Lysimachia christinae Hance with ice recrystallization inhibition activity and fermentation product and application thereof

Abstract

The invention discloses flammulina velutipes F9 with ice recrystallization inhibition activity as well as a fermentation product and application of the flammulina velutipes F9, and belongs to the technical field of food microorganisms. The longhairy fungus is preserved in China General Microbiological Culture Collection Center (CGMCC) on February 01, 2024, and the preservation number is CGMCC No.41162. The longhairy fungus has the advantages that the longhairy fungus is preserved in the China General Microbiological Culture Collection Center; fermentation supernate obtained after low-temperature cold induction of the longhairy funguses F9 has ice recrystallization inhibition activity and can effectively inhibit growth of ice crystals, and the average particle size area percentage (% MGS) of the generated ice crystals is only 55.29% of that of a control group. When the supernate is applied to frozen egg white gel, pore formation and water loss of the egg white gel in the freezing process can be remarkably reduced, the hardness, viscosity and chewiness of the egg white gel are enhanced, and the supernate has the potential of being developed into a natural food cryoprotectant. The method has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of food microbiology technology, specifically relating to *Flammulina velutipes* with ice recrystallization inhibitory activity, its fermentation products, and their applications. Background Technology

[0002] Freezing is the most common method of food preservation, protecting heat-sensitive substances and extending shelf life. In recent years, with the accelerated pace of life and changing lifestyles, the demand for frozen foods has been increasing year by year. During freezing, the formation of ice crystals and the resulting physicochemical changes can damage cell structure, leading to nutrient loss or a grainy texture. The low-temperature concentration effect caused by freezing, resulting in protein denaturation and lipid oxidation, negatively impacts the nutrition, texture, color, and flavor of food. Traditional chemical cryoprotectants such as sucrose, sorbitol, and phosphates are not only high in heat and salt but also pose risks of inducing celiac disease, gastrointestinal disorders, and diabetes. Therefore, the development of natural cryoprotectants has become a research hotspot in the frozen food industry in recent years.

[0003] Low-temperature chilling or freezing injury is lethal to most organisms, but cold-resistant organisms have evolved substances with antifreeze activity through adaptation to cold. Currently, scientists have discovered a variety of antifreeze-active substances in fish, insects, plants, bacteria, and fungi. These substances can effectively interact with ice crystals and limit their growth by inhibiting ice recrystallization (ice recrystallization inhibition activity), lowering the freezing point of solutions (thermal hysteresis activity), and modifying ice crystal morphology (dynamic ice-forming activity), thereby protecting organisms from low-temperature damage. These antifreeze-active substances have attracted considerable attention from scholars due to their unique functions and potential commercial value.

[0004] Compared to antifreeze-active substances from other sources, microbial antifreeze-active substances have low production costs, high yields, and are not limited by space, environment, or resources, making them an important approach for large-scale development of antifreeze-active substances. Macrofungi are an important component of microorganisms and a significant source for extracting natural active substances. *Flammulina velutipes* is one of the main cultivated edible fungi in my country and currently the mushroom species with the largest industrial production scale in the country. Currently, there are no literature reports on the selection methods for *Flammulina velutipes* with high recrystallization inhibition activity or the enrichment of high antifreeze-active substances from *Flammulina velutipes* using microbial fermentation. Summary of the Invention

[0005] Previous research in this invention has shown that the mycelium of *Flammulina velutipes* possesses high ice recrystallization inhibitory activity; however, the extraction process for antifreeze active substances from the mycelium is complex. To address the shortcomings of existing technologies and fully develop the antifreeze active substance resources of *Flammulina velutipes*, the primary objective of this invention is to provide a *Flammulina velutipes* strain with high ice recrystallization inhibitory activity. This *Flammulina velutipes* strain is *Flammulina velutipes* F9, with accession number CGMCC No. 41162, and was deposited at the China General Microbiological Culture Collection Center on February 1, 2024.

