Preparation method of lactic acid bacteria fermentation extract dairy product

Abstract

The invention discloses a preparation method of a lactic acid bacteria fermentation extract dairy product, and belongs to the technical field of dairy product processing. The method comprises the following steps: (1) mixing eclipta and glossy privet fruit according to a weight ratio of 1: (0.5-2), adding water, extracting, and concentrating to obtain an eclipta-glossy privet fruit extract; (2) mixing the skimmed milk with the herba ecliptae-glossy privet fruit extract, sterilizing, inoculating lactic acid bacteria, and fermenting until the pH value is 4.2-4.8 to obtain fermentation liquor; (3) sequentially carrying out two-step enzymolysis on the fermentation liquor through pepsin and trypsin to obtain enzymatic hydrolysate; (4) centrifuging the enzymatic hydrolysate, taking supernatant, enabling the supernatant to sequentially pass through ultrafiltration membranes with the molecular weight cut-off of 8kDa and 5kDa, and collecting components with the molecular weight of 5-8kDa; and (5) mixing the obtained components with the dairy product base material and the sweetening agent, homogenizing, and sterilizing to obtain the product. Through combination of fermentation, enzymolysis and ultrafiltration separation, active components in a specific molecular weight interval are enriched, and the prepared dairy product has the effects of inhibiting periodontal pathogenic bacteria and regulating inflammatory factors and can be used for daily prevention and adjuvant therapy of periodontitis.

Description

Technical Field

[0001] This invention relates to the field of dairy processing technology, specifically to a method for preparing dairy products from lactic acid bacteria fermentation extract. Background Technology

[0002] Periodontitis is a chronic infectious disease of the periodontal supporting tissues caused by dental plaque biofilm, and it is one of the leading causes of tooth loss in adults. Current clinical treatments mainly include mechanical debridement and drug therapy. However, mechanical debridement is difficult to completely remove deep-seated bacteria, long-term use of antibiotics easily leads to drug resistance, and nonsteroidal anti-inflammatory drugs (NSAIDs) have gastrointestinal side effects. Therefore, there is a practical need to develop a safe and suitable functional food for daily use for the prevention and adjunctive treatment of periodontitis.

[0003] Lactic acid bacteria fermented dairy products are widely used in the functional food industry due to their probiotic properties and good taste. Existing technologies already include solutions that combine traditional Chinese medicine extracts with lactic acid bacteria fermentation. For example, Chinese patent application CN112136892A discloses a lactic acid bacteria beverage containing daylily extract, and Chinese patent application CN117223809A discloses a technology for co-fermenting honeysuckle, dandelion, and lactic acid bacteria.

[0004] However, existing technologies still have the following shortcomings: First, the fermentation products are not separated and purified, the active ingredients are complex, the effective content is unclear, and it is difficult to guarantee the stability of the function.

[0005] Secondly, most of the products are liquid beverages that can be consumed directly, and their short residence time in the oral cavity makes it difficult for the active ingredients to reach an effective concentration in the periodontal tissues.

[0006] Third, existing technologies make rather general claims about the "anti-inflammatory" effects and lack functional optimization targeting the pathogenesis of periodontitis.

[0007] To address the aforementioned problems, this invention provides a method for preparing dairy products from lactic acid bacteria fermentation extract. By combining fermentation, enzymatic hydrolysis, and ultrafiltration separation, active components within a specific molecular weight range are enriched and then compounded with dairy product base materials to obtain dairy products suitable for daily consumption that have preventive and adjunctive therapeutic effects on periodontitis. Summary of the Invention

[0008] The purpose of this invention is to overcome the above-mentioned technical difficulties and provide a method for preparing dairy products from lactic acid bacteria fermentation extract.

