阅读说明：本技术 一种冻害芦苇草青贮方法 (Method for ensiling reed grass in freezing damage ) 是由 李平 陈良寅 白史且 苟文龙 肖冰雪 游明鸿 张玉 李达旭 于 2019-11-11 设计创作，主要内容包括：本发明公开了一种冻害芦苇草青贮方法,包括采摘芦苇草得到霜冻损坏的原料草料、分别切段、接种外源LAB混合均匀得到青贮材料及分份装袋抽真空继而青贮三个步骤。本发明还提供了一种该方法制备的青贮饲料。本发明通过接种外源LAB,特别是接种LB增加乙酸和丙酸含量,降低丁酸和氨氮含量,提高青贮期间的发酵速度、有氧稳定性、产品适口性及存放周期,整体优化冻害芦苇草青贮发酵过程,适宜在西部高原地区大规模生产,其成本低廉,大大提高青藏高原天然芦苇草利用率。(The invention discloses a method for ensiling freezing injury reed grass, which comprises the three steps of picking reed grass to obtain frost damaged raw material forage, respectively cutting into sections, inoculating exogenous LAB, uniformly mixing to obtain an ensiling material, bagging in parts, vacuumizing and then ensiling. The invention also provides the silage prepared by the method. According to the invention, exogenous LAB, especially LB is inoculated to increase the content of acetic acid and propionic acid, reduce the content of butyric acid and ammonia nitrogen, improve the fermentation speed, aerobic stability, product palatability and storage period during ensiling, integrally optimize the ensiling fermentation process of the freeze injury reed grass, be suitable for large-scale production in western plateau areas, have low cost and greatly improve the utilization rate of the natural reed grass in the Qinghai-Tibet plateau.)

1. A method for ensiling freeze injury reed grass is characterized by comprising the following steps:

(1) when the environmental temperature is lower than 0 ℃ for three continuous nights in 9 months, manually picking reed grass without seeds immediately in the morning of the fourth day to obtain raw material forage damaged by frost;

(2) dividing raw material forage into leaves, leaf sheaths L, stems S and whole crops W, respectively cutting L, S and W into sections of 0.5-1.0cm, and mixing uniformly;

(3) inoculating exogenous LAB into the fresh material obtained in the step (2) at a ratio of 90-120cfu/g, and thoroughly mixing to obtain an ensiling material;

(4) and (4) putting the silage material obtained in the step (3) into a polyester bag according to about 1.0 kg/part, vacuumizing, and then ensiling for 160-200 days in the environment with the ambient temperature lower than 20 ℃.

2. The method for ensiling the frozen reed weeds as claimed in claim 1, wherein: the exogenous LAB inoculated in the step (3) is one or two of Lactobacillus plantarum Chikuso-1 or L.

3. The method for ensiling the frozen reed weeds as claimed in claim 1, wherein: and (3) inoculating the fresh material obtained in the step (2) with exogenous LAB at a ratio of 106cfu/g, and thoroughly mixing to obtain the silage material.

4. The method for ensiling the frozen reed weeds as claimed in claim 1, wherein: the step (4) is ensiling for 180 days at the ambient temperature of less than 20 ℃ from 9 months to 3 months in the next year.

5. An ensilage prepared by the ensilage method of the frozen reed grasses according to any one of claims 1 to 4.

Technical Field

The invention relates to the technical field of silage, in particular to a method for optimizing freeze injury reed grass silage by inoculating exogenous LAB.

Background

Qinghai-Tibet plateaus, about 70% of which are high-altitude, cold pastures, few crops are suitable for planting due to natural, extreme, unstable climate and natural environment. One such alternative crop is Phragmites communis, such as Phalaris canariensis L, a high-yielding cold season grass species that is more productive than oats and other local grasses in the area. Due to bad weather conditions and poor management of grazing, when the environmental temperature is below 0 ℃ and lasts for 4 to 5 hours, the stems and leaves of the reed grass are frozen, resulting in rapid reduction of nutrients. Frost damaged Reed grass (RCG) is commonly used as a local animal feed ingredient, particularly for winter and early spring yaks. Many studies in japan, canada and the united states show that RCG can be stored as silage before being used in an anaerobic digester for methane production. However, these studies were conducted in the best growing season of RCG in summer, and there are few studies on ensiling fermentation of RCG harvested after frost death.

