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Effect of different silage storing conditions on oxygen concentration in the silo and fermentation quality of rice Ryuichi Uegaki, Kazuo Kawano, Ryo Ohsawa, Toshiyuki Kimura, and Kohji Yamamura J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 30 May 2017 Downloaded from http://pubs.acs.org on June 6, 2017

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Journal of Agricultural and Food Chemistry

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Effect of different silage storing conditions on oxygen concentration in the silo and

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fermentation quality of rice

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Ryuichi Uegaki*†, Kazuo Kawano❡, Ryo Ohsawa¶, Toshiyuki Kimura‡, Kohji

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Yamamura§

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†

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Animal Health, Kannondai 3-1-5, Tsukuba, Ibaraki, 305-0856 Japan

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❡

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Japan

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¶

National Agriculture and Food Research Organization (NARO), National Institute of

Nippon Kayaku Food Techno Co., Iwahana-cho 219, Takasaki, Gunma, 370-1208

Saitama Prefectural Agriculture and Forestry Research Center, Sugahiro 784,

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Kumagaya, Saitama, 360-0102 Japan

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‡

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Ibaraki, 305-8666 Japan *Present address, NARO, Food Research Institute, Kannondai

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2-1-12, Tsukuba, Ibaraki, 305-8642 Japan

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§

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305-8604 Japan

NARO, Central Region Agricultural Research Center, Kannondai 2-1-18, Tsukuba,

NARO, Institute for Agro-Environmental Sciences, Kannondai 3-1-3, Tsukuba, Ibaraki,

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1 ACS Paragon Plus Environment


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ABSTRACT: We investigated the effects of different silage storing conditions on

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oxygen concentration in the silo and fermentation quality of rice (Oryza sativa L.).

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Forage rice was ensiled in bottles (with or without space at the bottlemouth; with solid

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or pinhole cap; and with oxygen scavenger, ethanol transpiration agent, oxygen

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scavenger and ethanol transpiration agent, or no adjuvant) and stored for 57 d. Oxygen

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concentration decreased with the addition of oxygen scavenger and increased with that

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of ethanol transpiration agent. Oxygen scavenger facilitated silage fermentation and

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fungus generation, whereas ethanol transpiration agent suppressed silage fermentation

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and fungus generation. However, the combined use of oxygen scavenger and ethanol

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transpiration agent facilitated silage fermentation and also suppressed fungus generation.

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Overall, this study revealed the negative effects of oxygen on the internal silo and the

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positive effects of the combined use of oxygen scavenger and ethanol transpiration

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agent on silage fermentation quality.

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KEYWORDS:

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concentration, Oryza sativa, oxygen scavenger, silage fermentation

anaerobic

fermentation,

ethanol

transpiration

agent,

oxygen


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INTRODUCTION

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Silage is fermented stored fodder, which is used for feed. Silage fermentation, that is

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lactic acid fermentation, is one of the major storing methods that maintain the

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nutritional value of feed and is considered effective in the warm and humid areas of

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Japan. Silage is currently prepared using simple silos such as wrap silos, drum silos, and

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flexible containers. The use of facility type silos, such as tower silos, has become rare.

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One of the main problems in simple silos is the maintenance of stable anaerobic

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conditions, since the presence of oxygen negatively affects silage fermentation.1,

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Aerobic conditions negatively affect fermentation quality and the development of fungi,

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reducing the nutritional quality and hygiene of the feed. Previous studies on fungus

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prevention and the stabilization of silage fermentation quality showed that fungi can

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grow at oxygen concentrations as low as 0.1–0.4%.3, 4 Therefore, oxygen concentration

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needs to be maintained below 0.1% during silage storage. However, information on the

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changes in oxygen concentration during silage fermentation is limited.

2

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Oxygen scavenger and ethanol transpiration agents are commercially available for

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preventing fungal growth on food.3–6 Thus, they may be effective in improving and



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stabilizing the fermentation quality of silage. However, this has never been

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experimentally explored; the demonstration of such effects could also suggest the use of

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such agents for other types of anaerobic fermentation.

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Fungus development in silage storage usually occurs around the aperture of the silo7

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and can be suppressed by introducing some space at the aperture. However, the oxygen

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that remains in the space negatively affects silage fermentation. The relationship

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between the space at the aperture and silage fermentation has not been elucidated and

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thus, further research is needed.

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Rough rice (Oryza sativa L.) and rice grain are widely used for animal feed in Japan8

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and commonly stored in simple silos. In the present study, we aimed to investigate the

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effect of different silage storing conditions on the oxygen concentration in the silo and

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the fermentation quality of rice grain silage. To this end, we conducted a silage

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fermentation experiment using forage rice grain to investigate the effect of space at the

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top of the silo as well as the use of oxygen scavenger and ethanol transpiration agent on

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silage fermentation conditions and feed quality.

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MATERIALS AND METHODS

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Silage Preparation and Storage. The rough grain of forage rice (O. sativa L.

