Yam skin fermentation liquor as well as preparation method and application thereof

Abstract

The invention discloses Chinese yam skin fermentation liquor as well as a preparation method and application thereof. The invention provides a plant lactobacillus Z165 strain with good environmental tolerance and safety in order to overcome the defect of lack of a strain for Chinese yam skin fermentation at present. By utilizing the plant lactobacillus Z165 strain, the Chinese yam peel can be effectively fermented, the antioxidant activity and the probiotic property of the obtained Chinese yam peel fermentation liquor are improved, and compared with Chinese yam peel slurry before fermentation or single plant lactobacillus Z165 strain, the antibacterial range and the activity of the obtained Chinese yam peel fermentation liquor are increased, so that the anti-oxidative activity of the Chinese yam peel fermentation liquor is improved, and the anti-oxidative activity of the Chinese yam peel fermentation liquor is improved. Various pathogenic bacteria such as escherichia coli, staphylococcus aureus, listeria monocytogenes and vibrio parahaemolyticus can be inhibited. The method is not only beneficial to the development and utilization of waste Chinese yam peel resources, but also beneficial to the development of natural antibacterial products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine and food manufacturing. More particularly, it relates to a yam peel fermentation liquor and a preparation method and application thereof. BACKGROUND

[0002] Yam is the tuber of an annual or perennial herbaceous plant of the Dioscoreaceae family, and there are many varieties, such as Huaiyashanyao, Tiejunshanyao, Mashanyao and Huishanyao. As a traditional functional food, yam is listed as a medicine and food homologous substance, and its health care function is particularly outstanding, such as tonifying the middle and replenishing qi, beautifying and nourishing the skin, regulating the intestines and stomach, preventing diabetes, preventing cardiovascular diseases, antioxidant, enhancing human immunity, etc. At present, yam deep processing products are increasingly diversified, mainly into three categories: first, starch products, such as yam noodles, bread, biscuits, etc.; second, beverage products, including yam juice beverage and composite beverage with other fruits and vegetables; third, instant food, such as yam slices, yam cans, yam jelly, etc. However, in the yam processing industry chain, in order to improve the taste, remove the potential fishy smell, or meet the medicinal standards, the yam usually needs to be peeled before processing or eating, and the yam peel by-product generated thereby accounts for about 20% of the weight of the raw material. At present, only a small amount of yam peel is used as feed, and most of it is directly discarded or burned in the open air, causing resource waste and environmental pollution.

[0003] Fermentation processing is one of the important processing methods of medicine and food homologous substances, which can affect the biological transformation of the main active ingredients of medicine and food homologous substances through microbial fermentation or microbial metabolism, so as to improve the nutritional value and medicinal function of raw materials. Lactic acid bacteria is one of the commonly used fermentation strains, and appropriate lactic acid bacteria fermentation process can enhance the medicinal properties of medicine and food homologous substances, reduce toxicity, improve flavor characteristics and improve resource utilization. There are also reports in the prior art that lactic acid bacteria such as Streptococcus thermophilus can be used to ferment yam, and it is also reported that yam polysaccharide can promote the growth of Streptococcus thermophilus.

[0004] The main nutrient components of yam peel are similar to those of yam meat, and the content of some active ingredients is higher. However, it is found that the lactic acid bacteria capable of fermenting yam cannot necessarily be used to ferment yam peel, which makes the fermentation of yam peel lack of available strains and unable to realize the recycling of waste yam peel. SUMMARY

[0005] The present application provides a yam peel fermentation liquor and a preparation method and application thereof to overcome the deficiencies in the prior art.

[0006] The first object of the present application is to provide a yam peel fermentation liquor.

[0007] The second object of the present application is to provide a preparation method of the yam peel fermentation liquor.

[0008] A third object of the present application is to provide the use of the Dioscorea opposita Thunb. peel fermentation liquor in inhibiting bacteria.

[0009] A fourth object of the present application is to provide the use of the Dioscorea opposita Thunb. peel fermentation liquor in preparing a product for inhibiting bacteria.

[0010] A fifth object of the present application is to provide the use of the Dioscorea opposita Thunb. peel fermentation liquor in preparing an antioxidant product.

[0011] A sixth object of the present application is to provide the use of the Dioscorea opposita Thunb. peel fermentation liquor in preparing a product for prolonging life.

[0012] A seventh object of the present application is to provide the use of the Dioscorea opposita Thunb. peel fermentation liquor in preparing an anti-aging product.

[0013] An eighth object of the present application is to provide a Lactobacillus plantarum Z165 strain.

[0014] A ninth object of the present application is to provide the use of the Lactobacillus plantarum Z165 strain in fermenting Dioscorea opposita Thunb. peel or in preparing a preparation for fermenting Dioscorea opposita Thunb. peel.

[0015] A tenth object of the present application is to provide the use of the Lactobacillus plantarum Z165 strain in fermenting Dioscorea opposita Thunb. peel to prepare a bacteriostatic product.

[0016] The above objects of the present application are achieved by the following technical solutions: The present application provides a Lactobacillus plantarum Z165 strain with good environmental tolerance and safety to address the lack of strains available for Dioscorea opposita Thunb. peel fermentation. Fermentation of Dioscorea opposita Thunb. peel using the Z165 strain not only effectively improves the antioxidant activity and probiotic properties of the obtained Dioscorea opposita Thunb. peel fermentation liquor, but also increases the bacteriostatic range and activity of the obtained Dioscorea opposita Thunb. peel fermentation liquor compared to the Dioscorea opposita Thunb. peel slurry before fermentation or the Lactobacillus plantarum Z165 strain alone, which can inhibit various pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, and Vibrio parahaemolyticus. Therefore, the Z165 strain, the Dioscorea opposita Thunb. peel fermentation liquor, and related applications thereof are claimed in the present application.

