A high-efficiency degradation of tea saponin of serre and its application in fermented oil tea chaff feed

Abstract

The application provides a serreia that can degrade tea saponin efficiently and application thereof in fermentation of oil tea chaff feed, and relates to the field of microbial biotechnology and feed processing.The application discloses a serreia that can degrade tea saponin efficiently, namely, serreia NCUF-1, and application thereof.The strain is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC M 20253034.The serreia NCUF-1 is compounded with lactobacillus plantarum at a specific ratio, inoculated into oil tea chaff, and subjected to mixed bacteria solid state fermentation, so that tea saponin in the oil tea chaff can be degraded efficiently, the degradation rate of tea saponin in the oil tea chaff reaches 40% after three days of fermentation; meanwhile, the product quality is significantly improved, the protein content is stably above 8%, the total acid content is increased to more than 2.5 times of the raw material, at least 13 kinds of beneficial flavor substances such as linalool and phenylethanol are synthesized, and the palatability is effectively improved.The application provides a new strain that is efficient and safe for biological detoxification and high-value feed utilization of oil tea chaff, and provides an industrialized fermentation technical scheme.

Description

Technical Field

[0001] This invention relates to the fields of microbial biotechnology and feed processing, and in particular to a highly efficient Serratia marcescens bacterium that degrades tea saponins and its application in fermented camellia oil meal feed. Background Technology

[0002] my country boasts abundant camellia oil resources, with a planting area exceeding 67 million mu (approximately 4.8 million hectares) and an annual production of 900,000 tons of camellia oil meal, providing ample raw materials for animal feed. Camellia oil meal, a byproduct of camellia oil seed extraction, has a considerable yield, reaching 600,000 tons annually. Currently, camellia oil meal is commonly used as a pond cleaning agent, fuel, fertilizer, or for extracting active ingredients such as tea saponins, flavonoids, polyphenols, and polysaccharides for further applications in chemical, building materials, agriculture, pharmaceutical, and feed additive fields. However, its comprehensive utilization rate is low, resulting in significant resource waste. Research indicates that camellia oil meal contains rich nutrients and various functional active ingredients, which can promote the growth and development of livestock and poultry, enhance their disease resistance, and improve the quality of meat, eggs, and milk. Theoretically, it is a highly promising unconventional feed ingredient.

[0003] However, camellia oil meal contains a high content of tea saponin, a pentacyclic triterpenoid glycoside compound. Tea saponin has strong hemolytic toxicity and a bitter taste, which can damage the digestive tract mucosa of animals, affect the absorption of nutrients, and result in extremely poor palatability, severely limiting its direct and large-scale application in livestock and poultry feed.

[0004] Currently, the main methods for detoxifying and processing camellia seed meal into feed include physical, chemical, and biological methods. While physical methods (such as heating and puffing) and chemical methods (such as alkali treatment and organic solvent extraction) have some effectiveness, they generally suffer from high energy consumption, significant loss of nutrients, secondary safety risks caused by chemical reagent residues, or complex and costly processes, making them unsuitable for large-scale feed production.

[0005] Bio-fermentation is considered the most promising method for detoxifying and improving the quality of camellia seed meal due to its mild conditions, low energy consumption, good safety, and potential for enhancing nutritional value. While some research has been conducted in this field, significant shortcomings and bottlenecks remain: Highly efficient, specific strains suitable for feed fermentation are relatively scarce; many strains have limited degradation efficiency or insufficient stability (e.g., while fungi such as Aspergillus and Penicillium, and bacteria such as Bacillus and Pseudomonas in the natural environment possess the ability to degrade tea saponins, these strains generally suffer from limited degradation efficiency, insufficient stability, or demanding growth conditions); Research on the metabolic pathways and key enzyme systems involved in microbial degradation of tea saponins is not yet in-depth, limiting the precision of strain application; furthermore, systematic and applied research on mixed-culture fermentation parameters is limited, and a mature, stable, and scalable process scheme has not yet been formed.

[0006] Therefore, there is an urgent need to provide a solution to improve the above problems. Summary of the Invention

[0007] The purpose of this invention is to provide a Serratia marcescens strain that efficiently degrades tea saponins and its application in fermented camellia seed meal feed.

