 × 10 8 ±2.49×10 7 cfu / m.

[0082] Comparative Example 3: Comparative Example 3 is a blank control. Based on Example 1, RH15 and RH05 are removed from step b, and the fermentation medium is placed at 30±2°C for 60 hours to obtain a fermentation broth, and the rest remains unchanged. The test results are as follows:

[0083] The total antioxidant activity of chrysanthemum ferment (fraction I) = 9.8% ± 2.3%.

[0084] Postbiotic (component II) yield (bacterial density) = 6.50 × 10 3 ±1.33×10 2 cfu / mL.

[0085] Comparative Example 4: Based on Example 1, in step b, Angel-Yeast isolated from commercially available Angel yeast powder and Lactobacillus casei isolated from Yakult were used, and the inoculation density was 1×10 7 cfu / mL, the fermentation medium was placed at 30±2℃ for 60h to obtain the fermentation broth, and the rest remained unchanged. The test results are as follows:

[0086] The total antioxidant activity of chrysanthemum ferment (fraction I) = 35.5% ± 6.0%.

[0087] Postbiotic (component II) yield (bacterial density) = 3.77 × 10 8 ±1.48×10 8 cfu / mL.

[0088] Comparative Example 5: Based on Comparative Example 4, Angel-Yeast was omitted in step b, and Yakult-L.casei was activated and the inoculation density was 1×10 7 cfu / mL was inoculated into the fermentation medium and fermented at 30±2℃ for 60 hours to obtain the fermentation liquid, and the rest remained unchanged. The test results are as follows:

[0089] The total antioxidant activity of chrysanthemum ferment (fraction I) = 18.9% ± 5.3%.

[0090] Postbiotic (component II) yield (bacterial density) = 2.92 × 10 8 ±3.25×10 7 cfu / mL.

[0091] Comparative Example 6: Based on Comparative Example 4, Yakult-L.casei and Angel-Yeast activation were omitted in step b and the inoculation density was 1×10 7 cfu / mL was inoculated into the fermentation medium and fermented at 30±2℃ for 60h to obtain the fermentation broth, and the rest remained unchanged. The test results are as follows:

[0092] The total antioxidant activity of chrysanthemum ferment (fraction I) = 23.5% ± 3.9%.

[0093] Postbiotic (component II) yield (bacterial density) = 2.57 × 10 8 ±4.26×10 7 cfu / mL.

[0094] Test Example 1: Yield comparison and evaluation of synergistic effect between RH11 and RH05.

[0095] The chrysanthemum fermented products (component I) and postbiotics (component II) obtained in Example 1, Example 2, Example 3 and Comparative Example 1, Comparative Example 2, Comparative Example 3 were named as I-E1, I-E2, I-E3 and I-C1, I-C2, I-0, II-E1, II-E2, II-E3 and II-C1, II-C2, II-0, respectively. Their total antioxidant activities were compared in the following test.

[0096] Table 1 (Component II) yield comparison (characterized by bacterial density)

[0097]

[0098]

[0099] Note: a, b, and c are significantly different from each other (p<0.01).

[0100] Table 2 Total antioxidant activity of chrysanthemum fermentation product (component I) (U / mL)

[0101]

[0102] Note: a, b, and c are significantly different from each other (p<0.01).

[0103] The results are shown in Tables 1 and 2. The total antioxidant activity and the yield of postbiotics (component II) of the chrysanthemum fermentation (component I) obtained by co-fermentation of RH15 and RH05 (Example 1, Example 2, Example 3) were significantly higher than those of single-bacteria fermentation of RH15 or RH05 (Comparative Example 1, Comparative Example 2).

[0104] The microbial density of the co-fermentation of RH15 and RH05 (Example 1, Example 2, Example 3) is significantly higher than that of the single-bacteria fermentation of RH11 or RH05 (Comparative Example 1, Comparative Example 2). The Q value is calculated below to evaluate whether there is a synergistic effect between RH15 and RH05 (taking Example 3 as an example).

[0105] Total antioxidant activity of chrysanthemum fermentation products:

[0106] E I-C1 =(E I-C1 -E I-0 ) / E I-C1 =0.431,

[0107] E I-C2 =(E I-C2 -E I-0 ) / E I-C2 =0.462,

[0108] E I-E1 =(E I-E1 -E I-0 ) / E I-E1 =0.802,

[0109] E I-E2 =(E I-E2 -E I-0 ) / E I-E2 =0.802,

[0110] E I-E3 =(E I-E3 -E I-0 ) / EI-E3 =0.807,

[0111] Q I-E1 =E I-E1 / (E I-C1 +E I-C2 -E I-C1 ·E I-C2 )=1.156,

[0112] Q I-E2 =E I-E2 / (E I-C1 +E I-C2 -E I-C1 ·E I-C2 )=1.156,

[0113] Q I-E3 =E I-E3 / (E I-C1 +E I-C2 -E I-C1 ·E I-C2 )=1.163,

[0114] 0.85≤Q≤1.15, it is a simple additive effect, when Q>1.15, it is a synergistic effect, Q I-E1 , Q I-E2 , Q I-E3 All of them were greater than 1.15, indicating that when RH15 and RH05 were co-fermented, the total antioxidant activity of the chrysanthemum fermentation product was optimal, and there was a synergistic effect between the co-fermented RH15 and RH05.

