Application of luteolin in reducing generation of odor substance indole in or out of rumen of ruminant animal

Abstract

The invention discloses application of luteolin in reduction of indole production in ruminant rumen or in vitro. The invention also discloses a method for reducing or inhibiting the production of indole in a ruminant rumen or in an in-vitro fermentation system. It is found for the first time that luteolin can inhibit the activity of tryptophanase and reduce generation of indole. The invention has important application value in improving ruminant rumen microenvironment and reducing emission of malodorous substances.

Description

Technical Field

[0001] This invention belongs to the field of feed additives and ruminant nutrition and environmental control technology, specifically involving the application of luteolin in reducing the production of indole, an odor-causing substance, in the rumen or in vitro in ruminants. Background Technology

[0002] With the continued growth in global demand for dairy products and increasingly stringent consumer requirements for food safety and quality, the dairy farming industry faces the dual pressure of improving both production efficiency and product quality. The search for safe, efficient, and green natural feed additives has become an urgent issue. Flavonoids, as plant secondary metabolites, possess a wide range of biological activities and show great potential for application in ruminant nutrition. Luteolin, an important flavonoid, is widely found in various plants such as celery, chrysanthemum, and chamomile, and exhibits significant anti-inflammatory, antioxidant, antibacterial, and antiviral activities.

[0003] Several studies have demonstrated that flavonoids exhibit unique advantages in regulating rumen fermentation. Furthermore, milk flavor is a key factor influencing dairy product quality and consumer acceptance. Indole and skatole, produced by rumen microorganisms degrading tryptophan, are the main compounds causing off-odors in animal products. Therefore, effectively inhibiting indole production is of significant value in improving animal products.

[0004] However, no specific flavonoid has yet been identified that can inhibit indole production. Therefore, there is an urgent need to screen and elucidate a safe, natural flavonoid that can target and regulate rumen microbial tryptophanase activity, significantly inhibit indole production, and not affect the basic rumen fermentation process. However, this goal still faces significant technical uncertainties and difficulties in achieving under current technological conditions. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide the application of luteolin in inhibiting the production of indole in the rumen or in vitro in ruminants (preferably dairy cows) to reduce the level of odor substances in the rumen, improve nitrogen utilization efficiency, and improve the farming environment.

[0006] Technical solution: In order to achieve the above objectives, the present invention provides the application of luteolin in reducing the production of indole in the rumen or in vitro in ruminants.

[0007] The concentration of luteolin is 0.05~0.2mM.

[0008] The ruminant is a Holstein cow.

[0009] The present invention also provides a method for reducing or inhibiting indole production in the rumen or in vitro fermentation system of ruminants, the method comprising adding luteolin to the rumen or in vitro fermentation system of ruminants, thereby reducing the conversion of tryptophan to indole by inhibiting tryptophanase TnaA activity and / or inhibiting the growth of indole-producing bacteria.

[0010] The concentration of luteolin is 0.05~0.2mM, preferably 0.2mM, which significantly reduces the content of indole and skatole in the fermentation broth and improves the acetic acid / propionic acid ratio and nitrogen utilization efficiency in rumen fermentation.

[0011] The luteolin is added to the basal diet of ruminants in the form of a feed additive.

[0012] The fermentation system includes a substrate, fresh rumen fluid, a buffer solution, and tryptophan.

[0013] The substrate includes concentrate and roughage. The concentrate consists of corn and soybean meal, and the roughage consists of alfalfa and whole-plant silage corn.

[0014] The fermentation conditions are 39°C and anaerobic fermentation for 24 h. The indole content in the fermentation broth is reduced by more than 50% compared with the control group without luteolin, preferably by more than 70%.

[0015] The indole bacterium mentioned is Prevotella bryantii B14 (abbreviated as PB14).

[0016] Furthermore, the luteolin was purchased from Boson Biotechnology (Shanxi) Co., Ltd., with production batch number QK-2025102503 and a purity of 98.13%.

[0017] Furthermore, the luteolin is used to inhibit the growth and indole-producing ability of Prevotellabryantii B14 (PB14) derived from the rumen of ruminants, wherein the effective final concentration for inhibiting its growth is 0.2 mM, while a final concentration of 0.04 mM has virtually no adverse effect on its growth. Therefore, the optimal concentration of the luteolin for inhibiting the indole-producing ability of PB14 derived from the rumen is 0.04 mM.

