Lactobacillus rhamnosus and application thereof in preparation of composition with antioxidant and anti-inflammatory effects

Abstract

The invention relates to lactobacillus rhamnosus and application of the lactobacillus rhamnosus in preparation of a composition with antioxidant and anti-inflammatory effects, the lactobacillus rhamnosus RX-LA303 is separated from yoghourt, black truffle is subjected to solid state fermentation through the lactobacillus rhamnosus RX-LA303, then a fermentation product is subjected to ultrasonic extraction, centrifugation, concentration, precipitation and freeze-drying, and a black truffle fermentation extract is obtained. Experimental results show that the total polysaccharide yield of the prepared perigord truffle fermentation extract is 26% or above, is increased by 159% compared with the polysaccharide yield of an unfermented perigord truffle extract and is increased by 27.6% or higher compared with other strain fermentation, the polysaccharide content in the extract is remarkably increased, and therefore the oxidation resistance of the extract is improved. Besides, the perigord truffle fermentation extract can be combined and matched with other functional substances to prepare a composition with antioxidant and anti-inflammatory effects, and the composition can improve the nutrient absorption rate of a human body and has a good prospect in the functional food market.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation, and particularly relates to a lactobacillus rhamnosus and application thereof in preparation of a composition with antioxidant and anti-inflammatory effects. BACKGROUND

[0002] Black truffle is a rare and expensive fungus, which has unique and rich flavor. Black truffle contains dozens of active ingredients such as polysaccharides, polyphenols, sterols and amino acids. Black truffle polysaccharides have various health care effects such as antioxidant, immune regulation, blood sugar reduction, anti-tumor and the like. In addition, black truffle also contains rich nutritional ingredients and bioactive substances such as sphingolipids and triterpenoids, and therefore has great application potential in the fields of food, medicine and cosmetics.

[0003] At present, hot water extraction method is commonly used to extract polysaccharides from black truffle. In this method, black truffle powder is usually oscillated and extracted in hot water at 70-90°C for 2-3 hours, and then the crude polysaccharides are obtained through steps such as centrifugation, concentration and alcohol precipitation (usually using anhydrous ethanol). This method is simple in operation and low in cost, but also has obvious disadvantages: low extraction efficiency, resulting in low content of target components in the extract and unstable product quality. Ultrasonic-assisted extraction method can accelerate the dissolution of components in black truffle through cavitation effect of ultrasonic waves, and improve the extraction efficiency. However, if the intensity and time of ultrasonic waves are not properly controlled, not only the structure of black truffle polysaccharides may be damaged, but also the selectivity of the method is limited when used alone for the extraction of complex components, and the polysaccharide yield is even decreased due to more impurities in the extract.

[0004] In recent years, microbial fermentation technology plays an increasingly important role in the extraction of modern active substances. It is widely used in the enrichment and transformation of active ingredients of natural products due to its green and efficient characteristics. Microorganisms not only naturally produce various bioactive substances in the primary or secondary metabolic process, but also secrete rich enzymes that can convert substrates in the culture medium into more valuable active substances, and efficiently destroy the cell wall and cell membrane structure to release intracellular active ingredients, thereby greatly improving the extraction efficiency. SUMMARY

[0005] The present application aims to overcome the deficiencies of the prior art and provide a lactobacillus rhamnosus and application thereof in preparation of a composition with antioxidant and anti-inflammatory effects. The lactobacillus rhamnosus provided in the present application is used for solid-state fermentation of black truffle, and then the fermentation product is subjected to ultrasonic extraction, centrifugation, concentration and freeze-drying to obtain black truffle fermentation extract. The content of polysaccharides in the extract is significantly improved, and the black truffle fermentation extract can be combined with other functional substances to prepare a composition with antioxidant and anti-inflammatory effects, which can be used in the preparation of antioxidant and immunity-enhancing food.

[0006] To achieve the above object, the technical scheme adopted by the present application is: In a first aspect, the present application provides a Lacticaseibacillus rhamnosus RX-LA303, which has been deposited with the Guangdong Microbial Culture Collection Center on July 18, 2025, and has a deposit number of GDMCC No: 66713, a deposit address of No. 59, Building 5, 100, Martyrs' Road, Guangzhou, and a 16S rRNA gene sequence as shown in SEQ ID NO: 1.

