阅读说明：本技术 一种减少苜蓿青贮中叶绿素分解的方法 (Method for reducing chlorophyll decomposition in alfalfa silage ) 是由 曹阳 臧延青 李德军 张爱忠 姜宁 于 2020-05-29 设计创作，主要内容包括：本发明提出一种减少苜蓿青贮中叶绿素分解的方法。本方法包括以下步骤：苜蓿原料于避光通风处晾至半干,含水量50％左右,切碎至2-3cm,用1mol/L硫酸和2mol/L盐酸按体积比1:4混合进行制备混合酸,混合酸的添加量2～10％(质量百分比计),处理好样品后,分别称取200g装入规格为16×25cm黑色聚乙烯袋中,真空机密封包装,于室温避光环境下进行小规模发酵,分别在0、1、3、5、7、15、22、39、46、53、60、67、74天开封,每个时间开封3袋。通过本方法在青贮过程中叶绿素a和叶绿素b随着发酵时间的延长呈逐渐下降趋势,添加MA可以减缓苜蓿青贮后期的叶绿素损失。(The invention provides a method for reducing chlorophyll decomposition in alfalfa silage. The method comprises the following steps: airing the alfalfa raw materials to be half-dry in a dark and ventilated place, cutting the alfalfa raw materials into 2-3cm, mixing 1mol/L sulfuric acid and 2mol/L hydrochloric acid according to a volume ratio of 1:4 to prepare mixed acid, adding 2-10% (by mass percent) of the mixed acid, weighing 200g of the mixed acid respectively after processing a sample, putting the weighed mixed acid into black polyethylene bags with the specification of 16 x 25cm, sealing and packaging the black polyethylene bags by using a vacuum machine, performing small-scale fermentation in a dark environment at room temperature, unsealing the black polyethylene bags in 0, 1, 3, 5, 7, 15, 22, 39, 46, 53, 60, 67 and 74 days respectively, and unsealing the black polyethylene bags in 3 times. The chlorophyll a and the chlorophyll b gradually decline along with the prolonging of the fermentation time in the ensiling process by the method, and the addition of MA can slow down the chlorophyll loss in the later period of the alfalfa ensiling.)

1. A method of reducing chlorophyll decomposition in alfalfa silage, comprising the steps of: airing the alfalfa raw materials to be half-dry in a dark and ventilated place, cutting the alfalfa raw materials into 2-3cm, mixing 1-2 mol/L sulfuric acid and 1-2 mol/L hydrochloric acid according to the proportion of (1-2) to (1-4) to prepare mixed acid, adding 2-10 mass percent of the mixed acid, weighing 200g of the mixed acid respectively after processing a sample, filling the weighed mixed acid into black polyethylene bags with the specification of 16 multiplied by 25cm, sealing and packaging the mixed acid by a vacuum machine, performing small-scale fermentation in a dark environment at room temperature, unsealing the mixed acid in 0, 1, 3, 5, 7, 15, 22, 39, 46, 53, 60, 67 and 74 days, and unsealing 3 bags in each time.

2. The method of reducing chlorophyll decomposition in alfalfa silage as claimed in claim 1, wherein: airing the alfalfa raw materials to be half-dry in a dark and ventilated place, cutting the alfalfa raw materials into 2-3cm, mixing 1mol/L sulfuric acid and 2mol/L hydrochloric acid according to a ratio of 1:4 to prepare mixed acid, wherein the addition of the mixed acid is 8% (mass percent), weighing 200g of the mixed acid after processing a sample, respectively filling the weighed mixed acid into black polyethylene bags with the specification of 16 x 25cm, sealing and packaging the black polyethylene bags by using a vacuum machine, performing small-scale fermentation in a dark environment at room temperature, unsealing the black polyethylene bags in 0, 1, 3, 5, 7, 15, 22, 39, 46, 53, 60, 67 and 74 days, and unsealing the bags in 3 times.

The technical field is as follows:

the invention relates to the technical field of silage, in particular to a method for reducing chlorophyll decomposition in alfalfa silage.

