CN115886130A - Application of lonicera confusa in improving quality of alfalfa mixed silage - Google Patents
Application of lonicera confusa in improving quality of alfalfa mixed silage Download PDFInfo
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- CN115886130A CN115886130A CN202211582043.6A CN202211582043A CN115886130A CN 115886130 A CN115886130 A CN 115886130A CN 202211582043 A CN202211582043 A CN 202211582043A CN 115886130 A CN115886130 A CN 115886130A
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- China
- Prior art keywords
- alfalfa
- silage
- lonicera confusa
- mixed silage
- quality
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Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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- Fodder In General (AREA)
Abstract
The invention provides an application of lonicera confusa in improving the quality of alfalfa mixed silage, wherein the adding amount of the lonicera confusa is 5-25 g/kg of the weight of alfalfa silage raw materials, and the mixed silage also comprises soybean meal, DDGS (distillers dried grains with soluble) and lactic acid bacteria. According to the invention, the lonicera confusa is added into the alfalfa mixed silage, so that the fermentation quality, the oxidation resistance and the nutrition quality of the alfalfa mixed silage can be improved, and the increase of the pH value of the silage after unpacking can be inhibited.
Description
Technical Field
The invention belongs to the technical field of silage, and particularly relates to application of lonicera confusa in improving the quality of alfalfa mixed silage.
Background
Alfalfa (medicago sativa L.) is perennial high-quality leguminous forage, is rich in nutrients such as crude protein, multiple vitamins and multiple minerals and growth factors for promoting cell growth activity, has tender and delicious branches and leaves and good palatability, and is reputed as the king of forage. The alfalfa is mixed with auxiliary materials such as wheat bran, corn flour, corncobs and bran coat for ensiling, so that the alfalfa ensiling success rate can be improved, the overall nutritional value of the ensiling feed can be regulated, and the utilization efficiency of agricultural and sideline product feedingcan be promoted. At present, the fermented liquid feed mainly containing alfalfa can replace part of daily ration to be applied to pig breeding, and is deeply favored by farmers because the fermented liquid feed can improve the quality of pork and the health state of pigs.
The oxidative stress is directly or indirectly related to various diseases such as livestock and poultry immunity reduction, enteritis, pneumonia and the like, and exogenous antioxidants are usually added in the production to relieve the oxidative stress of organisms, but the oxidative stress is mostly in an excessive state and is easy to remain in the bodies of animals and animal products. The herbaceous plant rich in polyphenol, polysaccharide, alkaloid and other bioactive substances is a safe and efficient plant source antioxidant. The Lonicera confusa is a dried flower bud or a flower of Lonicera macranthoides (L.macroanthoides hand. -Mazz.), lonicera confusa (L.convusa DC.), lonicera fulvescens (L.fulvotomtosa Hsu et S.C.Cheng) and Lonicera hypoglauca (L.hygoglauca Miq.) (Caprifoliaceae). The flos Lonicerae bioactive components include volatile oil, flavonoids, organic acids, esters, etc. Wherein chlorogenic acid is the main effective pharmacological component of flos Lonicerae with antioxidant, antibacterial and antiviral effects. The flowers and stems of the lonicera confusa and the She Shenzhi roots all contain various bioactive components with different contents, such as chlorogenic acid, flavone and the like. Therefore, the further development of the lonicera confusa is particularly necessary, and no related research on the use of the lonicera confusa for ensiling exists at present.
Disclosure of Invention
In order to increase the health care function of the existing alfalfa mixed silage on livestock, the invention provides the application of the lonicera confusa in improving the quality of the alfalfa mixed silage.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
the invention provides application of lonicera confusa in improving the quality of alfalfa mixed silage.
As an alternative embodiment, in the application provided by the present invention, the application includes the application of lonicera confusa in improving the fermentation quality of alfalfa mixed silage.
As an alternative embodiment, in the application provided by the present invention, the application comprises the application of lonicera confusa in improving aerobic stability of alfalfa mixed silage.
As an alternative embodiment, in the application provided by the present invention, the application includes an application of lonicera confusa in improving the antioxidant capacity of the alfalfa mixed silage.
As an alternative embodiment, in the application provided by the present invention, the application includes the application of lonicera confusa in improving the nutritional quality of alfalfa mixed silage.
As an optional embodiment, in the application provided by the invention, the lonicera confusa is applied to improving the proliferation of the acid-resistant lactobacillus in the alfalfa mixed silage.
As an alternative embodiment, in the application provided by the present invention, the lonicera confusa is a pesticide-applying part of lonicera confusa, including whole plant or flower of lonicera confusa.
