Lactobacillus for increasing diacylglycerol content in oil and application thereof

Abstract

This invention discloses a lactobacillus that increases the diglyceride content in oils ( Lactobacillus sp. The lactobacillus (Lactobacillus sp. The accession number for this invention is CGMCC NO.37495. This invention also discloses a method for increasing the diglyceride content in oils. Furthermore, this invention discloses the application of using the *Lactobacillus* strain described above to increase the diglyceride content in oils. The *Lactobacillus* strain of this invention is the first strain capable of efficiently fermenting camellia seed oil and using it as a skincare matrix. This *Lactobacillus* strain exhibits good tolerance during the fermentation process, adapting to high-oil environments and weakly acidic fermentation conditions. The fermentation process is stable and controllable, requiring no chemical catalysts, thus avoiding solvent residues and byproduct problems that may arise from traditional chemical modification methods. It can serve as a high-performance biotransformation strain, playing a core role in the development of functional skincare ingredients.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and oil processing technology, specifically relating to a lactobacillus that increases the diglyceride content in oils and its application. Background Technology

[0002] Camellia seed oil is rich in unsaturated fatty acids such as oleic acid and linoleic acid, possessing excellent nutritional value and skincare benefits, and is widely used in the food and cosmetic industries. However, traditional camellia seed oil has a high triglyceride content and a low diglyceride content, limiting its application in functional oils and highly absorbable skincare products. Diglycerides, as a functional oil component, have better emulsifying properties, skin penetration, and bioavailability, and show great promise for application in functional foods and highly absorbable skincare products.

[0003] Currently, the main methods for increasing the diglyceride content in oils and fats include: 1. Enzymatic conversion: This method utilizes lipase to catalyze the partial hydrolysis or transesterification of triglycerides to produce diglycerides. However, it suffers from problems such as high enzyme costs, harsh reaction conditions, and complex product separation.

[0004] 2. Chemical synthesis: Diglycerides are synthesized through chemical catalysis, but this method has drawbacks such as numerous side reactions and questionable product safety.

[0005] 3. Microbial fermentation: This method utilizes specific microorganisms to transform oil components. It has advantages such as mild conditions, green and safe production, and natural products. However, most of the strains reported so far are focused on degrading oils (refer to CN110591952A), and there are few reports on their use for the efficient conversion of triglycerides into diglycerides.

[0006] Therefore, screening a microbial strain that can efficiently convert triglycerides into diglycerides and developing a corresponding fermentation process is of great significance for the preparation of high-quality camellia seed oil. Summary of the Invention

[0007] The purpose of this invention is to overcome the lack of strains that can convert triglycerides into diglycerides, thereby providing a microbial strain that can efficiently convert triglycerides into diglycerides.

[0008] To achieve the above objectives, the first invention of this invention provides a lactobacillus that increases the diglyceride content in oils (…). Lactobacillus sp. The lactobacillus (Lactobacillus sp. The accession number is CGMCCNO.37495.

[0009] A second aspect of the present invention provides a method for increasing the diglyceride content in oils, using Lactobacillus as described above ( Lactobacillus sp. Fermentation of oils.

[0010] According to a preferred embodiment of the present invention, the method includes the following steps: S1, the lactobacillus ( Lactobacillus sp. The seed culture was obtained by inoculating the seed culture in a culture medium and shaking it. S2. Inoculate the seed culture obtained in step S1 into the culture medium, mix it with the oil, and ferment to obtain the fermentation broth; S3. After centrifuging the fermentation broth obtained in step S2, the oil layer is separated, dehydrated, and then filtered to obtain fermented oil.

[0011] Preferably, the culture medium in step S1 is MRS liquid culture medium, and the shaking culture time is 24 hours.

[0012] Preferably, in step S2, the volume ratio of the oil to the culture medium is 1:2.

[0013] Preferably, the fermentation time in step S2 is 48 hours.

[0014] Furthermore, the filtration in step S3 uses a 0.22 μm microporous membrane.

[0015] Furthermore, the oil is camellia seed oil.

[0016] A third aspect of the present invention provides lactobacillus as described above. Lactobacillus sp. It is used to increase the diglyceride content in oils and fats.

[0017] According to a preferred embodiment of the present invention, the oil is camellia seed oil.