Background art:

in the production of silage, the common mineral acid additives are hydrochloric acid, sulfuric acid and phosphoric acid. In 1970, mixed acids (sulfuric acid and hydrochloric acid) as preservatives were proposed by german a.n. bnptahh to be used in ensilage to effectively inhibit proteolysis and respiration of plant cells. The industrial by-product hydrochloric acid is strong acid, but does not contain harmful components such as arsenic, chlorocarbon and the like, but can be used as a silage preservative in case of low concentration, has no adverse effect on animal organisms, and can also improve the intake of plant protein. When the beef cattle are fed with 6% alfalfa silage treated by mixed acid, the intratumoral pure protein of the acid treatment group is 21% higher than that of the control group, the protein utilization rate is 14% higher, and the amide is 21% lower. When the red clover silage treated by 4.5 percent mixed acid is fed to fattening pigs, the fact that 0.66-1.5kg of silage added into daily ration can meet the requirements of 15-20 percent of the energy requirement of the fattening pigs and 10-15 percent of the protein requirement is found. Research shows that the inorganic acid has better acidifying effect than organic acid and low cost. The research on the influence of the mixed inorganic acid treatment on the fermentation quality of the ensiling of the corn straws shows that the addition of the mixed acid (sulfuric acid and hydrochloric acid) can obviously reduce the pH value of the ensiling, inhibit the propagation of harmful microorganisms and reduce the content of crude fiber and ammoniacal nitrogen.

Chlorophyll in the alfalfa silage not only affects sensory scores of the fermentation quality of the alfalfa silage, but also is an important functional component in alfalfa silage, has the functions of hematopoiesis, vitamin supply, detoxification, disease resistance, oxidation resistance and the like, and can regulate insulin and promote fatty acid acidification after phytanic acid is converted in ruminants. The alfalfa ensiling with good fermentation quality keeps the color similar to that of green alfalfa raw materials, but the chlorophyll degradation and metabolism rules in the ensiling process are not clear.

The invention content is as follows:

the invention aims to provide a method for reducing chlorophyll decomposition in alfalfa silage. The application describes the chlorophyll degradation rule and the dynamic change of the related degrading enzyme activity in the mixed acid treatment alfalfa ensiling process in detail, and discloses the chlorophyll degradation metabolism rule in the alfalfa ensiling process. The chlorophyll a and the chlorophyll b gradually decline along with the prolonging of the fermentation time in the ensiling process by the method, and the addition of MA can slow down the chlorophyll loss in the later period of the alfalfa ensiling.

The technical scheme of the invention is as follows: a method of reducing chlorophyll decomposition in alfalfa silage, comprising the steps of: airing the alfalfa raw materials to be half-dry in a dark and ventilated place, cutting the alfalfa raw materials into 2-3cm, mixing 1mol/L sulfuric acid and 2mol/L hydrochloric acid according to a volume ratio of 1:4 to prepare mixed acid, wherein the addition of the mixed acid is 8% (by mass percent), weighing 200g of the mixed acid respectively after processing a sample, putting the weighed mixed acid into black polyethylene bags with the specification of 16 x 25cm, sealing and packaging the black polyethylene bags by using a vacuum machine, performing small-scale fermentation in a dark environment at room temperature, unsealing the black polyethylene bags in 0, 1, 3, 5, 7, 15, 22, 39, 46, 53, 60, 67 and 74 days respectively, and unsealing the bags in 3 times.

The invention has the following beneficial effects: (1) the addition of MA can improve the fermentation quality of alfalfa ensilage and increase the in vitro dry matter degradation rate. (2) Chlorophyll a and chlorophyll b show a gradual descending trend along with the extension of fermentation time in the ensiling process, and the addition of MA can slow down the chlorophyll loss in the later stage of alfalfa ensiling. (3) The activities of three important chlorophyll metabolizing enzymes Chlase, PPH and PAO in alfalfa ensilage show a trend of ascending at the initial stage and descending at the later stage by adding the mixed acid, which results in a degrading and metabolizing rule that the early stage and the later stage of rapid decomposition of chlorophyll in alfalfa ensilage tend to slow, but MA shows an inhibiting effect on chlorophyll degradation at the later stage of alfalfa fermentation, and further experiments are needed for verification.

Description of the drawings:

FIG. 1 is a schematic diagram of the technical scheme of the invention.

FIG. 2 is a graph showing the dynamic change of chlorophyll a content in alfalfa ensiling.

FIG. 3 is a graph showing the dynamic change of chlorophyll b content in alfalfa ensiling.

FIG. 4 is a graph of the dynamic change of Chlase activity level during alfalfa ensiling.

FIG. 5 is a graph showing the dynamic change of the PPH activity content during the ensiling of alfalfa.

FIG. 6 is a graph showing the dynamic change of PAO activity content during the ensiling of alfalfa.

