Lactic acid bacteria strains selected from fermented total mixed rations improve ensiling and in vitro rumen fermentation characteristics of corn stover silage

Huang Kailang,Chen Hongwei,Liu Yalu,Hong Qihua,Yang Bin,Wang Jiakun 아세아·태평양축산학회 2022 Animal Bioscience Vol.35 No.9

Objective: This study identified the major lactic acid bacteria (LAB) strains from different fermented total mixed rations (FTMRs) via metataxonomic analysis and evaluated the ability of their standard strain as ensiling inoculants for corn stover silage. Methods: The bacterial composition of eight FTMRs were analyzed by 16S rDNA sequencing. Corn stover was ensiled without LAB inoculation (control) or with 1×106 cfu/g LAB standard strain (Lactobacillus vaginalis, Lactobacillus reuteri, Lactobacillus helveticus, or Lactobacillus paralimentarius) selected from the FTMRs or 10 g/t commercial silage inoculant (CSI) around 25°C for 56 days. For each inoculation, a portion of the silage was sampled to analyze ensiling characteristics at time intervals of 0, 1, 3, 7, 14, 28, and 56 days, gas production (GP), microbial crude protein and volatile fatty acids as the measurements of rumen fermentation characteristics were evaluated in vitro with the silages of 56 days after 72 h incubation. Results: Lactobacillus covered >85% relative abundance of all FTMRs, in which L. pontis, L. vaginalis, L. reuteri, L. helveticus, and L. paralimentarius showed >4% in specific FTMRs. CSI, L. helveticus, and L. paralimentarius accelerated the decline of silage pH. Silage inoculated with L. paralimentarius and CSI produced more lactic acid the early 14 days. Silage inoculated with L. paralimentarius produced less acetic acid and butyric acid. For the in vitro rumen fermentation, silage inoculated with CSI produced more potential GP, isobutyric acid, and isovaleric acid; silage inoculated with L. helveticus produced more potential GP and isovaleric acid, silage inoculated with L. paralimentarius or L. reuteri produced more potential GP only. Conclusion: The standard strain L. paralimentarius (DSM 13238) is a promising ensiling inoculant for corn stover silage. The findings provide clues on strategies to select LAB to improve the quality of silage. Objective: This study identified the major lactic acid bacteria (LAB) strains from different fermented total mixed rations (FTMRs) via metataxonomic analysis and evaluated the ability of their standard strain as ensiling inoculants for corn stover silage.Methods: The bacterial composition of eight FTMRs were analyzed by 16S rDNA sequencing. Corn stover was ensiled without LAB inoculation (control) or with 1×106 cfu/g LAB standard strain (Lactobacillus vaginalis, Lactobacillus reuteri, Lactobacillus helveticus, or Lactobacillus paralimentarius) selected from the FTMRs or 10 g/t commercial silage inoculant (CSI) around 25°C for 56 days. For each inoculation, a portion of the silage was sampled to analyze ensiling characteristics at time intervals of 0, 1, 3, 7, 14, 28, and 56 days, gas production (GP), microbial crude protein and volatile fatty acids as the measurements of rumen fermentation characteristics were evaluated in vitro with the silages of 56 days after 72 h incubation.Results: Lactobacillus covered >85% relative abundance of all FTMRs, in which L. pontis, L. vaginalis, L. reuteri, L. helveticus, and L. paralimentarius showed >4% in specific FTMRs. CSI, L. helveticus, and L. paralimentarius accelerated the decline of silage pH. Silage inoculated with L. paralimentarius and CSI produced more lactic acid the early 14 days. Silage inoculated with L. paralimentarius produced less acetic acid and butyric acid. For the in vitro rumen fermentation, silage inoculated with CSI produced more potential GP, isobutyric acid, and isovaleric acid; silage inoculated with L. helveticus produced more potential GP and isovaleric acid, silage inoculated with L. paralimentarius or L. reuteri produced more potential GP only.Conclusion: The standard strain L. paralimentarius (DSM 13238) is a promising ensiling inoculant for corn stover silage. The findings provide clues on strategies to select LAB to improve the quality of silage.

