Method for extracting active ingredients of microbial fermentation plants for resisting wrinkles

Abstract

The invention relates to the technical field of extraction of active ingredients of fermented plants, in particular to an extraction method of active ingredients of microbial fermented plants for resisting wrinkles, which comprises the following steps: step S1, preparing materials; step S2, primary fermentation extraction; step S4, secondary fermentation extraction; step S4, cell lysis; and step S5, filtering. According to the method, a mixed fermentation mode of lactobacillus plantarum and saccharomyces cerevisiae is used for plants to be extracted, in the fermentation process, foam and bubbles generated by fermentation are monitored and analyzed in combination with an image recognition technology to represent the fermentation process and the fermentation rate, the stirring rotating speed is adaptively adjusted according to the foam output, or the foam is discharged in time, so that the extraction efficiency is improved. The needed fermentation extraction effect and the minimum foam output are achieved, whether the fermentation extraction process tends to be stable or not is represented through the bubble generation frequency, fermentation extraction nodes are automatically and accurately determined, the stability of target active ingredients is guaranteed, the extraction rate is increased, and the active ingredients of fermented and extracted plants are prevented from being damaged.

Description

Technical Field

[0001] The invention relates to the technical field of extracting active ingredients of fermented plants, and in particular to a method for extracting active ingredients of microbial fermented plants for anti-wrinkle. Background Art

[0002] Microbial fermentation refers to the process of using microorganisms to convert raw materials into products needed by humans through specific metabolic pathways under suitable conditions. Microorganisms contain rich enzyme systems, which can effectively destroy the cell walls of plants during the fermentation process and release the bound active ingredients and nutrients. At the same time, due to the action of the glycosyltransferase system, some active ingredients that are poorly soluble in water are combined with sugars to form glycosides, thereby increasing the water solubility of the active ingredients. Therefore, microbial fermentation is a process for extracting plant active ingredients and performing biotransformation. It has the characteristics of high active ingredient extraction rate, avoiding active ingredient destruction and diverse effects. Anthocyanins are a class of flavonoid compounds that are widely present in nature. They have antioxidant, anti-inflammatory, free radical scavenging, DPPH scavenging, anti-aging, and immune regulation functions. Therefore, anthocyanins have attracted much attention in the medical, beauty, food and other industries.

[0003] Chinese patent publication number: CN105061530B, discloses a process for extracting anthocyanins from bayberry, the technical point of which is to crush the bayberry and add acidified ethanol for reflux extraction; it can be seen that the existing anthocyanins are mostly extracted using organic solvents, the extraction rate is not high, and the introduction of organic solvents into the system is difficult to ensure health and safety. Therefore, a microbial fermentation extraction method is urgently needed, which uses lactic acid bacteria and yeast to ferment the whole plant to avoid the destruction of active ingredients, improve the extraction rate and efficiency of active ingredients, thereby preparing plant fermentation products with anti-wrinkle effects and applied to the cosmetics industry. Summary of the invention

[0004] To this end, the present invention provides a method for extracting active ingredients of plants from microbial fermentation for anti-wrinkle, so as to overcome the lack of real-time monitoring of bubbles generated during the fermentation process in the prior art, and automatically control the operating state of the fermentation tank according to the characteristics of the bubbles, so that the extraction rate of plant fermentation products is low, which leads to the problem of poor quality reproducibility of active substances extracted from different batches.

[0005] To achieve the above object, the present invention provides a method for extracting active ingredients of anti-wrinkle microbial fermented plants, comprising: Step S1, batching, crushing and sieving the raw materials, and mixing them with glucose and water to form a solution to be fermented; Step S2, primary fermentation extraction, after disinfecting the automatic fermentation tank, introducing carbon dioxide into the automatic fermentation tank to replace the air, injecting the solution to be fermented and the Lactobacillus plantarum seed solution into the automatic fermentation tank for anaerobic culture, obtaining foam characteristics according to the foam image, determining whether to adjust the operating state of the automatic fermentation tank according to the foam characteristics, obtaining bubble characteristics according to the bubble image, and determining a primary fermentation extraction node according to the bubble characteristics to obtain a fermentation liquid; The foam characteristics include real-time rising rate and real-time foam grayscale; The bubble characteristics include real-time floating rate; Step S3, secondary fermentation extraction, adding brewer's yeast seed liquid into the automatic fermentation tank, introducing oxygen into the automatic fermentation tank through a blower, aerobically culturing the fermentation liquid to generate a liquid to be lysed; Step S4, lysing, after adjusting the pH of the cell solution to be lysed, adding lysozyme to the automatic fermentation tank, so that the cell solution to be lysed and the lysozyme undergo a cell lysis reaction to obtain a fermentation product; Step S5, filtering, injecting the fermentation product into the filter layer for filtering, centrifuging the filtrate and taking the supernatant to obtain the target fermentation product.