In practice, it is still a difficult task to achieve the desired storability and stability of silage products due to improper handling procedures of silage or other factors. Poor fermentation of silage can be caused by insufficient exogenous bacteria, reduced number of beneficial primary bacteria (primary LAB), and more spoilage organisms (such as yeast and mold) on a frozen sample. Therefore, it is an important way to improve the fermentation rate and aerobic stability during the ensiling period by studying the type and amount of the exogenous bacterial species.

Disclosure of Invention

The invention mainly aims to solve the problems of low utilization rate, unstable ensiling process and product quality of the frozen reed weeds in the Qinghai-Tibet plateau, and provides the ensiling process of the frozen reed weeds. Meanwhile, the characteristics of reed grass silage, bacterial communities and other indexes under the conditions of no external source or external source in the Qinghai, Qinghai-Tibet plateau and other areas are researched, and the fermentation speed, aerobic stability, product palatability and storage period in the silage period are improved. Finally, the invention also provides the silage prepared by the method, which has the advantages of stable quality, high content of nutrient components, good palatability and long storage period.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

a method for ensiling freezing injury reed grass comprises the following steps:

(1) when the environmental temperature is lower than 0 ℃ for three continuous nights in 9 months, manually picking reed grass without seeds immediately in the morning of the fourth day to obtain raw material forage damaged by frost;

(2) dividing raw material forage into leaves, leaf sheaths L, stems S and whole crops W, respectively cutting L, S and W into sections of 0.5-1.0cm, and mixing uniformly;

(3) inoculating exogenous LAB into the fresh material obtained in the step (2) at a ratio of 90-120cfu/g, and thoroughly mixing to obtain an ensiling material;

(4) and (4) putting the silage material obtained in the step (3) into a polyester bag according to about 1.0 kg/part, vacuumizing, and then ensiling for 160-200 days in the environment with the ambient temperature lower than 20 ℃.

The exogenous LAB inoculated in the step (3) is one or two of Lactobacillus plantarum Chikuso-1 or L.

Lactobacillus plantarum is widely used in silage fermentation of other varieties of raw materials, and can rapidly increase the content of lactic acid to reduce the pH value of silage. Some studies have shown that silage material harvested after frost shows low levels of LAB and large amounts of yeast and moulds. On the other hand, frequent frost can destroy plant cells and release available substrates causing the plant microbiome to multiply rapidly. Obligate heterofermentative LAB, such as l.buchneri, can slowly convert lactic acid to acetic and propionic acids under anaerobic conditions. The final fermented silage product is influenced by the microbial population, and follow-up remedial adjustment measures for situations such as aerobic exposure can also be inspired by researching the microbial population.

Preferably, the step (3) is to inoculate the fresh material obtained in step (2) with exogenous LAB in a ratio of 106cfu/g and thoroughly mix to obtain silage material.

Through repeated tests and data statistical analysis, better fermentation results can be obtained by inoculating the silage material with exogenous LAB at a rate of 106cfu per gram of fresh material.

The step (4) is ensiling for 180 days at the ambient temperature of less than 20 ℃ from 9 months to 3 months in the next year.

The fermentation process is monitored to avoid the distribution of low beneficial microbial count and high undesirable microorganisms (including aerobic bacteria, yeast and mold) on the plant.

The invention makes full use of the natural environment of local (Qinghai-Tibet plateau and other western plateau in China) for fermentation, and the RCG silage can be well fermented due to the high distribution of lactobacillus, pediococcus and Weissella spp.

An silage prepared by the method for ensiling the reed grass with the freezing injury.

