A sunflower pollen ferment, its preparation method and application

Abstract

This invention, entitled "A Sunflower Pollen Fermentation Product, Its Preparation Method, and Its Application," belongs to the field of biotechnology. The technical problem to be solved is to improve the antioxidant, immunomodulatory, and intestinal protective capabilities of sunflower pollen. The key technical solution is the preparation method, which includes the following steps: adding sunflower pollen to a fermentation culture medium to obtain a fermentation substrate; inoculating the fermentation substrate with *Lactobacillus* for fermentation culture, separating the solid and liquid phases to obtain a supernatant; concentrating and drying the supernatant to obtain the final product. The *Lactobacillus* is selected from *Lactobacillus casei*, *Lactobacillus plantarum*, and *Lactobacillus rhamnosus*.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a sunflower pollen ferment, its preparation method, and its application. Background Technology

[0002] Sunflower pollen is rich in nutrients, containing vitamins B1, B2, and C, as well as dozens of essential amino acids such as leucine and glutamic acid, and various essential trace elements such as calcium, magnesium, and selenium. It also contains over 50 kinds of natural enzymes, coenzymes, growth hormones, antibiotics, alcohols, flavonoids, and other active substances. Linoleic acid and linolenic acid account for more than 60% of its unsaturated fatty acids, exhibiting certain antioxidant and uric acid-lowering physiological activities. Since sunflower pollen was approved as a common food ingredient in 2004, it has received widespread attention.

[0003] Relevant non-patent literature retrieved:

[0004] The journal title is *Food Industry Technology*, and the article title is "Study on the In Vitro Antioxidant Capacity of Water Extracts from 11 Pollen Species," published in issue 15 of 2014. This article discloses a study using 11 pollen species as research subjects and employing methods to scavenge ABTS. + The antioxidant capacity of pollen water extracts was evaluated using three in vitro indicators: ·OH and total reducing power. The contents of bioactive substances such as protein, polysaccharide, polyphenol, and flavonoid in the water extracts were also determined. The study found that 11 pollen species exhibited antioxidant activity against ABTS. + All pollen species possess scavenging abilities for ·OH, but these abilities differ. Analysis of the indicators shows that pollen with strong antioxidant capabilities includes Phellodendron amurense pollen, corn pollen, and pearl bush pollen.

[0005] Relevant patent documents retrieved: This document, published in China (CN120092932A) on June 6, 2025, discloses a method for fermenting sunflower bee pollen using a compound lactic acid bacteria fermentation process and its application. The fermentation method involves inoculating sunflower bee pollen with a compound lactic acid bacteria fermentation process. The compound lactic acid bacteria are prepared by mixing *Lactobacillus plantarum* and *Lactobacillus casei* at a volume ratio of 2:1 to 5:1. This invention addresses the shortcomings of existing bee pollen, such as poor palatability, low nutrient utilization, and insufficient biological activity. It establishes for the first time a compound lactic acid bacteria fermentation method suitable for sunflower bee pollen, providing a new pathway for the high-value utilization of bee pollen. It offers theoretical support for overcoming bottlenecks in bee pollen processing technology and developing innovative bee pollen products with high absorption rates, high activity, and pleasant flavor. This opens up new avenues for the high-value transformation of sunflower pollen and provides a theoretical basis for its in-depth development and utilization in multifunctional nutritional foods.

[0006] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The antioxidant capacity is relatively weak and needs improvement. Relevant evidence includes: a non-patent literature study on the in vitro antioxidant capacity of 11 pollen water extracts, which recorded the following order of antioxidant capacity: ABTS+ scavenging ability, in descending order: camellia > phellodendron bark > pearl bush > rose > corn > rapeseed > wild chrysanthemum > willow > kiwifruit > lotus > sunflower; and ·OH scavenging ability, in descending order: wild chrysanthemum > rapeseed > corn > phellodendron bark > rose > camellia > kiwifruit > pearl bush > lotus > willow > sunflower. This indicates that sunflower pollen has relatively weak antioxidant capacity. Patent literature CN120092932A records that the DPPH free radical scavenging activity of bee pollen increased from 32.28% to 51.29% after fermentation, which is 1.59 times that before fermentation.

[0007] The function is singular, and there is a lack of research on intestinal protection and immune regulation activities. The relevant evidence is that the patent document CN120092932A only discloses the effects of fermented bee pollen on anti-oxidation, anti-inflammation, hypoglycemia, whitening and antibacterial effects, but does not disclose that sunflower pollen or its extracts and active substances have intestinal protection, immune regulation and other effects. The functional application is obviously limited and cannot meet the multi-faceted health needs.

