Lactobacillus plantarum XQ1 and application thereof in storage, preservation and quality improvement of roxburgh rose pomace

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus plantarum XQ1 and application of the lactobacillus plantarum XQ1 in storage, preservation and quality improvement of roxburgh rose pomace. The lactobacillus plantarum XQ1 is preserved in the China Center for Type Culture Collection on December 30, 2024, and the preservation number of the lactobacillus plantarum XQ1 is CCTCC (China Center for Type Culture Collection) NO: M 20242955. The roxburgh rose pomace is directly adopted as a fermentation raw material, and lactobacillus plantarum XQ1 is directly mixed with the roxburgh rose pomace and then sealed and fermented. Under the aerobic condition, the roxburgh rose pomace can be stored for 2 months without rotting and deteriorating, and under the anaerobic condition, the roxburgh rose pomace can be stored for 6 months without rotting and deteriorating. The strain is high in acid resistance, cholate resistance, osmotic pressure resistance and ethanol resistance and can be directly and uniformly mixed with fresh roxburgh rose pomace, water addition for homogenization and sterilization are not needed in the fermentation, storage and preservation process, oxygen introduction is not needed in the fermentation process, and the fermentation method has the advantages of being simple, environmentally friendly and low in cost. When the strain is used for preserving fresh roxburgh rose pomace, the quality of the roxburgh rose pomace can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus plantarum XQ1 and application thereof in storage, freshness preservation and quality improvement of roxburghii pear pomace. Background Art

[0002] Guizhou's geology belongs to karst landform, with different weather every ten miles, large temperature difference between day and night, high cold and high humidity climate characteristics. This climatic condition is conducive to the accumulation of sugar and nutrients in sea buckthorn, so Guizhou sea buckthorn has a very high quality. At the same time, Guizhou is also the province with the highest production of sea buckthorn, and the sea buckthorn industry is a characteristic and advantageous industry in Guizhou agriculture. Sea buckthorn fruit is a medicinal and edible resource, and it has high nutritional value and medicinal value because it contains rich nutrients and active small molecules. However, the traditional processing of sea buckthorn mainly prepares juice by squeezing, and the discharge of sea buckthorn pomace is large, and the time of slag discharge of sea buckthorn pomace is concentrated (concentrated production from August to October each year), and it is in the high temperature and high humidity season, which makes the sea buckthorn pomace easy to rot and deteriorate, resulting in a waste of resources. Therefore, a method of energy-saving, simple, green, quality-improving and rapid large-scale treatment of sea buckthorn pomace is the basis for realizing the comprehensive utilization of sea buckthorn pomace.

[0003] Chinese patent CN201710444034.3 discloses a method for improving the comprehensive quality of roxburghii pear pomace by microbial fermentation. This invention patent utilizes two kinds of microorganisms (Lactobacillus brevis and Lactobacillus acidophilus) to jointly treat roxburghii pear pomace, which helps to improve the content of triterpenes and water-soluble dietary fiber in roxburghii pear pomace. However, the method needs to sterilize roxburghii pear pomace. However, for the characteristics of concentrated slag discharge time and large concentrated discharge volume of roxburghii pear pomace, sterilization treatment undoubtedly does not have the characteristics of energy saving, and it is also difficult to rapidly scale up the treatment of roxburghii pear pomace. In addition, the method is to ferment roxburghii pear homogenate, that is, it is necessary to add water to roxburghii pear pomace in addition, and the roxburghii pear pomace of homogenate increases moisture, which increases the difficulty of the comprehensive utilization of subsequent roxburghii pear pomace.

[0004] Chinese patent CN202110639233.6 discloses a preparation process of Rosa roxburghii pomace fermented with bacteria and enzymes. The patent combines three methods of physical treatment, enzyme treatment and fermentation treatment. After treatment, the soluble dietary fiber of the pomace is improved. However, because the acidity of Rosa roxburghii pomace is high and the pH is below 4, this method needs to adjust the pH before enzyme treatment, otherwise it will cause the added enzyme to be inactivated. In addition, it is mentioned in the content of the patent that continuous oxygenation is required during the treatment process. From these, we can see that this method does not have the characteristics of simple operation.

