A strain of *Lactobacillus plantarum* and its application in *Hydrangea macrophylla* fermentation products

Abstract

This invention discloses a strain of *Lactobacillus plantarum* and its application in *Hydrangea macrophylla* fermentation products. The *Lactobacillus plantarum* is *Lactobacillus plantarum* (… Lactiplantibacillus plants SCLP-8 was deposited on December 25, 2023, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 29380. This bacterium can stably and rapidly ferment fresh or dried *Hydrangea macrophylla* to form *Hydrangea macrophylla* fermented products. Fermentation with this bacterium can significantly improve the flavor of *Hydrangea macrophylla* and increase the solubility of polysaccharides, providing a new fermentation source for the production and development of *Hydrangea macrophylla* fermented beverages and functional foods.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of *Lactobacillus plantarum* and its application in *Hydrangea macrophylla* fermentation products. Background Technology

[0002] *Sparassis latifolia* YCDai & Zheng Wang belongs to the phylum Basidiomycota, class Agaricales, order Polyporaceae, and family Sprastrophaceae. It is a rare edible fungus with extremely high nutritional value. Modern research indicates that *Sparassis latifolia* can exert anti-cancer and cancer-preventive effects by enhancing human immunity and hematopoietic function. As a large edible fungus, its fruiting body contains a large amount of β-glucan, reaching 43.6g / 100g, which is 4-5 times higher than that of Ganoderma lucidum and Agaricus blazei. Although its β-glucan content is high, the proportion of soluble polysaccharides is not high, only about 10%. Furthermore, its fresh product has a distinctive mushroom-like odor, which sometimes leads to unpleasant consumption experiences for some consumers.

[0003] Lactic acid bacteria are important microorganisms in the fermentation industry. Lactic acid bacteria fermentation can improve the flavor of food, impart unique aromas, and, due to their probiotic function, can also enhance the nutritional value and health benefits of food ingredients. Currently, the development of fermented products using edible fungi and probiotics is still in its early stages, and there is currently no market for fermented products using *Hylocereus undatus* lactic acid bacteria.

[0004] The fruiting body of *Hylocereus undatus* is the edible part, mainly composed of the fungal cell wall. As a large edible fungus, *Hylocereus undatus* produces a large amount of fungal metabolites during its metabolism, some of which inhibit bacterial growth. Therefore, most lactic acid bacteria strains do not achieve ideal fermentation results on *Hylocereus undatus* fruiting bodies. Analysis of the fermentation effects of commercially available lactic acid bacteria starter cultures revealed common problems such as low substrate utilization, slow acid production, and high contamination rates.

[0005] To address the aforementioned issues, this invention aims to screen lactic acid bacteria strains capable of fermenting fresh Hydrangea macrophylla fruiting bodies or dried Hydrangea macrophylla powder into lactic acid bacteria fermentation products, thereby providing highly efficient fermentation strains for the production and development of Hydrangea macrophylla fermented products. Summary of the Invention

[0006] The purpose of this invention is to provide a strain of *Lactobacillus plantarum* and its application in *Hydrangea macrophylla* fermentation products. This *Lactobacillus plantarum* can be used to ferment and produce *Hydrangea macrophylla* fermented products. This invention provides a new and efficient fermentation strain for the production and development of *Hydrangea macrophylla* fermented products, and this strain can be applied to the fermentation of fresh or dried *Hydrangea macrophylla* fruiting bodies.

[0007] To achieve the above objectives, the technical solution provided by the present invention is as follows:

[0008] A strain of Lactiplantibacillus plantarum SCLP-8 was deposited on December 25, 2023, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 29380.

[0009] The screening method for *Lactiplantibacillus plantarum* SCLP-8 is as follows: Fresh *Lactiplantibacillus* was taken, and an equal volume of sterile glucose water (2%) was added. The mixture was homogenized using a sterile homogenizer and sealed in a culture flask. It was then fermented at 37°C for 30 days. 1 mL of the fermentation broth was taken and serially diluted. The diluted broth was then plated on MRS solid medium. Single, milky-white colonies suspected to be lactic acid bacteria were selected and further purified by streaking on MRS solid plates three times to obtain single-colony strains. Each single-colony strain was then incubated statically at 37°C for 24 hours in MRS liquid medium to obtain liquid inoculum. 10 mL of the inoculum was centrifuged at 3000 rpm for 5 minutes, the supernatant was discarded, and the bacterial sludge was collected. Add 10 ml of *Lactobacillus plantarum* slurry prepared by homogenizing 2% glucose solution and *Lactobacillus plantarum* fruiting bodies in a 1:1 ratio, mix thoroughly, and ferment at 37℃ for 24 h. Using pH, acidity, and the number of lactic acid bacteria colonies in the fermentation broth as indicators, strains capable of growing in this fermentation material, producing significant acid production, and showing a significant increase in colony count were selected. By analyzing the fermentation rate and contamination rate, the lactic acid bacteria strain that rapidly ferments *Lactobacillus plantarum* slurry, quickly produces acid, exhibits stable fermentation, and has minimal contamination was finally selected, preserved, and named SCLP-8. Through morphological observation, API bacterial identification system, 16S rDNA gene sequencing, and phylogenetic tree homology analysis, the strain was finally identified as *Lactobacillus plantarum*.