[0007] It should be noted that the International Committee on Systematics of Prokaryotes (ICSP) reclassified Lactobacillus in 2020, and some species were divided into new genera. The "Lacticaseibacillus rhamnosus" (formerly known as Lactobacillus rhamnosus) was classified into the new genus "Lacticaseibacillus", and the scientific name was updated to Lacticaseibacillus rhamnosus.

[0008] In a second aspect, the present application provides a microbial agent, which comprises the Lacticaseibacillus rhamnosus of the first aspect.

[0009] In a third aspect, the present application provides the use of the Lacticaseibacillus rhamnosus of the first aspect in the preparation of an antioxidant food.

[0010] In a fourth aspect, the present application provides a preparation method of a black truffle fermentation extract, comprising the following steps: S1, activating the Lacticaseibacillus rhamnosus RX-LA303 of the first aspect to obtain an RX-LA303 bacterial solution with a viable cell count of 1×10 8 -3×10 9 CFU / mL; S2, mixing black truffle powder and deionized water to obtain a solid fermentation material; wherein the mass ratio of the black truffle powder to the deionized water is 1: (0.4-0.8); S3, inoculating the RX-LA303 bacterial solution into the solid fermentation material at 3-5 wt% of the mass of the solid fermentation material for anaerobic fermentation culture, and obtaining the black truffle fermentation product after the fermentation is completed; wherein the temperature of the anaerobic fermentation is 35-37℃, and the time is 1-4d; S4, mixing the black truffle fermentation product with deionized water, then performing ultrasonic water extraction, centrifugation, collecting the supernatant, adding anhydrous ethanol after concentration, standing and precipitating, centrifugation, and freeze-drying the precipitate to obtain the black truffle fermentation extract; wherein the solid-liquid ratio of the black truffle fermentation product to the deionized water is 1g: (20-25)mL.

[0011] Preferably, in the step S1, the step of the activation treatment is: inoculating Lactobacillus rhamnosus RX-LA303 into the activation culture medium and culturing in a shaking flask at 35-37℃, 100-150r / min for 20-30h.

[0012] Preferably, in the step S4, the parameters of the ultrasonic treatment are: the frequency of the ultrasonic is 20-30kHz, the temperature is 80-90℃, the time is 20-40min, and the volume ratio of the supernatant and anhydrous ethanol is 1: (3-4).

[0013] In a fifth aspect, the present application provides a black truffle fermentation extract prepared by the method for preparing the black truffle fermentation extract in the fourth aspect.

[0014] In a sixth aspect, the present application provides a composition with antioxidant and anti-inflammatory effects, comprising the following components in parts by mass: 3-8 parts of collagen tripeptide, 5-10 parts of concentrated blackcurrant juice, 0.5-5 parts of concentrated blueberry juice, 0.1-1 part of hawthorn powder, 0.1-1 part of fishy grass powder, and 0.1-2 parts of the black truffle fermentation extract in the fifth aspect. The collagen tripeptide in the composition can repair the inflammatory damaged tissues (such as mucous membrane and connective tissue); the anthocyanins in the blackcurrant and blueberry can enhance the antioxidant capacity of the extract and further inhibit the inflammation mediated by oxidative stress; the organic acids in the hawthorn powder and the volatile oil (such as decanoyl acetaldehyde) in the fishy grass can assist in regulating immunity, while exerting antioxidant and anti-inflammatory effects, complementing the anti-inflammatory and antioxidant mechanisms of the black truffle fermentation extract, and ultimately improving the overall antioxidant effect.

[0015] In a seventh aspect, the present application provides the use of the composition with antioxidant and anti-inflammatory effects in the sixth aspect in the preparation of antioxidant food.

[0016] Compared with the prior art, the present application has the following beneficial effects: The present application relates to a lactobacillus rhamnosus and its application in preparing a composition with antioxidant and anti-inflammatory effects, the lactobacillus rhamnosus RX-LA303 of the present application is isolated from yogurt, through solid state fermentation of the bacteria on black truffle, and then the fermentation product is subjected to ultrasonic extraction, centrifugation, concentration, precipitation and freeze-drying to obtain a black truffle fermentation extract. The experimental results show that the yield of the black truffle fermentation extract prepared by the present application is more than 26%, which is 159% higher than that of the unfermented black truffle extract, and 27.6% or more higher than that of other strains, thus achieving a significant increase in the polysaccharide content in the extract, thereby improving the antioxidant performance of the extract. In addition, the black truffle fermentation extract can be combined with other functional substances to prepare a composition with antioxidant and anti-inflammatory effects, the composition can improve the absorption rate of nutrients by the human body, and has the effects of antioxidant and immune enhancement, and has a good prospect in the functional food market. DETAILED DESCRIPTION

[0017] In order to better illustrate the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific examples.