[0006] Furthermore, in step S2, when the solution to be fermented is subjected to anaerobically culture, a foam image above the solution to be fermented is obtained at a preset acquisition interval by a camera device, and the foam in the foam image is determined. If there is foam in the foam image, the real-time rising rate will be judged according to the standard rising rate to determine whether to adjust the initial stirring speed of the automatic fermentation tank; If there is no foam in the foam image, the bubble image on the surface of the solution to be fermented is obtained by a camera device, and the real-time floating rate is determined according to the standard floating rate to analyze the progress of a fermentation extraction.

[0007] Furthermore, in step S2, a standard rising rate is set, and when it is determined that there is foam in the foam image, the foam images corresponding to the two end points of the preset acquisition interval are obtained, and the total area of ​​the foam is obtained according to the foam image at the beginning of the acquisition interval to obtain the first foam area, and the total area of ​​the foam is obtained according to the foam image at the end of the acquisition interval to obtain the second foam area, and the difference between the second foam area and the first foam area is divided by the preset acquisition interval to obtain the real-time rising rate of the foam generated during the fermentation process, and the real-time rising rate is determined according to the standard rising rate. If the real-time rising rate is less than or equal to the standard rising rate, the real-time foam grayscale is determined according to the standard foam grayscale to select an adjustment method for the operating state of the automatic fermentation tank; If the real-time rising rate is greater than the standard rising rate, the real-time rising rate is compared with the maximum rising rate to select a method for eliminating the foam generated in the automatic fermentation tank.

[0008] Furthermore, in step S2, a maximum ascent rate is set, and when it is determined that the real-time ascent rate is greater than the standard ascent rate, the real-time ascent rate is compared with the maximum ascent rate. If the real-time rising rate is less than the maximum rising rate, adjust the initial stirring speed to Nc'=Nc×[1-(Vmax-Vs) / Vs]; If the real-time rising rate is greater than or equal to the maximum rising rate, a defoaming agent is injected into the automatic fermentation tank through the first delivery pipeline.

[0009] Furthermore, in step S2, a standard foam grayscale is set, and when it is determined that the real-time rising rate is less than or equal to the standard rising rate, the real-time foam color of the foam generated during the fermentation process is obtained, and the real-time foam grayscale is determined according to the standard foam grayscale. If the real-time foam grayscale is less than or equal to the standard foam grayscale, the real-time valve opening is calculated according to the real-time rising rate, and the foam is discharged to the second conveying pipeline with the exhaust valve opening.

[0010] Furthermore, in step S2, a standard foam grayscale is set. When it is determined that the real-time rising rate is less than or equal to the standard rising rate, the real-time foam color of the foam generated during the fermentation process is obtained, and the real-time foam grayscale is determined according to the standard foam grayscale. If the real-time foam grayscale is greater than the standard foam grayscale, the preset fermentation temperature is adjusted to Tc'=Tc×[1-(Vb-Vs) / Vs].

[0011] Furthermore, in step S2, when it is determined that there is no foam in the foam image, a bubble image on the surface of the solution to be fermented is obtained by a camera device, and bubbles in the bubble image are determined. If bubbles exist in the bubble image, the real-time floating rate will be determined according to the standard floating rate to analyze the fermentation and extraction process; If there are no bubbles in the bubble image, the pH value of the solution to be fermented is detected.

[0012] Furthermore, in step S2, a standard floating rate is set, and when it is determined that there are bubbles in the bubble image, the bubble images corresponding to the two end points of the preset acquisition interval are obtained, and the positions of each bubble and the corresponding area are obtained according to the bubble image at the beginning of the acquisition interval, and the sum of each area is calculated to obtain the initial bubble area, and the positions of newly added bubbles and the corresponding areas in the bubble image collected at the end of the acquisition interval are obtained, and the areas of each newly added bubble are added to obtain the real-time floating area of ​​the bubble, and the real-time floating area is divided by the initial bubble area to obtain the real-time floating rate of the bubbles generated during the fermentation process, and the real-time floating rate is determined according to the standard floating rate. If the real-time floating rate is less than or equal to the standard floating rate, the standard determination times are compared with the real-time determination times to determine whether a fermentation extraction is completed; If the real-time floating rate is greater than the standard floating rate, it is determined that one fermentation extraction is not completed.

[0013] Furthermore, in step S2, a standard determination number is set. When it is determined that the real-time floating rate is less than or equal to the standard floating rate, the real-time determination number that the real-time floating rate is less than or equal to the standard floating rate is obtained, and the standard determination number is compared with the real-time determination number. If the real-time determination number is less than the standard determination number, the fermentation extraction is continued.

[0014] Furthermore, in step S2, a standard judgment number is set. When it is determined that the real-time floating rate is less than or equal to the standard floating rate, the real-time judgment number that the real-time floating rate is less than or equal to the standard floating rate is obtained, and the standard judgment number is compared with the real-time judgment number. If the real-time judgment number is greater than or equal to the standard judgment number, it is determined that one fermentation extraction is completed, and a secondary fermentation extraction is performed.

