Combined application method of lactobacillus reuteri and EGCG (epigallocatechin gallate)

Abstract

The invention discloses a combined application method of lactobacillus reuteri and EGCG (epigallocatechin gallate), and belongs to the technical field of food. The invention provides a lactobacillus reuteri NDF46 (Lactobacillus reuteri) with high oxidation resistance, wherein the lactobacillus reuteri NDF46 can tolerate EGCG (Epigallocatechin Gallate) with the concentration of 5.0 mg / mL, and the lactobacillus reuteri NDF46 can tolerate the EGCG with the concentration of 5.0 mg / mL. The lactobacillus reuteri NDF46 and EGCG are jointly applied to fermentation of the siniperca chuatsi, it is found that loss of polyunsaturated fatty acid GLA can be remarkably inhibited, the TBARS value of the siniperca chuatsi in the fermentation process is reduced, the lipid oxidation reaction is better controlled, meanwhile, rising of the content of volatile basic nitrogen can be delayed, and the lactobacillus reuteri NDF46 and EGCG are of great significance in improving the nutritional quality of the siniperca chuatsi.

Description

Technical Field

[0001] The present invention relates to the technical field of food processing, and relates to a method for the combined application of Lactobacillus reuteri and epigallocatechin gallate (EGCG). Background Art

[0002] Fermentation is a traditional method for preserving meat and fish. In addition to extending the shelf life, it can also endow raw materials with unique flavors and textures, which is widely loved by people. Gamma-linolenic acid (GLA) is an essential unsaturated fatty acid for the human body and has various physiological functions, including anti-inflammatory effects, reducing triglyceride levels in the blood, and promoting cardiovascular health, etc. However, the fermentation time of fermented fish and fermented meat products is generally long. During the pickling and fermentation process, the unsaturated fatty acid gamma-linolenic acid is easily oxidized and degraded, resulting in a decrease in the nutritional value of the product. In addition, the fat and protein in meat will undergo complex hydrolysis and oxidation reactions under the action of microorganisms and enzymes, producing by-products such as malonaldehyde (MDA) and total volatile basic nitrogen (TVB-N). Excessive content of these by-products will have adverse effects on the flavor, safety, nutritional value, etc. of food.

[0003] Currently, people have adopted various methods to inhibit the oxidation reaction in fermented fish and fermented meat products, such as low-temperature treatment, inoculation of fermenting agents, addition of antioxidants, etc. Low temperature can slow down the oxidation reaction, but fermented fish and fermented meat products generally require specific temperatures to produce unique flavor characteristics. Long-term low-temperature treatment is not conducive to the fermentation process, and too low a temperature will also affect the texture and taste of the product, etc. Lactic acid bacteria are often used as fermenting agents in various fermented products, but the antioxidant capacity of some currently used lactic acid bacteria in the fermentation system is limited, and inoculating a single lactic acid bacteria may be difficult to relieve the complex oxidation reaction mechanism in fermented fish and fermented meat products. Some antioxidants have the effect of inhibiting the growth of microorganisms, which may have an adverse impact on the normal growth of beneficial fermenting microorganisms, thus affecting the fermentation process. In addition, some antioxidants have the characteristics of fast failure, and adding antioxidants alone may be difficult to protect the long-term fermentation process. Currently, the existing methods for inhibiting oxidation are relatively single and have their own defects. Therefore, it is necessary to develop some new technologies to better control the oxidation reaction in fermented fish and fermented meat products, inhibit the loss of GLA, reduce the content of malonaldehyde and total volatile basic nitrogen, and improve the nutritional value of fermented fish and fermented meat products. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for the combined application of Lactobacillus reuteri and epigallocatechin gallate (EGCG).

[0005] To achieve the above object, the present invention provides a method for inhibiting the loss of GLA, reducing the content of thiobarbituric acid reactive substances (TBARS) and volatile basic nitrogen by co-fermenting mandarin fish with Limosilactobacillus reuteri NDF46 and EGCG.

[0006] The first technical solution provided by the present invention is a strain of Limosilactobacillus reuteri NDF46, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 4, 2024, with the deposit number GDMCC NO. 65409.

[0007] The second technical solution provided by the present invention is a microbial preparation, which contains the Limosilactobacillus reuteri NDF46.

[0008] The third technical solution provided by the present invention is a starter containing the Limosilactobacillus reuteri NDF46, and the starter also contains epigallocatechin gallate EGCG or tea polyphenols.

[0009] The fourth technical solution provided by the present invention is a method for fermenting meat, adding the Limosilactobacillus reuteri NDF46 or the starter to the meat for fermentation; the meat includes mandarin fish meat, yellow croaker meat, perch meat or sausage.

[0010] In some embodiments, the addition amount of Limosilactobacillus reuteri NDF46 is 5.00×10 6 ~1.50×10 7 CFU / g, and the addition amount of EGCG is 250 - 400 mg / kg.

[0011] The fifth technical solution provided by the present invention is a method for fermenting mandarin fish, including the following steps:

[0012] S1. Pretreatment: Slaughter the mandarin fish and clean it thoroughly.

[0013] S2. Preparation of bacterial suspension: Activate Limosilactobacillus reuteri NDF46 and resuspend the bacterial cells with physiological saline to obtain a bacterial suspension.

[0014] S3. Preparation of fermentation broth: Weigh water equal in weight to the mandarin fish obtained in step S1, add seasonings, add the Limosilactobacillus reuteri NDF46 bacterial suspension obtained in step S2, and add EGCG to obtain a fermentation broth.

[0015] S4. Fermentation: Immerse the mandarin fish obtained in step S1 in the fermentation broth obtained in step S3 for fermentation to obtain fermented mandarin fish.

[0016] In some embodiments, in step S3, the fermentation temperature is 10 - 15 °C and the fermentation time is 7 - 11 days.

[0017] In some embodiments, the addition amount of Limosilactobacillus reuteri NDF46 is 5.00×10 6 ~1.50×10 7 CFU / g of meat, and the addition amount of EGCG is 250 - 400 mg / kg of meat.

[0018] The sixth technical solution provided by the present invention is the application of the Limosilactobacillus reuteri NDF46 or the microbial preparation or the starter in the preparation of fermented meat products, and the meat products include fish meat products or sausage products.

[0019] The seventh technical solution provided by the present invention is the application of the Limosilactobacillus reuteri NDF46 as an antioxidant.