The specific implementation mode is as follows:

the invention is further illustrated below with reference to experiments:

a method for reducing chlorophyll decomposition in alfalfa silage comprising the steps of: airing the alfalfa raw materials to be half-dry in a dark and ventilated place, cutting the alfalfa raw materials into 2-3cm, mixing sulfuric acid with different concentrations and hydrochloric acid with different concentrations according to a certain volume ratio to prepare mixed acid, adding a certain amount of the mixed acid into the alfalfa, weighing 200g of the mixed acid respectively after processing samples, putting the weighed mixed acid into black polyethylene bags with the specification of 16 multiplied by 25cm, sealing and packaging the black polyethylene bags by using a vacuum machine, performing small-scale fermentation at room temperature in a dark environment, unsealing the black polyethylene bags in 0, 1, 3, 5, 7, 15, 22, 39, 46, 53, 60, 67 and 74 days respectively, and unsealing the black polyethylene bags in 3 times.

The application screens out the best acid concentration, the addition amount and the volume ratio through earlier stage tests aiming at the influence (40 days) of the fermentation quality of the alfalfa caused by different concentrations of sulfuric acid and hydrochloric acid, the addition amount of mixed acid and the volume ratio of the sulfuric acid and the hydrochloric acid:

respectively preparing sulfuric acid and hydrochloric acid solutions with the molar concentrations of 1.0M and 2.0M, respectively adding the sulfuric acid and the hydrochloric acid solutions with the concentrations of 2%, 4%, 6%, 8% and 10% separately, and screening out the optimal concentration and the optimal addition amount; cutting herba Medicaginis into pieces of 2-3cm length, water content of 50%, mixing 2 kinds of acids with herba Medicaginis sample, weighing 200g, placing into transparent polyethylene bags of 16 × 25cm, vacuum sealing, and storing at room temperature for 40 days. The specific addition treatment method is shown in the following table a, and the result is shown in the following table b:

table a treatment method for the influence of the addition of sulfuric acid and hydrochloric acid on the fermentation quality of lucerne:

the results in Table b show that, taking into account the pH and lactic acid content of the alfalfa silage, the concentrations of sulfuric acid and hydrochloric acid were 1M and 2M, respectively, and the addition levels were 4% and 8%, respectively, for the best fermentation quality of the treated alfalfa. In addition, we also prepared 0.5M and 2.5M sulfuric acid and hydrochloric acid solutions, respectively, and the addition of 0.5M was effective, and the optimum effect was that the effective content of sulfuric acid or hydrochloric acid was just equivalent to the amount of 1M sulfuric acid and 2M hydrochloric acid, so we did not list in Table b in order to avoid the duplication of the effective amounts of 2 acids.

TABLE b influence of the addition of sulfuric acid and hydrochloric acid on the fermentation quality of Medicago sativa

According to the determined concentration and addition amount of 8% of the sulfuric acid 1M and the hydrochloric acid 2M, the influence of adding different proportions of sulfuric acid and hydrochloric acid on the quality of the alfalfa silage is studied, and the alfalfa material and the silage preparation method thereof are the same as above. Table c is a method of mixing ratios of 2 acids, and the results are shown in table d:

table c influence of the mixing ratio of sulfuric acid and hydrochloric acid on the fermentation quality of lucerne treatment method:

the results in Table d show that, taking into account the pH and lactic acid content of the alfalfa silage together, the concentrations of sulfuric acid and hydrochloric acid were 1mol/L and 2mol/L, respectively, and that the fermentation quality of the treated alfalfa was best with a 1:4 ratio of the 2 acids.

TABLE d Effect of different concentrations of sulfuric acid and hydrochloric acid and their ratios on the quality of alfalfa fermentation

From the above experiments it was determined that: the method for reducing chlorophyll decomposition in alfalfa silage comprises the following steps: airing the alfalfa raw materials to be half-dry in a dark and ventilated place, cutting the alfalfa raw materials into 2-3cm, mixing 1mol/L sulfuric acid and 2mol/L hydrochloric acid according to a volume ratio of 1:4 to prepare mixed acid, wherein the addition of the mixed acid is 8% (by mass percent), weighing 200g of the mixed acid respectively after processing a sample, putting the weighed mixed acid into black polyethylene bags with the specification of 16 multiplied by 25cm, sealing and packaging the mixed acid in a vacuum machine, performing small-scale fermentation in a dark environment at room temperature, and unsealing the mixed acid for 0, 1, 3, 5, 7, 15, 22, 39, 46, 53, 60, 67 and 74 days to perform experiments.

