A strain of Lactobacillus plantarum LWQ17 and its application

Abstract

The present invention discloses Lactobacillus plantarum LWQ17 and applications thereof, and belongs to the field of microbial technology. The deposit number of Lactobacillus plantarum LWQ17 is CCTCC NO:M 20231803. The present invention provides an application of Lactobacillus plantarum LWQ17 in the preparation of fungal inhibitors. In vitro antibacterial experiments show that its fermentation supernatant can significantly inhibit the growth of molds; Lactobacillus plantarum LWQ17 has good antioxidant capacity, is non-hemolytic, and has high biological safety. The Lactobacillus plantarum LWQ17 provided by the present invention is not mutually exclusive with yeast, and has no effect on yeast growth. It is used in sour dough to construct an in-situ preservation technology for steamed bread, which can effectively inhibit fungal contamination of steamed bread during storage at room temperature and extend the shelf life of steamed bread.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Lactobacillus plantarum LWQ17 and applications thereof. Background Art

[0002] Fungi are widely distributed in the natural environment, and food spoilage caused by fungal growth has become a major problem worldwide. In addition, some fungi can produce mycotoxins, which have a negative impact on public health and food safety.

[0003] Steamed bread is a kind of food made by mixing flour, yeast and water, kneading, shaping and steaming. The finished product is hemispherical or rectangular in shape. It is soft and delicious, rich in nutrition, and is one of the traditional Chinese noodles. However, steamed bread is easily infected by mold during production, transportation and storage, resulting in a short shelf life, which is only 1 to 3 days at room temperature. The main reasons for the spoilage of steamed bread are: 1. The business mode of steamed bread is mainly street stalls and small workshops, and the processing conditions and sanitary conditions are not guaranteed. 2. The dough contains various nutrients (carbohydrates, proteins, fats, minerals, vitamins, etc.) required for the growth of microorganisms, and has a high water content, which makes steamed bread very easy to spoil during storage, among which mold germination is the most difficult to control.

[0004] Sourdough is a fermentation agent with lactic acid bacteria and yeast as the main flora. Sourdough fermentation can change the food composition by utilizing the metabolism of various microorganisms (Research Progress on the Application of Sourdough in Steamed Bread Fermentation [J]. Food Industry Science and Technology, 2021, 42(15): 389-395.). Sourdoughs made from different raw materials or regions contain different lactic acid bacteria. Currently, the lactic acid bacteria isolated and identified from sourdough are mainly Lactobacillus. Lactic acid bacteria can produce different types of antibacterial substances during the fermentation process, such as bacteriocins, exopolysaccharides, organic acids, fatty acids and hydrogen peroxide. Applying them in flour products can extend the shelf life of food. Lactic acid bacteria can also improve the antioxidant properties of dough (How the sourdough may affect the functional features of leavened baked goods [J]. Food Microbiology, 2014, 37: 30-40).

[0005] Therefore, it is a technical problem that technicians in this field need to solve to screen lactic acid bacteria with inhibitory fungi activity and antioxidant capacity from sourdough and apply them to extend the shelf life of steamed bread at room temperature. Summary of the invention

[0006] The purpose of the present invention is to provide a lactic acid bacterium which can effectively inhibit the proliferation and growth of spoilage fungi in fermented pasta, and to apply the lactic acid bacterium to the development of food additives for inhibiting fungi.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] The invention separates a new lactic acid bacteria strain from sour dough, a steamed bread starter, and co-cultivates the fermentation supernatant of the isolated strain with different mold spores to screen out a strain with strong antibacterial activity against steamed bread spoilage mold. Through microbiological characteristics such as morphological characteristics, culture properties, physiological and biochemical characteristics, and 16S rRNA gene comparison analysis, it is identified that the strain belongs to the genus Lactobacillus and Lactobacillus plantarum, so it is named Lactobacillus plantarum LWQ17.

[0009] Lactobacillus plantarum LWQ17 was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on September 27, 2023, with the accession number: CCTCC NO: M 20231803.

[0010] In vitro antibacterial activity experiments showed that Lactobacillus plantarum LWQ17 exhibited excellent antibacterial activity against Aspergillus niger, Penicillium sp, Alternaria sp, Aspergillus flavus, Chaetomium globosum, Cladosporium sp, Penicillium italicum, and Rhizopus oryzae.

[0011] The study of the antibacterial mechanism found that the metabolites of Lactobacillus plantarum LWQ17 can destroy the mold cell membrane and change its permeability, causing the contents of the mold cells to leak out and exert a bactericidal effect. The antibacterial effect is produced by the action of organic acids in the metabolites.

[0012] The present invention provides application of the Lactobacillus plantarum LWQ17 in preparing fungal inhibitors.

[0013] The Lactobacillus plantarum LWQ17 or its metabolites or its inactivated bacteria can be used to prepare fungi-inhibiting drugs or food additives. The antibacterial activity of the Lactobacillus plantarum LWQ17 metabolites is not affected by temperature.

[0014] The Lactobacillus plantarum LWQ17 provided by the invention has no hemolytic property and has biological safety when used for preparing medicines or food additives.

[0015] Further, the fungus may be but not limited to mold, specifically, the fungus may be but not limited to any one or more of Aspergillus niger, Penicillium, Alternaria, Aspergillus flavus, Chaetomium globosum, Cladosporium, Penicillium italicum, and Rhizopus oryzae.

