Method for fermenting mulberry leaves through compound probiotics, mulberry leaf fermentation product and application

Abstract

The invention discloses a method for fermenting mulberry leaves through compound probiotics, a mulberry leaf fermentation product and application, and relates to the technical field of fermentation. The fermentation method comprises the following steps: carrying out compound fermentation on mulberry leaves by adopting staphylococcus xylosus and lactobacillus plantarum according to a volume ratio of (1-3): (1-3), wherein the total inoculum size is 2-10% of the volume of a fermentation culture medium; the mulberry leaf fermentation product is prepared by the method. The invention also provides application of the mulberry leaf fermentation product in preparation of whitening cosmetics and / or skin care products. The preparation method has the beneficial effects that the two probiotics including the lactobacillus plantarum and the staphylococcus xylosus are used for performing compound fermentation on the mulberry leaves, and the lactobacillus plantarum and the staphylococcus xylosus can achieve a synergistic effect during compound fermentation of the mulberry leaves, so that active substances for inhibiting tyrosinase in the mulberry leaves can be effectively released; active substances are all retained in the fermentation product, and the inhibition rate of the fermentation product on the tyrosinase activity is remarkably increased.

Description

Technical Field

[0001] The 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 medicinal material 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 anti-blood coagulation, blood pressure lowering, blood lipid lowering, cholesterol lowering, anti-thrombosis and anti-atherosclerosis, blood sugar lowering, 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] Studies have shown that various active substances such as flavonoids, polyphenols and polysaccharides in mulberry leaves have strong inhibitory ability to tyrosinase through competitive binding and antagonism. Due to the dense cellulose and wood structures in plant cells, it is difficult to release the active substances in the cells, and humans and animals lack the corresponding enzymes and are difficult to digest and hinder absorption. In order to solve this problem, the traditional method is to use water extraction, but the traditional water extraction method is inefficient, and the chemical extraction method for a single substance will cause serious waste and residue treatment problems. Studies have shown that the content of active substances in the product can be greatly increased by microbial fermentation, but due to the process reasons such as strains and fermentation conditions, while increasing the content of alkaloids, there is a loss of flavonoids and polyphenols. Therefore, optimizing the fermentation process of mulberry leaves based on the inhibition rate of tyrosinase and improving the extraction rate of active substances in mulberry leaves have positive significance for using mulberry leaves to develop efficient natural tyrosinase inhibitors. 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, and a mulberry leaf fermentation product and application.

[0006] The technical solution of the present invention is as follows: The first aspect of the present invention provides a method for fermenting mulberry leaves with composite probiotics, comprising the following steps: 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 1-3:1-3, and the total inoculation amount is 2%-10% of the volume of the fermentation medium.

[0007] Optionally, the xylosus Staphylococcus is xylosus Staphylococcus ( Staphylococcus xylosus ) ATCC29971; the plant lactobacillus is plant lactobacillus ( Lactobacillus plantarum )ATCC8014.

[0008] It should be noted that the above-mentioned Staphylococcus xylosus and Lactobacillus plantarum are both existing strains.

[0009] Optionally, the temperature of the composite fermentation is 30°C to 42°C, and the time of the composite fermentation is 12h to 60h.

[0010] Optionally, the pH of the composite fermentation is 5.5-7.5.

[0011] Optionally, the method specifically comprises the following steps: 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; 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. The fermentation culture is performed for 12 h to 60 h to obtain a fermentation product.

[0012] Optionally, the xylosus Staphylococcus is domesticated xylosus Staphylococcus, and the plant lactobacillus is domesticated plant lactobacillus; The domestication method of the xylose Staphylococcus and the plant lactobacillus comprises: firstly culturing the pre-domesticated xylose Staphylococcus and the pre-domesticated plant lactobacillus respectively in an MRS culture medium containing 0 mulberry leaves, then transferring them to MRS culture medium containing gradually increasing mulberry leaves for culturing respectively, and finally transferring them to a culture medium with only mulberry leaves as a substrate for culturing respectively.