[0005] Chinese Patent CN202311331985.1 discloses a method for fermenting Rosa roxburghii Tratt pomace using Lactobacillus plantarum and its application in the oxidative stress of ruminants. The fermented Rosa roxburghii Tratt pomace of this patent does not require sterilization, pH adjustment, or oxygen supply. Through the process of this patent, the original nutritional components and effective active components of Rosa roxburghii Tratt pomace are retained, the activity of oxidase in Rosa roxburghii Tratt pomace is increased, and the total antioxidant activity of Rosa roxburghii Tratt pomace is enhanced. The upgrading space of this patent is that if bacteria with better tolerance and fermentation performance than those used in this patent can be isolated, it will be more valuable and promising for the utilization of Rosa roxburghii Tratt pomace. Summary of the Invention

[0006] The first aspect of the present invention provides a Lactobacillus plantarum XQ1, which was deposited at the China Center for Type Culture Collection on December 30, 2024, with the deposit number CCTCC NO: M 20242955.

[0007] The Lactobacillus plantarum XQ1 has the following deposit information: Taxonomic name: Lactobacillus plantarum; Latin name: Lactobacillus plantarum; Full name of the unit depositing the biological material sample: China Center for Type Culture Collection; Address: Room 201, China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province; Deposit date: December 30, 2024, Deposit number: CCTCC NO: M 20242955.

[0008] The isolation and purification of Lactobacillus plantarum XQ1 includes the following steps: (1) Natural silage of Rosa roxburghii Tratt pomace: After collecting fresh Rosa roxburghii Tratt pomace produced in Guizhou Province, it is put into a polyolefin silage bag, compacted while filling, and then stored sealed for about 15 days to obtain naturally silaged Rosa roxburghii Tratt pomace. Take 1 g of the naturally silaged Rosa roxburghii Tratt pomace respectively, dilute it with sterile normal saline to 10 -4 、10 -5 、10 -6 , coat 100 μL of the bacterial solutions at three gradients on a plate, culture at 37 °C for 24 - 48 h, observe and record the colony morphology. Pick colonies with different morphologies on the plate for streak separation. After culturing at 37 °C for 48 h, pick different single colonies with different morphologies on the plate for streak separation again. Repeat this process multiple times until pure single colonies with consistent morphology are obtained.

[0009] Enlarged culture treatment of Lactobacillus plantarum XQ1: (1) Liquid seed culture medium: 20 g of glucose, 10 g of peptone, 10 g of beef extract, 2 g of diammonium citrate, 5 g of sodium acetate, 1 mL of Tween 80, 2 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate heptahydrate, 1.0 L of distilled water, natural pH, sterilized at 121 °C and 0.1 Mpa for 15 min, used for activation and enlarged culture of Lactobacillus plantarum XQ1. (2) Inoculate the XQ1 strain into the liquid medium and culture it on a shaker at 37 °C for 24 hours.

[0010] The second aspect of the present invention provides the application of Lactobacillus plantarum XQ1 in storing fresh Rosa roxburghii Tratt pomace or improving the quality of Rosa roxburghii Tratt pomace during storage.

[0011] The third aspect of the present invention provides a biological agent, which contains the above-mentioned Lactobacillus plantarum XQ1.

[0012] The fourth aspect of the present invention provides a method for fermenting Rosa roxburghii Tratt pomace, which is to mix the above-mentioned Lactobacillus plantarum XQ1 or biological agent with Rosa roxburghii Tratt pomace.

[0013] In a specific embodiment of the present invention, the addition amount of Lactobacillus plantarum XQ1 is 1×10 6 CFU / g.

[0014] In a specific embodiment of the present invention, the fermentation temperature is 20 - 40 °C.

[0015] The fifth aspect of the present invention provides a method for preserving Rosa roxburghii Tratt pomace, which is to mix the above-mentioned Lactobacillus plantarum XQ1 or biological agent with Rosa roxburghii Tratt pomace.

[0016] Furthermore, the preserved Rosa roxburghii Tratt pomace can be preserved aerobically or anaerobically.

[0017] Furthermore, the steps of aerobic preservation include: directly mixing Lactobacillus plantarum XQ1 with the fresh Rosa roxburghii Tratt pomace produced by pressing, and the inoculation amount of the strain is 1×10 6 -5×10 8 CFU / g. This method can preserve the fresh Rosa roxburghii Tratt pomace for 2 months without rotting or deteriorating.

[0018] Further, the steps of anaerobic preservation include: directly mixing Lactobacillus plantarum XQ1 with fresh Rosa roxburghii Tratt pomace produced by pressing, filling it into a high-density polyethylene bag, compacting it, and sealing it. This method can store fresh Rosa roxburghii Tratt pomace for more than 6 months without rotting or deteriorating. The inoculation amount is compounded and inoculated at 1×10 6 -5×10 8 CFU per gram of fresh fruit pomace.

