Fermentation agent and application thereof

Abstract

The invention relates to the field of microbial fermentation, and discloses a leavening agent and application thereof. The leavening agent comprises leuconostoc mesenteroides subsp. Mesenteroides and lactobacillus paracasei. The preservation number of the leuconostoc mesenteroides subsp. Mesenteroides is CGMCC (China General Microbiological Culture Collection Center) No.33433; the preservation number of the lactobacillus paracasei is CGMCC (China General Microbiological Culture Collection Center) No.15139. The leavening agent provided by the invention can be used for fermenting food, especially for fermenting dairy products, and further, the content of beneficial metabolites in the dairy products can be remarkably increased.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial fermentation, in particular to a fermentation agent and application thereof. BACKGROUND

[0002] In the production process of dairy products, fermentation agents play a crucial role. Traditional fermentation agents mainly focus on the production of specific target metabolites, such as lactic acid, acetic acid, etc., which have important influence on the flavor, texture and shelf life of dairy products. Current research on most fermentation agents mainly focuses on the optimization of specific target metabolites, while ignoring the comprehensive analysis and utilization of non-target metabolites. This limitation leads to the fact that many potential beneficial metabolites in dairy products are not fully developed and utilized, which limits the further development of dairy products in the field of health functional foods.

[0003] Therefore, it is necessary to conduct joint analysis of fermentation strains based on non-target metabolomics, study the significantly different metabolites produced during fermentation and preservation, and screen out a new type of fermentation agent that can significantly increase the content of beneficial metabolites in dairy products. SUMMARY

[0004] The purpose of the present application is to overcome the problems existing in the prior art, and to provide a fermentation agent and application thereof, which is composed of Leuconostoc mesenteroides subsp. mesenteroides and Lactobacillus paracasei, and can significantly increase the content of beneficial metabolites in dairy products when used for fermentation of dairy products.

[0005] To achieve the above-mentioned purpose, the first aspect of the present application provides a fermentation agent, which comprises Leuconostoc mesenteroides subsp. mesenteroides and Lactobacillus paracasei. The preservation number of the Leuconostoc mesenteroides subsp. mesenteroides is CGMCC No. 33433, and the preservation number of the Lactobacillus paracasei is CGMCC No. 15139.

[0006] The second aspect of the present application provides application of the fermentation agent as described above in fermented food.

[0007] The third aspect of the present application provides application of the fermentation agent as described above in increasing the content of beneficial metabolites in fermented dairy products. The beneficial metabolites include non-volatile metabolites and volatile metabolites.

[0008] The fourth aspect of the present application provides a fermented milk, and the preparation method thereof comprises inoculating the fermentation agent as described above into cow milk and / or goat milk.

[0009] Through the above technical solution, the present application can at least achieve the following beneficial effects: The fermenting agent provided by the present application can be used for fermenting food, in particular, fermenting dairy products, and further, can significantly increase the content of beneficial metabolites in dairy products.

[0010] Biological preservation Leuconostoc mesenteroides subsp. mesenteroides of the present application Leuconostoc mesenteroides subsp. mesenteroides The strain was preserved in China General Microbiological Culture Collection Center (CGMCC) (address: No. 1, Huayuancun, Beijing Economic-Technological Development Area, Beijing, China; mailbox: 100101) on January 15, 2025, and the accession number is CGMCC No. 33433. DETAILED DESCRIPTION

[0011] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as the exact dimensions are not considered critical for the present application. The endpoints of the ranges and any numerical values should be interpreted as approximately including values near the reported values. For numeric ranges, the endpoints of each range, the endpoints of each range and individual point values, and individual point values can be combined with each other to generate one or more new numeric ranges, which should be considered as specifically disclosed herein.

[0012] The present application provides a fermenting agent, which comprises Leuconostoc mesenteroides subsp. mesenteroides and Lactobacillus paracasei; The accession number of the Leuconostoc mesenteroides subsp. mesenteroides is CGMCC No. 33433, and the accession number of the Lactobacillus paracasei is CGMCC No. 15139.

