Composition, cosmetic and method for co-production of 1, 3-propylene glycol and lactic acid

Abstract

The invention provides a composition, cosmetics and a method for co-producing 1, 3-propylene glycol and lactic acid, the composition comprises 1, 3-propylene glycol and lactic acid, and the mass ratio of 1, 3-propylene glycol to lactic acid is (3-6): 1; the 1, 3-propylene glycol and the lactic acid are obtained through lactobacillus fermentation culture in the same fermentation system. The composition disclosed by the invention is strong in moisture retention, weak in skin irritation and low in stickiness, so that the quality of cosmetics containing the composition is improved, and the application is wide.

Description

Technical Field

[0001] This application relates to the field of biochemical engineering. Specifically, this application relates to compositions, cosmetics, and methods for the co-production of 1,3-propanediol and lactic acid. Background Technology

[0002] 1,3-Propanediol (1,3-PDO) and lactic acid (LA) are widely used in the cosmetics industry, commonly serving as solvents and moisturizers. Lactic acid (LA) is a fruit acid with a mild sour taste, found in human skin and hair, and is also widely used in daily cosmetics as a natural pH adjuster and moisturizer. The production methods for 1,3-propanediol and lactic acid include chemical and biological methods. Chemical methods often use non-renewable petrochemical resources as raw materials, resulting in high energy consumption, numerous and difficult-to-isolate byproducts, and immeasurable environmental pollution. Compared to chemical methods, biological methods for synthesizing 1,3-propanediol and lactic acid offer advantages such as simpler production steps, the use of renewable resources, and environmental friendliness.

[0003] Currently, how to better apply 1,3-propanediol and lactic acid synthesized by biological methods in cosmetics to improve cosmetic quality remains to be studied. Summary of the Invention

[0004] This invention aims to at least partially solve one of the technical problems existing in the prior art. To this end, this application proposes compositions, cosmetics, and methods for co-producing 1,3-propanediol and lactic acid. The compositions of this application have strong moisturizing properties, low skin irritation, and low stickiness, thereby improving the quality of cosmetics containing them and having wide applications.

[0005] In one aspect of the invention, this application provides a composition. According to an embodiment of this application, the composition comprises 1,3-propanediol and lactic acid, wherein the mass ratio of 1,3-propanediol to lactic acid is (3-6):1; the 1,3-propanediol and lactic acid are obtained by fermentation with Lactobacillus in the same fermentation system.

[0006] According to the embodiments of this application, the composition can be fermented and cultured with Lactobacillus to obtain a compound ratio of (3-6):1 by mass. This compound ratio can effectively improve the moisturizing properties of the composition and reduce its stickiness. It can be applied to cosmetics to improve their quality.

[0007] According to embodiments of this application, the above composition may also have the following additional technical features:

[0008] According to an embodiment of this application, the lactobacillus includes Lactobacillus reuteri, or the lactobacillus includes Lactobacillus mucinus reuteri, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 29, 2024, with accession number GDMCC NO: 64700;

[0009] And / or, the mass ratio of the 1,3-propanediol to lactic acid is (4-5):1.

[0010] According to an embodiment of this application, the initial culture medium for the fermentation culture contains glycerol and glucose.

[0011] According to embodiments of this application, the concentration of glycerol is 1 g / L to 50 g / L;

[0012] And / or, the concentration of the glucose is 1 g / L to 50 g / L.

[0013] According to embodiments of this application, the pH value of the composition is 4.5 to 5.0.

[0014] According to embodiments of this application, the composition further comprises: the lysate obtained from Lactobacillus fermentation culture.

[0015] In a second aspect of this application, a cosmetic product is provided. According to an embodiment of this application, the cosmetic product comprises the composition described in the first aspect of this application.

[0016] According to embodiments of this application, the content of the composition is 1% to 30% by mass, based on the total mass of the cosmetic.

[0017] In a third aspect of this application, a method for co-producing 1,3-propanediol and lactic acid is provided. According to an embodiment of this application, the method includes: fermenting and culturing Lactobacillus to obtain a fermentation broth containing 1,3-propanediol and lactic acid; the initial culture medium for the fermentation culture contains glycerol and glucose; and the mass ratio of 1,3-propanediol to lactic acid is (3-6):1.

[0018] According to embodiments of this application, the lactobacillus includes Lactobacillus reuteri;

[0019] And / or, the lactobacillus includes *Limosilactobacillus reuteri*, which was deposited on May 29, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC) with accession number GDMCC NO: 64700, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, postal code: 510070.

[0020] According to embodiments of this application, the concentration of glycerol is 1 g / L to 50 g / L;

[0021] And / or, the concentration of the glucose is 1 g / L to 50 g / L.

[0022] According to an embodiment of this application, the fermentation culture includes a fed-batch process;

[0023] The feed solution for the feed treatment includes at least one of glucose solution and reduced coenzyme solution and glycerol solution.

[0024] According to embodiments of this application, the concentration of the glycerol solution is 200 g / L to 1000 g / L, preferably 400 g / L to 600 g / L;

[0025] And / or, the concentration of the glucose solution is 100 g / L to 500 g / L, preferably 200 g / L to 400 g / L;

[0026] And / or, the concentration of the reduced coenzyme solution is 1 g / L to 10 g / L;

[0027] And / or, the reduced coenzyme includes at least one of reduced NADH coenzyme, reduced NADPH coenzyme, and reduced coenzyme Q10.