[0008] In summary, existing sunflower pollen-related products and preparation technologies suffer from drawbacks such as low antioxidant capacity, limited functional activity, and lack of intestinal protection and immune regulation, which restrict their further application in the fields of high-end functional foods and health products. Summary of the Invention

[0009] The purpose of this invention is to provide: A sunflower pollen ferment, and related technologies thereof, to address technical problems such as the weak antioxidant capacity of sunflower pollen, or a combination thereof.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms, the definition provided in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] Unless otherwise specified, conventional methods within the scope of the art, such as SOD enzyme activity, DPPH antioxidant capacity, polysaccharide content, and flavonoid content, shall be used for detection.

[0013] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0014] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0015] As used herein, the term "room temperature" refers to ambient temperature, ranging from about 10°C to about 40°C. In some embodiments, "room temperature" refers to a temperature ranging from about 20°C to about 30°C; in other embodiments, "room temperature" refers to a temperature ranging from about 25°C to about 30°C; and in still other embodiments, "room temperature" refers to 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, etc.

[0016] As used in this article, "sterilization" refers to the process of killing or removing all forms of microorganisms, including bacteria, fungi, viruses, mycoplasma, chlamydia, and highly resistant bacterial spores and fungal spores, from the environment, instruments, and reagents using physical or chemical methods, so as to bring the object to a sterile state. High-pressure steam sterilization, dry heat sterilization, filtration sterilization, or radiation sterilization are all conventional sterilization methods in this field.

[0017] As used in this article, "solid-liquid separation" refers to the process of separating a mixture containing solid particles and liquid components into independent solid and liquid phases using physical or mechanical methods. This process can remove solid impurities, cell debris, precipitates, microbial aggregates, and other solid substances from the mixture to obtain a clear liquid phase or collect the target solid product. It is a commonly used separation and purification method in cell culture, sample pretreatment, and reagent preparation. Centrifugation, filtration, sedimentation, and pressure filtration are all conventional solid-liquid separation methods in this field.

[0018] The term "inoculation" as used in this article refers to the core operation of transferring a target cell line, cell suspension, or cell-containing biological material into a suitable cell culture vessel and cell culture medium under strictly aseptic conditions, providing a suitable environment for cell growth, proliferation, differentiation, or expression, and initiating the in vitro cell culture process. Static plate inoculation, shake-flask suspension inoculation, and bioreactor inoculation are all routine cell inoculation methods in this field.

[0019] In a first aspect, the present invention provides a method for preparing sunflower pollen ferment, comprising the following steps: (4) Add sunflower pollen to the fermentation medium to obtain the fermentation substrate; (5) Inoculate the fermentation substrate with Lactobacillus for fermentation culture, separate the solid and liquid, and obtain the supernatant; (6) Concentrate and dry the supernatant.

[0020] The fermentation medium in step (1) contains 5-15 g / L glucose, 1-5 g / L potassium dihydrogen phosphate, and 0.5-2.5 g / L magnesium sulfate heptahydrate; preferably, it contains 8-15 g / L glucose, 2-4 g / L potassium dihydrogen phosphate, and 1-2 g / L magnesium sulfate heptahydrate; more preferably, it contains 9-11 g / L glucose, 2-4 g / L potassium dihydrogen phosphate, and 1-2 g / L magnesium sulfate heptahydrate; and even more preferably, it contains 10 g / L glucose, 3 g / L potassium dihydrogen phosphate, and 1.5 g / L magnesium sulfate heptahydrate.

[0021] In some embodiments, the pH of the fermentation medium is 4-8; preferably 5-7; more preferably 6.

[0022] In step (1), the amount of sunflower pollen added is 0.5%-2% of the mass of the fermentation medium, for example, it can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, or any range derived therefrom.

[0023] The lactobacillus mentioned in step (2) is selected from one of Lactobacillus casei, Lactobacillus plantarum and Lactobacillus rhamnosus.

[0024] In some embodiments, the lactobacillus is further preferably Lactobacillus casei or Lactobacillus plantarum; more preferably Lactobacillus plantarum.

[0025] In step (2), the inoculation amount of lactobacillus is 1%-10%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or any range derived therefrom.

[0026] The fermentation conditions described in step (2) are: temperature 30-37℃, time 24-48h.

[0027] The temperature can be selected from 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, or any range derived therefrom.

[0028] The time can be selected from 24h, 26h, 28h, 30h, 32h, 36h, 38h, 40h, 42h, 44h, 46h, 48h, or any range derived therefrom.

[0029] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first preferred approach: inoculating the fermentation substrate with Lactobacillus for fermentation culture. This approach, while addressing the technical problem of "improving the antioxidant capacity of sunflower pollen," further addresses the technical problem of "adding new intestinal protective effects."

[0030] The second priority option: Lactobacillus species. This technical solution, having already addressed the technical problem of "enhancing the antioxidant capacity of sunflower pollen," further addresses the technical problem of "increasing the efficacy of immune regulation while improving intestinal protection."

[0031] Secondly, the present invention provides sunflower pollen fermentation product obtained by the above preparation method.