[0010] The *Lactiplantibacillus plantarum* SCLP-8 described above can be used to prepare fermented products from *Hydrangea macrophylla*, using fresh *Hydrangea macrophylla* slurry or dried *Hydrangea macrophylla* powder as raw materials. For liquid fermentation using fresh *Hydrangea macrophylla* slurry, fresh *Hydrangea macrophylla* fruiting bodies are used as raw materials, and the slurry is prepared by colloid milling at a material-to-water ratio of 1:1. 1%-10% glucose is added, and the mixture is boiled for 5 minutes for sterilization. Then, 2 wt% of *Lactiplantibacillus plantarum* SCLP-8 mycelial slurry is inoculated, stirred evenly, and incubated at 37-42℃ for 24-48 hours. For solid-state fermentation using dried *Hydrangea macrophylla* powder, the *Lactiplantibacillus plantarum* SCLP-8 mycelial slurry is suspended in a 1%-5% glucose solution, and then mixed into the dried *Hydrangea macrophylla* powder at an inoculation rate of 2 wt% and stirred evenly. The material-to-water ratio is controlled at 1:3-1:5, and the mixture is incubated at 37-42℃ for 72-96 hours.

[0011] Lactobacillus plantarum SCLP-8 can ferment both fresh and dried *Hydrangea macrophylla* raw materials to obtain *Hydrangea macrophylla* fermented products. The liquid fermented *Hydrangea macrophylla* slurry has a mild taste and rich flavor. Because this bacterium can rapidly produce acid in the *Hydrangea macrophylla* fermentation substrate, the contamination rate is low and the fermentation success rate is high, making it suitable for large-scale production of *Hydrangea macrophylla* fermented beverages or enzymes. Solid-state fermentation of *Hydrangea macrophylla* dry powder yields *Hydrangea macrophylla* fermented products with reduced unpleasant odors and significantly improved flavor. Simultaneously, this bacterium can decompose the polysaccharide components in *Hydrangea macrophylla*, significantly increasing the dissolution rate of high molecular weight polysaccharides in the obtained *Hydrangea macrophylla* fermented products, making it a potential novel food ingredient for use in food and health food products.

[0012] The beneficial effects of this invention are as follows: It provides a strain of Lactiplantibacillus plantarum SCLP-8, which can stably and rapidly ferment fresh or dried Hydrangea macrophylla to form Hydrangea macrophylla fermented products. Furthermore, fermentation with this strain can significantly improve the flavor of Hydrangea macrophylla and increase the dissolution rate of high molecular weight polysaccharides, thus providing a new fermentation source for the production and development of Hydrangea macrophylla fermented beverages and functional foods. Attached Figure Description

[0013] Figure 1 This is a colony morphology diagram of strain SCLP-8.

[0014] Figure 2 This is a Gram-stained microscopic image of strain SCLP-8.

[0015] Figure 3 This is a map of the 16S rDNA amplification fragment of strain SCLP-8.

[0016] Figure 4 This is a phylogenetic tree diagram of strain SCLP-8.

[0017] Figure 5 This is the HPSEC spectrum of the molecular weight distribution of soluble crude polysaccharides before and after solid-state fermentation of *Hydrangea macrophylla* powder. Detailed Implementation

[0018] To make the content of this invention easier to understand, the technical solution of this invention will be further described below in conjunction with specific embodiments.