[0016] Furthermore, the fungal inhibitor includes the fermentation supernatant of Lactobacillus plantarum LWQ17 or its inactivated bacterial cells. The present invention utilizes the fermentation product of Lactobacillus plantarum LWQ17 or its inactivated bacterial cells to prepare the fungal inhibitor.

[0017] Further, Lactobacillus plantarum LWQ17 was inoculated into MRS solid culture medium, cultured at 37° C. for 48 h under an anaerobic environment, and then inoculated into MRS broth at a volume ratio of 2%, cultured at 37° C. for 24 h, taken out, centrifuged at 4° C. and 8000 r / min for 5 min, and the supernatant was filtered with a 0.22 μm filter membrane to obtain a cell-free fermentation supernatant, thereby obtaining the fermentation supernatant.

[0018] The preparation method of the inactivated bacterial body of Lactobacillus plantarum LWQ17 comprises the following steps: inoculating Lactobacillus plantarum LWQ17 into an MRS solid culture medium, culturing at 37°C for 48 hours under an anaerobic environment, inoculating into an MRS broth at a volume ratio of 2%, culturing at 37°C for 24 hours, taking out the broth, placing it in a 100°C water bath for 20 minutes, centrifuging it at 4°C and 8000 r / min for 5 minutes, discarding the supernatant, and obtaining the inactivated bacterial body.

[0019] When preparing the fungal inhibitor, in addition to the above-mentioned Lactobacillus plantarum LWQ17 or its metabolites or its inactivated bacteria as active ingredients, a carrier that helps stabilize the active ingredients and promotes body absorption is added. Specifically, when the fungal inhibitor is used as a drug, a pharmaceutically acceptable carrier is also included; when the fungal inhibitor is used as a food additive, a food-acceptable carrier is also included.

[0020] Further, the plant lactobacillus LWQ17 or its fermentation supernatant or its inactivated thalli is used as a fermented food additive. The plant lactobacillus LWQ17 provided by the present invention and yeast are not mutually exclusive, that is, they have no effect on yeast growth, therefore, the plant lactobacillus LWQ17 or its fermentation supernatant or its inactivated thalli can be used together with yeast to make fermented food. The fermented food can be, but is not limited to, steamed bread and bread.

[0021] The present invention also provides a method for extending the shelf life of steamed bread at room temperature, comprising the following steps:

[0022] (1) mixing the Lactobacillus plantarum LWQ17 with flour and water, and fermenting at 25 to 35° C. for 10 to 15 hours to obtain sourdough;

[0023] (2) Mix sourdough, flour, yeast and water to form dough, place it at room temperature for 4 to 6 hours, cut it into shapes, and ferment it under fermentation conditions; then steam it to form steamed buns.

[0024] In step (1), the ratio of Lactobacillus plantarum LWQ17 bacterial suspension to flour and water is 1 mL: 45-55 g: 45-55 g.

[0025] Furthermore, in step (1), a carbon source is added when making sourdough, and the carbon source is sucrose or glucose. Adding a carbon source can increase the biomass of Lactobacillus plantarum LWQ17 during sourdough fermentation, increase antibacterial active substances, and significantly delay the germination time of mold in steamed bread.

[0026] In step (2), the ratio of sourdough, flour, yeast and water is 90-110g: 140-160g: 1g: 45-55g. The raw materials are mixed and kneaded into dough, and then cut and shaped after proofing. The proofing conditions are 25-30℃, 4-6h. The proofed dough is divided into multiple dough pieces with a mass of 45-55g / piece, and the secondary proofing conditions are 25-30℃, 30-40min, and the proofed dough pieces are steamed to obtain the steamed bread.

[0027] Furthermore, in step (2), the steamed buns are cooled and then vacuum packed or a deoxidizer is added to the package. Studies of the present invention show that the germination time of mold in the steamed buns can be significantly delayed when the steamed buns are stored under deoxygenated conditions.

[0028] The present invention also provides the use of Lactobacillus plantarum LWQ17 in the preparation of antioxidants. The present invention studies show that the metabolites of Lactobacillus plantarum LWQ17 exhibit excellent antioxidant activity.

[0029] The present invention has the following beneficial effects:

[0030] (1) The present invention provides a new strain of Lactobacillus plantarum LWQ17. In vitro antibacterial experiments show that its fermentation supernatant can significantly inhibit the growth of mold; mechanism studies show that the fermentation supernatant of this strain has the ability to destroy the fungal cell membrane structure and change its permeability.

[0031] (2) The Lactobacillus plantarum LWQ17 provided by the present invention has no effect on yeast growth. Using it in sourdough to construct an in-situ preservation technology for steamed bread can effectively inhibit fungal contamination of steamed bread during storage at room temperature and extend the shelf life of steamed bread.

[0032] (3) The Lactobacillus plantarum LWQ17 provided by the present invention is non-hemolytic and has high biological safety.

[0033] (4) The Lactobacillus plantarum LWQ17 provided by the present invention has excellent antioxidant activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the growth curve of LWQ17 strain.

[0035] Figure 2 The plate growth image (A) and Gram staining microscope image (B) of the LWQ17 strain.

[0036] Figure 3 Homology analysis of LWQ17 strain.

[0037] Figure 4 This is the effect of LWQ17 strain on the growth of Aspergillus niger and Penicillium.

[0038] Figure 5 This is the hemolytic activity test of LWQ17 strain, where (A) is LWQ17 strain and (B) is Staphylococcus aureus.

