Limosilactobacillus fermentum producing exopolysaccharide and application thereof in hemp seed slurry fermented yogurt

Abstract

The present application relates to the technical field of microbial application, and in particular to Limosilactobacillus fermentum producing exopolysaccharide and application thereof in hemp seed slurry fermented yogurt. The microbial classification and naming of the Limosilactobacillus fermentum is Limosilactobacillus fermentum GXBMR4, and the preservation number is GDMCC No: 64791. The exopolysaccharide yield of the strain GXBMR4 reaches 288.33 mg / L, the viscosity of the fermentation broth reaches 40.66 mPa·s, and the strain GXBMR4 has safety characteristics such as bile salt tolerance, no hemolyticity, and high antibiotic sensitivity. In addition, the strain GXBMR4 can effectively solve the problems of unstable system and unique "fishy smell" existing in the hemp protein system in the development of hemp protein fermented food. Not only does it promote the development and utilization of hemp protein, but also has high industrial application value in the production of fermented food, and provides a new idea for the development of other plant proteins.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, and in particular to a Limoxicillin-producing Lactobacillus fermentum and its application in fermented yogurt made from hemp seed pulp. Background Technology

[0002] Extracellular polysaccharides (EPS) are capsular sugars (CPS) or mucopolysaccharides (SPS) secreted by bacteria and microalgae during their growth and metabolism. Based on their monosaccharide composition, they can be divided into homopolysaccharides (HoPS) composed of the same monosaccharide and heteropolysaccharides (HePS) composed of repeating units containing two or more different monosaccharides. Mucopolysaccharides are loosely structured polysaccharide components produced by lactic acid bacteria that easily enter the culture medium, while capsular polysaccharides are polysaccharides that adhere to the cell surface by forming an outer membrane with the cell wall through hydrogen bonds and non-covalent bonds. Currently, most known lactic acid bacteria that produce extracellular polysaccharides can produce mucopolysaccharides, and some can produce both mucopolysaccharides and capsular polysaccharides simultaneously. Lactic acid bacteria extracellular polysaccharides are non-toxic to the human body and are from safe and reliable sources. Furthermore, they can adsorb multiple milk proteins, forming electrostatic complexes that lead to protein aggregation and precipitation, resulting in stable milk gels. Therefore, they can replace exogenous thickeners to improve the texture and flavor of dairy products, and are widely used to enhance the physicochemical properties of various fermented foods. The application of lactic acid bacteria extracellular polysaccharides in food has, to some extent, replaced or reduced the amount of thickeners and stabilizers used, while also lowering production costs. Therefore, the development and utilization of lactic acid bacteria that produce extracellular polysaccharides has significant market potential.

[0003] Bama's unique natural environment has a profound impact on its flora and fauna, giving it a distinctive regional character. Hemp, a key specialty resource of Bama, has been shown in studies to possess antioxidant, antihypertensive, immune-regulating, cholesterol-lowering, and blood sugar-lowering properties. In particular, hemp protein is a high-quality plant protein with various beneficial physiological effects on human health. However, as a plant protein, hemp protein also suffers from poor processing characteristics, instability, and a distinctive "fishy" odor. This results in most hemp seed products being primary processed products, failing to fully realize its value and significantly hindering the development and utilization of hemp protein and the industrialization of hemp. Summary of the Invention

[0004] In view of this, the present invention proposes an exopolysaccharide-producing *Lactobacillus limnosus* and its application in fermented yogurt made from hemp seed pulp. This invention isolates an exopolysaccharide-producing lactic acid bacterium from naturally fermented foods in Bama and uses it to develop fermented foods containing hemp protein, thereby solving the problems of instability and a unique "fishy" taste inherent in hemp protein systems. This is of great significance for promoting the development and utilization of plant proteins such as hemp protein, especially for the development and utilization of Bama's unique resources.

[0005] The technical solution of this invention is implemented as follows:

[0006] In a first aspect, the present invention provides a Limosilactobacillus fermentum that produces extracellular polysaccharides, which is microbially classified as Limosilactobacillus fermentum GXBMR4 and has the accession number GDMCC No: 64791.

[0007] Furthermore, the *Limoxicillin-fermenting* bacterium possesses bile salt resistance; it inhibits *Escherichia coli*, *Staphylococcus aureus*, and *Salmonella typhimurium*; it is non-hemolytic; and it exhibits high antibiotic sensitivity.

[0008] Secondly, the present invention provides the application of the aforementioned *Lactobacillus limnosus* in the production of hemp protein fermented products.

[0009] Thirdly, the present invention provides the application of the aforementioned *Lactobacillus limoxifen* in the preparation of hemp seed paste fermented yogurt, wherein the yogurt is fermented using a mixed starter culture containing *Lactobacillus limoxifen* and *Lactobacillus bulgaricus*.

[0010] In some preferred embodiments, the inoculum ratio (volume percentage) of *Lactobacillus limnosus* and *Lactobacillus bulgaricus* is 1:1.

[0011] Fourthly, the present invention provides a bacterial culture containing the aforementioned *Lactobacillus limnoxicos*.

[0012] Fifthly, the present invention provides a fermented food, the preparation method of which includes contacting the Limoxicillin-fed Lactobacillus fermentum with a fermentation substrate.

[0013] In a sixth aspect, the present invention provides a method for extracting extracellular polysaccharides by fermentation, comprising the following steps: activating the Lactobacillus limnoxicamis, obtaining a seed culture after culturing, inoculating it into a fermentation medium at a volume ratio of 3-5% (v / v), culturing it at 32-37℃ for 18-30 h to obtain a fermentation broth, and extracting extracellular polysaccharides from the fermentation broth.

[0014] In some preferred embodiments, the *Lactobacillus limnoxicamis* is activated, cultured to obtain a seed culture, inoculated into a fermentation medium at a volume ratio of 4%, and cultured at 36.7°C for 24 hours to obtain a fermentation broth. The extracellular polysaccharides in the fermentation broth are then extracted.

[0015] In some preferred embodiments, the fermentation medium contains sucrose, yeast extract, dipotassium hydrogen phosphate, ammonium citrate, sodium acetate, magnesium sulfate, manganese sulfate, and Tween.

