Treatment method of pine pollen residues, pine pollen residue raw material, application of pine pollen residue raw material and gastrointestinal protection product

Abstract

The invention discloses a pine pollen residue treatment method, a pine pollen residue raw material, application of the pine pollen residue raw material and a gastrointestinal protection product, and belongs to the technical field of pine pollen residue treatment. The technical problems to be solved are high treatment cost and low treatment efficiency of the pine pollen residues. According to the technical scheme, the treatment method is characterized by comprising the following steps: (1) mixing pine pollen residues with water, and sterilizing to obtain a fermentation culture medium; (2) inoculating microbial probiotics into the fermentation culture medium for fermentation, and sterilizing to obtain fermentation liquor; and (3) homogenizing the fermentation liquor to obtain the pine pollen residue raw material.

Description

Technical Field

[0001] This invention belongs to the field of pine pollen residue treatment technology, specifically relating to a method for treating pine pollen residue, pine pollen residue raw materials and their applications, and gastrointestinal protection products. Background Technology

[0002] Pine pollen, the male gametophyte of pine trees, has a long history of use in my country as both food and medicine. As early as the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), its effects of "lightening the body, invigorating qi, and prolonging life" were recorded. Modern chemical analysis shows that pine pollen is rich in protein, vitamins, minerals, dietary fiber, and various bioactive substances such as flavonoids and unsaturated fatty acids.

[0003] Due to the excellent health benefits of pine pollen, many food factories extract and process its beneficial substances. However, the extraction rate is generally only 30-40%, resulting in a large amount of pine pollen residue. This residue is a wet, sticky paste that is difficult to store and easily spoils. Even after drying, it hardens and hardens; if further crushed, it easily absorbs moisture and clumps, leading to uneven composition and highly unstable properties. Currently, processing enterprises have not made comprehensive use of pine pollen residue, resulting in its large accumulation, resource waste, and environmental pollution.

[0004] Relevant patent documents retrieved: This document, published in China (CN118923850A) on August 26, 2024, discloses a method for the large-scale preparation of pine pollen residue treatment products. Using pine pollen residue discarded from a bioprocessing plant as raw material, the method involves first drying to stabilize the basic component content of the residue, then adding an amino acid solution for mixing, followed by a high-temperature Maillard reaction and a second drying. Next, water is added and stirred, and then continuous flow centrifugation is used at room temperature to wash away harmful substances produced by the Maillard reaction—acrylamide and 5-hydroxymethylfurfural. The centrifuged solid residue is then dried a third time, and finally, high-speed pulverization and sieving techniques are used to obtain the pine pollen residue treatment product (PPR). This invention essentially transforms pine pollen residue, which was originally discarded due to various defects and lack of usability, into PPR, a product with certain technological added value and good flavor activity. The product has excellent flavor, is suitable for consumption, exhibits superior antioxidant activity, and has good anti-caking properties.

[0005] The publication country is China, publication number CN121177166A, publication date December 23, 2025. This document discloses a pine pollen ferment with anti-wrinkle and anti-aging functions, which is obtained by fermentation with Lactobacillus plantarum. The preparation method is as follows: (1) Pine pollen is added to the fermentation medium, the pH value is adjusted, and then sterilized to obtain a sterilized medium; (2) Bacterial solution is inoculated into the sterilized medium and cultured at 37℃ for 24 hours to obtain the fermentation broth for later use; (3) The supernatant is taken after centrifugation of the fermentation broth, which is the pine pollen ferment. It directly adds Lactobacillus plantarum to pine pollen for fermentation, which is inexpensive and simple in process, and uses the simplest and most effective way to enhance its active ingredients and efficacy.

[0006] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: It is energy-intensive and costly. The relevant evidence is that the pine pollen residue treatment process in patent document CN118923850A requires three drying and heating processes, and additional amino acids need to be added. The process is complicated, energy-intensive, and costly.

