A strain of Lactobacillus johnsonii that efficiently utilizes honeysuckle residue and its application

Abstract

The application belongs to the technical field of microorganisms, and discloses a lactobacillus johnsonii capable of efficiently utilizing honeysuckle residues and an application thereof. The lactobacillus johnsonii has a preservation number of CCTCC NO: M 20261247. The strain can efficiently utilize honeysuckle residues, significantly reduce the content of acid detergent fiber and neutral detergent fiber in the residues, increase the content of functional active substances such as chlorogenic acid and flavone, and make the fermented residues have the activity of inhibiting salmonella and escherichia coli.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Lactobacillus johnsonii that efficiently utilizes honeysuckle residue and its applications. Background Technology

[0002] Traditional Chinese medicine residue is a major solid waste generated during the production of Chinese medicinal herbs. The extraction of honeysuckle extract alone produces a large amount of honeysuckle residue annually. Traditional treatment methods mainly involve landfilling and incineration, which not only result in a significant waste of biomass resources but also pose risks of soil, water, and air pollution. After steam distillation, the residue of honeysuckle is still rich in various nutrients and active ingredients such as crude protein, cellulose, chlorogenic acid, flavonoids, and polysaccharides, possessing the potential for high-value utilization as a substrate for microbial fermentation.

[0003] Current resource utilization of honeysuckle residue mainly focuses on direct use as feed additives or composting, which has low utilization efficiency and fails to effectively convert its fiber components and increase the content of functional active substances such as chlorogenic acid and flavonoids. In addition, fermentation broth often lacks the additional function of inhibiting pathogens (such as Salmonella and Escherichia coli), limiting its application in fields such as antibiotic alternatives.

[0004] Lactobacillus johnsonii is a symbiotic probiotic widely found in the intestines of mammals and is a safe, edible microorganism. This bacterium exhibits good acid and bile salt tolerance, and can regulate the balance of intestinal flora, enhance intestinal barrier function, and exert anti-inflammatory and antioxidant activities.

[0005] This invention screens a strain of Lactobacillus johnsonii from honeysuckle residue that can efficiently utilize the substrate. It can significantly reduce the content of acid detergent fiber and neutral detergent fiber in the residue, while simultaneously increasing the content of active substances such as chlorogenic acid and flavonoids. It also enables the fermented residue to inhibit Salmonella and Escherichia coli, providing a new solution for the resource utilization of traditional Chinese medicine residue and the fermentation of functional probiotics.

[0006] There are currently no research reports on the direct use of Lactobacillus johnsonii in the fermentation of traditional Chinese medicine residues to achieve fiber degradation and enrichment of active ingredients. Summary of the Invention

[0007] The purpose of this invention is to provide a strain of Lactobacillus johnsonii that efficiently utilizes honeysuckle residue, the preservation number of which is CCTCC NO: M 20261247.

[0008] Another object of the present invention is the application of Lactobacillus johnsonii.

[0009] To achieve the above objectives, the present invention adopts the following technical measures:

[0010] The applicant obtained a strain of Lactobacillus that can efficiently utilize honeysuckle residue through laboratory probiotic resource screening. It was identified as Lactobacillus johnsonii by 16S rDNA gene sequence and whole genome sequence. This strain was deposited at the China Center for Type Culture Collection on June 10, 2026, with the classification name: Lactobacillus johnsonii HBNK6, accession number: CCTCC NO: M20261247, address: Wuhan University, Wuhan, China.

[0011] The Lactobacillus johnsonii HBNK6 described is a Gram-positive bacillus, non-spore-forming, non-flagellated, and facultatively anaerobic. On MRS solid medium, colonies are milky white, smooth and moist, round and raised, with neat edges. Figure 1 The strain, after simple staining, appears as a rod shape under an optical microscope. Figure 2 This strain can efficiently utilize honeysuckle residue, significantly reducing the content of acidic and neutral detergent fibers in the residue, increasing the content of functional active substances such as chlorogenic acid and flavonoids, and giving the fermented residue inhibitory activity against Salmonella and Escherichia coli. The optimal growth temperature in the culture medium is 37℃.

[0012] The scope of protection of this invention also includes:

[0013] Fermentation broth of Lactobacillus johnsonii HBNK6.

[0014] A microbial preparation comprising Lactobacillus johnsonii HBNK6 and / or the above-mentioned fermentation broth.

[0015] The microbial preparations described above also include adjuvants.

[0016] Application of Lactobacillus johnsonii HBNK6, fermentation broth of Lactobacillus johnsonii HBNK6, or the above-mentioned microbial preparations in fermented honeysuckle residue.