[0016] In some preferred embodiments, the fermentation medium contains 35-55 g / L sucrose, 35-55 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L ammonium citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1.08 g / L Tween-80; the initial pH of the medium is 6.0.

[0017] In some preferred embodiments, the fermentation medium contains 55 g / L sucrose, 50 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 2 g / L ammonium citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, and 1.08 g / L Tween-80; the initial pH of the medium is 6.0.

[0018] In a seventh aspect, the present invention provides the application of extracellular polysaccharides extracted according to the fermentation extraction method described in any of the preceding claims in the preparation of hemp seed paste.

[0019] The beneficial effects of the present invention include at least the following:

[0020] (1) The extracellular polysaccharide yield and viscosity of the Limoxi fermenting Lactobacillus GXBMR4 provided by the present invention are high, with an extracellular polysaccharide yield of 288.33 mg / L and a fermentation broth viscosity of 40.66 mPa·s. It has high industrial application value in the production of fermented foods.

[0021] (2) The Limoxicillin-fermenting Lactobacillus GXBMR4 provided by this invention is a natural lactic acid bacterium with high safety. This strain is resistant to bile salts, does not have hemolytic activity, and has high antibiotic sensitivity. These characteristics ensure that its application in the food industry is safe and reliable.

[0022] (3) The Limoxicillin GXBMR4 and Lactobacillus bulgaricus provided by this invention can be used together to ferment hemp seed pulp into yogurt, which can reduce the whey separation rate of yogurt, increase the viscosity and water holding capacity of yogurt, give the product a better texture, and is conducive to the development of yogurt products.

[0023] (4) The Limoxicillin fermenting Lactobacillus GXBMR4 provided by this invention can effectively solve the problems of system instability and unique "fishy smell" of hemp protein system in the development of hemp protein fermented food; it not only promotes the development and utilization of hemp protein, but also provides new ideas for the development of other plant proteins.

[0024] Biological preservation of microbial strains

[0025] The Limosilactobacillus fermentum GXBMR4 strain provided by this invention was deposited at the Guangdong Microbial Culture Collection Center on June 24, 2024, at the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCCNo:64791. This strain was received and registered by the collection center on June 24, 2024. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The colony morphology (Fig. a) and cell morphology (Fig. b) of the strain GXBMR4 of the present invention are shown.

[0028] Figure 2 The effects of different types and amounts of carbon sources on the growth, extracellular polysaccharide production, and fermentation broth viscosity of strain GXBMR4 were investigated.

[0029] Figure 3 The effects of different nitrogen source types and amounts on the growth, extracellular polysaccharide production, and fermentation broth viscosity of strain GXBMR4 were investigated.

[0030] Figure 4 The effects of different inoculum amounts and initial pH on the growth, extracellular polysaccharide yield, and fermentation broth viscosity of strain GXBMR4 were investigated.

[0031] Figure 5 The effects of different culture temperatures and culture times on the growth, extracellular polysaccharide yield, and fermentation broth viscosity of strain GXBMR4 were investigated.

[0032] Figure 6 The effect of different amounts of exopolysaccharides added at different pH values ​​on the stability of the hemp seed paste system (the stability of the system was evaluated using different quantitative indicators AE); where A is the centrifugal sedimentation rate, B is the Zeta potential, C is the viscosity, D is the particle size distribution, and E is the evaluation particle size.

[0033] Figure 7 The results show the pH and viscosity of different strains in the fermentation broth of hemp seed pulp; where "R4" represents GXBMR4; "Bulgaria" represents Lactobacillus bulgaricus; "Thermophilic" represents Streptococcus thermophilus; and "Bifidobacterium" represents Bifidobacterium lactis; different letters indicate significant differences, p<0.05;

[0034] Figure 8 The fermentation effects of different strains in hemp seed paste are shown from left to right as follows: GXBMR4, GXBMR4+Streptococcus thermophilus, Streptococcus thermophilus, Bifidobacterium lactis, GXBMR4+Bifidobacterium lactis, GXBMR4+Lactobacillus bulgaricus, and Lactobacillus bulgaricus.

[0035] Figure 9 The results of pH and viscosity tests on different strains in the fermentation broth of whole milk powder;

[0036] Figure 10 The results of whey separation rate and water-holding capacity of different strains in whole milk powder fermentation broth;

[0037] Figure 11 The text describes the texture of fermented yogurt from different bacterial strains; where "R4" represents GXBMR4; "Bulgaria" represents Lactobacillus bulgaricus; "Thermophilic" represents Streptococcus thermophilus; and "Bifidobacterium" represents Bifidobacterium lactis. Different letters indicate significant differences, p<0.05. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Reagents not specifically described in detail herein are all conventional reagents and are commercially available; methods not specifically described in detail are all conventional experimental methods and can be learned from the prior art.

[0039] 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 limit the invention.

[0040] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0041] Culture medium formulations or sources in the examples

[0042] MRS medium: 10g peptone, 5g beef meal, 4g yeast powder, 20g glucose, 1mL Tween 80, 2g K2HPO4·7H2O, 5g sodium acetate·3H2O, 2g triammonium citrate, 0.2g MgSO4·7H2O, 0.05g MnSO4·4H2O, 15g agar, 1L distilled water. Adjust pH to 6.2 and autoclave at 121℃ for 20min.

[0043] MRS broth culture medium: 10g casein digest, 10g beef extract, 4g yeast extract, 2g triammonium citrate, 5g sodium acetate, 0.2g MgSO4·7H2O, 0.05g MnSO4·4H2O, 2g K2HPO4·7H2O, 20g glucose, 1.08g Tween-80, 1L distilled water. Adjust pH to 5.7 and autoclave at 121℃ for 20min.

[0044] MRS basal medium: yeast extract 20 g / L, glucose 20 g / L, dipotassium hydrogen phosphate 2 g / L, triammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween-80 1.08 g / L, adjust pH to 6.0, sterilize at 121℃ for 15 min.

[0045] Blood agar plates were purchased from Beijing Luqiao Technology Co., Ltd.