[0019] The sixth aspect of the present invention provides a method for improving the nutritional components of Rosa roxburghii Tratt pomace, which ferments Rosa roxburghii Tratt pomace with the above-mentioned Lactobacillus plantarum XQ1 or biological preparation.

[0020] The sixth aspect of the present invention provides the Rosa roxburghii Tratt pomace prepared by the above method.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. Lactobacillus plantarum XQ1 is a strain of Lactobacillus plantarum isolated and identified by the applicant's team from naturally sealed and fermented Rosa roxburghii Tratt pomace collected in Guizhou. It has a fast growth rate, good acid production ability, and characteristics such as acid resistance, bile salt resistance, and ethanol resistance. In addition, this bacterium has antibacterial effects against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, Mycobacterium smegmatis, and Pseudomonas aeruginosa.

[0023] 2. The present application directly uses Rosa roxburghii Tratt pomace as the fermentation raw material, and directly mixes Lactobacillus plantarum XQ1 with Rosa roxburghii Tratt pomace and then seals and ferments it. Under aerobic conditions, Rosa roxburghii Tratt pomace can be stored for 2 months without rotting or deteriorating. Under anaerobic conditions (compacting and sealing), Rosa roxburghii Tratt pomace can be stored for 6 months without rotting or deteriorating.

[0024] 3. Through experiments, the present application found that Lactobacillus plantarum XQ1 can emit the fragrance of sweet wine after storing and fermenting Rosa roxburghii Tratt pomace for seven days, and can improve the total phenol content of Rosa roxburghii Tratt pomace after 15 days of fermentation, improving the quality of Rosa roxburghii Tratt pomace.

[0025] 4. During the operation of preserving fresh Rosa roxburghii Tratt pomace or improving its quality, the present application does not require sterilization, homogenization, pH adjustment, enzyme addition, etc. It only needs to directly mix XQ1 with Rosa roxburghii Tratt pomace, and has the characteristics of simple operation and environmental protection. Description of the Drawings

[0026] Figure 1 It is a graph showing the growth curves and acid production abilities of Lactobacillus plantarum XQ1 and the control bacterium in Example 3.

[0027] Figure 2 It is a graph showing the acid resistance, bile salt resistance, ethanol resistance, and osmotic pressure resistance abilities of Lactobacillus plantarum XQ1 and the control bacterium in Example 3.

[0028] Figure 3 It is the result graph of Lactobacillus plantarum XQ1 and the control bacteria against six indicator pathogenic strains in Example 3.

[0029] Figure 4 It is the graph of the change in polyphenol content after storing Rosa roxburghii Tratt pomace using Lactobacillus plantarum XQ1 in Example 4.

[0030] Figure 5 It is the sensory evaluation picture of Rosa roxburghii Tratt pomace stored under aerobic conditions for 2 months in Example 4. On the left, A is the natural storage state of Rosa roxburghii Tratt pomace, which has become moldy and deteriorated. In the middle, B is the addition of a commercially purchased control bacteria, and mold can also be seen. On the right, C is the addition of XQ1, and there is no mildew or other phenomena in the Rosa roxburghii Tratt pomace, and the sensory evaluation is good.

[0031] Figures 6 - 8 It is the "Report on Strain Identification and Detection".

[0032] Figure 9 It is the colony morphology of the isolated Lactobacillus plantarum XQ1. On the left is the morphology of XQ1, and on the right is the Gram staining of XQ1.

[0033] Figure 10 It is the phylogenetic tree of the isolated Lactobacillus plantarum XQ1 of the present invention.

[0034] Figure 11 It is the picture of Rosa roxburghii Tratt pomace stored anaerobically for 6 months. Among them, on the left, CK is without adding any bacteria, and miscellaneous bacteria can be seen; in the middle, XQ1 is stored without mildew or rot; on the right, the commercially purchased control bacteria are stored. Although there is no mildew, the Rosa roxburghii Tratt pomace has serious browning and poor sensory quality evaluation. Detailed implementation mode

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

[0036] Example 1

[0037] This example provides a method for isolation and purification of Lactobacillus plantarum XQ1, including the following steps: respectively take 1 g of naturally ensiled Rosa roxburghii Tratt pomace (after collecting the fresh Rosa roxburghii Tratt pomace produced in Guizhou Province, put it into a polyethylene silage bag, compact it while filling, and then seal and store for about 15 days to obtain naturally ensiled Rosa roxburghii Tratt pomace.), and dilute it to 10 -4 、10 -5 、10 -6, 100 μL of the bacterial suspension with three gradients was spread on the plate and cultured at 37 °C for 24 - 48 h. Observe and record the colony morphology. Pick colonies with different morphologies on the plate for streak isolation. After culturing at 37 °C for 48 h, pick different single colonies on the plate for streak isolation again. Repeat this process multiple times until pure single colonies with consistent morphology are obtained.