The honeysuckle flower dregs and the alfalfa can be mixed for ensiling, and the same effect can be achieved. Meanwhile, the honeysuckle and the lonicera confusa have similar medicinal effects and can also be used for mixing with alfalfa for ensiling.
As an optional implementation manner, in the application provided by the present invention, the alfalfa mixed silage further includes soybean meal and DDGS.
The second aspect of the invention provides a method for improving the quality of alfalfa mixed silage.
As an optional implementation mode, in the method provided by the invention, the addition amount of the lonicera confusa is 5-25 g/kg of the weight of the alfalfa silage raw material.
As an optional implementation mode, in the method provided by the invention, the alfalfa mixed silage also comprises soybean meal and DDGS, and the mass ratio of the alfalfa, the soybean meal and the DDGS is (7-8): (0.5-1.5): (0.5-1.5).
The DDGS in the invention is a product obtained by using corn as a raw material, fermenting and distilling the corn to extract alcohol, and concentrating and drying at least three fourths of soluble solid matters in the alcohol and the residual liquid.
As an optional implementation mode, in the method provided by the invention, the alfalfa mixed silage further comprises lactic acid bacteria, and the viable count of the lactic acid bacteria is 1.0 × 10 11 cfu·g -1 The addition amount is 2-3 g.t -1 。
The third aspect of the invention provides alfalfa mixed silage prepared by the method.
Compared with the prior art, the invention has the beneficial effects that:
(1) According to the invention, after the lonicera confusa is added into the alfalfa silage, the fermentation quality of the silage is improved, and the fermentation quality specifically comprises the steps of improving the dry matter content, and reducing the pH value and ammoniacal nitrogen/total nitrogen of a fermentation product. The nutritional quality of the silage is improved, and specifically the soluble carbohydrate content is improved, and the neutral detergent fiber content, the acidic detergent fiber content and the coarse ash content are reduced. Improving the antioxidant capacity of the silage, specifically including improving DPPH free radical scavenging capacity, FRAP iron ion reduction/antioxidant capacity, ABTS cation free radical scavenging capacity, and total flavone and total phenol content, without affecting the aerobic stability of the silage.
(2) The lonicera confusa is used in the alfalfa silage, so that bioactive substances in the lonicera confusa can be stored and even improved in the lactic acid bacteria fermentation process, and harmful microorganisms in the lactic acid bacteria fermentation process can be inhibited and the silage antioxidant capacity can be improved due to the characteristics of oxidation resistance, bacteria resistance and the like of the lonicera confusa.
(3) The lonicera confusa alfalfa mixed silage can provide high-quality functional feed with nutrition and health care for livestock and poultry breeding, the product combines rich nutrition such as high-quality fiber and high protein of the alfalfa and multiple advantages of lonicera confusa antibacterial health care, and a new thought can be provided for reduction substitution work and antibiotic substitution work of protein feed resources such as soybean meal and the like in livestock and poultry breeding.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a graph of the results of temperature changes after aerobic exposure of mixed silage from example 3;
FIG. 2 is a graph showing the results of pH changes within 8d of aerobic exposure of mixed silage in example 3;
FIG. 3 is a graph of the relative abundance of total Lactobacillus mixed silage in example 5;
FIG. 4 is a graph of the variance analysis of the relative abundance of acid-fast Lactobacillus in mixed silage in example 5;
FIG. 5 is a heat map of the correlation of acid-tolerant Lactobacillus in mixed silage with mixed silage quality for example 5.
Detailed Description
In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
Example 1
Influence of flos lonicerae on fermentation quality of alfalfa mixed silage.
Experimental treatment and grouping: respectively adding 0 (CK), 5g/kg (SY-05), 10g/kg (SY-10), 15g/kg (SY-15), 20g/kg (SY-20) and 25g/kg (SY-25) of lonicera confusa in a raw material mixing ratio of fresh alfalfa (80%) + soybean meal (10%) + DDGS (10%), fixedly adding 2g/t of the composite lactic acid bacteria agent, and setting 5 treatment groups in total, wherein each treatment group is repeated.
Harvesting the alfalfa in the bud period, cutting and crushing the alfalfa by using a grass cutter, mixing the raw materials according to the mass ratio of 80% of the alfalfa, 10% of soybean meal and 10% of DDGS, adding lonicera confusa in different proportions, fully and uniformly mixing the raw materials again, filling the mixture into polyethylene vacuum silage bags, vacuumizing the bags, putting the bags into a storage chamber, and storing the bags in a dark place, wherein the silage bags are about 500g each silage bag. Opening the bag and sampling after 30d of ensiling, and analyzing each index.