[0017] The present application provides a Dioscorea opposita Thunb. peel fermentation liquor obtained by fermenting Dioscorea opposita Thunb. peel with a Lactobacillus plantarum (Lactobacillus plantarum) Z165 strain. Lactiplantibacillus plantarum The Lactobacillus plantarum Z165 strain is deposited at the Guangdong Microbial Culture Collection Center, with the accession number GDMCC NO: 67162, and the deposit date is October 28, 2025.

[0018] The present application also provides a preparation method of the Dioscorea opposita Thunb. peel fermentation liquor, comprising the following steps: S1. preparing a yam peel slurry; the yam peel slurry is prepared by mixing yam peel powder with water, or homogenizing yam peel with water after mixing; the ratio of yam peel powder or yam peel to water is 1:5-25 g / mL; S2. inoculating Lactobacillus plantarum Z165 strain into the yam peel slurry for fermentation; the concentration of Lactobacillus plantarum Z165 strain in the yam peel slurry is 1×10 6 -5×10 6 CFU / mL; the fermentation temperature is 30-42℃.

[0019] Preferably, in S1, the ratio of yam peel powder or yam peel to water is 1:10-20 g / mL.

[0020] Further preferably, in S1, the ratio of yam peel powder or yam peel to water is 1:10-15 g / mL.

[0021] More preferably, in S1, the ratio of yam peel powder or yam peel to water is 1:15 g / mL.

[0022] Specifically, the preparation method of the yam peel powder is: crushing the dried yam peel and passing through a screen.

[0023] More specifically, the screen is a 50-200 mesh screen.

[0024] Preferably, the screen is a 100 mesh screen.

[0025] Preferably, in S2, the concentration of Lactobacillus plantarum Z165 strain in the yam peel slurry is 2×10 6 -5×10 6 CFU / mL.

[0026] Further preferably, the concentration of Lactobacillus plantarum Z165 strain in the yam peel slurry is 2×10 6 -3×10 6 CFU / mL.

[0027] More preferably, the concentration of Lactobacillus plantarum Z165 strain in the yam peel slurry is 2×10 6 CFU / mL.

[0028] Preferably, in S2, the fermentation temperature is 37℃.

[0029] Specifically, in S2, the fermentation time is 48-96 h.

[0030] Preferably, the fermentation time is 60-72 h.

[0031] Further preferably, the fermentation time is 72 h.

[0032] The application also protects the use of the Dioscorea opposita Thunb peel fermentation liquor in the preparation of an anti-aging product.

[0033] The application also protects the use of the Dioscorea opposita Thunb peel fermentation liquor in the preparation of an anti-aging product.

[0034] Specifically, the bacteria are one or more of Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Klebsiella pneumoniae, Listeria monocytogenes, and Vibrio parahaemolyticus.

[0035] Preferably, the bacteria are one or more of Escherichia coli, Vibrio parahaemolyticus, Salmonella typhimurium, and Staphylococcus aureus.

[0036] Further preferably, the bacteria are one or more of Escherichia coli, Vibrio parahaemolyticus, and Salmonella typhimurium.

[0037] The application also protects the use of the Dioscorea opposita Thunb peel fermentation liquor in the preparation of an anti-aging product.

[0038] Specifically, the Dioscorea opposita Thunb peel fermentation liquor improves its antioxidant effect by improving the clearance rate of DPPH, ABTS + , hydroxyl radicals, and total reducing power.

[0039] The application also protects the use of the Dioscorea opposita Thunb peel fermentation liquor in the preparation of a product for prolonging life.

[0040] Specifically, the prolonging of life is the prolonging of the life of an animal.

[0041] In a specific embodiment of the application, the animal is Caenorhabditis elegans.

[0042] The application also protects the use of the Dioscorea opposita Thunb peel fermentation liquor in the preparation of an anti-aging product.

[0043] Specifically, the Dioscorea opposita Thunb peel fermentation liquor resists aging by reducing the level of active oxygen and fat content in the body, alleviating acute oxidative stress damage, and regulating oxidative stress.

[0044] More specifically, the Dioscorea opposita Thunb peel fermentation liquor regulates oxidative stress by up-regulating CAT, SOD, GSH-Px enzyme activity and down-regulating MDA content.

[0045] The application also protects the plant Lactobacillus plantarum (Lactobacillus plantarum) Z165 strain, which is deposited with the Guangdong Microbial Culture Collection Center on October 28, 2025, and has a deposit number of GDMCC NO: 67162. Lactiplantibacillus plantarum Further preferably, the fermentation time is 72 h.

[0032] The application also protects the use of the Dioscorea opposita Thunb peel fermentation liquor in the preparation of an anti-aging product.

[0033] The application also protects the use of the Dioscorea opposita Thunb peel fermentation liquor in the preparation of an anti-aging product.

[0034] Specifically, the bacteria are one or more of Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Klebsiella pneumoniae, Listeria monocytogenes, and Vibrio parahaemolyticus.

[0035] Preferably, the bacteria are one or more of Escherichia coli, Vibrio parahaemolyticus, Salmonella typhimurium, and Staphylococcus aureus.

[0036] Further preferably, the bacteria are one or more of Escherichia coli, Vibrio parahaemolyticus, and Salmonella typhimurium.

[0037] The application also protects the use of the Dioscorea opposita Thunb peel fermentation liquor in the preparation of an anti-aging product.

[0038] Specifically, the Dioscorea opposita Thunb peel fermentation liquor improves its antioxidant effect by improving the clearance rate of DPPH, ABTS + , hydroxyl radicals, and total reducing power.

[0039] The application also protects the use of the Dioscorea opposita Thunb peel fermentation liquor in the preparation of a product for prolonging life.

[0040] Specifically, the prolonging of life is the prolonging of the life of an animal.

[0041] In a specific embodiment of the application, the animal is Caenorhabditis elegans.

[0042] The application also protects the use of the Dioscorea opposita Thunb peel fermentation liquor in the preparation of an anti-aging product.

[0043] Specifically, the Dioscorea opposita Thunb peel fermentation liquor resists aging by reducing the level of active oxygen and fat content in the body, alleviating acute oxidative stress damage, and regulating oxidative stress.