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‘Bekoaoba’) grown in Tsukubamirai, Ibaraki, Japan in 2014 was used in this study.

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After harvest and threshing, rough rice was crushed to a particle size of 0.2–3 mm using

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a forage cutter (Toyohira Agricultural Machinery, Sapporo, Japan). To prepare silage

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material, 50 kg of crushed rough rice was mixed with 11.4 L of tap water to adjust

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moisture content to 30% (w/w) and 0.1 L of lactic acid bacteria (Lactobacillus

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plantarum chikuso-1, 103 cfu kg-1). Next, silage material was packed into 1-L

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polypropylene bottles (AS-One, Osaka, Japan) and sealed with caps. Two experimental

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groups (A, without space at the bottlemouth; B, with space at the bottlemouth [15%

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volume]) with two sub-groups (S, solid cap; P, pinhole cap [0.8 mm pinhole diameter])

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and four treatments each (1, no adjuvant; 2, oxygen scavenger [Moduran W-1000®,

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Nippon Kayaku Food Techno, Takasaki, Japan]; 3, ethanol transpiration agent [Oitec L

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5.0G ®, Nippon Kayaku Food Techno]; 4, oxygen scavenger and ethanol transpiration

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agent) were set up. Each treatment was applied in triplicate. The prepared bottles were

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placed in a stockyard at 13.4–21.4°C for 56 d. At d 57, samples were collected from the



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upper 3-cm surface (upper samples) and from the bottom after discarding 50% of the

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silage (internal samples).

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Measurement of Oxygen Concentration. Oxygen concentration was measured

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using a chip sensor type non-destructive oxygen concentration meter (Precision Sensing

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GmbH, Magdeburg, Germany). Chips were set at the back of the cap or right next to the

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top surface of the material. Measurements were carried out at 1–7 d intervals throughout

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the storage period. Oxygen concentration prior to measurements was calibrated to 21%.

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Analysis of Silage Fermentation Quality. Silage fermentation quality was

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determined by measuring pH, organic acid (lactic acid, acetic acid, propionic acid, and

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butyric acid) concentration, the number of living fungi, and the occurrence of visible

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fungi. A silage sample of 4 g from each treatment was homogenized with 40 ml of

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sterilized distilled water and used for quality analysis. The pH was measured using a

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glass electrode pH meter (Horiba Ltd., Kyoto, Japan). The content of lactic acid, acetic

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acid, propionic acid, and butyric acid was determined by high-performance liquid


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chromatography (HPLC; Shimadzu, Kyoto, Japan); this method is commonly used for

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measuring organic acids in silage.9 A homogenized sample of 1.5 ml was mixed with 20

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mg of cation exchange resin by shaking (Organo, Tokyo, Japan) and centrifuged at

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6,000 × g for 5 min. The supernatant was passed through a 0.45-µm membrane filter

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(Advantec, Tokyo, Japan), injected into the HPLC under the following conditions:

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column, Shim-pack SCR-102H (Shimadzu corporation); oven temperature, 40°C; eluent,

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5 mmol L-1 p-toluenesulfonic acid at 1.0 ml min-1; and post-column reaction reagent, 5

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mmol L-1 p-toluenesulfonic acid, 20 mmol L-1 Bis-Tris, and 100 mmol L-1

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ethylenediaminetetraacetic acid at 1.0 ml min-1. Components were detected using an

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electrical conductivity detector (CDD-10AVP, Shimadzu). The limit of qualification

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(LOQ) of each organic acid was 0.01 g kg-1 of fresh weight. Values lower than LOQ

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were treated as undetected. The number of living fungi was determined by the surface

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plate method using potato dextrose agar (Nihon Pharmaceutical, Tokyo, Japan) adjusted

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to pH 3.5 with 10% tartaric acid. A gradually diluted silage homogenized solution was

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placed on the medium, and after 48 h at 25°C under aerobic conditions, the number of

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generated fungi was counted. The occurrence of fungi was assessed at 1–7 d intervals



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throughout the storage period.

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Statistical Analysis. Key-factor/key-stage analysis (KFKSA)10 was performed for

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analyzing the changes in oxygen concentration. Analysis of variance (ANOVA) was

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then carried out to identify differences in pH, organic acid concentration, the ratio of

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lactic acid to acetic acid (L/A), and the microbial number, using Statistical Analysis

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(Esumi, Tokyo, Japan) and JMP 13 (SAS Institute, Cary, NC, USA). Box-Cox11

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transformation was used in the ANOVA to normalize the data, in which a variable y is

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given by z = (yλ – 1)/(λ Yλ-1), where Y is the geometric mean of y. The maximum

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likelihood estimates of λ obtained without random variables were λ = –2.0, 1.6, –0.2,

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and 0.2 for pH, lactic acid, acetic acid, and L/A, respectively.

