A method for high-efficiency degradation of cyanide by kaya silage fermentation and application of a compound lactic acid bacteria ferment

Abstract

The present application relates to the technical field of plant silage fermentation, and particularly relates to a high-efficiency cyanide-degrading cassava silage fermentation method and application of a compound lactic acid bacteria inoculum. 6 6 The silage fermentation method comprises the following steps: mixing cut cassava fresh samples with the compound lactic acid bacteria inoculum, vacuumizing, then sealing and anaerobically fermenting to obtain silage feed, wherein the addition amount of the compound lactic acid bacteria inoculum corresponding to 1 gram of the cassava fresh weight is 5x10 6 6 CFU, the compound lactic acid bacteria inoculum comprises lactobacillus plantarum and lactobacillus buchneri; after fermentation, the cyanide content in the cassava is reduced by 47-50%. The present application mixes the cassava fresh samples with a specific amount of the compound lactic acid bacteria inoculum, and then performs anaerobic fermentation. The method can maximize the retention of the crude protein content, significantly reduce the hydrogen cyanide content in the cassava to a safety threshold, thereby obtaining high-quality silage feed, and the silage feed can improve the digestibility of edible animals.

Description

Technical Field

[0001] This invention relates to the technical field of plant silage fermentation, specifically to a highly efficient cyanide-degrading Kaya silage fermentation method and the application of a compound lactic acid bacteria fermentation agent. Background Technology

[0002] Cnidoscolus chayamansa Mc Vaugh, commonly known as wood spinach, is a perennial shrub belonging to the Euphorbiaceae family, native to Mexico and Central America. It possesses both medicinal and edible value. Its biomass yield is significant (8-10 tons of fresh branches and leaves per acre, which can be processed into 2-2.5 tons of hay powder). Furthermore, its leaves are tender, exhibit strong regenerative ability, and nutritional analysis shows a crude protein content as high as 23.96%, demonstrating its great potential as a high-quality, high-protein forage.

[0003] However, fresh leaves of *Calamus lataniae* contain anti-nutritional factors such as cyanogenic glycosides (119.37 mg / kg), alkaloids, and steroidal saponins. Hydrogen cyanide (HCN), produced by the hydrolysis of cyanogenic glycosides by endogenous enzymes, can inhibit cytochrome oxidase activity, blocking the cellular respiratory chain and leading to acute asphyxiation and death in animals. This toxic risk severely limits its application in animal feed.

[0004] Currently, conventional physical or chemical detoxification methods often lead to nutrient loss. Therefore, there is an urgent need for a kalay silage method that can efficiently degrade cyanide while ensuring processing quality. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a highly efficient method for Kaya silage fermentation that degrades cyanide, and the application of a compound lactic acid bacteria fermentation agent, in order to solve the problem that existing Kaya fresh sample detoxification methods cannot simultaneously achieve high-efficiency degradation of cyanide while ensuring quality.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] A method for fermenting Kaya silage includes the following steps: mixing freshly cut Kaya samples with a compound lactic acid bacteria starter culture, vacuum sealing, and then sealing and anaerobic fermentation to obtain silage. The amount of compound lactic acid bacteria starter culture added per gram of fresh Kaya weight is 5 × 10⁻⁶. 6 ~5.5×10 6 CFU, the compound lactic acid bacteria starter contains Pediococcus lactis, Lactobacillus plantarum and Lactobacillus brunelli; after fermentation, the cyanide content in the carya is reduced by 47-50%.

[0008] This invention involves mixing fresh samples of Kayser's lactic acid bacteria with a compound lactic acid bacteria starter containing Pediococcus lactis, Lactobacillus plantarum, and Lactobacillus brucellosis, wherein the amount of the compound lactic acid bacteria starter added is 5 × 10⁻⁶.6 ~5.5×10 6 Anaerobic fermentation was carried out using CFU / g FM. This method significantly reduced the hydrocyanic acid content in silage to a safe threshold while maximizing the retention of crude protein, thus obtaining high-quality silage. Furthermore, this silage improves the digestibility of food-producing animals.

[0009] Preferably, compared with silage produced by fermentation without adding a fermenting agent, the cyanide content of silage produced by the compound lactic acid bacteria fermenting agent is reduced by 30-35%.

[0010] Preferably, the anaerobic fermentation time is 30-40 days. If the fermentation time is too short, fermentation will not be complete; if it is too long, bacteria will consume the nutrients in the raw materials.

