Application of Bifidobacterium animalis F1-7 in degradation of phenylalanine

Abstract

The present invention belongs to the field of microbial technology and specifically relates to the use of a strain of Bifidobacterium animalis F1-7 in degrading phenylalanine. The strain is used to degrade phenylalanine in vivo and in vitro, thereby improving inflammatory responses. The strain provided by the present invention is used to prepare a product or dairy product that degrades phenylalanine. The microbial preparation containing Bifidobacterium animalis F1-7 as an active ingredient can reduce phenylalanine content and serve as an auxiliary treatment for phenylketonuria.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to application of a strain of animal Bifidobacterium F1-7 in degrading phenylalanine. Background Art

[0002] Phenylketonuria (PKU) is an autosomal recessive genetic disease. Due to mutations in the Pah-enu2 gene, the activity of phenylalanine hydroxylase (PAH) in the patient's body is reduced or lost, resulting in the inability of phenylalanine to produce tyrosine through the normal metabolic pathway. The accumulation of phenylalanine and its bypass metabolites will have adverse effects on human organs, especially the nervous system.

[0003] The most common treatment for phenylketonuria (PKU) is dietary restriction, but this approach has drawbacks, including poor taste, nutrient deficiencies due to dietary restriction, high cost, and poor adherence. Numerous studies have demonstrated that probiotics can metabolize phenylalanine, enhance intestinal barrier function, and promote gut microbial balance, garnering increasing attention. Microbial degradation of phenylalanine may involve several different enzymes, including transaminases (ATases) (EC 2.6.1.5 and EC 2.6.1.58), decarboxylases (DCOOHases) (EC 4.1.1.25 and EC 4.1.1.53), and aromatic hydroxy acid dehydrogenases (EC 1.1.1.222). Using transcriptomic and metabolomics techniques, probiotics have been shown to metabolize phenylalanine via the phenylacetic acid, phenylpyruvic acid, and cinnamic acid pathways, thereby degrading phenylalanine.

[0004] It has been reported that nutritional bacterial powder can effectively reduce plasma phenylalanine levels in PUK patients, thereby preventing the accumulation of phenylalanine and its metabolites, which can damage the central nervous system and brain. Therefore, the development of safe and effective probiotic preparations and products to assist in the treatment of phenylketonuria is of great significance. However, developing a strain that can efficiently degrade phenylalanine and apply it to functional foods is an urgent challenge for those skilled in the art. Summary of the Invention

[0005] The purpose of the present invention is to solve the above problems existing in the prior art and proposes the use of a strain of Animal Bifidobacterium F1-7 in degrading phenylalanine. The strain has good ability to degrade phenylalanine in vivo and in vitro and improve inflammatory response.

[0006] The technical solution of the present invention is:

[0007] The present invention provides an application of Bifidobacterium animalis subsp. F1-7 in degrading phenylalanine. The Bifidobacterium animalis subsp. F1-7 was deposited in the China Center for Type Culture Collection on December 2, 2020, and the preservation address is the Collection Center of Wuhan University, Wuhan City, Hubei Province, and the preservation number is CCTCCNO: M2020833.

[0008] Furthermore, the application is the application of animal Bifidobacterium F1-7 in the in vitro degradation of phenylalanine; it has good in vitro phenylalanine degradation ability.

[0009] Furthermore, the application is the application of animal Bifidobacterium F1-7 in degrading phenylalanine in vivo; it has good ability to degrade phenylalanine in vivo and improve inflammatory response.

[0010] The present invention also provides the use of the animal Bifidobacterium F1-7 in preparing products that degrade phenylalanine.

[0011] Furthermore, the products include functional foods, medicines and health products.

[0012] The present invention also provides the use of the animal Bifidobacterium F1-7 in improving inflammatory response; Animal Bifidobacterium F1-7 can effectively improve the phenylalanine content in the serum and liver of mice and reduce the inflammatory response caused by high levels of phenylalanine.

[0013] The application of the animal Bifidobacterium F1-7 in the preparation of dairy products.

[0014] Furthermore, the dairy product is a fermented dairy product. The strain is used as an auxiliary starter in fermented milk to prepare a functional fermented milk that not only has a low phenylalanine content but also serves as a carrier for probiotics, which colonize the intestines and exert their probiotic functions.

[0015] The present invention further provides a use of a bacterial agent containing animal Bifidobacterium F1-7 in the adjuvant treatment of phenylketonuria. The bacterial agent is a microbial preparation with animal Bifidobacterium F1-7 as the main active ingredient, which can effectively and safely assist in the treatment of phenylketonuria.

[0016] Biomaterial sample deposit information:

[0017] Bifidobacterium animalis subsp. F1-7 is deposited in the China Center for Type Culture Collection, the collection address is the Collection Center of Wuhan University, Wuhan City, Hubei Province, the collection date is December 2, 2020, and the collection number is CCTCCNO: M2020833.

