Gluconobacter oxydans CY-1 for inhibiting harmful microorganisms and application thereof

Abstract

The invention discloses Gluconobacter oxydans CY-1 for inhibiting harmful microorganisms and application of the Gluconobacter oxydans CY-1, and belongs to the technical field of microbial engineering. The Gluconobacter oxydans CY-1 strain is preserved in the China General Microbiological Culture Collection Center, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation date is December 12, 2023, the preservation number is CGMCC NO.29299, and the Latin name is Gluconobacter oxydans. The Gluconobacter oxydans CY-1 for inhibiting harmful microorganisms, provided by the invention, has broad-spectrum antibacterial activity and can inhibit the growth of various pathogenic bacteria and putrefying bacteria; meanwhile, fermentation products contain propionic acid, malic acid, citric acid, acetic acid and other organic acids, and can be used for industrial acid production. The invention discloses that the Gluconobacter oxydans excavated in the aged yeast has higher mildew-proof and antibacterial characteristics for the first time, and is applied to the production of fermented flour products such as bread and steamed buns as a functional leavening agent.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial engineering, and relates to a new strain of Gluconobacter oxidans, in particular to a strain of Gluconobacter oxidans CY-1 capable of inhibiting harmful microorganisms and application thereof. Background Art

[0002] As traditional staple foods, steamed bread and bread are the most consumed flour-based foods, and their safety is closely related to the national health level. Traditional steamed bread and bread are made by fermenting sourdough (old leavener). With the changes in people's lifestyles, the production methods of modern steamed bread and bread have been transformed into fermentation with a single leavening agent, yeast. However, compared with products made by traditional sourdough fermentation, steamed bread and other products made by yeast fermentation have a shorter shelf life and often become corrupt and deteriorate due to mold contamination. Therefore, preventing mold contamination of fermented flour products such as steamed bread and bread is an effective strategy to improve their safety. In particular, steamed bread is a clean label food, and preservatives are not allowed to be added during production, so a "zero-addition" preservation method needs to be developed.

[0003] The microbial flora in the old leavener has undergone long-term natural domestication and has good microbial fermentation characteristics. Therefore, it is of great development value to explore functional leavening agents with antagonistic mold from my country's traditional old leaveners and extend the shelf life of products such as steamed bread and bread through in-situ preservation through fermentation.

[0004] Gluconobacter oxidans belongs to the genus Gluconobacter of the family Acetobacteraceae. Gluconobacter oxidans can utilize a variety of carbon sources, such as glucose, sorbitol, mannitol, etc., and because the dehydrogenase system bound to its cell membrane has the ability to rapidly and incompletely oxidize sugar alcohols, it is widely used in the industrial production of products such as gluconic acid and sorbitol. However, there are no research reports on Gluconobacter oxidans with antibacterial activity. The present invention discloses for the first time that Gluconobacter oxidans excavated from old leaveners has high anti-mildew and antibacterial properties, and has important application value as a functional leavening agent in fermented flour products such as bread and steamed buns. Summary of the invention

[0005] The purpose of the present invention is to provide a strain of Gluconobacter oxydans CY-1 that inhibits harmful microorganisms and its application, so as to solve the problem of short shelf life of products caused by contamination by harmful microorganisms such as molds.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] A strain of Gluconobacter oxydans CY-1 that inhibits harmful microorganisms. The Gluconobacter oxydans CY-1 strain is deposited in the China General Microbiological Culture Collection Center, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is December 12, 2023, the deposit number is CGMCC NO.29299, and the Latin name is Gluconobacter oxydans.

[0008] As a limitation, its 16S rDNA sequence is shown in SEQ ID NO.1, which is as follows:

[0009]

[0010] The present invention also provides an application of Gluconobacter oxydans CY-1 in inhibiting pathogenic bacteria and spoilage bacteria.

[0011] As a limitation, the pathogenic bacteria and spoilage bacteria include Gram-positive bacteria, Gram-negative bacteria and fungi;

[0012] The Gram-positive bacteria include Staphylococcus aureus, Bacillus cereus and Bacillus subtilis; the Gram-negative bacteria include Salmonella, Escherichia coli and Pseudomonas fluorescens; and the fungi include Penicillium expansum, Penicillium chrysogenum, Aspergillus niger and Aspergillus flavus.

[0013] The present invention also provides an application of Gluconobacter oxidans CY-1 in the preparation of propionic acid, malic acid, citric acid or acetic acid.