The quartering method comprises repeatedly taking 20g of fresh silage (sheared and mixed thoroughly) each time, placing into 250mL conical flask, adding 180mL deionized water, squeezing with a juice extractor (JYL-C012 multifunctional stirrer), filtering with 4 layers of gauze to obtain silage leaching solution, and measuring pH, lactic acid, acetic acid, propionic acid, and butyric acid with 3 times of indexes.
The pH value was measured using a Spectrum SI400 pH meter, and the measurement was repeated 3 times for each sample; the contents of lactic acid, acetic acid, propionic acid and butyric acid (column: agilent TC-C18 column (250 mm. Times.4.6 mm. Times.5 μm)) were measured by Agilent 1260Infinity II HPLC, and 20. Mu.L of the sample was injected at a flow rate of 0.7 mL/min -1 Column temperature 50 ℃, ultraviolet detection wavelength 210nm, running time 35min, mobile phase: a mobile phase A: methanol, mobile phase B:0.01 mol. L -1 Aqueous potassium dihydrogen phosphate (adjusted to pH 2.70 with phosphoric acid) and a gradient elution was set (0: B =3 at a: 00.
The results are shown in table 1:
table 1: mixed silage fermentation product of wild honeysuckle flower and alfalfaQualitative performance test (g.kg) -1 )
Different letters on the same row in table 1 indicate significant differences between treatments. As can be seen from Table 1, after 30 days of mixed silage, the dry matter content of the treated group is obviously increased along with the increase of the adding proportion of the lonicera confusa, and the dry matter loss is reduced along with the increase of the adding amount of the lonicera confusa. The pH value of the silage is an important index reflecting the quality of the silage, and generally the pH value of the high-quality alfalfa silage is below 4.5. The pH value of the lonicera confusa addition treatment group is below 4.50, and is obviously lower than that of the control group, and the pH value of the treatment group shows a trend of obviously reducing along with the increase of the lonicera confusa addition proportion.
Lactic acid is an important acid generated in the ensiling fermentation process, the pH of the ensiling environment is reduced due to the accumulation of the lactic acid, the propagation of harmful microorganisms is inhibited, and the content of the lactic acid in high-quality ensiling is generally higher than 40 g/kg -1 DM, the ratio of lactic acid/total acid of the high-quality silage is more than 75%, and the ratio of acetic acid/total acid is less than 15%. The ratio of lactic acid/acetic acid in the silage is higher than 3.0, the silage belongs to homolactic fermentation, and the silage is lower than 3.0, and the silage belongs to heterotypic lactic fermentation. Compared with heterotype fermentation, homotype fermentation can accumulate more nutrients, and acetic acid produced by heterotype fermentation has the effect of improving aerobic stability. The ratio of lactic acid/acetic acid in the high-quality silage should be higher than 3.0.
The alfalfa mixed silage formula used in the application is screened and obtained on the basis of earlier research, and has high-quality fermentation quality, nutritional quality, aerobic stability and the like. Therefore, as can be seen from Table 1, the lactic acid content of the alfalfa of different treatment groups after being mixed and ensiled for 30 days is 168.03-178.83 g.kg -1 The ratio range of lactic acid/total acid is 87.47-88.04%, the ratio range of acetic acid/total acid is 11.45-12.10%, butyric acid is not detected (not shown in the table), and the fermentation indexes are in a better range. However, as the adding proportion of the lonicera confusa increases, the ratio of lactic acid/total acid and lactic acid/acetic acid of the treatment group both increase, and homofermentation is dominantThe more obvious the advantage of (b).
The results show that compared with the control group, the dry matter loss rate in the alfalfa mixed silage added with the lonicera confusa is reduced, the pH value is obviously reduced, the ratio of lactic acid/total acid and lactic acid/acetic acid is increased, the yield of propionic acid with undefined effect in the silage is low, and harmful butyric acid is not generated. It can be seen that: the addition of flos lonicerae reduces the dry matter loss rate and improves the fermentation quality of the mixed silage of alfalfa.
Example 2
Influence of flos lonicerae on the nutrition quality of alfalfa mixed silage.