[0018] The beneficial effects of this invention are as follows: 1. The *Lactobacillus* strain of this invention is the first strain capable of efficiently fermenting camellia seed oil and using it as a skincare base. This *Lactobacillus* can degrade the triglyceride structure in camellia seed oil, breaking down large-molecule oils into small-molecule free fatty acids and glycerol through metabolically produced lipases, achieving a significant fermentation conversion rate. After fermentation, it can generate diglycerides, short-chain fatty acid esters, and small-molecule active substances with a better skin feel, significantly reducing the viscosity of the oil, making the oil texture lighter and more refreshing, easier to spread and penetrate than unfermented camellia seed oil, and significantly improving the skin feel.

[0019] 2. This lactobacillus exhibits good tolerance in the fermentation process, and can adapt to high-oil environments and weakly acidic fermentation conditions. The fermentation process is stable and controllable, and no chemical catalysts are required. This avoids the solvent residue and by-product problems that may be caused by traditional chemical modification methods. It can serve as a high-performance biotransformation strain and play a core role in the development of functional skin care ingredients. Attached Figure Description

[0020] Figure 1 This is a colony morphology diagram of the strain on MRS agar medium.

[0021] Figure 2 This image shows the growth results of the strain on a screening medium using camellia seed oil as the carbon source. Figure 2 A, Figure 2 B, Figure 2 C, Figure 2 Figure D shows the growth results of different Lactobacillus colonies on the screening medium.

[0022] Figure 3 This is a microscopic examination image of Lactobacillus WY-FCO-001.

[0023] Figure 4 Agarose gel electrophoresis image of the 16S rDNA PCR amplification product of Lactobacillus WY-FCO-001.

[0024] Figure 5 A phylogenetic tree of Lactobacillus WY-FCO-001 constructed based on the 16S rDNA sequence.

[0025] Preservation Matters The lactobacillus obtained in this invention ( Lactobacillus sp. WY-FCO-001 was deposited with the China General Microbiological Culture Collection Center (CGMCC) on January 21, 2026, with accession number CGMCCNO.37495. Detailed Implementation

[0026] The present invention will be described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0027] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are all available through conventional commercial channels.

[0028] The specific culture media and reagent formulations, as well as the test methods used in the following examples, are as follows: 1. Camellia seed oil used in the experiment.

[0029] The camellia seed oil used in the experiment was provided by Frog Prince (Fujian) Baby Care Products Co., Ltd.

[0030] 2. Experimental culture medium.

[0031] 2.1 MRS medium (1L): 20.0g glucose, 10.0g tryptone, 10.0g beef extract, 5.0g yeast extract, 2.0g dipotassium hydrogen phosphate, 2.0g triammonium citrate, 5.0g sodium acetate, 1mL Tween 80, 0.2g magnesium sulfate heptahydrate, 0.05g manganese sulfate, 15g agar, add distilled water to make up to 1L, and adjust pH to 7.0.

[0032] 2.2 Screening medium (1L): Based on MRS medium, glucose was removed, and 20g / L of camellia seed oil was added, namely 20.0g of camellia seed oil, 10.0g of tryptone, 10.0g of beef extract, 5.0g of yeast extract, 2.0g of dipotassium hydrogen phosphate, 2.0g of triammonium citrate, 5.0g of sodium acetate, 1mL of Tween 80, 0.2g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate, and 15g of agar. Distilled water was added to 1L, and the pH was adjusted to 7.0.

[0033] 2.3 Re-screening medium (1L): Based on MRS medium, remove the carbon sources such as glucose, beef extract, and yeast extract, add 20g / L of camellia seed oil (with camellia seed oil as the sole carbon source), and adjust the pH to 7.0.

[0034] 2.4 Fermentation medium: MRS basal medium (without agar) was used as the aqueous phase, and camellia seed oil was added to make the volume ratio of camellia seed oil to liquid medium 2:1, pH 7.0.