[0015] Compared with the prior art, the beneficial effect of the present invention lies in that, by using a mixed fermentation method of plant lactobacillus and brewer's yeast for the plants to be extracted, during the fermentation process, the foam and bubbles generated by the fermentation are monitored and analyzed in conjunction with image recognition technology to characterize the fermentation progress and fermentation rate, and the stirring speed is adaptively adjusted according to the foam output, or the foam is discharged in time to achieve the desired fermentation extraction effect and minimize the foam output, thereby improving the fermentation efficiency and extraction rate, and by monitoring the amount of bubble generation to characterize the amount of dissolved carbon dioxide, and if the microorganisms consume more non-target components in the plant, the fermentation efficiency is also correspondingly higher, and by characterizing whether the fermentation and extraction process tends to be stable by the frequency of bubble generation, it is possible to automatically and accurately determine a fermentation and extraction node, thereby improving the intelligence of the fermentation and extraction, ensuring the stability of the target active ingredients and improving the extraction rate, and avoiding the generation of dead bacterial cells or the destruction of plant active ingredients due to too long fermentation and extraction time.

[0016] Furthermore, by monitoring whether foam is generated above the liquid surface during the fermentation process, the amount of gas generated inside the automatic fermentation tank is analyzed. If it is determined that there is foam in the foam image, it means that the dissolved gas in the fermented solution has reached a saturated state. In order to avoid excessive bubble overflow, the real-time rising rate will be determined to accurately determine whether to adjust the operating state of the automatic fermentation tank according to the amount of foam generated, so that the fermentation extraction can be maintained in a stable state.

[0017] In particular, the state inside the fermentation tank is analyzed by calculating the rising rate of the foam. If the real-time rising rate is determined to be less than or equal to the standard rising rate, it means that the rising rate of the foam is slow. Then, the color of the foam is first determined to optimize the temperature conditions of the fermentation extraction. If the real-time rising rate is determined to be greater than the standard rising rate, it means that the foam is generated quickly and the foam needs to be eliminated in time to minimize the foam output.

[0018] Furthermore, by reducing the stirring speed, the generation of foam is reduced. When it is determined that the real-time rising rate is greater than or equal to the maximum rising rate, it means that the amount of foam generated is fast and large. In this case, a defoaming agent is added to the fermentation tank to prevent the foam from overflowing too quickly.

[0019] In particular, by judging the color of the foam, it is determined whether the temperature conditions in the fermentation and extraction process are optimized. If the real-time foam grayscale is determined to be less than or equal to the standard foam grayscale, it indicates that the fermentation and extraction process is normal. Then, by calculating the actual opening of the exhaust valve, the foam is discharged in time. If the real-time foam grayscale is determined to be greater than the standard foam grayscale, it indicates that the high temperature causes the fermentation liquid to change color. Then, the temperature conditions need to be optimized so that the environment inside the fermentation tank is always easy for microorganisms to destroy the plant cell walls, thereby promoting the release of active ingredients that are poorly soluble in water, namely flavonoid compounds, so that they can combine with sugars to form glycosides based on the action of the glycosyltransferase system to improve the water solubility of the active substances and thus improve the extraction rate.

[0020] Furthermore, by analyzing the fermentation situation based on the amount of bubbles generated during the fermentation process, when it is determined that the real-time flotation rate is less than or equal to the standard flotation rate, it indicates that the fermentation efficiency is tending to be stable, at this time, the fermentation process is tending to end; when it is determined that the real-time flotation rate is greater than the standard flotation rate, it indicates that the output of carbon dioxide is large, which causes a large amount of bubbles to be generated, and at this time, the fermentation efficiency is high.

[0021] Furthermore, by analyzing the frequency of the small amount of bubble generation, when the bubble generation does not continuously increase, the small amount of bubble generation is a continuous process, and the frequency is high, it is determined that one fermentation extraction is completed. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow chart of a method for extracting anti-wrinkle active ingredients from microbial fermented plants according to an embodiment of the present invention; Figure 2 This is a schematic diagram of the structure of an automatic fermentation tank according to an embodiment of the present invention; Figure 3 This is a logic determination diagram for regulating the operating state of an automatic fermentation tank according to a foam image in an embodiment of the present invention; Figure 4 It is a logic decision diagram for a fermentation extraction node in an embodiment of the present invention; Figure 5 This is a schematic diagram of the effects of the negative control, positive control and different concentrations of Morchella fermentation broth on the concentration of Collagen I in Example 1 of the present invention. DETAILED DESCRIPTION

[0023] In order to make the objects and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with 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.

[0024] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the protection scope of the present invention.

[0025] It should be noted that, in the description of the present invention, terms such as "up", "down", "left", "right", "inside" and "outside" indicating directions or positional relationships are based on the directions or positional relationships shown in the drawings. This is merely for the convenience of description and does not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation. Therefore, it cannot be understood as a limitation on the present invention.