[0009] To achieve the above objectives, the technical solution adopted is as follows: a method for preparing dairy products from lactic acid bacteria fermentation extract, comprising the following steps: (1) Mix Eclipta prostrata and Ligustrum lucidum in a weight ratio of 1:0.5-2, add 8-15 times the amount of water, extract at 80-95℃ 1-2 times, 1-2 hours each time, filter, concentrate the filtrate to a relative density of 1.05-1.15 at 60℃ to obtain Eclipta prostrata-Ligustrum lucidum extract. (2) Mix the skim milk with the Eclipta prostrata-Ligustrum lucidum extract obtained in step (1) at a weight ratio of 10:1-5, sterilize, inoculate with lactic acid bacteria, and ferment at 35-42℃ until the pH value is 4.2-4.8 to obtain the fermentation broth; (3) Adjust the pH of the fermentation broth obtained in step (2) to 2.0-2.5, add pepsin, and enzymatically hydrolyze at 35-40℃ for 2-4 hours; then adjust the pH to 7.0-7.5, add trypsin, and enzymatically hydrolyze at 45-50℃ for 2-5 hours; inactivate the enzyme to obtain the enzymatic hydrolysate; (4) Centrifuge the enzymatic hydrolysate obtained in step (3) and take the supernatant. Pass it through ultrafiltration membranes with molecular weight cutoffs of 8kDa and 5kDa in sequence to collect the active components with molecular weights of 5-8kDa. (5) Mix the active components obtained in step (4) with dairy product base and sweetener, homogenize and sterilize to obtain the lactic acid bacteria fermentation extract dairy product.

[0010] Furthermore, the dairy product base material in step (5) is fermented milk, sterilized milk, modified milk, or milk-containing beverage.

[0011] Furthermore, in step (5), the weight ratio of the active component to the dairy product base is 1:5-20.

[0012] Furthermore, the sweetener in step (5) is at least one of fructooligosaccharides, galactooligosaccharides, erythritol, xylitol, and steviol glycosides.

[0013] Furthermore, the amount of sweetener added in step (5) is 2-10% of the weight of the dairy product base.

[0014] Furthermore, the lactic acid bacteria in step (2) are at least one of Lactobacillus plantarum, Streptococcus thermophilus, and Lactobacillus bulgaricus.

[0015] Furthermore, the sterilization in step (2) is sterilization at 90-95℃ for 10-15 minutes.

[0016] Furthermore, in step (3), the amount of pepsin added is 0.1-0.5% of the weight of the fermentation broth, and the amount of trypsin added is 0.1-0.5% of the weight of the fermentation broth.

[0017] Furthermore, the centrifugation in step (4) is 8000-10000 r / min for 15-20 min.

[0018] Furthermore, in step (5), the homogenization pressure is 20-25 MPa and the homogenization time is 3-5 min; the sterilization is performed at 85-95℃ for 15-30 s.

[0019] The beneficial effects of adopting the above scheme are as follows: After the application of this method for preparing dairy products from lactic acid bacteria fermentation extract, (1) the components with a molecular weight of 5-8kDa are collected by two-stage ultrafiltration membrane separation, which realizes the directional enrichment of active ingredients and helps to ensure the stability of product function.

[0020] (2) Adding two enzymatic hydrolysis steps of pepsin and trypsin after fermentation can hydrolyze milk protein into small molecule peptides, while destroying the cell wall of lactic acid bacteria to release intracellular substances, and converting flavonoid glycosides in Eclipta prostrata-Ligustrum lucidum into aglycones, thereby improving the bioavailability of active ingredients.

[0021] (3) The final product is in the form of dairy products, has a good taste, and can be used as a functional food for daily consumption, for the prevention and adjunctive treatment of periodontitis.

[0022] (4) The selected Eclipta prostrata and Ligustrum lucidum are commonly used Chinese medicines listed in the pharmacopoeia, which have anti-inflammatory and antioxidant effects, providing theoretical support for the functional positioning of the present invention. Detailed Implementation

[0023] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. The described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Example

[0024] A method for preparing a dairy product from lactic acid bacteria fermentation extract includes the following steps: (1) Preparation of Eclipta prostrata-Ligustrum lucidum extract: Take 500g of Eclipta prostrata and 500g of Ligustrum lucidum, mix them, add 10 times the amount of water, extract twice at 90℃ for 1.5h each time, filter, combine the filtrates, concentrate under reduced pressure until the relative density is 1.10 at 60℃, and obtain Eclipta prostrata-Ligustrum lucidum extract.

