A method for preparing and applying Bifida Ferment Lysate and Fijora Ferment Lysate with whitening, anti-aging, and anti-inflammatory effects.

Abstract

This invention relates to a method for preparing and applying a fermented product of Bifida Ferment Lysate and feijoa fruit with whitening, anti-aging, and anti-inflammatory effects, belonging to the field of plant fermentation technology. This invention discloses a fruit fermentation product, which is a fermentation product of fruit fermented by *Bifidobacterium longum*, wherein the *Bifidobacterium longum* is at least one of *Bifidobacterium longum* HJ-005, *Bifidobacterium longum* subsp. *CGMCC* 1.2186, and *Bifidobacterium longum* subsp. *infant* CGMCC 1.15639. This invention utilizes multiple *Bifidobacterium longum* strains to ferment feijoa, passion fruit, custard apple, and avocado to prepare a fermented product that increases the content of proline, chlorogenic acid, ellagic acid, epicatechin, γ-aminobutyric acid, and total free polyphenols, and enhances its aromatic flavor. The effects are superior to other lactic acid bacteria, exhibiting whitening, anti-aging, and soothing anti-inflammatory effects.

Description

Technical Field

[0001] This invention relates to the field of plant fermentation technology, and in particular to a method for preparing and applying Bifida ferment lysate and feijoa ferment with whitening, anti-aging and anti-inflammatory effects. Background Technology

[0002] Feijoa (scientific name) Acca sellowiana Pineapple guava, also known as finio or pineapple guava, originated in South America. Its fruit combines the flavors of pineapple, strawberry, mango, and other fruits, and has high nutritional value. Its pulp and peel are rich in a variety of unique bioactive components.

[0003] Feijoa fruit is rich in polyphenols and flavonoids. Polyphenols mostly exist as glycosides bound to sugars (e.g., chlorogenic acid often exists as caffeoylquinic acid), and phenolic acids include chlorogenic acid and ellagic acid. Chlorogenic acid and its isomers are potent antioxidants that can scavenge free radicals and inhibit lipid peroxidation. They also possess anti-inflammatory, antibacterial, skin-whitening (inhibiting tyrosinase activity) and potential photoprotective effects. Ellagic acid is a potent skin-whitening and antioxidant that can inhibit melanin production and has anti-inflammatory and potential anti-cancer properties; it can be used as a raw material in high-end skin-whitening and spot-fading products.

[0004] Flavonoids are effective antioxidants that neutralize free radicals, reduce cellular oxidative stress, and thus protect the body from oxidative damage. Epicatechin is a natural flavonoid widely found in plants such as tea, cocoa, and grapes. It possesses antioxidant, anti-inflammatory, metabolic-regulating, cardiovascular- and nervous-system-protective effects, making it one of the important active ingredients for maintaining health.

[0005] Besides polyphenols, feijoa contains many other active ingredients such as amino acids. Among them, free proline can directly replenish glutamic acid, a raw material for collagen synthesis in the skin, which is decarboxylated to produce gamma-aminobutyric acid (GABA). GABA, as a neurotransmitter inhibitor, has soothing, anti-allergic, skin stress-reducing, and mood-improving effects.

[0006] Fermenting fruits with lactic acid bacteria can improve the flavor and quality of the fermentation products and enhance their nutritional value. The organic acids (such as lactic acid and acetic acid) produced by lactic acid bacteria fermentation can lower the pH value of the product, create an acidic environment, inhibit the growth and reproduction of harmful microorganisms, improve the safety and stability of the product, and extend its shelf life.

[0007] Chinese patent CN108576805A discloses an enzyme composition for improving constipation and its preparation method. Although the raw materials contain feijoa, they are also mixed with other kinds of fruits. Moreover, it is fermented by a variety of conventional compound fermentation bacteria. The content of each component in the fermentation product is not detected. The fermentation of feijoa and the strains of bacteria are not explored in depth, and it is difficult to further convert and utilize the fermentation product. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing and applying Bifida Ferment Lysate and Fiji Ferment, which have whitening, anti-aging and anti-inflammatory effects.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a fruit fermentation product, which is a fermentation product of fruit fermented by Bifidobacterium longum, wherein the Bifidobacterium longum includes at least one of Bifidobacterium longum HJ-005, Bifidobacterium longum subsp. longum CGMCC 1.2186, and Bifidobacterium longum subsp. infantum CGMCC 1.15639; wherein Bifidobacterium longum HJ-005 was deposited at the China Center for Type Culture Collection on July 23, 2025, with accession number: CCTCC NO: M 20251676.