Experimental treatment and grouping: the same as in example 1. The silage quartering method is used for 150g, drying for 48h at 65 ℃, and the dry sample is used for measuring soluble carbohydrate, crude fiber, crude ash, neutral detergent fiber, acidic detergent fiber and acidic detergent lignin, and each index is repeated for 3 times. The content of soluble carbohydrate is measured by adopting an anthrone colorimetric method, the crude fiber is measured by adopting an acid-base digestion method, and the neutral detergent fiber, the acidic detergent fiber and the acidic detergent lignin are measured by adopting a van der waals method. The results are shown in table 2:
table 2: performance detection of mixed silage nutrition quality (%) of alfalfa by lonicera confusa
Soluble carbohydrate in the feed is an important nutrient substance, coarse ash is a substance which can influence the digestibility of livestock, neutral detergent fiber, acid detergent fiber and acid detergent lignin are important fiber components in forage grass, the neutral detergent fiber can provide energy for the livestock, but the content of the neutral detergent fiber is too high to influence the digestibility of the livestock; neither ruminant nor monogastric livestock can digest acidic detergent fiber and acidic detergent lignin, as too high a content would seriously affect their digestibility.
The different letters in the same row in table 2 indicate that there is a significant difference between treatments. The soluble carbohydrate content of the lonicera confusa additive treatment group is increased, and the soluble carbohydrate content of SY-10, SY-15 and SY-25 is obviously higher than that of a control group (P < 0.05).
The contents of neutral detergent fiber, acidic detergent fiber, crude ash and acidic detergent lignin of the alfalfa mixed silage control group are 28.98%, 18.27%, 16.21%, 9.48% and 2.85% respectively, and the contents of the neutral detergent fiber, the acidic detergent fiber, the crude ash and the acidic detergent lignin of the lonicera confusa addition treatment group are all obviously lower than those of the control group (P < 0.05).
The results show that after the lonicera confusa is added, the content of soluble carbohydrate in the alfalfa mixed silage is increased, and the content of neutral washing fiber, acid washing fiber, crude ash and acid washing lignin is reduced. It can be seen that: the addition of the lonicera confusa promotes the retention of the content of soluble sugar in the alfalfa mixed silage, and meanwhile, the addition of the lonicera confusa can promote the degradation of fiber components and improve the nutritional quality of the silage.
Example 3
Influence of flos lonicerae on the oxygen stability of alfalfa mixed silage.
Experimental treatment and grouping: the same as in example 1.
After the silage bag is opened, silage is placed into a small plastic barrel, a sensor of an MDL-1048A high-precision temperature recorder is inserted into the silage bag, a barrel opening is covered by gauze to prevent cross contamination and reduce water loss, and then wiring of the sensor is orderly inserted into a recorder to observe and record. Blank bucket with sensor only put, end experiment 15 d.
During the period, the day of unpacking is taken as the 0 th day, the pH value of the silage is measured, then, the silage is sampled by a quartering method every other day to measure the pH value of the silage under the condition of aerobic exposure, and the experiment is ended after 8 days.
After the silage is exposed to the air, aerobic microorganisms such as saccharomycetes and mould fungi can decompose silage nutrients to generate heat energy, so that the temperature of the silage is increased, and the pH value of the silage is increased.
Aerobic stability refers to the time, in hours (h), during which the silage remains stable and does not deteriorate after exposure to air under certain conditions, and generally refers to the time, in hours (h), at which the core temperature of the silage exceeds 2 ℃. The aerobic stability can be maintained for several days, and the stability can be maintained for only several hours when the stability is poor. The high aerobic stability of silage means that the maximum recovery and utilization of dry matter, organic matter and energy is obtained.
The change in pH after aerobic exposure of silage is also a key indicator for the health and quality of the reaction feed. Researchers found that wild yeast started working after aerobic exposure, consuming sugars and fermentation acids. In addition, some enterohealth-destroying bacteria such as Clostridium (Clostridium spp.), listeria (Listeria spp.), salmonella (Salmonella spp.) and the like accelerate the growth of pathogenic bacteria after the pH is raised, and finally cause silage pollution. When the silage pH exceeds a threshold value of 4.8 to 5.0, moulds and pathogenic bacteria occupy the entire feed.
Under the aerobic exposure condition of the mixed silage, the aerobic stability of the control group and the lonicera confusa additive treatment group is more than 360h (the aerobic stability data is measured to be cut off after 15 d), wherein the temperature change after aerobic exposure and the pH value change within 8d are respectively shown in fig. 1 and fig. 2, the temperature change after aerobic exposure does not have obvious regularity in the control group and the treatment group, but the temperature change within the control group and the treatment group 15d is within +/-1.50 ℃, and the aerobic decay range is not more than or equal to 2 ℃. The pH change profile after aerobic exposure found: slightly increasing the pH value on day 2, but not exceeding 4.70, and then leading the pH value to change smoothly, wherein the pH value fluctuates between 4.40 and 4.70; however, the pH of the mixed silage of the honeysuckle additive treatment group after aerobic exposure is lower than that of the control group. It can be seen that: the addition of flos lonicerae does not affect the aerobic stability of the alfalfa mixed silage, but can keep lower pH during aerobic exposure to inhibit the breeding of harmful microorganisms.
