Phytobacterium plantarum and application thereof in fermentation of birch juice

Abstract

The invention relates to the field of food technology and probiotic fermentation, in particular to a plant lactobacillus and application thereof in fermentation of birch juice. The invention provides the plant lactobacillus with the preservation number of CGMCCNO.33553. Compared with other candidate lactic acid bacteria, the plant lactobacillus BB16 provided by the invention has the advantages that the growth performance of the plant lactobacillus BB16 in the birch juice is more excellent, and the metabolic activity is more outstanding. The beverage obtained by fermentation of the compound is richer in amino acid and volatile compound content, and the antioxidant activity is remarkably improved, so that higher-quality healthy beverage experience is brought to consumers.

Description

Technical Field

[0001] The present invention relates to the fields of food technology and probiotic fermentation, and particularly to a Lactiplantibacillus plantarum strain and its application in fermenting birch sap. Background Art

[0002] Birch sap is the sap flowing out of birch trunks, which is transparent, colorless or slightly yellowish, and has a faint birch fragrance. As a natural plant resource, it is rich in various nutrients beneficial to the human body, such as minerals, vitamins, amino acids, etc., and is known as the "guardian of human health". In recent years, with the increasing attention of people to healthy foods, the development and utilization of birch sap have gradually received attention. However, birch sap has problems such as a single taste and insufficient flavor.

[0003] In the food field, fermentation is an important means. Through fermentation, birch sap can be given a more rich and unique flavor level, significantly broadening its flavor dimension. The selection of strains plays a decisive role in the fermentation effect. Different strains have unique metabolic pathways and physiological characteristics, which enables them to produce completely different flavor substances when fermenting natural fruit juices. If the strain is not selected properly, it may not only lead to poor fermentation effects, but also cause the flavor of the juice to deviate from expectations and fail to meet market demands. Therefore, in order to obtain fermented beverages with excellent flavor and high quality, it is crucial to accurately screen suitable strains.

[0004] Lactic acid bacteria, as a type of probiotics, play an important role in the fermentation process of food and beverages. Lactic acid bacteria fermentation can not only endow the fermented products with unique flavors, but also improve the nutritional value of the products. Therefore, improving the flavor and nutrition of birch sap through lactic acid bacteria fermentation technology has become an important way to enhance its application value.

[0005] However, due to the significant differences in the contents of sugars, amino acids, and other components in birch sap compared with common fruit juices, lactic acid bacteria strains suitable for the fermentation of ordinary fruit juices are difficult to be directly applied to the fermentation of birch sap. The specific sugar ratio in birch sap may affect the metabolic pathway and growth rate of lactic acid bacteria; its unique amino acid composition may have special effects on the enzyme system and flavor substance synthesis of lactic acid bacteria. Therefore, in order to achieve high-quality fermentation of birch sap, it is necessary to screen out lactic acid bacteria strains that are compatible with it.

[0006] However, unfortunately, so far, there have been no reports on lactic acid bacteria strains suitable for the fermentation of birch sap. This gap not only restricts the development of birch sap fermented products, but also hinders the further expansion of birch sap in the fermented beverage market. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a Lactiplantibacillus plantarum strain and its application in fermenting birch sap.

[0008] The present invention provides Lactiplantibacillus plantarum with the preservation number of CGMCC NO. 33553.

[0009] In the present invention, Lactiplantibacillus plantarum BB16 was screened from 8 different lactic acid bacteria. Compared with other candidate lactic acid bacteria, Lactiplantibacillus plantarum BB16 of the present invention shows better growth performance and more prominent metabolic activity in birch sap. The drink fermented by it is richer in amino acids and volatile compounds, and its antioxidant activity is significantly improved.

[0010] The present invention also provides a bacterial agent, which comprises the aforementioned Lactiplantibacillus plantarum and auxiliary materials.

[0011] The bacterial agent provided by the present invention refers to a bacterial liquid, powder or tablet comprising the aforementioned Lactiplantibacillus plantarum.

