Method for improving biotransformation of conjugated linoleic acid by bifidobacterium breve

Abstract

The invention discloses a method for improving conjugated linoleic acid biotransformation of bifidobacterium breve. The method comprises the step of adding L-arginine into a system which takes linoleic acid as a substrate and utilizes bifidobacterium breve to biologically produce conjugated linoleic acid. According to the method, BBI expression regulation is realized through exogenous supplement of arginine, so that the yield of the bifidobacterium breve in biotransformation of CLA is increased, and complex gene editing operation or high screening cost of high-yield strains can be avoided.

Description

Technical Field

[0001] This application relates to the field of microbial biotechnology, specifically to a method for improving the biotransformation of conjugated linoleic acid by Bifidobacterium breve. Background Technology

[0002] Conjugated linoleic acid (CLA) belongs to the essential polyunsaturated fatty acid (PUFA) family, specifically referring to a series of positional and geometric isomers of linoleic acid (LA) containing conjugated double bonds. Since the 1980s, its physiological functions have been continuously expanded, including anti-obesity, antioxidant, anti-inflammatory, immunomodulatory, anti-cancer, anti-atherosclerotic, and growth-promoting and bone-forming effects. Given these physiological activities, CLA is widely used in medicine, food, and cosmetics.

[0003] Significant differences exist in the physiological functions of CLA isomers. Currently, the c9, t11-CLA and t10, c12-CLA isomers are the two most physiologically active. Among them, t10, c12-CLA typically plays a prominent role in improving lipid metabolism and reducing obesity, while c9, t11-CLA offers health benefits in inhibiting breast cancer cell proliferation, reducing neuronal β-amyloid secretion, and improving respiratory and intestinal inflammation. Therefore, obtaining pure CLA isomers has significant application value and strong market demand.

[0004] Currently, the commercial production of CLA typically involves the chemical isomerization of LA or LA-rich vegetable oils using methods such as alkaline treatment or transition metal ion catalysis. This process yields CLA in various isomer forms. Since only a few CLA isomers have been identified as possessing beneficial physiological activities, and mixtures of isomers may produce unknown physiological effects, safer isomer-selective processes or efficient purification / separation methods are urgently needed for the chemical synthesis of CLA.

[0005] Compared to chemical synthesis, biotransformation offers advantages such as milder reaction conditions and easier product separation and purification. It can selectively produce single CLA isomers and is more aligned with sustainable and green industrial production goals. Bifidobacterium is considered an ideal choice for producing single C9,T11-CLA isomers using food-grade microorganisms. Overall, Bifidobacterium strains with biotransformation capabilities exhibit two main characteristics: ① They are concentrated among four species: Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium pseudobifidum, and Bifidobacterium dendriticum, with Bifidobacterium breve showing the strongest biotransformation ability; ② Their biotransformation abilities differ significantly at both the species and strain levels. For example, Raimondi et al. used spectrophotometry to preliminarily screen 128 strains of 31 Bifidobacteria for the biotransformation of CLA, and used GC-MS to detect the optimal production strain—Bifidobacterium breve WC0421—which could convert LA (0.5 g / L) into 68.8% c9,t11-CLA and 25.1% t9,t11-CLA, with a total CLA conversion rate of 93.9%. However, the conversion rates of most strains were below 40%. Studies have also indicated that even within the same species, different Bifidobacteria exhibit significant differences in conversion rates; for example, Bifidobacterium breve CCFM683 had a conversion rate exceeding 90%, while Bifidobacterium breve UCC2003 had a conversion rate of only 45%.

[0006] However, the genus *Bifidobacterium* is highly diverse, and different species within the same genus exhibit significant differences in morphology, physiology, and metabolism. The biotransformation of CLA by *Bifidobacterium* involves a complex metabolic balance between strain growth and LA (substrate) stress. LA stress induces the expression of linoleic acid isomerase (BBI) in the strain, thereby converting LA into CLA.

[0007] To date, targeted screening of high-CLA-producing strains has been difficult to achieve. Furthermore, the regulatory mechanisms of Bifidobacterium biotransformation of CLA involve a complex metabolic balance between strain growth and LA (substrate) stress, and are not yet fully understood.

[0008] Therefore, low yield is currently the technical bottleneck in the production of CLA using Bifidobacterium breve. Summary of the Invention

[0009] Based on this, this application provides at least one method for improving the biotransformation of conjugated linoleic acid by Bifidobacterium breve.

