Nutritional and logarithmic fungal count of brewery spent grain in different conservation techniques and brewery factories’

The higher moisture level (70–80%) in wet brewer’s grain (WBG) poses two major difficulties when using it as a feed for different classes of animals. Firstly, transport of WBG is costly. Secondly, the rich polysaccharide and protein content and the high moisture content of WBG make it susceptible to microbial growth and spoilage. Therefore this study was planned to examine the effect of soaking, sun drying and ensiling on nutritional and fungal load dynamics of wet brewers’ grain. Wet brewers’ grain was collected from Meta Abo, Habesha, Dashen and Bedele breweries, Ethiopia. The sample was labeled and kept in a separate sterile bag and stored under − 20 °C. For soaking, 2 kg sample was uniformly treated with salt (3% on dry matter basis), placed in plastic container and covered with a lead for partial aerobic condition. Wet brewers’ grain (2 kg) was exposed to sun drying for the consecutive three days, eight hours per day. The sample (2 kg) with molasses (3% on dry matter basis) was ensiled in a plastic bottle. All samples except sun dried, were subjected to oven drying at 55 °C for 72 h. To evaluate insacco degradability, samples were incubated for 6, 12, 24, 48, 72 and 96 h in nylon bags (6.5 × 14 cm, 50 μm pore size) placed in ventral sac of three cannulated Boran-Friesian steers (550 ± 15 kg live weight). The samples (25 g) were dissolved in 225 ml of peptone water. Potato Dextrose agar medium was injected with 1 ppm per each 100 ml of agar with chloramphenicol and streptomycin to restrict bacterial growth. Plates were incubated aerobically at 28 ± 1 °C for 3 day and growing molds and yeast colonies were directly counted. The sample received from Meta Abo brewery factory had the higher (p < 0.05) acid detergent fiber, lignin and digestible organic matter but comparable dry matter (DM), ash, crude protein (CP) and neutral detergent fiber with other breweries. The minimal loss on DM and other nutrients, lower fungal, yeast and mold colony counts and the higher CP digestion kinetics was observed in ensiling techniques. If supply is not a constraint under local conditions, ensiling can be recommended as the best conservation practice.


Introduction
Fresh brewer's grain is a cheap, non-conventional protein source which is becoming increasingly available to urban and peri-urban dairy farms in Ethiopia. In the year 2016/2017, the annual total wet brewery spent grain (WBG) production from the twelve factories in Ethiopia was estimated at 26,723 tons DM (Kitaw et al. 2018). Wet brewery spent grain is a lignocellulosic material and the major constituents of the byproduct are fiber (hemicellulose and cellulose), protein and lignin (Xiros and Christakopoulos 2012).
It is a heterogeneous substance particularly with respect to inter-brewery variation. This is due to a number of factors, such as the cereal variety, type of hops added, the malting and mashing regime, and whether adjuncts were employed during brewing (Steiner et al. 2015). Fresh brewer's grain contains 70-80% water by weight (Kunze 2010). This high moisture level poses two major difficulties when using it as a feed for different classes of animals. Firstly, transport of wet WBG can be costly, this being a particular reason why supply to local farmers as cattle feed has primarily been the main outlet. Secondly, the rich polysaccharide and protein content and the high moisture content of WBG make it susceptible to microbial growth and spoilage, this being identified as a potential problem area which might restrict its successful exploitation as livestock feed. This being the case, improper storage of WBG results in a large loss of dry 1 3 120 Page 2 of 8 matter and nutrients, characterized by an unpleasant odor, and even stimulates mold to produce mycotoxins (Asurmendi et al. 2013). A number of methods includes: acid solutions such as lactic, acetic, formic and benzoic acids have been used, however, use of such chemicals under local context can be at odds with the livestock producers who not only had access to the chemicals but also lack proper knowledge and skill to handle such chemicals. Several physical methods of preservation have also been examined elsewhere, including oven-drying, freeze-drying, freezing and use of superheated steam (Bartolomé et al. 2002). Under local farmers conditions these are not affordable associated to initial procurement costs and costs of energy maintainace. In Ethiopia, farmers traditionally depend on either fresh wet brewery grain (WBG) or treated and soaked with a brine solution (Kitaw et al. 2018). According to same source sun drying and ensiling are also seldom used. The efficiency of these WBG conservation practices, particularly from perspectives of nutritional and fungal load dynamics have not so far been well documented. This study was aimed to investigating nutritional variations of WBG received from different brewery sources and to exploring the best WBG conservation practices based on nutritional merits and fungal load dynamics.