Effects of Lactic Acid Bacteria Inoculants on Fermentation of Low Moisture Fresh Rice Straw Silage at Different Storage Periods

Kuppusamy, Palaniselvam,Soundharrajan, Ilavenil,Park, Hyung Soo,Kim, Ji Hea,Kim, Won Ho,Jung, Jeong Sung,Choi, Ki Choon The Korean Society of Grassland and Forage Science 2019 한국초지조사료학회지 Vol.39 No.3

The purpose of this study was to analyze the effectiveness of different storage periods of lactic acid bacteria (LAB)-fermented low moisture fresh rice straw silage. The low moisture fresh rice straw sample was inculcated with LAB and stored for different storage periods such as 45, 90, and 365 days, respectively. The low moisture fresh rice straw (LMFRS) silage inoculated with LAB exhibited reduction in pH throughout the fermentation as compared with the control (P<0.05). The lactic acid content was increased at the late fermentation period (90 and 365 days, respectively) in LAB inoculated LMFRS silage as compared with the control (P<0.05). In contrast, the acetic acid and butyric acid concentrations were slightly reduced in the LAB inoculated LMFRS silage sample at 90 and 365 days fermentation, respectively. Meanwhile, the non-inoculated LMFRS silage showed higher amounts of acetic acid and butyric acid at an extended fermentation with low bacterial population as compared with the LAB inoculated LMFRS silage. However, lactic acid concentration was slightly high in the non-inoculated LMFRS silage at early 45 days fermentation. Additionally, the nutrient profile such as crude protein (CP), acid detergent fibre (ADF), neutral detergent fibre (NDF), and total digestibility nutrients (TDN) were not significantly different in control and LAB inculcated samples during all fermentation. Though, the microbial population was greater in the LAB inoculated LMFRS silage as compared with the control. However, the massive population was noted in the LAB inoculated LMFRS silage during all fermentation. It indicates that the inoculated LAB is the main reason for increasing fermentation quality in the sample through pH reduction by organic acids production. Overall results suggest that the LAB inoculums are the effective strain that could be a suitable for LMFRS silage fermentation at prolonged days.

Changes in fermentation pattern and quality of Italian ryegrass (Lolium multiflorum Lam.) silage by wilting and inoculant treatments

Liu, Chang,Zhao, Guo Qiang,Wei, Sheng Nan,Kim, Hak Jin,Li, Yan Fen,Kim, Jong Geun Asian Australasian Association of Animal Productio 2021 Animal Bioscience Vol.34 No.1

Objective: This study was conducted to investigate the effects of wilting and microbial inoculant treatment on the fermentation pattern and quality of Italian ryegrass silage. Methods: Italian ryegrass was harvested at heading stage and ensiled into vinyl bags (20 cm×30 cm) for 60d. Italian ryegrass was ensiled with 4 treatments (NWNA, no-wilting noadditive; NWA, no-wilting with additive; WNA, wilting no-additive; WA, wilting with additive) in 3 replications, wilting time was 5 hours and additives were treated with 106 cfu/g of Lactobacillus plantarum. The silages samples were collected at 1, 2, 3, 5, 10, 20, 30, 45, and 60 days after ensiling and analyzed for the ensiling quality and characteristics of fermentation patterns. Results: Wilting treatment resulted in lower crude protein and in vitro dry matter digestibility and there were no significant differences in acid detergent fiber (ADF), total digestible nutrient (TDN), water-soluble carbohydrate (WSC), ammonia content, and pH (p>0.05). However, wilting treatment resulted in higher ADF and neutral detergent fiber content of Italian ryegrass silage (p<0.05), and the WNA treatment showed the lowest TDN and in vitro dry matter digestibility. The pH of the silage was higher in the wilting group (WNA and WA) and lower in the additive treatment group. Meanwhile, the decrease in pH occurred sharply between the 3-5th day of storage. The ammonia nitrogen content was significantly lower in the additive treatment (p<0.05), and wilting had no effect. As fermentation progressed, the lactic and acetic acid contents were increased and showed the highest content at 30 days of storage. Conclusion: The wilting treatment did not significantly improve the silage fermentation, but the inoculant treatment improved the fermentation patterns and quality of the silage. So, inoculation before ensiling is recommended when preparing high quality of Italian ryegrass silage, and when wilting, it is recommended to combine inoculation for making high quality silage.