[0014] Among them, the output of organic acids can be adjusted according to the culture medium conditions. The output of propionic acid is 0-14g / L; the output of malic acid is 0-7g / L; the output of citric acid is 0-13g / L; and the output of acetic acid is 0-12g / L.

[0015] The present invention also provides a method for food preservation and freshness preservation, wherein the above-mentioned Gluconobacter oxidans CY-1 is added during the food preparation process.

[0016] As a limitation, the amount of Gluconobacter oxydans CY-1 added is 1×10 6 -2.16×10 9 CFU / g.

[0017] The invention also provides a flour-made food, to which the above-mentioned Gluconobacter oxidans CY-1 is added.

[0018] As a limitation, the flour-based food includes bread, steamed buns, cakes or biscuits.

[0019] Due to the adoption of the above technical solution, the present invention has the following technical advances compared with the prior art:

[0020] ① The present invention provides a strain of Gluconobacter oxydans CY-1 for inhibiting harmful microorganisms. The strain has a broad-spectrum antibacterial property and can inhibit the growth of various pathogenic bacteria and spoilage bacteria including Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Salmonella, Escherichia coli, Pseudomonas fluorescens, Penicillium expansum, Penicillium chrysogenum, Aspergillus niger and Aspergillus flavus;

[0021] ② The present invention provides a strain of Gluconobacter oxydans CY-1 that inhibits harmful microorganisms. The fermentation product contains organic acids such as propionic acid, malic acid, citric acid, and acetic acid, and can be used for industrial acid production;

[0022] ③ The Gluconobacter oxydans CY-1 provided by the present invention, which inhibits harmful microorganisms, can be used as a functional leavening agent for bread and other flour-based foods, and can significantly reduce the number of microorganisms during food storage, thereby extending the shelf life of the food, and providing a new strain resource for the development of new nutritional and safe foods, health products, agricultural products or daily chemical products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a colony morphology diagram of Gluconobacter oxydans CY-1 in Example 2 of the present invention;

[0024] Figure 2 This is the Gram staining image of Gluconobacter oxydans CY-1 in Example 2 of the present invention;

[0025] Figure 3 This is a schematic diagram of the phylogenetic tree of Gluconobacter oxydans CY-1 in Example 2 of the present invention;

[0026] Figure 4 This is a diagram showing the antiseptic effect of bread in Example 5 of the present invention. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below by specific examples. It should be understood that the described examples are only used to explain the present invention, and are not intended to limit the present invention.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0029] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0030] Example 1

[0031] This embodiment provides a strain of Gluconobacter oxydans CY-1 that inhibits harmful microorganisms. The strain is deposited in the China General Microbiological Culture Collection Center, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is December 12, 2023, the deposit number is CGMCC NO.29299, and the Latin name is Gluconobacter oxydans.

[0032] The 16SrDNA sequence of the above-mentioned Gluconobacter oxydans CY-1 that inhibits harmful microorganisms is shown in SEQ ID NO.1, which is:

[0033]

[0034] Example 2

[0035] This embodiment provides a method for separating and purifying a strain of Gluconobacter oxydans CY-1 that inhibits harmful microorganisms. The method is carried out according to the following steps:

[0036] S1. Sample collection and strain purification

[0037] The traditional old yeast from Chengcheng, Shaanxi Province, my country was selected as the sample. After diluting it with sterile saline, a suitable dilution was selected and spread on a YPD solid culture medium plate. The plate was left to stand and cultured at 30°C for 24 hours. The colonies were selected according to their morphology for microscopic observation. The results were as follows: Figure 1 As shown;

[0038] Depend on Figure 1 It can be observed that the colonies are mostly white, round, transparent, with a raised middle, neat edges, uniform texture, consistent color on both sides, sticky, and easy to pick up;

[0039] The colonies were gram-stained and the results were Figure 2 As shown;

[0040] Depend on Figure 2 It can be seen that the colonies are Gram-negative, short-rod to oval, small in size, and appear in pairs;

[0041] The initially identified colonies were streaked several times on a YPD solid medium plate to further purify them, and then a single colony was picked out and cultured in a YPD medium at 30°C until the logarithmic phase, and a glycerol tube was made and stored at -20°C for later use;

[0042] S2. Identification of strains

[0043] The 16SrDNA sequence of the strain was identified by 16SrDNA. The sequencing result was compared with the 16SrDNA sequence in GenBank, and then a phylogenetic tree was constructed to determine the species relationship of the strain. The results were as follows: Figure 3 As shown;

[0044] Depend on Figure 3 The results of homology analysis showed that the strain was most closely related to Gluconobacter oxydans 621H (LT900338.1), belonging to Gluconobacter oxydans and named Gluconobacter oxydans CY-1.

