A method for fermenting mulberry leaves with composite probiotics, mulberry leaf fermentation products and applications

Abstract

The present invention discloses a method for composite probiotic fermentation of mulberry leaves, a mulberry leaf fermentation product and an application, and relates to the field of fermentation technology. The fermentation method comprises: using Staphylococcus xylosus and Lactobacillus plantarum to composite ferment mulberry leaves in a volume ratio of 1-3:1-3, and the total inoculation amount is 2%-10% of the volume of the fermentation medium; a mulberry leaf fermentation product is prepared by the above method; the present invention also provides the application of the above mulberry leaf fermentation product in the preparation of whitening cosmetics and / or skin care products. The beneficial effect of the present invention is to composite ferment mulberry leaves using two probiotics, Lactobacillus plantarum and Staphylococcus xylosus, and Lactobacillus plantarum and Staphylococcus xylosus can play a synergistic role in composite fermentation of mulberry leaves, and can achieve the effective release of active substances that inhibit tyrosinase in mulberry leaves. The active substances are all retained in the fermentation product, and the inhibition rate of the fermentation product on tyrosinase activity is significantly increased.

Description

Technical Field

[0001] The present invention relates to the technical field of fermentation, and in particular to a method for fermenting mulberry leaves with composite probiotics, and mulberry leaf fermentation products and applications. Background Art

[0002] Mulberry leaf is a traditional Chinese medicine that can be used as both medicine and food. It is rich in various flavonoids, alkaloids, γ-hydroxybutyric acid, polysaccharides and other active substances that are beneficial to human health. It has anticoagulant, blood pressure lowering, blood lipid lowering, cholesterol lowering, anti-thrombosis and anti-atherosclerosis effects, blood sugar lowering effects, antibacterial and anti-inflammatory effects.

[0003] Tyrosinase is a key enzyme in the formation of melanin and is widely present in organisms. Its inhibitors are used in the medical field to treat and prevent pigmentation diseases such as melanoma and chloasma, in the cosmetics field for human skin whitening, and in the food industry to prevent fruits and vegetables from losing their freshness and spoiling due to browning. In the agricultural field, tyrosinase is a key enzyme for the survival of insects and is also one of the most promising biological insecticides.

[0004] Research has shown that various active substances in mulberry leaves, such as flavonoids, polyphenols, and polysaccharides, exhibit strong inhibitory activity against tyrosinase through competitive binding and antagonism. The dense cellulose and woody structures of plant cells hinder the release of active substances within the cells, making digestion difficult and hindering absorption in humans and animals due to the lack of the appropriate enzymes. To address this issue, aqueous extraction has traditionally been used, but this method is inefficient, and chemical extraction of single substances results in significant waste and waste disposal issues. Studies have shown that microbial fermentation can significantly increase the content of active substances in the product. However, due to process factors such as the strain and fermentation conditions, while the alkaloid content is increased, flavonoids and polyphenols are lost. Therefore, optimizing the fermentation process of mulberry leaves to improve the extraction rate of active substances from mulberry leaves by targeting tyrosinase inhibition has positive implications for the development of highly effective natural tyrosinase inhibitors using mulberry leaves. Summary of the Invention

[0005] The purpose of the present invention is to solve at least one of the technical problems existing in the prior art and to provide a method for fermenting mulberry leaves with composite probiotics, a mulberry leaf fermentation product and an application thereof.

[0006] The technical solutions of the present invention are as follows:

[0007] A first aspect of the present invention provides a method for fermenting mulberry leaves with composite probiotics, comprising the following steps:

[0008] Inoculating Staphylococcus xylosus and Lactobacillus plantarum into a fermentation medium containing mulberry leaves for composite fermentation;

[0009] The volume ratio of the Staphylococcus xylosus to the Lactobacillus plantarum is 1-3:1-3, and the total inoculation amount is 2%-10% of the volume of the fermentation medium.

[0010] Optionally, the xylosus Staphylococcus is xylosus Staphylococcus ( Staphylococcus xylosus ) ATCC29971; the plant lactobacillus is plant lactobacillus ( Lactobacillus plantarum )ATCC8014.

[0011] It should be noted that the above-mentioned Staphylococcus xylosus and Lactobacillus plantarum are both existing strains.

[0012] Optionally, the temperature of the composite fermentation is 30° C. to 42° C., and the time of the composite fermentation is 12 h to 60 h.

[0013] Optionally, the pH of the composite fermentation is 5.5-7.5.