[0115] Test Example 2: Yield comparison and evaluation of the synergistic effect between common lactic acid bacteria and brewer's yeast.

[0116] The chrysanthemum fermentation products (component I) and postbiotics (component II) obtained in Comparative Examples 4, 5, 6 and 3 were named I-C4, I-C5, I-C6 and I-0, II-C4, II-C5, II-C6 and II-0, respectively. Their total antioxidant activities and yields were compared in the following test examples.

[0117] Table 3 (Component II) yield comparison (characterized by bacterial density)

[0118]

[0119] Note: a, b, and c are significantly different from each other (p<0.01).

[0120] Table 4 Total antioxidant activity of chrysanthemum fermentation product (component I) (U / mL)

[0121]

[0122] Note: a, b, and c are significantly different from each other (p<0.01).

[0123] The results are shown in Tables 3 and 4. The total antioxidant activity and postbiotic yield of the chrysanthemum fermentation product (component I) obtained by co-fermentation of Yakult-L.casei and Angel-Yeast (Comparative Example 4) were significantly higher than those of Yakult-L.casei or Angel-Yeast single-strain fermentation (Comparative Example 5, Comparative Example 6).

[0124] The microbial density of the co-fermentation of Yakult-L.casei and Angel-Yeast (Comparative Example 4) was significantly higher than that of the single-bacteria fermentation of Yakult-L.casei or Angel-Yeast (Comparative Example 5, Comparative Example 6). The Q value was calculated below to evaluate whether there was a synergistic effect between Yakult-L.casei and Angel-Yeast.

[0125] Total antioxidant activity of chrysanthemum fermentation products:

[0126] E I-C4 =(E I-C4 -E I-0 ) / E I-C4 =0.639,

[0127] E I-C5 =(E I-C5 -E I-0 ) / E I-C5 =0.323,

[0128] E I-C6 =(E I-C6 -E I-0 ) / E I-C6 =0.455,

[0129] Q I-C4 =E I-C4 / (E I-C5 +E I-C6 -E I-C5 ·E I-C6 )=1.01,

[0130] 0.85≤Q≤1.15 is a simple additive effect, and when Q>1.15, it is a synergistic effect. The above Q value analysis shows that 0.85≤Q I-C4 ≤1.15, indicating that when Yakult-L.casei and Angel-Yeast were co-fermented, although the total antioxidant activity of chrysanthemum fermentation product (component I) was the best, there was no synergistic effect between Yakult-L.casei and Angel-Yeast, but only a simple additive effect.

[0131] Test Example 3: Yield Comparison.

[0132] Table 5

[0133]

[0134] Note: There is a significant difference between a and b (p<0.01).

[0135] Table 6

[0136]

[0137] Note: There is a significant difference between a and b (p<0.01).

[0138] The total antioxidant activity and the yield of postbiotics (component II) of the chrysanthemum fermentation product (component I) of Example 1, Example 2, Example 3 were compared with those of Comparative Example 4. The results are shown in Tables 5 and 6. The total antioxidant activity and the yield of postbiotics (component II) of the chrysanthemum fermentation product (component I) of Example 1, Example 2, and Example 3 were significantly higher than those of Comparative Example 4, indicating that due to the synergistic effect between RH15 and RH05, it is significantly better than the combination of ordinary lactic acid bacteria and brewer's yeast when co-fermenting chrysanthemum.

[0139] Test Example 4: Tyrosinase and Hyaluronidase Inhibition Assay.

[0140] According to the steps described in Example 1, the chrysanthemum fermentation product (component I) and the postbiotic (component II) of each embodiment (Example 1, Example 2, Example 3) and the comparative example (Comparative Example 1, Comparative Example 2, Comparative Example 3) were obtained, and compounded into a composition, wherein the compounding ratio of the component of Example 1 to component II was 1:10 (w / w), the compounding ratio of the component of Example 2 to component II was 1:20 (w / w), and the compounding ratio of the components of Example 3 and Comparative Example 1, Comparative Example 2, and Comparative Example 3 to component II was 1:15 (w / w). Each composition was named Z-E1, Z-E2, Z-E3, Z-C1, Z-C2, and Z-C0 in the order of Example 1, Example 2, and Comparative Example 1, Comparative Example 2, and Comparative Example 3, respectively, and Z-C0 was a blank control. At the same time, kojic acid and sodium cromoglycate were used as positive controls, and the tyrosinase inhibition rate and the hyaluronidase inhibition rate were determined at 48 hours. The results are shown in Tables 7 and 8.