[0006] The present invention also provides a fermentation product of *Flammulina velutipes* as described above, wherein the fermentation product is a cold-induced fermentation supernatant of *Flammulina velutipes*, and has high ice recrystallization inhibitory activity.

[0007] The present invention also provides a method for preparing the fermentation product as described above, the method comprising the following steps:

[0008] (1) Inoculate the *Flammulina velutipes* onto PDA medium and culture it. Then, scrape off *Flammulina velutipes* mycelial blocks, inoculate them into seed culture medium, and place them in a shaker at 18-20°C. Culture at 180-200 r / min (preferably 180 r / min) for 6-8 days (preferably 7 days) to obtain seed liquid.

[0009] (2) The seed liquid obtained in step (1) is inoculated into the fermentation medium at an inoculation rate of 8%-15% (preferably 10%) and cultured in a shaker at 18-20°C at 180-200 r / min (preferably 180 r / min) for 4-6 days (preferably 5 days) to obtain the fermentation broth;

[0010] (3) Add 2.5 to 7.5 mg / L of abscisic acid to the fermentation broth obtained in step (2) and cold induce it at 4°C for 5 to 8 days (preferably 6 days) to obtain the cold-induced fermentation broth;

[0011] (4) The cold-induced fermentation broth obtained in step (3) is centrifuged at 4°C and 8000-10000 rpm (preferably 8500 rpm) for 10-20 min (preferably 15 min) to obtain the cold-induced fermentation supernatant, which is the fermentation product. The fermentation product is then stored at -20°C for later use.

[0012] In one specific embodiment, the method for preparing the fermentation product is as follows:

[0013] (1) Inoculate the *Flammulina velutipes* strain onto potato dextrose agar (PDA) plates and culture until the mycelium fully colonizes the medium; scoop out *Flammulina velutipes* blocks, inoculate them into seed culture medium, and culture them in a shaker at 18-20°C at 180-220 r / min in the dark for 6-8 days to obtain *Flammulina velutipes* seed liquid.

[0014] (2) The seed liquid of *Flococcus truncatula* obtained in step (1) is inoculated into 50 mL of fermentation medium at a 10% inoculation rate, and then cultured in a shaker at 18-20°C and 180-220 r / min in the dark for 4-6 days to obtain *Flococcus truncatula* fermentation broth.

[0015] (3) Add a certain amount of abscisic acid to the fermentation broth obtained in step (2), place it at 4°C, and perform cold induction for a certain period of time to obtain the cold-induced fermentation broth.

[0016] (4) Centrifuge the cold-induced fermentation broth obtained in step (3) at 4°C and 8000-10000 rpm for 10-20 min. The supernatant obtained is the cold-induced fermentation supernatant, which is the fermentation product. Then, store the fermentation product at -20°C for later use.

[0017] In step (1), the PDA culture medium consists of: 180-220 g / L potato, 1-3 g / L yeast powder, 1-2 g / L potassium dihydrogen phosphate, 0.5-1 g / L magnesium sulfate heptahydrate, 18-22 g / L glucose, 18-22 g / L agar, and the remainder is distilled water.

[0018] In step (1), the components per liter of the seed culture medium are as follows:

[0019] Potato 180-220 g / L, glucose 18-22 g / L, yeast extract 1.8-2.2 g / L, potassium dihydrogen phosphate 0.8-1.2 g / L, magnesium sulfate heptahydrate 0.45-0.55 g / L, defoamer 0.3-0.4 g / L, vitamin B1 0.04-0.06 g / L, with the remainder being water.

[0020] In step (1), the size of the *Flammulina velutipes* mycelium block is 4–9 mm. 2 The number is 4 to 6.

[0021] In step (2), the components of the fermentation medium per liter are as follows:

[0022] Soluble starch 50-60 g / L, potassium dihydrogen phosphate 1.0-2.0 g / L, yeast peptone 5-10 g / L, defoamer 0.3-0.4 g / L, VB1 0.04-0.06 g / L, the remainder being water.