[0035] Example 1: Isolation, screening and identification of diglyceride-producing Lactobacillus WY-FCO-001 1. Isolation and initial screening of bacterial strains Soil samples were collected from Wuyishan Mountain in Fujian Province, sealed in sterile sampling bags, and promptly transported back to the laboratory for processing. Under aseptic conditions, 10 g of soil sample was accurately weighed and added to an Erlenmeyer flask containing 90 mL of sterile physiological saline. The sample was thoroughly shaken to ensure uniform dispersion; this constitutes 10 g of the soil sample. - ¹Dilution buffer. Continue with 10-fold serial dilutions using sterile physiological saline to prepare 10... -4 10 -5 10 -6 Diluted bacterial suspension.

[0036] Take 100 μL of each of the above dilutions of bacterial culture and spread it evenly on the surface of the screening medium. Camellia seed oil was added to this medium as an inducing carbon source to selectively screen for lactobacilli with lipid-decomposing capabilities. After spreading, the culture was incubated at 37°C under anaerobic conditions for 48 h.

[0037] After the culture is completed, observe the colony growth. Figure 1 This is a colony morphology diagram of the strain on MRS agar medium, such as... Figure 1 As shown, select a typical single colony growing on the plate. It is milky white, round, with neat edges, smooth and moist surface, and about 1-2 mm in diameter. Figure 2 This is a diagram showing the growth results of the strain on a screening medium with camellia seed oil as the sole carbon source, as shown below. Figure 2 As shown, under harsh conditions where oil is the sole carbon source, only a few strains (such as...) Figure 2 A, Figure 2 The bacteria (B colonies) can grow, which initially suggests that they have the ability to degrade and utilize oils.

[0038] 2. Purification and rescreening of strains The colonies obtained from the initial screening were inoculated onto fresh MRS agar plates and purified using a repeated streak plating method until single colonies with consistent morphology and good purity were obtained. The purified strain was then inoculated again into a liquid secondary screening medium with camellia seed oil as the sole carbon source to verify its growth ability. Finally, a well-growing target strain was obtained, designated WY-FCO-001.

[0039] 3. Identification of strain WY-FCO-001 3.1 Morphological identification of the strain The purified strain WY-FCO-001 was examined under a microscope after methylene blue staining. Figure 3 This is a microscopic image of the strain, such as... Figure 3 As shown, under a microscope, this strain appears as short rods, arranged singly or in pairs, without spores, and is non-motile, consistent with the typical morphological characteristics of lactobacillus.

[0040] 3.2 Molecular biological identification Genomic DNA was extracted from bacterial strain WY-FCO-001 using the Ezup column-based bacterial genomic DNA extraction kit. PCR amplification was performed using universal primers for bacterial 16S rDNA. Figure 4 This is an agarose gel electrophoresis image of PCR-amplified 16S rDNA, as shown below. Figure 4 As shown, a clear and bright specific band is visible at approximately 1500 bp. After purification and recovery of the PCR product, it was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, yielding a sequence of approximately 1480 bp.

[0041] The obtained sequences were submitted to the NCBI database for BLAST homology comparison. Figure 5 It is a phylogenetic tree of strains constructed based on 16S rDNA sequences, such as Figure 5 As shown, by constructing a phylogenetic tree for evolutionary analysis and combining it with morphological results, strain WY-FCO-001 was finally identified as Lactobacillus.

[0042] This strain was deposited on January 21, 2026, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 37495.

[0043] Example 2: Preparation of diglycerides from camellia seed oil by fermentation with Lactobacillus WY-FCO-001 This embodiment provides a specific process for fermenting camellia seed oil using Lactobacillus WY-FCO-001 screened and identified in Example 1 to significantly increase the diglyceride content in camellia seed oil. The specific steps are as follows: 1. Activation of microbial strains The Lactobacillus WY-FCO-001 obtained in Example 1 was inoculated into MRS liquid culture medium and cultured with shaking at 37°C and 150 rpm for 24 hours to obtain activated seed culture.

[0044] 2. Fermentation culture The activated seed culture was inoculated into the fermentation medium at an inoculation rate of 3% (v / v). The inoculated fermentation system was then placed at 37°C and 150 rpm for 48 hours.

[0045] 3. Separation and purification of fermentation products After fermentation, the fermentation broth was centrifuged at 8000 rpm for 10 minutes. Due to the low density of the oil, the upper oil layer could be clearly separated. This oil layer was collected, and anhydrous sodium sulfate was added to dehydrate it and remove trace amounts of water. Then, it was filtered through a 0.22 μm microporous membrane to obtain clear and transparent camellia seed fermented oil.