[0044] More specifically, the Dioscorea opposita Thunb peel fermentation liquor regulates oxidative stress by up-regulating CAT, SOD, GSH-Px enzyme activity and down-regulating MDA content.

[0045] The application also protects the plant Lactobacillus plantarum (Lactobacillus plantarum) Z165 strain, which is deposited with the Guangdong Microbial Culture Collection Center on October 28, 2025, and has a deposit number of GDMCC NO: 67162. Lactiplantibacillus plantarum

[0046] The present invention also claims protection for the use of the *Lactobacillus plantarum* Z165 strain in fermenting yam peel or in preparing formulations for fermenting yam peel.

[0047] This invention also seeks protection for the use of the *Lactobacillus plantarum* Z165 strain in the preparation of antibacterial products from fermented yam peel, and the use of the *Lactobacillus plantarum* Z165 strain in the preparation of formulations for the preparation of antibacterial products from fermented yam peel should also be within the scope of protection of this invention.

[0048] Specifically, the antibacterial product can inhibit one or more of Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Klebsiella pneumoniae, Listeria monocytogenes, and Vibrio parahaemolyticus.

[0049] The present invention has the following beneficial effects: This invention addresses the current lack of strains suitable for yam peel fermentation by providing a *Lactobacillus plantarum* strain Z165 with good environmental tolerance and safety. Using this *Lactobacillus plantarum* strain Z165 not only effectively ferments yam peels, enhancing the antioxidant activity and probiotic properties of the resulting fermented yam peel broth, but also, compared to the unfermented yam peel slurry or *Lactobacillus plantarum* strain Z165 alone, the resulting fermented yam peel broth exhibits increased antibacterial spectrum and activity, inhibiting various pathogens such as *Escherichia coli*, *Staphylococcus aureus*, *Listeria monocytogenes*, and *Vibrio parahaemolyticus*. This invention not only benefits the development and utilization of waste yam peel resources but also facilitates the development of natural antibacterial products. Attached Figure Description

[0050] Figure 1 The graph shows the test results of the fermentation effect of different lactic acid bacteria on yam peel; A in the graph represents the number of viable bacteria in the fermentation broth after different fermentation times; B in the graph represents the pH value of the fermentation broth after different fermentation times.

[0051] Figure 2 Figure A shows the acid resistance and bile salt tolerance test results of different lactic acid bacteria; Figure B shows the bile salt tolerance test results of strain Z165; Figure C shows the bile salt tolerance test results of strain M4; Figure D shows the bile salt tolerance test results of strain W1.

[0052] Figure 3 The graph shows the results of the gastrointestinal fluid tolerance test for different lactic acid bacteria; Figure A shows the survival rate of different lactic acid bacteria in artificial simulated gastric fluid; Figure B shows the survival rate of different lactic acid bacteria in artificial simulated intestinal fluid; different lowercase letters in the graph represent significant differences within groups.

[0053] Figure 4 The image shows the hemolytic test results for strain Z165.

[0054] Figure 5 Figure 1 shows the optimization results of the material-to-liquid ratio, inoculum size, and fermentation temperature in yam peel fermentation. Figure A shows the optimization results of the material-to-liquid ratio; Figure B shows the optimization results of the inoculum size; and Figure C shows the optimization results of the fermentation temperature.

[0055] Figure 6 The graph shows the changes in viable cell count and pH value of strain Z165 in the fermentation broth of yam peel over fermentation time; in the graph, A represents the change in viable cell count over fermentation time; in the graph, B represents the change in pH value over fermentation time; different lowercase letters in the graph represent significant differences within groups.

[0056] Figure 7 This is a graph showing the results of the in vitro antioxidant capacity test of yam peel fermentation broth; ** in the graph represent significant differences between groups. P <0.01).

[0057] Figure 8 The graph shows the effect of different concentrations of yam peel fermentation broth and yam peel slurry on the lifespan of nematodes.

[0058] Figure 9 The graph shows the effect of different concentrations of yam peel fermentation liquid and yam peel slurry on the motility of nematodes; A in the graph is a statistical graph of the number of times nematodes bend; B in the graph is a statistical graph of the number of times nematodes shake their heads; C in the graph is a statistical graph of the number of times nematodes swallow; different lowercase letters in the graph represent significant differences within groups, and different uppercase letters represent significant differences between groups.

[0059] Figure 10 The figure shows the effect of different concentrations of yam peel fermentation broth and yam peel slurry on the ROS level in nematodes.

[0060] Figure 11 The figure shows the effect of different concentrations of yam peel fermentation broth and yam peel slurry on the lipid content of nematodes; Figure A is the staining result of nematodes, and the scale bar in the figure is 100 μm; Figure B is a statistical graph of the staining results of nematodes using ImageJ software.

[0061] Figure 12 The figure shows the effect of different concentrations of yam peel fermentation broth and yam peel slurry on the survival rate of nematodes in an antioxidant stress experiment.

[0062] Figure 13 The figure shows the effect of different concentrations of yam peel fermentation broth and yam peel slurry on oxidative stress indices in the nematode antioxidant stress experiment; A in the figure is the CAT enzyme activity statistics; B in the figure is the SOD enzyme activity statistics; C in the figure is the GSH-Px enzyme activity statistics; D in the figure is the MDA content statistics; different lowercase letters in the figure represent significant differences within the group. Detailed Implementation

[0063] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0064] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0065] The plant lactobacillus used in this invention ( Lactiplantibacillus plantarum Z165, Lactobacillus fermentum ( Limosilactobacillus fermentum W1, Streptococcus thermophilus ( Streptococcus thermophilus Z63, Lactobacillus pentosus ( Lactobacillus pentosus Z51 and Lactobacillus paracasei ( Lacticaseibacillus paracasei All M4 strains were isolated, purified, identified, and preserved in the inventor's laboratory; the *E. coli* used were... Escherichia coli ATCC-25922, Staphylococcus aureus ( Staphylococcus aureus ATCC-25923, Salmonella Typhimurium ( Salmonella typhi ATCC-14028, Klebsiella pneumoniae ( Klebsiella pneumoniae ATCC-13883, Listeria monocytogenes ( Listeria monocytogenes ATCC-19115 and Vibrio parahaemolyticus ( Vibrio parahaemolyticus All strains of ATCC-14028 were purchased from the Guangdong Institute of Microbiology's Microbial Culture Collection Center. Among them, *Lactobacillus plantarum* strain Z165 is also deposited at the Guangdong Microbial Culture Collection Center (GDMCC), with accession number GDMCC NO: 67162, deposit date October 28, 2025, and address 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province.