[0010] In a first aspect of this application, an application of L-arginine in the production of conjugated linoleic acid is provided.

[0011] In a second aspect of this application, a method is provided for improving the bioproduction of conjugated linoleic acid from Bifidobacterium breve, the method comprising:

[0012] L-arginine was added to a system that uses linoleic acid as a substrate and utilizes Bifidobacterium breve to bioproduce conjugated linoleic acid.

[0013] In a third aspect of this application, a product portfolio for producing conjugated linoleic acid is provided, the product portfolio comprising Bifidobacterium breve, L-arginine, and a culture medium suitable for culturing Bifidobacterium breve.

[0014] This application provides an exemplary and simple method to increase the yield of CLA biotransformed by Bifidobacterium breve by regulating BBI expression through exogenous arginine supplementation, which can avoid complex gene editing operations or the high screening costs of high-yield strains. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without creative effort. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in illustrating this application.

[0016] Figure 1 This is a growth curve of Bifidobacterium SHMB8001 under different culture conditions in one embodiment of this application.

[0017] Figures 2A to 2F This is a chromatogram of free fatty acids in a culture during the logarithmic growth phase and stationary phase, as analyzed by gas chromatography-mass spectrometry (GC-MS) in one embodiment of this application.

[0018] Figure 3 This invention illustrates the analysis of conjugated linoleic acid (CLA) content produced by Bifidobacterium breve SHMB8001 at different time points in one embodiment of this application.

[0019] Figure 4 This diagram illustrates the changes in the relative activity of Bifidobacterium breve SHMB8001 under different culture conditions for CLA biotransformation in one embodiment of this application.

[0020] Figure 5A and Figure 5B This diagram illustrates the results of arginine promoting the expression of linoleic acid isomerase BBI and transcription factor ArgR in Bifidobacterium breve according to one embodiment of this application.

[0021] Figure 6This diagram illustrates the result of arginine promoting the binding of transcription factors and the BBI gene promoter in one embodiment of this application.

[0022] Figures 7A to 7C This diagram illustrates the formation of two CLA isomers when the substrate LA is 1.0 g / L, according to one embodiment of this application. Detailed Implementation

[0023] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0025] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and other instances that otherwise indicate "one or more" shall be understood in the same way unless otherwise specified.

[0026] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0027] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method" etc., shall be defined as being able to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0028] In this application, terms such as "further," "even more," "particularly," "for example," "like," "example," and "exemplary" are used for descriptive purposes to indicate that different technical solutions preceding and following each other are related in terms of their coverage, but should not be construed as limiting the preceding technical solution or restricting the scope of protection of this application. In this application, unless otherwise specified, A (e.g., B) indicates that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0029] The terms “containing,” “comprising,” and “including” as used in this application are synonyms and are inclusive or open-ended, not excluding additional, uncited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions under which actions occur, timing, states, etc.

[0030] In this application, the technical features or solutions described in open-ended language include both closed-ended technical features or solutions consisting of the listed contents and open-ended technical features or solutions that include the listed contents.

[0031] In this application, the exemplary descriptions such as "in some implementations (or embodiments)" and "in one implementation (or embodiment)" may cover, but are not limited to, the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0032] In this application, the terms "first aspect," "second aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first aspect," "second aspect," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0033] In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the distribution of selectable numerical values ​​within the numerical interval is considered continuous, and includes the two endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. The term "numerical interval" can be broadly included to include numerical interval types such as percentage intervals, ratio intervals, and proportion intervals.

[0034] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated herein, there is no strict order restriction on the execution of these steps; they can be executed in any order other than those described. Moreover, any step may include multiple sub-steps or multiple stages, which are not necessarily completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be performed alternately or simultaneously with other steps or parts of the sub-steps or stages of other steps.

[0035] Bifidobacterium breve directly converts LA to CLA by expressing a single gene encoding the linoleic acid isomerase BBI. Under the same conditions, the CLA conversion rate of Bifidobacterium breve catalyzed by the single enzyme (BBI) is typically 40-60%. However, in the four common Bifidobacterium species, including Bifidobacterium breve, the biotransformation of CLA generally occurs during the growth stage of the strain. The mainstream hypothesis suggests that the CLA biotransformation of Bifidobacteria is a defense mechanism against LA stress. Therefore, the delicate balance between free LA stress and Bifidobacterium growth and metabolism is a crucial factor determining CLA conversion during the process. This balance not only affects CLA yield but may also determine the survival and metabolic activity of the strain.