[0007] The processing efficiency is low, and pine pollen residues are still involved after processing, failing to achieve full utilization of raw materials. Relevant evidence is: Patent document CN121177166A uses pine pollen as fermentation raw material, and the supernatant after fermentation is pine pollen fermentation product, which still contains insoluble residues (pine pollen residues), failing to achieve full utilization of raw materials and resulting in low processing efficiency.

[0008] The patent document CN121177166A is unsuitable for processing pine pollen residues. The relevant evidence is that the patent document CN121177166A uses pine pollen as a fermentation raw material and relies on a specific fermentation culture medium (10 g / L glucose, 3 g / L potassium dihydrogen phosphate and 1.5 g / L magnesium sulfate heptahydrate). The conditions are harsh and it is not applicable to the processing of pine pollen residues after extraction and processing. Summary of the Invention

[0009] The purpose of this invention is to provide: A method for treating pine pollen residues, and related technologies, to solve technical problems such as high treatment costs and low treatment efficiency of pine pollen residues, or a combination thereof.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. 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 in this document, the definitions 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] Definitions of standard chemical terms can be found in reference textbooks or reference books.

[0013] Unless otherwise stated, conventional methods within the scope of the art, such as methods for detecting protein content, fat content, DPPH, hydroxyl radicals, and pancreatic lipase inhibition capacity, shall be used.

[0014] 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.

[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 processing pine pollen residue, comprising the following steps: (1) Add water to the pine pollen residue, sterilize, and obtain a fermentation culture medium; (2) Inoculate the fermentation medium with probiotic microorganisms, ferment, sterilize, and obtain fermentation broth; (3) Homogenize the fermentation liquid to obtain pine pollen residue raw material.

[0020] In some implementations, the pine pollen residue in step (1) is the precipitate obtained by solid-liquid separation after water extraction of pine pollen.

[0021] The preferred method of water extraction is: decoction, reflux extraction, or ultrasonic extraction; more preferably: reflux extraction or ultrasonic extraction; and even more preferably: ultrasonic extraction.

[0022] The temperature of the water extraction is selected from 80-90℃. For example, the temperature is selected from 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 88℃, 89℃, 90℃, or any range derived therefrom.

[0023] The amount of water added in the water extract is 8-18 times the mass of pine pollen, for example, it can be selected from 8 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, or any range derived therefrom.

[0024] The water extraction is performed at least once, preferably 1-5 times, more preferably 2-4 times, and even more preferably 2-3 times.

[0025] The water extraction time is 0.5-3 hours per extraction, for example, it can be selected from 0.5 hours per extraction, 1 hour per extraction, 1.5 hours per extraction, 2 hours per extraction, 2.5 hours per extraction, 3 hours per extraction, or any range derived therefrom.

[0026] In some embodiments, the concentration of pine pollen residue in the fermentation medium in step (1) is 3wt%-10wt%, for example, it can be selected from 3wt%, 4wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9%, 10%, or any range derived therefrom; preferably 3wt%-8wt%; more preferably 4wt%-6wt%.

[0027] In some embodiments, the microbial probiotic in step (2) is at least one of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus acidophilus; preferably Lactobacillus plantarum.

[0028] In some implementations, the inoculation amount of the probiotic microorganisms in step (2) is 5%-10%, for example 5%, 6%, 7%, 8%, 9%, 10%, or any range derived therefrom.

[0029] In some implementations, the fermentation conditions in step (2) are: temperature 30-37°C, time 24-48h.

[0030] The temperature can be selected from 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, or any range derived therefrom.

[0031] The time can be selected from 24h, 26h, 28h, 30h, 32h, 36h, 38h, 40h, 42h, 44h, 46h, 48h, or any range derived therefrom.

[0032] 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 solution involves adding water and sterilizing pine pollen residue to obtain a fermentation medium. This solution not only addresses the technical problems of "low processing efficiency and high cost in the treatment of pine pollen residue," but also further solves the technical problem of "adding new anti-inflammatory effects."

