Effects of Apple Vinegar Addition on Aerobic Deterioration of Fermented High Moisture Maize Using Infrared Thermography as an Indicator

This study was carried out to determine the effects of apple vinegar and sodium diacetate addition on the aerobic stability of fermented high moisture maize grain (HMM) silage after opening. In the study, the effect of three different levels (0%, 0.5% and 1%) of apple vinegar (AV) and sodium diacetate (SDA) supplementation to fermented HMM at two different storage conditions (27–29 °C, 48% Humidity; 35–37 °C, 26% Humidity) were investigated. The material of the study was fermented rolled maize grain with 62% moisture content stored for about 120 days. Silage samples were subjected to aerobic stability test with three replicates for each treatment group. Wendee and microbiological analyses were made at 0, 2, 4, 7, and 12 days. Meanwhile, samples were displayed in the T200 IR brand thermal camera. According to the thermogram results, 1% SDA addition positively affected HMM silages at the second and fourth days of aerobic stability at both storage conditions (p < 0.05). Aerobic stability and infrared thermography analysis indicated that 1% AV, 0.5%, and 1% SDA additions to HMM silages had promising effects. Due to our results, we concluded that thermal camera images might be used as an alternative quality indicator for silages in laboratory conditions.


Introduction
Conservation of forage and cereal grains as silage is a very important, common source of ruminant nutrition around the world [1,2]. The silage process might be divided in four stages: (1) The aerobic stage in the silo immediately after harvest, (2) the fermentation stage, (3) the stable storage stage in the silo, and (4) the feed-out stage when the silo face is opened and exposed to air. Producing high-quality silage, and also avoiding dry matter losses as much as possible, is a challenge, and quality problems can occur any time during the entire silage process [3].
High moisture maize grain (HMM) is an indispensable part of the total mixed ration (TMR), especially in dairy cattle diets; TMR might comprise 18-20% HMM [4]. However, the high moisture and starch contents of HMM poses a significant risk in terms of its susceptibility to aerobic degradation during the feeding period [5][6][7][8][9]. However, to reduce the labor in field conditions, ensilaged HMM is taken from the silo in the amount that would not meet the daily requirements of animals but instead meet the requirements for 5-7 days. In addition, sometimes the ensilaged HMM is transferred from one farm to another to meet their needs. These situations lead to undesirable consequences in terms of aerobic deterioration. The objective of the study was to improve the aerobic stability of HMM silage

Silage Material
High moisture (62%) maize grain crushed and fermented in a plastic sausage silo for approximately 120 days was used as the research material. At the end of the silage fermentation process, approximately 40 kg of silage sample was brought to the laboratory. Samples from freshly opened silage were taken and analyzed to evaluate the initial material. Meanwhile, the fermented high moisture maize samples were then divided into 12 treatment groups with 3 replicates in each. Treatment groups consist of two different supplements (apple vinegar and sodium diacetate) with three different levels (0%, 0.5% and 1%) and stored at two different storage conditions (a room and incubator). Each treatment group was weighed as 1000 gr and put in plastic bags. Then additives were sprayed on the silage material and mixed for homogeneity. In the control groups, 20 mL of purified water were added as an equivalent dose to the treatment groups. After supplementation of additives, silage samples were stored for 12 days to evaluate the aerobic stability changes in room (27-29 • C; 48% humidity) and incubator (35-37 • C; 26% humidity) conditions.