Example 4
Influence of flos Lonicerae on antioxidant activity of herba Medicaginis mixed silage.
Experimental treatment and grouping: the same as in example 1.
Preparation of sample solution: weighing 1g of sample sieved by a 40-mesh sieve, adding 20ml of 70% ethanol, and usingOscillating for 12h with constant temperature culture oscillator at 300 r.min -1 Standing for 30min, placing in high speed centrifuge for 6000 rpm, centrifuging at 25 deg.C for 10min, collecting supernatant to 100ml Erlenmeyer flask, repeatedly extracting for 2 times, mixing, placing in sample tube, and storing at-20 deg.C.
(1) Determination of chlorogenic acid content
Establishment of a standard curve: prepared into 100 ug/mL by 70 percent ethanol -1 The chlorogenic acid standard mother liquor (chlorogenic acid is purchased from Beijing Solebao science and technology Co., ltd.) is respectively absorbed with a certain amount of chlorogenic acid standard mother liquor to be prepared into 0, 3.297, 6.522, 9.677, 12.766, 15.789, 18.75 and 21.649 mu g.mL -1 Measuring absorbance of the stock solution of chlorogenic acid at the wavelength of 329nm of ultraviolet to obtain a linear regression equation, wherein y =0.0561x +0.0419, R 2 =0.9998, which shows that the standard curve of the content of chlorogenic acid has good linear relation.
Measuring the content of chlorogenic acid in the sample: weighing 0.5g of a sample sieved by a 40-mesh sieve, adding 30ml of 70% ethanol, uniformly mixing for 5min by using a vortex mixer, performing ultrasonic extraction for 25min at 25 ℃ (ultrasonic frequency is 40KHz and power is 100W), uniformly mixing for 5min by using the vortex mixer again, standing for half an hour, putting the mixture into a high-speed centrifuge for 6000 revolutions, centrifuging for 10min at 25 ℃, taking supernatant liquid to a 100ml conical flask, and repeatedly extracting for 2 times. Adjusting pH of the extractive solution to 3 with hydrochloric acid, adding activated carbon at a ratio of 1.4%, and decolorizing at 60 deg.C for 1 hr. The liquid was transferred to a 100ml volumetric flask to be constant volume, appropriately diluted and measured at 329nm wavelength, and the measurement was repeated 3 times for each sample.
(2) DPPH radical scavenging Capacity determination
Establishment of a standard curve: prepared into 0.5 mg/mL by absolute ethyl alcohol -1 Respectively absorbing a certain amount of Trolox standard mother liquor to prepare 0, 19.23, 37.04, 53.57, 68.97, 83.33 and 109.38 mu g/mL -1 The absorbance of the Trolox standard stock solution is measured at the wavelength of ultraviolet 517nm to obtain a linear regression equation shown in the figure, y = -0.0037x +0.592, R 2 =0.9988, accounting for this total DPPH radical scavengingThe linear relation of the capability standard curve is good.
Determination of DPPH radical scavenging Capacity of samples: preparation of 0.1 mmol. L -1 The DPPH radical ethanol solution (DPPH radical is purchased from Shanghai Yuanye science and technology Co., ltd.) was put in a brown volumetric flask and placed in a cool place, and the solution was prepared as it is. Then 0.05mL of sample solution and 0.6mL of 0.1 mmol.L are taken -1 The DPPH free radical ethanol solution is fully and uniformly mixed, the reaction is carried out for 30min in a dark place at normal temperature, the absorbance is measured at the wavelength of 517nm, and each sample is repeatedly measured for 3 times.
(3) FRAP iron ion reduction/oxidation resistance measurement
Establishment of a standard curve: weighing 27.8mg of ferrous sulfate heptahydrate (the ferrous sulfate heptahydrate is purchased from Nanjing Bin Yuntian biotechnology Co., ltd.) to prepare 100mM ferrous sulfate heptahydrate standard mother liquor, sucking a certain amount of ferrous sulfate heptahydrate standard mother liquor to prepare 0.1757, 0.3509, 0.6994, 1.0457 and 1.3896mM ferrous sulfate heptahydrate standard stock solution, measuring absorbance at the wavelength of 593nm to obtain a linear regression equation, wherein y =0.2956x +0.0552 2 =0.9999, indicating that the standard curve of FRAP iron ion reduction/oxidation capacity has a good linear relationship.