[0020] The present invention has the following beneficial effects:

[0021] The present invention provides a method for the combined application of Limosilactobacillus reuteri and EGCG. Among them, Limosilactobacillus reuteri NDF46 is isolated from the fermented food dairy tofu, has high antioxidant activity, and can tolerate EGCG at a concentration of 5.0 mg / mL. Its combined use with the natural antioxidant EGCG inhibits the loss of GLA during the fermentation of mandarin fish, reduces the TBARS value, better controls the lipid oxidation reaction, and at the same time can delay the increase in the content of volatile basic nitrogen and improve the nutritional quality of mandarin fish.

[0022] Biological preservation material

[0023] A strain of Limosilactobacillus reuteri NDF46, classified and named Limosilactobacillus reuteri, has been deposited in the Guangdong Provincial Microbial Culture Collection Center, with the deposit number GDMCC No: 65409, the deposit date is November 4, 2024, and the deposit address is the 5th floor of Building 59, No. 100, Xianlie Middle Road, Guangzhou, Guangdong Province. Description of the drawings

[0024] Figure 1 It is a heat map of the DPPH and ABTS free radical scavenging abilities of the supernatant of sarcoplasmic protein fermented by 42 lactic acid bacteria.

[0025] Figure 2 It is a heat map of the average value of the DPPH and ABTS free radical scavenging abilities of the supernatant of sarcoplasmic protein fermentation measured twice. Note: The data in the figure represents the result of taking the average value after normalizing the two original data (0 - 1).

[0026] Figure 3 Growth of Lactobacillus mucosae NDF46 at different EGCG concentrations.

[0027] Figure 4 Growth of Lactobacillus mucosae CICC6118 at different EGCG concentrations. Detailed implementation method

[0028] The following are the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0029] Raw materials used in the embodiments:

[0030] 1. Lactobacillus mucosae CICC6118, Lactobacillus mucosae CICC6123, Lactobacillus plantarum CICC6253, purchased from China Center for Industrial Culture Collection.

[0031] 2. MRS liquid medium: Peptone 10.0 g / L, Beef extract powder 8.0 g / L, Yeast extract powder 4.0 g / L, Glucose 20.0 g / L, Dipotassium hydrogen phosphate 2.0 g / L, Ammonium citrate dibasic 2.0 g / L, Sodium acetate 5.0 g / L, Magnesium sulfate 0.2 g / L, Manganese sulfate 0.04 g / L, Tween 80 1.0 g / L, pH 5.7±0.2.

[0032] 3. MRS solid medium: Peptone 10.0 g / L, Beef extract powder 8.0 g / L, Yeast extract powder 4.0 g / L, Glucose 20.0 g / L, Dipotassium hydrogen phosphate 2.0 g / L, Ammonium citrate dibasic 2.0 g / L, Sodium acetate 5.0 g / L, Magnesium sulfate 0.2 g / L, Manganese sulfate 0.04 g / L, Agar 14.0 g / L, Tween 80 1.0 g / L, pH 6.5±0.2.

[0033] 4. Myogen (sterile) fermentation medium: Weigh 100 g of the back muscle of mandarin fish (skinned), add 400 mL of phosphate buffer solution (10 mM, pH = 7.0), and homogenize. Centrifuge at 9000 rpm / min at 4°C for 15 min. Collect the supernatant, remove insoluble impurities with filter paper, measure the myogen concentration by the biuret method, adjust the protein concentration to 3.0 mg / mL after passing through the membrane with phosphate buffer solution, and add 1% glucose. Then filter and sterilize with a 0.22 μm aqueous filter membrane. Check with PCA plate coating to ensure sterility before use.

[0034] 5. EGCG (purity ≥ 98%) was purchased from Shaanxi Dannaisi Biotechnology Co., Ltd., with the product number 989-51-5; tea polyphenols (purity ≥ 98%, EGCG content 50%) were purchased from Jiangsu Dehe Biotechnology Co., Ltd., with the product number TP98.

[0035] Example 1: Screening of Strains

[0036] Alternative starter culture sources: Search for existing lactic acid bacteria strains in the laboratory, all of which are from fermented foods, and a total of 42 available lactic acid bacteria strains were obtained.

[0037] Among them, Limosilactobacillus reuteri NDF46 was isolated from milk tofu. Screening process: Take 1.0 g of milk tofu, add it to 10 mL of physiological saline, transfer it into a homogenization bag, beat for 30 min, centrifuge at 500×g for 5 min to remove residues, and aseptically transfer the turbid liquid part containing bacteria to a 50 mL centrifuge tube. After appropriately diluting the culture solution, spread it on MRS solid medium. After the liquid is completely absorbed, incubate it upside down at 37°C for 24 - 48 h. Then pick single colonies, streak them on MRS solid medium to isolate single colonies. Continuously purify for three generations. After determining that there is no contamination, inoculate them into MRS liquid medium for expanded culture, and freeze and preserve the cultured strains, named NDF46. Take another part of the cultured bacterial liquid for identification.

[0038] Extract the genome of NDF46 for 16s rDNA identification. The genomic extraction method is carried out according to the glass bead method in the "Concise Molecular Biology Experiment Guide" and perform Blastn analysis. It is found that the homology of this bacterium with Limosilactobacillus reuteri is the highest, reaching 100%. Therefore, this strain is identified as Limosilactobacillus reuteri and named Limosilactobacillus reuteri NDF46. The Limosilactobacillus reuteri NDF46 grows well in MRS solid medium. After culturing at 37°C for 24 h, the colonies formed by Lactobacillus brevis are round, with a smooth surface and a milky yellow color.

[0039] After morphological and 16s rDNA identification, this strain is named Limosilactobacillus reuteri NDF46 and was deposited in the Guangdong Provincial Culture Collection of Microorganisms on November 4, 2024, with the deposit number: GDMCC NO: 65409. Deposit address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, Guangdong Province, Guangdong Provincial Culture Collection of Microorganisms.