Laboratory Analysis
Dry matter (DM), pH, lactic acid (LA), water soluble carbohydrate (WSC), ammonianitrogen (NH3-N), lactic acid bacteria, yeast, and mould counts were determined at 0th, 2nd, 4th, 7th, and 12th days of storage to evaluate the aerobic stability of silage samples. Temperature changes in the storage ambient and inside the silage samples have been recorded by data logger devices (Hobo pendant, Bourne, MA, USA) every 30 min during the experiment period.
Chemical analyses were performed on triplicate samples. DM was determined by oven drying for 48 h at 60 • C. The pH in fresh material and silage samples was measured according to the British standard method [24]. The ammonia nitrogen (NH3-N) content of silages was determined, according to Jackson et al. [24]. The WSC content of silages was determined by spectrophotometer (Shimadzu UV-1201, Kyoto, Japan); after a reaction with the antron reagent [25]. Lactic acid (LA) was determined by the spectrophotometric method [26]. Microbiological evaluation included enumeration of lactobacilli on pour-plate Rogosa agar (Oxoid CM627, Oxoid, Basingstoke, UK). Yeast and moulds were determined by pour-plating in malt extract agar (Oxoid CM59) that had been acidified, after autoclaving, by the addition of 85% LA at a concentration of 0.5% vol/vol. Plates were incubated aerobically at 32 • C for 48-72 h.

Infrared Thermal Camera Imaging
Thermal camera imaging was recorded by using a Fluke Ti9 IR (IR Sensor Size: 160 × 120 Focal Plane Array, WA, USA) thermal camera from a 1-m distance with a surface size of 20 × 30 cm (with two replicates from each sample; n = 24). Thermography images were taken in laboratory conditions (ambient temperature: 22 • C) without direct sunlight and air velocity. The average, minimum, and maximum temperatures of the silage surface were calculated using SmartView ® software program in which each pixel of the image was allocated to one temperature value.

Statistical Analysis
The research was conducted according to the 2 × 2 × 3 factorial trial design. To reveal the effects of treatment and temperature, the data were evaluated according to the variance analysis technique. If the difference between groups was found to be significant, Duncan's range test was applied [27]. The applied mathematical model was as follows (Equation (1)): Yijk = µ + Ai + Bj+ Ck + (AB)ij + (AC)ik + (BC)jk + (ABC)ijk + eijkl. (1) Yijk: Observation applying the i th supplement with j th inclusion level and stored at k th temperature' µ: Overall average; Ai: Effect of i th supplement; Bj: Effect of j th inclusion level of supplement; Ck: Effect of k th storage temperature; (AB)ij: Interaction effect of i th supplement × j th inclusion level; (AC)ik: Interaction effect of i th supplement × k th storage temperature; (BC)jk: Interaction effect of j th inclusion level × k th storage temperature; (ABC)ijk: Interaction effect of i th supplement × j th inclusion level × k th storage temperature; eijkl: Error associated with each observation.

Results and Discussion
This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.
In Table 1, the analysis results of the beginning material of fermented maize silage are given. Mould has not been detected in the initial materials. As a result of thermal camera footage carried out on day zero of aerobic stability, the average temperature of fermented maize silage was 30.62 • C, while the background temperature was 22 • C. Demirel et al. [28] stated that there is an inverse relationship between the DM and pH levels of the raw material to be ensilaged. Their results showed that the pH value of corn silage (with 23.48% DM) harvested during the milking phase was 4.15. The pH value of HMM silage was found to be low in the intial material (such as 3.900). These results might be evaluated as a harmonious result with Demirel et al. [28].

2nd Day of Aerobic Deterioration
Second-day aerobic stability results of HMM silages are presented in Table 2. In the LAB count results, the lowest value was observed in the groups that has 0.5% SDA added and were stored in the incubator, and the highest value was observed in the groups that had 1% AV added and were stored at room temperature. In addition, significant reductions in LAB numbers were observed in all silage samples stored in the incubator (35-37 • C, 26% humidity; p < 0.001). High temperatures with low humidity values might be responsible for the change in LAB counts. However, no yeast could be detected in groups with 0% and 1% AV and 1% SDA supplemented and stored in room conditions (p < 0.001). According to the mould count results, the highest value was found in a SDA control group stored in incubator conditions with 3.310 log 10 cfu/g, while the lowest value was 2.220 log 10 cfu/g in 0.5% vinegar added and stored under room conditions (p < 0.001).