[0028] According to the embodiments of this application, the fermentation culture time is 36h to 60h, preferably 42h to 54h;

[0029] And / or, the initial feeding time of the glycerol solution is 1h to 24h, preferably 1h to 4h;

[0030] And / or, the feeding rate of the glycerol solution is 10 mL / h to 50 mL / h, preferably 15 mL / h to 30 mL / h;

[0031] And / or, the initial feeding time of the glucose solution is 2h to 24h, preferably 5h to 15h;

[0032] And / or, the feeding rate of the glucose solution is 10 mL / h to 50 mL / h, preferably 20 mL / h to 30 mL / h;

[0033] And / or, the initial feeding time of the NADH coenzyme solution is 0h to 30h, preferably 10h to 24h;

[0034] And / or, the feeding rate of the NADH coenzyme solution is 10 mL / h to 50 mL / h, preferably 25 mL / h to 40 mL / h.

[0035] According to embodiments of this application, the reduced coenzyme includes at least one of reduced NADH coenzyme, reduced NADPH coenzyme, and reduced coenzyme Q10.

[0036] According to embodiments of this application, the fermentation medium further contains at least one of the following: peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, sodium acetate, diammonium hydrogen citrate, magnesium sulfate, manganese sulfate, and Tween-80, and water.

[0037] And / or, the pH of the fermentation medium is 4 to 6.

[0038] According to embodiments of this application, the fermentation culture satisfies at least one of the following conditions:

[0039] The fermentation treatment temperature is 35℃~40℃;

[0040] The fermentation process is carried out at a speed of 100 rpm to 500 rpm.

[0041] The dissolved oxygen level during the fermentation process is 0%–20%.

[0042] The OD of the fermentation broth 600 The value ranges from 0.6 to 3.0;

[0043] The fermentation process takes 36 to 60 hours.

[0044] According to an embodiment of this application, during the fermentation process, culture medium is added every 10 to 14 hours, and the added culture medium includes the initial culture medium but does not contain glycerol.

[0045] According to an embodiment of this application, the method further includes:

[0046] The fermentation broth containing 1,3-propanediol and lactic acid obtained from the fermentation culture was centrifuged, and the supernatant was collected.

[0047] The supernatant was subjected to ultrafiltration, and the filtrate was collected.

[0048] The filtrate was subjected to distillation to obtain a composition containing 1,3-propanediol and lactic acid.

[0049] According to an embodiment of this application, the ultrafiltration membrane used in the ultrafiltration treatment has a molecular weight cutoff of no more than 1000 Da;

[0050] And / or, the temperature of the distillation process is 60°C to 90°C.

[0051] In a fourth aspect of this application, a ferment is provided. According to an embodiment of this application, the ferment is obtained by the method described in the third aspect above for the co-production of 1,3-propanediol and lactic acid.

[0052] In a fifth aspect of this application, a cosmetic product is proposed. According to an embodiment of this application, the cosmetic product comprises the fermentation product described in the fourth aspect above.

[0053] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0054] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0055] Figure 1 A graph showing the metabolism of 1,3-propanediol and lactic acid in a microorganism according to an embodiment of this application is displayed. Detailed Implementation

[0056] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0057] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0058] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this application.

[0059] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0060] In one aspect of the invention, this application provides a composition. According to an embodiment of this application, the composition comprises 1,3-propanediol and lactic acid, wherein the mass ratio of 1,3-propanediol to lactic acid is (3-6):1; the 1,3-propanediol and lactic acid are obtained by fermentation with Lactobacillus in the same fermentation system.

[0061] Currently, although studies have disclosed that both 1,3-propanediol and lactic acid have moisturizing properties, their ratio affects the moisturizing effect. When the mass ratio of 1,3-propanediol to lactic acid is (3-6):1, for example, 3:1, 3.5:1, 4:1, 4.5:1, 5:1, 5.5:1, 6:1, etc., the composition exhibits better moisturizing effects, with (4-5):1 being preferred. Furthermore, highly moisturizing cosmetics often have a sticky feel, primarily due to the following reasons: moisturizing ingredients lock in moisture by absorbing moisture from the air or forming a protective layer on the skin surface to prevent evaporation. This protective layer may increase the product's viscosity, resulting in a sticky feeling. Many moisturizing ingredients, such as glycerin, hyaluronic acid, and plant oils, have high viscosity or stickiness; while providing moisturizing effects, these ingredients may also cause the product to leave a sticky feeling on the skin. The composition of this application contains 1,3-propanediol and lactic acid in the above-mentioned proportions. While improving moisturizing properties, it reduces stickiness, thus allowing the skin to maintain a refreshing feel while providing high hydration and comfort. Furthermore, the 1,3-propanediol and lactic acid in the above proportions can be obtained through lactobacillus fermentation in the same fermentation system. Compared to chemical synthesis methods, the bio-fermentation method has advantages such as simple production steps, use of renewable resources, and environmental friendliness.

[0062] In this application, the term "1,3-propanediol and lactic acid obtained by fermentation with Lactobacillus in the same fermentation system" means that 1,3-propanediol and lactic acid coexist in the same fermentation broth. In other words, after fermentation culture, microorganisms simultaneously produce 1,3-propanediol and lactic acid, which are obtained by fermentation in the same fermentation system.

[0063] Currently, the main microorganisms used for fermenting 1,3-propanediol include Klebsiella pneumoniae, Escherichia coli, Lactobacillus, and Citrate bacteria. Among these, Klebsiella pneumoniae and Escherichia coli, as pathogenic bacteria, face numerous limitations in strain transportation and preservation, fermentation processes, separation and purification processes, and applications in cosmetics and food. Furthermore, their separation and purification processes are complex, energy-intensive, and involve high costs for waste treatment. Lactobacillus, on the other hand, is a naturally occurring probiotic found in the human gut, offering strong safety. As an anaerobic bacterium, it is less susceptible to contamination by other microorganisms during production, resulting in higher efficiency and lower costs. Moreover, Lactobacillus makes it relatively easier to obtain 1,3-propanediol and lactic acid in the specific ratios required.