[0008] In a first aspect, the present invention provides a highly efficient Serratia sp. NCUF-1 for degrading tea saponins, wherein the Serratia sp. NCUF-1 is deposited at the China Center for Type Culture Collection, with accession number CCTCC M 20253034, deposit date of December 29, 2025, and classified as Serratia sp. NCUFSP1.

[0009] Secondly, the present invention provides an application of the above-mentioned Serratia marcescens NCUF-1, including applying the Serratia marcescens NCUF-1 to the production of camellia seed meal biological detoxification and / or fermented feed.

[0010] Optionally, the application of Serratia marcescens NCUF-1 is to use Serratia marcescens NCUF-1 together with Lactobacillus plantarum in the mixed fermentation of camellia seed cake.

[0011] Optionally, the inoculation ratio of Serratia marcescens NCUF-1 to Lactobacillus plantarum is (0.5-2):1.

[0012] Optionally, the inoculation ratio of Serratia marcescens NCUF-1 to Lactobacillus plantarum is 1:1.

[0013] Optionally, the total inoculum amount for the mixed fermentation is 4%-8% of the dry weight of the camellia seed meal.

[0014] Optionally, the total inoculum amount for the mixed fermentation is 6% of the dry weight of the camellia seed cake.

[0015] Optionally, the initial material-to-water ratio for the mixed-culture fermentation is 1:1.

[0016] Optionally, the temperature for the mixed-culture fermentation is 30℃-38℃.

[0017] Optionally, the fermentation time of the mixed bacteria is 3-10 days.

[0018] Optionally, the temperature for the mixed-culture fermentation is 36°C.

[0019] Optionally, after 3 days of mixed fermentation, the degradation rate of tea saponins in the camellia oil meal is ≥40%.

[0020] Optionally, after 3 days of mixed-culture fermentation, the crude protein content is ≥8%.

[0021] Optionally, after 3 days of mixed fermentation, the total acid content is ≥ 2.5 times the total acid content of the camellia seed meal before fermentation.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention provides a novel strain of Serratia marcescens NCUF-1, which can grow efficiently with tea saponin as the sole carbon source, providing a new strain resource with strong specificity and high activity for the biological detoxification of camellia oil meal, and solving the problem of the lack of efficient, stable and feed-fermentation-appropriate strains in the prior art. (2) The *Serratia marcescens* strain provided by this invention has certain application value in aquaculture under specific conditions, especially showing potential in biological control and growth promotion. *Serratia marcescens* NCUF-1 is γ-hemolytic, indicating that it does not have hemolytic activity. *Serratia marcescens* NCUF-1 has no inhibitory effect on Gram-negative *Escherichia coli* and *Salmonella*, but it has a significant inhibitory effect on Gram-positive *Staphylococcus aureus*. *Serratia marcescens* NCUF-1 has a 100% resistance rate to cefazolin, nitrofurantoin, cefotetan, cefoxitin, cefuroxime, tigecycline, and colistin; however, it has no resistance to levofloxacin, norfloxacin, and minocycline. Therefore, this strain can be used to prepare disease control drugs for loach farming or as a feed additive, reducing antibiotic use. (3) The mixed fermentation method provided by the present invention can significantly accelerate the degradation process of tea saponins through the specific combination and synergistic effect of Serratia marcescens NCUF-1 and Lactobacillus plantarum. The degradation rate can reach more than 40% in 3 days of fermentation. It has high detoxification efficiency and short cycle, which is better than single-strain fermentation or known strain combination. (4) The mixed fermentation method provided by the present invention can significantly improve the comprehensive nutritional value of the fermentation product while efficiently removing tea saponins, so that the protein content is kept stable at more than 8% and the total acid content is increased to more than 2.5 times that of the raw material, thus achieving multiple goals of detoxification, nutrient preservation and anti-corrosion and acidification. (5) The mixed fermentation method provided by the present invention can improve the flavor of the product in a targeted manner. It not only effectively degrades undesirable aldehydes such as isovaleraldehyde, but also specifically synthesizes at least 13 beneficial flavor substances such as linalool, phenylethanol and a series of lactones, which fundamentally improves the palatability of fermented camellia seed meal feed. This is an effect that is difficult to achieve with existing simple fermentation processes. (6) The whole-process biological fermentation method adopted in this invention is mild, does not add chemical reagents, and has no toxic or harmful substance residues. The final product is highly safe and environmentally friendly, providing an effective way for the green and high-value feed utilization of camellia seed meal. Attached Figure Description