[0040] Screen lactic acid bacteria with high antioxidant capacity according to the following method:

[0041] (1) Fermentation of sarcoplasmic protein

[0042] Take out the lactic acid bacteria stored in the -80 °C refrigerator, streak and activate them on the MRS solid medium, incubate them in an incubator at 37 °C for 24 h, pick a single colony and inoculate it into the MRS liquid medium for 24 h. Centrifuge the bacterial liquid, and adjust the OD with physiological saline 600nm to 0.8, and inoculate it into the sarcoplasmic protein (sterile) fermentation medium at an inoculation amount of 2%, and culture it at 37 °C for 3 d. Use the sarcoplasmic protein (sterile) fermentation medium without inoculation as the control group. After fermentation, centrifuge the fermentation broth at 15000×g, 4 °C for 20 min, and collect the supernatant for testing.

[0043] (2) Determination of DPPH free radical scavenging ability: Add 1 mL of the supernatant to an equal volume of DPPH solution (0.2 mmol / L, dissolved in absolute ethanol) as the sample group, add 1 mL of the supernatant to an equal volume of ethanol as the control group, and add 1 mL of ethanol and an equal volume of DPPH solution (0.2 mmol / L, dissolved in absolute ethanol) as the blank group. After reacting in the dark at room temperature for 30 min, measure the OD 517nm , and calculate the DPPH free radical scavenging ability according to the following formula: DPPH free radical scavenging ability (%) = 1 - [(OD 517nm sample - OD 517nm control) / OD 517nm blank] × 100%.

[0044] (3) Determination of ABTS free radical scavenging ability: Measure the ABTS free radical scavenging ability of the fermentation supernatant. React 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution in equal volume in the dark for 12 - 16 h. Dilute the solution with deionized water to adjust the OD 734nm to 0.7 to obtain the ABTS working solution. Take 0.1 mL of the sample solution and add 0.9 mL of the ABTS working solution, use 0.1 mL of deionized water as the blank, react in the dark for 15 min, and measure the OD 734nm . Calculate the ABTS free radical scavenging ability according to the following formula: ABTS free radical scavenging ability (%) = (OD 734nm blank - OD 734nm sample) / OD 734nm blank × 100%.

[0045] The measurement results are as Figure 1 , 2As shown, after measurement, the DPPH and ABTS radical scavenging abilities of the control group were 58.60±0.84% and 40.50±5.61% respectively. Compared with the control group, after Limosilactobacillus reuteri NDF13, Limosilactobacillus reuteri NDF14, and Limosilactobacillus reuteri NDF46 fermented sarcoplasmic protein, the DPPH radical scavenging ability of the fermentation supernatant reached more than 95%, and the ABTS radical scavenging ability reached more than 65%, which was significantly higher than that of the control group. After repeating the measurement of 14 lactic acid bacteria with strong antioxidant ability and normalizing and averaging the results of the two measurements, it was found that Limosilactobacillus reuteri NDF46 had the highest antioxidant ability ( Figure 2 ). Therefore, based on the comprehensive judgment of the results of the two measurements, Limosilactobacillus reuteri NDF46 was selected and applied to the fermentation of mandarin fish.

[0046] Example 2: Determination of colony count and growth curve

[0047] After sterilizing MRS broth, prepare 9 mL of broth solutions containing 0, 0.5, 5.0, and 10.0 mg / mL EGCG respectively, add 1 mL of Limosilactobacillus reuteri NDF46 at the end of the logarithmic growth phase (initial inoculation concentration is 10 8 CFU / mL), mix well and culture at 37°C and 150 r / min for 24 h, then spread and count. Use MRS broth without adding EGCG in equal volume as the control.

[0048] After sterilizing MRS broth, prepare 9 mL of broth solutions containing 0, 0.1, 0.2, and 0.3 mg / mL EGCG respectively, add 1 mL of the bacterial suspension of Limosilactobacillus reuteri NDF46 and CICC62118 at the end of the logarithmic growth phase (initial inoculation concentration is 10 8 CFU / mL), mix well, take 300 μL and put it into a special plate for growth curve culture, and culture in a full-automatic growth curve instrument for 48 h. Use MRS broth without adding EGCG in equal volume as the control.

[0049] It can be seen from Figure 3 (A) that there was no significant difference in the growth status of Limosilactobacillus reuteri NDF46 under the conditions of 0, 0.1, 0.2, and 0.3 mg / mL EGCG. With the increase of the EGCG concentration, the growth of Limosilactobacillus reuteri NDF46 in the MRS medium with a concentration of 5.0 mg / mL EGCG was not affected, and the growth was significantly inhibited at a concentration of 10.0 mg / mL EGCG ( Figure 3B), while Lactobacillus reuteri CICC6118 is sensitive to EGCG, and its growth is affected under the condition of 0.1 mg / mL EGCG. Moreover, as the concentration of EGCG increases, the growth of Lactobacillus reuteri CICC6118 becomes slower and slower( Figure 4 ). It shows that Lactobacillus reuteri NDF46 has excellent EGCG tolerance compared with other strains of the same species, and the concentration of EGCG it can tolerate is 5.0 mg / mL.

[0050] Example 3: Treatment of mandarin fish with Lactobacillus reuteri NDF46 and EGCG in combination

[0051] This example involves the application of Lactobacillus reuteri inoculum at 10 7 CFU / g of meat and the addition amount of EGCG at 250, 275, 300, 325, 350, 375, 400 mg / kg in mandarin fish.

[0052] S1. Pretreatment: Remove the internal organs of the mandarin fish and weigh 3 kg;

[0053] S2. Preparation of the bacterial suspension: Prepare the Lactobacillus reuteri NDF46 bacterial suspension in the following manner: Pick a single colony of Lactobacillus reuteri NDF46 and inoculate it into MRS liquid medium. Culture at 37 °C and 200 rpm / min for 24 h. Take the bacterial liquid and centrifuge it at 8000 rpm for 5 min to discard the supernatant. Dilute the bacterial cells with a 0.9% (mass fraction) sodium chloride aqueous solution to 10 9 CFU / mL for standby.

[0054] S3. Preparation of the fermentation broth: Weigh water equal to the weight of the mandarin fish obtained in step S1. Based on the weight of water being 100%, add 6 wt% salt, 0.3 wt% ginger, 0.15 wt% pepper, 0.15 wt% Chinese prickly ash, and 0.02 wt% fennel. Add the Lactobacillus reuteri NDF46 bacterial suspension obtained in step S2 so that the amount of bacterial cells and the addition amount of EGCG in the fermentation broth are as shown in the following table to obtain the fermentation broth;

[0055] Table 1 Combinations of different addition amounts of EGCG

[0056]

[0057] S4. Fermentation: Immerse the mandarin fish obtained in step S1 in the fermentation broth obtained in step S3. The fermentation temperature is 15 °C and the fermentation time is 7 days to obtain the fermented mandarin fish.