[0017] Application of Lactobacillus johnsonii HBNK6, fermentation broth of Lactobacillus johnsonii HBNK6, or the above-mentioned microbial preparations in the preparation of chlorogenic acid and / or flavonoids from fermented honeysuckle residue.

[0018] Application of Lactobacillus johnsonii HBNK6, fermentation broth of Lactobacillus johnsonii HBNK6, or the above-mentioned microbial preparations in the preparation of antibacterial microbial preparations from fermented honeysuckle residue.

[0019] The above-described applications include the inhibition of Escherichia coli and / or Salmonella.

[0020] The above-described application involves fermenting honeysuckle residue by mixing it with MRS culture medium.

[0021] A microbial fermentation broth is a fermentation broth made by Lactobacillus johnsonii HBNK6 fermenting honeysuckle residue.

[0022] The honeysuckle residue mentioned in the above applications refers to the residue after extracting honeysuckle dew.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The Lactobacillus johnsonii HBNK6 (accession number CCTCCNO: M 20261247) provided by this invention can directly use honeysuckle extract residue as a substrate for efficient fermentation without the need for complex acid, alkali or enzymatic pretreatment of the residue, which significantly reduces the cost of resource utilization of traditional Chinese medicine residue.

[0025] This strain can significantly reduce the content of acid detergent fiber and neutral detergent fiber in honeysuckle residue during fermentation, effectively degrade lignocellulose components, and thus improve the digestibility and utilization rate of the residue.

[0026] A more unique characteristic is that this strain can simultaneously increase the content of functional active substances such as chlorogenic acid and flavonoids in the residue, achieving a synergistic effect of fiber degradation and active ingredient enrichment.

[0027] Furthermore, the honeysuckle residue fermented by this strain exhibits inhibitory activity against Salmonella and Escherichia coli, endowing the fermentation broth with additional antibacterial properties. This invention provides a green, economical, and efficient new solution for the high-value utilization of traditional Chinese medicine residues and the fermentation of functional probiotics. It not only solves the environmental pollution problem caused by the dumping of traditional Chinese medicine residues but also yields fermented products with anti-inflammatory, antioxidant, and antibacterial potential. Attached Figure Description

[0028] Figure 1 This is a colony morphology diagram of Lactobacillus johnsonii HBNK6.

[0029] Figure 2 This is a simple staining microscopic image of Lactobacillus johnsonii HBNK6.

[0030] Figure 3 This is a growth curve diagram of Lactobacillus johnsonii HBNK6.

[0031] Figure 4 This is a graph showing the changes in neutral detergent fibers in honeysuckle residue after fermentation by Lactobacillus johnsonii HBNK6 and in the unfermented residue.

[0032] Figure 5 This is a graph showing the changes in acid detergent fibers in honeysuckle residue after fermentation by Lactobacillus johnsonii HBNK6 and in the unfermented residue.

[0033] Figure 6 The Oxford Cup experiment results show the inhibitory effect of Lactobacillus johnsonii HBNK6 fermentation broth on the pathogen.

[0034] Figure 7 This is a graph showing the results of the Oxford Cup experiment on the inhibitory effect of antibiotics on pathogens. Detailed Implementation

[0035] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field; unless otherwise specified, the reagents or materials described are all from commercial sources.

[0036] Example 1:

[0037] Obtaining Lactobacillus Johannesburg HBNK6, which can synthesize chlorogenic acid and flavonoids from honeysuckle residue:

[0038] 1) The applicant selected nine probiotic strains from its laboratory's probiotic resource bank, including *Lactobacillus amyloliquefaciens*, *Pediococcus pentosaceus*, *Lactobacillus reuteri*, *Lactobacillus johnsonii*, *Lactobacillus amyloliquefaciens*, and *Myxobacillus reuteri*, to evaluate their ability to enhance the functional active substances such as chlorogenic acid and flavonoids in honeysuckle residue after fermentation. The nine probiotic strains were inoculated from glycerol tubes into MRS liquid medium for activation, and then transferred to MRS liquid medium for static incubation for 18 hours to obtain the seed culture. 1 mL of the seed culture (approximately 1 × 10⁻⁶) was used as the seed culture. 9 CFU (chlorogenic acid) was inoculated into 60 ml MRS medium containing 10 g of honeysuckle residue powder at 37°C for fermentation for 96 hours. A blank control was prepared using 60 ml MRS medium containing 10 g of honeysuckle residue powder without inoculation. The effects of different strains on increasing the content of chlorogenic acid, flavonoids, and other active substances after fermentation were determined using a chlorogenic acid and flavonoid detection kit.