Experimental location, sampling procedures and measurements
The trials were conducted on samples collected from three districts in the Amhara and Oromia Regional states of Ethiopia. Specific locations include Bedele, Debereberehan and Sebeta districts. Bedele is district in south-western Ethiopia, Oromia regional state of Ethiopia. It is located at 8° 27′ 14.74'' N latitude and 36° 21′ 18.82'' E longitude and at an average altitude of 2053 m above sea level (masl). The average temperature is 18.48 °c and the area receives 1850 mm precipitation annually. Debere Birhan is district in North Shewa zone, Amhara regional state of Ethiopia. It is located at 9° 40′ 35.97'' N latitude and 39°31′58.79''E longitude and at an average altitude of 920 m above sea level (masl).The average temperature is 13 °c. The district has two breweries: Dashen and Habesha breweries factory. Sebeta is district in the special Oromia zone of Oromia regional state of Ethiopia. It is located at 8° 54′ 40.45'' N latitude and 38° 37′ 36.35'' E longitude and at an average altitude of 1073 m above sea level (masl).The average temperature is 17.4 °c. The two experiments were conducted at Holetta agricultural research center in animal nutrition research and dairy microbiology research laboratories. For the first experiment, about a kilogram of fresh samples (two days' sampling) were collected from Meta Abo, Dashen, Bedele and Habesha breweries factory. The collected samples were stored in an ice box. In each day about 500 g of pooled fresh sample was taken from three batches. The sample was labeled and kept in a separate sterile bag and stored under − 20 °C temperatures. In the second laboratory trial, three WBG conservation practices including soaking, sun drying and ensiling were compared to a control, freeze-dried sample. After sub-sampling 40 kg of fresh sample, 2 kg fresh WBG sample in five replications was immediately subjected to frozen inside the freeze dry system (Lyph Lock 4.5 L (77,510-00) between temperatures − 50 °C and − 80 °C, pressure lowered at 1.33 × 10 -3 mbar. Samples were allowed to dry until the moisture in WBG reached 4% by removing the ice in the frozen WBG through the sublimation process (Freeze Dry Systems Catalog 2004). The remaining 30 kg of fresh sample was divided in to three equal parts and subjected to the first local storage treatment option called soaking in which 2 kg fresh WBG sample was uniformly treated with salt at roughly 3% on DM basis and placed in cold water submerged plastic container of 2 kg capacity, finally covered with a lead for partial aerobic storage. For sun drying technique, 2 kg fresh WBG sample was exposed to sun for three days, eight hours per day. Sun dried brewery spent grin was prepared by using a clean sterile plastic sheet with the recommendation Boessinger et al. (2005). Another 2 kg fresh WBG sample was ensiled in a plastic bottle of 2 kg capacity. Molasses was added at the rate of 3% on DM basis to enhance proper fermentation and compaction inside the silo, sealed with caps and left to properly ferment under anaerobic conditions for about 42 days. In each conservation technique, samples were replicated in five times.

In-situ CP degradability characteristics of brewer's grain conserved under different conservation
The crude protein degradability was carried out through Ørskov and McDonald (1979) procedure. Three cannulated Boran-Friesian steers (550 ± 15 kg live weight) were used to measure the degradability of the feed. The steers were fed with maintenance diet i.e., adlibitum natural pasture hay (6.8% CP) and 2 kg concentrate (20% CP) head −1 day −1 on DM basis. All samples were ground to pass through 2 mm sieve size. Three-gram sample was placed into nylon bags (6.5 × 14 cm, 50 μm pore size) and incubated for 6, 12, 24, 48, 72 and 96 h. All bags were subjected to hand washing using tap water until clean water comes out of the washing. The bags with residues were dried at 55 °C for 72 h in air forced oven, hot weighed and finally the residues recovered for further CP analysis. Ruminal CP degradability characteristics were calculated as the difference of CP in the residues and original samples.