[0014] Optionally, the method specifically includes the following steps:

[0015] Optionally, 5 g to 25 g of mulberry leaves are mixed with 50 mL to 200 mL of water and 0.5 g to 2 g of glucose to obtain a fermentation medium; and the pH of the fermentation medium is adjusted to 5.5 to 7.5;

[0016] The fermentation medium is inoculated with the Staphylococcus xylosus and the Lactobacillus plantarum, and the medium is placed in an incubator at 30° C. to 42° C. for fermentation for 12 h to 60 h to obtain a fermentation product.

[0017] Optionally, the Staphylococcus xylosus is an acclimated Staphylococcus xylosus, and the Lactobacillus plantarum is an acclimated Lactobacillus plantarum;

[0018] The acclimation method of the xylosus Staphylococcus and the plant lactobacillus comprises: firstly culturing the pre-acclimated xylosus Staphylococcus and the pre-acclimated plant lactobacillus respectively in an MRS culture medium containing 0 mulberry leaves, then transferring them respectively to MRS culture medium containing gradually increasing mulberry leaves for culturing, and finally transferring them respectively to a culture medium with only mulberry leaves as a substrate for culturing.

[0019] Optionally, the acclimation method of Staphylococcus xylosus specifically comprises the following steps:

[0020] 14-16 g of mulberry leaves and 100 mL of water were mixed to prepare a mulberry leaf culture medium, and the mulberry leaf culture medium was mixed with an MRS culture medium according to a volume ratio to prepare an acclimation culture medium, wherein the volume content of the MRS culture medium in the acclimation culture medium was 0%-100%;

[0021] The unacclimated Staphylococcus xylosus was inoculated into an acclimation medium containing 100% MRS medium at a volume percentage of 1-3%, and cultured at 36-38°C.

[0022] Waiting for OD 600 When the concentration reaches 1.9-2.1 for the first time, take the culture medium and transfer it to an acclimation medium containing 75%-85% MRS medium, and continue to culture at 36-38°C.

[0023] Waiting for OD 600 When the concentration reaches 1.9-2.1 for the second time, take the culture medium and transfer it to an acclimation medium containing 55%-65% MRS medium, and continue to culture at 36-38°C.

[0024] Waiting for OD 600 When the concentration reaches 1.9-2.1 for the third time, take the culture medium and transfer it to an acclimation medium containing 35%-45% MRS medium, and continue to culture at 36-38°C.

[0025] Waiting for OD 600 When the concentration reaches 1.9-2.1 for the fourth time, take the culture medium and transfer it to an acclimation medium containing 15%-25% MRS medium, and continue to culture at 36-38°C.

[0026] Waiting for OD 600 When the concentration reached 1.9-2.1 for the fifth time, the culture solution was taken and transferred to an acclimation medium containing 0% MRS culture medium, and the culture was continued at 36-38°C to obtain acclimated Staphylococcus xylosus.

[0027] Optionally, the domestication method of the plant lactobacillus specifically comprises the following steps:

[0028] 14-16 g of mulberry leaves and 100 mL of water were mixed to prepare a mulberry leaf culture medium, and the mulberry leaf culture medium was mixed with an MRS culture medium according to a volume ratio to prepare an acclimation culture medium, wherein the volume content of the MRS culture medium in the acclimation culture medium was 0%-100%;

[0029] The Lactobacillus plantarum before acclimation is inoculated into an acclimation medium with a volume content of 100% of the MRS medium at a volume percentage of 1-3%, and statically cultured at 36-38°C;

[0030] Waiting for OD 600 When the concentration reaches 1.9-2.1 for the first time, take the culture medium and transfer it to an acclimation medium containing 75%-85% of MRS medium by volume, and continue to culture at 36-38°C.

[0031] Waiting for OD 600When the concentration reaches 1.9-2.1 for the second time, take the culture medium and transfer it to an acclimation medium containing 55%-65% of MRS medium by volume, and continue to culture at 36-38°C.

[0032] Waiting for OD 600 When the concentration reaches 1.9-2.1 for the third time, take the culture medium and transfer it to an acclimation medium containing 35%-45% of MRS medium by volume, and continue to culture at 36-38°C;

[0033] Waiting for OD 600 When the concentration reaches 1.9-2.1 for the fourth time, take the culture medium and transfer it to an acclimation medium containing 15%-25% of MRS medium by volume, and continue to culture at 36-38°C;

[0034] Waiting for OD 600 When the concentration reaches 1.9-2.1 for the fifth time, the culture solution is taken and transferred to an acclimation medium containing 0% MRS medium by volume, and the culture is continued at 36-38° C. to obtain the acclimated Lactobacillus plantarum.

[0035] The second aspect of the present invention provides a mulberry leaf fermentation product prepared by the above method.

[0036] Optionally, the mulberry leaf fermentation product of the present invention can inhibit tyrosinase activity by up to 82.23%.