[0046] Example 1

[0047] Isolation and purification of bacterial strains

[0048] During the research process, the inventors used the dilution plating method to isolate a strain numbered GXBMR4 from a naturally fermented kimchi sample purchased from a farmers' market in Bama Yao Autonomous County, Hechi City, Guangxi Zhuang Autonomous Region, China.

[0049] Physicochemical properties of the strain

[0050] (1) The Gram staining result of strain GXBMR4 was positive, and the catalase test result was negative.

[0051] (2) This strain has strong bile salt tolerance, with growth lag periods of 0.65 h and 2.50 h in environments with bile salt concentrations (m / v) of 0.03% and 0.05%, respectively.

[0052] (3) Based on the criteria for determining the diameter of the inhibition zone of the Clinica Land Laboratory Standards Institute (CLSI), strain GXBMR4 showed extremely high sensitivity to eight common antibiotics (tetracycline, gentamicin, erythromycin, penicillin, polymyxin B, ampicillin, cefazolin and lincomycin).

[0053] (4) The inhibition zones of strain GXBMR4 against Escherichia coli, Staphylococcus aureus, and Salmonella were all greater than 6 mm, indicating a strong inhibitory effect on the three pathogenic bacteria. The experimental strains were sourced from the China General Microbiological Culture Collection Center: Staphylococcus aureus (CGMCC14519), Escherichia coli (CGMCC112252), and Salmonella (CGMCC11190).

[0054] (5) After streaking the strain onto a blood agar plate and incubating at 37°C for 2 days, no green or transparent areas appeared around the colony, indicating γ-hemolysis, which means the test strain has no hemolytic ability.

[0055] Identification of bacterial strains

[0056] (1) Morphological characteristics

[0057] The colony morphology and cell morphology of strain GXBMR4 are as follows: Figure 1 As shown, the colonies are oval or round, milky white, raised, moist and glossy, with neat edges, which conforms to the morphological characteristics of lactic acid bacteria. The bacteria are rod-shaped, scattered and not connected.

[0058] (2) 16S rDNA gene sequencing

[0059] Extracellular polysaccharide-producing lactic acid bacteria were activated twice in MRS broth (i.e., cultured twice in liquid medium). The activated strains were streaked onto MRS medium and incubated at 37°C for 24 hours. The 16S rDNA gene was amplified by PCR using universal primers 27F-1492R. The forward primer was 27F (sequence: TACGYTACCTTGTTACGACTT), and the reverse primer was 1492R (sequence: AGAGTTTGATCMTGGCTCAG). The PCR product was sequenced at Nanning Guotuo Biotechnology Co., Ltd., and the sequencing results are shown in SEQ ID NO: 1. The sequencing results were compared and analyzed using the Basic Local Alignment Search (BLAST) tool in the EzBioCloud and NCBI databases. The sequence of the type strain with high homology was selected as a reference, and a phylogenetic tree of the strain was constructed using MEGA 7.0. The strain GXBMR4 has the highest similarity of 99.21% to the existing published species Limosilactobacillusfermentum strain CIP 102980 in the Lactobacillus family, and is on the same branch as Limosilactobacillus fermentum in the phylogenetic tree, therefore it belongs to the genus Limosilactobacillus fermentum.

[0060] (3) Preservation of microbial strains

[0061] The Limosilactobacillus fermentum GXBMR4 strain provided by this invention was deposited at the Guangdong Microbial Culture Collection Center on June 24, 2024, at the address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCCNo:64791. This strain was received and registered by the collection center on June 24, 2024.

[0062] Example 2: Extraction and determination of extracellular polysaccharides

[0063] (1) Extraction of extracellular polysaccharides

[0064] The *Lactobacillus limnoxicamis* GXBMR4 was prepared into a bacterial suspension and the absorbance value (OD) was adjusted. 600nmThe inoculum was 0.8 ± 0.1, and 2% (v / v) was inoculated into MRS broth medium and cultured at 37℃ for 24 h to obtain the fermentation broth. The broth was incubated in a 100℃ water bath for 20 min to inactivate any enzymes that might degrade polysaccharides. The supernatant was collected by centrifuging the fermentation broth at 7000 r / min, 4℃ for 20 min after the boiling water bath. Trichloroacetic acid solution with a mass fraction of 800 g / L was added to a final mass fraction of 40 g / L. The mixture was allowed to stand at 4℃ for 12 h, then centrifuged at 7000 r / min, 4℃ for 20 min to remove cells and proteins. The supernatant was collected, and three volumes of 95% ethanol were added. The mixture was allowed to stand at 4℃ for 12 h, then centrifuged at 7000 r / min, 4℃ for 20 min. The supernatant was discarded, and the precipitate was reconstituted with deionized water and placed in a dialysis bag (molecular weight cutoff of 14 kDa) for dialysis for 48 h, changing the water every 8 h. The dialysate was refrigerated for later use.

[0065] (2) Determination of extracellular polysaccharide production

[0066] Pipette 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of glucose standard solution (40 μg / mL) into graduated test tubes, respectively, and add water to a final volume of 1.0 mL. Then add 0.6 mL of 60 g / L phenol solution and 3.0 mL of concentrated sulfuric acid, shake well, heat in an 80°C water bath for 15 min, and rapidly cool to room temperature. Using the zero tube as a blank, measure the absorbance at a wavelength of 490 nm to plot a glucose standard curve and obtain the standard curve regression equation. Accurately measure 1.0 mL of dialysate and follow the same steps, measure the absorbance, and substitute it into the regression equation to calculate the extracellular polysaccharide yield in the sample.

[0067] (3) Determination of extracellular polysaccharide properties

[0068] Accurately weigh an appropriate amount of the extracted extracellular polysaccharide, prepare a 10 mg / mL polysaccharide solution with deionized water, and determine the apparent viscosity of the extracellular polysaccharide using a rheometer.

[0069] Measurement results The extracellular polysaccharide yield of strain GXBMR4 was 95.73±4.37 mg / L, and the viscosity of the extracellular polysaccharide was 4.88±0.1 mPa·s.

