Study on the bacterial community structure and fermentation characteristics of fresh and ensiled paper mulberry

Two experiments were conducted to investigate the bacterial community of fresh and ensiled paper mulberry prepared with or without lactic acid bacteria (LAB) inoculants in South China. In Experiment 1, the bacterial community, chemical composition, and fermentation products of paper mulberry were analyzed. The results showed that fresh paper mulberry had high crude protein content, buffering capacity value, and amounts of uncultured bacteria. Ensiled paper mulberry showed poor fermentation with high pH value, ammonia–N content, and butyric acid content. In addition, Enterobacter was the dominant genus in silage, followed by Lactobacillus and Enterococcus. Water-soluble carbohydrates, ammonia–N, propionic acid, pH, and lactic acid (LA) were the main factors affecting bacterial community of silage. In Experiment 2, the BP17 (Lactobacillus plantarum) from natural fermented paper mulberry silage and two commercial inoculants (Silage-help [SH] and Chikuso-1 [CH]) were used as additives. Compared with other treatments, BP17 inoculant decreased (p < 0.05) pH and ammonia–N content and increased (p < 0.05) LA content of silage. Inoculation of BP17 also increased the dominance of desirable Lactobacillus and inhibited the growth of harmful bacteria in silage. These results confirmed that paper mulberry could be ensiled and epiphytic LAB inoculant can improve its fermentation quality.     

Fermentation quality and chemical composition of shrub silage treated with lactic acid bacteria inoculants and cellulase additives

Effects of lactic acid bacteria (LAB) inoculants and cellulase additives on fermentation quality and chemical compositions of shrub silages were studied by using a small‐scale fermentation system. Two LAB inoculants of Qingbao (Lactobacillus plantarum, Pediococcus acidilacticii, Lactobacillus casei and Clostridium phage) and Caihe (Lactobacillus plantarum, Lactobacillus brevis and Pediococcus acidilactici) and a commercial cellulase made from Trichoderma reesei were used as additives for intermediate pea‐shrub, rush bushclover, arborescent ceratoides and shrubby silage preparation. The crude protein, neutral detergent fiber and water‐soluble carbohydrate contents of the four shrub materials were 10.1–14.2, 62.6–67.2 and 1.9–3.5% on a dry matter basis, respectively. All shrub silages had pH 3.40–4.43, ammonia‐N 0.1–0.2% g/kg and lactic acid 1.3–2.9% on a fresh matter basis. The silage quality of LAB‐inoculated silages did not have a greater effect than control silages, except shrubby silage preparation. Silages treated with the cellulase, the pH of rush bushclover and shrubby sweetvetch silage were significantly (P < 0.05) lower and the lactic acid content were significantly (P < 0.05) higher than the control silages. The results confirmed that shrub contained a relatively high content of crude protein; its silages can be preserved in good quality, and they are new potential resources for livestock feed.     

Characteristics of isolated lactic acid bacteria and their effectiveness to improve stylo (Stylosanthes guianensis Sw.) silage quality at various temperatures

Two lactic acid bacteria (LAB) strains, Pediococcus pentosaceus SC1 and Lactobacillus paraplantarum SC2 isolated from king grass silage, were characterized and their effectiveness to improve the silage fermentation quality of stylo (Stylosanthes guianensis Sw.) was studied. Strain SC1 was able to grow at a high temperature of 45°C, while SC2 did not. SC2 normally grew at a low pH of 4.0, while SC1 could not. These two strains and a commercial inoculant of LAB (L. plantarum, LP) were used as additives to stylo silage preparation at various temperatures (20°C, 30°C and 40°C). All LAB inoculants significantly (P < 0.05) reduced the pH value and ammonia-N content, and increased the ratio of lactic acid to acetic acid and quality score compared with the control. In addition, inoculating LAB strains markedly (P < 0.05) reduced butyric acid content at the temperatures of 30°C and 40°C. Compared to SC2 and LP strains, strain SC1 was the most effective for improving stylo silage quality at 20°C, indicated by the increase in lactic acid, ratio of lactic acid to acetic acid and quality score. At 30°C and 40°C, there were no significant differences among SC1, SC2 and LP treatments in pH values, contents of acetic acid, butyric acid and ammonia-N (P > 0.05).     