[0018] The raw materials used in the following examples and comparative examples are as follows: Black truffle: purchased from Yunnan Huomu Trade Co., Ltd., black truffle dry slices; Other materials, reagents, etc. used in the examples can be obtained from commercial channels unless otherwise specified.

[0019] Example 1 Screening and isolation of strains 1. Strain screening and purification (1) Strain source: traditional fermented milk was collected in Dali, Yunnan in April 2023 and placed in a sterile test tube for cold chain transportation back to the laboratory for standby use.

[0020] (2) Preparation of sample: ① 8 mL of sterilized normal saline (0.85%) was placed in a sterile conical flask, then 2 mL of fermented milk obtained in step (1) was added, and the mixture was stirred and oscillated at 4°C for 30 min to obtain a sample original solution, which was labeled as No. 1. ② The sample original solution of step ① was diluted to prepare samples with different concentration gradients, which were 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , respectively, labeled as No. 2, 3, 4, 5, 6, 7, respectively, for standby use.

[0021] (3) Preparation of MRS culture medium: Casein peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, glucose 5.0 g, sodium acetate 5.0 g, diamine citric acid 2.0 g, Tween 80 1.0 g, K2HPO4 2.0 g, MgSO4·7H2O 0.2 g, MnSO4·H2O 0.05 g, CaCO3 20.0 g, agar 13.0 g, distilled water 1.0 L; after mixing the above raw materials, adjust the pH to 6.8, stir the bacterial solution, sterilize at 121℃, 0.1 MPa for 20 min, pour the sterilized medium into a flat dish, and cool it down before use.

[0022] (4) Culturing strains: 1-7 solutions were respectively coated on MRS medium using a coater, and cultured at 37℃ under anaerobic conditions for 48h; (5) The colonies were selected according to the following colony characteristics: Diameter 1-2mm, round colony, neat edge, slightly white with convex in the middle, calcium-dissolving ring above 5mm.

[0023] (6) Preliminary screening: the single colonies meeting the conditions of step (5) were inoculated into MRS medium using streaking method for isolation and purification culture (cultured at 37℃ under anaerobic conditions for 48h, repeated for 2-3 times), and 6 strains were obtained, named as J01, J02, J03, J04, J05, J06, and stored at -20℃ for standby.

[0024] (7) Rescreening: ①Preparation of black truffle fermentation extract, the specific steps are as follows: S1, the above stored strains J01, J02, J03, J04, J05, J06 were activated respectively, and 6 groups of bacterial solution with viable count of 2×10 9 CFU / mL were obtained; S2, dry black truffle was crushed through a 60 mesh sieve to obtain black truffle powder, and the black truffle powder was mixed with deionized water at a mass ratio of 1:0.6 to obtain a solid screening medium, which was sterilized for standby; S3, the 6 groups of bacterial solution obtained in step S1 were inoculated at an inoculation amount of 4wt% (based on the mass of solid fermentation material) and evenly sprayed on the solid screening medium obtained in step S2, and fermented and cultured at a temperature of 37℃ for 40h to obtain 6 groups of black truffle fermentation products; S4, the fermentation extracts of each group of black truffle were mixed with deionized water, then ultrasonic water extraction was performed, centrifuged, the supernatant was collected, and water extraction was repeated 3 times, then the supernatant of multiple extractions was mixed and concentrated, 60°C reduced pressure concentration was performed to 1 / 4 of the original volume, then 4 times the volume of anhydrous ethanol was added to the concentrated solution, 4°C was placed for 24h to produce a precipitate, 6000r / min centrifugation was performed for 30min, the precipitate was collected and freeze-dried to obtain 6 groups of black truffle fermentation extracts; wherein, the solid-liquid ratio of black truffle fermentation and deionized water was 1g:20mL, the ultrasonic frequency was 20kHz, the temperature was 85°C, and the time was 30min; the centrifugal speed after ultrasonic water extraction was 15000rpm, and the time was 5min.