수산물 가공부산물의 이용에 관한 연구 I -가다랭이 내장 발효 silage 제조를 위한 유산균주의 배양특성

윤호동,이두석,지청일,서상복,YOON Ho-Dong,LEE Doo-Seog,JI Cheong-Il,SUH Sang-Bok 한국수산과학회 1997 한국수산과학회지 Vol.30 No.1

가다랭이 통조림을 제조할 때 대량 폐기되는 내장을 유효하게 이용할 목적으로 어류 내장 silage 제조를 위한 유산균주의 배양특성 및 저장기간에 따른 원료의 성상변화를 조사한 결과, MRS 배지에서 배양한 L. bulgaricus 및 L. plantarum 균주는 $pH\;5.5\~6.5$, 배양온도 $35^{\circ}C$ 부근에서 산생성 및 증식이 양호하였으며, 가다랭이 내장 자숙액에 사용된 당질원인 당밀 dextrose의 적정 첨가농도는 가다랭이 내장 중량 당 각각 $10\%$와 $7\%$ 정도인 것으로 나타났다. 저장기간에 따라 산첨가 silage의 pH는 초기 4.0에서 저장 42일째 pH 4.5로 다소 증가하였으며, 유산균 발효 silage는 초기 pH 5.9에서 점차 감소하여 pH 약 4.0까지 저하되었다. silage의 초기 VBN 함량은 약 $62\~65\;mg/100g$에서 저장기간에 따라 점차 증가하여 저장 42일후 산첨가 silage는 112.6\;mg/100g이었고, L. bulgarius 및 L. plantarum 발효 silage는 각각 139.9 mg/100g와 155.0 mg/100g 이었다. L. plantarum 발효 silage의 저장중 경시별 아미노질소 함량은 저장 4일째 1391.3 mg/100g으로서 약 $81\%$의 가수분해율을 나타내었다. 또한 L. plantarum을 이용하여 제조한 발효 silage의 초기 생균수는 $2.7\times10^8/g$에서 저장 35일째는 $2.3\times10^7/g$으로 나타났다. In order to utilize fish by-products from the skipjack tuna (Katsuwonus pelamis) canning manufactures Lactobacillus buigaricus KCTC 3188 and L. piantarum KCTC 1048 were used as a starter culture for the preparation of fermented fish silage with skipjark tuna viscera. The optimum temperature and pH on barterial growth and lactic acid production of L. bulgaricus and L. plantarum in MRS broth were $35^{\circ}C$ and around pH 6.0, respectively. And the optimum concentrations of the carbohydrate sources added to the broths were $7\%$ for dextrose and $10\%$ for molasses on the basis of total weights of skipjack tuna viscera. The pH of acid treated skipjack tuna viscera silage (ASS) slightly increased from 4.0 to 4.5, while that of fermented skipjack tuna viscera silages by the use of lactic acid bacterias (FSS) was significantly declined from 5.9 to about 40 after 42 days of storage at $35^{\circ}C$. Though the content of volatile basie nitrogen (VBN) in ASS was lower than those of FSS after 42 days of storage at $35^{\circ}C$, VBN content in silages slightly increased from an initial value of $62\~65{\cdot}mg/100g$ to final value of $113\~155\;mg/100g$ over 42 days. The fermented silage by L. piantarum reached a maximum concentration of amino nitrogen and showed $81\%$ of hydrolysis degree after 4 days of storage at $35^{\circ}C$.

