Paenibacillus splendidus and application thereof
A brown algae oligosaccharide vegetable and fruit preservative was prepared by fermentation with Bacillus splendidus LZY-12138, which solved the problem of blueberries being prone to spoilage in hot and rainy seasons, achieving a safe and effective preservation effect and extending the shelf life of blueberries.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-24
- Publication Date
- 2026-04-07
AI Technical Summary
Blueberries are prone to softening or breaking when ripening in the hot and rainy summer, resulting in a short shelf life for fresh produce. Existing chemical preservatives pose safety risks, necessitating the development of a safe and effective preservation method.
Brown algae oligosaccharides with a degree of polymerization of 2-3 were prepared by fermentation using Bacillus Brilliantus LZY-12138. These oligosaccharides were then used to prepare fruit and vegetable preservatives containing brown algae oligosaccharides. By soaking blueberries in the solution and storing them under specific conditions, the shelf life of the fruits was extended.
It effectively slows down the spoilage rate of blueberries, extends their shelf life, increases their firmness and soluble solids content, reduces titratable acidity, and significantly extends shelf life by more than 3-5 days. It is safe and non-toxic.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of strain development and fruit and vegetable preservation and processing technology, specifically relating to a Bacillus splendidus and its applications. Background Technology
[0002] blueberry( Vacciniccm spp. Blueberries, scientifically known as Vaccinium bracteatum, have various health benefits, including protecting eyesight, anti-cancer and antioxidant properties, and slowing aging. They are also popular among consumers due to their rich nutrients and unique flavor. The World Health Organization has listed blueberries as one of the healthiest foods in the world. Blueberry cultivation is expanding rapidly in Northeast my country, becoming a significant industry in the region. However, blueberries typically ripen during the hot and rainy summer months, making the fruit prone to softening and breakage during harvesting and storage, resulting in a very short shelf life for fresh blueberries. While many fruit and vegetable preservation methods exist, the use of chemical preservatives poses safety risks for blueberries, which are normally consumed directly. Therefore, developing a safe and effective blueberry preservation method is urgently needed.
[0003] Fucoidosaccharides are oligosaccharide molecules with a degree of polymerization below 20, obtained by degrading sodium alginate through certain methods. Increasing research confirms that fucoidan oligosaccharides possess antioxidant, antibacterial, antiseptic, and water-retaining activities, and are gradually being applied to postharvest fruit preservation. Studies have shown that treating postharvest fruit with fucoidan oligosaccharides can alter cell membrane permeability, reduce oxidation and water loss, increase the activity of antioxidant enzymes in the fruit, and enhance the ability to scavenge free radicals; it can effectively extend the storage period of fruit and improve its commercial value. Common methods for preparing fucoidan oligosaccharides include physical degradation, chemical degradation, and enzymatic hydrolysis. Among these, enzymatic hydrolysis is widely used due to its mild reaction and simple operation. Summary of the Invention
[0004] The purpose of this invention is to provide a Bacillus splendens and its applications.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A species of *Bacillus splendidus*, *Bacillus splendidus* LZY-12138, was deposited on December 18, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.29352, and classified as *Bacillus splendidus* (…). Paenibacilius lautus The address of the collection is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0006] An application of the aforementioned Bacillus splendens, specifically the application of Bacillus splendens LZY-12138 in the degradation of sodium alginate.
[0007] The application of the Lysinibacillus sphaericus LZY-12138 in degrading sodium alginate to prepare brown algae oligosaccharide with a polymerization degree of 2-3.
[0008] The application of the Lysinibacillus sphaericus LZY-12138 in preparing a fruit and vegetable fresh-keeping agent containing brown algae oligosaccharide.
[0009] A fruit and vegetable fresh-keeping agent containing brown algae oligosaccharide, which contains the Lysinibacillus sphaericus and is prepared by enzymolysis of sodium alginate as a substrate to obtain the fruit and vegetable fresh-keeping agent containing brown algae oligosaccharide.
[0010] Further, the content of the brown algae oligosaccharide in the fruit and vegetable fresh-keeping agent is 50 mg / L-250 mg / L, preferably 200 mg / L-250 mg / L.