[0026] In addition, it should be noted that in the description of the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium, or it can be the internal communication of two components. For those skilled in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0027] See also Figure 1 As shown, it is a flow chart of a method for extracting active ingredients of anti-wrinkle microbial fermented plants according to an embodiment of the present invention. The present invention provides a method for extracting active ingredients of anti-wrinkle microbial fermented plants, comprising: Step S1, batching, crushing and sieving the raw materials, and mixing them with glucose and water to form a solution to be fermented; Step S2, primary fermentation extraction, after disinfecting the automatic fermentation tank, introducing carbon dioxide into the automatic fermentation tank to replace the air, injecting the solution to be fermented and the Lactobacillus plantarum seed solution into the automatic fermentation tank for anaerobic culture, obtaining foam characteristics according to the foam image, determining whether to adjust the operating state of the automatic fermentation tank according to the foam characteristics, obtaining bubble characteristics according to the bubble image, and determining a primary fermentation extraction node according to the bubble characteristics to obtain a fermentation liquid; The foam characteristics include real-time rising rate and real-time foam grayscale; The bubble characteristics include real-time floating rate and real-time determination times; Step S3, secondary fermentation extraction, adding brewer's yeast seed liquid into the automatic fermentation tank, introducing oxygen into the automatic fermentation tank through a blower, aerobically culturing the fermentation liquid to generate a liquid to be lysed; Step S4, lysing, after adjusting the pH of the lysate to be lysed, adding lysozyme to the automatic fermentation tank, so that the lysate to be lysed and the lysozyme undergo a lysing reaction to obtain a fermentation product; Step S5, filtering, injecting the fermentation product into the filter layer for filtering, centrifuging the filtrate and taking the supernatant to obtain the target fermentation product.

[0028] See also Figure 2 As shown, it is a structural schematic diagram of an automatic fermentation tank 6 according to an embodiment of the present invention. The present invention also provides an automatic fermentation tank 6, including a camera device 1, a blower 2, an exhaust valve 3, a first conveying pipeline 4 and a second conveying pipeline 5.

[0029] In this embodiment, deep fermentation is adopted. Deep fermentation refers to the process in which microorganisms grow and reproduce in a free-flowing liquid. The growth of microorganisms produces a variety of hydrolases. These enzymes catalyze biological reactions during the fermentation process to decompose plants. Microorganisms can consume non-target components in plants during the growth and reproduction process, thereby increasing the concentration of target active ingredients and concentrating the target components in plants. By using a mixed fermentation method of plant lactobacillus and brewer's yeast for the plants to be extracted, during the fermentation process, the foam and bubbles generated by the fermentation are monitored and analyzed by combining image recognition technology to characterize the fermentation process and fermentation rate. The stirring speed is adaptively adjusted according to the foam output, or it is discharged in time to achieve the desired fermentation extraction effect and minimize the foam output, thereby improving the fermentation efficiency and extraction rate. The amount of bubbles generated is monitored to characterize the amount of carbon dioxide dissolved. If the microorganism consumes more non-target components in the plant, The fermentation efficiency is also correspondingly high. By using the frequency of bubble generation to characterize whether the fermentation and extraction process tends to be stable, the primary fermentation and extraction node can be automatically and accurately determined, the intelligence of the fermentation and extraction can be improved, the stability of the target active ingredients can be guaranteed, and the extraction rate can be improved. The fermentation and extraction time can be too long, which can easily produce dead bacterial cells or destroy the plant active ingredients. The secondary fermentation and extraction is an aerobic culture, which requires sufficient oxygen. The blower 2 and stirring are used to increase the dissolved oxygen. Oxygen is introduced into the automatic fermentation tank 6 through the blower 2 to increase the gas-liquid contact area, thereby promoting the dissolution of oxygen. The raw materials can be plants containing flavonoids such as honeysuckle, rhodiola rosea, and raspberry, or fungi such as morels. The active ingredients in the raw materials are extracted through microbial fermentation, that is, flavonoids and polyphenols are extracted as target fermentation products. The target fermentation products are used in the cosmetics industry as raw materials for skin care products because of their anti-wrinkle effects.

[0030] See also Figure 3 As shown, it is a logic determination diagram for regulating the operating state of the automatic fermentation tank according to the foam image according to an embodiment of the present invention; Specifically, in step S2, when the fermentation solution is subjected to anaerobically culture, the foam image above the fermentation solution is acquired at a preset acquisition interval by the camera device 1, and the foam in the foam image is determined. If there is foam in the foam image, the real-time rising rate will be judged according to the standard rising rate to determine whether to adjust the initial stirring speed of the automatic fermentation tank 6; If there is no foam in the foam image, the bubble image on the surface of the solution to be fermented is obtained by the camera device 1, and the real-time floating rate is determined according to the standard floating rate to analyze the fermentation and extraction process.