The test adopts two-factor test design, and is divided into 2 treatment groups (each treatment group is 50 repeated to prevent damage from influencing the test), wherein the treatment groups comprise a Control group (no addition, Control) and a mixed acid treatment group (MA represents mixed acid, the addition is 8 percent), and the addition is a fresh material basis.

Firstly, the sensory quality, general chemical components, organic acid, ammonia nitrogen, microbial composition and the change of in vitro rumen fermentation of the acid treatment and control group alfalfa ensilage along with the extension of the fermentation time are evaluated.

1. The sensory quality evaluation method comprises the following steps:

the alfalfa ensilage was opened and then subjected to quality assessment for its color, odor, texture and presence or absence of mildew according to the specifications of the ensilage quality assessment criteria.

2. General chemical composition analysis:

the measurement items include Dry Matter (DM), Organic Matter (OM), crude fat (EE), Crude Protein (CP), Acid Detergent Fiber (ADF), Neutral Detergent Fiber (NDF), and Total Nitrogen (TN) content. And (3) putting a part of the unsealed sample into an electric heating constant-temperature air-blowing drying oven, and drying the sample to constant weight at the temperature of 65 ℃, thereby obtaining the free water content. Crushing the sample with constant weight by a miniature plant crusher, sieving the crushed sample by a 1mm sieve, carrying out general chemical component analysis,

OM, EE, CP and TN were measured by methods 934.01, 976.05 and 920.39 in (AOAC), respectively, and NDF and ADF were measured by the Van soest method.

3. And (3) organic acid determination:

weighing 20g of the unsealed sample, placing the sample in a polyethylene bag, adding 180mL of sterilized distilled water, and beating the sample for 90s by using a homogenizer to prepare the silage leaching liquor. The filtrate was filtered through quick qualitative filter paper and the pH of the filtrate was measured. 1.5mL of the filtrate obtained was taken over 12000 rmin^-1Centrifuging for 5min, filtering with 0.22 μm microporous membrane, and analyzing the content of Lactic Acid (LA), Acetic Acid (AA), Propionic Acid (PA) and Butyric Acid (BA) by high performance gas chromatography.

The chromatographic conditions are as follows: the parameters of the vaporization chamber are carrier gas nitrogen (N)₂) The split ratio is 40: 1, the sample injection amount is 0.4 mu L, and the temperature is 220 ℃; the chromatographic column parameters are HP-INNOWAx capillary chromatographic column constant flow mode, the flow is 2.0mL/min, and the average linear speed is 38 cm/s; the column oven parameters are programmed to raise the temperature to 120 ℃ for 3min, and then raise the temperature to 180 ℃ at a speed of 10 ℃/min for 1 min; the detector parameter is hydrogen (H)₂) The flow rate is 40mL/min, the air flow is 450mL/min, the column flow and the tail gas blowing flow are 45mL/min, and the temperature of a Flame Ion Detector (FID) is 250 ℃.

4. Measurement of Ammonia Nitrogen:

according to von Zong Ci^[108]Measuring ammoniacal Nitrogen (NH)₃-N). Preparing a standard solution by a spectrophotometer method: (1) 0.955g of ammonium chloride was accurately weighed and dissolved in 0.2mol/L hydrochloric acid solution to a constant volume of 250mL to obtain solution A. (2) And taking 25mL of the solution A, diluting the solution A with distilled water to reach the constant volume of 250mL to obtain solution B. (3) Taking 0, 1, 2, 4 and 6mL of the solution B in turn, respectively placing the solution B in a 50mL volumetric flask, then adding 10, 9, 8, 6 and 4mL of distilled water in turn, and then adding 0.2mol/L of hydrochloric acid to reach 50mL of constant volume, which is 5 standard solutions. And C, liquid C: accurately weighed 0.2g of sodium nitroferricyanide was dissolved in 250mL of 14% sodium salicylate solution and mixed for use. And (3) liquid D: 5mL of sodium hypochlorite solution is mixed with 250mL of 0.3mol/L sodium hydroxide solution for later use.

Calculation and color comparison: (1) measuring 0.8mL of 5 standard solutions in turn, placing the standard solutions in 20mL glass stopper test tubes respectively, adding 4mL of C solution and 4mL of D solution in turn into each tube, shaking up, standing for 10min, measuring OD values of the 5 standard solutions by using a 0.5cm quartz cuvette under the wavelength of 700nm, and deriving a regression equation by using a reference group as a nitrogen-free standard solution. (2) Sample treatment: 20mL of the silage leach filtrate was centrifuged at 3500r/min for 10 min. 4mL of the supernatant was placed in a 20mL tube, and 16mL of 0.2mol/L hydrochloric acid was added to prepare a treatment solution. 0.8mL of the treated solution was weighed into a 10mL test tube, 4mL of the solution C and 4mL of the solution D were added, and the mixture was shaken well and then allowed to stand for 10min to determine the OD value.