[0066] The yam peel used in this invention is the peel of iron yam, which is produced in Jiaozuo, Henan.

[0067] Data analysis and processing methods: The experiment was repeated three times. Excel, Origin 2024, Graphpad Prism 10.4.1, and SPSS 16.0 software were used for data processing and analysis. Different lowercase letters in the graphs and tables represent significant differences within groups, different uppercase letters represent significant differences between groups, and ** represents... P <0.01, * represents 0.01 < P <0.05.

[0068] Example 1: Test on the fermentation effect of different lactic acid bacteria on yam peel This invention uses five strains of lactic acid bacteria (Lactobacillus plantarum Z165, Lactobacillus fermentum W1, Streptococcus thermophilus Z63, Lactobacillus pentosus Z51, and Lactobacillus paracasei M4) preserved in the laboratory to ferment yam peel, and prepare corresponding yam peel fermentation broths. The fermentation effect of the lactic acid bacteria on yam peel is analyzed by detecting the number of viable bacteria and pH value in the obtained fermentation broth.

[0069] Preparation of yam peel fermentation broth: 1. Activation of bacterial strains: The preserved Lactobacillus plantarum Z165, Lactobacillus fermentum W1, Streptococcus thermophilus Z63, Lactobacillus pentosus Z51 and Lactobacillus paracasei M4 were inoculated from glycerol tubes into MRS liquid medium (Guangzhou Huankai Biotechnology Co., Ltd.), and cultured statically at 37℃ for 24 h. After activation twice, the seed culture of each lactic acid bacteria was obtained for later use.

[0070] 2. Preparation of yam peel slurry After drying, the yam peel is crushed and passed through a 100-mesh sieve. The yam peel powder is mixed with water at a ratio of 1:15. The mixture is then heated in a 95°C water bath for 20 minutes to sterilize it. After cooling, the yam peel slurry is obtained.

[0071] 3. Fermentation culture The activated seed cultures of each lactic acid bacteria were inoculated into yam peel slurry, with the inoculation concentration controlled at 1×10⁻⁶. 6 The cells were incubated at 37°C with CFU / mL and fermented statically. Samples were taken at 0, 36, and 60 h of fermentation to detect the viable cell count and pH value in the fermentation broth.

[0072] The test results of the fermentation effects of different lactic acid bacteria on yam peel are as follows: Figure 1 As shown; Figure 1 In this context, A represents the number of viable bacteria in the fermentation broth after different fermentation times. Figure 1 In this context, B represents the pH value of the fermentation broth after different fermentation times. Figure 1 It can be seen that the *Lactobacillus plantarum* strain Z165 has the best fermentation effect on yam peel, and the viable bacteria count in the fermentation broth after 60 h of fermentation of yam peel can reach 1.07 × 10⁻⁶. 8 The fermentation broth concentration was reduced to CFU / mL, and the pH value also dropped to 3.79. Meanwhile, Streptococcus thermophilus strain Z63 showed virtually no fermentation ability on yam peel, with no significant changes in viable cell count or pH value in the fermentation broth. The fermentation ability of the strains was ranked as follows: Z165 > M4 > W1 > Z51 > Z63. The first three lactic acid bacteria strains were used for subsequent experiments.

[0073] Example 2: Environmental tolerance and safety analysis of lactic acid bacteria This invention analyzes the environmental tolerance and safety of *Lactobacillus plantarum* Z165, *Lactobacillus fermentum* W1, and *Lactobacillus paracasei* M4.

[0074] 1. Analysis of the acid resistance and bile salt resistance of lactic acid bacteria Acid tolerance test: MRS liquid culture medium with pH values ​​of 1, 2, 3, 4, and 5 was prepared, and lactic acid bacteria were inoculated at a ratio of 5% (v / v). After static incubation at 37℃ for 24 h, the OD of each group was measured. 600nm The positive control group was MRS medium at pH 6.

[0075] Bile salt tolerance test: MRS liquid medium containing 0.05, 0.1, 0.15, 0.2, 0.25, and 0.3 wt% porcine bile salts was prepared, and lactic acid bacteria were inoculated at a ratio of 5% (v / v). After static incubation at 37°C for 0, 3, 8, and 24 h, the OD of each group was measured. 600nm The control group was MRS medium without porcine bile salts.

[0076] The results of acid resistance and bile salt resistance tests of different lactic acid bacteria are as follows: Figure 2 As shown; Figure 2 In the figure, A represents the acid resistance test results of different lactic acid bacteria; Figure 2 Figures B through D in the graphs show the bile salt tolerance test results for strains Z165, M4, and W1, respectively. Figure 2 It was found that the three strains showed almost no growth at pH ≤ 2, increased growth at pH ≥ 3, and good growth at pH 5, essentially on par with the control group (pH 6). The order of acid tolerance was: Z165 > W1 > M4. Furthermore, the growth ability of the strains was inversely proportional to the bile salt concentration; all three strains ceased growth when the bile salt concentration was ≥ 0.15%. At a bile salt concentration of 0.05%, the OD values ​​of strains Z165, M4, and W1 after 24 h of culture were [not specified in the original text]. 600nm The values ​​were 0.950, 0.792, and 0.791, respectively, which were 85.59, 73.70, and 68.5% of the control group. The order of growth capacity was: Z165 > M4 > W1.