[0023] Figure 1 This is a colony characteristic diagram of Serratia marcescens NCUF-1 on LB solid medium according to the present invention; Figure 2 This is a phylogenetic tree diagram of Serratia marcescens NCUF-1 of the present invention; Figure 3 The growth curve of Serratia marcescens NCUF-1 in liquid culture medium with tea saponin as the sole carbon source is shown in this invention. Figure 4 This is a graph showing the changes in tea saponin content during fermentation in the control group, single-strain group, and mixed-strain group in Example 3 of the present invention; Figure 5 This is a graph showing the changes in protein content and total acid content during fermentation in the control group, single-strain group, and mixed-strain group in Example 3 of the present invention; wherein, Figure 5 (A) in the graph shows the change in protein content; Figure 5 (B) in the graph shows the change in total acid content; Figure 6 This is a graph showing the changes in flavor substance content during fermentation in the control group, single-strain group, and mixed-strain group (inoculated with Serratia marcescens NCUF-1 and Lactobacillus plantarum in a 1:1 ratio) in Example 3 of the present invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains.

[0025] This invention provides a Serratia marcescens NCUF-1 strain that efficiently degrades tea saponins. This Serratia marcescens NCUF-1 strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 20253034.

[0026] In fact, this strain was screened from camellia seed cake and its surrounding environment and can grow using tea saponin as the sole carbon source.

[0027] The present invention also provides an application of the above-mentioned Serratia marcescens NCUF-1, including applying Serratia marcescens NCUF-1 with preservation number CCTCC M20253034 to the production of biodetoxification and / or fermented feed from camellia seed meal.

[0028] In some embodiments, Serratia marcescens NCUF-1 is used by combining Serratia marcescens NCUF-1 with Lactobacillus plantarum in the mixed fermentation of camellia seed cake.

[0029] In fact, this invention isolates and domesticates the highly efficient microbial strain Serratia marcescens NCUF-1, which degrades tea saponins, and combines it with Lactobacillus plantarum in a specific ratio. This mixture is then inoculated into camellia oil meal for mixed-culture solid-state fermentation, which can efficiently degrade tea saponins in camellia oil meal while significantly improving product quality and palatability. This results in the development of a safe and nutritious new protein feed ingredient. Pilot-scale production of this ingredient is being conducted to explore its feasibility. This invention helps promote the resource utilization of camellia oil by-products, alleviate feed shortages, and promote the extension and sustainable development of the camellia oil industry chain, thus having significant economic, ecological, and social benefits.

[0030] In some embodiments, the inoculation ratio of Serratia marcescens NCUF-1 to Lactobacillus plantarum is (0.5-2):1.

[0031] In some embodiments, the inoculation ratio of Serratia marcescens NCUF-1 to Lactobacillus plantarum is 1:1.

[0032] In some embodiments, the total inoculum amount of Serratia marcescens NCUF-1 and Lactobacillus plantarum for mixed fermentation is 4%-8% of the dry weight of camellia seed cake.

[0033] In some embodiments, the total inoculum amount of Serratia marcescens NCUF-1 and Lactobacillus plantarum for mixed fermentation is 6% of the dry weight of camellia oil meal.

[0034] In some embodiments, the initial feed-to-water ratio for mixed fermentation of Serratia marcescens NCUF-1 and Lactobacillus plantarum was 1:1.

[0035] In some embodiments, the fermentation temperature of Serratia marcescens NCUF-1 and Lactobacillus plantarum was 30°C-38°C.

[0036] In some embodiments, the fermentation time of mixed culture of Serratia marcescens NCUF-1 and Lactobacillus plantarum is 3-10 days.