[0010] This invention utilizes different types of Bifidobacterium longum to ferment feijoa, passion fruit, custard apple, and avocado. Compared to other lactic acid bacteria, it significantly increases the content of proline, chlorogenic acid, ellagic acid, epicatechin, γ-aminobutyric acid, and total free polyphenols in the fermentation products, and enhances their aromatic flavor.

[0011] In a specific embodiment of the present invention, the fruit includes at least one of feijoa, passion fruit, custard apple, and avocado.

[0012] Secondly, the present invention provides a method for preparing the fruit fermentation product, wherein the Bifidobacterium longum is inoculated into a fermentation system and fermented to obtain the fruit fermentation product; the fermentation system contains fruit homogenate, milk powder, sugar and water.

[0013] Furthermore, the fruit homogenate has a mass concentration of 5% to 20%, the milk powder has a mass concentration of 1% to 3%, and the white sugar has a mass concentration of 3% to 6%.

[0014] Furthermore, the fruit homogenate has a mass concentration of 10%, the milk powder has a mass concentration of 2%, and the sugar has a mass concentration of 5%.

[0015] Furthermore, the pH value of the fermentation system is 6.0~6.5, preferably 6.2.

[0016] Furthermore, the volume concentration of the inoculum of Bifidobacterium longum is 1% to 10%, preferably 5%.

[0017] Furthermore, the fermentation temperature is 36~37.5℃, preferably 37℃.

[0018] Furthermore, the fermentation time is 18-48 hours, preferably 24 hours.

[0019] Thirdly, the present invention provides the application of the fruit fermentation product in the preparation of whitening, anti-aging and / or anti-inflammatory products.

[0020] In a specific embodiment of the present invention, the fermentation product of Fiji fruit fermented by Bifidobacterium longum HJ-005 can significantly inhibit the production of melanin in zebrafish embryos, thus having a whitening effect; it can significantly promote the expression of zebrafish type I collagen gene and Elna gene, promote the regeneration of collagen and elastin, thus having an anti-aging effect; and it can also significantly inhibit the aggregation of neutrophils in zebrafish embryos, thus having a soothing and anti-inflammatory effect.

[0021] Furthermore, the products include cosmetics.

[0022] Fourthly, the present invention provides a cosmetic product containing the aforementioned fruit fermentation product.

[0023] Fifthly, the present invention provides a method for increasing the content of active ingredients in fruit fermentation products, characterized in that the fruit fermentation products are obtained by the preparation method described above; the active ingredients include at least one of polyphenolic compounds, flavonoid compounds, and amino acid compounds.

[0024] Furthermore, the polyphenolic compounds include at least one of chlorogenic acid, ellagic acid, and total free polyphenols; the flavonoid compounds include epicatechin; and the amino acid compounds include proline and / or γ-aminobutyric acid.

[0025] In a specific embodiment of the present invention, the fermentation product of feijoa fermented by Bifidobacterium longum HJ-005 showed that, compared with the fermentation product, the proline content was 26.6 times, the chlorogenic acid content was 6.7 times, the ellagic acid content was 11.5 times, the epicatechin content was 11.3 times, the γ-aminobutyric acid content was 89.1 times, and the total free polyphenol content was 12.6 times.

[0026] Sixthly, the present invention provides a method for improving the aroma of fruit fermentation products, wherein the fruit fermentation products are obtained by the preparation method described above.

[0027] Furthermore, the aromatic odorant includes at least one of esters, aldehydes, and terpenoids.

[0028] Furthermore, the ester compounds include ethyl acetate and / or ethyl butyrate, the aldehyde compounds include hexanal and / or geraniol, and the terpenoid compounds include limonene and / or linalool.

[0029] In a seventh aspect, the present invention provides a Bifidobacterium longum, which is Bifidobacterium longum HJ-005, and was deposited at the China Center for Type Culture Collection on July 23, 2025, with accession number: CCTCC NO: M 20251676.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes various *Bifidobacterium longum* strains (HJ-005, *Bifidobacterium longum* subsp. *CGMCC* 1.2186, and *Bifidobacterium longum* subsp. *infant* CGMCC 1.15639) to ferment feijoa, passion fruit, custard apple, and avocado, preparing fermentation products that increase the content of proline, chlorogenic acid, ellagic acid, epicatechin, γ-aminobutyric acid, and total free polyphenols, while enhancing their aromatic flavor. The effects are superior to other lactic acid bacteria, significantly inhibiting melanin production in zebrafish embryos, resulting in a whitening effect; significantly promoting the expression of zebrafish type I collagen and Elna genes, promoting collagen and elastin regeneration, thus exhibiting anti-aging effects; and significantly inhibiting neutrophil aggregation in zebrafish embryos, providing a soothing and anti-inflammatory effect, making it suitable as a new raw material for cosmetics. Attached Figure Description

[0031] Figure 1 Phylogenetic tree of Bifidobacterium longum HJ-005.