[0037] The third aspect of the present invention provides the use of the above-mentioned mulberry leaf fermentation product in the preparation of whitening cosmetics and / or skin care products.

[0038] The mulberry leaf fermentation product of the present invention can achieve an inhibition rate of 82.23% on tyrosinase activity, thereby having a good inhibitory effect on the expression of melanin, and can be used in the preparation of whitening cosmetics and / or whitening skin care products.

[0039] The present invention has at least one of the following beneficial effects:

[0040] 1, method of the present invention utilizes plant lactobacillus and two kinds of probiotics of xylosus staphylococcus to carry out composite fermentation to mulberry leaf, plant lactobacillus and xylosus staphylococcus can play synergistic effect when composite fermentation mulberry leaf, can realize the effective release of the active substance that suppresses tyrosinase in mulberry leaf, active substance all remains in fermented product.Compared plant lactobacillus and xylosus staphylococcus ferment separately, composite fermentation has significantly improved the effect of mulberry leaf fermentation, after testing, xylosus staphylococcus and plant lactobacillus ferment separately mulberry leaf obtain the mulberry leaf fermented product to the inhibition rate of tyrosinase activity and are only respectively 33.47% and 53.27%, and the mulberry leaf fermented product that composite fermentation obtains is to tyrosinase activity inhibition rate reach 82.23%, thus showing that the mulberry leaf fermented product that composite fermentation obtains raises the inhibition rate of tyrosinase activity.

[0041] 2. The present invention also domesticates Staphylococcus xylosus and Lactobacillus plantarum to adapt them to a culture environment with mulberry leaves as substrates. After testing, the inhibition rates of mulberry leaf fermentation products obtained by fermenting mulberry leaves by Staphylococcus xylosus and Lactobacillus plantarum alone on tyrosinase activity were only 25.81% and 49.55% respectively before domestication. After domestication, the inhibition rates of mulberry leaf fermentation products obtained by fermenting mulberry leaves by Staphylococcus xylosus and Lactobacillus plantarum alone on tyrosinase activity were 33.47% and 53.27% respectively. This shows that the fermentation performance of Staphylococcus xylosus and Lactobacillus plantarum can be improved by domestication, thereby increasing the inhibition rate of tyrosinase activity of the fermentation products.

[0042] 3. Both the plant lactobacillus and the xylose Staphylococcus of the present invention are probiotics, which are safe and reliable. The product has a unique flavor and is safe and has no toxic or side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 The morphological characteristics of the domesticated Lactobacillus plantarum in Example 1 are shown in Figure 1, where A is the Gram staining result and B is the colony characteristic diagram.

[0044] Figure 2 The morphological characteristics of Staphylococcus xylosus after acclimation in Example 1, wherein A is the Gram staining result; B is the colony characteristic diagram.

[0045] Figure 3 1 is the growth curve of plant lactobacillus after acclimation in Example 1.

[0046] Figure 4 This is the growth curve of Staphylococcus xylosus after acclimation in Example 1.

[0047] Figure 5 This is a diagram showing the effect of pH on the composite fermentation results in Example 1.

[0048] Figure 6 This is a diagram showing the effect of inoculation amount on the composite fermentation results in Example 1.

[0049] Figure 7 This is a diagram showing the effect of fermentation time on the composite fermentation results in Example 1.

[0050] Figure 8 This is a diagram showing the effect of fermentation temperature on the composite fermentation results in Example 1.

[0051] Figure 9 This is a graph showing the effect of the bacterial inoculation ratio on the composite fermentation results in Example 1.

[0052] Figure 10 This is a photo showing the effects of Lactobacillus plantarum and Staphylococcus xylosus on the growth and development of silkworm larvae in Example 2.

[0053] Figure 11 This is a comparison chart of the effects of Lactobacillus plantarum and Staphylococcus xylosus on the growth and development of silkworm larvae in Example 2. DETAILED DESCRIPTION

[0054] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0055] The present invention is further described in detail below with reference to specific examples, but the present invention is not limited to the following specific examples.

[0056] It should be noted that the plant lactobacillus used in the following examples and comparative examples is plant lactobacillus ( Lactobacillus plantarum ) ATCC8014, the xylosus Staphylococcus used was xylosus Staphylococcus ( Staphylococcus xylosus ) ATCC29971, were purchased from Hunan Fenghui Biotechnology Co., Ltd.

[0057] The MRS medium used in the following examples is a commercially available medium.

[0058] In the following examples and comparative examples, the mushroom tyrosinase dopa rate oxidation method was used to determine the tyrosinase inhibition rate. For the specific operation of the determination method, refer to the following literature: Zhang Zhoumei, Xie Chunliang, Yan Shaowei, et al. Comparative analysis of the differences in antioxidant and whitening activities of Ganoderma lucidum fermentation broths from different strains [J]. Chinese Edible Fungi, 2021, 40(08): 43-48+56.