[0039] Figure 6 This is the effect of LWQ17 strain on yeast.

[0040] Figure 7 The effect of LWQ17 fermentation supernatant on the radial growth of Aspergillus niger.

[0041] Figure 8 The effect of different temperature treatments on the antibacterial activity of LWQ17 fermentation supernatant.

[0042] Fig. 9 The effect of different proteases on the antibacterial activity of LWQ17 fermentation supernatant.

[0043] Fig.10 The effect of LWQ17 fermentation supernatant treated with different pH conditions on the antibacterial activity of fungi.

[0044] Fig.11 Treatment of LWQ17 fermentation supernatant with fungal OD 260nm impact.

[0045] Fig.12 The effect of LWQ17 on the integrity of fungal cell membrane.

[0046] Fig.13 The following is a comparison of the appearance of steamed buns treated with LWQ17 and the steamed buns in the control group when stored at room temperature for one month, where (A) is the steamed bun in the control group and (B) is the steamed bun treated with LWQ17. DETAILED DESCRIPTION

[0047] The present invention will be further described below in conjunction with specific embodiments. The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of application of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the inventive method, steps or conditions all belong to the scope of the present invention.

[0048] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0049] In the following examples, MRS broth, potato dextrose water (PDB) medium, PDA solid medium, blood plate, and agar powder are all conventional culture media / liquids in the art.

[0050] In the following examples, flour is a product of Wudeli, with a product number of Q / WDL 0016S; dry yeast is a product of Angel Yeast Co., Ltd., with a product number of GB / T 20886.1; and food deoxidizer is a product of Shanghai Fengwei Industrial Co., Ltd., with a product number of SB / T 10514.

[0051] Aspergillus niger, Penicillium sp, Alternaria sp, Aspergillus flavus, Chaetomium globosum, Cladosporium sp, Penicillium italicum, and Rhizopus oryzae in the following examples are isolated from steamed bread samples. The specific separation method is as follows: Place the moldy steamed bread in a clean bench, remove the rotten part of the steamed bread with a sterile scalpel and place it on a PDA plate, and place it in a 28°C mold incubator for 2-4 days. Classify the mold according to its microbiological characteristics such as colony morphology and color and physiological and biochemical characteristics, and identify different molds by ITS sequencing. Then cut different types of mold spores and place them on a PDA plate, culture them at 28°C until spores are formed, expand the culture again, and set aside.

[0052] Example 1: Screening and identification of lactic acid bacteria that resist spoilage mold

[0053] 1. Experimental Materials

[0054] Lactic acid bacteria were isolated and screened from sour dough (a traditional leavening agent used for making steamed bread in the north), and the present invention used samples from Shandong.

[0055] 2. Experimental methods

[0056] 2.1 Isolation of strains

[0057] Weigh 10 g of the sample and place it in a homogenizing bag and add 95 mL of sterilized saline to mix well. Then prepare a 10-fold gradient series of sample dilutions. -5 , 10 -6 , 10 -7 Three gradients, each gradient is coated on MRS solid culture medium, three in parallel, and placed in an anaerobic incubator at 37°C for 24-48h. After a single colony grows, select the colonies with obvious differences and separate them by streaking on MRS solid culture medium, and place them in an anaerobic incubator at 37°C for 24-48h. After repeating the streaking 3-5 times, pick a single colony and inoculate it into the hydrogen peroxide solution (5%) dropped on the slide and mix it. Observe that the bubbles are positive and the ones without bubbles are negative. The catalase test result is a negative strain. One of the suspected lactic acid strains was named LWQ17 for subsequent performance testing.

[0058] 2.2 Mold inhibition activity detection

[0059] In order to explore the antibacterial activity of LWQ17 against steamed bread spoilage mold, the fermentation supernatant of strain LWQ17 was co-cultured with spores of different molds (Cladosporium, Penicillium italicum, Aspergillus flavus, Aspergillus niger, Penicillium, Rhizopus oryzae, Chaetomium globosum and Alternaria alternata isolated from moldy steamed bread) in 96-well plates for 48 h, and the inhibition rate was calculated.

[0060] 2.2.1 Preparation of fermentation supernatant

[0061] Experimental method: The strain LWQ17 was inoculated into MRS solid culture medium. After anaerobically culturing at 37°C for 48 hours, the bacterial liquid was inoculated into MRS broth at a volume ratio of 2% (v / v). After anaerobically culturing at 37°C for 24 hours, the culture was taken out and centrifuged at 4°C and 8000r / min for 5 minutes. The supernatant was filtered with a 0.22μm filter membrane to obtain the cell-free fermentation supernatant, which was placed in a 4°C refrigerator for use.

[0062] 2.2.2 Preparation of indicator bacteria spore suspension

[0063] Experimental method: The mold isolated from the moldy steamed bread was inoculated on a PDA plate and cultured at 28°C for 3-4 days until spores were formed. The spores on the surface of the culture medium were scraped with sterile physiological water and filtered through 4 layers of sterile gauze to remove the mycelium to obtain a spore suspension. The spore count was determined by hemocytometer and the spore concentration was adjusted to 10 with sterile physiological saline. 6 pcs / mL for future use.