[0032] Figure 2 The results of Gram staining of Bifidobacterium longum HJ-005 are shown in the microscopic examination.

[0033] Figure 3 A bar chart showing melanin inhibition in zebrafish.

[0034] Figure 4 The results represent the whitening efficacy test results; where A is the blank control group; B is the positive control group; C is the sample treatment group; and D is the model group.

[0035] Figure 5 This is a bar chart showing the relative expression levels of type I collagen genes in zebrafish.

[0036] Figure 6 This is a bar chart showing the relative expression levels of the elastin gene in zebrafish.

[0037] Figure 7 This represents the number of neutrophil aggregates in zebrafish embryos. Detailed Implementation

[0038] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, other materials and reagents used in the embodiments are commercially available.

[0039] Example 1: Screening and Identification of Fermentation Strains 1. Isolation and Purification: Samples from the intestinal contents (feces) of healthy individuals and traditional fermented food (pickled vegetables) were collected. Modified MRS and M17 media were used, with separate culture conditions set for different bacterial strains, followed by serial dilutions and streaking. The cultures were incubated at 37°C, and single colonies were picked for repeated purification to obtain pure cultures. Specifically, *Lactobacillus*, *Bifidobacterium*, and *Leuconostoc* strains were cultured on modified MRS medium, with *Lactobacillus* and *Bifidobacterium* requiring anaerobic incubation for 48–72 h; *Lactococcus* and *Streptococcus* strains were cultured on M17 medium under aerobic or microaerobic conditions for 24–48 h.

[0040] The modified MRS medium (per liter) contains: 10.0 g peptone, 10.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 1.0 g Tween 80, 2.0 g K2HPO4·3H2O, 5.0 g sodium acetate trihydrate, 2.0 g triammonium citrate, 0.2 g MgSO4·7H2O, 0.05 g MnSO4·4H2O, and 0.5 g L-cysteine, adjusted to pH 6.2±0.1.

[0041] M17 medium (per liter) contains: 5.0 g peptone, 5.0 g soybean peptone, 2.5 g beef extract, 2.5 g yeast extract, 5.0 g glucose, 5.0 g lactose, 0.5 g ascorbic acid, 0.25 g MgSO4·7H2O, 2.5 g K2HPO4, and pH adjusted to 6.8±0.1.

[0042] 2. Identification: Gram staining, microscopic examination and basic physiological and biochemical tests were performed on the pure cultures obtained in step 1. The physiological and biochemical characteristics of different strains were different.

[0043] Lactobacillus, Bifidobacterium, Lactococcus, and Streptococcus are all catalase-negative. Lactobacillus ferments glucose to produce lactic acid without producing gas. Bifidobacterium ferments glucose to produce lactic acid and acetic acid (molar ratio 2:3). Lactococcus ferments glucose to produce lactic acid without producing gas. Streptococcus strains are hemolytic.

[0044] Following the method described in the literature (Xu Chao, Zhang Shaobing, Meng Jun, et al. Preliminary study on antioxidant activity and cholesterol degradation capacity of different lactic acid bacteria [J]. Food and Fermentation Industries, 2023, 49(02):152-158+165.DOI:10.13995 / j.cnki.11-1802 / ts.031305.), the antioxidant capacity of pure cultures with potential probiotic functions was compared, and strains with strong antioxidant capacity were obtained.

[0045] Genomic DNA was further extracted from each lactic acid bacteria strain, and PCR amplification was performed using universal primers for the 16S rRNA gene. The primer sequences were: upstream primer 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and downstream primer 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). The amplification system (25 μL) consisted of: 2.5 μL of 10×PCR Buffer, 2 μL of dNTPs mixture (2.5 mmol / L), 0.5 μL each of upstream and downstream primers (10 μmol / L), 0.2 μL of Taq DNA polymerase (5 U / μL), 1 μL of template DNA, and sterile double-distilled water to a total amplification volume of 25 μL. The amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1.5 min, for a total of 35 cycles; 72℃ final extension for 10 min, and incubation at 4℃. After sequencing the amplified products, the obtained sequences were compared with the NCBI database to classify and identify the selected strains.