[0011] Preferably, the anaerobic fermentation temperature is 25~30℃, and the fermentation is carried out in the dark.

[0012] Preferably, the length of the cut fresh sample is 2-5 cm.

[0013] Preferably, the fresh Kaya sample is a Kaya plant that has grown to 1.20-1.50m. Kaya plants at this growth stage are rich in nutrients and suitable for use as animal feed.

[0014] Preferably, vacuuming involves placing the cut fresh Kayak sample and the compound lactic acid bacteria fermentation agent into a silage bag and then vacuuming it.

[0015] Preferably, the Kaya silage fermentation method of the present invention includes the following steps:

[0016] Step 1: Cut the Kaya plants that have grown to 1.20~1.50m and remove the soil and impurities;

[0017] Step 2: Cut the harvested fresh Calabash stalks into 2-3 cm lengths, mix with the compound lactic acid bacteria starter culture, pack into silage bags, and vacuum-seal the silage bags. The dosage of compound lactic acid bacteria starter culture is 5 × 10⁻⁶ per gram of fresh Calabash stalks. 6 ~5.5×10 6 CFU;

[0018] Step 3: Seal the vacuum-sealed silage bags and place them in a dark environment at room temperature of 25-30℃ for anaerobic fermentation for 30-40 days.

[0019] The second technical solution of the present invention is:

[0020] Application of a compound lactic acid bacteria starter in the efficient degradation of cyanide: the dosage of compound lactic acid bacteria starter per gram of fresh weight of cyanide is 5 × 10⁻⁶. 6 ~5.5×106 CFU, the compound lactic acid bacteria starter contains Pediococcus lactis, Lactobacillus plantarum and Lactobacillus brunelli; after fermentation, the cyanide content in the carya is reduced by 47-50%.

[0021] The technical solution of the present invention achieves the following beneficial technical effects:

[0022] 1. Highly Effective Detoxification: Anaerobic fermentation with a compound of lactic acid bacteria including Pediococcus lactis, Lactobacillus plantarum, and Lactobacillus brunelli significantly reduced the cyanide content in kaya. Experimental data showed that the cyanide content in the experimental group (LAB group) was reduced by 47.42% and 30.35% compared to before fermentation and the control group (fermentation without adding starter culture), respectively, effectively eliminating potential feed safety hazards.

[0023] 2. Nutritional Retention: The method of this invention can effectively retain the high protein content of Kaya. Compared with the control group, the dry matter (DM) and crude protein (CP) content of the experimental group were significantly higher, and the decrease in CP content (12.12%) was significantly lower than that of the control group (19.37%).

[0024] 3. Improved fermentation quality: Inoculation with compound lactic acid bacteria significantly reduced the pH value of silage (down to 3.87) and significantly increased the content of lactic acid, acetic acid and propionic acid, inhibited butyric acid fermentation, and reduced protein degradation (ammonia nitrogen / TN was significantly lower than the control group).

[0025] 4. Improved digestibility: In vitro rumen fermentation experiments show that the Kaya silage prepared by this invention has good in vitro digestibility and can optimize the rumen fermentation mode (such as increasing the proportion of propionic acid). Attached Figure Description

[0026] Figure 1 The values ​​represent the carboxycyanide content under different treatments in the experimental examples of this invention, where lowercase letters a, b, and c represent the significance level of P < 0.05. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to specific embodiments.

[0028] Example 1

[0029] The efficient cyanide-degrading silage fermentation method of this embodiment adopts the following steps:

[0030] Step 1: Raw material preparation: When the Kaya grows to 1.20~1.50m, it is harvested to remove soil and impurities;

[0031] Step 2, Cutting: Use an automatic grass cutter to cut the harvested Kaya grass sample to a length of 2-3 cm, and thoroughly mix the stems and leaves;

[0032] Step 3, Inoculation: Add compound lactic acid bacteria starter culture (LAB) to the chopped Kaya according to fresh weight, at a rate of 5 × 10⁻⁶. 6 CFU / g FM (colony forming units / fresh weight in grams); Compound lactic acid bacteria fermentation agent (Zhengzhou Haowangnong Biotechnology Co., Ltd., mixed feed additive (OKH4601)) contains Pediococcus lactis, Lactobacillus plantarum, and Lactobacillus buchneri.

