Study of the influence of exogenous fibrolytic enzyme additive on chemical composition, fermentation characteristics, and nutritional value of brewer’s spent grain

Abstract This study explores the influence of different doses of two exogenous fibrolytic enzyme (EFE) additives (liquid (EFE1: 1, 2, and 4 μΙ/g DM (dry matter)) and powder (EFE2: 1, 2, and 4 mg/g DM)) on the chemical composition, fermentation characteristics, and nutritional value of brewer's spent grain (BSG). The results indicate that EFE1 at low doses does not affect the chemical composition, fermentation characteristics, and the nutritional value of BSG. The medium dose EFE1 decreases the fiber compound but increases the nonfiber carbohydrates (NFC) and soluble dry matter. Also, this dose modified the fermentation of BSG by increasing the amount of gas and its fermentation rate and decreasing the time between the inoculation and start of fermentation. Therefore, it increases the digestibility, metabolizable energy, net energy‐lactation (NEL), total volatile fatty acids, and the microbial crude protein production of BSG. The high dose of EFE1 decreases the fiber compound and increases the nonfiber carbohydrates and soluble dry matter; however, it also decreases the potential of gas production and does not affect the nutritional value of BSG. For EFE2, all the doses do not modify the chemical composition, fermentation characteristics, and the nutritional value of BSG. These results suggest that the effectiveness of EFE varied, depending on the type of EFE and dose. Increase in the nutritional value of BSG by EFE1 at the medium dose can encourage breeders to use these wastes as feed at low cost in cow nutrition.

problems (Lazarevich & Lesnov, 2010). This solid by-product is a mixture of insoluble malted seed and seed coat-pericarp husk-of barley grain (Lynch et al., 2016). Fibers and proteins compounds are the principal compound of this agro-industrial wastes (Lynch et al., 2016;Mussatto et al., 2006). Due to their relatively low price, approximately 39 USD•per ton, BSG is used for as nutrition for ruminants ((Lynch et al., 2016). It has been found to increases milk yield, without affecting animal fertility (Mussatto et al., 2006) and also provides a wide variety of amino acids that are essential in the diet of cows (Lynch et al., 2016). Although the capacity of ruminants to degrade fibrous feed by the rumen microbes is inherent, this digestion is partial in most cases as only 10%-35% of gross energy is used as net energy (Varga & Kolver, 1997). To ameliorate the nutritional value of fiber feed, several approaches had been used, such as the chemical additive and biological additive. The biological treatments are more effective than the chemical treatments due to their greater substrate specificity and their lower pollution effects (Misra et al., 2006). Several studies show that the use of EFE enhanced digestibility of fibrous feed (Abid et al., , 2020, growth performance, and milk production and economic returns in the dairy industry (Abid et al., 2020;Lunsin et al., 2021;Mohamed et al., 2013).
The aim of this work is to explore the influence of different doses of two EFEs on the chemical composition, fermentation characteristics, and nutritional value of BSG.

| Sampling and treatment
Fresh BSG was collected from the beer industry located in Tunis (North of Tunisia). BSG was treated with two exogenous fibrolytic enzymes (EFEs) (EFE 1 : 1, 2, and 4 μΙ/g DM (dry matter) and EFE 2 : 1, 2, and 4 mg/g DM) at 26°C and for 12 h before the in vitro fermentation. EFE 1 is a liquid mixture (50:50) of Cellulase Plus and Xylanase Plus (Dyadic International Inc. Jupiter, Florida) from T. longibrachiatum. The EFE 2 is a powder preparation (MAXFIBER−I ® , Shaumann GmbH, Wahlstedt, Germany) from A. niger, A. tubingensis, A. orzyae, A. sojae, and N. intermedia. These enzyme activities were measured at a pH of 6.6 and a temperature of 39°C, which imitate a cow rumen environment. The endoglucanase and exoglycanase were analyzed using cellulose and carboxymethylcellulose sodium salt as the substrate, conferring to the methods defined by Wood and Bhat (1988).
Xylanase activity was tested using oat spelt xylan as the substrate according to the methods of Bailey et al., 1992. The enzyme activity of EFE1 and EFE2 is noted in Table 1.