The sample FRAP iron ion reduction/oxidation resistance capacity is measured: TPTZ diluent, TPTZ solution and detection buffer solution in a FRAP method total oxidation resistance detection kit (the FRAP method total oxidation resistance detection kit is purchased from Nanjing Biyun biotechnology Limited) are fully and uniformly mixed in sequence according to the proportion of 10. Then 0.005mL of the sample solution and 0.18mL of FRAP working solution were incubated at 37 ℃ for 5 minutes, and absorbance was measured at a wavelength of 593nm, and the measurement was repeated 3 times for each sample.
(4) Determination of the cationic radical scavenging Capacity of ABTS
Establishing a standard curve: absorbing a certain amount of 10mM Trolox standard mother liquor (Trolox is purchased from Nanjing Bintian biotechnology limited) to prepare 0, 0.146, 0.283, 0.536 and 0.763mM Trolox standard stock solution, measuring the absorbance at the wavelength of 734nm ultraviolet to obtain a linear regression equation, wherein y = is﹣0.8524x+0.6906，R 2 =0.9999, indicating that the ABTS cation radical scavenging ability standard curve has a good linear relationship.
Determination of ABTS cationic radical scavenging Capacity of samples: the ABTS solution in an ABTS method total oxidation resistance detection kit (the ABTS method total oxidation resistance detection kit is purchased from Nanjing Biyuntian biotechnology limited) and an oxidant solution are fully and uniformly mixed according to the proportion of 1:1 to prepare ABTS working mother liquor, the ABTS working mother liquor can be used after being stored for 12-16 hours at room temperature in a dark place, the ABTS working mother liquor is used within 3 days, and the ABTS working mother liquor is diluted by a proper time when the ABTS working mother liquor is used. Then 0.01mL of the sample solution and 0.2mL of ABTS working mother liquor were incubated at room temperature for 5min, and absorbance was measured at 734nm wavelength, and the measurement was repeated 3 times for each sample.
(5) Determination of total flavone content
Establishment of a standard curve: prepared into 0.5 mg/mL by using 70% ethanol -1 The rutin standard mother liquor (rutin is purchased from Shanghai leaf science and technology Co., ltd.) is respectively prepared into 0, 0.0313, 0.0625, 0.125, 0.1875 and 0.25 mg/mL by absorbing a certain amount of rutin standard mother liquor -1 The absorbance of the rutin standard product stock solution is measured at the wavelength of 410nm of ultraviolet to obtain a linear regression equation, y =2.2093x +0.0419, R 2 =1, which shows that the standard curve of the total flavone content has good linear relation.
And (3) determining the total flavone content of the sample: 2ml of the sample solution was aspirated, and 5ml of the sample solution was added thereto at a concentration of 1 mol. L -1 The potassium acetate solution (potassium acetate available from chemical reagents of national medicine group Co., ltd.) was thoroughly mixed, and 2ml of the mixture was added thereto at a concentration of 0.1 mol. L -1 The aluminum trichloride solution (aluminum trichloride purchased from national pharmaceutical group chemical reagent Co., ltd.) was mixed thoroughly and uniformly again, reacted at 20 ℃ for 20min, absorbance was measured at a wavelength of ultraviolet 410nm, and each sample was repeatedly measured 3 times.
(6) Determination of Total phenol content
Establishment of a standard curve: prepared into 0.5 mg/mL by using 70% ethanol -1 Respectively extracting a certain amount of mother liquor of gallic acid standard (gallic acid is from Shanghai-sourced leaf science and technology Co., ltd.) to obtain solutions of 0, 0.0151 and 0.0294、0.0429、0.0758、0.0882mg·mL -1 Measuring the absorbance of the gallic acid standard stock solution at the wavelength of 760nm ultraviolet to obtain a linear regression equation, wherein y =18.939x +0.2336, R 2 =0.9991, which shows that the standard curve for the total phenol content is linear.
And (3) determining the total phenol content of the sample: sucking 1ml of sample solution, adding 0.5ml of sample solution with a concentration of 0.5 mol.L -1 The folin-phenol reagent (the folin-phenol reagent is purchased from national medicine group chemical reagent Co., ltd.) is fully mixed and reacted for 5min, and then 2.5ml of 75 g.L is added -1 The sodium carbonate solution (sodium carbonate is purchased from national chemical reagent, inc.) is fully and uniformly mixed again, the mixture is reacted for 2 hours in a dark place at room temperature, the absorbance is measured at the wavelength of ultraviolet 760nm, and each sample is repeatedly measured for 3 times.