[0141] Table 7 Tyrosinase inhibition rate

[0142]

[0143]

[0144] Note: a, b, and c are significantly different from each other (p<0.01).

[0145] Table 8 Hyaluronidase inhibition rate

[0146]

[0147] Note: a, b, and c are significantly different from each other (p<0.01).

[0148] Q value analysis of tyrosinase inhibition rate:

[0149] E Z-C1 =(E Z-C1 -EZ-C0) / E Z-C1 =0.362,

[0150] E Z-C2 =(E Z-C2 -EZ-C0) / E Z-C2 =0.465,

[0151] E Z-E1 =(E Z-E1 -EZ-C0) / E Z-E1 =0.765,

[0152] E Z-E2 =(E Z-E2 -EZ-C0) / E Z-E2 =0.761,

[0153] E Z-E3 =(E Z-E3 -EZ-C0) / E Z-E3 =0.784,

[0154] Q Z-E1 =E Z-E1 / (E Z-C1 +E Z-C2 -E Z-C1 ·E Z-C2 )=1.161,

[0155] Q Z-E2 =E Z-E2 / (E Z-C1 +E Z-C2 -E Z-C1 ·E Z-C2 )=1.155,

[0156] Q Z-E3 =E Z-E3 / (E Z-C1 +E Z-C2 -E Z-C1 ·E Z-C2 )=1.190,

[0157] 0.85≤Q≤1.15, it is a simple additive effect, when Q>1.15, it is a synergistic effect, Q Z-E1 , Q Z-E12 and Q Z-E3 All were >1.15, showing a synergistic effect.

[0158] Q value analysis of hyaluronidase inhibition rate:

[0159] E Z-C1 =(E Z-C1 -EZ-C0) / E Z-C1 =0.335,

[0160] E Z-C2 =(E Z-C2 -EZ-C0) / E Z-C2 =0.461,

[0161] E Z-E1 =(E Z-E1 -EZ-C0) / E Z-E1 =0.757,

[0162] E Z-E2 =(E Z-E2 -EZ-C0) / E Z-E2 =0.759,

[0163] E Z-E3 =(E Z-E3 -EZ-C0) / E Z-E3 =0.771,

[0164] Q Z-E1 =E Z-E1 / (E Z-C1 +E Z-C2 -E Z-C1 ·E Z-C2 )=1.179,

[0165] Q Z-E2 =E Z-E2 / (E Z-C1 +E Z-C2 -E Z-C1 ·E Z-C2 )=1.182,

[0166] Q Z-E3 =E Z-E3 / (E Z-C1 +E Z-C2 -E Z-C1 ·E Z-C2 )=1.201,

[0167] 0.85≤Q≤1.15, it is a simple additive effect, when Q>1.15, it is a synergistic effect, Q Z-E1 , Q Z-E2 and Q Z-E3 All were >1.15, showing a synergistic effect.

[0168] It shows that the composition of "component I + component II" obtained after co-fermentation of RH15 and RH05 not only performs best in inhibiting hyaluronidase and tyrosinase, but also there is a synergistic effect between the co-fermented RH15 and RH05.

[0169] The fermentation broth obtained in Example 3 was directly freeze-dried without separation of the chrysanthemum fermentation product (component I) and the postbiotics (component II) and compounding in a specific ratio to become a mixture of chrysanthemum fermentation product (component I) and postbiotics (component II), named H-E3, and the hyaluronidase inhibition rate and tyrosinase inhibition rate were determined. The results are shown in Tables 9 and 10, and the Q value was calculated.

[0170] Table 9 Tyrosinase inhibition rate

[0171]

[0172] Note: a, b, and c are significantly different from each other (p<0.01).

[0173] Table 10 Hyaluronidase inhibition rate

[0174]

[0175] Note: a, b, c, and d are significantly different from each other (p<0.01).

