阅读说明：本技术 一种冻害芦苇草青贮方法及制备的青贮饲料 (Method for ensiling reed grass in freezing damage and ensiling feed prepared by method ) 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,还提供了一种检测RCG鲜样及青贮饲料的方法,最后还提供了一种描述RCG鲜样及青贮饲料细菌生态学的下一代测序技术。本发明描述了在青藏高原上无外源条件下制备的霜冻RCG青贮饲料的青贮特征和细菌群落,从而进一步改善和控制青贮发酵工艺,实现较好的、稳定的青贮发酵过程,提高了青藏高原霜冻损坏芦苇草的利用率。(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 and uniformly mixing to obtain ensiling material, bagging in parts, vacuumizing and then ensiling. The invention also provides the silage prepared by the method, a method for detecting the RCG fresh sample and the silage, and finally a next generation sequencing technology for describing the ecology of the RCG fresh sample and the silage bacteria. The invention describes the ensiling characteristics and bacterial community of the frost RCG ensilage prepared on the Qinghai-Tibet plateau without external sources, thereby further improving and controlling the ensilage fermentation process, realizing a better and stable ensilage fermentation process and improving the utilization rate of the frost damage to the reed grass on 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, picking up reed grass manually 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 a whole crop W, respectively cutting L, S and W into sections, and uniformly mixing to obtain an ensiling material;

(3) the ensiling material is divided into portions of about 1.0 kg/portion, put into a polyester bag, vacuumized and then ensiled for 160-200 days in an environment with the ambient temperature lower than 20 ℃.

2. The method for ensiling the frozen reed weeds as claimed in claim 1, wherein: the environment in the step (1) is a Qinghai-Tibet plateau, and the reed grass is seed-free reed grass with seeds harvested in 8 months.

3. The method for ensiling the frozen reed weeds as claimed in claim 1, wherein: cutting into sections of 0.5-1.0cm in the step (2).

4. The method for ensiling the frozen reed weeds as claimed in claim 1, wherein: the step (3) 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 belongs to the technical field of silage, and particularly relates to a method for ensiling reed grass in freezing injury.

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 lower than 0 ℃ and lasts for 4 to 5 hours, the stems and leaves of the reed grass are frozen, so that the nutrient substances are rapidly reduced. Frost damaged Reed grass (RCG) is commonly used as a local animal feed ingredient, particularly for winter and early spring yaks. Currently, the study on Phragmites communis is performed in the summer season where it is best grown, and there are few studies on ensiling fermentation of RCG harvested after frost.

Furthermore, over the past decades, the microbial population of silage has been detected primarily by culture-based techniques and PCR-based Sanger sequencing techniques. At present, the flora before and after RCG ensilage harvested after freezing injury is hardly researched.

Disclosure of Invention

The main purpose of the invention is to research the ensiling characteristics and bacterial community of the frost RCG ensilage prepared on the Qinghai-Tibet plateau under the condition of no external source, thereby further improving the ensilage fermentation process and improving the utilization rate of the reed grass. Meanwhile, the invention also provides the silage prepared by the method, the quality of the silage is stable, and the contents of various beneficial components are high. The invention also provides a method for detecting the RCG fresh sample and the silage. The invention also provides a next generation sequencing technology for describing the ecology of RCG fresh samples and silage bacteria.

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, picking up reed grass manually 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 a whole crop W, respectively cutting L, S and W into sections, and uniformly mixing to obtain an ensiling material;

(3) the ensiling material is divided into portions of about 1.0 kg/portion, put into a polyester bag, vacuumized and then ensiled for 160-200 days in an environment with the ambient temperature lower than 20 ℃.

Further, the environment in the step (1) is a Qinghai-Tibet plateau, and the reed grass is seed-free reed grass with seeds harvested in 8 months.

RCG continued greening after 8 months of seed harvest, which would affect ensiling fermentation due to its special structure and components, if not picked and separated.

Cutting into sections of 0.5-1.0cm in the step (2).

Experiments have shown that a section of 0.5-1.0cm will be most advantageous for ensiling fermentation. Too long will cause too many voids, which are detrimental to the anaerobic reaction, and too short will cause the silage material to be too dense, not fully fermented or partially rotten.

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

Generally, in the Qinghai-Tibet plateau area, the environmental temperature is lower from 9 months to 3 months in the next year, and the ensiling 180 is more suitable.

The invention makes full use of the plateau and cold natural environment in the western region of China and can carry out large-scale production in local places. Mainly uses local natural microbial colony for fermentation. Lactobacillus plantarum is widely used in silage, and it can rapidly increase the lactic acid content to lower the pH of the silage. However, in actual production, the exogenous bacterial strain for accelerating the fermentation of silage contains one or more lactic acid bacteria, which may cause the exogenous bacterial strain to dominate the original bacterial strain in the silage process, change the microbial community structure, and increase unstable factors of fermentation. The technology provided by the invention pays attention to the influence of the original strain in the local environment on the fermentation characteristic and aerobic stability of the silage in special crop varieties and special conditions in cold regions.

