The Fermentation Process Improves the Nutritional Value of Rapeseed Cake for Turkeys—Effects on Performance, Gut Bacterial Population and Its Fermentative Activity

Simple Summary Rapeseed cake (RC) could be valuable raw material in turkey diets, but its wide use is limited by the presence of anti-nutritional factors that are detrimental to gut function. The fermentation process contributes to the degradation of over 80% of carbohydrates, 30% of lignin, and 45% of total glucosinolates in RC, which are harmful to birds. Our research showed that fermentation can improve the nutritional value of RC, enabling good performance and maintainenance of a healthy gut in birds. Therefore, FRC appears to be highly promising in commercial turkey nutrition. Abstract This experiment investigated the potential inclusion of fermented rapeseed cake (FRC) in turkey diets. The turkeys received diets either not supplemented (C) or supplemented with raw rapeseed cake (RRC) or FRC at 150 g/kg diet. In comparison with RRC, turkeys receiving FRC achieved significantly higher final BW comparable with that noted in the control group. The dietary inclusion of FRC increased the concentrations of propionic and valeric acid in the cecal digesta compared with the control group, and increased the proportion of butyric acid in SCFA profile compared with RRC group. The activities of glycolytic bacterial enzymes in the cecal digesta, were lowest in turkeys fed FRC. Experimental diets did not cause a shift in the relative abundances of the main bacterial phyla or orders in the cecal digesta. FRC increased the abundance of Bacteroidaceae at the family level, but decreased the abundance of Lactobacillus at the genus level compared with birds fed RRC. In conclusion, the dietary inclusion of FRC at 150 g/kg did not compromise bird performance, did not excessively stimulate bacterial activity, and did not cause shifts in the bacterial composition in the cecum. Actually, FCR exerted several beneficial effects that contributed to maintaining gut health in turkeys, which points to its advantage over RRC.


Introduction
Rapeseed cake (RC), a by-product of oil production, could be a valuable component of poultry diets because it contains up to 400 g/kg crude protein (CP) and high concentrations of metabolizable energy [1,2]. Unfortunately, wide use of RC is limited by the presence of non-starch polysaccharides (NSP) and other anti-nutritional factors such as glucosinolates, phytic acid, sinapine, and tannins [3].

Animals and Experimental Design
The experimental material consisted of one-day-old female Hybrid Converter turkeys raised until 112 days of age. Turkeys were raised in pens on litter in a building with a controlled environment. Every pen was equipped with an automatic feeder and a bell-type drinker, and both water and feed were provided ad libitum. The temperature and lighting programs were consistent with the recommendations of Hybrid Turkeys [19].
The experiment was carried out on 1350 turkeys assigned to three experimental groups of 450 birds each (9 replications of 50 individuals each), which differed in the source of feed protein. In the control group, soybean meal was the main source of dietary protein, whereas the experimental groups were fed diets containing 15% of RRC or FRC. All diets were isocaloric and isonitrogenous, and contained similar amounts of major amino acids (including lysine, methionine with cysteine and threonine), minerals (including calcium and available phosphorus), and vitamins. The nutritional value of diets was consistent with the nutrient requirements of turkeys [19]. The compositions of control and experimental diets prepared in successive 4-week feeding periods are shown in Table 1. Table 1. Composition and calculated analysis of control (C) and experimental diets containing raw rapeseed cake (RRC) or fermented rapeseed cake (FRC) fed during four feeding periods to turkeys aged 1-112 days, in g/kg as-fed basis (unless indicated otherwise). The diets for the first period (days 1 to 28) were offered in crumble form, and the diets offered in the subsequent periods (days 29-112) were fed in pelleted form. At the end of each 4-week period, the body weight (BW) of turkeys, feed intake (FI) and mortality rates were recorded, and each pen of 50 birds was considered as an experimental unit. Feed conversion ratio (FCR) was calculated for each group based on BW gain and feed consumption.