SILAGE FERMENTATION AND SILAGE ADDITIVES - Review -

Bolsen, K.K.,Ashbell, G.,Weinberg, Z.G. Asian Australasian Association of Animal Productio 1996 Animal Bioscience Vol.9 No.5

Advances in silage technology, including precision chop forage harvesters, improved silos, polyethylene sheeting, shear cutting silo unloaders, and the introduction of total mixed rations, have made silage the principal method of forage preservation. A better understanding of the biochemistry and microbiology of the four phases of the ensiling process has also led to the development of numerous silage additives. Although acids and acid salts still are used to ensile low-DM forages in wet climates, bacterial inoculants have become the most widely used silage additives in the past decade. Commercial inoculants can assure a rapid and efficient fermentation phase; however, in the future, these products also must contribute to other areas of silage management, including the inhibition of enterobacteria, clostridia, and yeasts and molds. Nonprotein nitrogen additives have the problems of handling, application, and reduced preservation efficiency, which have limited their wide spread use. Aerobic deterioration in the feedout phase continues to be a serious problem, especially in high-DM silages. The introduction of competitive strains of propionic acid-producing bacteria, which could assure aerobically stable silages, would improve most commercial additives. New technologies are needed that would allow the farmer to assess the chemical and microbial status of the silage crop on a given day and then use the appropriate additive(s).

Effects of New Inoculants on In vitro Digestibility and Fermentation Indices of High Moisture Rye Silage

Seong Shin Lee,Dimas H. V. Paradhipta,Young Ho Joo,Hyuk Jun Lee,Hyun Son,Ouk Kyu Han,Dong Hyeon Kim,Sam Churl Kim 경상대학교 농업생명과학연구원 2018 농업생명과학연구 Vol.52 No.5

This study was conducted to examine the effect of new inoculants on in vitro digestibility and fermentation characteristics of high moisture rye silage. Rye was harvested at heading stage and divided into 5 treatments, following: No additives(CON); L. plantarum R48-27(NI1); L. buchneri R4-26(NI2); mixture of NI1 and NI2 at 1:1 ratio(MIX); and L. buchneri(LB). The rye forage was ensiled into 10 L bucket silo for 100 days. In vitro digestibility of dry matter and neutral detergent fiber were highest(p<0.05) in NI2 silage. The pH in NI2 and LB silages were lower(p<0.05) than CON silage. Lactate concentration was highest(p<0.05) in NI1 silage. While concentrations of acetate and propionate were highest(p<0.05) in MIX silage. Lactates : acetate ratio was highest(p<0.05) in NI1 silage, but lowest in LB silage. Butyrate concentrations of NI2 and LB silages were lower(p<0.05) than that in CON and NI1 silages. Lactic acid bacteria (LAB) count in all inoculated silages was higher(p<0.05) than that in CON silage, while yeast count in LB silage was lower than in CON, NI1, and MIX silages. In conclusion, application of NI2 inoculant could improve potentially fermentation quality and digestibility of high moisture rye silage.

Effects of Microbial and Organic Additives on Fermentation Quality and Aerobic Stability of Barley Silage

Young-Ho Joo,Dong-Hyeon Kim,Hyuk-Jun Lee,Seong-Shin Lee,Dimas Hand Vidya Paradhipta,In-Hag Choi,Sam-Churl Kim 경상대학교 농업생명과학연구원 2020 농업생명과학연구 Vol.54 No.1

The goal of this study was to evaluate effects of various microbial and organic additives on chemical compositions, fermentation indices, and aerobic stability of barley silage. Youngyang barley harvested at 31.5% dry matter (DM), and ensiled into 20 L bucket silo for 0, 1, 3, 7, 48, and 100 d in quadruplicates with four additives following: sterile destilled water at 1% of fresh forage (CON); Lactobacillus plantarum at 1.2 x 105 cfu/g of fresh forage (CL); Lactobacillus buchneri at rate of 1.2 x 105 cfu/g fresh forage (LB); Fermented Persimmon Extract at 1% of fresh forage (FPE); and Essential Oil at 1% of fresh forage (EO). On 100 d of ensiling, CL and FPE silages had lower (p<0.05) DM than CON silage. Additionally, FPE silage had higher (p<0.05) crude protein than CON silage. All silages with additives, except EO, had higher (p<0.05) neutral detergent fiber (NDF) than CON silage. Silage treated with CL, LB, and FPE had lower in vitro DM digestibility than CON silage, and silages treated with LB and FPE had higher in vitro NDF digestibility (IVNDFD) on 100 d of ensiling. The PFE silage produced the highest (p<0.05) lactate during ensiling period, while LB silage produced the highest (p<0.05) acetate. All inoculated silages had higher (p<0.05) LAB count than control silage. Only CL silage had higher (p<0.05) yeast count than control silage, while the other silages were not differ compared to CON silage. The aerobic stability was higher (p<0.05) in LB and FPE silages than in CON silage. In conclusion, FPE could be an alternative additive to increase IVNDFD, fermentation indices, and aerobic stability of barley silage.

