Lactobacillus salivarius and application thereof in fermentation of bee pollen

Abstract

The invention discloses combined lactobacillus salivarius and application of the combined lactobacillus salivarius in fermentation of bee pollen. The invention relates to a strain of combined lactobacillus salivarius XIF24, and the preservation number of the combined lactobacillus salivarius XIF24 is CCTCC (China Center for Type Culture Collection) NO: M 20252062. When the saliva is combined with the lactobacillus XIF24 to generate bee pollen, characteristic flavor substances including hexanoic acid and ethyl hexanoate can be produced, and due to the coexistence of hexanoic acid and residual ethyl hexanoate, the fermented bee pollen is endowed with attractive fruity flavor by enhancing the volatility and smell perception intensity of aroma substances; the acetoin is high in yield, and the fermented bee pollen is endowed with attractive sweet taste and milk taste; the bee pollen is endowed with attractive rose fragrance, so that the bee pollen has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbiology, in particular to a combination of lactobacillus salivarius and its use in fermented bee pollen. BACKGROUND

[0002] Bee pollen is a natural mixture composed of nectar, pollen, enzymes and honeybee saliva secretions. In recent years, the research on bee pollen at home and abroad mainly focuses on the development of nutritional components and bioactive substances in bee pollen. Studies have shown that bee pollen is rich in carbohydrates, proteins, amino acids, vitamins, unsaturated fatty acids, polyphenols and flavonoids, etc., and has a variety of biological activities. It is worth noting that bee pollen, as a natural source of food ingredients, does not contain any additives or artificial ingredients, and is a good choice as a kitchen seasoning or food processing raw material. However, the alcohol and phenolic acid compounds contained in bee pollen make it have a bitter and sour taste in addition to the umami and sweet taste, which reduces the acceptance of consumers, and this limitation seriously restricts the application of bee pollen in high value-added products. Therefore, it is of great significance to develop an efficient flavor improvement technology to improve the flavor substances of bee pollen.

[0003] Lactobacillus is a gram-positive bacterium with good acid and bile salt tolerance and acid production capacity, which widely exists in the gastrointestinal tract of humans and animals. Lactobacillus fermentation, as a simple and efficient means to improve food flavor, has great potential in the production of biological activity and flavor in food. It is worth noting that lactobacillus salivarius belongs to the genus lactobacillus, which is a safe strain allowed to be used in food by the National Health Commission of China. However, the improvement of food flavor by lactobacillus salivarius has not been studied.

[0004] Probiotic fermentation, as a common food processing method, has great potential in the preservation and safety improvement, nutritional property modification and flavor characteristic improvement of food. It has been reported that proteins, carbohydrates and fats are important substrates for probiotic fermentation, which can be decomposed into various small molecular flavor substances by probiotics. Studies have found that transamination is the first step in the process of producing aroma by microorganisms using proteins. Amino acids generate α-keto acids under the action of transaminase, and α-keto acids further metabolize to produce fatty acids, alcohols and aldehydes, etc. The enzymes involved in this process mainly include branched-chain transaminase (BCTA ilvE gene encoding), aspartate transaminase (ASPA aspC gene encoding) and aromatic transaminase (ARO tyrB gene encoding). In addition, the production of flavor substances in fermented food is closely related to the carbohydrate content. Pyruvate produced by glycolysis can participate in the conversion of various flavor substances. Among them, pyruvate can be converted into acetyl-CoA by acetyl-CoA synthase (acs ilvBAcetol is produced by the action of gene encoding acetol, which is then chemically oxidized and decarboxylated to produce diacetyl, an important food flavor compound. Furthermore, acetol can also be decarboxylated by α-acetolactate decarboxylase (…). alsD Under the catalytic action of gene encoding, another important food flavor compound—acetoin—is produced. Therefore, the pathways by which probiotic fermentation improves food flavor compounds can be understood. ilvB and alsD Gene expression levels were confirmed. Summary of the Invention

[0005] In view of the shortcomings of the prior art described above, the object of the present invention is to provide a *Lactobacillus salivarius* and its use in fermented bee pollen.

[0006] To achieve the above and other related objectives, the present invention is obtained through the following technical solution.

[0007] The first aspect of this invention protects a strain of Lactobacillus salivarius XIF24, which has the accession number CCTCC NO:M20252062.

[0008] In some embodiments, the *Lactobacillus saliva-associated* XIF24 is yellow, with medium-sized, raised, slightly white, moist colonies with neat edges and round shape.