[0011] A preparation method of the fruit and vegetable fresh-keeping agent containing brown algae oligosaccharide, comprising the following steps: Step S1: inoculating the Lysinibacillus sphaericus into a liquid culture medium at an inoculation amount of 1%-3% v / v, and oscillating culture for 12-36 h; then transferring the Lysinibacillus sphaericus into a fermentation culture medium at an inoculation amount of 1%-3% v / v, and oscillating culture at 28-32 DEG C and 180-200 r / min for 24-36 h, and obtaining a crude enzyme liquid by centrifugation, (NH4)2SO4 precipitation and dialysis under the condition of 4 DEG C; Step S2: mixing sodium alginate, the crude enzyme liquid obtained in S1 and distilled water, and degrading at 25-30 DEG C in a constant-temperature water bath for 24-48 h; then boiling in a water bath for 15-30 min to inactivate the enzyme, and centrifuging at 10,000 r / min for 10-15 min, and obtaining the fruit and vegetable fresh-keeping agent containing brown algae oligosaccharide from the supernatant.
[0012] Preferably, in step S1, the Lysinibacillus sphaericus is inoculated into a liquid culture medium at an inoculation amount of 3% v / v, and oscillating culture for 36 h; then the Lysinibacillus sphaericus is transferred into a fermentation culture medium at an inoculation amount of 3% v / v, and oscillating culture at 32 DEG C and 200 r / min for 36 h, and a crude enzyme liquid is obtained by centrifugation, (NH4)2SO4 precipitation and dialysis under the condition of 4 DEG C; Step S2: mixing sodium alginate, the crude enzyme liquid obtained in S1 and distilled water, and degrading at 25-30 DEG C in a constant-temperature water bath for 24-48 h; then boiling in a water bath for 15-30 min to inactivate the enzyme, and centrifuging at 10,000 r / min for 10-15 min, and obtaining the fruit and vegetable fresh-keeping agent containing brown algae oligosaccharide from the supernatant.
[0013] The liquid culture medium in step S1 is composed of peptone 1.0 g, sodium chloride 5.0 g, sodium alginate 1.0 g, ammonium chloride 5.0 g, potassium chloride 2.0 g, magnesium sulfate 1.0 g, and distilled water 1 L; and the fermentation medium is composed of peptone 1.0 g, sodium chloride 5.0 g, sodium alginate 5.0 g, potassium phosphate dibasic 2.0 g, magnesium sulfate 1.0 g, calcium chloride 0.2 g, and distilled water 1 L.
[0014] In step S2, the mass ratio of sodium alginate, crude enzyme solution and distilled water is (0.8-1.2):(2.5-3.5):(90-110). Preferably, it is 1:3:100.
[0015] The fruit and vegetable fresh-keeping agent containing alginate oligosaccharide is used for post-harvest blueberry preservation.
[0016] The specific application method is that the fresh blueberries without visible mechanical damage on the skin are soaked in the fruit and vegetable fresh-keeping agent containing alginate oligosaccharide for 10 min, then the treated blueberry fruits are placed on water-absorbing paper and naturally dried at room temperature. After drying, the blueberries are placed in a plastic box and stored at 20±2℃ and 80±2% relative humidity.
[0017] The advantages of the present application are: (1) The present application provides a method for preparing alginate oligosaccharide biological fresh-keeping agent by fermentation of Paenibacillus lautus, which can slow down the decay rate of blueberries to the maximum extent, prolong the preservation and storage period of blueberries, and extend the shelf life of post-harvest blueberries. The biological fresh-keeping agent is safe and non-toxic, and does not have the potential safety hazards of chemical fresh-keeping agents, and can expand the application of oligosaccharides from marine sources.
[0018] (2) The Paenibacillus lautus of the present application can degrade sodium alginate with high efficiency to prepare alginate oligosaccharide with a degree of polymerization of 2-3, and the content of alginate oligosaccharide in the product is high. The strain has stable performance, superior performance in large-scale production, stable and controllable fermentation process, short fermentation period, and is suitable for large-scale production.