[0058] The above product is named Example 3 - number.

[0059] The contents of GLA, TBARS value, and TVB - N in the above examples are shown in Table 2.

[0060] Table 2 GLA content, TBARS value and TVB-N content at the end of fermented mandarin fish with stink

[0061]

[0062] As can be seen from Table 2, when the inoculation amount of Lactobacillus mucosae is 10 7 CFU / g of meat, under the condition of different EGCG addition amounts, the GLA content, TBARS value and TVB-N content at the end of fermented mandarin fish with stink show different changing trends. Compared with other examples, Example 3-3 shows the best effect, with the highest GLA content, and at the same time, the TBARS value and TVB-N content are significantly reduced, indicating that when the inoculation amount of Lactobacillus mucosae is 10 7 CFU / g and the EGCG addition amount is 300 mg / kg, the best antioxidant effect can be achieved.

[0063] Example 4: Treatment of mandarin fish with stink by the combination of Lactobacillus mucosae NDF46 and EGCG

[0064] This example relates to the application of Lactobacillus mucosae with an inoculation amount of 5.00×10 6 ~1.50×10 7 CFU / g of meat and an EGCG addition amount of 300 mg / kg in mandarin fish with stink. For the specific implementation method, refer to Example 3, the difference is that in step S3, the addition amounts of Lactobacillus mucosae NDF46 and EGCG are shown in Table 3, and the obtained product is named Example 4-number.

[0065] Table 3 Different combinations of inoculation amounts

[0066]

[0067] The GLA content, TBARS value and TVB-N content in the above examples are shown in Table 4.

[0068] Table 4 GLA content, TBARS value and TVB-N content at the end of fermented mandarin fish with stink

[0069]

[0070] As can be seen from Table 4, with the increase of the inoculation amount, the GLA content shows a trend of first increasing and then decreasing. When the inoculation amount is 10 7 CFU / g of meat, the GLA content reaches the highest, while the TBARS value and TVB-N value show a trend of first decreasing and then increasing, and reach the lowest value when the inoculation amount is 10 7 CFU / g of meat. Too low or too high inoculation amount is not conducive to the combined action of lactic acid bacteria and EGCG. The above results are similar to those of Example 3-3, indicating that when the inoculation amount of Lactobacillus mucosae is 107 CFU / g of meat, and the best effect can be achieved when the addition amount of EGCG is 300 mg / kg.

[0071] Example 5: Treatment of mandarin fish with Limosilactobacillus reuteri NDF46 and EGCG in combination

[0072] This example involves an inoculation amount of Limosilactobacillus reuteri NDF46 of 10 7 CFU / g of meat and the application of an addition amount of 300 mg / kg of EGCG in mandarin fish.

[0073] S1. Pretreatment: Remove the internal organs of the mandarin fish and weigh 3 kg.

[0074] S2. Preparation of the bacterial suspension: Prepare the Limosilactobacillus reuteri NDF46 bacterial suspension in the following manner: Pick a single colony of Limosilactobacillus reuteri NDF46 and inoculate it into MRS liquid medium. Culture at 37 °C and 200 rpm / min for 24 h. Take the bacterial liquid and centrifuge it at 8000 rpm / min for 5 min, discard the supernatant, and dilute the bacterial cells with a 0.9% (mass fraction) aqueous sodium chloride solution to 10 9 CFU / mL for standby.

[0075] S3. Preparation of the fermentation broth: Weigh water with the same weight as the mandarin fish obtained in step S1. Based on the weight of water being 100%, add 6 wt% salt, 0.3 wt% ginger, 0.15 wt% pepper, 0.15 wt% Chinese prickly ash, and 0.02 wt% fennel. Add the Limosilactobacillus reuteri NDF46 bacterial suspension obtained in step S2 so that the fermentation broth contains 3.00×10 10 CFU of bacterial cells, add 900 mg of EGCG, and obtain the fermentation broth.

[0076] S4. Fermentation: Immerse the mandarin fish obtained in step S1 in the fermentation broth obtained in step S3. The fermentation temperature is 15 °C and the fermentation time is 7 days to obtain the fermented mandarin fish.

[0077] The above product is named Example 5.

[0078] Example 6: Treatment of mandarin fish with Limosilactobacillus reuteri NDF46 and EGCG in combination

[0079] This example involves an inoculation amount of Limosilactobacillus reuteri NDF46 of 10 7 CFU / g of meat, an addition amount of EGCG of 300 mg / kg, a fermentation temperature of 12 °C, and a fermentation time of 9 days in the application of mandarin fish.

[0080] The specific implementation method refers to Example 5, with the difference that the fermentation temperature in step S4 is replaced from 15 °C to 12 °C, and the fermentation time of 7 days is replaced by 9 days.

[0081] Example 7: Treatment of stinky yellow croaker with the combination of Lactobacillus reuteri NDF46 and EGCG

[0082] This example involves the application of Lactobacillus reuteri NDF46 with an inoculation amount of 10 7 CFU / g of meat and an addition amount of 300 mg / kg of EGCG in stinky yellow croaker.

[0083] For the specific implementation method, refer to Example 5, with the difference that the mandarin fish in step S1 is replaced by yellow croaker, and the above product is named Example 7.

[0084] Example 8: Treatment of stinky perch with the combination of Lactobacillus reuteri NDF46 and EGCG

[0085] This example involves the application of Lactobacillus reuteri NDF46 with an inoculation amount of 10 7 CFU / g of meat and an addition amount of 300 mg / kg of EGCG in stinky perch.

[0086] For the specific implementation method, refer to Example 5, with the difference that the mandarin fish in step S1 is replaced by perch, and the above product is named Example 8.

[0087] Example 9: Treatment of dried sausage with the combination of Lactobacillus reuteri NDF46 and EGCG

[0088] This example involves the application of Lactobacillus reuteri NDF46 with an inoculation amount of 10 7 CFU / g of meat and an addition amount of 300 mg / kg of EGCG in dried sausage.