[0009] A second aspect of the present invention protects a culture obtained by culturing *Lactobacillus saliva-associated* XIF24 as described above.

[0010] A third aspect of the present invention protects a microbial agent comprising the above-described Lactobacillus salivarius XIF24 or the above-described culture.

[0011] The fourth aspect of the present invention protects the use of the above-described Lactobacillus salivae XIF24 or the above-described culture or the above-described microbial agent in at least one of the following: 1) fermenting bee pollen; 2) enhancing the flavor of bee pollen; 3) preparing fermented food.

[0012] The fifth aspect of the present invention protects a method for enhancing the flavor of bee pollen, which involves fermenting bee pollen using Lactobacillus saliva-associated XIF24, the culture described above, or the microbial agent described above.

[0013] In some embodiments, the bee pollen is selected from one or more of rapeseed pollen, corn pollen, sunflower pollen, milkvetch pollen, buckwheat pollen, sesame pollen, and sorghum pollen.

[0014] In some implementations, glucose is also added during fermentation.

[0015] In some embodiments, the mass ratio of bee pollen to glucose is (0.5-2):1. Preferably, it is 25:22.

[0016] In some embodiments, the fermentation temperature is 32–40°C. Preferably, it is 37°C.

[0017] In some embodiments, the fermentation time is 7 to 30 hours. Preferably, it is 37°C.

[0018] The sixth aspect of this invention protects bee pollen obtained by the method described above.

[0019] The seventh aspect of this invention protects the use of bee pollen as described above in the preparation of functional bee pollen products.

[0020] Compared with the prior art, the present invention has the following beneficial effects: To address the problems of poor palatability, low nutrient utilization, and insufficient biological activity in existing bee pollen, this invention screens a strain of *Lactobacillus salivarius* from infant feces. Ligilactobacillus salivarius XIF24, used as a fermentation strain, significantly improved the sensory quality and functional properties of bee pollen. During fermentation, it produced high yields of the characteristic flavor compound acetoin, along with 10 new volatile compounds. Furthermore, the increased lactic acid content and decreased pH inhibited the growth of putrefactive bacteria. In addition, *Lactobacillus salivarius* XIF24, through the secretion of specific enzymes and the production of metabolites, chemically decomposed cell wall components, opening up some channels (germination pores and furrows) in the inner and outer walls of the pollen, causing the pollen wall to rupture and releasing nutrients from the inner wall, thus improving the digestibility and utilization rate of bee pollen. This invention establishes for the first time a fermentation method suitable for bee pollen, providing a technical solution for the development of functional bee pollen products that combines nutritional fortification and flavor enhancement. It breaks through the bottleneck of bee pollen processing technology and provides theoretical support for the development of innovative bee pollen products with high absorption rates, high activity, and pleasant flavor. Attached Figure Description

[0021] Figure la This is a colony growth morphology diagram of strain XIF24 in Example 1 of the present invention.

[0022] Figure lb This is a diagram showing the Gram staining results of the strain in Example 1 of the present invention.

[0023] Figure lc This is a microscopic morphological image of strain XIF24 in Example 1 of the present invention at 10,000x magnification.

[0024] Figure ld This is a microscopic morphological image of strain XIF24 in Example 1 of the present invention at 20,000x magnification.

[0025] Figure leThis is the phylogenetic tree of Lactobacillus saliva-associated with XIF24 in Example 1 of the present invention.

[0026] Figure 2 This is an electron microscope image of the bee pollen fermentation product during the fermentation process in Example 3 of the present invention.

[0027] Figure 3a This refers to the change in the electronic nose response value of the bee pollen fermentation product during the fermentation process in Example 3 of the present invention.

[0028] Figure 3b This is a graph showing the changes in compound content during the fermentation process of bee pollen fermentation product in Example 3 of the present invention.

[0029] Figure 4 This is a statistical chart showing the types and contents of volatile substances detected in bee pollen fermentation products in Example 3 of the present invention.

[0030] Figure 5 This is a principal component analysis diagram of the bee pollen fermentation product detected in Example 3 of the present invention.

[0031] Figure 6 This is a bar chart showing the changes in glucose and lactic acid content in bee pollen fermentation product over time in Example 3 of the present invention. Detailed Implementation

[0032] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0033] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0034] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. 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. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0035] Example 1: Strain Screening Weigh out infant feces and dilute with an appropriate amount of physiological saline to obtain a fecal dilution. Use a sterile inoculation loop to pick up the enriched culture medium and streak it continuously on an MRS solid medium plate. After 24 h of incubation, scattered single colonies will appear at the end of the streak. Select single colonies with consistent morphology (color, edge, elevation, etc.), pick them up with an inoculation loop, and transfer them to fresh MRS solid medium (Guangdong Huankai Microbial Technology Co., Ltd.) for purification and culture. The culture conditions are 37℃ with aerobic conditions.