The invention also provides:

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

The raw material tests show that the chemical components of the reed grass are not changed greatly before and after freezing injury, the stems have the highest content of water-soluble carbohydrate (WSC) and Total Alkaloid (TA), while the leaves show opposite trends, and the Dry Matter (DM) and Neutral Detergent Fiber (NDF) content of the reed grass are the lowest. 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. Frequent frost can destroy plant cells and release available substrates to rapidly propagate the plant microbiome.

WSC content decreases after ensiling, while NDF content of leaves increases after ensiling, due to the conversion of a large amount of available substrates into metabolism (mainly organic acids, such as lactic acid and acetic acid) by the plants themselves and by exogenous microorganisms under anaerobic conditions. The presence of tannins is considered to be an advantageous component as they protect feed proteins from degradation by inhibiting plant and microbial enzymes and/or by forming complexes with proteins. The total tannins remaining after ensiling continue to be at relatively high levels.

The invention also provides a method for detecting the RCG fresh sample and the silage, and also provides a next generation sequencing technology (NGS) for describing the fresh sample and the prepared silage bacteria ecology. Aims to research the characteristics of reed grass silage, bacterial communities and other indexes under the condition of no external source (or external source) in the Qinghai, Qinghai-Tibet plateau and other areas.

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

the invention provides a method for ensiling frost reed grass, aiming at the problems in the prior art, the ensiling characteristics and bacterial community of frost RCG ensilage prepared on the Qinghai-Tibet plateau under the condition of no external source are tested and researched, thereby further improving and controlling the ensilage fermentation process and realizing a better ensilage fermentation process. The invention also provides the silage prepared by the method, which has stable quality and higher content of various beneficial components. The invention also provides a method for detecting the RCG fresh sample and the silage. The invention also provides a next generation sequencing technology for describing the ecology of RCG fresh samples and silage bacteria. At present, the method of the invention is used for scale-up production in western plateau areas, the fermentation process is stable, the yield is higher, and the utilization rate of the reed grass damaged by frost in the Qinghai-Tibet plateau is improved in a more natural environment.

Drawings

FIG. 1 is a schematic representation of the ambient temperature before and after ensiling in examples 1-3 of the present invention;

FIG. 2 is a graph showing the relative abundance of bacteria at the genus level in test example 1 of the present invention;

FIG. 3 is a diagram of the genus RCG lactobacillus of fresh sample and silage in detection example 1 of the present invention;

FIG. 4 is a drawing showing fresh-like and ensiled RCG Pediococcus in detection example 1 of the present invention;

FIG. 5 is a diagram of Weissella of RCG fresh and ensiled in detection example 1 of the present invention;

FIG. 6 is a drawing showing fresh samples and ensiled RCG butyric acid bacteria in detection example 1 of the present invention;

FIG. 7 is a graph showing analysis of the colony composition of fresh samples and ensiled RCG in detection example 1 of the present invention;

FIG. 8 is a diagram showing the pH and bacterial community composition under the influence of fermentation products of fresh samples and ensiled RCG in test example 1 according to the present invention;

FIG. 9 is a graph showing the relative abundance of bacteria at the genus level in detection example 2 of the present invention;

FIG. 10 is a diagram of the genus RCG lactobacillus of fresh sample and silage in detection example 2 of the present invention;

FIG. 11 is a drawing showing fresh-like and ensiled RCG Pediococcus in detection example 2 of the present invention;

FIG. 12 is a diagram of Weissella of RCG fresh and ensiled in detection example 2 of the present invention;

FIG. 13 is a drawing showing fresh samples and ensiled RCG butyric acid bacteria in detection example 2 of the present invention;

FIG. 14 is a graph showing analysis of the colony composition of fresh samples and ensiled RCG in detection example 2 of the present invention;

FIG. 15 is a diagram showing the bacterial community composition under the influence of pH and fermentation products of fresh samples and ensiled RCG in test example 2 of the present invention;

FIG. 16 is a graph showing the relative abundance of bacteria at the genus level in detection example 3 of the present invention;

FIG. 17 is a diagram of the genus RCG lactobacillus of fresh sample and silage in detection example 3 of the present invention;

FIG. 18 is a drawing showing fresh-like and ensiled RCG Pediococcus in detection example 3 of the present invention;

FIG. 19 is a diagram of Weissella of RCG fresh and ensiled in detection example 3 according to the present invention;

FIG. 20 is a drawing showing fresh samples and ensiled RCG butyric acid bacteria in detection example 3 of the present invention;

FIG. 21 is a graph showing analysis of the colony composition of fresh samples and ensiled RCG in detection example 3 of the present invention;

FIG. 22 is a graph showing the bacterial community composition under the influence of pH and fermentation products of fresh-samples and ensiled RCG in test example 3 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.