[0033] The second priority solution is to address the concentration of pine pollen residue in the fermentation medium. This solution, in addition to solving the technical problems of "low processing efficiency, high cost, and poor physical quality of pine pollen residue," further addresses the technical problem of "improving the chemical quality of pine pollen residue, including increasing protein content and enhancing antioxidant capacity."

[0034] Secondly, the present invention provides pine pollen residue raw material obtained by the above-mentioned processing method.

[0035] The protein content of the pine pollen residue raw material is preferably above 2 mg / mL, and more preferably 3-10 mg / mL, 4-10 mg / mL, 5-10 mg / mL, 6-10 mg / mL, 7-10 mg / mL or 6-8 mg / mL.

[0036] The preferred fat content of the pine pollen residue raw material is below 10%, more preferably 1%-2%, 1%-3%, 1%-4%, 1%-5%, 1%-6%, 1%-7%, 1%-8%, 1%-9%, 1%-10%, 2%-3%, 2%-4%, 2%-5%, 2%-6%, 2%-7%, 2%-8%, 2%-9%, 2%-10%, 3%-4%, 3%-5%, 3%-6%, 3%. -7%, 3%-8%, 3%-9%, 3%-10%, 4%-5%, 4%-6%, 4%-7%, 4%-8%, 4%-9%, 4%-10%, 5%-6%, 5%-7%, 5%-8%, 5%-9%, 5%-10%, 6%-7%, 6%-8%, 6%-9%, 6%-10%, 7%-8%, 7%-9%, 7%-10%, 8%-9%, 8%-10%, or 9%-10%.

[0037] Thirdly, the present invention provides the application of the above-mentioned processing method in improving the quality of pine pollen residue.

[0038] In some implementations, the quality includes at least one of chemical quality and physical quality.

[0039] The chemical quality mentioned herein includes at least one of the following: (1) Increase protein content; (2) Improve antioxidant capacity; (3) Enhance the degradation capacity of pancreatic lipase; (4) Reduce fat content.

[0040] The antioxidant capacity includes DPPH free radical scavenging capacity and hydroxyl free radical scavenging capacity.

[0041] The physical quality mentioned above includes at least one of the following: (1) Texture; (2) Structure; (3) Liquidity.

[0042] Fourthly, the present invention provides the application of the above-mentioned treatment method in improving the anti-inflammatory properties of pine pollen residue.

[0043] Among them, the inflammatory factors with anti-inflammatory properties include IL-6.

[0044] Fifthly, the present invention provides the application of the above-mentioned pine pollen residue raw material in the preparation of gastrointestinal protection products.

[0045] The gastrointestinal protection mentioned herein is selected from the treatment of gastritis and / or enteritis.

[0046] The products mentioned include food, health products, or medicines.

[0047] In a sixth aspect, the present invention provides a gastrointestinal protection product made from pine pollen residue raw material obtained by the aforementioned processing method and acceptable excipients.

[0048] The acceptable excipients are food-grade, health-grade, or pharmaceutically acceptable excipients.

[0049] In this invention, Examples 1-4 and Comparative Example 1 at least support the addition of water and sterilization of pine pollen residues to obtain the protection range of the fermentation culture medium.

[0050] The pine pollen residue was mixed with water and sterilized to obtain a fermentation medium, which is summarized from the above explanation and / or the corresponding examples 1-4 and Comparative Example 1, such as "the pine pollen residue was mixed with water and sterilized at 115°C for 20 min to obtain a fermentation medium containing 5 wt% pine pollen residue", "the pine pollen residue was mixed with water and sterilized at 115°C for 20 min to obtain a fermentation medium containing 6 wt% pine pollen residue", "the pine pollen residue was mixed with water and sterilized at 115°C for 20 min to obtain a fermentation medium containing 8 wt% pine pollen residue", "the pine pollen residue was mixed with water and sterilized at 115°C for 20 min to obtain a fermentation medium containing 3 wt% pine pollen residue", and "the pine pollen residue was mixed with water and sterilized at 115°C for 20 min to obtain a fermentation medium containing 10 wt% pine pollen residue", etc., based on the common feature "the pine pollen residue was mixed with water and sterilized to obtain a fermentation medium". Therefore, those skilled in the art can reasonably presume that the process of adding water and sterilizing pine pollen residue to obtain a fermentation culture medium, its subordinate concepts, its essentially equivalent technical means, and technical means that can replace the process of adding water and sterilizing pine pollen residue to obtain a fermentation culture medium based on existing technology and conventional technical means and common knowledge, should all fall within the scope of protection of this invention.