[0013] The inventors of the present application accidentally isolated a strain of Leuconostoc mesenteroides subsp. mesenteroides from a Kefir particle, and ingeniously found that the strain, when combined with Lactobacillus paracasei (K56, accession number: CGMCC No. 15139) as a fermenting agent, can effectively increase the content of beneficial metabolites in fermented dairy products.

[0014] In the present application, the sequence of 16S rDNA of the Leuconostoc mesenteroides subsp. mesenteroides is shown in SEQ ID NO: 1.

[0015] SEQ ID NO: 1:

[0016] According to the present invention, preferably, the ratio of the number of viable cells of Leuconostoc mesenteroides subsp. mesenteroides to Lactobacillus paracasei is 1:0.5-1.5. For example, the ratio of the number of viable cells of Leuconostoc mesenteroides subsp. mesenteroides to Lactobacillus paracasei can be 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:0.95, 1:1, 1:1.05, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, or a range consisting of any two of the above values, or any intermediate value within the range.

[0017] In some particularly preferred embodiments of the present invention, the ratio of the viable cell counts of Leuconostoc mesenteroides subsp. mesenteroides to Lactobacillus paracasei is 1:0.9-1.1.

[0018] According to the present invention, preferably, the number of viable bacteria in the starter is 1×10 6 -1×10 8 CFU / mL. For example, the number of viable bacteria in the bacterial agent can be 1×10 6 , 2×10 6 , 3×10 6 , 4×10 6 , 5×10 6 , 6×10 6 , 7×10 6 , 8×10 6 , 9×10 6 , 9.5×10 6 , 1×10 7 , 1.5×10 7 , 2×10 7 , 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , or it can be a range consisting of any two of the above values, or any intermediate value in the range.

[0019] According to the present invention, in order to further reduce the curdling time of fermented milk, the starter also includes Lactobacillus bulgaricus and / or Streptococcus thermophilus.

[0020] The second aspect of the present invention provides use of the aforementioned starter in fermented foods.

[0021] According to the present invention, preferably, the fermented food is a fermented dairy product.

[0022] In the present application, the fermenting agent can be used for fermentation of various types of dairy products, preferably, the fermented dairy products include but are not limited to yogurt, cheese and / or sour cream.

[0023] The third aspect of the present application provides use of the fermenting agent as described above for increasing the content of beneficial metabolites in fermented dairy products. The beneficial metabolites include non-volatile metabolites and volatile metabolites.

[0024] In the present application, the beneficial metabolites refer to various types of metabolites produced by microbial metabolism during the fermentation or storage of dairy products, which are helpful to improve the flavor of dairy products, increase the nutritional value of dairy products and prolong the shelf life of dairy products. The beneficial metabolites include non-volatile metabolites and volatile metabolites, in the present application, the volatile metabolites are determined by solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC / MS), and the non-volatile metabolites are determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0025] In the present application, the volatile metabolites include but are not limited to at least one of 1-heptanol, 1-octanol, 2-heptanol, 2-nonanol, 3-methyl-1-pentanol, benzoic acid, cis-5-octen-1-ol and hexyl formate.

[0026] In the present application, the non-volatile metabolites include but are not limited to at least one of α-hederin, citrusin I, D-3-hydroxyphenyl lactic acid, dopamine, endomorphin-1, liguliatine, L-tyrosine, phenylacetic acid, proteinase-activated receptor-4, camellia saponinogen E and withaferin A.

[0027] The fourth aspect of the present application provides a fermented milk, and the preparation method of the fermented milk comprises: inoculating the fermenting agent as described above into cow milk and / or goat milk.

[0028] In the present application, the inoculation amount can be selected according to actual conditions, preferably, the inoculation amount of the fermenting agent is 1×10 9 -1×10 12 CFU per kilogram of cow milk and / or goat milk.

[0029] Through the above technical solution, the present application can at least achieve the following beneficial effects: The fermenting agent provided by the present application can be used for fermented food, especially fermented dairy products, and further can significantly increase the content of beneficial metabolites in dairy products.

[0030] The present application will be described in detail through the following examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present application, and are not used to limit the present application.

[0031] The reagents and materials used in the following examples are purchased from regular chemical reagent suppliers, and the purity is analytical pure if not specially stated.