[0032] Thirdly, the present invention provides the application of the above-mentioned preparation method in improving the quality of sunflower pollen.

[0033] The quality includes at least one of the following: total polysaccharide content, flavonoid content, antioxidant capacity, intestinal protection, and immune regulation.

[0034] In some implementations, the antioxidant capacity includes DPPH antioxidant capacity.

[0035] Fourthly, the present invention provides a method for improving the quality of sunflower pollen, using sunflower pollen as raw material and processing it using the aforementioned preparation method.

[0036] Fifthly, the present invention provides the application of the above-mentioned sunflower pollen ferment in the preparation of products with intestinal protection and / or immunomodulatory effects.

[0037] The products mentioned include food, health products, or medicines.

[0038] In a sixth aspect, the present invention provides a product having intestinal protection and / or immune-modulating effects, made from sunflower pollen ferment obtained by the aforementioned preparation method and acceptable excipients.

[0039] The acceptable excipients are food-grade, health-grade, or pharmaceutically acceptable excipients.

[0040] In this invention, Examples 1-5 at least support the protection range of "fermentation medium containing 5-15 g / L glucose, 1-5 g / L potassium dihydrogen phosphate, and 0.5-2.5 g / L magnesium sulfate heptahydrate".

[0041] "Fermentation medium, comprising 5-15 g / L glucose, 1-5 g / L potassium dihydrogen phosphate, and 0.5-2.5 g / L magnesium sulfate heptahydrate," is summarized by the common feature "limiting the range of core nutrients and inorganic salt components and contents in the fermentation medium" as explained above and / or in Examples 1-5. Examples 1-3 use 10 g / L glucose, 3 g / L potassium dihydrogen phosphate, and 1.5 g / L magnesium sulfate heptahydrate; Example 4 uses 8 g / L glucose, 4 g / L potassium dihydrogen phosphate, and 2 g / L magnesium sulfate heptahydrate; and Example 5 uses 15 g / L glucose, 2 g / L potassium dihydrogen phosphate, and 1 g / L magnesium sulfate heptahydrate. These correspond to the middle, near-lower limit, and upper limit of the protection range of this technical feature, respectively, and comprehensively cover the range of 5-15 g / L glucose, 1-5 g / L potassium dihydrogen phosphate, and 0.5-2.5 g / L magnesium sulfate heptahydrate. Therefore, those skilled in the art can reasonably presume that "fermentation culture medium containing 5-15 g / L glucose, 1-5 g / L potassium dihydrogen phosphate, and 0.5-2.5 g / L magnesium sulfate heptahydrate", its subordinate concepts, essentially equivalent technical means, and technical means that can be replaced by conventional technical means and common knowledge based on the existing technical level, still fall within the scope of protection claimed by this invention.

[0042] In this invention, Examples 1-5 at least support the protection scope of "the amount of sunflower pollen added is 0.5%-2% of the mass of the fermentation medium".

[0043] The statement that "the amount of sunflower pollen added is 0.5%-2% of the mass of the fermentation medium" is derived from the foregoing explanation and / or the corresponding sunflower pollen addition parameters in Examples 1-5, summarized by the common feature "limiting the range of mass addition ratio of sunflower pollen in the fermentation medium." Examples 1 use 1.5%, Examples 2 use 1.5%, Examples 3 use 1.5%, Examples 4 use 0.5%, and Examples 5 use 2%, respectively corresponding to the middle, lower, and upper limits of the protection scope of this technical feature, comprehensively covering the addition ratio range of 0.5%-2%. Therefore, those skilled in the art can reasonably infer that "the amount of sunflower pollen added is 0.5%-2% of the mass of the fermentation medium," its subordinate concepts, substantially equivalent technical means, and technical means that can be replaced within the scope of conventional and common knowledge based on the existing technical level, still fall within the protection scope claimed by this invention.

[0044] Examples 1-5 of this invention at least support the protection range of "the inoculation amount of the lactobacillus is 1%-10%".

[0045] The statement "the inoculation amount of Lactobacillus is 1%-10%" is derived from the foregoing explanation and / or the corresponding Lactobacillus inoculation amount parameters in Examples 1-5, summarized by the common feature "limiting the range of inoculation proportions of Lactobacillus in the fermentation substrate." Examples 1 use 10%, Examples 2 use 10%, Examples 3 use 10%, Examples 4 use 5%, and Examples 5 use 5%, respectively corresponding to the upper and middle values ​​of the protection range of this technical feature, covering the inoculation amount range of 1%-10%. Therefore, those skilled in the art can reasonably infer that "the inoculation amount of Lactobacillus is 1%-10%", its subordinate concepts, substantially equivalent technical means, and technical means that can be replaced within the scope of conventional and common knowledge based on the existing technical level, should all fall within the protection scope claimed by this invention.

[0046] In this invention, Examples 1-5 at least support the protection range of "fermentation culture conditions: temperature 30-37℃, time 24-48h".