[0036] To address the problem of low CLA yield from Bifidobacterium breve biotransformation, this application provides a simple method to increase the yield of CLA during the preparation process by the strain.

[0037] One aspect of this application provides the use of L-arginine in the production of conjugated linoleic acid.

[0038] Another aspect of this application provides a method for improving the bioproduction of conjugated linoleic acid from Bifidobacterium breve, the method comprising:

[0039] L-arginine was added to a system that uses linoleic acid as a substrate and utilizes Bifidobacterium breve to bioproduce conjugated linoleic acid.

[0040] In some embodiments, the concentration of L-arginine in the system is 0.2 g / L to 5 g / L. Exemplary values ​​include, for example, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, or any range or value between two values. For example, 0.5 g / L to 2.5 g / L.

[0041] The Bifidobacterium shortis used in the methods described in this regard may be any common Bifidobacterium shortis known in the art, including but not limited to Bifidobacterium shortis SHMB8001 with accession number CGMCC No.32608, Bifidobacterium shortis C11 (CCFM683) with accession number CGMCC No.11828, and Bifidobacterium shortis FBJCP2M1 with accession number GDMCC No.60934.

[0042] In some embodiments, the concentration of linoleic acid in the system is 0.5 g / L to 1 g / L. Exemplary values ​​include, for example, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, or any range or value between two such values.

[0043] In some embodiments, the conjugated linoleic acid includes or is c9,t11-CLA.

[0044] In some embodiments, the system described in the method is used for anaerobic culture. The anaerobic culture time is, for example, less than 72 hours.

[0045] In some implementations, for example, when the concentration of arginine in the system is 2.5 g / L, a large accumulation of CLA can be observed after 15 h, with a total CLA conversion rate reaching 78.4%, of which c9,t11-CLA accounts for the highest proportion of 95%, and t9,t11-CLA accounts for only 5%. With the extension of culture time, the increase in total CLA conversion rate tends to stabilize, reaching a peak at 32 h to 48 h, with the proportion of c9,t11-CLA showing a decreasing trend while the proportion of t9,t11-CLA shows an increasing trend.

[0046] In some implementations, the anaerobic culture time is, for example, more than 15 hours and less than 72 hours, such as 15 hours to 32 hours.

[0047] In some implementations, the anaerobic culture temperature is exemplarily 37°C.

[0048] In some embodiments, the method for improving the bioproduction of conjugated linoleic acid by Bifidobacterium breve includes the following steps:

[0049] Seed culture of *Bifidobacterium breve* was inoculated into mMRS broth medium containing 0.5 g / L–1 g / L linoleic acid and 0.5 g / L–2.5 g / L L-arginine, and cultured anaerobically to prepare the culture; and,

[0050] Conjugated linoleic acid was isolated from the culture.

[0051] The inoculation amount of seed liquid can be 2% (v / v) for example.

[0052] In another aspect of this application, a product portfolio for the production of conjugated linoleic acid is provided, the product portfolio comprising Bifidobacterium breve, L-arginine, and a culture medium suitable for culturing Bifidobacterium breve.

[0053] In some embodiments, the product portfolio also includes linoleic acid.

[0054] As mentioned above, the Bifidobacterium breve can be a conventional Bifidobacterium breve in the art, such as Bifidobacterium breve SHMB8001 with accession number CGMCC No. 32608, Bifidobacterium breve C11 (CCFM683) with accession number CGMCC No. 11828, and Bifidobacterium breve FBJCP2M1 with accession number GDMCC No. 60934.

[0055] In some embodiments, the L-arginine in the product combination is dissolved in a culture medium at a concentration of 0.2 g / L to 5 g / L. Exemplary values ​​include, for example, 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L, 1 g / L, 1.5 g / L, 2 g / L, 2.5 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, or any range or value between two values. For example, 0.5 g / L to 2.5 g / L.

[0056] In some embodiments, the product combination contains linoleic acid and is dissolved in a culture medium at a concentration, for example, 0.5 g / L to 1 g / L.