[0045] Example 3

[0046] This example studies the characteristics of Gluconobacter oxydans CY-1 in inhibiting pathogenic bacteria and spoilage bacteria, and specifically includes the following steps:

[0047] S1. Sample preparation

[0048] The indicator strains included bacteria (Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 11778, Bacillus subtilis ATCC 6051, Escherichia coli ATCC 44752, Pseudomonas fluorescens ATCC 13525, Salmonella ATCC14028) and fungi (Penicillium expansum CICC 40658, Penicillium chrysogenum ATCC 10106, Aspergillus niger ATCC 16404, Aspergillus flavus ATCC 9643);

[0049] The above indicator strains are stored in the Food and Biotechnology Laboratory of Hebei University of Science and Technology and can be obtained by purchase;

[0050] The above bacteria and fungi were cultured in nutrient broth (NB) and potato dextrose agar (PDA), respectively. After the bacteria were cultured in a shaking incubator to the logarithmic phase, the concentration of the bacterial suspension was adjusted to 10 using an ultraviolet spectrophotometer. 8 CFU / mL, set aside; after the fungus was cultured on the PDA plate for 4 days, the spores were washed with 0.85% saline and the spore count was adjusted to 10 on a hemocytometer. 6 CFU / mL, stored at 4°C for future use;

[0051] S2. Antibacterial experiment

[0052] After the above bacteria and fungi were inoculated into NA and PDA culture media respectively, the plates were poured and the final concentration of the bacterial plate was 10 6 CFU / mL, the final concentration of fungal plate is 10 4 CFU / mL;

[0053] The preserved Gluconobacter oxydans CY-1 strain was inoculated into an activation medium containing 50 g / L sorbitol and 5 g / L yeast extract powder, and activated at 30°C for 24 hours. Then, the inoculum was added into a fermentation medium containing 30 g / L glucose, 46.7 g / L yeast extract powder, 0.1 g / L manganese sulfate tetrahydrate and 0.05 g / L L-proline at a volume percentage of 2%. After culturing at 30°C for 24 hours, the fermentation liquid was centrifuged through a membrane, and the supernatant was injected into an Oxford cup. After the plate was placed in a 4°C refrigerator for pre-diffusion, the bacteria were cultured in a 37°C constant temperature incubator for 24 hours, and the fungi were cultured in a 25°C constant temperature incubator for 2 days. The antibacterial effect was observed, and the diameter of the inhibition zone was measured and recorded. The results are shown in Table 1:

[0054] Table 1 Antibacterial effect of Gluconobacter oxidans CY-1 strain

[0055]

[0056] As shown in Table 1, Gluconobacter oxydans CY-1 has a broad-spectrum antibacterial activity and can inhibit the growth of various pathogenic bacteria and spoilage bacteria including Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Salmonella, Escherichia coli, Pseudomonas fluorescens, Penicillium expansum, Penicillium chrysogenum, Aspergillus niger and Aspergillus flavus.

[0057] Example 4

[0058] This example studies the acid production characteristics of Gluconobacter oxydans CY-1, and specifically includes the following steps:

[0059] The preserved Gluconobacter oxydans CY-1 strain was inoculated into an activation medium containing 50 g / L sorbitol and 5 g / L yeast extract, and activated at 30°C for 24 hours. Then, an inoculum of 2% by volume was added into a fermentation medium containing 30 g / L glucose, 46.7 g / L yeast extract, 0.1 g / L manganese sulfate tetrahydrate and 0.05 g / L L-proline, and cultured at 30°C for 24 hours. The fermentation liquid was centrifuged and filtered to obtain a supernatant. The content of organic acids in the supernatant of Gluconobacter oxydans CY-1 was analyzed by high performance liquid chromatography. The results are shown in Table 2:

[0060] Table 2 Organic acid production of Gluconobacter oxidans CY-1

[0061] Types of organic acids Yield (g / L) Propionic acid 12.14±1.85 Malic Acid 6.15±0.87 Citric Acid 11.47±1.54 Acetic acid 10.27±1.71