4th Day of Aerobic Deterioration
Aerobic stability analysis results of HMM silage on the fourth day of storage were given at Table 3. LAB counts were intended to be lower at SDA added groups and also lower at groups stored at 35-37 • C (p < 0.001). In addition, the highest LA level was observed at 0.5% SDA supplemented and stored at room conditions (27-29 • C; 48% Humidity; p < 0.05). In addition, the lowest yeast counts were observed at 0.5 and 1% SDA added and stored at high temperatures (p < 0.001). However, WSC levels were not affected by additive sources (p > 0.05). In order to avoid the deterioration of the ensilaged material, there must be LAB in the silage and also sufficient amount of WSC. Thus, LAB can produce LA required for silage fermentation by using WSC in the medium [29]. However, Alçiçek and Özkan [30] reported that LA content should not be higher than 2% for good silage quality. Fermentation properties of silages are also effective on aerobic deterioration. Unused sugars and high levels of LA in the silage reduce aerobic stability. Some yeasts and moulds might cause CO 2 production in silages by using the remaining sugars and LA as nutrients.
As a result, an increase occurs in ambient pH and temperature [31]. The data obtained from the study support the previous research results [32][33][34].

7th Day of Aerobic Deterioration
The effects of additives on HMM silage on the seventh day of aerobic stability are presented in Table 4. When pH, NH3-N, WSC analysis results, LAB, and yeast numbers are evaluated, it has seen that the highest values belong to the SDA control group under room conditions (27-29 • C; 48% Humidity; p < 0.001). However, the highest LA value (13.740 g/kg DM) was found in silages stored in the incubator with 0.5% SDA, while the lowest LA (1.147 g/kg) was observed in silages stored in room conditions with 1% AV addition (p < 0.001). The effects of the silage additive, addition level, storage temperature, their double and triple interactions on pH, NH3-N, LA, WSC, and LAB values were found to be statistically different (p < 0.001). Pahlow et al. [17] reported that the number of yeast in high-moisture maize silages was 3-5 log 10 cfu/g and that a high yeast count reduced aerobic stability, especially at high temperatures. Teller et al. [35] stated that physical damage to the grain might cause substrate formation for microorganisms and might cause the high yeast content in highmoisture maize silages. Considering the results of the research, the high yeast content in the control group, particularly at high temperatures supports previous studies on this subject. Besides, there was a decrease in the yeast and LAB numbers of silages stored in the incubator. In addition, mould counts were found to be zero in silages stored in incubator conditions for all additives, and additional levels (p < 0.001). In the study, silage samples stored at 35-37 • C had relatively low humidity (26%). That might have affected microbial growth negatively. However, a 1% SDA addition has been found effective in both room and incubator conditions on LAB and Yeast counts.

12th Day of Aerobic Deterioration
The 12th day analysis results of aerobic stability are given in Table 5. According to the pH results, it was observed that the lowest values were found in silages stored at high temperatures and the effect of storage temperatures was statistically significant (p < 0.001). Similarly, a decrease was observed in NH3-N values under high temperature storage conditions (p < 0.01). However, the NH3-N value was found to be lower in the group containing 1% SDA and stored in room conditions (p < 0.01). In addition, WSC contents were found to be lower in all groups stored at room conditions, too. However, one of the lowest values were detected at the 0.5%-AV added and incubator-stored group. McDonald [15] has stated that WSC are the most important energy source used by lactic acid bacteria. Some researchers observed an increase in the amount of WSC due to increasing SDA supplementation. Researchers attributed this increase to the antifungal properties of additives [36][37][38]. They prevent the growth of unwanted microorganisms and caused a reduction of DM and nutrition losses. In the study, all parameters (additives, addition ratios, and storage conditions) revealed significant effects on WSC (p < 0.001).
LA levels demonstrated an increase at higher storage temperatures except for 1.0% SDA-added groups. Besides, the highest value (11.407 g/kg) was determined in the 1% AV-added groups. Reeves et al. [39] reported that the amount of LA in dry matter of maize silage varied between 1.58% and 8.57%. In addition, Deswysen et al. [40] reported that the amount of lactic acid on dry matter in maize silage was 6.31%, while Phillip and Hidalgo [41] reported that it was 5%. In the present study, LA levels ranged between 0.881-1.469% on the second day, and 0.030-1.141% on the 12th day of aerobic stability (Tables 2 and 5).
According to the results of the 12th day of aerobic stability, there was an increase in LAB counts of silage samples stored in the incubator compared to those in the chamber, but a decrease in yeast count was observed (except vinegar control and 0.5% SDA supplemented groups) (p < 0.001). In addition to these results, mould counts were found to be 0 in all treatment groups on the 12th day of aerobic stability.