[0064] 2.2.3 Microplate method

[0065] Experimental method: 100 μL of lactic acid bacteria fermentation supernatant and 100 μL of 106 Spore indicator bacteria suspension of spores / mL. Mix them evenly, culture them at 28℃ for 48h, and measure their absorbance at 600nm with a microplate reader. MRS broth is used as the control group. The antibacterial activity of lactic acid bacteria can be expressed by the antibacterial rate:

[0066] Inhibition rate (%) = 100-(OD LAB ×100 / OD 对照 ) (1)

[0067] Where: OD LAB It indicates the OD value of mold spores cultured in lactic acid bacteria fermentation supernatant for 48 hours. 600nm OD 对照 Indicates the OD value of mold spores cultured in MRS broth for 48 hours 600nm .

[0068] As shown in Table 1, LWQ17 has the best inhibitory effect on Penicillium italicum, Aspergillus flavus, Aspergillus niger and Rhizopus oryzae, with an inhibition rate of more than 90%, followed by Cladosporium, Chaetomium globosum and Alternariae, with an inhibition rate of more than 80%, and an inhibition rate of more than 70% on Penicillium. The results show that the fermentation supernatant of LWQ17 has different degrees of inhibitory effect on various spoilage molds in steamed bread.

[0069] Table 1. Inhibitory effect of strains on spoilage fungi

[0070] Inhibition rate of LWQ17 % Cladosporium 83.79 Penicillium italicum 94.24 Aspergillus flavus 94.92 Aspergillus niger 94.80 Penicillium 72.74 Rhizopus oryzae 93.56 Chaetomium globosum 84.73 Alternaria 86.83

[0071] 2.3 Plotting of growth curve

[0072] Experimental method: Inoculate the strain LWQ17 isolated in 2.1 into MRS broth, culture anaerobically at 37℃ for 24h, inoculate into MRS solid medium with an inoculation loop, culture for 48h, pick a single colony and inoculate into MRS broth, culture at 37℃ for 24h. Adjust the absorbance at 600nm to 0.1 with new MRS broth. Take 1mL of the bacterial solution and place it in a microbial growth analyzer to determine its growth curve, and do 3 sets of parallel.

[0073] Experimental results: Figure 1 As shown in the figure, the absorbance of the strain first increased rapidly and then stabilized during the measurement time. At about 2.5 hours, the lactic acid bacteria entered the logarithmic growth phase and began to multiply in large numbers, and the absorbance at 600nm increased; at about 14 hours, the lactic acid bacteria entered the stable period, and the absorbance remained stable during 14 to 48 hours.

[0074] 2.4 Strain identification

[0075] 2.4.1 Morphological identification

[0076] Experimental method: The strain was streaked and cultured, and a single bacterium was selected and cultured in MRS broth at 37°C for 24 hours. The plate morphology was observed and Gram staining was performed.

[0077] The results are as follows Figure 2 As shown in the figure, the colony morphology of LWQ17 on the MRS culture dish is round, convex, and slightly white. After Gram staining, it appears blue-purple, rod-shaped, and uniform in morphology, and is determined to be a Gram-positive bacterium.

[0078] 2.4.2 16S rRNA gene sequence analysis

[0079] Experimental method: Pick a single colony from the LWQ17 plate and inoculate it into MRS broth, culture it anaerobically at 37℃ for 24h, pick the bacterial solution with an inoculation loop and streak it onto MRS solid medium, and culture it anaerobically at 37℃ for 48h. After the colony is streaked and purified twice, pick a single colony and culture it anaerobically at 37℃ into MRS broth medium for 24h, use a DNA extraction kit to extract the DNA of the bacterial solution, and then amplify it after measuring its DNA concentration.

[0080] Reaction system: ultrapure water 7.0 μL, DNA template 1.0 μL, 2×M5 Hiper plus Taq HiFi PCR mix 10.0 μL, upstream primer and downstream primer 1 μL each.

[0081] Table 2. 16S rRNA primers

[0082]

[0083] Reaction procedure: pre-denaturation at 95°C for 2 min, 95°C for 30 s, 56°C for 30 s, 72°C for 1 min, 36 cycles; finally, incubation at 72°C for 10 min.

[0084] The PCR amplification products that met the conditions were sent to Bioengineering Company for sequencing to obtain the amplified gene sequence of the LWQ17 strain, and its 16S rDNA sequence is shown in SEQ ID NO. 1. The processed gene sequence was subjected to BLAST homology comparison analysis in the NCBI database, and the analysis obtained the known strains with the highest homology in the database, such as Figure 3 As shown, the homology with the known strain Lactiplantibacillus plantarum reached more than 99%.

[0085] Combined with the above analysis, LWQ17 was identified as Lactiplantibacillus and Lactiplantibacillus plantarum, and therefore was named Lactiplantibacillus plantarum LWQ17.

[0086] Lactobacillus plantarum LWQ17 was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on September 27, 2023, with the deposit number: CCTCC NO: M20231803, and was tested on October 4, 2023 and identified as alive.

[0087] Example 2: Antibacterial activity detection of Lactobacillus plantarum LWQ17

[0088] 1. Experimental strains

[0089] Example 1 Isolation and identification of Lactobacillus plantarum LWQ17.

[0090] 2. Experimental methods and conclusions

[0091] 2.1 Fungal inhibition assay

[0092] Experimental method: MRS solid medium was used as the lower medium, 10 μL of strain LWQ17 bacterial solution was spotted in the center of the plate, and cultured anaerobically at 37°C for 48 h. The concentration of mold (Aspergillus niger, Penicillium) spores was adjusted to 10 6 After the mixture reached 100 μg / mL, it was fully mixed with melted PDA medium, poured onto MRS solid medium, and cultured at 28°C for 3 days before observing fungal activity.

