A process for improving the content and activity of antioxidant substances in pine pollen by fermenting with Rhizopus oryzae

Abstract

The present invention discloses a process for improving the content and activity of antioxidant substances in pine pollen by fermentation with Rhizopus oryzae. The process specifically includes the following steps: preparation of a liquid medium for pine pollen, activation and inoculation of Rhizopus oryzae, and fermentation to obtain a fermentation product. The method provided by the present invention can improve the polyphenol and flavonoid contents and antioxidant properties of pine pollen through fermentation, thereby enhancing the efficacy of pine pollen, and providing theoretical and technical support for the further development of new functional pine pollen products.

Description

Technical Field

[0001] The present invention relates to a process for improving the content and activity of antioxidant substances in pine pollen by fermentation with Rhizopus oryzae, belonging to the field of food bioprocessing. Background Art

[0002] Pine pollen (English: POLLEN PINI), also known as pine flower and pine yellow, generally refers to the dried pollen produced by the stamens of pine plants such as Pinus massoniana, Pinus tabuliformis, Pinus koraiensis, Pinus armandii, and Pinus sylvestris var. mongolica. It was first recorded in "Shennong Ben Cao Jing" in the Eastern Han Dynasty. It is a light yellow or yellowish powder, with a sweet and flat taste and non-toxic. It is a traditional Chinese food. In addition to general nutrients such as protein, fat, and carbohydrates, it also contains more than 200 nutritional components and bioactive substances such as various vitamins, trace elements, polyphenols, flavonoids, enzymes, fatty acids, and coenzymes, having broad development and application prospects. However, most active substances, such as insoluble bound polyphenols, usually form covalent complexes with the structural components of cell walls such as cellulose, hemicellulose, lignin, protein, polysaccharide, pectin, etc., and are not easily released. Microbial fermentation produces enzymes to break covalent bonds and promote the release of active ingredients.

[0003] Rhizopus oryzae Went et Pr.Geerl. is one of the important molds in Chinese medicine and koji, and is an edible and safe strain. The fermentation process is green and mild, and can promote the dissociation and release of active substances after fermentation, while improving the nutritional value and efficacy of food. Summary of the Invention

[0004] According to the deficiencies of the above prior art, the purpose of the present invention is to provide a process for improving the content and activity of antioxidant substances in pine pollen by fermentation with Rhizopus oryzae.

[0005] To achieve the above purpose, the technical solution adopted is:

[0006] A process for improving the content and activity of antioxidant substances in pine pollen by fermentation with Rhizopus oryzae, comprising the following steps:

[0007] (1) Take wall-broken pine pollen and dissolve it in a citric acid buffer solution, and stir evenly;

[0008] (2) Heat the pine pollen culture medium obtained in step (1) 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 immediately cool it to room temperature with ice water after the insulation time;

[0009] (3) Adjust the pH of the cooled pine pollen culture medium obtained in step (2) to 6.6;

[0010] (4) adding Rhizopus oryzae to the pine pollen culture medium obtained in step (3), and fermenting at 30° C. and 180 r / min for 1, 2, 4, 6, or 8 days to obtain a fermentation product.

[0011] Furthermore, the Rhizopus oryzae is 3010 Rhizopus oryzae.

[0012] Furthermore, the concentration of the citric acid buffer used in step (1) is 0.1 mol / L.

[0013] Furthermore, in step (3), the pH is adjusted to 6.6 using 0.2 mol / L Na2HPO4 solution.

[0014] Furthermore, the step (1) further comprises the following steps: adjusting the initial pH to 4.3 using 0.2 mol / L Na2HPO4 solution.

[0015] Furthermore, the weight ratio of the broken pine pollen: citric acid buffer solution + Na2HPO4 solution in step (1) and step (3) is 1:20.

[0016] Furthermore, the sterilization conditions of the Na2HPO4 solution are 121°C, 20 min.

[0017] Furthermore, in step (4), the volume ratio of Rhizopus oryzae to pine pollen culture medium is 1:20.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention discloses a new process for increasing the free active substances in pine pollen, i.e., polyphenols and flavonoids, and improving the antioxidant activity by fermenting with Rhizopus oryzae. The active substances combined with macromolecules in pine pollen cells are released by fermenting and producing enzymes, thereby increasing the content of free polyphenols and flavonoids in broken-wall pine pollen and improving the antioxidant capacity of broken-wall pine pollen. DETAILED DESCRIPTION

[0020] The present invention is described below in conjunction with examples, which are only used to explain the present invention and are not used to limit the scope of the present invention.