[0018] Beneficial effects of the present invention:

[0019] The present invention provides the use of Bifidobacterium animalis subsp. F1-7 in degrading phenylalanine. The strain F1-7 can efficiently degrade phenylalanine and improve or reduce the inflammatory response caused by high levels of phenylalanine. The strain is used in the preparation of phenylalanine-degrading products or dairy products, which can reduce the phenylalanine content and assist in the treatment of phenylketonuria. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the color development results of ferric chloride in mouse urine provided in an embodiment of the present invention;

[0021] Figure 2 This is a graph showing the results of mouse serum phenylalanine content provided by an embodiment of the present invention;

[0022] Figure 3 A graph showing the phenylalanine content in mouse liver according to an embodiment of the present invention;

[0023] Figure 4 This is a graph showing the results of mouse serum IL-6 levels provided in an embodiment of the present invention;

[0024] Figure 5 This is a graph showing the results of mouse serum IL-1β content provided by an embodiment of the present invention;

[0025] Figure 6 This is a graph showing the TNF-α content in mouse liver provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0026] To further understand the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0027] Example 1

[0028] Screening and Cultivation of Bifidobacterium animalis F1-7

[0029] This strain was isolated from infant feces. The specific method is as follows: A 10-fold dilution of the fecal sample is used for strain isolation, and 100 μL is spread onto a semi-selective plate using MRS-cys supplemented with X-gal. The plate is incubated at 37°C for 48 hours, then allowed to stand in air for 4 hours. Blue colonies are picked, purified, and then identified for 16S rRNA. Genomic DNA from the bacteria is extracted using a kit. PCR is then performed using the genomic DNA as a template and universal primers to amplify the 16S rRNA. After the reaction is complete and the target fragment is confirmed by agarose gel electrophoresis, the PCR amplification product is sent to Shanghai Bioengineering for sequencing. The sequence was submitted to the GenBank database, and a homology search was performed using BLAST to find the 16SrDNA of the known taxonomic species with the highest homology to the target sequence. The bacterium was determined to be animal Bifidobacterium F1-7 strain, which is consistent with the strain in the patent with application number 202011586443.5, entitled A strain of animal Bifidobacterium subspecies F1-7, a composition, a feces drying compound preparation, and a fruit-flavored dairy product.

[0030] The screened animal Bifidobacterium F1-7 was inoculated at a 2% (v / v) inoculum into 10 ml of MRS liquid medium and incubated at 37°C for 24 hours. The activated strain was washed with 25 mmol / L Tris-HCl (pH 7.4), prepared as a suspension, inoculated into L-phenylalanine-specific selective medium, and incubated at 37°C for 72 hours. The suspension was then centrifuged at 6000 rpm for 15 minutes, and the supernatant was collected.

[0031] Example 2 Determination of phenylalanine content

[0032] Prepare the ninhydrin colorimetric reagent by diluting 0.5g of ninhydrin, 0.3g of fructose, 11.1g of Na₂HPO₄.12H₂O, and 6.0g of KH₂PO₄ to 100mL. Dissolve 0.1000g of dry glycine in distilled water to 100mL, pipette 2.00mL into 100mL to obtain a 20μg / mL solution. Dilute the solution to 2-20μg / mL. Add 2.00mL of the diluent to a test tube and 1.00mL of the ninhydrin colorimetric reagent. After mixing, heat the dilution in a boiling water bath for 15 minutes. Perform a blank test. Immediately cool the solution in cold water, add 5.00mL of 40% ethanol solution, and shake vigorously for several minutes until the brown-red color returns. Let the solution stand at room temperature for 15 minutes, adjust the concentration to zero with distilled water, and measure the absorbance at 570nm. Construct a standard curve with the molar number of glycine as the horizontal axis and the absorbance as the vertical axis. Take 2.00 mL of the supernatant culture medium of the bacteria, dilute it appropriately, and repeat the above steps. The phenylalanine degradation rate of the strain is calculated based on the change in phenylalanine concentration in the system before and after the inoculation of the strain.

[0033] According to the above determination steps, the ninhydrin colorimetric method was used to determine the phenylalanine content in the culture medium before and after inoculation of F1-7, and then the phenylalanine degradation rate of F1-7 in vitro was calculated. The results are shown in Table 1 below.

[0034] Table 1 Phenylalanine degradation rate data table

[0035] Strain name Phenylalanine degradation rate % Bifidobacterium animalis F1-7 <![CDATA[10.0918±1.7963 a ]]> LGG <![CDATA[5.7501±0.1609 b ]]>

[0036] The phenylalanine degradation rate of animal Bifidobacterium F1-7 was 10.0918%, which was significantly higher than that of Lactobacillus rhamnosus LGG, whose degradation rate was 5.7501%.