Subsequent tests on fresh materials, RCG materials and silage products show that compared with the fresh materials, the content of water-soluble carbohydrate (WSC) of the RCG materials is reduced, and the content of Neutral Detergent Fiber (NDF) after silage is increased, because a large amount of available substrates are converted into metabolites (mainly organic acids such as lactic acid and acetic acid) by plants and exogenous microorganisms existing in the plants under the anaerobic condition, the total content of the organic acids in the silage is effectively increased, the content of ammoniacal nitrogen is reduced, and therefore the palatability of the silage is favorably improved, and the nutritional value is increased. The presence of tannins is considered to be an advantageous component because they protect feed proteins from degradation by inhibiting plant and microbial enzymes and/or by forming complexes with proteins, also reducing the content of ammoniacal nitrogen. The total tannins remaining after ensiling continue to be at relatively high levels. However, the total tannin content of the silage after exogenous LAB inoculation was significantly reduced compared to the control (P < 0.05). Theoretically, the Crude Protein (CP) content (TN × 6.25) is not directly affected by fermentation (ammonia nitrogen included in TN), but decreases linearly with increasing gas and wastewater flow losses in silage (Ferreira et al, 15 al., 2013; Santos et al, 2014). In the present invention, the CP content of the frost RCG after ensiling fermentation is reduced. LB inoculation increases (P <0.05) acetic acid and propionic acid content, reduces (P <0.05) butyric acid and ammonia nitrogen content, and enhances preservation of Crude Protein (CP) and Ether Extract (EE) in the whole crop. Therefore, the inoculation of the exogenous LAB can enhance the preservation of the nutrient components in the silage, can also obviously reduce the content of ethanol, inhibit the growth of fungi in the silage, and improve the aerobic stability of the silage, thereby being beneficial to prolonging the preservation period of the silage.

In terms of the impact of exogenous LAB on silage pH and fermentation products, previous studies showed that frost-damaged silage exhibits poorer fermentation characteristics, reduced lactic acid content, increased pH, increased butyric acid and ammonia nitrogen content, compared to normal silage (Mohammadzadeh et al, 2014). The main objective of exogenous LAB inoculation is to produce large amounts of lactic acid by consuming WSC to achieve the effect of enhancing silage pH reduction. The lowest pH values observed for exogenous LAB-inoculated silage (3.93, mean) indicate a higher efficiency of sugar utilization by these microorganisms. The recommended content of acetic acid in the silage is 10-30g/kg DM. High levels of acetic acid (>30-40g/kg DM) may result in low DM recovery and energy (Kung and Shaver, 2001; Kung et al, 2018). However, too low an acetic acid concentration can produce an unstable silage state when exposed to air. In the present invention, the acetic acid content (4.8 to 7.3g/kg DM) in the control and LP inoculated silage indicate a poor possibility to maintain aerobic stability. However, inoculation of LB did increase (P <0.05) the concentrations of acetic and propionic acids and decrease the levels of butyric, ethanol and ammonia-N in silage (P <0.05) compared to the control. pH of frost damaged RCG silage (3.88-4.16) and lactic acid (34.9-40.3g/kg DM) and acetic acid (4.8-14.3g/kg DM) concentrations versus (Contreras-Govea) data lower et al, 2009) they reported that normal silage RCG harvested from stalk elongation to early ear in the United states normally has a pH of 4.58-4.87, lactic acid of 45.8-63.4g/k DM and acetic acid of 13.2-28.1g/kg DM. (Kung et al, 2018) reviewed and concluded that well fermented forage silage has undetectable characteristics of butyric acid, propionic acid, less than 1g/kg DM and ammonia nitrogen content of 80-120g/kg DM, etc., when the silage dry matter is 25-35%. The contents of propionic acid (1.0-4.7g/kg DM), butyric acid (4.6-8.0g/kg DM) and ammonia nitrogen (104.2-127.0g/kg TN) in the silage are higher, and the fermentation effect of the silage is poorer. Therefore, the improvement effect of the outdoor source inoculation on the freezing damage RCG silage fermentation is limited.