[0089] S1. Preparation of bacterial suspension: Prepare the Lactobacillus reuteri NDF46 bacterial suspension in the following manner: Pick a single colony of Lactobacillus reuteri NDF46 and inoculate it into MRS liquid medium, culture it at 37°C and 200 rpm / min for 24 h, take the bacterial liquid, centrifuge it at 8000 rpm / min for 5 min, discard the supernatant, and dilute the bacterial cells with a 0.9% sodium chloride aqueous solution to 10 9 CFU / mL for standby.

[0090] S2. Preparation of dried sausage: Select lean pork (fat - to - lean ratio of 2:8), cut it into dices, add auxiliary materials, add the bacterial liquid and EGCG for pickling, enema, fermentation and drying, packaging, and finished product.

[0091] The auxiliary materials include: salt: 1 wt%, monosodium glutamate: 0.5 wt%, white wine: 1.5 wt%, white sugar: 1 wt%, soy sauce: 1.5 wt%;

[0092] The inoculation amount of Lactobacillus reuteri NDF46 is 10 7CFU / g of meat, and the addition amount of EGCG is 300 mg / kg;

[0093] Among them, the fermentation and drying conditions are: the temperature is generally set at (12 ± 2) °C, the relative humidity is 70% - 75%, and the time is 12 days.

[0094] The above product is named Example 9.

[0095] Example 10: Treatment of mandarin fish with mucus-producing Lactobacillus reuteri NDF46 combined with tea polyphenols

[0096] The specific implementation method refers to Example 5, the difference is that in step S3, 1800 mg of tea polyphenols (purchased from Jiangsu Dehe Biotechnology Co., Ltd., the content ratio of EGCG is 50%, the same below) is used to replace EGCG. The above product is named Example 10.

[0097] Comparative Example 1: Naturally fermented mandarin fish

[0098] The specific steps are as follows:

[0099] S1. Pretreatment: Remove the internal organs of the mandarin fish and weigh 3 kg;

[0100] S2. Preparation of the fermentation broth: Weigh water equal to the weight of the mandarin fish obtained in step S1. Based on the weight of water being 100%, add 6 wt% of table salt, 0.3 wt% of ginger, 0.15 wt% of chili peppers, 0.15 wt% of Chinese prickly ash, and 0.02 wt% of fennel.

[0101] S4. Fermentation: Immerse the mandarin fish obtained in step S1 in the fermentation broth obtained in step S2, the fermentation temperature is 15 °C, and the fermentation time is 7 days to obtain the fermented mandarin fish.

[0102] The above product is named Comparative Example 1.

[0103] Comparative Example 2: Naturally fermented yellow croaker

[0104] The specific implementation method refers to Comparative Example 1, the difference is that the mandarin fish is replaced by yellow croaker, and the above product is named Comparative Example 2.

[0105] Comparative Example 3: Naturally fermented sea bass

[0106] The specific implementation method refers to Comparative Example 1, the difference is that the mandarin fish is replaced by sea bass, and the above product is named Comparative Example 3.

[0107] Comparative Example 4: Naturally fermented dried sausage

[0108] The specific steps are as follows:

[0109] Preparation of dried sausage: Select tenderloin (fat-to-lean ratio of 2:8), cut and minced into dices, add auxiliary materials for marinating, stuff into casings, ferment and dry, package, and obtain the finished product.

[0110] Among them, the auxiliary materials include: table salt: 1 wt%, monosodium glutamate: 0.5 wt%, liquor: 1.5 wt%, white sugar: 1 wt%, soy sauce: 1.5 wt%.

[0111] Among them, the fermentation and drying conditions are: the temperature is generally set at (12 ± 2) °C, the relative humidity is 70% - 75%, and the time is 12 days.

[0112] The above product is named Comparative Example 4.

[0113] Comparative Example 5: Single-strain fermentation of stinky mandarin fish by Lactobacillus reuteri NDF46

[0114] The inoculation amount of Lactobacillus reuteri NDF46 is 10 7 CFU / g of meat, and the specific steps are as follows:

[0115] S1. Pretreatment: Remove the internal organs of the mandarin fish and weigh 3 kg;

[0116] S2. Preparation of bacterial suspension: Prepare the bacterial suspension of Lactobacillus reuteri NDF46 in the following manner: Pick a single colony of Lactobacillus reuteri NDF46 and inoculate it into MRS liquid medium, culture it at 37 °C and 200 rpm / min for 24 h, take the bacterial liquid, centrifuge it at 8000 rpm / min for 5 min, discard the supernatant, and dilute the thallus with 0.9% sodium chloride aqueous solution to 10 9 CFU / mL for standby.

[0117] S3. Preparation of fermentation broth: Weigh water equal to the weight of the mandarin fish obtained in step S1. Based on the weight of water being 100%, add 6 wt% table salt, 0.3 wt% ginger, 0.15 wt% pepper, 0.15 wt% Chinese prickly ash, and 0.02 wt% fennel. Add the bacterial suspension of Lactobacillus reuteri NDF46 obtained in step S2 to make the fermentation broth contain 3.00×10 10 CFU to obtain the fermentation broth;

[0118] S4. Fermentation: Immerse the mandarin fish obtained in step S1 in the fermentation broth obtained in step S3, the fermentation temperature is 15 °C, and the fermentation time is 7 days to obtain stinky mandarin fish.

[0119] The above product is named Comparative Example 5.

[0120] Comparative Example 6: Single-strain fermentation of stinky mandarin fish by Lactobacillus reuteri CICC 6118

[0121] For the specific implementation method, refer to Comparative Example 5, the difference is that Lactobacillus reuteri NDF46 is replaced by Lactobacillus reuteri CICC 6118, and the above product is named Comparative Example 6.

[0122] Comparative Example 7: Single-strain fermentation of fermented mandarin fish with Lactobacillus mucosae CICC 6123

[0123] The specific implementation method refers to Comparative Example 5, with the difference that Lactobacillus mucosae NDF46 is replaced by Lactobacillus mucosae CICC 6123, and the above product is named Comparative Example 7.

[0124] Comparative Example 8: Treatment of fermented mandarin fish with the combination of Lactobacillus mucosae CICC 6118 and EGCG

[0125] The specific implementation method refers to Comparative Example 5, with the difference that Lactobacillus mucosae NDF46 is replaced by Lactobacillus mucosae CICC 6118, and 900 mg of EGCG is added to the fermentation broth. The above product is named Comparative Example 8.