Silage preparation and fermentation quality of kudzu,sugarcane top and their mixture treated with lactic acid bacteria,molasses and cellulase

We studied silage fermentation of kudzu (KZ), sugarcane top (ST) and their mixtures treated with additives to be able to effectively use available local feed resources. The silages were prepared using KZ, ST, KZ 90% + ST 10%, KZ 80% + ST 20%, KZ 70% + ST 30% and KZ 60% + ST 40%, based on fresh matter (FM). These silages were treated with 10⁸ colony‐forming units/g epiphytic lactic acid bacteria (LAB), 5% molasses and 0.02% cellulase of FM. The KZ contained higher crude protein (CP; 14.52%) content and lower levels of neutral detergent fiber (NDF; 62.15%) than those of ST (6.84% CP and 64.93% NDF) based on dry matter (DM). The KZ 60% + ST 40% silage fermented well with a higher (P < 0.05) lactic acid content and lower (P < 0.05) pH than those of the other mixed silages. Silages treated with molasses had lower (P < 0.05) ammonia‐N, NDF, acid detergent fiber, and hemi‐cellulose contents as well as pH, but higher (P < 0.05) DM, water‐soluble carbohydrate, and lactic acid contents, than those of the control, LAB and cellulase treatments. The results confirmed that KZ 60% + ST 40% was the best mixing ratio to prepare silage, and molasses improved silage fermentation and fiber degradation.     

添加糖蜜与乳酸菌对燕麦秸杆和黑麦草混合青贮品质的影响

为探讨西藏地区燕麦（Avena sativa L.）秸秆和多年生黑麦草（Lolium perenne L.）混合青贮使用添加剂改善发酵品质的措施,进一步提高燕麦秸秆利用效率,将收获籽粒后的燕麦秸秆和抽穗期刈割的多年生黑麦草以4∶6混合青贮,分别添加1×10⁶ cfu·g^-1 FW的乳酸菌制剂、4% FW糖蜜和乳酸菌+糖蜜,青贮60 d后取样分析青贮饲料发酵品质。结果表明：单独或组合添加糖蜜和乳酸菌均不同程度的改善了混合青贮发酵品质,显著降低了pH值、氨态氮/总氮、丙酸和丁酸含量（P<0.05）,显著提高了乳酸含量和乳酸/乙酸（P<0.05）。添加糖蜜组发酵品质优于添加乳酸菌组,显示较高的乳酸和水溶性碳水化合物含量（P<0.05）,且单独添加糖蜜与组合添加组各发酵指标无显著差异。综合考虑,在燕麦秸秆和多年生黑麦草（4∶6）混合青贮时添加4%糖蜜较为适宜。     

糖蜜和乳酸菌对去穗玉米秸秆青贮发酵品质的影响

为研究糖蜜和乳酸菌对去穗玉米（Zea mays L.）秸秆青贮饲料发酵品质的影响,试验设对照（C）、糖蜜（M）、乳酸菌（LAB）、糖蜜+乳酸菌（M+LAB）4个处理,发酵60天后测定发酵品质。结果表明：添加糖蜜或乳酸菌均提高了去穗玉米秸秆青贮饲料的发酵品质,M+LAB组的V-Score评分最高。因此,在去穗玉米秸秆青贮实际生产过程中,建议使用糖蜜和乳酸菌组合添加剂。     

Effect of grape pomace on fermentation quality and aerobic stability of sweet sorghum silage

The objective of this study was to evaluate the effect of grape pomace (GP) with different adding levels (0%, 5%, 10% and 15%, fresh matter basis), alone (GP‐LAB) or in combine with an inoculant LAB (GP+LAB), on the fermentation quality and aerobic stability of sweet sorghum silage. After 90 days of ensiling in vacuumized mini‐silos, silages were subject to a 7‐day aerobic stability test, in which chemical, microbial and polyphenol composition were measured. In the GP‐LAB group, adding GP decreased (P < 0.05) concentrations of water‐soluble carbohydrate (WSC) and butyric acid in silage. In the GP+LAB group, adding GP increased (P < 0.05) concentrations of lactic acid, WSC and crude protein, decreased (P < 0.05) final pH value, NH₃‐N ratio and butyric acid concentration in silage. Polyphenol level was reduced (P < 0.05) after silage fermentation. During aerobic exposure, the fungi count, pH value and silage temperature increased (P < 0.05), the levels of lactic acid, acetic acid and polyphenols (quercetin 3‐O‐glucoside and quercetin 3‐O‐glucuronid) decreased (P < 0.05) in silage. GP+LAB treated silage had a lag phase for aerobic spoilage. When the fermentation products, microbial counts, chemical and polyphenol composition were considered, the use of 10% GP+LAB at ensiling could provide a valuable source for improved fermentation quality and aerobic stability of sweet sorghum silage.     