[0036] like Figure la As shown, the colonies on MRS solid medium are yellow, medium-sized, raised, slightly white, moist, with neat edges, and round in shape. They are named strain XIF24.

[0037] The grown bacterial strains were identified by Gram staining and sequencing. The specific identification process is as follows.

[0038] Gram staining: XIF24 bacterial cells were stained using a Gram staining kit from Beijing Solarbio Science & Technology Co., Ltd. In short, XIF24 bacterial suspension was evenly smeared, dried, and fixed by flame 1-2 times. The staining procedure was as follows: first, crystal violet staining for 1 min, followed by washing with water; then iodine mordanting for 1 min, followed by washing with water; then destaining with destaining solution, shaking the slide for 30 s depending on the smear thickness, followed by washing with water and abstaining off excess water; then counterstaining with safranin for 1 min, followed by washing with water; finally, abstaining dry or air-drying, and examining under a 100× oil immersion microscope. Activated Lactobacillus saliva-associated bacterial suspension XIF24 was centrifuged, and the bacterial pellet was collected. The pellet was washed with 0.1 mol / L phosphate-buffered saline (PBS) and fixed with 2.5% glutaraldehyde. Subsequently, the microscopic morphology of Lactobacillus saliva-associated bacterial suspension XIF24 was observed using a scanning electron microscope at magnifications of 10.0kx and 20.0kx.

[0039] like Figure lb As shown, Gram staining results indicate that strain XIF24 is a Gram-positive bacterium, and its arrangement is mainly in pairs or short chains.

[0040] like Figure lc and1d As shown, the morphological characteristics under different microscopes indicate that XIF24 is a short rod-shaped, unbranched rod with blunt, rounded ends. In summary, strain XIF24 conforms to the morphological characteristics of *Lactobacillus salivarius*.

[0041] Sequencing was performed by Shanghai Meiji Biotechnology Co., Ltd. The genome of *Lactobacillus salivarius* XIF24 was sequenced using Illumina and PacBio. Based on comparison with Meiji Biotechnology's local database, 20 strains most closely related at the species level were selected using the 16S rRNA sequence. A phylogenetic tree was constructed using the Neighbor-Joining (NJ) method with MEGA 6.0 software. The results are shown below. Figure le .

[0042] from Figure le It can be seen that strain XIF24 is mainly composed of Lactobacillus assemblica ( Ligilactobacillus Branching occurs, particularly with Lactobacillus GCF_000615845.1, which is associated with the host of Hayarai Town. Ligilactobacillus_hayakitensis|GCF_ 000615845.1 They belong to the same phylogenetic branch.

[0043] MRS solid medium: 10g peptone, 5g beef extract powder, 4g yeast extract powder, 20g glucose, 1.0mL Tween-80, 2.0g dimethyl hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 15g agar, final pH 6.2±0.2.

[0044] Based on Gram staining results, strain morphology, and 16S rRNA phylogenetic tree, strain XIF24 was identified as *Lactobacillus salivarius*. Furthermore, *Lactobacillus salivarius* XIF24 is a bacterium rich in carbohydrate metabolism-related genes, with proteins exhibiting active molecular functions, and its genome-encoded proteins demonstrating high efficiency in biochemical processes such as catalytic reactions, substance binding, and signal transduction.

[0045] The strain was named *Lactobacillus salivarius*. Ligilactobacillus salivarius XIF24, with accession number CCTCC NO: M 20252062, was deposited on September 18, 2025, at the China Center for Type Culture Collection, located at Luojia Mountain, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0046] Example 2: Fermentation of pollen by bacterial strain In this Example 2, the strain XIF24 from Example 1 is used to ferment bee pollen. The steps include: 2.1 Preparation of Rapeseed and Bee Pollen Culture Medium Accurately weigh 25g of commercially available rapeseed bee pollen (meeting the requirements of GB / T 30359-2021 bee pollen standard, with moisture 1.38g / 100g, ash 2.7g / 100g, protein 26.7g / 100g, fat 10g / 100g, single-variety pollen rate 98%, acidity 5.5, no lead detected, total bacterial count <10 CFU / g, coliform bacteria <0.30 MPN / g, mold <10 CFU / g) and 22g of glucose, and dissolve them in 1000mL of distilled water to form a bee pollen culture medium. Homogenize using a high-pressure homogenizer at 20MPa for 2min. After homogenization, dispense the medium into clean conical flasks and seal them with breathable sealing film. The packaged bee pollen culture medium is pasteurized by placing it in a 72°C constant temperature water bath for 30 seconds. After sterilization, the bee pollen culture medium is quickly removed and cooled to 30°C at room temperature before use.