[0126] Comparative Example 9: Treatment of fermented mandarin fish with the combination of Lactobacillus mucosae CICC 6123 and EGCG

[0127] The specific implementation method refers to Comparative Example 5, with the difference that Lactobacillus mucosae NDF46 is replaced by Lactobacillus mucosae CICC 6123, and 900 mg of EGCG is added to the fermentation broth. The above product is named Comparative Example 9.

[0128] Comparative Example 10: Fermentation of fermented mandarin fish by adding EGCG alone

[0129] S1. Pretreatment: Remove the internal organs of the mandarin fish and weigh 3 kg;

[0130] S2. Preparation of the fermentation broth: Weigh water equal to the weight of the mandarin fish obtained in step S1. Based on the weight of water being 100%, add 6 wt% salt, 0.3 wt% ginger, 0.15 wt% pepper, 0.15 wt% Chinese prickly ash, and 0.02 wt% fennel, and add 900 mg of EGCG (the addition amount is 300 mg / kg) to obtain the fermentation broth.

[0131] S4. Fermentation: Immerse the mandarin fish obtained in step S1 in the fermentation broth obtained in step S2. The fermentation temperature is 15°C and the fermentation time is 7 days to obtain fermented mandarin fish.

[0132] The above product is named Comparative Example 10.

[0133] Comparative Example 11: Fermentation of fermented yellow croaker by adding EGCG alone

[0134] The specific implementation method refers to Comparative Example 10, with the difference that the mandarin fish is replaced by yellow croaker. The above product is named Comparative Example 11.

[0135] Comparative Example 12: Fermentation of fermented perch by adding EGCG alone

[0136] For the specific implementation manner, refer to Comparative Example 10, with the difference that mandarin fish is replaced by perch, and the above product is named Comparative Example 12.

[0137] Comparative Example 13: Adding EGCG alone to fermented and dried sausage

[0138] The specific steps are as follows:

[0139] Preparation of dried sausage: Select tenderloin (fat-to-lean ratio 2:8), cut and minced into dices, add auxiliary materials, add EGCG for marinating, stuff into casings, ferment, dry, package, and obtain the finished product.

[0140] The auxiliary materials include: salt: 1 wt%, monosodium glutamate: 0.5 wt%, white liquor: 1.5 wt%, white sugar: 1 wt%, soy sauce: 1.5 wt%; the addition amount of EGCG is 300 mg / kg;

[0141] The fermentation and drying conditions are: the temperature is generally set at (12 ± 2) °C, the relative humidity is 70% - 75%, and the time is 12 days.

[0142] The above product is named Comparative Example 13.

[0143] Comparative Example 14: Using Lactobacillus plantarum CICC6253 and tea polyphenols in combination to treat stinky mandarin fish

[0144] S1. Pretreatment: Remove the internal organs of the mandarin fish and weigh 3 kg;

[0145] S2. Preparation of bacterial suspension: The preparation of Lactobacillus plantarum CICC6253 bacterial suspension is as in S1 of Example 5.

[0146] S3. Preparation of fermentation broth: Weigh water equal to the weight of the mandarin fish obtained in step S1. Based on the weight of water being 100%, add 6 wt% salt, 0.3 wt% ginger, 0.15 wt% pepper, 0.15 wt% Chinese prickly ash, and 0.02 wt% fennel. Add the Lactobacillus plantarum CICC6253 bacterial suspension obtained in step S2 so that the fermentation broth contains 3.00×10 10 CFU of bacteria, and add 1800 mg of tea polyphenols to obtain the fermentation broth;

[0147] S4. Fermentation: Immerse the mandarin fish obtained in step S1 in the fermentation broth obtained in step S3, ferment at a temperature of 15 °C for 7 days to obtain stinky mandarin fish.

[0148] The above product is named Comparative Example 14.

[0149] Comparative Example 15: Adding tea polyphenols alone to treat stinky mandarin fish

[0150] S1. Pretreatment: Remove the internal organs of the mandarin fish and weigh 3 kg;

[0151] S2. Preparation of fermentation liquid: weigh water of the same weight as the mandarin fish obtained in step S1, add 6wt% of salt, 0.3wt% of ginger, 0.15wt% of chili, 0.15wt% of prickly ash and 0.02wt% of fennel, and add 1800mg of tea polyphenols to obtain fermentation liquid, taking the weight of water as 100%.

[0152] S4, fermentation: soaking the mandarin fish obtained in step S1 in the fermentation liquid obtained in step S2, the fermentation temperature is 15° C., the fermentation time is 7 days, and the smelly mandarin fish is obtained.

[0153] The above product is named Comparative Example 15.

[0154] Comparative Example 16: Treatment of smelly mandarin fish with Lactobacillus reuteri NDF46 and grape seed extract

[0155] Specific implementation method Referring to Example 5, the difference is that in step S3, EGCG is replaced by 900 mg of grape seed extract (purchased from Shanghai Yuanye Biotechnology Co., Ltd., item number: Y70767). The above product is named Comparative Example 16.

[0156] Embodiment 11

[0157] The products obtained from the embodiments of the present invention and the comparative examples were tested:

[0158] 1. Determination of γ-linolenic acid, arachidonic acid and oleic acid content.

[0159] (1) Extraction of oil: Weigh 5.00 g of fish sample, add 8 mL of chloroform and 4 mL of methanol, homogenize, make up to 30 mL with chloroform-methanol mixed solution, let stand at 4 °C for 1 h, filter with filter paper, add 0.2 times 0.85% saline to the filtrate, shake and mix, centrifuge at 4000 rpm / min for 15 min, discard the upper layer of liquid, blow dry the lower layer of solution with nitrogen, and obtain the oil sample.

[0160] (2) Fatty acid methyl esterification: 5 mg of oil sample, 200 μL of internal standard solution (1 mg / mL 11-carbon triglyceride dissolved in chloroform) and 2 mL of 0.5 mol / L sodium hydroxide-methanol solution were added to a round-bottom flask. After thorough mixing, the round-bottom flask was refluxed in a water bath at 80°C for 5 min. Then, 2 mL of boron trifluoride-methanol solution (14%, w / w) was added through the upper inlet of the condenser and the mixture was reacted at 80°C for another 2 min. The round-bottom flask was then cooled to room temperature and 1.5 mL of n-hexane was added to fully shake and extract fatty acid methyl esters. After standing, the upper n-hexane organic layer was collected and an appropriate amount of anhydrous sodium sulfate was added to stand for 1 h to remove excess water. Filtered with a 0.22 μm microporous organic membrane. 10 mg of γ-linolenic acid methyl ester, arachidonic acid methyl ester and oleic acid methyl ester standard products were weighed and dissolved in 5 mL of n-hexane to prepare standard stock solutions for use.