[0057] In some embodiments, the method includes the following steps:

[0058] A strain of Bifidobacterium breve isolated from breast milk, SHMB8001 (deposited at the China General Microbiological Culture Collection Center, CGMCC No. 32608), was activated using mMRS broth medium to prepare seed culture.

[0059] mMRS broth (control group) and mMRS broth supplemented with L-arginine at final concentrations of 0.5 g / L and 2.5 g / L (experimental group) were prepared separately.

[0060] The seed culture was inoculated into the above culture medium at a 2% inoculation rate, and free linoleic acid substrate was added at a final concentration of 0.5 g / L. The culture was anaerobic at 37℃ for 0, 5, 8, 15, 24, 32, 48, and 72 h. After the culture was completed, the fermentation supernatant was collected for fatty acid extraction and detection.

[0061] When the amount of L-arginine added was 2.5 g / L and the fermentation time was 32 h, the CLA conversion rate of the strain increased from 57% to 83%. The product was mainly c9,t11-CLA, accounting for 93% of the total CLA, and contained a small amount of t9,t11-CLA isomers, accounting for 7% of the total CLA.

[0062] The following are some examples.

[0063] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, reference should be made to the guidelines given in this application, or to experimental manuals or conventional conditions in the art, or to the conditions recommended by the manufacturer, or to experimental methods known in the art.

[0064] Example 1

[0065] Preparation of mMRS broth medium (1L): De Man, Rogosa, and Sharpe (MRS) medium containing cysteine, with the following formulation: 10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g glucose, 1 mL Tween 80, 0.2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g diamine citrate, 0.02 g magnesium sulfate, 0.01 g manganese sulfate, 0.5 g L-cysteine;

[0066] Preparation of mMRS solid culture medium (1L): 10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g glucose, 1 mL Tween 80, 0.2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g diamine citrate, 0.02 g magnesium sulfate, 0.01 g manganese sulfate, 0.5 g L-cysteine, 20 g agar powder; Free linoleic acid (LA) stock solution (30 g / L, 20 mL): 600 mg LA, 400 mg Tween 80, diluted to 20 mL with sterile water, vortexed at room temperature for 20 min until fully emulsified, filtered through a sterile 0.22 μm aqueous filter membrane for sterilization, dispensed into 1 mL portions, and stored at -20°C protected from light. To avoid demulsification after freeze-thaw cycles, vortex for 5 min before use. L-arginine stock solution (150 g / L): 1.5 g L-arginine, diluted to 10 mL with sterile water, vortexed at room temperature for 5 min until the fixed powder is fully dissolved.

[0067] Reagents related to fatty acid detection, such as hexane, isopropanol, and methanol, were all analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd.; methyl esterification reagents, such as diazomethane, were purchased from Bailingwei Chemical Reagent Co., Ltd.; pentadecanoic acid, linoleic acid (LA), and conjugated linoleic acid (CLA) with a purity >99.9% were all purchased from Nu-chek, Inc., USA; and reagent kits and enzyme-free consumables related to qpCR detection were purchased from Nanjing Novizan Biotechnology Co., Ltd.

[0068] To evaluate the biotransformation capacity of Bifidobacterium acnes (CLA) by biotransformation, a glycerol-preserved bacterial strain was taken from a -80°C freezer and streaked onto mMRS solid medium using a 5 μL disposable inoculation loop. The culture was then anaerobic at 37°C for 24–48 h until single colonies appeared. Single colonies were picked and anaerobically cultured in mMRS broth at 37°C for 20 h to activate the strain. This process was repeated to prepare a seed culture. The seed culture was then inoculated at a 2% inoculation rate into mMRS broth containing L-arginine at final concentrations of 0, 0.5, and 2.5 g / L, respectively, with linoleic acid added as a substrate at a final concentration of 0.5 g / L. The cultures were then statically incubated at 37°C for 72 h under anaerobic conditions. After incubation, the cultures were centrifuged at 12000g for 5 min, and the fermentation supernatant and bacterial precipitate were collected at different time points for fatty acid detection and bacterial density analysis.

[0069] Bacterial density analysis

[0070] 1 mL of the collected bacterial pellet was resuspended in 1 mL of PBS, and the bacterial culture was transferred to a 96-well plate. The absorbance at 600 nm was measured using a microplate reader. This was used to evaluate the growth of the strain under different culture conditions. The bacterial density (OD) of *Bifidobacterium breve* under different treatments was also measured. 600 The values ​​were used to plot the corresponding growth curves. For example... Figure 1 As shown, supplementation with exogenous L-arginine (final concentrations of 0.5 g / L and 2.5 g / L) significantly increased the bacterial density of *Bifidobacterium breve* during the stable growth phase, reaching 0.62 (p<0.05) and 0.71 (p<0.001), respectively. This indicates that exogenous arginine supplementation helps *Bifidobacterium breve* cope with free LA stress in a concentration-dependent manner.