5. The microbial composition is as follows:

the MRS, BLB, NA and PDA culture media are defined as 1 × 10^-1Double, 1 × 10^-3Double, 1 × 10^-5Doubling of 3 culture areas, in which CCA and NA (Heat resistant) medium demarcated 1 × 10^-1 Multiple sum 1 × 10^-2Multiplying 2 culture areas, diluting the silage leaching liquor with sterile water in a gradient way, and continuously diluting the silage leaching liquor to 1 × 10^-1Double liquid, 1 × 10^-2Double liquid, 1 × 10^-3Double liquid, 1 × 10^-4And 1 × 10^-5Pipetting 20 μ L of the diluent with pipette, dropping the diluent into the culture medium according to pre-divided regions of the culture medium, spreading the diluent evenly with a spreading stick, and spreading the diluent with a spreading stick of 1 × 10^-1And 1 × 10^-2Heating the diluent in water bath (75 deg.C, 15min), rapidly cooling, and applying CCA and NA (heat-resistant) culture medium. Wherein MRS and CCA are placed in an anaerobic incubator (37 ℃) for culture, and the rest are placed in a constant temperature incubator (37 ℃) for culture for 48 hours, and then taken out for counting the colony number.

6. In vitro rumen fermentation:

using male large-tail Han sheep with permanent rumen fistula at both ends, weighing 37 + -5 kg (animal feeding laboratory of university of eight agricultural cultivations in Heilongjiang), extracting rumen fluid from rumen fistula 2h after feeding in the morning (all tools used must be preheated to 39 deg.C in advance), filtering with gauze, and introducing CO₂Keeping anaerobic environment, and mixing with artificial saliva and rumen at ratio of 4: 1. Artificial saliva is prepared according to McDougll's buffer method^[109](potassium chloride 0.57g/L, sodium bicarbonate 9.80g/L, calcium chloride 0.04g/L, disodium hydrogen phosphate dodecahydrate 9.30g/L, magnesium chloride 0.06g/L, sodium chloride 0.47g/L, L-cysteine hydrochloride 0.25g/L, and sodium celosite 0.01g/L) and the pH value is 6.9. Accurately weighing 0.50g (dry matter basis) of the silage crushed after opening the seal, filling the silage into a serum bottle with the capacity of 125mL, injecting 50mL of mixed culture solution, quickly sealing the bottle mouth, placing the mixed culture solution at 39 ℃ and an air bath oscillator with the speed of 100r/min, and culturing for 48 h.

After the culture was completed, the serum bottle was placed in ice water to stop fermentation, the scalp needle was inserted into the serum bottle and sealed with rubber, and the amount of gas produced was recorded by passing through a 100mL glass syringe connected thereto. Filtering the culture solution by using constant-weight rapid qualitative filter paper, measuring the pH value, putting the filtered culture solution residues into an electric heating constant-temperature air blast drying oven, and drying at 102 ℃ to constant weight, thereby calculating the dry matter degradation rate.

Taking 10ml of culture solution filtered by rapid qualitative filter paper, placing in a centrifuge tube, and high-speed freezing centrifuge 2200 r.min^-1Centrifuging for 5min, and removing supernatant for use. 1mL of the supernatant was taken out, and an equal amount of a 10% metaphosphoric acid solution (metaphosphoric acid dissolved in 1mol/L sulfuric acid solution, 10% w/v) was added thereto and mixed well (deproteinization). Then passing through a high-speed refrigerated centrifuge at 6000 r.min^-1Centrifuging for 30min to obtain supernatant. Taking 0.5ml of supernatant, adding 0.5ml of 20mmol/L butenoic acid standard substance, and mixing thoroughly. The concentration of crotonic acid in the mixed solution was 10mmol/L (same as that of the standard solution). Then, 0.01ml of pure phosphoric acid was added to the mixture and mixed well (adjusted to acidity), and the mixed solution was left at 4 ℃ for 12 hours. The liquid after standing is processed by a high-speed refrigerated centrifuge at 6000 r.min^-1Centrifuging for 30min, collecting supernatant, filtering with 0.45 μm microporous membrane, and analyzing AA, PA and BA content (with the same chromatographic conditions) and NH with high performance gas chromatograph₃the-N determination was as described above.