[0064] According to an embodiment of this application, the lactobacillus includes *Lactobacillus reuteri*, which was deposited on May 29, 2024, at the Guangdong Provincial Center for Microbial Culture Collection with accession number GDMCC NO: 64700.

[0065] The metabolic pathway for microbial production of 1,3-propanediol and lactic acid mainly includes: First, glycerol is converted to 3-hydroxypropanal (3-HPA) by glycerol dehydratase (GDHt) within the microorganism; then, 3-hydroxypropanal is reduced to 1,3-propanediol by 1,3-propanediol oxidoreductase (PDOR) under the action of the reducing coenzyme NADH, releasing NAD. + Meanwhile, glycerol dehydrogenases in microorganisms oxidize 3-hydroxypropanal to lactic acid. The pathways of 3-hydroxypropanal reduction to 1,3-propanediol and 3-hydroxypropanal oxidation to lactic acid are in competition with each other; when the yield of 1,3-propanediol increases, the yield of lactic acid tends to decrease.

[0066] Currently, existing technologies primarily produce 1,3-propanediol through microbial fermentation. Because there is competitive inhibition between the biosynthetic pathways of 1,3-propanediol and lactic acid, researchers typically increase 1,3-propanediol yield by inhibiting lactic acid growth through genetic modification or fermentation process optimization. Few researchers have attempted to co-produce 1,3-propanediol and lactic acid using biosynthesis, and even fewer have attempted to simultaneously increase the yields of both. This represents a significant technological barrier in the field.

[0067] A strain of *Limosilactobacillus reuteri* PDO / LA-3, capable of simultaneously producing high levels of 1,3-propanediol and lactic acid, was obtained through UV-plasma combined mutagenesis. Furthermore, after multiple subcultures, it maintains good growth and metabolic capabilities, as well as high production capacity of 1,3-propanediol and lactic acid, demonstrating strong stability. It also readily yields 1,3-propanediol and lactic acid in a mass ratio of (3–6):1. In this application, the terms "Limosilactobacillus reuteri" and "Lactobacillus reuteri" are used interchangeably.

[0068] According to embodiments of this application, the initial culture medium for fermentation contains glycerol and glucose. Glycerol provides a carbon source for microbial fermentation and can be converted to 1,3-propanediol and lactic acid via 3-hydroxypropanal. NADH is produced simultaneously with the conversion of glucose to lactic acid. The metabolic pathway from glycerol to 3-hydroxypropanal to 1,3-propanediol consumes NADH, and the NADH generated from the glucose to lactic acid pathway can compensate for the NADH consumed in the glycerol to 1,3-propanediol conversion, thereby promoting the production of 1,3-propanediol. The simultaneous addition of glycerol and glucose to the fermentation medium is beneficial for the growth and metabolism of Lactobacillus, achieving high production of 1,3-propanediol and lactic acid, with a mass ratio of 1,3-propanediol to lactic acid of (3-6):1.

[0069] According to embodiments of this application, the concentration of glycerol is 1 g / L to 50 g / L. Exemplarily, the concentration of glycerol is 1 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, etc. This can increase the yield of 1,3-propanediol and lactic acid, and also allow the mass ratio of the two to be (3-6):1.

[0070] According to embodiments of this application, the concentration of glucose is 1 g / L to 50 g / L, exemplarily 1 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, etc. This can increase the yield of 1,3-propanediol and lactic acid, and also allow the mass ratio of the two to be (3-6):1.

[0071] According to embodiments of this application, the pH value of the composition is 4.5 to 5.0, for example, it can be 4.5, 4.6, 4.7, 4.8, 4.9, and 5.0. Therefore, the composition has low skin irritation and is suitable for different skin types.

[0072] According to embodiments of this application, the composition further comprises: the lysate obtained from Lactobacillus fermentation culture, also known as "Lactobacillus lysate". The substance released after the cells of Lactobacillus fermentation culture rupture is called "Lactobacillus lysate," which is rich in active ingredients such as polypeptides, nucleic acids, polysaccharides, organic acids, vitamins, and amino acids, and has moisturizing effects with low irritation. Therefore, combining Lactobacillus lysate with 1,3-propanediol and lactic acid can further improve the moisturizing properties of the composition, reduce irritation, decrease stickiness, and provide a better skin feel after use.

[0073] According to embodiments of this application, a method for obtaining the lysate includes:

[0074] Lactobacillus was fermented to obtain a fermentation broth;

[0075] The fermentation broth was centrifuged, and the precipitate was collected.

[0076] The precipitate was lysed to obtain the lysate of the lactobacillus.

[0077] Furthermore, the method for obtaining the lysate may further include:

[0078] The pyrolysis solution obtained from the pyrolysis process is subjected to ultrafiltration, and the filtrate is collected.

[0079] The filtrate was subjected to distillation to obtain the lysate of the lactobacillus.

[0080] In some embodiments, the ultrafiltration membrane used in the ultrafiltration treatment has a molecular weight cutoff of no more than 1000 Da;

[0081] And / or, the temperature of the distillation process is 60°C to 90°C.

[0082] In a second aspect of this application, the cosmetic product of this application comprises the composition described in the first aspect. The cosmetic product of this application has high moisturizing properties and low irritation, low stickiness, and is suitable for different skin types.

[0083] According to embodiments of this application, the content of the composition is 1% to 30% by mass, based on the total mass of the cosmetic. The composition of this application can effectively improve the moisturizing properties of cosmetics at low addition levels (1% to 10%) and reduce the stickiness at high addition levels (10% to 30%), resulting in a more refreshing and comfortable feel on the skin.