[0070] Example 3: Optimization of fermentation conditions for extracting extracellular polysaccharides

[0071] The fermentation conditions of strain GXBMR4 were investigated using single-factor experiments combined with response surface methodology to explore the effects of nitrogen source type and concentration, carbon source type and concentration, pH, culture time, culture temperature, and inoculum size on the production of extracellular polysaccharides, cell growth, and fermentation broth viscosity. Based on the single-factor experiments, the fermentation conditions were optimized using response surface methodology. Plackett-Burman and Box-Behnken designs were employed, and the data were processed using Design-Expert 13 software to establish and analyze mathematical models relating each factor to extracellular polysaccharide yield.

[0072] Fermentation conditions settings

[0073] Fermentation medium optimization: Based on the MRS basal medium, the carbon source in the medium was replaced with lactose, sucrose, fructose, and glucose (concentration 20 g / L), respectively; the carbon source with the highest extracellular polysaccharide yield was selected, and concentrations (5, 15, 25, 35, 45, 55 g / L) were prepared. Based on the MRS basal medium and its optimized carbon source, the nitrogen source in the medium was replaced with different nitrogen sources (20 g / L each of soybean peptone, tryptone, casein peptone, peptone, yeast extract, hemp protein, and skim milk powder); the nitrogen source with the highest extracellular polysaccharide yield was selected, and fermentation mediums with different concentrations (5, 10, 15, 20, 25, 30 g / L) were prepared, the pH was adjusted to 6.0, sterilized at 121℃ for 15 min, and after cooling, the inoculum was inoculated at a rate of 2% (V / V) (OD of inoculum suspension). 600nm The value was 0.8, and the mixture was incubated at 37℃ for 24 hours.

[0074] Optimization of inoculum size, initial pH, culture temperature, and culture time: Based on the optimized carbon and nitrogen sources, 50 mL of liquid culture medium was prepared, and the bacterial suspension (OD) was prepared at different inoculum sizes (1%, 2%, 3%, 4%, 5% v / v). 600nm The culture medium (value 0.8) was inoculated into sterilized, optimized fermentation medium. Based on optimized carbon source, nitrogen source, and inoculum size, 50 mL of liquid culture medium was prepared with initial pH values ​​of 4.5, 5.0, 5.5, 6.0, 6.5, and 7.0. Based on optimized carbon source, nitrogen source, inoculum size, and initial pH, 50 mL of liquid culture medium was prepared, and different incubation temperatures (28℃, 31℃, 34℃, 37℃, 40℃, and 43℃) were set. Based on optimized carbon source, nitrogen source, inoculum size, initial pH, and incubation temperature, 50 mL of liquid culture medium was prepared, and different incubation times (6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, and 48 h) were set.

[0075] Measurement methods

[0076] Fermentation broth OD 600 Assay: Using uninoculated culture medium as a blank, 250 μL of the mixed fermentation broth was transferred to a 96-well plate, and the absorbance was measured at 600 nm using an ELISA reader.

[0077] Determination of extracellular polysaccharide content in fermentation broth: The fermentation broth cultured to the endpoint was boiled in a water bath for 20 min, then transferred to a centrifuge tube and centrifuged at 7000 r / min for 20 min. The supernatant was collected, and 800 g / L trichloroacetic acid solution was added to a mass fraction of 40 g / L. After mixing, the mixture was allowed to stand at 4℃ for 12 h, centrifuged at 7000 r / min for 20 min, and the supernatant was collected. An appropriate amount of the supernatant was placed in a dialysis bag (molecular weight cutoff of 14 kDa) and dialyzed for 48 h to remove small molecule impurities such as monosaccharides and pigments. The extracellular polysaccharide content of the fermentation broth was determined according to the extracellular polysaccharide yield determination method (sulfuric acid-phenol method) in Example 2.

[0078] Fermentation broth viscosity determination: The viscosity of the fermentation broth (mPa·s) was determined using a rapid viscosity analyzer. The amount of hemp seed paste added was 25 mL, the measurement temperature was 25℃, and the viscosity was measured at a rotation speed of 80 r / min.

[0079] Experimental results

[0080] (1) Optimization of carbon source type and concentration (amount added): such as Figure 2 As shown, different carbon sources affect the production of extracellular polysaccharides by the strain. Although the strain can grow well with glucose, lactose, and sucrose as carbon sources, sucrose is more conducive to the production of extracellular polysaccharides by strain GXBMR4. Changing the carbon source affects the viscosity of the fermentation broth, with sucrose being more effective in increasing the viscosity. As the amount of sucrose added increases, the growth of the strain also increases. When the sucrose addition exceeds 45 g / L, the growth of the strain is inhibited, and the growth rate decreases. There is a certain correlation between the extracellular polysaccharide content and the viscosity of the fermentation broth; both increase with the addition of sucrose. When the sucrose addition exceeds 45 g / L, the production of extracellular polysaccharides decreases due to the inhibition of strain growth. The continued increase in viscosity at this point is due to excessive sucrose addition, which the strain cannot fully utilize. A sucrose addition of 45 g / L is most conducive to the growth of the strain, and the extracellular polysaccharide production is the highest. A sucrose addition of 45 g / L was selected for further optimization.

[0081] (2) Optimization of nitrogen source type and concentration: such as Figure 3As shown, different nitrogen sources have varying effects on the growth, extracellular polysaccharide content, and fermentation broth viscosity of strain GXBMR4. Yeast extract is most beneficial for strain growth, extracellular polysaccharide production, and increasing fermentation broth viscosity. There is a certain correlation between extracellular polysaccharide production and strain growth status; better strain growth is more conducive to extracellular polysaccharide production. However, there is no perfect correlation between fermentation broth viscosity and extracellular polysaccharide yield, as fermentation broth viscosity is a result of factors such as extracellular polysaccharide content, nitrogen source type and content, and strain yield. Different amounts of yeast extract added had varying effects on the growth, extracellular polysaccharide production, and fermentation broth viscosity of strain GXBMR4. Strain growth increased with increasing yeast extract concentration, reaching a maximum at 45 g / L; concentrations exceeding this level inhibited growth. Extracellular polysaccharide production correlated with strain growth, increasing with increasing growth; however, concentrations above 45 g / L inhibited growth and reduced polysaccharide production. Fermentation broth viscosity correlated with polysaccharide content, increasing with increasing polysaccharide content; however, even at concentrations above 45 g / L, viscosity continued to increase due to the excess yeast extract. Microbial growth requires a limited amount of nitrogen; both excessively low and high concentrations are detrimental to growth. A yeast extract concentration of 45 g / L was selected for further optimization.