[0012] In the present invention, the auxiliary materials in the bacterial agent include but are not limited to nutritional auxiliary materials, protective auxiliary materials, regulatory auxiliary materials or filling auxiliary materials. As a feasible case, the nutritional auxiliary materials include but are not limited to glucose, sucrose, milk powder, yeast extract, peptone, polypeptide, vitamin; the protective auxiliary materials include but are not limited to ascorbic acid, trehalose; the regulatory auxiliary materials include but are not limited to buffer solution or sodium carboxymethylcellulose, xanthan gum; the filling auxiliary materials include but are not limited to maltodextrin or corn starch.

[0013] Furthermore, the present invention also provides a preparation method of the aforementioned bacterial agent, which comprises culturing the aforementioned Lactiplantibacillus plantarum, and then concentrating or drying in the presence of auxiliary materials to obtain the bacterial agent.

[0014] In the present invention, the culture medium for culturing is MRS medium.

[0015] In the present invention, the auxiliary materials include at least one of milk powder, sucrose, trehalose, maltodextrin or corn starch.

[0016] Even further, the present invention also provides the application of the aforementioned Lactiplantibacillus plantarum or the aforementioned bacterial agent in fermenting birch sap.

[0017] Even further, the present invention also provides a preparation method of fermented birch sap, which comprises: activating the aforementioned Lactiplantibacillus plantarum or the aforementioned bacterial agent, and then fermenting the birch sap.

[0018] In the present invention, the inoculation amount for fermentation is 10 5 CFU / mL~10 7CFU / mL, and the fermentation conditions include fermenting at 28 - 32°C for 48 h. Preferably, the inoculation amount for fermentation is 10 6 CFU / mL, and the fermentation conditions include fermenting at 30°C for 48 h.

[0019] In the present invention, the activation includes inoculating the Lactiplantibacillus plantarum as described above or the bacterial agent as described above in MRS medium, activating at 35 - 40°C, subculturing every 8 - 15 h, and removing the medium after 30 - 40 h and resuspending the bacterial cells with physiological saline. Preferably, the activation conditions include activating at 37°C, subculturing every 12 h, removing the medium after 36 h and resuspending the bacterial cells with physiological saline to obtain a seed solution.

[0020] Furthermore, the present invention also provides fermented birch sap prepared by the preparation method as described above.

[0021] Furthermore, the present invention also provides a food or beverage, which includes acceptable ingredients in the food or beverage and fermented birch sap prepared by the preparation method as described above.

[0022] As a feasibility case, in the food or beverage, the addition amount of the fermented birch sap as described above is 1% - 99%. For example, the food or beverage includes birch sap yogurt, pastries, compound fruit juices, candies, jellies or instant powders.

[0023] The present invention provides Lactiplantibacillus plantarum with the preservation number of CGMCC NO. 33553. Compared with other candidate lactic acid bacteria, the Lactiplantibacillus plantarum BB16 of the present invention has better growth performance and more prominent metabolic activity in birch sap. The beverage fermented by it has richer amino acid and volatile compound contents, and significantly improved antioxidant activity, thus bringing a higher-quality healthy beverage experience to consumers.

[0024] Biological preservation description

[0025] The biological material Lactiplantibacillus plantarum BB16, classified and named: Lactiplantibacillus plantarum, was preserved in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 15, 2025. The address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. The preservation number is CGMCC NO. 33553. Description of the drawings

[0026] Figure 1 shows the viable count of 8 different lactic acid bacteria in fermented birch sap;

[0027] Figure 2 shows the pH value of the birch sap fermented beverage prepared by 8 different lactic acid bacteria;

[0028] Figure 3 Total acid content in birch sap fermented beverages prepared with 8 different lactic acid bacteria;

[0029] Figure 4 Reducing sugar content in birch sap fermented beverages prepared with 8 different lactic acid bacteria;

[0030] Figure 5 Antioxidant activity in birch sap fermented beverages prepared with 8 different lactic acid bacteria. Detailed implementation manners

[0031] The present invention provides a Lactiplantibacillus plantarum strain and its application in fermenting birch sap. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate alterations and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0032] The test materials used in the present invention are all ordinary commercially available products and can be purchased in the market. The following further elaborates the present invention in conjunction with embodiments:

[0033] Example 1 Screening of lactic acid bacteria for fermenting birch sap

[0034] The present invention uses 8 lactic acid bacteria strains (the specific strain numbers and sources are shown in Table 1). Among them, 4 strains are isolated from fruits and identified as Lactiplantibacillus plantarum, and the other 4 strains are commercially sourced strains, namely Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus casei, and Lactobacillus acidophilus. The above-mentioned 8 lactic acid bacteria strains are used to ferment birch sap respectively. The lactic acid bacteria with the most excellent growth performance and the most prominent vitality are selected. The growth of lactic acid bacteria is represented by the viable count of lactic acid bacteria after fermentation in birch sap, that is, the more viable lactic acid bacteria, the better the growth of lactic acid bacteria in birch sap; the activity of lactic acid bacteria is reflected by the total acid content in birch sap after fermentation by lactic acid bacteria, that is, the higher the total acid content, the better the fermentation situation, that is, the higher the activity of lactic acid bacteria. Strains with good growth and high activity are more suitable for fermenting birch sap.

[0035] The specific process is as follows:

[0036] Inoculate lactic acid bacteria in liquid MRS medium, place it in an incubator at 37°C for activation, subculture every 12 h, remove the medium after 36 h, wash and dissolve it with physiological saline, and finally obtain a lactic acid bacteria seed solution. The lactic acid bacteria seed solution is inoculated into birch sap at a ratio of 10 6Inoculate the birch sap at an inoculation concentration of CFU / mL, and ferment it at 30 °C for 48 h to obtain fermented birch sap.

[0037] Determine the viable count of lactic acid bacteria, pH, and total acid in the obtained 8 different fermented birch saps.

[0038] Determination of viable count of lactic acid bacteria: After appropriately diluting the fermentation broth with sterile physiological saline in gradient, take 1 mL of the diluted solution and add it to a petri dish. Then, use the pour plate method to add MRS agar medium to the petri dish containing the diluted sample solution, shake well, let it solidify, and place it in an incubator at 37 °C for cultivation. After 48 h, count the colonies.

[0039] pH value determination: Use a pH meter to directly measure the pH value of the fermentation broth.

[0040] Total acid determination: The total acid is determined with reference to the national standard GB 12456-2021. Accurately pipette 10 ml of the sample, use phenolphthalein as an indicator, and titrate it with 0.05 mol / L NaOH solution to the end point, and calculate the total acid content.

[0041] From Figures 1-3 It was found from the results that after 48 h of fermentation in birch sap, the viable count of Lactiplantibacillus plantarum BB16 in birch sap was the highest, reaching 8.0 lg CFU / ml, and the viable counts of other strains were between 7.4 lg and 8.0 lg. Compared with the viable count of 6 lg (CFU / mL) at the time of inoculation, the viable counts of all strains increased by at least one order of magnitude after 48 h, indicating that lactic acid bacteria can adapt to birch sap and utilize its nutrients for growth and metabolism, and at the same time, there are differences in the growth ability of different strains in birch sap. After fermentation by BB16, the pH value decreased to 3.37, and the total acid content was the highest, exceeding 0.6 g / L, which was about 16 times higher than that of the unfermented birch sap, while the total acid content of the birch sap fermented by other strains was about between 0.2 and 0.6 g / L. This shows that the Lactiplantibacillus plantarum BB16 of the present invention has excellent growth performance and outstanding activity in birch sap, demonstrating strong fermentation ability and being the best strain for fermenting birch sap.

[0042] Table 1 Sources and numbers of 8 strains of lactic acid bacteria

[0043]

[0044] Example 2 Preparation of fermented birch sap drink

[0045] 1. Preparation method

[0046] Inoculate Lactobacillus BB16 in liquid MRS medium, place it in an incubator at 37 °C for activation, subculture it every 12 h, and after 36 h, remove the medium, wash and dissolve it with physiological saline, and finally obtain a Lactobacillus seed solution. Take the Lactobacillus seed solution at 106 Inoculate into birch sap at an inoculation concentration of CFU / mL, and ferment at 30 °C for 48 h to obtain fermented birch sap. Measure the pH, total acid, reducing sugar, antioxidant activity, volatile compounds and amino acids of the fermented birch sap, with unfermented birch sap as the control.