[0025] ②Test the polysaccharide yield of each group of black truffle fermentation extracts 1g of each group of black truffle fermentation extracts was added to 5 times the mass of deionized water to mix, to obtain a test solution, 1.0ml of the test solution was accurately measured, 1.0ml of 5% phenol solution and 5.0ml of concentrated sulfuric acid solution were added under light-proof conditions, and mixed uniformly. After standing in a 30°C water bath for 30min, the absorbance value of the solution at 490nm was tested by an enzyme marker. The absorbance values at different concentrations were determined by the above method using glucose as the standard, and a standard curve was prepared. The polysaccharide concentration and polysaccharide yield in the test solution were calculated according to the standard curve. The polysaccharide yield calculation method is as follows: ; In the formula, x is the polysaccharide yield (%), C is the polysaccharide concentration contained in the test solution, V is the volume of the test solution, N is the total mass of the black truffle fermentation extract, and m is the mass of the dried black truffle. The polysaccharide yield of each group of black truffle fermentation extracts is shown in Table 1.

[0026] Table 1 Polysaccharide yield of each group of black truffle fermentation extracts Group / Performance Polysaccharide yield / % J01 10.32 J02 9.70 J03 14.24 J04 17.93 J05 21.41 J06 12.62 As can be seen from Table 1, the polysaccharide yield of the black truffle fermentation extract fermented by J05 is the highest, and the J05 strain selected is named RX-LA303.

[0027] Example 2 Identification of strain Morphological identification: the above strain RX-LA303 was inoculated in MRS medium and cultured at 37°C for 48h, the strain grew well, formed a milky white, round, opaque colony, the edge was neat, and the colony surface was moist, the bacterial morphology was short rod-shaped, no spores, and the gram staining reaction was positive.

[0028] Molecular biology identification: the 16S rRNA sequence of the strain RX-LA303 was amplified, and the amplification product was sequenced, and the 16S rRNA sequence was obtained as shown in SEQ ID NO. 1:

[0029] The 16S rRNA sequence information (SEQ ID NO. 1) obtained by sequencing was subjected to sequence alignment, homology analysis in the NCBI database, and a phylogenetic tree was constructed. The results showed that the strain RX-LA303 was 99.86% homologous to Lactobacillus rhamnosus Lacticaseibacillus rhamnosus in gene sequence, and combined with the morphological characteristics, the strain RX-LA303 was determined to be Lactobacillus rhamnosus, and was classified and named as Lactobacillus rhamnosus RX-LA303. Lacticaseibacillus rhamnosus It was preserved by the patent procedure, and the preservation date was July 18, 2025, the preservation address was No. 59, Building 5, 100, Martyrs' Road, Guangzhou, Guangdong Province, and the preservation number was GDMCC No: 66713.

[0030] Example 3 Test of antioxidant effect of truffle fermentation extract (1) Preparation of truffle fermentation extract Experimental group: application example 1, comparative application example 1-2; Application example 1: Lactobacillus rhamnosus RX-LA303 was used to ferment truffles, and the specific preparation method was as follows: S1, inoculate Lactobacillus rhamnosus RX-LA303 into the activated culture medium for activation treatment, and obtain the bacterial liquid with a viable count of 2×10 9 CFU / mL; wherein the activated culture medium is a liquid culture medium prepared from 10 g casein peptone, 10 g beef extract, 5 g yeast extract, 5 g glucose, 5 g sodium acetate, 2 g citric acid diamine, 1 g Tween 80, 2 g K2HPO4, 0.2 g MgSO4·7H2O, 0.05 g MnSO4·H2O and 1.0 L deionized water; the activation culture conditions are 37℃, 120r / min shaking flask culture for 24h; S2, crush the dried truffles through a 60 mesh sieve to obtain truffle powder, mix the truffle powder with deionized water at a mass ratio of 1:0.6 to obtain solid state fermentation material, sterilize and reserve; S3, the Lactobacillus rhamnosus RX-LA303 bacterial liquid obtained in step S1 is inoculated at an inoculation amount of 4wt% (based on the mass of the solid state fermentation material) and uniformly sprayed into the solid state fermentation material, and the fermentation is carried out under the condition of 37℃ without oxygen for 3d to obtain the truffle fermentation product; S4, the obtained black truffle fermentation was mixed with deionized water, and then ultrasonic water extraction was performed, centrifuged, the supernatant was collected, and water extraction was repeated for 3 times, then the supernatant of multiple extractions was mixed and concentrated, 60℃ reduced pressure concentration to 1 / 4 of the original volume, then 4 times the volume of anhydrous ethanol was added to the concentrated solution, 4℃ standing for 24h to produce a precipitate, 6000r / min centrifugation for 30min, the precipitate was collected and freeze-dried to obtain the black truffle fermentation extract; wherein the solid-liquid ratio of the black truffle fermentation and deionized water was 1g:20mL, the frequency of ultrasonic was 20kHz, the temperature was 85℃, and the time was 30min; the speed of centrifugation after ultrasonic water extraction was 15000rpm, and the time was 5min.

