Exploring Lactobacillus plantarum on Fermentation Quality, Gas Emissions, and In Vitro Digestibility of Different Varieties of Litchi Leaves Silage
1
Guangdong Key Laboratory for Innovative Development and Utilization of Forest Plant Germplasm, State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangdong Research and Development Center of Modern Agriculture (Woody Forage) Industrial Technology, College of Forestry and Landscape Architecture, Guangdong Province Research Center of Woody Forage Engineering Technology, South China Agricultural University, Guangzhou 510000, China
2
Maoming Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Maoming 525000, China
*
Author to whom correspondence should be addressed.
Fermentation 2023, 9(7), 651; https://doi.org/10.3390/fermentation9070651
Submission received: 22 May 2023
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Revised: 23 June 2023
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Accepted: 30 June 2023
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Published: 11 July 2023
(This article belongs to the Section Industrial Fermentation)
Abstract
:To investigate the feasibility of developing litchi leaves as silage, we determined the fermentation quality of four varieties of litchi leaves (including “Wanpu”, “Wuyejiu”, “Tongzai” and “Zhuangyuanhong”) ensiled with or without Lactobacillus plantarum on day 3, 7, 14 and 30. The in vitro dry matter digestibility and gas production of litchi leaves silages were also determined after 30 days of ensiling. The results showed that Lactobacillus plantarum significantly reduced pH value (p < 0.01), inhibited coliform bacteria, and reduced the production of ammonia nitrogen (p < 0.01) in all the four kinds of litchi leaves silage. Moreover, Lactobacillus plantarum treated litchi leaves (“Wanpu” and “Zhuangyuanhong”) had lower yeasts than the untreated litchi leaves during ensiling. The number of molds in Lactobacillus plantarum treated groups (“Tongzai” and “Zhuangyuanhong”) was below the detected level after 30 days ensiling, which was lower than that of the untreated groups. The addition of Lactobacillus plantarum also contributed to improving IVDMD and markedly reduced (p < 0.01) gas production of all litchi leaves silages. Conclusions: Lactobacillus plantarum can improve the fermentation quality and in vitro digestion characteristics of litchi leaves silage. Developing litchi leaves as silage material is a feasible way to recycle litchi leaves.
1. Introduction
The lack of local forage resources is the primary factor limiting the development of animal husbandry in the tropic [1]. Finding new locally available feed sources, to cope with feed shortages may be one of the effective solutions. Therefore, extensive research was used to estimate the feeding value of natural biomass, and attention has gradually shifted to the processing of woody plant feed [2]. Litchi (Litchi chinensis Sonn.) is a nutrient-rich fruit with high commercial value. It is mainly planted in countries like China, Vietnam, India, and Thailand, with a total planting area of around 800,000 hm2 [3]. Litchi leaf, as a by-product, is abundantly produced when pruning fruit trees every year. These abandoned litchi leaves will not only affect the management of orchards but also breed diseases and pests. A few studies showed that litchi leaves are often used in traditional medicine and tea infusions [4,5]. But the utilization rate of agricultural litchi leaves is not high. In this case, the reasonable utilization of litchi leaves is particularly important to achieve waste recycling and maximum economic benefits. The related research has shown that litchi leaf contains multiple active ingredients (including flavonoid, polysaccharides, phenolic compounds, etc) [6,7]. Due to the presence of those compounds, litchi leaf extract has shown strong anti-inflammatory, bactericidal, and anti-oxidative effects. Previous studies proved that phenolic compounds isolated from litchi leaves have high antioxidant activity and can effectively inhibit the growth of undesirable microorganisms, such as yeasts, coliform bacteria, aspergillus niger, etc [8,9,10]. In addition, Wen et al. (2014) found that flavonoids and sesquilignans isolated from ethyl acetate extract of litchi leaves had antibacterial activity and better antioxidant activity, respectively [9,11]. And Castellain et al. (2014) also found that litchi leaf extract has antinociceptive properties and could relieve the pain of mice [5]. In a word, litchi leaf is a good resource of natural active substances with excellent pharmacological and antibacterial effects. Its extract is also commonly used as feed additives [10]. But there is little research on directly applying litchi leaves as silage.