[0046] Bifidobacterium longum was finally screened from the feces. Bifidobacterium longum Streptococcus thermophilus was screened from kimchi. Streptococcus thermophilus Lactobacillus plantarum ( Lactiplantibacillus plantarum ) and lactococci ( Lactococcus sp.).

[0047] The 16S rRNA sequence of *Bifidobacterium longum* is shown in SEQ ID NO: 1, and it is similar to... Bifidobacterium longum subsp. longum The ATCC 15707 strain showed high homology; comparison results from the NCBI Blast database showed a similarity of over 95%, and the phylogenetic tree is as follows. Figure 1 As shown, the microscopic results of Gram staining are as follows: Figure 2 As shown. The obtained Bifidobacterium longum (also known as Bifida ferment lysate) was named Bifidobacterium longum (as shown). Bifidobacterium longum HJ-005 was deposited at the China Center for Type Culture Collection (CCTCC) on July 23, 2025, with accession number CCTCC NO: M 20251676, at Wuhan University, China.

[0048] The obtained thermophilic streptococcus was named thermophilic streptococcus ( Streptococcus thermophilus HJA09, the obtained Lactobacillus plantarum was named Lactobacillus plantarum ( Lactiplantibacillus plantarum HJB73, the obtained lactococcus was named Lactococcus lactis ( Lactococcus sp.)HJG02.

[0049] Example 2: Fermentation of fruit with Bifidobacterium longum HJ-005 and detection of active ingredients in the fermentation products. I. Experimental Methods 1. Select fresh, unrotten, unblemished, and pest-free feijoa, passion fruit, custard apple, or avocado. Rinse repeatedly with deionized water 3-5 times to remove surface dirt, dust, and residual impurities. Place them on clean filter paper to absorb surface moisture. Separate the pulp from the peel, removing inedible parts such as passion fruit seeds, custard apple core, and avocado pit. Place the separated pulp in a high-speed tissue homogenizer, add sterile deionized water (1 mL:1 g ratio of sterile deionized water to pulp), and homogenize until a fine pulp (particle size ≤1 mm) is formed. Pass the pulp through an 80-mesh sieve to remove coarse residue and collect the sieved homogenate for later use.

[0050] The homogenate was 10% (final mass concentration). 2.0% (final mass concentration) of milk powder and 5.0% (final mass concentration) of white sugar were added as the carbon and nitrogen source for fermentation. 0.1% (mass concentration) of baking soda was added to adjust the initial pH to 6.2. Sterile deionized water was added to make up to 1 L to prepare the fermentation system. The mixture was dispensed into 2 L shake flasks, with the liquid volume controlled at 50% (volume concentration) to allow for fermentation space.

[0051] The dispensed fermentation system was placed in a high-pressure steam sterilizer and sterilized at 121℃ and 0.1 MPa for 20 min. After sterilization, it was quickly placed in a clean bench to cool to 37℃. After the fermentation broth cooled down, it was inoculated with Bifidobacterium longum HJ-005 seed culture (OD500) activated to the logarithmic phase at an inoculation rate of 5% (volume concentration). 600 A value of 8 indicates a colony count greater than 1×10⁻⁶. 8 (1 / mL), after inoculation, gently shake to fully mix the seed liquid with the fermentation system.

[0052] After inoculation, the shake flasks were placed in a constant temperature incubator and allowed to ferment at 37°C under anaerobic conditions for 24 hours. During the fermentation process, samples were taken every 8 hours to monitor changes in pH value and viable cell count. After fermentation, the precipitate was removed by centrifugation (8000 r / min, 10 min), and the supernatant was collected as the fermentation product.

[0053] 2.2. Detect the content of proline, chlorogenic acid, ellagic acid, gallic acid, γ-aminobutyric acid and total free polyphenols in fruits before fermentation and fermentation products after fermentation.