[0018] Furthermore, the effective concentration of luteolin for inhibiting tryptophanase TnaA activity in vitro is 10 μM.

[0019] Furthermore, the optimal final concentration of luteolin for inhibiting indole production in the fermentation broth during rumen in vitro fermentation is 0.2 mM.

[0020] Beneficial Effects: Compared with the prior art, the present invention has the following advantages: The present invention is the first to discover that luteolin can inhibit the activity of tryptophanase and reduce the production of indole. The luteolin of the present invention can also inhibit the growth of indole-producing bacteria from the rumen of ruminants, thereby inhibiting indole production. The present invention has important application value in improving the rumen microenvironment of ruminants and reducing the emission of malodorous substances. Attached Figure Description

[0021] Figure 1 This figure shows the in vitro validation results of candidate plant extracts with inhibitory effects. A represents the OD of PB14 after 12 h of treatment with different candidate plant extracts. 600 B shows the growth curves of PB14 at different concentrations after 24 h of treatment. C shows the substrate residue after 1 min of reaction between TnaA and each candidate plant extract (LU: luteolin; PA: paeoniflorin; VE: tocopherol; RH: emodin). Data are expressed as percentages, and statistical significance is indicated by an asterisk (ns = no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). D shows the indole content of 0.04 mM luteolin after 0, 4, 12, and 24 h of treatment with PB14. Each test was independently repeated four times, and the significance was as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ns = no significance.

[0022] Figure 2 The effect of luteolin on the content of indole and skatole in the fermentation broth after 24 h of fermentation was investigated. Each test was independently repeated four times. Different lowercase letters indicated significant differences between treatments at the same time point (or in the same row / column) (p < 0.05); those with the same letter did not indicate significant differences. Detailed Implementation

[0023] The embodiments of the present invention will be described in detail below with reference to the examples. The following examples are only for illustrating the present invention. Unless otherwise specified, the conditions in the examples are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all commercially available conventional products.

[0024] The method for detecting indole content in this invention refers to the following literature (Schreurs N, Tavendale M, Lane G, Barry T, Marotti D, McNabb W. Postprandial indole and skatole formation in the rumen when feeding white clover, perennial ryegrass and Lotuscorniculatus, 2003). The method for detecting volatile fatty acid content in rumen fluid refers to the following literature (Jin W, Meng ZX, Wang J, Cheng YF, Zhu WY. Effect of nitrooxy compounds with different molecular structures on the rumen methanogenesis, metabolic profile, and methanogenic community. Current Microbiology, 2017). The method for detecting ammonia nitrogen content in rumen fluid refers to the following literature (Feng Zongci, Gao Min. Improvement of the method for determining ammonia nitrogen content in rumen fluid by colorimetry [J]. Animal Husbandry and Feed Science, 2010, (6):37.).

[0025] Luteolin was purchased from Boson Biotechnology (Shanxi) Co., Ltd., with production batch number QK-2025102503 and a purity of 98.13%.

[0026] Indole-producing bacteria: Prevotella bryantii B14, abbreviated as PB14, purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product number B260991.

[0027] Example 1: Test on the inhibitory effect of luteolin on the indole production and tryptophanase activity of Prevotella.

[0028] 1. Preparation of working solution for candidate inhibitors

[0029] Weigh 20 mg of luteolin (Shanghai Taoshu Biotechnology Co., Ltd., product number: T1027) and dissolve it in 698.7 μL DMSO, then vortex to prepare a 100 mM stock solution.

[0030] Weigh 25 mg of paeoniflorin (Shanghai Taoshu Biotechnology Co., Ltd., product number: T2230) and dissolve it in 520.3 μL DMSO. Vortex the solution to prepare a 100 mM stock solution.

[0031] Weigh 20 mg of tocopherol (Shanghai Shenze Chemical Technology Co., Ltd., product number: T914084-10 mg) and dissolve it in 464.3 μL of DMSO. Vortex the solution to prepare a 100 mM stock solution.