[0093] Experimental results: Figure 4 As shown in the figure, an obvious inhibition zone is formed around the strain spot. Aspergillus niger is white at first, then turns bright yellow and finally black and thick. As time goes by, the control group plate is full of Aspergillus niger, and an obvious inhibition zone is formed around the LWQ17 group spot.

[0094] As for the growth of Penicillium, it was white at first and then gradually turned green. As the culture time prolonged, the control group plate was full of Penicillium, and an obvious inhibition zone was formed around the LWQ17 group spot.

[0095] The above results showed that LWQ17 could significantly inhibit the growth of Aspergillus niger and Penicillium.

[0096] 2.2 Hemolytic activity assay

[0097] Hemolysis is a phenomenon in which the hemolytic toxins contained in the strain are secreted, causing cells to rupture and dissolve. The hemolysis circles formed are divided into grass-green hemolysis circles of potentially pathogenic bacteria (α-type hemolysis), colorless and transparent hemolysis circles of strongly pathogenic bacteria (β-type hemolysis) and no hemolysis.

[0098] Experimental method: Streak the strain onto a blood plate and culture at 37°C for 48 hours. Use Staphylococcus aureus as a positive control and observe whether a hemolytic zone is formed around the colony.

[0099] Experimental results: Figure 5 As shown, a transparent hemolytic zone is formed around the streak of Staphylococcus aureus, but no hemolytic zone appears around the colony of LWQ17, indicating that LWQ17 is non-hemolytic.

[0100] 2.3 Interaction assay with yeast

[0101] Experimental method: Draw two 2.5 mm lines on MRS solid medium with the bacterial liquid of strain LWQ17, and culture anaerobically at 37℃ for 48 hours. Pour the PDA medium containing yeast on the upper layer, wait for it to solidify, culture at 28℃ for 48 hours, and observe the results.

[0102] Experimental results: Figure 6 As shown, no inhibition zone was formed around the strain streaking line, indicating that LWQ17 and yeast were not mutually exclusive, that is, it had no effect on the growth of yeast.

[0103] 2.4 Antioxidant capacity determination

[0104] 2.4.1 The ability to scavenge 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals can be used to evaluate its antioxidant capacity.

[0105] Experimental method: The method for preparing the fermentation supernatant is the same as 2.2.1 of Example 1. Take 1.0mL of LWQ17 fermentation supernatant and add it to 1.0mL of DPPH-ethanol solution, mix it, and place it in the dark at room temperature (25°C) for 30 minutes. The control group is PBS and DPPH-ethanol solution. The blank group contains sample and ethanol. After centrifugation at 8000r / min for 10 minutes, take the supernatant and measure the absorbance at 517nm with a multifunctional microplate reader, with 3 parallels in one group. The scavenging activity against DPPH is calculated according to the following formula.

[0106] Scavenging activity (%) = [1-(OD LAB -OD 空白 ) / OD 对照 ]×100% (2)

[0107] Where: OD LAB It indicates the OD after the lactic acid bacteria react with DPPH-ethanol. 517nm OD 空白 Indicates the OD after the sample reacts with ethanol 517nm OD 对照 Represents the OD after the reaction of PBS and DPPH-ethanol 517nm .

[0108] 2.4.2 Total antioxidant activity was determined using the Nanjing Jiancheng Total Antioxidant Capacity Assay Kit (ABTS method). ABTS is oxidized to green ABTS in the presence of an appropriate oxidant. In the presence of antioxidants, the production of ABTS will be inhibited. The total antioxidant capacity of the sample can be determined and calculated by measuring the absorbance of ABTS at 405 nm.

[0109] Experimental results: It can be seen from Table 3 that the LWQ17 fermentation supernatant had high DPPH clearance rate and total antioxidant capacity detection. The results showed that the LWQ17 fermentation product had a certain antioxidant capacity.

[0110] Table 3. Antioxidant capacity of LWQ17 fermentation supernatant

[0111]

[0112] Example 3: Analysis of the antibacterial mechanism of Lactobacillus plantarum LWQ17

[0113] 1. Experimental strains

[0114] Example 1 Isolation and identification of Lactobacillus plantarum LWQ17.

[0115] 2. Experimental methods and conclusions

[0116] 2.1 Preparation of fermentation supernatant and concentrate

[0117] Experimental method: The preparation of fermentation supernatant was the same as in 2.2.1 of Example 1.

[0118] 500 mL of LWQ17 fermentation supernatant was freeze-dried and dissolved in 50 mL of sterile water to obtain a 10-fold concentrated LWQ17 concentrate; 500 mL of MRS broth was freeze-dried and dissolved in 50 mL of sterile water to obtain a 10-fold concentrated MRS concentrate.

[0119] 2.2 Preparation of indicator bacteria spore suspension

[0120] Experimental method: Inoculate the mold on PDA medium and culture at 28℃ for 3-4 days until spores are formed. Scrape the spores on the surface of the medium with sterile physiological water, filter through 4 layers of sterile gauze, and remove the mycelium to obtain the spore suspension. Use the hemocytometer to determine the number of spores, and adjust the spore concentration to 10 with sterile physiological saline. 6 pcs / mL for future use.