[0013] Optionally, the domestication method of Staphylococcus xylosus specifically comprises the following steps: 14-16 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 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 is 0%-100%; The acclimated Staphylococcus xylosus was inoculated into an acclimated medium containing 100% MRS medium at a volume percentage of 1-3%, and statically cultured at 36-38°C; Waiting for OD 600When 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. 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. 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; 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; Waiting for OD 600 When the pH value reached 1.9-2.1 for the fifth time, the culture solution was taken and transferred to an acclimated medium containing 0% MRS medium, and static culture was continued at 36-38°C to obtain acclimated Staphylococcus xylosus.

[0014] Optionally, the domestication method of plant lactobacillus specifically comprises the following steps: 14-16 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 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 is 0%-100%; The Lactobacillus plantarum before domestication is inoculated into a domestication 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.; 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 with a volume content of 75%-85% of MRS medium, and continue to culture at 36-38°C; Waiting for OD 600 When the second time reaches 1.9-2.1, take the culture medium and transfer it to an acclimation medium with a volume content of 55%-65% of MRS medium, and continue to culture at 36-38°C; Waiting for OD 600 When the value reaches 1.9-2.1 for the third time, take the culture medium and transfer it to an acclimation medium containing 35%-45% of the volume of MRS medium, 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 the volume of MRS medium, 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 static culture is continued at 36-38° C. to obtain the acclimated Lactobacillus plantarum.

[0015] The second aspect of the present invention provides a mulberry leaf fermentation product prepared by the above method.

[0016] Optionally, the inhibition rate of the mulberry leaf fermentation product of the present invention on tyrosinase activity can reach 82.23%.

[0017] 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.

[0018] 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.

[0019] The present invention has at least one of the following beneficial effects: 1, the method of the present invention is to utilize plant lactobacillus and two kinds of probiotics of xylose staphylococcus to carry out composite fermentation to mulberry leaf, plant lactobacillus and xylose staphylococcus can play synergistic effect when composite fermentation mulberry leaf, can realize the effective release of the active substance that inhibits tyrosinase in mulberry leaf, and active substance is all retained in fermentation product.Compared with plant lactobacillus and xylose staphylococcus fermentation alone, composite fermentation significantly improves the effect of mulberry leaf fermentation, after testing, xylose staphylococcus and plant lactobacillus fermentation alone mulberry leaf obtain mulberry leaf fermentation product to the inhibition rate of tyrosinase activity only respectively 33.47% and 53.27%, and the mulberry leaf fermentation product obtained by composite fermentation to the inhibition rate of tyrosinase activity reaches 82.23%, thus it can be seen that the inhibition rate of tyrosinase activity of mulberry leaf fermentation product obtained by composite fermentation increases.

[0020] 2. The present invention also domesticates Staphylococcus xylosus and Lactobacillus plantarum to adapt them to a culture environment with mulberry leaves as substrates. According to testing, the inhibition rates of mulberry leaf fermentation products obtained by fermenting mulberry leaves with Staphylococcus xylosus and Lactobacillus plantarum alone on tyrosinase activity were only 25.81% and 49.55%, respectively, before domestication. The inhibition rates of mulberry leaf fermentation products obtained by fermenting mulberry leaves with Staphylococcus xylosus and Lactobacillus plantarum alone on tyrosinase activity were 33.47% and 53.27%, respectively, after domestication. It can be seen 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.

[0021] 3. Both the plant lactobacillus and the xylose Staphylococcus of the present invention are probiotics, which are safe and reliable, and the product has a unique flavor and is safe and has no toxic side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The morphological characteristics of the domesticated Lactobacillus plantarum in Example 1, wherein A is the Gram staining result; and B is the colony characteristic diagram.

[0023] Figure 2 The morphological characteristics of Staphylococcus xylosus after domestication in Example 1, wherein A is the Gram staining result; and B is the colony characteristic diagram.

[0024] Figure 3 This is the growth curve of Lactobacillus plantarum after domestication in Example 1.

[0025] Figure 4 This is the growth curve of Staphylococcus xylosus after domestication in Example 1.

[0026] Figure 5 This is a diagram showing the effect of pH on the composite fermentation results in Example 1.

[0027] Figure 6 This is a diagram showing the effect of inoculation amount on the composite fermentation results in Example 1.

[0028] Figure 7 This is a diagram showing the effect of fermentation time on the composite fermentation results in Example 1.