[0031] In this embodiment, a threshold segmentation method is used to extract foam features from a foam image, that is, a watershed algorithm is used to segment the foam edge, which compares the maximum grayscale value to a peak and the minimum grayscale value to a valley, and realizes areas separated by peaks according to different grayscale values, so as to process superimposed foam. At the same time, in order to avoid over-segmentation by segmenting the noise into small basins when the image has more noise and texture, limited contrast adaptive histogram equalization is used before segmentation to perform preprocessing operations on the enhanced image, so as to improve the contrast of the image, avoid amplification of image noise, and improve the accuracy of bubble recognition and processing; the preset acquisition interval is set to be between 3 minutes and 10 minutes, and is selected and adjusted according to the actual total fermentation time and the needs of the fermentation process; the preset acquisition interval is a time interval, the initial moment of the acquisition interval represents the start moment of the preset acquisition interval, and the final moment of the acquisition interval represents the end moment of the preset acquisition interval.

[0032] By monitoring whether foam is generated above the liquid surface during the fermentation process, the amount of gas generated inside the automatic fermentation tank 6 is analyzed. If it is determined that there is foam in the foam image, it means that the dissolved gas in the fermented solution has reached a saturated state. In order to avoid excessive bubble overflow, the real-time rising rate will be determined to accurately determine whether to adjust the operating state of the automatic fermentation tank 6 according to the amount of foam generated, so that the fermentation extraction is maintained in a stable state.

[0033] Specifically, in step S2, a standard rising rate is set. When it is determined that there is foam in the foam image, the foam images corresponding to the two end points of the preset acquisition interval are obtained, and the total area of ​​the foam is obtained according to the foam image at the beginning of the acquisition interval to obtain the first foam area. The total area of ​​the foam is obtained according to the foam image at the end of the acquisition interval to obtain the second foam area. The difference between the second foam area and the first foam area is divided by the preset acquisition interval to obtain the real-time rising rate of the foam generated during the fermentation process. The real-time rising rate is determined according to the standard rising rate. If the real-time rising rate is less than or equal to the standard rising rate, the real-time foam grayscale will be determined according to the standard foam grayscale to select the adjustment method for the operating state of the automatic fermentation tank 6; If the real-time rising rate is greater than the standard rising rate, the real-time rising rate is compared with the maximum rising rate to select a method for eliminating the foam generated in the automatic fermentation tank 6 .

[0034] The standard rising rate set in this embodiment is related to the diameter of the automatic fermentation tank 6, the viscosity of the solution to be fermented and the number of bacterial species. Generally, it is set at 1 cm per second and adjusted according to the actual fermentation conditions. The foam image is an image taken in the direction of the side wall above the liquid surface of the fermentation tank, and the rising rate represents the rate of change of the height of the generated foam.

[0035] By calculating the rising rate of the foam, the internal state of the fermentation tank is analyzed. If the real-time rising rate is determined to be less than or equal to the standard rising rate, it means that the rising rate of the foam is slow. Then, the color of the foam is first determined to optimize the temperature conditions of the fermentation extraction. If the real-time rising rate is determined to be greater than the standard rising rate, it means that the foam is generated quickly and the foam needs to be eliminated in time to minimize the foam output.

[0036] Specifically, in step S2, a maximum ascent rate is set. When it is determined that the real-time ascent rate is greater than the standard ascent rate, the real-time ascent rate is compared with the maximum ascent rate. If the real-time rising rate is less than the maximum rising rate, adjust the initial stirring speed to Nc'=Nc×[1-(Vmax-Vs) / Vs]; If the real-time rising rate is greater than or equal to the maximum rising rate, a defoaming agent is injected into the automatic fermentation tank 6 through the first delivery pipeline 4 .

[0037] Wherein, Nc' represents the adjusted initial stirring speed, Nc represents the set initial stirring speed when the fermenter is running, Vs represents the calculated real-time rising rate, and Vmax represents the set maximum rising rate; When the foam reaches a certain liquid level, the automatic fermentation tank 6 opens the discharge valve to discharge the foam; By reducing the stirring speed, the generation of foam can be reduced. When it is determined that the real-time rising rate is greater than or equal to the maximum rising rate, it means that the amount of foam generated is fast and large. In this case, defoaming agent is added to the fermentation tank to prevent the foam from overflowing too quickly.

[0038] Specifically, in step S2, a standard foam grayscale is set. When it is determined that the real-time rising rate is less than or equal to the standard rising rate, the real-time foam color of the foam generated during the fermentation process is obtained, and the real-time foam grayscale is determined according to the standard foam grayscale. If the real-time foam grayscale is less than or equal to the standard foam grayscale, the real-time valve opening is calculated according to the real-time rising rate, and the foam is discharged to the second delivery pipeline 5 according to the opening of the exhaust valve 3; If the real-time foam grayscale is greater than the standard foam grayscale, the preset fermentation temperature is adjusted to Tc'=Tc×[1-(Vb-Vs) / Vs]; Wherein, Ks=Kmax×[1-(Vb-Vs) / Vb], Tc' represents the adjusted preset fermentation temperature, Tc represents the set preset fermentation temperature, Vb represents the set standard rising rate, Vs represents the calculated real-time rising rate, Kmax represents the maximum opening of the exhaust valve 3, and Ks represents the calculated real-time valve opening of the exhaust valve 3; the preset fermentation temperature is set to 24°C, and the minimum value of the preset fermentation temperature is 23°C; The standard foam grayscale set in this embodiment represents the average grayscale value of the foam pixels in the foam image. Generally, the standard foam grayscale is the average grayscale value of the pixels of the image obtained by taking a picture of the fermentation solution. The maximum valve opening is 80%, and the maximum valve opening meets the demand for gas discharge during the fermentation process, while ensuring that no external pollutants are introduced and maintaining a suitable pressure in the tank.