Sample Collection
After 112 days of feeding, nine birds representing the average BW of each group were slaughtered by cervical dislocation to collect the test material. The ceacums were removed, emptied, and digesta was homogenized. Subsequently, samples were collected and used for the analysis of SCFAs and bacterial enzymes activity. The remaining portion of cecal digesta was transferred to test tubes and stored at −70 • C until needed.

Chemical Analysis
For chemical analyses, samples of RC were ground to pass through a 0.5-mm sieve. The samples were analyzed in duplicate for the content of DM, CP, and crude fiber (CF) according to AOAC [21] methods 934.01, 976.05 and 978.10, respectively. Gross energy (GE) was determined with an adiabatic bomb calorimeter (KL 12 Mn, Precyzja-Bit PPHU, Poland) standardized with benzoic acid. Phytate-phosphorus was determined as described by Haugh and Lantzsch [22]. Non-starch polysaccharides (NSP) were determined by gas-liquid chromatography (constituent neutral sugars) using an SP-2340 column and a Varian CP3380 gas chromatograph (Varian Inc., Palo Alto, CA, USA), and by colorimetry (uronic acids) using a Biochrom Ultrospec 50 (Biochrom Ltd., Cambridge, UK), according to the procedure described by Englyst andCummings (1984, 1988) with modifications [23]. Uronic acids were determined as described by Scott [24]. Sugars (glucose-GLU, fructose, sucrose, raffinose, and stachyose) were determined as described by Slominski et al. [25]. Glucosinolates were determined by gas-liquid chromatography as described by Slominski and Campbell [26].
SCFAs were analyzed by gas chromatography (Shimadzu GC-2010, Kyoto, Japan) equipped with a capillary column (SGE, BP21, 30 m × 0.53 mm, SGE Europe Ltd., Kiln Farm Milton Keynes, UK). Samples of digesta (0.2 g) were mixed with 0.2 mL of formic acid, diluted with deionized water and centrifuged at 7211 g for 10 min. The supernatant was loaded onto a capillary column (SGEBP21, 30 m × 0.53 mm) using an on-column injector.
The activities of bacterial enzymes (α-and β-glucosidase, αand β-galactosidase, β-glucuronidase, α-arabinopyranosidase, α-arabinofuranosidase β-xylosidase, β-cellobiosidase) released into the cecal environment were measured as the rate of p-or o-nitrophenol release from their respective nitrophenylglucosides [27]. The reaction mixture contained 0.3 mL of a substrate solution (5 mM) and 0.2 mL of a 1:10 (v/v) dilution of the cecal digesta sample in 100 mM phosphate buffer (pH 7.0) after centrifugation at 7211 g for 15 min. Incubation was carried out at 39 • C, and p-nitrophenol was quantified at 400 nm and at 420 nm (o-nitrophenol concentration) after the addition of 2.5 mL of 0.25 M cold sodium carbonate. Enzyme activity was expressed as µmol of the product formed per h per g of digesta. For sequencing the individual libraries, they were diluted to 4 nM, denatured with 10 mM Tris pH 8.5 and spiked with 20% (v/v) of PhiX. An aliquot of 5 µL of diluted DNA was mixed to prepare pooled libraries for the MiSeq (Illumina, San Diego, CA, USA) run. > 100,000 reads were performed per sample. The sequencing data were clustered into operational taxonomic units (OTUs), and were classified at several taxonomic levels, kingdom, phylum, class, order, family, genus and species. The Greengenes database was used for metagenomic analysis.

Statistical Analysis
For a statistical analysis of performance parameters, a single pen (n = 9) was considered as a replicate experimental unit. For analyses of the physiological and microbiological parameters of the GIT, individual birds were considered as experimental units. All analyses were performed on 27 birds representing 9 replicates from each of the 3 experimental groups. One-way analysis of variance (ANOVA) was performed with the use of Statistica 10.0 software (StatSoft, Krakow, Poland). When a significant treatment effect was noted, the post-hoc Tukey test was used to determine differences between treatment groups. Data were presented as means±SEM, and the value of p < 0.05 was considered statistically significant. The post-hoc Tukey HSD test did not reveal significant differences between group means with regard to the BW of birds and butyric acid, although p-value reached 0.04. Therefore, Duncan's test was used, and the observed significant differences were considered as tendencies. The significance of differences between livability data was determined by the Kruskal-Wallis test by ranks (non-parametric ANOVA on ranks).