[0084] In a third aspect of this application, a method for the co-production of 1,3-propanediol and lactic acid is provided. According to an embodiment of this application, the method includes: fermenting and culturing *Lactobacillus* to obtain a fermentation broth containing 1,3-propanediol and lactic acid; the initial culture medium for the fermentation culture contains glycerol and glucose; and the mass ratio of 1,3-propanediol to lactic acid is (3-6):1. As previously described, fermenting and culturing *Lactobacillus* in an initial culture medium containing glycerol and glucose can yield 1,3-propanediol and lactic acid with a mass ratio of (3-6):1. It should be noted that the features and advantages described for the composition above also apply to this method for the co-production of 1,3-propanediol and lactic acid, and will not be repeated here.

[0085] According to embodiments of this application, the lactobacillus includes *Lactobacillus reuteri*. Exemplarily, the lactobacillus includes *Lactobacillus reuteri*, which was deposited on May 29, 2024, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC NO: 64700), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510070, China. *Lactobacillus reuteri* PDO / LA-3 exhibits good growth and metabolic capabilities, 1,3-propanediol and lactic acid production capacity, strong stability, and readily obtains 1,3-propanediol and lactic acid in a mass ratio of (3–6):1.

[0086] According to embodiments of this application, the fermentation medium further contains at least one of: peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, sodium acetate, diammonium hydrogen citrate, magnesium sulfate, manganese sulfate, and Tween-80, and water. This is suitable for cell growth and metabolism, increasing the yield of 1,3-propanediol and lactic acid.

[0087] According to embodiments of this application, the pH value of the fermentation medium is 4 to 6, for example, 4, 4.5, 5, 5.5, 6, etc. This is suitable for cell growth and metabolism, increasing the yield of 1,3-propanediol and lactic acid.

[0088] According to an embodiment of this application, the fermentation culture includes a fed-batch treatment, wherein the fed-batch solution includes at least one of a glucose solution and a reduced coenzyme solution and a glycerol solution.

[0089] Figure 1 The diagram illustrates the metabolism of 1,3-propanediol and lactic acid within microorganisms. Glycerol is converted to 3-hydroxypropanal (3-HPA) by glycerol dehydratase (GDHt) within the microorganisms. Then, 3-hydroxypropanal is reduced to 1,3-propanediol by 1,3-propanediol oxidoreductase (PDOR) in the presence of NADH coenzyme, releasing NAD+. + Meanwhile, glycerol dehydrogenase in microorganisms oxidizes 3-hydroxypropionaldehyde to lactic acid, and this oxidation pathway inhibits the reduction pathway that produces 1,3-propanediol to some extent.

[0090] During fermentation, the NADH-coenzyme cycle and the NADH-coenzyme fed-batch pathway can not only increase the yield of 1,3-propanediol and lactic acid, but also achieve a mass ratio of 1,3-propanediol to lactic acid of (3-6):1. Specifically, the NADH-coenzyme cycle involves the simultaneous addition of glucose and glycerol, allowing glucose to generate lactic acid. Glycerol can then be converted to 1,3-propanediol and lactic acid via 3-hydroxypropanal. Furthermore, NADH is produced during the glucose-to-lactic acid conversion process, while the glycerol-to-3-hydroxypropanal-to-1,3-propanediol metabolic pathway consumes NADH. The NADH generated from the glucose-to-lactic acid pathway can compensate for the NADH consumed in the glycerol-to-1,3-propanediol conversion, thereby promoting the production of 1,3-propanediol and achieving a mass ratio of 1,3-propanediol to lactic acid of (3-6):1.

[0091] The NADH-coenzyme-fed supplementation pathway can be implemented in two ways: one is by adding glycerol and NADH coenzyme, and the other is by adding glycerol, glucose, and NADH coenzyme. Glycerol consumption during conversion can effectively replenish NADH, thus promoting 1,3-propanediol synthesis. Simultaneously, glycerol supplementation also contributes to the production of 1,3-propanediol and lactate. Glucose addition not only promotes lactose production, but the NADH produced during its metabolism can also replenish NADH consumed during glycerol conversion, further promoting 1,3-propanediol synthesis. Furthermore, the NADH-coenzyme-fed supplementation pathway can achieve a 1,3-propanediol to lactate mass ratio of (3–6):1.

[0092] It should be noted that the feeding solution of this application may contain any two or three of the following: glucose solution, NADH coenzyme solution, and glycerol solution. Alternatively, these three solutions may be stored and fed separately. The method of storing and feeding them separately is preferred, as this allows for flexible control of the timing and amount of feeding based on the metabolic status of each substance.

[0093] According to embodiments of this application, the reduced coenzyme includes at least one of reduced NADH coenzyme, reduced NADPH coenzyme, and reduced coenzyme Q10. Among these, the highest yields of 1,3-propanediol and lactic acid are obtained by supplementing with the reducing coenzyme NADH.

[0094] According to embodiments of this application, the concentration of the glycerol solution is 200 g / L to 1000 g / L, for example, 200 g / L, 300 g / L, 400 g / L, 500 g / L, 600 g / L, 700 g / L, 800 g / L, 900 g / L, 1000 g / L, etc., preferably 400 g / L to 600 g / L. This results in good bacterial growth and metabolism, and can increase the yield of 1,3-propanediol and lactic acid while reducing the formation of byproducts. In some embodiments, the glycerol solution further includes a solvent, which includes one or more of water, PBS buffer, physiological saline, and ethanol.