[0032] Weigh 25 mg of emodin (Shanghai Taoshu Biotechnology Co., Ltd., catalog number: T5S0384) and dissolve it in 594.7 μL of DMSO. Vortex the solution to prepare a 100 mM stock solution.

[0033] 2. In vitro verification of the ability of luteolin to inhibit indole production by Prevotella.

[0034] The Prevotella PB14 strain used in the experiment was purchased from Ningbo Mingzhou Biotechnology Co., Ltd., catalog number B260991. The complete genome of strain PB14 can be obtained and downloaded from NCBI (https: / / www.ncbi.nlm.nih.gov / nuccore / NZ_FOEM00000000).

[0035] The culture medium for this strain consisted of: 4.5 g / L of standard Medium 159 (Shandong Top Biotechnology Co., Ltd., catalog number: MD043B); 1 g / L of maltose; 1 g / L of soluble starch; and 1 g / L of cellobiose. These substances were dissolved in 700 mL of distilled water, mixed thoroughly, and then 300 mL of clear, cell-free rumen fluid and 1 mL of 0.1% resazurin solution were added. The mixture was heated to boiling, and CO2 was immediately introduced. Before dispensing the culture medium, 1.0 g of L-cysteine ​​hydrochloride was added. Once the medium color changed from red to colorless, it was dispensed into anaerobic fermentation tubes, 10 mL per tube. The tubes were quickly sealed with rubber stoppers, capped with aluminum caps, and then autoclaved at 121℃ and 103 kPa for 20 min. The preparation of the clear, cell-free rumen fluid involved collecting fresh bovine rumen fluid, filtering it through four layers of sterile gauze, and centrifuging it at 4℃ and 12000 rpm / min for 20 min.

[0036] Initial screening: The culture medium was preheated in a 39℃ incubator before use, and then transferred to a clean bench. 0.2% tryptophan solution (0.2 mL / 10 mL culture medium) was added to each tube. The experiment was divided into six groups: control group (CON, 0.2 mL culture medium), DMSO group (DMSO, 0.2 mL DMSO), luteolin group (LU, 0.2 mL luteolin stock solution), paeoniflorin group (PA, 0.2 mL paeoniflorin stock solution), tocopherol group (VE, 0.2 mL tocopherol stock solution), and rhein group (RH, 0.2 mL rhein stock solution), with four replicates per group. 3% bacterial culture (12 h incubated) was inoculated into anaerobic tubes, shaken well, and incubated in a 39℃ incubator. Growth curves were measured every 2 h for the first 12 h, and then every 6 h thereafter, for a total monitoring period of 24 h. The absorbance (OD value) of bacterial cultures in 96-well plates was measured at 600 nm using a DEL-100 microplate reader (MIU Instrument Co. Ltd., Hangzhou, China). A blank control (containing only culture medium) was used for absorbance calibration before measurement to accurately analyze the growth curve of PB14. Indole content was measured at 4 h, 12 h, and 24 h.

[0037] Preliminary screening results showed that luteolin significantly inhibited the growth of PB14 after 12 h of anaerobic culture (P < 0.05). Figure 1 A).

[0038] Secondary screening: Luteolin, which showed inhibitory potential in the initial screening, was selected. Its OD600 and indole content were also measured under anaerobic conditions. Inhibitor concentration gradients were set, with final inhibitor concentrations of 0.04 mM and 0.2 mM, respectively. Four biological replicates were performed for each gradient. Further secondary screening results showed that 0.2 mM luteolin significantly inhibited the growth of PB14, but 0.04 mM had almost no effect. Figure 1 B) suggests that the antibacterial effect of luteolin on this strain is dose-dependent.

[0039] 3. In vitro verification of luteolin's inhibition of tryptophanase activity.