Laboratory analysis
Brewers' spent grain samples from the different brewery factory and conservation practices were dried in a forcedventilation oven (55 °C for 72 h) and ground to pass through 1 mm, Cyclotec sample mill screen (Tecator 1093, Tecator AB, Hoganas, Sweden). All samples and residues after degradability were analyzed for DM, total ash and crude protein (CP) by using AOAC (1990) procedure. Neutral detergent fiber (NDFom-NDF), acid detergent fiber (ADFom-ADF) and lignin (pm-Lignin determined by oxidation of lignin with permanganate) were determined by Van Soest and Robertson (1985) procedure. Tilley and Terry (1963) twostage in-vitro digestibility technique was employed to analyze and calculate the digestible organic matter in the dry matter. Metabolizable energy (ME) was estimated as ME (MJ/kg) = 0.16 × DOMD (dry matter digestibility) (McDonald et al. 2002). Yeasts and molds were counted by pour plating. The samples (25 g) were dissolved in 225 ml of peptone water. Potato Dextrose agar medium was injected with 1 ppm per each 100 ml of agar with chloramphenicol and streptomycin to restrict bacterial growth. Plates were incubated aerobically at 28 ± 1 °C for 3 day and growing molds and yeast colonies were directly counted (MoH 2010).

Statistical analyses
The model used for lab trial was a completely randomized design with five replications: Y ijk = + C i + e ij, Where; = Overall mean; C i = Effect of brewery source (experiment-1) and/ or effect of local storage practices (experiment-2); e ij = Random error.
In situ CP degradability trial was fitted into the exponential models of Ørskov and McDonald (1979) as: P = a + b (1 − e −ct ); where; P = CP disappearance in rumen at time t; a = the washing loss fraction; b = the slowly but potentially degradable fraction; c = the rate at which the "b" fraction degraded (in % h −1 ). Similarly, effective CP degradability was calculated applying the equation of Ørskov and McDonald (1979) as ED = a + b[c / (c + k)], Where; k = the rumen outflow rate (3% per h). Data from all trials were subjected to analysis of variance using the general linear model (GLM) procedures of statistical analysis system, version 9.3 (SAS 2014). Mean separations were made using least significant differences (LSD) analysis at p ≤ 0.05.

Nutritional variations of brewers' grain collected from different breweries
The nutritional composition of brewery spent grain is significantly affected (p < 0.05) by brewery factory and presented in Table 1. The spent grain from Meta Abo and Dashen brewery factory had the greater dry matter (DM) but lower (p < 0.05) neutral detergent fiber (NDF) content than the Habesha D/B and Bedele breweries. Ash content of the brewery spent grains was in the order of Meta Abo = Habesha D/B > Dashen > Bedele; while the CP content was in the order of Meta Abo = Habesha D/B > Dashen > Bedele (p < 0.05). The highest (p < 0.05) acid detergent fiber content of BSG was observed in Habesha brewery factory than the other factory while the lignin content was highest in Habesha and Bedele brewery factories. The digestible organic matter in the dry matter was differ (p < 0.05) among breweries being in the order of Meta Abo > Dashen > Bedele, while the value for Habesha differ only with Meta Abo.

Chemical composition of brewer's grain stored under different conservation practices
The effects of conservation practices on the chemical composition and logarithmic fungal count are presented Table 2. The dry matter content of brewery spent grain (BSG) was in the order of control-freeze-dried sample > ensiled > sundried > soaked, while crude protein content of BSG was in the order of control-freeze-dried sample = ensiled > sundried > soaked (p < 0.05). The values of digestible organic matter were similar between the control and ensiling

Fungal load dynamics of brewery spent gain conserved under different practices
Fungal load dynamics of brewery spent grain was affected by the different conservation practices and presented in Table 3. Yeast count in brewery spent grain was in the order of soaking > sun drying > ensiling = control-freeze-dried sample (p < 0.05). The minimum number of mold count (p < 0.05) was observed in the control group and the maximum number of mold count was also recorded in soaking than the other conservation practices. The total fungal count in brewery spent grain was lowest in the control than the other conservation practices.