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RESULTS AND DISCUSSION

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Changes in Oxygen Concentration. Changes in oxygen concentrations during silage

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fermentation are shown in Figure 1. The average value of three replications is shown in

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a logarithmic scale, in which the average was discrete-adjusted in the form of x + (1/6),


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since the discrete width was 1/3 (average of three observations).12 At d 1, oxygen

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concentration in AS1–AP4, in which no space was left at the top of the bottle, were less

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than 0.1%, except for that in AP1 (0.13%) and AP3 (0.20%). Oxygen concertation in

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AS1 and AS2 was less than 0.1% until d 56; that in AS3 was higher than 0.1% at d 6–17,

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whereas that in AS4 was less than 0.1% until d 52 and approximately 0.1–0.30% until d

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56. AP1–AP4 contained pinholes and simulated damaged silos. In AP1, oxygen

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concertation was 0.13% at d 1 and reached a maximum concentration of 1.73% after 10

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d; subsequently, oxygen concentration reduced and was maintained at or less than 0.1%

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at d 21–56. In AP1, AP3, and AP4, oxygen concertation was less than 0.13% at d 1 and

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then fluctuated throughout the storage period (increased at d 2–13, decreased at d 14–20,

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and increased at d 21–56). Oxygen concentration in AP4 was 5.7% at d 56, whereas that

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in AP2 was less than 0.5% until d 56.

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AS1–AS4, using pinhole-free bottles, simulated ideal conditions for silage

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fermentation. The oxygen concentration was less than 0.1% at d 1, similar to that

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reported in previous studies at d 1–3. 2, 13, 14 The consumption of the initial oxygen is

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attributed to plant respiration and the growth of aerobic microorganisms. 2, 13, 14 In the



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present study, the breathing silage material was considered to be close to zero, and thus,

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the existing oxygen in the bottle was rapidly consumed by aerobic microorganisms.

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Oxygen concentration in BS1–BP4 was less than 0.1% after d 1, except for that in

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BP1 (0.17%) and BP3 (0.30%). Oxygen concentration in BS1–BS4 remained at less

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than 0.1% until d 56. Oxygen concentration in BP1 reached 0.2% after d 1, increased to

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2.1% after d 10, gradually decreased until d 29, and remained at 0.2% until d 56.

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Oxygen concentration in BP2 and BP4 was 0.1% after d 1, approximately 0.2% until d

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30, and then gradually increased, reaching 0.5% in BP2 and 1.7% in BP4 at d 56.

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Oxygen concentration in BP3 decreased to 0.3% at d 1, but then, increased to more than

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10% at d 13, and reached a maximum of 15% at d 56.

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Figure 2 shows the results of KFKSA regarding the effects of space at the

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bottlemouth, pinhole cap, and adjuvant (oxygen scavenger, ethanol transpiration agent,

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or combined use) on oxygen concentration. Variance components that affected oxygen

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concentration were plotted against time; a positive value indicated that the factor

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increased variance and thus strongly influenced oxygen concentration, a negative value

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indicated that the factor reduced variance, the ‘residual’ indicated variance due to


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unknown factors, and ‘adjuvant’ indicated the combined effects of ethanol transpiration

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agent and oxygen scavenger. Most factors had a high positive effect on oxygen

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concentration at d 3–13, but a negative effect at d 15–29. Thus, further analysis needs to

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be performed during the first 13 d.

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The effect of space at the bottlemouth on oxygen concentration was moderate during

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d 1–6, increased gradually after d 6, and was relatively strong at d 10–d 13. The effect

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of the pinhole cap on oxygen concentration followed the same pattern as that of the

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adjuvant; it was moderate at d 1–3, but then increased, reaching a maximum at d 9.

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Unknown components (residual) had also a highly positive effect on oxygen

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concertation at d 6–13 and d 30–56. At d 1–15, the unknown components were probably

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related to the action of microorganisms, especially of lactic acid bacteria, that became

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more active, leading to fluctuations from d 3 to d 15. At d 30–56, the unknown

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components were probably related to the development of new microbial flora that

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affected oxygen concentration. Kuikui et al.

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ryegrass, alfalfa, and corn silage by denaturing gradient gel electrophoresis (DGGE) and

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next generation sequencing and showed that different microbiota were dominant in each

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assessed the microbial flora of Italian



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crop species, especially lactic acid bacteria, Agrobacterium spp., and Methylobacterium

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spp. Furthermore, the microbial flora of silage differed considerably from the

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microbiota present before silage fermentation. Wang et al.16 analyzed the microbial flora

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of rice straw silage by DGGE and reported that it varies depending on the silage

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additive. However, further investigation is needed to identify associations between

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silage fermentation and microbial flora.

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Oxygen concentration is the result of the balance between invading oxygen and

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oxygen consumed by aerobic microorganisms. Oxygen concentration initially decreased

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owing to the activity of rapidly growing aerobic microorganisms, but then gradually

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increased owing to a decrease in the activity of these organisms. However, the reason

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for the increase in oxygen concentration at approximately d 13 remained unknown.

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ANOVA results of oxygen concentration at d 13 are shown in Table 1. Oxygen

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concentration was significantly increased by adding the ethanol transition agent (P

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