7. And (3) test results:

(1) sensory quality

TABLE 1 Effect of acid treatment on the organoleptic qualities of alfalfa ensilage

As can be seen from Table 1: the green color of the alfalfa gradually changes into yellow brown along with the prolonging of the fermentation time in the ensiling process, the alfalfa ensiling of the group MA shows the trend of fading the green color at first, the acid fragrance of the group MA is stronger than that of the group Control, and the ensiling feed after fermentation treatment has soft texture, proper moisture, clear stem leaves and no mildew.

(2) General chemical composition

TABLE 2 Effect of acid treatment on the general chemical composition of alfalfa ensilage

Note: the difference of the same column data with the shoulder letters indicates significant difference (P < 0.05). Dm (dry matter), representing a dry matter basis.

As can be seen from Table 2: the DM content in the alfalfa silage is not remarkably different along with the extension of the fermentation days (P is more than 0.05), the CP, OM, ADF and NDF content is remarkably reduced along with the extension of the fermentation days (P is less than 0.05), and the EE content is remarkably increased along with the extension of the fermentation days (P is less than 0.05); compared with the Control group, the content of DM, EE, OM and NDF in the MA group is not significantly different (P >0.05), the content of CP is significantly different from that in the Control group (P <0.05), and the content of ADF is significantly different from that in the Control group; the differences in DM content were not significant (P >0.05), and the differences in CP, EE, OM, ADF and NDF content were significant (P <0.05) with the interaction of fermentation days and treatments.

(3) Organic acid and NH₃-N/TN

TABLE 3 acid treatment on alfalfa ensilage organic acids and NH₃Influence of-N/TN

Note: the difference of the same column data with the shoulder letters indicates significant difference (P < 0.05). Nd (not detected), indicating that no colonies were detected. "-" indicates not statistically. FM (fresh mate), which represents the fresh material basis.

As can be seen from Table 3: the pH value in alfalfa ensilage is obviously reduced along with the prolonging of fermentation days (P)<0.05)，NH₃-N/TN, LA and AA content increased significantly with increasing number of days of fermentation (P)<0.05); AA content of MA group was not significantly different from that of Control group (P)>0.05), pH, NH₃N/TN is significantly lower than that of the Control group (P)<0.05), the LA content is significantly higher than that of Control group (P)<0.05); pH, NH in the interaction of fermentation days and treatment₃The content of-N/TN, LA and AA is obviously different (P)<0.05); no PA and BA were detected.

(4) Microbial composition

TABLE 4 Effect of acid treatment on the microbial composition of alfalfa ensilage

Note: the difference of the same column data with the shoulder letters indicates significant difference (P < 0.05). Nd (not detected), indicating that no colonies were detected. "-" indicates not statistically. FM (fresh mate), which represents the fresh material basis.

As can be seen from Table 4: the number of LAB in alfalfa silage increases significantly with the increase of fermentation days (P <0.05), and the number of Bacill, Coliform, Aerobic and Yeasts decreases significantly with the increase of fermentation days (P < 0.05); the number of LAB and Bacillus of the MA group is not significantly different from that of the Control group (P >0.05), and the number of Coliform, Aerobic and Yeasts is significantly lower than that of the Control group (P < 0.05); under the interaction of fermentation days and treatments, the number of LAB, Bacill, Coliform, Aerobic and Yeasts differed significantly (P < 0.05); clostridium and Molds were not detected.

(5) In vitro rumen fermentation

TABLE 5 Effect of acid treatment on rumen digestion dry matter degradation rate in vitro of alfalfa ensilage

Note: the difference of the same column data with the shoulder letters indicates significant difference (P < 0.05). Dm (dry matter), representing a dry matter basis.

As can be seen from Table 5: the pH value of the alfalfa ensilage in vitro digestion is remarkably reduced along with the prolonging of the fermentation days (P <0.05), and the gas production and the dry matter degradation rate are remarkably increased along with the prolonging of the fermentation days (P < 0.05); compared with the Control group, the pH value of the MA group has no significant difference (P >0.05), and the gas production rate and the dry matter degradation rate are significantly higher than those of the Control group (P < 0.05); under the interaction of fermentation days and treatment, the differences of pH value, gas production rate and dry matter degradation rate are obvious (P < 0.05).

TABLE 6 Effect of acid treatment on VFA digestion in vitro of alfalfa ensilage

Note: the difference of the same column data with the shoulder letters indicates significant difference (P < 0.05).