[0046] Example 3: High-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) analysis of glycerol ester composition in fermented camellia seed oil This embodiment performs qualitative and quantitative comparative analysis on the glycerol ester composition of fermented camellia seed oil prepared in Example 2 and unfermented raw camellia seed oil to verify the transformation effect of strain WY-FCO-001.

[0047] 1. Sample pretreatment Accurately weigh 10 mg each of camellia seed crude oil and camellia seed fermented oil samples and place them separately in 10 mL volumetric flasks. Dissolve them in a methanol-isopropanol mixed solution containing 5 mmol / L ammonium acetate (methanol-isopropanol volume ratio 1:1) and dilute to the mark. Vortex vigorously until the samples are completely dissolved. Filter the solution through a 0.22 μm polytetrafluoroethylene (PTFE) microporous membrane and store at -20℃ for later use.

[0048] 2. HPLC-HRMS analytical conditions Chromatographic conditions: A Phenomenex Kinetex C18 column (100 mm × 2.1 mm, 2.6 μm) was used; mobile phase A was a water-methanol-acetonitrile mixture (1:1:1 v / v, containing 5 mM ammonium acetate), and mobile phase B was isopropanol (containing 5 mM ammonium acetate); flow rate was 0.3 mL / min; column temperature was 45 °C; injection volume was 1 μL. A gradient elution program was used.

[0049] Mass spectrometry conditions: High-resolution mass spectrometry was used, with an electrospray ionization (ESI) source and positive ion mode scanning. Data acquisition employed frequency sweep (i-DIA) mode to improve the accuracy and sensitivity of qualitative analysis.

[0050] 3. Results Analysis By comparing the retention times and characteristic ion peaks of triglyceride and diglyceride standards, the glycerides in the sample can be accurately quantified.

[0051] The quantitative results are shown in the table below: Table 1. Glyceryl ester composition of camellia seed oil before and after fermentation As shown in Table 1, the main component of unfermented camellia seed oil is triglycerides, with a low content of diglycerides. However, after fermentation with Lactobacillus WY-FCO-001 for 48 hours, the glyceride composition of the fermented camellia seed oil changed significantly: the diglyceride content increased dramatically to 90.0%, while the triglyceride content decreased significantly to 10.0%. This indicates that the Lactobacillus WY-FCO-001 provided by this invention can efficiently and specifically convert triglycerides in camellia seed oil into diglycerides, with extremely high conversion efficiency.

[0052] The above description is merely a preferred embodiment for explaining the present invention and is not intended to limit the present invention in any way. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included within the scope of protection intended by the present invention.

Claims

1. A type of lactobacillus that increases the diglyceride content in oils ( Lactobacillus sp. ), characterized in that, The lactobacillus (Lactobacillus sp. The accession number for this item is CGMCC NO.37495.

2. A method for increasing the diglyceride content in oils, characterized in that, Using the lactobacillus described in claim 1 ( Lactobacillus sp. Fermentation of oils.

3. The method according to claim 2, characterized in that, Includes the following steps: S1, the lactobacillus ( Lactobacillus sp. The seed culture was obtained by inoculating the seed culture in a culture medium and shaking it. S2. Inoculate the seed culture obtained in step S1 into the culture medium, mix it with the oil, and ferment to obtain the fermentation broth; S3. After centrifuging the fermentation broth obtained in step S2, the oil layer is separated, dehydrated, and then filtered to obtain fermented oil.

4. The method according to claim 3, characterized in that, The culture medium in step S1 is MRS liquid culture medium, and the shaking culture time is 24 hours.

5. The method according to claim 3, characterized in that, In step S2, the volume ratio of the oil to the culture medium is 1:

2.

6. The method according to claim 3, characterized in that, The fermentation time in step S2 is 48 hours.

7. The method according to claim 3, characterized in that, The filtration in step S3 uses a 0.22 μm microporous membrane.

8. The method according to any one of claims 2 to 7, characterized in that, The oil in question is camellia seed oil.

9. The lactobacillus as described in claim 1 ( Lactobacillus sp. It is used to increase the diglyceride content in oils and fats.

10. The application according to claim 9, characterized in that, The oil in question is camellia seed oil.

Images