[0029] Figure 8 This is a diagram showing the effect of fermentation temperature on the composite fermentation results in Example 1.

[0030] Fig. 9 This is a graph showing the effect of the bacterial inoculation ratio on the composite fermentation results in Example 1.

[0031] Fig.10 This is a physical picture of the effects of Lactobacillus plantarum and Staphylococcus xylosus on the growth and development of silkworm larvae in Example 2.

[0032] Fig.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

[0033] 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 in conjunction with 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 used to limit the present invention.

[0034] The present invention is further described in detail below with specific examples, but the present invention is not limited to the following specific examples.

[0035] It should be noted that the plant lactobacillus used in the following examples and comparative examples is plant lactobacillus ( Lactobacillus plantarum ) ATCC8014, the Staphylococcus xylosus used was Staphylococcus xylosus ( Staphylococcus xylosus ) ATCC29971, were purchased from Hunan Fenghui Biotechnology Co., Ltd.

[0036] The MRS medium used in the following examples is a commercially available medium.

[0037] 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.

[0038] Example 1 This embodiment provides a method for fermenting mulberry leaves with composite probiotics, and the specific method is as follows: 1. Determination of bacterial species 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 now called "Lactobacillus plantarum ( Lactiplantibacillus plantarum )".

[0039] The xylosus Staphylococcus used in this example is xylosus Staphylococcus ( Staphylococcus xylosus ) ATCC29971, purchased from Hunan Fenghui Biotechnology Co., Ltd.

[0040] 2. Recovery and activation of strains (1) Domestication of Lactobacillus plantarum 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 volume contents of MRS culture medium of 100%, 80%, 60%, 40%, 20% and 0% respectively in a volume ratio of 10~0:0~10. The culture medium was sterilized by high pressure at 121°C for 15 min and set aside.

[0041] Inoculate 2% (2% of the volume of the acclimation medium) of Lactobacillus plantarum into the acclimation medium with a volume content of 100% of the MRS medium, and incubate 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 containing 80% of the volume of MRS medium, and culture it at 37°C.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 volume of MRS medium and culture at 37°C. 600 When the OD reached about 2.0 for the fourth time, the cells were transferred to an acclimation medium containing 20% ​​of the volume of MRS medium and cultured at 37°C. 600 When the fifth time reached about 2.0, it was transferred to an acclimation medium with a volume content of 0% MRS medium (i.e., 100% mulberry leaf medium) and cultured at 37°C. By gradually reducing the content of MRS in the culture medium, plant lactobacillus can adapt to the culture environment with only mulberry leaves as the substrate, and strains with good fermentation performance were screened out. 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 bacteria.

[0042] 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 domestication, where A is the Gram staining result and B is the colony characteristic diagram.

[0043] (2) Domestication of Staphylococcus xylosus 15 g of mulberry leaves and 100 mL of water were prepared into mulberry leaf culture medium. The mulberry leaf culture medium and MRS culture medium were mixed according to volume ratio to prepare acclimation culture medium with MRS culture medium volume contents of 100%, 80%, 60%, 40%, 20% and 0%. The culture medium was sterilized by high pressure at 121°C for 15 min and set aside.

[0044] Inoculate 2% of Staphylococcus xylosus (2% of the volume of the acclimation medium) into the acclimation medium with MRS medium content of 100%, and culture 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 containing 80% of the volume of MRS medium, and culture it at 37°C. 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 volume of MRS medium and culture at 37°C. 600 When the OD reached about 2.0 for the fourth time, the cells were transferred to an acclimation medium containing 20% ​​of the volume of MRS medium and cultured at 37°C. 600When the fifth time reached about 2.0, it was transferred to an acclimation medium with a volume content of 0% MRS medium (i.e., 100% mulberry leaf medium) and cultured at 37°C. By gradually reducing the content of MRS in the culture medium, Staphylococcus xylosus can adapt to the culture environment with only mulberry leaves as substrates, and strains with good fermentation performance were screened out. 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 bacteria.

[0045] 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; B is the colony characteristic diagram.