[0059] Example 1

[0060] This embodiment provides a method for fermenting mulberry leaves with composite probiotics, and the specific method is as follows:

[0061] 1. Determination of bacterial species

[0062] The plant lactobacillus used in this embodiment is plant lactobacillus ( Lactobacillus plantarum ) ATCC8014, purchased from Hunan Fenghui Biotechnology Co., Ltd. According to the updated announcement of the "List of Bacteria that can be used in food" (No. 4 of 2022), "Lactobacillus plantarum ( Lactobacillus plantarum )" is updated to "Lactobacillus plantarum ( Lactiplantibacillus plantarum )".

[0063] The xylosus Staphylococcus used in this example is xylosus Staphylococcus ( Staphylococcus xylosus ) ATCC29971, purchased from Hunan Fenghui Biotechnology Co., Ltd.

[0064] 2. Recovery and activation of strains

[0065] (1) Domestication of Lactobacillus plantarum

[0066] 15 g of mulberry leaves and 100 mL of water were prepared into mulberry leaf culture medium. Mulberry leaf culture medium and MRS culture medium were prepared into acclimated culture medium with a volume content of 100%, 80%, 60%, 40%, 20% and 0% of MRS culture medium according to a volume ratio of 10~0:0~10, respectively. The culture medium was sterilized at 121°C for 15 min and set aside.

[0067] Plant Lactobacillus was inoculated into the acclimation medium with a volume content of 100% of the MRS medium at 2% (2% of the volume of the acclimation medium), and cultured at 37°C. 600 When the OD reaches about 2.0 for the first time, take the culture medium and transfer it to an acclimation medium with a volume content of 80% of MRS medium, and culture it at 37℃. 600 When the OD reaches about 2.0 for the second time, transfer to an acclimation medium containing 60% of the volume of MRS medium and culture at 37°C. 600 When the OD reaches about 2.0 for the third time, transfer to an acclimation medium containing 40% of the MRS medium volume and culture at 37°C. 600 When the OD reaches about 2.0 for the fourth time, transfer to an acclimation medium containing 20% ​​of the MRS medium volume and culture at 37°C. 600 When the fifth fermentation reached approximately 2.0, the culture was transferred to an acclimation medium containing 0% MRS medium (i.e., 100% mulberry leaf medium) and incubated at 37°C. By gradually reducing the MRS content in the culture medium, Lactobacillus plantarum was able to adapt to the culture environment using only mulberry leaves as a substrate. Strains with good fermentation performance were screened, and combined with sensory evaluation, the optimal strain with the potential to ferment mulberry leaves was selected and maintained in the form of glycerol strains at -20°C.

[0068] The plant lactobacillus adapted to 0% MRS medium was re-streaked on the plate to isolate single colonies, and the morphological characteristics of the isolated single colonies were observed. Figure 1 The morphological characteristics of Lactobacillus plantarum after acclimation, where A is the Gram staining result and B is the colony characteristic diagram.

[0069] (2) Acclimation of Staphylococcus xylosus

[0070] 15 g of mulberry leaves and 100 mL of water were prepared into mulberry leaf culture medium, and the mulberry leaf culture medium was mixed with MRS culture medium according to the volume ratio to prepare acclimation culture medium with MRS culture medium volume content of 100%, 80%, 60%, 40%, 20% and 0%, and sterilized at 121°C for 15 min.

[0071] Inoculate 2% of Staphylococcus xylosus (2% of the volume of the acclimation medium) into the acclimation medium with 100% MRS medium content and culture it at 37°C. 600 When the OD reaches about 2.0 for the first time, take the culture medium and transfer it to an acclimation medium with a volume content of 80% of MRS medium, and culture it at 37℃. 600 When the OD reaches about 2.0 for the second time, transfer to an acclimation medium containing 60% of the volume of MRS medium and culture at 37°C. 600 When the OD reaches about 2.0 for the third time, transfer to an acclimation medium containing 40% of the MRS medium volume and culture at 37°C. 600 When the OD reaches about 2.0 for the fourth time, transfer to an acclimation medium containing 20% ​​of the MRS medium volume and culture at 37°C. 600 When the fifth dilution reached approximately 2.0, the culture was transferred to an acclimation medium containing 0% MRS medium (i.e., 100% mulberry leaf medium) and incubated at 37°C. By gradually reducing the MRS content in the culture medium, the xylosus Staphylococcus aureus was able to adapt to the culture environment using only mulberry leaves as the substrate. Strains with good fermentation performance were screened, and combined with sensory evaluation, the best strains with the potential to ferment mulberry leaves were selected and maintained at -20°C in the form of glycerol strains.