[0019] (3) The alginate oligosaccharide biological fresh-keeping agent obtained by enzymatic hydrolysis is safe, has no potential danger, and has good fresh-keeping effect on post-harvest blueberries. It can slow down the decay of blueberries to the maximum extent, significantly improve the indicators of Vc, soluble solids and hardness of blueberries, and effectively extend the shelf life of blueberries by more than 3-5 days. BRIEF DESCRIPTION OF DRAWINGS
[0020] Figure 1 The phylogenetic tree of the Paenibacillus lautus strain provided in the embodiments of the present application. Paenibacillus lautus
[0021] Figure 2 The picture of the Paenibacillus lautus strain provided in the embodiments of the present application.
[0022] Figure 3 A comparison chart of the hardness of blueberries after different treatments over time.
[0023] Figure 4 A comparison chart of the soluble solids content of blueberries after different treatments over time.
[0024] Figure 5 A comparison chart of the titratable acid content of blueberries after different treatments over time.
[0025] Figure 6 A comparison chart of the rot rate of blueberries after different treatments over time. DETAILED DESCRIPTION
[0026] In addition to the objects, features, and advantages described above, the present application has other objects, features, and advantages. The present application will be described in further detail below.
[0027] The following describes embodiments of the present application in detail, but the present application can be implemented in various different ways as defined and covered by the claims.
[0028] Example 1 Isolation and identification of Sporosarcina splendidus strain Paenibacillus lautus 1. Isolation of the strain 1. Isolation of the strain The rotten sargassum was collected from the intertidal zone, soaked in sterile water, and then inoculated in a liquid seawater medium for enrichment after soaking, and the liquid seawater medium had the following components: 10 g of proteose peptone, 5 g of yeast extract powder, and 1 L of seawater. Then, it was spreaded / streaked on a sodium alginate medium, and the sodium alginate medium had the following components: 10 g of sodium alginate, 5 g of ammonium sulfate, 2 g of dipotassium hydrogen phosphate, 1 g of magnesium sulfate, 0.01 g of ferrous sulfate heptahydrate, and 1 L of ultrapure water. The medium was cultured at 32°C for 24 h, and the single colonies that grew well on the sodium alginate plate were numbered and transferred. Then, the original plate was dyed with Gram iodine solution, and whether transparent circles appeared in each numbered strain was observed. The appearance of a transparent circle indicated the ability to degrade sodium alginate. These strains with the ability to degrade sodium alginate were purified and separated (through several plate streaking until consistent single colonies grew), and Sporosarcina splendidus was obtained. Paenibacillus lautus
[0029] 2. Identification of the strain 2.1. Morphological identification For strains in the logarithmic growth phase with stable colony size, the isolated and purified strains obtained in the above steps were described as single colony characteristics, mainly including colony size, color, transparency, moisture content, colony surface condition, and colony edge condition. On the other hand, for strains in the logarithmic growth phase, the morphology of the cells was observed using an optical microscope after smear staining.
[0030] The results showed that the strain was a Gram-negative bacterium.
[0031] 2.2 Homology analysis of 16S rDNA sequences After colony formation, 16S rDNA was analyzed to obtain the spliced sequence, which was then subjected to BLAST analysis at NCBI (website: https: / / blast.ncbi.nlm.nih.gov / Blast.cgi#alnHdr_559795384). The identified strain typically matches the strain with the highest BLAST similarity (usually >97%) (see [link to BLAST analysis]). Figure 1 ).
[0032] 2.3 Identification of Physiological and Biochemical Characteristics The physiological and biochemical characteristics of the strains were determined with reference to the "Handbook of Systematic Identification of Common Bacteria" (Dong Xiuzhu, Cai Miaoying. Handbook of Systematic Identification of Common Bacteria. Beijing: Science Press, 2011.) and "Microbiology Experiments" (Shen Ping, Fan Xiurong, Li Guangwu. Microbiology Experiments (Third Edition). Beijing: Higher Education Press, 1999.).
[0033] Specific colony morphology as follows: Figure 2 The colonies are yellow with irregular edges and obvious ridges.
[0034] Based on the above morphological, physiological and biochemical characteristics analysis and 16S rDNA sequence homology analysis results, the strain obtained by isolation and purification in step 1 was identified as Paenibacillus lautus.
[0035] This *Paenibacillus lautus* was deposited on December 18, 2023, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC NO.29352, and classified as *Paenibacillus lautus*. Paenibacillus lautus The address of the collection is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
[0036] The strain sequence is:
[0037] Example 2 The above-mentioned Bacillus splendens ( Paenibacillus lautus LZY-12138 was used to prepare a vegetable and fruit preservative containing brown algae oligosaccharides.