[0039] The honeysuckle residue powder is prepared by the following method: the residue after extracting honeysuckle dew is dried to constant weight, placed in an ultra-micro pulverizer and pulverized, then passed through a 60-mesh copper mesh sieve, and the sieve-passing material is taken to obtain the honeysuckle residue powder.

[0040] As shown in Table 1, Lactobacillus johnsonii HBNK6 had the highest yields of chlorogenic acid and flavonoids, at 1.4651 mg / mL and 3.0893 mg / mL, respectively. These values ​​were 29.45% higher than those in the unfermented honeysuckle residue (blank control group) and 56.13% higher than those in the blank control group.

[0041] Table 1 Results of chlorogenic acid and flavonoid production by fermentation of honeysuckle residue using different probiotics

[0042] .

[0043] Identification and preservation of Lactobacillus johnsonii HBNK6:

[0044] The selected target strain HBNK6 was simply stained with crystal violet and observed under an optical microscope. It was found that this strain is a Gram-positive bacillus, non-spore-forming, non-flagellated, and facultatively anaerobic. On MRS solid medium, colonies are milky white, smooth, moist, round, raised, with regular edges, and a diameter generally 1.0-2.0 mm. Figure 1 The strain, after simple staining, appears as a rod shape under an optical microscope. Figure 2 The optimal growth pH is 6.0-6.5; the optimal growth temperature is 34℃.

[0045] Genome sequencing of *Lactobacillus johnsonii* HBNK6 yielded a full-length 16S rDNA, which was confirmed as a *Lactobacillus johnsonii* strain by comparison with the NCBI database. This strain was deposited on June 10, 2026, at the China Center for Type Culture Collection (CCTCC), with the classification name *Lactobacillus johnsonii* HBNK6, accession number CCTCC NO: M 20261247, address: Wuhan University, Wuhan, China.

[0046] Example 2:

[0047] Growth curve determination of Lactobacillus johnsonii HBNK6

[0048] Lactobacillus johnsonii HBNK6 glycerol-preserved bacterial culture was transferred to MRS liquid medium for activation, and then transferred to fresh MRS liquid medium at a 1% inoculum. The culture was incubated at 37°C for 18 h to obtain a seed culture. The turbidity of the seed culture was adjusted to 0.5 (equivalent to approximately 1 × 10⁻⁶) using a McFarland turbidimeter. 8 CFU / mL), serially diluted 10-fold with MRS broth to 1×10⁻⁶ CFU / mL. 6 CFU was added to 200 μL of diluted bacterial culture into a 96-well plate. The culture was continuously monitored for 24 h using a multi-mode microplate reader (Victor Nivo, PerkinElmer), with the program set to 37℃ and OD600 values ​​measured every 2 h. A growth curve was plotted with culture time on the x-axis and OD600 values ​​on the y-axis. The results are shown below. Figure 3 As shown, the fermentation time is 14-16 hours, and the highest effective bacterial concentration of 3*10⁻⁶ can be achieved within 24 hours. 9 cfu / ml.

[0049] Example 3:

[0050] Lactobacillus johnsonii HBNK6 reduces the content of neutral detergent fiber in honeysuckle residue.

[0051] Preparation of neutral detergent (3% sodium lauryl sulfate):

[0052] Accurately weigh 18.6g of disodium ethylenediaminetetraacetate and 6.8g of sodium tetraborate into a 100mL graduated beaker. Add a small amount of distilled water and heat to dissolve. Then add 30g of sodium dodecyl sulfate and 10mL of ethylene glycol ether. Weigh 4.56g of anhydrous disodium hydrogen phosphate into another beaker, add a small amount of distilled water and heat gently to dissolve. Pour the solution into the first beaker and then transfer it to a volumetric flask to dilute to 1000ml. The pH of this solution should be between 6.9 and 7.1.

[0053] Preparation of fermentation broth:

[0054] Lactobacillus johnsonii HBNK6 was inoculated from a glycerol tube into MRS liquid medium to activate the inoculum, and then transferred to MRS liquid medium and incubated statically for 18 h to create a seed culture. The seed culture (approximately 1 × 10⁻⁶) was then... 9 CFU was inoculated into a 60ml MRS medium container containing 10g of honeysuckle residue powder at 37℃ for fermentation. After fermentation for 96 hours, the fermentation broth was air-dried.

[0055] A 60 ml MRS medium containing 10 g of honeysuckle residue powder without inoculation was used as a control.