[0033] Step 4: Vacuuming: Pack the processed material into silage bags and use a vacuum machine to vacuum the silage bags.

[0034] Step 5: Sealed fermentation: Seal the vacuum-sealed silage bags and place them in a dark environment (25-30℃) for anaerobic fermentation. The fermentation cycle is 30 days.

[0035] Experimental Example

[0036] In this experiment, uniformly grown Calacatta mushroom samples were cut to 2-3 cm. Two treatment groups were established: a control group (no additives added); and an experimental group, i.e., Example 1 (with added compound lactic acid bacteria, 5 × 10⁻⁶). 6 (CFU / g FM); each group had 3 replicates, for a total of 24 bags. After vacuum sealing, the samples were fermented at room temperature in the dark for 30 days, and then samples were taken. The samples without fermentation were used. The fermentation quality, nutrient composition and hydrocyanic acid content of the samples were determined.

[0037] The methods for measuring each indicator are as follows:

[0038] 1. Fermentation quality determination

[0039] After opening the silage bags, a representative sample of 20 g was weighed, mixed thoroughly with 180 mL of distilled water, and extracted at 4℃ for 24 h. The mixture was then filtered through four layers of gauze and qualitative filter paper to obtain the silage extract, which was stored at -20℃ for fermentation quality determination. The pH value was measured using a precision pH meter; the ammonia nitrogen content was determined using the phenol-hypochlorite colorimetric method; and the contents of lactic acid, acetic acid, propionic acid, and butyric acid were determined using high-performance liquid chromatography (HPLC) according to DB15 / T1458.

[0040] 2. Nutritional composition determination

[0041] Approximately 200 g of raw materials and silage samples were dried in an oven at 65℃ to constant weight, pulverized, and passed through a 40-mesh sieve. The samples were then stored in resealable bags for nutrient analysis. Dry matter (DM) content was determined using the drying method according to GB / T 6435. Neutral detergent fiber (NDF) content was determined using the Panthen method according to GB / T 20806, and acid detergent fiber (ADF) content was determined according to NY / T 1459. Crude protein (CP) content was determined using the Kjeldahl method according to GB / T 6432. Water-soluble carbohydrate (WSC) content was determined using the anthrone-sulfuric acid colorimetric method.

[0042] 3. Rumen in vitro fermentation

[0043] The in vitro fermentation experiment was conducted using the Menke in vitro gas production method. Rumen fluid from three healthy Leizhou black goats of similar weight was extracted, filtered through four layers of gauze, and mixed with buffer solution (with carbon dioxide continuously bubbled through before use to ensure an anaerobic environment) at a ratio of 1:2. The mixture was then placed in a 39°C water bath. 40 ml of the culture medium was transferred to a fermentation tube containing 0.3 g of sample, excess air was expelled, and the tube was incubated at 39°C for 48 h. Gas chromatography was used to determine the contents of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid, and valeric acid.

[0044] 4. Detection of cyanide

[0045] Weigh 10-20g of the sample (accurate to 0.001g) into a 250mL distillation flask, add approximately 200mL of water, seal tightly, and let stand at room temperature for 2-4 hours for hydrolysis. Add 20mL of zinc acetate solution and 1-2g of tartaric acid, connect the distillation apparatus, and insert the lower end of the condenser into a 100mL volumetric flask containing 5mL of sodium hydroxide solution below the liquid surface. Distill with steam until approximately 100mL is reached, and then dilute to the mark. Take 10 mL of distillate into a 25 mL colorimetric tube and operate simultaneously with the potassium cyanide standard series (0 μg~1.5 μg hydrogen cyanide): add 1 mL of sodium hydroxide solution and 1 drop of phenolphthalein, adjust with acetic acid until the red color just disappears, add 5 mL of phosphate buffer solution, add 0.25 mL of chloramine T solution at 37℃, mix well and let stand for 5 min, then add 5 mL of isonicotinic acid-pyrazolone solution, add water to 25 mL, develop color at 25℃~40℃ for 40 min, and measure the absorbance at 638 nm using a 2 cm cuvette.

[0046] (1) Changes in nutritional composition

[0047] As shown in Table 1, compared with the direct fermentation control group without additives, the addition of lactic acid bacteria (Example 1) significantly improved the dry matter (DM) and crude protein (CP) retention rates of Kaya silage (P<0.05, the same below).