Although the nutritional components of litchi leaves were also confirmed to be beneficial to agricultural application and production, the litchi leaves as raw materials have not been well recycled [12]. Developing litchi leaf as woody feed may be a good method to improve animal health, enhance the sustainability of feed resources and alleviate the shortage of roughage resources in production. Ensiling, considered a complex process, involves vastly epiphytic microorganisms (mainly lactic acid bacteria) fermentation in fresh forage, which is widely used to preserve nutrient of forage [13]. However, microbial fermentation is usually unstable. It may change the nutritive aspects of forage and lead to poor fermentation quality [14]. Inoculating lactic acid bacteria is conducive to the rapid production of organic acids (such as lactic acid and acetic acid), and then the pH reduction inhibits microbial activities, thereby achieving the preservation of the nutritional of the fresh forage. In particular, Lactobacillus plantarum, as a strain of LAB popularly used as a bacterial additive for ensiling various types of raw materials intended to feeding, has been effectively used to reduce pH and inhibit undesirable microorgansims by rapidly metabolizing water-soluble carbohydrates (WSC) to lactic acid (LA). Thus, we added Lactobacillus plantarum to different varieties of litchi leaves to make litchi leaves silages, and then determined their fermentation quality. The methods of in vitro digestion (IVDMD) and gas production have been widely used to evaluate the nutritional and applied values of ruminant feed. Their analysis contributes to understanding the digestion characteristics of litchi leaves in the rumen. After 30 ensiling, we determined 72 h gas production and 48 h IVDMD of litchi leaves. According to the above results, we evaluated the feasibility of litchi leaves as silage materials.
2. Materials and Methods
2.1. Silage Preparation
In February 2019, four varieties of potted litchi seeding were planted in Litchi Garden of the South China Agricultural University, including “Wanpu”, “Wuyejiu”, “Tongzai” and “Zhuangyuanhong”. A total of twenty trees (five trees per variety) were selected and cultivated at 3 m × 6 m spacing, each in a 2 m × 1.5 m bottomless “soil pit”, 1.5 m deep and lined with plastic film. Irrigation was supplied regularly. Fertilizers (mixed fertilizers and compound fertilizers) were applied around 25 kg/m. The amount of fertilizer application was reduced during the growth period and the application frequency is four times a year according to needs. In September 2021, these trees were pruned by fruit tree administrators. Immediately, we picked the leaves from different parts of a tree. Respectively, different varieties of fresh litchi leaves were crushed into small pieces (2–3 cm) and mixed evenly. Subsequently, litchi leaves were ensiled with (1) no addtive (CK) and (2) 106 colony forming units (CFU)/g of fresh material (FM) Lactobacillus plantarum, respectively. After thorough mixing, about 180 g of each sample was vacuum sealed in a polyethylene plastic bag. A total of 96 bags (4 varieties of litchi leaves × 2 treatments × 4 ensiling time points × 3 replicates) were packed and placed in a large cardboard box (50 cm × 25 cm × 30 cm). Subsequently, they were covered with black film and were conserved at room temperature (26–31 °C). Three bags of each variety were randomly opened for analysis of fermentation characteristics on day 3, 7, 14 and 30.
2.2. Analysis of Microbial Population, Organic Acid and Chemical Composition
The procedures of this section were based on Wang et al. (2019), unless otherwise specified [15]. First all of 20 g silage samples were mixed with 180 mL sterilized saline water, and serially diluted. The lactic acid bacteria and coliform bacteria were cultured on MRS agar plate and VRBA agar plate, respectively; yeasts and molds were counted on Rose Bengal Agar after being incubated at 28 °C for 2 days under aerobic condition [16,17]. To determine fermentation parameters, the sample (20 g) was mixed with 180 mL sterile water and kept at 4 °C overnight and then filtered via filter paper. The filtrate was used to measure pH, the concentrations of ammonia-N and organic acids. A pH meter was used to measure the pH value. The organic acids concentration was measured using HPLC. Ammonia-N was determined using the phenol-hypochlorite colorimetric method [16]. The remaining silages were dried in a forced-air oven at 65 °C for 48 h to determine dry matter (DM) content and then crushed it into particle with a diameter 1.0 mm for chemical analysis. Crude protein and true protein were measured using a Kjeldahl nitrogen analyzer, while non-protein nitrogen was calculated by their difference [18]. After the reaction with anthrone, the content of WSC was determined via colorimetry [19]. In addition, the contents of neutral detergent fiber and acid detergent fiber were analyzed using an A220 fiber analyzer [20].
2.3. In Vitro Dry Matter Digestibility and Gas Production
Rumen inoculum was collected from the rumen of three black Angus cattle (600 kg body weight). The cattle were fed 8 kg DM everyday with a diet containing 2.2 kg silage, 2.2 kg wheat straw, 1 kg bean crud and 4 kg concentrate, and they had free access to water.
In vitro incubations were performed using the Ankom Gas Monitoring System (Ankom Technology, Macedon, NY, UAS) to determine DM digestibility and gas production. Roughly, 0.5 g of each dried sample was mixed with 75 mL of incubation solution (25 mL of rumen fluid was taken from three rumen-fistulated dairy cows + 50 mL of buffer solution) in a glass bottle [21]. After purging by N2 for 5 s, all the bottles were placed at 39 °C, and gas emissions during the whole fermentation process were constantly detected using an automated system. The slurry in each bottle was filtered through a 42-μm-pore-size nylon bag [22]. The in vitro dry matter digestibility (IVDMD) was counted later [23].
2.4. Statistical Analyses
All statistical analyses were completed using SPSS 23.0 software. Two-way analysis of variance and Duncan’s multiple range tests below 5% significance levels were used for comparisons. Figures are exported in Graphpad prism 8.