[0038] Enlarged culture treatment of Lactobacillus plantarum XQ1: (1) Liquid seed medium: 20 g of glucose, 10 g of peptone, 10 g of beef extract, 2 g of diammonium citrate, 5 g of sodium acetate, 1 mL of Tween 80, 2 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate heptahydrate, 0.05 g of manganese sulfate heptahydrate, 1.0 L of distilled water, natural pH, sterilized at 121 °C and 0.1 Mpa for 15 min, used for the activation and enlarged culture of Lactobacillus plantarum XQ1. (2) Inoculate the XQ1 strain into the liquid medium and culture it on a shaker at 37 °C for 24 hours.

[0039] Example 2

[0040] This example provides the identification of Lactobacillus plantarum XQ1.

[0041] (1) Genomic DNA extraction: Extracted by column method

[0042] 1) Take a 2 ml centrifuge tube, add 200 μL of pretreatment solution and a little grinding beads, then add an appropriate amount of Lactobacillus plantarum XQ1 sample, and grind it in a grinder until sufficient.

[0043] 2) Add 20 μL of Proteinase K, add 200 μL of lysis solution, mix well by inverting thoroughly, and place at 70 °C for 10 min. 3) Add 200 μL of absolute ethanol, mix well by inverting thoroughly, and centrifuge briefly to remove the liquid droplets on the inner wall of the tube cap.

[0044] 4) Pass through the adsorption column, wash once with the washing solution and twice with the rinsing solution.

[0045] 5) Place the adsorption column at room temperature for 3 - 5 minutes to thoroughly dry the residual rinsing solution in the adsorption material.

[0046] 6) Transfer the adsorption to a new centrifuge tube, suspend and drop 50 - 100 μL of ddH2O in the middle of the adsorption membrane, place at room temperature for 3 - 5 min, and centrifuge at 12000 rpm for 2 min to collect the solution into the centrifuge tube.

[0047] (2) 16S / 18S amplification

[0048] (3) Detection and purification of PCR products

[0049] (4) Sequencing

[0050] (5) Result comparison

[0051] For the specific experimental steps and conclusions of steps (2)-(5), please refer to the "Strain Identification Test Report".

[0052] Judging from the phylogenetic tree picture, XQ1 is located on the branch related to Lactiplantibacillus plantarum, clustering with Lactiplantibacillus plantarum strains numbered MT229370.1 and NR115605.1. The bootstrap value of the branch is 100, indicating that this clustering relationship has a high credibility. And the NCBI BLAST results show that the similarity between XQ1 and them is 100% and 99.93% respectively. Therefore, XQ1 is identified as Lactiplantibacillus plantarum.

[0053] Example 3

[0054] This example provides the verification of the physicochemical properties of Lactiplantibacillus plantarum XQ1.

[0055] Growth ability: Take 20 mL of activated bacterial liquid (OD600 = 1) and inoculate it into 200 mL of MRS broth. Cultivate it at 37 °C and 160 r / min, and take samples every 5 hours until the stationary phase to evaluate the growth and reproduction ability of its bacteria. Growth ability determination: Take 5 mL of the culture solution, and use the culture medium at the 0th hour as a control to measure the OD600 value of the culture solution.

[0056] It can be seen from Figure 1 that the growth ability of Lactiplantibacillus plantarum XQ1 is equivalent to that of the control bacterium (Lactiplantibacillus plantarum used in CN202311331985.1), but its acid production ability is stronger.

[0057] Acid tolerance, bile salt tolerance, ethanol tolerance, and osmotic pressure tolerance:

[0058] After activating the XQ1 strain in MRS broth for 40 h, inoculate 1 μL of it alone into the corresponding MRS broth with different concentrations of additives. Acid tolerance: pH (1.5, 2.5, 3.5, 4.5, 5.5), adjust the pH with 1 mmol / L HCl; Bile salt tolerance: porcine bile salt (0.05%, 0.1%, 0.5%, 1%, 1.5%); Ethanol tolerance: ethanol (2%, 4%, 6%, 8% and 10%); Osmotic pressure tolerance: NaCl (3%, 4%, 5%, 6%, 7%, 8%). After culturing for 24 h, measure the growth situation by the absorbance at 600 nm

[0059] It can be seen from Figure 2It can be seen that Lactobacillus plantarum XQ1 has stronger acid tolerance, bile salt tolerance, ethanol tolerance, and osmotic pressure tolerance than the control strain (Lactobacillus plantarum used in CN202311331985.1).