[0054] (1) Pretreatment of fermentation products for detection: Accurately measure 10 mL of fermentation product and place it in a centrifuge tube. Centrifuge at 8000 r / min for 15 min, discard the precipitate (to remove Bifidobacterium longum cells and undegraded pulp particles), and collect the supernatant. Add 5% (v / v) trichloroacetic acid solution to the supernatant to a final concentration of 1%, shake well, and let stand for 30 min to precipitate proteins. Centrifuge again at 8000 r / min for 15 min, collect the supernatant, and adjust the pH to neutral with sodium hydroxide solution. Add 50 mL of 2 mol / L hydrochloric acid solution, shake well, and reflux in an 85℃ water bath for 2 h. Cool to room temperature, adjust the pH to neutral with sodium hydroxide solution, and transfer to a separatory funnel. Add 50 mL of petroleum ether (60~90℃), shake to extract for 15 min, let stand to separate the layers, and collect the upper organic phase. Repeat the extraction 3 times and combine the organic phases. The combined organic phases were dehydrated by passing them through an anhydrous sodium sulfate column, and the filtrate was collected. The filtrate was concentrated to dryness by rotary evaporation at 45°C. The residue was dissolved in methanol and transferred to a 10 mL volumetric flask. The volume was adjusted to the mark with methanol and filtered through a 0.45 μm organic phase filter membrane to obtain the post-fermentation sample solution for subsequent analysis.

[0055] (2) Pretreatment of pulp for testing The homogenate prepared in step 1 was centrifuged at 8000 r / min for 15 min, and the precipitate was discarded (to remove coarse particulate impurities such as fibers and lignin that are not easily broken in the pulp). The supernatant was collected and transferred to a clean beaker. Then the supernatant was treated according to the pretreatment method for fermentation product detection in step (1) to obtain the sample solution to be tested before fermentation for subsequent detection.

[0056] Following the method described in the literature (Rapid Determination of 18 Free Amino Acids in Fruits Based on High-Resolution Mass Spectrometry using Ultra-High Performance Liquid Chromatography-Quadrupole-Electrostatic Field Orbital Trap, Food Industry Technology, 2021, Vol. 42, No. 5, pp. 243-249), the proline content in fruits before fermentation and the proline content in fermentation products after fermentation were determined.

[0057] Following the method described in the literature (HPLC method for simultaneous determination of chlorogenic acid, luteolin and isochlorogenic acid A in luteolin ester granules, Special Products Research 2024 Vol.46 No.4 pp.110-115), the chlorogenic acid content in the fruit before fermentation and the chlorogenic acid content in the fermentation product after fermentation were determined.

[0058] The content of ellagic acid in the fruit before fermentation and the content of ellagic acid in the fermentation product after fermentation were determined according to the method in the literature (HPLC simultaneous determination of gallic acid, corilagin and ellagic acid in the aqueous extract of three fruits, China Journal of Information on Traditional Chinese Medicine, 2017, Vol. 24, No. 9, pp. 76-79).

[0059] Following the method described in the literature (Comparison of colorimetric and HPLC methods for determining the content of γ-aminobutyric acid in mulberry leaf tea, Food Science 2018 Vol. 39 No. 24 pp. 256-260), the γ-aminobutyric acid content in the fruit before fermentation and the γ-aminobutyric acid content in the fermentation products after fermentation were determined by HPLC.

[0060] Following the method described in the literature (Research on the content and antioxidant activity of main functional components of purple passion fruit from different origins, China Fruit Trees 2023, Vol. 5, pp. 76-83), the total free polyphenol content in the fruit before fermentation and the total free polyphenol content in the fermentation products after fermentation were determined.

[0061] 3. Detect the aroma substances in the fermentation products. To clarify the aroma characteristics of fruits before and after fermentation, this invention uses headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) to detect its volatile components. A 50 / 30 μm DVB / CAR / PDMS fiber tip (activated at 250℃ for 30 min) was used. 10.0 g of the sample solution before and after fermentation was accurately weighed and placed in a 20 mL headspace vial. 2.0 g of anhydrous sodium sulfate and 50 μL of 100 μg / mL n-hexane internal standard were added. After sealing and shaking, the solution was equilibrated at 45℃ for 15 min, followed by adsorption at the same temperature for 30 min (stirring speed 300 r / min). The fiber tip was then inserted into the 250℃ gas chromatograph injection port for splitless desorption for 5 min. Gas chromatography was performed using an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm) with helium of ≥99.999% purity as the carrier gas (flow rate 1.0 mL / min). The column temperature program was: 40℃ for 3 min, ramping up to 150℃ at 5℃ / min and holding for 2 min, ramping up to 230℃ at 10℃ / min and holding for 5 min. Mass spectrometry was performed using an EI ion source (230℃, 70 eV), a quadrupole temperature of 150℃, a mass scan range of m / z 35~450, and full scan mode to record the total ion chromatogram (solvent delay 3 min). Qualitative analysis was performed using the NIST17 mass spectrometry library (match ≥80%), and quantification was performed using the internal standard method. Each sample was tested in triplicate (RSD ≤ 5%), with a blank control included to ensure accurate and reliable results. Aroma pleasantness was quantitatively analyzed using sensory evaluation (9-point scale) and aroma activity value (OAV).

