Method for improving antioxidant activity of wall-broken pine pollen through lactobacillus fermentation

Abstract

The invention belongs to the technical field of food biological processing, and relates to a method for improving the antioxidant activity of wall-broken pine pollen by lactobacillus fermentation, which comprises the following steps: preparing a wall-broken pine pollen culture medium, adding glucose, heating and preserving heat, inoculating lactobacillus, and fermenting to obtain a fermentation product. The lactic acid bacteria are used for fermentation to improve the level of phenolic components with antioxidant activity and the antioxidant activity of the phenolic components in the wall-broken pine pollen, protease and pectinase secreted by the lactic acid bacteria are used for destroying combination of the phenolic components and macromolecules such as glycoprotein in cell walls, free phenolic components are released, and the antioxidant activity of the wall-broken pine pollen is improved. Therefore, the antioxidant activity of the wall-broken pine pollen fermentation product is improved; meanwhile, by adjusting the pH value of the culture medium, adding glucose, controlling the heating and cooling conditions and optimizing the fermentation time, it is ensured that the fermentation process is efficiently carried out, and therefore the yield of polyphenol in the wall-broken pine pollen is remarkably increased, and the antioxidant activity of the wall-broken pine pollen is enhanced.

Description

Technical Field

[0001] The present invention belongs to the technical field of food bioprocessing, and relates to a method for improving the antioxidant activity of broken-wall pine pollen by lactic acid bacteria fermentation. Background Art

[0002] Broken-wall pine pollen is a pollen variety that is both a medicine and a food in China, mainly derived from the male reproductive cells of Pinus massoniana Lamb. of the Pinaceae family or plants of the same genus. It is rich in active ingredients such as polyphenols, sterols, and polysaccharides, and has broad prospects for development and application, attracting extensive attention in the health food industry. However, many small-molecule active substances exist in a bound state with macromolecular substances such as starch and cellulose, resulting in low bioavailability and difficulty in being absorbed and utilized by the body.

[0003] Fermentation technology can disrupt the binding of small-molecule active substances to macromolecules through enzymes such as proteases, cellulases, and glycosidases secreted during the microbial metabolism process, converting them from a bound state to a free state, thereby increasing the content of small-molecule bioactive substances and enhancing their biological activities.

[0004] Lactic acid bacteria have been widely used in the fields of food, brewing, healthcare, and bioengineering. The fermentation products of lactic acid bacteria are pure biological products, free of chemical components, and have the characteristics of high digestibility and low cost. Currently, the fermentation of pine pollen by lactic acid bacteria is mainly aimed at supplementing the efficacy of probiotics themselves, and some technologies aim to improve the antioxidant ability in vitro, but their efficacy still cannot meet the requirements of functional foods. There is a need for a method that can effectively increase the content of phenolic components in broken-wall pine pollen and thereby enhance its antioxidant ability. Summary of the Invention

[0005] In view of the above deficiencies in the existing technology, the present invention provides a method for improving the antioxidant activity of broken-wall pine pollen by lactic acid bacteria fermentation. The specific technical solution is as follows:

[0006] A method for improving the antioxidant activity of broken-wall pine pollen by lactic acid bacteria fermentation, comprising the following steps:

[0007] (1) Place the broken-wall pine pollen in deionized water, stir evenly, and then adjust the pH value to 5-7 to obtain a broken-wall pine pollen culture medium;

[0008] (2) Add a glucose solution with a mass fraction of 1-9% to the broken-wall pine pollen culture medium in step (1);

[0009] (3) Heat the broken-wall pine pollen culture medium in step (2) to a central temperature of 80-90 °C, and keep it in a water bath at 80-90 °C for 25-40 minutes. After the insulation is completed, quickly cool it to room temperature;

[0010] (4) Inoculate the lactic acid bacteria liquid into the broken-wall pine pollen culture medium in step (3), and ferment at 30-40 °C under static conditions for 1-9 days to obtain a fermentation product; the lactic acid bacteria include Lacticaseibacillus paracasei 6244), Lactiplantibacillus lantarum 21793), Lactococcus lactis 23609), any one or more of them.