[0095] According to embodiments of this application, the concentration of the glucose solution is 200 g / L to 1000 g / L, for example, 200 g / L, 300 g / L, 400 g / L, 500 g / L, 600 g / L, 700 g / L, 800 g / L, 900 g / L, 1000 g / L, etc., preferably 400 g / L to 600 g / L. This results in good bacterial growth and metabolism, and can increase the yield of 1,3-propanediol and lactic acid while reducing the formation of byproducts. In some embodiments, the glucose solution further includes a solvent, which includes one or more of water, PBS buffer, physiological saline, and ethanol.

[0096] According to embodiments of this application, the concentration of the reduced coenzyme solution is 1 g / L to 10 g / L, for example, it can be 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, etc. This results in good bacterial growth and metabolism, and increases the production of 1,3-propanediol and lactic acid while reducing byproduct formation. In some embodiments, the reduced coenzyme solution further includes a solvent, which includes one or more of water, PBS buffer, physiological saline, and ethanol.

[0097] According to embodiments of this application, the fermentation culture time is 36h to 60h, for example, 36h, 42h, 46h, 50h, 54h, 58h, 60h, etc., preferably 42h to 54h. This results in higher yields of 1,3-propanediol and lactic acid, fewer byproducts, and helps maintain microbial stability and metabolic activity during fermentation, thus making the entire fermentation process efficient and stable.

[0098] According to embodiments of this application, the initial feeding time of the glycerol solution is 1h to 24h, for example, it can be 1h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, 24h, etc., preferably 1h to 4h. Therefore, the feeding time is appropriate, increasing the yield of 1,3-propanediol and lactic acid and ensuring they meet the required ratio, reducing the risk of insufficient carbon source addition due to late initial feeding time and excessively high glycerol concentration due to early initial feeding time, which could lead to strain intolerance.

[0099] According to embodiments of this application, the feeding rate of the glycerol solution is 10 mL / h to 50 mL / h, for example, it can be 10 mL / h, 15 mL / h, 20 mL / h, 25 mL / h, 30 mL / h, 35 mL / h, 40 mL / h, 45 mL / h, 50 mL / h, etc., preferably 15 mL / h to 30 mL / h. This avoids substrate inhibition and excessive consumption, increases the yield of 1,3-propanediol and lactic acid, and reduces the formation of byproducts.

[0100] According to embodiments of this application, the initial feeding time of the glucose solution is 2h to 24h, for example, 2h, 4h, 6h, 8h, 10h, 12h, 24h, etc., preferably 5h to 15h. This allows for a balance between the glycerol and glucose metabolic pathways, ensuring an appropriate overall glucose addition amount, thereby increasing the yield of 1,3-propanediol and lactic acid and achieving the required ratio between them. This reduces NADH deficiency caused by late initial feeding and competition between the glucose glycolysis and glycerol metabolic pathways caused by early initial feeding.

[0101] According to embodiments of this application, the glucose solution feed rate is 10 mL / h to 50 mL / h, for example, 10 mL / h, 15 mL / h, 20 mL / h, 25 mL / h, 30 mL / h, 35 mL / h, 40 mL / h, 45 mL / h, 50 mL / h, etc., preferably 20 mL / h to 30 mL / h. This avoids substrate inhibition and excessive consumption, increases the yield of 1,3-propanediol and lactic acid, and reduces the formation of byproducts.

[0102] According to embodiments of this application, the initial feeding time of the reduced coenzyme solution is 0h to 30h, for example, 0h, 6h, 12h, 24h, 36h, 47h, etc., preferably 10h to 24h. This effectively replenishes the NADH consumed in the production of 1,3-propanediol from 3-hydroxypropanal, promoting full reaction of the materials and increasing the yield of 1,3-propanediol and lactic acid.

[0103] According to embodiments of this application, the feeding rate of the reduced coenzyme solution is 10 mL / h to 50 mL / h, for example, 10 mL / h, 20 mL / h, 30 mL / h, 40 mL / h, 50 mL / h, etc., preferably 25 mL / h to 40 mL / h. This avoids substrate inhibition and excessive consumption, increases the yield of 1,3-propanediol and lactic acid, and reduces the formation of byproducts.

[0104] According to embodiments of this application, the fermentation culture satisfies at least one of the following conditions:

[0105] The fermentation temperature is 35℃~40℃, for example, it can be 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, etc.;

[0106] The fermentation speed is 100 rpm to 500 rpm, for example, it can be 100 rpm, 200 rpm, 300 rpm, 400 rpm, 500 rpm, etc.;

[0107] The dissolved oxygen in the fermentation process is 0% to 20%, for example, it can be 0%, 1%, 2%, 5%, 10%, 15%, 20%, etc.;

[0108] The OD of the fermentation broth 600 The value ranges from 0.6 to 3.0, for example, it can be 0.6, 1.0, 1.5, 2.0, 2.5, 3.0, etc., "OD". 600 "Value" refers to the absorbance of the fermentation broth at a wavelength of 600 nm, which can be measured using a UV spectrophotometer, etc.

[0109] The fermentation time is 36h to 60h, for example, 36h, 42h, 48h, 54h, 60h, etc.

[0110] The above fermentation conditions are suitable for cell growth and metabolism, increasing the yield of 1,3-propanediol and lactic acid, and the mass ratio of 1,3-propanediol to lactic acid satisfies (3-6):1.

[0111] It should be noted that this application does not impose strict limitations on the fermentation culture container or device; it can be cultured in either a culture flask or a fermenter, and the specific choice can be made flexibly according to the actual situation.