3. Results
3.1. Characteristics of Fresh Litchi Leaves Prior to Ensiling
Chemical composition and microbial population in different varieties of litchi leaves are presented in Table 1. Among the four varieties of litchi leaves, the DM content of “Wanpu”, “Wuyejiu” and “Tongzai” litchi leaves were close to 50% (49.21%, 49.29%, and 49.87%, respectively), and the DM content of “Wuyejiu” litchi leaves was the highest (49.29%). The crude protein content of “Wanpu”, “Wuyejiu”, “Tongzai”, and “Zhuangyuanhong” litchi leaves was 9.92%, 9.73%, 8.79% and 9.29%, respectively. In this study, the WSC content was low in four kinds of litchi leaves, the range of which was from 1.62% to 1.86%. The microorganisms attached to the surface of litchi leaves were observed. The number of lactic acid bacteria (LAB) was comparable with that of coliform bacteria, which exceeded 4.00 log10 CFU·g−1 FM). It is worth noting that the amounts of yeast and mold were detected between 2 and 3 log10 CFU·g−1 FM.
3.2. Fermentation Quality
Ensiling is an effective method to preserve plant nutrients. The fermentation parameters and nitrogen fractions of different varieties of litchi leaves ensiled with or without Lactobacillus plantarum are shown in Table 2, Table 3, Table 4 and Table 5. The addition of Lactobacillus plantarum significantly reduced (p < 0.01) the pH of litchi leaves silage. The dry matter content of all litchi leaves was stable and did not exhibit a significant difference after 3 and 30 days ensiling. Among the four kinds of litchi leaves silage, lactic acid bacteria with an apparent increase was observed by inoculating Lactobacillus plantarum, while coliform bacteria were the opposite. Moreover, “Wanpu” and “Zhuangyuanhong” litchi leaves treated with Lactobacillus plantarum had lower yeast counts than those of control during ensiling. During anaerobic fermentation, there were almost no yeasts in “Wuyejiu” and “Tongzai” silages. The number of molds in Lactobacillus plantarum-treated groups (“Tongzai” and “Zhuangyuanhong”) was undetected after 30 days ensiling, which was lower than that of the untreated groups. With the prolonged fermentation time, the molds in “Wanpu” silages were reduced, which were invisible after 14 days ensiling. Even they were undetected in “Wuyejiu” silages in the whole process of ensiling. Compared with the control, the addition of Lactobacillus plantarum tended to enhance the contents of lactic acid and acetic acid with prolonged ensiling time. There was no obvious difference in crude protein between the control and Lactobacillus plantarum treated groups in the four kinds of litchi leaves. However, Lactobacillus plantarum application notably reduced (p < 0.01) the content of ammonia nitrogen in all litchi leaves silage significantly.
3.3. In Vitro Dry Matter Digestibility and Gas Production
As described in Figure 1, the in vitro gas production of all litchi leaves silages increased with time. The range of which was 8.10–11.33 mL after 72 h. The untreated “Wanpu” litchi leaves had the highest content of in vitro gas production. On the contrary, the Lactobacillus plantarum treated “Zhuangyuanhong” litchi leaves had the lowest content of in vitro gas production. Four varieties of litchi leaves ensiled with Lactobacillus plantarum were lower than that of the control.
The 48 h in vitro dry matter digestibility of different varieties of litchi leaves silage is presented in Figure 2. Different IVDMD levels can be observed between different varieties of litchi leaves. And the IVDMD of litchi leaves was 11.14~13.17% in this study. Among all treatments, the “Wanpu” litchi leaves silage inoculated with Lactobacillus plantarum had the highest IVDMD (13.17%). Compared with the control, the application of Lactobacillus plantarum markedly increased the IVDMD of litchi leaves silage.
4. Discussion
4.1. Characteristics of Fresh Litchi Leaves
The DM content of raw materials is an important index [24,25]. The reason is that it affects fermentation characteristics, pH value, and microbial structure [26]. In general, the ideal range is 30–40% FM, which can keep the vigorous growth of LAB and prevent undesirable microorganisms (clostridial or yeasts) fermentation [27]. The DM content of litchi leaves is relatively higher than that of ideal value which might make it difficult to avoid the nutrient loss caused by yeast metabolism at the initial stage of fermentation. However, to the best of our knowledge, high DM content contributes to nitrogen conversion during forage fermentation. Muck et al. (1987) investigated the relationship between dry matter content and protein distribution in alfalfa silage, and found that the hydrolysis rate of protein decreased linearly with the increase in DM content [28]. Therefore, protein preservation of litchi leaves may be related to their DM content. Although the CP content of litchi leaves was lower than that of alfalf, the protein preservation of litchi leaves may be also related to their DM content [29]. To note, corn is the most widely planted silage material in the world, and its protein content typically is lower than 9% DM [25,30]. The protein content of litchi leaves was higher than that of corn, which indicated that litchi leaves may be suitable as an unconventional feed. Silage is a microbial fermentation product. Epiphytic LAB is essential for spontaneous fermentation [31,32]. It produces amounts of organic acid (mainly lactic acid) by transforming water-soluble carbohydrates (WSC) and then accelerates acidification to inhibit microbial activities [33]. However, untreated litchi leaves may be hard to obtain good fermentation quality. On the one hand, LAB was below 5 log10 CFU·g−1 FM in litchi leaves except for “Tongzai”. Coliform bacteria, meanwhile, were high in number. On the other hand, although the WSC content was different among the litchi leaves, all of them cannot meet the basic requirement (>38 g/kg) [27]. Therefore, effective additives are needed to promote lactic acid bacteria fermentation and reduce nutrient losses.