[0037] In some embodiments, the fermentation temperature of Serratia marcescens NCUF-1 and Lactobacillus plantarum was 36°C.

[0038] In some embodiments, after 3 days of mixed fermentation, the degradation rate of tea saponins in camellia seed cake was ≥40%.

[0039] In some embodiments, after 3 days of mixed fermentation, the crude protein content in the camellia seed cake is ≥8%.

[0040] In some embodiments, after 3 days of mixed fermentation, the total acid content in the camellia seed meal was ≥ 2.5 times that of the total acid content before fermentation.

[0041] Example 1: Screening and identification of Serratia marcescens NCUF-1 Screening method: Take 1.0 g of camellia seed meal sample, add physiological saline to make a suspension, and inoculate into enrichment medium (LB + 1.0 g tea saponin), and incubate at 32℃ with shaking for 3 days. Then, transfer the culture medium to basal salt medium with tea saponin concentrations of 1.0 g / L to 5.0 g / L for gradual concentration acclimatization. Finally, spread on solid plates containing 5.0 g / L tea saponin to isolate single colonies.

[0042] Strain characteristics: The selected strain NCUF-1 forms round, translucent, moist colonies on LB agar plates, 1-2 mm in diameter; Gram staining is negative. The colony characteristics of Serratia marcescens NCUF-1 on LB solid medium are shown in the figure below. Figure 1 As shown.

[0043] Molecular identification: Genomic DNA was extracted, and the 16S rDNA sequence was amplified and sequenced (GenBank ID OL677875). BLAST alignment and phylogenetic analysis showed that NCUF-1 is most closely related to Serratiasp. It was named Serratiasp. NCUF-1.

[0044] Example 2: Growth characteristics and performance determination of Serratia marcescens NCUF-1 Growth characteristics of Serratia marcescens NCUF-1: The purified Serratia marcescens NCUF-1 was inoculated into LB liquid medium containing 5 g / L tea saponin, and the growth curve was determined. Figure 3 The data chart shown. Figure 3 The results showed that the strain was in the delayed phase before 12 hours; after 12 hours, it entered the logarithmic growth phase, and the cell concentration increased rapidly; after 96 hours, it entered the stationary phase. This indicates that the strain has a strong ability to utilize tea saponins for metabolic growth.

[0045] Safety analysis of Serratia marcescens NCUF-1 strain: The purified Serratia marcescens NCUF-1 was streaked onto blood agar plates and incubated at 37°C for 24 hours. The experimental results were observed and recorded, as shown in Table 1. Staphylococcus aureus hemolytic activity was used as a positive control to observe whether hemolysis occurred. Table 1 shows that Staphylococcus aureus exhibited β-hemolysis, while Serratia marcescens NCUF-1 showed γ-hemolysis, indicating that Serratia marcescens NCUF-1 does not possess hemolytic activity.

[0046] Table 1: Safety analysis data of Serratia marcescens NCUF-1 strain

[0047] Analysis of the antibacterial properties of Serratia marcescens NCUF-1 strain: Table 2 shows the antagonistic effects of Serratia marcescens NCUF-1 against pathogenic Escherichia coli, Salmonella, and Staphylococcus aureus. As the data in Table 2 indicate, Serratia marcescens NCUF-1 showed no inhibitory effect on Gram-negative Escherichia coli and Salmonella, but exhibited significant inhibitory effect on Gram-positive Staphylococcus aureus.

[0048] Table 2: Data on the antibacterial performance of Serratia marcescens NCUF-1 strain

[0049] Antibiotic susceptibility analysis of Serratia marcescens NCUF-1 strain: The results of the susceptibility analysis of Serratia marcescens NCUF-1 to 10 antibiotics are shown in Table 3. As shown in Table 3, Serratia marcescens NCUF-1 showed 100% resistance to cefazolin, nitrofurantoin, cefotetan, cefoxitin, cefuroxime, tigecycline, and colistin; while showing no resistance to levofloxacin, norfloxacin, and minocycline.