[0023] In step (3), the amount of abscisic acid added is 2.5 to 7.5 mg / L, the temperature of cold induction is 4°C, and the time of cold induction is 5 to 8 days; preferably, the amount of abscisic acid added is 5 mg / L, the temperature of cold induction is 4°C, and the time of cold induction is 6 days.

[0024] The present invention also provides the application of the above-mentioned *Flammulina velutipes*, or the above-mentioned fermentation product, or the above-mentioned preparation method in the cryopreservation of egg white gel, natural cryoprotectants, frozen foods, frozen cells, etc.

[0025] The applications include using the cold-induced fermentation supernatant of *Flammulina velutipes* as a cryoprotectant and using the cold-induced fermentation supernatant of *Flammulina velutipes* for cryopreservation.

[0026] In this invention, the high-ice recrystallization inhibitor is preferably the cold-induced fermentation supernatant of the above-mentioned *Flammulina velutipes* which has high ice recrystallization inhibitory activity.

[0027] Preferably, the application in the cryopreservation of egg white gel involves cryopreserving the egg white gel using the cold-induced fermentation supernatant of *Flammulina velutipes*.

[0028] As a preferred embodiment, the application in the cryopreservation of egg white gel specifically includes: adding the cold-induced fermentation supernatant to egg white liquid at a volume concentration of 0.1% to 5%, and then freezing the prepared egg white gel at -20°C for 0 days, 3 days, and 7 days.

[0029] This invention provides a strain of *Flammulina velutipes* F9 that exhibits cryoprotective properties against agar gels and can be used for the cryopreservation of egg white gels. After 7 days of cryopreservation, the control group's egg white gels showed obvious pores and uneven surfaces, while the egg white gels containing 1% of the cold-induced fermentation broth of *Flammulina velutipes* from this invention inhibited the formation of large ice crystals during freezing, thus maintaining a smooth surface. The centrifugal water loss rate decreased from 58.2% in the control group to 48.7%. The hardness, viscosity, and chewiness of the egg white gels increased from 598.9, 532.7, and 524.5 in the control group to 660.1, 595.1, and 587.9, respectively.

[0030] The present invention has the following advantages and effects compared with the prior art:

[0031] Previous research in our laboratory has shown that the mycelial extract of *Flammulina velutipes* possesses high ice recrystallization inhibitory activity, but this requires a complex process involving water extraction, dilute alkali extraction, and concentrated alkali extraction under high temperature and pressure. Therefore, this invention simplifies the extraction process by obtaining antifreeze active substances from the fermentation broth and enriches the source of microbial antifreeze active substances. This invention also investigates the cryoprotection of the cold-induced fermentation supernatant using agarose gel, demonstrating that the supernatant can effectively inhibit the freezing degradation of agarose gel and has the potential to be developed into a food-grade antifreeze additive, possessing significant application value. Attached Figure Description

[0032] Figure 1 This is a colony morphology diagram of the *Flammulina velutipes* F9 strain provided by this invention.

[0033] Figure 2 The results of the ice recrystallization inhibition activity of the fermentation supernatant of *Flammulina velutipes* in Example 2 and Comparative Examples 1 and 2 of this invention are as follows: Figure 2 A shows the ice crystal images generated at 0 min and 30 min; Figure 2 B represents the percentage of the average diameter and average area of ​​ice crystals at 30 min (%MGS).

[0034] Figure 3 These are photographs taken after adding different amounts of egg white gel and freezing for different times, as shown in Example 3 of this invention.

[0035] Figure 4 The effect of *Flammulina velutipes* fermentation broth on the properties of frozen egg white gel in Example 3 of this invention: A: centrifugal water loss rate; B: hardness; C: viscosity; D: chewiness. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to the specific embodiments and accompanying drawings. The principles and features of the present invention are described below, and the examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0037] All variations and advantages that can be conceived by those skilled in the art are included in this invention and are protected by the appended claims. The processes, conditions, reagents, experimental methods, etc., for carrying out this invention are all common knowledge and general knowledge in the art, and this invention does not have any particular limitations.

[0038] The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the invention or its application or use. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0039] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may have different values.