[0062] The liquid chromatography analysis conditions were as follows: the chromatographic column was an Aminex HPX-87H column (300 mm × 7.8 mm), the UV detection wavelength was 210 nm, the injection volume was 10 μL, the column temperature was 50 °C, the mobile phase was 5 mmol / L sulfuric acid solution, the flow rate was 0.5 mL / min, and the elution was isocratic;

[0063] As shown in Table 2, under the culture medium conditions, the production of propionic acid by Gluconobacter oxydans CY-1 was 12.14 g / L, the production of malic acid was 6.15 g / L, the production of citric acid was 11.47 g / L, and the production of acetic acid was 10.27 g / L. This strain can be used for industrial acid production.

[0064] Example 5

[0065] This example is an application of Gluconobacter oxydans CY-1, which is used to prepare bread and test the preservation of bread products, specifically including the following steps:

[0066] S1. Preparation of bread

[0067] Blank group: wheat flour 100%, water 60%, white sugar 10%, butter 7.5%, salt 1%, yeast 0.5% (the percentage of each component is based on the weight of dry wheat flour);

[0068] 7.20×10 8 CFU / g mixed bacteria group: wheat flour 100%, water 60%, white sugar 10%, butter 7.5%, salt 1%, yeast 0.5% (the percentage of each component is based on the weight of dry wheat flour), and 7.20×10 8 CFU / g of Gluconobacter oxydans CY-1;

[0069] 1.44×10 9 CFU / g mixed bacteria group: wheat flour 100%, water 60%, white sugar 10%, butter 7.5%, salt 1%, yeast 0.5% (the percentage of each component is based on the weight of dry wheat flour), and 1.44×10 9 CFU / g of Gluconobacter oxydans CY-1;

[0070] 2.16×10 9 CFU / g mixed bacteria group: wheat flour 100%, water 60%, white sugar 10%, butter 7.5%, salt 1%, yeast 0.5% (the percentage of each component is based on the weight of dry wheat flour), and 2.16×10 9 CFU / g of Gluconobacter oxydans CY-1;

[0071] The components in the above groups were mixed evenly, and proofed in a constant temperature and humidity incubator at 30°C for 5 hours. The dough of each group was taken to prepare bread, which was baked in an oven at 150°C for 30 minutes. After cooling to room temperature, the bread was sliced ​​into equal sizes using a slicer.

[0072] S2. Effect of Gluconobacter oxydans CY-1 on the antiseptic properties of bread

[0073] 50 spores of Penicillium expansum were added to the middle of each bread slice and placed in a ziplock bag and stored in an incubator at 25°C. The preservation effect of the bread during storage was as follows: Figure 4 As shown:

[0074] Depend on Figure 4 It can be seen that Gluconobacter oxydans CY-1 can inhibit the growth of various microorganisms and extend the shelf life of bread.

[0075] Three slices of bread were randomly selected every 2 days, sterile physiological saline (0.85%, w / v) was added, patted for homogenization, serially diluted, and spread on plate count medium (PCA) and Bengal red plate, respectively. Bacteria were cultured at 37°C for 48 hours and colonies were counted, and fungi were cultured at 25°C for 72 hours and counted. The growth of microorganisms is shown in Table 3:

[0076] Table 3 Effect of the addition amount of Gluconobacter oxydans CY-1 on the colony count during bread storage

[0077]

[0078] As shown in Table 3, the total number of colonies in the bread with Gluconobacter oxydans CY-1 was significantly lower than that in the yeast fermentation group during storage. 9 The mixed bacteria group with a storage capacity of 1.544 Lg CFU / g had the best antibacterial effect. On the 8th day of storage, the total colony counts of bacteria and fungi were 4.74 Lg CFU / g and 2.98 Lg CFU / g, respectively, which were significantly lower than those of the yeast fermentation group.

[0079] The results showed that the addition amount of Gluconobacter oxidans CY-1 was 1×10 6 -2.16×10 9 CFU / g, it can have a good effect on the preservation and freshness of food.

Claims

1. A strain of glucobacterium oxidized by inhibiting harmful microorganisms, It is characterized in that The CY-1 strain of glucobacterium oxidized is deposited in the China Common Microbial Sprout Preservation and Management Center. The deposit address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is December 12, 2023, and the deposit number is CGMCC NO.29299, Latin name is Gluconobacter oxygendans .