[0047] The conditions for fermentation cultivation are: temperature 30-37℃, time 24-48h. This is derived from the aforementioned explanation and / or the corresponding fermentation temperature and time parameters in Examples 1-5, summarized by the common feature "limiting the temperature and time range for *Lactobacillus sunfloweris* fermentation." Example 1 uses 37℃ for 24h, Example 2 uses 37℃ for 24h, Example 3 uses 37℃ for 24h, Example 4 uses 30℃ for 48h, and Example 5 uses 35℃ for 30h. These correspond to the upper, lower, and middle values ​​of temperature and the lower, upper, and middle values ​​of time within the protection range of this technical feature, comprehensively covering the temperature range of 30-37℃ and the time range of 24-48h. Therefore, those skilled in the art can reasonably infer that the conditions for fermentation cultivation are: temperature 30-37℃, time 24-48h, its subordinate concepts, substantially equivalent technical means, and technical means that can be replaced within the conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope claimed by this invention.

[0048] In this invention, Examples 1-3 at least support the protection scope of "Lactobacillus, selected from Lactobacillus casei, Lactobacillus plantarum and Lactobacillus rhamnosus".

[0049] "Lactobacillus, selected from one of Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus rhamnosus," is summarized by the common feature "limited to a single species of Lactobacillus for sunflower pollen fermentation" from the foregoing explanation and / or the corresponding Lactobacillus species selection parameters in Examples 1-5. Example 1 uses Lactobacillus rhamnosus, Example 2 uses Lactobacillus plantarum, and Example 3 uses Lactobacillus casei, completely covering the range of three single species: Lactobacillus rhamnosus, Lactobacillus plantarum, and Lactobacillus casei. Therefore, those skilled in the art can reasonably presume that "Lactobacillus, selected from one of Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus rhamnosus," its subordinate concepts, substantially equivalent technical means, and technical means that can be replaced within the scope of conventional technical means and common knowledge based on the existing technical level should all fall within the protection scope claimed by this invention.

[0050] In this invention, test examples 1-3 at least support the protection scope of "the application of the preparation method described herein in improving the quality of sunflower pollen".

[0051] The "application of the preparation method in improving the quality of sunflower pollen" is summarized from the aforementioned explanation and / or the corresponding verification data on antioxidant capacity, intestinal protection, and immune regulation efficacy in Test Examples 1-3, based on the common feature "using a single strain of Lactobacillus to ferment sunflower pollen, significantly increasing its total polysaccharide and flavonoid content, and enhancing its antioxidant, intestinal protection, and immune regulation qualities." Therefore, those skilled in the art can reasonably presume that the "application of the preparation method in improving the quality of sunflower pollen," its subordinate concepts, substantially equivalent technical means, and technical means that can be replaced within the scope of conventional and common knowledge based on the existing technical level should all fall within the protection scope of this invention. For example, if the application scenario is changed to the quality improvement application of functional foods, health products, or pharmaceuticals in the same field while other technical features remain unchanged, it still falls within the protection scope of this invention.

[0052] The beneficial effects of this invention are as follows: 1. According to experimental tests, the sunflower pollen fermentation product prepared by Lactobacillus fermentation in this invention has a DPPH free radical scavenging capacity that is more than twice that of unfermented sunflower pollen, and its DPPH antioxidant capacity is significantly improved compared with the prior art.

[0053] 2. According to experimental tests, compared with unfermented pollen, the sunflower pollen ferment of the present invention can significantly enhance GES1 cell activity, reduce the level of inflammatory factor IL-6, effectively repair mucosal damage, reduce inflammatory response, and endow sunflower pollen with a brand-new intestinal protection function.

[0054] 3. According to experimental tests, compared with unfermented pollen, the sunflower pollen ferment of the present invention can significantly promote macrophage proliferation, enhance cell phagocytic activity, and has a better immune-enhancing effect.

[0055] In summary, this invention utilizes Lactobacillus fermentation to enhance the antioxidant, immunomodulatory, and intestinal protective capabilities of sunflower pollen, thereby achieving high-value utilization of sunflower pollen.