[0047] 2.2 Fermentation In Example 1, *Lactobacillus salivarius* XIF24, selected from the strain, was preserved in 50% glycerol. Before use, 120 μl of the frozen XIF24 strain was aseptically transferred to MRS liquid medium and activated by incubation at the optimal temperature of 37°C for 24 h. After activation, the aseptic streaked broth was incubated in MRS solid medium, inverted, for 12 h. Typical single colonies were then inoculated into MRS liquid medium and incubated for 12 h to form a seed culture. The OD of the seed culture was... 600 The value was 0.921, and the viable bacteria content was 1.18 × 10⁻⁶. -9 The seed culture was inoculated at a volume ratio of 2% (CFU / mL) into a 2.5 wt% bee pollen culture medium and fermented in a shaker at 37°C for 24 h to obtain bee pollen fermentation product. Quantitative samples were taken every 6 h for subsequent testing.

[0048] Example 3: Testing of fermentation products In Example 3, the fermentation product obtained in Example 2 was measured. The steps included: 3.1 Analysis of Surface Morphology Changes The surface morphology of bee pollen fermentation samples at different fermentation times was observed using a scanning electron microscope (TM3030Plus, HITACHI, Japan). After centrifugation, the samples were freeze-dried using a vacuum freeze dryer, and the surface morphology of the freeze-dried samples was observed at 1000×. Results are shown below. Figure 2 .

[0049] The pollen wall is a complex wall system comprising three main structural domains: the pollen sheath, the exine (in a microretic pattern), and the inner wall. Beneath the exine, the inner wall encloses the protoplast of the male gametophyte. The pollen sheath extends directly around the exine layer, often penetrating deep into the highly sculpted spaces of the exine.

[0050] from Figure 2 It was observed that in the control group (CON), unfermented bee pollen grains partially lost their pollen coats, exposing sculpted outer walls and larger internal areas. After 6 hours of fermentation, the bee pollen fermentation product (L6 group) mostly consisted of empty outer shells and broken pollen wall fragments. As fermentation time increased, intact empty outer shells were no longer observed; only broken pollen wall fragments remained, and the pollen grains were highly fragmented. Bee pollen is rich in nutrients, but the pollen wall hinders the dissolution of nutrients and the full utilization of functional components by the human body. During fermentation, *Lactobacillus salivarius* XIF24, through secreting specific enzymes and producing metabolites, chemically decomposes cell wall components, opening some channels (germination pores, furrows) between the inner and outer walls of the pollen, causing the pollen wall to rupture and releasing nutrients from the inner wall.

[0051] 3.2 Electronic nose analysis Take 5 mL of bee pollen fermentation products from different fermentation periods in Example 2 into a 50 mL headspace vial, seal and let stand for 30 min. The electronic nose (PEN 3, AIRSENSE Analytics, Germany) was used for testing for 120 s, and the washing time was 120 s. Bee pollen fermentation products at different fermentation times.

[0052] A radar chart was generated using Prism to reflect changes in the electronic nose's response value. Figure 3a Among them, -20, 020, 40, 60, 80, 100, 120, 140, and 160 represent the signal values ​​of the electronic nose sensor, respectively; W1C is the aromatic component - benzene, W1S is the methyl component, W1W is the sulfide, W2W is the organic sulfide, W2S is the aldehyde and ketone, W3C is the aromatic component - ammonia, W3S is the long-chain alkane, W5C is the short-chain alkane, W5S is the nitrogen oxide, and W6S is the hydride.

[0053] Changes in compound content during the fermentation of bee pollen fermentation products are shown in the figure. Figure 3b In this context, CON represents unfermented bee pollen culture medium, L-6, L-12, L-18, and L-24 represent bee pollen fermented for 6, 12, 18, and 24 hours, respectively; W1W represents sulfides, W2W represents organosulfides, W6S represents hydrides, W5C represents short-chain alkanes, and W5S represents nitrogen oxides.

[0054] Meanwhile, bee pollen that was not inoculated with any strains was used as a control group, labeled CON.