[0161] (3) The gas chromatographic separation of fatty acid methyl esters was carried out using an FID detector and a Supelco SP 2560 capillary column (100 m × 0.25 mm inner diameter, 0.2 μm). GC parameters: The initial column oven temperature was maintained at 120 °C for 9 min, then increased to 200 °C at a rate of 20 °C / min and held for 5 min, and then increased to 230 °C at a rate of 3 °C / min and held for 10 min. The injection port temperature was set at 220 °C, the injection volume was 1 μL, and the split ratio was 20:1. The constant carrier gas (N 2 ) flow rate was set at 2.0 mL / min, and the FID temperature was set at 260 °C.

[0162] II. Determination of TBARS value.

[0163] (1) Preparation of solutions:

[0164] Malondialdehyde standard stock solution (100 μg / mL): Accurately pipette 0.315 g (accurate to 0.001 g) of 1,1,3,3-tetraethoxypropane and make up the volume to 1000 mL.

[0165] Malondialdehyde standard working solution (10 μg / mL): Accurately pipette 10 mL of the malondialdehyde standard stock solution and make up the volume to 100 mL for standby.

[0166] 7.2% BHT solution (2,6-di-tert-butyl-p-cresol): Accurately weigh 7.2 g of BHT and dissolve it in 100 mL of absolute ethanol.

[0167] 30% TCA solution (trichloroacetic acid): Accurately weigh 30 g of BHT and dissolve it in 100 mL of deionized water.

[0168] 30 mM TBA solution (thiobarbituric acid): Accurately weigh 0.4325 g of TBA and make up the volume to 100 mL. Ultrasonic treatment can be used to accelerate dissolution.

[0169] (2) Determination of the standard curve:

[0170] Accurately pipette 0 mL, 0.2 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL, 2.5 mL of the malondialdehyde standard working solution and make up the volume to 10 mL. The concentrations of this standard solution series are 0 μg / mL, 0.2 μg / mL, 0.5 μg / mL, 1.0 μg / mL, 1.5 μg / mL, 2.0 μg / mL, 2.5 μg / mL. Pipette 1 mL of the above standard solution and mix it with 2 mL of TBA / TCA solution (1:1), heat it in a boiling water bath for 20 min for color development, cool it in an ice bath for 10 min, and measure the absorbance at 532 nm. Use the mass concentration of the standard series solution as the abscissa and the absorbance value as the ordinate to plot the standard curve.

[0171] (3) Determination of the sample:

[0172] Take 5 g of the sample, cut it into pieces, mix it with 15 mL of deionized water in a centrifuge tube, use a homogenizer to break and mix it evenly, and immediately place the homogenized slurry in an ice bath for later use. Transfer 1 mL of the slurry to a 10 mL EP tube, add 50 μL of BHT solution, and mix with 2 mL of TBA / TCA solution (1:1). Color development is carried out in a boiling water bath for 20 min, and then cooled in an ice bath for 10 min. Centrifuge the mixed solution and take the supernatant, and measure the absorbance at 532 nm. Blank control: Use 1 mL of deionized water instead of the slurry as the blank, and measure the absorbance according to the above treatment. The experiment is carried out in triplicate.

[0173] III. Determination of the content of volatile basic nitrogen.

[0174] Determination is carried out according to the automatic Kjeldahl method in GB 5009.228—2016 "National Food Safety Standard Determination of Volatile Basic Nitrogen in Foods". The specific method is as follows:

[0175] (1) Sample treatment: After removing fat, bone, skin and tendon from the sample, it is minced and stirred evenly. Weigh 5 g of the sample (accurate to 0.001 g) into a 50 mL centrifuge tube, add 25 mL of distilled water, homogenize for 1 min, seal the bottle mouth with plastic wrap, soak at 4 °C for 30 min, and then centrifuge for 5 min. Take the supernatant for standby.

[0176] (2) Determination: Take 10 mL of the supernatant into a digestion tube, add 5 mL of magnesium oxide suspension (10 g / L), and immediately distill for 6 min. Add 3 drops of mixed indicator to a 250 mL distillation flask, and immediately titrate the absorption solution after absorbing ammonia with a 0.01 mol / L hydrochloric acid standard solution. The end point is when the solution changes from blue-green to gray-red. At the same time, carry out a reagent blank determination (titrate the blank first), 0.1 M (NH 4 ) 2 SO 4 is used as a positive control. Calculate the content of TVB-N according to the amount of hydrochloric acid consumed.

[0177] The contents of GLA, TBARS and TVB-N in the examples and comparative examples are shown in Table 5.

[0178] Table 5 Contents of GLA, TBARS and TVB-N at the end of the fermentation of marinated mandarin fish

[0179]

[0180] Table 6 Contents of arachidonic acid, oleic acid and TVB-N at the end of the fermentation of marinated mandarin fish

[0181]

[0182]

[0183] It was found through experiments that the combined use of Lactobacillus mucosae NDF46 and EGCG could significantly inhibit the loss of GLA during the fermentation of mandarin fish, reduce the TBARS value and TVB-N content, inhibit the oxidation reaction, and when the inoculum amount was 10 7 CFU / g of meat and the addition amount of EGCG was 300 mg / kg, the effect was the best (Example 5), the GLA content reached 0.90 g / 100 g of oil, the TBARS value was 0.92 mg MDA / kg, and the TVB-N content was 27.92 mg / 100 g. In addition, the combined use of Lactobacillus mucosae NDF46 and EGCG to treat mandarin fish, sea bass and dried sausage (Examples 7-9) also had significant effects, such as the GLA content increased from 0.19 - 0.41 g / 100 g of oil to more than 0.70 g / 100 g of oil.