[0112] It is understood that the fermentation culture in this application is actually an expanded fermentation culture. Before fermentation, the Lactobacillus is activated in advance. The activation culture conditions are not strictly limited and can be the activation culture conditions of conventional Lactobacillus or Lactobacillus reuteri in the art, and can be flexibly selected according to the actual situation. In addition, this application does not strictly limit the number of expanded fermentation cultures. It can be carried out only once, or it can be carried out multiple times to further increase the bacterial concentration and activity, thereby increasing the yield of 1,3-propanediol and lactic acid and ensuring that the ratio of the two meets the aforementioned conditions. Each expanded fermentation culture involves inoculating the culture broth obtained from the previous culture into fresh culture medium, and the feeding treatment is carried out during the last expanded fermentation culture.

[0113] According to an embodiment of this application, during the fermentation process, culture medium is added every 10-14 hours. The added culture medium includes the initial culture medium but does not contain glycerol. This provides the nutrients required for the growth and metabolism of the microorganisms.

[0114] According to an embodiment of this application, the method further includes:

[0115] The fermentation broth containing 1,3-propanediol and lactic acid obtained from the fermentation culture was centrifuged, and the supernatant was collected.

[0116] The supernatant was subjected to ultrafiltration, and the filtrate was collected.

[0117] The filtrate was subjected to distillation to obtain a composition containing 1,3-propanediol and lactic acid.

[0118] Centrifugation removes cell debris and large particulate impurities from the fermentation broth, while ultrafiltration of the collected supernatant effectively retains target molecules and further removes small molecule impurities and some soluble macromolecules. Distillation utilizes the boiling point differences of different components to extract high-purity products, ultimately obtaining a high-purity composition containing 1,3-propanediol and lactic acid, wherein the mass ratio of 1,3-propanediol to lactic acid in this composition is (3-6):1.

[0119] According to embodiments of this application, the ultrafiltration membrane used in the ultrafiltration process has a molecular weight cutoff of no more than 1000 Da, such as 1000 Da, 800 Da, 600 Da, 500 Da, 100 Da, 80 Da, 50 Da, 20 Da, 10 Da, 5 Da, 1 Da, etc. Therefore, 1,3-propanediol and lactic acid can permeate through the ultrafiltration membrane, while large molecules are retained, thereby achieving further enrichment of 1,3-propanediol and lactic acid.

[0120] According to embodiments of this application, the distillation temperature is 60°C to 90°C, for example, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, etc. Utilizing the boiling point difference between 1,3-propanediol and lactic acid and other impurities, by setting the above-mentioned distillation temperature, these two substances can be preferentially evaporated, and subsequently recovered by condensation, thereby effectively separating pure 1,3-propanediol and lactic acid from the mixture, while removing unwanted impurities and reducing the loss of these two substances. The distillation time can be calculated based on the volume of the feed liquid to be distilled; for example, the distillation time for every 200 mL of feed liquid to be distilled is 50 minutes to 70 minutes.

[0121] It should be noted that the features and advantages described above with respect to the composition of the first aspect also apply to the cosmetic of the second aspect and the method of co-producing 1,3-propanediol and lactic acid of the third aspect, and will not be repeated here.

[0122] In a fourth aspect, this application discloses a fermentation product. According to an embodiment of this application, the fermentation product is obtained by the method described in the third aspect above for the co-production of 1,3-propanediol and lactic acid. The fermentation product of this application is rich in 1,3-propanediol and lactic acid, has few impurities, and the mass ratio of 1,3-propanediol to lactic acid is (3-6):1. The composition has high moisturizing properties and low stickiness, and can be used in cosmetics to improve their quality.

[0123] In a fifth aspect of this application, a cosmetic product is provided. According to an embodiment of this application, the cosmetic product comprises the fermentation product described in the fourth aspect above.

[0124] It should be noted that the features and advantages described above for the method of co-producing 1,3-propanediol and lactic acid in the third aspect also apply to the fermentation product in the fourth aspect and the cosmetic in the fifth aspect, and will not be repeated here.

[0125] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0126] Example 1

[0127] 1. Prepare compositions containing different ratios of 1,3-propanediol and lactic acid, as shown in Table 1.

[0128] 2. Test the moisturizing properties, irritation, and viscosity of the composition separately. The specific test methods are as follows:

[0129] (1) Moisturizing test

[0130] Twenty participants were selected, aged 18-65 years (excluding those who are pregnant or breastfeeding); those without serious systemic diseases, immunodeficiency, or autoimmune diseases; those whose test sites had not undergone skin treatments, cosmetic procedures, or other tests that might affect the results; those without active allergic diseases; those without highly sensitive constitutions; those who had not used hormonal drugs or immunosuppressants within the past month; and those whose test sites were not currently or within the past three months participating in other clinical trials.

[0131] Skin moisture content testing was conducted on the forearms of the subjects in a room at 20–22°C and 40%–60% relative humidity. Volunteers were instructed not to expose their forearms to water for 3 hours prior to the test and to wipe the flexor surfaces of their forearms with a tissue before the test. Three 3×3cm square test areas were marked on each of the left and right forearms, spaced 1cm apart. The test area was required to be exposed to the testing environment for at least 20 minutes before the test.

[0132] Before applying the sample, the skin moisture content of different areas of the arm was measured using a Corneometer CM825 (Courage+Khazaka, Germany), and the raw values ​​were recorded.

[0133] 100 μL of test sample was transferred directly to the test area on the subject's arm using a pipette, and rubbed in a circular motion 30 times with the fingers (wearing rubber gloves). Four hours after application, the skin moisture content of the test area was measured using a Corneometer CM825, and the moisturizing coefficient W was calculated. Each test sample was tested 10 times on the skin of different subjects, and the W value was calculated using the average value.