[0037] Example 3 Construction of a high phenylalanine mouse model and in vivo efficacy verification

[0038] Thirty mice were acclimated for 7 days with free access to food and water during the experiment. Each group consisted of 10 mice; all three groups were fed a standard diet and allowed to eat freely 30 minutes after gavage. Model establishment: From days 1 to 28, mice in the model and F1-7 intervention groups were gavaged once daily with a 4.2 mmol / kg Phe solution. Urine was collected for a ferric chloride colorimetric assay to assess model success. Simultaneously with model establishment, mice were treated with F1-7 intervention: a negative control group (gavage with 100 μL / 10 g PBS) and an experimental group (gavage with a suspension of 109 CFU / mL prepared from the activated strain F1-7 described in Example 1). During this period, mice were allowed free access to food. Mice were sacrificed on day 28 of gavage. Blood and liver tissue were collected. Blood was centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant was carefully aspirated with a pipette and frozen at -80°C for later use. Weighed liver tissue was minced and homogenized at a ratio of 1:9 to prepare a 10% homogenate. The homogenate was then centrifuged at 3000 rpm for 10 minutes at 4°C. The supernatant was collected and stored in an ice-water bath for subsequent analysis. Phenylalanine levels in mouse serum and liver homogenate were measured using commercial kits (Nanjing Jiancheng, China). IL-6 and IL-1β levels in mouse serum and TNF-α levels in liver homogenate were measured using commercial kits (Nanjing Jiancheng, China).

[0039] (1) In vivo efficacy verification of Bifidobacterium animalis F1-7

[0040] Depend on Figure 2 It can be seen that the serum phenylalanine content of the model group mice (Mod group) increased by 103.79%, while the liver phenylalanine content increased by 113.11%. Figure 1 The urine ferric chloride color development result was positive, which met the diagnostic criteria for phenylketonuria, preliminarily indicating that the model was successfully constructed. The results are shown in Figure 2 .

[0041] Depend on Figure 2 and Figure 3 The results showed that after intervention with animal Bifidobacterium F1-7, the phenylalanine levels in the serum and liver of mice were significantly reduced. Although there was still a gap with the levels of normal mice, the serum phenylalanine level of mice in the animal Bifidobacterium F1-7 intervention group was significantly reduced by 23.35% compared with the model group, and the liver phenylalanine level was significantly reduced by 31.64% compared with the model group. The results of animal experiments show that animal Bifidobacterium F1-7 has the prebiotic function of efficiently degrading phenylalanine, which lays the foundation for the application of animal Bifidobacterium F1-7.

[0042] (2) Determination of inflammatory factors in mouse serum and liver tissue homogenate

[0043] In the high phenylalanine mouse model, a large amount of IL-6, IL-1β, and TNF-ɑ are released, which triggers an inflammatory response in mice. Figure 4 、 Figure 5 and Figure 6 As shown in the results, compared with the blank group, the levels of IL-6, IL-1β, and TNF-ɑ in the model group were significantly increased (p<0.05). This indicates that the modeling led to the production of pro-inflammatory factors in the mouse blood. Compared with the model group, the serum levels of cytokines IL-6 and IL-1β in mice treated with F1-7 were significantly reduced, by 19% and 7%, respectively. At the same time, the level of cytokine TNF-ɑ in the mouse liver decreased by about 5%, indicating that F1-7 can indeed alleviate the inflammatory response of mice caused by phenylalanine.

[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein with equivalents. Any modifications, equivalent replacements, and modifications made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. The application of a strain of Bifidobacterium animalis subsp. F1-7 in the degradation of phenylalanine. The Bifidobacterium animalis F1-7 was deposited in the China Center for Type Culture Collection on December 2, 2020. The preservation address is the Collection Center of Wuhan University, Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M 2020833.

2. The use according to claim 1, characterized in that The application is the application of animal Bifidobacterium F1-7 in degrading phenylalanine in vitro.

3. The use according to claim 1, characterized in that The application is the application of animal Bifidobacterium F1-7 in degrading phenylalanine in vivo.

4. Use of the animal Bifidobacterium F1-7 according to claim 1 in the preparation of a product for degrading phenylalanine.

5. The use according to claim 4, characterized in that The products include functional foods, medicines and health products.

6. Use of the animal Bifidobacterium F1-7 according to claim 1 in improving inflammatory response.

7. Use of the animal Bifidobacterium F1-7 according to claim 1 in the preparation of dairy products.

8. The use according to claim 7, characterized in that The dairy product is a fermented dairy product.

9. Use of a bacterial agent containing Bifidobacterium animalis F1-7 in the adjuvant treatment of phenylketonuria.

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