[0056] Determination of neutral detergent fiber (NDF) content:

[0057] Accurately weigh 1.0 g (accurate to 0.0002 g) of the air-dried sample that has passed through a 40-mesh sieve and place it in a tall beaker. If the sample has a fat content ≥10%, it needs to be pre-treated with petroleum ether for defatting. Add 100 mL of neutral detergent, 2 mL of decahydronaphthalene, and 0.5 g of anhydrous sodium sulfite. Attach a condenser and immediately place the beaker on an electric furnace to boil for 5-10 minutes. After the solution boils, adjust the furnace to maintain a gentle boil, frequently shaking the beaker to ensure thorough mixing of the sample and solution. Reflux for 1 hour (adding water during this time to maintain the liquid level at 100 mL). Remove from heat and cool for 10 minutes. Install a pre-weighed glass crucible on a vacuum filtration flask. Transfer all the residue into the flask, filter, and rinse with boiling water until the filtrate is neutral. Rinse the residue with 20 mL of acetone, repeating until the filtrate is colorless (usually 3 times). Transfer the glass crucible to a 105℃ forced-air drying oven and dry until constant weight (approximately 3-4 hours). Then, transfer it to a desiccator and cool for 30 minutes before weighing. Repeat the drying and weighing process until the difference between two weighings does not exceed 0.001g. Calculate using the following formula:

[0058] NDF (%) = (Cruise weight + Residue constant weight - Crucible constant weight) / Sample mass × 100%

[0059] The results showed that the neutral detergent fiber content of honeysuckle residue fermented by Lactobacillus johnsonii HBNK6 strain was 29.762%, which was 5.91% lower than the neutral detergent fiber content of 35.672% in unfermented honeysuckle residue. Figure 4 This indicates that the Lactobacillus Johannesburg strain HBNK6 can effectively secrete cellulase, hemicellulase and other related enzyme systems during fermentation, thereby degrading and utilizing the cellulose and hemicellulose components in the honeysuckle residue.

[0060] Example 4:

[0061] Lactobacillus johnsonii HBNK6 reduces the acid detergent fiber content in honeysuckle residue.

[0062] Preparation of acidic detergent (2% hexadecanetrimethylammonium bromide):

[0063] Weigh 20 g of cetyltrimethylammonium bromide (CTAB), dissolve it in 1000 mL of 1.00 mol / L sulfuric acid solution, stir to dissolve, and filter if necessary.

[0064] Preparation of fermentation broth:

[0065] Lactobacillus johnsonii HBNK6 was inoculated from a glycerol tube into MRS liquid medium to activate the inoculum, and then transferred to MRS liquid medium and incubated statically for 18 h to create a seed culture. The seed culture (approximately 1 × 10⁻⁶) was then... 9 CFU was inoculated into a 60ml MRS medium container containing 10g of honeysuckle residue powder at 37℃ for fermentation. After fermentation for 96 hours, the fermentation broth was air-dried.

[0066] Determination of Acid Detergent Fiber (ADF) Content:

[0067] After pulverizing the sample through a 40-mesh sieve, accurately weigh approximately 1g, add 100mL of acidic detergent (sulfuric acid solution containing hexadecyltrimethylammonium bromide) and a few drops of defoamer, reflux and digest under gentle boiling for 60 minutes, then filter while hot and wash repeatedly with boiling water until no foam remains, then wash with acetone until the filtrate is colorless and dry under vacuum, finally place the crucible in a 105℃ oven to dry to constant weight, and calculate the ADF content based on the weight of the residue.

[0068] ADF (%) = (Cruise weight + Residue constant weight - Crucible constant weight) / Sample mass × 100%

[0069] The results showed that the acid detergent fiber content of honeysuckle residue fermented by Lactobacillus johnsonii HBNK6 strain was 23.206%, which was 8.01% lower than the 31.214% medium-acid detergent fiber content of unfermented honeysuckle residue. Figure 5This indicates that *Lactobacillus johnsonii* strain HBNK6 can effectively secrete cellulase, hemicellulase, and other related enzyme systems during fermentation, effectively degrading and utilizing acidic detergent fiber components such as cellulose and lignin in honeysuckle residue. This result demonstrates that *Lactobacillus johnsonii* strain HBNK6 has good development potential in the biotransformation and feed utilization of traditional Chinese medicine residue, effectively reducing the content of anti-nutritional factors in honeysuckle residue and significantly enhancing its application value as a functional roughage ingredient.