| Chemical analyses
Samples of BSG untreated and treated with the appropriate EFE and in various doses were oven-dried to constant weight aimed to determine dry matter (DM) content and then were ground to 1 mm (Cyclotec 1093 Sample Mill;Tecator, Höganäs, Sweden). Before that, the sample was examined to analyze crude protein (CP), ether extracts (EE), and ash conferring to the procedures described by the

| In vitro ruminal fermentation
Rumen fluid for fermentation studies was collected from two Holstein cows (650 ± 20 kg) fed twice daily with a diet of 8 kg of grass hay and 2 kg of concentrate. The rumen content was filtered through four layers of cheesecloth, mixed with a buffer solution (1:2 v/v), and held under a continuous flow of CO 2 (Menke & Steingass, 1988).
Samples of 200 mg DM of BSG (untreated and treated with the appropriate EFE and doses) and 30 ml of the incubation inoculum were incubated in 120-ml volume serum bottles. Blank samples (negative controls) were used to remove for gas production from the fermentation of residues. The bottles were instantly closed with rubber stoppers and placed in a shaking water bath at 39°C for 96 hr. This research was realized at three runs and three repeats per run. Gas pressure was recorded at 2, 4, 6, 8, 12, 24, 48, 72, and 96 hr by a pressure transducer related to a visual display transducer.
Kinetic gas production was estimated according to the model of where GP is the cumulative gas produced at the time t (ml/g DM), t is the incubation time (h), B is the potential of gas production (ml/g DM), C is the rate of gas production (ml/h), and Lag is time between inoculation and commencement of gas production (h).
The organic matter degradability (OMD), metabolizable energy (ME) values, and net energy-lactation (NE L ) were calculated with Equations 3, 4, and 5, obtained from the work of Menke and Steingass (1988). The total volatile fatty acids (VFA) were estimated with Equation 6, which was referred from Getachew et al. (1998): where OMD is organic matter degradability in %, ME is the metabo- where PF 24 is the partitioning factor of incubation at 24 hr, GP is the net gas production in milliliters (ml) from 1 g of DM at 24 hr of fermentation, and aDMD is the amount of dry matter digestibility in grams at the end of incubation.
where MCP is the microbial crude protein in mg/g dry matter, GP is the net gas production (ml) from 200 mg of DM of substrate at 24 hr of fermentation, aDMD is the amount of dry matter digestibility in g at the end of incubation, and the 2.2 mg/ml is a stoichiometric factor (Blümmel et al., 1997). where μ is the overall mean, D i is the effect of the dose (i = 0, 1, 2, and 4), EFE j is the effect of the EFE (j = 1, 2), (D × EFE) ij is the interaction between the dose and the EFE, and E jik is the error term.

| Statistical analysis
The orthogonal contrasts were performed to study the linear and quadratic properties of doses for each EFE. The difference between the mean was evaluated by using Duncan's tests with p value<0.05 (Duncan, 1955).

| RE SULTS
The influence of EFF on the chemical composition of BSG is presented in  (Table 3). Only the medium dose of EFE1 modified the kinetics (improved B (potential of gas production (ml/g DM) and C (rate of gas production (ml/h), decreased lag) and improved ME,