Results
Fermentation increased the content of DM, CP, and CF, and decreased the content of anti-nutritional factors in RC (Table 2). In comparison with RRC, FRC had nearly ten-fold lower glucosinolates content, nearly two-fold lower carbohydrates content and nearly twenty-fold lower content of phytate-phosphorus. Fermentation had no effect on NSP concentrations in RC. The inclusion of 15% of RRC or FRC in turkey diets had no influence on the growth performance parameters of birds, including DFI, FCR, and livability (Table 3). However, the average final BW of turkeys receiving RRC tended to decrease in comparison with the remaining groups. Fermentation increased the final BW of turkeys, which was approximately 1.3% higher in the FRC group than in the RRC group, and comparable with that in the control group (p = 0.043). Table 3. Growth performance of turkeys fed a control diet (C) and diets containing raw rapeseed cake (RRC) or fermented rapeseed cake (FRC). x,y means within a row with different superscripts were considered as a near-significant trend; DFI-daily feed intake; BW-body weight; FCR-feed conversion ratio.

Effect of Diets Containing RRC or FRC on SCFA Concentrations and the Activities of Bacterial Enzymes in the Cecal Digesta
The inclusion of RRC or FRC in turkey diets slightly affected SCFA concentrations in the cecal digesta (Table 4). Neither total SCFA concentrations nor the total pool or the total concentrations of putrefactive SCFAs differed between the treatments (p > 0.05). In the SCFA profile, acetic acid predominated, followed by butyric acid and propionic acid. The proportion of butyric acid in the SCFA profile was higher in birds fed FRC than in those fed RRC (p = 0.047). The concentrations of propionic acid and valeric acid were higher in FRC-fed birds compared with the control treatment (p = 0.028 and p = 0.048, respectively).
An analysis of the activities of bacterial enzymes in the cecal digesta (Table 4) indicated that only α-glucosidase and β-galactosidase were not affected by the dietary treatments (p > 0.05). More specifically, the activities of β-glucosidase (p = 0.026), α-galactosidase (p = 0.024), α-arabinopyranosidase (p < 0.001), α-arabinofuranosidase (p < 0.001), β-xylosidase (p < 0.001), and β-celobiosidase (p < 0.001) were lower in birds fed diets with FRC vs. RRC. In comparison with the control treatment, the administration of RRC or FRC resulted in lower activity of β-glucuronidase in the cecal digesta (p < 0.001). Table 4. Concentrations of short-chain fatty acids (SCFAs) and activities of selected bacterial enzymes in the cecal digesta of turkeys fed a control diet (C) and diets containing raw rapeseed cake (RRC) or fermented rapeseed cake (FRC) from 1 to 112 days of age 1 .

Effect of Diets Containing RRC or FRC on the Microbial Composition in the Cecal Digesta
A metagenomic analysis of the cecal digesta revealed that the predominant bacterial phyla were Firmicutes, Bacteroidetes, and Proteobacteria (Table 5). Their relative abundances were as follows: Firmicutes-around 50% (49.42201003350.33%), Bacteroidetes-around 20% (19.15-28.64%), Proteobacteria-around 5% (4.68-5.33%), and Cyanobacteria-around 2.5% (1.35-3.22%) of OTUs. Unclassified bacterial 16SrRNA sequences accounted for 6.44 to 7.04%. The inclusion of RRC or FRC in turkey diets did not affect the bacterial populations (OTUs) at the phylum or order levels. The dietary treatments had a minor effect on the relative abundances of bacteria at the family and genus levels. Fermented rapeseed cake caused a significant increase in the relative abundance of Bacteroidaceae (p = 0.036 vs. C). The relative abundance of Lactobacillus decreased significantly in the FRC group, and increased in the RRC group (p = 0.004 vs. C). Table 5. Relative abundance of bacterial communities in the cecal digesta of turkeys fed a control diet (C) and diets containing raw rapeseed cake (RRC) or fermented rapeseed cake (FRC) 1 .