[0046] (3) Determination of the growth curve of Lactobacillus plantarum after acclimation Take the domesticated Lactobacillus plantarum strain stored at -20℃, inoculate it into 10 mL of MRS liquid culture medium, and place it in a 37℃ constant temperature incubator for 18 h to 24 h for recovery; take 0.2 mL of the recovered strain, inoculate it into 10 mL of MRS liquid culture medium, and culture it at 37℃. At 0h, 3h, 6h, 9h, 12h, 15h, 18h, 21h and 24h, measure the absorbance of the culture solution at 600nm. 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.

[0047] 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, 6h-12h is in the logarithmic phase, 12h-18h is the stable phase, and gradually enters the plateau phase after 18h.

[0048] (4) Determination of the growth curve of Staphylococcus xylosus after acclimation Take the acclimated xylose Staphylococcus strain stored at -20℃, inoculate it in 10 mL of MRS liquid culture medium, and place it in a 37℃ constant temperature incubator for 18 h to 24 h for recovery; take 0.2 mL of the recovered strain, inoculate it in 10 mL of MRS liquid culture medium, and culture it at 37℃. At 0h, 3h, 6h, 9h, 12h, 15h, 18h, 21h and 24h, measure the absorbance of the culture solution at 600 nm. 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.

[0049] Depend on Figure 4It 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.

[0050] 3. Investigate the effects of pH, inoculation size, fermentation time, fermentation temperature and bacterial strain ratio on composite fermentation results The following steps all use domesticated Lactobacillus plantarum and domesticated Staphylococcus xylosus.

[0051] (1) Effect of pH on tyrosinase activity inhibition rate Prepare 5 portions of culture medium containing 15g mulberry leaves, 100mL water and 0.5g glucose, adjust the initial pH to 5.5, 6.0, 6.5, 7.0, 7.5 with 1.0 mol / L NaOH solution and 1.0 mol / L HCl solution respectively, inoculate the strain volume ratio of xylose Staphylococcus: plantarum Lactobacillus = 1:2, inoculate 5% of the volume of the culture medium, and place in a 37 ℃ incubator for 48h for fermentation. When the pH is 6.5, the fermentation product has the highest inhibition rate of tyrosinase activity, which is 69.54% ( Figure 5 ), so 6.5 was determined to be the optimal fermentation pH.

[0052] (2) Effect of inoculation amount on tyrosinase activity inhibition rate Prepare 5 portions of culture medium containing 15g mulberry leaves, 100mL water and 0.5g glucose, adjust the pH value to 6.5, inoculate the strains in a volume ratio of 1:2 of Staphylococcus xylosus: Lactobacillus plantarum, and inoculate 2.0%, 4.0%, 6.0%, 8.0% and 10.0% of the volume of the culture medium, respectively, and place in a 37 ℃ incubator for fermentation for 48h. Increasing the inoculation amount can significantly increase the inhibition rate. When the inoculation amount is 6%, the fermentation effect is the best ( Figure 6 ), but too high an inoculation amount may cause the bacteria to consume too much nutrients during proliferation and affect the fermentation effect.

[0053] (3) Effect of fermentation time on tyrosinase activity inhibition rate Prepare 5 portions of culture medium containing 15g mulberry leaves, 100mL water and 0.5g glucose, adjust the pH value to 6.5, inoculate the strains in a ratio of 1:2 of Staphylococcus xylosus: Lactobacillus plantarum, and inoculate 5% of the volume of the culture medium. Ferment in a 37°C incubator 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, so 48 hours was determined to be the optimal fermentation time.

[0054] (4) Effect of fermentation temperature on tyrosinase activity inhibition rate Prepare 5 portions of culture medium containing 15g mulberry leaves, 100mL water and 0.5g glucose, adjust the pH value to 6.5, inoculate the strains with a volume ratio of 1:2 of Staphylococcus xylosus: Lactobacillus plantarum, and inoculate 6% of the volume of the culture medium, and ferment for 48h in incubators at 30℃, 33℃, 36℃, 39℃, and 42℃, respectively. As the fermentation temperature increases, the inhibition rate increases first and then decreases ( Figure 8 ), the highest at 36 ℃, reaching 77.34%. Although fermentation temperature can improve the activity of bacteria, as the bacteria proliferate too fast, it will also increase the consumption of nutrients in the fermentation product, and 36 ℃ is the best growth temperature for bacteria, and finally determined to be the best fermentation temperature.