[0021] Example 1

[0022] A process for increasing the content and activity of antioxidant substances in pine pollen by fermentation with Rhizopus oryzae, comprising the following steps:

[0023] (1) Dissolve the broken pine pollen in 0.1 mol / L pH 4.3 citric acid buffer and stir evenly;

[0024] (2) Pasteurize the pine pollen medium by heating it 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 ends;

[0025] (3) Mix 0.2 mol / L Na2HPO4 solution with the pine pollen medium in a laminar flow hood and adjust the pH to 6.6. Among them, the weight ratio of wall-broken pine pollen to the medium (citrate buffer + Na2HPO4 solution) is 1:20;

[0026] (4) Inoculate Rhizopus oryzae 3010 at an inoculum size of 5%, and ferment for 1 d at 30 °C and 180 r / min;

[0027] (5) After fermentation, perform solid-liquid separation, freeze-dry the pine pollen substrate, and extract polyphenols and flavonoids from the pine pollen with ethanol;

[0028] (6) Using unfermented pine pollen as a control, after the experiment ends, measure and compare the total phenol, total flavonoid contents, ABTS radical scavenging ability, and ferric ion reducing ability (FRAP) before and after fermentation.

[0029] Example 2

[0030] A process for improving the content and activity of antioxidant substances in pine pollen by fermentation with Rhizopus oryzae is as follows:

[0031] (1) Take wall-broken pine pollen and dissolve it in 0.1 mol / L citrate buffer with a pH of 4.3, and stir evenly;

[0032] (2) Pasteurize the pine pollen medium by heating it 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 ends;

[0033] (3) Mix 0.2 mol / L Na2HPO4 solution with the pine pollen medium in a laminar flow hood and adjust the pH to 6.6. Among them, the weight ratio of wall-broken pine pollen to the medium (citrate buffer + Na2HPO4 solution) is 1:20;

[0034] (4) Inoculate Rhizopus oryzae 3010 at an inoculum size of 5%, and ferment for 2 d at 30 °C and 180 r / min;

[0035] (5) After fermentation, perform solid-liquid separation, freeze-dry the pine pollen substrate, and extract polyphenols and flavonoids from the pine pollen with ethanol;

[0036] (6) Using unfermented pine pollen as a control, after the experiment ends, measure and compare the total phenol, total flavonoid contents, ABTS radical scavenging ability, and ferric ion reducing ability (FRAP) before and after fermentation.

[0037] Example 3

[0038] A process for improving the content and activity of antioxidant substances in pine pollen by fermentation with Rhizopus oryzae is as follows:

[0039] (1) Take the broken-wall pine pollen and dissolve it in 0.1 mol / L citric acid buffer solution with a pH of 4.3, and stir evenly;

[0040] (2) Pasteurize the pine pollen culture medium, heat it 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 when the holding time is up;

[0041] (3) Mix 0.2 mol / L Na2HPO4 solution with the pine pollen culture medium in a laminar flow hood and adjust the pH to 6.6; among them, the weight ratio of broken-wall pine pollen to the culture medium (citric acid buffer solution + Na2HPO4 solution) is 1:20;

[0042] (4) Inoculate Rhizopus oryzae 3010 at an inoculation amount of 5% and ferment for 4 d at 30 °C and 180 r / min;

[0043] (5) After fermentation, separate the solid and liquid, freeze-dry the pine pollen substrate, and extract polyphenols and flavonoids in the pine pollen with ethanol;

[0044] (6) Using the unfermented pine pollen as a control, after the experiment, measure and compare the total phenol, total flavonoid content, ABTS free radical scavenging ability and ferric ion reducing ability (FRAP) before and after fermentation.

[0045] Example 4

[0046] A process for improving the content and activity of antioxidant substances in pine pollen by fermentation with Rhizopus oryzae is as follows:

[0047] (1) Take the broken-wall pine pollen and dissolve it in 0.1 mol / L citric acid buffer solution with a pH of 4.3, and stir evenly;

[0048] (2) Pasteurize the pine pollen culture medium, heat it 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 when the holding time is up;

[0049] (3) Mix 0.2 mol / L Na2HPO4 solution with the pine pollen culture medium in a laminar flow hood and adjust the pH to 6.6; among them, the weight ratio of broken-wall pine pollen to the culture medium (citric acid buffer solution + Na2HPO4 solution) is 1:20;

[0050] (4) Inoculate Rhizopus oryzae 3010 at an inoculum size of 5% and ferment for 6 days at 30 °C and 180 r / min;

[0051] (5) After fermentation, perform solid-liquid separation. Freeze-dry the pine pollen substrate and extract polyphenols and flavonoids from the pine pollen with ethanol;

[0052] (6) Using unfermented pine pollen as a control, after the experiment, measure and compare the total phenol, total flavonoid contents, ABTS radical scavenging ability, and ferric ion reducing ability (FRAP) before and after fermentation.