[0071] Fatty acid detection:

[0072] (1) Fatty acid extraction

[0073] Take 1 mL of the collected fermentation supernatant into a 5 mL glass serum bottle, add 100 μL of 2 g / L internal standard stock solution (n-pentadecanoic acid C15:0), an equal volume of isopropanol, and 2 volumes of n-hexane, and extract thoroughly by shaking at 2000 rpm for 30 s; use a pipette to transfer the upper layer (containing the n-hexane layer of fatty acids) into a new glass serum bottle.

[0074] (2) Fatty acid methyl esterification and GC-MS sample preparation

[0075] The hexane liquid in the serum vial was dried using a nitrogen evaporator. 400 mL of methanol and 60 μL of (trimethylsilyl) diazomethane were added to induce fatty acid methyl esterification. After standing at room temperature for 15 min, the methyl esterification was complete if the yellow color of the solution did not fade. Finally, the liquid in the vial was dried again with nitrogen, and 1 mL of hexane was added to fully dissolve the oily residue adhering to the vial walls. The solution was then transferred to a 2 mL sample vial and stored at -20 °C or directly analyzed by GC-MS.

[0076] (3) GC-MS detection conditions

[0077] According to the literature description [1] GC-MS analysis was performed on methylated fatty acid samples. An RT-5MS column was used with helium as the carrier gas and split injection was employed. The column temperature program was as follows: initial column temperature 180 ℃, held for 3 min; increased to 190 ℃ at 10 ℃ / min, held for 3 min; then increased to 220 ℃ at 5 ℃ / min, held for 1 min; finally increased to 230 ℃ at 2 ℃ / min, held for 18 min. The injection well and detector temperatures were maintained at 240 ℃, the ion source temperature was 230 ℃, and the electron energy was 70 eV. The concentrations (C) of the substrate LA and product CLA were calculated based on the ratio of the peak area of ​​different target analytes to the peak area of ​​the internal standard. CLA conversion rate was used as an indicator of the biotransformation ability of Bifidobacterium CLA, and the conversion rate was calculated using the following formula: CLA conversion rate (%) = C CLA / C (CLA+LA) ×100. For example... Figures 2A to 2F As shown, the chromatograms display the peaks of typical fatty acids in each group. The results show that Bifidobacterium breve SHMB8001 converts LA into two CLA isomers, c9,t11-CLA and t9,t11-CLA.

[0078] The CLA generation rate for each group was calculated using the internal standard method, and the results are as follows: Figure 3As shown, the strain exhibits rapid CLA accumulation after entering the logarithmic phase, followed by a plateau in CLA accumulation after entering the stationary phase. When the L-arginine addition is 2.5 g / L and the fermentation time is 32 h, the CLA conversion rate of the strain increases from 57% to 83%, with c9,t11-CLA being the predominant product, accounting for 93% of the total CLA, and containing a small amount of t9,t11-CLA isomers, accounting for 7% of the total CLA. These are the optimal conditions for the preparation of conjugated linoleic acid. Furthermore, as... Figure 4 As shown, the relative conversion activity of CLA (OD) 233 / OD 600 The same trend was also observed, with the exogenous arginine group showing the highest value, with its relative activity increasing by about 65% compared to the control group.