[0072] The xylose Staphylococcus adapted to 0% MRS medium was re-streaked on the plate to isolate single colonies, and the morphological characteristics of the isolated single colonies were observed. Figure 2 Shown are the morphological characteristics of Staphylococcus xylosus after acclimation, where A is the Gram staining result and B is the colony characteristic diagram.

[0073] (3) Determination of the growth curve of Lactobacillus plantarum after acclimation

[0074] Take the acclimated Lactobacillus plantarum strain stored at -20℃, inoculate it into 10 mL of MRS liquid medium, and place it in a 37℃ incubator for 18 h to 24 h to recover; take 0.2 mL of the recovered strain and inoculate it into 10 mL of MRS liquid medium, and incubate it at 37℃. The absorbance of the culture medium at 600 nm was measured at 0 h, 3 h, 6 h, 9 h, 12 h, 15 h, 18 h, 21 h, and 24 h. With time (h) as the horizontal axis, OD 600 The value is the vertical axis, and the growth curve is drawn, such as Figure 3 shown.

[0075] Depend on Figure 3 It can be seen that after acclimation, Lactobacillus plantarum grows slowly in the first 6 h and is in the lag phase, is in the logarithmic phase from 6 h to 12 h, is in the stable phase from 12 h to 18 h, and gradually enters the plateau phase after 18 h.

[0076] (4) Determination of the growth curve of Staphylococcus xylosus after acclimation

[0077] Take the acclimated Staphylococcus xylosus strain stored at -20℃, inoculate it into 10 mL of MRS liquid medium, and place it in a 37℃ incubator for 18 h to 24 h to recover; take 0.2 mL of the recovered strain and inoculate it into 10 mL of MRS liquid medium, and incubate it at 37℃. The absorbance of the culture medium at 600 nm was measured at 0h, 3h, 6h, 9h, 12h, 15h, 18h, 21h and 24h. With time (h) as the horizontal axis, OD 600 The value is the vertical axis, and the growth curve is drawn, such as Figure 4 shown.

[0078] Depend on Figure 4 It can be seen that after acclimation, Staphylococcus xylosus is in the lag phase for the first 3 hours, enters the logarithmic phase around the 3rd hour, is in the logarithmic phase from 3h to 12h, is in the stable phase from 12h to 18h, and gradually enters the plateau phase after 18h.

[0079] 3. Investigate the effects of pH, inoculation size, fermentation time, fermentation temperature and strain ratio on compound fermentation results

[0080] The following steps all use domesticated Lactobacillus plantarum and domesticated Staphylococcus xylosus.

[0081] (1) Effect of pH on tyrosinase activity inhibition rate

[0082] Five culture media containing 15g of mulberry leaves, 100mL of water, and 0.5g of glucose were prepared. The initial pH was adjusted to 5.5, 6.0, 6.5, 7.0, and 7.5 using 1.0 mol / L NaOH solution and 1.0 mol / L HCl solution, respectively. The inoculation volume ratio of Staphylococcus xylosus to Lactobacillus plantarum was 1:2, and the inoculum volume was 5% of the culture medium volume. The culture was placed in a 37°C incubator and incubated for 48 hours for fermentation. When the pH was 6.5, the fermentation product had the highest inhibition rate on tyrosinase activity, reaching 69.54% ( Figure 5 ), so 6.5 was determined to be the optimal fermentation pH.

[0083] (2) Effect of inoculation amount on tyrosinase activity inhibition rate

[0084] Prepare 5 portions of culture medium containing 15g of mulberry leaves, 100mL of water, and 0.5g of glucose. Adjust the pH to 6.5 and inoculate with a volume ratio of 1:2 of Staphylococcus xylosus to Lactobacillus plantarum. The inoculation amounts are 2.0%, 4.0%, 6.0%, 8.0%, and 10.0% of the culture volume, respectively. Fermentation is carried out in a 37°C incubator for 48 hours. Increasing the inoculation amount significantly improves the inhibition rate. The best fermentation effect is achieved when the inoculation amount is 6%. Figure 6 ), but too high an inoculation amount may cause the bacteria to consume too many nutrients during proliferation and affect the fermentation effect.