[0039] By judging the color of the foam, it is determined whether the temperature conditions in the fermentation and extraction process are optimized. If it is determined that the real-time foam grayscale is less than or equal to the standard foam grayscale, it means that the fermentation and extraction process is normal. Then, by calculating the actual opening of the exhaust valve 3, the foam is discharged in time. If it is determined that the real-time foam grayscale is greater than the standard foam grayscale, it means that the high temperature causes the fermentation liquid to change color. Then, it is necessary to optimize and adjust the temperature conditions in time so that the environment inside the fermentation tank is always easy for microorganisms to destroy the plant cell walls, thereby promoting the release of active ingredients that are difficult to dissolve in water, namely flavonoid compounds, so that they can be combined with sugars to form glycosides based on the action of the glycosyltransferase system to improve the water solubility of the active substances and thus improve the extraction rate.

[0040] See also Figure 4 As shown, it is a logic decision diagram for a primary fermentation extraction node in an embodiment of the present invention; Specifically, in step S2, when it is determined that there is no foam in the foam image, the bubble image on the surface of the solution to be fermented is obtained by the camera device 1, and the bubbles in the bubble image are determined. If bubbles exist in the bubble image, the real-time floating rate will be determined according to the standard floating rate to analyze the fermentation and extraction process; If there are no bubbles in the bubble image, detecting the pH value of the solution to be fermented; In this embodiment, if it is determined that there are no bubbles in the bubble image, the pH value of the fermented solution is detected to see if it is an acidic condition, because anthocyanins or other active ingredients can only remain stable under acidic conditions, and the pH value is prevented from affecting the fermentation extraction efficiency. The optimal pH value is 5.8.

[0041] Specifically, in step S2, a standard floating rate is set. When it is determined that there are bubbles in the bubble image, the bubble images corresponding to the two end points of the preset acquisition interval are obtained, and the positions of each bubble and the corresponding areas are obtained according to the bubble image at the beginning of the acquisition interval. The sum of each area is calculated to obtain the initial bubble area, and the positions of newly added bubbles and the corresponding areas in the bubble image collected at the end of the acquisition interval are obtained. The areas of each newly added bubble are added to obtain the real-time floating area of ​​the bubble, and the real-time floating area is divided by the initial bubble area to obtain the real-time floating rate of the bubbles generated during the fermentation process. The real-time floating rate is determined according to the standard floating rate. If the real-time floating rate is less than or equal to the standard floating rate, the standard determination times are compared with the real-time determination times to determine whether a fermentation extraction is completed; If the real-time floating rate is greater than the standard floating rate, it is determined that one fermentation extraction is not completed, and another fermentation extraction is continued.

[0042] The standard floating rate set in this embodiment indicates the floating rate of bubbles in the fermentation liquid, which represents the output of carbon dioxide. The set value is related to the design of the fermentation tank, the fermentation conditions and the viscosity of the fermentation liquid. Generally, the standard floating rate is set at 30 mm per second and is selected according to the actual volume of the fermentation liquid. By analyzing the fermentation situation based on the amount of bubbles generated during the fermentation process, when the real-time floating rate is determined to be less than or equal to the standard floating rate, it means that the fermentation efficiency is slow and tends to be stable. At this time, the fermentation process tends to end. When the real-time floating rate is determined to be greater than the standard floating rate, it means that the output of carbon dioxide is large, which makes the amount of bubbles generated larger. At this time, the fermentation efficiency is high.

[0043] Specifically, in step S2, a standard determination number is set. When it is determined that the real-time floating rate is less than or equal to the standard floating rate, the real-time determination number of the real-time floating rate is obtained, and the standard determination number is compared with the real-time determination number. If the real-time determination times are less than the standard determination times, continue to perform fermentation extraction once; If the real-time determination times are greater than or equal to the standard determination times, it is determined that the first fermentation extraction is completed, and the second fermentation extraction is performed.

[0044] The standard number of determinations set in this embodiment is 3 to 5 times; By analyzing the frequency of the small amount of bubble generation, when the bubble generation does not continuously increase, the small amount of bubble generation is a continuous process, and the frequency is high, it is determined that one fermentation extraction is completed.