[0062] II. Experimental Results Table 1. Changes in various functional components in the fermentation products of fruits fermented with Bifidobacterium longum HJ-005. Table 2. Changes in various active ingredients in the fermentation products of fruits fermented with Bifidobacterium longum HJ-005 over time. As shown in Tables 1 and 2, the fermentation of passion fruit, custard apple, and avocado by *Bifidobacterium longum* HJ-005 increased the content of most active ingredients. However, the levels of chlorogenic acid and total polyphenols in passion fruit and ellagic acid in avocado decreased after fermentation compared to the unfermented state. This indicates that the enzyme system secreted by *Bifidobacterium longum* HJ-005 and its endogenous enzymes can promote the release and transformation of some active ingredients.

[0063] After fermentation, the levels of proline, chlorogenic acid, ellagic acid, epicatechin, γ-aminobutyric acid, and total free polyphenols in feijoa increased most significantly, with increases exceeding those of passion fruit, custard apple, and avocado. This indicates that the fermentation of feijoa by *Bifidobacterium longum* HJ-005 does not simply "consume" its nutrients, but rather promotes their release. For example, many polyphenols in feijoa exist in the form of glycosides bound to sugars (such as chlorogenic acid, which often exists in the form of caffeoylquinic acid). *Bifidobacterium longum* HJ-005 secretes enzymes such as β-glucosidase to cleave glycosidic bonds, converting bound polyphenols into free polyphenol monomers. Furthermore, the significant 89.1-fold increase in GABA conversion rate is likely related to the decarboxylation of abundant glutamate in feijoa mediated by *Bifidobacterium longum* HJ-005.

[0064] The feijoa and its fermentation product of Bifidobacterium longum HJ-005 (5% inoculum, anaerobic fermentation at 37℃ for 24 h) have a complex fruity aroma of pineapple, strawberry and mango, with a fresh and sweet fragrance.

[0065] GC-MS analysis showed that before fermentation, feijoa contained esters (48.2% ± 1.5%), such as ethyl acetate (1256 ± 48 μg / kg) and ethyl butyrate (873 ± 35 μg / kg); aldehydes (22.5 ± 0.8%), such as acetaldehyde (386 ± 16 μg / kg) and geraniol (214 ± 9 μg / kg); and terpenes (20.3 ± 1.2%), such as D-limonene (987 ± 39 μg / kg) and linalool (562 ± 23 μg / kg), among other aroma compounds. After fermentation, esters increased to 55.7 ± 1.8%, with ethyl acetate increasing to 1892 ± 63 μg / kg and ethyl butyrate increasing to 1158 ± 42 μg / kg; terpenes increased to 23.8 ± 1.4%, with D-limonene increasing to 1123 ± 45 μg / kg and linalool increasing to 689 ± 28 μg / kg; aldehydes decreased to 15.3 ± 0.6%, with acetaldehyde decreasing to 253 ± 11 μg / kg and geraniol decreasing to 147 ± 7 μg / kg, resulting in a more harmonious and pleasant flavor.

[0066] Sensory evaluation on a 9-point scale (n=50) showed that feijoa had an aroma pleasantness score of 7.8 ± 0.5 before fermentation, which rose to 8.5 ± 0.4 after fermentation, significantly higher than passion fruit (6.5 ± 0.6). P <0.001), significantly better than avocado (3.2 ± 0.4 points) and custard apple (4.8 ± 0.5 points). This effect is related to the high content of pleasurable esters (ethyl butyrate r=0.89, P <0.001) and terpenes (linalool r=0.82, P <0.001) is closely related.

[0067] Taking all factors into consideration, feijoa is a more suitable fruit for fermentation by Bifidobacterium longum HJ-005 compared to passion fruit, custard apple, and avocado, which may be related to its unique composition of active ingredients.

[0068] Example 3: Effects of different lactic acid bacteria on the active ingredients of feijoa fermentation products I. Experimental Methods Bifidobacterium longum HJ-005 was replaced with Streptococcus thermophilus HJA09, Lactobacillus plantarum HJB73, Lactococcus lactis HJG02, Bifidobacterium longum subsp. longum (CGMCC 1.2186), and Bifidobacterium longum subsp. infantum (CGMCC 1.15639), respectively. After fermenting feijoa according to the method of Example 2, the changes of each active ingredient were detected.