[0025] (2) Fermentation: Take 1000g of skim milk and 200g of Eclipta prostrata-Ligustrum lucidum extract obtained in step (1), mix them evenly, sterilize at 95℃ for 10min, cool to 37℃, inoculate with Lactobacillus plantarum, ferment at 37℃ until the pH value is 4.5, and obtain fermentation liquid.

[0026] (3) Enzymatic hydrolysis: Adjust the pH of the fermentation broth obtained in step (2) to 2.2, add pepsin (0.3% of the weight of the fermentation broth), and hydrolyze at 37°C for 3 hours; then adjust the pH to 7.2, add trypsin (0.3% of the weight of the fermentation broth), and hydrolyze at 48°C for 3 hours; inactivate the enzyme at 90°C for 10 minutes to obtain the hydrolysate.

[0027] (4) Separation: Centrifuge the enzymatic hydrolysate obtained in step (3) at 8000 r / min for 15 min and take the supernatant; pass it through ultrafiltration membranes with molecular weight cutoffs of 8 kDa and 5 kDa in sequence to collect the components with molecular weights of 5-8 kDa.

[0028] (5) Compounding: Mix the component with a molecular weight of 5-8kDa obtained in step (4) with the fermented milk base material at a weight ratio of 1:10, add fructooligosaccharides (the amount added is 5% of the weight of the fermented milk base material), homogenize (pressure 22MPa, time 3min), sterilize at 85℃ for 15s, fill, and obtain lactic acid bacteria fermented extract fermented milk. Example

[0029] A method for preparing a dairy product from lactic acid bacteria fermentation extract includes the following steps: (1) Preparation of Eclipta prostrata-Ligustrum lucidum extract: Take 400g of Eclipta prostrata and 600g of Ligustrum lucidum, mix them, add 12 times the amount of water, extract twice at 85℃ for 2 hours each time, filter, combine the filtrates, concentrate under reduced pressure until the relative density is 1.08 at 60℃, and obtain Eclipta prostrata-Ligustrum lucidum extract.

[0030] (2) Fermentation: Take 1000g of skim milk and 150g of Eclipta prostrata-Ligustrum lucidum extract obtained in step (1), mix them evenly, sterilize at 95℃ for 12min, cool to 40℃, inoculate with Streptococcus thermophilus, ferment at 40℃ until the pH value is 4.4, and obtain fermentation broth.

[0031] (3) Enzymatic hydrolysis: Adjust the pH of the fermentation broth obtained in step (2) to 2.0, add pepsin (0.2% of the weight of the fermentation broth), and hydrolyze at 38℃ for 4 hours; then adjust the pH to 7.0, add trypsin (0.4% of the weight of the fermentation broth), and hydrolyze at 45℃ for 4 hours; inactivate the enzyme at 90℃ for 10 minutes to obtain the hydrolysate.

[0032] (4) Separation: Centrifuge the enzymatic hydrolysate obtained in step (3) at 9000 r / min for 12 min and take the supernatant; pass it through ultrafiltration membranes with molecular weight cutoffs of 8 kDa and 5 kDa in sequence to collect the components with molecular weights of 5-8 kDa.

[0033] (5) Compounding: The component with a molecular weight of 5-8kDa obtained in step (4) is mixed with the sterilized milk base material at a weight ratio of 1:15, erythritol is added (the amount added is 4% of the weight of the sterilized milk base material), homogenized (pressure 20MPa, time 4min), sterilized at 90℃ for 20s, and filled to obtain sterilized milk of lactic acid bacteria fermentation extract. Example

[0034] A method for preparing a dairy product from lactic acid bacteria fermentation extract includes the following steps: (1) Preparation of Eclipta prostrata-Ligustrum lucidum extract: Take 600g of Eclipta prostrata and 400g of Ligustrum lucidum, mix them, add 8 times the amount of water, extract once at 95℃ for 2 hours each time, filter, concentrate the filtrate under reduced pressure until the relative density is 1.12 at 60℃, and obtain Eclipta prostrata-Ligustrum lucidum extract.