[0079] Extraction and RT-qPCR analysis of Bifidobacterium breve RNA

[0080] Cultured Bifidobacterium cells were collected by centrifugation (4℃, 6000g, 5 min). Total RNA was extracted from Bifidobacterium according to the instructions of the bacterial RNA extraction kit (Novizan R403-01), and the RNA content was detected using a NanoDrop micro-spectrophotometer. 1 μg of RNA was used to prepare a reaction system for first-strand cDNA synthesis according to the instructions of the genomic-detyped one-step reverse transcription kit (Novizan R333-01); real-time quantitative PCR (RT-qPCR) analysis was performed according to the instructions of the SYBR Mix kit (Novizan Q712-02). Reference Methods [2] Using 16S rRNA as the housekeeping gene, and utilizing 2 -ΔΔCt The transcriptional levels of the bbi and argR genes in *Bifidobacterium breve* were assessed using a method. The corresponding primers are listed in Table 1. RT-qPCR analysis was performed using a Bio-Rad CFX96 multicolor real-time PCR detection system. The RT-qPCR conditions were as follows: 95℃ for 3 min; 95℃ for 15 s, 50℃ for 30 s, 40 cycles; melting curves were acquired using the instrument's default program. Results are shown below. Figure 5A and Figure 5B As shown, arginine upregulated the gene expression of Bifidobacterium breve linoleic acid isomerase BBI and transcription factor ArgR in a concentration-dependent manner; when the L-arginine concentration was 2.5 g / L, the expression levels of the two were upregulated by approximately 8-fold and 3-fold, respectively.

[0081] Table 1. List of primers used in the experiment

[0082]

[0083] Promoter prediction and P bbi Probe preparation

[0084] Based on the genome of *Bifidobacterium breve* SHMB8001, the CD region and upstream and downstream sequences of the *bbi* gene were determined by BLAST alignment. These sequences were then uploaded to the Softberry-BPROM server (http: / / www.softberry.com) for promoter prediction. Based on the prediction results, specific primers (see Table 1) were designed for high-fidelity enzyme PCR amplification and product purification to obtain the *bbi* promoter (P...). bbi The probe was tested for correctness using 1% gel electrophoresis and Sanger sequencing.

[0085] Heterologous expression and purification of recombinant protein ArgR-his

[0086] Following our previously established protocol, purified recombinant protein ArgR-his, fused with a 6×his-tag at the C-terminus, was obtained using the *E. coli* system and nickel affinity chromatography. In short, specific primers were designed based on the argR coding sequence (Table 1). Using the *Bifidobacterium breve* SHMB8001 genome as a template, the target fragment was amplified by high-fidelity enzyme PCR. The recombinant plasmid pET28a-argR-his was constructed by double enzyme digestion and transformed into *E. coli* DH5α competent cells. Positive colonies were selected based on kanamycin resistance, and after verification by Sanger sequencing, the strain was expanded and the plasmid was extracted. This was to facilitate the transformation of pET28a-argR-his into the protein expression host *E. coli* BL21(DE). Induction culture was performed under the following conditions: LB medium (50 μg / mL kanamycin), 37°C, 200 rpm, until OD500. 600 =0.6, then add 0.2mM IPTG and induce culture at 16℃ and 180 rpm for 16h. The pET28a empty vector plasmid was used as a control. After induction culture, the bacterial cell pellet was harvested by centrifugation (12000 × g, 10 min, 4°C). The bacterial cells were reconstituted with PBS (1:10 m / v), sonicated (3 s on, 2 s off, 15 min), and then analyzed for protein expression by SDS-PAGE and Western Blot. Subsequently, protein purification was performed using a nickel ion affinity chromatography column. After elution with an imidazole gradient, the purified protein was analyzed by SDS-PAGE and Western Blot.

[0087] Electrophoretic Mobility Shift Assay (EMSA)

[0088] As previously described, the purified protein was exchanged into EMSA buffer (10 mM Tris-HCl, 50 mM NaCl, 2.5 mM MgCl2·6H2O, 0.1 mM EDTA-Na2, 0.1 mM dithiothreitol, 10% (v / v) glycerol, pH 7.4). 100 nM Pbbi probe was mixed with the purified protein at different molar ratios in the EMSA buffer and incubated at 25°C for 1 h. Electrophoresis, SYPRO staining, and imaging analysis were performed according to the EMSA kit (Thermo Fisher Scientific, USA) instructions. Briefly: Electrophoretic separation was performed using a 6% TBE-polyacrylamide gel at a constant voltage of 120 V for 1 h in pre-chilled 0.5×TBE electrophoresis buffer (89 mM Tris, 89 mM boric acid, 1 mM EDTA-Na2, pH 8.0).

[0089] like Figure 6 As shown, ArgR specifically binds to bbi, and L-arginine can significantly promote the binding between the two.

[0090] The changes in gene transcription levels and the results of EMSA analysis together indicate that the reason why arginine enhances the biotransformation of CLA by Bifidobacterium breve is related to the transcriptional regulation of bbi, that is, arginine promotes the production of CLA by upregulating the expression level of transcription factors.