[0031] Comparative Application Example 1: Compared with Application Example 1, the difference lies in that Lactobacillus rhamnosus CCFM1161 (GDMCC No: 61714) is used instead of Lactobacillus rhamnosus RX-LA303 to ferment black truffles (i.e. both the activation and fermentation steps use Lactobacillus rhamnosus CCFM1161), and the rest of the steps and parameters are the same as those of Application Example 1, thereby preparing the black truffle fermentation extract of Comparative Application Example 1.

[0032] Comparative Application Example 2: Compared with Application Example 1, the difference lies in that the J04 strain in Example 1 is used instead of Lactobacillus rhamnosus RX-LA303 to ferment black truffles (i.e. both the activation and fermentation steps use the J04 strain), and the rest of the steps and parameters are the same as those of Application Example 1, thereby preparing the black truffle fermentation extract of Comparative Application Example 2.

[0033] Control group: The dried black truffles were crushed through a 60-mesh sieve to obtain black truffle powder, the black truffle powder was mixed with deionized water, and then ultrasonic water extraction was performed, centrifuged, the supernatant was collected, and water extraction was repeated for 3 times, then the supernatant of multiple extractions was mixed and concentrated, 60℃ reduced pressure concentration to 1 / 4 of the original volume, then 4 times the volume of anhydrous ethanol was added to the concentrated solution, 4℃ standing for 24h to produce a precipitate, 6000r / min centrifugation for 30min, the precipitate was collected and freeze-dried to obtain the black truffle extract; wherein the solid-liquid ratio of the black truffle fermentation and deionized water was 1g:20mL, the frequency of ultrasonic was 20kHz, the temperature was 85℃, and the time was 30min; the speed of centrifugation after ultrasonic water extraction was 15000rpm, and the time was 5min.

[0034] (2) Polysaccharide extraction rate test of black truffle fermentation extract / black truffle extract The polysaccharide yield of the black truffle fermentation extract in step ② of Reference Example 1 was tested, and the polysaccharide yields of Application Example 1, Comparative Application Examples 1-2, and the control group were obtained. The specific data are shown in Table 2.

[0035] Table 2 Polysaccharide yield of extract Group / Performance Polysaccharide yield / % Example 1 26.29 Comparative Example 1 8.46 Comparative Example 2 20.60 Control group 10.15 According to the results in Table 2, it can be seen that the L. rhamnosus RX-LA303 screened in the application can significantly improve the polysaccharide yield of the extract; and the microorganism has adaptability with the substrate. For example, although the L. rhamnosus CCFM1161 in the prior art has been proved to be able to significantly improve the content of polygonatum polysaccharide after fermentation, the polysaccharide yield of the extract is actually reduced when the black truffle is used as the substrate for fermentation, which also indicates that the fermentation of microorganisms in different substrates is unpredictable.

[0036] (3) Test of antioxidant performance of black truffle fermentation extract In a test tube, 1 mL of salicylic acid-ethanol solution (5 mmol / L), 1 mL of FeSO4 solution (5 mmol / L), 1 mL of H2O2 solution (3 mmol / L) and 1 mg of black truffle fermentation extract / black truffle extract (application example 1, comparative application example 1-2 and control group) were mixed uniformly, then distilled water was added to 10 mL, 37°C water bath for 15 min, 4°C, 6000 rpm centrifugation for 10 min, the supernatant was taken and the absorbance was measured at 510 nm (distilled water as reference). HO· clearance rate (%) = [(Ao-As) / Ao] x 100% (Ao is the OD value without sample; As is the OD value with sample), the higher the HO· clearance rate, the stronger the active oxygen scavenging effect, that is, the stronger the antioxidant capacity. The test results are shown in Table 3.