[0047] 2. Experimental results

[0048] (1) Measure the pH, total acid and reducing sugar of birch sap before and after fermentation.

[0049] pH value measurement: Use a pH meter to directly measure the pH value of the fermentation broth.

[0050] Total acid measurement: The total acid content is measured with reference to the national standard GB 12456-2021. Precisely pipette 10 ml of the sample, use phenolphthalein as an indicator, and titrate to the end point with 0.05 mol / L NaOH solution to calculate the total acid content.

[0051] Reducing sugar measurement: After diluting the sample 10 times, measure it according to the national standard GB NYT2742-2015. Take the glucose concentration as the abscissa and the absorbance value as the ordinate to draw a standard curve, and calculate the reducing sugar content according to the standard curve.

[0052] Figures 2-4 The results show that compared with unfermented birch sap, the total acid of fermented birch sap increased significantly, while the pH and reducing sugar decreased significantly. Among them, the total acid content of the birch sap fermented by BB16 inoculation was the highest, about 0.6 g / L, and the corresponding pH value was the lowest. The reducing sugar content generally decreased, around 3 - 4.6 g / L. Lactic acid bacteria consumed sugars during the growth, reproduction and metabolism process. Therefore, lactic acid bacteria can adapt to the fermentation environment of birch sap, utilize the carbon source in the fermentation system to convert it into organic acids, making the fermented birch sap drink sour and sweet, with more flavor.

[0053] (2) Evaluate the antioxidant activity of birch sap before and after fermentation. The antioxidant activity of the fermentation broth was evaluated by the ABTS radical scavenging rate and measured according to the kit method.

[0054] Figure 5 The results show that the ability of birch sap to scavenge free radicals increased significantly after fermentation. The scavenging rate after fermentation was between 3% - 13%, and the scavenging rate of BB16 was the highest, at 13%.

[0055] This indicates that different strains have different effects on the antioxidant properties of birch sap. BB16 showed outstanding performance in the ABTS radical scavenging rate, indicating strong antioxidant properties.

[0056] (3) Use HS-SPME-GC-MS technology to separate and identify volatile compounds in the fermented birch sap drink prepared by Lactiplantibacillus plantarum BB16.

[0057] Qualitative and quantitative analysis: Under the same chromatographic and mass spectrometric conditions, the n-alkane series was analyzed to calculate the retention indices (RIs) of volatile compounds. The volatile compounds were identified by comparing their mass spectrometric information and retention indices with those of the standards. For volatile compounds without available standards, their retention indices and mass spectrometric information were compared with NIST14 to identify the volatile compounds. The content (Cj) of each volatile compound was represented by multiplying the peak area ratio of the volatile compound (Aj) to the internal standard 4-methyl-2-pentanol (Ais) by the internal standard concentration (Cis, 0.2 g / L), as shown in the following formula (1).

[0058] Cj = Aj × Cis / Ais (1)

[0059] As shown in Table 2, a total of 43 volatile compounds were identified, including 13 alcohols, 5 aldehydes, 12 ketones, 6 acids, 4 esters, and 3 others. 22 were found in unfermented birch sap, and after fermentation with BB16, decanal, ethyl octanoate, 2-pentylfuran, and phenol, as well as 5 alcohols, 8 ketones, and 4 acids were added. Ethyl octanoate has a sweet, fruity, and buttery flavor, and 2-pentylfuran has a vegetable-like aroma. Compared with unfermented sap, the content of volatile compounds in birch sap after fermentation with BB16 increased, except for dodecanol, n-hexanal, 2-butenal, benzaldehyde, and 6,10-dimethyl-5,9-undecadien-2-one. This indicates that the metabolic activity of Lactobacillus plantarum BB16 had a significant impact on the composition of volatile compounds in birch sap, thereby endowing birch sap with unique flavor characteristics.

[0060] Table 2 Composition of volatile compounds in unfermented birch sap and birch sap fermented with BB16

[0061]

[0062]

[0063] Note: Different lowercase letters in the same row indicate significant differences (p < 0.05), and "ND" indicates not detected

[0064] (4) High-performance liquid chromatography (HPLC) technology was used for qualitative and quantitative analysis of amino acids in the birch sap fermented beverage prepared with Lactobacillus plantarum BB16.