[0011] In the present invention, the level of phenolic components with antioxidant activity and their antioxidant activity in broken-wall pine pollen are improved by fermentation with lactic acid bacteria. The protease and pectinase secreted by lactic acid bacteria are used to destroy the binding of phenolic components to macromolecules such as glycoproteins in the cell wall, releasing free phenolic components, thereby enhancing the antioxidant activity of the broken-wall pine pollen fermentation product. At the same time, by adjusting the pH value of the culture medium, adding glucose, controlling the heating and cooling conditions, and optimizing the fermentation time, the fermentation process is ensured to proceed efficiently, thereby significantly increasing the yield of polyphenols in broken-wall pine pollen and enhancing its antioxidant activity.

[0012] Further, in the step (1), the weight ratio of broken-wall pine pollen to deionized water is 1:10-30.

[0013] Further, in the step (1), a 4 mol / L NaOH solution and / or a 4 mol / L HCl solution are used to adjust the pH value.

[0014] Further, in the step (2), a 3% glucose solution by mass fraction is added to the broken-wall pine pollen culture medium in step (1).

[0015] Further, in the step (4), the volume ratio of the lactic acid bacteria liquid to the culture medium is 1:5-20, and the strain concentration of the lactic acid bacteria liquid is 1-2×10 8 cfu / mL.

[0016] Further, in the step (4), the volume ratio of the lactic acid bacteria liquid to the culture medium is 1:20.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention significantly improves the antioxidant activity of broken-wall pine pollen by lactic acid bacteria fermentation, and has the advantages of fermentation strain advantages, enhanced competitiveness and high comprehensive utilization value. Specifically, lactic acid bacteria fermentation significantly increases the content of phenolic components in broken-wall pine pollen and its antioxidant capacity, enhances its bioavailability and health care functions; lactic acid bacteria are probiotics with a wide range of applications, high yields and suitable for food development, and have the advantages of high activity, easy cultivation, low fermentation cost, etc.; compared with Rhizopus oryzae, lactic acid bacteria are strains in the List of Strains That Can Be Used in Foods announced by the National Health Commission, while Rhizopus oryzae is mainly used for traditional brewing such as soy sauce, white wine, and fermented bean curd. Lactic acid bacteria have a wider range of applications, a shorter fermentation cycle, do not require stirring, lower labor and energy consumption, and are more environmentally friendly; compared with Poria cocos fermentation, lactic acid bacteria are more effective in increasing the total phenolic content and antioxidant activity, and can develop more competitive fermentation products in the market; at the same time, the present invention not only improves the utilization value of broken-wall pine pollen, but also provides a theoretical basis and technical support for the development of functional foods. Detailed Embodiments

[0019] The principles and features of the present invention are described below in conjunction with the embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0020] Example 1:

[0021] A method for improving the antioxidant activity of broken-wall pine pollen by lactic acid bacteria fermentation, condition optimization experiment:

[0022] 1. Fermentation strain optimization:

[0023] (1) Place the broken-wall pine pollen in deionized water, stir evenly, and adjust the pH value to 6.5 with 4 mol / L NaOH solution and 4 mol / L HCl solution to obtain the broken-wall pine pollen culture medium; wherein, the weight ratio of broken-wall pine pollen to deionized water is 1:20;

[0024] (2) Heat the broken-wall pine pollen culture medium in a boiling water bath until the central temperature reaches 85 °C, keep it in a water bath at 85 °C for 30 minutes, and then quickly cool it to room temperature;

[0025] (3) Inoculate Lacticaseibacillus paracasei 6244), Ganoderma lingzhi 14048) and Rhizopus chinensis 41051) Ferment for 3 days under the optimal temperature of each strain and the optimal rotation speed of each strain. The specific conditions are as follows: the Lactobacillus casei - like group at 37°C, static; the Ganoderma lucidum group at 26°C, 120 r / min; the Rhizopus chinensis group at 30°C, 200 r / min. Among them, the concentration of each bacterial liquid is 1.5×10 8 cfu / mL;

[0026] (4) Store the fermentation product at -20°C.

[0027] Using the unfermented water extract of broken - wall pine pollen as a control, after the experiment is completed, measure and compare the total phenol content and ABTS free - radical scavenging ability before and after fermentation.