[0051] Examples 1-4 and Comparative Example 1 in this invention at least support the protection range of the concentration of pine pollen residue in the fermentation medium.

[0052] The concentration of pine pollen residue in the fermentation medium is 3wt%-10wt%, which is summarized by the common characteristics "5wt%, 6wt%, 8wt%, 3wt%, and 10wt%" in the foregoing explanation and / or Examples 1-4 and Comparative Example 1, as well as the corresponding "fermentation medium yielding 5wt% pine pollen residue," "fermentation medium yielding 6wt% pine pollen residue," "fermentation medium yielding 8wt% pine pollen residue," "fermentation medium yielding 3wt% pine pollen residue," and "fermentation medium yielding 10wt% pine pollen residue." Therefore, those skilled in the art can reasonably infer that the concentration of pine pollen residue in the fermentation medium of 3wt%-10wt%, its subordinate concepts, its substantially equivalent technical means, and technical means that can replace the concentration of pine pollen residue in the fermentation medium of 3wt%-10wt% based on existing technology and conventional technical means and common knowledge should all fall within the protection scope of this invention.

[0053] Examples 1-4 and Comparative Example 1 in this invention at least support the protection scope of fermentation.

[0054] The protection of fermentation includes the inoculum size, fermentation temperature, and time. This is based on the foregoing explanations and / or the corresponding descriptions in Examples 1-4 and Comparative Example 1 of "adding *Lactobacillus plantarum* to the fermentation medium (inoculum size 10 v / v, viable count approximately 1 × 10⁻⁶)". 9 Fermentation was carried out at 37℃ for 24 hours, followed by sterilization at 115℃ for 20 minutes to obtain the fermentation broth. *Lactobacillus plantarum* (inoculum size 5 v / v%) was added to the fermentation medium, with a viable count of approximately 1 × 10⁻⁶. 9 Fermentation was carried out at 30℃ for 48 hours, followed by sterilization at 115℃ for 20 minutes to obtain the fermentation broth. *Lactobacillus plantarum* (inoculum size 8 v / v%) was added to the fermentation medium, with a viable count of approximately 1 × 10⁻⁶. 9 Fermentation was carried out at 32℃ for 42 hours, followed by sterilization at 115℃ for 20 minutes to obtain the fermentation broth. *Lactobacillus plantarum* (inoculum size 7 v / v%, viable count approximately 1 × 10⁻⁶) was added to the fermentation medium. 9The fermentation broth was obtained by fermenting at 35°C for 36 hours (CFU / g) and then sterilizing at 115°C for 20 minutes. This is summarized by common features 1 (inoculation amounts of 10v / v%, 5v / v%, 8v / v%, and 7v / v%), 2 (37°C, 30°C, 32°C, and 35°C), and 3 (fermentation times of 24, 48, 42, and 36 hours). Therefore, those skilled in the art can reasonably infer that the inoculation amount, fermentation temperature and time, their subordinate concepts, their essentially equivalent technical means, and technical means that can replace the inoculation amount, fermentation temperature, and time within the scope of conventional techniques and common knowledge based on existing technology should all fall within the protection scope of this invention.

[0055] Examples 1-4 of this invention at least support the scope of sterilization protection.

