Dynamics of proteolysis, protease activity and bacterial community of Neolamarckia cadamba leaves silage and the effects of formic acid and Lactobacillus farciminis

doi:10.1016/j.biortech.2019.122127

Bioresource Technology

Volume 294, December 2019, 122127

https://doi.org/10.1016/j.biortech.2019.122127 Get rights and content

Highlights

•
Over 85% protein in N. cadamba leaves (NCL) was well preserved after 60 days ensiling.
•
Protease activity and bacterial diversity in NCL silage were very low.
•
Exiguobacterium dominated and increased during the ensiling of NCL.
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Low protease and bacterial activities assured protein well-preserved in NCL silage.

Abstract

To investigate the reason for well preservation of protein in Neolamarckia cadamba leaves (NCL) during ensiling, fresh NCL were ensiled with or without addition of 2.0 mL/kg formic acid (FA) or 1.0 × 10⁹ CFU/kg Lactobacillus farciminis (LF), and the dynamics of protease activity and microbial community were analyzed. Nonprotein-N, free amino acid, ammonia-N, the activities of carboxypeptidase and aminopeptidase, and bacterial diversity were low during NCL ensiling. Exiguobacterium dominated in NCL silage and its relative abundance increased while Enterobacter abundance decreased during ensiling. FA lowered (P < 0.05) pH and coliform bacteria number, while LF increased (P < 0.05) lactic acid bacteria number, lactic acid content and Lactobacillus abundance at the early stage of fermentation. In summary, protein in NCL can be well preserved during ensiling likely due to its low protease and bacterial activity, and FA and LF improve the quality of NCL silage in different ways.

Introduction

As a popular and traditional method of fresh forage preservation, ensiling not only reduces weather risk and harvest loss compared with haymaking, but also supplies year-round availability of nutritious and palatable feed for livestock. Forages fed as silage often allow greater efficiency of feed mixing and handling, and reduce sorting behavior and bunk-staying time of animals relative to dry forages (Grant and Ferraretto, 2018). Moreover, life cycle assessment reveals that silage production seems to have better benefit to cost ratio with less energy use relative to haymaking (Fathollahi et al., 2018). Ensiling has been becoming a most prevailing form of feedstock preservation in livestock production, even in biorefineries (Wilkinson and Rinne, 2018). However, a major concern of ensiling is the degradation of protein. During ensiling, proteins are extensively degraded to peptides, free amino acid and ammonia-N, which always results in inefficient microbial N synthesis in rumen (Tabacco et al., 2006). The poor N utilization may lead to economic loss and potential environmental pollution.

Neolamarckia cadamba, which previously named Anthocephalus chinensis, is a semi-deciduous, evergreen and tall tree of the Rubiaceae family and widely distributed throughout tropical and subtropical regions. Owing to high yield, nutritive value and antibacterial property, N. cadamba leaves (NCL) can be used for woody forage production to relieve the pressure of forage supply (Wang et al., 2017). Our previous study found that there were relatively low nonprotein-N and ammonia-N contents in NCL silage after 60 days fermentation (He et al., 2018). Furthermore, mixing NCL could reduce the nonprotein-N and ammonia-N contents in legume silage substantially (Wang et al., 2019a). It is known that the degradation of protein during ensiling is mainly caused by plant enzymes and microbial activities. Plant enzymes catalyze the hydrolysis of proteins into peptides and free amino acids, whereas microbial activities degrade them to amides, amines, and ammonia further (Albrecht and Muck, 1991). However, little information about dynamics of protein degradation, protease activity and microbial community of NCL silage is available, and the reason for well preservation of protein in NCL during ensiling is still unknown. The analyses of such items will elucidate the mechanism of NCL protein well-preserved during ensiling and help us to improve forage storage.

On the other hand, formic acid and lactic acid bacteria inoculants are two different kinds of additives which are commonly used at ensiling. Formic acid inhibits fermentation by direct acidification and antimicrobial activity, while lactic acid bacteria inoculants accelerate fermentation by increasing lactic acid bacteria number of the forage. They are proved to inhibit the activities of undesirable organisms and reduce proteolysis during ensiling (Muck et al., 2018). Therefore, to investigate the reason for little proteolysis in NCL silage, fresh NCL were ensiled either with or without addition of formic acid or Lactobacillus farciminis (LF), and then protein fraction, protease activity and bacterial diversity of the silages were analyzed after 1, 3, 7, 14, 30 and 60 days of ensiling, respectively.

Section snippets

Raw materials and silage preparation

Neolamarckia cadamba was cultivated in the experimental field of South China Agricultural University (23°19′ latitude N, 113°34′ longitude E, Guangdong, China, Guangzhou, China) and the leaves were manually collected in August 2018. The fresh leaves (moisture content: 883 g/kg fresh matter) were chopped to about 2 cm by a handy paper cutter. Then a given amount of leaves (about 170 g, based on the capacity of mini silo bag) were treated with (i) no additive control (CK), (ii) 2.0 mL/kg formic

Characteristics of fresh NCL

The dry matter content of fresh NCL was 217 g/kg fresh matter. The crude protein content (107 g/kg DM) was comparable with the data reported by He et al. (2018) but far lower than that of Wang et al. (2018b). The difference might be because the forage quality could be influenced by factors like climate, fertilization (Van Soest et al., 1978) and harvest time (Zhang et al., 2016). The relatively high content of crude protein, true protein-N (887 g/kg total N) and low content of fiber (237,

Conclusions

Overall, the protein in N. cadamba leaves could be well preserved during ensiling. As proteolysis during ensiling mainly caused by plant enzymes and microbial activities, the relatively low contents of nonprotein-N, free amino acid and ammonia-N might be due to the low protease activity, bacterial diversity and relative abundance of undesirable microorganism in NCL silage. Formic acid lowered pH and coliform bacteria number rapidly, while Lactobacillus farciminis LF increased lactic acid

Acknowledgements

This work was financially supported by 2018 Big Pig-producing County Reward Funds (Research and promotion of key technologies for healthy feeding of pigs and resource utilization of manure pollution), National Key R&D Projects (Grant No. 2017YFD0502102-02) and Guangzhou Science Forestry Technology and Innovation Commission (Grant No. 2018KJCX001).

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¹: L.H. and C.W. contributed equally to this article.

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Dynamics of proteolysis, protease activity and bacterial community of Neolamarckia cadamba leaves silage and the effects of formic acid and Lactobacillus farciminis

Highlights

Abstract

Introduction

Section snippets

Raw materials and silage preparation

Characteristics of fresh NCL

Conclusions

Acknowledgements

Anim. Feed Sci. Technol.

J. Clean. prod.

J. Dairy Sci.

J. Dairy Sci.

J. Dairy Sci.

Anim. Feed Sci. Tech.

Bioresour. Technol.

J. Dairy Sci.

Anim. Feed Sci. Tech.

J. Dairy Sci.

J. Dairy Sci.

Bioresour. Technol.

J. South China Agri. Uni.

Bioresour. Technol.

Bioresour. Technol.

J. Dairy Sci.

Proteolysis in ensiled forage legumes that vary in tannin concentration

Crop Sci.

Influence of Lactobacillus spp. from an inoculant and of Weissella and Leuconostoc spp. from forage, crops on silage fermentation

Appl. Environ. Microbiol.

Leaf proteolytic activities and senescence during grain development of field-grown corn (Zea mays L.)

Plant Physiol.

Microbiological basis of phosphate removal in the activated sludge process for treatment wastewater

Microb. Ecol.

Changes in the distribution of nitrogen and plant enzymatic activity during ensilage of lucerne treated with different additives

Grass Forage Sci.