[0040] This invention discloses a Flammulinavelutipes strain with ice recrystallization inhibitory activity, its fermentation products, and their applications, belonging to the field of food microbiology technology. The Flammulinavelutipes strain was deposited on February 1, 2024, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 41162. The fermentation supernatant of Flammulinavelutipes F9 strain after low-temperature cold induction exhibits ice recrystallization inhibitory activity, effectively inhibiting ice crystal growth and reducing the MGS (Medium-to-Saturated Grain) ice crystal formation to only 55.29% of the control group. When applied to frozen egg white gel, this supernatant significantly improves the large pores and uneven surface morphology of thawed egg white gel, reducing the centrifugal water loss rate from 58.2% in the control group to 48.7%; the gel hardness, viscosity, and chewiness increase from 598.9, 532.7, and 524.5 in the control group to 660.1, 595.1, and 587.9, respectively. This invention has the potential to be developed into a natural food cryoprotectant and has broad application prospects.

[0041] Unless otherwise specified, the experimental materials used in the examples are all conventional biochemical reagents.

[0042] Example 1: Screening and ITS sequence analysis of *Flammulina velutipes* with ice recrystallization inhibition activity

[0043] (1) Screening and isolation of strains

[0044] Fifteen strains of *Gynostemma pentaphyllum* preserved in the laboratory were inoculated into 50 mL of fermentation medium and cultured at 180 rpm and 18 °C for 5 days. After cold induction, the ice recrystallization inhibition activity of the supernatant of the cold-induced fermentation was determined according to the following “(2) Screening Method”, and strains with high ice recrystallization inhibition activity were screened out.

[0045] (2) Screening method

[0046] The cold-induced fermentation supernatant obtained in (1) was mixed with an equal volume of 46% sucrose. The extract was mixed with the 46% sucrose solution at a ratio of 1:1 (v / v). 1 μL was dropped into the center of a circular glass slide placed on a hot-cold stage. The temperature of the hot-cold stage was cooled from room temperature to -40°C at a cooling rate of 80°C / min, and then heated to -6°C at the same rate and held for 30 min. The hot-cold stage was placed on the stage of an optical microscope and the ice crystal size was recorded using a 10× objective lens. The average diameter of the ice crystals was calculated using Image Pro Plus software. The percentage of average ice crystal diameter (%MGS) = (average diameter of ice crystals in the treatment group / average diameter of ice crystals in the control group) × 100. The ice recrystallization activity of the strain is inversely proportional to the size of the generated ice crystals. The smaller the ice crystals, the higher the ice recrystallization activity of the strain. Based on this, the strain F9 with the highest recrystallization inhibition activity of the fermentation supernatant was selected. Its colony morphology is shown in the figure below. Figure 1 As shown.

[0047] (3) ITS sequence analysis of the strain

[0048] The F9 strain obtained in step (2) underwent ITS sequence analysis. Genomic DNA was extracted using the TIANGEN fungal DNA extraction kit, and PCR amplification was performed using universal fungal primers ITS1 and ITS4 to obtain its ITS sequence, which was then sequenced. The determined ITS sequence is shown below. BLAST homology comparison of this sequence in GenBank revealed a 98% similarity to a previously reported strain of *Flammulina velutipes* (accession number: KY686284.1) in GenBank.

[0049] The ITS sequence SEQ ID NO.1 of *Fructus styracifolium* F9 is as follows:

[0050]

[0051]

[0052] Example 2: Preparation of fermentation supernatant of cold-induced *Flammulina velutipes*

[0053] (1) Preparation of seed culture: Take 4-6 F9 strains of *Flammulina velutipes*, each about 4-9 mm in size, from the PDA plate culture medium. 2 The bacterial clumps were inoculated into 50 mL of seed culture medium (2 g / L yeast extract, 20 g / L glucose, 200 g / L potato, 1 g / L potassium dihydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.33 g / L antifoaming agent, and 0.05 g / L vitamin B1), and then placed in a shaker at 180 rpm for 7 days at 180 °C to obtain the seed culture.