[0053] Example 5

[0054] A process for improving the content and activity of antioxidant substances in pine pollen by fermentation with Rhizopus oryzae, the steps are as follows:

[0055] (1) Take wall-broken pine pollen and dissolve it in 0.1 mol / L citric acid buffer solution with a pH of 4.3, and stir evenly;

[0056] (2) Pasteurize the pine pollen medium. Heat it 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 when the holding time is up;

[0057] (3) Mix 0.2 mol / L Na2HPO4 solution with the pine pollen medium in a laminar flow hood, and adjust the pH to 6.6. Among them, the weight ratio of wall-broken pine pollen to the medium (citric acid buffer solution + Na2HPO4 solution) is 1:20;

[0058] (4) Inoculate Rhizopus oryzae 3010 at an inoculum size of 5% and ferment for 8 days at 30 °C and 180 r / min;

[0059] (5) After fermentation, perform solid-liquid separation. Freeze-dry the pine pollen substrate and extract polyphenols and flavonoids from the pine pollen with ethanol;

[0060] (6) Using unfermented pine pollen as a control, after the experiment, measure and compare the total phenol, total flavonoid contents, ABTS radical scavenging ability, and ferric ion reducing ability (FRAP) before and after fermentation.

[0061] Analyze and organize the measurement results of Examples 1-5, and the specific results are shown in the following table.

[0062]

[0063] Note: Each condition has three replicates, and the results are expressed as mean ± standard deviation. The letters in the upper right corner indicate significance.

[0064] The determination results of total phenols, total flavonoids, ABTS radical scavenging ability and ferric ion reducing ability showed that, compared with before fermentation, the total phenols, total flavonoids, ABTS radical scavenging ability and ferric ion reducing ability of pine pollen after fermentation were all significantly improved. With the increase of fermentation time, the overall trend was first to increase and then to decrease. When the fermentation time was 6 days, the total phenol content was the highest, reaching 14.5 mg / g, higher than that before fermentation (7.81 mg / g). When the fermentation time was 4 days, the total flavonoid content was the highest, reaching 8.44 mg / g, higher than that before fermentation (5.26 mg / g). When the fermentation time was 6 days, the ABTS radical scavenging ability was the highest, reaching 301.58 μM / g, significantly higher than that before fermentation (158.50 μM / g). When the fermentation time was 4 days, the ferric ion reducing ability was the highest, reaching 55.92 μM / g, significantly higher than that before fermentation (28.36 μM / g).

[0065] Thus, it can be seen that the process method provided by the present invention for improving the polyphenol and flavonoid contents and antioxidant properties of pine pollen by fermentation can effectively improve the polyphenol and flavonoid contents and antioxidant properties. This method is mild, green, highly safe, low-cost and convenient for large-scale industrial production. The material changes after fermentation are rich, not only limited to active substances, but also some basic components such as proteins and polysaccharides may change accordingly.

[0066] It is worth mentioning that through a large number of experiments and creative work, the applicant of the present invention analyzed that the increase in the contents of polyphenols and flavonoids may be due to the production of protease, amylase and glucoamylase by Rhizopus oryzae fermentation, which releases the polyphenol substances bound to the cell wall. And the trend changes of antioxidant properties are basically the same as those of polyphenols and flavonoids, probably because polyphenol-flavonoid substances have strong antioxidant properties.

[0067] 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 content and activity of antioxidant substances in pine pollen by fermenting with Rhizopus oryzae, characterized in that, It includes the following steps: (1) Take broken-wall pine pollen and dissolve it in a citric acid buffer solution, and stir evenly; (2) Heat the pine pollen culture medium obtained in step (1) in a boiling water bath until the central temperature reaches 85 °C, place it in a water bath at 85 °C and keep it warm for 30 min. Immediately cool it to room temperature with ice water after the insulation time; (3) Adjust the pH of the cooled pine pollen culture medium obtained in step (2) to 6.6; (4) Add Rhizopus oryzae to the pine pollen culture medium obtained in step (3), and ferment it at 30 °C and 180 r / min for 1, 2, 4, 6, 8 d to obtain a fermentation product; The Rhizopus oryzae is Rhizopus oryzae 3010.

2. The method according to claim 1, wherein The concentration of the citric acid buffer solution used in step (1) is 0.1 mol / L.

3. The method according to claim 1, characterized in that, In step (3), adjust the pH to 6.6 with a 0.2 mol / L Na2HPO4 solution.

4. The method according to claim 1, characterized in that, The following steps are also included in step (1): Adjust the initial pH to 4.3 with a 0.2 mol / L Na2HPO4 solution.

5. The method according to claim 4, characterized in that The weight ratio of the broken-wall pine pollen: citric acid buffer solution + Na2HPO4 solution in steps (1) and (3) is 1:

20.

6. The method according to any one of claims 3 to 5, characterized in that The sterilization conditions of the Na2HPO4 solution are 121 °C and 20 min.

7. The method according to claim 1, wherein In step (4), the volume ratio of Rhizopus oryzae to the pine pollen culture medium is 1:20.

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