[0077] 2. Gastrointestinal fluid tolerance analysis of lactic acid bacteria The method described in the reference (Cao Haipeng, Xu Xingna, Wen Xiaofei. Isolation and probiotic properties of a strain of *Lactobacillus plantarum* with strong antibacterial activity [J], China Brewing, 2021, 40(06): 141-146.) was used to prepare artificial gastric and intestinal fluids. The activated lactic acid bacteria seed culture was inoculated into the artificial gastric and intestinal fluids at a ratio of 5% (v / v). After static incubation at 37℃ for 0 and 3 h, the bacterial culture was diluted and spread on MRS plates and counted. The viable bacteria count at 0 and 3 h was used to determine the gastrointestinal fluid tolerance of the lactic acid bacteria. The survival rate was calculated using the following formula: ; In the formula,X Survival rate (%) A A, viable cell count after 3 h of incubation, CFU / mL; B, viable cell count after 0 h of incubation, CFU / mL.

[0078] Results of gastrointestinal fluid tolerance tests for different lactic acid bacteria are as follows: Figure 3 As shown; Figure 3 In this context, A represents the survival rate of different lactic acid bacteria in artificial simulated gastric juice; Figure 3 In the figure, B represents the survival rate of different lactic acid bacteria in simulated intestinal fluid. After incubation in simulated gastric fluid for 3 hours, the survival rates of the three lactic acid bacteria were all above 75%, ranked from strongest to weakest as follows: W1 > Z165 > M4, with survival rates of 88.22%, 84.58%, and 79.05%, respectively. The survival rates of the three strains in simulated intestinal fluid were all above 60%, ranked from strongest to weakest as follows: Z165 > W1 > M4, with survival rates of 89.52%, 86.58%, and 60.60%, respectively. Since strains Z165 and W1 showed similar tolerance to gastrointestinal fluid, and considering other properties of strain Z165, it was selected as the subject of subsequent research.

[0079] 3. Hemolytic activity analysis of strain Z165 Single colonies of the Z165 strain to be tested were streaked onto Columbia blood agar plates and incubated upside down at 37°C for 48 h. Hemolysis was then observed. The positive control group consisted of Listeria monocytogenes. Hemolytic bacteria can pose certain health risks to humans, and the Food and Agriculture Organization of the United Nations stipulates that probiotics in food should not exhibit hemolysis. Hemolysis can be classified into three types: α-hemolysis, characterized by a greenish-yellow hemolytic zone around the colony; β-hemolysis, characterized by a clear hemolytic zone around the colony; and γ-hemolysis, characterized by no obvious changes around the colony, i.e., no hemolysis.

[0080] The hemolytic test results of strain Z165 are as follows: Figure 4 As shown. By Figure 4 It can be seen that the positive control (Listeria monocytogenes) is β-hemolytic, while the Z165 strain is γ-hemolytic, indicating that the Z165 strain is not hemolytic.

[0081] 4. Antimicrobial susceptibility analysis of strain Z165 The method described in the reference (Huang Fang, Liu Xintong, Huang Jiarong, et al. Screening and safety and probiotic properties of cholesterol-lowering lactic acid bacteria in sour tea [J]. Chinese Journal of Food Science, 2025, 25(03): 82-92.) was used to conduct drug susceptibility tests on strain Z165 using the agar diffusion method. The antibiotic susceptibility was determined according to the latest standards of the American CLSI (Clinical and Laboratories Standards Institute). The results are shown in Table 1.

[0082] Table 1. Antibiotic susceptibility analysis of strain Z165

[0083] Note: In the table, S represents "sensitive"; I represents "intermediate"; and R represents "drug resistance".

[0084] As shown in Table 1, strain Z165 is sensitive to most antibiotics, but not to aminoglycosides and quinolones. Current research has found that lactic acid bacteria exhibit natural resistance to aminoglycosides and quinolones, possibly because lactic acid bacteria produce aminoglycoside-inactivating enzymes during their growth and metabolism, leading to the inactivation of aminoglycoside antibiotics.

[0085] In summary, the Z165 strain described in this invention exhibits good environmental tolerance and safety.

[0086] Example 3: Optimization of fermentation conditions for yam peel To optimize the fermentation process of yam peel using lactic acid bacteria, this invention tested three key parameters: the material-to-liquid ratio, the inoculum size, and the fermentation temperature. A single-factor experimental method was used, with samples taken at specific time points during fermentation. Viable cell counts were then performed using MRS plate dilution and plating to assess the impact of each parameter on cell growth.

[0087] 1. Optimization of the feed-liquid ratio The activated seed culture of strain Z165 was resuspended in PBS and the concentration was adjusted to 1×10⁻⁶. 8 CFU / mL was inoculated into yam peel slurry (prepared in the same way as in Example 1) at an inoculation rate of 2% (v / v) and a material-to-liquid ratio of 1 / 5, 1 / 10, 1 / 15, 1 / 20, and 1 / 25 g / mL, respectively, and incubated at 37°C for 72 h. Samples were taken at 0, 12, 24, 36, 48, 60, and 72 h, respectively, and the viable count was determined by the dilution plating method.

[0088] 2. Optimization of vaccination volume Based on the determined optimal material-to-liquid ratio, seed culture of strain Z165 was inoculated at inoculation rates of 1%, 2%, 3%, 4%, and 5% (v / v) and cultured at 37°C for 72 h. During this period, samples were taken at 0, 12, 24, 36, 48, 60, and 72 h, and the viable cell count was determined by the dilution plating method to determine the optimal inoculation rate.

[0089] 3. Optimization of fermentation temperature Using the determined optimal material-to-liquid ratio and inoculum size, the cultures were statically incubated at 30, 37, and 42°C for 72 h. Samples were taken at 0, 12, 24, 36, 48, 60, and 72 h during this period, and the viable cell count was determined using the dilution-spreading method to determine the optimal fermentation temperature.