[0040] The amino acid sequence of tryptophanase (TnaA) was obtained from the whole genome of PB14. The sequence is: MVEPIKKSTREQREKWLNEAHQNVFMLKSDYVYIDLLTDSGTGAMSDRQWAAMMMGDESYGGARSFYHMKDTITELTGFEYVIPTHQGRAAENVLFSYLVKPGMIIPGNAHFDTTKGHIESRKAFAIDVTVKEAYDTQLEVPFKGNVDLAKLEKVLQENKGNVPFMVLTVTNNTVGGQPVSMQNIRETCELCHKYGVPVNVDSARF IENAYFIKTREKGYENKTLREIALEMFSYADSMTMSAKKDGLVNMGGFIATNKQEWYEGAKMFCIPFEGFLTYGGMNGRDMDALAVGLKENIEFEMVETRIKQVHYLAAKLDEYGIPYQR PAGGHAIFVDADKVLTNIPKEEFPAQTLTCELYLEAGIRACEIGYVLADRDPVTRENRFGGNDFVRLCIPRRVYTNNHMDVIAAALKNVYDRRNTIKRGLEITWEAELMRHFTCQFKRLG.

[0041] The gene sequence encoding tryptophanase (TnaA) is as follows:

[0042]

[0043] Expression and purification of TnaA: Plasmid construction was commissioned to General Biotechnology (Anhui) Co., Ltd., specifically as follows: The target gene sequence of tryptophanase (TnaA) containing Xho I and Not I restriction sites was synthesized. The pET28a vector was digested with Xho I and Not I restriction enzymes to obtain a linear pET28a vector. The target gene sequence was ligated to the linearized pET28a vector using T4 DNA ligase. The ligation product was transformed into BL21(DE3) E. coli competent cells to obtain recombinant strains. The recombinant strains were inoculated onto LB agar plates containing kana-resistant bacteria. Single colonies were picked and cultured in kana-resistant LB medium, and positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequence identification. Prokaryotic expression plasmids with correct sequencing were inoculated into kana-resistant LB solid medium to screen for positive strains and cultured overnight at 37°C. Pick a single colony and place it in a test tube. Incubate overnight at 37°C with shaking at 215 rpm. Then, inoculate 1% of the colony into LB liquid medium and incubate with shaking for about 4 hours (until the bacterial OD of the culture medium is reached). 600 (Value 0.5), continue culturing at 18℃ until OD 600 The concentration was 0.8. Expression was induced overnight with IPTG to a final concentration of 0.1 mM. After induction, the cells were collected by centrifugation at 4500 × g for 20 min at 4 °C. The cells were resuspended in buffer A [0.1 M potassium phosphate buffer (pH 7.5), 50 mM imidazole, 50 μM pyridoxal phosphate (PLP)], and the supernatant was collected after sonication. The supernatant was loaded onto a Ni-NTA HP column (GE Healthcare) and purified on an AKTApress FPLC system (Amersham Bioscience), followed by washing with buffer A. The target protein was eluted with buffer B [0.1 M potassium phosphate buffer (pH 7.5), 350 mM imidazole, 50 μM PLP]. The eluted protein was stored at -80 °C for long-term storage.

[0044] In vitro inhibition assay: S-(o-nitrophenyl)-L-cysteine ​​(SOPC) was purchased in solid form (Beijing Kaiguo Technology Co., Ltd., catalog number: B191972), dissolved in DMSO at a concentration of 20 mM. The experiment was conducted in 96-well black transparent plates and incubated at 37°C for 5 min. The reaction system consisted of 5 μL TnaA (final concentration 604.3 nM), 50 μL reaction buffer (0.1 M potassium phosphate, pH 7.5), and 5 μL luteolin (10 μM and 100 μM). The control group was replaced with buffer instead of the inhibitor. Subsequently, 45 μL of 1.1 mM SOPC was added, and the absorbance of substrate degradation (370 nm) was measured using a CLARIOstar Plus microplate reader (BMG Labtech). SOPC concentration was determined using a standard curve (10–900 μM). The initial rate curve was fitted using MATLAB linear regression, and the rate was taken as the mean ± standard error (SEM) of four biological replicates.

[0045] In vitro assays showed that 10 μM luteolin effectively inhibited TnaA activity (P < 0.01). Figure 1 C). Furthermore, compared to the control group, the indole content in the bacterial culture of the 0.04 mM luteolin group was significantly lower after 12 h of culture, and the indole content also showed a decreasing trend at 4 h and 24 h, but the differences were not statistically significant (P < 0.01). Figure 1 D).