[0085] (3) Effect of fermentation time on tyrosinase activity inhibition rate

[0086] Five culture media containing 15g of mulberry leaves, 100mL of water, and 0.5g of glucose were prepared. The pH was adjusted to 6.5, and the inoculation volume ratio of Staphylococcus xylosus to Lactobacillus plantarum was 1:2, with an inoculum size of 5% of the culture volume. The culture was placed in a 37°C incubator for fermentation, and the culture was incubated for 12h, 24h, 36h, 48h, and 60h, respectively. The tyrosinase inhibition rate of the fermentation product reached a maximum of 79.15% at 48h. Figure 7 ), as the fermentation time prolonged, the inhibition rate began to decrease, indicating that the microorganisms in the fermentation liquid began to consume the nutrients released from the mulberry leaves, suggesting that the fermentation should be terminated in time. Therefore, 48 hours was determined to be the optimal fermentation time.

[0087] (4) Effect of fermentation temperature on the inhibition rate of tyrosinase activity

[0088] Prepare 5 portions of culture medium containing 15g of mulberry leaves, 100mL of water, and 0.5g of glucose. Adjust the pH to 6.5, inoculate with a volume ratio of 1:2 of Staphylococcus xylosus to Lactobacillus plantarum, and inoculate at 6% of the culture volume. Ferment for 48h in incubators at 30°C, 33°C, 36°C, 39°C, and 42°C, respectively. As the fermentation temperature increases, the inhibition rate shows a trend of first increasing and then decreasing ( Figure 8 ), reaching a peak of 77.34% at 36°C. While fermentation temperature can enhance bacterial activity, rapid bacterial proliferation also increases nutrient consumption in the fermentation product. 36°C is the optimal growth temperature for bacterial strains, ultimately determining the optimal fermentation temperature to be 36°C.

[0089] (5) Effect of bacterial species ratio on tyrosinase activity inhibition rate

[0090] Five culture media containing 15g of mulberry leaves, 100mL of water, and 0.5g of glucose were prepared at pH 6.5. Staphylococcus xylosus and Lactobacillus plantarum were inoculated at volume ratios of 3:1, 2:1, 1:1, 1:2, and 1:3, respectively. The inoculum volume was 5% of the culture medium volume and the cells were incubated at 37°C for 48 hours. As the proportion of Lactobacillus plantarum in the strain increased, the inhibition rate increased rapidly and significantly, reaching a maximum of 83.61% ( Figure 9 ), and finally determined the optimal fermentation ratio to be 1:2.

[0091] 4. Compound Fermentation Process

[0092] 5 g of mulberry leaves, 100 mL of water, and 0.5 g of glucose were prepared into a fermentation medium, and the pH of the fermentation medium was adjusted to 6.5.

[0093] The fermentation medium was inoculated with acclimated Staphylococcus xylosus and acclimated Lactobacillus plantarum at a volume ratio of 1:2, with a total inoculation amount of 6% of the volume of the medium. The culture medium was placed in a 36° C. incubator for fermentation for 48 hours to obtain a fermentation product.

[0094] In this example, the optimal combined bacterial strain ratio (Staphylococcus xylosus:Lactobacillus plantarum) of 1:2 was optimized for the fermentation of mulberry leaves. The inoculum size was 6%. 15g of mulberry leaves were added to 150mL of water and 5g of glucose, and the mixture was allowed to ferment at 36°C for 48h. The fermentation broth after composite bacterial fermentation was brown, with a slightly bitter taste, a tea aroma, and a sweet flavor. The inhibition rate of the fermentation product on tyrosinase activity was 82.23% as determined by the mushroom tyrosinase DOPA rate oxidation method.

[0095] Comparative Example 1

[0096] The acclimated Lactobacillus plantarum and the acclimated Staphylococcus xylosus are used to ferment mulberry leaves separately, and the specific method is as follows:

[0097] (1) Fermentation of mulberry leaves with Staphylococcus xylosus after acclimation

[0098] The acclimated xylose Staphylococcus was inoculated into a culture medium consisting of 15 g fresh mulberry leaves, 0.5 g glucose and 100 mL water at a 2% inoculation rate, and fermented at 37° C. for 48 hours. The fermentation was shaken every 4-6 hours to ensure full contact between the fermentation products to obtain a fermentation product.

[0099] The inhibition rate of the fermentation product on tyrosinase activity was detected by mushroom tyrosinase dopa rate oxidation method, which was 33.47%. It had a fresh grassy smell with a slight fishy smell.

[0100] (2) Fermentation of mulberry leaves with Lactobacillus plantarum after acclimation

[0101] The acclimated Lactobacillus plantarum was inoculated into a culture medium consisting of 15 g fresh mulberry leaves, 0.5 g glucose and 100 mL water at a 2% inoculation rate, and fermented at 37° C. for 48 hours. The fermentation was shaken every 4 to 6 hours to ensure full contact of the fermentation to obtain a fermentation product.

[0102] The inhibition rate of tyrosinase activity of the fermentation product was 53.27% detected by mushroom tyrosinase dopa-rate oxidation method, and it had a distinct lactic acid taste.