[0121] 2.3 Effect of LWQ17 on the radial growth of Aspergillus niger

[0122] Experimental method: LWQ17 stock solution (LWQ17 fermentation supernatant), LWQ17 concentrate, control stock solution (MRS broth) and control concentrate (MRS concentrate) were added to PDA medium at a volume ratio of 10%, mixed and poured into culture dishes. After the culture medium was condensed, 5 μL of Aspergillus niger spore suspension was inoculated at the center of the plate. The Aspergillus niger colony diameter was observed at 28°C to verify the antibacterial activity.

[0123] Experimental results: Figure 7 As shown in the figure, as time goes by, the radial growth of Aspergillus niger in the LWQ17 stock solution group is slower than that in the control stock solution group. In the concentrated group, LWQ17 completely inhibited the radial growth of Aspergillus niger after concentration, while the radial growth process of Aspergillus niger was accelerated after concentration in the control group. The results show that the fermentation supernatant of LWQ17 can inhibit the radial growth rate of Aspergillus niger.

[0124] 2.4 Effect of different temperatures on the inhibitory fungal activity of fermentation supernatant

[0125] Experimental method: The fermentation supernatant was treated at -20℃, 4℃, 60℃, 80℃, 100℃ and 121℃ for 20min, and then kept for later use after the temperature dropped to room temperature.

[0126] 100 μL of fungal spore suspension (10 6 / mL) and 100 μL of fermentation supernatant or MRS broth treated at different temperatures, with untreated fermentation supernatant as the control group. After culturing at 28°C for 48 h, the absorbance was measured at 600 nm by a microplate reader, and the antibacterial rate of the fermentation supernatant treated at different temperatures was calculated according to the following formula.

[0127] Antibacterial rate (%) = [(OD1-OD2) / OD2] × 100% (3)

[0128] Where: OD1 represents the OD value of mold spores cultured in lactic acid bacteria fermentation supernatant for 48 hours. 600nm OD2 represents the OD of mold spores cultured in MRS broth for 48 h 600nm .

[0129] Experimental results: Figure 8 As shown, after the fermentation supernatant was treated at different temperatures, the inhibition rate against Aspergillus niger and Penicillium was not significantly different from that of the untreated fermentation supernatant. The results showed that the antibacterial substances in the LWQ17 fermentation supernatant were stable in the range of -20-121°C.

[0130] 2.5 Effects of different enzymes on the inhibitory fungal activity of fermentation supernatant

[0131] Experimental method: The fermentation supernatant of LWQ17 was adjusted to the optimum pH of pepsin 2, trypsin 7, proteinase K 7, and catalase 7 with 1 mol / L NaOH and 1 mol / L lactic acid, and then the enzymes were added to make the final concentration 1 mg / mL. After reacting in a 37°C water bath for 2 hours, it was placed in a 100°C water bath for 5 minutes to inactivate the enzymes, and the pH was adjusted to the initial pH after the temperature dropped to room temperature.

[0132] 100 μL of fungal spore suspension (10 6 / mL) and 100 μL of fermentation supernatant or MRS broth after different enzyme treatments were added, and the untreated fermentation supernatant was used as the control group. The cells were cultured at 28°C for 48 h, and the absorbance was measured at 600 nm using a microplate reader. The antibacterial rate of the fermentation supernatant after different enzyme treatments was calculated according to formula (3).

[0133] Experimental results: Fig. 9 As shown in the figure, the inhibition rate of LWQ17 fermentation supernatant against Aspergillus niger decreased significantly after being treated with pepsin and catalase. There was no significant effect on the inhibition rate of Penicillium. The results showed that the LWQ17 fermentation supernatant may contain hydrogen peroxide or protein substances that have a certain inhibitory effect on Aspergillus niger.

[0134] 2.6 Effect of different pH values ​​on the fungal inhibition activity of fermentation supernatant

[0135] Experimental method: The pH value of the fermentation supernatant was adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 with 1 mol / L NaOH and 1 mol / L lactic acid, and 100 μL of mold spore suspension (10 6 The untreated fermentation supernatant was used as the control group and cultured at 28 °C for 48 h. The absorbance was measured at 600 nm using a microplate reader. The antibacterial rate of the fermentation supernatant after different enzyme treatments was calculated according to formula (3).

[0136] Experimental results: Fig.10 As shown in the results, pH treatment had a great influence on the antibacterial rate of the fermentation supernatant of LWQ17. When the pH was 3 and 4, the antibacterial rate of the fermentation supernatant against Aspergillus niger and Penicillium was not significantly different from that of the control. When the pH was ≥ 5, the antibacterial rate of the fermentation supernatant against Aspergillus niger and Penicillium was significantly reduced. The inhibition of Aspergillus niger and Penicillium was the lowest when the pH was 5. The results showed that the organic acids in the fermentation supernatant of LWQ17 were the main antibacterial substances.

[0137] 2.7 Effect of fermentation supernatant on fungal nucleic acid leakage

[0138] Experimental method: The concentration of 106 100 ml / mL of mold spore suspension was inoculated into PDB medium, cultured at 180 r / min and 28 °C for 48 h, centrifuged at 8000 r / min for 5 min, discarded the supernatant, and washed with sterile water several times to obtain mycelial balls. Weigh 0.5 g of mycelial balls into a 10 mL centrifuge tube, add 5 mL of fermentation supernatant to it, and an equal volume of sterile saline as the control group. Cultured at 180 r / min and 28 °C, aspirated the solution at 0, 2, 4, and 6 h, centrifuged at 4 °C and 8000 r / min for 5 min, and measured the absorbance at 260 nm with an ultra-micro spectrophotometer. Three parallels were used in each group.