[0037] Table 3 HO· clearance rate data of each group Group / Performance HO· clearance rate / % Example 1 82.72 Comparative Example 1 40.43 Comparative Example 2 71.09 Control group 46.14 As can be seen from Table 3, the antioxidant performance of each group is positively correlated with the polysaccharide yield of the black truffle fermentation extract, which indicates that the extract obtained by fermenting black truffle with L. rhamnosus RX-LA303 (i.e. J05) screened in the application has a higher polysaccharide yield and more excellent antioxidant activity than other strains.

[0038] Example 4 Preparation of antioxidant and anti-inflammatory composition and test of antioxidant effect thereof 1 g of black truffle fermentation extract obtained in application example 1, 5 g of collagen tripeptide, 8 g of concentrated blackcurrant juice, 3 g of concentrated blueberry juice, 0.6 g of hawthorn powder, 0.3 g of fishwort powder and 82.1 g of distilled water were mixed uniformly, and the antioxidant and anti-inflammatory composition of application example 2 was obtained by centrifugation to take the supernatant; 1 g of black truffle fermentation extract obtained in comparative application example 2, 5 g of collagen tripeptide, 8 g of concentrated blackcurrant juice, 3 g of concentrated blueberry juice, 0.6 g of hawthorn powder, 0.3 g of fishwort powder and 82.1 g of distilled water were mixed uniformly, and the antioxidant and anti-inflammatory composition of comparative application example 3 was obtained by centrifugation to take the supernatant; The black truffle extract (control group) 1 g, collagen tripeptide 5 g, concentrated black currant juice 8 g, concentrated blueberry juice 3 g, hawthorn powder 0.6 g, fishy grass powder 0.3 g obtained from Example 3 were mixed with 82.1 g of distilled water, and the supernatant was obtained by centrifugation to obtain the antioxidant and anti-inflammatory composition of Comparative Application Example 4.

[0039] Antioxidant test of the composition 1) The hydroxyl radical HO· scavenging rate of the antioxidant and anti-inflammatory compositions of Application Example 2 and Comparative Application Examples 3-4 was determined according to the antioxidant performance test procedure in Example 3. 2) DPPH· scavenging rate determination The antioxidant and anti-inflammatory compositions of Application Example 2 and Comparative Application Examples 3-4 were mixed with anhydrous ethanol to prepare a sample solution of 5 mg·mL -1 ; 2.0 mL of DPPH· ethanol solution with a concentration of 0.2 mmol·L -1 was accurately taken into a 10 mL test tube, and 2 mL of the sample solution of Application Example 2 and Comparative Application Examples 3-4 was added, respectively, and mixed quickly. After being placed in a 37℃ constant temperature water bath under dark and static conditions, the reaction was allowed to proceed for 30 min, and the absorbance A1 was determined. 2 mL of anhydrous ethanol was mixed with 2 mL of DPPH· ethanol (0.2 mmol·L -1 ), and the absorbance A0 was determined. At the same time, the absorbance A2 of the mixture of 2 mL of anhydrous ethanol and 2 mL of the sample solution was determined. The DPPH· scavenging rate was calculated according to the following formula: DPPH· scavenging rate = [1-(A 1- A2) / A0] × 100%. The higher the DPPH· scavenging rate, the stronger the DPPH· scavenging effect, i.e., the stronger the antioxidant capacity. The test results are shown in Table 4.

[0040] Table 4 Antioxidant data of each group Group / Performance HO· clearance rate / % DPPH· clearance rate / % Example 2 88.96 94.35 Comparative Example 3 76.32 85.93 Comparative Example 4 59.48 72.61 (2) Anti-inflammatory test of the composition ① Materials and reagents The composition of Application Example 2; ICR male mice with a body weight of 20±2 g; TNF-α, IL-6 and IL-1β detection kits; ② Animal experiment conditions Preparation: The mice were raised in an environment with a temperature of 25±5℃ and a humidity of 30%-40%, and the experimental mice could freely eat and drink water. After the mice were adaptively fed for 7 d, the experiment was formally started; Grouping: 30 mice were randomly divided into 3 groups, 10 mice in each group, namely a normal group, a model group and a sample group; The mice in the normal group were given 200 μL of sterile distilled water by gavage every day; The model group mice were given 200 μL of sterile distilled water by gavage every day; The sample group mice were given 200 μL of the composition of Application Example 2 by gavage every day; The intervention was continued for 14 days. After the last intervention, the model group and sample group mice were injected intraperitoneally with LPS at 5 mg / kg (prepared in normal saline) to establish an acute inflammatory model of LPS mice, and the blank control group was injected with the same volume of normal saline. Two hours later, the mice were sacrificed and blood was collected.