Thermal Camera Imaging Results of Aerobic Stability
Temperature data obtained from dataloggers for 12 days were summarized in Figures 1-4. The temperatures of sensor data and thermal camera images in the study showed compatible results with each other. The finding was parallel with two field studies [42,43].
Mean, maximum, minimum, and standard deviation values of temperature measurements of aerobic stability period were determined by thermal camera with the results presented in Tables 6-9. Arithmetic means was subsequently created on the basis of all values. Data logger records at the time of thermograms taken were also given in the tables. Additionally, one of the thermograms captured from each treatment (n = 12; same samples during the experiment) are shown at Figures 5-8. They might help to visualize the heat differences in the silage samples. According to the results of the 12th day of aerobic stability, there was an increase in LAB counts of silage samples stored in the incubator compared to those in the chamber, but a decrease in yeast count was observed (except vinegar control and 0.5% SDA supplemented groups) (p < 0.001). In addition to these results, mould counts were found to be 0 in all treatment groups on the 12th day of aerobic stability.

Thermal Camera Imaging Results of Aerobic Stability
Temperature data obtained from dataloggers for 12 days were summarized in Figures 1-4. The temperatures of sensor data and thermal camera images in the study showed compatible results with each other. The finding was parallel with two field studies [42,43].    According to the results of the 12th day of aerobic stability, there was an increase in LAB counts of silage samples stored in the incubator compared to those in the chamber, but a decrease in yeast count was observed (except vinegar control and 0.5% SDA supplemented groups) (p < 0.001). In addition to these results, mould counts were found to be 0 in all treatment groups on the 12th day of aerobic stability.