[0032] In the following examples, the solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC / MS) is used to determine volatile metabolites, Sample preparation process: take 2 g of sample into a 20 mL headspace bottle, add 2.5 μL of internal standard naphthalene-D8 (concentration of 20 μg / mL) and 4 mL of saturated sodium chloride aqueous solution, immediately seal the headspace bottle for testing.

[0033] The quality control sample is prepared by mixing all the samples to be tested, and one quality control sample is inserted every 5 samples.

[0034] Detection conditions: VF-WAXms capillary column (25 mm, 0.25 mm x 0.2 μm) is used for separation, temperature program: initial temperature is 40℃, no holding time for equilibrium, then increase to 120℃ at a rate of 8℃ / min, then increase to 230℃ at a rate of 20℃ / min, and keep at this temperature for 4.5 min, the total running time is 20 min. The mass spectrometer uses electron impact source (EI), the scanning range is m / z 35-500. HS-SPME uses headspace solid phase microextraction fiber, the extraction temperature is 80℃, and the extraction time is 10 min.

[0035] In the following examples, the liquid chromatography-tandem mass spectrometry (LC-MS / MS) is used to determine non-volatile metabolites, Sample preparation process: take 100 μL of sample into a 1.5 mL centrifuge tube, add 300 μL of mixed solution of methanol and acetonitrile (volume ratio of 1:1), and internal standard L-2-chlorophenylalanine with a final concentration of 0.02 mg / mL. Extract at 5℃, 40 kHz for 30 minutes, freeze the sample at -20℃ for 30 min, then centrifuge at 4℃, 13000 x g for 15 min. Take the supernatant and dry it completely by nitrogen blowing, add 100 μL of mixed solution of acetonitrile and water (volume ratio of 1:1) for re-dissolution, extract at 5℃, 40 kHz for 5 minutes, then centrifuge at 4℃, 13000 x g for 10 min, take the supernatant for testing.

[0036] The quality control sample is prepared by mixing all the samples to be tested, and one quality control sample is inserted every 5 samples.

[0037] Detection conditions: ACQUITY UPLC HSS T3 column (1.8 μm 2.1 x 100 mm) was used, mobile phase A was 95 wt% water and 5 wt% acetonitrile (containing 0.1 wt% formic acid), mobile phase B was 47.5 wt% acetonitrile, 47.5 wt% isopropanol and 5 wt% water (containing 0.1 wt% formic acid), injection volume was 3 μL, column temperature was 40 °C. Mass spectrometer used electrospray ion source (ESI), scan range was m / z 70-1050.

[0038] The commercial starter used in the following examples was purchased from Denmark company Hansen, product number YoFlex® Premium 5.0.

[0039] Lactobacillus paracasei (K56) was purchased from Inner Mongolia Yili Industrial Co., Ltd., and the accession number was CGMCC No. 15139.

[0040] Leuconostoc mesenteroides subsp. mesenteroides (G12S) was isolated from kefir grains, and the accession number was CGMCC No. 33433.

[0041] The medium components used in the following examples were as follows: MRS liquid medium: 10 g of proteose peptone, 8 g of beef powder, 4 g of yeast powder, 20 g of glucose, 5 g of sodium acetate trihydrate, 0.2 g of magnesium sulfate heptahydrate, 2 g of dipotassium hydrogen phosphate trihydrate, 2 g of ammonium citrate, 0.05 g of manganese sulfate heptahydrate, 1 mL of Tween 80, 1 L of distilled water.

[0042] Example 1 S1, Leuconostoc mesenteroides subsp. mesenteroides G12S (2 vol%) was inoculated into MRS liquid medium, and activated at 30 °C, 150 rpm for 16 h; Lactobacillus paracasei K56 (2 vol%) was inoculated into MRS liquid medium, and activated at 37 °C, 180 rpm for 16 h.

[0043] S2, K56 and G12S were mixed according to the ratio of 1:1 of viable bacteria, and the total viable bacteria were 1 x 10 7 CFU / mL was added to cow milk, and 0.00004 g / mL of commercial starter was added at the same time, and fermented at 37 °C for 5 h as the experimental group; only the commercial starter was added and fermented under the same conditions as the control group.