[0055] (5) Effect of bacterial strain ratio on tyrosinase activity inhibition rate Five culture media containing 15g mulberry leaves, 100mL water and 0.5g glucose were prepared at pH 6.5. Staphylococcus xylosus and Lactobacillus plantarum were inoculated at a volume ratio of 3:1, 2:1, 1:1, 1:2 and 1:3, respectively. The inoculation amount was 5% of the volume of the culture medium and the culture was kept at 37°C for 48 hours. As the proportion of Lactobacillus plantarum in the strain increased, the inhibition rate increased rapidly and the increase was large, reaching an extreme value of 83.61% (1:2). Fig. 9 ), and finally determined the optimal fermentation ratio to be 1:2.

[0056] 4. Composite fermentation process 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.

[0057] The fermentation medium was inoculated with domesticated Staphylococcus xylosus and domesticated Lactobacillus plantarum at a volume ratio of 1:2, with a total inoculation amount of 6% of the volume of the medium. The medium was placed in a 36° C. incubator for fermentation for 48 hours to obtain a fermentation product.

[0058] In this embodiment, the optimal combined strain volume ratio (xylose Staphylococcus: plantarum Lactobacillus) obtained by optimizing the composite strain fermentation of mulberry leaves was 1:2, the inoculation amount was 6%, 15g mulberry leaves, 150mL water, 5g glucose were inoculated, and the fermentation was allowed to stand at 36°C for 48h. The fermentation broth after composite bacteria fermentation showed a brown color, a light bitter taste, a tea fragrance and a sweet taste. The inhibition rate of the fermentation product on tyrosinase activity detected by mushroom tyrosinase dopa rate oxidation method reached 82.23%.

[0059] Comparative Example 1 The domesticated plant lactobacillus and the domesticated xylose Staphylococcus are used to ferment mulberry leaves separately, and the specific method is as follows: (1) Fermentation of mulberry leaves with Staphylococcus xylosus after domestication The domesticated Staphylococcus xylosus 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 fermented material was shaken every 4-6 hours to ensure full contact between the fermented materials, thereby obtaining a fermentation product.

[0060] The inhibition rate of tyrosinase activity by the fermentation product was 33.47% using the mushroom tyrosinase DOPA rate oxidation method. It was fresh like grass with a slight fishy smell.

[0061] (2) Fermentation of mulberry leaves with Lactobacillus plantarum after domestication The domesticated 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 between the fermentation products, thereby obtaining a fermentation product.

[0062] 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.

[0063] 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 used to perform composite fermentation on mulberry leaves in a volume ratio of 1:2, the inhibition rate of the fermentation product on tyrosinase reaches 82.23%; while when the domesticated Staphylococcus xylosus and the domesticated Lactobacillus plantarum are used to ferment mulberry leaves separately, 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.

[0064] Comparative Example 2 Untamed Lactobacillus plantarum and untamed Staphylococcus xylosus are used to ferment mulberry leaves separately, and the specific method is as follows: (1) Fermentation of mulberry leaves with untamed Staphylococcus xylosus The fermentation method is the same as that of Comparative Example 1, except that untamed Staphylococcus xylosus is used instead of tamed Staphylococcus xylosus.

[0065] The inhibition rate of the fermentation product on tyrosinase activity was detected by mushroom tyrosinase DOPA rate oxidation method and was 25.81%. The fermentation product was fresh like grass with a slight fishy smell.

[0066] (2) Fermentation of mulberry leaves with untamed Lactobacillus plantarum The fermentation method is the same as that of Comparative Example 1, except that untamed Lactobacillus plantarum is used instead of tamed Lactobacillus plantarum.

[0067] The inhibition rate of the fermentation product on tyrosinase activity was detected by mushroom tyrosinase DOPA rate oxidation method, which was 49.55%, with a distinct lactic acid taste.