[0054] (2) Fermentation culture: The seed liquid obtained in step (1) was inoculated into 50 mL of fermentation culture medium (soluble starch 55.2 g / L, potassium dihydrogen phosphate 1.5 g / L, yeast peptone 7.8 g / L, defoamer 0.33 g / L, VB1 0.05 g / L) and cultured at 180 r / min for 5 days in a shaker at 18℃.

[0055] (3) Cold induction treatment: Add abscisic acid to the fermentation broth obtained in step (2) at a ratio of 5 mg / L and cold induction at 4℃ for 6 days.

[0056] (4) Centrifugation: Centrifuge the cold-induced fermentation broth obtained in step (3) at 4℃ and 8500rpm for 15min to obtain the cold-induced fermentation supernatant.

[0057] Comparative Example 1

[0058] The difference between this comparative example and Example 2 is that the fermentation supernatant prepared in this example was obtained by fermentation culture for 5 days, but it was not subjected to cold induction treatment at 4°C.

[0059] Comparative Example 2

[0060] The difference between this comparative example and Example 2 is that abscisic acid was not added during the 6-day cold induction process.

[0061] Determination of recrystallization inhibition activity of fermentation supernatant: Equal volumes of fermentation supernatants prepared in Example 2, Comparative Example 1, and Comparative Example 2 were mixed with 46% sucrose solution, with 23% sucrose solution as a control. 1 μL of the solution was dropped into the center of a circular slide on a hot-cold stage, which was placed on the stage of an optical microscope beforehand. The temperature control program for the hot-cold stage was as follows: first, the temperature was lowered to 0°C at a rate of 10°C / min, then cooled to -40°C at a rate of 80°C / min, and then raised to -6°C at the same rate, maintaining this temperature for 30 min. The ice crystal size of each fermentation supernatant was photographed under a microscope at 0 min and 30 min, and the average diameter and average surface area percentage (%MGS) of ice crystals for each group of samples at 30 min were calculated. Images and sizes of ice crystals generated in each fermentation supernatant at 0 min and 30 min are shown below. Figure 2 As shown, compared with Comparative Examples 1 and 2, the ice crystals generated in the fermentation supernatant of Example 2 were smaller, with an average diameter of only 15.93 μm at 30 min and an ice crystal %MGS of only 55.29%. This indicates that low temperature induction and the addition of abscisic acid can give the fermentation supernatant of *Fragaria scabra* high ice recrystallization inhibition activity.

[0062] Example 3: Cryoprotection of egg white gel by fermentation supernatant of cold-induced *Flammulina velutipes*

[0063] (1) The method for preparing the fermentation supernatant of cold-induced Fecundactylum styracifolium is the same as in Example 2 of this invention.

[0064] (2) Add 0%, 0.1%, 0.5%, 1%, 3%, and 5% of cold-induced fermentation supernatant of *Flammulina velutipes* to the egg white solution.

[0065] (3) Mix the fermentation supernatant of cold-induced Fecundida styracifolium with egg white liquid, heat in a constant temperature water bath at 90℃ for 15 min to prepare egg white gel, and cool at 4℃ for 5 h.

[0066] (4) Store at -20℃ for 0 days, 3 days, or 7 days.

[0067] (5) Egg white gels frozen for different times were thawed at 25℃ for 5 hours and then photographed. The results are as follows. Figure 3 As shown: During freezing, the control group egg white gel (CK, referring to egg white gel with 0% cold-induced *Flammulina velutipes* fermentation supernatant) showed more obvious porosity and unevenness with prolonged freezing time, while the sample with the cold-induced *Flammulina velutipes* fermentation supernatant of this invention maintained better appearance. After 7 days of freezing, the egg white gel with 1.0% cold-induced *Flammulina velutipes* fermentation supernatant of this invention still had a relatively smooth cut surface, with small and fewer pores. Further testing of the centrifugal water loss rate, hardness, viscosity, and chewiness of each group of egg white gels yielded the following results: Figure 4 As shown: After freezing for 7 days, the centrifugal water loss rate of the group with 1.0% of the cold-induced *Flammulina velutipes* fermentation supernatant added according to this invention decreased from 58.2% in the control group to 48.7%; the hardness, viscosity, and chewiness of the egg white gel increased from 598.9, 532.7, and 524 in the control group to 660.1, 595.1, and 587.9, respectively. These experimental results collectively confirm that the cold-induced *Flammulina velutipes* fermentation supernatant prepared according to this invention has a significant cryoprotective effect on egg white gel.