[0082] (3) Optimization of inoculum size and initial pH: such as Figure 4 As shown, different inoculum amounts did not significantly affect the growth of strain GXBMR4, but they did significantly affect the extracellular polysaccharide yield and fermentation broth viscosity. Extracellular polysaccharide yield increased with increasing inoculum amount, reaching a maximum at 4% (v / v). Above this value, extracellular polysaccharide yield decreased with further increases in inoculum amount. Fermentation broth viscosity showed a certain correlation with polysaccharide content, reaching a maximum at approximately 3% inoculum amount. Above 4%, viscosity decreased with decreasing polysaccharide content. A 4% inoculum amount was selected for further optimization. Different pH values ​​had varying effects on the growth, extracellular polysaccharide yield, and fermentation broth viscosity of strain GXBMR4. The strain's growth increased with increasing pH, reaching its maximum at pH 6.0; pH values ​​above 6.0 inhibited growth. Extracellular polysaccharide yield correlated with growth, increasing with pH; however, growth was inhibited and polysaccharide yield decreased at pH values ​​above 6.0. Fermentation broth viscosity also correlated with extracellular polysaccharide content, with the highest viscosity observed at pH 6.0. pH 6.0 was selected for further optimization.

[0083] (4) Optimization of culture temperature and culture time: such as Figure 5As shown, the growth rate of the strain increased with increasing culture temperature, reaching a maximum at 37℃. Above 37℃, the growth rate decreased with further increases in culture temperature. There was a correlation between extracellular polysaccharide content and strain growth rate; the content increased with increasing growth rate, but strain growth was inhibited and extracellular polysaccharide production decreased at 40℃. A correlation also existed between fermentation broth viscosity and extracellular polysaccharide content; both were highest at 37℃. Therefore, a culture temperature of 37℃ was selected for further optimization. The bacterial growth rate increased with increasing culture time, reaching a maximum at 30 hours. Extracellular polysaccharide content correlated with bacterial growth rate, increasing with culture time, reaching a maximum at 24 hours and then remaining relatively stable. Fermentation broth viscosity also correlated with extracellular polysaccharide content, increasing with polysaccharide content, reaching a maximum at 24 hours, and then slightly decreasing at 30 hours without significant further change. This slight decrease in viscosity is due to the depletion of the fermentation substrate, indicating that bacterial growth and polysaccharide production had ceased to increase. Therefore, a culture time of 24 hours was chosen.

[0084] (5) Finally, after optimization using Design-Expert 13 software, the optimal fermentation conditions for producing extracellular polysaccharides were obtained as follows: sucrose addition 55 g / L, yeast extract addition 50 g / L, dipotassium hydrogen phosphate 2 g / L, ammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween-80 1.08 g / L, initial pH of the culture medium 6.0, inoculum size 4% (v / v), culture temperature 36.7℃, and culture time 24 h. Under these conditions, the predicted yield of extracellular polysaccharides was 290.86 mg / L. Using the above conditions as the optimal fermentation conditions, three replicate experiments were conducted for verification. The actual yield of extracellular polysaccharides was measured to be 288.33 ± 12.58 mg / L. The absolute relative error between the actual and predicted values ​​was 0.88% (<0.05), indicating that the optimized fermentation conditions for producing extracellular polysaccharides were basically accurate and reliable.

[0085] Under optimized fermentation conditions, the extracellular polysaccharide yield of strain GXBMR4 was 288.33 mg / L, and the viscosity of the fermentation broth reached 40.66 mPa·s, which were 3 times and 2 times higher than before optimization, respectively.

[0086] Example 4: Application of extracellular polysaccharides from strain GXBMR4 in the preparation of hemp seed paste

[0087] (1) Steps

[0088] Weigh out clean and whole shelled hemp seeds (purchased locally in Bama), soak them in 3 times their volume of 30°C pure water for 3.5 hours, rinse them twice with pure water and drain them, grind them into a pulp at a material-to-liquid ratio of 1:33, filter the pulp through a 500-mesh filter cloth, and the filtrate is the hemp seed pulp.

[0089] The strain GXBMR4 was cultured using the optimized culture medium in Example 3, and crude polysaccharide was extracted according to the method in Example 2. The polysaccharide was then placed in a dry, sealed container and stored at room temperature for later use.

[0090] The pH of the hemp seed slurry was adjusted to 3.5, 4.0, 4.5, and 5.0 using 1 mol / L HCl and NaOH. Different masses of extracellular polysaccharides were added to the hemp seed slurry at different pH values ​​to achieve concentrations of 0.05 mg / mL, 0.10 mg / mL, 0.15 mg / mL, 0.20 mg / mL, and 0.25 mg / mL, respectively. The mixtures were thoroughly mixed. Hemp seed slurry without added extracellular polysaccharides was used as a blank control. The centrifugal sedimentation rate, Zeta potential, viscosity, particle size distribution, and particle size of the system under different conditions were measured. (The amount of extracellular polysaccharides required to significantly change the stability indicators of the hemp seed slurry system at pH 3.5 was determined by increasing the maximum amount added from 0.25 mg / mL to 0.35 mg / mL.)

[0091] (2) Measurement method

[0092] Centrifugation sedimentation rate determination: Take a certain amount of the mixed sample into a centrifuge tube, centrifuge at 7000 r / min for 20 min, remove the floating oil layer and clear liquid, invert the centrifuge tube to drain the water, accurately weigh the mass of the sediment, and calculate the centrifugation sedimentation rate.

[0093] Zeta potential determination: Before the determination, dilute the emulsion with deionized water to a suitable concentration, take an appropriate amount of sample in a potentiometric cuvette, and measure it with a potentiometer. The refractive index of the substance is 1.46, the refractive index of the medium is 1.333, the measurement temperature is 25℃, and the equilibration time is 1 min.

[0094] Particle size and distribution determination: The particle size and distribution of the sample were determined using a particle size analyzer. The refractive index of the substance was 1.46, the refractive index of the medium was 1.333, the measurement temperature was 25℃, and the shading rate was 8% to 15%.