[0041] 3. Measurement of cell inflammatory factors After the blood was collected from the mice in each group, the blood was left to stand at room temperature for 30 min, and then centrifuged at 3000 g for 30 min. The serum was carefully aspirated and used to measure the concentrations of TNF-α, IL-6 and IL-1β. The results are shown in Table 5.

[0042] Table 5. Anti-inflammatory test data for the antioxidant and anti-inflammatory compositions of each group Group / Performance TNF-α (pg / mL) IL-6 (pg / mL) IL-1β (pg / mL) Example 2 82.4 ± 3.6 # ]] 70.7 ± 1.9 # ]] 32.7 ± 2.3 # ]] Blank group 16.1±1.5 38.9±2.4 18.3±1.2 Model group 121.3 ± 8.3 ▲ ]] 146.4 ± 5.4 ▲ ]] 79.1 ± 4.8 ▲ ]] In Table 5, ▲ indicates that the model group is significantly different from the blank group (p<0.05); and # indicates that the sample group is significantly different from the model group (p<0.05).

[0043] As can be seen from Tables 4-5, the antioxidant and anti-inflammatory composition of Application Example 2 has significantly better antioxidant performance than the compositions of Comparative Application Examples 3-4, and the composition also has significant anti-inflammatory performance; and is suitable as a nutritional supplement.

[0044] Finally, it should be noted that the above examples are merely used to illustrate the technical solutions of the present application and do not limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application.

Claims

1. A strain of Lactobacillus rhamnosus RX-LA303, with accession number GDMCC No: 66713.

2. A microbial inoculant, characterized in that, The microbial agent comprises Lactobacillus rhamnosus as described in claim 1.

3. Use of the Lactobacillus rhamnosus of claim 1 in the preparation of antioxidant and anti-inflammatory compositions.

4. A method for preparing a fermented extract of black truffle, characterized in that, Includes the following steps: S1. Activate the *Lactobacillus rhamnosus* RX-LA303 as described in claim 1 to obtain a viable count of 1 × 10⁻⁶. 8 -3×10 9 RX-LA303 bacterial suspension at CFU / mL; S2. Mix black truffle powder with deionized water to obtain solid fermentation material; wherein the mass ratio of black truffle powder to deionized water is 1:(0.4-0.8). S3. Inoculate the solid fermentation material with RX-LA303 bacterial solution at 3-5 wt% of the solid fermentation material and carry out anaerobic fermentation culture. After the fermentation is completed, the black truffle fermentation product is obtained. The anaerobic fermentation temperature is 35-37℃ and the time is 1-4 days. S4. After mixing the black truffle ferment with deionized water, perform ultrasonic water extraction, centrifuge, collect the supernatant, concentrate, add anhydrous ethanol, let stand to precipitate, centrifuge, take the precipitate and freeze dry to obtain the black truffle ferment extract; wherein, the ratio of black truffle ferment to deionized water is 1g:(20-25)mL.

5. The method for preparing the fermented black truffle extract as described in claim 4, characterized in that, In step S1, the activation process involves inoculating Lactobacillus rhamnosus RX-LA303 into an activation medium and culturing it in a shake flask at 35-37°C and 100-150 r / min for 20-30 hours.

6. The method for preparing the fermented black truffle extract as described in claim 4, characterized in that, In step S4, the parameters of the ultrasonic treatment are: ultrasonic frequency of 20-30kHz, temperature of 80-90℃, time of 20-40min, and volume ratio of concentrated solution to anhydrous ethanol of 1:(3-4).

7. A fermented black truffle extract, characterized in that, It is prepared by the method for preparing black truffle fermented extract according to any one of claims 4-6.

8. A composition having antioxidant and anti-inflammatory effects, characterized in that, It comprises the following components by weight: 3-8 parts collagen tripeptide, 5-10 parts concentrated blackcurrant juice, 0.5-5 parts concentrated blueberry juice, 0.1-1 parts hawthorn powder, 0.1-1 parts houttuynia cordata powder and 0.1-2 parts black truffle fermented extract as described in claim 6.

9. The use of the composition with antioxidant and anti-inflammatory effects as described in claim 8 in the preparation of antioxidant foods.