乳酸菌和糖蜜对构树叶青贮品质和细菌多样性的影响

为改善构树(Broussonetia papyrifera)叶青贮发酵品质,探究乳酸菌和糖蜜对构树叶青贮发酵指标和细菌多样性的影响,本实验以构树叶为原料,设置对照、添加乳酸菌(L)、糖蜜(M)和乳酸菌+糖蜜混合(ML)4个处理,贮藏30天后,测定其营养成分、发酵品质并及细菌群落结构。结果显示,除了添加乳酸菌组,其他添加剂处理pH值均显著降低(P<0.05),乳酸含量提高(P<0.05),乙酸(P<0.05)和丙酸含量下降。微生物分析表明与CK组相比,M和ML组均提高了细菌群落多样性,另外,添加剂处理改变了细菌群落结构,与CK相比,糖蜜处理组(M)乳杆菌属(Lactobacillus)相对丰度显著提升,魏斯氏菌属(Weissella)和肠杆菌属(Enterobacter)相对丰度降低。综上,构树叶青贮添加糖蜜可提高青贮饲料的发酵品质,并改变青贮细菌多样性。     

Microbial population,chemical composition and silage fermentation of cassava residues

In order to effectively use the cassava (Manihot esculenta Crantz) residues, including cassava leaves, peel and pulp for livestock diets, the chemical and microbiological composition, silage preparation and the effects of lactic acid bacteria (LAB) inoculants on silage fermentation of cassava residues were studied. These residues contained 10⁴ to 10⁵ LAB and yeasts, 10³ to 10⁴ coliform bacteria and 10⁴ aerobic bacteria in colony forming units (cfu) on a fresh matter (FM) basis. The molds were consistently at or below the detectable level (10² cfu of FM) in three kinds of cassava residues. Dry matter (DM), crude protein (CP) and neutral detergent fiber (NDF) content of cassava residues were 17.50‐30.95%, 1.30‐16.41% and 25.40–52.90% on a DM basis, respectively. The silage treatments were designed as control silage without additive (CO) or with LAB inoculants Chikuso‐1 (CH, Lactobacillus plantarum) and Snow Lacto (SN, Lactobacillus rhamnosus) at a rate of 5 mg/kg of FM basis. All silages were well preserved with a low pH (below 4.0) value and when cassava residues silage treated with inoculants CH and SN improved fermentation quality with a lower pH, butyric acid and higher lactic acid than control silage.     

添加乳酸菌、糖蜜和无机酸对羊草青贮饲料发酵品质及体外干物质消失率的影响

本试验旨在探讨添加乳酸菌、糖蜜和无机酸对羊草青贮饲料发酵品质及体外干物质消失率的影响.采用单因子完全随机设计,在羊草青贮时设置如下5个处理:1)无添加(对照组,C组);2)添加0.00025％乳酸菌(LAB组);3)添加4％糖蜜(M组);4)添加4％无机酸(IA组);5)添加0.00025％乳酸菌+4％糖蜜(LAB+M...     

Effects of chlorpyrifos and chlorantraniliprole on fermentation quality of alfalfa (Medicago sativa L.) silage inoculated with or without Lactobacillus plantarum LP

The effects of pesticides and Lactobacillus plantarum (LP) on fermentation quality of alfalfa (Medicago sativa L.) silage were investigated. Chlorpyrifos and chlorantraniliprole were sprayed on the surface of alfalfa plants at 658.6 and 45.0 g active ingredient/ha, respectively. Alfalfa plants were harvested on day 5 post‐application and ensiled with or without LP. Chlorpyrifos and chlorantraniliprole decreased the yeast count of alfalfa material (P < 0.05). Both pesticides increased the butyric acid content of alfalfa silage (P < 0.001). Chlorpyrifos increased pH and decreased lactic acid, acetic acid and short‐chain fatty acid contents (P < 0.05). LP decreased pH and butyric acid content, and increased lactic acid and short‐chain fatty acid contents of alfalfa silage treated with pesticides (P < 0.05). LP increased the concentration of chlorpyrifos residue in alfalfa silage (P < 0.05). Chlorpyrifos and chlorantraniliprole affected the microbial communities of the material before ensiling, especially coliform bacteria and yeast; the two pesticide residues were reduced after the fermentation of alfalfa silage and affected the fermentation process, whereas LP improved the fermentation quality of pesticides‐contaminated alfalfa silage and slowed down the dissipation of chlorpyrifos.     