There is a strong correlation between microbial populations and silage fermentation in terms of the effect of exogenous LAB on the microbial population of silage plate cultures (McDonald et al, 1991). Compared with fresh pasture, the amount of LAB is increased by 74.60-82.93%, while the amount of aerobic bacteria and yeast of the silage is reduced by 19.30-65.22% after 180 days of silage. Inoculation with LB increased the amount of LAB in silage compared to the control. Similar results are obtained (Da 11Silva et al, 2018). The effect associated with grass species and the inoculation rate of lactobacillus buchneri will cause different results. Previous studies have shown that high levels of acetic acid can inhibit the activity of yeast in silage, as it is able to penetrate the cell membrane in a non-dissociated form, subsequently releasing H + in the cytoplasm, and then destroying DNA structures in the nucleus (Romero et al, 2017; tabaco et al, 2011). However, in this study, the high yeast count (3.2-4.6cfu/g FM) of the culture-based technology was still in all ensilages. Similar yeast counts were observed in other ensilages (Zhou et al, 2016; 19Xing et al, 2009). This is probably because (1) the relatively low acetic acid (<20g/kg 20DM, silage moisture of about 57%) does not inhibit the activity of certain yeast species, particularly for lactic acid assimilating yeasts; (2) when volatile fatty acids (in particular acetic acid and propionic acid) are present in the silage, the production of yeast spores can be inhibited; (3) then certain yeasts, such as Acetobacter spp, may be stimulated and propagated during ensiling due to the production of acetic acid by l. Leaf silage has a lower LAB number and a higher number of yeasts and moulds compared to silage, indicating a high probability of deterioration of exposure. (3) Then certain yeasts, such as Acetobacter spp, may be stimulated and propagated during ensiling due to the production of acetic acid by l. Leaf silage has a lower LAB number and a higher number of yeasts and moulds compared to silage stalk feed, indicating a high probability of deterioration of exposure.

In terms of the effect of exogenous LAB on the composition of the ensiled bacterial community, a coverage rate >0.99 indicates that most of the bacterial community composition at the time of sampling has been deeply captured. Relatively high bacterial OTU (72 to 117), abundance (Chao 1,70 to 119; ACE, 76 to 129) and diversity (Shannon, 3.69 to 5.51; Simpson, 0.86 to 0.96) were observed in fresh pasture and these were reduced by 35.10% to 64.10%, 32.77% to 64.71%, 44.96% to 64.34%, 31.50% to 55.17% and 7.29% to 36.46% after ensiling in whole crops. Similar results were reported (Guan et al, 2018; Li et al, 2019), but not in other reports (Ni et al, 2017; ZHao et al, 2017). This can be explained by the rate and extent of pH reduction during ensiling. Inoculation with exogenous LAB reduced the bacterial abundance index of ACE and Chao1 in both whole crop and stem silage compared to controls (P < 0.05). Lower bacterial diversity was also observed in corn silage treated with LAB compared to the control (Ogunade et al, 2018). In particular, inoculation of LB reduced (P <0.05) the diversity index of bacteria OTU and silage Shannon and Simpson. This is consistent with the immediate production of acetic acid and propionic acid, which can effectively destroy bacterial DNA. Overall, the abundance index and diversity index of silage leaves are highest, but this can be reduced by inoculating exogenous LAB. The relative abundance of bacteria at the genus level shows that Massilia is the major microorganism, followed by lactobacilli in fresh forage after freezing injury. The bacterial community composition changed greatly after ensiling. Lactobacillus (60.97-90.50%), Pediococcus (1.57-16.99%) and Clostridium (2.10-5.46%) predominate in silage with relative abundances of 60.97-90.50%, 1.57-16.99% and 2.10-5.46%, respectively. High abundance lactobacilli have also been reported in our previous studies on annual ryegrass, clover and mixtures thereof silage in southern china (Li et al, 2019). No difference in the relative abundance of lactobacilli was observed between exogenous LAB inoculated silage and control silage. Studies from (Ogunade et al, 2018) show that inoculation with Lactobacillus plantarum can reduce the abundance of Chlorella and Weissella. (Romero et al, 2017) reported that inoculation with l.buchneri enhanced the presence of Weissella in oat silage. The main distribution of clostridium indicates that the silage is poor in fermentation effect and high in butyric acid level. Inoculation with exogenous LAB (especially LB) can reduce the relative abundance of clostridia. This is probably because clostridium is less tolerant to high osmotic pressures and low pH values, whereas lactic acid is produced relatively fast. Leaf silage exhibits a higher (P <0.05) relative abundance of pediococcus, Clostridium, caprolipids and Bacillus, but a lower relative abundance of lactobacilli (P < 0.05). The composition of the different bacterial communities in silage can be clearly identified by NMDS plots. Samples from fresh forage were significantly separated from samples from silage. This observation confirms that fermentation has a significant effect on the structure of the silage bacterial community. After LB inoculation, the bacterial community composition of the whole plant, stem and leaf in the control silage is changed, and the change is very obvious, which shows that L.buchneri inoculation plays an important role in the change of the bacterial community composition.