[0139] Experimental results: Fig.11 As shown, as time went on, the absorbance difference at 260nm in the LWQ17 group was significantly higher than that in the control group, and continued to increase. The results showed that the cell membranes of Aspergillus niger and Penicillium were damaged after treatment, resulting in the leakage of nucleic acids in the cells. The nucleic acids penetrated through the cell membrane into the culture solution, causing the nucleic acid content in the solution to increase rapidly.

[0140] 2.8 Effects of fermentation supernatant on mycelial cell membrane

[0141] Experimental method: After the mycelial pellets were cultured as in 2.7 of Example 3 and washed, they were placed in a 10 mL centrifuge tube and resuspended with the fermentation supernatant to a final mold concentration of 10 6 / mL, with no fermentation supernatant added as the control group, and cultured at 180r / min and 28°C for 48h. Collect the mycelium, rinse twice with distilled water, centrifuge at 4000r / min for 5min, discard the supernatant, add 1mL of 10μg / mL PI staining solution, mix well, and stain at room temperature in the dark for 30min, wash the excess dye with PBS, resuspend in PBS buffer, observe under a laser confocal microscope, and take pictures and record.

[0142] Experimental results: PI staining solution can pass through damaged cell membranes and specifically bind to DNA, emitting red fluorescence, but it cannot pass through intact cell membranes. Fig.12 As shown in the figure, Aspergillus niger and Penicillium cultured with LWQ17 fermentation supernatant were easily stained by PI, and more hyphae in the LWQ17 group were stained and emitted red fluorescence compared with the control group, indicating that LWQ17 treatment caused Aspergillus niger and Penicillium to lose cell viability, and a considerable proportion of cell membranes lost integrity.

[0143] The results showed that LWQ17 has the ability to destroy the fungal cell membrane structure and change its permeability.

[0144] Example 4: Application of Lactobacillus plantarum LWQ17 in the preservation of steamed bread at room temperature

[0145] 1. Experimental Materials

[0146] The strain used was Lactobacillus plantarum LWQ17 isolated and identified in Example 1. The flour used was Wudeli Baxing flour.

[0147] 2. Experimental methods and conclusions

[0148] 2.1 Experiment on the preservation of steamed bread at room temperature

[0149] Experimental method: The strain was cultured in MRS broth for 24 hours and then collected by centrifugation. After pouring out the supernatant, the bacteria were washed and resuspended in sterile saline. 4 mL of cell suspension was mixed with 200 g of flour and 200 mL of water, and fermented at 30 ° C for 12 hours to prepare sourdough; the same volume of sterile water was used as the control group. After fermentation, 300 g of sourdough was taken out and mixed with 450 g of flour, 3 g of yeast and 150 mL of water to form dough, and the dough was fermented at room temperature for 5 hours. The dough was smoothed with a small amount of flour, cut into 50 g dough pieces and shaped, and fermented in a fermentation box at 27-30 ° C for 30-40 minutes. After heating the steamer to boil, put the dough pieces in and steam for 40 minutes. Put the steamed buns on the cooling rack to cool, put them in a food plastic bag and heat seal them, and store them at room temperature.

[0150] Storage conditions: average temperature 23-26°C, humidity 68-75% RH.

[0151] Observe the changes in the LWQ17 group and the control group, and record the germination time of steamed bread mold within one month.

[0152] Experimental results: As shown in Table 4, mold germination was observed in 10 steamed buns of the control group within 7 days, and mold was observed in 3 steamed buns of the LWQ17 group on the 7th day. Fig.13 As shown in the figure, when the steamed buns were placed for one month, mold germination was serious in the control group, while the surface of the steamed buns in the LWQ17 group was smooth and mold-free. The results show that steamed buns fermented with LWQ17 can significantly extend the shelf life of steamed buns at room temperature.

[0153] Table 4. Mold germination of steamed bread at room temperature

[0154]

[0155] Note: - indicates that no mold germination was found on the outer skin of the steamed buns during the one-month observation period.

[0156] 2.2 Experiment on preservation of steamed bread in vacuum packaging at room temperature

[0157] Experimental method: The experimental method is the same as 2.1 of Example 4. After the steamed buns are cooled, they are packaged in vacuum packaging bags and vacuumized. The changes of steamed buns in the LWQ17 group and the control group are observed, and the germination time of mold in the steamed buns is recorded within one month.

[0158] Storage conditions: Average temperature 28-33°C, humidity 80-86% RH.

[0159] Experimental results: As shown in Table 5, mold germination was observed in 6 steamed buns of the control group within 4 days, and mold germination was observed in 3 steamed buns of the LWQ17 group on the 4th day, and mold germination was observed in 1 steamed bun on the 13th day and 15th day respectively. The results show that vacuum treatment of LWQ17 steamed buns in high temperature and high humidity weather can extend the shelf life of steamed buns at room temperature to a certain extent.

[0160] Table 5. Mold germination of vacuum-packed steamed bread at room temperature

[0161]

[0162] Note: - indicates that no mold germination was found on the outer skin of the steamed buns during the one-month observation period.