[0056] Sterilization, as explained above, refers to the process of using physical or chemical methods to kill or remove all forms of microorganisms in the environment, instruments, and reagents, including bacteria, fungi, viruses, mycoplasma, chlamydia, and highly resistant bacterial spores and fungal spores, to bring the treated object to a sterile state. High-pressure steam sterilization, dry heat sterilization, filtration sterilization, or radiation sterilization are all conventional sterilization methods in the art. This is summarized by the common characteristic of "sterilization, removing microorganisms, and bringing the fermentation medium to a sterile state," as well as the "sterilization at 115°C for 20 minutes" in Examples 1-4. Therefore, those skilled in the art can reasonably presume that sterilization, its subordinate concepts, its essentially equivalent technical means, and technical means that can replace sterilization within the scope of conventional and common knowledge based on the existing level of technology should all fall within the protection scope of this invention.

[0057] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: Compared with existing technologies, the present invention has better technical effects in terms of processing efficiency, cost and quality.

[0058] The specific analysis is as follows: This invention directly uses the residue after water extraction of pine pollen as a culture medium for fermentation, without adding any other substances. After fermentation, all raw materials are utilized, and no residue is generated. The process is simple and low-cost. Compared to existing technologies, this invention does not require the addition of glucose, potassium dihydrogen phosphate, or sulfatase, nor does it require pH adjustment. The fermentation process is simple, low-cost, and achieves full utilization of raw materials with high processing efficiency.

[0059] This invention greatly improves the physical quality of pine pollen residue compared to unfermented pine pollen residue. Specifically, unfermented pine pollen residue has a fine texture, but is dense and prone to clumping, with a firm structure, poor flowability, and easy formation of hard lumps. In contrast, the pine pollen residue of this invention has a loose and soft texture, a sponge-like structure, good flowability, and is loose and easy to disperse.

[0060] According to experimental tests, this invention increases the protein content in pine pollen residue raw materials from about 1.58 mg / mL in unfermented pine pollen residue to more than 7.5 mg / mL; and reduces the fat content from about 12.6% in unfermented pine pollen residue to about 4%.

[0061] According to experimental tests, the antioxidant capacity and pancreatic lipase inhibition capacity of the pine pollen residue raw material of the present invention are greatly improved compared with unfermented pine pollen residue.

[0062] According to experimental tests, the pine pollen residue raw material of the present invention has produced a new effect - anti-inflammatory, compared with unfermented pine pollen residue, achieving unexpected technical results.

[0063] Furthermore, based on the present invention: Based on Examples 1-4 and Comparative Examples 1 and 2, it can be seen that the product obtained by conventional fermentation of pine pollen residue has a substantial difference in anti-inflammatory activity compared with the pine pollen residue raw material prepared by the combination of specific fermentation culture medium and microbial probiotic fermentation in this invention. This further verifies that the processing method claimed in this invention has achieved unexpected technical effects in improving the anti-inflammatory activity of the raw material.

[0064] The pine pollen residue raw material prepared by the processing method of this invention has good nutritional quality and biological activity, providing technical support for the high-value development of pine pollen residues. Detailed Implementation

[0065] 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.

[0066] 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.

[0067] 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.

[0068] Unless otherwise specified, percentages in this invention refer to mass percentages, temperatures refer to room temperature, and solvents refer to water.

[0069] For example, the pine pollen residue used in this embodiment is prepared by mixing pine pollen and water at a mass ratio of 1:10, ultrasonically extracting twice at 80-90℃ for 30 minutes each time, centrifuging at 8000 rpm, and the precipitate is the pine pollen residue. *Lactobacillus plantarum* CICC 22195 was purchased from the China Industrial Microbial Culture Collection Center.

[0070] Example 1 Methods for treating pine pollen residue (1) After adding water to the pine pollen residue and mixing evenly, sterilize at 115℃ for 20 min to obtain a fermentation medium containing 5wt% pine pollen residue; (2) Add Lactobacillus plantarum to the fermentation medium (inoculation amount 10 v / v, viable count approximately 1 × 10⁻⁶). 9 (CFU / g), fermented at 37℃ for 24h, and then sterilized at 115℃ for 20min to obtain fermentation broth; (3) After the fermentation liquid is homogenized by a homogenizer (5000 rpm, 5 min), the pine pollen residue raw material is obtained.