Effect of Mediterranean saltbush (Atriplex halimus) ensilaging with two developed enzyme cocktails on feed intake,nutrient digestibility and ruminal fermentation in sheep

The aim of this study was to assess the effects of feeding Atriplex halimus (AH) silage treated with two developed enzyme cocktails to sheep on feed intake, nutrient digestibility and ruminal fermentation. The AH silage was treated without or with 2 L of ZAD1^® or ZAD2^®/1000 kg with 5% molasses and ensiled for 30 days. Barley grain (300 g/head/day) was fed as an energy supplement once daily at 10.00 hours and AH silage with or without enzyme treatment was offered ad libitum to animals twice daily at 09.00 and 16.00 hours. Sheep were fed on four experimental forage diets comprised of AH silage and barley (D1), AH silage treated with ZAD1^® and barley (D2), AH silage treated with ZAD2^® and barley (D3) and AH silage treated with a combination of ZAD1^® and ZAD2^® (1:1) and barley (D4). Ensiling AH with enzymes reduced its contents of neutral detergent fiber and acid detergent fiber. The dry matter intake of AH of D2, D3 and D4 decreased (P < 0.001) as compared to D1. However, enzyme‐treated diets had greater total digestible nutrients intake (P < 0.001) as compared to D1. The nutrients digestibility for D2, D3 and D4 were higher than those for D1 (P < 0.001), and were higher for D3 as compared to both D2 and D4. Sheep fed on D3 had highest (P < 0.001) ruminal total volatile fatty acids concentration, ammonia nitrogen concentration and microbial protein yield. It could be concluded that AH silage treated with ZAD1^® or ZAD2^® improved digestibility and rumen fermentation in sheep.     

Effect of microbial inoculants on the fermentation characteristics,nutritive value,and in vitro digestibility of various forages

This study was aimed to examine the characteristics of three isolated lactic acid bacteria (LAB) strains (HG24, M1, and TG1), and to evaluate their effects on fermentation characteristics, nutritive value, and in vitro digestibility in a variety of substrates. The experiments were conducted with Italian ryegrass (Lolium multiflorum Lam.), tall fescue (Festuca arundinacea Schred.), and oat (Avena sativa L.) in laboratory silo (1 L) for ensiling 30 days. All the treated silages had significantly (p < 0.05) higher in vitro dry matter digestibility (IVDMD), and lower undesirable microorganism populations compared with controls. In Italian ryegrass and oat silages, all the inoculants improved the fermentation quality, evidenced by distinctly (p < 0.05) higher LA contents and ratios of LA to acetic acid, and lower pH, butyric acid and ammonia nitrogen contents. Strains HG24 and commercial inoculant G (Lactobacillus plantarum MTD‐1) performed better than strains M1 and TG1, indicated by obviously (p < 0.05) higher LA contents, LAB numbers, and IVDMD. In tall fescue silage, all the isolated LAB strains (HG24, M1 and TG1) markedly (p < 0.05) increased IVDMD compared with strain G. Strain HG24 is recommended as a good LAB inoculant for ensiling in various forages.     