[0056] Furthermore, based on the present invention: Some experiments have shown that altering the composition and dosage of the culture medium is outside the scope of protection claimed in this invention, resulting in a deterioration or even loss of intestinal protection in the prepared sunflower pollen fermentation. Therefore, this invention successfully solves the technical problems of existing sunflower pollen's weak antioxidant properties, limited functionality, and lack of intestinal protection and immunomodulatory activity by fermenting sunflower pollen with a specific single lactobacillus in a specific culture medium. Attached Figure Description

[0057] Figure 1 The effect of sunflower pollen fermentation on the proliferation activity of GES1 cell enteritis model; Figure 2The effect of sunflower pollen fermentation on IL-6 levels in a GES1 cell enteritis model; Figure 3 The effect of sunflower pollen fermentation product on the proliferation activity of Raw264.7 cells; Figure 4 The effect of sunflower pollen fermentation on the phagocytic activity of Raw264.7 cells; Wherein, A is unfermented sunflower pollen, B is the sunflower pollen fermentation product prepared in Example 1, C is the sunflower pollen fermentation product prepared in Example 2, D is the sunflower pollen fermentation product prepared in Example 3, E is the sunflower pollen fermentation product prepared in Comparative Example 1, and ## indicates that compared with the CK group, P < 0.01. This indicates that P < 0.05 compared to the model group. This indicates that P < 0.01 compared to the model group. Detailed Implementation

[0058] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0059] All numerical values ​​or expressions relating to component amounts, process conditions, etc., used in this invention shall be understood to be modified by the word "about" in all cases. When referring to a quantity or range of values, the quantity or range is an approximation within experimental variability (or within statistical experimental error). In this invention, the term "about" shall have the meaning of being within 10%, preferably within 5%, of the specified value or range.

[0060] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0061] The Lactobacillus plantarum has the accession number CICC 22195, Lactobacillus casei has the accession number CICC 23184, and Lactobacillus rhamnosus has the accession number CICC 23119. All of them are deposited at the China Industrial Microbial Culture Collection Center (CICC) and were purchased by Ningbo Mingzhou Biotechnology Co., Ltd.

[0062] Example 1: A sunflower pollen ferment The method for preparing the sunflower pollen ferment includes the following steps: (1) Sunflower pollen was added to the fermentation medium (10 g / L glucose, 3 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate, pH 6, sterilized at 121℃ for 10 min) to obtain the fermentation substrate. The amount of sunflower pollen added was 1.5% of the mass of the fermentation medium. The fermentation medium without sunflower pollen was used as the control.

[0063] (2) Inoculate the fermentation substrate with Lactobacillus rhamnosus (10 v / v%, viable count approximately 1 × 10⁻⁶). 9 (CFU / g), fermented at 37℃ for 24h, the sample was centrifuged at 4000 rpm for 15min and the supernatant was collected.

[0064] (3) The collected supernatant was evaporated in a rotary evaporator at 50°C for 4 hours until the liquid became thick. The evaporated sample was then freeze-dried in a freeze dryer (vacuum drying at -80°C for 24-48 hours), and the freeze-dried powder was collected for testing.

[0065] Example 2 The only difference between this embodiment and Embodiment 1 is that the bacterial strain is Lactobacillus plantarum.

[0066] Example 3 The only difference between this embodiment and Embodiment 1 is that the bacterial strain is Lactobacillus casei.

[0067] Example 4 The method for preparing the sunflower pollen ferment includes the following steps: (1) Sunflower pollen was added to the fermentation medium (8 g / L glucose, 4 g / L potassium dihydrogen phosphate, 2 g / L magnesium sulfate heptahydrate, pH 4, sterilized at 121℃ for 10 min) to obtain the fermentation substrate. The amount of sunflower pollen added was 0.5% of the mass of the fermentation medium. The fermentation medium without sunflower pollen was used as the control.

[0068] (2) Inoculate the fermentation substrate with Lactobacillus plantarum (5 v / v%, viable count approximately 1 × 10⁻⁶). 9 The sample was fermented at 30℃ for 48 hours (CFU / g), and then centrifuged at 4000 rpm for 15 minutes. The supernatant was then collected.

[0069] (3) The collected supernatant was evaporated in a rotary evaporator at 50°C for 4 hours until the liquid became thick. The evaporated sample was then freeze-dried in a freeze dryer (vacuum drying at -80°C for 24-48 hours), and the freeze-dried powder was collected for testing.

[0070] Example 5 The method for preparing the sunflower pollen ferment includes the following steps: (1) Sunflower pollen was added to the fermentation medium (15 g / L glucose, 2 g / L potassium dihydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, pH 8, high temperature and high pressure sterilization at 121℃ for 10 min) to obtain the fermentation substrate. The amount of sunflower pollen added was 2% of the mass of the fermentation medium. The fermentation medium without sunflower pollen was used as the control.

[0071] (2) Inoculate the fermentation substrate with Lactobacillus plantarum (5 v / v%, viable count approximately 1 × 10⁻⁶). 9 (CFU / g), fermented at 35℃ for 30h, the sample was centrifuged at 4000 rpm for 15min and the supernatant was collected.

[0072] (3) The collected supernatant was evaporated in a rotary evaporator at 50°C for 4 hours until the liquid became thick. The evaporated sample was then freeze-dried in a freeze dryer (vacuum drying at -80°C for 24-48 hours), and the freeze-dried powder was collected for testing.

[0073] Comparative Example 1 The only difference between this embodiment and Embodiment 1 is that the bacterial strains are Lactobacillus plantarum and Lactobacillus casei, with a mass ratio of 3:1.