[0069] II. Experimental Results Table 3. Changes in various functional components in the fermentation products of Fijora fermented with different Bifidobacterium longum strains. Table 4. Changes in various functional components in the fermentation products of feijoa fermented with other lactic acid bacteria. As shown in Table 3, the levels of all active substances were increased after fermentation of Bifidobacterium longum HJ-005, Bifidobacterium longum subsp. longum (CGMCC 1.2186), and Bifidobacterium longum subsp. infantum (CGMCC 1.15639), with the increase being more than 6 times. However, the fermentation effect of Bifidobacterium longum HJ-005 was better.

[0070] As shown in Table 4, after fermentation of Fiji fruit by Streptococcus thermophilus HJA09, Lactobacillus plantarum HJB73 and Lactococcus lactis HJG02, the detection levels of a small number of active substances were promoted to some extent, but the content of most active substances changed by less than 4 times (except for epicatechin, which increased by 5.7 times after fermentation by Streptococcus thermophilus HJA09), and different strains had different specificities.

[0071] The fermentation of feijoa by *Bifidobacterium longum* is essentially a biotransformation and biocatalysis process. *Bifidobacterium longum* utilizes its secreted enzyme system to break down the macromolecules (such as proteins, polysaccharides, and polyphenol-glycoside complexes) already present in feijoa, converting precursor substances into new, or more abundant, and more bioactive metabolites. It is most likely to significantly enrich and activate those components most directly and effectively beneficial to skin health, such as free polyphenols, lactic acid, GABA, and amino acids. The inherent compositional characteristics of feijoa and the metabolic properties of *Bifidobacterium longum* align well, which is a likely reason for the significant increase in the content of these various beneficial components.

[0072] Example 4: Efficacy test of Bifidobacterium longum HJ-005 Fijora fermentation product I. Experimental Methods 1. Whitening effect (zebrafish embryo melanin inhibition test) Twenty 8-hour zebrafish embryos were exposed to a sample solution (sample treatment group). The sample solution was prepared by fermentation of Bifidobacterium longum HJ-005 feijoa for 24 hours. The fermentation product was centrifuged, filtered for sterilization, and then diluted to a volume concentration of 90% with embryo culture medium. A blank control group (fish embryo culture medium), a 100% melanin inhibition model group (30 mg / L phenylthiourea), and a positive control group (2.5 g / L kojic acid) were set up. After 48 hours of exposure, the fish embryos were photographed under a microscope to measure the melanin signal intensity and perform statistical analysis.

[0073] Preparation of fish embryo culture medium: Weigh 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate, and 55 mg of potassium chloride and dissolve them in 10 L of water to prepare the medium with a pH of 6.5~8.5.

[0074] 2. Anti-aging effects (zebrafish elastin and type I collagen test) Thirty-six 6-day-old zebrafish were divided into three groups and exposed to sample solutions (sample treatment group). A blank control group (fish embryo culture medium) was set up at the same time. After 24 h of exposure, RNA was extracted from the zebrafish and reverse transcribed into cDNA. β-actin was used as the housekeeping gene and real-time PCR amplification was performed. The Ct value was used as the amplification result. The relative expression levels of col1a1a, col1a1b, col1a2 and Elna were calculated and statistically analyzed.

[0075] PCR amplification system (20 μL): 10 μL SYBR Green Master Mix, 0.6 μL forward primer (10 μM), 0.6 μL reverse primer (10 μM), 2 μL cDNA template, 6.8 μL RNase-free water. Primer sequences are shown in Table 5.

[0076] PCR program: Pre-denaturation: 95℃ for 3 min; Cyclic reaction: 95℃ for 10 s, annealing temperature adjusted according to primer Tm value (60℃ for β-actin, col1a1a, and col1a2, 62℃ for col1a1b), 30 s, for a total of 40 cycles; Melting curve: 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s (to verify primer specificity).

[0077] Table 5 3. Anti-inflammatory efficacy test (zebrafish embryo neutrophil assay method) A model of neutrophil aggregation induced by damage to neurothalamic cells in the lateral line region of zebrafish embryos using copper sulfate was tested. Twenty-four zebrafish embryos were exposed to 10 µM anhydrous copper sulfate and sample solution (sample treatment group). A blank control group (fish embryo culture medium + 10 µM anhydrous copper sulfate), a positive control group (zebrafish embryo culture medium + 10 µM anhydrous copper sulfate + 10 µM indomethacin), and a model control group (zebrafish embryo culture medium + 10 µM anhydrous copper sulfate, used to induce an inflammatory response) were also established. After 40 min of exposure, the embryos were fixed and stained with Sudan Black. The number of neutrophils in the lateral line region was counted and statistically analyzed.