[0035] (2) Fermentation: Take 1000g of skim milk and 100g of Eclipta prostrata-Ligustrum lucidum extract obtained in step (1), mix them evenly, sterilize at 90℃ for 15min, cool to 35℃, inoculate with Lactobacillus bulgaricus, ferment at 35℃ until the pH value is 4.6, and obtain fermentation broth.

[0036] (3) Enzymatic hydrolysis: Adjust the pH of the fermentation broth obtained in step (2) to 2.5, add pepsin (0.4% of the weight of the fermentation broth), and hydrolyze at 40°C for 2 hours; then adjust the pH to 7.5, add trypsin (0.2% of the weight of the fermentation broth), and hydrolyze at 50°C for 2 hours; inactivate the enzyme at 90°C for 10 minutes to obtain the hydrolysate.

[0037] (4) Separation: Centrifuge the enzymatic hydrolysate obtained in step (3) at 10000 r / min for 10 min and take the supernatant; pass it through ultrafiltration membranes with molecular weight cutoffs of 8 kDa and 5 kDa in sequence to collect the components with molecular weights of 5-8 kDa.

[0038] (5) Compounding: Mix the component with a molecular weight of 5-8kDa obtained in step (4) with the base material of the milk beverage at a weight ratio of 1:5, add galactooligosaccharides (the amount added is 6% of the weight of the base material of the milk beverage), homogenize (pressure 25MPa, time 3min), sterilize at 95℃ for 15s, and fill to obtain a milk beverage containing lactic acid bacteria fermentation extract.

[0039] Comparative Example 1: The difference from Example 1 is that the enzymatic hydrolysis in step (3) is not performed, and the fermentation broth is directly subjected to ultrafiltration separation in step (4).

[0040] Comparative Example 2: The difference from Example 1 is that the ultrafiltration separation in step (4) is not performed, and the enzymatic hydrolysate is directly mixed in step (5).

[0041] Comparative Example 3: The difference from Example 1 is that in step (4), an ultrafiltration membrane with a molecular weight cutoff of 3kDa is used to collect components with a molecular weight less than 3kDa.

[0042] Comparative Example 4: The difference from Example 1 is that in step (4), an ultrafiltration membrane with a molecular weight cutoff of 10 kDa is used to collect components with a molecular weight greater than 10 kDa.

[0043] Comparative Example 5: The difference from Example 1 is that: in step (2), no Eclipta prostrata-Ligustrum lucidum extract is added, and only skim milk is used for fermentation. Example

[0044] The applicant conducted a sensory evaluation of the lactic acid bacteria fermentation extract dairy products prepared in Examples 1-3 (n=20), and the results are shown in Table 1 (sensory evaluation results).

[0045] project Example 1 Example 2 Example 3 Color Milky white, uniform Milky white, uniform Milky white, uniform taste The sweet and sour flavors are well-balanced, with no unpleasant odors. The sweet and sour flavors are well-balanced, with no unpleasant odors. The sweet and sour flavors are well-balanced, with no unpleasant odors. odor Normal fermented frankincense normal frankincense normal frankincense Organizational status Even and smooth, without layering Even and smooth, without layering Even and smooth, without layering Table 1 The applicant used HPLC to detect the content of wedelia lactone and oleanolic acid in each group of samples. The results are shown in Table 2 (results of active ingredient content detection).

[0046] Group Wedelia lactone (μg / g) Oleanolic acid (μg / g) Example 1 12.5-15.0 8.0-10.0 Example 2 10.0-12.5 10.0-12.0 Example 3 15.0-18.0 6.0-8.0 Comparative Example 1 6.0-8.0 4.0-5.5 Comparative Example 2 5.0-7.0 3.5-5.0 Comparative Example 5 Not detected Not detected Table 2 The applicant used high performance liquid chromatography to detect the molecular weight distribution of the components obtained in step (4) of Example 1. The results showed that the molecular weight was mainly distributed in the 5-8 kDa range, which accounted for more than 70% of the total peak area.