[0048] Table 1. Nutrient composition (% DM) of Kaya silage fermentation under different treatments

[0049]

[0050] (2) Fermentation quality

[0051] As shown in Table 2, compared with the control group of direct fermentation, the pH value of the experimental group (i.e., Example 1) was significantly lower, the lactic acid and acetic acid content was significantly higher, and the ammonia nitrogen / total nitrogen (AN / TN) was significantly lower than that of the control group, indicating that the fermentation quality was excellent and the protein decomposition was less.

[0052] Table 2. Fermentation quality of Kaya under different treatments (% DM)

[0053]

[0054] (3) Cyanide content

[0055] like Figure 1 As shown, the differences in cyanide content among the treatments were significant (P<0.05). The experimental group (i.e., Example 1) had the lowest cyanide content, which was reduced by 47.42% and 30.35% compared with the pre-fermentation and control groups, respectively.

[0056] (4) In vitro digestibility

[0057] The results of the in vitro simulated incubator test (see Table 3) showed that the contents of propionic acid and valeric acid in the rumen fluid of the experimental group (i.e., Example 1) were significantly higher than those of the raw material (P<0.05), and the acetic acid / propionic acid ratio was significantly reduced, indicating that the silage can provide more energy.

[0058] Table 3. Organic acids in rumen fluid of Carya under different treatments (mmol / L)

[0059]

[0060] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A highly efficient method for Kaya silage fermentation to degrade cyanide, characterized in that, Includes the following steps: Freshly cut Kayak samples were mixed with a compound lactic acid bacteria starter culture, vacuum-sealed, and then anaerobic fermented to produce silage. The dosage of compound lactic acid bacteria starter culture added per gram of fresh Kayak sample was 5 × 10⁻⁶. 6 ~5.5×10 6 CFU, wherein the compound lactic acid bacteria starter contains Pediococcus lactis, Lactobacillus plantarum and Lactobacillus bruneri; After fermentation, the cyanide content in Kaya decreased by 47-50%.

2. The efficient cyanide-degrading silage fermentation method according to claim 1, characterized in that, Compared with silage produced by fermentation without adding a fermenting agent, the cyanide content of silage produced by the compound lactic acid bacteria fermenting agent is reduced by 30-35%.

3. The efficient cyanide-degrading kaya silage fermentation method according to claim 1, characterized in that, The anaerobic fermentation time is 30-40 days.

4. The efficient cyanide-degrading silage fermentation method according to claim 3, characterized in that, The anaerobic fermentation is carried out at a temperature of 25-30°C in the dark.

5. The efficient cyanide-degrading silage fermentation method according to claim 1, characterized in that, The length of the cut fresh Kaya sample is 2-5 cm.

6. The efficient cyanide-degrading silage fermentation method according to claim 5, characterized in that, The fresh samples of Kaya were Kaya plants that had grown to 1.20~1.50m.

7. The efficient cyanide-degrading silage fermentation method according to any one of claims 1-6, characterized in that, Vacuuming involves placing the cut fresh Kayak silage samples and compound lactic acid bacteria starter into silage bags and then vacuuming them.

8. The efficient cyanide-degrading silage fermentation method according to any one of claims 1-6, characterized in that, Includes the following steps: Step 1: Cut the Kaya plants that have grown to 1.20~1.50m and remove the soil and impurities; Step 2: Cut the harvested fresh Calabash stalks into 2-3 cm lengths, mix with the compound lactic acid bacteria starter culture, pack into silage bags, and vacuum-seal the silage bags. The dosage of compound lactic acid bacteria starter culture is 5 × 10⁻⁶ per gram of fresh Calabash stalks. 6 ~5.5×10 6 CFU; Step 3: Seal the vacuum-sealed silage bags and place them in a dark environment at room temperature of 25-30℃ for anaerobic fermentation for 30-40 days.

9. The application of a compound lactic acid bacteria starter in the efficient degradation of carbamate, characterized in that, The dosage of compound lactic acid bacteria starter culture per gram of fresh weight of Cayenne is 5 × 10⁻⁶. 6 ~5.5×10 6 CFU, wherein the compound lactic acid bacteria starter contains Pediococcus lactis, Lactobacillus plantarum and Lactobacillus bruneri; After fermentation, the cyanide content in Kaya decreased by 47-50%.

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