4.2. Fermentation Quality
Although the chemical components of litchi leaves (neutral detergent fiber, acid detergent fiber and crude protein) has only changed a little before and after ensiling, the other indices in different treatments had obviously differed after ensiling. Silage pH is a basic index to evaluate the fermentation quality of silage [34]. After ensiling 30 days, pH values were >4.2 for all treatments under 40% DM content. The pH of all treatments was more than 4.7, which might be related with the low WSC content and high buffer capacity of litchi leaves. Notably, the addition of Lactobacillus plantarum significantly reduced the pH of litchi leaves silage, which was in accordance with the abundant accumulation of organic acid content. Lactic acid and acetic acid, as key metabolites of lactic acid bacteria, can rapidly acidize environment and inhibit the activities of microorganisms [35]. The popular narrative is that the application of Lactobacillus plantarum is conducive to lactic acid bacteria quickly occupying the dominant position with the competition of undesirable microorganisms in the early fermentation stage and accelerating organic acid product, thereby reaching a bacteriostatic effect [36]. Compared with control groups, LAB became the predominant bacteria in Lactobacillus plantarum groups, while coliform bacteria were inhibited. Moreover, the addition of Lactobacillus plantarum also restricted the growth of yeasts in litchi leaves (“Wanpu”, “Wuyejiu”, and “Zhuangyuanhong”) silages during ensiling. As for “Tongzai”, the count of yeasts still remained below 2 log10 CFU/g−1 FM during the whole ensiling period, regardless of whether Lactobacillus plantarum was added or not. It might be because that “Tongzai” had active substances with antibacterial effect. It is worth mentioning that the number of molds in all treatments was still maintained at a low level with the prolonged fermentation time. And the application of Lactobacillus plantarum in litchi leaves (“Wanpu”, “Tongzai”, and “Zhuangyuanhong”) tended to inhibit the molds after 30 days ensiling. The above-mentioned microorganisms change reasonably explained the more effective preservation of nutrients and protein in all Lactobacillus plantarum treated litchi leaves than that control groups. As is known, the content of ammonia-N is influenced by the activities of undesirable microorganisms during ensiling process [37]. Although ruminants could utilize ammonia-N in the rumen, the utilization efficiency is low and the accumulation of ammonia-N is generally considered as the indicator of protein degradation. Therefore, ammonia-N is usually recognized as undesirable in silage on livestock production and animal excretion of ammonia-N will also negatively influence the environment [38]. However, the content of ammonia-N is influenced by the activities of undesirable microorganisms. Inoculation of Lactobacillus plantarum could reduce the content of ammonia nitrogen in all litchi leaves silage, which was consistent with the decline of coliform bacteria count. The above results indicate that Lactobacillus plantarum played a crucial role in decreasing nutrition loss and inhibiting undesirable microorgansims, thereby improving fermentation quality of all litchi leaves silage.
4.3. In Vitro Dry Matter Digestibility and Gas Production
Gas production and in vitro dry matter digestibility can effectively simulate the dynamic fermentation process of feed in rumen, which is widely used to determine the nutritional value of silage because of its convenience and representativeness [39]. Fermentation gas is produced by rumen microorganisms using various nutrients. So, it is generally related to the level of nutrients in the rumen and the composition of the diet. In this study, there was a difference in GP72 (cumulative gas production at 72 h) among different varieties of litchi leaves silage. This might be related to the chemical composition of litchi leaves. Lactobacillus plantarum had positive effects on GP72 of all litchi leaves silage, and it might reduce the degradation of DM fractions and nutrients [14]. To improve the fermentation quality of silage, preparation of feed is often required to reduce the gas production. On one hand, gas production means the increased nutrient loss during ensiling. On the other hand, the main components of gas production were H2, CO2, and CH4. The emission of these gas may aggravate the greenhouse effect, which is detrimental to the sustainable development of the ecological environment [40]. Therefore, low gas production is desirable.