[0134] W = (Skin moisture content 4 hours after sample application - Skin moisture content before sample application) / Skin moisture content before sample application × 100%

[0135] (2) Stimulation test

[0136] (2-1) pH value detection

[0137] (2-2) Using cultured cells from rabbit cornea, determine the concentration (IC50) of each sample required to inhibit 50% cell growth, and assess skin irritation according to the following criteria:

[0138] Mild irritation: IC50 not less than 500 ppm;

[0139] Moderate irritation: IC50 not less than 200 ppm, but less than 500 ppm;

[0140] Strong irritant: IC50 below 200ppm.

[0141] (3) Viscosity test

[0142] The viscosity of the samples was measured using a rotational viscometer, with each sample measured three times and the average value taken.

[0143] The results are shown in Table 1. When the mass ratio of 1,3-propanediol to lactic acid is (3-6):1, the composition has strong moisturizing properties, low viscosity, a more refreshing feel, and weak irritation. Among them, when the mass ratio of 1,3-propanediol to lactic acid is (4-5):1, the overall quality of the composition is better.

[0144] Table 1

[0145]

[0146] Example 2

[0147] Experimental group 1:

[0148] 1. Inoculate 5 mL of seed culture medium with *Lactobacillus reuteri* PDO / LA-3 and incubate statically at 37°C for 48 h until OD (Organic Difference) is reached. 600nm =3.0.

[0149] Seed liquid culture medium: 10 g / L peptone, 8 g / L beef extract powder, 4 g / L yeast extract powder, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L diammonium hydrogen citrate, 0.2 g / L magnesium sulfate (MgSO4·7H2O), 0.04 g / L manganese sulfate (MnSO4·4H2O), 1 mL / L Tween-80, with the remainder being water. After preparing the solution, the pH was adjusted to 5.5 using dilute hydrochloric acid and ammonia.

[0150] 2. Add initial culture medium containing 30 g / L glycerol and MRS medium to a 2.5 L fermenter. Before inoculation, purge with nitrogen to create anaerobic conditions. Then, inoculate with 10% of Lactobacillus reuteri (preservation number GDMCCNO: 64700) PDO / LA-3 for scale-up fermentation. During the fermentation process, monitor the anaerobic culture at 37℃, pH 5.5 (adjusted by dilute hydrochloric acid and ammonia) and 300 rpm for 48 h.

[0151] MRS medium contains the following components: 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L diammonium hydrogen citrate, 0.2 g / L magnesium sulfate (MgSO4·7H2O), 0.04 g / L manganese sulfate (MnSO4·4H2O), 1 mL / L Tween-80, and the remainder is water. After preparing the solution, the pH is adjusted to 5.5 using dilute hydrochloric acid and ammonia. The pH adjuster is 0.1 M dilute hydrochloric acid and 28% ammonia.

[0152] 3. Separation and purification

[0153] (1) Centrifuge the mixture in the fermenter at 8000 rpm and 4℃ for 10 min. Observe the OD of the fermentation supernatant after one centrifugation. 600 =0.2, color is brownish-yellow;

[0154] (2) The fermentation supernatant after the first centrifugation was placed in a centrifuge equipped with an ultrafiltration membrane with a molecular weight cutoff of 1000 Da, and centrifuged for 20 min at a speed of 10000 rpm and a temperature of 4℃. The OD of the fermentation filtrate after the second centrifugation and filtration was... 600 =0.05, clear and transparent in color, pH=4.8;

[0155] (3) The fermentation filtrate was concentrated by distillation at 90℃. The distillation time for each 200mL of fermentation filtrate was 60 minutes to remove impurities such as water, ethanol, and acetic acid, and to obtain a composition containing 1,3-propanediol and lactic acid.

[0156] The differences between experimental groups 2-8 and experimental group 1 are shown in Table 2.

[0157] Experimental group 9:

[0158] 1. Same as experimental group 1.

[0159] 2. Add initial culture medium containing 30 g / L glycerol and MRS medium to a 2.5 L fermenter. Before inoculation, purge with nitrogen to create anaerobic conditions. Then, inoculate with 10% of Lactobacillus reuteri (preservation number GDMCCNO: 64700) PDO / LA-3 for scale-up fermentation. During the fermentation process, monitor the anaerobic culture at 37℃, pH 5.5 (adjusted by dilute hydrochloric acid and ammonia) and 300 rpm for 48 h.

[0160] Feeding methods: 500 g / L glycerol solution and 250 g / L glucose solution were used as feed solutions. The glycerol solution was fed at a rate of 21 mL / h for 1 hour initially, continuing until fermentation ended. The glucose solution was fed at a rate of 25 mL / h for 8 hours initially, continuing until fermentation ended. During fermentation, 250 g / L MRS medium was added every 12 hours.

[0161] The differences between experimental groups 10-32 and experimental group 9 are shown in Table 2.

[0162] Test case

[0163] The compositions prepared in experimental groups 1 to 32 were tested respectively, and the specific test methods are as follows:

[0164] 1. Content of 1,3-propanediol and lactic acid

[0165] The contents of 1,3-propanediol and lactic acid in the sample were tested using a gas chromatography-mass spectrometry system. A DB-WAX polar column was used, with the column oven temperature at 250℃, the injector temperature at 250℃, the detector temperature at 275℃, the heating rate at 1–30℃ / min, and nitrogen purging.

[0166] 2. Moisturizing test, irritation test, and viscosity test:

[0167] Same as Example 1.