[0074] Detection Example 1 1. Determination of SOD enzyme activity Enzyme activity was detected using an SOD enzyme activity assay kit (Nanjing Jiancheng).

[0075] 2. DPPH antioxidant capacity determination Accurately weigh 2.5 mg of DPPH, dissolve it in anhydrous ethanol in a 100 mL volumetric flask, dilute to volume and shake well to obtain its standard solution. It should be prepared fresh for each test.

[0076] Samples were prepared into different concentration gradients. 2 mL of each gradient was placed in a 10 mL centrifuge tube, and 2 mL of DPPH solution was added. The mixture was vortexed and reacted at room temperature in the dark for 30 min. The absorbance was measured at 517 nm. Anhydrous ethanol + DPPH was used as the blank group, anhydrous ethanol + sample as the solvent group, and sample + DPPH as the experimental group.

[0077] Clearance rate (%) = (A1 - A2 + A3) / A1 × 100% In the formula: A1, A2, and A3 are the absorbance values ​​of the blank group, experimental group, and solvent group, respectively.

[0078] 3. Determination of polysaccharide content (1) Sample processing Transfer 2 ml of sample to a 15 mL centrifuge tube. Add 10 mL of 80% anhydrous ethanol and vortex to mix thoroughly. Sonicate for 30 min. After washing, centrifuge at 3000 rpm for 10 min and discard the supernatant. Wash the insoluble matter again with 10 mL of 80% ethanol solution and centrifuge. Dissolve the insoluble matter in 5 mL of water and sonicate for 30 min until the precipitate is completely dissolved. This solution is the sample assay solution.

[0079] (2) Preparation of standard curve Pipette 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of standard glucose working solutions into 10 mL test tubes, respectively, and bring the volume to 1.0 mL with distilled water. Add 1.0 mL of phenol solution to the test solution, followed by 5.0 mL of sulfuric acid, and let stand for 10 min. Vortex the reaction solution to ensure thorough mixing, then place the test tubes in a 30 ℃ water bath for 20 min, and measure the absorbance at 490 nm. Plot the glucose mass concentration on the x-axis and the absorbance value on the y-axis to construct a standard curve.

[0080] (3) Measurement method Pipette 1 mL of the sample solution into a 10 mL test tube and follow step (2) to measure the absorbance.

[0081] (4) Calculation The polysaccharide content in the sample is expressed as a mass fraction ω, in mg / mL, and is calculated as follows.

[0082] ; Where: m1: Sugar content (μg) in the sample solution calculated from the standard curve. V1: Sample final volume (mL) V2: Volume (mL) of sample solution transferred during absorbance measurement. m2: Sample volume (mL) 0.9: Correction factor for converting glucose to dextran 4. Determination of flavonoid content The second method for determining total flavonoids in health food products was used for detection.

[0083] The results are shown in Table 1.

[0084] Table 1

[0085] The results showed that, compared with unfermented sunflower pollen, the total polysaccharide content, flavonoid content, SOD enzyme activity, and DPPH free radical scavenging rate of the sunflower pollen ferments prepared in Examples 1-5 and Comparative Example 1 were significantly increased. This indicates that fermentation of sunflower pollen with *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, *Lactobacillus casei*, or a mixture of *Lactobacillus plantarum* and *Lactobacillus casei* can significantly increase the content of active ingredients such as polysaccharides and flavonoids in sunflower pollen, and significantly enhance its antioxidant capacity.

[0086] Evaluation of the intestinal protective efficacy of cells in Case 2 GES1 gastric mucosal epithelial cells were used for the experiment. Cell viability was detected by the CCK-8 assay, and interleukin-6 (IL-6) content was detected by the human interleukin-6 (IL-6) ELISA kit (Elabscience).

[0087] 1. Experimental Methods (1) Sample processing All samples were prepared using ultrapure water, filtered through a 0.22 μM filter for sterilization, and then serially diluted with culture medium before the experiments.

[0088] (2) Cell culture GES1 culture conditions: cultured in DMEM high glucose medium (containing double antibiotics) + fetal bovine serum (10%), and grown in a 37℃, 5% carbon dioxide incubator.

[0089] (3) Assay of cell proliferation activity Cell seeding: Logarithmic phase cells were seeded into 96-well plates; Sample preparation: 5% ethanol was used for modeling, and normal control group, model group, and sample groups with different concentrations were set up, with 6 replicates in each group; Cell viability assay and calculation: Cells were treated with different concentrations of samples for 24 h, the supernatant was discarded, and each well was incubated with 110 μL of culture medium containing 10% CCK-8 solution for 2 h. The OD values ​​of each well were measured at 450 nm and calculated using the following formula:

[0090] .