[0055] from Figure 3a and Figure 3bIt can be seen that the odor profiles of bee pollen fermentation products are basically similar at different fermentation time points. However, as fermentation progresses, the response values ​​of five types of substances—nitrogen oxides, short-chain alkanes, hydrides, sulfides, and organic sulfides—show significant differences from the control group, and these differences are positively correlated with fermentation time.

[0056] 3.3 Extraction of volatile components from bee pollen fermentation products using solid-phase microextraction optical fiber. Separation and detection were performed using a gas chromatography-mass spectrometry (GC-MS, SHIMADZU, Japan) system equipped with a DB-WAX column. GC conditions: DB-WAX capillary column (30 m × 0.25 mm × 0.25 µm); injection port temperature: 250 ℃, split injection; carrier gas (He, 99.999%) pressure: 100 kPa, total flow rate: 50 mL / min. MS conditions: ion source temperature: 230 ℃; interface temperature: 210 ℃.

[0057] The specific method is as follows: Weigh 3 mL of bee pollen fermentation sample and 0.5 g of sodium chloride into a headspace vial, and add 1-octanol as an internal standard to the headspace vial. First, age the solid-phase microextraction fiber at 250 °C for 30 min using GC-MS. Then, insert the solid-phase microextraction fiber into the headspace vial, incubate it in a water bath at 55 °C for 30 min, and then place it in the injection port for extraction at 260 °C for 5 min. The column temperature was initially set at 40 °C and held for 3 min, then increased to 100 °C at a rate of 6 °C / min. Subsequently, the temperature was increased to 230 °C at a rate of 10 °C / min and held for 7 min. Finally, the concentration of the internal standard was compared by searching the NIST standard spectral library, and the substances were qualitatively and quantitatively analyzed using the peak area normalization method.

[0058] The Pca plot was constructed using the Lianchuan Bio platform, and the main variation features of flavor compounds in bee pollen fermentation products were extracted through dimensionality reduction to achieve data simplification and visualization. Experimental results are shown below. Figure 4 Each group has three replicates. Here, nd indicates not detected, and Benzal dehyde, 2,5-bis[(trimethylsilyl)oxy]-Benzyl nitrile indicates 2,5-bis[(trimethylsilyl)oxy]benzonitrile.

[0059] from Figure 4It was found that 26 volatile components were detected in the bee pollen fermentation product, including 9 esters, 6 alcohols, 4 acids, 2 ketones, 1 benzene compound, and 4 other substances, totaling 6 categories of compounds. The richer the variety of volatile compounds produced during fermentation and the more suitable the content of characteristic components, the more complex, rich, and unique the flavor of the food. Ten new volatile substances were produced during the bee pollen fermentation process, including tert-butyl isosulfate, methyl N-hydroxybenzoimide, ethyl DL-2-hydroxy-4-methylvalerate, CH3C(O)OCH(CH3)C(O)CH3, 1-nonanol, phenylethanol, 2-ethylbutyric acid, acetic acid, hexanoic acid, and 2-methylbutyric acid. The content of 11 volatile substances decreased, mainly including 3-butenyl isothiocyanate, butyl isothiocyanate, cyclopentyl isothiocyanate, ethyl octanoate, ethyl hexanoate, 1,1'-[1-(1,1-dimethylethyl)-2-methyl-1,3-propadiyl]-2-methyl-propionate, Benzyl nitrile, 5-hexanonitrile, 3,5-octadien-2-one, methylheptenone, and 2-butoxyethanol. Among them, 3-butenyl isothiocyanate, butyl isothiocyanate, and cyclopentyl isothiocyanate all exhibit a strong pungent flavor, so it is speculated that they are the source of unpleasant odor in bee pollen.

[0060] from Figure 5 Principal component analysis revealed that regional locations indicate fermentation time influences the aroma characteristics of each component, with flavor differences becoming more significant with increasing fermentation time. Notably, acetoin concentration significantly increased during fermentation. Acetylin, possessing a sweet and milky flavor, is a widely present pleasant flavor component in fermented foods. Phenylene alcohol, a pleasant flavor component combining rose and natural fruit aromas, is also a characteristic metabolite of fermented foods. Furthermore, hexanoic acid and ethyl hexanoate enhance sweetness perception through aroma-taste interaction, producing a rich fruity aroma. These changes in the content and types of volatile components, as well as their synergistic effects, play a crucial role in altering the flavor of bee pollen.

[0061] 3.4 Analysis of changes in nutritional components The glucose and lactic acid contents in bee pollen fermentation products at different fermentation times were determined using a glucose-lactic acid analyzer. The results are shown below. Figure 6 .