添加糖蜜和乳酸菌制剂对西藏青稞秸秆和多年生黑麦草混合青贮发酵品质的影响

为探究不同添加剂对青稞秸秆与多年生黑麦草(4∶6)混合青贮发酵品质的影响,试验设对照组(C)、乳酸菌制剂组(LAB)、糖蜜组(M)、乳酸菌制剂+糖蜜组(LAB+M)4个处理,青贮后第7, 24和45天打开,测定青贮发酵品质。结果表明,添加乳酸菌制剂加速了青贮早期乳酸发酵进程,但由于底物不足,青贮后期丁酸含量和氨态氮/总氮值有所升高;添加糖蜜补充了发酵底物,促进了乳酸发酵,显著(P<0.05)提高了乳酸含量,降低了pH值(P<0.05),从而有效地抑制了有害微生物的活性,减少了丙酸和丁酸的生成。乳酸菌制剂+糖蜜组与单独添加乳酸菌制剂组相比显著提高了青贮发酵品质, 但与单独添加糖蜜组相比差异不显著,表明发酵底物不足是限制青稞秸秆与多年生黑麦草混合青贮发酵品质的主要因素。     

乳酸菌和纤维素酶对直穗鹅观草青贮的改善效果

本试验以直穗鹅观草(Roegneria turczaninovii)为材料,研究了乳酸菌和纤维素酶单独添加或以不同比例混合添加对鹅观草青贮发酵品质和营养价值的改善效果。处于抽穗期的直穗鹅观草经刈割切短后,分别添加纤维素酶0.06 g·kg-1 (CF),乳酸菌0.01 g·kg-1 (LD1)、0.02 g·kg-1 (LD2),纤维素酶+乳酸菌(0.06+0.01) g·kg-1 (CF+LD1)和(0.06+0.02) g·kg-1 (CF+LD2),并设置无添加剂组为对照(CK),每个处理3个重复。真空密封条件下贮藏90 d后,所有添加剂处理组pH值、丙酸、丁酸和氨态氮含量均显著低于对照组（P<0.05）,乳酸、乙酸、粗蛋白（CP）和可溶性糖水化合物(WSC)含量均显著高于对照组（P<0.05）,且CF、CF+LD1和CF+LD2处理的中性洗涤纤维（NDF）和酸性洗涤纤维（ADF）含量显著低于对照（P<0.05）。由此可见,添加乳酸菌和纤维素酶使直穗鹅观草青贮饲料的发酵品质和营养价值均得到了改善,且以混合添加0.06 g·kg-1的纤维素酶和0.02 g·kg-1的乳酸菌制剂的效果最好。     

Effects of inoculants and environmental temperature on fermentation quality and bacterial diversity of alfalfa silage

To investigate the effects of inoculants and environmental temperature on fermentation quality and bacterial diversity of alfalfa silage, first‐cut alfalfa was ensiled with or without two screened lactic acid bacteria (LAB) strains, Lactobacillus plantarum, LP, and Lactobacillus casei, LC. Each treatment was divided into three parts and stored at 20°C, 30°C, 40°C, respectively. After 60 days ensiling, fermentation characteristics were measured and bacterial diversity was investigated by 16S ribosomal RNA gene sequencing using Illumina MiSeq platform. LP and LC decreased pH, coliform bacteria counts and increased lactic acid content at 20°C, and the two strains decreased pH, ammonia‐N concentration, coliform bacteria counts at 30°C. When the environmental temperature was 40°C, silage treated with LC showed lower LAB and coliform bacteria counts and higher lactic acid content than the untreated and LP treated silages. Butyric acid mainly appeared in silages stored at 40°C. The relative abundance of Lactobacillus in alfalfa silages stored at 20°C and 30°C was highest and increased after LP and LC were added. Garciella was another dominant genus in silages stored at 40°C. In conclusion, LP and LC improved fermentation quality of alfalfa silage by increasing Lactobacillus proportions at 20°C and 30°C; ensiling alfalfa at 40°C was difficult because of Garciella.     

5株乳酸菌复合物与CaCO3,酶及尿素不同组合对全株玉米青贮品质影响

为探讨前期筛选出的5株对好氧细菌、酵母菌和霉菌具有优良抑菌活性乳酸菌的复合物及其与CaCO₃、酶及尿素不同组合对全株玉米青贮发酵品质及营养成分的影响,进一步筛选优质复合青贮生物制剂,共设置7个不同组合添加物进行全株玉米青贮,并设空白对照组,测定青贮3、10和30 d时其发酵品质和营养成分的变化情况。结果表明,添加5株乳酸菌混合剂有提高全株玉米青贮发酵品质和营养品质的趋势;同时添加CaCO₃和复合乳酸菌能进一步增加青贮饲料的乳酸(LA)和乙酸(AA)产量,尤其是AA产量,防止pH值过分降低,也有提高青贮营养品质的趋势;同时添加尿素和复合乳酸菌能进一步提高青贮的AA和氨态氮的含量,防止pH值过分降低,显著提高粗蛋白(CP)含量,从而提高青贮营养品质;纤维素酶和淀粉酶复合物可以提高发酵产物的水溶性碳水化合物(WSC)含量及LA和AA产量,改善青贮发酵品质;复合CaCO₃、尿素、纤维素酶、淀粉酶和复合乳酸菌的添加对提高全株玉米青贮的发酵品质和营养品质的效果最好;全株玉米青贮发酵品质和营养品质在前3 d变化最快,发酵不同阶段不同添加组合的变化不尽一致。     