Effects of Combined Treatment of Lactic Acid Bacteria and Cell Wall Degrading Enzymes on Fermentation and Composition of Rhodesgrass (Chloris gayana Kunth.) Silage

Ridla, M.,Uchida, S. Asian Australasian Association of Animal Productio 1998 Animal Bioscience Vol.11 No.5

This experiment was conducted to study the effects of lactic acid bacteria (LAB) inoculation either alone or in combination with cell wall degrading enzymes on the fermentation characteristics and chemical compositions of Rhodesgrass silage. Over to 1 kg of fresh Rhodesgrass sample a treatment of inoculant LAB with or without addition of an enzyme of Acremoniumcellulase (A) or Meicelase (M) or a mixture of both enzymes (AM) was applied. The treatments were control untreated, LAB-treated (application rate $1.0{\times}10^5cfu/g$ fresh sample), LAB+A 0.005%, LAB+A 0.01%, LAB+A 0.02%, LAB+M 0.005%, LAB+M 0.01%, LAB+M 0.02 %, LAB+AM 0.005%, LAB+AM 0.01%, and LAB+AM 0.02%. The sample was ensiled into 2-L vinyl bottle silo, with 9 silages of each treatment were made. Three silages of each treatment were incubated at 20, 30 and $40^{\circ}C$ for 2-months of storage period. All silages were well preserved with their fermentation quality has low pH values (3.91-4.26) and high lactic acid concentrations (4.11-9.89 %DM). No differences were found in fermentation quality and chemical composition of the control untreated silage as compared to the LAB-treated silage. Combined treatment of LAB+cellulases improved the fermentation quality of silages measured in terms of lower (p < 0.01) pH values and higher (p < 0.05) lactic concentrations than those of LAB-treated silages. Increasing amount of cellulase addition resulted in decrease (p < 0.05) of pH value and increase (p < 0.05) of lactic acid concentration. LAB + cellulase treatments (all cellulase types) reduced (p < 0.01) NDF, ADF and in vitro dry matter digestibility of silages compared with the control untreated silages. The fermentation quality and the rate of cell wall reduction were higher (p < 0.01) in the silages treated with LAB + cellulase A than in the silages treated with either LAB+cellulase M or LAB + cellulase AM. Incubation temperature of $40^{\circ}C$ was likely to be more appropriate environment for stimulating the fermentation of Rhodesgrass silages than those of 20 and $30^{\circ}C$.

옥수수 사일리지에서 미생물 첨가제와 저장기간이 발효 품질과 미생물 군집에 미치는 영향

정승민,오미래 한국초지조사료학회 2024 한국초지조사료학회지 Vol.44 No.2

This study was conducted to find a way to improve quality by observing changes in quality and microbial communities according to whether corn silage was treated with additives and the storage period, and to utilize them as basic research results. The experimental design was performed by 2˟4 factor desigh, and the untreated (CON), and the additive inoculated (ADD) silage were stored and fermented for 30 (TH), 60 (ST), 90 (NT), and 120 (OHT) days, with each condition repeated 3 times. There was no change in the nutrient content of corn silage according to additive treatment and storage period (p>0.05). However, the change in DM and the increase in the relative proportions of lactic acid content and Lactobacillales according to the storage period (p<0.05) indicate that continuous fermentation progressed until OHT days of fermentation. Enterobacterales (33.0%), Flavobacteriales (14.4%), Sphingobacteriales (12.7%), Burkholderiales (9.28%) and Pseudomonadales (6.18%) dominated before fermentation of corn silage, but after fermentation, the diversity of microorganisms decreased sharply due to the dominance of Lactobacillales (69.4%) and Bacillales (11.5%), Eubacteriales (7.59%). Therefore, silage maintained good fermentation quality with or without microbial additives throughout all fermentation periods, but considering the persistence of fermentation even in long-term storage and the aerobic stability, it would be advantageous to use microbial additives.