Thermal Camera Imaging Results of Aerobic Stability
Temperature data obtained from dataloggers for 12 days were summarized in Figures 1-4. The temperatures of sensor data and thermal camera images in the study showed compatible results with each other. The finding was parallel with two field studies [42,43].     Mean, maximum, minimum, and standard deviation values of temperature measurements of aerobic stability period were determined by thermal camera with the results presented in Tables 6-9. Arithmetic means was subsequently created on the basis of all values. Data logger records at the time of thermograms taken were also given in the tables. Additionally, one of the thermograms captured from each treatment (n = 12; same samples during the experiment) are shown at Figures 5-8. They might help to visualize the heat differences in the silage samples.   Mean, maximum, minimum, and standard deviation values of temperature measurements of aerobic stability period were determined by thermal camera with the results presented in Tables 6-9. Arithmetic means was subsequently created on the basis of all values. Data logger records at the time of thermograms taken were also given in the tables. Additionally, one of the thermograms captured from each treatment (n = 12; same samples during the experiment) are shown at Figures 5-8. They might help to visualize the heat differences in the silage samples. On the 2nd and 4th day of aerobic stability, the lowest temperature values were observed in the 1% SDA added HMM silages at both storage temperatures (Tables 6 and 7). In the thermal camera measurements conducted on the 7th day of storage, the lowest temperature values were observed in the SDA control group kept under room conditions ( Table 8). Thermograms of the 12th day of aerobic stability showed that the lowest values were found in the groups that had AV added and were kept under room conditions (Table 9). These results supported the importance of storage conditions and additives on quality properties of silages even after opening (p < 0.01).
Thermal camera imaging results of aerobic stability approved that the temperature of silage samples stored in 35-37 • C was higher compared to the stored at 27-29 • C. However, when the temperature differences between the silages and storage environment were considered, it is seen that the temperature differences increase in the room conditions (27-29 • C; 48% humidity), heat rises in the samples, and therefore deterioration occurs in stored silages. Similarly Kaya and Polat [44] reported that more than 2 • C of the temperature difference between the ambient and silage may indicate deterioration. Koc et al. [45] also indicated that heat differences are major signs of deterioration.      Heat differences in maize silages stored at 27-29 • C, started to increase at day 4. However, on day 7 differences had maximized and started to decline at day 12. Due to this increase on the seventh day, pH values, yeast, and mould counts were found higher for all treatments in room conditions. Similarly, Junga and Trávníček [13] have found a connection between infrared thermography images with chemical, and microbial analysis results in the field conditions. Samples from surfaces with a higher temperature have resulted with an increase in pH values, mould, and yeast counts. Santos et al. [18] indicated that feeding silages with high concentrations of yeasts from aerobic spoilage is often implicated as a cause of poor animal performance on dairy animals.   Borreani and Tabacco [46] evaluated the temperature at 11 locations on 54 silos and correlated the temperature with chemical composition and microbial count. They also concluded that temperature is linked to microbial activity and might be an important indicator of the early stages of aerobic degradation.
Addah et al. [21] used an infrared thermal imaging technique to assess that the heating of barley silages stored in large cylindrical bag silos could serve as a simple and rapid method of directly measuring and visualizing heat distribution over the feed-out face of silos in real-time.   Alsaaod et al. [23] reported that environmental factors such as airflow, environment temperature, humidity, sunlight, and motion could be affected by the thermograms. The preferred environment conditions appear to be in the neutral zone temperature, without direct sunlight and detectable airflow. In addition, infrared thermal imaging technique does not need light or shadow to capture the frame accurately [47].   Researchers suggested that infrared thermography might be used for quick temperature detection of silo surface, of silage layers, and also might be a practical method for assessing the aerobic stability of silages on field and laboratory conditions [13,21,43,48]. According to the results of study, thermal camera imaging technique might be used to detect temperature differences and to reveal deterioration in the silage.

Conclusions
Fermented high moisture maize grain (HMM) is an important part of the TMR for dairy cattle rations with 18-20% inclusion levels. However, low dry matter and high starch contents of HMM increase aerobic deterioration risks during the feeding period. The objective of the study was to improve and reveal aerobic stability duration after opening HMM silage by easily prepared and supplemented additives.
In the light of the study results, it was seen that the silage additives and their usage levels had a positive effect on dry matter, NH3-N, LA production, WSC amount, LAB, and yeast counts. In addition to these results, it was observed that the storage temperature also had a significant effect on pH, NH3-N, LA production, WSC, the numbers of LAB, and yeast. The additive usage (%) and their effects on aerobic stability parameters were not in line with the study. Differences in this regard may be due to the chemical, physical, and microbiological properties of the beginning material.
When the thermal camera images were evaluated, the temperature differences between the silage and storage environment might be able to become more visible. Due to the increase in temperature differences at room conditions on the fourth day of aerobic stability, deterioration was observed in HMM silage. The similarity and relation between chemical analysis and microbial composition results are also noteworthy. The results support that the infrared thermography method might be an effective tool in the early detection of silage deterioration in laboratory conditions, by determining temperature differences and heat spots in silage.
It was concluded that supplementing different doses of apple vinegar and sodium diacetate to HMM silages after silo opening improved aerobic stability. However, further studies were needed to determine effective dosages of apple vinegar on field conditions. and also requires support with in vivo studies for feed palatability.