添加剂对苜蓿青贮料品质和乳酸菌群的影响

以紫花苜蓿（Medicago sativa）为原料,分别添加植物乳杆菌WN5（Lactobacillus plantarum WN5,保藏号为CGMCC NO.7469）和干酪乳杆菌SN3（L.casei SN3,CGMCC NO.7470）的菌液,以及菌液与蔗糖、纤维素酶的混合液后青贮,探讨添加剂对苜蓿青贮料品质和乳酸菌群的影响。结果表明：苜蓿直接青贮发酵品质差;添加剂可改善苜蓿青贮料的发酵品质;添加乳酸菌和蔗糖对于苜蓿青贮饲料的营养价值和乳酸菌群有更明显的改善,青贮饲料中乳酸菌群占绝对优势,植物乳杆菌WN5和其他乳酸菌存活,但乳酸菌种类因蔗糖浓度不同而有差异;添加10 mL乳酸菌和2.0 g蔗糖的效果最好,其苜蓿青贮饲料的pH最低,粗蛋白、甲酸、乙酸和丙酸的含量最高,乳酸菌群的遗传差异大。乳酸菌和蔗糖作为优质添加剂,在苜蓿青贮发酵过程中可以保留大量不同种类的乳酸菌群,从而有利于形成品质良好的青贮苜蓿。     

Identification of lactic acid bacteria isolated from corn stovers

One hundred and twenty‐six strains were isolated from corn stover in Henan Province, China, of which 105 isolates were considered to be lactic acid bacteria (LAB) according to Gram‐positive, catalase‐negative and mainly metabolic lactic acid product. Analysis of the 16S ribosomal DNA sequence of 21 representative strains was used to confirm the presence of the predominant groups and to determine the phylogenetic affiliation of isolates. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank type strains between 99.4% and 100%. The prevalent LAB, predominantly Lactobacillus (85.6%), consisted of L. plantarum (33.3%), L. pentosus (28.6%) and L. brevis (23.7%). Other LAB species as Leuconostoc lactis (4.8%), Weissella cibaria (4.8%) and Enterococcus mundtii (4.8%) also presented in corn stover. The present study is the first to fully document corn stover‐associated LAB involved in the silage fermentation. The identification results revealed LAB composition inhabiting corn stover and enabling the future design of appropriate inoculants aimed at improving the fermentation quality of silage.     

The effects of wilting and storage temperatures on the fermentation quality and aerobic stability of stylo silage

In order to clarify the ensiling characteristics of stylo (Stylosanthes guianensis Swartz), the effects of wilting (no wilting, light wilting and heavy wilting) and storage temperatures (10°C, 20°C, 30°C and 40°C) on the fermentation quality and aerobic stability of stylo silage were investigated. Wilting had no significant influence on the contents of crude protein, ether extract and acid detergent fiber, and numbers of lactic acid bacteria, aerobic bacteria, yeasts and mold (P > 0.05). Heavy wilted material, wilted for 12 h, had higher neutral detergent fiber content and lower water‐soluble carbohydrate content than unwilted and light wilted materials (P < 0.05). Wilting and storage temperatures had significant effects on pH value, acetic acid, butyric acid and NH₃‐N contents of stylo silage (P < 0.01 or P < 0.05). Wilting tended to reduce acetic acid and NH₃‐N contents and improve the fermentation quality of stylo silage. In all the silages, no wilting silage ensiled at 30°C had the highest butyric acid content (P < 0.05). High temperature of 40°C markedly restricted the growth of lactic acid bacteria and aerobic bacteria in silage, irrespective of wilting. The wilted silage or silage stored at low temperature had poor aerobic stability.