[0028] Table 1 Optimization of fermentation strains

[0029]

[0030] According to the experimental data in Table 1, the total phenol content of the broken - wall pine pollen fermented by Lactobacillus casei - like reaches 3.91 mg / g, which is significantly higher than that of the unfermented water extract (2.49 mg / g), as well as the fermentation by Ganoderma lucidum (2.56 mg / g) and Rhizopus chinensis (2.48 mg / g), which are commonly used strains in food. At the same time, the ABTS free - radical scavenging ability of the broken - wall pine pollen fermented by Lactobacillus casei - like is 72.57 μmol / g, which is much higher than that of the water extract (21.23 μmol / g), Ganoderma lucidum fermentation (45.77 μmol / g) and Rhizopus chinensis fermentation (35.65 μmol / g). Compared with the examples in the existing patents on Poria cocos fermentation, the improvement effect of the total phenol content in the fermentation by Lactobacillus casei - like is more significant (the highest total phenol content in the examples of Poria cocos fermentation patents is 2.74 mg / g). However, due to the advantages of Lactobacillus casei - like, such as a short growth cycle, strong fermentation ability, and easy large - scale cultivation, choosing it as the fermentation strain can more effectively improve the total phenol content and antioxidant ability of broken - wall pine pollen, thereby enhancing its physiological activity.

[0031] 2. Optimization of fermentation inoculum amount:

[0032] (1) Dissolve broken - wall pine pollen in deionized water, stir evenly, and then use 4 mol / L NaOH and 4 mol / L HCl to adjust the pH value to 6.5 to obtain the broken - wall pine pollen medium. Among them, the weight ratio of broken - wall pine pollen to deionized water is 1:20;

[0033] (2) Heat the broken - wall pine pollen medium in a boiling water bath until the central temperature reaches 85°C, place it in a water bath at 85°C for 30 min, and immediately cool it to room temperature with ice water after the insulation time is up;

[0034] (3) Inoculate Lactiplantibacillus paracasei 6244 at the inoculum ratios of 1%, 3%, 5%, 7%, and 9%, and ferment for 3 days at 37°C under static conditions; among them, the concentration of the bacterial liquid is 1.5×10 cfu / mL; 8 cfu / mL;

[0035] (4) Store the fermentation product at -20°C.

[0036] Taking the unfermented aqueous extract of broken-wall pine pollen as a control, after the experiment, measure and compare the total phenol content and ABTS free radical scavenging ability before and after fermentation.

[0037] Table 2 Optimization of fermentation inoculum amount

[0038]

[0039] As can be seen from Table 2, when the inoculum amount is 5%, the total phenol content and ABTS free radical scavenging ability increase most significantly. Therefore, an inoculum amount of 5% is selected for fermentation.

[0040] 3. Optimization of exogenous glucose addition amount:

[0041] (1) Place the broken-wall pine pollen in deionized water, stir evenly, and adjust the pH value to 6.5 using 4 mol / L NaOH and 4 mol / L HCl to obtain the broken-wall pine pollen culture medium; among them, the weight ratio of broken-wall pine pollen to deionized water is 1:20;

[0042] (2) Add glucose with mass fractions of 0%, 1%, 2%, 3%, 5%, 7%, and 9% to the broken-wall pine pollen culture medium;

[0043] (3) Heat the broken-wall pine pollen culture medium in a boiling water bath until the central temperature reaches 85°C, keep it in a water bath at 85°C for 30 min, and immediately cool it to room temperature with ice water after the holding time;

[0044] (4) Inoculate Lactiplantibacillus paracasei 6244 at an inoculum ratio of 10%, and ferment for 3 days at 37°C under static conditions; among them, the concentration of the bacterial liquid is 1.5×10 cfu / mL; 8 cfu / mL;

[0045] (5) Store the fermentation product at -20°C.

[0046] Taking the unfermented aqueous extract of broken-wall pine pollen as a control, after the experiment, measure and compare the total phenol content and ABTS free radical scavenging ability before and after fermentation.

[0047] Table 3 Optimization of exogenous glucose addition amount

[0048]

[0049] According to the experimental data in Table 3, the addition amount of exogenous glucose has a significant effect on the total phenol content and antioxidant capacity of broken-wall pine pollen. When the glucose addition amount is 3%, the total phenol content reaches the highest value of 3.91 mg / g, which is significantly higher than that of the water extract (2.49 mg / g) and other addition amount groups. At the same time, the ABTS free radical scavenging ability at 3% glucose addition amount is also relatively high, being 72.57 μmol / g. In contrast, when the glucose addition amount is 1%, the total phenol content slightly decreases to 2.03 mg / g, while when the glucose addition amount is 5% or more, both the total phenol content and antioxidant capacity decrease. This indicates that an appropriate amount of glucose can effectively promote the growth and metabolism of microorganisms, thereby increasing the total phenol content and antioxidant capacity.