[0091] (4) Measurement of IL-6 content in cells Cell seeding: Logarithmic phase cells were seeded into 96-well plates; Sample processing: TNF-α at 5 ng / mL was used to establish the model. Normal control group, model group, and sample group were set up, with 6 replicates in each group. Cellular IL-6 content determination and calculation: Cells were treated with 10% sample for 24 h, and the supernatant was collected. The IL-6 concentration in each well was determined and calculated according to the human interleukin-6 (IL-6) ELISA kit.

[0092] 2. Experimental Results Evaluation of the proliferation capacity of the GES1 cell enteritis model, results as follows: Figure 1 As shown.

[0093] The results showed that, compared with the normal control group (CK), the activity of GES1 cells in the model group was significantly reduced after ethanol modeling (P < 0.01), indicating that the enteritis injury model was successfully established. Compared with the model group, unfermented sunflower pollen and the sunflower pollen fermentation product prepared in Comparative Example 1 had no significant effect on improving cell activity (P > 0.05) (as shown in Figures 1-1). Figure 1 (As shown in A and E in the figure). The sunflower pollen ferments prepared in Examples 1, 2, and 3 all significantly enhanced the relative activity of GES1 cells in a concentration-dependent manner, showing significant differences from the model group (P < 0.05 or P < 0.01) (as shown in Figures 1-3). Figure 1 (As shown in B, C and D), and the sunflower pollen ferment prepared in Example 2 had the best effect.

[0094] In summary, fermentation of sunflower pollen by Lactobacillus rhamnosus, Lactobacillus plantarum, or Lactobacillus casei can significantly enhance the cell proliferation activity of sunflower pollen and repair gastric mucosal epithelial cells damaged by enteritis, producing new effects compared to unfermented sunflower pollen or fermentation by Lactobacillus plantarum and Lactobacillus casei.

[0095] IL-6 levels were measured in the GES1 cell colitis model, and the results were as follows: Figure 2 As shown.

[0096] The results showed that, compared with the normal control group (CK), the IL-6 content in the model group was significantly increased after modeling (P < 0.01), indicating that the inflammation model was successfully established. Compared with the model group, unfermented sunflower pollen had no significant inhibitory effect on IL-6 (P > 0.01), while the sunflower pollen fermentation products prepared in Examples 1, 2, 3, and Comparative Example 1 all significantly reduced the IL-6 secretion level in the enteritis model cells (P < 0.01). This indicates that fermentation of sunflower pollen with Lactobacillus rhamnosus, Lactobacillus plantarum, or Lactobacillus casei can significantly enhance the anti-inflammatory and intestinal protective capabilities of sunflower pollen, producing new efficacy compared to unfermented sunflower pollen.

[0097] Evaluation of Cellular Immunotherapy in Case 3 Raw264.7 mouse mononuclear macrophages were used in the experiment. Cell viability was detected by the CCK-8 assay, phagocytic activity was detected by the neutral red assay, and NO was detected by a nitric oxide kit (Beijing Beyotime).

[0098] 1. Experimental Methods (1) Sample processing All liquid samples were sterilized by filtration using a 0.22 μM filter and stored at 4°C for later use.

[0099] (2) Cell culture Raw264.7 culture conditions: cultured in DMEM high glucose medium (containing double antibiotics) + serum (10%), and grown in a 37℃, 5% CO2 incubator.

[0100] (3) Cell viability assay Cell seeding: Logarithmic phase cells were seeded into 96-well plates; Sample preparation: A normal control group and sample groups with different concentrations were set up, with 6 replicates in each group; Cell viability assay and calculation: Cells were treated with different concentrations of samples for 24–48 h, the supernatant was discarded, and 110 μL of culture medium containing 10% CCK-8 solution was added to each well for 2 h. The OD values ​​of each well were measured at 450 nm and calculated using the following formula:

[0101] .

[0102] (4) Assay of cellular phagocytic activity Cell seeding: Logarithmic phase cells were seeded into 96-well plates; Sample preparation: Set up a normal control group, a blank group, and sample groups with different concentrations, with 6 replicates in each group; Cell phagocytic activity assay and calculation: Cells were treated with different concentrations of samples for 24–48 h. The supernatant was discarded, and the cells were washed once with PBS. Neutral red solution was added, and the cells were incubated in the dark for 10 min. The neutral red solution was discarded, and the cells were washed three times with PBS. Prepared cell lysis buffer (acetic acid: anhydrous ethanol, volume ratio = 1:1) was added to each well, and the cells were incubated in the dark for 30 min. The OD value of each well at 570 nm was measured and calculated using the following formula: Relative phagocytic activity (%) = [(sample well OD value - blank well OD value) / (normal well OD value - blank well OD value) - 1] × 100%.