[0090] The optimization results of the material-to-liquid ratio, inoculum size, and fermentation temperature in yam peel fermentation are as follows: Figure 5 As shown, Figure 5 In the figures, A through C represent the optimized results for the substrate-to-liquid ratio, inoculum size, and fermentation temperature, respectively. Figure 5 As shown in A, the viable cell count of Z165 is positively correlated with the concentration of yam peel slurry. When the material-to-liquid ratio increases from 1 / 25 to 1 / 5 g / mL, the viable cell count increases from 9.4 × 10⁻⁶ g / mL. 7 CFU / mL increased to 2.7 × 10⁻⁶ 8 CFU / mL, with a material-to-liquid ratio of 1 / 15 g / mL, the viable count was 2.0 × 10⁻⁶. 8 CFU / mL, a high material-to-liquid ratio results in a higher viable cell count, but the yam peel slurry is too viscous. Considering both material cost and fermentation efficiency, 1 / 15 g / mL was determined to be the optimal choice. Figure 5 From B, we know that a 2% (v / v) bacterial suspension (i.e., a concentration of 2 × 10⁻⁶) 6 An inoculum size of 2 × 10⁻⁶ CFU / mL offers the best cost-effectiveness while ensuring a high viable count. Subsequent experiments selected an inoculum size of 2 × 10⁻⁶ CFU / mL. 6 The inoculation concentration was CFU / mL. Figure 5 According to C, 37℃ is the optimal fermentation temperature.

[0091] 4. Changes in viable cell count and pH value in fermentation broth over fermentation time. This invention tested the changes in viable cell count and pH value of strain Z165 in the fermentation broth under determined optimal material-to-liquid ratio, inoculum amount and fermentation temperature conditions, thereby determining the fermentation endpoint.

[0092] Under the determined optimal material-to-liquid ratio, inoculum size, and fermentation temperature, the changes in viable cell count and pH value of strain Z165 in the fermentation broth with fermentation time are as follows: Figure 6 As shown. By Figure 6 It was observed that the viable cell count of strain Z165 reached its peak around 60 h and remained at a high level for the following 12 h, indicating a stable growth phase. After 96 h, the viable cell count decreased significantly, and a large number of lactic acid bacteria began to die. Within the first 36 h, the pH of the fermentation broth dropped sharply; this stage represents the exponential growth phase and the period of vigorous primary metabolism of the lactic acid bacteria. After 36 h, the rate of pH decrease slowed, and the pH reached 3.48 at 144 h. Based on these findings, the fermentation endpoint for yam peel fermentation was determined to be 72 h.

[0093] Example 4 Activity detection of yam peel fermentation broth This invention is applicable to a feed-to-liquid ratio of 1 / 15 g / mL and a concentration of 2×10⁻⁶ g / mL. 6 The antioxidant and other activities of the fermented yam peel obtained by fermentation under the conditions of CFU / mL bacterial suspension inoculum concentration, fermentation temperature of 37℃ and fermentation time of 72 h were detected.

[0094] 1. In vitro antioxidant capacity test of yam peel fermentation broth The reference method (Chen L, Sun Y, Cai H, et al. Simultaneous determination of eleven bioactive constituents in honey-processed licorice by high-performance liquid chromatography-diode array detector and its application from the perspective of processing influence under orthogonal design[J]. World Journal of Traditional Chinese Medicine, 2022,8(3). and Meng X, Zheng S, Yin Z, et al. Apigenin ameliorates imiquimod-induced psoriasis in C57BL / 6Jmice by inactivating STAT3 and NF-KB[J]. Food Science and Human Wellness,2024,13(1): 211-224.) was used to determine the effect of fermented yam peel dilution on DPPH and ABTS. + The ability to scavenge hydroxyl radicals and the total reducing power.

[0095] Among them DPPH, ABTS + The formula for calculating the hydroxyl radical scavenging rate is shown below:

[0096] In the formula, X represents the clearance rate (%), A1 represents the absorbance of the experimental group (sample solution + working solution), A2 represents the sample control (sample solution + distilled water), and A3 represents the blank control (distilled water + working solution).

[0097] The total reducing power is expressed as a solution of vitamin C at the corresponding concentration. Using vitamin C as a standard, the regression equation for the standard curve is: y = 4.3124x + 0.0562, R0 2 =0.99.

[0098] Undiluted yam peel fermentation broth exhibits strong free radical scavenging ability. It was diluted five times before its antioxidant capacity was measured. Simultaneously, unfermented yam peel slurry was also diluted five times as a control. The in vitro antioxidant capacity test results of the yam peel fermentation broth are as follows: Figure 7 As shown, the antioxidant properties of the fermented yam peel broth are significantly higher than those of the unfermented yam peel slurry group. The diluted fermented broth showed better antioxidant activity against DPPH and ABTS. + The hydroxyl radical scavenging rates reached 48.07%, 93.79%, and 54.40%, respectively, compared to 31.31%, 82.85%, and 31.93% in the unfermented group. Compared to the unfermented group, the fermented group diluted solution significantly reduced DPPH and ABTS. + The antioxidant capacity of yam peel increased by 53.53%, 13.20%, and 70.37%, respectively. At this point, the total reducing power of the fermented group and the diluted unfermented yam peel slurry were equivalent to vitamin C solutions with concentrations of 85.83 and 45.27 μg / mL, respectively, with the total reducing power increasing by 89.60% after fermentation. In summary, fermentation significantly enhances the antioxidant capacity of yam peel, with the increase in each indicator from largest to smallest being: total reducing power > hydroxyl radical > DPPH > ABTS. + .

[0099] 2. Antibacterial activity test of yam peel fermentation broth After centrifuging the yam peel fermentation broth at 8000 r / min for 10 min, the supernatant was collected for later use. The antibacterial activity of the fermented yam peel supernatant was tested using the Oxford cup method with *Escherichia coli*, *Staphylococcus aureus*, *Salmonella*, *Vibrio parahaemolyticus*, *Listeria monocytogenes*, and *Klebsiella pneumoniae* as indicator bacteria. A lactic acid solution with the same pH as the fermentation supernatant was prepared for the antibacterial test to eliminate the influence of pH on the indicator bacteria. Unfermented yam peel slurry served as a negative control group.