[0071] Example 2 Methods for treating pine pollen residue (1) After adding water to the pine pollen residue and mixing evenly, sterilize at 115℃ for 20 min to obtain a fermentation medium containing 6wt% pine pollen residue; (2) Add Lactobacillus plantarum to the fermentation medium (inoculation amount 5 v / v, viable count approximately 1 × 10⁻⁶). 9 (CFU / g), fermented at 30℃ for 48h, and sterilized at 115℃ for 20min after fermentation to obtain fermentation broth; (3) After the fermentation liquid is homogenized by a homogenizer (5000 rpm, 5 min), the pine pollen residue raw material is obtained.

[0072] Example 3 Methods for treating pine pollen residue (1) After adding water to the pine pollen residue and mixing evenly, sterilize at 115℃ for 20 min to obtain a fermentation medium containing 8wt% pine pollen residue; (2) Add Lactobacillus plantarum to the fermentation medium (inoculation amount 8 v / v, viable count approximately 1 × 10⁻⁶).9 (CFU / g), fermented at 32℃ for 42h, and sterilized at 115℃ for 20min after fermentation to obtain fermentation broth; (3) After the fermentation liquid is homogenized by a homogenizer (5000 rpm, 5 min), the pine pollen residue raw material is obtained.

[0073] Example 4 Methods for treating pine pollen residue (1) After adding water to the pine pollen residue and mixing evenly, sterilize at 115℃ for 20 min to obtain a fermentation medium containing 3wt% pine pollen residue; (2) Add Lactobacillus plantarum to the fermentation medium (inoculation amount 7v / v, viable count approximately 1×10⁻⁶). 9 (CFU / g), fermented at 35℃ for 36h, and sterilized at 115℃ for 20min after fermentation to obtain fermentation broth; (3) After the fermentation liquid is homogenized by a homogenizer (5000 rpm, 5 min), the pine pollen residue raw material is obtained.

[0074] Comparative Example 1 Methods for treating pine pollen residue (1) After adding water to the pine pollen residue and mixing evenly, sterilize at 115℃ for 20 min to obtain a fermentation medium containing 10wt% pine pollen residue; (2) Add Lactobacillus plantarum to the fermentation medium (inoculation amount 10 v / v, viable count approximately 1 × 10⁻⁶). 9 (CFU / g), fermented at 37℃ for 48h, and sterilized at 115℃ for 20min after fermentation to obtain fermentation broth; (3) After the fermentation liquid is homogenized by a homogenizer (5000 rpm, 5 min), the pine pollen residue raw material is obtained.

[0075] Comparative Example 2 Methods for treating pine pollen residue (1) Fermentation medium: 10 g / L glucose, 3 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate heptahydrate, 5 wt% pine pollen residue, pH=6, sterilized at 115℃ for 20 min to obtain sterilized medium; (2) Add 10 v / v% of Lactobacillus plantarum seed culture (accession number CICC22195, viable count approximately 1×10⁻⁶) to the culture medium obtained in step (1). 9 (CFU / g), cultured at 37℃ for 24h, sterilized at 115℃ for 20min after fermentation, and homogenized in a homogenizer (5000rpm, 5min) to obtain the final product.

[0076] Detection Example 1 1. Method for detecting protein content: micro-protein analyzer.

[0077] 2. Method for detecting fat content: Fully automated fat analyzer.

[0078] 3. DPPH Detection Method Determination of DPPH free radical antioxidant capacity: Accurately weigh 2.5 mg of DPPH and dilute to 100 mL with anhydrous ethanol to prepare a solution with a mass concentration of 2.5 × 10⁻⁶ mg / mL. -2 The standard solution at mg / mL was stored in a refrigerator for later use (prepared fresh each time). 2.0 mL of each sample solution was placed in a 10 mL centrifuge tube, and 2.0 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 background color group, sample + DPPH as the experimental group, and Vc + DPPH as the positive group.