[0103] In summary, by comparing Example 1 with Comparative Example 1, it can be seen that when the domesticated Staphylococcus xylosus and the domesticated Lactobacillus plantarum are fermented in a volume ratio of 1:2 on mulberry leaves, the inhibition rate of the fermentation product on tyrosinase reaches 82.23%; when the domesticated Staphylococcus xylosus and the domesticated Lactobacillus plantarum are fermented on mulberry leaves alone, the inhibition rates of the fermentation products on tyrosinase activity are only 33.47% and 53.27%, respectively. Therefore, the inhibition rate of the fermentation product on tyrosinase activity indicates that the two bacteria have a significant synergistic effect during the fermentation process.

[0104] Comparative Example 2

[0105] Untamed Lactobacillus plantarum and untamed Staphylococcus xylosus were used to ferment mulberry leaves separately. The specific method is as follows:

[0106] (1) Fermentation of mulberry leaves by undomesticated Staphylococcus xylosus

[0107] The fermentation method is the same as that of Comparative Example 1, except that unacclimated Staphylococcus xylosus is used instead of acclimated Staphylococcus xylosus.

[0108] The inhibition rate of the fermentation product on tyrosinase activity was detected by mushroom tyrosinase dopa rate oxidation method, which was 25.81%. The fermentation product was fresh like grass with a slight fishy smell.

[0109] (2) Fermentation of mulberry leaves with undomesticated Lactobacillus plantarum

[0110] The fermentation method is the same as that of Comparative Example 1, except that untamed Lactobacillus plantarum is used instead of tamed Lactobacillus plantarum.

[0111] The inhibition rate of the fermentation product on tyrosinase activity was detected by mushroom tyrosinase dopa rate oxidation method, which was 49.55%, and it had a distinct lactic acid taste.

[0112] In summary, by comparing Comparative Example 2 with Comparative Example 1, it can be seen that by acclimating Staphylococcus xylosus and Lactobacillus plantarum, the inhibition rate of the product after fermentation of Staphylococcus xylosus on tyrosinase activity changes from 25.81% before acclimation to 33.47% after acclimation, and the inhibition rate of the product after fermentation of Lactobacillus plantarum on tyrosinase activity changes from 49.55% before acclimation to 53.27% after acclimation. This shows that by acclimating Staphylococcus xylosus and Lactobacillus plantarum, they can adapt to the culture environment with only mulberry leaves as the substrate, thereby improving the fermentation performance of Staphylococcus xylosus and Lactobacillus plantarum.

[0113] Example 2

[0114] This example evaluates the safety of Staphylococcus xylosus and Lactobacillus plantarum, and the evaluation method is as follows:

[0115] Treatment group: 3-year-old silkworms were fed with high concentration (1×10 8 CFU / mL) of the test bacteria (acclimated Staphylococcus xylosus and acclimated Lactobacillus plantarum) were added, and the growth and survival rate of the fifth-instar larvae were observed and counted.

[0116] Blank group: The test bacteria (Staphylococcus xylosus and Lactobacillus plantarum) were not fed, and other conditions were the same as those of the treatment group.

[0117] The test results are as follows Figure 10-11 As shown by Figure 10-11 It can be seen that the weight of silkworm larvae in the group fed with Staphylococcus xylosus increased by 12.97% compared with the blank group, with a significant difference of p<0.05; the larvae were more robust in body shape, and there was no significant difference in the actual measured body length and the survival rate of the 5th instar larvae compared with the blank group.

[0118] In summary, it can be seen from Example 2 that the Staphylococcus xylosus and Lactobacillus plantarum used in the present invention are safe and reliable, so the mulberry leaf fermentation product obtained by composite fermentation of Staphylococcus xylosus and Lactobacillus plantarum is safe and has no toxic side effects, and can be used to prepare whitening cosmetics and / or whitening skin care products.

[0119] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A method for fermenting mulberry leaves with composite probiotics, characterized in that: The following steps are involved: Inoculating Staphylococcus xylosus and Lactobacillus plantarum into a fermentation medium containing mulberry leaves for composite fermentation; The volume ratio of the Staphylococcus xylosus to the Lactobacillus plantarum is 1:2, and the total inoculation amount is 2% to 10% of the volume of the fermentation medium; The xylosus Staphylococcus is xylosus Staphylococcus ( Staphylococcus xylosus ) ATCC29971 domesticated strain, the plant lactobacillus is plant lactobacillus ( Lactobacillus plantarum ) ATCC8014 domesticated strain; The domestication method of Staphylococcus xylosus and Lactobacillus plantarum includes: firstly culturing the pre-domesticated Staphylococcus xylosus and Lactobacillus plantarum respectively using an MRS culture medium containing 0 mulberry leaves, then transferring them respectively to MRS culture medium with gradually increasing mulberry leaf content for culturing, and finally transferring them respectively to a culture medium with only mulberry leaves as a substrate for culturing.