[0046] In summary, the results show that luteolin, on the one hand, directly inhibits TnaA activity, blocking the conversion of tryptophan to indole at the enzymatic level, and on the other hand, has a certain antibacterial effect on PB14, thus reducing the potential for indole formation at the microbial community level. This provides experimental evidence and theoretical support for its application in reducing the production of indole, an odor-causing substance in the rumen of ruminants.

[0047] Example 2: Assay of luteolin inhibiting indole formation during rumen fermentation

[0048] 1. Preparation of luteolin working solution

[0049] Weigh 0.2862 g of purchased luteolin powder (Boson Biotechnology, purity 98.13%). Dissolve in 5 mL DMSO to prepare a 200 mM luteolin stock solution.

[0050] 2. Rumen in vitro fermentation test

[0051] The experimental animals were housed at the dairy cattle experimental base of the College of Animal Science and Technology, Nanjing Agricultural University (Nanjing, Jiangsu Province). Three multiparous Holstein dairy cows with permanent rumen fistulas and an average weight of 613±35 kg were used. On the day of the experiment, rumen fluid was collected half an hour before morning feeding and quickly placed into a pre-insulated bottle filled with carbon dioxide and brought back to the laboratory. The collected fresh rumen fluid was filtered through four layers of gauze, and the rumen fluid was mixed with buffer solution at a ratio of 1:2 (v / v) to prepare a mixed artificial rumen culture medium. The composition of the buffer solution is shown in Table 1. The mixed artificial rumen culture medium was dispensed into 180 mL fermentation bottles at a rate of 100 mL each. The temperature was maintained at 39℃ throughout the process, and carbon dioxide was continuously introduced to maintain an anaerobic environment. 1.0 g of substrate (a concentrate-to-roughage ratio of 6:4, with concentrate consisting of corn and soybean meal at a mass ratio of 7:3, and roughage consisting of alfalfa and whole-plant silage corn at a mass ratio of 4:6), a final concentration of 5 mM tryptophan solution, and different doses of luteolin solution were pre-added to the fermentation flasks. All fermentation flasks were fermented at 39℃ and 80 r / min for 24 hours. The experiment was divided into four groups: 1) Control group: no additive, but supplemented with 100 μL DMSO; 2) Low-dose group: 25 μL of 200 mM luteolin stock solution was added, and 75 μL DMSO was added; 3) Medium-dose group: 50 μL of 200 mM luteolin stock solution was added, and 50 μL DMSO was added; 4) High-dose group: 100 μL of 200 mM luteolin stock solution was added, without additional DMSO.

[0052] Table 1 Buffer composition (1 L)

[0053] Specific solution preparation:

[0054] (1) Trace element solution (solution A)

[0055] Add 13.2 g CaCl2·2H2O, 10.0 g MnCl2·4H2O, 1.0 g CoCl2·6H2O, and 8.0 g FeCl3·6H2O to deionized water to a final volume of 100 mL and mix well.

[0056] (2) Buffer solution (Solution B)

[0057] Add 4.0 g NH4HCO3 and 35.0 g NaHCO3 to deionized water to a final volume of 1000 mL and mix well.

[0058] (3) Solution of constant elements (C solution)

[0059] Add 9.45 g Na2HPO4·12H2O, 6.2 g KH2PO4, and 0.6 g MgSO4·7H2O to deionized water to a final volume of 1000 mL and mix well.

[0060] (4) 0.1% resazurin solution (D solution)

[0061] Add 100 mg of resazurin to 80 mL of deionized water, dissolve completely, and then bring the volume to 100 mL. Mix well.

[0062] (5) Reducing agent solution (E solution)

[0063] Add 625 mg L-cysteine ​​hydrochloride, 4.0 mL 1M NaOH, 625 mg Na2S 9H2O, and 95 mL distilled water. Mix well.