In terms of the relationship of the composition of the bacterial colony of silage bacteria to fermentation variables, the analysis shows that the diversity index of silage bacteria α is positively correlated to the ammonia nitrogen concentration (observed species r 0.686, P0.041; Shannon, r 0.711, P0.032; Chao1, r 0.669, P0.049) and ethanol (observed species r 0.767, P0.016; Shannon, r 0.750; P0.002; Chao1, r 0.750; P0.002) in the observed cold axis of lactobacillus strain, the typical correlation (CCA) plot also serves to detect the correlation between the composition of bacteria at the level of the genus strain and the colony fermentation variables, which is likely to be found to be more negative than the strain of lactobacillus during the initial fermentation phase of the lactobacillus strain, which is more likely to be responsible for the growth of the lactobacillus strain of the lactobacillus under the cold axis of fermentation, which is more likely to be responsible for the growth of the cold lactobacillus strain, but which is more likely to be responsible for the growth of lactobacillus strain of the cold strain of lactobacillus under the cold strain, which the cold strain of lactobacillus which is likely to be responsible for the growth under the initial fermentation phase of the growth of lactobacillus strain of the cold strain of lactobacillus strain of the lactobacillus under the cold strain of the lactobacillus strain of the lactobacillus under the lactobacillus strain of the lactobacillus under the strain of the lactobacillus strain of the lactobacillus under the strain of the lactobacillus strain of the strain of lactobacillus strain of the lactobacillus under the strain of the lactobacillus under the strain of the lactobacillus under the strain of the lactobacillus under the strain of the lactobacillus under the strain of the lactobacillus under the strain of the lactobacillus under the strain of the lactobacillus under the strain of the lactobacillus.

Compared with the prior art, the invention has the beneficial effects that:

the inventor provides a method for ensiling the reed grass in the frozen plateau, and the method reduces (P <0.05) diversity of bacteria α and shift of bacterial community composition (P <0.05) by inoculating exogenous LAB, but does not change (P >0.05) relative abundance of lactobacillus as a dominant genus in the ensiling feed, especially inoculates LB (lactobacillus plantarum L. buchneri) to increase the content of acetic acid and propionic acid, reduce the content of butyric acid and ammonia nitrogen, improve the fermentation speed, aerobic stability, product palatability and storage period in the ensiling period, integrally optimizes the ensiling fermentation process of the frozen reed grass, is suitable for large-scale production in western plateau areas, has low cost and greatly improves the utilization rate of the natural reed grass in the Tibetan plateau.