Discussions
In the present study, RC fermentation marginally affected its chemical composition, including the content of CP, CF, and GE. These results corroborate our previous findings [28]. However, the fermentation process decreased the concentrations of anti-nutritional factors and indigestible substances including glucosinolates and phytate phosphorus, which are detrimental to poultry. Therefore, our data indicate that RC fermentation may be beneficial since the predictable value of raw materials after processing is one of the key determinants of their use as feedstuffs in turkey diets.
The present report demonstrated that diets containing 150 g/kg of FRC were well utilized by birds, as their growth performance was marginally affected in the experimental groups and remained within the limits typical of female hybrid turkeys of the same age [19]. The fermentation process was beneficial because RRC decreased the final BW of turkeys, relative to the control group, whereas this effect was not observed in birds fed FRC. These data are consistent with the findings of Rad-Spice et al. [29] who noted adverse effects of the dietary inclusion of raw rapeseed in the amount of 150 g/kg or higher on the performance of broiler chickens. Therefore, fermentation enables to supplement turkey diets with RC at 150 g/kg without compromising bird performance.
The nutritional value of different feedstuffs is largely determined by their influence on the physiological status of the gut. In the current study, diets containing either RRC or FRC had a significant effect on cecal parameters, as selected SCFAs concentration and activities of bacterial enzymes. Dietary substrates, including NSP and protein, which are not fully digested and absorbed in the upper GIT of birds, may accumulate in the distal part of the gut, thus altering the profile and activity of microbiota, which manifests as changes in SCFA concentrations. Short-chain fatty acids are an important energy source for the intestinal epithelium, and they can also regulate mucin production and intestinal immune responses, which play a key role in the development of microbiota and in the prevention of pathogen colonization [13,30]. In the cecum, which is the main site of fermentation in birds, the digesta is fermented for a longer period of time, and the concentrations of SCFAs are higher than in the ileum. The SCFAs produced in the cecum also play an important role in maintaining bird health [31]. In our study, diets containing RC did not induce excessive fermentation in the cecum since total SCFA concentrations were similar in turkeys fed experimental and control diets. Acetic, butyric and propionic acids are the most abundant SCFAs in the cecal digesta of birds fed different diets. In the current experiment, the above acids were also present in the highest concentrations in the cecal digesta of turkeys, which indicates that experimental diets containing RC did not disturb cecal fermentation [4]. Interestingly, our findings revealed that the replacement of RRC with FRC in turkey diets caused a beneficial shift in the concentration of propionic acid and in the proportion of butyric acid in the SCFA profile. The beneficial metabolic exerted by propionic acid is suggested to increase insulin sensitivity and glucose tolerance as sensed in the portal vein [32], whereas butyric acid is the preferred energy source for enterocytes, it exerts bacteriostatic effects on selected enteric bacteria, and may reduce the abundance of Enterobacteriaceae including Salmonella [14]. Furthermore, butyrate can also affect the hormonal and nervous systems, and therefore affect host physiology, including protection against colorectal cancer, inflammation, and appetite regulation [33]. Branched SCFAs such as isobutyrate and isovalerate are the end-products of protein fermentation, whereas acetic acid, propionic acid, and butyric acid are generated through the fermentation of dietary fibers [34,35]. Thus, in the light of our findings (a minor effect of RC on the SCFA profile and the concentrations of individual fatty acids), the inclusion of RRC and, in particular, FRC in turkey diets did not lead to an excess supply of fermentative protein and fiber substrates. Therefore, RC is unlikely to cause a negative shift in cecal fermentation. The above results indicate that FRC was well tolerated by birds. The present findings, unlike those reported by Mikulski et al. [6], confirm that rapeseed cultivars have been significantly improved in recent years. Therefore, rapeseed products can be included in turkey diets, especially that additional processing such as fermentation can further improve their nutritional value.