[0050] However, when the glucose addition amount exceeds 3%, the excessively high glucose concentration may lead to an increase in the osmotic pressure of the microbial growth environment, thereby inhibiting the growth and reproduction of microorganisms. The increase in osmotic pressure will cause the loss of water in microbial cells, affecting their normal physiological functions, and further inhibiting certain key metabolic pathways, resulting in a decrease in the total phenol content and antioxidant capacity. Therefore, a 3% glucose addition amount is the best choice for enhancing the physiological activity of broken-wall pine pollen, which can not only promote the growth and metabolism of microorganisms but also not inhibit their growth due to excessive osmotic pressure.

[0051] Example 2:

[0052] A method for enhancing the antioxidant activity of broken-wall pine pollen by lactic acid bacteria fermentation, comprising the following steps:

[0053] (1) Place the broken-wall pine pollen in deionized water, stir evenly, and use 4 mol / L NaOH and 4 mol / L HCl to adjust the pH value to 6.5 to obtain a broken-wall pine pollen culture medium; wherein, the weight ratio of broken-wall pine pollen to deionized water is 1:15;

[0054] (2) Add glucose with a mass fraction of 3% to the broken-wall pine pollen culture medium;

[0055] (3) Heat the broken-wall pine pollen culture medium in a boiling water bath until the central temperature reaches 85°C, keep it in a water bath at 85°C for 30 min, and immediately cool it to room temperature with ice water after the holding time;

[0056] (4) Inoculate Lacticaseibacillus paracasei 6244 at an inoculation amount of 5% of the strain ratio, and ferment for 1 day under static conditions at 37°C; wherein, the concentration of the bacterial liquid is 1.5×10 6244), and ferment for 1 day under static conditions at 37°C; wherein, the concentration of the bacterial liquid is 1.5×10 8cfu / mL;

[0057] (5) The fermentation product was stored at -20 °C.

[0058] Using the unfermented water extract of broken-wall pine pollen as a control, after the experiment, the total phenolic content and ABTS radical scavenging ability before and after fermentation were measured and compared.

[0059] In this example, the total phenolic content of the broken-wall pine pollen fermentation broth reached 3.70 mg / g, and that of the control group was 2.49 mg / g; the ABTS radical scavenging ability was 77.10 μmol / g, and that of the control group was 21.23 μmol / g. The total phenolic content and antioxidant ability of the fermented broken-wall pine pollen increased significantly.

[0060] Example 3:

[0061] A process for improving the antioxidant activity of broken-wall pine pollen by lactic acid bacteria fermentation is as follows:

[0062] (1) The broken-wall pine pollen was placed in deionized water. After stirring evenly, the pH value was adjusted to 5 using 4 mol / L NaOH and 4 mol / L HCl; the broken-wall pine pollen culture medium was obtained. Among them, the weight ratio of broken-wall pine pollen to deionized water was 1:20;

[0063] (2) Glucose with a mass fraction of 3% was added to the broken-wall pine pollen culture medium.

[0064] (3) The broken-wall pine pollen culture medium was heated in a boiling water bath until the central temperature reached 85 °C, and then kept in a water bath at 85 °C for 30 min. After the holding time, it was immediately cooled to room temperature with ice water.

[0065] (4) Inoculate Lactiplantibacillus plantarum (Lactiplantibacillus lantarum 21793) at an inoculation amount of 5% of the strain ratio, and ferment at 37 °C under static conditions for 1 day. Among them, the concentration of the bacterial liquid was 1.5×10 8 cfu / mL;

[0066] (5) The fermentation product was stored at -20 °C.;

[0067] Using the unfermented water extract of broken-wall pine pollen as a control, after the experiment, the total phenolic content and ABTS radical scavenging ability before and after fermentation were measured and compared.

[0068] In this example, the total phenolic content of the broken-wall pine pollen fermentation broth reached 3.73 mg / g, and that of the control group was 2.49 mg / g; the ABTS radical scavenging ability was 59.07 μmol / g, and that of the control group was 21.23 μmol / g. The total phenolic content and antioxidant ability of the fermented broken-wall pine pollen increased significantly.