[0100] The results are shown in Table 2. The fermented yam peel broth has a broad antibacterial spectrum and is more effective than lactic acid solution at the same pH value, while the unfermented yam peel slurry has no antibacterial activity. This indicates that yam peel can acquire antibacterial activity through lactic acid bacteria fermentation. The fermented broth showed the best inhibitory effect on Escherichia coli, with an inhibition zone diameter of up to 31.83 mm. The inhibitory effect on Listeria monocytogenes was weaker, with an inhibition zone diameter of only 13.80 mm. The inhibitory ability of the yam peel fermented broth against various pathogenic bacteria was ranked as follows: Escherichia coli > Vibrio parahaemolyticus > Salmonella typhimurium > Staphylococcus aureus > Klebsiella pneumoniae > Listeria monocytogenes.

[0101] Table 2 Diameter of the inhibition zone in the yam peel fermentation broth

[0102] 3. The beneficial properties of fermented yam peel on nematodes (1) Grouping of nematodes Under aseptic conditions, nematodes were inoculated onto the coating. E. coli OP50 nematode growth medium (NGM) was used, and the plates were rotated every 4 days. Nematodes in the oviposition stage were collected after rotation for synchronization. The NGM plates were washed multiple times with M9 buffer to collect nematodes. The nematodes were lysed using sodium hypochlorite-sodium hydroxide lysis buffer to obtain a large number of eggs. After lysis, a large amount of M9 buffer was quickly added to wash the eggs. The purified eggs were then inoculated onto plates coated with... E. coli Nematodes were cultured on OP50 NGM medium at 20°C for 48–54 h to obtain highly uniform L4-stage nematodes for subsequent experiments. To prevent nematode oviposition from interfering with the experimental results, 100 μmol / L FUDR solution was added to the NGM medium in subsequent experiments. The nematode groups are shown in Table 3. Synchronized nematodes were inoculated onto NGM medium coated with... E. coli OP50 bacterial suspensions were cultured in NGM medium containing different components and divided into CK, T1, T2, K1, and K2 groups. After being cultured in an incubator at 20°C for a period of time, subsequent experiments were conducted.

[0103] Table 3. Grouped experiments of wild-type Caenorhabditis elegans N2 nematode.

[0104] (2) Effect of yam peel fermentation liquid on the lifespan of nematodes According to the grouping in Table 3, synchronized nematodes were collected using a nematode-picking needle and placed in NGM culture media with different compositions. The number of surviving nematodes was recorded daily until all nematodes died, with at least 30 nematodes per group. Survival curves were plotted based on the nematode lifespan measurement data. Nematode lifespan is an important indicator related to lipid-lowering and antioxidant effects. Figure 8 As shown in Table 4, the lifespan of nematodes was ranked as follows: T2>K2>T1>K1>CK group. Compared with the CK group, the maximum lifespan of nematodes increased by 14.63%, 24.84%, 6.59%, and 15.72%, respectively. The fermented yam peel liquid was more effective than the unfermented yam peel slurry in prolonging the lifespan of nematodes, and the high-dose experimental group was more effective than the low-dose group.

[0105] Table 4. Lifespan of wild-type Caenorhabditis elegans N2 from different groups

[0106] (3) Effects of yam peel fermentation liquid on the motility of nematodes Nematodes cultured to days 3 and 9 were randomly selected from each group. The number of times the nematodes bent within 30 seconds, the number of times they swayed their heads within 1 minute, and the number of times they swallowed within 30 seconds were measured. Each group had at least 10 nematodes. Nematode motility is one of the core health indicators of nematodes, directly reflecting their nervous system function, muscle condition, and overall energy metabolism level. Figure 9 It can be seen that the motility of nematodes decreased significantly with prolonged culture time, but there were still significant differences in motility levels among different groups. On day 3, compared with the CK group, the number of bends, head swings, and swallows in the T1 group increased by 14.23%, 16.86%, and 22.01%, respectively; the number of bends, head swings, and swallows in the T2 group increased by 17.79%, 20.85%, and 36.50%, respectively; the number of bends, head swings, and swallows in the K1 group increased by 5.55%, 10.17%, and 2.59%, respectively; and the number of bends, head swings, and swallows in the K2 group increased by 13.45%, 15.69%, and 28.12%, respectively. On day 9, the motility of all groups decreased, but was still higher than that of the CK group, with the T2 group still showing the best motility level.

[0107] (4) Effect of yam peel fermentation broth on ROS levels in nematodes ROS (Reactive Oxygen Species) is a free radical that is activated under oxidative stress and aging conditions, leading to damage to macromolecules such as lipids, proteins, and DNA in organisms, thereby accelerating disease and aging processes. Therefore, measuring the ROS level in nematodes can directly assess their health status and environmental stress. Approximately equal amounts of nematodes cultured for 3 days were collected from each group, rinsed, centrifuged, and then stained with an appropriate amount of DCFH-DA working solution (final concentration 20 μmol / L) for 3 h in the dark. Fluorescence intensity was measured using a continuous wavelength multi-functional microplate at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. The ROS level of the CK group was used as a standard control, and the results are expressed as a relative percentage of the CK group. Results are as follows: Figure 10 As shown, by Figure 10 It can be seen that the T2 group had the lowest ROS level in the nematodes. Compared with the CK group, the reactive oxygen species levels in the T1, T2, K1 and K2 groups decreased by 18.43%, 43.10%, 11.52% and 33.72% respectively. This indicates that both fermented yam peel liquid and unfermented yam peel slurry can reduce the reactive oxygen species level in the nematodes, but the effect of fermented yam peel liquid is more superior.