[0062] from Figure 6It is evident that the glucose content significantly decreases with fermentation time. This is because glucose is an important carbon and energy source for *Lactobacillus salivarius* XIF24, which it can continuously utilize for metabolic activities. The significant increase in lactic acid content indicates that the metabolic pathway of *Lactobacillus salivarius* XIF24 is mainly lactic acid fermentation, and its products continuously accumulate, indicating ongoing fermentation activity. Furthermore, the accumulation of lactic acid and other organic acids leads to a decrease in the system's pH, which can enhance the solubility of volatile substances such as esters and alcohols, while simultaneously lowering the perception threshold of bitter substances.

[0063] Fermentation to improve the aroma of bee pollen is a highly complex biological process involving microbial metabolic regulation, targeted transformation of raw material components, and dynamic interactions within the fermentation environment. This invention discovers that *Lactobacillus salivarius* XIF24, through the synergistic effect of its own metabolic network and substrate-specific modification, on the one hand, directionally synthesizes characteristic flavor compounds, and on the other hand, degrades undesirable flavor components, ultimately achieving overall flavor optimization through the synergistic effect of multiple flavor compounds. Taking the biosynthesis of the key aroma component acetoin as an example, *Lactobacillus salivarius* XIF24 uses fermentable sugars in bee pollen culture medium as a carbon source, generating pyruvate via the glycolysis pathway (EMP pathway). Then, α-acetolactate synthase (… AlsS Under the catalysis of α-acetyllactone, two molecules of pyruvate undergo a condensation reaction, losing one molecule of CO2 to generate α-acetyllactone; subsequently, α-acetyllactone is decarboxylated by α-acetyllactone decarboxylase (α-acetyllactone decarboxylase). AlsD Under the action of esterase, ethyl hexanoate is further decarboxylated, ultimately forming acetoin (3-hydroxy-2-butanone). This metabolic pathway achieves efficient conversion through a two-step enzymatic decarboxylation reaction, significantly improving the synthesis efficiency of acetoin. Meanwhile, the regulation of ester metabolism in the fermentation system plays an important role in flavor optimization. Studies show that the content of ethyl hexanoate dynamically decreases during fermentation, and its degradation is mainly through esterases (…). holA The hydrolysis reaction is catalyzed to produce hexanoic acid and ethanol. Notably, the interaction between acid and ester compounds significantly lowers the odor perception threshold of esters, creating a synergistic effect: the coexistence of hexanoic acid and residual ethyl hexanoate enhances the volatility and olfactory intensity of aroma compounds, promoting the accumulation of fruity aroma characteristics and playing a key role in shaping the characteristic aroma profile of bee pollen fermentation products. Therefore, the saliva-based fermentation of bee pollen with Lactobacillus XIF24 according to this invention can enhance the flavor of bee pollen.

[0064] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A strain of *Lactobacillus salivarius* ( Ligilactobacillus salivarius XIF24, with accession number CCTCC NO: M 20252062.

2. A culture, characterized in that, The culture was obtained by culturing *Lactobacillus saliva-associated* XIF24 as described in claim 1.

3. A microbial inoculant, characterized in that, The microbial agent comprises the Lactobacillus saliva-associated XIF24 of claim 1 or the culture of claim 2.

4. The use of the Lactobacillus salivae XIF24 of claim 1, the culture of claim 2, or the microbial agent of claim 3 in at least one of the following: 1) fermenting bee pollen; 2) enhancing the flavor of bee pollen; 3) preparing fermented food.

5. A method for enhancing the flavor of bee pollen, characterized in that, Bee pollen is fermented using the Lactobacillus saliva-associated XIF24 as described in claim 1, the culture as described in claim 2, or the microbial agent as described in claim 3.

6. The method according to claim 5, characterized in that, Fermentation also includes the addition of glucose; And / or, the bee pollen is selected from one or more of rapeseed pollen, corn pollen, sunflower pollen, milkvetch pollen, buckwheat pollen, sesame pollen, and sorghum pollen.

7. The method according to claim 6, characterized in that, The mass ratio of bee pollen to glucose is (0.5-2):

1.

8. The method according to claim 5, characterized in that, The fermentation temperature is 32–40°C; And / or, the fermentation time is 7 to 30 hours.

9. Bee pollen obtained according to the method of any one of claims 5-8.

10. The use of bee pollen as described in claim 9 in the preparation of functional bee pollen products.

Images