[0095] Viscosity determination: The viscosity of the fermentation broth (mPa·s) was determined using a rapid viscosity analyzer. The amount of hemp seed paste added was 25 mL, the measurement temperature was 25℃, and the viscosity was measured at a rotation speed of 80 r / min.

[0096] (3) Results: The test results are as follows Figure 6 As shown.

[0097] (4) Conclusion

[0098] The extracellular polysaccharides of strain GXBMR4 can improve the instability of hemp seed paste system caused by pH decrease. At constant pH, within a certain range of extracellular polysaccharide concentration, with increasing extracellular polysaccharide addition, the absolute value of the zeta potential gradually increases, the electrostatic repulsion between particles gradually strengthens, particles are less prone to aggregation, particle dispersibility is better, the average particle size gradually decreases, the system becomes more stable, and the centrifugal sedimentation rate gradually decreases. When pH changes, the effect of extracellular polysaccharides in improving system stability is weakened with decreasing pH; therefore, increasing the amount of extracellular polysaccharides added is necessary to significantly improve system stability.

[0099] Therefore, the extracellular polysaccharide of Lactobacillus limnosus GXBMR4 provided by this invention can not only increase the viscosity of the hemp seed paste system, but also increase the absolute value of the zeta potential of the system, reduce the average particle size, improve particle dispersibility, and make the system more stable.

[0100] Example 5: Application of strain GXBMR4 in hemp seed pulp fermentation and yogurt fermentation

[0101] In this embodiment, Lactobacillus bulgaricus, Streptococcus thermophilus, and Bifidobacterium lactis were all purchased from Beijing Chuanxiu Technology Co., Ltd.

[0102] (1) Preparation of hemp seed paste

[0103] Weigh a certain amount of dehulled hemp seeds and soak them in 3 times their volume of 30℃ pure water for 3.5 hours. After washing and draining twice, mix the dehulled hemp seeds with water and grind them into a slurry (material-to-liquid ratio of 1:33). Perform coarse grinding to form whole grains, followed by two fine grindings. Filter the slurry through a 500-mesh filter cloth to obtain hemp seed slurry.

[0104] (2) Fermentation effect in hemp seed paste

[0105] The hemp seed paste prepared in step (1) was sterilized at 80℃ for 30 min. It was then inoculated with strains GXBMR4, Streptococcus thermophilus, Lactobacillus bulgaricus, Bifidobacterium lactis, GXBMR4 + Streptococcus thermophilus (inoculation ratio 1:1), GXBMR4 + Lactobacillus bulgaricus (1:1), and R4 + Bifidobacterium lactis (1:1) at a constant temperature of 37℃. After fermentation, the pH, coagulation time, whey separation rate, water holding capacity, viscosity, texture, and flavor of the fermentation broth were evaluated to determine the suitable strains.

[0106] (3) Fermentation effect in whole milk powder emulsion

[0107] Prepare whole milk powder emulsion (whole milk powder dissolved in water, with a protein content of 4.0%), sterilize at 80℃ for 30 min, and follow the same steps as step (2) for inoculation and fermentation effect detection.

[0108] (4) Detection method

[0109] pH determination: After the sample is cooled to room temperature, it is stirred evenly with a glass rod, and the pH value is measured directly with a pH meter.

[0110] Curdling time determination: After the emulsion has fermented for a certain period of time, the liquid in the emulsion turns into a solid state. When the container is tilted, the emulsion no longer flows. The fermentation time at this point is the curdling time.

[0111] Viscosity determination: After the refrigerated sample was taken out and allowed to stand at room temperature, it was stirred evenly and the viscosity of the fermentation broth (mPa·s) was measured using a rapid viscosity analyzer. The sample addition amount was 25 mL, and the viscosity of the fermentation broth was measured at a temperature of 25℃ and a rotation speed of 80 r / min.

[0112] Whey separation rate determination: Take the sample out of the 4℃ refrigerator and weigh it, place it at room temperature at an angle for 1 hour, discard the whey and weigh it again, and calculate the whey separation rate according to the following formula.

[0113]

[0114] In the formula: m0 is the total weight of the yogurt cup and the fermented milk; m1 is the mass of the yogurt cup; m2 is the total weight remaining after removing the whey.

[0115] Water-holding capacity determination: Take an appropriate amount of fermented milk sample and weigh it, put it into a centrifuge tube, centrifuge at 6000 r / min for 10 min, remove the supernatant and weigh it, and calculate the water-holding capacity (WHC) according to the following formula.

[0116]

[0117] In the formula: m4 is the mass of the precipitate after centrifugation, g; m3 is the mass of the yogurt sample, g.

[0118] Sensory evaluation: The fermented sample was taken out and cooled to room temperature. An evaluation team of ten food professionals evaluated the sample from four aspects: color, texture, flavor, and taste, in accordance with the sensory evaluation standard of GB19302-2010 for fermented milk.

[0119] All experiments were conducted in triplicate. Experimental data were analyzed using SPSS statistical analysis.

[0120] (5) Detection results of fermentation of different strains in hemp seed paste

[0121] The pH value and viscosity of the fermentation broth after fermentation of hemp seed pulp by different strains are as follows: Figure 7As shown, the pH value of the fermentation broth was significantly lower when strain GXBMR4 was used in combination with *Lactobacillus bulgaricus* or *Bifidobacterium lactis* compared to when strain GXBMR4 was used alone. This indicates that the combined use of strains can compensate for the insufficient acid-producing capacity of strain GXBMR4 in hemp seed pulp. Therefore, compared to using strain GXBMR4, *Lactobacillus bulgaricus*, *Streptococcus thermophilus*, or *Bifidobacterium lactis* alone, using strain GXBMR4 in combination with these strains can increase the viscosity of the fermentation broth.

[0122] The effects of different strains on hemp seed paste fermentation for 20 hours are as follows: Figure 8 As shown, none of the fermentation broths from any of the strains could form a stable emulsion, nor could they coagulate to form a viscous or coagulated consistency similar to yogurt. All exhibited severe fat bubbling and a small amount of sediment. This is because the protein content of the hemp seed slurry (approximately 0.61%) is very low. The fermentation process causes the pH of the system to continuously decrease, but it cannot form a yogurt-like coagulated consistency. This disrupts the system's stability, leading to fat bubbling and sedimentation.