Effects of different nitrogen doses and cultivars on fermentation quality and nutritive value of Italian ryegrass (Lolium multiflorum Lam.) silages

Ertekin Ibrahim,Atis Ibrahim,Aygun Yusuf Ziya,Yilmaz Saban,Kizilsimsek Mustafa 아세아·태평양축산학회 2022 Animal Bioscience Vol.35 No.1

Objective: The fermentation profile and silage quality of 3 Italian ryegrass (Lolium multiflorum Lam.) cultivars (cvs. Devis, Hellen, and Trinova) treated with 5 nitrogen doses (0, 50, 100, 150, and 200 kg/ha) were evaluated. Methods: The experiment was laid out in split plot in randomized complete block design with three replications. Annual ryegrass cultivars used in this study have been commonly grown in Turkey. Nitrogen doses were set in main plot and cultivars in split plot in the field. Plants were harvested at full-flowering stage with dry matter content about 220 g/kg for first cutting and 260 g/kg for second cutting. Harvested plants were chopped theoretically into 2 to 3 cm lengths for ensiling. Chopped fresh materials were ensilaged by compressing in 2 L plastic jars about 3±0.1 kg. Results: Effects of N doses on dry matter, neutral detergent fiber, acid detergent fiber, dry matter digestibility, relative feed value, crude protein, pH, ammonia nitrogen, lactic acid, acetic acid, and lactic acid/acetic acid were statistically significant while water soluble carbohydrate, ash and organic matter were not statistically different. Ammonia nitrogen, crude protein, ash, organic matter, lactic acid, and lactic acid/acetic acid were affected by cultivars, but the other parameters were not. Increasing nitrogen applications positively affected the chemical composition of annual ryegrass silage. The significant increase in protein content was remarkable, however, silage fermentation properties were adversely affected by the increasing nitrogen dose. Conclusion: It can be recommended 150 kg/ha nitrogen dose for annual ryegrass harvested at full blooming stage. Even though the silage fermentation properties of the used cultivars were similar, cv. Devis gave better results than the others in terms of silage pH and relative feed value. Objective: The fermentation profile and silage quality of 3 Italian ryegrass (Lolium multiflorum Lam.) cultivars (cvs. Devis, Hellen, and Trinova) treated with 5 nitrogen doses (0, 50, 100, 150, and 200 kg/ha) were evaluated.Methods: The experiment was laid out in split plot in randomized complete block design with three replications. Annual ryegrass cultivars used in this study have been commonly grown in Turkey. Nitrogen doses were set in main plot and cultivars in split plot in the field. Plants were harvested at full-flowering stage with dry matter content about 220 g/kg for first cutting and 260 g/kg for second cutting. Harvested plants were chopped theoretically into 2 to 3 cm lengths for ensiling. Chopped fresh materials were ensilaged by compressing in 2 L plastic jars about 3±0.1 kg.Results: Effects of N doses on dry matter, neutral detergent fiber, acid detergent fiber, dry matter digestibility, relative feed value, crude protein, pH, ammonia nitrogen, lactic acid, acetic acid, and lactic acid/acetic acid were statistically significant while water soluble carbohydrate, ash and organic matter were not statistically different. Ammonia nitrogen, crude protein, ash, organic matter, lactic acid, and lactic acid/acetic acid were affected by cultivars, but the other parameters were not. Increasing nitrogen applications positively affected the chemical composition of annual ryegrass silage. The significant increase in protein content was remarkable, however, silage fermentation properties were adversely affected by the increasing nitrogen dose.Conclusion: It can be recommended 150 kg/ha nitrogen dose for annual ryegrass harvested at full blooming stage. Even though the silage fermentation properties of the used cultivars were similar, cv. Devis gave better results than the others in terms of silage pH and relative feed value.