| DISCUSS IONS
The chemical compositions of BSG were relatively similar for the chemical composition of BSG in the review realized by Lynch et al. (2016). This waste was characterized by high CP (>28%), which was corrected protein-deficiency ruminants' ratio, high NDF compound (>40%), and fat content (>9%). The high fat content may decrease the digestibility of carbohydrates and limit the connection of cellulosic bacteria to feed particles (Clinquart et al., 1995).
The effectiveness of the EFE depends on the type of EFE preparation. Indeed, EFE 2 did not have any significant impact on the chemical composition, parameters of kinetics of fermentation, and nutritional value of BSG. The lack of effect of EFE2 can also be explained by the fact that the enzyme activity of this preparation was incompatible with the substrate used, or/and the doses used are not efficient, or/ and the form of this enzyme (powder) was not very active. According to Beauchemin et al. (2004) and Beauchemin and Holtshausen (2010) water services, the diffusion of enzymes is vital for the hydrolysis of of stay of feed in the rumen and induce greater dry matter intakes.
Also, it decreases the time between inoculation and the beginning of fermentation from 1.01 to 0.12 h. A similar result was found by Yang et al. (1999) and Wang et al. (2001). On the other hand, this additive at medium dose increased the degradability of organic matter by 11%. A similar result was found by  on almond hull. Moreover, this treatment at medium dose increases the ME from 6.5 to 7.2 MJ/kg DM. Therefore, this treatment makes the ME of this by-product acceptable for feeding cattle (ME >MJ/kg DM) (NRC, 2001). Besides, it increases the net energy-lactation (NE L ) by 12%. In vivo study confirmed that EFE improves milk production (Lunsin et al., 2021;Mohamed et al., 2013). Also, at this dose, the EFE1 increased in microbial production of crude protein by 7%. This effect may be due to a better use of nutrients by rumen microbes (Getachew et al., 2004), and harmonization between ME and CP in ruminal fermentation (Kaur et al., 2009). This effect is similar to the result of Salem et al. (2015) who noted an increase of microbial crude protein production of corn silage and concentrate supplemented TA B L E 2 Influence of exogenous fibrolytic enzymes (EFEs) on the chemical composition of Brewer's spent grain (BSG) after preincubation (% dry matter) with EFE. VFA are the principal source of energy in ruminants and they reflect the metabolic activity in the rumen (Lee et al., 2018). In this study, EFE1 at medium dose increases the production of volatile fatty acids by 21%. A similar effect was demonstrated in vitro (Sujani et al., 2015) and in vivo (Arriola et al., 2011). The high dose of EFE1 decreases NDF and NDF contents, increases NFC and SDM of BSG.
TA B L E 3 Effect of exogenous fibrolytic enzymes' (EFEs') treatment on parameters of kinetics of fermentation and nutritional value of brewer's spent grain (BSG) Abbreviations: B, potential of gas production (ml/g DM); C, rate of gas production (ml/h); DMD, dry matter degradability (%); Lag, time between inoculation and commencement of gas production (h); MCP, microbial crude protein (mg/g DM); ME, metabolizable energy value (MJ/kg DM); NE L , net energy-lactation (MJ/kg DM); NS, not specified; OMD, organic matter degradability (%); PF24, partitioning factor of incubation at 24 hr (mg substrate truly degraded/mL gas);SEM, standard error of the mean; VFA, ruminal total volatile fatty acids (mmol/200 mg DM). a,b,c Means in the same row with different superscripts differed (p < .05).
However, it decreases the potential of gas production. The inhibitory effect of EFE on high dose has also been proven in vivo (Lunsin et al., 2021) and in vitro b). This phenomenon might be explained by the fact that excessive doses of enzymes mask the surface of feed particles, which reduce the microorganisms' adhesion to the substrate and subsequent fermentation ). Nsereko et al. (2000) assumed that the extreme doses of this additive can liberate sugar, which remains linked to the fiber, which will possibly trap the places of the action of the enzyme.

| CON CLUS ION
These results showed clearly that the mode of action of EFE varies depending on the type of EFE, dose of EFE, and their interactions, which highlights the importance of determining the dose compatibility of each EFE product. In fact, only the EFE 1 at 2 µl/g dry matter hydrolyzed cell-wall components, increased solubilized dry matter, stimulated the in vitro fermentation, and increased the nutritional value of the BSG. At 4 µl/ g dry matter, it hydrolyzes cell-wall components but decreases nutritional value of the BSG.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.