[0163] 2.3 Experiment on the preservation of steamed bread at room temperature after deoxygenation packaging

[0164] When the growth environment of mold reaches a certain temperature and humidity, the growth rate of mold is faster. As the temperature rises, the fungal inhibition effect of strain LWQ17 on steamed bread is affected, so deoxygenated packaging (deoxidizer) is tried for storage.

[0165] Experimental method: The experimental method is the same as 2.1 of Example 4. When the steamed buns are cooled and packaged, deoxidizer is added and sealed together. The changes of steamed buns in the LWQ17 group and the control group are observed, and the germination time of mold in the steamed buns is recorded within one month.

[0166] Storage conditions: Same as 2.2 of Example 4.

[0167] Experimental results: As shown in Table 6, when stored in food plastic bags (ordinary packaging), mold germination was observed in the control group on the 2nd day and in the LWQ17 group on the 4th day; when deoxygenated packaging (adding deoxidizer) was used, mold germination was observed in the control group on the 9th day and in the LWQ17 group on the 30th day, and the remaining 4 could be stored for 1 month. The results show that the steamed bread made with the LWQ17 strain can significantly extend the shelf life of the steamed bread at room temperature in hot and humid weather with the deoxidizer.

[0168] Table 6. Mold germination of steamed bread with deoxidizer at room temperature

[0169]

[0170] Note: - indicates that no mold germination was found on the outer skin of the steamed buns during the one-month observation period.

[0171] 2.4 Experiment on the preservation of steamed bread with carbon source at room temperature

[0172] In order to obtain a large amount of LWQ17 bacteria during sourdough fermentation, a certain amount of carbon source (sugar) was added when making sourdough to promote the biomass of LWQ17 during the fermentation process.

[0173] Experimental method: The experimental method is the same as 2.1 of Example 4, and 1.8% soft sugar is added to the total volume of sourdough (400g) to make sourdough. The changes of steamed bread in the LWQ17 group and the control group are observed, and the mold germination time is recorded within one month.

[0174] Storage conditions: Same as 2.2 of Example 4.

[0175] Experimental results: As shown in Table 7, mold germination was observed in 5 steamed buns of the unsugared control group on the 2nd day. Mold germination was observed in 2 steamed buns of the unsugared LWQ17 group on the 4th day, and mold germination was observed in 1 steamed bun on the 6th and 7th days respectively, and the remaining 1 steamed bun could be stored for 1 month; mold germination was observed in 5 steamed buns of the sugared control group within 3 days. Mold germination was observed in 1 steamed bun of the sugared LWQ17 group on the 10th and 13th days respectively, and the remaining 3 steamed buns could be stored for 1 month. The results show that supplementing carbon sources when making sourdough can significantly extend the shelf life of steamed buns in high temperature and high humidity seasons.

[0176] Table 7. Germination of mold on steamed bread with carbon source at room temperature

[0177]

[0178] Note: - indicates that no mold germination was found on the outer skin of the steamed buns during the one-month observation period.

[0179] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A strain of Lactobacillus plantarum ( Lactiplantibacillus plantarum ) LWQ17, characterized in that, The preservation number of this plant lactobacillus is CCTCC NO: M 20231803.

2. The use of Lactobacillus plantarum LWQ17 according to claim 1 in the preparation of fungal inhibitors or antioxidants, characterized in that: The fungus is Aspergillus niger ( Aspergillus niger )、Penicillium( Penicillium sp )、Alternaria Alternaria sp )、Aspergillus flavus( Aspergillus flavus ), Chaetomium globosum ( Chaetomium globosum )、Cladosporium( Cladosporium sp )、Penicillium italicum( Penicillium italicum ) Rhizopus oryzae ) Any one or more of .

3. The use according to claim 2, characterized in that The fungal inhibitor includes the fermentation supernatant of Lactobacillus plantarum LWQ17.

4. The use according to claim 3, characterized in that The preparation method of the fermentation supernatant of Lactobacillus plantarum LWQ17 comprises: inoculating Lactobacillus plantarum LWQ17 in an MRS solid culture medium, culturing at 37°C for 48 hours under an anaerobic environment, inoculating into an MRS broth at a volume ratio of 2%, culturing at 37°C for 24 hours, taking out, centrifuging at 4°C and 8000 r / min for 5 minutes, taking out the supernatant and filtering it with a 0.22 μm filter membrane to obtain a cell-free fermentation supernatant, and obtaining the fermentation supernatant.

5. The use according to claim 2, characterized in that The fungus inhibitor also includes a food-acceptable carrier.

6. The use according to claim 5, characterized in that The plant lactobacillus LWQ17 or its fermentation supernatant is used as a fermented food additive.

7. A method for extending the shelf life of steamed bread at room temperature, characterized in that: The following steps are involved: (1) mixing the Lactobacillus plantarum LWQ17 as claimed in claim 1 with flour and water, and fermenting at 25-35° C. for 10-15 h to obtain sourdough; (2) Mix sourdough, flour, yeast and water to form dough, place it at room temperature for 4 to 6 hours, cut it into shapes, ferment it under fermentation conditions, and then steam it to form steamed buns.

8. The method for extending the shelf life of steamed bread at room temperature as claimed in claim 7, characterized in that: In step (1), a carbon source is added when making sourdough, and the carbon source is sucrose or glucose.

9. The method for extending the shelf life of steamed bread at room temperature as claimed in claim 7, characterized in that: In step (2), the steamed buns are cooled and then vacuum packed or a deoxidizer is added into the packaging.

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