2. The strain of glucobacterium oxidized by claim 1 is inhibited by harmful microorganisms, It is characterized in that Its 16SrDNA sequence is as follows: TCAGAGCGAACGCTGGCGCATGCTTAACACATGCAAGTCGCACGAAGGTTTCGGCCTTAGTGGCGACGGGTGAGTAACGCGTAGGGATCTATCACGGGTGGGGACAACTTCGGGAAACTGGAGCTAATACCTGAGGGTCAAAGGCGCAAGTCGCCTGTGGAGGGGGGG AACCTGCGTTCGATTAGCTAGTTGGTGGGTAAAGGCCTACCAAGGCGATGATCGATAGCTGGTTTGAGAGGATGATCAGCCACACACGGCCCAGACTCCCTACGGGAGGCAGCAGTGGGGAATTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGGT GTGAAGAAGGTCTTCGGATTGTAAAGCACTTTCGACGGGACGATGATGACGGTACCGTAGAAGAAGCCCCGGCTAACTTCGTGCCAGCAGCCGGTAATACGAAGGGGGCTAGCGTTGCTCGGAATGACTGGGCGTAAAGGGCGTAGGTTGCTCGGAATGACTGGGCGTAAAGGGCGTAGGCGTAGGTTGTTACAGTCAGATGTGAAA TCCCCGGGCTTAACCTGGGAACTGCATTTGATACGTGACGACTAGAGTTCGAGAGAGGGTTGTGGAATTCCCAGTGTAGAGGTGAAATTCGTAGATTGGGAAGAACACCGGTGGCGAAGGCGCAACCTGGCTCGATACTGACGCTGAGGCGAAAGCGTGGGAGCAAACAAGCGTGGGAGCAAACAAAGG ATTAGATACCCTGGTAGTCCACGCTGTAAACGATGTGTGCTGGATGTTGGGAAACTTAGTTTCTCAGTGTCGAAGCTAACGCGCTAAGCACACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGGGCCGCACAAGCGGTGGAGCATGTGGTTTAATTCG AAGCAACGCGCAGAACCTTACCAGGGCTTGCATGGGGAGGACCGGTTCAGAGATGGACCTTTCTTCGGACCTCCCGCACAGGTGCTGCATGGCTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTTGTCTTTAGTTGCCAGCACTTTCAGGTG GGCACTCTAGAGAGACTGCCGGTGACAAGCCGGAGGAAGGTGGGATGACGTCAAGTCCTCATGGCCCTTATGTCCTGGGCTACACACGTGCTACAATGGCGGTGACAGTGGGAAGCTACATGGTGACATGGTGCTGATCTCTAAAAGCCGTCTCAGTTCGGATTGTACTCTGCAAC TCGAGTACATGAAGGTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGTGAATACGTTCCCGGGCCTTGTACACACCGCCGTCACACCATGGGAGTTGGTTCGACCTTAAGCCGGTGAGCGAACCGCAAGGACGCAGCCGACCACGGGGTCAGCGACTGGGGTGAAGTC.

3. The use of glucobacterium oxidized CY-1 as claimed in claim 1 or 2 in inhibiting pathogenic bacteria and spoilage bacteria.

4. The application of claim 3, It is characterized in that The pathogenic bacteria and spoilage bacteria include Gram-positive bacteria, Gram-negative bacteria and fungi; The Gram-positive bacteria include Staphylococcus aureus, Bacillus cereus and Bacillus subtilis; the Gram-negative bacteria include Salmonella, E. coli and Pseudomonas fluorescent; the fungus includes Penicillium, Produce Penicillium chlorella, Aspergillus niger and Aspergillus flavus.

5. Use of glucobacterium oxidized CY-1 according to claim 1 or 2 in the preparation of propionic acid, malic acid, citric acid or acetic acid.

6. A method for preserving foods, It is characterized in that The glucobacterium oxide CY-1 of claim 1 or 2 is added during the food preparation process.

7. The method of claim 6, It is characterized in that The amount of the addition of the glucobacterium oxidized CY-1 is 1×10 6 -2.16×10 9 CFU / g.

8. A kind of noodle food, It is characterized in that The glucobacterium oxide CY-1 of claim 1 or 2 is added.

9. The noodle food according to claim 8, It is characterized in that The noodle foods include bread, steamed buns, cakes or biscuits.

Images