[0064] 3. Results Analysis

[0065] The results are shown in Table 2. Luteolin significantly promoted rumen fermentation kinetics in the early stages of fermentation. Compared with the control group, the addition of luteolin significantly increased the cumulative gas production at 6 h and 12 h (P < 0.05). The addition level of 0.2 mM luteolin showed the highest gas production at 6 h, significantly higher than the CON group. However, by 24 h, there was no significant difference in cumulative gas production among the groups, suggesting that luteolin mainly accelerated the substrate degradation rate and early microbial activation process during the fermentation initiation stage, without adversely affecting the overall fermentation endpoint. Meanwhile, the rumen fluid pH in all groups remained within the normal physiological range at 24 h of fermentation, indicating that at the addition level in this experiment, luteolin did not disrupt the acid-base homeostasis of the rumen environment and had good safety for the rumen microbiota.

[0066] Table 2. Effects of luteolin on gas production (cumulative gas production) and pH in rumen in vitro fermentation

[0067] The differences between groups labeled with different letters (a, b, c, d) were statistically significant (P < 0.05).

[0068] Regarding the composition of volatile fatty acids, compared with the control group, the addition of 0.2 mM luteolin significantly increased the acetic acid concentration (P < 0.001) and significantly decreased the concentrations of propionic and butyric acids (P < 0.01), resulting in a significant increase in the acetic acid / propionic acid ratio (A / P). Simultaneously, the concentrations of branched-chain fatty acids such as isobutyric acid and isovaleric acid were significantly decreased in the 0.2 mM group (P < 0.01). Combined with nitrogen metabolism indicators, the addition of 0.05–0.2 mM luteolin significantly reduced the ammonia nitrogen concentration at 24 h of fermentation (P < 0.001), exhibiting a clear dose-dependent effect, with the 0.2 mM group showing the most significant inhibitory effect. These results collectively indicate that luteolin can effectively inhibit the excessive degradation and deamination of dietary proteins and amino acids by rumen microorganisms without inhibiting overall fermentation capacity, optimize the rumen fermentation acidity, and reshape the rumen nitrogen metabolism pathway (Table 3).

[0069] Table 3 Effects of luteolin supplementation in high-concentrate diets on VFA and NH3-N in vitro fermentation (24 h)

[0070] Furthermore, luteolin had a significant regulatory effect on the formation of odor precursors in the fermentation broth. Compared with the control group, the addition of luteolin at a final concentration of 0.2 mM significantly reduced the indole content in the fermentation broth (P < 0.01), with a reduction of 71.9%. The indole content in the low-dose groups with final concentrations of 0.05 mM and 0.1 mM was numerically higher than that in the control group, but the difference was not statistically significant (P > 0.05). Regarding skatole, the addition of luteolin at final concentrations of 0.05 mM and 0.2 mM both significantly reduced the skatole content in the fermentation broth (P < 0.05). Figure 2 In conclusion, under the conditions of this experiment, luteolin can improve rumen nitrogen utilization efficiency while ensuring normal rumen fermentation function, and significantly reduce the formation of malodorous precursors such as indole and skatole. Therefore, it is expected to be used to optimize rumen fermentation patterns in ruminants and improve the breeding environment, and has good application prospects and promotion value.

Claims

1. Application of luteolin in reducing indole production in the rumen or in vitro in ruminants.

2. Use according to claim 1, characterized in that, The concentration of luteolin is 0.05~0.2mM.

3. Use according to claim 1, characterized in that, The ruminant is a Holstein cow.

4. A method of reducing or inhibiting indole production in a ruminant's rumen or in a fermentation system in vitro, characterized in that, The method involves adding luteolin to the rumen of ruminants or to an in vitro rumen fermentation system, thereby reducing the conversion of tryptophan to indole by inhibiting tryptophanase TnaA activity and / or inhibiting the growth of indole-producing bacteria.

5. The method of claim 4, wherein, The concentration of luteolin is 0.05~0.2mM.

6. The method of claim 4, wherein, The luteolin is added to the basal diet of ruminants in the form of a feed additive.

7. The method according to claim 4, characterized in that, The fermentation system includes substrate, fresh rumen fluid, buffer solution, and tryptophan.

8. The method according to claim 7, characterized in that, The substrates include concentrate and roughage, with the concentrate consisting of corn and soybean meal, and the roughage consisting of alfalfa and whole-plant silage corn.

9. The method according to claim 4, characterized in that, The fermentation conditions were 39°C and anaerobic conditions for 24 hours.

10. The method according to claim 4, characterized in that, The indole bacterium is Prevotella bryantii B14.

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