[0079] Clearance rate (%) = [Blank group - (Experimental group - Background group)] / Blank group 100% 4. Detection method for hydroxyl radicals: Hydroxyl radical scavenging ability test kit (brand: Abbkine) 5. Detection methods for pancreatic lipase inhibition capacity Pancreatic lipase was dissolved in 50 mmol / L Tris buffer (pH 8.0), centrifuged (10000×g, 10 min), and the supernatant was collected to obtain a 1000 IU / mL pancreatic lipase solution. The reaction substrate 4-nitrophenyl palmitate (pNPP) was dissolved in Tris buffer containing 5% isopropanol to prepare a 2.0 mmol / L pNPP solution.

[0080] Sample solutions of different concentrations (50 μL) and pancreatic lipase solution (50 μL) were mixed thoroughly and incubated at 37 °C for 10 min. Immediately afterwards, 100 μL of pNPP solution was added, and the mixture was incubated at 37 °C for another 10 min. After the reaction was completed, the mixture was immediately placed in a water bath at 100 °C for 5 min to terminate the reaction. The terminated reaction solution was centrifuged (5000 × g, 10 min) to remove the precipitate, and the absorbance of each reaction system was measured at a wavelength of 405 nm.

[0081] The test results are shown in Table 1.

[0082] Table 1

[0083] Detection Example 2 Evaluation of gastrointestinal protective efficacy: GES-1 cells (human immortalized gastric mucosal epithelial cells) and HT-29 cells (human colorectal adenocarcinoma cells) were selected using TNFα modeling agent, and interleukin-6 (IL-6) was used as the indicator.

[0084] Specific methods for evaluating gastritis (enteritis) protective capacity: A. Cell viability assay Collect cells in the logarithmic growth phase at a concentration of 2.0 × 10⁻⁶. 5 Cells were seeded at a density of 100 μL / mL into 96-well plates and cultured for 24 h. The supernatant was discarded, and the plates were randomly divided into control wells and sample wells. 100 μL of complete culture medium was added to the control wells, and 100 μL of complete culture medium containing different concentrations of the sample was added to the sample wells. Each concentration was repeated in 5 replicates. The plates were cultured for 2 h. The culture medium was discarded, and 100 μL of culture medium containing 10% CCK-8 reagent was added to each well. After incubation for 2 h, the absorbance of the sample was measured at 450 nm.

[0085] Calculate cell viability using the following formula: Cell viability (%) = OD value of sample well / OD value of control well × 100%.

[0086] B. Measurement of IL-6 content in cell supernatant Collect cells in the logarithmic growth phase at a concentration of 2.0 × 10⁻⁶. 5 Cells were seeded at a density of 100 μL / mL into 96-well plates and cultured for 24 h. The plates were then randomly divided into normal control wells, model wells, and sample wells. 100 μL of complete culture medium was added to the normal control wells, 100 μL of complete culture medium containing 5 ng / mL TNF-α was added to the model wells, and 5 ng / mL TNF-α and evaluation sample complete culture medium was added to the sample wells. After 24 h of culture, the IL-6 content in each well was measured and calculated according to the instructions of the high-sensitivity human interleukin-6 (IL-6) enzyme-linked immunosorbent assay kit (purchased from Elite Biotech).

[0087] The results are shown in Table 2.

[0088] Table 2

[0089] Note: Compared with the blank control group, ##P<0.01, compared with the model group, P < 0.05.

[0090] Verification of technical effectiveness and / or analysis of technical problem solving Table 1 shows that, compared with unfermented pine pollen residues, the protein content of the pine pollen residues in Examples 1-4 and Comparative Example 1 was significantly increased and the fat content was significantly decreased after fermentation treatment, which optimized the nutritional structure of pine pollen residues and improved the bioavailability of protein.