[0103] (5) Cellular nitric oxide assay Cell seeding: Logarithmic phase cells were seeded into 96-well plates; Sample preparation: Modeling was performed using 1 μg / ml LPS. Normal control group, model group, and sample groups with different concentrations were set up, with 4 replicates in each group. Cell NO content determination and calculation: Cells were treated with different concentrations of samples for 24-48 h, and the supernatant was collected. The NO concentration in each well was determined and calculated according to the nitric oxide kit (Beijing Beyotime).

[0104] 2. Experimental Results Evaluation of Raw264.7 cell proliferation capacity, results are as follows: Figure 3 As shown.

[0105] The results showed that, compared with the control group, unfermented sunflower pollen, and the sunflower pollen fermentation products prepared in Examples 1, 2, and 1 (Comparative Example 1) did not promote the proliferation of Raw264.7 cells, and there was no statistically significant difference in relative cell activity (P>0.05). Figure 3 (As shown in A, B, C, and E); the sunflower pollen fermentation product prepared by only the third step significantly promoted the proliferation of Raw264.7 cells, and the relative cell activity was significantly different from that of the control group (P < 0.01) (as shown in Figure 3). Figure 3 (As shown in D).

[0106] This indicates that Lactobacillus casei fermentation of sunflower pollen can enhance the proliferation capacity of sunflower pollen macrophages.

[0107] Evaluation of phagocytic activity in Raw264.7 cells, results are as follows: Figure 4 As shown.

[0108] The results showed that the concentrations of unfermented sunflower pollen, sunflower pollen fermentation products prepared in Example 1 and Comparative Example 1 were below 25 mg / mL (as shown in Figures 1-1). Figure 4 As shown in A, B, and E in the figures, it had no effect on enhancing macrophage phagocytic activity, and there was no significant difference compared with the control group (P > 0.05); while the sunflower pollen fermentation products prepared in Examples 2 and 3, at concentrations of 0.78 mg / mL and 3.13 mg / mL respectively (as shown in A, B, and E in the figures), had no effect on enhancing macrophage phagocytic activity, and there was no significant difference compared with the control group (P > 0.05); Figure 4 When (as shown in C and D in the figure), the phagocytic activity of macrophages is significantly enhanced, with a highly significant difference compared to the control group (P < 0.01), and the effect of Example 2 is significantly better than that of Example 3.

[0109] This indicates that fermenting sunflower pollen with Lactobacillus plantarum or Lactobacillus casei can significantly enhance the immunomodulatory effects of sunflower pollen in boosting macrophage phagocytic activity, with Lactobacillus plantarum showing better performance in enhancing immune phagocytic activity.

[0110] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing sunflower pollen ferment, characterized in that, Includes the following steps: (1) Add sunflower pollen to the fermentation medium to obtain the fermentation substrate; (2) Inoculate the fermentation substrate with Lactobacillus for fermentation culture, separate the solid and liquid, and obtain the supernatant; (3) Concentrate and dry the supernatant; The fermentation medium described in step (1) contains 5-15 g / L glucose, 1-5 g / L potassium dihydrogen phosphate, and 0.5-2.5 g / L magnesium sulfate heptahydrate.

2. The preparation method according to claim 1, characterized in that, The amount of sunflower pollen added in step (1) is 0.5%-2% of the mass of the fermentation medium.

3. The preparation method according to claim 1, characterized in that, The lactobacillus mentioned in step (2) is selected from one of Lactobacillus casei, Lactobacillus plantarum and Lactobacillus rhamnosus.

4. The preparation method according to claim 1, characterized in that, The lactobacillus is either Lactobacillus casei or Lactobacillus plantarum.

5. The preparation method according to claim 1, characterized in that, The inoculation amount of lactobacillus in step (2) is 1%-10%.

6. The preparation method according to claim 1, characterized in that, The fermentation conditions described in step (2) are: temperature 30-37℃, time 24-48h.

7. The sunflower pollen ferment obtained by the preparation method according to any one of claims 1-6.

8. The application of the preparation method according to any one of claims 1-6 in improving the quality of sunflower pollen.

9. The application according to claim 8, characterized in that, The quality includes at least one of the following: total polysaccharide content, flavonoid content, antioxidant capacity, intestinal protection, and immune regulation.

10. The application according to claim 8, characterized in that, The qualities mentioned include at least one of antioxidant capacity, intestinal protection, and immune regulation.

11. A method for improving the quality of sunflower pollen, characterized in that, Using sunflower pollen as raw material, the sunflower pollen is processed using the preparation method described in any one of claims 1-6.

12. The use of the sunflower pollen ferment as described in claim 7 in the preparation of products with intestinal protection and / or immunomodulatory effects.

13. The application according to claim 12, characterized in that, The products include food, health products, or medicines.

14. A product having intestinal protection and / or immune-modulating effects, characterized in that, It is made from sunflower pollen ferment obtained by the preparation method according to any one of claims 1-6 and acceptable excipients.

15. The product according to claim 14, characterized in that, The acceptable excipients are food-grade, health-grade, or pharmaceutically acceptable excipients.

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