[0176] Q value analysis of tyrosinase inhibition rate:

[0177] E Z-C1 =(E Z-C1 -EZ-C0) / E Z-C1 =0.362,

[0178] E Z-C2 =(E Z-C2 -EZ-C0) / E Z-C2 =0.465,

[0179] E H-E3 =(E H-E3 -EZ-C0) / E H-E3 =0.661,

[0180] Q H-E3 =E H-E3 / (E Z-C1 +E Z-C2 -E Z-C1 ·EZ-C2 )=1.009,

[0181] Q value analysis of hyaluronidase inhibition rate:

[0182] E Z-C1 =(E Z-C1 -EZ-C0) / E Z-C1 =0.335,

[0183] E Z-C2 =(E Z-C2 -EZ-C0) / E Z-C2 =0.461,

[0184] E H-E3 =(E H-E3 -EZ-C0) / E H-E3 =0.706,

[0185] Q H-E3 =E H-E3 / (E Z-C1 +E Z-C2 -E Z-C1 ·E Z-C2 )=1.100,

[0186] 0.85≤Q≤1.15 is a simple additive effect, and when Q>1.15, it is a synergistic effect. Regardless of the hyaluronidase inhibition rate or the tyrosinase inhibition rate, Q H-E3 All of them were between 0.85 and 1.15, and none was greater than 1.15, indicating that H-E3 did not exhibit a synergistic effect but only an additive relationship.

[0187] Therefore, it is necessary to separate and prepare chrysanthemum fermentation product (component I) and postbiotics (component II) and then compound them in a suitable proportion, which can fully reflect the synergy between the two components.

[0188] In summary, there is a synergistic effect between the Lactobacillus fermentum ZJU2303-RH15 (CGMCC1.60150) and the Saccharomyces cerevisiae ZJU2014-RH05 (CGMCC 2.6673) of the present invention, which is significantly superior to the combination of common lactic acid bacteria and Saccharomyces cerevisiae in co-fermentation of chrysanthemum.

[0189] RH15 and RH05 not only have synergy when co-fermenting chrysanthemum, but the generated chrysanthemum fermentation product (component I) and postbiotics (component II) also show synergistic effects in efficacy. The composition prepared by the two in a suitable proportion can play a role in skin care and beauty.

Claims

1. A chrysanthemum product co-fermented by lactic acid bacteria and yeast, characterized in that: It includes chrysanthemum fermented products and postbiotics produced by co-fermenting chrysanthemum with lactic acid bacteria and yeast, and the ratio of chrysanthemum fermented products to postbiotics is 1:10 to 1:

20.

2. The chrysanthemum product co-fermented by lactic acid bacteria and yeast according to claim 1, characterized in that: The ratio of chrysanthemum fermentation product to postbiotics is 1:

15.

3. The chrysanthemum product co-fermented by lactic acid bacteria and yeast according to claim 1, characterized in that: The lactic acid bacteria is Lactobacillus fermentum ZJU2303-RH15, with a preservation number of CGMCC 1.60150; the yeast is Saccharomyces cerevisiae ZJU2014-RH05, with a preservation number of CGMCC 2.6673.

4. The method for preparing a chrysanthemum product by co-fermentation of lactic acid bacteria and yeast according to claim 1, 2 or 3, characterized in that: The following steps are included: a. First, crush the chrysanthemum, add water and mix well, and then add brown sugar to obtain a fermentation medium. Each liter of the fermentation medium contains 4-6 parts of chrysanthemum and 1-3 parts of brown sugar, each part of which is 10g; b. Activate lactic acid bacteria and yeast separately and inoculate them into the fermentation medium at a density of (0.5-1.5)×10 7 cfu / mL, ferment at 30±2℃ for 48-72h to obtain fermentation broth; c. The fermentation liquid is centrifuged to obtain a supernatant and a residual portion, the supernatant is concentrated under reduced pressure to 15% to 25% of the original volume, and then freeze-dried to obtain a chrysanthemum fermentation product; d. Ultrasonic treatment and spray drying of the remaining portion of step c are performed to obtain postbiotics; e. Compounding the chrysanthemum fermentation product and postbiotics in proportion to obtain a finished product.

5. The method for preparing a chrysanthemum product by co-fermentation of lactic acid bacteria and yeast according to claim 4, characterized in that: In the step a, each liter of fermentation medium contains 5 parts of chrysanthemum and 2 parts of brown sugar.

6. The method for preparing a chrysanthemum product by co-fermentation of lactic acid bacteria and yeast according to claim 4, characterized in that: In step b, the inoculation density is 1×10 7 cfu / mL, fermentation for 60h.

7. The method for preparing a chrysanthemum product by co-fermentation of lactic acid bacteria and yeast according to claim 4, characterized in that: In the step c, the supernatant is concentrated and freeze-dried under the conditions of a vacuum degree of 65-75 KPa and a temperature of 55-60° C.

8. The method for preparing a chrysanthemum product by co-fermentation of lactic acid bacteria and yeast according to claim 4, characterized in that: In the step d, the remaining part of step c is treated with ultrasound to break the cells in the remaining part and then spray-dried.

9. Use of the chrysanthemum product co-fermented by lactic acid bacteria and yeast as claimed in claim 1, 2 or 3 as a whitening and anti-wrinkle functional ingredient in cosmetics, characterized in that: The added ratio in cosmetics is 0.5% to 1.5%.

10. The use according to claim 9, characterized in that: The addition ratio is 1%.

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