[0050] Table 3: Data on antibiotic susceptibility analysis of Serratia marcescens NCUF-1 strain

[0051] Example 3: Application effect of Serratia marcescens NCUF-1 in the fermentation of camellia seed meal feed Fermentation process: The sterilized camellia seed cake is bottled, with a total inoculum quantity of 6%. The following groups are set up: Control group: No inoculation or conventional fermentation.

[0052] Single-strain group: Serratia marcescens NCUF-1 only; or Lactobacillus plantarum only.

[0053] Mixed culture group: Inoculated with Serratia marcescens NCUF-1 and Lactobacillus plantarum (at inoculation ratios of 1:1, 2:1, and 1:2, respectively). Fermentation conditions: material-to-water ratio 1:1, temperature 36℃, fermentation time 10 days.

[0054] Tea saponin degradation effect: During fermentation, the tea saponin content was measured periodically in the control group, single-strain groups (inoculated only with Serratia marcescens NCUF-1, inoculated only with Lactobacillus plantarum), and mixed-strain groups (inoculated with Serratia marcescens NCUF-1 and Lactobacillus plantarum at a ratio of 1:1, 2:1, and 3:1). Results are as follows: Figure 4 As shown, the single-strain fermentation group showed a slower decline in tea saponin content, while the mixed-strain group showed the fastest decline, with a tea saponin degradation rate of over 40% after 3 days of fermentation, demonstrating a significant degradation effect.

[0055] Physicochemical index analysis: Results are as follows Figure 5 As shown, where, Figure 5 (A) in the graph shows the change in protein content; Figure 5 (B) in the graph shows the change in total acid content. From... Figure 5 Data analysis shows that the physicochemical properties of the fermented feed are best when the inoculation ratio is 1:1. This significantly improves the nutritional value and palatability of the fermented products. The protein content remains stable at over 8%, and the total acid content increases significantly to more than 2.5 times that of the raw material. This demonstrates that Serratia marcescens NCUF-1 and Lactobacillus plantarum have a good synergistic effect in camellia oil meal.

[0056] Flavor index analysis: The graph shows the changes in flavor substance content during fermentation in the control group, single-strain group, and mixed-strain group (inoculated with Serratia marcescens NCUF-1 and Lactobacillus plantarum at a 1:1 ratio) in Example 3 of this invention. It can be seen that mixed-strain fermentation effectively degraded various undesirable aldehydes and specifically synthesized 13 beneficial flavor substances, including linalool, phenylethanol, and a series of lactones, significantly increasing the total amount of aldehydes, alcohols, acids, and esters.

[0057] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A highly efficient Serratia marcescens NCUF-1 strain for degrading tea saponins, characterized in that, The Serratia marcescens NCUF-1 strain is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC M 20253034.

2. An application of Serratia marcescens NCUF-1 as described in claim 1, characterized in that, The Serratia marcescens NCUF-1 is used in the biological detoxification of camellia seed meal and / or the production of fermented feed.

3. The application according to claim 2, characterized in that, The application involves using Serratia marcescens NCUF-1 and Lactobacillus plantarum together in the mixed fermentation of camellia seed cake.

4. The application according to claim 3, characterized in that, The inoculation ratio of Serratia marcescens NCUF-1 to Lactobacillus plantarum is (0.5-2):

1.

5. The application according to claim 4, characterized in that, The inoculation ratio of Serratia marcescens NCUF-1 to Lactobacillus plantarum is 1:

1.

6. The application according to claim 3, characterized in that, The total inoculum amount for the mixed fermentation is 4%-8% of the dry weight of the camellia seed meal.

7. The application according to claim 6, characterized in that, The total inoculum amount for the mixed fermentation is 6% of the dry weight of the camellia seed cake.

8. The application according to claim 3, characterized in that, The initial material-to-water ratio of the mixed-culture fermentation is 1:1; and / or, the temperature of the mixed-culture fermentation is 30℃-38℃; and / or, the fermentation time is 3d-10d.

9. The application according to claim 8, characterized in that, The temperature for the mixed-culture fermentation was 36°C.

10. The application according to claim 3, characterized in that, After 3 days of mixed-culture fermentation, the degradation rate of tea saponins in camellia seed cake was ≥40%.

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