As can be seen from Table 6: the contents of AA, PA and BA in the alfalfa silage digested in vitro are obviously increased along with the prolonging of the fermentation days (P)<0.05)，NH₃The N content decreases significantly with increasing number of fermentation days (P)<0.05); AA, PA and BA in the MA group are significantly higher than in the Control group (P)<0.05)，NH₃N content significantly lower than that of Control group (P)<0.05); AA, PA, BA and NH in the interaction of fermentation days and treatments₃Significant difference in-N content (P)<0.05)。

8. Conclusion

The addition of mixed acid in alfalfa ensilage can protect CP from being degraded into NH to a certain extent₃-N, reduced ADF and NDF content, increased volatile fatty acid content and in vitro fermented dry matter degradation rate. The pH value can be rapidly reduced without LA accumulation in the initial stage of ensiling fermentation, the growth and the propagation of harmful microorganisms can be effectively inhibited, and unnecessary fermentation loss is reduced. In conclusion, the fermentation quality of the MA group was superior to that of the Control group.

And secondly, analyzing the dynamic change rule of the chlorophyll content in the alfalfa ensiling process along with the content change of chlorophyll a and chlorophyll b in the alfalfa ensiling process of the acid treatment and control group along with the prolonging of the fermentation time. And the effect of acid treatment on alfalfa ensilage chlorophyll-related degrading enzymes.

The experimental design is the same as that of the test,

1. measuring the chlorophyll content:

reference Yan vibration^[134]And the content of the chlorophyll a and the chlorophyll b is measured by adopting a spectrophotometer method, and the extraction solution is acetone: ethanol (1: 1; V/V). Weighing about 2g of sample, putting the sample into a 1.5mL centrifugal tube, adding a little calcium carbonate and silicon dioxide, adding a proper amount of extracting solution, and grinding the mixture by using a tissue grinder until the tissue is whitish. Filtering with quick qualitative filter paper, diluting to 25mL, and mixing. OD values at 665nm and 649nm absorbance were measured with a spectrophotometer, respectively.

The calculation formula is as follows:

chlorophyll a ═ 12.72 × A₁-2.59×A₂)×V/1 000×m

Chlorophyll b ═ 22.88 × A₁-4.67×A₂)×V/1 000×mA₁665OD value; a. the₂649OD value; v ═ liquid volume (mL); sample mass (g)

2. Test results

TABLE 7 Effect of acid treatment on alfalfa ensilage chlorophyll

Note: the difference of the same column data with the shoulder letters indicates significant difference (P < 0.05).

As can be seen from Table 7: the contents of chlorophyll a and chlorophyll b in the alfalfa silage are obviously reduced along with the prolonging of the fermentation days (P is less than 0.05); the chlorophyll a and chlorophyll b content of the MA group is obviously lower than that of the Control group (P is less than 0.05); under the interaction of fermentation days and treatment, the content difference of chlorophyll a and chlorophyll b is obvious (P is less than 0.05).

FIG. 2 is a graph showing the dynamic change of chlorophyll a content during the course of alfalfa ensiling, and it can be seen from FIG. 2 that chlorophyll a gradually decreases as the number of days for alfalfa ensiling fermentation increases. Chlorophyll a of the MA group rapidly decreased in the initial stage of fermentation, and was always lower than that of the Control group at the first 53d of fermentation, but was higher than that of the Control group after 53d of fermentation.

FIG. 3 is a graph showing the dynamic change of chlorophyll b content during alfalfa ensiling. As can be seen from FIG. 3, chlorophyll b gradually decreased as the number of days of alfalfa ensiling increased. Chlorophyll b of group MA rapidly decreased in the early stage of fermentation, and was lower than that of Control throughout the fermentation.

3. And (4) conclusion:

the degradation speed of chlorophyll b in the MA group shows a trend of continuously decreasing and being lower than that of the Control group in the ensiling process, the degradation speed of chlorophyll a shows a trend of rapidly decreasing in the early stage of fermentation but gradually decreasing in the later stage of fermentation, and is higher than that of the Control group from 53d to the end of fermentation, which shows that the addition of MA can delay the degradation speed of chlorophyll a to a certain extent.

4. The results of the acid treatment effect on alfalfa ensilage chlorophyll-associated degrading enzymes are shown in table 8:

TABLE 8 Effect of acid treatment on alfalfa ensilage chlorophyll-related degrading enzymes

Note: the difference of the same column data with the shoulder letters indicates significant difference (P < 0.05).