[0068] In summary, by comparing Comparative Example 2 with Comparative Example 1, it can be seen that by domesticating Staphylococcus xylosus and Lactobacillus plantarum, the inhibition rate of the product after fermentation of Staphylococcus xylosus on tyrosinase activity changes from 25.81% before domestication to 33.47% after domestication, and the inhibition rate of the product after fermentation of Lactobacillus plantarum on tyrosinase activity changes from 49.55% before domestication to 53.27% after domestication, which shows that by domesticating Staphylococcus xylosus and Lactobacillus plantarum, they can adapt to the culture environment with mulberry leaves as the substrate only, thereby improving the fermentation performance of Staphylococcus xylosus and Lactobacillus plantarum.

[0069] Example 2 In this example, the safety of Staphylococcus xylosus and Lactobacillus plantarum was evaluated, and the evaluation method was as follows: 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), and the growth and survival rate of the 5th instar larvae were observed and counted.

[0070] 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.

[0071] The test results are as follows Figure 10~Figure 11 As shown by Figure 10~Figure 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 the actual measured body length and the survival rate of the 5th instar larvae were not significantly different from those of the blank group.

[0072] 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 that the mulberry leaf fermentation product obtained by the 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.

[0073] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope 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 1-3:1-3, and the total inoculation amount is 2%-10% of the volume of the fermentation medium.

2. The method according to claim 1, characterized in that The xylosus Staphylococcus is a xylosus Staphylococcus ( Staphylococcus xylosus ) ATCC29971; the plant lactobacillus is plant lactobacillus ( Lactobacillus plantarum )ATCC8014.

3. 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.

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

5.

5. The method according to claim 1, characterized in that The method specifically comprises the following steps: Mix 5 g to 25 g of mulberry leaves with 50 mL to 200 mL of water and 0.5 g to 2 g 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 h to 60 h to obtain a fermentation product.

6. The method according to claim 1, characterized in that The xylose Staphylococcus is domesticated xylose Staphylococcus, and the plant lactobacillus is domesticated plant lactobacillus; The domestication method of the xylose Staphylococcus and the plant lactobacillus comprises: firstly culturing the pre-domesticated xylose Staphylococcus and the pre-domesticated plant lactobacillus respectively in an MRS culture medium containing 0 mulberry leaves, then transferring them to MRS culture medium containing gradually increasing mulberry leaves for culturing respectively, and finally transferring them to a culture medium with only mulberry leaves as a substrate for culturing respectively.

7. The method according to claim 6, 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 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 is 0% to 100%; The acclimated Staphylococcus xylosus was inoculated into an acclimated medium with a volume content of 100% of the MRS medium at a volume percentage of 1% to 3%, and 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 with a volume content of 75%-85% of MRS medium, and continue to culture at 36℃-38℃; Waiting for OD 600 When the second time reaches 1.9-2.1, take the culture medium and transfer it to an acclimation medium with a volume content of 55%-65% of MRS medium, and continue to culture at 36℃-38℃; Waiting for OD 600 When the value 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°C-38°C; Waiting for OD 600 When the pH value reaches 1.9-2.1 for the fourth time, take the culture medium and transfer it to an acclimation medium containing 15%-25% of the volume of MRS medium, and continue to culture at 36°C-38°C; Waiting for OD 600 When the pH value reached 1.9-2.1 for the fifth time, the culture solution was taken and transferred to an acclimation medium containing 0% of the volume of MRS medium, and static culture was continued at 36°C-38°C to obtain acclimated Staphylococcus xylosus.

8. The method according to claim 6, 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 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 is 0% to 100%; The Lactobacillus plantarum before domestication is inoculated into a domestication 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 with a volume content of 75%-85% of MRS medium, and continue to culture at 36℃-38℃; Waiting for OD 600 When the second time reaches 1.9-2.1, take the culture medium and transfer it to an acclimation medium with a volume content of 55%-65% of MRS medium, and continue to culture at 36℃-38℃; Waiting for OD 600 When the value 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°C-38°C; Waiting for OD 600 When the pH value reaches 1.9-2.1 for the fourth time, take the culture medium and transfer it to an acclimation medium containing 15%-25% of the volume of MRS medium, and continue to culture at 36°C-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 static culture is continued at 36° C.-38° C. to obtain the acclimated Lactobacillus plantarum.

9. A mulberry leaf fermentation product, characterized in that: The method is prepared by any one of claims 1 to 8.

10. Use of the mulberry leaf fermentation product according to claim 9 in the preparation of whitening cosmetics and / or skin care products.

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