Crude protein degradability of brewer's grain conserved under different storage practices
In-situ crude protein (CP) degradability of brewery spent grain stored under different conservation techniques are presented in Table 4 and Fig. 1. The higher (p < 0.05) "a" fraction of CP was obtained in soaking than the other conservation methods. However, the "b" fraction for CP was lower in ensiling than soaking and sun drying but was comparable to the control. The rate at which "b" fraction degraded (c) was greater (p < 0.05) in soaking and was lower in ensiled and sun dried than the other preservation techniques. The brewery spent grain conserved by using the ensiling method had the least (p < 0.05) effective CP degradability (ED) followed by sun drying, control and soaking treatments. Calculated ED in soaking were 10.1, 26.73 and 18.42% higher (p < 0.05) than control-freeze-dried sample, ensiling and sun-dried, respectively. Potential degradability (PD) of crude protein (CP) was higher (p < 0.05) in the soaked brewery spent grain than other preservation methods. As indicted in Fig. 1, at early phase of ruminal incubation period (≤ 48 h.), CP disappearance of the grain in the all treatments were higher, and the rate slowed down afterward. The trend of ruminal CP disappearance of the brewery spent grain at any incubation period was in the order of soaking > controlfreeze-dried sample > sun drying > ensiling.

Nutritional variations of brewers' grain collected from different breweries
All chemical composition and in vitro digestibility with exception of some nutritional parameters of wet brewery grain from the present study were found within the ranges of a recent finding (Heuzé et al. 2017). However, slight deviations were noted against NRC (2001) for all measured parameters. The acid detergent fiber and neutral detergent fiber components of wet brewery grain in this study were differed with the report of Westendorf and Wohlt (2002) and dry matter was also different with the report of Senthilkumar et al. (2010). The reasons for this variations maybe related with a variety of factors that include: grain and/or varietal difference among the malt grain used as foundation grain, harvesting time and the conditions under which it was cultivated; the conditions used for malting and mashing and the amount and type of the adjuncts added in mixture with the barley malt during the process of wort production. In addition, period of fermentation, processing techniques and analytical procedures followed may be partly responsible for the observed variations.
In agreement to the present finding Waters et al. (2012) found nutritional variations among wet brewery grain (WBG) derived from brewing processes without addition of adjuncts (i.e. using 100% barley malt), but the former used Brazilian barley malt while the latter used barley malt from Ireland. Differences can also be observed when comparing the results of these authors with those reported by Meneses et al. (2013) who used WBG derived from a process using barley malt with adjuncts obtained from two different Portuguese breweries. The variations observed in the results of these authors suggest that the differences in the source of malt grain and the brewing process conditions affected the composition of the residual WBG material. In fact, the conditions used for the brewing process (i.e., heat applied during the malting and mashing process, and the type of procedure followed for starch extraction during the wort filtration process etc.) were not reported in any of the studies reported and this is probably another factor with significant influence on the results indicated in Table 2.

Chemical composition of brewer's grain stored under different conservation practices
Soaking is the most prominently used wet brewery grain conservation practice under on-farm conditions despite extensive losses in feed and microbial quality compared to the other storage methods evaluated in the current study. Sodium chloride is reported to have good anti-microbial property and often considered as "fermentation inhibitor" in feed preservation process (McDonald et al. 1991). However, sufficient scientific literature was not found to substantiate this with the result obtained from the soaking method in the present study. In line to this, the level of salt used in the soaking process and the storage duration that optimizes proper storage of fresh wet brewery grain need further research. Lower dry matter (DM) loss for the ensiled sample was a good sign of the absence of any significant degradation of nutrients during the ensiling process as compared to the other two local WBG conservation practices. Higher DM loss in the soaked and sun-dried brewery grain compared to the control (freeze-dried) and ensiled brewery grain could probably be associated to the relatively higher fungal colony count that arise from the slow sun drying process mainly attributed to the lower solar energy and to the frequent opening of the storage container for feed removal that often induces aerobic deterioration in the soaked sample.
In general, during sun drying and soaking process, residual soluble carbohydrates in brewery grain possibly have been converted into ethanol, CO 2 and water in the presence of large colonies of yeast and mold (McDonald et al. 1991) thereby leading to excessive loss of dry matter (DM) and other nutrients from the WBG. Moreover, in agreement to same author, the lower CP observed for brewery grain conserved using these two preservation practices could probably be associated to the extensive proteolysis that might have occurred during the open air storage conditions. The greater values of neutral detergent fiber, acid detergent fiber and permanganate lignin across the preservation practices except ensiling indicated that there were undesirable microbial activities which can be witnessed from the larger fungal colony counts in the present study, as soluble nutrients have been degraded, the proportion of fiber components tended to have proportionally increased. Likewise, lower values of digestible organic matter in sun-dried and soaked sample, can be possibly linked to the loss in DM and other soluble nutrients and a sharp increase in cell wall fractions. In a related study Baskett et al. (2009) also reported losses in DM equivalent to 8.6% and 9.6% for wet distiller grains stored in aerobic and anaerobic storage conditions in bunker silos.