[0047] The applicant used the micro-broth dilution method to determine the minimum inhibitory concentration (MIC) of each group of samples against Porphyromonas gingivalis. The results are shown in Table 3 (MIC detection results for Porphyromonas gingivalis).

[0048] Group MIC (mg / mL) Example 1 0.2-0.3 Example 2 0.2-0.4 Example 3 0.2-0.3 Comparative Example 1 0.5-0.7 Comparative Example 2 0.6-0.8 Comparative Example 3 0.8-1.0 Comparative Example 4 0.9-1.2 Comparative Example 5 1.5-2.0 Table 3 The applicant used an LPS-induced RAW264.7 macrophage model and used ELISA to detect the inhibition rate of TNF-α secretion in each group of samples. The results are shown in Table 4 (TNF-α inhibition rate detection results).

[0049] Group TNF-α inhibition rate (%) Example 1 75-85 Example 2 70-80 Example 3 72-82 Comparative Example 1 40-50 Comparative Example 2 35-45 Comparative Example 3 30-40 Comparative Example 4 25-35 Comparative Example 5 10-20 Table 4 As shown in Table 4, the TNF-α inhibition rate of the example group was higher than that of each comparative group, indicating that the product prepared by the method of the present invention has better anti-inflammatory activity. Example

[0050] Based on the applicant's previous research, this embodiment further verifies the regulatory effect of the lactic acid bacteria fermentation extract dairy products prepared in this invention on the TGF-β signaling pathway.

[0051] (1) Preparation of Eclipta prostrata-Ligustrum lucidum extract: Take 500g of Eclipta prostrata and 500g of Ligustrum lucidum, mix them, add 10 times the amount of water, extract twice at 90℃ for 1.5h each time, filter, combine the filtrates, and concentrate under reduced pressure until the relative density is 1.10 at 60℃ to obtain Eclipta prostrata-Ligustrum lucidum extract.

[0052] (2) Fermentation: Take 1000g of skim milk and 200g of Eclipta prostrata-Ligustrum lucidum extract obtained in step (1), mix them evenly, sterilize at 95℃ for 10min, cool to 37℃, inoculate with Lactobacillus plantarum, ferment at 37℃ until the pH value is 4.5, and obtain fermentation broth.

[0053] (3) Enzymatic hydrolysis: Adjust the pH of the fermentation broth obtained in step (2) to 2.2, add pepsin (0.3% of the weight of the fermentation broth), and enzymatically hydrolyze at 37°C for 3 hours; then adjust the pH to 7.2, add trypsin (0.3% of the weight of the fermentation broth), and enzymatically hydrolyze at 48°C for 3 hours; inactivate the enzyme at 90°C for 10 minutes to obtain the enzymatic hydrolysate.

[0054] (4) Separation: Centrifuge the enzymatic hydrolysate obtained in step (3) at 8000 r / min for 15 min and take the supernatant; pass it through ultrafiltration membranes with molecular weight cutoffs of 8 kDa and 5 kDa in sequence to collect the fraction with molecular weight of 5-8 kDa.

[0055] (5) Compound preparation: The component with a molecular weight of 5-8 kDa obtained in step (4) is mixed with the fermented milk base material at a weight ratio of 1:10. Fructooligosaccharides are added (the amount added is 5% of the weight of the fermented milk base material), homogenized (pressure 22 MPa, time 3 min), sterilized at 85℃ for 15 s, and filled to obtain lactic acid bacteria fermented extract fermented milk.