The results are shown in Table 3:
table 3: antioxidant capacity (mg.g) of flos Lonicerae to alfalfa mixed silage -1 ) Detection of (2)
As can be seen from Table 3, the DPPH radical scavenging ability, FRAP iron ion reducing/antioxidant ability and ABTS cation radical scavenging ability of the alfalfa mixed silage are 2.79, 13.83, 72.41mg g -1 The DPPH free radical scavenging capacity and FRAP iron ion reduction/oxidation resistance of the lonicera confusa adding treatment group are obviously higher than those of a control group, and the DPPH free radical scavenging capacity and the FRAP iron ion reduction/oxidation resistance of the lonicera confusa are increased along with the increase of the lonicera confusa adding amount to show a rising trend. The ABTS cation free radical scavenging capacity of the lonicera confusa additive treatment group is larger than that of the control group, but no significant difference exists. The chlorogenic acid content of the lonicera confusa adding treatment group is higher than that of the control group, and the chlorogenic acid content tends to increase along with the increase of the adding proportion of the lonicera confusa. The contents of total flavone and total phenol in the lonicera confusa additive treatment group are obviously higher than those of a control group.
The results show that after the lonicera confusa is added, the DPPH free radical scavenging capacity, the FRAP iron ion reduction/oxidation resistance capacity and the ABTS cation free radical scavenging capacity, the chlorogenic acid content, the total flavone content and the total phenol content of the silage are all increased, and the oxidation resistance of the silage is improved.
Example 5
Influence of flos Lonicerae on fermentation microorganism of mixed silage of herba Medicaginis.
Experimental treatment and grouping: the same as in example 1.
In the quartering method, 20g of silage per repetition, 50g of a sample at-80 ℃ is freeze-dried and ground to allow it to pass through a 4mm sieve. Then, 5g of the sample was ground with a grinding ball at room temperature for 1min, and total DNA was extracted using TGuide S96 bacterial DNA isolation kit (DP 812, tiangen, beijing, china) according to the instructions. DNA concentration and purity were assessed using NanoDrop2000, while DNA quality was assessed using 1% agarose gel electrophoresis. The 16S rRNA full-length gene was amplified using 27F and 1492R primers and sequenced using real-time sequencing technology (SMRT). The PCR procedure was as follows: amplifying at 95 ℃ for 2min, amplifying at 98 ℃ for 10s, amplifying at 55 ℃ for 30s, amplifying at 72 ℃ for 90s, finally amplifying at 72 ℃ for 2min, and circulating for 25 times. Amplicon sequencing was performed by the PacBio sequence sequencing platform (pacfic Biosciences, menlo Park, CA, USA). Original reads were assembled using SMRT linked Cycle Consensus Sequencing (CCS) software. CCS was determined using Lima (v1.7.0 software), raw-CCS sequence data was generated using barcodes, primer sequences were identified and removed using Cutadapt (1.9.1 software), and length filtered to give clear-CCS sequences, excluding primers. Then, the CCS of each sample was examined, and chimeric reads were removed by UCHIME (v 4.2 software) to obtain an optimized sequence. Comparisons were made with representative sequences using Usearch software to generate classification information (using a 0.97bootstrap threshold). Prior to subsequent statistical analysis, unknown species were filtered at each species level (genus and species) and the filtered species abundances were classified as other species. QIIME software (version 1.7.0) was used to determine the Shannon and Simpson diversity index, chao1 and ACE diversity index.
The results of the tests are shown in the following tables 4 and 5 and fig. 3 and 4:
table 4: detection of diversity of mixed silage bacteria of alfalfa by lonicera confusa
The Chao1 index, ACE index, shannon index and Simpson index of the bacterial community of the lonicera confusa additive treatment group are lower than those of the control group and are obviously reduced along with the increase of the lonicera confusa additive amount, which indicates that the diversity and the richness of the bacterial community in alfalfa mixed silage are obviously reduced by adding the lonicera confusa (table 4).
Fig. 3 shows that Lactobacillus (Lactobacillus) is absolutely predominant after alfalfa mixed ensiling, the relative abundance content exceeds 96.95%, lactobacillus bacteria in the lonicera confusa adding and treating group are significantly higher than those in the control group, and the Lactobacillus bacteria tend to increase with the increase of the lonicera confusa adding amount.