[0070] Example 5:

[0071] Determination of the antibacterial effect of fermentation broth from honeysuckle residue fermented with Lactobacillus johnsonii HBNK6

[0072] Preparation of fermentation broth:

[0073] Lactobacillus johnsonii HBNK6 was inoculated from a glycerol tube into MRS liquid medium to activate the inoculum, and then transferred to MRS liquid medium and incubated statically for 18 h to create a seed culture. The seed culture (approximately 1 × 10⁻⁶) was then... 9 CFU was inoculated into a container at 37°C containing 10 g of honeysuckle residue powder and 60 ml of MRS medium for fermentation. The fermentation time was 96 hours to obtain the fermentation broth. Oxford Cup Antibacterial Experiment:

[0074] MHA culture medium was autoclaved at 121℃ for 15 minutes and then kept warm in an oven for later use. The bacterial suspensions of *E. coli* K88, K99, 987P, ATCC25922, and *Salmonella* S.Tm15E475 were adjusted to 0.5 McFarland turbidity (equivalent to 1×10⁻⁶) using a McFarland turbidimeter. 8 (CFU / mL), take 200 μL of bacterial culture at a ratio of 1:100 and add it to 20 mL of MHA. Mix well and pour into a 90 mm plate. Let it stand for 20-30 minutes to solidify. Place a sterile Oxford cup (the inner diameter of the Oxford cup is 8 mm) in the center of the plate and add 200 μL of the fermentation broth to be tested. At the same time, set up an antibiotic (streptomycin 32 μg / mL) as a positive control. After incubation, measure the diameter of the inhibition zone.

[0075] The results showed that the Oxford cup antibacterial experiment demonstrated stable antibacterial activity against a variety of pathogenic bacteria in 200 μL of honeysuckle fermentation broth. Specifically, the inhibition zone diameter against Salmonella 15E475 was 1.4 cm, against Escherichia coli ATCC25922 was 1.4 cm, against Escherichia coli K99 was 1.4 cm, and against Escherichia coli K88 and 987P were 1.3 cm and 1.3 cm, respectively. Figure 6The inhibition zone diameter of the antibiotic against Salmonella 15E475 was 2.6 cm, against Escherichia coli ATCC25922 was 1.8 cm, and against Escherichia coli K88, K99, and 987P were 2.5 cm, 2.3 cm, and 3.9 cm, respectively. Figure 7 ).

[0076] Overall, the inhibition zone diameters of the tested strains ranged from 1.3 to 1.4 cm, indicating that the honeysuckle fermentation broth exhibited a broad-spectrum and relatively consistent inhibitory effect on Gram-negative bacteria, showing good antibacterial efficacy against both standard strains and clinical isolates. These results suggest that the metabolites produced by *Lactobacillus johnsonii* HBNK6 after fermenting honeysuckle residue contain broad-spectrum antibacterial active substances, providing data support for its potential application as a feed additive to replace antibiotics.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. An isolated strain of Lactobacillus johnsonii ( Lactobacillus johnsonii HBNK6, the preservation number of the Lactobacillus johnsonii is CCTCC NO: M20261247.

2. The fermentation broth of Lactobacillus Johannesburg HBNK6 as described in claim 1.

3. A microbial preparation comprising Lactobacillus johnsonii HBNK6 as described in claim 1 and / or the fermentation broth as described in claim 2.

4. The application of Lactobacillus johnsonii HBNK6 as described in claim 1, the fermentation broth of Lactobacillus johnsonii HBNK6 as described in claim 2, or the microbial preparation as described in claim 3 in fermented honeysuckle residue.

5. The use of Lactobacillus johnsonii HBNK6 as described in claim 1, the fermentation broth of Lactobacillus johnsonii HBNK6 as described in claim 2, or the microbial preparation as described in claim 3 in the preparation of chlorogenic acid and / or flavonoids from fermented honeysuckle residue.

6. The application of Lactobacillus johnsonii HBNK6 as described in claim 1, the fermentation broth of Lactobacillus johnsonii HBNK6 as described in claim 2, or the microbial preparation as described in claim 3 in the preparation of antibacterial microbial preparations from fermented honeysuckle residue, wherein the antibacterial effect is the inhibition of Escherichia coli and / or Salmonella.

7. The application according to any one of claims 4-6, wherein the fermented honeysuckle residue is fermented by mixing honeysuckle residue with MRS culture medium.

8. A microbial fermentation broth, which is the fermentation broth of honeysuckle residue fermented by Lactobacillus johnsonii HBNK6 as described in claim 1.

9. The application according to any one of claims 4-6, or the microbial fermentation broth according to claim 8, wherein the honeysuckle residue is the residue after extracting honeysuckle dew.

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