As can be seen from Table 8: the activity contents of Chlase and PPH in the alfalfa silage are obviously increased along with the prolonging of the fermentation days (P <0.05), and the activity contents of PAO are not obviously different along with the prolonging of the fermentation days (P > 0.05); the Chlase activity content of the MA group is obviously higher than that of the Control group (P <0.05), and the activity contents of PPH and PAO are not obviously different from that of the Control group (P > 0.05); the differences in Chlase, PPH and PAO activity levels were significant (P <0.05) with the interaction of fermentation days and treatment.

As can be seen from FIG. 4, the Chlast activity level of the Control group gradually increased with the number of days of fermentation, while the MA group showed a tendency of increasing first and then decreasing, but the decrease was not large. Chlase of the MA group rapidly increased in activity content at 0-5d of fermentation and was significantly higher than that of the Control group, and slightly higher than that of the Control group at 5-46 d. The peak occurs at 39d, and the Chlast activity level of the MA group is lower than that of the Control group after 53d until the end of fermentation.

As can be seen from FIG. 5, the PPH activity content increased first and then decreased with the increase of the fermentation time of alfalfa ensilage. The PPH activity content of the MA group rapidly increased and was significantly higher than that of the Control group from 0 to 22d of the fermentation, and remained equal to that of the Control group from 22 to 39d, but after 39d until the end of the fermentation, the PPH activity content of the MA group was lower than that of the Control group.

As can be seen from FIG. 6, the PAO activity content of the Control group gradually increased with the increase of the number of days of fermentation, the PAO activity content of the MA group rapidly increased from 0 to 5 days and was significantly higher than that of the Control group, and the PAO activity content of the MA group was lower than that of the Control group after 5 days until the end of fermentation.

5. And (4) conclusion: the active contents of Chlase, PPH and PAO are in the trend of increasing and then decreasing in the alfalfa ensiling process, and the active contents of Chlase, PPH and PAO in the MA group are higher than those in the Control group before 53d, 46d and 7d, and lower than those in the Control group after. Therefore, the addition of MA to alfalfa silage increases the enzyme activity content during the early fermentation period, but has an inhibitory effect during the late fermentation period.

The above experiments show that:

(1) the green color of the alfalfa is gradually changed into yellow brown color in the ensiling process, the alfalfa ensiling of the MA group shows the trend of fading first, the acid fragrance of the MA group is stronger than that of the Control group, and the two treatment groups have soft texture, clear stem leaves, proper water content and no mildew; with the prolongation of fermentation time, the degradation rate of CP, ADF in general chemical components of MA group, LA in organic acid, AA, PA, BA, gas production and dry matter in-vitro rumen fermentation is significantly higher than that of Control group (P)<0.05); pH value, NH₃-N/TN, Co in microbial compositionliform, Aerobic, Yeasts, NH in rumen fermentation in vitro₃N is significantly lower than in Control group (P)<0.05); DM, EE, OM, NDF in general chemical components, AA in organic acid, LAB and Bacillus in microbial composition are not obviously different from those in Control group (P)>0.05)。

(2) With the extension of fermentation days, the contents of chlorophyll a and chlorophyll b in the two treatment groups gradually decrease; the chlorophyll a content of the MA group is lower than that of the Control group at 0-53d of fermentation, but the chlorophyll a content of the MA group is higher than that of the Control group after 53d until the fermentation is finished; chlorophyll b content of MA group decreased rapidly in the early stage of fermentation, significantly lower than Control group throughout the fermentation process (P < 0.05).

(3) The Chlase activity content of the MA group rapidly increases and is obviously higher than that of the Control group (P <0.05) at 0-5d of fermentation, is slightly higher than that of the Control group at 5-39d, but is lower than that of the Control group after 39d until the end of fermentation; the PPH activity content of the MA group rapidly rises and is obviously higher than that of the Control group (P <0.05) at 0-22d of fermentation, is equal to that of the Control group at 22-39d, but is lower than that of the Control group after 39d until the end of fermentation; the PAO activity content of the MA group rapidly increases and is remarkably higher than that of the Control group (P <0.05) at 0-5 days, and the PAO activity content of the MA group is lower than that of the Control group after 5 days until the end of fermentation.

In conclusion, the addition of MA can effectively improve the fermentation quality of the alfalfa ensilage, and inhibit the activity of chlorophyll-related degradation enzymes after 30 days of fermentation, thereby reducing the degradation and decomposition of chlorophyll.