[0078] II. Experimental Results 1. For example Figure 3 , Figure 4 A, Figure 4 B Figure 4 C and Figure 4 As shown in D, the sample solution inhibited melanin production in zebrafish embryos by 7.72% ( p =0.0073); It can significantly inhibit melanin production in zebrafish embryos and has a whitening effect. The whitening effect of the blank control group was not significant (0% melanin inhibition rate), the whitening effect of the positive control group was significant (30% melanin inhibition rate), and the whitening effect of the model control group was significant (100% melanin inhibition rate).

[0079] 2. For example Figure 5 As shown, the sample treatment group promoted the expression of zebrafish col1a1a, col1a1b, and col1a2 genes by 125% ( p =0.00009), 56% p =0.0032) and 32% ( p =0.0016), p <0.05. This indicates that the sample treatment group can significantly promote the expression of type I collagen gene in zebrafish, and has the effect of promoting type I collagen regeneration and anti-aging.

[0080] like Figure 6As shown, the sample promoted the expression of the Elna gene in zebrafish by 174% ( p =0.0015), which can significantly promote the expression of the Elna gene in zebrafish, promote elastin regeneration, and has anti-aging effects.

[0081] 3. For example Figure 7 As shown, the sample inhibited neutrophil aggregation in zebrafish embryos by 48% ( p =0.00029), which significantly inhibited neutrophil aggregation in zebrafish embryos and had a soothing and anti-inflammatory effect. The positive control group showed an inhibition rate of 30% on neutrophil aggregation in zebrafish embryos ( p =0.00072), with a significant soothing effect.

[0082] This embodiment uses a zebrafish model to confirm that the fermented Fiji fruit product of Bifidobacterium longum HJ-005 has significant whitening, anti-aging and anti-inflammatory effects, providing experimental evidence for its application in cosmetics, health products and other fields.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A fruit fermentation product, characterized in that, The fermentation product is a fermentation product of fruit fermented with Bifidobacterium longum, wherein Bifidobacterium longum includes at least one of Bifidobacterium longum HJ-005, Bifidobacterium longum subsp. longum CGMCC 1.2186, and Bifidobacterium longum subsp. infantum CGMCC 1.15639; wherein Bifidobacterium longum HJ-005 was deposited at the China Center for Type Culture Collection on July 23, 2025, with accession number: CCTCC NO: M 20251676.

2. The fruit fermentation product as described in claim 1, characterized in that, The fruit includes at least one of feijoa, passion fruit, custard apple, and avocado.

3. The method for preparing the fruit fermentation product according to claim 1 or 2, characterized in that, The *Bifidobacterium longum* as described in claim 1 is inoculated into a fermentation system and fermented to obtain the fruit fermentation product; the fermentation system contains fruit homogenate, milk powder, sugar and water.

4. The preparation method according to claim 3, characterized in that, The fruit homogenate has a mass concentration of 5% to 20%, the milk powder has a mass concentration of 1% to 3%, and the white sugar has a mass concentration of 3% to 6%.

5. The use of the fruit fermentation product according to claim 1 or 2 in the preparation of whitening, anti-aging and / or anti-inflammatory products.

6. A cosmetic product, characterized in that, The cosmetic contains the fruit fermentation product as described in claim 1 or 2.

7. A method for increasing the content of active ingredients in fruit fermentation products, characterized in that, Fruit is fermented using the preparation method described in claim 3 to obtain a fruit fermentation product; the active ingredients include at least one of polyphenolic compounds, flavonoid compounds, and amino acid compounds.

8. The method as described in claim 7, characterized in that, The polyphenolic compounds include at least one of chlorogenic acid, ellagic acid, and total free polyphenols; the flavonoid compounds include epicatechin; and the amino acid compounds include proline and / or γ-aminobutyric acid.

9. A method for enhancing the aroma of fruit fermentation products, characterized in that, Fruit is fermented using the preparation method described in claim 3 to obtain fruit fermentation products.

10. A type of Bifidobacterium longum, characterized in that, The Bifidobacterium longum is Bifidobacterium longum HJ-005, which was deposited at the China Center for Type Culture Collection on July 23, 2025, with accession number: CCTCC NO: M 20251676.

Images