[0019] Example 1

[0020] Isolation, screening and identification of strains

[0021] 1. Isolation and screening of strains

[0022] Fresh *Hydrangea macrophylla* was taken and an equal volume of sterile glucose solution (2%) was added. The mixture was homogenized using a sterile homogenizer and sealed in a culture flask. It was then incubated at 37°C for 30 days. One mL of the fermentation broth was taken and serially diluted. The diluted broth was then plated on MRS solid medium. Single, milky-white colonies suspected to be lactic acid bacteria were selected and further purified by streaking on MRS solid plates three times to obtain single-colony strains. Each single-colony strain was then incubated statically at 37°C for 24 hours in MRS liquid medium to obtain liquid inoculum. 10 mL of this inoculum was centrifuged at 3000 rpm for 5 minutes, the supernatant was discarded, and the bacterial sludge was collected. Add 10 ml of *Hydrangea macrophylla* slurry prepared by homogenizing 2% glucose solution and *Hydrangea macrophylla* fruiting bodies in a 1:1 ratio, mix well, and ferment at 37℃ for 24 h. Using the pH, acidity, and number of lactic acid bacteria colonies in the fermentation broth as indicators, strains capable of growing in this fermentation material, producing significant acid production, and showing a significant increase in colony count were selected. By analyzing the fermentation rate and contamination rate, the lactic acid bacteria strain that rapidly ferments *Hydrangea macrophylla* slurry, quickly produces acid, exhibits stable fermentation, and has minimal contamination was finally selected, preserved, and named SCLP-8.

[0023] 2. Identification of strains

[0024] (1) Morphological observation of bacteria

[0025] Colony morphology of SCLP-8 cells was observed. Figure 1 Gram staining microscopic observation Figure 2 The main morphological characteristics of SCLP-8 are as follows: the colonies are round, milky white and opaque on MRS medium, and Gram stain purple; the bacterial cells are rod-shaped.

[0026] (2) The API bacterial identification system is used for bacterial identification and physiological and biochemical analysis.

[0027] The carbohydrate utilization and enzyme production of bacterial SCLP-8 were analyzed using the API50 CHL V5.1 kit, and the results are shown in Table 1.

[0028] Table 1. Carbohydrate Utilization in the API Bacterial Identification System

[0029]

[0030]

[0031] The test results were compared with those of standard strains using software. The results showed that after 24 and 48 hours of culture, SCLP-8 had a 99.9% identification rate with *Lactiplantibacillus plantarum*, with a confidence level (T-value) of 0.98, while its identification rate with the next lower taxonomic unit, *Lactiplantibacillus pentosus*, was only 0.1%, with a T-value of only 0.51. Using the API bacterial identification system, SCLP-8 was identified as *Lactiplantibacillus plantarum*.

[0032] (3) Molecular biological identification of SCLP-8 bacteria

[0033] The 16S rDNA sequence of the bacterial strain was determined by PCR using universal microbial primers 27F and 1492R.

[0034] DNA extraction: Follow the instructions for the bacterial genomic DNA extraction kit.

[0035] PCR amplification and sequencing of 16S rDNA sequence:

[0036] The primers used to amplify the 16S rDNA gene sequence were:

[0037] Upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0038] Downstream primer 1492R: 5'-TACGGCTACCTTGTTACGACTT-3';

[0039] PCR reaction system: Total volume 40 μL. DNA 1.0 μL, 2×Taq Master Mix 20 μL, primers 2.0 μL each, ddH2O 15 μL.

[0040] PCR amplification program: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, annealing at 56℃ for 30 s; 72℃ extension for 1.5 min; 30 cycles in total, followed by a final extension at 72℃ for 20 min, and storage at 4℃.

[0041] The PCR amplification products were subjected to agarose gel electrophoresis. Observation using a gel imaging system revealed that the amplified 16S rDNA target fragment was approximately 1500 bp in length. Figure 3 As shown.

[0042] Sequencing of the PCR amplification product of the strain revealed a sequence length of 1475 bp, as shown in SEQ ID NO.1. BLAST alignment analysis of this sequence in NCBI data showed over 99% homology with the sequence of *Lactiplantibacillus plantarum*. Developmental tree construction analysis was performed using MEGA 7.0. Figure 4 This strain is genetically closest to *Lactiplantibacillus plantarum*. Based on morphological observation and physiological and biochemical detection using the API bacterial identification system, the strain was identified as *Lactiplantibacillus plantarum*, named SCLP-8, and deposited at the China General Microbiological Culture Collection Center (CGMCC) on December 25, 2023, with accession number CGMCC NO: 29380.

[0043] Example 2

[0044] Liquid fermentation of fresh Hydrangea mycelium slurry was carried out using Lactobacillus plantarum SCLP-8.

[0045] Step 1: Inoculate Lactobacillus plantarum SCLP-8 into 500ml MRS liquid medium and ferment at 37℃ for 24h. Centrifuge the fermentation broth, discard the supernatant, collect the bacterial sludge precipitate, and suspend it in 20ml of 2% glucose solution as the inoculum.