[0123] As shown in Table 1, regardless of whether GXBMR4, Streptococcus thermophilus, Bifidobacterium lactis, or GXBMR4 combined with Streptococcus thermophilus or Bifidobacterium lactis fermented hemp seed pulp alone, the fermented pulp had a distinct sour and off-flavor, resulting in poor flavor. However, when Lactobacillus bulgaricus alone or Lactobacillus bulgaricus combined with GXBMR4 fermented hemp seed pulp, although the fermented pulp still had a sour and off-flavor, it was not prominent and there was no off-flavor. This indicates that Lactobacillus bulgaricus can improve the flavor defects of GXBMR4-fermented hemp seed pulp.

[0124] Table 1. Tissue state and flavor of fermentation samples from different strains

[0125] GXBMR4 The solution showed significant fat floating and a small amount of sediment. It has a strong sour and rancid smell Bulgaria The solution showed significant fat floating and a small amount of sediment. Slightly sour and rancid smell Thermophilic The solution showed significant fat floating and a small amount of sediment. It has a strong sour and rancid smell Bifid The solution showed significant fat floating and a small amount of sediment. It has a sour and rancid smell GXBMR4+ Bulgaria The solution showed significant fat floating and a small amount of sediment. Slightly sour and rancid smell GXBMR4+ thermophilic The solution showed significant fat floating and a small amount of sediment. It has a strong sour and rancid smell GXBMR4+ Bifidobacteria The solution showed significant fat floating and a small amount of sediment. It has a sour and rancid smell

[0126] (6) Detection results of fermentation of different bacterial strains in whole milk powder emulsion

[0127] Table 2 shows the coagulation time of whole milk powder emulsion fermented by different strains. Under the fermentation conditions, strain GXBMR4 had very poor coagulation ability and a coagulation time as long as 21 hours. Lactobacillus bulgaricus had the best coagulation ability. When Lactobacillus bulgaricus, Streptococcus thermophilus, or Bifidobacterium lactis were co-fermented with strain GXBMR4, the coagulation time did not change.

[0128] Table 2. Curd time of yogurt fermented with different bacterial strains

[0129] GXBMR4 21 Bulgaria 10 Thermophilic 12 Bifid 14 GXBMR4+ Bulgaria 10 GXBMR4+ thermophilic 12 GXBMR4+ Bifidobacteria 14

[0130] Depend on Figure 9It is known that microbial fermentation lowers the pH of the system. Strain GXBMR4 and Bifidobacterium lactis exhibit poor acid-producing performance in whole milk powder emulsions, while Lactobacillus bulgaricus demonstrates the best acid-producing performance. Combining strain GXBMR4 with Lactobacillus bulgaricus or Streptococcus thermophilus can significantly lower the pH of yogurt, compensating for the insufficient acid-producing performance of GXBMR4. Yogurt fermented using Lactobacillus bulgaricus or a combination of Lactobacillus bulgaricus and GXBMR4 yields the highest viscosity. Compared to using commercially available yogurt starter alone, combining it with GXBMR4 can increase the viscosity of yogurt, especially the combination of GXBMR4 with Lactobacillus bulgaricus or Streptococcus thermophilus.

[0131] The whey separation rate and water-holding capacity of yogurt obtained by fermenting whole milk powder emulsion with different strains of bacteria are as follows: Figure 10 As shown, yogurt fermented with Lactobacillus bulgaricus had the lowest whey separation rate. This is because Lactobacillus bulgaricus has outstanding acid-producing properties, enabling it to better coagulate and form a gel network structure. Fermentation of strain GXBMR4 with Lactobacillus bulgaricus, Streptococcus thermophilus, or Bifidobacterium lactis significantly reduced the whey separation rate of yogurt. The water-holding capacity of yogurt produced by fermenting Lactobacillus bulgaricus and Streptococcus thermophilus alone or separately with GXBMR4 was significantly higher than that of yogurt produced by fermenting Bifidobacterium lactis alone or in combination with GXBMR4. Moreover, compared with single-strain fermentation, fermentation of Lactobacillus bulgaricus, Streptococcus thermophilus, or Bifidobacterium lactis with GXBMR4 can significantly improve the water-holding capacity of yogurt.

[0132] Depend on Figure 11 It is known that yogurt fermented with Lactobacillus bulgaricus has a firmer, cleaner texture with no water droplets adhering to the surface, while yogurt fermented with Streptococcus thermophilus has a looser texture with a lot of water adhering to the surface. Yogurt fermented with Bifidobacterium lactis has a soft, loose texture with a large amount of water adhering to it, while yogurt fermented with strain GXBMR4 has an extremely loose texture, is easily broken, and does not clump together. Yogurt fermented with GXBMR4 in combination with Lactobacillus bulgaricus, Streptococcus thermophilus, or Bifidobacterium lactis has a firmer, finer, and more moist texture with a prominent viscosity. Fermentation of GXBMR4 in combination with other strains can compensate for the insufficient curdling ability of GXBMR4 and give the yogurt a better texture.

[0133] As shown in Table 3, the yogurt fermented with Lactobacillus bulgaricus has a better flavor than the yogurt fermented with GXBMR4, Streptococcus thermophilus, or Bifidobacterium lactis. Furthermore, the yogurt fermented with Lactobacillus bulgaricus and GXBMR4 has a significantly better flavor than the yogurt fermented with Streptococcus thermophilus or Bifidobacterium lactis and GXBMR4. The combined fermentation of Lactobacillus bulgaricus and GXBMR4 can better compensate for the poor flavor of the yogurt fermented with GXBMR4.