[0056] (6) Verification of the regulatory effect of TGF-β pathway Human periodontal ligament fibroblasts (hPDLFs) were used as a model. A control group, an LPS stimulation group, and a sample group (concentration 0.05 mg / mL) from Example 4 were set up. After culturing for 48 h, the protein expression levels of TGF-β1, Smad2, and Smad3 were detected by Western blotting, and the mRNA expression levels of TGF-β1, Smad2, and Smad3 were detected by qRT-PCR. As can be seen from the table above, LPS stimulation can inhibit the expression of TGF-β signaling pathway-related factors. After the samples were treated in Example 5 of this invention, the expression levels of TGF-β1, Smad2, and Smad3 significantly increased, indicating that the lactic acid bacteria fermentation extract dairy products prepared in this invention have the effect of activating the TGF-β signaling pathway.

[0057] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0058] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing a dairy product from a lactic acid bacteria fermentation extract, characterized in that: Includes the following steps: (1) Mix Eclipta prostrata and Ligustrum lucidum in a weight ratio of 1:0.5-2, add 8-15 times the amount of water, extract at 80-95℃ 1-2 times, 1-2 hours each time, filter, concentrate the filtrate to a relative density of 1.05-1.15 at 60℃ to obtain Eclipta prostrata-Ligustrum lucidum extract. (2) Mix the skim milk with the Eclipta prostrata-Ligustrum lucidum extract obtained in step (1) at a weight ratio of 10:1-5, sterilize, inoculate with lactic acid bacteria, and ferment at 35-42℃ until the pH value is 4.2-4.8 to obtain the fermentation broth; (3) Adjust the pH of the fermentation broth obtained in step (2) to 2.0-2.5, add pepsin, and enzymatically hydrolyze at 35-40℃ for 2-4 hours; then adjust the pH to 7.0-7.5, add trypsin, and enzymatically hydrolyze at 45-50℃ for 2-5 hours; inactivate the enzyme to obtain the enzymatic hydrolysate; (4) Centrifuge the enzymatic hydrolysate obtained in step (3) and take the supernatant. Pass it through ultrafiltration membranes with molecular weight cutoffs of 8kDa and 5kDa in sequence to collect the active components with molecular weights of 5-8kDa. (5) Mix the active components obtained in step (4) with dairy product base and sweetener, homogenize and sterilize to obtain the lactic acid bacteria fermentation extract dairy product.

2. The method for preparing dairy products from lactic acid bacteria fermentation extract according to claim 1, characterized in that: The dairy product base material mentioned in step (5) is fermented milk, sterilized milk, modified milk or milk-containing beverage.

3. The method for preparing dairy products from lactic acid bacteria fermentation extract according to claim 1, characterized in that: The weight ratio of the active component to the dairy product base in step (5) is 1:5-20.

4. The method for preparing dairy products from lactic acid bacteria fermentation extract according to claim 1, characterized in that: The sweetener mentioned in step (5) is at least one of fructooligosaccharides, galactooligosaccharides, erythritol, xylitol, and steviol glycosides.

5. The method for preparing dairy products from lactic acid bacteria fermentation extract according to claim 1, characterized in that: The amount of sweetener added in step (5) is 2-10% of the weight of the dairy product base.

6. The method for preparing dairy products from lactic acid bacteria fermentation extract according to claim 1, characterized in that: The lactic acid bacteria mentioned in step (2) are at least one of Lactobacillus plantarum, Streptococcus thermophilus, and Lactobacillus bulgaricus.

7. The method for preparing dairy products from lactic acid bacteria fermentation extract according to claim 1, characterized in that: The sterilization in step (2) is sterilization at 90-95℃ for 10-15 minutes.

8. The method for preparing dairy products from lactic acid bacteria fermentation extract according to claim 1, characterized in that: In step (3), the amount of pepsin added is 0.1-0.5% of the weight of the fermentation broth, and the amount of trypsin added is 0.1-0.5% of the weight of the fermentation broth.

9. The method for preparing dairy products from lactic acid bacteria fermentation extract according to claim 1, characterized in that: The centrifugation in step (4) is 8000-10000 r / min for 15-20 min.

10. The method for preparing dairy products from lactic acid bacteria fermentation extract according to claim 1, characterized in that: The homogenization pressure in step (5) is 20-25 MPa, and the homogenization time is 3-5 min; the sterilization is performed at 85-95℃ for 15-30 s.