[0044] S3, the fermented milk of the experimental group and the control group was stored in a 4 °C refrigerator, and the fermented milk stored for 1 d, 14 d and 28 d was taken for metabolite analysis, respectively. The results are shown in Table 1.

[0045] Table 1

[0046] For volatile metabolites, the starter culture provided by the present application can increase the content of alcohol substances (1-heptanol, 1-octanol, 2-heptanol, 2-nonanol and 3-methyl-1-pentanol), which can impart more rich flavor levels to yogurt, possibly bringing floral, fruity or sweet aroma, and improving the delicacy and complexity of the taste; cis-5-octen-1-ol has the aroma of watermelon or melon, and its increased content can add fruitiness to yogurt; hexyl formate can show significant advantages in the later stage of storage, and can make the fruitiness and flavor of yogurt more persistent; benzoic acid can effectively inhibit the growth of molds, yeasts and bacteria, and its increased content can prolong the shelf life of yogurt and reduce the risk of spoilage during storage; in addition, the content of metabolic product 2-acetylfuran is also reduced, which usually causes a bitter taste of yogurt, and its reduced content helps to improve the overall taste of yogurt.

[0047] For non-volatile metabolites, the starter culture provided by the present application increases the content of D-3-phenyllactic acid, dopamine, L-tyrosine and phenylacetic acid, which have potential physiological activities such as antioxidant, neurotransmitter (dopamine) regulation or immune function enhancement, thereby improving the nutritional value of yogurt. Endomorphin-1 is an endogenous opioid peptide that may have a soothing or analgesic effect; citrusin I, camellia saponinogen E and withaferin A are plant-derived active ingredients (such as anti-inflammatory and antioxidant) that enhance the health benefits of yogurt. Alpha-hederin and proteinase-activated receptor-4 play a role in the later stage of storage, enhancing the stability or functionality of yogurt (such as anti-inflammatory and digestive enzyme activity regulation); the production of harmful substance castanospermine is inhibited, which has a negative impact on the human body (such as inhibition of glycosidase), and its reduced content indicates that the strain provided by the present application can reduce the accumulation of harmful metabolites. The dairy product prepared by the starter culture provided by the present application has a significantly higher content of beneficial metabolites than that prepared by using a commercial starter culture after long-term storage.

[0048] The above describes the preferred embodiments of the present application, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be considered as disclosed by the present application, and all fall within the protection scope of the present application.

Claims

1. A leavening agent, characterized in that The fermentation agent includes Leuconostoc mesenteroides subspecies mesenteroides and Lactobacillus paracasei; The preservation number of the Leuconostoc mesenteroides subspecies mesenteroides is CGMCC No. 33433; the preservation number of the Lactobacillus paracasei is CGMCC No. 15139.

2. The fermentation agent according to claim 1, wherein The live cell count ratio of Leuconostoc mesenteroides subspecies mesenteroides to Lactobacillus paracasei is 1:0.5-1.

5.

3. The fermentation agent according to claim 1, wherein The number of viable bacteria in the fermentation agent is 1×10 6 -1×10 8 CFU / mL.

4. The fermentation agent according to claim 1, wherein The fermentation agent further comprises Lactobacillus bulgaricus and / or Streptococcus thermophilus.

5. Use of the starter according to any one of claims 1 to 4 in fermented foods.

6. The use according to claim 5, wherein: The fermented food is a fermented dairy product.

7. The use according to claim 6, wherein: The fermented dairy products include yogurt, cheese and / or sour cream.

8. Use of the starter culture according to any one of claims 1 to 4 for increasing the content of beneficial metabolites in fermented dairy products; The beneficial metabolites include non-volatile metabolites and volatile metabolites.

9. A fermented milk, characterized in that: The method for preparing the fermented milk comprises: inoculating the starter according to any one of claims 1 to 4 into cow's milk and / or goat's milk.

10. The fermented milk according to claim 9, wherein The inoculation amount of the starter is 1×10 9 -1×10 12 CFU.

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