[0045] Specifically, the extraction method of this embodiment is used, and the target fermentation product obtained under the culture conditions of temperature 24° C. and pH value 5.8 includes Morchella fermentation liquid. The Morchella fermentation liquid prepared by the present invention is used as a test sample to carry out an anti-wrinkle experiment. Fibroblasts are used as a test system. The content change of the main type of Collagen I is used to test whether the test sample has anti-wrinkle and firming effects. The experimental method, experimental data, and experimental results are as follows: According to 8×10 per well 4 The cells were seeded into 24-well plates at a density of 10 cells / well and placed in an incubator at 37°C and 5% CO. 2Incubate overnight under the conditions of ; When the spreading rate of cells in the 24-well plate reached 40% to 60%, start adding corresponding treatment solutions to different groups, among which, Experimental group: 1 mL of culture medium containing the test substance at corresponding concentration was added to each well. The test substances were divided into three groups, with corresponding concentrations of 1.25 (%, V / V), 2.5 (%, V / V) and 5.0 (%, V / V); Blank control group and negative control group: 1 mL of cell culture medium was added to each well; Positive control group: 1 mL of culture medium containing 100 μg / mL vitamin C and 7 μg / mL vitamin E was added to each well; After the administration, the cells in the negative control group, positive control group and sample group were exposed to UVA radiation with a total dose of 9 J / cm² to simulate the UV damage that the cells may suffer. The blank control group was not exposed to UVA radiation to serve as a control without radiation effects. After radiation, all cells continued to be incubated in the incubator for 48 hours. After the incubation, the cell culture supernatant was collected. The liquid contained proteins and other substances secreted by the cells. After collection, it was frozen in a refrigerator at -80°C. The content of Collagen I (collagen type I) in the collected supernatant was detected and analyzed using an enzyme-linked immunosorbent assay (ELISA) kit according to its operating instructions.

[0046] Collagen Ⅰ is an important extracellular matrix protein, and changes in its content can reflect the synthetic function of cells and the potential effects of samples.

[0047] Experimental results refer to Figure 5 As shown, Figure 5 This is a schematic diagram of the effects of negative control, positive control and different concentrations of Morchella fermentation broth on Collagen I concentration in Example 1 of the present invention; Compared with the BC group, the NC group fibroblasts received a total dose of 9 J / cm 2 After UVA irradiation, the content level of CollagenⅠ decreased significantly (p<0.01), indicating that the irradiation stimulation was effective. Compared with the NC group, vitamin C and vitamin E in the PC group could significantly increase the content level of CollagenⅠ (p<0.01), indicating that the positive control test was effective. Compared with the NC group, the fermented morel liquid at 2.50% and 5.0% (V / V) concentrations showed a significant effect on the content of CollagenⅠ in UVA-induced fibroblasts (p<0.01), indicating that the fermented morel liquid at this concentration has anti-wrinkle and firming effects on cells.

[0048] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for extracting active ingredients from microbial fermented plants for anti-wrinkle, characterized in that: include: Step S1, batching, crushing and sieving the raw materials, and mixing them with glucose and water to form a solution to be fermented; Step S2, primary fermentation extraction, after disinfecting the automatic fermentation tank, introducing carbon dioxide into the automatic fermentation tank to replace the air, injecting the solution to be fermented and the Lactobacillus plantarum seed solution into the automatic fermentation tank for anaerobic culture, obtaining foam characteristics according to the foam image, determining whether to adjust the operating state of the automatic fermentation tank according to the foam characteristics, obtaining bubble characteristics according to the bubble image, and determining a primary fermentation extraction node according to the bubble characteristics to obtain a fermentation liquid; The foam characteristics include real-time rising rate and real-time foam grayscale; The bubble characteristics include real-time floating rate; Step S3, secondary fermentation extraction, adding brewer's yeast seed liquid into the automatic fermentation tank, introducing oxygen into the automatic fermentation tank through a blower, aerobically culturing the fermentation liquid to generate a liquid to be lysed; Step S4, lysing, after adjusting the pH of the lysate to be lysed, adding lysozyme to the automatic fermentation tank, so that the lysate to be lysed and the lysozyme undergo a lysing reaction to obtain a fermentation product; Step S5, filtering, injecting the fermentation product into the filter layer for filtering, centrifuging the filtrate and taking the supernatant to obtain the target fermentation product.

2. The method for extracting active ingredients from microbial fermented plants for anti-wrinkle according to claim 1, characterized in that: In step S2, when the fermentation solution is subjected to anaerobically culture, a foam image above the fermentation solution is acquired at a preset acquisition interval by a camera device, and the foam in the foam image is determined. If there is foam in the foam image, the real-time rising rate is determined according to the standard rising rate to determine whether to adjust the initial stirring speed of the automatic fermentation tank; If there is no foam in the foam image, the bubble image on the surface of the solution to be fermented is obtained by a camera device, and the real-time floating rate is determined according to the standard floating rate to analyze the progress of a fermentation extraction.