The activities of bacterial enzymes in the cecum are indicative of dietary ingredient-host interactions. The physicochemical structure of the substrates that escaped digestion in the upper GIT affects the activities of bacterial enzymes. In the current study, the use of FRC, but not RRC, downregulated microbial activity in the cecal ecosystem; the activities of most enzymes (in particular β-glucuronidase, α-arabinopyranosidase and α-arabinofuranosidase) were lower in birds fed diets with FRC than in those fed the control diet. The decreased activity of β-glucuronidase may be indicative of reduced E. coli and Clostridium populations, and a lower risk of glucuronide hydrolysis in the gut lumen, which generates toxic and carcinogenic substances from nontoxic glycosides [36]. Similarly, the decreased activities of α-arabinopyranosidase, α-arabinofuranosidase and β-xylosidase suggest that there was no shift in the abundance of residing bacteria, which can release arabinose, xylose, arabinose, and galactose from dietary substrates [37].
The microbiome of the intestinal tract plays a fundamental role in gut health by modulating the fermentation of polysaccharides and the metabolism of nitrogen, fatty acids, and lipids. The diversity of gastrointestinal microbiome is believed to have a critical effect on the overall health status of birds. Despite the fact that the interaction between gut microbiota and health status has not been fully elucidated to date, it can be used as a reliable indicator of birds' response to dietary treatment. 16S rRNA next-generation sequencing enables to describe all detected microbial populations in one analysis. The results of metagenomic bacterial analyses do not depend on bacterial culture conditions and stand in opposition to direct DNA-DNA hybridization methods. The metagenomic data of 16S rRNA sequencing provide important information about the genetic material of the analyzed bacteria. In previous studies [38,39] the bacterial phyla of Firmicutes and Bactroidates were most abundant in the ceca of tukeys, which is consistent with our findings. In the present experiment, the above phyla accounted for 53.5% and 24.0%, respectively, of the cecal microbiota in turkeys. Moreover, the experimental diets had no significant influence on the relative abundances of the main phyla of bacteria residing in the ceca. However, they significantly affected the abundance of the family Bacteroidaceae in the ceca, which was greater in birds fed FRC than in those fed RRC, whereas the opposite was noted for the genus Lactobacillus. Since the abundance of Bacteroidaceae in birds fed FRC did not differ significantly from that noted in the control birds, their shift should not be attributed to the adverse effect of FRC inclusion. Regarding Lactobacillus bacteria, which are defined as microorganisms exerting a beneficial effect on the host, their significantly lower abundance in turkeys fed FRC than in those fed RRC could be considered undesirable. According the Józefiak et al. [40], an increase in the counts of Bacteroides and a decrease in the counts of Lactobacilli are negatively correlated with intestinal health. However, it should be noted that in the present study, Lactobacillus abundance was similar in turkeys fed FRC and in control birds, thus suggesting that the experimental diets were unlikely to exert an adverse effect on gut microbiota by causing a shift in the abundance of bacteria residing in the cecum. The significantly higher abundance of Lactobacillus in birds fed RRC compared with the remaining treatments, could result from the fact that more substrates escaped digestion in the upper GIT of turkeys and were able to enter their ceca [41], thus supporting Lactobacillus proliferation. This speculation was also supported by the fact that the performance of birds fed FRC was not compromised relative to the RRC group, indicating that the reduction in Lactobacillus abundance in the FRC treatment was marginal in terms of the gut health status. These findings, as well as the responses of SCFAs and bacterial enzymes reported above, appear to confirm that FRC exerted a negligible but generally beneficial effect on gut physiology in turkeys.

Conclusions
The results of this study indicate that FRC can be included in turkey diets at 150 g/kg without compromising the growth performance of turkeys and maintaining a healthy gut. Our findings also suggest that in comparison with unprocessed RC, the fermentation process improved the nutritional value of RC and exerted beneficial effects on SCFA concentrations and the activities of bacterial glycolytic enzymes. Therefore, fermentation represents an interesting approach to increasing the efficacy of RC in commercial turkey nutrition.