[0069] Example 4:

[0070] A process for enhancing the antioxidant activity of broken-wall pine pollen by lactic acid bacteria fermentation, the steps are as follows:

[0071] (1) Place the broken-wall pine pollen in deionized water, stir evenly, and adjust the pH value to 6.5 using 4 mol / L NaOH and 4 mol / L HCl; obtain the broken-wall pine pollen culture medium; wherein, the weight ratio of broken-wall pine pollen to deionized water is 1:20;

[0072] (2) Add glucose with a mass fraction of 3% to the broken-wall pine pollen culture medium;

[0073] (3) Heat the broken-wall pine pollen culture medium in a boiling water bath until the central temperature reaches 85°C, keep it in a water bath at 85°C for 30 min, and immediately cool it to room temperature with ice water after the insulation time;

[0074] (4) Inoculate Lactococcus lactis 23609) at an inoculation amount of 10% of the strain ratio, and ferment for 1 day under static conditions at 37°C; wherein, the concentration of the bacterial liquid is 1.5×10 8 cfu / mL;

[0075] (5) Store the fermentation product at -20°C.

[0076] Taking the water extract of unfermented broken-wall pine pollen as a control, after the experiment, measure and compare the total phenol content and ABTS free radical scavenging ability before and after fermentation.

[0077] In this example, the total phenol content of the broken-wall pine pollen fermentation broth can reach 3.90 mg / g, and the control group is 2.17 mg / g; the ABTS free radical scavenging ability is 70.67 μmol / g, and the control group is 21.23 μmol / g. The total phenol content and antioxidant capacity of the fermented pollen increase significantly. Compared with the highest total phenol content of 2.74 mg / g and the highest ABTS free radical scavenging ability of 47.01 μmol / g in the examples of the existing Poria cocos fermentation patents, the improvement effect of this example on the total phenol and antioxidant activity is better.

[0078] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for improving the antioxidant activity of broken-wall pine pollen by lactic acid bacteria fermentation, characterized in that, It includes the following steps: (1) Place the wall-broken pine pollen in deionized water, stir evenly, and then adjust the pH value to 6-7 to obtain a wall-broken pine pollen culture medium; (2) Add a glucose solution with a mass fraction of 1-9% to the wall-broken pine pollen culture medium in step (1); (3) Heat the wall-broken pine pollen culture medium in step (2) to a central temperature of 80-90 °C, and keep it warm in a water bath at 80-90 °C for 25-40 minutes. After the insulation is completed, quickly cool it to room temperature; (4) Add lactic acid bacteria liquid to the broken-wall pine pollen culture medium in step (3), and ferment at 30-40 °C under static conditions for 1-9 days to obtain a fermentation product; the lactic acid bacteria include Lacticaseibacillus paracasei 6244), Lactiplantibacillus lantarum 21793), Lactococcus lactis 23609), any one or more of them.

2. The method for enhancing the antioxidant activity of broken wall pine pollen by lactic acid bacteria fermentation according to claim 1, wherein In step (1), the weight ratio of the wall-broken pine pollen to the deionized water is 1:10-30.

3. The method for improving the antioxidant activity of broken pine pollen by lactic acid bacteria fermentation according to claim 1, characterized in that In step (1), use a 4 mol / L NaOH solution and / or a 4 mol / L HCl solution to adjust the pH value.

4. The method for enhancing the antioxidant activity of broken pine pollen by lactic acid bacteria fermentation according to claim 1, characterized in that, In step (2), add a glucose solution with a mass fraction of 3% to the wall-broken pine pollen culture medium in step (1).

5. The method for enhancing the antioxidant activity of broken pine pollen by lactic acid bacteria fermentation according to claim 1, characterized in that In the step (4), the volume ratio of the lactic acid bacteria liquid to the culture medium is 1:5 to 20, and the strain concentration of the lactic acid bacteria liquid is 1 to 2×10 8 cfu / mL.

6. The method for enhancing the antioxidant activity of broken wall pine pollen by lactic acid bacteria fermentation according to claim 1, characterized in that, In step (4), the volume ratio of the lactic acid bacteria liquid to the culture medium is 1:20.

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