In addition, Lactobacillus plantarum played an essential role in improving the IVDMD of all litchi leaves. A potential explanation was that the application of Lactobacillus plantarum promoted the enzymatic saccharification reaction, where sugars and proteins were enzymatically generated to glycoproteins, increasing the rumen-fermentable fraction of silage. Therefore, Lactobacillus plantarum is considered as an effective additive to increase the IVDMD of silage. The study of Yi et al. (2023) supports this result [41,42].
5. Conclusions
This study shows that ensiling is an effective method for preservation of litchi leaves. The addition of Lactobacillus plantarum significantly reduced pH value, inhibited harmful microorganisms (coliform bacteria), and reduced the production of ammonia nitrogen in litchi leaves silage. The number of molds in Lactobacillus plantarum-treated groups (“Tongzai” and “Zhuangyuanhong”) was undetected after 30 days ensiling, which was lower than that of the untreated groups. With the prolonged fermentation time, the molds in “Wanpu” silages were reduced, which were invisible after 14 days ensiling. Moreover, Lactobacillus plantarum can improve the in vitro dry matter digestibility and markedly reduce gas production of litchi leaves silage. In the future, utilization of litchi leaves can be improved by using it for mixed silage or for extracting effective components that can be used as a feed additive.
Author Contributions
D.C.: Investigation, Data curation, Writing original draft. Y.Z.: Investigation, Data curation. D.Y.: Software. W.Z.: Investigation, Data curation and Software. X.C.: Supervision, Validation. Q.Z.: Methodology, Project administration. All authors have read and agreed to the published version of the manuscript.
Funding
The Central Government Guides Local Funds for Science and Technology Development–Basic Research Projects with Free Exploration (2022ZY0152). Independent Research and Development Projects of Maoming Laboratory (Grant No. 2022ZD002); Guangdong Provincial Science and Technology Special Foundation (210723106900762 and 2021020103-2).
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
Not applicable.
Acknowledgments
This work was financially supported by The Central Government Guides Local Funds for Science and Technology Development–Basic Research Projects with Free Exploration (2022ZY0152), Independent Research and Development Projects of Maoming Laboratory (Grant No. 2022ZD002), Guangdong Provincial Science and Technology Special Foundation (210723106900762 and 2021020103-2).
Conflicts of Interest
The authors declare no confict of interest.
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Figure 1.
In vitro gas production of different varieties of litchi leaves ensiled with or without Lactobacillus plantarum in 72 h.
Figure 1.
In vitro gas production of different varieties of litchi leaves ensiled with or without Lactobacillus plantarum in 72 h.
Figure 2.
In vitro dry matter digestibility of different varieties of litchi leaves ensiled with or without Lactobacillus plantarum in 48 h. a and b, the differences between Control and Lactobacillus plantarum of the same variety of litchi leaves.
Figure 2.
In vitro dry matter digestibility of different varieties of litchi leaves ensiled with or without Lactobacillus plantarum in 48 h. a and b, the differences between Control and Lactobacillus plantarum of the same variety of litchi leaves.
Table 1.
Chemical composition and microorganisms counts in leaves of different varieties of Litchi (±SD, n = 3).
Table 1.
Chemical composition and microorganisms counts in leaves of different varieties of Litchi (±SD, n = 3).
| Item | Wanpu | Wuyejiu | Tongzai | Zhuangyuanhong |
|---|---|---|---|---|
| Dry matter (%FM) | 49.21 ± 0.27 | 49.29 ± 0.19 | 49.87 ± 0.12 | 44.96 ± 0.25 |
| Crude protein ( % DM) | 9.92 ± 0.25 | 9.73 ± 0.23 | 8.79 ± 1.14 | 9.29 ± 0.40 |
| Neutral detergent fiber (% DM) | 64.89 ± 1.60 | 63.36 ± 0.64 | 66.14 ± 3.63 | 59.54 ± 0.12 |
| Acid detergent fiber (% DM) | 51.21 ± 2.41 | 51.27 ± 2.71 | 52.24 ± 2.35 | 46.36 ± 1.19 |
| Water soluble carbohydrate (% DM) | 1.62 ± 0.15 | 1.72 ± 0.12 | 1.67 ± 0.04 | 1.86 ± 0.06 |
| Lactic acid bacteria (LAB, log10 CFU·g−1 FM) | 4.69 ± 0.24 | 4.67 ± 0.42 | 5.11 ± 0.74 | 4.88 ± 0.37 |
| Yeasts (log10 CFU·g−1 FM) | 2.92 ± 0.26 | 2.15 ± 0.21 | 2.49 ± 0.20 | 2.42 ± 0.10 |
| Molds (log10 CFU·g−1 FM) | 2.63 ± 0.31 | 2.30 ± 0.30 | 2.36 ± 0.39 | 2.46 ± 0.41 |
| Coliform bacteria (CB, log10 CFU·g−1 FM) | 4.53 ± 0.63 | 4.26 ± 0.94 | 4.75 ± 0.16 | 4.44 ± 0.11 |
FM, fresh matter; CFU, colony forming units.
Table 2.