Table 5 shows that the top 10 of the alfalfa mixed silage microorganisms belong to lactic acid bacteria, and the bacteria of the genus Lactobacillus (Lactobacillus) account for the dominant bacteria, and the relative abundance exceeds 93%. Acid-resistant lactobacillus (l.acetotolerans) in lactobacillus bacteria is absolutely predominant, and the relative abundance of acid-resistant lactobacillus (l.acetotolerans) in the lonicera confusa addition treatment group is significantly higher than that of the control group, and linearly increases with the increase of the lonicera confusa addition amount (fig. 4).
Table 5: detection of relative abundance (%) of dominant lactic acid bacteria added to lonicera confusa before 10 ranks of alfalfa mixed silage
The results show that after the lonicera confusa is added, the beneficial microorganisms of the silage, namely Lactobacillus acidophilus, are propagated in a large quantity, wherein Lactobacillus acidophilus is an absolute dominant strain.
Drawing a Spearman correlation heat map of indexes of fermentation quality, nutrition quality and oxidation resistance of the lactobacillus acidophilus (L.acetotolerans) and lonicera confusa-added alfalfa mixed silage by utilizing Vegan software. The results are shown in FIG. 5. As can be seen from fig. 5, the acid-resistant lactobacillus (l.acetotolerans) is in a very significant negative correlation with pH and acetic acid content, in a very significant negative correlation with lactic acid content, in a very significant positive correlation with dry matter content, in a very significant positive correlation with soluble sugar content, in a very significant negative correlation with neutral detergent fiber and acidic detergent fiber content, and in a very significant positive and negative correlation with total phenol content, chlorogenic acid content, and FRAP iron ion reduction/oxidation resistance. The results show that the key fermentation indexes (pH and acetic acid) and fiber indexes (neutral detergent fiber and acid detergent fiber), antioxidant (total phenol, chlorogenic acid) and FRAP iron ion reduction/oxidation resistance are positively influenced by the dominant bacterium, namely acid-resistant Lactobacillus (Lactobacillus acidoterrens).
In combination with the foregoing results, it can be found that: the lonicera confusa improves the fermentation quality, the fiber quality and the oxidation resistance (oxidation resistance) of the alfalfa mixed silage by regulating and controlling the accumulation of acid-resistant Lactobacillus (Lactobacillus acetotartolorans) in the alfalfa mixed silage.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and is not intended to limit the practice of the invention to these embodiments. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (9)
1. Application of flos Lonicerae in improving quality of mixed silage of herba Medicaginis.
2. The application of the lonicera confusa in improving the quality of the alfalfa mixed silage as claimed in claim 1, characterized by comprising at least one of the following applications:
the application of the lonicera confusa in improving the fermentation quality of the alfalfa mixed silage; or,
the application of the lonicera confusa in improving the aerobic stability of the alfalfa mixed silage feed; or,
the application of flos lonicerae in improving the oxidation resistance of the alfalfa mixed silage feed; or,
the application of the lonicera confusa in improving the nutritional quality of the alfalfa mixed silage feed; or,
application of flos Lonicerae in improving acid-resistant Lactobacillus proliferation in mixed silage of herba Medicaginis.
3. The application of the lonicera confusa in improving the quality of the alfalfa mixed silage feed according to claim 1 or 2, wherein the lonicera confusa is a part of lonicera confusa used as a medicine, and comprises the whole plant or flower of lonicera confusa.
4. The application of lonicera confusa to improving the quality of alfalfa mixed silage according to claim 1 or 2, wherein the alfalfa mixed silage further comprises soybean meal and DDGS.
5. A method for improving the quality of alfalfa mixed silage is characterized in that lonicera confusa is added into the alfalfa mixed silage.
6. The method for improving the quality of the alfalfa mixed silage feed according to claim 5, wherein the lonicera confusa is added in an amount of 5-25 g/kg of weight of alfalfa silage raw materials.
7. The method for improving the quality of the alfalfa mixed silage as claimed in claim 5, wherein the alfalfa mixed silage further comprises soybean meal and DDGS, and the mass ratio of the alfalfa, the soybean meal and the DDGS is (7-8): (0.5-1.5): (0.5-1.5).
8. The method for improving the quality of the alfalfa mixed silage as claimed in claim 5, wherein the alfalfa mixed silage further comprises lactic acid bacteria, the number of viable lactic acid bacteria is 1.0 x 10 11 cfu·g -1 The addition amount is 2-3 g per ton.
9. A alfalfa mixed silage, prepared by the method of any of claims 5-8.
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