Drawings

FIG. 1 is a schematic representation of the ambient temperature before and after ensiling in examples 1-6 of the present invention;

FIG. 2 is a graph showing the relative abundance of bacteria at the genus level in detection example 3 of the present invention;

FIG. 3 is a diagram of RCG Lactobacilli of fresh samples, blanks and addition of exogenous LAB silage in test example 3 according to the present invention;

FIG. 4 is a photograph of RCG Pediococcus of the present invention in test example 3 with fresh sample, blank and added exogenous LAB silage;

FIG. 5 is a diagram of RCG Weissella in test example 3 of the present invention with fresh samples, blanks and addition of exogenous LAB silage;

FIG. 6 is a diagram of RCG butyric acid bacteria of fresh sample, blank and added exogenous LAB silage in detection example 3 of the present invention;

FIG. 7 is a diagram showing the analysis of the colony composition of fresh sample, blank and RCG added with exogenous LAB ensilage in detection example 3 according to the present invention;

FIG. 8 is a diagram showing bacterial community composition under the influence of pH and fermentation products of fresh samples, blanks and added exogenous LAB silage in detection example 3 according to the present invention;

FIG. 9 is a graph showing the relative abundance of bacteria at the genus level in test example 4 of the present invention;

FIG. 10 is a diagram of RCG Lactobacilli of fresh samples, blanks and addition of exogenous LAB silage in test example 4 of the present invention;

FIG. 11 is a photograph of RCG Pediococcus of the present invention in test example 4 with fresh sample, blank and added exogenous LAB silage;

FIG. 12 is a diagram of RCG Weissella in fresh sample, blank and with exogenous LAB ensilage in test example 4 according to the present invention;

FIG. 13 is a diagram of RCG butyric acid bacteria of fresh sample, blank and added exogenous LAB silage in detection example 4 of the present invention;

FIG. 14 is a diagram showing the analysis of the colony composition of fresh sample, blank and RCG added with exogenous LAB ensilage in detection example 4 according to the present invention;

FIG. 15 is a diagram showing the bacterial community composition under the influence of the pH and fermentation products of fresh samples, blanks and RCG added with exogenous LAB silage in test example 4 according to the present invention;

FIG. 16 is a graph showing the relative abundance of bacteria at the genus level in detection example 5 of the present invention;

FIG. 17 is a diagram of RCG Lactobacilli of fresh samples, blanks, and addition of exogenous LAB silage in test example 5 of the present invention;

FIG. 18 is a photograph of RCG Pediococcus of the present invention in test example 5 with fresh sample, blank and added exogenous LAB silage;

FIG. 19 is a diagram of RCG Weissella in fresh sample, blank and with exogenous LAB ensilage in test example 5 according to the present invention;

FIG. 20 is a drawing of RCG butyric acid bacteria of fresh sample, blank and added exogenous LAB silage in detection example 5 of the present invention;

FIG. 21 is a diagram showing analysis of the colony composition of fresh sample, blank and RCG added with exogenous LAB ensilage in detection example 5 according to the present invention;

FIG. 22 is a diagram showing the bacterial community composition under the influence of the pH and fermentation products of fresh samples, blanks and RCG added with exogenous LAB silage in test example 5 according to the present invention;

FIG. 23 is a graph showing the relative abundance of bacteria at the genus level in detection example 6 of the present invention;

FIG. 24 is a diagram of RCG Lactobacilli of fresh samples, blanks, and addition of exogenous LAB silage in test example 6 of the present invention;

FIG. 25 is a photograph of RCG Pediococcus of the present invention in test example 6 with fresh sample, blank and added exogenous LAB silage;

FIG. 26 is a diagram of RCG Weissella in fresh sample, blank and with exogenous LAB ensilage in test example 6 according to the present invention;

FIG. 27 is a drawing of RCG butyric acid bacteria of fresh sample, blank and added exogenous LAB silage in detection example 6 according to the present invention;

FIG. 28 is a diagram showing analysis of the colony composition of fresh sample, blank and RCG ensiled with addition of exogenous LAB in test example 6 according to the present invention;

FIG. 29 is a diagram showing the bacterial community composition under the influence of the pH and fermentation product of fresh sample, blank and RCG ensiled with added exogenous LAB in test example 6 of the present invention.

Detailed Description

The foregoing summary of the invention is described in further detail below with reference to specific embodiments.

It should not be understood that the scope of the above-described subject matter of the present invention is limited to the following examples. Various substitutions and alterations can be made without departing from the technical idea of the invention as described above, according to the common technical knowledge and conventional means in the field, and the scope of the invention is covered.