[0038] Step S1: Inoculate Paenibacillus lautus strain LZY-12138 into liquid culture medium at an inoculation rate of 3% (v / v) and culture with shaking for 36 h; then transfer to fermentation medium at an inoculation rate of 3% (v / v) and culture with shaking at 32℃ and 200 r / min for 36 h; centrifuge the obtained bacterial solution at 10000 r / min at 4℃, take the supernatant and add (NH4)2SO4 to saturate it to 40%, incubate at 4℃ overnight to allow for complete precipitation, centrifuge at 12000 r / min at 4℃ the next day, take the supernatant and add (NH4)2SO4 to saturate it to 60%, incubate at 4℃ overnight to allow for complete precipitation, centrifuge again at 12000 r / min at 4℃ the next day, dialyze the obtained precipitate with phosphate buffer at pH=7.2 for 12-24 h, and the substance obtained in the dialyzing bag is the crude enzyme solution; Step S2: Mix sodium alginate, crude enzyme solution, and distilled water at a mass ratio of 1:3:100, degrade in a constant temperature water bath at 30℃ for 24 hours, then inactivate the enzyme by boiling in a water bath for 30 minutes, centrifuge at 10000 r / min for 10 minutes, and the resulting supernatant is the fruit and vegetable preservative containing 50 mg / L of alginate oligosaccharides.
[0039] The liquid culture medium consists of 1.0 g peptone, 5.0 g sodium chloride, 1.0 g sodium alginate, 5.0 g ammonium chloride, 2.0 g potassium chloride, 1.0 g magnesium sulfate, and 1 L distilled water. The fermentation medium consists of 1.0 g peptone, 5.0 g sodium chloride, 5.0 g sodium alginate, 2.0 g dipotassium hydrogen phosphate, 1.0 g magnesium sulfate, 0.2 g calcium chloride, and 1 L distilled water.
[0040] Example 3 Mature blueberries were harvested from a blueberry plantation in Changbai Mountain City, Jilin Province. Selected blueberries were fresh, with no visible mechanical damage to the skin, and of uniform ripeness. The selected blueberries were rinsed with sterile water and then randomly grouped. The blank control group used distilled water (CK) as a preservative, while the positive control group used chitosan (150 mg / L COS) as a preservative. Preservatives with different contents of alginate oligosaccharides (200, 250 mg / L AOS) were prepared according to the method in Example 2. The blueberries were soaked in each preservative for 10 minutes, then placed on absorbent paper and air-dried at room temperature. After drying, they were placed in conventional commercial plastic boxes and stored for 8 days at 20±2℃ and 80±2% relative humidity. Thirty blueberries were collected every 48 hours. Ten of these were used for hardness, TSS, and TA content analysis, while the remaining 20 were wrapped in aluminum foil and immediately immersed in liquid nitrogen at -80℃ for further analysis.
[0041] (1) Fruit firmness Ten blueberries were randomly selected from each group and placed on a flat tray. The firmness of the fruits in each group was then measured using a GY-4 fruit firmness tester. For puncture, a P / 2 stigma (Φ=2mm) was selected, the testing speed was 2.0mm / s, and the puncture depth was 10mm. This value is used to represent the firmness (N) of the blueberries in each treatment group. Results are as follows: Figure 3 As shown. By Figure 3 It is evident that the blueberries in the experimental group had higher firmness than those in the control group and the positive control group, indicating that the fruit and vegetable preservative treatment containing alginate oligosaccharide (AOS) can maintain the firmness of blueberries and extend their storage period.
[0042] (2) Soluble solids (TSS) content After homogenization, samples from each treatment group were filtered through four layers of gauze. 0.3 mL of the filtrate was then placed on an ATAGO-PAL-1 handheld refractometer calibrated with distilled water for measurement. Each treatment was repeated three times, and the average value was taken. Results are as follows: Figure 4 As shown. By Figure 4 It is evident that the soluble solids content of blueberries in the experimental group was higher than that in the control group and the positive control group, indicating that fruit and vegetable preservatives containing alginate oligosaccharides (AOS) are beneficial for maintaining fruit quality.