[0091] In addition, the inventors also tested the formation of two CLA isomers at a substrate LA concentration of 1.0 g / L:

[0092] At a substrate LA concentration of 1.0 g / L, compared to the control group, simultaneous supplementation with arginine and LA resulted in a greater delay in both the lag phase and logarithmic phase of the strain's growth. This may be due to metabolic overload caused by both substances during the early stages of the strain's growth. (Based on OD...) 233 The cumulative CLA value is evaluated, and OD is used as the OD value. 233 / OD 600 Assess the relative CLA transformation activity per unit cell.

[0093] The results showed that at a substrate concentration of 1.0 g / L, the production of CLA was delayed until 48 h due to metabolic overload; however, the supplementation of exogenous arginine (0.5 and 2.5 g / L) still enhanced the relative activity of Bifidobacterium breve in the biotransformation of CLA; for example, at the fermentation endpoint (72 h), the relative activity OD of the arginine (2.5 g / L) group was significantly higher. 233 / OD 600 =22.6, compared to the control group (OD 233 / OD 600 =12.1) is approximately 1.87 times higher (see Figures 7A to 7C ).

[0094] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0095] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

[0096] References

[0097] [1] Gao He. Study on the mechanism of biotransformation of conjugated linoleic acid by Bifidobacterium [D]. Jiangnan University, 2020.

[0098] [2]Mei Y, Chen H, Yang B, et al. Computational Analysis and Heterologous Expression of BBI-like Proteins from Food-Grade BifidobacteriumSpecies Reveal Possibly a Key Factor in Conjugated Linoleic AcidBioconversion[J]. J Agric Food Chem, 2023,71(21):8093-8103.

Claims

1. Application of L-arginine in the production of conjugated linoleic acid.

2. A method for improving the bioproduction of conjugated linoleic acid by *Bifidobacterium breve*, characterized in that, The method includes: L-arginine was added to a system that uses linoleic acid as a substrate and utilizes Bifidobacterium breve to bioproduce conjugated linoleic acid.

3. The method for improving the bioproduction of conjugated linoleic acid by Bifidobacterium breve as described in claim 2, characterized in that, The concentration of L-arginine in the system is 0.2 g / L to 5 g / L.

4. The method for improving the bioproduction of conjugated linoleic acid by Bifidobacterium breve as described in claim 3, characterized in that, The concentration of L-arginine in the system is 0.5 g / L to 2.5 g / L.

5. The method for improving the bioproduction of conjugated linoleic acid by *Bifidobacterium breve* as described in any one of claims 2 to 4, characterized in that, The Bifidobacterium breve was selected from: Bifidobacterium breve SHMB8001 with accession number CGMCC No. 32608, Bifidobacterium breve C11 (CCFM683) with accession number CGMCC No. 11828, and Bifidobacterium breve FBJCP2M1 with accession number GDMCC No. 60934.

6. The method for improving the bioproduction of conjugated linoleic acid by *Bifidobacterium breve* as described in any one of claims 2 to 4, characterized in that, The concentration of linoleic acid in the system is 0.5 g / L to 1 g / L.

7. The method for improving the bioproduction of conjugated linoleic acid by *Bifidobacterium breve* as described in any one of claims 2 to 4, characterized in that, The conjugated linoleic acid includes C9,T11-CLA.

8. The method for improving the bioproduction of conjugated linoleic acid by *Bifidobacterium breve* as described in any one of claims 2 to 4, characterized in that, The method includes the following steps: Seed culture of *Bifidobacterium breve* was inoculated into mMRS broth containing 0.5 g / L–1 g / L linoleic acid and 0.5 g / L–2.5 g / L L-arginine, and cultured anaerobically to prepare the culture; and, Conjugated linoleic acid was isolated from the culture.

9. The method for improving the bioproduction of conjugated linoleic acid by Bifidobacterium breve as described in claim 8, characterized in that, The method satisfies any one of the following conditions: (1) The inoculum size of the seed solution is 2%; (2) The anaerobic culture temperature is 37℃; (3) The anaerobic culture time is less than 72 h.

10. A product portfolio for the production of conjugated linoleic acid, characterized in that, The product portfolio includes Bifidobacterium breve, L-arginine, a culture medium suitable for culturing Bifidobacterium breve, and optionally linoleic acid.

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