[0108] (5) Effect of yam peel fermentation liquid on the fat content of nematodes Due to its transparent body wall, *Caenorhabditis elegans* can be stained with Oil Red O dye, allowing for the visualization and detection of lipids under a microscope. Observing the staining intensity and distribution characteristics under a microscope provides a quantitative assessment of the organism's nutritional status and lipid metabolism dynamics, and serves as an indirect indicator of aging and health in nematodes.

[0109] Ten nematodes from each group, cultured for 6 days, were randomly selected using a nematode picking needle. After fixation with paraformaldehyde for 15 min, they were centrifuged and dehydrated for 10 min with 60% isopropanol solution. The mixture was centrifuged again, retaining a small amount of isopropanol solution. The nematodes underwent repeated freeze-thaw cycles in liquid nitrogen to remove their chitinous exoskeleton. 200 μL of Oil Red O dye working solution was added, and staining was performed in the dark for 2 h. The supernatant was discarded after centrifugation. The nematodes were transferred to 2% agarose gel slides, and the staining results were observed and photographed using an optical microscope. ImageJ software was used to analyze and statistically analyze the staining results. The results are as follows: Figure 11 As shown, the CK group of nematodes had the deepest dye color, while the staining depth of other groups decreased to varying degrees. The order of fat density was: CK>K2>T1>K2>T2 group. This indicates that the fermented yam peel liquid can effectively reduce the fat content in nematodes and has a positive effect on regulating fat metabolism.

[0110] (6) Effects of yam peel fermentation broth on the acute oxidative stress capacity of nematodes Twenty nematodes from each group, cultured for three days, were transferred to NGM liquid medium containing 20 mmol / L hydrogen peroxide. The number of surviving nematodes was recorded every 40 minutes until all nematodes died. Survival curves were plotted based on the nematode antioxidant stress experiment lifespan data. The experimental results are as follows: Figure 12 As shown in Table 5, under acute oxidative stress, all nematodes died within 240 minutes. The average lifespan of nematodes varied considerably among different groups. The average lifespan of nematodes in group T2 reached 140 minutes, while that in group CK was only 101.33 minutes. Other treatment groups showed varying degrees of increase in lifespan. The lifespan was ranked as follows: T2>K2>T1>K1>CK. This indicates that both fermented yam peel liquid and unfermented yam peel slurry can alleviate acute oxidative stress damage in nematodes to some extent, but fermented yam peel liquid is more effective.

[0111] Table 5 Lifespan of wild-type Caenorhabditis elegans under acute oxidative stress N2.

[0112] Note: The table uses letter notation to represent differences between groups: the same letter indicates that the difference between groups is not significant. P >0.05), different letters indicate significant differences between groups ( P <0.05).

[0113] (7) Effects of yam peel fermentation broth on oxidative stress indicators of nematodes According to the grouping in Table 3, the nematodes were repeatedly washed with M9 buffer and centrifuged. Following the kit instructions, the activities of CAT, SOD, GSH-Px enzymes, and MDA content in the supernatant were measured, and the results were normalized to the protein level. Experimental results are as follows: Figure 13 As shown, with the CK group as the control, the CAT, SOD, and GSH-Px enzyme activities in each group were higher than those in the control group, and the trends were similar. In addition, the MDA content in the nematodes showed a decreasing trend. It is suggested that the fermented yam peel broth can regulate the oxidative stress of nematodes by upregulating the CAT, SOD, and GSH-Px enzyme activities and downregulating the MDA content in the nematodes. Its effect is closely related to the concentration and whether fermentation is carried out. Moreover, the oxidative stress regulation effect of the unfermented yam peel slurry is significantly weaker than that of the fermented broth.

[0114] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A fermented yam peel liquid, characterized in that, The yam peel fermentation liquid is produced by *Lactobacillus plantarum* (… Lactiplantibacillus plantarum The Z165 strain was obtained by fermenting yam peel; the Z165 strain of *Lactobacillus plantarum* was deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67162 and deposit date of October 28, 2025.

2. The method for preparing the yam peel fermentation liquid according to claim 1, characterized in that, Includes the following steps: S1. Preparation of yam peel slurry; the yam peel slurry is prepared by mixing yam peel powder with water, or by mixing yam peel with water and then beating it into a homogenate; the ratio of yam peel powder or yam peel to water is 1:5-25 g / mL; S2. Inoculate the *Lactobacillus plantarum* Z165 strain into the yam peel slurry for fermentation; the concentration of *Lactobacillus plantarum* Z165 strain in the yam peel slurry is 1×10⁻⁶. 6 ~5×10 6 CFU / mL; fermentation temperature: 30℃~42℃.

3. The application of the yam peel fermentation liquid according to claim 1 in inhibiting bacteria, characterized in that, The bacteria are one or more of the following: Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Klebsiella pneumoniae, Listeria monocytogenes, and Vibrio parahaemolyticus.

4. The use of the yam peel fermentation broth according to claim 1 in the preparation of products for inhibiting bacteria, characterized in that, The bacteria are one or more of the following: Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Klebsiella pneumoniae, Listeria monocytogenes, and Vibrio parahaemolyticus.

5. The application of the yam peel fermentation liquid according to claim 1 in the preparation of antioxidant products.

6. The use of the yam peel fermentation liquid according to claim 1 in the preparation of products for extending shelf life.

7. The application of the yam peel fermentation liquid according to claim 1 in the preparation of anti-aging products.

8. A type of *Lactobacillus plantarum* ( Lactiplantibacillus plantarum Z165 strain, characterized in that, The *Lactobacillus plantarum* strain Z165 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO: 67162, and the deposit date is October 28, 2025.

9. The use of the Lactobacillus plantarum Z165 strain according to claim 8 in fermenting yam peel or in preparing formulations for fermenting yam peel.

10. The application of the *Lactobacillus plantarum* Z165 strain according to claim 8 in the preparation of antibacterial products from fermented yam peel, characterized in that... The antibacterial product can inhibit one or more of the following bacteria: Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Klebsiella pneumoniae, Listeria monocytogenes, and Vibrio parahaemolyticus.

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