[0134] Table 3. Texture and flavor of yogurt obtained from fermentation by different strains

[0135] GXBMR4 The curd was quite thin, and the whey separated significantly. The flavor is not pure; the yogurt taste is weak, and there is a slight off-odor. Bulgaria The curd is firm, with a small amount of whey separating out. It has a pure and refreshing flavor with a prominent yogurt taste. Thermophilic The curd was quite firm, and whey separation was severe. The flavor is impure, the yogurt taste is weak, and the off-flavor is prominent. Bifid The curd is thick and the whey separates significantly. The flavor is not pure; the yogurt taste is weak, and there is a slight off-odor. GXBMR4+ Bulgaria The curd is firm, with a small amount of whey separating out. It has a light and fragrant aroma with a distinct yogurt flavor. GXBMR4+ thermophilic The curd is quite firm, and a significant amount of whey separates out. The flavor is impure, the yogurt taste is weak, and the off-flavor is prominent. GXBMR4+ Bifidobacteria The curd is quite firm, and a significant amount of whey separates out. The flavor is not pure; the yogurt taste is weak and there is an off-odor.

[0136] In summary, the experimental results show that strain GXBMR4 exhibits poor acid-producing and aroma-producing abilities in both hemp seed pulp and whole milk powder emulsion. Combining strain GXBMR4 with *Lactobacillus bulgaricus*, *Streptococcus thermophilus*, or *Bifidobacterium lactis* can compensate for the poor acid-producing ability of GXBMR4 in hemp seed pulp or whole milk powder emulsion. Furthermore, compared to combining with *Streptococcus thermophilus* or *Bifidobacterium lactis*, the hemp seed pulp fermentation liquid or yogurt obtained by combining strain GXBMR4 with *Lactobacillus bulgaricus* has a better flavor, thus compensating for the insufficient aroma-producing ability of GXBMR4. In addition, the combined fermentation of GXBMR4 with *Lactobacillus bulgaricus*, *Streptococcus thermophilus*, or *Bifidobacterium lactis* can reduce the whey separation rate of yogurt, increase the viscosity and water-holding capacity of yogurt, and give the yogurt a better texture. The combined effect of *Lactobacillus bulgaricus* and GXBMR4 is the most prominent compared to other strains.

[0137] Example 6: Application of strain GXBMR4 in the preparation of hemp seed fermented yogurt

[0138] (1) Preparation of hemp seed paste

[0139] Weigh a certain amount of dehulled hemp seeds and soak them in 3 times their volume of 30℃ pure water for 3.5 hours. After washing and draining twice, mix the dehulled hemp seeds with water and grind them into a slurry (material-to-liquid ratio of 1:33). Perform coarse grinding to form whole grains, followed by two fine grindings. Filter the slurry through a 500-mesh filter cloth to obtain hemp seed slurry.

[0140] (2) The hemp seed pulp and the reconstituted emulsion were mixed at a ratio of 6:4. The inoculated starter culture was strain GXBMR4 and Lactobacillus bulgaricus (1:1). A three-factor, four-level orthogonal experiment was set up with the amount of starter culture added, fermentation time, and fermentation temperature respectively. The experiment was conducted according to L9(3 4 An orthogonal experiment was conducted using an orthogonal array. Sucrose was added at 3%. The factors and levels are shown in Table 4. The pH, viscosity, whey separation rate, and water holding capacity of the product were measured and sensory evaluation was performed (measurement method is the same as in Example 5) to determine the optimal fermentation conditions.

[0141] Table 4. Factor Level Table for Orthogonal Experiment

[0142] 1 37 4 1 2 39 6 2 3 41 8 3 4 43 10 4

[0143] The experimental results showed that the yogurt prepared under conditions A2B2C1 had the highest sensory score, but its pH value was relatively high (pH=4.84±0.07), and its viscosity was only 146.67±10.26 mPa·s, which was significantly lower than that of the yogurt prepared under conditions A4B4C1 (676.33±9.07 mPa·s). Furthermore, the whey separation rate of the yogurt was relatively high (3.58±0.60%), which was not significantly different from that of the group with the highest whey separation rate, A1B3C3 (4.24±0.69%). Yogurt prepared under conditions A4B2C3 had a higher sensory score (73.40±10.36 points) and viscosity (578.33±9.02 mPa·s), a lower whey separation rate (0.05±0.05%), better water holding capacity (51.91±2.03%), and a suitable pH (4.38±0.02). The fermentation time was 6 hours, resulting in the best overall product performance.

[0144] Taking all factors into consideration, a fermentation temperature of 43℃, a fermentation time of 6 hours, and an inoculum amount of 3% (v / v) were selected as the optimal fermentation conditions for fermenting hemp seed paste into yogurt. Under these conditions, the yogurt produced is milky white, moist and glossy, with the flavors of both yogurt and hemp seeds, moderate viscosity, and a good taste.

[0145] In summary, the extracellular polysaccharide of *Lactobacillus limnosus* GXBMR4 provided by this invention not only increases the viscosity of the hemp seed paste system, increases the absolute value of the system's zeta potential, reduces the average particle size, improves particle dispersibility, and makes the system more stable, but also allows for the co-fermentation of hemp seed paste-fermented yogurt with common commercially available yogurt starter cultures, especially with *Lactobacillus bulgaricus*. This reduces the whey separation rate of the yogurt, increases its viscosity and water retention, and imparts a better texture to the product. Furthermore, the optimal fermentation conditions for hemp seed paste-fermented yogurt are: fermentation temperature 43℃, fermentation time 6 hours, and inoculum size 3% (v / v).

[0146] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A Limoxicillin-producing Lactobacillus fermentum (Lactobacillus rimoxime) Limosilactobacillus fermentum The application of GXBMR4 in the preparation of hemp seed pulp fermented yogurt is characterized by, Includes the following steps: (1) Weigh out the shelled hemp seeds, add 3 times the volume of pure water at 30℃ and soak for 3.5 h, add water at a material-to-liquid ratio of 1:33 (g:mL) to grind the seeds into a paste, filter, and collect the filtrate to obtain hemp seed paste; (2) Mix the hemp seed paste and the reconstituted emulsion at a volume ratio of 6:4, and inoculate the Limoxifen fermenting Lactobacillus and Lactobacillus bulgaricus at a volume ratio of 1:

1. The total inoculation amount of the Limoxifen fermenting Lactobacillus and Lactobacillus bulgaricus is 3% v / v. Ferment at 43°C for 6 hours to obtain the hemp seed paste fermented yogurt. The preservation number of the *Lactobacillus limnoxicamella* is GDMCC No: 64791; the *Lactobacillus bulgaricus* was purchased from Beijing Chuanxiu Technology Co., Ltd.

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