[0046] Step 2: Add 500g of fresh *Hydrangea macrophylla* to 500ml of water and grind into a slurry using a colloid mill. Then add 5% glucose at wt%, boil and sterilize for 5 minutes to obtain *Hydrangea macrophylla* slurry. Bottle the slurry into a fermentation tank, inoculate the *Hydrangea macrophylla* slurry with the inoculum prepared in Step 1, stir well, and let it ferment at 37℃ for 48 hours to obtain *Hydrangea macrophylla* slurry liquid fermentation product. Store the harvested fermentation product in a refrigerator at 4℃ for later use.

[0047] Step 3: The viable cell count, soluble solids, soluble crude polysaccharide content, total acid, and pH value of the *Hydrangea macrophylla* slurry before and after fermentation were determined (Table 2). The results showed that SCLP-8 could rapidly multiply in the *Hydrangea macrophylla* slurry, with the viable cell count in the fermentation product reaching 7.5 × 10⁻⁶. 6With cfu / ml, moderate acidity, and smooth texture, this strain significantly increases the soluble solids and soluble crude polysaccharide content of the *Hydrangea macrophylla* pulp after fermentation. This indicates that SCLP-8 can be used as a fermentation strain in the fermentation production of fresh *Hydrangea macrophylla* pulp, which can significantly improve the flavor and taste of the product and enhance its nutritional value.

[0048] Table 2 Comparison of data on indicators before and after fermentation of Hydrangea mycelium pulp

[0049]

[0050]

[0051] Example 3

[0052] Solid-state fermentation of *Hydrangea macrophylla* powder using *Lactobacillus plantarum* SCLP-8.

[0053] Step 1: Inoculate Lactobacillus plantarum SCLP-8 into 100ml MRS liquid medium and ferment at 37℃ for 24h. Centrifuge the fermentation broth, discard the supernatant, collect the bacterial sludge precipitate, and suspend it in 200ml of 2% glucose solution as the inoculum.

[0054] Step 2: Crush the dried *Hydrangea macrophylla* and pass it through a 100-mesh sieve to obtain *Hydrangea macrophylla* dry powder. Place 50g of *Hydrangea macrophylla* dry powder in a fermentation box, add 200ml of the inoculum prepared in Step 1, stir until it becomes a paste, flatten the surface, seal the box, and place it in a 37℃ fermentation incubator for 96 hours to obtain *Hydrangea macrophylla* powder solid fermentation product. The harvested fermentation product is freeze-dried in a vacuum freeze dryer and then crushed to obtain *Hydrangea macrophylla* fermentation powder.

[0055] Step 3: The polysaccharide content and molecular weight distribution of soluble crude polysaccharides in *Hylocereus undatus* powder before and after fermentation were analyzed. The contents of α-glucan and β-glucan in *Hylocereus undatus* powder before and after fermentation were determined using an enzymatic assay kit. The results are shown in Table 3. After fermentation, the β-glucan content in *Hylocereus undatus* powder decreased significantly, while the α-glucan content increased significantly, indicating that SCLP-8 can degrade and utilize β-glucan in *Hylocereus undatus*. The molecular weight distribution of soluble polysaccharides in *Hylocereus undatus* powder before and after fermentation was analyzed using HPSEC. The results showed that ( Figure 5 After fermentation with SCLP-8, the dissolution rate of medium molecular weight polysaccharides with a molecular weight of approximately 45 kDa in the *Hydrangea hydrangea* sample was significantly improved. Among the dissolved polysaccharide components, the peak area ratio of this component increased from 18.59% to 62.13%. Generally, the molecular weight and solubility of polysaccharides are important factors affecting their biological activity, and better solubility is conducive to the exertion of biological activity.

[0056] Table 3 Comparison of data on indicators before and after fermentation of Hydrangea macrophylla powder

[0057]

Claims

1. A strain of *Lactobacillus plantarum*, characterized in that, It is Lactobacillus plantarum ( Lactiplantibacillus plantarum SCLP-8 was deposited on December 25, 2023, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 29380.

2. The application of the *Lactobacillus plantarum* strain as described in claim 1 in *Hydrangea macrophylla* fermentation products.

3. The application according to claim 2, characterized in that, The slurry made from fresh Hydrangea macrophylla was used as raw material, inoculated with Lactobacillus plantarum SCLP-8, and then fermented.

4. The application according to claim 2, characterized in that, The dried and pulverized *Hydrangea macrophylla* powder was used as raw material, inoculated with *Lactobacillus plantarum* SCLP-8, and then fermented.

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