[0168] The results are shown in Tables 2 and 3. Experimental groups 1 and 5 show that adding glycerol to the initial fermentation medium allows the 1,3-propanediol and lactic acid in the fermentation broth to achieve a mass ratio of (3-6):1. Furthermore, experimental groups 1-4 show that using *Lactobacillus reuteri* as the fermentation strain yields a 1,3-propanediol and lactic acid content that meets the (3-6):1 mass ratio, while using a non-lactobacillus—*Citrobacter freundii*—as the fermentation strain fails to produce the required 1,3-propanediol and lactic acid. Experimental groups 1 and 9-32 demonstrate that fed-batch fermentation can increase the yield of 1,3-propanediol and lactic acid.

[0169]

[0170]

[0171]

[0172]

[0173] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0174] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A composition characterized in that, Comprising: 1,3-propanediol and lactic acid, a mass ratio of (3-6):1; The 1,3-propanediol and lactic acid are obtained by fermentation culture of Lactobacillus in the same fermentation system.

2. The composition of claim 1, wherein, The Lactobacillus comprises Lactobacillus reuteri, or the Lactobacillus comprises Lactobacillus reuteri mucosus, which is preserved in Guangdong Microbial Culture Collection Center on May 29, 2024, with a preservation number of GDMCC NO: 64700. And / or, the mass ratio of the 1,3-propanediol and lactic acid is (4-5):1; And / or, the pH value of the composition is 4.5-5.

0.

3. The composition of claim 1, wherein Further comprising: The cell lysate obtained by the fermentation culture of Lactobacillus.

4. A cosmetic product, characterized by Comprising: The composition of any one of claims 1-3.

5. The cosmetic product according to claim 4, characterized in that, The content of the composition is 1 mass% to 30 mass% based on the total mass of the cosmetic.

6. A process for the co-production of 1,3-propanediol and lactic acid, characterized in that, Comprising: fermentation culture of Lactobacillus to obtain a fermentation broth containing 1,3-propanediol and lactic acid; The initial culture medium of the fermentation culture contains glycerol and glucose; The mass ratio of the 1,3-propanediol and lactic acid is (3-6):

1.

7. The method of claim 6, wherein, The Lactobacillus comprises Lactobacillus reuteri; And / or, the Lactobacillus comprises Lactobacillus reuteri mucosus, which is preserved in Guangdong Microbial Culture Collection Center on May 29, 2024, with a preservation number of GDMCC NO: 64700.

8. The method of claim 6, wherein, The concentration of the glycerol is 1 g / L to 50 g / L; And / or, the concentration of the glucose is 1 g / L to 50 g / L.

9. The method of claim 6, wherein, The initial culture medium of the fermentation culture further contains at least one of peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, sodium acetate, diammonium hydrogen citrate, magnesium sulfate, manganese sulfate, Tween-80, and water; And / or, the pH value of the fermentation culture medium is 4 to 6.

10. The method of claim 6, wherein, The fermentation culture comprises a fed-batch process; The feed solution of the fed-batch process comprises at least one of a glucose solution and a reduced coenzyme solution, and a glycerol solution.

11. The method of claim 10, wherein, The concentration of the glycerol solution is 200 g / L to 1000 g / L, preferably 400 g / L to 600 g / L; And / or, the concentration of the glucose solution is 100 g / L to 500 g / L, preferably 200 g / L to 400 g / L; And / or, the concentration of the reduced coenzyme solution is 1 g / L to 10 g / L; And / or, the reduced coenzyme comprises at least one of reduced NADH coenzyme, reduced coenzyme NADPH, and reduced coenzyme Q10.

12. The method according to claim 10 or 11, characterized in that, The fermentation culture time is 36 h to 60 h, preferably 42 h to 54 h; And / or, the initial feeding time of the glycerol solution is 1 h to 24 h, preferably 1 h to 4 h; And / or, the feeding rate of the glycerol solution is 10 mL / h to 50 mL / h, preferably 15 mL / h to 30 mL / h; And / or, the initial feeding time of the glucose solution is 2 h to 24 h, preferably 5 h to 15 h; And / or, the feeding rate of the glucose solution is 10 mL / h to 50 mL / h, preferably 20 mL / h to 30 mL / h; And / or, the start feeding time of the reduced coenzyme solution is 0h-30h, preferably 10h-24h; And / or, the feeding rate of the reduced coenzyme solution is 10mL / h-50mL / h, preferably 25mL / h-40mL / h.

13. The method of claim 10 or 11, wherein, The fermentation culture satisfies at least one of the following conditions: The temperature of the fermentation treatment is 35℃-40℃; The rotation speed of the fermentation treatment is 100rpm-500rpm; The dissolved oxygen of the fermentation treatment is 0%-20%; OD value of the fermentation broth 600 0.6 to 3.0; The time of the fermentation treatment is 36h-60h.

14. The method of claim 6 or 10, wherein, During the fermentation culture, medium is added at intervals of 10h-14h, and the added medium comprises the initial medium and does not contain glycerol.

15. The method of claim 10 or 11, wherein, Further comprising: The fermentation liquid containing 1,3-propanediol and lactic acid obtained by the fermentation culture is subjected to centrifugal treatment, and the supernatant is collected; The supernatant is subjected to ultrafiltration treatment, and the filtrate is collected; The filtrate is subjected to rectification treatment to obtain a composition containing 1,3-propanediol and lactic acid.

16. The method of claim 15, wherein, The ultrafiltration membrane used in the ultrafiltration treatment has a molecular weight cut-off of not more than 1000Da; And / or, the temperature of the rectification treatment is 60℃-90℃.

17. A fermentate, characterized in that, The fermentation product is obtained by the method for co-producing 1,3-propanediol and lactic acid according to any one of claims 6-16.

18. A cosmetic product, characterized by, The cosmetic product comprises the fermentation product according to claim 17.

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