[0060] Anti-pathogenic bacteria:

[0061] The inhibitory activity of XQ1 against indicator strains (pathogenic bacteria) was detected by the agar diffusion method. 1% (v / v) of the indicator strain was configured in nutrient agar medium and solidified. After punching holes, 100 μL of the activated XQ1 bacterial liquid was added. After incubating the plate at 37 °C for 24 hours, the diameter of the inhibition zone was measured. The indicator strains were: Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, Mycobacterium smegmatis, and Pseudomonas aeruginosa.

[0062] From Figure 3 it can be seen that Lactobacillus plantarum XQ 1 showed excellent performance against six indicator pathogenic strains (Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Mycobacterium smegmatis), and was superior to the control strain (the control strain was purchased from the China Center for Agricultural Culture Collection, and the preservation number was ACCC11016).

[0063] Example 4

[0064] This example provides a verification of the effect of Lactobacillus plantarum XQ1 on preserving Rosa roxburghii Tratt pomace. The specific steps are as follows:

[0065] Aerobic preservation: Fresh Rosa roxburghii Tratt pomace was directly mixed with XQ1 and then loaded into a glass bottle. The inoculation amount of XQ1 strain was 1×10 6 CFU / g

[0066] Figure 5 Figure A shows the picture of Rosa roxburghii Tratt pomace preserved under aerobic conditions for 2 months, where Figure 5 A: Natural preservation (without inoculating any strain), B: Preservation with the inoculation of the control strain (the control strain was purchased from the China Center for Agricultural Culture Collection, and the preservation number was ACCC11016) (the red circle in B is the mildewed part), C: Preservation with the inoculation of XQ1. From Figure 5 it can be seen that the Rosa roxburghii Tratt pomace preserved naturally has rotted and deteriorated. The Rosa roxburghii Tratt pomace in the control strain group is of better quality than the natural preservation group, but partial mildew has also started to appear. There is no mildew phenomenon in the group of Rosa roxburghii Tratt pomace treated with XQ1, and the quality is well preserved.

[0067] Anaerobic preservation: Fresh Rosa roxburghii Tratt pomace was directly mixed with XQ1 and then loaded into an airtight bag. It was compacted while loading (the purpose was to exclude oxygen). After loading, the bag mouth was sealed to prevent air from entering.

[0068] From Figure 4It can be seen that after storing the Rosa roxburghii Tratt pomace with Lactobacillus plantarum XQ1, the polyphenol content in the Rosa roxburghii Tratt pomace increased on the 15th day.

[0069] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A Lactobacillus plantarum XQ1, characterized in that The Lactobacillus plantarum XQ1 (Lactobacillus plantarum) was deposited in the China Center for Type Culture Collection on December 30, 2024, and its deposit number is CCTCC NO: M 20242955.

2. Application of Lactobacillus plantarum XQ1 according to claim 1 in storage or fermentation of roxburghii pomace.

3. A biological agent, characterized in that: The biological preparation contains the Lactobacillus plantarum XQ1 (Lactobacillus plantarum) according to claim 1.

4. A method for fermenting roxburghii pomace, characterized in that: The Lactobacillus plantarum XQ1 (Lactobacillus plantarum) according to claim 1 or the biological preparation according to claim 3 is mixed with roxburghii pomace.

5. The method according to claim 4, characterized in that The amount of Lactobacillus plantarum XQ1 added is 1×10 6 -5×10 8 CFU / g.

6. The method according to claim 4, characterized in that The fermentation temperature is 20-40℃.

7. A method for preserving roxburghii pomace, characterized in that: The Lactobacillus plantarum XQ1 (Lactobacillus plantarum) according to claim 1 or the biological preparation according to claim 3 is mixed with roxburghii pomace.

8. A method for improving the nutritional content of roxburghii pomegranate, characterized in that: The Lactobacillus plantarum XQ1 (Lactobacillus plantarum) according to claim 1 or the biological preparation according to claim 3 is used to ferment the roxburghii pomegranate residue.

9. The roxburghii pomace prepared by the method according to any one of claims 4 to 8.

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