[0065] Qualitative and quantitative analysis: Under the same chromatographic conditions, 21 kinds of amino acid standards were analyzed. The retention times of the standards were compared with those of the amino acids in the samples to identify the types of amino acids in the samples. The amino acid standards were gradually diluted to 5 - 7 concentration points, and the chromatographic peaks were measured. With the peak area as the ordinate and the amino acid concentration as the abscissa, a standard curve was plotted, and the amino acid concentration in the samples was calculated according to the standard curve.

[0066] As can be seen from Table 4, a total of 14 kinds of amino acids were identified, 12 in unfermented birch sap and 12 in BB16 - fermented birch sap, among which there were 6 essential amino acids. Alanine and tryptophan were only detected in trace amounts in unfermented birch sap and not detected after BB16 fermentation. Only trace amounts of cysteine could be detected after BB16 fermentation as its content decreased. In unfermented birch sap, the main amino acid components were histidine (28.91 μg / L), isoleucine (17.98 μg / L), and cysteine (15.55 μg / L). After fermentation, they were mainly aspartic acid (72.21 μg / L), isoleucine (27.73 μg / L), and proline (19.61 μg / L). The total amount of amino acids increased from 78.29 μg / L to 148.18 μg / L.

[0067] During the fermentation process, BB16 grew and metabolized, producing new amino acids or transforming the original amino acids, which led to changes in the types of amino acids. At the same time, the metabolic activities also promoted the synthesis of amino acids, resulting in an increase in the total amount. This shows the potential of fermentation technology in enhancing the nutritional value of natural products. BB16 fermentation can optimize the nutritional components of birch sap and endow the product with various health - promoting potentials.

[0068] Table 3 Standard curve of 21 kinds of amino acid standards

[0069]

[0070] Table 4 Amino acid composition in unfermented birch sap and BB16 - fermented birch sap

[0071]

[0072] Note: Different lowercase letters in the same row indicate significant differences (P < 0.05). "ND" means not detected, and "TR" means trace amount.

[0073] In summary, Lactiplantibacillus plantarum BB16 in the present invention has excellent growth characteristics and metabolic activities in birch sap. The birch sap beverage fermented by it is richer in amino acids and volatile compounds, and its antioxidant activity is improved, enhancing the nutritional value, biological activity, and drinking experience of the birch sap beverage.

[0074] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. Lactobacillus plantarum with a deposit number of CGMCC NO.33553.

2. A bacterial agent comprising the plant lactobacillus according to claim 1 and auxiliary materials.

3. The method for preparing the bacterial agent according to claim 2, comprising culturing the plant lactobacillus according to claim 1, and then concentrating or drying in the presence of auxiliary materials to obtain the bacterial agent.

4. The preparation method according to claim 3, characterized in that: The culture medium is MRS medium. The auxiliary material comprises at least one of milk powder, sucrose, trehalose, maltodextrin or corn starch.

5. Use of the plant lactobacillus according to claim 1 or the bacterial agent according to any one of claims 2 to 4 in fermenting birch sap.

6. A method for preparing fermented birch sap, comprising: After activating the plant lactobacillus according to claim 1 or the bacterial agent according to any one of claims 2 to 4, the birch sap is fermented.

7. The preparation method according to claim 6, characterized in that: The inoculum size of the fermentation was 10 5 CFU / mL~10 7 CFU / mL, and the fermentation conditions include fermentation at 28-32°C for 48h.

8. The preparation method according to claim 6 or 7, characterized in that: The activation comprises inoculating the plant lactobacillus according to claim 1 or the bacterial agent according to any one of claims 2 to 4 into an MRS culture medium, activating at 35 to 40° C., subculturing once every 8 to 15 hours, removing the culture medium after 30 to 40 hours, and resuspending the bacteria with physiological saline to obtain a seed solution.

9. Fermented birch sap obtained by the preparation method according to any one of claims 6 to 8.

10. Food or drink comprising an ingredient acceptable in food or drink and fermented birch sap obtained by the preparation method according to any one of claims 6 to 8.

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