[0068] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0069] As used in this invention, the term "comprising" is an open-ended expression, meaning it includes the contents specified in this invention but does not exclude other aspects.

[0070] As used in this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0071] The scope of protection of this invention is not limited to the above embodiments. Any variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A type of *Flococcus filamentosa*, characterized in that, The *Flammulina velutipes* species exhibits ice recrystallization inhibition activity. *Flammulina velutipes* F9 was deposited on February 1, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41162.

2. A fermentation product of *Flococcus spp.* as described in claim 1, characterized in that, The fermentation product is the fermentation supernatant of cold-induced *Flammulina velutipes*, which has ice recrystallization inhibitory activity.

3. A method for preparing the fermentation product as described in claim 2, characterized in that, The preparation method includes the following steps: (1) Inoculate the fungus *Flammulina velutipes* onto PDA medium and culture it. Then, scrape off the fungal blocks of *Flammulina velutipes*, inoculate them into seed culture medium, and place them in a shaker at 18-20°C. Culture at 180-200 r / min for 6-8 days to obtain seed liquid. (2) The seed liquid obtained in step (1) is inoculated into the fermentation medium at an inoculation rate of 8%-15%, and then cultured in a shaker at 180-200 r / min for 4-6 days at 18-20℃ to obtain the fermentation broth; (3) Add 2.5 to 7.5 mg / L of abscisic acid to the fermentation broth obtained in step (2) and cold induce it at 4°C for 5 to 8 days to obtain the cold-induced fermentation broth; (4) Centrifuge the cold-induced fermentation broth obtained in step (3) at 4°C and 8000-10000 rpm for 10-20 min to obtain the cold-induced fermentation supernatant, which is the fermentation product. Then store the fermentation product at -20°C for later use.

4. The preparation method according to claim 3, characterized in that, In step (1), the seed culture medium comprises the following components per liter: 180-220 g / L potato, 18-22 g / L glucose, 1.8-2.2 g / L yeast extract, 0.8-1.2 g / L potassium dihydrogen phosphate, 0.45-0.55 g / L magnesium sulfate heptahydrate, 0.3-0.4 g / L defoamer, 0.04-0.06 g / L vitamin B1, and the remainder is water.

5. The preparation method according to claim 3, characterized in that, In step (2), the fermentation medium comprises the following components per liter: soluble starch 50-60 g / L, potassium dihydrogen phosphate 1.0-2.0 g / L, yeast peptone 5-10 g / L, defoamer 0.3-0.4 g / L, VB1 0.04-0.06 g / L, and the remainder is water.

6. The use of the *Flammulina velutipes* as described in claim 1, or the fermentation product as described in claim 2, or any one of claims 3-5 in the preparation method described herein in the cryopreservation of egg white gel, natural cryoprotectants, frozen foods, and frozen cells.

7. The application as described in claim 6, characterized in that, The applications include using the cold-induced fermentation supernatant of the *Flammulina velutipes* as a cryoprotectant; and / or, The supernatant of the cold-induced fermentation of the *Flammulina velutipes* was cryopreserved.

8. The application as described in claim 7, characterized in that, The application in the cryopreservation of egg white gel involves using the supernatant of cold-induced fermentation of *Flammulina velutipes* to cryopreserve the egg white gel.

9. The application according to claim 8, characterized in that, The specific application in the cryopreservation of egg white gel includes: adding the volume concentration of the cold-induced fermentation supernatant to egg white liquid at 0.1%, 0.5%, 1%, 3%, and 5%, and then freezing the resulting egg white gel at -20°C.

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