Fermentation quality of “Wanpu” litchi leaves ensiled with or without Lactobacillus plantarum on day 3, 7, 14 and 30.
Table 2.
Fermentation quality of “Wanpu” litchi leaves ensiled with or without Lactobacillus plantarum on day 3, 7, 14 and 30.
| Items | Treatment | Day | SEM | p-Value | |||||
|---|---|---|---|---|---|---|---|---|---|
| D3 | D7 | D14 | D30 | T | D | T*D | |||
| pH value | CK | 5.71 a | 5.60 abA | 5.47 abA | 5.39 bA | 0.05 | ** | ** | NS |
| LP | 5.18 a | 4.99 bB | 4.97 bB | 4.93 bB | 0.03 | ||||
| DM(% FM) | CK | 48.4 ab | 48.0 ab | 47.8 b | 49.0 a | 0.20 | * | * | NS |
| LP | 48.3 b | 48.9 ab | 48.6 ab | 49.3 a | 0.14 | ||||
| LAB (log10 CFU·g−1 FM) | CK | 7.87 bB | 8.42 aB | 7.49 cB | 7.88 b | 0.10 | ** | ** | ** |
| LP | 8.57 bA | 9.06 aA | 8.54 bA | 8.08 c | 0.11 | ||||
| Yeasts (log10 CFU·g−1 FM) | CK | 2.60 | 2.50 | 2.41 | 2.15 | 0.55 | ND | ND | ND |
| LP | 2.45 | 2.15 | ND | ND | 0.12 | ||||
| Molds (log10 CFU·g−1 FM) | CK | 2.50 | 2.40 | ND | ND | 0.13 | ND | ND | ND |
| LP | 2.57 | 2.26 | ND | ND | 0.14 | ||||
| Coliform bacteria (log10 CFU·g−1 FM) | CK | 7.38 abA | 7.72 aA | 7.20 bA | 7.18 bA | 0.09 | ** | * | NS |
| LP | 6.66 B | 6.58 B | 6.08 B | 6.22 B | 0.11 | ||||
| Lactic acid (%DM) | CK | 1.57 | 0.71 | 0.51 | 0.39 | 0.21 | NS | NS | NS |
| LP | 1.84 | 1.87 | 1.40 | 1.00 | 0.30 | ||||
| Acetic acid (%DM) | CK | ND | ND | ND | 0.11 | ND | ND | ND | ND |
| LP | 0.11 | 0.09 | 0.10 | 0.15 | 0.01 | ||||
| Crude protein ( %DM) | CK | 10.00 ab | 10.00 ab | 10.70 a | 9.55 b | 0.18 | * | NS | NS |
| LP | 10.10 | 10.00 | 10.30 | 10.00 | 0.12 | ||||
| Ammonia-N (%TN) | CK | 0.29 b | 0.55 b | 0.74 b | 1.99 aA | 0.20 | ** | ** | ** |
| LP | 0.25 c | 0.40 c | 0.69 b | 0.95 aB | 0.09 | ||||
Means in the same row (a–c) or column (A–B) followed by different letters differ; CK, control; LP: Lactobacillus plantarum treatment; **, p < 0.01; *, p < 0.05 (differ significantly); NS, not significant; ND, not detect; TN, total nitrogen; D, ensiling days effect; T, treatments effect; D*T, the interaction effect of treatments and ensiling days; CFU, colony forming units; FM, fresh matter; SEM, standard error of the mean.
Table 3.
Silage fermentation quality of “Wuyejiu” litchi leaves ensiled with or without Lactobacillus plantarum on day 3, 7, 14 and 30.
Table 3.
Silage fermentation quality of “Wuyejiu” litchi leaves ensiled with or without Lactobacillus plantarum on day 3, 7, 14 and 30.