Fungal load dynamics of brewery spent gain conserved under different practices
The higher fungal count in the soaked brewery spent grain (WBG) compared to the other two conservation practices and the control can be speculated to the aerobic exposure of WBG during feed removal for routine feeding on the one hand and the proportion of salt to water that might not have optimized proper fermentation partly due to lack of research recommendations for the soaking technique. Longer storage durations may also hold responsible for the larger fungal contamination seen in the soaking process. Nutrient loss from the sun drying brewery grain in the present study was also relatively higher since drying was done under a low temperature that increased risks of mold growth and mycotoxin production (Chulze 2010). According to Woolford (1990) and Dairy one (2017) the presence of yeasts and mold in a stored feed greater than 5.00 CFU/g DM, is considered undesirable leading to higher losses of DM and other essential nutrients. Hence, based on these recommendations both yeast and mold colony counts from the present study were considerably lower implying such feeds can safely be fed to dairy cattle. The average yeast and mold forming colony units (log CFU/g DM) obtained from the current study were slightly higher than the values of 2.7 and 1.8 which was reported earlier in aerobically and anaerobically conserved WBG using organic acids (lactic acid strains) (Marston et al. 2009), the variation being attributed to the strong inhibitory effect of the organic acid used in the latter case.

Crude protein degradability of brewer's grain conserved under different storage practices
The result of degradability constants excluding water-soluble crude protein fraction from the current study is in line with the previous report (NRC 2001;Kazemi et al. 2014;Heuzé et al. 2017). The variation of the "a" fraction in crude protein degradability can related with differences in the type of WBG conservational practices employed and differences in the type of washing methods (hand Vs machine). In agreement to the present finding, Gao et al. (2015) noted low repeatability for rapidly soluble ruminal and post-ruminal nitrogen and amino acids fractions of three supplemental protein sources. The ''c'' and ''ED'' values of CP fraction varied greatly among the conservation practices and the control sample. This variation could be related in the differences modes of dry, fresh or ensilage (Nocek 1985). In agreement to the present finding, Kamalak et al. (2005) noted that in situ DM disappearance after 48 and 96 h to be negatively correlated with neutral detergent fiber of the WBG stored under different preservation practices. The poorest 'ED' and rate constant 'c' values in CP degradability of the ensiled wet brewery grain than the control, soaked and dried grain in the present study provided a clear evidence of ensiling have relatively lower rumen fermentable protein. Similar observations have been reported earlier by NRC (2001). The estimated rumen undegradable protein (RUP) in this study was 53.8, 43.7, 58.2 and 56.4% in control, soaked, ensiled and sun-dried samples, respectively. This estimated RUP percentage for the fresh and the remaining local storage techniques from the present study was slightly higher than the mean values reported by NRC (1989) for cottonseed meal (41%) and sunflower meal (26%) but was lower than the content of RUP for dried distillers' grain (67.1%) (Kelzer et al. 2010). Lower potential CP degradability (a + b) values was found for the control, ensiled and sun-dried samples compared to WBG preserved using the soaking method. Similar results were noted from related research work by Promkot et al. (2007). In-situ degradability results have been affected by different factors such as feed particle and sample size, bag material pore size; test animal diet and washing procedures (Nocek 1985;Gao et al. 2015).

Conclusions
Brewery spent grain from Meta Abo brewery factory had the higher nutritional values than the remaining beer factories. The minimal loss of dry matter and other nutrients, lower fungal, yeast and mold colony counts and the higher crude protein digestion kinetics was observed in ensiling techniques. If supply is not a constraint under local conditions, ensiling can be recommended as a best wet brewery spent grain conservation practice.