[0184] Compared with the combined use method, the treatment of mandarin fish with EGCG alone had a lower level of TVB-N content (24.83 mg / 100 g), but the GLA content was only 0.79 g / 100 g of oil, and the arachidonic acid and oleic acid contents (Table 6) were 2.57 g / 100 g of oil and 34.96 g / 100 g of oil respectively, which were significantly lower than those of the combined treatment group (p < 0.05), and the TBARS value of 1.06 mg MDA / kg was significantly higher than that of the combined treatment group (p < 0.05), indicating that the method of adding EGCG alone did not reach the optimal level in inhibiting the loss of unsaturated fatty acids and controlling lipid oxidation. In addition, the treatment of mandarin fish with EGCG alone had a certain effect compared with natural fermentation (Control Example 1), but the treatment of mandarin fish, sea bass and dried sausage with EGCG alone (Control Examples 11-13) did not have obvious effects in all aspects. For example, in Control Example 11, there was no obvious difference in the GLA content (0.43 g / 100 g of oil), TBARS value (2.09 mg MDA / kg), and TVB-N content (41.21 mg / 100 g) compared with Control Example 2 (p > 0.05), indicating that adding EGCG alone may not be a stable control method for most products.

[0185] In addition, when the stinky mandarin fish was treated with the single inoculation of strain NDF46 (Comparative Example 5), the GLA content was 0.64 g / 100 g oil, showing no significant difference compared with Comparative Example 1 (0.63 g / 100 g oil) (p > 0.05). The TVB-N content reached 36.87 mg / 100 g, which was significantly higher than that of Comparative Example 1 (p < 0.05). However, the TBARS value was 1.02 mg MDA / kg, which was significantly lower than that of Comparative Example 1 and Comparative Example 10 (p < 0.05). The contents of arachidonic acid and oleic acid (Table 6) were 2.99 g / 100 g oil and 38.46 g / 100 g oil respectively, which were significantly higher than those of Comparative Example 1 and Comparative Example 10. In the combined treatment group (Example 5), they reached 3.53 g / 100 g oil and 40.52 g / 100 g oil respectively. Generally, the single inoculation of strain NDF46 could significantly inhibit lipid oxidation and showed a good effect in controlling the loss of arachidonic acid and oleic acid. However, there was still a certain gap compared with the combined use group. The combination of NDF46 and EGCG could exert a better antioxidant effect. The above results indicate that the method of adding EGCG alone or inoculating Lactobacillus mucosae NDF6 alone is not the optimal choice. In addition, when other Lactobacillus mucosae were used to treat stinky mandarin fish (Comparative Examples 6 - 9), whether inoculated alone or in combination with EGCG, good effects could not be achieved, and even negative effects occurred. The GLA content was as low as 0.36 g / 100 g oil, far lower than that of Comparative Example 1, and the TVB-N content was up to 45.89 mg / 100 g, which was significantly higher than that of Comparative Example 1 (p < 0.05).

[0186] In addition, the main component of tea polyphenols is EGCG, and its content ratio can reach 50%. Both tea polyphenols and EGCG have antioxidant capabilities. Therefore, adding tea polyphenols during the fermentation of stinky mandarin fish may have a similar effect to adding EGCG. The experimental results show that, similar to the result of adding EGCG alone, adding tea polyphenols alone (Comparative Example 15) could not achieve good results. The GLA content was only 0.68 g / 100 g oil, and the TBARS value and TVB-N content were significantly higher than those of Example 5. The combined use of a certain amount of tea polyphenols and Lactobacillus mucosae NDF46 (Example 10) could effectively inhibit the loss of GLA and reduce the TVB-N content. The experimental results were similar to those of Example 5. In addition, replacing tea polyphenols or EGCG with grape seed extract and combining it with strain NDF46 had a poor effect. To sum up, the combined use of Lactobacillus mucosae NDF46 and EGCG can play a synergistic promoting role in the various characteristics of fermented stinky mandarin fish, and also has an obvious effect on stinky perch, stinky yellow croaker, and dried sausage. It can be used as an important means to effectively improve the nutritional quality value of fermented fish and fermented meat products, and EGCG can be replaced by tea polyphenols.

[0187] Although the present invention has been disclosed above in its preferred embodiments, it is not intended to limit the present invention. Anyone skilled in this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. Limosilactobacillus reuteri NDF46, which was deposited in Guangdong Provincial Microbiological Culture Collection Center on November 4, 2024, with the deposit number GDMCCNO.65409.

2. A microbial preparation containing the Lactobacillus reuteri NDF46 according to claim 1.

3. A starter containing Lactobacillus reuteri NDF46 according to claim 1, characterized in that: The leavening agent also contains epigallocatechin gallate EGCG or tea polyphenols.

4. A method for fermenting meat, characterized in that: The Lactobacillus reuteri NDF46 described in claim 1, or the microbial preparation described in claim 2, or the starter described in claim 3 is added to meat for fermentation; the meat includes mandarin fish meat, yellow croaker meat, sea bass meat or sausage.

5. The method according to claim 4, characterized in that The amount of Lactobacillus reuteri NDF46 added was 5×10 6 ~1.5×10 7 CFU / g meat, the amount of EGCG added is 250-400 mg / kg meat.

6. A method for fermenting smelly mandarin fish, characterized in that: The following steps are involved: S1. Pretreatment: Slaughter and clean the mandarin fish; S2. Preparation of bacterial suspension: activating Lactobacillus reuteri NDF46, and resuspending the bacteria with physiological saline to obtain a bacterial suspension; S3, preparation of fermentation liquid: weigh water of the same weight as the mandarin fish obtained in step S1, add seasoning, add the bacterial suspension of Lactobacillus reuteri NDF46 obtained in step S2, and add EGCG to obtain fermentation liquid; S4, fermentation: the mandarin fish obtained in step S1 is immersed in the fermentation liquid obtained in step S3 for fermentation to obtain smelly mandarin fish.

7. The method according to claim 6, characterized in that The fermentation temperature is 10-15°C and the fermentation time is 7-11 days.

8. The method according to claim 6 or 7, characterized in that: The addition amount of Lactobacillus reuteri NDF46 was 5.00×10 6 ~1.50×10 7 CFU / g meat, the amount of EGCG added is 250-400 mg / kg meat.

9. Use of Lactobacillus reuteri NDF46 according to claim 1 or the microbial preparation according to claim 2 or the starter culture according to claim 3 in the preparation of fermented meat products, characterized in that: The meat product includes fish product or sausage product.

10. Use of the Lactobacillus reuteri NDF46 according to claim 1 as an antioxidant.

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