| Items | Treatment | Day | SEM | p-Value | |||||
|---|---|---|---|---|---|---|---|---|---|
| D3 | D7 | D14 | D30 | T | D | T*D | |||
| pH value | CK | 5.75 bA | 5.75 bA | 5.89 aA | 5.69 bA | 0.02 | ** | ** | ** |
| LP | 5.20 aB | 5.03 bB | 5.00 bB | 4.82 cB | 0.04 | ||||
| DM (%FM) | CK | 48.3 | 47.6 | 47.4 | 48.1 | 0.15 | * | NS | NS |
| LP | 48.7 | 48.9 | 48.0 | 48.5 | 0.21 | ||||
| LAB (log10 CFU·g−1 FM) | CK | 7.48 bB | 8.10 aB | 7.96 a | 7.40 b | 0.10 | ** | ** | ** |
| LP | 8.47 aA | 8.71 aA | 7.87 b | 7.85 b | 0.13 | ||||
| Yeasts (log10 CFU·g−1 FM) | CK | 2.44 | ND | ND | ND | ND | ND | ND | ND |
| LP | ND | ND | ND | ND | ND | ||||
| Molds (log10 CFU·g−1 FM) | CK | ND | ND | ND | ND | ND | ND | ND | ND |
| LP | ND | ND | ND | ND | ND | ||||
| Coliform bacteria (CB, log10 CFU·g−1 FM) | CK | 7.45 bA | 7.81 aA | 7.50 bA | 7.49 bA | 0.05 | ** | ** | ** |
| LP | 6.54 aB | 6.10 abB | 5.79 bB | 5.07 cB | 0.17 | ||||
| Lactic acid (LA, %DM) | CK | 1.16 | 0.59 a | 0.60 | 0.52 B | 0.11 | ** | NS | ** |
| LP | 0.65 b | 1.57 ab | 1.23 ab | 2.12 aA | 0.20 | ||||
| Acetic acid (AA,%DM) | CK | 0.08 | ND | ND | ND | ND | ND | ND | ND |
| LP | ND | 0.09 ab | 0.06 b | 0.12 a | 0.01 | ||||
| Crude protein (CP, %DM) | CK | 9.51 | 10.10 | 9.74 | 9.31 | 0.17 | NS | NS | NS |
| LP | 9.65 | 10.00 | 10.30 | 9.21 | 0.18 | ||||
| Ammonia-N (%TN) | CK | 0.34 c | 0.36 cB | 0.70 b | 1.50 aA | 0.14 | ** | ** | ** |
| LP | 0.41 b | 0.47 bB | 0.54 ab | 0.71 aB | |||||
Means in the same row (a–c) or column (A–B) followed by different letters differ; CK, control; LP: Lactobacillus plantarum treatment; **, p < 0.01; *, p < 0.05 (differ significantly); NS, not significant; ND, not detect; TN, total nitrogen; D, ensiling days effect; T, treatments effect; D*T, the interaction effect of treatments and ensiling days; CFU, colony forming units; FM, fresh matter; SEM, standard error of the mean.
Table 4.
Silage fermentation quality of “Tongzai” litchi leaves ensiled with or without Lactobacillus plantarum on day 3, 7, 14 and 30.
Table 4.
Silage fermentation quality of “Tongzai” litchi leaves ensiled with or without Lactobacillus plantarum on day 3, 7, 14 and 30.
| Items | Treatment | Day | SEM | p-Value | |||||
|---|---|---|---|---|---|---|---|---|---|
| D3 | D7 | D14 | D30 | T | D | T*D | |||
| pH value | CK | 5.54 bA | 5.64 a | 5.57 abA | 5.42 cA | 0.03 | ** | ** | * |
| LP | 5.24 bB | 5.55 a | 5.00 bB | 4.96 bB | 0.08 | ||||
| DM (%FM) | CK | 49.1 | 48.4 | 48.6 | 49.2 | 0.18 | NS | * | NS |
| LP | 49.6 a | 48.5 b | 48.8 b | 49.0 ab | 0.16 | ||||
| LAB (log10 CFU·g−1 FM) | CK | 7.68 bB | 8.15 aB | 7.80 bB | 7.24 cB | 0.11 | ** | ** | NS |
| LP | 8.45 bA | 8.88 aA | 8.33 bA | 7.80 cA | 0.12 | ||||
| Yeasts (log10 CFU·g−1 FM) | CK | ND | ND | ND | ND | ND | ND | ND | ND |
| LP | ND | ND | ND | ND | ND | ||||
| Molds (log10 CFU·g−1 FM) | CK | ND | ND | ND | 3.15 | ND | ND | ND | ND |
| LP | ND | ND | ND | ND | ND | ||||
| Coliform bacteria (CB, log10 CFU·g−1 FM) | CK | 7.82 a | 7.73 a | 7.64 aA | 7.09 bA | 0.10 | ** | ** | * |
| LP | 7.46 a | 7.59 a | 6.65 bB | 5.91 bB | 0.23 | ||||
| Lactic acid (LA, %DM) | CK | 1.58 a | 1.29 abB | 0.69 ab | 0.37 b | 0.20 | ** | NS | NS |
| LP | 3.07 | 4.70 A | 2.32 | 2.11 | 0.59 | ||||
| Acetic acid (AA,%DM) | CK | ND | ND | ND | 0.06 | ND | NS | NS | NS |
| LP | 0.13 | 0.11 | 0.10 | 0.07 | 0.02 | ||||
| Crude protein (CP, %DM) | CK | 9.02 | 8.68 | 8.45 | 9.00 | 0.16 | NS | NS | NS |
| LP | 9.04 | 8.45 | 9.21 | 9.29 | 0.15 | ||||
| Ammonia-N (%TN) | CK | 0.34 c | 0.42 c | 0.73 b | 1.16 aA | 0.10 | NS | ** | * |
| LP | 0.32 b | 0.66 a | 0.73 a | 0.80 aB | 0.07 | ||||
Means in the same row (a–c) or column (A–B) followed by different letters differ; CK, control; LP: Lactobacillus plantarum treatment; **, p < 0.01; *, p < 0.05 (differ significantly); NS, not significant; ND, not detect; TN, total nitrogen; D, ensiling days effect; T, treatments effect; D*T, the interaction effect of treatments and ensiling days; CFU, colony forming units; FM, fresh matter; SEM, standard error of the mean.