[0043] (3) Titratable acid content The determination was performed using the standard NaOH titration method. The results are as follows: Figure 5 As shown. By Figure 5 It is evident that the titratable acid content of blueberries in the experimental group was consistently higher than that in the control group and the positive control group, indicating that fruit and vegetable preservatives containing alginate oligosaccharides (AOS) can effectively slow down the decrease in titratable acidity and maintain their good flavor during storage.
[0044] (4) Rot rate The number of rotten blueberries in each treatment group was counted each time. Rotten fruit was identified by the appearance of lesions and mold on the skin. The rot rate was calculated using the formula: Rot Rate / % = Number of Rotten Fruits / Total Number of Fruits × 100. Results are as follows: Figure 6 As shown. By Figure 6 It is evident that the rot rate of blueberries in the experimental group was consistently lower than that in the control group and the positive control group, indicating that the fruit and vegetable preservative containing alginate oligosaccharides (AOS) had a better anti-rot effect on the fruit.
[0045] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.
Claims
1. A type of Bacillus brilliance, characterized in that: Bacillus splendidus LZY-12138 was deposited on December 18, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.29352, and is classified as Bacillus splendidus (…). Paenibacilius lautus The address of the collection is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.
2. An application of the *Bacillus brilliance* as described in claim 1, characterized in that: The application of the Bacillus Brilliantus LZY-12138 in the degradation of sodium alginate.
3. The application of *Bacillus scintillans* according to claim 2, characterized in that: The application of Bacillus Brilliantus LZY-12138 in the degradation of sodium alginate to prepare alginate oligosaccharides with a degree of polymerization of 2-3.
4. The application of *Bacillus scintillans* according to claim 3, characterized in that: Application of the Bacillus Brilliantus LZY-12138 in the preparation of a brown algae oligosaccharide preservative for vegetables and fruits.
5. A fruit and vegetable preservative containing fucoidan oligosaccharides, characterized in that: The fruit and vegetable preservative containing the Bacillus splendens as described in claim 1 is prepared by enzymatic hydrolysis using sodium alginate as a substrate.
6. The fruit and vegetable preservative containing fucoidan oligosaccharides according to claim 5, characterized in that: The content of brown algae oligosaccharides is 50mg / L-250mg / L.
7. A method for preparing a fruit and vegetable preservative containing fucoidan oligosaccharides as described in claim 5, characterized in that: Includes the following steps: Step S1: Inoculate the *Bacillus Brilliantus* as described in claim 1 into a liquid culture medium at an inoculation rate of 1%-3% v / v and culture with shaking for 12-36 h; then transfer the inoculation to a fermentation culture medium at an inoculation rate of 1%-3% v / v and culture with shaking at 28-32℃ and 180-200 r / min for 24-36 h. The resulting bacterial solution is obtained by centrifugation, precipitation with (NH4)2SO4, and dialyzing at 4℃ to obtain crude enzyme solution. In step S2, sodium alginate is mixed with the crude enzyme solution obtained in S1 and distilled water, and degraded in a constant temperature water bath at 25-30℃ for 24-48 hours. Then, the enzyme is inactivated by boiling in a water bath for 15-30 minutes. After centrifugation at 10000r / min for 10-15 minutes, the resulting supernatant is the fruit and vegetable preservative containing alginate oligosaccharides.
8. The preparation method according to claim 7, characterized in that: In step S1, the liquid culture medium consists of 1.0 g peptone, 5.0 g sodium chloride, 1.0 g sodium alginate, 5.0 g ammonium chloride, 2.0 g potassium chloride, 1.0 g magnesium sulfate, and 1 L of distilled water; the fermentation culture medium consists of 1.0 g peptone, 5.0 g sodium chloride, 5.0 g sodium alginate, 2.0 g dipotassium hydrogen phosphate, 1.0 g magnesium sulfate, 0.2 g calcium chloride, and 1 L of distilled water.
9. The preparation method according to claim 7, characterized in that: In step S2, the mass ratio of sodium alginate to crude enzyme solution and distilled water is (0.8~1.2):(2.5~3.5):(90~110).
10. A fruit and vegetable preservative containing brown algae oligosaccharides as described in claim 5, used for blueberry preservation.