2. The method according to claim 1, characterized in that The temperature of the composite fermentation is 30° C. to 42° C., and the time of the composite fermentation is 12 h to 60 h.

3. The method according to claim 1, characterized in that The pH of the composite fermentation is 5.5-7.

5.

4. The method according to claim 1, wherein The method specifically comprises the following steps: Mix 5g to 25g of mulberry leaves with 50mL to 200mL of water and 0.5g to 2g of glucose to obtain a fermentation medium; adjust the pH of the fermentation medium to 5.5 to 7.5; The fermentation medium is inoculated with the Staphylococcus xylosus and the Lactobacillus plantarum, and the medium is placed in an incubator at 30° C. to 42° C. for fermentation for 12 hours to 60 hours to obtain a fermentation product.

5. The method according to claim 1, characterized in that The domestication method of Staphylococcus xylosus specifically comprises the following steps: 14 g to 16 g of mulberry leaves and 100 mL of water are prepared into a mulberry leaf culture medium, and the mulberry leaf culture medium is mixed with an MRS culture medium in a volume ratio to prepare an acclimation culture medium, wherein the volume content of the MRS culture medium in the acclimation culture medium is 0% to 100%; The unacclimated Staphylococcus xylosus was inoculated into an acclimation medium containing 100% of the volume of MRS medium at a volume percentage of 1% to 3%, and the culture was statically cultured at 36°C to 38°C. Waiting for OD 600 When the concentration reaches 1.9-2.1 for the first time, take the culture medium and transfer it to an acclimation medium containing 75%-85% of MRS medium by volume, and continue to culture at 36-38°C. Waiting for OD 600 When the concentration reaches 1.9-2.1 for the second time, take the culture medium and transfer it to an acclimation medium containing 55%-65% of MRS medium by volume, and continue to culture at 36-38°C. Waiting for OD 600 When the concentration reaches 1.9-2.1 for the third time, take the culture medium and transfer it to an acclimation medium containing 35%-45% of MRS medium by volume, and continue to culture at 36-38°C. Waiting for OD 600 When the concentration reaches 1.9-2.1 for the fourth time, take the culture medium and transfer it to an acclimation medium containing 15%-25% of MRS medium by volume, and continue to culture at 36-38°C. Waiting for OD 600 When the concentration reached 1.9-2.1 for the fifth time, the culture solution was taken and transferred to an acclimation medium containing 0% MRS medium by volume, and the culture was continued at 36°C-38°C to obtain acclimated Staphylococcus xylosus.

6. The method according to claim 1, characterized in that The domestication method of the plant lactobacillus specifically comprises the following steps: 14 g to 16 g of mulberry leaves and 100 mL of water are prepared into a mulberry leaf culture medium, and the mulberry leaf culture medium is mixed with an MRS culture medium in a volume ratio to prepare an acclimation culture medium, wherein the volume content of the MRS culture medium in the acclimation culture medium is 0% to 100%; The Lactobacillus plantarum before acclimation is inoculated into an acclimation medium with a volume content of 100% of the MRS medium at a volume percentage of 1-3%, and statically cultured at 36° C.-38° C. Waiting for OD 600 When the concentration reaches 1.9-2.1 for the first time, take the culture medium and transfer it to an acclimation medium containing 75%-85% of MRS medium by volume, and continue to culture at 36-38°C. Waiting for OD 600 When the concentration reaches 1.9-2.1 for the second time, take the culture medium and transfer it to an acclimation medium containing 55%-65% of MRS medium by volume, and continue to culture at 36-38°C. Waiting for OD 600 When the concentration reaches 1.9-2.1 for the third time, take the culture medium and transfer it to an acclimation medium containing 35%-45% of MRS medium by volume, and continue to culture at 36-38°C. Waiting for OD 600 When the concentration reaches 1.9-2.1 for the fourth time, take the culture medium and transfer it to an acclimation medium containing 15%-25% of MRS medium by volume, and continue to culture at 36-38°C. Waiting for OD 600 When the pH value reaches 1.9-2.1 for the fifth time, the culture solution is taken and transferred to an acclimation medium containing 0% of the volume of MRS medium, and the culture is continued at 36° C.-38° C. to obtain the acclimated Lactobacillus plantarum.

7. A mulberry leaf fermentation product, characterized in that: The method according to any one of claims 1 to 6 is used for preparation.

8. Use of the mulberry leaf fermentation product according to claim 7 in the preparation of whitening cosmetics and / or skin care products.

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