[0091] Meanwhile, compared with unfermented pine pollen residues, the pine pollen residues in Examples 1-4 and Comparative Example 1 showed significantly improved antioxidant capacity and pancreatic lipase inhibition capacity after fermentation treatment, exhibiting better biological activity.

[0092] In summary, the pine pollen residue raw material prepared by the processing method of the present invention has good nutritional quality and biological activity, providing technical support for the high-value development of pine pollen residues.

[0093] Table 2 shows that at a concentration of 1 μg / mL, the IL-6 content in the cells of the pine pollen residue raw materials obtained in Examples 1-4 was significantly lower than that in the model group (P < 0.05), while the IL-6 content in the cells of the unfermented pine pollen residue was not statistically significant compared with that in the model group (P > 0.05). This indicates that the pine pollen residue raw material prepared by the present invention produces a new efficacy—anti-inflammatory—compared to the unfermented pine pollen residue, achieving unexpected technical effects.

[0094] Meanwhile, it was found that the pine pollen residue raw materials obtained in Comparative Examples 1 and 2 had no anti-inflammatory activity at a concentration of 1 μg / mL, and the IL-6 content in their cells was not statistically significant compared with the model group (P > 0.05). This further verifies that the treatment method claimed in this invention has achieved unexpected technical effects in improving the anti-inflammatory activity of the raw materials.

[0095] 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 treating pine pollen residue, characterized in that, Includes the following steps: (1) Mix the pine pollen residue with water, sterilize, and obtain the fermentation culture medium; (2) Inoculate the fermentation medium with probiotic microorganisms, ferment, sterilize, and obtain fermentation broth; (3) Homogenize the fermentation liquid to obtain pine pollen residue raw material.

2. The processing method according to claim 1, characterized in that, The pine pollen residue mentioned in step (1) is the precipitate obtained by solid-liquid separation after water extraction of pine pollen.

3. The processing method according to claim 2, characterized in that, The water extraction method described in step (1) is decoction, heating and reflux, or ultrasonic extraction.

4. The processing method according to claim 1, characterized in that, The concentration of pine pollen residue in the fermentation medium described in step (1) is 3wt%-10wt%.

5. The processing method according to claim 1, characterized in that, The concentration of pine pollen residue in the fermentation medium described in step (1) is 3wt%-8wt%.

6. The processing method according to claim 1, characterized in that, The microbial probiotics mentioned in step (2) are at least one of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus rhamnosus, and Lactobacillus acidophilus.

7. The processing method according to claim 1, characterized in that, The inoculation amount of the probiotics in step (2) is 5%-10%.

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

9. Pine pollen residue raw material obtained by the processing method according to any one of claims 1-8.

10. The application of the processing method according to any one of claims 1-8 in improving the quality of pine pollen residue, characterized in that, The quality includes at least one of chemical quality and physical quality; The chemical quality includes at least one of the following: (1) Increase protein content; (2) Improve antioxidant capacity; (3) Enhance the degradation capacity of pancreatic lipase; (4) Reduce fat content; The physical quality includes at least one of the following: (1) Texture; (2) Structure; (3) Liquidity.

11. The application of the treatment method according to any one of claims 1-8 in improving the anti-inflammatory properties of pine pollen residue.

12. The application according to claim 11, characterized in that, The inflammatory factors with anti-inflammatory properties include IL-6.

13. The use of the pine pollen residue raw material according to claim 9 in the preparation of gastrointestinal protection products.

14. The application according to claim 13, characterized in that, The gastrointestinal protection mentioned is selected from the improvement or treatment of gastritis and / or enteritis.

15. The application according to claim 14, characterized in that, The products include food, health products, or medicines.

16. A gastrointestinal protection product, characterized in that, It is made from pine pollen residue raw material obtained by the processing method according to any one of claims 1-8 and acceptable excipients.

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