Table 5.
Silage fermentation quality of “Zhuangyuanhong” litchi leaves ensiled with or without Lactobacillus plantarum on day 3, 7, 14 and 30.
Table 5.
Silage fermentation quality of “Zhuangyuanhong” litchi leaves ensiled with or without Lactobacillus plantarum on day 3, 7, 14 and 30.
| Items | Treatment | Day | SEM | p-Value | |||||
|---|---|---|---|---|---|---|---|---|---|
| D3 | D7 | D14 | D30 | T | D | T*D | |||
| pH value | CK | 5.82 bA | 6.64 aA | 5.65 cA | 5.53 dA | 0.13 | ** | ** | NS |
| LP | 5.05 bB | 5.66 aB | 4.88 bB | 4.87 bB | 0.10 | ||||
| DM (% FM) | CK | 44.4 a | 43.8 b | 44.1 ab | 44.7 a | 0.12 | ** | ** | NS |
| LP | 44.6 ab | 44.4 b | 44.3 b | 45.0 a | 0.11 | ||||
| LAB (log10 CFU·g−1 FM) | CK | 7.72 bB | 8.24 aB | 7.68 bB | 7.08 cB | 0.13 | ** | ** | NS |
| LP | 8.64 bA | 8.99 aA | 8.51 bA | 7.88 cA | 0.13 | ||||
| Yeasts (log10 CFU·g−1 FM) | CK | 3.13 | 3.06 | 2.38 | 2.66 | 0.14 | ND | ND | ND |
| LP | 2.64 | ND | ND | ND | ND | ||||
| Molds (log10 CFU·g−1 FM) | CK | ND | ND | ND | 2.15 | ND | ND | ND | ND |
| LP | ND | ND | ND | ND | ND | ||||
| Coliform bacteria (CB, log10 CFU·g−1 FM) | CK | 7.75 aA | 7.95 aA | 7.69 aA | 6.76 bA | 0.15 | ** | ** | * |
| LP | 6.81 aB | 6.60 aB | 5.76 bB | 5.21 bB | 0.21 | ||||
| Lactic acid (LA, %DM) | CK | 0.86 | 0.13 cB | 0.58 b | 0.49 | 0.15 | NS | * | NS |
| LP | 0.27 c | 0.65 A | 0.82 | 1.32 a | 0.15 | ||||
| Acetic acid (AA,%DM) | CK | 0.06 | 0.10 A | 0.09 | 0.08 | 0.01 | NS | NS | NS |
| LP | ND | 0.04 bB | 0.12 a | 0.09 ab | 0.01 | ||||
| Crude protein (CP, %DM) | CK | 8.26 | 9.12 | 8.65 | 8.83 | 0.27 | NS | NS | NS |
| LP | 9.03 | 8.99 | 9.16 | 9.09 | 0.14 | ||||
| Ammonia-N (%TN) | CK | 0.29 c | 0.57 b | 0.59 b | 1.12 aA | 0.34 | * | ** | NS |
| LP | 0.33 b | 0.49 ab | 0.38 ab | 0.69 aB | 0.06 | ||||
Means in the same row (a–c) or column (A–B) followed by different letters differ; CK, control; LP: Lactobacillus plantarum treatment; **, p < 0.01; *, p < 0.05 (differ significantly); NS, not significant; ND, not detect; TN, total nitrogen; D, ensiling days effect; T, treatments effect; D*T, the interaction effect of treatments and ensiling days; CFU, colony forming units; FM, fresh matter; SEM, standard error of the mean.
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Chen, D.; Zhou, Y.; Yang, D.; Zhou, W.; Chen, X.; Zhang, Q. Exploring Lactobacillus plantarum on Fermentation Quality, Gas Emissions, and In Vitro Digestibility of Different Varieties of Litchi Leaves Silage. Fermentation 2023, 9, 651. https://doi.org/10.3390/fermentation9070651
AMA Style
Chen D, Zhou Y, Yang D, Zhou W, Chen X, Zhang Q. Exploring Lactobacillus plantarum on Fermentation Quality, Gas Emissions, and In Vitro Digestibility of Different Varieties of Litchi Leaves Silage. Fermentation. 2023; 9(7):651. https://doi.org/10.3390/fermentation9070651
Chicago/Turabian StyleChen, Dandan, Yuxin Zhou, Dan Yang, Wei Zhou, Xiaoyang Chen, and Qing Zhang. 2023. "Exploring Lactobacillus plantarum on Fermentation Quality, Gas Emissions, and In Vitro Digestibility of Different Varieties of Litchi Leaves Silage" Fermentation 9, no. 7: 651. https://doi.org/10.3390/fermentation9070651
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