3. The method for extracting active ingredients from microbial fermented plants for anti-wrinkle according to claim 2, characterized in that: In step S2, a standard rising rate is set. When it is determined that there is foam in the foam image, the foam images corresponding to the two end points of the preset acquisition interval are obtained, and the total area of ​​the foam is obtained according to the foam image at the beginning of the acquisition interval to obtain the first foam area. The total area of ​​the foam is obtained according to the foam image at the end of the acquisition interval to obtain the second foam area. The difference between the second foam area and the first foam area is divided by the preset acquisition interval to obtain the real-time rising rate of the foam generated during the fermentation process. The real-time rising rate is determined according to the standard rising rate. If the real-time rising rate is less than or equal to the standard rising rate, the real-time foam grayscale will be judged according to the standard foam grayscale to select the adjustment method for the operating state of the automatic fermentation tank; If the real-time rising rate is greater than the standard rising rate, the real-time rising rate is compared with the maximum rising rate to select a method for eliminating the foam generated in the automatic fermentation tank.

4. The method for extracting active ingredients from microbial fermented plants for anti-wrinkle according to claim 3, characterized in that: In step S2, a maximum ascent rate is set. When it is determined that the real-time ascent rate is greater than the standard ascent rate, the real-time ascent rate is compared with the maximum ascent rate. If the real-time rising rate is less than the maximum rising rate, the initial stirring speed is adjusted to Nc'=Nc×[1-(Vmax-Vs) / Vs]; If the real-time rising rate is greater than or equal to the maximum rising rate, a defoaming agent is injected into the automatic fermentation tank through the first delivery pipeline.

5. The method for extracting active ingredients from microbial fermented plants for anti-wrinkle according to claim 4, characterized in that: In step S2, a standard foam grayscale is set. When it is determined that the real-time rising rate is less than or equal to the standard rising rate, the real-time foam color of the foam generated during the fermentation process is obtained, and the real-time foam grayscale is determined according to the standard foam grayscale. If the real-time foam grayscale is less than or equal to the standard foam grayscale, the real-time valve opening is calculated according to the real-time rising rate, and the foam is discharged to the second delivery pipeline at the exhaust valve opening.

6. The method for extracting active ingredients from microbial fermented plants for anti-wrinkle according to claim 4, characterized in that: In step S2, a standard foam grayscale is set. When it is determined that the real-time rising rate is less than or equal to the standard rising rate, the real-time foam color of the foam generated during the fermentation process is obtained, and the real-time foam grayscale is determined according to the standard foam grayscale. If the real-time foam grayscale is greater than the standard foam grayscale, the preset fermentation temperature is adjusted to Tc'=Tc×[1-(Vb-Vs) / Vs].

7. The method for extracting active ingredients from microbial fermented plants for anti-wrinkle according to claim 2, characterized in that: In step S2, when it is determined that there is no foam in the foam image, a bubble image on the surface of the solution to be fermented is obtained by a camera device, and bubbles in the bubble image are determined. If bubbles exist in the bubble image, the real-time floating rate will be determined according to the standard floating rate to analyze the fermentation and extraction process; If there are no bubbles in the bubble image, the pH value of the solution to be fermented is detected.

8. The method for extracting active ingredients from microbial fermented plants for anti-wrinkle according to claim 7, characterized in that: In step S2, a standard floating rate is set. When it is determined that there are bubbles in the bubble image, the bubble images corresponding to the two end points of the preset acquisition interval are obtained, the positions of each bubble and the corresponding area are obtained according to the bubble image at the beginning of the acquisition interval, the sum of each area is calculated to obtain the initial bubble area, the position of the newly added bubble in the bubble image collected at the end of the acquisition interval and the corresponding area are obtained, the areas of each newly added bubble are added together to obtain the real-time floating area of ​​the bubble, the real-time floating area is divided by the initial bubble area to obtain the real-time floating rate of the bubbles generated during the fermentation process, and the real-time floating rate is determined according to the standard floating rate. If the real-time floating rate is less than or equal to the standard floating rate, the standard determination times are compared with the real-time determination times to determine whether a fermentation extraction is completed; If the real-time floating rate is greater than the standard floating rate, it is determined that one fermentation extraction is not completed.

9. The method for extracting active ingredients from microbial fermented plants for anti-wrinkle according to claim 8, characterized in that: In step S2, a standard determination number is set. When it is determined that the real-time floating rate is less than or equal to the standard floating rate, the real-time determination number that the real-time floating rate is less than or equal to the standard floating rate is obtained, and the standard determination number is compared with the real-time determination number. If the real-time determination number is less than the standard determination number, the fermentation extraction is continued.

10. The method for extracting active ingredients from microbial fermented plants for anti-wrinkle according to claim 8, characterized in that: In step S2, a standard judgment number is set. When it is determined that the real-time floating rate is less than or equal to the standard floating rate, the real-time judgment number that the real-time floating rate is less than or equal to the standard floating rate is obtained, and the standard judgment number is compared with the real-time judgment number. If the real-time judgment number is greater than or equal to the standard judgment number, it is determined that one fermentation extraction is completed, and a secondary fermentation extraction is performed.

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